Datasets:

Jingmei

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Pandemic_PMC

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Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3036466/

The Evaluation of Lower-Extremity Ulcers

ABSTRACT Lower-limb ulceration is prevalent in Western countries. There are many different types of ulcers with several causes. The most prevalent are those due to vascular disease, of which venous is the most common, accounting for over two-thirds of all types of ulcers. There are also many other causes for ulceration such as malignancy, infections, and skin, drug-induced, and autoimmune diseases. The ulcers have different characteristics, which may be differentiated by the history and clinical examination of the patients. However, objective documentation for the ulcer etiology is necessary prior to instigating treatment. The methods for diagnosing the causes for the ulcers include plethysmography, ultrasound, angiography, computer tomography, magnetic resonance imaging, and skin biopsy. All these tests should be used in conjunction with the clinical presentation of the patient. They should be performed in a cost-effective manner to avoid delays in diagnosis and reduce costs and usage of resources.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453464/

Public-Private Partnership Policy in Primary Health Care: A Scoping Review

Background: Given the challenges of governments to deliver primary health care (PHC), engaging private sector in the form of public-private partnership (PPP) can be effective policy. The aim of present study is to review the experiences of implementing PPP policy in PHC. Methods: This scoping review study was conducted in 2019 using the framework proposed by Arkesy and O'Malley. Required data were collected through search the related keywords in databases, manual search of some journals, websites, and other sources of information and through references check, from January 2000 to May 2019. All studies, which focused on the results of PPP in PHC, and published in English or Persian were included in the study. Results: A total of 108 articles were included in the study. The studies were mostly conducted in low- and middle-income countries. The quantitative studies have demonstrated the success of this policy in improving PHC indicators. Based on the qualitative studies PPP in PHC has many benefits, including access improvement, economic benefits, and service quality enhancement. Conclusions: The present study provides useful information on the experiences of different countries in the field of PPP in PHC that can be used by experts and decision makers to decide whether to engage the private sector in the form of PPP model. Background: Given the challenges of governments to deliver primary health care (PHC), engaging private sector in the form of public-private partnership (PPP) can be effective policy. The aim of present study is to review the experiences of implementing PPP policy in PHC. Methods: This scoping review study was conducted in 2019 using the framework proposed by Arkesy and O'Malley. Required data were collected through search the related keywords in databases, manual search of some journals, websites, and other sources of information and through references check, from January 2000 to May 2019. All studies, which focused on the results of PPP in PHC, and published in English or Persian were included in the study. Results: A total of 108 articles were included in the study. The studies were mostly conducted in low- and middle-income countries. The quantitative studies have demonstrated the success of this policy in improving PHC indicators. Based on the qualitative studies PPP in PHC has many benefits, including access improvement, economic benefits, and service quality enhancement. Conclusions: The present study provides useful information on the experiences of different countries in the field of PPP in PHC that can be used by experts and decision makers to decide whether to engage the private sector in the form of PPP model. Introduction The ultimate goal of health system in each country is to improve the health of people to be able to participate actively in economic and social activities while enjoying health. 1 Undoubtedly, today primary health care (PHC) is the main strategy of countries for achieving this goal. 2 In a 2008 report, the World Health Organization states, "There is no longer any doubt that by changing the previous practice (focusing solely on clinical services) and emphasizing the PHC, the cost-effectiveness of health services can be improved." 3 In fact, to achieve the values of PHC including equity, people-centeredness, and community participation, service delivery structure should be changed and the challenges of PHC, as the gateway for people to enter the health system, must be met. 4 Till now, different approaches to address the health system' challenges are used by countries, of which one of the most effective is public-private partnership (PPP), which as a bilateral partnership and win-win policy, makes use of the capacities of both public and private parties to achieve the goals. 5 - 7 In general, PPP is a mechanism whereby the public sector (government and other governmental entities) in order to provide the infrastructure services (water and wastewater, transportation, health, education, etc) utilizes the capacity of the private sector (cooperatives, private companies, charities, and nongovernmental organizations [NGOs]), including knowledge, experience, and financial resources. In PPP, a contract would conclude between the public and private sector to share the risk, responsibility and benefits, and to synchronize resources and expertise of both sectors in providing infrastructure services. 5 In PPP, the role of government changes from investor, implementer, and beneficiary of infrastructure projects to policy maker, regulator, and supervisor of the quality and quantity of provided services. 8 - 10 The private sector considers the PPP as a viable solution and opportunity for market growth and profit making, which also provide adequate facilities and innovative management for the public sector. 5 , 9 Governments use PPP as an efficient and cost-effective mechanism in implementing their goals and policies. 11 If PPP plans are implemented, insurance companies can increase people's satisfaction at a lower cost and also allow better and more accurate monitoring of the quality of service provision in the private sector. Under such conditions, people are expected to receive more diverse services with more coverage, higher quality, better access, and even lower costs, which will in turn improve their health and satisfaction. 12 Given the importance of PHC to promote the health of the community and due to the problems and shortcomings in this area, the use of private sector capacities and capabilities, which can be realized in the form of PPP, can be effective and useful in this regard. That is why there is a need to examine the performance and achievements of PPP in the field of PHC worldwide to be used in policy making and designing proper structures and patterns in each country, especially in low- and middle-income countries (LMICs). Therefore, the aim of the present study is to study the experiences of implementation PPP policy in PHC through scoping review. Materials and Methods In this study, Arkesy and O'Malley framework was used for scoping review, which include 6 steps of identification of the research question, identification of relevant studies, study selection, data charting, data analysis, and reporting the results and consultation exercise. 13 Step 1: Identification of Relevant Studies The main research question of the present study is "What are the results and achievements of PPP in the provision of PHC in different countries?" which specifically include following questions: In which countries is PPP in the provision of PHC prevalent? How is the time trend of using PPP in the provision of PHC worldwide? In which settings (urban, rural, suburbs, etc) PPP in the provision of PHC is most prevalent? Which models of PPP are used to provide PHC? What are the role and responsibilities of the public and private sectors in the PPP in PHC plans? What are the goals of using PPP in the provision of PHC in different countries? For what tasks and services PPP has been applied in the provision of PHC? What are the achievements of implementing PPP policy in the provision of PHC worldwide? Inclusion and Exclusion Criteria All articles and reports on using PPP in the PHC projects published in Persian or English between 2000 and 2019 were included. Studies written in English or Persian (which could be translated by a member of our team) were included. The articles and studies that report the experience of implementing PPP plans in other health sectors (other than PHC) were excluded. Step 2: Identification of Relevant Studies In this phase, required data were extracted from PubMed, Scopus, Science Direct, Web of Science, and Embase databases. The keywords were searched in different areas of primary health care, including primary health care, tuberculosis, vaccination, and so on. Persian equivalent of the above searches was performed in Persian databases like MagIran and SID. The time frame selected for searching the articles was January 2000 to May 2019 ( Appendix 1 in the Supplementary Material). Table 1 shows the search strategy proposed for PubMed. Table 1. Search Strategy for the PubMed Database. Search number Areas Search string #1 private OR "public private partnership" OR "public private participation" OR "public-private mix" OR "public-private cooperation" OR "mixed system" OR privatiz* OR "public-private coordination" OR "public-private collaboration" OR "contract* out" OR outsource* OR "Servic* contract*" OR "management contract*" OR "lease* contract*" OR "Private Finance Initiative Contract*" OR "concession contract*" OR "divesture contract*". #2 Primary health care "primary health care OR "primary health services" OR PHC OR "basic health care" OR "primary care" #3 Tuberculosis TB OR tuberculosis #4 Vaccination Immunization OR vaccination OR vaccine #5 Maternal and child care maternity OR child* OR maternal OR mother* OR midwifery OR pregnancy #6 Screening Screening #7 Case finding "Case find*" OR "find* case" OR "case detection" OR "detect* case" #8 Health education "Health education" OR "health training" OR "health teaching" OR "health promotion" #9 Mental health "Mental health*" #10 Occupational health "Occupational health*" OR "occupational hygiene" #11 Environmental health "Environmental health*" OR "environmental hygiene" OR "water hygiene" OR "wastewater hygiene" OR "water health" OR "wastewater health" #12 Oral health "Oral health*" OR "tooth health*" OR "dental health*" OR "oral hygiene" OR "tooth hygiene" OR "dental hygiene" #13 Congenital Anomalies "Thalassemia*" OR "hemophilia*" #14 Family medicine "Family physician" OR "family medicine" OR "family doctor" #15 Elderly health "Elderly health*" OR "aging health*" OR "aged health*" OR "older health*" OR "elderly care" OR "aging care" OR "aged care" OR "older care" #16 School health "School health" OR "student health" #17 Surveillance "Surveillance" #18 Diabetes Diabet* #19 Hypertension "Blood pressure" OR "high blood pressure" OR hypertension #20 Asthma "Asthma" #21 Cancer "Cancer" #22 Noncommunicable diseases (general) "Non-communicable diseases" OR "noncommunicable diseases" #23 HIV "HIV" OR "AIDS" OR "Human immunodeficiency virus infection" OR "acquired immune deficiency syndrome" #24 Hepatitis "Hepatitis" #25 STD "Sexually transmit*" OR "STD" OR "STI" #26 Malaria "Malaria" #27 Pediculosis "Pediculosis" OR "lice" OR "phthiriasis" #28 Fecal-oral diseases "Water and food borne" OR "fecal-oral" OR "diarrheal" OR "diarrhea" OR "food poisoning" OR "Water intoxication" OR parasite OR "Gastrointestinal" OR cholera #29 Influenza "Influenza" OR "flu" #30 Communicable diseases (general) "Communicable diseases" #31 Rabies Rabies OR rabid #32 Malta fever "Malta fever" OR brucellosis OR "brucella Infection" OR "brucella fever" #33 Anthrax Anthrax #34 Ebola Ebola #35 Neglected disease "Neglected disease*" #36 #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #28 OR #29 OR #30 OR #31 OR #32 OR #33 OR #34 OR #35 #37 #1 AND #36 To identify and cover of further published articles, after searching databases, selected journals and articles references were manually searched. In addition, Google Scholar, published organizational reports and government documents, websites, and other available information sources were also searched. Step 3: Study Selection The entire process of selection and screening of studies were performed independently by 2 members of the research team. Disagreements have been resolved through discussion in the first instance, otherwise were referred to a third party with more knowledge and experience. At first, the titles of all articles were reviewed, and those that were not consistent with the study objectives were excluded. Subsequently, abstracts and full texts of the articles were studied in order to identify and exclude studies that met exclusion criteria and had poor correlation with study objectives. Given that the structure of PHC delivery varies across countries, to determine whether the provided services are PHC, 2 researchers examined the information provided in each study and determined whether they were PHC or not. Thus, the types of services were determined based on the information provided and the agreement between the researchers. In case of disagreement, the agreement was reached through discussion between the 2 researchers. If no agreement was reached through discussion between the 2 researchers, cases were referred to a third party, who had higher expertise and experience in the field of PHC. Endnote X5 was used to organize and identify duplicates, as well as read the titles and abstracts. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flowchart 14 - 16 was used to report the results of screening and selection process ( Figure 1 ). Figure 1. Selection of sources of evidence. Step 4: Data Charting To extract data from quantitative and qualitative studies, at first 2 data extraction forms were manually designed using the Microsoft Word 2010 software. At first, the data of 3 articles were extracted tentatively using these forms and the deficiencies and problems in the original form were solved. The information was extracted independently by 2 researchers and the ambiguities were resolved in consultation with other members of the research team. Information extractable using quantitative study form included: author, year of publication, country of study, study context (city, village, settlers, etc), aim of study, study design, participants (private and public sector), the subject of the study, the interval between the implementation of the PPP plan and the evaluation, the role of the public and private sector, the applied public-private partnership models, the studied indicators, the results, the overall outcome of implementation of PPP plan, and the conclusions. Data extractable using the qualitative studies form included the following: author and year of publication, country of study, aim of study, participants, method of data collection and results. As defined by the Canadian Council for Public-Private Partnerships (2011), 17 PPP models include design-build, design-build-maintain, design-build-operate, lease-operate-maintain, design-build-operate-maintain, build-own-operate-transfer, concession, and build-own-operate. Therefore, outsourcing is not part of PPP, but in some studies, it has been classified as part of PPP projects. Articles that categorized this method as one of the PPP models were reviewed by the research team and the data obtained showed that these studies met all PPP criteria, such as long-term contract, significant financial risk for private sector, and so on that provided by the World Bank for PPP projects, 18 and consequently entered into the study as PPP plans based on the research team's decision. On the other hand, in case of studies that did not directly refer to the PPP model used, 2 researchers determined the PPP model based on the information provided by the study. Cases where disagreement between the 2 researchers were not resolved through discussion were referred to the third person. The results of the studies were reviewed by 2 researchers and categorized into 4 categories: positive/effective, somewhat positive/effective, neutral (no effect), and negative/bad effect. Disagreements were discussed between the 2 researchers and in cases where the dispute was not resolved it was referred to a third party. In order to determine the study design, the information provided in each study were reviewed by 2 researchers and based on the agreement between the researchers, the type of study design determined. In case of disagreement, the disputes were referred to a third researcher with more experience and expertise. Step 5: Data Analysis and Reporting the Results After extracting the data by data extraction form, the extracted data were manually analyzed, summarized, and reported using the content-analysis method. Content analysis is a method for identifying, analyzing, and reporting patterns (themes) within the text and is widely used in qualitative data analysis. 19 - 22 Data were coded independently by 2 researchers. The steps for analyzing and coding the data were: Familiarity with the text of articles (immersion in article results), identifying and extracting primary themes (identifying and extracting studies related to primary themes), placing articles in determined themes, reviewing and completing the results of each theme with the use of results of the articles, and ensure the reliability of the themes and the results extracted in each theme. In cases of disagreement between the 2 coders, the dispute was resolved through discussion and if an agreement was not obtained, the disagreement was referred to a third researcher. Step 6: Consultation Exercise After extracting the results, based on the results obtained and the opinions of the research team's members, tips and suggestions were presented in the form of article discussion. Step 1: Identification of Relevant Studies The main research question of the present study is "What are the results and achievements of PPP in the provision of PHC in different countries?" which specifically include following questions: In which countries is PPP in the provision of PHC prevalent? How is the time trend of using PPP in the provision of PHC worldwide? In which settings (urban, rural, suburbs, etc) PPP in the provision of PHC is most prevalent? Which models of PPP are used to provide PHC? What are the role and responsibilities of the public and private sectors in the PPP in PHC plans? What are the goals of using PPP in the provision of PHC in different countries? For what tasks and services PPP has been applied in the provision of PHC? What are the achievements of implementing PPP policy in the provision of PHC worldwide? Inclusion and Exclusion Criteria All articles and reports on using PPP in the PHC projects published in Persian or English between 2000 and 2019 were included. Studies written in English or Persian (which could be translated by a member of our team) were included. The articles and studies that report the experience of implementing PPP plans in other health sectors (other than PHC) were excluded. Inclusion and Exclusion Criteria All articles and reports on using PPP in the PHC projects published in Persian or English between 2000 and 2019 were included. Studies written in English or Persian (which could be translated by a member of our team) were included. The articles and studies that report the experience of implementing PPP plans in other health sectors (other than PHC) were excluded. Step 2: Identification of Relevant Studies In this phase, required data were extracted from PubMed, Scopus, Science Direct, Web of Science, and Embase databases. The keywords were searched in different areas of primary health care, including primary health care, tuberculosis, vaccination, and so on. Persian equivalent of the above searches was performed in Persian databases like MagIran and SID. The time frame selected for searching the articles was January 2000 to May 2019 ( Appendix 1 in the Supplementary Material). Table 1 shows the search strategy proposed for PubMed. Table 1. Search Strategy for the PubMed Database. Search number Areas Search string #1 private OR "public private partnership" OR "public private participation" OR "public-private mix" OR "public-private cooperation" OR "mixed system" OR privatiz* OR "public-private coordination" OR "public-private collaboration" OR "contract* out" OR outsource* OR "Servic* contract*" OR "management contract*" OR "lease* contract*" OR "Private Finance Initiative Contract*" OR "concession contract*" OR "divesture contract*". #2 Primary health care "primary health care OR "primary health services" OR PHC OR "basic health care" OR "primary care" #3 Tuberculosis TB OR tuberculosis #4 Vaccination Immunization OR vaccination OR vaccine #5 Maternal and child care maternity OR child* OR maternal OR mother* OR midwifery OR pregnancy #6 Screening Screening #7 Case finding "Case find*" OR "find* case" OR "case detection" OR "detect* case" #8 Health education "Health education" OR "health training" OR "health teaching" OR "health promotion" #9 Mental health "Mental health*" #10 Occupational health "Occupational health*" OR "occupational hygiene" #11 Environmental health "Environmental health*" OR "environmental hygiene" OR "water hygiene" OR "wastewater hygiene" OR "water health" OR "wastewater health" #12 Oral health "Oral health*" OR "tooth health*" OR "dental health*" OR "oral hygiene" OR "tooth hygiene" OR "dental hygiene" #13 Congenital Anomalies "Thalassemia*" OR "hemophilia*" #14 Family medicine "Family physician" OR "family medicine" OR "family doctor" #15 Elderly health "Elderly health*" OR "aging health*" OR "aged health*" OR "older health*" OR "elderly care" OR "aging care" OR "aged care" OR "older care" #16 School health "School health" OR "student health" #17 Surveillance "Surveillance" #18 Diabetes Diabet* #19 Hypertension "Blood pressure" OR "high blood pressure" OR hypertension #20 Asthma "Asthma" #21 Cancer "Cancer" #22 Noncommunicable diseases (general) "Non-communicable diseases" OR "noncommunicable diseases" #23 HIV "HIV" OR "AIDS" OR "Human immunodeficiency virus infection" OR "acquired immune deficiency syndrome" #24 Hepatitis "Hepatitis" #25 STD "Sexually transmit*" OR "STD" OR "STI" #26 Malaria "Malaria" #27 Pediculosis "Pediculosis" OR "lice" OR "phthiriasis" #28 Fecal-oral diseases "Water and food borne" OR "fecal-oral" OR "diarrheal" OR "diarrhea" OR "food poisoning" OR "Water intoxication" OR parasite OR "Gastrointestinal" OR cholera #29 Influenza "Influenza" OR "flu" #30 Communicable diseases (general) "Communicable diseases" #31 Rabies Rabies OR rabid #32 Malta fever "Malta fever" OR brucellosis OR "brucella Infection" OR "brucella fever" #33 Anthrax Anthrax #34 Ebola Ebola #35 Neglected disease "Neglected disease*" #36 #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #28 OR #29 OR #30 OR #31 OR #32 OR #33 OR #34 OR #35 #37 #1 AND #36 To identify and cover of further published articles, after searching databases, selected journals and articles references were manually searched. In addition, Google Scholar, published organizational reports and government documents, websites, and other available information sources were also searched. Step 3: Study Selection The entire process of selection and screening of studies were performed independently by 2 members of the research team. Disagreements have been resolved through discussion in the first instance, otherwise were referred to a third party with more knowledge and experience. At first, the titles of all articles were reviewed, and those that were not consistent with the study objectives were excluded. Subsequently, abstracts and full texts of the articles were studied in order to identify and exclude studies that met exclusion criteria and had poor correlation with study objectives. Given that the structure of PHC delivery varies across countries, to determine whether the provided services are PHC, 2 researchers examined the information provided in each study and determined whether they were PHC or not. Thus, the types of services were determined based on the information provided and the agreement between the researchers. In case of disagreement, the agreement was reached through discussion between the 2 researchers. If no agreement was reached through discussion between the 2 researchers, cases were referred to a third party, who had higher expertise and experience in the field of PHC. Endnote X5 was used to organize and identify duplicates, as well as read the titles and abstracts. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flowchart 14 - 16 was used to report the results of screening and selection process ( Figure 1 ). Figure 1. Selection of sources of evidence. Step 4: Data Charting To extract data from quantitative and qualitative studies, at first 2 data extraction forms were manually designed using the Microsoft Word 2010 software. At first, the data of 3 articles were extracted tentatively using these forms and the deficiencies and problems in the original form were solved. The information was extracted independently by 2 researchers and the ambiguities were resolved in consultation with other members of the research team. Information extractable using quantitative study form included: author, year of publication, country of study, study context (city, village, settlers, etc), aim of study, study design, participants (private and public sector), the subject of the study, the interval between the implementation of the PPP plan and the evaluation, the role of the public and private sector, the applied public-private partnership models, the studied indicators, the results, the overall outcome of implementation of PPP plan, and the conclusions. Data extractable using the qualitative studies form included the following: author and year of publication, country of study, aim of study, participants, method of data collection and results. As defined by the Canadian Council for Public-Private Partnerships (2011), 17 PPP models include design-build, design-build-maintain, design-build-operate, lease-operate-maintain, design-build-operate-maintain, build-own-operate-transfer, concession, and build-own-operate. Therefore, outsourcing is not part of PPP, but in some studies, it has been classified as part of PPP projects. Articles that categorized this method as one of the PPP models were reviewed by the research team and the data obtained showed that these studies met all PPP criteria, such as long-term contract, significant financial risk for private sector, and so on that provided by the World Bank for PPP projects, 18 and consequently entered into the study as PPP plans based on the research team's decision. On the other hand, in case of studies that did not directly refer to the PPP model used, 2 researchers determined the PPP model based on the information provided by the study. Cases where disagreement between the 2 researchers were not resolved through discussion were referred to the third person. The results of the studies were reviewed by 2 researchers and categorized into 4 categories: positive/effective, somewhat positive/effective, neutral (no effect), and negative/bad effect. Disagreements were discussed between the 2 researchers and in cases where the dispute was not resolved it was referred to a third party. In order to determine the study design, the information provided in each study were reviewed by 2 researchers and based on the agreement between the researchers, the type of study design determined. In case of disagreement, the disputes were referred to a third researcher with more experience and expertise. Step 5: Data Analysis and Reporting the Results After extracting the data by data extraction form, the extracted data were manually analyzed, summarized, and reported using the content-analysis method. Content analysis is a method for identifying, analyzing, and reporting patterns (themes) within the text and is widely used in qualitative data analysis. 19 - 22 Data were coded independently by 2 researchers. The steps for analyzing and coding the data were: Familiarity with the text of articles (immersion in article results), identifying and extracting primary themes (identifying and extracting studies related to primary themes), placing articles in determined themes, reviewing and completing the results of each theme with the use of results of the articles, and ensure the reliability of the themes and the results extracted in each theme. In cases of disagreement between the 2 coders, the dispute was resolved through discussion and if an agreement was not obtained, the disagreement was referred to a third researcher. Step 6: Consultation Exercise After extracting the results, based on the results obtained and the opinions of the research team's members, tips and suggestions were presented in the form of article discussion. Results Of the 6933 studies and experiences found through searching the databases and other information sources, 1889 studies were excluded in duplicate articles screening, and 4915 articles were excluded with regard to title and abstract screening. Also 21 studies were excluded due to lack of enough information. Finally, 108 articles (including 85 quantitative and 23 qualitative) were included in the study ( Figure 1 ) ( Appendix 2 in the Supplementary Material). Sample Population of Included Studies Based on the findings of present study from all quantitative articles found, most studies have examined PHC centers as study unit, of which mostly examining the performance of PPP centers exclusively (without comparing with the public centers). In the studies that surveyed individuals, the sample population was mostly selected from those who had referred to public centers to receive PHC services ( Figure 2 ). Figure 2. Sample population of studies on implementation of public-private partnerships in primary health care based on studied units. Geographical Distribution of Included Studies In total, included studies have been conducted in 35 countries. Most studies have been conducted in India. Based on the latest World Bank Classification in 2019-2020, 23 10 studies were conducted in low-income countries, 65 studies in lower-middle income countries, 15 studies in upper-middle income countries and 22 studies in high-income countries ( Figure 3 ). It should be noted that the difference between the total number of studies in different countries and the total number of included studies is because of 2 studies were conducted in 3 countries. Figure 3. Geographical distribution of studies on implementation of public-private partnerships in primary health care by country. Time Trend of Included Studies Included articles were conducted from 2001 to 2019. The results show that the trend of studies' publication is incremental ( Figure 4 ). Figure 4. Time scattering of studies on implementation of public-private partnerships in primary health care by publication date. Study Design of Included Studies Only 2 studies had not indicated the type of study design and also could not been determined based on the information provided in these studies. Most type of the studies design were cross-sectional and then comparative studies. The least type of study design found in the included studies was population-based longitudinal studies and cohort studies ( Figure 5 ) ( Appendix 3 in the Supplementary Material). Figure 5. Study design of studies on implementation of public-private partnerships in primary health care. Study Context of Included Quantitative Studies According to the information presented in the included studies, 13 studies were conducted in urban context, 16 in rural and 2 studies were implemented in both of urban and rural area. Other studies (54 studies) had not specified the study setting. Duration of PPP Implementation Based on the results, studies were conducted on average 26 months after the implementation of the PPP plan. The maximum time was 122 months and the shortest was 2 months after PPP implementation. In total, the time after initiation of PPP plans in included studies, was calculated 255 years and 11 months. Public-Private Partnership Models in Included Studies The results of the review of PPP models indicated that except 2 studies, the other studies had not highlighted the PPP model used in the plan. Furthermore, in most studies the type of used PPP model had not been identifiable based on the information provided. In most other studies, the PPP model used in the plan was service contract and management contract ( Figure 6 ). Figure 6. Type of public-private partnership (PPP) models that had been used in studies on implementation of PPP in primary health care. Role of Public and Private Sectors in PPP Plans In the public sector, the highest frequency was related to supportive tasks and the least to stewardship tasks. Based on the information provided in the included articles, service provision with the most repetitive and financing and management with the least repetitive were among the tasks which had been assigned to the private sector. In 25 studies, public sector roles were not specified and in 19 studies, private sector roles were not mentioned ( Table 2 ). Table 2. The Role of Public and Private Sectors in Public-Private Partnership Plans in Primary Health Care Based on Included Studies. Governmental Number Nongovernmental Number Supporting 58 a Service provision 110 Monitoring 45 Support 11 Financing 37 Financing 3 Service provision 31 Management 3 Stewardship 14 Support 5 Not available 25 Not available 19 a Because of the multiple roles in a study, the number of roles exceeds the total number of included studies. Supporting roles included activities such as organizing, training, licensing, providing drugs, providing some equipment, providing infrastructure, support, providing incentives, providing physical space, and providing human resources. The financing roles included tasks such as financing and reimbursement to the private sector. The service provision role also included activities such as drug and vaccine distribution, vaccination, giving feedback on each patient referred, diagnostic and treatment services, health education, referral of patients to higher levels, recording of patient information and diseases screening. On the other hand, the role of monitoring included activities such as monitoring and evaluation, control, treatment process monitoring, accreditation, quality assessment, and disease monitoring. The role of stewardship also included tasks such as policy making, providing treatment guidelines, providing programmatic assistance, managing of the research activities, and governing the project. Finally, the management role that had been played by the private sector involved organizing and managing a health services center. Distribution of Included Studies by Aim of the Study The most common aim of the included studies was to measure the utilization improvement and the least was to measure the impact of PPP implementation on service quality ( Figure 7 ). Figure 7. Distribution of studies on implementation of public-private partnerships in primary health care by study aim. Indicators Examined to Determine the Project Success Rate In the included studies, the case detection and service coverage, more than other indicators, had been used as criteria to measure the success of the PPP project. On the other hand, the least used indicators to measure PPP plans success were quality of care, equity, and satisfaction of service providers and service receivers ( Figure 8 ) ( Appendix 4 in the Supplementary Material). Figure 8. Indicators used to determine the success rate of public-private partnership in primary health care plans and their results. Results of Implementation of PPP in PHC by the Type of Assigned Services Based on the results, in only one study the results of PPP implementation were negative. In most studies, the results of PPP implementation in PHC were evaluated as positive. The results in some studies were "somewhat positive" and in a few studies the project implementation evaluated as neutral ( Figure 9 ). Figure 9. Results of implementation of public-private partnership in primary health care by type of assigned services. Results of Qualitative Studies The results of qualitative studies are presented in 3 sections: advantages and disadvantages (from the experts' perspective), achievements and failures (results from actual experiences/project implementation), and barriers and challenges ( Table 3 ). Table 3. Results of Included Qualitative Studies on Implementation of Public-Private Partnership (PPP) in Primary Health Care (PHC). Main theme Theme Subtheme Advantages/benefits/strengths (in the viewpoint of experts) Organizational/managerial • Committed relationship over a long time • Closeness with community • Increase in department focus (core activities) • To develop innovative and more effective health programs and modes of service delivery (flexibility) • Diversity in partner expertise • High-quality monitoring and evaluation • Strong leadership • Understanding and agreeing with the national plan Access • Assurance of services access (improving access to diagnosis and treatment to many people, who could have been missed out, because of involvement of more service providers) less waiting time by patients at the facilities Supply PPP is better significantly in infrastructure, availability of essential medicines, basic medical appliances, mini-lab facilities and vehicles for referrals Economic • Reducing costs to both government and private party • Governments could reinvest savings from reduced hospitalizations to strengthen publicly funded PHC. Sustainable investment Social • Social obligation • Community awareness Community involvement Quality of care • Preventing hospitalizations • Improving quality of care in general practitioner (GP) services through measures such as improved multidisciplinary coordinated care programs, training, increased nursing workforce capacity, new administrative tools or additional funding. • Improving punctuality of doctors Care coordination and continuity of care Human resource Learning and teaching opportunity for employees Disadvantages/weakens (in the viewpoint of experts) Organizational/managerial: • More difficult accountability • Loss of control (less control, harder to control, and lack of information required for control) • Use of nonprofessional administrators Quality of care • Conflicts of interest • Negative impacts on quality of patient care Successes/achievements (results of real experience/implemented plans) Health outcomes • Improved health outcomes (in a very instance) • Iachieved targeted treatment outcomes (like more effective patient retention) Quality of care • Enhanced patient-provider relationships • Good or better continuity of care • Quality improvement • Strong health management information system (HMIS) • IRigorous follow-up Economic • Cost saving • IThere were gain in cost effectiveness, efficiency or revenue Access • Improving access to diagnosis and treatment • Providing access to training materials • ITo involve in new services Equity Increased access to services (including expanded hours, more service locations and improved access for vulnerable populations) Organizational/managerial • Relief of overburdened public sector resources • Strong integration with national goals • Sufficient number of trained clinical staff • Securing commitment and ownership from all parties involved • Positive effect on the practice of health education • Better monitoring and evaluation • Increased professional opportunities • Improved risk management • IIncreased efficiency Social • Providing information skills training (to patients) in the community • Raised awareness • IStrong communication channels Satisfaction • More service receivers' satisfaction • IPrivate practitioners' confidence in the quality and sustainability of the public–private partnership Supply • having a positive experience with regard to support mechanism of the project • IAccess to specialized equipment Failures (results of real experience/implemented plans) Supply • Inefficient procurement • Failure to harmonize public-private health management information system • IIrregular resource provision (in terms of material resources) Access • Under-utilized treatment support services • IPoorer access to services Organizational/managerial • Weak communication channels at the district level • (Autonomy) inability of manager to determine the proportion of employees in enabler's package • Weak evaluation • IIncreased level of administration Equity • Inequities in training opportunities • Inequities in higher facility utilization • IHigher out-of-pocket (driven by steeper transport costs and user charges for additional diagnostics) Economic • Funding challenges at the national program level • Irregular funding • Private sector is less affordable places • Increased monitoring cost • Increased administration cost because of increased level of administration • IHigher cost of provide some services privately (some director reported initial saving, but then increase in cost several years after privatization) Quality of care Loss of quality Barriers/limitations (before or after implementation of PPP plans) Organizational/managerial • Lacking governance framework • Lacking capacity within the public sector • Uncertainty in roles and responsibilities • Bureaucratic processes • Limited representation from ministries of health in governance structure • Overburden because of extra work needed to implement the partnership activities • Shortage of expert staff within the public sector • To maintain the progress of the partnership, and to ensure joint ownership of decisions and collective responsibility for the direction and activities of the partnership. • High expectations of private providers • Unclear administrative procedures for providers • Poor relationship between GPs and hospital doctors • Differences between the primary care and hospital systems • A limited ability to negotiate prices Organizational culture • Difficulties of to bring a heterogeneous group under one umbrella • Perceived power inequities between partners Access • Physical distances • Limited time at clinic Infrastructural • Inadequate transport • Lack of equipment and medicines • Inefficiency of local officials • The shortage of trained human resources Public culture Low demand for facility-based care in nonemergency settings Service provider The inability of GPs to cope with patients' needs Sample Population of Included Studies Based on the findings of present study from all quantitative articles found, most studies have examined PHC centers as study unit, of which mostly examining the performance of PPP centers exclusively (without comparing with the public centers). In the studies that surveyed individuals, the sample population was mostly selected from those who had referred to public centers to receive PHC services ( Figure 2 ). Figure 2. Sample population of studies on implementation of public-private partnerships in primary health care based on studied units. Geographical Distribution of Included Studies In total, included studies have been conducted in 35 countries. Most studies have been conducted in India. Based on the latest World Bank Classification in 2019-2020, 23 10 studies were conducted in low-income countries, 65 studies in lower-middle income countries, 15 studies in upper-middle income countries and 22 studies in high-income countries ( Figure 3 ). It should be noted that the difference between the total number of studies in different countries and the total number of included studies is because of 2 studies were conducted in 3 countries. Figure 3. Geographical distribution of studies on implementation of public-private partnerships in primary health care by country. Time Trend of Included Studies Included articles were conducted from 2001 to 2019. The results show that the trend of studies' publication is incremental ( Figure 4 ). Figure 4. Time scattering of studies on implementation of public-private partnerships in primary health care by publication date. Study Design of Included Studies Only 2 studies had not indicated the type of study design and also could not been determined based on the information provided in these studies. Most type of the studies design were cross-sectional and then comparative studies. The least type of study design found in the included studies was population-based longitudinal studies and cohort studies ( Figure 5 ) ( Appendix 3 in the Supplementary Material). Figure 5. Study design of studies on implementation of public-private partnerships in primary health care. Study Context of Included Quantitative Studies According to the information presented in the included studies, 13 studies were conducted in urban context, 16 in rural and 2 studies were implemented in both of urban and rural area. Other studies (54 studies) had not specified the study setting. Duration of PPP Implementation Based on the results, studies were conducted on average 26 months after the implementation of the PPP plan. The maximum time was 122 months and the shortest was 2 months after PPP implementation. In total, the time after initiation of PPP plans in included studies, was calculated 255 years and 11 months. Public-Private Partnership Models in Included Studies The results of the review of PPP models indicated that except 2 studies, the other studies had not highlighted the PPP model used in the plan. Furthermore, in most studies the type of used PPP model had not been identifiable based on the information provided. In most other studies, the PPP model used in the plan was service contract and management contract ( Figure 6 ). Figure 6. Type of public-private partnership (PPP) models that had been used in studies on implementation of PPP in primary health care. Role of Public and Private Sectors in PPP Plans In the public sector, the highest frequency was related to supportive tasks and the least to stewardship tasks. Based on the information provided in the included articles, service provision with the most repetitive and financing and management with the least repetitive were among the tasks which had been assigned to the private sector. In 25 studies, public sector roles were not specified and in 19 studies, private sector roles were not mentioned ( Table 2 ). Table 2. The Role of Public and Private Sectors in Public-Private Partnership Plans in Primary Health Care Based on Included Studies. Governmental Number Nongovernmental Number Supporting 58 a Service provision 110 Monitoring 45 Support 11 Financing 37 Financing 3 Service provision 31 Management 3 Stewardship 14 Support 5 Not available 25 Not available 19 a Because of the multiple roles in a study, the number of roles exceeds the total number of included studies. Supporting roles included activities such as organizing, training, licensing, providing drugs, providing some equipment, providing infrastructure, support, providing incentives, providing physical space, and providing human resources. The financing roles included tasks such as financing and reimbursement to the private sector. The service provision role also included activities such as drug and vaccine distribution, vaccination, giving feedback on each patient referred, diagnostic and treatment services, health education, referral of patients to higher levels, recording of patient information and diseases screening. On the other hand, the role of monitoring included activities such as monitoring and evaluation, control, treatment process monitoring, accreditation, quality assessment, and disease monitoring. The role of stewardship also included tasks such as policy making, providing treatment guidelines, providing programmatic assistance, managing of the research activities, and governing the project. Finally, the management role that had been played by the private sector involved organizing and managing a health services center. Distribution of Included Studies by Aim of the Study The most common aim of the included studies was to measure the utilization improvement and the least was to measure the impact of PPP implementation on service quality ( Figure 7 ). Figure 7. Distribution of studies on implementation of public-private partnerships in primary health care by study aim. Indicators Examined to Determine the Project Success Rate In the included studies, the case detection and service coverage, more than other indicators, had been used as criteria to measure the success of the PPP project. On the other hand, the least used indicators to measure PPP plans success were quality of care, equity, and satisfaction of service providers and service receivers ( Figure 8 ) ( Appendix 4 in the Supplementary Material). Figure 8. Indicators used to determine the success rate of public-private partnership in primary health care plans and their results. Results of Implementation of PPP in PHC by the Type of Assigned Services Based on the results, in only one study the results of PPP implementation were negative. In most studies, the results of PPP implementation in PHC were evaluated as positive. The results in some studies were "somewhat positive" and in a few studies the project implementation evaluated as neutral ( Figure 9 ). Figure 9. Results of implementation of public-private partnership in primary health care by type of assigned services. Results of Qualitative Studies The results of qualitative studies are presented in 3 sections: advantages and disadvantages (from the experts' perspective), achievements and failures (results from actual experiences/project implementation), and barriers and challenges ( Table 3 ). Table 3. Results of Included Qualitative Studies on Implementation of Public-Private Partnership (PPP) in Primary Health Care (PHC). Main theme Theme Subtheme Advantages/benefits/strengths (in the viewpoint of experts) Organizational/managerial • Committed relationship over a long time • Closeness with community • Increase in department focus (core activities) • To develop innovative and more effective health programs and modes of service delivery (flexibility) • Diversity in partner expertise • High-quality monitoring and evaluation • Strong leadership • Understanding and agreeing with the national plan Access • Assurance of services access (improving access to diagnosis and treatment to many people, who could have been missed out, because of involvement of more service providers) less waiting time by patients at the facilities Supply PPP is better significantly in infrastructure, availability of essential medicines, basic medical appliances, mini-lab facilities and vehicles for referrals Economic • Reducing costs to both government and private party • Governments could reinvest savings from reduced hospitalizations to strengthen publicly funded PHC. Sustainable investment Social • Social obligation • Community awareness Community involvement Quality of care • Preventing hospitalizations • Improving quality of care in general practitioner (GP) services through measures such as improved multidisciplinary coordinated care programs, training, increased nursing workforce capacity, new administrative tools or additional funding. • Improving punctuality of doctors Care coordination and continuity of care Human resource Learning and teaching opportunity for employees Disadvantages/weakens (in the viewpoint of experts) Organizational/managerial: • More difficult accountability • Loss of control (less control, harder to control, and lack of information required for control) • Use of nonprofessional administrators Quality of care • Conflicts of interest • Negative impacts on quality of patient care Successes/achievements (results of real experience/implemented plans) Health outcomes • Improved health outcomes (in a very instance) • Iachieved targeted treatment outcomes (like more effective patient retention) Quality of care • Enhanced patient-provider relationships • Good or better continuity of care • Quality improvement • Strong health management information system (HMIS) • IRigorous follow-up Economic • Cost saving • IThere were gain in cost effectiveness, efficiency or revenue Access • Improving access to diagnosis and treatment • Providing access to training materials • ITo involve in new services Equity Increased access to services (including expanded hours, more service locations and improved access for vulnerable populations) Organizational/managerial • Relief of overburdened public sector resources • Strong integration with national goals • Sufficient number of trained clinical staff • Securing commitment and ownership from all parties involved • Positive effect on the practice of health education • Better monitoring and evaluation • Increased professional opportunities • Improved risk management • IIncreased efficiency Social • Providing information skills training (to patients) in the community • Raised awareness • IStrong communication channels Satisfaction • More service receivers' satisfaction • IPrivate practitioners' confidence in the quality and sustainability of the public–private partnership Supply • having a positive experience with regard to support mechanism of the project • IAccess to specialized equipment Failures (results of real experience/implemented plans) Supply • Inefficient procurement • Failure to harmonize public-private health management information system • IIrregular resource provision (in terms of material resources) Access • Under-utilized treatment support services • IPoorer access to services Organizational/managerial • Weak communication channels at the district level • (Autonomy) inability of manager to determine the proportion of employees in enabler's package • Weak evaluation • IIncreased level of administration Equity • Inequities in training opportunities • Inequities in higher facility utilization • IHigher out-of-pocket (driven by steeper transport costs and user charges for additional diagnostics) Economic • Funding challenges at the national program level • Irregular funding • Private sector is less affordable places • Increased monitoring cost • Increased administration cost because of increased level of administration • IHigher cost of provide some services privately (some director reported initial saving, but then increase in cost several years after privatization) Quality of care Loss of quality Barriers/limitations (before or after implementation of PPP plans) Organizational/managerial • Lacking governance framework • Lacking capacity within the public sector • Uncertainty in roles and responsibilities • Bureaucratic processes • Limited representation from ministries of health in governance structure • Overburden because of extra work needed to implement the partnership activities • Shortage of expert staff within the public sector • To maintain the progress of the partnership, and to ensure joint ownership of decisions and collective responsibility for the direction and activities of the partnership. • High expectations of private providers • Unclear administrative procedures for providers • Poor relationship between GPs and hospital doctors • Differences between the primary care and hospital systems • A limited ability to negotiate prices Organizational culture • Difficulties of to bring a heterogeneous group under one umbrella • Perceived power inequities between partners Access • Physical distances • Limited time at clinic Infrastructural • Inadequate transport • Lack of equipment and medicines • Inefficiency of local officials • The shortage of trained human resources Public culture Low demand for facility-based care in nonemergency settings Service provider The inability of GPs to cope with patients' needs Discussion Study results were categorized and reported under 12 main themes (sample population of included studies, geographical distribution, time scattering of included studies' publication date, study design of included studies, study context of included quantitative studies, duration of PPP implementation, PPP models, role of public and private sector, distribution of included studies by aim of the study, indicators examined to determine the project success rate, results of implementation of PPP in PHC plans and results of qualitative studies). Geographical Distribution of Included Studies The results of the study showed that most studies have been conducted in low-income and lower-middle income countries. One of the possible reasons for this could be the economic situation of these countries, where the government utilize resources and the ability of the private sector to provide PHC services due to lack of financial resources. On the other hand, the inability of the government in providing quality services, providing trained human resources, expanding services in remote areas, and so on can be other reasons for using the private sector to cover the weaknesses in the public sector. Another possible reason is the lack of infrastructures and backgrounds to full privatization. Given the limited resources of the health system, especially in LMICs, the use of private sector resources seems inevitable. In the Lei et al study, 24 which was a systematic review of PPP projects in the field of tuberculosis also most included studies were conducted in LMICs. The study also mostly included studies in South Asia and India. 24 Unlike previous study, in the study by Grochtdreis et al, 25 which conducted a systematic review study examining the cost-effectiveness of providing treatment services for depression disorders using PPPs, most studies were conducted in high-income countries. This may be due to the lack of access to cost information in low- and middle-income countries, whereas in developed countries this data may be available due to better information infrastructure. Time Scattering of Included Studies Since the present study was conducted in the fifth month of 2019, the number of included studies in 2018 and 2019 is lower than in previous years. Despite some fluctuations in the number of studies published in reviewed years, time scattering of included studies indicate that the publication trend of studies in the field of PPP implementation in PHC is incremental. One reason for this finding could be that the publication of articles on preliminary positive results has convinced governments that implementing PPP in PHC can be beneficial and promote this field, thereby governments have moved to implementing such plans. The findings of the study by Roehrich et al, 26 which reviewed and analyzed about 1400 articles published during the past 2 decades, show that during this time, public sector policymakers have paid particular attention to the capabilities of the private sector in development, financing, and provision of services and infrastructure of the health system, so that, since 2006 there has been a significant jump in the number of articles published in the field of PPP. On the other hand, another reason for the increase in tendency to PPP may be the weakness of governments in providing services and the economic problems. Study Design of Included Studies According to the results, the most used study designs were cross-sectional, comparative, and before and after study. It seems, because of the nature of studies, which aimed at examining the results of PPP implementation, the above study designs are more appropriate. In the study by Roehrich et al, 26 the case study approach was the main method of collecting data on PPP plans and comparing organizations. Meanwhile survey method was used in a small number of articles. Given the long-term nature of most PPP plans, it seems limited evidence of results in the form of longitudinal or time series studies have been published. In order to better reflect the success of PPP plans, it is recommended to use more longitudinal, process, and case-control studies. Also, based on the results of the study, few economic evaluation studies have been conducted to assess the PPP success. As one of the drivers of PPP projects implementation is economic issues, the use of economic evaluation studies to determine the success of these types of projects can provide useful and effective information for researchers and executives. The number of mix method studies is also relatively low. Since mixed-method studies address different aspects of a subject and can better and more efficiently evaluate the success or failure of a project, it is better to use these studies to evaluate the success of PPP projects. On the other hand, the studies' design was mostly cross-sectional and pre- and poststudy without control group, and few of studies were conducted as quasi-experimental (controlled). Since studies with controlled group are the best type of studies to accurately determine the impact of PPP implementation, it is recommended that researchers mostly use quasi-experimental (controlled) studies in future studies. Duration of PPP Implementation In some included studies, shortly after implementation of the PPP plan its success had been evaluated, which it is not possible to make a fair judgment about the success or failure of the plan after such short time period. When a PPP plan is implemented, a major change occurs in the system and adapting with new structure will take time. The public and private sectors need time to adjust and stabilize with new conditions, at such point, the actual performance can be measured. For this reason, it is better to make adequate time interval between project implementation and its evaluation. In the study of Grochtdreis et al (2015), 25 the average time interval between implementation and evaluation the success of PPP projects, using the cost-effectiveness index, were ranged from 6 to 24 months. Public-Private Partnership Models in Included Studies One of the most important things that determine the assignment method, assigned functions, monitoring and evaluation method, how to pay to the private sector, and so on is the PPP model. Also, the used PPP model can be very helpful in conveying the experience gained from the plan and identifying its strengths and weaknesses. The results of present study showed that the model of PPP is less addressed in the included studies. One possible reason could be the authors' purpose of writing the articles or reports, which may be to just provide a report of the results of an experience and therefore have paid less attention to detail. Another possible reason may be the lack of experience and technical knowledge of the authors in this field. Role of Public and Private Sectors in Public-Private Partnership The roles that the private sector can take on are varied and largely depend on the type of service that should be provided by this sector. In some cases, PPP plans are described based on roles that are assigned to the private sector. As expected, the results indicated that the role of private sector in implementation of PPP in PHC mostly is service provision. But the role of the public sector in service provision is still significant, and the main role of this sector, which is stewardship, has largely been ignored. Public sector need to reduce energy to provide services, and assign it to the private sector, and spend most of their time and energy on stewardship as its main task. In a review study by Dewan et al, 27 which examines PPP plans implemented in India to control tuberculosis, stated that the role of government is to educate and supervise private companies providing tuberculosis-related care, while the role of the private sector is to provide services in accordance with public sector standards and criteria. 27 The authors' proposed pattern for implementing PPP plans in the health sector is that the government should focus more on governance tasks such as stewardship and supporting, and assign services provision to the private sector ( Figure 10 ). According to this model, as we move from tasks such as service provision to governance tasks, such as stewardship, supporting and supervision, the role of the private sector becomes less and the role of the public sector becomes more prominent. In order for the government to perform its tasks properly, it must assign the lower-level duties to the private sector and spend most of its time and energy on stewardship and governance tasks. Also, under this model, some specific services can be provided by the public sector, which may decrease or increase depending on the capacity of the private sector and the existence of required infrastructures for providing these specific services in this sector, as well as the capacity of the public sector for monitoring and supporting. In some included studies, it is found that in implementation of PPP plans this pattern have not followed, which is not consistent with the nature of PPP. Figure 10. Pattern of responsibilities in public-private partnerships in health sector. Distribution of Included Studies by Study Context and Aim of the Study The most important achievements of PPP are improving the quality of service, service receivers' satisfaction, access, utilization, and equity. Because it is expected that in areas such as the affluent and congested areas where the public sector lacks the willingness or ability for providing health services, through assigning this responsibility to the private sector access and utilization, and consequently, public satisfaction increased in these areas. Assigning health care provision in the areas of interest of private sector will increase the capacity and potential of the public sector to provide health services in deprived areas, which is a clear indication of equity in the health system. Likely, because governments, particularly in LMICs, have difficulties in providing services to urban marginalized areas and deprived and remote areas, health indicators are low in these areas, in implementation of PPP plans these areas have been mostly addressed, and may for this reason, indicators such as utilization, access and case detection were used to evaluate the success of PPP in PHC plans. On the other hand, these indicators are much easier to evaluate and manage, and more compatible with the nature of PHC. These indicators have also been widely used in various studies evaluating PPP in PHC plans. 28 - 33 Therefore, it is suggested that researchers compare the rural and deprived areas with urban areas, or compare the status of these areas with the preplan conditions to evaluate the success rate of PPP plans. Indicators Examined to Determine the Project Success Rate Based on the results of this study, in most cases, the effect of project implementation on improvement of studied indicators was effective. As well, because the changes in primary health care could have significant impact on case detection rate and service delivery indicators, and because these indicators are easier and more objective to measure, these indicators are mostly used to monitor the PPP plans' success. In the study of Lei et al, 24 the indicators of DOTS utilization, case detection, treatment outcome, case management, costs, access and equity were used to measure the effectiveness of PPP plans, all of which were extracted in the present study. Also, in the study of Dewan et al 27 in India, the case detection and treatment success rate has considered as the measures of effectiveness of provided services through PPP plan. The impact of implementation of PPP plans on improving the cost analysis index is somewhat less than its impact on other indicators. This probably indicates that reviewed studies did not specify the costs of before implementation of plan precisely, and also the extraction of this information after implementation of the plan may has been erroneous. Another reason could be not choosing the right metrics to measure this indicator. It is also important how to collect data and calculate these indices. A study by Hatcher et al 34 in Pakistan examined the unit cost of 2 rural primary health centers that had been assigned to a private company. The results of the study showed that the unit costs of these centers were higher than expected. 34 It is suggested that accurate indicators that reflect changes in this sector be selected for accurate monitoring of health system performance, and the data on these indicators routinely collected, not just for research or cross-sectional review. The results also show that indicators of service quality, satisfaction of service receivers and providers, and equity have less used to evaluate the success of PPP plans. Indicators of service quality and satisfaction are predictors of people's utilization of PHC, which can lead to equity. Therefore, because these indicators are more in line with the nature of PHC, it is recommended that in the future these indicators be used more to evaluate the impact of PPP implementation. Results of Implementation of PPP in PHC by the Type of Assigned Services According to the results of the study, most of the PPP projects has implemented in the field of TB related services delivery, which has greatly led to improve these services. The lowest rates of PPP plan were implemented in the field of disease surveillance, nutrition, reproductive health, gastroenteritis, elderly care, and mental health, which should be given more attention due to the importance of these areas in PHC. One of the possible reasons for less implementation of PPP plans in these areas could be the inability or unwillingness of the private sector to participate in these areas, where the public sector required to provide the preconditions (such as incentive packages, private sector empowerment, precise monitoring and evaluation criteria, etc) for assigning these services. In the study of Lei et al, 24 the success rate of PPP projects in the field of tuberculosis services was about 65%, which is lower than that obtained in the present study. One reason for this may be that in the mentioned study, tuberculosis-related services have been examined in all sectors of the health system (laboratories, inpatient services, imaging, etc), but in the present study services that provided in PHC have been examined. As pointed out in the time scattering section, it seems that due to the positive results of implementation of PPP projects, the acceptance of these plans has increased. The results have also shown that most of these projects have had a significant positive impact on PHC services provision. The effectiveness of these plans in the field of maternal and child care and vaccination was less than in other areas, which may be due to the lack of public awareness or cultural barriers to receiving these services. Results of Qualitative Studies The results showed that PPP plans can have different benefits. To take advantage of these benefits, it is recommended that the executors of these plans make the necessary arrangements, such as studying the existing conditions and contexts, and providing the necessary infrastructure before the implementation. Notification and informing the public about these benefits can also get the support of the community and authorities. In a review study by Hernandez-Aguado and Zaragoza, 35 the benefits and requisites of using PPP in health promotion have been examined and it has been concluded that PPP enhances the capacity and potential of health services provision, increases attention to health in all policies, increases self-control, and improves the quality of services. 35 In included qualitative studies, disadvantages of the PPP plans were less than the benefits, which may indicate a positive outlook to such projects, which has resulted from the positive results of these plans in recent years. The results of the present study showed that the achievements of PPP in PHC plans were also positively evaluated, but some failures were noted. In a qualitative study conducted by Gasparinene et al 36 in Lithuania with the aim of examining the experts' views on the factors influencing the use of outsourcing in public health services, and the factors affecting cost savings after outsourcing, the results showed that the main factors that lead to the selection of outsourcing in public health care are lower cost, access to new technologies, and better quality of service. The results also indicated that outsourcing could lead to a significant reduction in the cost of noncore activities and the cost of investing in resources and storage. 36 One of the most important issues to consider in PPP plans, is the obstacles and challenges of implementing such plans. The results of present study illustrate different challenges in implementing PPP in PHC projects which one of the most important of them is organizational barriers. One possible reason for the organizational barriers is the lack of readiness in public sector to implement such plans. To eliminate these barriers, it is recommended that all aspects that influence the PPP implementation be considered before implementing these plans so that through proper planning barriers can be solved before or during the implementation of the plan. For example, the resources needed to execute the plan, including human resources, finance, physical space, experts training in the field of PPP plans, and son, should be provided before implementation. Also, one of the solutions to identifying and addressing challenges is to implement the project in a smaller, pilot scale, because doing so will help identify many of the challenges and barriers and take action to address them before the project is widely implemented. It seems that all sections of society and public organizations should be involved in the process of implementing PPP in PHC plans in order to remove the existing challenges such as cultural and infrastructural barriers. Some of the qualitative results showed different and sometimes conflicting views on PPP plans. One of the possible reasons for this may be the implementation of these projects in different countries with different context. Therefore, after studying the experiences of different countries and regions, countries should design a PPP plan tailored to their national and local conditions. Application in LMICs The results of the present study show that the PPP policy in PHC is more considered in LMICs than in high-income countries. Therefore, the results of this study are inherently more applicable to LMICs. The present study provides a comprehensive overview of PPP experiences in PHC, and provides relatively comprehensive information, evidence, and information for health authorities and policymakers in LMICs. One of the challenges of PPP plans, especially in developing countries, is the lack of rich information to judge about the success of PPP plans. To overcome this, it is better to define some indexes for determining the success rate of the plan, and design the mechanisms to collect the relevant data, or even include it in the PPP contracts as one of the private sector' duties. On the other hand, good design and implementation of a PPP plan does not ensure its success, but the success of such plans requires continuous monitoring and evaluation to identify possible barriers and challenges as well as facilitating factors. Because of political unsustainability that sometimes lies in developing countries, it seems that applying civil pressures is critical to prevent reforms from stopping. For applying civil pressures, attracting the support of people and stakeholders seems to be essential. Creating management capability in public sector for monitoring and evaluating the performance of private sector is one of the most important requirements for implementing PPP plans in developing countries. This requires development of proper indexes and objectives, establishment of a targeted and accurate monitoring and evaluation program, development of high-precision tools, and finally pay-for-performance based on the results of monitoring and evaluations. One of the necessary and useful tools in this area is implementation and use of information systems that can help improve the accuracy and precision of monitoring and evaluation, and payment based on it. Based on developing such a structure, specialized and independent private companies' capacity can be applied to measure and improve quality. It should be noted that PPP's success in health sector is largely influenced by its design and the context in which it implemented. To overcome this, each country should design and implement its own PPP models which is adapted to country's national and local conditions or context. Recommendations The results show that while PPP plans is regarded as an essential and effective way to provide social justice, implement family practice plan, and achieve universal health care, challenges such as political and financial unsustainability, delay in reimbursements, lack of stakeholder cooperation, and lack of continuous quality of services evaluation have had negative effect on the PPP plans. Overcoming challenges, more success and expanding this plan require accepting the private sector as one of the main sources of the health system, and require more support from authorities of health, national and local government. It is recommended that designers and executers of these plans adopt strategies in order to ensure adequate and timely funding. One of the solutions is attracting the cooperation of insurance companies. On the other hand, successful formulation and start-up of the plan does not guarantee its success, so a mechanism should be designed to monitor and modify the plan. The last proposal is to involve stakeholders in the process of managing and organizing the implementation of the plan, so that a common language create among all stakeholders. Limitations Overall, the information provided in the reviewed studies was incomplete. For example, many studies did not address the project implementation context (urban or rural), PPP model, role of the public and private sector, sample size, study design, and so on. It is better to refer to all aspects when conducting a study in the field of PPP plans (including setting (urban/rural/immigrant/suburb/etc), aim of study, study design, participants (governmental and nongovernmental), Period after implementation of PPP plan, Roles/task of parties (public and private), PPP model, Indicators have used to evaluate the success of PPP plan, data collection method), to better evaluate the validity of the results and the reasons for success or failure. Since most included studies were not clear on the methodology of the study, there were 2 ways for the authors: either the methodology should not be reported or the method of study should be determined by the research team based on the information presented in the article. If these cases were not reported, the final report would be incomplete, so the research team inevitably reviewed the studies and determined the study design based on the provided information, which may have caused errors in some cases. Geographical Distribution of Included Studies The results of the study showed that most studies have been conducted in low-income and lower-middle income countries. One of the possible reasons for this could be the economic situation of these countries, where the government utilize resources and the ability of the private sector to provide PHC services due to lack of financial resources. On the other hand, the inability of the government in providing quality services, providing trained human resources, expanding services in remote areas, and so on can be other reasons for using the private sector to cover the weaknesses in the public sector. Another possible reason is the lack of infrastructures and backgrounds to full privatization. Given the limited resources of the health system, especially in LMICs, the use of private sector resources seems inevitable. In the Lei et al study, 24 which was a systematic review of PPP projects in the field of tuberculosis also most included studies were conducted in LMICs. The study also mostly included studies in South Asia and India. 24 Unlike previous study, in the study by Grochtdreis et al, 25 which conducted a systematic review study examining the cost-effectiveness of providing treatment services for depression disorders using PPPs, most studies were conducted in high-income countries. This may be due to the lack of access to cost information in low- and middle-income countries, whereas in developed countries this data may be available due to better information infrastructure. Time Scattering of Included Studies Since the present study was conducted in the fifth month of 2019, the number of included studies in 2018 and 2019 is lower than in previous years. Despite some fluctuations in the number of studies published in reviewed years, time scattering of included studies indicate that the publication trend of studies in the field of PPP implementation in PHC is incremental. One reason for this finding could be that the publication of articles on preliminary positive results has convinced governments that implementing PPP in PHC can be beneficial and promote this field, thereby governments have moved to implementing such plans. The findings of the study by Roehrich et al, 26 which reviewed and analyzed about 1400 articles published during the past 2 decades, show that during this time, public sector policymakers have paid particular attention to the capabilities of the private sector in development, financing, and provision of services and infrastructure of the health system, so that, since 2006 there has been a significant jump in the number of articles published in the field of PPP. On the other hand, another reason for the increase in tendency to PPP may be the weakness of governments in providing services and the economic problems. Study Design of Included Studies According to the results, the most used study designs were cross-sectional, comparative, and before and after study. It seems, because of the nature of studies, which aimed at examining the results of PPP implementation, the above study designs are more appropriate. In the study by Roehrich et al, 26 the case study approach was the main method of collecting data on PPP plans and comparing organizations. Meanwhile survey method was used in a small number of articles. Given the long-term nature of most PPP plans, it seems limited evidence of results in the form of longitudinal or time series studies have been published. In order to better reflect the success of PPP plans, it is recommended to use more longitudinal, process, and case-control studies. Also, based on the results of the study, few economic evaluation studies have been conducted to assess the PPP success. As one of the drivers of PPP projects implementation is economic issues, the use of economic evaluation studies to determine the success of these types of projects can provide useful and effective information for researchers and executives. The number of mix method studies is also relatively low. Since mixed-method studies address different aspects of a subject and can better and more efficiently evaluate the success or failure of a project, it is better to use these studies to evaluate the success of PPP projects. On the other hand, the studies' design was mostly cross-sectional and pre- and poststudy without control group, and few of studies were conducted as quasi-experimental (controlled). Since studies with controlled group are the best type of studies to accurately determine the impact of PPP implementation, it is recommended that researchers mostly use quasi-experimental (controlled) studies in future studies. Duration of PPP Implementation In some included studies, shortly after implementation of the PPP plan its success had been evaluated, which it is not possible to make a fair judgment about the success or failure of the plan after such short time period. When a PPP plan is implemented, a major change occurs in the system and adapting with new structure will take time. The public and private sectors need time to adjust and stabilize with new conditions, at such point, the actual performance can be measured. For this reason, it is better to make adequate time interval between project implementation and its evaluation. In the study of Grochtdreis et al (2015), 25 the average time interval between implementation and evaluation the success of PPP projects, using the cost-effectiveness index, were ranged from 6 to 24 months. Public-Private Partnership Models in Included Studies One of the most important things that determine the assignment method, assigned functions, monitoring and evaluation method, how to pay to the private sector, and so on is the PPP model. Also, the used PPP model can be very helpful in conveying the experience gained from the plan and identifying its strengths and weaknesses. The results of present study showed that the model of PPP is less addressed in the included studies. One possible reason could be the authors' purpose of writing the articles or reports, which may be to just provide a report of the results of an experience and therefore have paid less attention to detail. Another possible reason may be the lack of experience and technical knowledge of the authors in this field. Role of Public and Private Sectors in Public-Private Partnership The roles that the private sector can take on are varied and largely depend on the type of service that should be provided by this sector. In some cases, PPP plans are described based on roles that are assigned to the private sector. As expected, the results indicated that the role of private sector in implementation of PPP in PHC mostly is service provision. But the role of the public sector in service provision is still significant, and the main role of this sector, which is stewardship, has largely been ignored. Public sector need to reduce energy to provide services, and assign it to the private sector, and spend most of their time and energy on stewardship as its main task. In a review study by Dewan et al, 27 which examines PPP plans implemented in India to control tuberculosis, stated that the role of government is to educate and supervise private companies providing tuberculosis-related care, while the role of the private sector is to provide services in accordance with public sector standards and criteria. 27 The authors' proposed pattern for implementing PPP plans in the health sector is that the government should focus more on governance tasks such as stewardship and supporting, and assign services provision to the private sector ( Figure 10 ). According to this model, as we move from tasks such as service provision to governance tasks, such as stewardship, supporting and supervision, the role of the private sector becomes less and the role of the public sector becomes more prominent. In order for the government to perform its tasks properly, it must assign the lower-level duties to the private sector and spend most of its time and energy on stewardship and governance tasks. Also, under this model, some specific services can be provided by the public sector, which may decrease or increase depending on the capacity of the private sector and the existence of required infrastructures for providing these specific services in this sector, as well as the capacity of the public sector for monitoring and supporting. In some included studies, it is found that in implementation of PPP plans this pattern have not followed, which is not consistent with the nature of PPP. Figure 10. Pattern of responsibilities in public-private partnerships in health sector. Distribution of Included Studies by Study Context and Aim of the Study The most important achievements of PPP are improving the quality of service, service receivers' satisfaction, access, utilization, and equity. Because it is expected that in areas such as the affluent and congested areas where the public sector lacks the willingness or ability for providing health services, through assigning this responsibility to the private sector access and utilization, and consequently, public satisfaction increased in these areas. Assigning health care provision in the areas of interest of private sector will increase the capacity and potential of the public sector to provide health services in deprived areas, which is a clear indication of equity in the health system. Likely, because governments, particularly in LMICs, have difficulties in providing services to urban marginalized areas and deprived and remote areas, health indicators are low in these areas, in implementation of PPP plans these areas have been mostly addressed, and may for this reason, indicators such as utilization, access and case detection were used to evaluate the success of PPP in PHC plans. On the other hand, these indicators are much easier to evaluate and manage, and more compatible with the nature of PHC. These indicators have also been widely used in various studies evaluating PPP in PHC plans. 28 - 33 Therefore, it is suggested that researchers compare the rural and deprived areas with urban areas, or compare the status of these areas with the preplan conditions to evaluate the success rate of PPP plans. Indicators Examined to Determine the Project Success Rate Based on the results of this study, in most cases, the effect of project implementation on improvement of studied indicators was effective. As well, because the changes in primary health care could have significant impact on case detection rate and service delivery indicators, and because these indicators are easier and more objective to measure, these indicators are mostly used to monitor the PPP plans' success. In the study of Lei et al, 24 the indicators of DOTS utilization, case detection, treatment outcome, case management, costs, access and equity were used to measure the effectiveness of PPP plans, all of which were extracted in the present study. Also, in the study of Dewan et al 27 in India, the case detection and treatment success rate has considered as the measures of effectiveness of provided services through PPP plan. The impact of implementation of PPP plans on improving the cost analysis index is somewhat less than its impact on other indicators. This probably indicates that reviewed studies did not specify the costs of before implementation of plan precisely, and also the extraction of this information after implementation of the plan may has been erroneous. Another reason could be not choosing the right metrics to measure this indicator. It is also important how to collect data and calculate these indices. A study by Hatcher et al 34 in Pakistan examined the unit cost of 2 rural primary health centers that had been assigned to a private company. The results of the study showed that the unit costs of these centers were higher than expected. 34 It is suggested that accurate indicators that reflect changes in this sector be selected for accurate monitoring of health system performance, and the data on these indicators routinely collected, not just for research or cross-sectional review. The results also show that indicators of service quality, satisfaction of service receivers and providers, and equity have less used to evaluate the success of PPP plans. Indicators of service quality and satisfaction are predictors of people's utilization of PHC, which can lead to equity. Therefore, because these indicators are more in line with the nature of PHC, it is recommended that in the future these indicators be used more to evaluate the impact of PPP implementation. Results of Implementation of PPP in PHC by the Type of Assigned Services According to the results of the study, most of the PPP projects has implemented in the field of TB related services delivery, which has greatly led to improve these services. The lowest rates of PPP plan were implemented in the field of disease surveillance, nutrition, reproductive health, gastroenteritis, elderly care, and mental health, which should be given more attention due to the importance of these areas in PHC. One of the possible reasons for less implementation of PPP plans in these areas could be the inability or unwillingness of the private sector to participate in these areas, where the public sector required to provide the preconditions (such as incentive packages, private sector empowerment, precise monitoring and evaluation criteria, etc) for assigning these services. In the study of Lei et al, 24 the success rate of PPP projects in the field of tuberculosis services was about 65%, which is lower than that obtained in the present study. One reason for this may be that in the mentioned study, tuberculosis-related services have been examined in all sectors of the health system (laboratories, inpatient services, imaging, etc), but in the present study services that provided in PHC have been examined. As pointed out in the time scattering section, it seems that due to the positive results of implementation of PPP projects, the acceptance of these plans has increased. The results have also shown that most of these projects have had a significant positive impact on PHC services provision. The effectiveness of these plans in the field of maternal and child care and vaccination was less than in other areas, which may be due to the lack of public awareness or cultural barriers to receiving these services. Results of Qualitative Studies The results showed that PPP plans can have different benefits. To take advantage of these benefits, it is recommended that the executors of these plans make the necessary arrangements, such as studying the existing conditions and contexts, and providing the necessary infrastructure before the implementation. Notification and informing the public about these benefits can also get the support of the community and authorities. In a review study by Hernandez-Aguado and Zaragoza, 35 the benefits and requisites of using PPP in health promotion have been examined and it has been concluded that PPP enhances the capacity and potential of health services provision, increases attention to health in all policies, increases self-control, and improves the quality of services. 35 In included qualitative studies, disadvantages of the PPP plans were less than the benefits, which may indicate a positive outlook to such projects, which has resulted from the positive results of these plans in recent years. The results of the present study showed that the achievements of PPP in PHC plans were also positively evaluated, but some failures were noted. In a qualitative study conducted by Gasparinene et al 36 in Lithuania with the aim of examining the experts' views on the factors influencing the use of outsourcing in public health services, and the factors affecting cost savings after outsourcing, the results showed that the main factors that lead to the selection of outsourcing in public health care are lower cost, access to new technologies, and better quality of service. The results also indicated that outsourcing could lead to a significant reduction in the cost of noncore activities and the cost of investing in resources and storage. 36 One of the most important issues to consider in PPP plans, is the obstacles and challenges of implementing such plans. The results of present study illustrate different challenges in implementing PPP in PHC projects which one of the most important of them is organizational barriers. One possible reason for the organizational barriers is the lack of readiness in public sector to implement such plans. To eliminate these barriers, it is recommended that all aspects that influence the PPP implementation be considered before implementing these plans so that through proper planning barriers can be solved before or during the implementation of the plan. For example, the resources needed to execute the plan, including human resources, finance, physical space, experts training in the field of PPP plans, and son, should be provided before implementation. Also, one of the solutions to identifying and addressing challenges is to implement the project in a smaller, pilot scale, because doing so will help identify many of the challenges and barriers and take action to address them before the project is widely implemented. It seems that all sections of society and public organizations should be involved in the process of implementing PPP in PHC plans in order to remove the existing challenges such as cultural and infrastructural barriers. Some of the qualitative results showed different and sometimes conflicting views on PPP plans. One of the possible reasons for this may be the implementation of these projects in different countries with different context. Therefore, after studying the experiences of different countries and regions, countries should design a PPP plan tailored to their national and local conditions. Application in LMICs The results of the present study show that the PPP policy in PHC is more considered in LMICs than in high-income countries. Therefore, the results of this study are inherently more applicable to LMICs. The present study provides a comprehensive overview of PPP experiences in PHC, and provides relatively comprehensive information, evidence, and information for health authorities and policymakers in LMICs. One of the challenges of PPP plans, especially in developing countries, is the lack of rich information to judge about the success of PPP plans. To overcome this, it is better to define some indexes for determining the success rate of the plan, and design the mechanisms to collect the relevant data, or even include it in the PPP contracts as one of the private sector' duties. On the other hand, good design and implementation of a PPP plan does not ensure its success, but the success of such plans requires continuous monitoring and evaluation to identify possible barriers and challenges as well as facilitating factors. Because of political unsustainability that sometimes lies in developing countries, it seems that applying civil pressures is critical to prevent reforms from stopping. For applying civil pressures, attracting the support of people and stakeholders seems to be essential. Creating management capability in public sector for monitoring and evaluating the performance of private sector is one of the most important requirements for implementing PPP plans in developing countries. This requires development of proper indexes and objectives, establishment of a targeted and accurate monitoring and evaluation program, development of high-precision tools, and finally pay-for-performance based on the results of monitoring and evaluations. One of the necessary and useful tools in this area is implementation and use of information systems that can help improve the accuracy and precision of monitoring and evaluation, and payment based on it. Based on developing such a structure, specialized and independent private companies' capacity can be applied to measure and improve quality. It should be noted that PPP's success in health sector is largely influenced by its design and the context in which it implemented. To overcome this, each country should design and implement its own PPP models which is adapted to country's national and local conditions or context. Recommendations The results show that while PPP plans is regarded as an essential and effective way to provide social justice, implement family practice plan, and achieve universal health care, challenges such as political and financial unsustainability, delay in reimbursements, lack of stakeholder cooperation, and lack of continuous quality of services evaluation have had negative effect on the PPP plans. Overcoming challenges, more success and expanding this plan require accepting the private sector as one of the main sources of the health system, and require more support from authorities of health, national and local government. It is recommended that designers and executers of these plans adopt strategies in order to ensure adequate and timely funding. One of the solutions is attracting the cooperation of insurance companies. On the other hand, successful formulation and start-up of the plan does not guarantee its success, so a mechanism should be designed to monitor and modify the plan. The last proposal is to involve stakeholders in the process of managing and organizing the implementation of the plan, so that a common language create among all stakeholders. Limitations Overall, the information provided in the reviewed studies was incomplete. For example, many studies did not address the project implementation context (urban or rural), PPP model, role of the public and private sector, sample size, study design, and so on. It is better to refer to all aspects when conducting a study in the field of PPP plans (including setting (urban/rural/immigrant/suburb/etc), aim of study, study design, participants (governmental and nongovernmental), Period after implementation of PPP plan, Roles/task of parties (public and private), PPP model, Indicators have used to evaluate the success of PPP plan, data collection method), to better evaluate the validity of the results and the reasons for success or failure. Since most included studies were not clear on the methodology of the study, there were 2 ways for the authors: either the methodology should not be reported or the method of study should be determined by the research team based on the information presented in the article. If these cases were not reported, the final report would be incomplete, so the research team inevitably reviewed the studies and determined the study design based on the provided information, which may have caused errors in some cases. Conclusions The results of the present study provide useful information on the experiences of different countries, and on the aspects and dimensions of PPP in the of PHC services provision. Given the significant achievements of PPP in PHC, limited access to countries' experiences, increasing importance of PHC, as well as weaknesses of governments to provide these services, policy makers and authorities of health system in different countries, especially LMICs, could use this strategy as an appropriate approach to improve PHC performance. Supplemental Material Annex_1 – Supplemental material for Public-Private Partnership Policy in Primary Health Care: A Scoping Review Click here for additional data file. Supplemental material, Annex_1 for Public-Private Partnership Policy in Primary Health Care: A Scoping Review by Jafar Sadegh Tabrizi, Saber Azami-aghdash and Hojatolah Gharaee in Journal of Primary Care & Community Health Annex_2 – Supplemental material for Public-Private Partnership Policy in Primary Health Care: A Scoping Review Click here for additional data file. Supplemental material, Annex_2 for Public-Private Partnership Policy in Primary Health Care: A Scoping Review by Jafar Sadegh Tabrizi, Saber Azami-aghdash and Hojatolah Gharaee in Journal of Primary Care & Community Health Annex_3 – Supplemental material for Public-Private Partnership Policy in Primary Health Care: A Scoping Review Click here for additional data file. Supplemental material, Annex_3 for Public-Private Partnership Policy in Primary Health Care: A Scoping Review by Jafar Sadegh Tabrizi, Saber Azami-aghdash and Hojatolah Gharaee in Journal of Primary Care & Community Health Annex_4 – Supplemental material for Public-Private Partnership Policy in Primary Health Care: A Scoping Review Click here for additional data file. Supplemental material, Annex_4 for Public-Private Partnership Policy in Primary Health Care: A Scoping Review by Jafar Sadegh Tabrizi, Saber Azami-aghdash and Hojatolah Gharaee in Journal of Primary Care & Community Health Biographic_sketch – Supplemental material for Public-Private Partnership Policy in Primary Health Care: A Scoping Review Click here for additional data file. Supplemental material, Biographic_sketch for Public-Private Partnership Policy in Primary Health Care: A Scoping Review by Jafar Sadegh Tabrizi, Saber Azami-aghdash and Hojatolah Gharaee in Journal of Primary Care & Community Health

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832758/

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin. Introduction Bacterial toxins endowed with enzymatic activity generally have targets, or require co-factors, that reside in the cytoplasm of the target cell. Such is the case for the anthrax toxin produced by Bacillus anthracis . It is composed of three independent polypeptide chains, 2 of which have an enzymatic activity–edema factor (EF) and lethal factor (LF)–and one, the protective antigen (PA), which has the ability to interact with the target cell. EF is a calmodulin dependent adenylate cyclase that must thus reach the cytoplasm to become active. LF is a metalloprotease that cleaves MAP kinase kinases, all of which are cytoplasmic [1] , [2] . EF and LF reach their final destination through the help of PA. PA binds specifically to two identified anthrax toxin receptors, tumor endothelial marker 8 (TEM8, also called ANTXR1) and capillary morphogenesis 2 (CMG2, or ANTXR2), two type I membrane proteins that share extensive sequence similarity both in their extracellular and intracellular domains [3] , [4] . PA, which is produced by the bacterium as an 83 kDa form (PA83), is processed into an 63 kDa form, by host enzymes such as the endoprotease furin [5] . The thus generated PA63 has the capacity to heptamerize (PA 7mer ) into a ring-like structure, which serves as the receptor of EF and LF [6] . The hetero oligomeric complex–i.e. PA 7mer -EF/LF and receptors–is then internalized by the cell and delivered to early endosomes, where PA 7mer undergoes a conformational change that leads to its membrane insertion and pore-formation (pPA 7mer ) [2] . EF and LF are also sensitive to the acidic pH of endosomes, which leads to their partial unfolding, allowing them to be translocated through the lumen of the PA channel to the other side of the membrane [7] . Considering that PA 7mer is the receptor for EF and LF, one crucial point is that PA should not undergo endocytosis in its monomeric form, i.e. the enzymatic subunits would fail to be delivered to the cytoplasm. This is indeed what has been observed by us and others [8] , [9] leading to the notion that the anthrax toxin times its entry into the cells, in particular by triggering the activation of src -like kinases [10] . We have previously shown that processing of PA leads to a relocalization of the toxin from the glycerolipid region of the plasma membrane to lipid rafts [8] , where the receptors encounter the E3 ubiquitin ligase Cbl that modifies a juxtamembranous lysine of the cytoplasmic tail of the receptors [11] . Ubiquitination in turn promotes endocytosis of the toxin through a mechanism that requires the large GTPase dynamin [8] , [12] –involved in vesicles fission, but not the caveolar proteins caveolin-1, pointing towards a role for clathrin in anthrax toxin endocytosis [8] . We here sought to identify other molecular players involved in anthrax toxin uptake. We show that clathrin is, as predicted, involved in endocytosis but that the anthrax toxin follows a non-canonical clathrin-dependent route that depends of β-arrestins and the heterotetrameric adaptor complex AP-1. Moreover we show that endocytosis is strongly actin dependent, in contrast to endocytosis of another bacterial toxin, diphtheria toxin. Interestingly we found that actin also promotes the heptamerization of PA63, but only when it is bound to TEM8, not when bound to CMG2. Results Endocytosis of the anthrax toxin is clathrin mediated Previous studies of our laboratory indicated that anthrax toxin endocytosis in HeLa cells is independent of caveolin-1 but was affected by the overexpression of dominant negative mutants of dynamin 2 or of the accessory protein Eps15 [8] . Here we sought to confirm these findings using an independent method, namely gene silencing by RNAi, to more precisely define the molecular players involved in anthrax toxin endocytosis. As mentioned, PA63 heptamerizes at the cell surface in to PA 7mer , which is an SDS-sensitive complex. Upon arrival in endosomes, PA 7mer converts to an SDS-resistant form (pPA 7mer ), which is transmembrane. Although formation of pPA 7mer is a late event, which occurs only after sorting of the toxin-receptor complex into the intraluminal vesicles of multivesicular endosomes [13] , monitoring formation of pPA 7mer as a function of time is a convenient read-out to identify factors that affect toxin endocytosis. As shown in Fig. 1A , RNAi against either CHC or dynamin 2 strongly delayed the formation of the pPA 7mer in HeLa cells, whereas RNAi against Eps15 had no effect, despite efficient silencing of protein expression ( Fig. S1A ). We believe that the discrepancy between our present findings based on RNAi and our previous conclusions based on over-expression of dominant negative Eps15 is due to the fact that inhibition of either transferrin or PA endocytosis required strong over-expression, which might have been somewhat toxic [8] . Moreover, the effect of Eps15 over-expression was by no means as strong as the one observed for dynamin, in parallel experiments [8] . Hela cells express mainly TEM8. The above experiments therefore indicate that PA is internalized in a clathrin dependent manner when bound to TEM8. To investigate whether CMG2-mediated PA uptake is also clathrin dependent, we first made use of Baby Hamster Kidney (BHK) cells–which strongly express CMG2 ( Fig. 1B ) but have undetectable levels of TEM8 messenger (not shown)–and for which a cell line is available that allows the inducible expression of clathrin heavy chain (CHC) antisense RNA [14] . Formation of pPA 7mer was strongly delayed in CHC antisense RNA-expressing BHK cells ( Fig. 1C ). Since formation of pPA 7mer is a rather late event, we used a recently established FACS-based assay to monitor the initial step of endocytosis of the toxin [10] . PA was pre-bound to BHK cells at 4°C. Cells were then incubated at 37°C for different times prior to PA labeling at 4°C and FACS analysis. Incubation at 37°C led to a decrease of surface fluorescence for control cells, indicating that PA had been internalized but not for the CHC antisense RNA-expressing BHK cells ( Fig. S1B ). 10.1371/journal.ppat.1000792.g001 Figure 1 Anthrax toxin enter cells by clathrin mediated endocytosis. A: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the dynamin 2 (Dyn-2) or Epidermal growth factor receptor pathway substrate 15 (Eps 15). Cells were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA monomer and SDS-resistant heptamer (pPA 7mer ). B: Cell extracts (40 µg of protein) from Raw, BHK and Hela–transfected or not with mouse CMG2-4–cells were analyzed by SDS-PAGE and Western blotting for the expression of CMG2. C: Stable BHK21-tTA/anti-CHC cells maintained in tetracycline (control cells) or not (CHC AS) were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA63 and SDS-resistant heptamer (pPA 7mer ). DE: Hela cells were transfected 72 hrs with human CMG2-V5 and with control siRNAs or siRNAs against CHC. Cells were then treated as in A. Cells extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and the N-terminus of the LF target MEK1 (MEK1 (N)). To independently confirm the involvement of clathrin in CMG2-mediated PA endocytosis, we ectopically expressed CMG2 in Hela cells and performed RNAi against CHC. As shown in Fig. 1D , ectopic expression of CMG2 markedly accelerated and increased the formation of pPA 7mer , indicating that the transfected receptor was mediating PA uptake. This was confirmed by the fact that immunoprecipitation of V5-tagged CMG2 led to the co-immunoprecipitation of PA63 and pPA 7mer ( Fig. S1C , left panel). We next performed RNAi against CHC in these cells and found that formation of pPA7mer was abolished during the first 45 min ( Fig. 1E ), indicating that CHC was implicated not only in TEM8 mediated PA uptake but also CMG2-mediated uptake. Clathrin mediated toxin uptake is AP1 and β-arrestin dependent Clathrin coated vesicles are composed of three layers: the inner layer consists of the cargo, the outer layer is composed by clathrin and the middle layer is composed of adaptor and accessory/regulatory molecules that link the cargo to the clathrin coat [15] . We here investigated the possible involvement of the heterotetrameric adaptor complex AP-2–arguably the most widely used adaptor in the formation of clathrin-coated vesicles at the plasma membrane [16] –, of AP-1–which mostly operates on the Trans-Golgi Network and endosomes–, and the adaptor proteins Grb2, Dab2 and β-arrestins. β-arrestins have been shown to act as adaptors, linking cargo receptors to clathrin, either directly, or via AP2 [17] . Their involvement has been shown in particular during the endocytosis of G-coupled receptors but also single-transmembrane receptors, such as the tyrosine kinase IGF-1R [18] or the Drosophila Notch receptor [19] . Expression of these different adaptors/accessory proteins was silenced by RNAi, and formation of pPA 7mer , the SDS-resistant heptameric PA pore, was monitored as a function of time. As a second read-out, we also monitored the cleavage of the LF target MEK1, which reveals the delivery of LF to the cytoplasm. RNAi against CHC and the E3 ligase Cbl were used as positive controls. All RNAi duplexes were efficient at lowering the expression levels of the corresponding proteins ( Fig. S1A ). Due to the high degree of similarity between β-arrestin-1 and 2, silencing one gene led to a decrease in the levels of the other. Our studies can therefore not discriminate between the two forms. Silencing of Grb2, Dab2 and the µ subunit of the AP-2 complex had no effect on anthrax toxin entry into Hela cells ( Fig. 2AB ). The lack of involvement of AP-2 was somewhat surprising, raising the possibility that AP2 was insufficiently knocked down to see an effect on endocytosis. We therefore monitored the entry of a second bacterial toxin, namely diphtheria toxin, which has also been shown to enter cells via clathrin-mediated endocytosis [20] as confirmed here ( Fig. S2A ). Diphtheria toxin is an ADP-ribosylating toxin that modifies elongation factor 2 thus leading to the inhibition of protein synthesis [21] . In contrast to the anthrax toxin, entry of diphtheria toxin was delayed in AP-2, but not AP-1, RNAi treated cells, when compared to cells treated with an irrelevant RNAi ( Fig. S2B ), indicating that silencing of AP-2 was efficient. It has been previously reported for the EGF-receptor that the temperature at which the ligand is added to the cells affects the apparent AP-2 dependence of the process, i.e. EGF-R endocytosis in AP-2 RNAi treated cells still occurred when the ligand was pre-bound at 4°C, but not when the ligand was added to the cells at 37°C [22] . We therefore repeated our knockdown experiments, omitting the pre-binding of PA63 at 4°C and adding it directly to cells at 37°C. In contrast to what was observed for EGF receptor, we still found that AP-2 depletion had no effect on the formation of pPA 7mer ( Fig. S3A ). 10.1371/journal.ppat.1000792.g002 Figure 2 Endocytosis of PA is β-arrestin and AP-1 dependent. A-B: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the ubiquitin ligase E3 Cbl (Cbl), Grb2, AP-1, AP-2, β-arrestin-1 (β-Arr-1), β-arrestin-2 (β-Arr-2) or Dab2. The efficiency of siRNAs was analyzed on cell extracts (40 µg of proteins) by SDS-PAGE and western blotting ( Fig. S1 ). Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA SDS-resistant heptamer (pPA 7mer ) and N-terminus of MEK1 (MEK1 (N)). B: Levels of pPA 7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. C: Hela cells were transfected 72 hours with CMG2-V5 and control siRNAs or siRNAs against AP-1, AP-2 or β-arrestin-2 (β-Arr-2). Cell extracts were subsequently analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and N-terminus of MEK1 (MEK1 (N)). In marked contrast to the knock down of AP-2, silencing the µ subunit of the AP-1 adaptor drastically delayed the formation of the SDS-resistant pPA 7mer and LF-mediated MEK1 cleavage in Hela cells Fig. 2AB . This was not due to an off-target effect of the RNAi duplexes, since a similar effect was observed upon silencing of the γ subunit of the AP-1 complex ( Fig. S3B ). The lack of pPA 7mer formation was not due to an effect of AP-1 silencing on endosome acidification–due for example to altered trafficking of vATPase components–since diphtheria toxin, which as the anthrax toxin requires transport to an acidic compartment for cytoplasmic delivery of the enzymatic subunit, retained full activity in AP-1 depleted cells ( Fig. S2B ). In addition to a requirement for AP-1, we found that silencing β-arrestins strongly delayed formation of pPA 7mer Fig. 2AB . Knowing that Hela cells mainly express TEM8, the above experiments indicate that TEM8-mediated uptake depends on AP1 and β-arrestins but not on AP-2. To investigate whether CMG2-mediated uptake had the same requirements, we repeated the above experiments in Hela cells transfected with CMG2. As shown in Fig. 2C , formation of pPA7mer was not detected within the first 45 min when silencing AP-1 or β-arrestin, but occurred normally when silencing AP-2, showing that both receptors have the same adaptor requirements. We next investigated whether the delay in pPA 7mer formation observed in Hela cells depleted in AP-1 or β-arrestins was due to an effect on the initial step of uptake from the plasma membrane. We included the Cbl silencing in this analysis since the role of ubiquitination in the initial uptake of plasma membrane proteins is somewhat unclear in mammalian cells [23] . Using our FACS based endocytosis assay, PA was found to remain at the cell surface upon silencing of the µ subunit of AP-1, of β-arrestins as well as of Cbl, while silencing of the µ subunit of AP-2 had no effect ( Fig. 3A ). 10.1371/journal.ppat.1000792.g003 Figure 3 Endocytosis of PA involves the recruitment of Cbl by β-arrestin. A: Hela cells were transfected 72 hours with control siRNAs or siRNAs against Cbl, AP-1, AP-2, β-arrestin-1 (β-Arr-1), or β-arrestin-2 (β-Arr-2). Cells were treated with 1 µg/ml of PA63 for 1 hr at 4°C (red) and 30 min at 37°C (blue) and subsequently submitted to FACS analysis. B: Hela cells were transfected 72 hrs with TEM8/1-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, TEM8-HA and PA. C: Hela cells were transfected or not for 24 hrs with TEM8/1-HA. Immunoprecipitates against β-arrestin were analyzed by SDS-PAGE and western blotting against Cbl, TEM8-HA and β-arrestin. D: Hela cells were transfected 72 hours with human CMG2-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against CMG2-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, CMG2-HA and PA. As has been well established, endocytosis of PA requires heptamerization [1] , [9] . Therefore the lack of endocytosis of PA upon RNAi silencing of genes could be due either to an effect on heptamerization or on endocytosis itself. Heptameric PA at the cell surface is sensitive to SDS and thus migrates as a monomer on SDS gels. It can however be converted to an SDS resistant form by submitting cell extracts to a pH 4.5 treatment. Using this treatment, we could rule out that silencing of CHC, β-arrestins or AP-1 affected formation of surface PA 7mer in Hela cells ( Fig. S3C ), altogether showing that the endocytosis process itself was affected upon silencing of these genes. While the role of AP adaptor complexes in recruiting clathrin to cargo molecules has been well established, the role of β-arrestins is less clear [17] . β-arrestins have been proposed to be adaptors either for clathrin or for E3 ubiquitin ligases to the cargo receptor [24] . We therefore investigated whether silencing β-arrestins would affect ubiquitination of TEM8 or CMG2. Two isoforms of TEM8 have been reported, TEM8-1, which has a long 200 amino acid cytoplasmic tail, and TEM8-2 which has a short truncated tail ( Fig. S7A ). The long form is thought to be the more ubiquitous form. While silencing of AP-1 had no significant effect on TEM8-1 ubiquitination ( Fig. S4 ), silencing β-arrestins strongly diminished ubiquitination ( Fig. 3B , note that the apparent lack of ubiquitination in the 40 min is not due to the lower about of TEM8, since even at higher levels of TEM8, as shown in Fig. S4 , no ubiquitination signal can be detected upon β-arrestin silencing). That β-arrestins are involved in recruiting Cbl to the anthrax receptors was further supported by immunoprecipitation experiments. Immunoprecipitation of endogenous β-arrestin indeed led to the co-immunoprecipitation of Cbl and of TEM8-1-HA, when transfected ( Fig. 3C ). Cbl was not detected in immunoprecipitations against an irrelevant protein (not shown). It is worth mentioning that another E3 ligase known to be involved in endocytosis of certain transmembrane proteins such as the epithelial sodium channel [25] , namely Nedd4, is not involved in anthrax toxin endocytosis ( Fig. S3B ), underlying the specificity of the E3 ligase Cbl. We similarly found that silencing of β-arrestin, prevented PA induced ubiquitination of CMG2 upon ectopic expression in Hela cells ( Fig. 3D ). Intracellular delivery of anthrax toxin is actin dependent To further dissect the mechanisms governing anthrax toxin endocytosis, we investigated the role of actin. It has previously been reported that production of cAMP triggered by anthrax edema toxin is actin dependent [26] . More recently, Mogridge and colleagues have shown that TEM8-1, but not TEM8-2, interacts with actin and have proposed that interaction with actin modulates the affinity for PA, TEM8-2 having a higher affinity for PA than TEM8-1 [27] . During the course of the present studies, Garlick et al. identified a 33 residue stretch in the cytoplasmic tail of TEM8-1 responsible for actin binding and found that a synthetic peptide encompassing this region could trigger actin bundling in vitro [27] . We here sought to investigate what the exact role of actin in anthrax toxin endocytosis could be. Using our FACS based PA internalization assay, we monitored the effect of the G-actin sequestering drug Latrunculin A. Whereas PA was rapidly internalized in controls cells, endocytosis was completely blocked in latrunculin treated Hela cells ( Fig. 4 ). This was confirmed by the fact that the SDS-resistant pPA 7mer failed to form and MEK1 remained intact ( Fig. 5A and Fig. S5 ). 10.1371/journal.ppat.1000792.g004 Figure 4 Latrunculin A inhibits anthrax toxin uptake. Hela cells were treated 45 min at 37°C with or without Latrunculin A prior to addition of 1 µg/ml of PA63 for 1 hr at 4°C (red) followed by different incubation times at 37°C. A: Cells were then submitted to FACS analysis. B: The plot represents the mean of the percentage of PA at the cell surface for 4 experiments. Errors represent standard deviations. 10.1371/journal.ppat.1000792.g005 Figure 5 Latrunculin A prevents transport of PA to endosomes and subsequent cleavage of MEK1. A: Hela cells were treated 45 min at 37°C with or without Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal pPA 7mer , PA63 and the N-terminus of MEK1 (MEK1 (N)). Tubulin is the equal loading control. B: Cell extracts (40 µg of proteins) described in A were treated 10 min at room temperature with acid buffer pH 4.5 and analyzed by SDS-PAGE and western blotting to reveal the total heptameric PA63 (PA 7mer ) population and PA63 monomer. Involvement of actin in heptamerization of TEM8-bound PA As mentioned above, endocytosis of PA requires heptamerization [1] . To test whether latrunculin treatment affected heptamerization of PA at the cell surface, cell extracts were submitted to a pH 4.5 treatment, prior to SDS-PAGE. To our surprise, heptamerization was severely inhibited in latrunculin treated Hela cells, indicating that the drug had blocked the oligomerization process ( Fig. 5B ). This was not due to an effect of latrunculin on PA heptamerization per se , as will become apparent later. The involvement of actin in PA63 heptamerization at the surface of Hela cells was further confirmed by treating cells with blebbistatin, an inhibitor of myosin II ATPase activity [28] . Although the effect was not as strong as with latrunculin, blebbistatin significantly inhibited both the appearance of endosomal pPA 7mer (SDS resistant) and the surface PA 7mer (converted to SDS resistant by an acid treatment) ( Fig. S6 ). The above experiments show that the actin cytoskeleton promotes toxin oligomerization in Hela cells. We next wished to confirm the findings of Go et al. on the differential ability of TEM8 isoforms 1 and 2 to interact with actin [29] . Actin could be readily detected upon immuno-precipitation of HA-tagged TEM8-1 expressed in Hela cells. We could also co-immunoprecipitate the actin nucleating protein talin, its interacting protein vinculin ( Fig. 6A ) and the blebbistatin sensitive myosin II heavy chain 9, MyH9 [30] ( Fig. S7B ). Since the interaction of TEM8-1 with actin appears to be direct [27] , talin, vinculin and myosin II might be involved in regulating the dynamics of the process, as suggested by the here-observed effects of blebbistatin. This will however require further investigation. 10.1371/journal.ppat.1000792.g006 Figure 6 Binding of PA leads to the release of the actin-interacting complex form the cytosolic tail of TEM8. Hela cells were transfected 24 hrs with TEM8/1-HA (ABC) or TEM8/2-HA (B) or CMG2 (B) or constructs CTT (B) or CCT (B) or TEM8/1 Y383C-HA (C). Cells were then treated or not with 1 µg/ml of PA83 WT (ABC) or mutant resistant to furin cleavage (C) for 1 hr at 4°C. A: Cells were subsequently incubated 5 or 20 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, Talin, Vinculin, TEM8-HA and PA. B: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against HA were analyzed by SDS-PAGE and western blotting against Vinculin, Actin and HA. C: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, TEM8-HA and PA. In contrast to the observations reported by Go et al. [29] , we could also detect actin in TEM8-2-HA immunoprecipitates, albeit at far lower levels than for TEM8-1-HA. In agreement with Go et al. [29] , the Y383C mutation, which mimics a mutation found in Hyaline fibromatosis syndrome [31] , [32] , affected actin binding, but in our hands did not abolish it ( Fig. 6C ). We were however unable to detect any co-immunoprecipitation of actin with CMG2 ( Fig. 6B ). This was somewhat surprising considering the high degree of similarity between the cytoplasmic tails of CMG2 and TEM8-1 ( Fig. S7A ). Since the tail of TEM8-1 is some 50 residues longer than that of CMG2, we generated a truncated TEM8-1 matching the length of CMG2, and found that the ability to immunoprecipitate actin was unaffected (not shown). We also constructed chimeric proteins in which the transmembrane and/or cytosolic tail of CMG2 were replaced by that of TEM8-1 and found that the cytosolic tail of TEM8-1 is sufficient to confer actin-binding ability to the proteins ( Fig. 6B ). Garlick et al. have narrowed down the actin interacting domain of TEM8-1 with actin to residues 379 to 411 (DASYYGGRGVGGIKRMEVRWG E KGSTEEGAKLE) [27] , a stretch that is absent in TEM8-2 but fully conserved, with the exception of a single residue (bold underlined), in CMG2. Combined, the observations of Mogridge and ours thus suggest that direct binding of the 379–411 amino acid stretch to actin must be prevented in CMG2, through regions that differ from the tail of TEM8-1 ( Fig. S7A ). The observation that TEM8-2 can, albeit to a far lesser extend, also interacts with actin suggest that TEM8 might be able to interact with actin by more than one way, possibly directly and indirectly via the interaction with talin, vinculin and myosin II, an interaction that would also be prevented in CMG2. The effect of latrunculin A on PA heptamerization prompted us to evaluate the effect of PA binding on the interaction of TEM8 with actin. Interestingly, when TEM8-1 (WT or Y383C mutant) was immuno-precipitated from PA83 treated cells, the interaction with actin, talin and vinculin was strongly diminished Fig. 6AC . The decreased interaction with actin did not require the oligomerization of PA63 since it was equally observed when treating cells with a mutant of PA that is resistant to furin cleavage and thus remains monomeric ( Fig. 6C ) [8] . These observations show that TEM8-1, as well as TEM8-2 albeit to a far lesser extent, interact with the actin cytoskeleton, but that binding of PA83 releases this interaction. These findings provide a mechanistic explanation for the observations of Mogridge and colleagues that the association of TEM8 with the cytoskeleton correlates with weakened binding to PA [27] , [29] . They also provide the first evidence that binding of the toxin to TEM8-1 on the outside of the cell leads to a conformational change on the cytosolic side, corresponding to a form of outside-in signaling. Go et al. recently described inside-out signaling where by actin might govern the affinity of TEM8 for its ligand [29] . Thus TEM8 is, as integrins, capable of both inside-out and outside-in signaling. Receptor mobility and actin dependence Using fluorescence recovery after photobleaching (FRAP), Go et al. [29] have shown that the motilities of TEM8-1 and TEM8-2 at the cell surface differ, TEM8-1 being less mobile with a 25% immobile fraction. The difference between the two isoforms was however abolished after latrunculin treatment [29] . We made the same observations after expression of GFP-fusions of these proteins in Hela cells and FRAP analysis ( Fig. 7A for TEM8-1; TEM8-2 not shown). More specifically, the recovery half-life (t 1/2 ) of TEM8-1 was 27.4±5.4 s in control cells, with an immobile fraction of 30±9%. After latrunculin treatment, the immobile fraction dropped to 16±7% and t 1/2 decreased to 12.2±4.9 s. We extended these analyses to CMG2. As predicted from the biochemical observations, we found no effect of latrunculin A treatment on the mobility of CMG2 using FRAP: t 1/2 was 13.3±4.2 s with 9±9% immobile fraction in control cells vs. t 1/2 = 11±4.2 s in latrunculin treated cell, p = 0.21 ( Fig. 7B ). 10.1371/journal.ppat.1000792.g007 Figure 7 Effect of latrunculin A on the surface mobility of TEM8-1 and CMG2. Hela cells were transfected 48 hrs with TEM8-1-GFP (AC) or CMG2-GFP (BD) and treated (red curves) or not (blue curves) with Latrunculin A. AB: Cells were submitted to FRAP analysis in the absence of toxin treatment. CD: Cells were incubated with 3 µg/ml of mutant PA, resistant to furin cleavage and thus defective in heptamerization, at 4°C for 1 hr and subsequently warmed back to room temperature, in the presence or absence of latrunculin A, for FRAP analysis. Each curve is the average of at least 9 different cells. Error bars represent standard deviations of the mean at each time point. FRAP analysis also confirmed the observation that PA binding alleviates the interaction with actin. When cells were incubated with the PA, the latrunculin A dependence of t 1/2 for TEM8-1 was indeed strongly reduced ( Fig. 7C ) and in certain experiments even abolished. These experiments were performed using the mutant PA that cannot heptamerize, in order to focus on the effect of toxin binding, without the complications of oligomerization and internalization. The residual effect of latrunculin A could be due to the fact that not all TEM8-1 molecules are occupied by PA, and this may also account for the variability between experiments performed on transiently transfected cells, where expression levels are not tightly controlled. PA binding had not effect on the latrunculin A dependence of CMG2 mobility as expected from the biochemical observations ( Fig. 7D ). Differential receptor-dependent requirement for actin in toxin uptake The differential abilities of TEM8-1, TEM8-2 or CMG2 to interact with actin suggested that the requirement for actin in toxin uptake might depend on the receptor used. To test this, we made use of a CHO mutant cell lines that is devoid of anthrax toxin receptors (CHO ΔATR ) and thus defective in toxin binding [8] . These cells were recomplemented with TEM8-1, TEM8-2 or CMG2 and the formation of the SDS-resistant pPA 7mer was monitored. To follow the heptamerization process, we also, in a parallel set of gels, submitted cell extracts to the pH 4.5 treatment mentioned above, that converts all PA 7mer to an SDS-resistant form. As expected, recomplementation with TEM8-1 led to the same observation as in HeLa cells: latrunculin A treatment prevented heptamerization of PA, thus even after acid treatment, heptamers could not be detected ( Fig. 8A ). In cells expressing TEM8-2, formation of pPA 7mer was also completely blocked by latrunculin A. When monitoring the heptamerization process however, it became apparent that latrunculin A affected the process, but much less than for TEM8-1 (≈40% residual oligomerization, Fig. 8B ). Finally, in cells recomplemented with CMG2, formation of pPA 7mer was again drastically affected by latrunculin, but in striking contrast to the observations made for TEM8, surface heptamerization was unaltered (indicating that latrunculin does not inhibit the heptamerization process per se ) ( Fig. 8C ). The absence of pPA 7mer , despite normal heptamerization, indicates that latrunculin A blocked the endocytic process. To test this directly, we monitored endocytosis of PA in cells recomplemented with CMG2-GFP using our FACS assay, and found that indeed latrunculin A blocked endocytosis ( Fig. S8 ). 10.1371/journal.ppat.1000792.g008 Figure 8 Receptor dependent actin requirements for PA heptamerization and endocytosis. CHO ΔATR cells were transfected 48 hrs with TEM8/1-HA (A) or TEM8/2-HA (B) or CMG2/4-V5 (C) or empty pCDNA3 plasmid (control). Cells were then treated 45 min at 37°C with (filled symbols) or without (open symbols) Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Total cell extracts were either analyzed directly by SDS-PAGE and western blotting against PA or first treated 10 min at room temperature with acid buffer to reveal the total PA63 heptameric population (surface + intracellular: dashed lines). PA 7mer and pPA 7mer levels were quantified using the Typhoon scanner and normalized to 1 at the level at time 0 (1 hour at 4°C). The plot represents the mean of 3 independent experiments. Errors represent standard deviations. Altogether these observations show that 1) actin is required for efficient heptamerization of PA when bound to TEM8-1, to a lesser extent when bound to TEM8-2, but not when bound to CMG2; 2) actin is required for the endocytosis of heptameric PA, probably irrespective of the receptor, as observed for CMG2 and TEM8-2. While actin was shown to play an active role in the formation of endocytic vesicles in yeast [33] , the situation is less clear in mammalian cells. To test whether actin is systematically required for clathrin-mediated endocytosis, we investigated the effect of latrunculin on diphtheria toxin entry. As shown in Fig. S2C , latrunculin had no effect on the kinetics of diphtheria mediated ADP-ribosylation of EF-2, illustrating that clathrin-dependent endocytosis can be actin independent. Endocytosis of the anthrax toxin is clathrin mediated Previous studies of our laboratory indicated that anthrax toxin endocytosis in HeLa cells is independent of caveolin-1 but was affected by the overexpression of dominant negative mutants of dynamin 2 or of the accessory protein Eps15 [8] . Here we sought to confirm these findings using an independent method, namely gene silencing by RNAi, to more precisely define the molecular players involved in anthrax toxin endocytosis. As mentioned, PA63 heptamerizes at the cell surface in to PA 7mer , which is an SDS-sensitive complex. Upon arrival in endosomes, PA 7mer converts to an SDS-resistant form (pPA 7mer ), which is transmembrane. Although formation of pPA 7mer is a late event, which occurs only after sorting of the toxin-receptor complex into the intraluminal vesicles of multivesicular endosomes [13] , monitoring formation of pPA 7mer as a function of time is a convenient read-out to identify factors that affect toxin endocytosis. As shown in Fig. 1A , RNAi against either CHC or dynamin 2 strongly delayed the formation of the pPA 7mer in HeLa cells, whereas RNAi against Eps15 had no effect, despite efficient silencing of protein expression ( Fig. S1A ). We believe that the discrepancy between our present findings based on RNAi and our previous conclusions based on over-expression of dominant negative Eps15 is due to the fact that inhibition of either transferrin or PA endocytosis required strong over-expression, which might have been somewhat toxic [8] . Moreover, the effect of Eps15 over-expression was by no means as strong as the one observed for dynamin, in parallel experiments [8] . Hela cells express mainly TEM8. The above experiments therefore indicate that PA is internalized in a clathrin dependent manner when bound to TEM8. To investigate whether CMG2-mediated PA uptake is also clathrin dependent, we first made use of Baby Hamster Kidney (BHK) cells–which strongly express CMG2 ( Fig. 1B ) but have undetectable levels of TEM8 messenger (not shown)–and for which a cell line is available that allows the inducible expression of clathrin heavy chain (CHC) antisense RNA [14] . Formation of pPA 7mer was strongly delayed in CHC antisense RNA-expressing BHK cells ( Fig. 1C ). Since formation of pPA 7mer is a rather late event, we used a recently established FACS-based assay to monitor the initial step of endocytosis of the toxin [10] . PA was pre-bound to BHK cells at 4°C. Cells were then incubated at 37°C for different times prior to PA labeling at 4°C and FACS analysis. Incubation at 37°C led to a decrease of surface fluorescence for control cells, indicating that PA had been internalized but not for the CHC antisense RNA-expressing BHK cells ( Fig. S1B ). 10.1371/journal.ppat.1000792.g001 Figure 1 Anthrax toxin enter cells by clathrin mediated endocytosis. A: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the dynamin 2 (Dyn-2) or Epidermal growth factor receptor pathway substrate 15 (Eps 15). Cells were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA monomer and SDS-resistant heptamer (pPA 7mer ). B: Cell extracts (40 µg of protein) from Raw, BHK and Hela–transfected or not with mouse CMG2-4–cells were analyzed by SDS-PAGE and Western blotting for the expression of CMG2. C: Stable BHK21-tTA/anti-CHC cells maintained in tetracycline (control cells) or not (CHC AS) were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA63 and SDS-resistant heptamer (pPA 7mer ). DE: Hela cells were transfected 72 hrs with human CMG2-V5 and with control siRNAs or siRNAs against CHC. Cells were then treated as in A. Cells extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and the N-terminus of the LF target MEK1 (MEK1 (N)). To independently confirm the involvement of clathrin in CMG2-mediated PA endocytosis, we ectopically expressed CMG2 in Hela cells and performed RNAi against CHC. As shown in Fig. 1D , ectopic expression of CMG2 markedly accelerated and increased the formation of pPA 7mer , indicating that the transfected receptor was mediating PA uptake. This was confirmed by the fact that immunoprecipitation of V5-tagged CMG2 led to the co-immunoprecipitation of PA63 and pPA 7mer ( Fig. S1C , left panel). We next performed RNAi against CHC in these cells and found that formation of pPA7mer was abolished during the first 45 min ( Fig. 1E ), indicating that CHC was implicated not only in TEM8 mediated PA uptake but also CMG2-mediated uptake. Clathrin mediated toxin uptake is AP1 and β-arrestin dependent Clathrin coated vesicles are composed of three layers: the inner layer consists of the cargo, the outer layer is composed by clathrin and the middle layer is composed of adaptor and accessory/regulatory molecules that link the cargo to the clathrin coat [15] . We here investigated the possible involvement of the heterotetrameric adaptor complex AP-2–arguably the most widely used adaptor in the formation of clathrin-coated vesicles at the plasma membrane [16] –, of AP-1–which mostly operates on the Trans-Golgi Network and endosomes–, and the adaptor proteins Grb2, Dab2 and β-arrestins. β-arrestins have been shown to act as adaptors, linking cargo receptors to clathrin, either directly, or via AP2 [17] . Their involvement has been shown in particular during the endocytosis of G-coupled receptors but also single-transmembrane receptors, such as the tyrosine kinase IGF-1R [18] or the Drosophila Notch receptor [19] . Expression of these different adaptors/accessory proteins was silenced by RNAi, and formation of pPA 7mer , the SDS-resistant heptameric PA pore, was monitored as a function of time. As a second read-out, we also monitored the cleavage of the LF target MEK1, which reveals the delivery of LF to the cytoplasm. RNAi against CHC and the E3 ligase Cbl were used as positive controls. All RNAi duplexes were efficient at lowering the expression levels of the corresponding proteins ( Fig. S1A ). Due to the high degree of similarity between β-arrestin-1 and 2, silencing one gene led to a decrease in the levels of the other. Our studies can therefore not discriminate between the two forms. Silencing of Grb2, Dab2 and the µ subunit of the AP-2 complex had no effect on anthrax toxin entry into Hela cells ( Fig. 2AB ). The lack of involvement of AP-2 was somewhat surprising, raising the possibility that AP2 was insufficiently knocked down to see an effect on endocytosis. We therefore monitored the entry of a second bacterial toxin, namely diphtheria toxin, which has also been shown to enter cells via clathrin-mediated endocytosis [20] as confirmed here ( Fig. S2A ). Diphtheria toxin is an ADP-ribosylating toxin that modifies elongation factor 2 thus leading to the inhibition of protein synthesis [21] . In contrast to the anthrax toxin, entry of diphtheria toxin was delayed in AP-2, but not AP-1, RNAi treated cells, when compared to cells treated with an irrelevant RNAi ( Fig. S2B ), indicating that silencing of AP-2 was efficient. It has been previously reported for the EGF-receptor that the temperature at which the ligand is added to the cells affects the apparent AP-2 dependence of the process, i.e. EGF-R endocytosis in AP-2 RNAi treated cells still occurred when the ligand was pre-bound at 4°C, but not when the ligand was added to the cells at 37°C [22] . We therefore repeated our knockdown experiments, omitting the pre-binding of PA63 at 4°C and adding it directly to cells at 37°C. In contrast to what was observed for EGF receptor, we still found that AP-2 depletion had no effect on the formation of pPA 7mer ( Fig. S3A ). 10.1371/journal.ppat.1000792.g002 Figure 2 Endocytosis of PA is β-arrestin and AP-1 dependent. A-B: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the ubiquitin ligase E3 Cbl (Cbl), Grb2, AP-1, AP-2, β-arrestin-1 (β-Arr-1), β-arrestin-2 (β-Arr-2) or Dab2. The efficiency of siRNAs was analyzed on cell extracts (40 µg of proteins) by SDS-PAGE and western blotting ( Fig. S1 ). Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA SDS-resistant heptamer (pPA 7mer ) and N-terminus of MEK1 (MEK1 (N)). B: Levels of pPA 7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. C: Hela cells were transfected 72 hours with CMG2-V5 and control siRNAs or siRNAs against AP-1, AP-2 or β-arrestin-2 (β-Arr-2). Cell extracts were subsequently analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and N-terminus of MEK1 (MEK1 (N)). In marked contrast to the knock down of AP-2, silencing the µ subunit of the AP-1 adaptor drastically delayed the formation of the SDS-resistant pPA 7mer and LF-mediated MEK1 cleavage in Hela cells Fig. 2AB . This was not due to an off-target effect of the RNAi duplexes, since a similar effect was observed upon silencing of the γ subunit of the AP-1 complex ( Fig. S3B ). The lack of pPA 7mer formation was not due to an effect of AP-1 silencing on endosome acidification–due for example to altered trafficking of vATPase components–since diphtheria toxin, which as the anthrax toxin requires transport to an acidic compartment for cytoplasmic delivery of the enzymatic subunit, retained full activity in AP-1 depleted cells ( Fig. S2B ). In addition to a requirement for AP-1, we found that silencing β-arrestins strongly delayed formation of pPA 7mer Fig. 2AB . Knowing that Hela cells mainly express TEM8, the above experiments indicate that TEM8-mediated uptake depends on AP1 and β-arrestins but not on AP-2. To investigate whether CMG2-mediated uptake had the same requirements, we repeated the above experiments in Hela cells transfected with CMG2. As shown in Fig. 2C , formation of pPA7mer was not detected within the first 45 min when silencing AP-1 or β-arrestin, but occurred normally when silencing AP-2, showing that both receptors have the same adaptor requirements. We next investigated whether the delay in pPA 7mer formation observed in Hela cells depleted in AP-1 or β-arrestins was due to an effect on the initial step of uptake from the plasma membrane. We included the Cbl silencing in this analysis since the role of ubiquitination in the initial uptake of plasma membrane proteins is somewhat unclear in mammalian cells [23] . Using our FACS based endocytosis assay, PA was found to remain at the cell surface upon silencing of the µ subunit of AP-1, of β-arrestins as well as of Cbl, while silencing of the µ subunit of AP-2 had no effect ( Fig. 3A ). 10.1371/journal.ppat.1000792.g003 Figure 3 Endocytosis of PA involves the recruitment of Cbl by β-arrestin. A: Hela cells were transfected 72 hours with control siRNAs or siRNAs against Cbl, AP-1, AP-2, β-arrestin-1 (β-Arr-1), or β-arrestin-2 (β-Arr-2). Cells were treated with 1 µg/ml of PA63 for 1 hr at 4°C (red) and 30 min at 37°C (blue) and subsequently submitted to FACS analysis. B: Hela cells were transfected 72 hrs with TEM8/1-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, TEM8-HA and PA. C: Hela cells were transfected or not for 24 hrs with TEM8/1-HA. Immunoprecipitates against β-arrestin were analyzed by SDS-PAGE and western blotting against Cbl, TEM8-HA and β-arrestin. D: Hela cells were transfected 72 hours with human CMG2-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against CMG2-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, CMG2-HA and PA. As has been well established, endocytosis of PA requires heptamerization [1] , [9] . Therefore the lack of endocytosis of PA upon RNAi silencing of genes could be due either to an effect on heptamerization or on endocytosis itself. Heptameric PA at the cell surface is sensitive to SDS and thus migrates as a monomer on SDS gels. It can however be converted to an SDS resistant form by submitting cell extracts to a pH 4.5 treatment. Using this treatment, we could rule out that silencing of CHC, β-arrestins or AP-1 affected formation of surface PA 7mer in Hela cells ( Fig. S3C ), altogether showing that the endocytosis process itself was affected upon silencing of these genes. While the role of AP adaptor complexes in recruiting clathrin to cargo molecules has been well established, the role of β-arrestins is less clear [17] . β-arrestins have been proposed to be adaptors either for clathrin or for E3 ubiquitin ligases to the cargo receptor [24] . We therefore investigated whether silencing β-arrestins would affect ubiquitination of TEM8 or CMG2. Two isoforms of TEM8 have been reported, TEM8-1, which has a long 200 amino acid cytoplasmic tail, and TEM8-2 which has a short truncated tail ( Fig. S7A ). The long form is thought to be the more ubiquitous form. While silencing of AP-1 had no significant effect on TEM8-1 ubiquitination ( Fig. S4 ), silencing β-arrestins strongly diminished ubiquitination ( Fig. 3B , note that the apparent lack of ubiquitination in the 40 min is not due to the lower about of TEM8, since even at higher levels of TEM8, as shown in Fig. S4 , no ubiquitination signal can be detected upon β-arrestin silencing). That β-arrestins are involved in recruiting Cbl to the anthrax receptors was further supported by immunoprecipitation experiments. Immunoprecipitation of endogenous β-arrestin indeed led to the co-immunoprecipitation of Cbl and of TEM8-1-HA, when transfected ( Fig. 3C ). Cbl was not detected in immunoprecipitations against an irrelevant protein (not shown). It is worth mentioning that another E3 ligase known to be involved in endocytosis of certain transmembrane proteins such as the epithelial sodium channel [25] , namely Nedd4, is not involved in anthrax toxin endocytosis ( Fig. S3B ), underlying the specificity of the E3 ligase Cbl. We similarly found that silencing of β-arrestin, prevented PA induced ubiquitination of CMG2 upon ectopic expression in Hela cells ( Fig. 3D ). Intracellular delivery of anthrax toxin is actin dependent To further dissect the mechanisms governing anthrax toxin endocytosis, we investigated the role of actin. It has previously been reported that production of cAMP triggered by anthrax edema toxin is actin dependent [26] . More recently, Mogridge and colleagues have shown that TEM8-1, but not TEM8-2, interacts with actin and have proposed that interaction with actin modulates the affinity for PA, TEM8-2 having a higher affinity for PA than TEM8-1 [27] . During the course of the present studies, Garlick et al. identified a 33 residue stretch in the cytoplasmic tail of TEM8-1 responsible for actin binding and found that a synthetic peptide encompassing this region could trigger actin bundling in vitro [27] . We here sought to investigate what the exact role of actin in anthrax toxin endocytosis could be. Using our FACS based PA internalization assay, we monitored the effect of the G-actin sequestering drug Latrunculin A. Whereas PA was rapidly internalized in controls cells, endocytosis was completely blocked in latrunculin treated Hela cells ( Fig. 4 ). This was confirmed by the fact that the SDS-resistant pPA 7mer failed to form and MEK1 remained intact ( Fig. 5A and Fig. S5 ). 10.1371/journal.ppat.1000792.g004 Figure 4 Latrunculin A inhibits anthrax toxin uptake. Hela cells were treated 45 min at 37°C with or without Latrunculin A prior to addition of 1 µg/ml of PA63 for 1 hr at 4°C (red) followed by different incubation times at 37°C. A: Cells were then submitted to FACS analysis. B: The plot represents the mean of the percentage of PA at the cell surface for 4 experiments. Errors represent standard deviations. 10.1371/journal.ppat.1000792.g005 Figure 5 Latrunculin A prevents transport of PA to endosomes and subsequent cleavage of MEK1. A: Hela cells were treated 45 min at 37°C with or without Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal pPA 7mer , PA63 and the N-terminus of MEK1 (MEK1 (N)). Tubulin is the equal loading control. B: Cell extracts (40 µg of proteins) described in A were treated 10 min at room temperature with acid buffer pH 4.5 and analyzed by SDS-PAGE and western blotting to reveal the total heptameric PA63 (PA 7mer ) population and PA63 monomer. Involvement of actin in heptamerization of TEM8-bound PA As mentioned above, endocytosis of PA requires heptamerization [1] . To test whether latrunculin treatment affected heptamerization of PA at the cell surface, cell extracts were submitted to a pH 4.5 treatment, prior to SDS-PAGE. To our surprise, heptamerization was severely inhibited in latrunculin treated Hela cells, indicating that the drug had blocked the oligomerization process ( Fig. 5B ). This was not due to an effect of latrunculin on PA heptamerization per se , as will become apparent later. The involvement of actin in PA63 heptamerization at the surface of Hela cells was further confirmed by treating cells with blebbistatin, an inhibitor of myosin II ATPase activity [28] . Although the effect was not as strong as with latrunculin, blebbistatin significantly inhibited both the appearance of endosomal pPA 7mer (SDS resistant) and the surface PA 7mer (converted to SDS resistant by an acid treatment) ( Fig. S6 ). The above experiments show that the actin cytoskeleton promotes toxin oligomerization in Hela cells. We next wished to confirm the findings of Go et al. on the differential ability of TEM8 isoforms 1 and 2 to interact with actin [29] . Actin could be readily detected upon immuno-precipitation of HA-tagged TEM8-1 expressed in Hela cells. We could also co-immunoprecipitate the actin nucleating protein talin, its interacting protein vinculin ( Fig. 6A ) and the blebbistatin sensitive myosin II heavy chain 9, MyH9 [30] ( Fig. S7B ). Since the interaction of TEM8-1 with actin appears to be direct [27] , talin, vinculin and myosin II might be involved in regulating the dynamics of the process, as suggested by the here-observed effects of blebbistatin. This will however require further investigation. 10.1371/journal.ppat.1000792.g006 Figure 6 Binding of PA leads to the release of the actin-interacting complex form the cytosolic tail of TEM8. Hela cells were transfected 24 hrs with TEM8/1-HA (ABC) or TEM8/2-HA (B) or CMG2 (B) or constructs CTT (B) or CCT (B) or TEM8/1 Y383C-HA (C). Cells were then treated or not with 1 µg/ml of PA83 WT (ABC) or mutant resistant to furin cleavage (C) for 1 hr at 4°C. A: Cells were subsequently incubated 5 or 20 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, Talin, Vinculin, TEM8-HA and PA. B: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against HA were analyzed by SDS-PAGE and western blotting against Vinculin, Actin and HA. C: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, TEM8-HA and PA. In contrast to the observations reported by Go et al. [29] , we could also detect actin in TEM8-2-HA immunoprecipitates, albeit at far lower levels than for TEM8-1-HA. In agreement with Go et al. [29] , the Y383C mutation, which mimics a mutation found in Hyaline fibromatosis syndrome [31] , [32] , affected actin binding, but in our hands did not abolish it ( Fig. 6C ). We were however unable to detect any co-immunoprecipitation of actin with CMG2 ( Fig. 6B ). This was somewhat surprising considering the high degree of similarity between the cytoplasmic tails of CMG2 and TEM8-1 ( Fig. S7A ). Since the tail of TEM8-1 is some 50 residues longer than that of CMG2, we generated a truncated TEM8-1 matching the length of CMG2, and found that the ability to immunoprecipitate actin was unaffected (not shown). We also constructed chimeric proteins in which the transmembrane and/or cytosolic tail of CMG2 were replaced by that of TEM8-1 and found that the cytosolic tail of TEM8-1 is sufficient to confer actin-binding ability to the proteins ( Fig. 6B ). Garlick et al. have narrowed down the actin interacting domain of TEM8-1 with actin to residues 379 to 411 (DASYYGGRGVGGIKRMEVRWG E KGSTEEGAKLE) [27] , a stretch that is absent in TEM8-2 but fully conserved, with the exception of a single residue (bold underlined), in CMG2. Combined, the observations of Mogridge and ours thus suggest that direct binding of the 379–411 amino acid stretch to actin must be prevented in CMG2, through regions that differ from the tail of TEM8-1 ( Fig. S7A ). The observation that TEM8-2 can, albeit to a far lesser extend, also interacts with actin suggest that TEM8 might be able to interact with actin by more than one way, possibly directly and indirectly via the interaction with talin, vinculin and myosin II, an interaction that would also be prevented in CMG2. The effect of latrunculin A on PA heptamerization prompted us to evaluate the effect of PA binding on the interaction of TEM8 with actin. Interestingly, when TEM8-1 (WT or Y383C mutant) was immuno-precipitated from PA83 treated cells, the interaction with actin, talin and vinculin was strongly diminished Fig. 6AC . The decreased interaction with actin did not require the oligomerization of PA63 since it was equally observed when treating cells with a mutant of PA that is resistant to furin cleavage and thus remains monomeric ( Fig. 6C ) [8] . These observations show that TEM8-1, as well as TEM8-2 albeit to a far lesser extent, interact with the actin cytoskeleton, but that binding of PA83 releases this interaction. These findings provide a mechanistic explanation for the observations of Mogridge and colleagues that the association of TEM8 with the cytoskeleton correlates with weakened binding to PA [27] , [29] . They also provide the first evidence that binding of the toxin to TEM8-1 on the outside of the cell leads to a conformational change on the cytosolic side, corresponding to a form of outside-in signaling. Go et al. recently described inside-out signaling where by actin might govern the affinity of TEM8 for its ligand [29] . Thus TEM8 is, as integrins, capable of both inside-out and outside-in signaling. Receptor mobility and actin dependence Using fluorescence recovery after photobleaching (FRAP), Go et al. [29] have shown that the motilities of TEM8-1 and TEM8-2 at the cell surface differ, TEM8-1 being less mobile with a 25% immobile fraction. The difference between the two isoforms was however abolished after latrunculin treatment [29] . We made the same observations after expression of GFP-fusions of these proteins in Hela cells and FRAP analysis ( Fig. 7A for TEM8-1; TEM8-2 not shown). More specifically, the recovery half-life (t 1/2 ) of TEM8-1 was 27.4±5.4 s in control cells, with an immobile fraction of 30±9%. After latrunculin treatment, the immobile fraction dropped to 16±7% and t 1/2 decreased to 12.2±4.9 s. We extended these analyses to CMG2. As predicted from the biochemical observations, we found no effect of latrunculin A treatment on the mobility of CMG2 using FRAP: t 1/2 was 13.3±4.2 s with 9±9% immobile fraction in control cells vs. t 1/2 = 11±4.2 s in latrunculin treated cell, p = 0.21 ( Fig. 7B ). 10.1371/journal.ppat.1000792.g007 Figure 7 Effect of latrunculin A on the surface mobility of TEM8-1 and CMG2. Hela cells were transfected 48 hrs with TEM8-1-GFP (AC) or CMG2-GFP (BD) and treated (red curves) or not (blue curves) with Latrunculin A. AB: Cells were submitted to FRAP analysis in the absence of toxin treatment. CD: Cells were incubated with 3 µg/ml of mutant PA, resistant to furin cleavage and thus defective in heptamerization, at 4°C for 1 hr and subsequently warmed back to room temperature, in the presence or absence of latrunculin A, for FRAP analysis. Each curve is the average of at least 9 different cells. Error bars represent standard deviations of the mean at each time point. FRAP analysis also confirmed the observation that PA binding alleviates the interaction with actin. When cells were incubated with the PA, the latrunculin A dependence of t 1/2 for TEM8-1 was indeed strongly reduced ( Fig. 7C ) and in certain experiments even abolished. These experiments were performed using the mutant PA that cannot heptamerize, in order to focus on the effect of toxin binding, without the complications of oligomerization and internalization. The residual effect of latrunculin A could be due to the fact that not all TEM8-1 molecules are occupied by PA, and this may also account for the variability between experiments performed on transiently transfected cells, where expression levels are not tightly controlled. PA binding had not effect on the latrunculin A dependence of CMG2 mobility as expected from the biochemical observations ( Fig. 7D ). Differential receptor-dependent requirement for actin in toxin uptake The differential abilities of TEM8-1, TEM8-2 or CMG2 to interact with actin suggested that the requirement for actin in toxin uptake might depend on the receptor used. To test this, we made use of a CHO mutant cell lines that is devoid of anthrax toxin receptors (CHO ΔATR ) and thus defective in toxin binding [8] . These cells were recomplemented with TEM8-1, TEM8-2 or CMG2 and the formation of the SDS-resistant pPA 7mer was monitored. To follow the heptamerization process, we also, in a parallel set of gels, submitted cell extracts to the pH 4.5 treatment mentioned above, that converts all PA 7mer to an SDS-resistant form. As expected, recomplementation with TEM8-1 led to the same observation as in HeLa cells: latrunculin A treatment prevented heptamerization of PA, thus even after acid treatment, heptamers could not be detected ( Fig. 8A ). In cells expressing TEM8-2, formation of pPA 7mer was also completely blocked by latrunculin A. When monitoring the heptamerization process however, it became apparent that latrunculin A affected the process, but much less than for TEM8-1 (≈40% residual oligomerization, Fig. 8B ). Finally, in cells recomplemented with CMG2, formation of pPA 7mer was again drastically affected by latrunculin, but in striking contrast to the observations made for TEM8, surface heptamerization was unaltered (indicating that latrunculin does not inhibit the heptamerization process per se ) ( Fig. 8C ). The absence of pPA 7mer , despite normal heptamerization, indicates that latrunculin A blocked the endocytic process. To test this directly, we monitored endocytosis of PA in cells recomplemented with CMG2-GFP using our FACS assay, and found that indeed latrunculin A blocked endocytosis ( Fig. S8 ). 10.1371/journal.ppat.1000792.g008 Figure 8 Receptor dependent actin requirements for PA heptamerization and endocytosis. CHO ΔATR cells were transfected 48 hrs with TEM8/1-HA (A) or TEM8/2-HA (B) or CMG2/4-V5 (C) or empty pCDNA3 plasmid (control). Cells were then treated 45 min at 37°C with (filled symbols) or without (open symbols) Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Total cell extracts were either analyzed directly by SDS-PAGE and western blotting against PA or first treated 10 min at room temperature with acid buffer to reveal the total PA63 heptameric population (surface + intracellular: dashed lines). PA 7mer and pPA 7mer levels were quantified using the Typhoon scanner and normalized to 1 at the level at time 0 (1 hour at 4°C). The plot represents the mean of 3 independent experiments. Errors represent standard deviations. Altogether these observations show that 1) actin is required for efficient heptamerization of PA when bound to TEM8-1, to a lesser extent when bound to TEM8-2, but not when bound to CMG2; 2) actin is required for the endocytosis of heptameric PA, probably irrespective of the receptor, as observed for CMG2 and TEM8-2. While actin was shown to play an active role in the formation of endocytic vesicles in yeast [33] , the situation is less clear in mammalian cells. To test whether actin is systematically required for clathrin-mediated endocytosis, we investigated the effect of latrunculin on diphtheria toxin entry. As shown in Fig. S2C , latrunculin had no effect on the kinetics of diphtheria mediated ADP-ribosylation of EF-2, illustrating that clathrin-dependent endocytosis can be actin independent. Discussion The present work provides a more complete view of anthrax toxin endocytosis and uncovers an interesting and unexpected role for actin. In the absence of toxin, the two anthrax toxin receptors, TEM8-1 and CMG2, reside in somewhat different environments. TEM8 appears to be actively organized in an actin dependent manner, while CMG2 is either more randomly distributed or organized in an actin independent manner. Following toxin binding, processing by furin and oligomerization, the receptor tail undergoes src -dependent phosphorylation [10] and subsequent β-arrestin-dependent Cbl-mediated ubiquitination. Ubiquitination in turn allows endocytosis via a pathway that appears to be independent of AP-2, Eps15, Grb2, Dab2 (this paper) or AP180 (not shown) but requires the multimeric adaptor AP-1, dynamin, clathrin and actin. β-arrestins in clathrin-dependent anthrax toxin endocytosis Our previous findings that anthrax toxin endocytosis is dependent on dynamin, while independent of caveolin [8] , pointed towards a requirement for clathrin. We now show that silencing of CHC inhibits endocytosis of the toxin. Clathrin-mediated endocytosis however does not correspond to a unique entry route but encompassed a collection of internalization pathways that are linked by the use of the clathrin coat protein. The last years have indeed revealed a highly flexible system where each membrane cargo protein recruits, via "sorting signals" in its cytoplasmic domain, a specific set of adaptors and accessory proteins that then interact with clathrin [15] . In addition, some "add on" modules [15] maybe allow and modulate interactions with the actin cytoskeleton (see below). We had previously shown that ubiquitination of anthrax toxin receptors is necessary for the formation of pPA7 mers in endosomes [11] . We had however not determined the step at which ubiquitination is required, i.e. removal from the plasma membrane or sorting into intraluminal vesicles of multivesicular endosomes. Here we show that ubiquitination by Cbl is at least required for the initial step of endocytosis, much like what has been observed in yeast. Interestingly, we found that ubiquitination of the receptors depends on β-arrestins. These adaptor molecules have been implicated in the endocytosis of G coupled receptors, as well as single-transmembrane receptors, such as the tyrosine kinase receptor IGF-1R [18] , the Drosophila Notch receptor [19] and others (for review see [34] ). Since the tails of β-arrestins contain clathrin and AP-2 interaction motifs, they have been proposed to act as adaptors to bring clathrin to the cargo receptor [17] . Recent studies in yeast identified a family of arrestin related proteins–so called ART (arrestin-related trafficking adaptors) proteins–that mediate endocytosis of specific plasma membrane proteins. Instead of acting as an adaptor between the receptor and AP-2, ARTs were shown to act as adaptors for the Nedd4-like E3 ubiquitin ligase Rsp5 [24] , [35] . Such an adaptor function of arrestins to recruit ubiquitin ligases has also been proposed for the β2-adrenergic receptor [17] . It is likely that β-arrestin is important for recruitment of RING domain containing ligase Cbl to the cytoplasmic tail of anthrax toxin receptors. β-Arrestin antibodies indeed precipitated both TEM8-1 and Cbl and depletion of β-arrestin prevented the Cbl-mediated ubiquitination of TEM8-1. Combined with the observations in yeast, our findings illustrate that β-arrestins are able to recruit E3 ligases both of RING (here) and HECT (in yeast) domain containing E3 ligases. Role of AP-1 in clathrin-dependent anthrax toxin endocytosis Both TEM8-1 and CMG2 contain two YXXF motifs (F: bulky hydrophobic, http://elm.eu.org/ ) in their cytoplasmic tails that are potential interaction sites with heterotetrameric clathrin adaptors. The surprise was to find a requirement for AP-1, but not AP-2. The best-described role of AP-1 is in budding of clathrin coated vesicles from the Trans-Golgi network and endosomes [36] . Therefore the apparent requirement of AP-1 in anthrax endocytosis could be due to an indirect effect, such as the surface delivery of a required component. AP-1 silencing however had no effect on the level of the anthrax receptors at the cell surface–toxin binding was unaffected–, nor on endocytosis of diphtheria toxin or acidification of endosomes. Moreover, this is not the first report for a role of AP-1 at the cell surface. AP-1 was found to be required for phagocytosis in macrophages and in Dictyostelium, and was detected on nascent phagosomes [37] possibly to deliver membrane. Similarly a requirement for AP-1, but not AP-2, was found for clathrin-mediated entry of the human bacterial pathogen Listeria monocytogenes and AP-1 localized to the entering bacterium [38] again possibly to deliver membrane. Finally, AP-1 was recently found to be able to functionally compensate for AP-2 in mediating the recycling of synaptic vesicles [39] . Although we cannot fully exclude an indirect effect of AP-1 silencing, our data combined with that of recent literature do suggest that AP-1 could play a role in specific types on endocytosis. Role of actin in anthrax toxin endocytosis When analyzing the role of actin in anthrax toxin endocytosis, we found that TEM8-1 driven heptamerization of PA was strongly affected by actin depolymerizing drugs or inhibitors of the myosin II motor. Intriguingly, whereas TEM8-1 was found to interact with actin in control cells, this interaction was lost upon toxin binding. Our interpretation of these findings is that the cortical actin cytoskeleton actively organizes TEM8-1 at the cell surface, in a manner that favors the oligomerization process. Similarly Mayor and coworker recently found that actin actively organizes GPI-anchored proteins into domains [40] . We are not insinuating that GPI-anchored proteins and TEM8-1 reside in similar domains, was it only because GPI-anchored are well established to associate with lipid rafts, which is not the case for TEM8-1 at steady state [8] , [11] . The similarities between TEM8-1 and GPI-anchored proteins in terms of actin dependence do however illustrate the capacity of the cortical cytoskeleton to organize protein domains within membranes. A second surprising observation was that whereas latrunculin led to an increase in the 2 dimensional diffusion coefficient of TEM8-1 in the plasma membrane (FRAP experiments), it inhibited heptamerization. The efficiency of oligomerization depends on the collision probability between receptor bound PA monomers. Therefore, one would expect that increased motility would lead to accelerated oligomerization. This was however not the case, indicating that the actin dependent localization/organization of TEM8-1 on the membrane is more important for efficient oligomerization than the ability of this receptor to rapidly diffuse at the cell surface. Despite the high degree of similarity between the cytoplasmic tail of CMG2 and that of TEM8, and in particular the conservation of the various potential motifs for binding of actin or actin interacting proteins (multiple potential SH3 binding motifs, profiling binding motifs and possibly a distant WASP interacting motif, Fig. S7A ), we could not detect any interaction of CMG2 with actin using three totally independent methods (immunoprecipitation, FRAP and PA heptamerization experiments). Since CMG2 also contains the stretch of residues found by Garlick et al. [27] to mediate direct binding of TEM8-1 to actin, it appears that regions of the cytoplasmic tail of CMG2, that are not conserved in TEM8-1, might be involved in preventing actin binding at steady state, possibly to ensure high ligand binding affinity. The observation that actin promotes PA heptamerization when the toxin is bound to TEM8-1, but not when bound to CMG2 provides an possible explanation for an issue that has remained mysterious to us. In vitro studies have shown that the affinity of PA for the von Willebrand domain of CMG2 is some 3 orders of magnitude higher than that of PA for the von Willebrand domain of TEM8 [3] , [41] . Interestingly, Liu et al. recently reported that the apparent functional affinity of PA for its receptor is only 10 times higher for CMG2 than TEM8 [42] . These functional affinities were obtained by performing toxicity tests. It is interesting to note that in these assays performed on living cells, the apparent difference in binding is 100 lower than expected from experiments on purified von Willebrand domains. Finally when analyzing endocytosis of the toxin in cellular systems–i.e. in the absence of competition with an inactive PA mutant, we have not observed any drastic difference in the kinetics of PA heptamerization or of MEK1 cleavage between cells expressing TEM8 vs. CMG2. One speculation to explain these anomalies is that actin promoted oligomerization partially compensates for the lower affinity of PA for the von Willebrand domain of TEM8. In addition to its role in PA heptamerization, we found that actin is required for the endocytic process, irrespective of the receptor usage. For large cargoes such as the Vesicular Stomatitis virus, it has been proposed that since the transport vesicles is only incompletely coated by clathrin, actin is required to complete the process [43] . Considering the small size of the anthrax toxin, even taking into account heptamerization of PA and binding of EF and LF, partial coating seems somewhat unlikely. In this case, actin could accelerate the pinching off and detachment of the clathrin coated vesicle as observed in yeast [15] . Clarifying the role of actin in clathrin mediated endocytosis in mammalian cells will clearly require further studies using multiple systems and cargoes. Concluding remarks Combined with previous reports, our present findings provide the following sequence of events leading to endocytosis of the anthrax toxin ( Fig. 9 ). In the absence of toxin, TEM8-1 interacts with the actin cytoskeleton. This reduces its mobility and leads to some form of surface organization, all of which is not observed for CMG2. Upon secretion by the bacterium and diffusion towards target cells, PA binds to the receptors with an affinity that depends of the receptor identity (higher affinity for CMG2 than TEM8) and on the inside-out signaling, mediated by the actin cytoskeleton, that affects the conformation of the ectodomain of TEM8-1 [29] , similar to what is observed for integrins. Upon toxin binding, interaction between TEM8 and the actin cytoskeleton is released. Despite this lost interaction, the actin dependent surface organization/clustering of TEM8-1 favors the heptamerization of PA63. Heptamerization leads to the activation of src -like kinases which in turn phosphorylate the cytoplasmic tail of CMG2 [10] , as step that is required for the β-arrestin mediated Cbl-dependent ubiquitination of the receptors. The modifications finally allow the recruitment of AP-1 and clathrin, leading to clathrin coated pit formation, which could pinch off and detach through the action of the GTPase dynamin and of actin. 10.1371/journal.ppat.1000792.g009 Figure 9 Schematic representation of the endocytosis of the anthrax toxin. TEM8-1 is pre-organized at the cell surface by the cortical actin cytoskeleton while CMG2 is not. Upon PA binding, processing and oligomerization, the toxin receptor complex moves to lipid rafts. There, β-arrestin mediates the recruitment of the E3 ligase Cbl to the cytoplasmic tail of the receptor. The ubiquinated receptor subsequently recruits the heterotetrameric adaptor AP-1 and finally clathrin. Completion of the endocytic process and pinching off of the toxin containing clathrin-coated vesicle requires both actin and dynamin. β-arrestins in clathrin-dependent anthrax toxin endocytosis Our previous findings that anthrax toxin endocytosis is dependent on dynamin, while independent of caveolin [8] , pointed towards a requirement for clathrin. We now show that silencing of CHC inhibits endocytosis of the toxin. Clathrin-mediated endocytosis however does not correspond to a unique entry route but encompassed a collection of internalization pathways that are linked by the use of the clathrin coat protein. The last years have indeed revealed a highly flexible system where each membrane cargo protein recruits, via "sorting signals" in its cytoplasmic domain, a specific set of adaptors and accessory proteins that then interact with clathrin [15] . In addition, some "add on" modules [15] maybe allow and modulate interactions with the actin cytoskeleton (see below). We had previously shown that ubiquitination of anthrax toxin receptors is necessary for the formation of pPA7 mers in endosomes [11] . We had however not determined the step at which ubiquitination is required, i.e. removal from the plasma membrane or sorting into intraluminal vesicles of multivesicular endosomes. Here we show that ubiquitination by Cbl is at least required for the initial step of endocytosis, much like what has been observed in yeast. Interestingly, we found that ubiquitination of the receptors depends on β-arrestins. These adaptor molecules have been implicated in the endocytosis of G coupled receptors, as well as single-transmembrane receptors, such as the tyrosine kinase receptor IGF-1R [18] , the Drosophila Notch receptor [19] and others (for review see [34] ). Since the tails of β-arrestins contain clathrin and AP-2 interaction motifs, they have been proposed to act as adaptors to bring clathrin to the cargo receptor [17] . Recent studies in yeast identified a family of arrestin related proteins–so called ART (arrestin-related trafficking adaptors) proteins–that mediate endocytosis of specific plasma membrane proteins. Instead of acting as an adaptor between the receptor and AP-2, ARTs were shown to act as adaptors for the Nedd4-like E3 ubiquitin ligase Rsp5 [24] , [35] . Such an adaptor function of arrestins to recruit ubiquitin ligases has also been proposed for the β2-adrenergic receptor [17] . It is likely that β-arrestin is important for recruitment of RING domain containing ligase Cbl to the cytoplasmic tail of anthrax toxin receptors. β-Arrestin antibodies indeed precipitated both TEM8-1 and Cbl and depletion of β-arrestin prevented the Cbl-mediated ubiquitination of TEM8-1. Combined with the observations in yeast, our findings illustrate that β-arrestins are able to recruit E3 ligases both of RING (here) and HECT (in yeast) domain containing E3 ligases. Role of AP-1 in clathrin-dependent anthrax toxin endocytosis Both TEM8-1 and CMG2 contain two YXXF motifs (F: bulky hydrophobic, http://elm.eu.org/ ) in their cytoplasmic tails that are potential interaction sites with heterotetrameric clathrin adaptors. The surprise was to find a requirement for AP-1, but not AP-2. The best-described role of AP-1 is in budding of clathrin coated vesicles from the Trans-Golgi network and endosomes [36] . Therefore the apparent requirement of AP-1 in anthrax endocytosis could be due to an indirect effect, such as the surface delivery of a required component. AP-1 silencing however had no effect on the level of the anthrax receptors at the cell surface–toxin binding was unaffected–, nor on endocytosis of diphtheria toxin or acidification of endosomes. Moreover, this is not the first report for a role of AP-1 at the cell surface. AP-1 was found to be required for phagocytosis in macrophages and in Dictyostelium, and was detected on nascent phagosomes [37] possibly to deliver membrane. Similarly a requirement for AP-1, but not AP-2, was found for clathrin-mediated entry of the human bacterial pathogen Listeria monocytogenes and AP-1 localized to the entering bacterium [38] again possibly to deliver membrane. Finally, AP-1 was recently found to be able to functionally compensate for AP-2 in mediating the recycling of synaptic vesicles [39] . Although we cannot fully exclude an indirect effect of AP-1 silencing, our data combined with that of recent literature do suggest that AP-1 could play a role in specific types on endocytosis. Role of actin in anthrax toxin endocytosis When analyzing the role of actin in anthrax toxin endocytosis, we found that TEM8-1 driven heptamerization of PA was strongly affected by actin depolymerizing drugs or inhibitors of the myosin II motor. Intriguingly, whereas TEM8-1 was found to interact with actin in control cells, this interaction was lost upon toxin binding. Our interpretation of these findings is that the cortical actin cytoskeleton actively organizes TEM8-1 at the cell surface, in a manner that favors the oligomerization process. Similarly Mayor and coworker recently found that actin actively organizes GPI-anchored proteins into domains [40] . We are not insinuating that GPI-anchored proteins and TEM8-1 reside in similar domains, was it only because GPI-anchored are well established to associate with lipid rafts, which is not the case for TEM8-1 at steady state [8] , [11] . The similarities between TEM8-1 and GPI-anchored proteins in terms of actin dependence do however illustrate the capacity of the cortical cytoskeleton to organize protein domains within membranes. A second surprising observation was that whereas latrunculin led to an increase in the 2 dimensional diffusion coefficient of TEM8-1 in the plasma membrane (FRAP experiments), it inhibited heptamerization. The efficiency of oligomerization depends on the collision probability between receptor bound PA monomers. Therefore, one would expect that increased motility would lead to accelerated oligomerization. This was however not the case, indicating that the actin dependent localization/organization of TEM8-1 on the membrane is more important for efficient oligomerization than the ability of this receptor to rapidly diffuse at the cell surface. Despite the high degree of similarity between the cytoplasmic tail of CMG2 and that of TEM8, and in particular the conservation of the various potential motifs for binding of actin or actin interacting proteins (multiple potential SH3 binding motifs, profiling binding motifs and possibly a distant WASP interacting motif, Fig. S7A ), we could not detect any interaction of CMG2 with actin using three totally independent methods (immunoprecipitation, FRAP and PA heptamerization experiments). Since CMG2 also contains the stretch of residues found by Garlick et al. [27] to mediate direct binding of TEM8-1 to actin, it appears that regions of the cytoplasmic tail of CMG2, that are not conserved in TEM8-1, might be involved in preventing actin binding at steady state, possibly to ensure high ligand binding affinity. The observation that actin promotes PA heptamerization when the toxin is bound to TEM8-1, but not when bound to CMG2 provides an possible explanation for an issue that has remained mysterious to us. In vitro studies have shown that the affinity of PA for the von Willebrand domain of CMG2 is some 3 orders of magnitude higher than that of PA for the von Willebrand domain of TEM8 [3] , [41] . Interestingly, Liu et al. recently reported that the apparent functional affinity of PA for its receptor is only 10 times higher for CMG2 than TEM8 [42] . These functional affinities were obtained by performing toxicity tests. It is interesting to note that in these assays performed on living cells, the apparent difference in binding is 100 lower than expected from experiments on purified von Willebrand domains. Finally when analyzing endocytosis of the toxin in cellular systems–i.e. in the absence of competition with an inactive PA mutant, we have not observed any drastic difference in the kinetics of PA heptamerization or of MEK1 cleavage between cells expressing TEM8 vs. CMG2. One speculation to explain these anomalies is that actin promoted oligomerization partially compensates for the lower affinity of PA for the von Willebrand domain of TEM8. In addition to its role in PA heptamerization, we found that actin is required for the endocytic process, irrespective of the receptor usage. For large cargoes such as the Vesicular Stomatitis virus, it has been proposed that since the transport vesicles is only incompletely coated by clathrin, actin is required to complete the process [43] . Considering the small size of the anthrax toxin, even taking into account heptamerization of PA and binding of EF and LF, partial coating seems somewhat unlikely. In this case, actin could accelerate the pinching off and detachment of the clathrin coated vesicle as observed in yeast [15] . Clarifying the role of actin in clathrin mediated endocytosis in mammalian cells will clearly require further studies using multiple systems and cargoes. Concluding remarks Combined with previous reports, our present findings provide the following sequence of events leading to endocytosis of the anthrax toxin ( Fig. 9 ). In the absence of toxin, TEM8-1 interacts with the actin cytoskeleton. This reduces its mobility and leads to some form of surface organization, all of which is not observed for CMG2. Upon secretion by the bacterium and diffusion towards target cells, PA binds to the receptors with an affinity that depends of the receptor identity (higher affinity for CMG2 than TEM8) and on the inside-out signaling, mediated by the actin cytoskeleton, that affects the conformation of the ectodomain of TEM8-1 [29] , similar to what is observed for integrins. Upon toxin binding, interaction between TEM8 and the actin cytoskeleton is released. Despite this lost interaction, the actin dependent surface organization/clustering of TEM8-1 favors the heptamerization of PA63. Heptamerization leads to the activation of src -like kinases which in turn phosphorylate the cytoplasmic tail of CMG2 [10] , as step that is required for the β-arrestin mediated Cbl-dependent ubiquitination of the receptors. The modifications finally allow the recruitment of AP-1 and clathrin, leading to clathrin coated pit formation, which could pinch off and detach through the action of the GTPase dynamin and of actin. 10.1371/journal.ppat.1000792.g009 Figure 9 Schematic representation of the endocytosis of the anthrax toxin. TEM8-1 is pre-organized at the cell surface by the cortical actin cytoskeleton while CMG2 is not. Upon PA binding, processing and oligomerization, the toxin receptor complex moves to lipid rafts. There, β-arrestin mediates the recruitment of the E3 ligase Cbl to the cytoplasmic tail of the receptor. The ubiquinated receptor subsequently recruits the heterotetrameric adaptor AP-1 and finally clathrin. Completion of the endocytic process and pinching off of the toxin containing clathrin-coated vesicle requires both actin and dynamin. Materials and Methods Cells Hela cells were grown in complete Modified Eagle's medium (MEM) (Gibco) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, penicillin and streptomycin. The anthrax toxin receptor–deficient CHO (here designated as CHO ΔATR ) cells were grown in F12 medium as described previously [44] , [45] . Stable BHK21-tTA/anti-CHC cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 200 ng ml–1 puromycin and 2 µg ml–1 tetracycline [46] . To induce CHC antisense RNA expression, tetracycline was removed from the medium for 48 hours. Toxins, antibodies and reagents Anthrax toxin subunits and diphtheria toxin were a gift from S. Leppla, prepared as described [47] , including wild type PA, PA U7 in which the furin cleavage site RKKR is changed to PGG, LF. PA63 corresponds to trypsin-nicked PA83 [8] . Antibodies against anthrax PA were from the Leppla laboratory; the aerolysin mutant (G202C-I445C) named ASSP was produced in our lab as described [48] . The rat anti-mouse CMG2 was generated by genetic immunization with the mouse CMG2 construct (outsourced to Genovac). The antibody against the N-terminal peptide of MEK1 was produced in our laboratory; anti-HA, anti-GFP monoclonals and anti-HA-agarose conjugated beads from Roche (Applied Science, IN); anti-tubulin, anti-AP1, anti-vinculin, anti-talin, anti-MYH9 and anti-β arrestin from Sigma; anti-actin from Millipore; anti-EF2, anti-Cbl, anti-AP2, anti-caveolin, anti-Ubiquitin from Santa Cruz; anti-GRB2 from BD Transduction Laboratories; anti-Dab2 from Abcam laboratories, anti-CHC from Affinity Bioreagents, anti-transferrin receptor from Zymed, anti-caveolin from Santa Cruz, anti-rab 5 was kindly provided by J. Gruenberg. Mouse and rabbit HRP secondary antibodies were from Pierce, IL, rat HRP secondary antibodies were from Sigma and Alexa-conjugated secondary antibodies from Molecular Probes. Latrunculin A was purchased from Invitrogen and used at a final concentration of 0.4 µg/ml for 45 min in medium without serum at 37°C. Blebbistatin was purchased from Sigma and used at a final concentration of 50 µM for 1 hr in medium without serum at 37°C. Plasmids and transfection Human TEM8-HA/1, TEM8-HA/2 and CMG2-4-HA were cloned in pIREShyg2 as described [11] . The human TEM8/1-GFP (isoform 1) and the human TEM8/2-GFP (isoform 2) were cloned in pHS003-EGFP, kindly provided by J. Young. Human TEM8-HA/1 Y383C was generated by mutagenesis using Quickchange (Stratagene) reagent with the following primers: 5′- G GTA GAC GCC TCT TAT TGT GGT GGG AGA GGC GTT GG-3′ . The human and mouse CMG2 (isoform 4) gene tagged with a V5 epitope was cloned in pcDNA3.1/V5-HIS-TOPO expression vector. The human CMG2-GFP (isoform 1) was cloned in pHS003-EGFP as described [49] . Synthetic genes were synthesized by Geneart: CTT for extracellular part of CMG2 (amino acids 1 to 318) and transmembrane and cytosolic regions of TEM8 (amino acids 321 to 573), CCT for extracellular and transmembrane regions of CMG2 (amino acids 1 to 341) and cytosolic part of TEM8 (amino acids 344 to 573) and cloned in pIREShyg2 with a HA tag at the C-terminus. The human Eps15 gene was cloned in pEGFP-C2, kindly provided by A. Dautry-Varsat (Pasteur Institute). Plasmids were transfected into Hela cells for 48 or 72 hrs (2 µg cDNA/9.6 cm 2 plate) using Fugene (Roche Diagnostics Corporation). RNAi experiments siRNA target sequences were the following: human CHC: GGCCCAGGT GGTAATCATTTT , Grb2: AAGTTTGGAAACGATGTGCAG , NEDD4: ATGGAGTTGATTAGATTACAA , Dynamin II: CTGCAGCTCATCTTCTCAAAA , EPS15: GTGGACCAACATAATATTAAA , AP1 µ subunit: AAGGCATCAAGTATCGGAAGA , AP1 γ subunit: AACGAATGTTCGGATGACTTT , AP2 µ subunit: GGAAAACATCAAGAACAATTT , β-arrestin 1: AAAGCCTTCTGCGCGGAGAAT , β-arrestin 2: AAGGACCGCAAAGTGTTTGTG , DAB2: AAGGTTGGCCTTAGTAGTCAA , were purchased from Qiagen. Cbl siRNA was purchased from Santa Cruz (sc-29242). As control siRNA we used the following target sequence of the viral glycoprotein VSV-G: ATTGAACAAACGAAACAAGGA . To do silencing, Hela cells were transfected for 72 hours with 100 pmol/9.2 cm 2 dish of siRNA using oligofectamine (Invitrogen) transfection reagent. Total cell extracts, in vitro acid pulse and western blot analysis Hela cells were harvested, washed with PBS and homogenized by passage through a 22G injection needle in HB (HB: 2.9 mM imidazole and 250 mM sucrose, pH 7.4) containing the Roche mini tablet protease inhibitors cocktail following manufacturer's instructions. To convert surface PA 7mer to an SDS-resistant form, cell extracts were incubated at room temperature for 10 min with 145 mM NaCl and 20 mM MES-Tris, pH 4.5. Protein quantification was done with Pierce BCA kit. Proteins were loaded at 40 µg prot/lane and separated on a 4–20% acrylamide precast Novex gel (Invitrogen) under reducing conditions for PA and native condition for diphtheria toxin and transferred to nitrocellulose membranes (Schleicher and Schuell). Immunoprecipitation For immunoprecipitations, cells were lysed 30 min at 4°C in IP buffer (0.5%NP40, 500 mM NaCl, 500 mM Tris-HCl pH 7.4, 20 mM EDTA, 10 mM NaF, 2 mM benzamidine, and a cocktail of protease inhibitors, Roche), centrifuged 3 min at 2000 g and supernatants were incubated 16 h at 4°C with antibodies and beads. To follow protein Ubiquitination, 1 mM of NEM was added in the lysis buffer described above. Flow cytometric analysis BHK or Hela cells were incubated one hour at 4°C with 1 ug/ml PA63, washed and incubated different times at 37°C, washed at 4°C and incubated 5 min on ice with cold trypsin. Loosely attached cells were harvested by pipetting and stained for 30 min on ice with anti-PA antibodies, followed stained for 30 min on ice with secondary fluorescent antibodies, washed in PBS+1%FCS and then evaluated on a FACSCalibur™ (Becton Dickinson). FACS data were analyzed using FlowJo software (FlowJo, LLC). Fluorescence recovery after photobleaching (FRAP) HeLa cells were seeded and transfected with GFP-tagged receptors in 35 mm glass bottom dishes (MatTek). Samples were analyzed on a Leica SP2 confocal scanning microscope using a 63x oil immersion objective. After 10 scans of a chosen region a rectangle of 1×3 µm on the edge of a cell was irreversibly bleached using the full power of the 488, 458 and 476 nm laser lines. The bleaching resulted in a ∼80% depletion of fluorescence. Recovery of fluorescence was monitored over 80 seconds with a 2 second interval by scanning the region with a low 488 nm laser power to minimize photobleaching during sampling. The fluorescence of the bleached area was normalized at each time point using a non-bleached control area. Recovery kinetics were fitted to an exponential function. Cells Hela cells were grown in complete Modified Eagle's medium (MEM) (Gibco) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, penicillin and streptomycin. The anthrax toxin receptor–deficient CHO (here designated as CHO ΔATR ) cells were grown in F12 medium as described previously [44] , [45] . Stable BHK21-tTA/anti-CHC cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 200 ng ml–1 puromycin and 2 µg ml–1 tetracycline [46] . To induce CHC antisense RNA expression, tetracycline was removed from the medium for 48 hours. Toxins, antibodies and reagents Anthrax toxin subunits and diphtheria toxin were a gift from S. Leppla, prepared as described [47] , including wild type PA, PA U7 in which the furin cleavage site RKKR is changed to PGG, LF. PA63 corresponds to trypsin-nicked PA83 [8] . Antibodies against anthrax PA were from the Leppla laboratory; the aerolysin mutant (G202C-I445C) named ASSP was produced in our lab as described [48] . The rat anti-mouse CMG2 was generated by genetic immunization with the mouse CMG2 construct (outsourced to Genovac). The antibody against the N-terminal peptide of MEK1 was produced in our laboratory; anti-HA, anti-GFP monoclonals and anti-HA-agarose conjugated beads from Roche (Applied Science, IN); anti-tubulin, anti-AP1, anti-vinculin, anti-talin, anti-MYH9 and anti-β arrestin from Sigma; anti-actin from Millipore; anti-EF2, anti-Cbl, anti-AP2, anti-caveolin, anti-Ubiquitin from Santa Cruz; anti-GRB2 from BD Transduction Laboratories; anti-Dab2 from Abcam laboratories, anti-CHC from Affinity Bioreagents, anti-transferrin receptor from Zymed, anti-caveolin from Santa Cruz, anti-rab 5 was kindly provided by J. Gruenberg. Mouse and rabbit HRP secondary antibodies were from Pierce, IL, rat HRP secondary antibodies were from Sigma and Alexa-conjugated secondary antibodies from Molecular Probes. Latrunculin A was purchased from Invitrogen and used at a final concentration of 0.4 µg/ml for 45 min in medium without serum at 37°C. Blebbistatin was purchased from Sigma and used at a final concentration of 50 µM for 1 hr in medium without serum at 37°C. Plasmids and transfection Human TEM8-HA/1, TEM8-HA/2 and CMG2-4-HA were cloned in pIREShyg2 as described [11] . The human TEM8/1-GFP (isoform 1) and the human TEM8/2-GFP (isoform 2) were cloned in pHS003-EGFP, kindly provided by J. Young. Human TEM8-HA/1 Y383C was generated by mutagenesis using Quickchange (Stratagene) reagent with the following primers: 5′- G GTA GAC GCC TCT TAT TGT GGT GGG AGA GGC GTT GG-3′ . The human and mouse CMG2 (isoform 4) gene tagged with a V5 epitope was cloned in pcDNA3.1/V5-HIS-TOPO expression vector. The human CMG2-GFP (isoform 1) was cloned in pHS003-EGFP as described [49] . Synthetic genes were synthesized by Geneart: CTT for extracellular part of CMG2 (amino acids 1 to 318) and transmembrane and cytosolic regions of TEM8 (amino acids 321 to 573), CCT for extracellular and transmembrane regions of CMG2 (amino acids 1 to 341) and cytosolic part of TEM8 (amino acids 344 to 573) and cloned in pIREShyg2 with a HA tag at the C-terminus. The human Eps15 gene was cloned in pEGFP-C2, kindly provided by A. Dautry-Varsat (Pasteur Institute). Plasmids were transfected into Hela cells for 48 or 72 hrs (2 µg cDNA/9.6 cm 2 plate) using Fugene (Roche Diagnostics Corporation). RNAi experiments siRNA target sequences were the following: human CHC: GGCCCAGGT GGTAATCATTTT , Grb2: AAGTTTGGAAACGATGTGCAG , NEDD4: ATGGAGTTGATTAGATTACAA , Dynamin II: CTGCAGCTCATCTTCTCAAAA , EPS15: GTGGACCAACATAATATTAAA , AP1 µ subunit: AAGGCATCAAGTATCGGAAGA , AP1 γ subunit: AACGAATGTTCGGATGACTTT , AP2 µ subunit: GGAAAACATCAAGAACAATTT , β-arrestin 1: AAAGCCTTCTGCGCGGAGAAT , β-arrestin 2: AAGGACCGCAAAGTGTTTGTG , DAB2: AAGGTTGGCCTTAGTAGTCAA , were purchased from Qiagen. Cbl siRNA was purchased from Santa Cruz (sc-29242). As control siRNA we used the following target sequence of the viral glycoprotein VSV-G: ATTGAACAAACGAAACAAGGA . To do silencing, Hela cells were transfected for 72 hours with 100 pmol/9.2 cm 2 dish of siRNA using oligofectamine (Invitrogen) transfection reagent. Total cell extracts, in vitro acid pulse and western blot analysis Hela cells were harvested, washed with PBS and homogenized by passage through a 22G injection needle in HB (HB: 2.9 mM imidazole and 250 mM sucrose, pH 7.4) containing the Roche mini tablet protease inhibitors cocktail following manufacturer's instructions. To convert surface PA 7mer to an SDS-resistant form, cell extracts were incubated at room temperature for 10 min with 145 mM NaCl and 20 mM MES-Tris, pH 4.5. Protein quantification was done with Pierce BCA kit. Proteins were loaded at 40 µg prot/lane and separated on a 4–20% acrylamide precast Novex gel (Invitrogen) under reducing conditions for PA and native condition for diphtheria toxin and transferred to nitrocellulose membranes (Schleicher and Schuell). Immunoprecipitation For immunoprecipitations, cells were lysed 30 min at 4°C in IP buffer (0.5%NP40, 500 mM NaCl, 500 mM Tris-HCl pH 7.4, 20 mM EDTA, 10 mM NaF, 2 mM benzamidine, and a cocktail of protease inhibitors, Roche), centrifuged 3 min at 2000 g and supernatants were incubated 16 h at 4°C with antibodies and beads. To follow protein Ubiquitination, 1 mM of NEM was added in the lysis buffer described above. Flow cytometric analysis BHK or Hela cells were incubated one hour at 4°C with 1 ug/ml PA63, washed and incubated different times at 37°C, washed at 4°C and incubated 5 min on ice with cold trypsin. Loosely attached cells were harvested by pipetting and stained for 30 min on ice with anti-PA antibodies, followed stained for 30 min on ice with secondary fluorescent antibodies, washed in PBS+1%FCS and then evaluated on a FACSCalibur™ (Becton Dickinson). FACS data were analyzed using FlowJo software (FlowJo, LLC). Fluorescence recovery after photobleaching (FRAP) HeLa cells were seeded and transfected with GFP-tagged receptors in 35 mm glass bottom dishes (MatTek). Samples were analyzed on a Leica SP2 confocal scanning microscope using a 63x oil immersion objective. After 10 scans of a chosen region a rectangle of 1×3 µm on the edge of a cell was irreversibly bleached using the full power of the 488, 458 and 476 nm laser lines. The bleaching resulted in a ∼80% depletion of fluorescence. Recovery of fluorescence was monitored over 80 seconds with a 2 second interval by scanning the region with a low 488 nm laser power to minimize photobleaching during sampling. The fluorescence of the bleached area was normalized at each time point using a non-bleached control area. Recovery kinetics were fitted to an exponential function. Supporting Information Figure S1 Endocytosis of the anthrax toxin is clathrin mediated. A: The efficiency of the different siRNAs used in the study was analyzed on cell extracts (40 µg of proteins) by SDS-PAGE and western blotting. B: Stable BHK21-tTA/anti-CHC cells were maintained in 2 µg/ml tetracycline (control cells). To induce CHC antisense RNA expression (CHC AS), tetracycline was removed from the medium for 48 hrs. Control and CHC AS cells were treated with 1 µg/ml of PA63 for 1 hr at 4°C (red) and 5 min at 37°C (green). Cells were prepared for FACS analysis. C: Hela cells were transfected 72 hrs with human CMG2-V5 and with control siRNAs or siRNAs against CHC and incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C. Immunoprecipitation was performed against CMG2-V5 and samples were analyzed by SDS-PAGE and western blotting to reveal the different forms of PA. (0.27 MB PDF) Click here for additional data file. Figure S2 Diphtheria toxin enters cells via a clathrin and AP-2 dependent route that does not require actin. A-B: Hela cells were or not transfected for 72 hrs with the pSuper vector containing the human CHC (A) or with RNAi oligonucleotide control siRNAs or siRNAs against the µ chains of AP-1 or AP-2 (B). Cells were incubated with 500 ng/ml of trypsin-nicked DT different times at 37°C in serum free medium and extracts (40 µg of proteins) were analyzed by Native or SDS-PAGE and western blotting to reveal EF2. C: Hela cells were treated 45 min at 37°C or not with Latrunculin A. Cells were then treated in the presence or absence of Latrunculin A with 500 ng/ml of trypsin-nicked DT different times at 37°C in serum free medium. Cell extracts were prepared and the modification of EF2 analyzed on native or SDS-PAGE (40 µg of proteins). (0.48 MB PDF) Click here for additional data file. Figure S3 Endocytosis of the anthrax toxin depends on AP1 but not Nedd4 and AP2. A: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against µ chains of AP-1 or AP-2. Cells were incubated directly at 37°C with 500 ng/ml PA63 and 100 ng/ml LF for different times. 40 µg of proteins were analyzed by SDS-PAGE and western blotting to reveal PA63 and SDS-resistant heptamer (pPA 7mer ). B: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Nedd4, µ chains of AP-1 or γ chains of AP-1. Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C. 40 µg of proteins were analyzed by SDS-PAGE and western blotting to reveal PA63 and SDS-resistant heptamer (pPA 7mer ). C: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against CHC, µ chains of AP-1 or β-arrestin1 (β-Arr1). Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C and cell extracts were submitted to a pH 4.5 treatment to convert all heptameric PA to an SDS-resistant form detectable by SDS PAGE. 40 µg of proteins were analyzed by SDS-PAGE and western blotting to reveal PA. (0.60 MB PDF) Click here for additional data file. Figure S4 β-arrestin affects ubiquitination of TEM8-1. Hela cells were transfected 72 hrs with control siRNAs or siRNAs against AP-1 and β-arrestin-1 (β-Arr-1) and with TEM8/1-HA. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, TEM8-HA and PA. (0.38 MB PDF) Click here for additional data file. Figure S5 Latrunculin A prevents transport of PA to endosomes and subsequent cleavage of MEK1. Hela cells were treated 45 min at 37°C with or without Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal pPA 7mer , PA63 and the N-terminus of MEK1 (MEK1(N)) (see main Fig. 6 ) Levels of pPA 7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. (0.40 MB PDF) Click here for additional data file. Figure S6 The myosin II inhibitor blebbistatin inhibits PA heptamerization and endocytosis in Hela cells. A-C: Hela cells were treated 45 min at 37°C with or without blebbistatin, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. A: Cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal pPA 7mer and PA63. B: Levels of pPA 7mer and N-terminus of MEK1 (MEK1(N)). Levels of pPA 7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. C: Cell extracts obtained in A were submitted to a pH 4.5 treatment to convert all heptameric PA63 to an SDS-resistant form prior to SDS-PAGE and western blotting against PA. (0.36 MB PDF) Click here for additional data file. Figure S7 TEM8-1 interacts with talin, vinculin and myosin II. A: Alignment of the cytoplasmic tails of human TEM8 isoforms 1 and 2 and human CMG2 isoform 4 using the SIM software of the EXPASY server ( www.expasy.ch ). Regions of identity are shown in yellow. B: Hela cells were transfected 24 hrs with TEM8/1-HA or TEM8/2-HA. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Talin, Vinculin, TEM8-HA and the blebbistatin sensitive myosin II heavy chain 9, MyH9. C: Hela cells were transfected 24 hrs with TEM8/1-HA. Cells were treated 45 min at 37°C with or without Latrunculin A, prior to the addition or not of 500 ng/ml of PA63 for 1 hr at 4°C and 10 minutes at 37°C. Cells were solubilized in 1% Triton-X-100 at 4°C, loaded at the bottom of an Optiprep gradient, and 6 fractions were collected from the top and analyzed by SDS-PAGE and western blotting against Tem8-HA, PA, Transferrin Receptor (Trf-R) and Caveolin 1 (Cav-1). (0.24 MB PDF) Click here for additional data file. Figure S8 CMG2 mediated PA endocytosis is latrunculin dependent. CHO ΔATR cells were transfected 48 hrs with CMG2/1-GFP. Cells were treated 45 min at 37°C or not with Latrunculin A in serum free medium. Cells were then treated in the presence or absence of Latrunculin A with 1 ug/ml of PA63 for 1 hr at 4°C (red) and incubated 20 minutes at 37°C (blue), and subsequently submitted to FACS analysis. Only GFP positive cells were gated. (0.21 MB PDF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724637/

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α 1 PDX (α 1 -Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras *

Background: α 1 -Protease inhibitor Portland (α 1 PDX) and serpin B8 are proprotein convertase (PC) inhibitors whose specificity and selectivity for PCs are not understood. Results: α 1 PDX-serpin B8 and furin-PC chimeras revealed new serpin and protease (re)active-site and exosite determinants of reactivity. Conclusion: α 1 PDX reactive-site and exosite determinants may be exploited for engineering specificity and selectivity for inhibiting PCs. Significance: Specific PC inhibitors will advance understanding of PC function and therapeutics.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144527/

Sequential ubiquitination of NLRP3 by RNF125 and Cbl-b limits inflammasome activation and endotoxemia

Hyper-activation of NLRP3 inflammasomes contributes to the development of endotoxemia, but the molecular mechanisms are poorly defined. Tang et al. demonstrate that sequential ubiquitination of NLRP3 is crucial to keep NLRP3 inflammasomes in check and limits endotoxemia. Introduction Sepsis, a systemic inflammatory response syndrome (SIRS) in patients following infection or injury, causes millions of deaths globally each year ( Angus et al., 2001 ; Deutschman and Tracey, 2014 ; Hutchins et al., 2014 ; Martin et al., 2003 ). Sepsis caused by gram-negative bacteria is thought to be largely due to the host's response to LPS or endotoxins ( Bosmann and Ward, 2013 ). IL-1, particularly IL-1β, is one of several pro-inflammatory cytokines produced during SIRS that serves to initiate the host inflammatory response and to integrate nonspecific immunity. Many of IL-1's effects are beneficial in times of stress, but when produced for extended periods of time or in excessive quantities, IL-1β contributes to host morbidity and mortality ( Bosmann and Ward, 2013 ; Pruitt et al., 1995 ; Wiersinga et al., 2014 ). In addition, pyroptosis mediated by caspase-11 (Casp-11; Kayagaki et al., 2015 ; Shi et al., 2015 ), which has been shown to be a direct cytosolic receptor for LPS ( Shi et al., 2014 ), also contributes to the mortality of high-dose LPS-induced endotoxemia ( Kayagaki et al., 2011 ). The release of IL-1β and the induction of pyroptosis during sepsis are tightly controlled by a multi-protein complex, termed the nucleotide-binding oligomerization domain (NOD)–, leucine-rich repeat (LRR)–, and pyrin domain-containing protein 3 (NLRP3) inflammasome. This complex consists of NLRP3, apoptosis-associated speck-like protein (ASC), and Casp-1 ( Davis et al., 2011 ; Martinon et al., 2009 ; Martinon and Tschopp, 2004 ), or in some instances a noncanonical inflammasome involving Casp-11/gasdermin D (GSDMD; Kayagaki et al., 2015 ; Shi et al., 2015 ) rather than Casp-1. Nlrp3 −/− , Casp11 −/− , and Casp1 −/− mice are protected from LPS-induced endotoxemia under various conditions ( Kayagaki et al., 2011 ; Li et al., 1995 ; Mao et al., 2013 ; Sarkar et al., 2006 ; Wang et al., 1998 ), while a naturally occurring polymorphism for human Casp-12, a putative regulator of Casp-1, or mice deficient for Casp-12 have been linked to sepsis in both humans and mice ( Saleh et al., 2006 ; Saleh et al., 2004 ). Moreover, NLRP3 polymorphisms have been linked to sepsis morbidity ( Zhang et al., 2011 ). Thus, NLRP3 inflammasome activation appears to be a prerequisite for a competent immune response during endotoxemia. However, the mechanisms for negatively regulating the activation of the NLRP3 inflammasomes during endotoxemia are largely unknown. Casitas-B-lineage lymphoma protein-b (Cbl-b) is a RING finger E3 ubiquitin ligase that plays a crucial role in T cell activation, tolerance induction, and differentiation ( Bachmaier et al., 2000 ; Chiang et al., 2000 ; Guo et al., 2012 ; Harada et al., 2010 ; Heissmeyer et al., 2004 ; Jeon et al., 2004 ; Li et al., 2004 ; Qiao et al., 2008 ; Qiao et al., 2014 ; Qiao et al., 2013 ; Zhang et al., 2002 ), but little is known about Cbl-b's role in the innate immune response. NLRP3 undergoes polyubiquitination when BRCC3, a deubiquitinating enzyme, is inhibited ( Py et al., 2013 ), suggesting that ubiquitination is one of the major mechanisms regulating NLRP3 inflammasome activity. It was reported that tripartite motif 31 (TRIM31) may ubiquitinate NLRP3 and induce NLRP3 degradation during LPS priming ( Song et al., 2016 ), suggesting that TRIM31 may mainly affect the expression of NLRP3 during the priming phase. Therefore, the E3 ubiquitin ligase(s) specifically responsible for NLRP3 ubiquitination induced by NLRP3 inflammasome activators and the biological relevance of NLRP3 ubiquitination have not been fully characterized. Interestingly, although Cbl-b does not inhibit the signaling derived from TLRs ( Xiao et al., 2016 ), we show here that it specifically inhibits IL-1β production by macrophages induced by canonical and noncanonical NLRP3 inflammasome stimuli. Therefore, we hypothesized that Cbl-b may be the E3 ubiquitin ligase that negatively regulates NLRP3. In this study, we show that the ubiquitin-associated region (UBA) of Cbl-b binds to the K63 ubiquitin chains that attach to the NLRP3 LRR domain upon NLRP3 inflammasome stimulation. Cbl-b then targets NLRP3 for K48-linked polyubiquitination. We also found that RNF125, a RING finger E3 ubiquitin ligase, initially induces K63-linked polyubiquitination of NLRP3 within the LRR domain, which is required for the recruitment of Cbl-b. Therefore, our data collectively indicate that NLRP3 undergoes sequential K63- and K48-linked polyubiquitination mediated by RNF125 and Cbl-b, respectively, which is essential for controlling its activation and ultimately endotoxemia and polymicrobial sepsis. Results Cbl-b selectively dampens NLRP3 inflammasomes in vitro There are conflicting published views about the involvement of Cbl-b in TLR4 or MyD88-mediated signaling in monocytes or macrophages ( Bachmaier et al., 2007 ; Han et al., 2010 ; Yee and Hamerman, 2013 ). We therefore revisited whether Cbl-b plays a significant role in the production of pro-inflammatory cytokines in response to TLR ligands. We stimulated WT and Cblb −/− bone marrow–derived macrophages (BMDMs) with various TLR ligands and measured the production of TNF-α and IL-6 in cell culture supernatants. Consistent with recent published findings ( Xiao et al., 2016 ; Yee and Hamerman, 2013 ), we detected no difference in the production of these pro-inflammatory cytokines by BMDMs generated from WT and Cblb −/− mice ( Fig. 1 A ). We failed to observe any reduction in TLR4 or MyD88 degradation in BMDMs lacking Cbl-b in response to LPS stimulation ( Fig. 1 B ). Furthermore, phosphorylation of IκBα and p65 and induction of NLRP3 and pro–IL-1β were comparable between WT and Cblb −/− BMDMs in response to LPS stimulation ( Fig. 1 C ). Taken together, our data suggest that Cbl-b does not regulate TLR signaling. Figure 1. Cbl-b selectively inhibits canonical and noncanonical NLRP3 inflammasomes . (A) TNF-α and IL-6 production by BMDMs from WT and Cblb −/− mice stimulated with TLR ligands for 24 h. Med, medium; CpG, CpG oligodeoxynucleotides. (B) Immunoblot analysis of TLR4, MyD88, and CBLB of WT and Cblb −/− BMDMs stimulated with LPS (500 ng/ml) at indicated time points. (C) Immunoblot analysis of phospho-IκBα, phospho-p65, NLRP3, pro–IL-1β, and NLRP3 of WT and Cblb −/− BMDMs stimulated with LPS at various time points. (D) IL-1β production by LPS-primed BMDMs stimulated with ATP (2.5 mM) for 30 min, nigericin (20 µM) for 3 h, CTB (40 µg/ml) for 16 h, anthrax LT (500 µg/ml) for 6 h, poly(dA:dT) (1 µg/10 6 cells) for 6 h, and flagellin (6.25 µg/10 6 cells) for 4 h. (E) Immunoblots of pro–Casp-1, pro–IL-1β, NLRP3, and CBLB expression in cell extracts and Casp-1 activation and IL-1β maturation in the supernatants of LPS-primed WT and Cblb −/− BMDMs stimulated with ATP (2.5 mM) for 30 min and CTB (40 µg/ml) for 16 h. CL, cell lysate; SN, supernatants. (F) IL-1β production by LPS-primed WT and Cblb −/− BMDMs after infection with EHEC (MOI = 25:1), P. aeruginosa (MOI = 30:1), and F. novicida (MOI = 100:1) for the indicated periods of time. (G) IL-1β production by LPS-primed BMDMs from WT, Cblb −/− , Nlrp3 −/− , Cblb −/− Nlrp3 −/− , Casp11 −/− , and Cblb −/− Casp11 −/− mice stimulated with ATP (2.5 mM) for 30 min or CTB (40 µg/ml) for 16 h or infected with EHEC (MOI = 25:1) for 12 h. (H) Lactate dehydrogenase (LDH) release from LPS-primed BMDMs from WT, Cblb −/− , Nlrp3 −/− , Cblb −/− Nlrp3 −/− , Casp11 −/− , and Cblb −/ − Casp11 −/ − mice infected with EHEC (MOI = 25:1) for 12 h. Data are shown as mean ± SD. Data are representative of three independent experiments. **, P 70%. In addition to Cbl-b, we identified several additional E3 ubiquitin ligases including TRIM213, TRIM21, TRIM47, Ring finger protein 125 (RNF125), and RNF213 also binding to NLRP3 ( Fig. S4 B ). Note that among these candidates, Cbl-b was the only E3 ubiquitin ligase that bound to NLRP3 upon ATP stimulation ( Fig. S4 B ). Figure S4. Identification of E3 ubiquitin ligases that bind to NLRP3. Related to Fig. 5 . (A) Silver staining of GST or GST-NLRP3–binding proteins eluted from lysates of B6 BMDMs primed with LPS and stimulated with ATP for 5 min. (B) LC-MS/MS analysis of GST-NLRP3–binding E3 ubiquitin ligases eluted from lysates of B6 BMDMs primed with LPS and stimulated with ATP, captured, and trypsin digested. The top six hits were selected from three independent experiments. To further verify the association of these E3 ubiquitin ligases with NLRP3 and the role that they play in NLRP3 inflammasome activation, we performed Co-IP experiments, with the focus on the above E3 ubiquitin ligases identified by LC-MS/MS. We also included TRIM31 since it is reported that TRIM31 is a potential E3 ubiquitin ligase for NLRP3 ( Song et al., 2016 ). These experiments revealed that TRIM14, TRIM21, TRIM47, RNF213, and RNF125 bound to NLRP3 in WT BMDMs upon LPS priming and ATP stimulation ( Fig. 5 A ). In contrast, we could not detect an interaction between NLRP3 and TRIM31 by LC-MS/MS and Co-IP assays at the time points that we tested ( Fig. 5 A and Fig. S4 B ). To examine whether any of these E3 ubiquitin ligases are involved in NLRP3 inflammasome activation, we silenced TRIM14, TRIM21, TRIM47, TRIM31, RNF213, and RNF125 in WT BMDMs by specific siRNA. We found that silencing RNF125, but not TRIM family members and RNF213, resulted in heightened IL-1β production upon LPS priming and ATP stimulation ( Fig. 5 B ). The failure to reproduce the TRIM31–NLRP3 interaction reported by Song et al. (2016) is currently unknown. Nevertheless, our data collectively indicate that RNF125 is the initial E3 ubiquitin ligase that induces NLRP3 K63-linked polyubiquitination within its LRR domain. We should also note that HEK293T cells expressed detectable RNF125 ( Fig. 5 C ), which may explain why NLRP3 underwent both K63- and K48-linked polyubiquitination without transfection with exogenous RNF125. Figure 5. Identification of RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked polyubiquitination of NLRP3 . (A) Immunoblot analysis of NLRP3 immunoprecipitates (IP) and total cell lysates (CL) from WT BMDMs primed with LPS and stimulated with ATP (2.5 mM) for 5 and 15 min with antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, RNF213, CBLB, and NLRP3. (B) IL-1β production by BMDMs from WT mice ( n = 3) that were transfected with siRNAs specific for Trim14 , Trim21 , Trim31 , Trim47 , Rnf125 , and Rnf213 , primed with LPS, and stimulated with ATP. Data are represented as mean ± SD. P R), Myc-tagged RNF125, or Myc-RNF125 RM mutant together with His-tagged K63 ubiquitin. (D) Anti-K63 ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (E) Anti-K63 and K48 immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3, HA-tagged Cbl-b, Myc-RNF125, and His-tagged ubiquitin treated with or without AMSH. (F) Anti-Myc immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Myc-tagged RNF125, RNF125 (1–76), RNF125 (Δ1-76), or RNF126 (ZF), together with Flag-tagged NLRP3. (G) Anti-HA immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 LRR or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b UBA, Myc-RNF125 or RNF125 RM, and His-tagged K63 ubiquitin in the presence of MG132 (10 µM, overnight). (H) Anti–K48-ubiquitin immunoblot of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b, Myc-RNF125, and His-tagged K63 ubiquitin. (I) Anti-RNF125 immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT and Cblb −/− BMDMs that were primed with LPS and stimulated with ATP. (J) Anti-NLRP3 immunoblot analysis of Cbl-b immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (K) Anti-NLRP3 and anti-RNF125 immunoblot analysis of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (L) Anti-NLRP3 and anti–pro-IL-1β immunoblot analysis of WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS for 1, 2, and 4 h. Data are representative of three independent experiments. Actin was used as a loading control. To further determine whether RNF125 is the E3 ubiquitin ligase to induce K63-linked polyubiquitination of NLRP3 within its LRR domain, we knocked down endogenous RNF125 in HEK293T cells by Rnf125 siRNA and then transfected these cells with Myc-tagged RNF125 or RNF125 Ring mutant (RM), which contains C37R and A40M mutations within the RNF125 Ring finger domain ( Kim et al., 2015 ), together with Flag-tagged NLRP3 or NLRP3 LRR K/R mutant in the presence of K63 ubiquitin. Indeed, RNF125 induced NLRP3 K63-linked polyubiquitination, and this ubiquitination was abrogated in HEK293T cells expressing RNF125 RM or an NLRP3 LRR K/R mutation ( Fig. 5 C and Fig. S5 A ). To further confirm these data, we silenced the Rnf125 gene in WT BMDMs by Rnf125 siRNA, primed them with LPS, and stimulated with ATP. Silencing Rnf125 abrogated K63-linked polyubiquitination of NLRP3 ( Fig. 5 D and Fig. S5 B ). To verify this result, we employed the K63-specific deubiquitinating enzyme AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule), which has been shown to specifically remove K63-ubiquitin chains ( McCullough et al., 2004 ). We cotransfected HEK293T cells with HA-tagged Cbl-b, Flag-tagged NLRP3, Myc-tagged RNF125, and His-tagged ubiquitin. The cell lysates were immunoprecipitated with anti-Flag and then treated with or without the K63-specific deubiquitinating enzyme AMSH. As shown in Fig. 5 E , treating the Flag immunoprecipitates with AMSH abrogated K63- but not K48-linked polyubiquitination of NLRP3. These data indicate that RNF125 is the initiating E3 ubiquitin ligase by directing K63-linked polyubiquitination of the NLRP3 LRR domain. In support of these data, we found that RNF125 N-terminal 1–76 fragment interacted with NLRP3 ( Fig. 5 F ). Figure S5. Verification of RNF125 as an additional E3 ubiquitin ligase to initiate K63-linked polyubiquitination of NLRP3 and confirmation of K496 as the ubiquitination site of NLRP3. Related to Figs. 5 and 6 . (A) Anti-His and anti-Flag immunoblot analysis of Flag immunoprecipitates (IP) of lysates from Rnf125 gene–silenced HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K>R), Myc-tagged RNF125, or Myc-RNF125 RM mutant together with His-tagged K63 ubiquitin under the denaturing condition. CL, cell lysate. (B) Anti-K63 ubiquitin and anti-NLRP3 immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control (Ctr) siRNA, primed with LPS, and stimulated with ATP under the denaturing condition. (C) Anti–K48-ubiquitin and anti-Flag immunoblot of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b, Myc-RNF125, and His-tagged K63 ubiquitin under the denaturing condition. (D) Anti-His and anti-Flag immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with HA-tagged Cbl-b and His-tagged K48 ubiquitin together with Flag-tagged NLRP3 K/R mutants (K324R, K430R, K437R, K496R, and K510R) under the denaturing condition. (E) Anti–K48 ubiquitin and anti-NLRP3 immunoblot analysis of NLRP3 immunoprecipitates of lysates from Nlrp3 −/− BMDMs reconstituted with pCMV6, Flag-tagged NLRP3, or NLRP3 K496R plasmid under the denaturing condition (upper panel). Anti-NLRP3 and anti-actin immunoblots of lysates from Nlrp3 −/− BMDMs reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP under the denaturing condition (lower panel). Data are representative of two independent experiments (A, B, C, and E) and representative of three independent experiments (D). Since NLRP3 undergoes both K63- and K48-linked polyubiquitination, we hypothesized that RNF125 targets NLRP3 LRR domain for K63-linked polyubiquitination, which subsequently recruits Cbl-b to NLRP3 via its UBA domain. To test this, we transfected HEK293T cells with RNF125 or RNF125 RM, HA-tagged Cbl-b or Cbl-b UBA, Flag-tagged NLRP3 or NLRP3 LRR K/R mutant, and His-tagged K63 ubiquitin. We found that K63 ubiquitin chains were required for the binding of Cbl-b UBA to NLRP3 and for K48-linked polyubiquitination of NLRP3 ( Fig. 5, G and H and Fig. S5 C ). In further support of this notion, knocking down Rnf125 in WT BMDMs abrogated the interaction between Cbl-b and NLRP3, whereas RNF125–NLRP3 interaction remained intact in the absence of Cbl-b ( Fig. 5, I and J ). Furthermore, silencing the Rnf125 gene abrogated NLRP3 degradation ( Fig. 5 K ). Note that silencing the Rnf125 gene led to an increase in the expression of NLRP3 and pro–IL-1β during LPS priming ( Fig. 5 L ), suggesting that RNF125 inhibits the priming process. Taken together, our data collectively indicate that NLRP3 undergoes sequential K63- and K48-linked polyubiquitination mediated by RNF125 and Cbl-b, respectively. Lysine 496 is the site for the attachment of K48-linked ubiquitin chains to NLRP3 To identify which lysine residue(s) of NLRP3 is the potential ubiquitination site(s) mediated by Cbl-b, we transfected HEK293T cells with HA-tagged Cbl-b, Flag-tagged NLRP3, and His-tagged ubiquitin and then stimulated with nigericin and lysed in radioimmunoprecipitation assay (RIPA) buffer. The Flag-tagged NLRP3 was affinity purified using the FLAG M Purification Kit. The enriched fractions of NLRP3 and ubiquitinated NLRP3 were resolved on SDS-PAGE and were Coomassie blue stained. The bands corresponding to NLRP3 and ubiquitinated NLRP3 were cut into small slices in gel digested with trypsin and were processed for mass spectrometric analysis. Although we failed to identify any lysine residues within the NLRP3 LRR domain that were ubiquitinated, we did identify that 491 NHGLQK(GG) 496 ADVSAFLR 504 within the NLRP3 nucleotide-binding domain (NBD) was ubiquitinated ( Fig. 6 A ). The reason for the failure to identify the ubiquitination sites within the NLRP3 LRR domain might be due to the higher affinity of ubiquitin chains attached to K496 compared with the ubiquitin chains attached to lysine residues within the NLRP3 LRR domain. Alternatively, attachment of large ubiquitin chains to the NLRP3 LRR domain may also affect fragmentation. Furthermore, the protein mobility smear caused by polyubiquitin chains will reduce NLRP3 protein concentrations across the gel slices, which can adversely affect detection. Figure 6. Lysine 496 is the site for the attachment of K48-linked ubiquitin chains to NLRP3 . (A) Mass spectrometric analysis of Flag-tagged NLRP3 showed that K496 [ 491 NHGLQK(GG) 496 ADVSAFLR 504 ] was ubiquitinated. (B) Anti-His immunoblot analysis of Flag immunoprecipitates (IP) of lysates from HEK293T cells transfected with HA-tagged Cbl-b and His-tagged K48 ubiquitin together with Flag-tagged NLRP3 lysine (K)/arginine (R) mutants (K324R, K430R, K437R, K496R, and K510R). CL, cell lysate. (C) Anti-K48 ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from Nlrp3 −/− BMDMs reconstituted with pCMV6, Flag-tagged NLRP3, or NLRP3 K496R plasmid (upper panel). Anti-NLRP3 and anti-actin immunoblots of lysates from Nlrp3 −/− BMDMs reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP (lower panel). (D) IL-1β production by BMDMs from Nlrp3 −/− mice ( n = 3) reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP for 30 min. Data are represented as mean ± SD. **, P 70%. In addition to Cbl-b, we identified several additional E3 ubiquitin ligases including TRIM213, TRIM21, TRIM47, Ring finger protein 125 (RNF125), and RNF213 also binding to NLRP3 ( Fig. S4 B ). Note that among these candidates, Cbl-b was the only E3 ubiquitin ligase that bound to NLRP3 upon ATP stimulation ( Fig. S4 B ). Figure S4. Identification of E3 ubiquitin ligases that bind to NLRP3. Related to Fig. 5 . (A) Silver staining of GST or GST-NLRP3–binding proteins eluted from lysates of B6 BMDMs primed with LPS and stimulated with ATP for 5 min. (B) LC-MS/MS analysis of GST-NLRP3–binding E3 ubiquitin ligases eluted from lysates of B6 BMDMs primed with LPS and stimulated with ATP, captured, and trypsin digested. The top six hits were selected from three independent experiments. To further verify the association of these E3 ubiquitin ligases with NLRP3 and the role that they play in NLRP3 inflammasome activation, we performed Co-IP experiments, with the focus on the above E3 ubiquitin ligases identified by LC-MS/MS. We also included TRIM31 since it is reported that TRIM31 is a potential E3 ubiquitin ligase for NLRP3 ( Song et al., 2016 ). These experiments revealed that TRIM14, TRIM21, TRIM47, RNF213, and RNF125 bound to NLRP3 in WT BMDMs upon LPS priming and ATP stimulation ( Fig. 5 A ). In contrast, we could not detect an interaction between NLRP3 and TRIM31 by LC-MS/MS and Co-IP assays at the time points that we tested ( Fig. 5 A and Fig. S4 B ). To examine whether any of these E3 ubiquitin ligases are involved in NLRP3 inflammasome activation, we silenced TRIM14, TRIM21, TRIM47, TRIM31, RNF213, and RNF125 in WT BMDMs by specific siRNA. We found that silencing RNF125, but not TRIM family members and RNF213, resulted in heightened IL-1β production upon LPS priming and ATP stimulation ( Fig. 5 B ). The failure to reproduce the TRIM31–NLRP3 interaction reported by Song et al. (2016) is currently unknown. Nevertheless, our data collectively indicate that RNF125 is the initial E3 ubiquitin ligase that induces NLRP3 K63-linked polyubiquitination within its LRR domain. We should also note that HEK293T cells expressed detectable RNF125 ( Fig. 5 C ), which may explain why NLRP3 underwent both K63- and K48-linked polyubiquitination without transfection with exogenous RNF125. Figure 5. Identification of RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked polyubiquitination of NLRP3 . (A) Immunoblot analysis of NLRP3 immunoprecipitates (IP) and total cell lysates (CL) from WT BMDMs primed with LPS and stimulated with ATP (2.5 mM) for 5 and 15 min with antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, RNF213, CBLB, and NLRP3. (B) IL-1β production by BMDMs from WT mice ( n = 3) that were transfected with siRNAs specific for Trim14 , Trim21 , Trim31 , Trim47 , Rnf125 , and Rnf213 , primed with LPS, and stimulated with ATP. Data are represented as mean ± SD. P R), Myc-tagged RNF125, or Myc-RNF125 RM mutant together with His-tagged K63 ubiquitin. (D) Anti-K63 ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (E) Anti-K63 and K48 immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3, HA-tagged Cbl-b, Myc-RNF125, and His-tagged ubiquitin treated with or without AMSH. (F) Anti-Myc immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Myc-tagged RNF125, RNF125 (1–76), RNF125 (Δ1-76), or RNF126 (ZF), together with Flag-tagged NLRP3. (G) Anti-HA immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 LRR or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b UBA, Myc-RNF125 or RNF125 RM, and His-tagged K63 ubiquitin in the presence of MG132 (10 µM, overnight). (H) Anti–K48-ubiquitin immunoblot of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b, Myc-RNF125, and His-tagged K63 ubiquitin. (I) Anti-RNF125 immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT and Cblb −/− BMDMs that were primed with LPS and stimulated with ATP. (J) Anti-NLRP3 immunoblot analysis of Cbl-b immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (K) Anti-NLRP3 and anti-RNF125 immunoblot analysis of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (L) Anti-NLRP3 and anti–pro-IL-1β immunoblot analysis of WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS for 1, 2, and 4 h. Data are representative of three independent experiments. Actin was used as a loading control. To further determine whether RNF125 is the E3 ubiquitin ligase to induce K63-linked polyubiquitination of NLRP3 within its LRR domain, we knocked down endogenous RNF125 in HEK293T cells by Rnf125 siRNA and then transfected these cells with Myc-tagged RNF125 or RNF125 Ring mutant (RM), which contains C37R and A40M mutations within the RNF125 Ring finger domain ( Kim et al., 2015 ), together with Flag-tagged NLRP3 or NLRP3 LRR K/R mutant in the presence of K63 ubiquitin. Indeed, RNF125 induced NLRP3 K63-linked polyubiquitination, and this ubiquitination was abrogated in HEK293T cells expressing RNF125 RM or an NLRP3 LRR K/R mutation ( Fig. 5 C and Fig. S5 A ). To further confirm these data, we silenced the Rnf125 gene in WT BMDMs by Rnf125 siRNA, primed them with LPS, and stimulated with ATP. Silencing Rnf125 abrogated K63-linked polyubiquitination of NLRP3 ( Fig. 5 D and Fig. S5 B ). To verify this result, we employed the K63-specific deubiquitinating enzyme AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule), which has been shown to specifically remove K63-ubiquitin chains ( McCullough et al., 2004 ). We cotransfected HEK293T cells with HA-tagged Cbl-b, Flag-tagged NLRP3, Myc-tagged RNF125, and His-tagged ubiquitin. The cell lysates were immunoprecipitated with anti-Flag and then treated with or without the K63-specific deubiquitinating enzyme AMSH. As shown in Fig. 5 E , treating the Flag immunoprecipitates with AMSH abrogated K63- but not K48-linked polyubiquitination of NLRP3. These data indicate that RNF125 is the initiating E3 ubiquitin ligase by directing K63-linked polyubiquitination of the NLRP3 LRR domain. In support of these data, we found that RNF125 N-terminal 1–76 fragment interacted with NLRP3 ( Fig. 5 F ). Figure S5. Verification of RNF125 as an additional E3 ubiquitin ligase to initiate K63-linked polyubiquitination of NLRP3 and confirmation of K496 as the ubiquitination site of NLRP3. Related to Figs. 5 and 6 . (A) Anti-His and anti-Flag immunoblot analysis of Flag immunoprecipitates (IP) of lysates from Rnf125 gene–silenced HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K>R), Myc-tagged RNF125, or Myc-RNF125 RM mutant together with His-tagged K63 ubiquitin under the denaturing condition. CL, cell lysate. (B) Anti-K63 ubiquitin and anti-NLRP3 immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control (Ctr) siRNA, primed with LPS, and stimulated with ATP under the denaturing condition. (C) Anti–K48-ubiquitin and anti-Flag immunoblot of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b, Myc-RNF125, and His-tagged K63 ubiquitin under the denaturing condition. (D) Anti-His and anti-Flag immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with HA-tagged Cbl-b and His-tagged K48 ubiquitin together with Flag-tagged NLRP3 K/R mutants (K324R, K430R, K437R, K496R, and K510R) under the denaturing condition. (E) Anti–K48 ubiquitin and anti-NLRP3 immunoblot analysis of NLRP3 immunoprecipitates of lysates from Nlrp3 −/− BMDMs reconstituted with pCMV6, Flag-tagged NLRP3, or NLRP3 K496R plasmid under the denaturing condition (upper panel). Anti-NLRP3 and anti-actin immunoblots of lysates from Nlrp3 −/− BMDMs reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP under the denaturing condition (lower panel). Data are representative of two independent experiments (A, B, C, and E) and representative of three independent experiments (D). Since NLRP3 undergoes both K63- and K48-linked polyubiquitination, we hypothesized that RNF125 targets NLRP3 LRR domain for K63-linked polyubiquitination, which subsequently recruits Cbl-b to NLRP3 via its UBA domain. To test this, we transfected HEK293T cells with RNF125 or RNF125 RM, HA-tagged Cbl-b or Cbl-b UBA, Flag-tagged NLRP3 or NLRP3 LRR K/R mutant, and His-tagged K63 ubiquitin. We found that K63 ubiquitin chains were required for the binding of Cbl-b UBA to NLRP3 and for K48-linked polyubiquitination of NLRP3 ( Fig. 5, G and H and Fig. S5 C ). In further support of this notion, knocking down Rnf125 in WT BMDMs abrogated the interaction between Cbl-b and NLRP3, whereas RNF125–NLRP3 interaction remained intact in the absence of Cbl-b ( Fig. 5, I and J ). Furthermore, silencing the Rnf125 gene abrogated NLRP3 degradation ( Fig. 5 K ). Note that silencing the Rnf125 gene led to an increase in the expression of NLRP3 and pro–IL-1β during LPS priming ( Fig. 5 L ), suggesting that RNF125 inhibits the priming process. Taken together, our data collectively indicate that NLRP3 undergoes sequential K63- and K48-linked polyubiquitination mediated by RNF125 and Cbl-b, respectively. Lysine 496 is the site for the attachment of K48-linked ubiquitin chains to NLRP3 To identify which lysine residue(s) of NLRP3 is the potential ubiquitination site(s) mediated by Cbl-b, we transfected HEK293T cells with HA-tagged Cbl-b, Flag-tagged NLRP3, and His-tagged ubiquitin and then stimulated with nigericin and lysed in radioimmunoprecipitation assay (RIPA) buffer. The Flag-tagged NLRP3 was affinity purified using the FLAG M Purification Kit. The enriched fractions of NLRP3 and ubiquitinated NLRP3 were resolved on SDS-PAGE and were Coomassie blue stained. The bands corresponding to NLRP3 and ubiquitinated NLRP3 were cut into small slices in gel digested with trypsin and were processed for mass spectrometric analysis. Although we failed to identify any lysine residues within the NLRP3 LRR domain that were ubiquitinated, we did identify that 491 NHGLQK(GG) 496 ADVSAFLR 504 within the NLRP3 nucleotide-binding domain (NBD) was ubiquitinated ( Fig. 6 A ). The reason for the failure to identify the ubiquitination sites within the NLRP3 LRR domain might be due to the higher affinity of ubiquitin chains attached to K496 compared with the ubiquitin chains attached to lysine residues within the NLRP3 LRR domain. Alternatively, attachment of large ubiquitin chains to the NLRP3 LRR domain may also affect fragmentation. Furthermore, the protein mobility smear caused by polyubiquitin chains will reduce NLRP3 protein concentrations across the gel slices, which can adversely affect detection. Figure 6. Lysine 496 is the site for the attachment of K48-linked ubiquitin chains to NLRP3 . (A) Mass spectrometric analysis of Flag-tagged NLRP3 showed that K496 [ 491 NHGLQK(GG) 496 ADVSAFLR 504 ] was ubiquitinated. (B) Anti-His immunoblot analysis of Flag immunoprecipitates (IP) of lysates from HEK293T cells transfected with HA-tagged Cbl-b and His-tagged K48 ubiquitin together with Flag-tagged NLRP3 lysine (K)/arginine (R) mutants (K324R, K430R, K437R, K496R, and K510R). CL, cell lysate. (C) Anti-K48 ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from Nlrp3 −/− BMDMs reconstituted with pCMV6, Flag-tagged NLRP3, or NLRP3 K496R plasmid (upper panel). Anti-NLRP3 and anti-actin immunoblots of lysates from Nlrp3 −/− BMDMs reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP (lower panel). (D) IL-1β production by BMDMs from Nlrp3 −/− mice ( n = 3) reconstituted with Flag-tagged NLRP3 or NLRP3 K496R plasmid, primed with LPS, and stimulated with ATP for 30 min. Data are represented as mean ± SD. **, P < 0.01 ( Nlrp3 −/− BMDMs transfected with WT NLRP3 vs. with NLRP3 K496R); Student's t test. Data are representative of two independent experiments (A) and representative of three independent experiments (B–D). Since the NLRP3 LRR domain underwent K63-linked polyubiquitination, we reasoned that Cbl-b mediates K48-linked polyubiquitination within the NBD domain of NLRP3. To confirm whether K496 is the ubiquitination site of NLRP3, we generated NLRP3 K496R mutant. We also used the UbPred, a random forest-based predictor of potential ubiquitination sites in proteins ( http://www.ubpred.org ) to predict the potential ubiquitination sites within the NLRP3 NBD domain. We identified four other lysine residues within the NBD domain with low to medium confidence (K324, K430, and K510) by the UbPred, of which K324 and K510 were also detected by LS-MS/MS analysis ( Fig. 6 A ). We also included K437, which is within the NLRP3 NBD domain, although UbPred shows this lysine residue with no confidence. We generated NLRP3 K324R, K430R, K437R, and K510R mutants and transfected HEK293T cells with HA-tagged Cbl-b, Flag-tagged NLRP3 or NLRP3 K/R mutants, and His-tagged K48 ubiquitin. Mutation of NLRP3 at K496R completely abrogated K48-linked polyubiquitination of NLRP3 induced by Cbl-b ( Fig. 6 B and Fig. S5 D ). Reconstituting Nlrp3 −/− BMDMs with WT NLRP3, but not NLRP3 K496R, rescued K48-linked ubiquitination and subsequent degradation of NLRP3 ( Fig. 6 C and Fig. S5 E ), which correlated with heightened IL-1β production by Nlrp3 −/− BMDMs reconstituted with NLRP3 K496R ( Fig. 6 D ). Therefore, our data collectively indicate that lysine 496 within the NLRP3 NBD domain is the site for K48-linked ubiquitin chain attachment. In vivo silencing of Rnf125 increases the susceptibility of mice to septic shock induced by a sub-lethal dose of LPS Since RNF125 is required for the initiation of K63-linked polyubiquitination of the NLRP3 LRR domain, which recruits Cbl-b, it is likely that RNF125-deficient mice should also be highly susceptible to LPS-induced endotoxemia as shown in Cblb −/− mice. Recently, we established a protocol that silences the Cblb gene in WT mice in vivo ( Xiao et al., 2016 ). To test this, we silenced the Rnf125 gene by in vivo delivery of Rnf125 -specific siRNA via tail vein injection. As expected, all the mice receiving Rnf125 siRNA died within 40 h after injection with a sub-lethal dose of LPS, whereas 60% of mice receiving control siRNA survived ( Fig. 7 A ), which correlated with heightened IL-1β and TNF-α in the sera ( Fig. 7 C ). These data indicate that the phenotype of mice with Rnf125 gene silencing recapitulated the one observed in Cblb −/− mice. Figure 7. Systemic in vivo delivery of Rnf125 -specific siRNA renders mice susceptible to endotoxemia induced by a sub-lethal dose of LPS. (A) Kaplan-Meier survival curve of C57BL/6 mice treated with in vivo–grade Rnf125 -specific siRNA or a nonsense siRNA (2 mg/kg/mouse) via tail vein injection for 24 h before LPS injection (5 mg/kg). *, P < 0.05 by log-rank test. (B) Immunoblot analysis for RNF125 in spleen cells from C57BL/6 mice that were treated with the control siRNA or Rnf125- specific siRNA. Actin was used as a loading control (Ctr). (C) Serum IL-1β and TNF-α levels from C57BL/6 mice (five mice per group) that were treated with the control siRNA or Rnf125- specific siRNA before LPS injection (1 mg/kg). Data are shown as mean ± SD. *, P < 0.05; **, P < 0.01; Student's t test. Data are representative of three independent experiments (A and C) and representative of two independent experiments (B). Discussion It has been shown that NLRP3 ubiquitination negatively regulates NLRP3 inflammasome in vitro ( Py et al., 2013 ), but the E3 ubiquitin ligase or ligases that ubiquitinate NLRP3 are not fully defined. Furthermore, the biological relevance of in vivo NLRP3 ubiquitination remains to be established. Here, we first report that NLRP3 undergoes sequential K63- and K48-linked polyubiquitination, which is mediated by E3 ubiquitin ligases RNF125 and Cbl-b respectively ( Figs. 4 and 5 ). Consistent with this, Cbl-b deficiency or inactivation leads to hyper-activation of canonical and noncanonical NLRP3 inflammasomes ( Fig. 1, D–H ). Mice deficient for Cbl-b are extremely sensitive to a sub-lethal dose of LPS, which is rescued by IL-1RA treatment or introduction of NLRP3 deficiency ( Fig. 2, A and E ). Furthermore, mice with Rnf125 gene silencing are also highly susceptible to a sub-lethal dose of LPS-induced endotoxemia ( Fig. 7 A ). Therefore, our data demonstrate that RNF125 and Cbl-b coordinately regulate NLRP3 inflammasome activation by targeting NLRP3 for ubiquitination in vitro and in vivo ( Fig. 8 ). Figure 8. Schematic model for RNF125 and Cbl-b in LPS-induced endotoxemia. When mice receive a sub-lethal dose of LPS, LPS triggers (1) TLR4 to initiate a MyD88-dependent signaling pathway, which activates NF-κB and induces the expression of NLRP3 and pro–IL-1β and (2) TRIF-dependent pathway that induces the expression of Casp-11. LPS also undergoes endocytosis and binds its cytosolic sensor Casp-11 to induce oligomerizations of Casp-11, which cleaves GSDMD to liberate its N-terminal domain (NTD). NTD may trigger the activation of the NLRP3 inflammasome, leading to the release of IL-1β without inducing pyroptosis. RNF125 targets the NLRP3 LRR domain for K63-linked polyubiquitination. The K63 ubiquitin chains attached to the NLRP3 LRR domain recruit Cbl-b by binding its UBA region. Cbl-b then ubiquitinates NLRP3 at K496 within its NBD and targets NLRP3 to the proteasome for degradation, thus keeping the NLRP3 inflammasome in check. In the absence or inactivation of Cbl-b, mice are highly susceptible to LPS-induced endotoxemia. However, when mice receive a lethal dose of LPS exposure, LPS binds to Casp-11 and mainly triggers Casp-11–mediated pyroptosis via GSDMD. Cbl-b is unable to control this noncanonical inflammasome-induced lethality. CARD, caspase recruitment domain; PYD, pyrin domain; TKB; protein tyrosine kinase-binding; L, linker region; RF, Ring finger; P, peptide; PR, proline-rich region; Toll/IL‐1R domain–containing adaptor‐inducing IFN‐β. Red arrows indicate activation or induction, whereas the blue dashed arrow indicates possible activation. It has been shown that inhibiting BRCC3, a deubiquitinating enzyme, results in NLRP3 ubiquitination, thus suppressing NLRP3 inflammasome activity ( Py et al., 2013 ). However, the E3 ubiquitin ligases responsible for ubiquitinating NLRP3 are not fully characterized. It is reported that TRIM31 may be the E3 ubiquitin ligase for NLRP3 ( Song et al., 2016 ), but the in vivo relevance of this report has not been established because of a lack of TRIM31 E3 ubiquitin ligase dead knock-in mice. Furthermore, loss of TRIM31 also only moderately slows the kinetics of NLRP3 degradation induced by LPS in the presence of cycloheximide ( Song et al., 2016 ). In addition, none of the NLRP3 inflammasome stimuli were used to induce NLRP3 degradation ( Song et al., 2016 ). Therefore, whether NLRP3 degradation observed in this study is induced by NLRP3 inflammasome stimuli is unknown. In our GST-NLRP3 pull-down assay coupled with LC-MS/MS analysis, we found that TRIM14, TRIM21, TRIM47, RNF125, and RNF213 but not TRIM31 associated with NLRP3 ( Fig. 5 A , upper panel, and Fig. S4 B ). Furthermore, silencing the Trim31 gene in WT BMDMs does not affect LPS-primed, ATP-stimulated production of IL-1β ( Fig. 5 B ). Therefore, TRIM31 may not be the E3 ubiquitin ligase that targets NLRP3 for ubiquitination. Intriguingly, we found that Cbl-b binds to the NLRP3 LRR domain via its UBA region ( Fig. 3, D–F ), suggesting that Cbl-b binds to the ubiquitin chains that attach to the NLRP3 LRR domain. Consistent with this notion, mutating the 11 lysine residues to arginine within the LRR domain abrogates Cbl-b–NLRP3 interaction ( Fig. 3 G ). Reconstituting Nlrp3 −/− BMDMs with the NLRP3 K/R mutant results in heightened IL-1β production in response to LPS priming and ATP stimulation ( Fig. 3 H ). The binding of Cbl-b to the ubiquitin chains attached to the NLRP3 LRR domain is critical for NLRP3 degradation in the proteasome because NLRP3 degradation is abrogated in macrophages expressing inactive Cbl-b upon ATP stimulation or EHEC infection ( Fig. 4 E ). Through a GST-NLRP3 pull-down assay coupled with LC-MS/MS and Co-IP experiments, we identified RNF125 as an initial E3 ubiquitin ligase to induce K63-linked NLRP3 ubiquitination within its LRR domain ( Fig. 5 and Fig. S5, A–C ). This results in the recruitment of Cbl-b via its UBA region ( Figs. 3 and 5 ), enabling Cbl-b to target NLRP3 NBD for K48-linked polyubiquitination at K496 ( Fig. 6 and Fig. S5, D and E ) and proteasome-mediated degradation ( Fig. 4 ). To our knowledge, our data are the first to identify a novel sequential ubiquitination event mediated by two E3 ubiquitin ligases, RNF125 and Cbl-b. Therefore, our data also indicate that ubiquitin chains can function as an adaptor to recruit E3 ubiquitin ligases to their substrates. It was recently shown that Casp-11 is a cytosolic sensor for LPS ( Shi et al., 2014 ) and Casp-11 mediates noncanonical inflammasome activation independently of TLR4 ( Hagar et al., 2013 ; Kayagaki et al., 2013 ). Further analysis indicates that GSDMD, a substrate of Casp-11 and Casp-1, is a key player of pyroptosis ( Kayagaki et al., 2015 ; Shi et al., 2015 ). It was reported that LPS-induced lethality in mice is mediated by Casp-11 because mice deficient for Casp-11 are rescued from death induced by a lethal dose of LPS ( Kayagaki et al., 2011 ). We showed that mice lacking Cbl-b or expressing Cbl-b C373A die from an injection of a sub-lethal dose of LPS (5 mg/kg), which is rescued by deficiency of NLRP3 or by IL-1RA treatment, which blocks both IL-1α and IL-1β ( Fig. 2, A, B, and G ). These data suggest that Cblb −/− or Cblb C373A mice are likely to die from aberrant production of IL-1β and subsequent TNF-α rather than pyroptosis. Indeed, blocking TNF-α by an anti–TNF-α neutralizing antibody also prevents LPS-induced mortality in Cblb −/− mice ( Fig. 2 E ). In support of this, a lethal dose of LPS-induced mortality in Cblb −/− mice is not rescued by NLRP3 deficiency but by Casp-11 deficiency ( Fig. 2 J ). These are further supported by our in vitro data that loss of Casp-11, but not NLRP3, impairs EHEC -triggered pyroptosis in WT and Cblb −/− macrophages ( Fig. 1 H ). Although it has been suggested that the cleavage of GSDMD by both Casp-1 and Casp-11 is a key executive event in pyroptosis, macrophages deficient for Casp-11, but not Casp-1, display defective pyroptosis triggered by cytosolic LPS ( Napier et al., 2016 ). This suggests that noncanonical inflammasomes mainly trigger Casp-11–mediated pyroptosis independently of Casp-1. Our data are further supported by a recent report that GSDMD is required for IL-1 secretion in living macrophages without pyroptosis ( Evavold et al., 2018 ). Collectively, our data suggest that gram-negative bacteria-induced sepsis may activate both canonical and noncanonical inflammasome pathways, while Cbl-b mainly regulates the release of IL-1β ( Fig. 8 ). It is important to note that we did not observe any difference in IL-1β in the sera of WT and Cblb −/− mice infected with Candida albicans , suggesting that Cbl-b does not regulate Casp-8–mediated inflammasomes ( Xiao et al., 2016 ). Previous studies suggest that Cbl-b may target TLR4 or MyD88 for ubiquitination ( Bachmaier et al., 2007 ; Han et al., 2010 ). However, TNF-α and IL-6 production by macrophages lacking Cbl-b stimulated with TLR ligands 1–9 is not increased ( Fig. 1 A ). Furthermore, LPS-induced expression of TLR4, MyD88, NLRP3, and pro–IL-1β and activation of NF-κB are also not augmented in macrophages lacking Cbl-b ( Fig. 1, B and C ). Therefore, our data collectively indicate that Cbl-b does not regulate TLR signaling. In support of these data, it was reported that Cblb −/− BMDMs are not hypersensitive to TLR stimulation ( Yee and Hamerman, 2013 ). Although LPS-induced endotoxemia is mainly mediated by innate immune cells, T cells do respond to LPS via TLR4 ( Zanin-Zhorov et al., 2007 ). However, Rag1 −/− Cblb −/− mice injected with a sub-lethal dose of LPS ( Fig. S2 A ) recapitulate the phenotype observed in Cblb −/− mice, suggesting that Cbl-b deficiency in innate immune cells is responsible for the phenotype observed. Likewise, mice lacking Cbl-b in myeloid cells but not T cells are also highly sensitive to a sub-lethal dose of LPS ( Fig. S2 C ). Therefore, our data indicate that Cbl-b expression in innate immune cells is crucial for the suppression of LPS-induced endotoxemia. In summary, we have identified RNF125 and Cbl-b as the key E3 ubiquitin ligases that keep NLRP3 inflammasomes in check by targeting NLRP3 for sequential K63- and K48-linked polyubiquitination. Thus, NLRP3 inflammasome-mediated endotoxemia is prevented in the presence of RNF125 and Cbl-b ( Fig. 8 ). Materials and methods Mice C57BL/6J, B6.129S7- Rag1 tm1Mom /J ( Rag1 −/− ), and B6.129S6- Nlrp3 tm1Bhk /J ( Nlrp3 −/− ) mice were purchased from the Jackson Laboratory. Cblb −/− mice were kindly provided by Dr. Josef M. Penninger (University of Toronto, Toronto, ON, Canada). Casp11 −/− mice ( Wang et al., 1998 ) were kindly provided by Dr. Junying Yuan (Harvard University, Cambridge, MA). Cblb −/− mice were crossed with Rag1 −/− , Nlrp3 −/− , and Casp11 −/− mice to generate Cblb −/− Rag1 −/− , Cblb −/− Nlrp3 −/− , and Cblb −/− Casp11 −/− mice. Cblb C373A mice were described previously ( Oksvold et al., 2008 ). The mouse strain carrying the Cblb tm1a(KOMP)Wtsi allele was generated at The Ohio State University Genetically Engineered Mouse Modeling Core Facility by standard embryonic stem (ES) cell technology ( Piovan et al., 2014 ). The ES clone EPD0703_2_B11 was acquired from the International Knockout Mouse Phenotyping Consortium (project #79117; http://www.mousephenotype.org/ ). Prior to microinjection, the identity of the targeted ES cells was verified by 5′ long-range PCR using a primer external to the targeting vector. Chimeric males were bred to C57BL/6 Albino females, and germline transmission was verified by PCR to detect the mutant together with the WT allele in the F1 heterozygous mice. Prior to utilization of the strain for experiments, mice were crossed to a Flipe ubiquitous strain (ACTB:FLPe B6J, JAX strain #005703; Rodríguez et al., 2000 ) to eliminate the lacZ/neo cassette and obtain the clean tm1c allele according to the breeding schemes recommended by the International Knockout Mouse Phenotyping Consortium. The LoxP flanked Cblb allele ( Cblb f/f ) was crossed to LysM Cre knock-in allele to specifically delete Cbl-b in myeloid cells including macrophages. All mice were 8–12 wk of age when used, and both male and female mice were used in this study. All animal experimentation involving LPS injection, CLP, and in vivo delivery of nonsense and Rnf 125-specific siRNA was approved by the Institutional Animal Care and Use Committees of The Ohio State University and the University of Iowa. Reagents Antibodies against CBLB (G-1; sc-8006), HA (Y-11; sc-805), GST (sc-33613), Casp-1 (P10; M20; sc-514), TLR4 (sc-10741), and MyD88 (sc-11356) were purchased from Santa Cruz Biotechnology, Inc. Anti-NLRP3 (D4D8T; #15101 and 15101S), anti–IL-1β (3A6; #12242), anti–K48 linkage–specific polyubiquitin (#4289), anti–K63 linkage–specific polyubiquitin (#12930), anti–phospho-NF-κB p65 (S536; #3031), and anti–phospho-IκBα (5A5; #9246) were purchased from Cell Signaling Technology, Inc. Anti-ubiquitin (05–944) was purchased from EMD Millipore. Anti-Flag (M2; F3165 and F2555) and anti-actin (AC74; A2228) were obtained from Sigma-Aldrich. Anti-RNF125 (LS-B11326) was purchased from LSBio. Anti–c-Myc (9E10; MA1-980) was purchased from Invitrogen. Antibody against TRIM31 (OWL-A48647) was obtained from One World Lab. Antibodies against TRIM14 (PA5-50806), TRIM21 (PA5-52178), TRIM47 (PA5-50892), and RNF213 (PA5-51902) were obtained from Thermo Fisher Scientific. ELISA kits for mouse IL1β (432606), TNF-α (430906), and IL-6 (431306) were purchased from BioLegend. HRP-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG were purchased from Kirkegaard & Perry Laboratories. Protein G-sepharose was purchased from GE Healthcare. Human recombinant AMSH was purchased from Boston Biochem, Inc. Bacteria The growth conditions for EHEC (700927 strain), F. novicida (JSG1819), and P. aeruginosa (PAO1) were described previously ( Bell et al., 2010 ; Dyszel et al., 2010 ; Wyckoff and Wozniak, 2001 ). Plasmids and transfection Flag-tagged NLRP3 plasmid and its truncated fragments were provided by Dr. Fabio Martinon (University of Lausanne, Lausanne, Switzerland). HA-tagged Cbl-b and its truncated fragments were described previously ( Davies et al., 2004 ; Xiao et al., 2015 ). Flag-tagged RNF125 and its truncated fragments were from Dr. Ze'ev A. Ronai (Sanford-Burnham Medical Research Institute, La Jolla, CA), in which the Flag tag was replaced with a Myc tag at Mutagenex Inc. His-tagged WT ubiquitin and K48 and K63 ubiquitin were purchased from Boston Biochem. HEK293T cells were transfected with various plasmids by calcium precipitation ( Qiao et al., 2014 ). Generation of BMDMs and activation of inflammasomes BM cells from WT and various knockout mouse strains were harvested from the femurs and tibias of mice. Cells were cultured in DMEM containing 10% FBS and 30% conditioned medium from L929 cells expressing M-CSF as described ( Xiao et al., 2016 ). After 1 wk of culture, nonadherent cells were removed, and adherent cells were 80%–90% F4/80 + CD11b + as determined by flow cytometric analysis. LPS-primed BMDMs were infected with P. aeruginosa (multiplicity of infection [MOI] = 30:1), EHEC (MOI = 25:1), and F. novicida (MOI = 100:1) for 1.5 h and then cultured in 100 µg/ml gentamycin. Other stimulations included ATP (2.5 mM, 30 min), monosodium urate (200 µg/ml, 4 h), CTB (40 µg/ml, 16 h), nigericin (20 µM, 3 h), and anthrax LT (500 µg/ml, 6 h). Poly(dA:dT) (1 µg/10 6 cells, 6 h) and flagellin (6.25 µg/10 6 cells, 4 h) were transfected using Lipofectamine (Invitrogen). Generation of MDMs, silencing of the Cblb gene, and activation of NLRP3 inflammasome Human MDMs were generated as previously described ( Rajaram et al., 2011 ). In brief, peripheral blood mononuclear cells from healthy donors were isolated from heparinized blood on Ficoll-sodium diatrizoate gradients and then cultured for 5 d in RPMI containing 20% autologous serum (2.0 × 10 6 mononuclear cells/ml) at 37°C. On day 5, human MDMs were transfected with control siRNA or Accell human CBLB siRNA (100 or 200 nM; Dharmacon RNA Technologies) by using Lonza nucleofector reagent and plated in RPMI 1640 containing 20% autologous serum. After 36 h, the MDMs were washed, primed with LPS, and stimulated with ATP or CTB or infected with EHEC . The protocol was approved by The Ohio State University Institutional Review Board. ELISA and cell death assay Cell culture supernatants and serum were assayed by ELISA for IL-1β, IL-6, and TNF-α. Cell death was measured by a lactate dehydrogenase assay kit from Sigma-Aldrich. LPS-induced endotoxemia Various groups of mice at 8–12 wk of age were injected i.p. with a sub-lethal dose of LPS (5 mg/kg; E. coli 0111:B4) and monitored for survival rate for 24 h. Some Cblb −/− mice were injected with IL-1RA (25 mg/kg) or TNF-α neutralization antibody (50 µg/mouse) before LPS injection. For serum pro-inflammatory cytokines, 1 mg/kg of LPS was injected i.p., and serum was collected every 4 h for 24 h. In some experiments, mice were injected i.p. with a lethal dose of LPS (54 mg/kg) and monitored every 4 h for 14–28 h. To determine the serum IL-1β levels, the mice were injected i.p. with 20 mg/kg of LPS, and serum was collected at 12 h after LPS injection. Induction of polymicrobial sepsis by CLP Polymicrobial sepsis was induced via CLP as previously described ( Moreno et al., 2006 ). In brief, mice were anesthetized with aerosol isoflurane (3%–4% in 100% O 2 flow at 2 liter/min). A 1-cm midline incision was made on the anterior abdomen. The cecum was ligated with a 2–0 vicryl suture at a point ∼1 cm from the cecal tip, and the cecum was punctured with a 21-gauge needle three times. The cecum was gently squeezed to express a small amount of fecal material and then returned to the central abdominal cavity. In sham-operated control animals, the cecum was isolated but neither ligated nor punctured. The abdominal incision was closed in two layers with 6–0 vicryl sutures. The muscle layer was closed with vicryl suture by applying simple running sutures, and the skin was closed with a 5–0 vicryl suture by applying simple interrupted sutures. The mice were monitored for survival rate for 14–28 h. Approximately 50–100 µl of blood was obtained via submandibular vein bleeding at the time of and 12 h after surgery. Detection of NLRP3 inflammasome activation by Western blot analysis BMDMs that were primed with LPS and stimulated with various inflammation activators or infected with various pathogens were lysed in RIPA buffer. The supernatants collected from the BMDM cultures were chloroform-methanol precipitated. The cell lysates or the supernatants were blotted with antibodies to Casp-1 p10 (1:1,000), IL-1β p17 (1:1,000), and actin (1:3,000). Immunoprecipitation For Co-IP assays, WT BMDMs were primed with LPS (100 ng/ml) for 4 h and stimulated with ATP (2.5 mM) for various times. Cells were lysed in 0.5% NP-40 lysis buffer. The cell lysates were immunoprecipitated with anti-NLRP3 (1:200) and blotted with anti-TRIM14 (1:500), anti-TRIM21 (1:500), anti-TRIM31 (1:500), anti-TRIM47 (1:500), anti-CBLB (1:500), anti-RNF213 (1:500), and anti-RNF125 (1:500). For Co-IP assays in HEK293T cells transfected with various HA-tagged Cbl-b or Cbl-b mutants or Flag-tagged NLRP3 or NLRP3 mutants, the cell lysates were immunoprecipitated with anti-Flag (1:200) and blotted with anti-HA (1:1,000) or, alternatively, immunoprecipitated with anti-HA and blotted with anti-Flag (1:1,000). To detect NLRP3 ubiquitination, BMDMs from WT and Cblb C373A mice were primed with LPS (100 µg/ml) for 4 h and stimulated with ATP for 5 min and then lysed in RIPA buffer containing 2% SDS, sonicated, and diluted to 0.5% of SDS using RIPA buffer without SDS before immunoprecipitation in order to disrupt the proteins associated with NLRP3. In some experiments, a denaturing step was used by heating to 95°C for 10 min before sonication. The cell lysates were immunoprecipitated with anti-NLRP3 and blotted with anti-ubiquitin (1:1,000) or with anti–K48- or anti–K63-specific ubiquitin antibodies (1:1,000). To assess the protein stability of NLRP3, BMDMs from WT, Cblb −/− , or Cblb C373A mice were primed with LPS and stimulated with ATP or CTB or infected with EHEC at indicated time points and blotted with antibodies against NLRP3 (1:1,000). To determine whether NLRP3 undergoes proteasome- or lysosome-mediated degradation, WT BMDMs were primed with LPS, pretreated with MG-132 (5 µM) or E-64 (10 µM) for 30 min, and then stimulated with ATP, CTB, or EHEC at various times and lysed. The cell lysates were blotted with anti-NLRP3. Deubiquitination assay To determine whether NLRP3 ubiquitination occurs sequentially by RNF125 and then Cbl-b, we transfected HEK293T cells with Myc-tagged RNF125, HA-tagged Cbl-b, Flag-tagged NLRP3, and His-tagged ubiquitin, lysed in RIPA buffer, and immunoprecipitated with anti-Flag. The Flag immunoprecipitates were treated with or without AMSH (200 nM) in deubiquitinating enzyme buffer (50 mM Tris-HCl, pH 7.2, 25 mM KCl, 5 mM MgCl 2 , and 1 mM dithiothreitol) at 37°C for 30 min ( McCullough et al., 2004 ). The reaction was terminated by addition of SDS sample buffer. The ubiquitination and deubiquitination of NLRP3 was detected by immunoblotting with anti-K63 ubiquitin and anti-K48 ubiquitin antibodies, respectively. Identification of E3 ubiquitin ligases that initiate K63-linked polyubiquitination of NLRP3 To identify potential NLRP3-binding E3 ubiquitin ligases, GST and GST–NLRP3 protein were incubated with lysates from WT BMDMs primed by LPS and stimulated by ATP for 5 min, followed by incubation with glutathione-sepharose beads. Suspension trapping filters for cleanup and digestion Homemade suspension trapping filters were fashioned from 8 × 3–mm punches of Munktell MK 360 Circles 9.0 cm (3600-0900) discs (Ahlstrom Laboratory Filters) M, stacked above two layers of C18 resin (Empore C18; 3M) in a 200-µl pipette tip ( Zougman et al., 2014 ). This was filled with 100 mM TRIS-HCl (pH 7.4) in methanol. The sample was solubilized with SDS (4% wt/vol) and 20 mM dithiothreitol and incubated at 95°C for 5 min, then alkylated with chloroacetamide in the dark for 30 min. Phosphoric acid was added to 3%, and the sample was transferred to the trapping pipette. Proteins were captured on quartz fibers as a fine dispersion while mass spectrometry–incompatible materials passed through the tip. The stack was then rinsed and refilled with cold trypsin in 50 mM AmBiC and set to digest at 47°C for 2 h. Peptides were eluted with 300 μl of 50% ACN and 0.1% TFA, lyophilized and stored at −20°C. LC-MS/MS Full MS1 profile data were acquired on a Q-Exactive hf (Thermo Fisher Scientific) from 380–1,700 m/z at a resolution of 60,000 ( Yu et al., 2015 ). The 10 most abundant precursors were selected with a mass window of 2 m/z thomson and subjected to higher-energy collisional dissociation at 37% activation efficiency. A 30-s dynamic exclusion improved selection of lower abundant ions. Fragmentation data were acquired in centroid at 30,000 resolution. Initial spectral searches were performed with both Mascot version 2.6.2 (MatrixScience) and Byonic search engines (Protein Metrics ver. 2.8.2) against the 2/06/2016 UniprotKB and reverted entry database for mouse. With 5 and 10 ppm tolerance for precursor and fragments, respectively, searches allowed accepted fixed Cys mods of 57 D, as well as variable mods of 16 (M), 80 (S, T), and 114 (K) Th. Final discriminant scores were determined by Scaffold Q + S ver. 4.7 (Proteome Software) at 1.2% false discovery rate. Identification of potential NLRP3 ubiquitination sites by mass spectrometry NLRP3 protein was purified by a FLAG Purification Kit from HEK293T cells cotransfected with Flag-tagged NLRP3, HA-tagged Cbl-b, and His-tagged ubiquitin, which was treated with nigericin. The lysates were separated by SDS-PAGE. The bands corresponding to NLRP3 and ubiquitinated NLRP3 were cut, washed, and digested with trypsin overnight after reduction and alkylation. Peptides were extracted from the gel pieces and dried in a vacufuge, and peptides were resuspended in 20 μl of 50 mM acetic acid for LC-MS/MS analysis. Capillary-liquid chromatography–nanospray tandem mass spectrometry (capillary–LC/MS/MS) performed on a Thermo Fisher Scientific LTQ orbitrap mass spectrometer equipped with a microspray source (Michrom Bioresources Inc.) operated in positive ion mode. Samples were separated on a capillary column (0.2 × 150 mm Magic C18AQ 3µ 200A; Bruker Daltonics) using an UltiMate 3000 HPLC system from Thermo Fisher Scientific. Mobile phase A was 50 mM acetic acid in water, and acetonitrile was used as mobile phase B. Flow rate was set at 2 µl/min. Tandem mass spectrometry data were acquired with a spray voltage of 2.2 kV, and a capillary temperature of 175°C was used. The scan sequence of the mass spectrometer was based on the preview mode data-dependent TopTen method. Sequence information from the tandem mass spectrometry data were processed by converting the raw files into a merged file (.mgf) using MS convert (ProteoWizard). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.3.2. The fragment mass tolerance was set to 0.5 D. Considered variable modifications were Ubiquitinylation (K), oxidation (Met), deamidation (N and Q), and carbamidomethylation (Cys). Identified ubiquitinylated peptides were manually checked for validation. Generation of NLRP3 LRR K/R mutants and RNF125 truncated fragments Single and multiple lysine-to-arginine (K-to-R) encoding mutations of NLRP3 (NLRP3 K324R, K430R, K437R, K496R, 3 K570R, and NLRP3 LRR 11K/R) and Myc-tagged RNF125, N-terminal 1–76 fragment, zinc finger, and Δ1-76 were generated by site-directed mutagenesis at Mutagenex. Nlrp3 − / − BMDM reconstitution Nlrp3 − / − BMDMs were transfected with constructs expressing Flag-tagged NLRP3, NLRP3 LRR K/R, or NLRP3 K496R by Lipofectamine 2000 according to the manufacturer's instructions. In vitro knockdown experiments Rnf 125 Accell siRNA, Rnf 213 Accell siRNA, Trim 21 Accell siRNA, Trim3 1 Accell siRNA, Trim 14 Accell siRNA, Trim 47 Accell siRNA, or nonsense siRNA was obtained from Dharmacon. BMDMs or HEK293T cells (for Rnf 125 siRNA) were plated in 12 wells and were transiently transfected with 2 µg of siRNAs plus 4 µl Lipofectamine 2000 according to the manufacturer's instruction. 36 h later, cells were harvested. The cell lysates were blotted for specific antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, and RNF213, respectively. In vivo delivery of Rnf 125 siRNA WT mice were treated with Rnf 125 Accell siRNA (2 mg/kg/mouse) or a nonsense siRNA intravenously. 24 h later, mice were i.p. injected with LPS (5 mg/kg) for the survival study. In a parallel experiment, the spleen cells from WT mice received Rnf 125 Accell siRNA, or the control siRNA were collected at day 2 and lysed in RIPA buffer. The cell lysates were blotted with anti-RNF125 and anti-actin, respectively. Data analysis and statistical analysis ELISA data were analyzed by using the Student's t test. Survival data were analyzed by using the Kaplan-Meier log-rank test. Differences were considered significant at P < 0.05. No animals were excluded from the analysis. Mice were allocated to experimental groups based on their genotypes and were randomized within their sex- and age-matched groups. No statistical method was used to predetermine sample size. It was assumed that normal variance occurred between the experimental groups. Online supplemental material Fig. S1 shows that silencing the CBLB gene in human macrophages leads to heightened IL-1β and TNF-α production upon LPS priming and ATP, EHEC, and CTB stimulation. Fig. S2 shows that Cblb −/− Rag1 −/− and LysM Cre-Cblb f/f mice are hypersensitive to septic shock induced by a sub-lethal dose of LPS. Fig. S3 shows that Cbl-b targets NLRP3 for K48-linked polyubiquitination in RIPA buffer containing SDS under the denaturing condition. Fig. S4 shows the identification of potential E3 ubiquitin ligases that bind to NLRP3 by LC-MS/MS analysis. Fig. S5 shows the confirmation of RNF125 as the initial E3 ubiquitin ligase to target NLRP3 LRR for K63-linked polyubiquitination and verification of K496 as the ubiquitination site of NLRP3 under the denaturing condition. Mice C57BL/6J, B6.129S7- Rag1 tm1Mom /J ( Rag1 −/− ), and B6.129S6- Nlrp3 tm1Bhk /J ( Nlrp3 −/− ) mice were purchased from the Jackson Laboratory. Cblb −/− mice were kindly provided by Dr. Josef M. Penninger (University of Toronto, Toronto, ON, Canada). Casp11 −/− mice ( Wang et al., 1998 ) were kindly provided by Dr. Junying Yuan (Harvard University, Cambridge, MA). Cblb −/− mice were crossed with Rag1 −/− , Nlrp3 −/− , and Casp11 −/− mice to generate Cblb −/− Rag1 −/− , Cblb −/− Nlrp3 −/− , and Cblb −/− Casp11 −/− mice. Cblb C373A mice were described previously ( Oksvold et al., 2008 ). The mouse strain carrying the Cblb tm1a(KOMP)Wtsi allele was generated at The Ohio State University Genetically Engineered Mouse Modeling Core Facility by standard embryonic stem (ES) cell technology ( Piovan et al., 2014 ). The ES clone EPD0703_2_B11 was acquired from the International Knockout Mouse Phenotyping Consortium (project #79117; http://www.mousephenotype.org/ ). Prior to microinjection, the identity of the targeted ES cells was verified by 5′ long-range PCR using a primer external to the targeting vector. Chimeric males were bred to C57BL/6 Albino females, and germline transmission was verified by PCR to detect the mutant together with the WT allele in the F1 heterozygous mice. Prior to utilization of the strain for experiments, mice were crossed to a Flipe ubiquitous strain (ACTB:FLPe B6J, JAX strain #005703; Rodríguez et al., 2000 ) to eliminate the lacZ/neo cassette and obtain the clean tm1c allele according to the breeding schemes recommended by the International Knockout Mouse Phenotyping Consortium. The LoxP flanked Cblb allele ( Cblb f/f ) was crossed to LysM Cre knock-in allele to specifically delete Cbl-b in myeloid cells including macrophages. All mice were 8–12 wk of age when used, and both male and female mice were used in this study. All animal experimentation involving LPS injection, CLP, and in vivo delivery of nonsense and Rnf 125-specific siRNA was approved by the Institutional Animal Care and Use Committees of The Ohio State University and the University of Iowa. Reagents Antibodies against CBLB (G-1; sc-8006), HA (Y-11; sc-805), GST (sc-33613), Casp-1 (P10; M20; sc-514), TLR4 (sc-10741), and MyD88 (sc-11356) were purchased from Santa Cruz Biotechnology, Inc. Anti-NLRP3 (D4D8T; #15101 and 15101S), anti–IL-1β (3A6; #12242), anti–K48 linkage–specific polyubiquitin (#4289), anti–K63 linkage–specific polyubiquitin (#12930), anti–phospho-NF-κB p65 (S536; #3031), and anti–phospho-IκBα (5A5; #9246) were purchased from Cell Signaling Technology, Inc. Anti-ubiquitin (05–944) was purchased from EMD Millipore. Anti-Flag (M2; F3165 and F2555) and anti-actin (AC74; A2228) were obtained from Sigma-Aldrich. Anti-RNF125 (LS-B11326) was purchased from LSBio. Anti–c-Myc (9E10; MA1-980) was purchased from Invitrogen. Antibody against TRIM31 (OWL-A48647) was obtained from One World Lab. Antibodies against TRIM14 (PA5-50806), TRIM21 (PA5-52178), TRIM47 (PA5-50892), and RNF213 (PA5-51902) were obtained from Thermo Fisher Scientific. ELISA kits for mouse IL1β (432606), TNF-α (430906), and IL-6 (431306) were purchased from BioLegend. HRP-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG were purchased from Kirkegaard & Perry Laboratories. Protein G-sepharose was purchased from GE Healthcare. Human recombinant AMSH was purchased from Boston Biochem, Inc. Bacteria The growth conditions for EHEC (700927 strain), F. novicida (JSG1819), and P. aeruginosa (PAO1) were described previously ( Bell et al., 2010 ; Dyszel et al., 2010 ; Wyckoff and Wozniak, 2001 ). Plasmids and transfection Flag-tagged NLRP3 plasmid and its truncated fragments were provided by Dr. Fabio Martinon (University of Lausanne, Lausanne, Switzerland). HA-tagged Cbl-b and its truncated fragments were described previously ( Davies et al., 2004 ; Xiao et al., 2015 ). Flag-tagged RNF125 and its truncated fragments were from Dr. Ze'ev A. Ronai (Sanford-Burnham Medical Research Institute, La Jolla, CA), in which the Flag tag was replaced with a Myc tag at Mutagenex Inc. His-tagged WT ubiquitin and K48 and K63 ubiquitin were purchased from Boston Biochem. HEK293T cells were transfected with various plasmids by calcium precipitation ( Qiao et al., 2014 ). Generation of BMDMs and activation of inflammasomes BM cells from WT and various knockout mouse strains were harvested from the femurs and tibias of mice. Cells were cultured in DMEM containing 10% FBS and 30% conditioned medium from L929 cells expressing M-CSF as described ( Xiao et al., 2016 ). After 1 wk of culture, nonadherent cells were removed, and adherent cells were 80%–90% F4/80 + CD11b + as determined by flow cytometric analysis. LPS-primed BMDMs were infected with P. aeruginosa (multiplicity of infection [MOI] = 30:1), EHEC (MOI = 25:1), and F. novicida (MOI = 100:1) for 1.5 h and then cultured in 100 µg/ml gentamycin. Other stimulations included ATP (2.5 mM, 30 min), monosodium urate (200 µg/ml, 4 h), CTB (40 µg/ml, 16 h), nigericin (20 µM, 3 h), and anthrax LT (500 µg/ml, 6 h). Poly(dA:dT) (1 µg/10 6 cells, 6 h) and flagellin (6.25 µg/10 6 cells, 4 h) were transfected using Lipofectamine (Invitrogen). Generation of MDMs, silencing of the Cblb gene, and activation of NLRP3 inflammasome Human MDMs were generated as previously described ( Rajaram et al., 2011 ). In brief, peripheral blood mononuclear cells from healthy donors were isolated from heparinized blood on Ficoll-sodium diatrizoate gradients and then cultured for 5 d in RPMI containing 20% autologous serum (2.0 × 10 6 mononuclear cells/ml) at 37°C. On day 5, human MDMs were transfected with control siRNA or Accell human CBLB siRNA (100 or 200 nM; Dharmacon RNA Technologies) by using Lonza nucleofector reagent and plated in RPMI 1640 containing 20% autologous serum. After 36 h, the MDMs were washed, primed with LPS, and stimulated with ATP or CTB or infected with EHEC . The protocol was approved by The Ohio State University Institutional Review Board. ELISA and cell death assay Cell culture supernatants and serum were assayed by ELISA for IL-1β, IL-6, and TNF-α. Cell death was measured by a lactate dehydrogenase assay kit from Sigma-Aldrich. LPS-induced endotoxemia Various groups of mice at 8–12 wk of age were injected i.p. with a sub-lethal dose of LPS (5 mg/kg; E. coli 0111:B4) and monitored for survival rate for 24 h. Some Cblb −/− mice were injected with IL-1RA (25 mg/kg) or TNF-α neutralization antibody (50 µg/mouse) before LPS injection. For serum pro-inflammatory cytokines, 1 mg/kg of LPS was injected i.p., and serum was collected every 4 h for 24 h. In some experiments, mice were injected i.p. with a lethal dose of LPS (54 mg/kg) and monitored every 4 h for 14–28 h. To determine the serum IL-1β levels, the mice were injected i.p. with 20 mg/kg of LPS, and serum was collected at 12 h after LPS injection. Induction of polymicrobial sepsis by CLP Polymicrobial sepsis was induced via CLP as previously described ( Moreno et al., 2006 ). In brief, mice were anesthetized with aerosol isoflurane (3%–4% in 100% O 2 flow at 2 liter/min). A 1-cm midline incision was made on the anterior abdomen. The cecum was ligated with a 2–0 vicryl suture at a point ∼1 cm from the cecal tip, and the cecum was punctured with a 21-gauge needle three times. The cecum was gently squeezed to express a small amount of fecal material and then returned to the central abdominal cavity. In sham-operated control animals, the cecum was isolated but neither ligated nor punctured. The abdominal incision was closed in two layers with 6–0 vicryl sutures. The muscle layer was closed with vicryl suture by applying simple running sutures, and the skin was closed with a 5–0 vicryl suture by applying simple interrupted sutures. The mice were monitored for survival rate for 14–28 h. Approximately 50–100 µl of blood was obtained via submandibular vein bleeding at the time of and 12 h after surgery. Detection of NLRP3 inflammasome activation by Western blot analysis BMDMs that were primed with LPS and stimulated with various inflammation activators or infected with various pathogens were lysed in RIPA buffer. The supernatants collected from the BMDM cultures were chloroform-methanol precipitated. The cell lysates or the supernatants were blotted with antibodies to Casp-1 p10 (1:1,000), IL-1β p17 (1:1,000), and actin (1:3,000). Immunoprecipitation For Co-IP assays, WT BMDMs were primed with LPS (100 ng/ml) for 4 h and stimulated with ATP (2.5 mM) for various times. Cells were lysed in 0.5% NP-40 lysis buffer. The cell lysates were immunoprecipitated with anti-NLRP3 (1:200) and blotted with anti-TRIM14 (1:500), anti-TRIM21 (1:500), anti-TRIM31 (1:500), anti-TRIM47 (1:500), anti-CBLB (1:500), anti-RNF213 (1:500), and anti-RNF125 (1:500). For Co-IP assays in HEK293T cells transfected with various HA-tagged Cbl-b or Cbl-b mutants or Flag-tagged NLRP3 or NLRP3 mutants, the cell lysates were immunoprecipitated with anti-Flag (1:200) and blotted with anti-HA (1:1,000) or, alternatively, immunoprecipitated with anti-HA and blotted with anti-Flag (1:1,000). To detect NLRP3 ubiquitination, BMDMs from WT and Cblb C373A mice were primed with LPS (100 µg/ml) for 4 h and stimulated with ATP for 5 min and then lysed in RIPA buffer containing 2% SDS, sonicated, and diluted to 0.5% of SDS using RIPA buffer without SDS before immunoprecipitation in order to disrupt the proteins associated with NLRP3. In some experiments, a denaturing step was used by heating to 95°C for 10 min before sonication. The cell lysates were immunoprecipitated with anti-NLRP3 and blotted with anti-ubiquitin (1:1,000) or with anti–K48- or anti–K63-specific ubiquitin antibodies (1:1,000). To assess the protein stability of NLRP3, BMDMs from WT, Cblb −/− , or Cblb C373A mice were primed with LPS and stimulated with ATP or CTB or infected with EHEC at indicated time points and blotted with antibodies against NLRP3 (1:1,000). To determine whether NLRP3 undergoes proteasome- or lysosome-mediated degradation, WT BMDMs were primed with LPS, pretreated with MG-132 (5 µM) or E-64 (10 µM) for 30 min, and then stimulated with ATP, CTB, or EHEC at various times and lysed. The cell lysates were blotted with anti-NLRP3. Deubiquitination assay To determine whether NLRP3 ubiquitination occurs sequentially by RNF125 and then Cbl-b, we transfected HEK293T cells with Myc-tagged RNF125, HA-tagged Cbl-b, Flag-tagged NLRP3, and His-tagged ubiquitin, lysed in RIPA buffer, and immunoprecipitated with anti-Flag. The Flag immunoprecipitates were treated with or without AMSH (200 nM) in deubiquitinating enzyme buffer (50 mM Tris-HCl, pH 7.2, 25 mM KCl, 5 mM MgCl 2 , and 1 mM dithiothreitol) at 37°C for 30 min ( McCullough et al., 2004 ). The reaction was terminated by addition of SDS sample buffer. The ubiquitination and deubiquitination of NLRP3 was detected by immunoblotting with anti-K63 ubiquitin and anti-K48 ubiquitin antibodies, respectively. Identification of E3 ubiquitin ligases that initiate K63-linked polyubiquitination of NLRP3 To identify potential NLRP3-binding E3 ubiquitin ligases, GST and GST–NLRP3 protein were incubated with lysates from WT BMDMs primed by LPS and stimulated by ATP for 5 min, followed by incubation with glutathione-sepharose beads. Suspension trapping filters for cleanup and digestion Homemade suspension trapping filters were fashioned from 8 × 3–mm punches of Munktell MK 360 Circles 9.0 cm (3600-0900) discs (Ahlstrom Laboratory Filters) M, stacked above two layers of C18 resin (Empore C18; 3M) in a 200-µl pipette tip ( Zougman et al., 2014 ). This was filled with 100 mM TRIS-HCl (pH 7.4) in methanol. The sample was solubilized with SDS (4% wt/vol) and 20 mM dithiothreitol and incubated at 95°C for 5 min, then alkylated with chloroacetamide in the dark for 30 min. Phosphoric acid was added to 3%, and the sample was transferred to the trapping pipette. Proteins were captured on quartz fibers as a fine dispersion while mass spectrometry–incompatible materials passed through the tip. The stack was then rinsed and refilled with cold trypsin in 50 mM AmBiC and set to digest at 47°C for 2 h. Peptides were eluted with 300 μl of 50% ACN and 0.1% TFA, lyophilized and stored at −20°C. LC-MS/MS Full MS1 profile data were acquired on a Q-Exactive hf (Thermo Fisher Scientific) from 380–1,700 m/z at a resolution of 60,000 ( Yu et al., 2015 ). The 10 most abundant precursors were selected with a mass window of 2 m/z thomson and subjected to higher-energy collisional dissociation at 37% activation efficiency. A 30-s dynamic exclusion improved selection of lower abundant ions. Fragmentation data were acquired in centroid at 30,000 resolution. Initial spectral searches were performed with both Mascot version 2.6.2 (MatrixScience) and Byonic search engines (Protein Metrics ver. 2.8.2) against the 2/06/2016 UniprotKB and reverted entry database for mouse. With 5 and 10 ppm tolerance for precursor and fragments, respectively, searches allowed accepted fixed Cys mods of 57 D, as well as variable mods of 16 (M), 80 (S, T), and 114 (K) Th. Final discriminant scores were determined by Scaffold Q + S ver. 4.7 (Proteome Software) at 1.2% false discovery rate. Identification of potential NLRP3 ubiquitination sites by mass spectrometry NLRP3 protein was purified by a FLAG Purification Kit from HEK293T cells cotransfected with Flag-tagged NLRP3, HA-tagged Cbl-b, and His-tagged ubiquitin, which was treated with nigericin. The lysates were separated by SDS-PAGE. The bands corresponding to NLRP3 and ubiquitinated NLRP3 were cut, washed, and digested with trypsin overnight after reduction and alkylation. Peptides were extracted from the gel pieces and dried in a vacufuge, and peptides were resuspended in 20 μl of 50 mM acetic acid for LC-MS/MS analysis. Capillary-liquid chromatography–nanospray tandem mass spectrometry (capillary–LC/MS/MS) performed on a Thermo Fisher Scientific LTQ orbitrap mass spectrometer equipped with a microspray source (Michrom Bioresources Inc.) operated in positive ion mode. Samples were separated on a capillary column (0.2 × 150 mm Magic C18AQ 3µ 200A; Bruker Daltonics) using an UltiMate 3000 HPLC system from Thermo Fisher Scientific. Mobile phase A was 50 mM acetic acid in water, and acetonitrile was used as mobile phase B. Flow rate was set at 2 µl/min. Tandem mass spectrometry data were acquired with a spray voltage of 2.2 kV, and a capillary temperature of 175°C was used. The scan sequence of the mass spectrometer was based on the preview mode data-dependent TopTen method. Sequence information from the tandem mass spectrometry data were processed by converting the raw files into a merged file (.mgf) using MS convert (ProteoWizard). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.3.2. The fragment mass tolerance was set to 0.5 D. Considered variable modifications were Ubiquitinylation (K), oxidation (Met), deamidation (N and Q), and carbamidomethylation (Cys). Identified ubiquitinylated peptides were manually checked for validation. Generation of NLRP3 LRR K/R mutants and RNF125 truncated fragments Single and multiple lysine-to-arginine (K-to-R) encoding mutations of NLRP3 (NLRP3 K324R, K430R, K437R, K496R, 3 K570R, and NLRP3 LRR 11K/R) and Myc-tagged RNF125, N-terminal 1–76 fragment, zinc finger, and Δ1-76 were generated by site-directed mutagenesis at Mutagenex. Generation of NLRP3 LRR K/R mutants and RNF125 truncated fragments Single and multiple lysine-to-arginine (K-to-R) encoding mutations of NLRP3 (NLRP3 K324R, K430R, K437R, K496R, 3 K570R, and NLRP3 LRR 11K/R) and Myc-tagged RNF125, N-terminal 1–76 fragment, zinc finger, and Δ1-76 were generated by site-directed mutagenesis at Mutagenex. Nlrp3 − / − BMDM reconstitution Nlrp3 − / − BMDMs were transfected with constructs expressing Flag-tagged NLRP3, NLRP3 LRR K/R, or NLRP3 K496R by Lipofectamine 2000 according to the manufacturer's instructions. In vitro knockdown experiments Rnf 125 Accell siRNA, Rnf 213 Accell siRNA, Trim 21 Accell siRNA, Trim3 1 Accell siRNA, Trim 14 Accell siRNA, Trim 47 Accell siRNA, or nonsense siRNA was obtained from Dharmacon. BMDMs or HEK293T cells (for Rnf 125 siRNA) were plated in 12 wells and were transiently transfected with 2 µg of siRNAs plus 4 µl Lipofectamine 2000 according to the manufacturer's instruction. 36 h later, cells were harvested. The cell lysates were blotted for specific antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, and RNF213, respectively. In vivo delivery of Rnf 125 siRNA WT mice were treated with Rnf 125 Accell siRNA (2 mg/kg/mouse) or a nonsense siRNA intravenously. 24 h later, mice were i.p. injected with LPS (5 mg/kg) for the survival study. In a parallel experiment, the spleen cells from WT mice received Rnf 125 Accell siRNA, or the control siRNA were collected at day 2 and lysed in RIPA buffer. The cell lysates were blotted with anti-RNF125 and anti-actin, respectively. Data analysis and statistical analysis ELISA data were analyzed by using the Student's t test. Survival data were analyzed by using the Kaplan-Meier log-rank test. Differences were considered significant at P < 0.05. No animals were excluded from the analysis. Mice were allocated to experimental groups based on their genotypes and were randomized within their sex- and age-matched groups. No statistical method was used to predetermine sample size. It was assumed that normal variance occurred between the experimental groups. Online supplemental material Fig. S1 shows that silencing the CBLB gene in human macrophages leads to heightened IL-1β and TNF-α production upon LPS priming and ATP, EHEC, and CTB stimulation. Fig. S2 shows that Cblb −/− Rag1 −/− and LysM Cre-Cblb f/f mice are hypersensitive to septic shock induced by a sub-lethal dose of LPS. Fig. S3 shows that Cbl-b targets NLRP3 for K48-linked polyubiquitination in RIPA buffer containing SDS under the denaturing condition. Fig. S4 shows the identification of potential E3 ubiquitin ligases that bind to NLRP3 by LC-MS/MS analysis. Fig. S5 shows the confirmation of RNF125 as the initial E3 ubiquitin ligase to target NLRP3 LRR for K63-linked polyubiquitination and verification of K496 as the ubiquitination site of NLRP3 under the denaturing condition.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077800/

Development of CMX001 for the Treatment of Poxvirus Infections

CMX001 (phosphonic acid, [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl] ester) is a lipid conjugate of the acyclic nucleotide phosphonate, cidofovir (CDV). CMX001 is currently in Phase II clinical trials for the prophylaxis of human cytomegalovirus infection and under development using the Animal Rule for smallpox infection. It has proven effective in reduction of morbidity and mortality in animal models of human smallpox, even after the onset of lesions and other clinical signs of disease. CMX001 and CDV are active against all five families of double-stranded DNA (dsDNA) viruses that cause human morbidity and mortality, including orthopoxviruses such as variola virus, the cause of human smallpox. However, the clinical utility of CDV is limited by the requirement for intravenous dosing and a high incidence of acute kidney toxicity. The risk of nephrotoxicity necessitates pre-hydration and probenecid administration in a health care facility, further complicating high volume CDV use in an emergency situation. Compared with CDV, CMX001 has a number of advantages for treatment of smallpox in an emergency including greater potency in vitro against all dsDNA viruses that cause human disease, a high genetic barrier to resistance, convenient oral administration as a tablet or liquid, and no evidence to date of nephrotoxicity in either animals or humans. The apparent lack of nephrotoxicity observed with CMX001 in vivo is because it is not a substrate for the human organic anion transporters that actively secrete CDV into kidney cells. The ability to test the safety and efficacy of CMX001 in patients with life-threatening dsDNA virus infections which share many basic traits with variola is a major advantage in the development of this antiviral for a smallpox indication. 1. Introduction Variola virus, the etiologic agent of smallpox, is a CDC Category A pathogen. Smallpox is one of the most pestilent diseases feared by mankind since antiquity, and historically has been associated with the periodic occurrence of widespread, often devastating epidemics. The fatality rate associated with the disease was reported to be roughly 30%, and a significant number of survivors were disfigured and/or blinded. However, historical data were derived from a population in which the disease was endemic. The last naturally acquired case of smallpox occurred in 1977 and the last laboratory acquired infection in 1978 [ 1 ]. In 1980, the World Health Organization (WHO) reported that, due to their aggressive worldwide vaccination program, smallpox had been eradicated [ 2 ]. Subsequent to this declaration, the worldwide vaccination campaign was stopped (the last vaccinations of the general population in the United States were in 1972 and in the rest of the world in 1984) because of side effects associated with smallpox vaccine [ 2 ]. As a result, herd immunity has been lost in the ensuing 30+ years, leaving most of the world's population highly vulnerable to morbidity and mortality from variola infection. In the wake of the terrorist attacks on September 11, 2001, and the anthrax attacks using the U.S. Postal Service to deliver letters containing Bacillus anthracis in October of 2001, the United States began in earnest to consider the impact of reintroduction of smallpox via a deliberate release into a highly susceptible population. Within weeks of September 11, 2001, the U.S. government decided to purchase adequate smallpox vaccine, 300 million doses, to immunize the entire population of the United States. If the public health infrastructure is prepared for the release of smallpox into the general population, many people could indeed be protected by vaccination. However, if vaccination is delayed by as few as three to four days after exposure, there may be little benefit. As in the case of mortality figures, the impact of delay on the development of a protective immune response after vaccination is based on observations made during the WHO eradication campaign under natural transmission conditions in a population in which the disease was endemic [ 3 ]. The impact of infection with a large inoculum of variola virus on the window of opportunity for vaccinating primary cases is unknown in the current highly susceptible population. In any event, many secondary cases of smallpox will likely occur and the disease could become widespread before primary cases of smallpox are identified, in part due to the lack of familiarity of disease signs and symptoms among the current generation of healthcare professionals. Diagnosis for public health response purposes will most likely be made after the onset of lesional disease. Consequently there is a need for antiviral drugs to treat patients who are diagnosed with smallpox after the appearance of lesions. Ultimately the same drugs will be used prophylactically/preemptively in exposed but asymptomatic populations to delay the progression of infection and increase the window of opportunity for vaccination and the development of protective immunity. In response to the threat of variola release, the Interagency Working Group on smallpox, a U.S. government group made up of officials at the Assistant Secretary level, recommended in 1998 that two antiviral drugs for treating smallpox with different mechanisms of action be developed. At least one of these drugs was to be orally available [ 4 ]. However, in addition to the indication for the treatment of smallpox in the general population, the drug would be the first line of defense for the tens of millions of people who are at risk of life-threatening vaccinia infections if vaccinated with a live attenuated virus vaccine such as the currently stockpiled ACAM2000. These populations include pregnant women and many groups of patients whose immune systems are weakened (e.g., AIDS patients, organ transplant recipients and patients on cancer therapy). Another possible threat is suggested by the demonstration that genetically engineered interleukin (IL)-4-positive strains of mousepox virus are lethal in mice, despite prior vaccination [ 5 ]. This modification, if applied to variola virus, could produce a strain that causes severe illness or death even in the vaccinated population. It is clear that the complete defense of the country against a terrorist attack using variola virus, whether modified or not, requires safe, easily administered antiviral drugs ready for use in the Strategic National Stockpile. 2. The Ideal Product Profile for Use in a Public Health Emergency The Department of Health and Human Services through the Biomedical Advanced Research and Development Authority (BARDA) issued a request for proposals in March of 2009 (RFP-BARDA-09-35) to provide a medical countermeasure that could specifically treat symptomatic individuals exposed to smallpox. Beyond stating that the product acquired under this contract must be able to "support a public health emergency", no specific properties, performance criteria or therapeutic profile in humans were specified. In the absence of specific guidance on product profile we identified the properties listed below as being essential for a drug to be highly effective in a public health emergency. Potency Clinically, potency is best defined as the relationship between the concentration of drug achieved in the plasma and the intensity of the therapeutic effect of the drug [ 6 ]. In the case of a medical countermeasure to treat an acute viral infection in a public health emergency, the potency needs to be such that at a level of drug that is easily and reproducibly achieved in the plasma of sick patients, the antiviral effect is adequate to quickly reduce viral burden thereby reducing the morbidity of the infection and, owing to reduced viral shedding, limiting infectivity. Schedule The course of therapy necessary to suppress the virus long enough for the host adaptive immune response to develop should be as short as possible and the dosing regimen should be simple and easy to follow to ensure compliance in a public health emergency. Risk of Resistance Emergence of a resistant virus post attack may allow the unchecked spread of disease through the population. The probability that a rapidly replicating virus circulating through a highly susceptible population during an epidemic would develop resistance is high. Resistance has developed to every widely used antiviral drug to date and has had a profound impact on the development of new antiviral agents [ 7 ]. The best approach to limiting viral breakthrough has been to develop potent drugs that target key replication proteins (enzymes) with a high genetic barrier to resistance. In addition, the resistant virus that ultimately emerges from such targeted drugs may be non-virulent, especially if mutations in the active site of a critical enzyme are required for the development of clinical resistance. Drug-Drug Interaction Antiviral therapy during a smallpox attack would be used for the treatment/prophylaxis of: (1) immunosuppressed patients who were exposed and are symptomatic with smallpox and, (2) those who received smallpox vaccination, developed complications from the vaccine and consequently need antiviral therapy. These groups of people will typically require significant medical management and be on multiple medications. The ideal smallpox antiviral will need to have minimal potential for drug interactions and will not require dosage adjustment in patients taking multiple concomitant medications. Formulation Characteristics The manufacturing process for the drug needs to be robust with the potential to accommodate surge capacity if necessary. The dose formulation should be easy to produce, and stable for long-term storage under a range of conditions. These properties represent the context in which we are developing CMX001 as a potential medical countermeasure for use in a smallpox attack. Potency Clinically, potency is best defined as the relationship between the concentration of drug achieved in the plasma and the intensity of the therapeutic effect of the drug [ 6 ]. In the case of a medical countermeasure to treat an acute viral infection in a public health emergency, the potency needs to be such that at a level of drug that is easily and reproducibly achieved in the plasma of sick patients, the antiviral effect is adequate to quickly reduce viral burden thereby reducing the morbidity of the infection and, owing to reduced viral shedding, limiting infectivity. Schedule The course of therapy necessary to suppress the virus long enough for the host adaptive immune response to develop should be as short as possible and the dosing regimen should be simple and easy to follow to ensure compliance in a public health emergency. Risk of Resistance Emergence of a resistant virus post attack may allow the unchecked spread of disease through the population. The probability that a rapidly replicating virus circulating through a highly susceptible population during an epidemic would develop resistance is high. Resistance has developed to every widely used antiviral drug to date and has had a profound impact on the development of new antiviral agents [ 7 ]. The best approach to limiting viral breakthrough has been to develop potent drugs that target key replication proteins (enzymes) with a high genetic barrier to resistance. In addition, the resistant virus that ultimately emerges from such targeted drugs may be non-virulent, especially if mutations in the active site of a critical enzyme are required for the development of clinical resistance. Drug-Drug Interaction Antiviral therapy during a smallpox attack would be used for the treatment/prophylaxis of: (1) immunosuppressed patients who were exposed and are symptomatic with smallpox and, (2) those who received smallpox vaccination, developed complications from the vaccine and consequently need antiviral therapy. These groups of people will typically require significant medical management and be on multiple medications. The ideal smallpox antiviral will need to have minimal potential for drug interactions and will not require dosage adjustment in patients taking multiple concomitant medications. Formulation Characteristics The manufacturing process for the drug needs to be robust with the potential to accommodate surge capacity if necessary. The dose formulation should be easy to produce, and stable for long-term storage under a range of conditions. These properties represent the context in which we are developing CMX001 as a potential medical countermeasure for use in a smallpox attack. Potency Clinically, potency is best defined as the relationship between the concentration of drug achieved in the plasma and the intensity of the therapeutic effect of the drug [ 6 ]. In the case of a medical countermeasure to treat an acute viral infection in a public health emergency, the potency needs to be such that at a level of drug that is easily and reproducibly achieved in the plasma of sick patients, the antiviral effect is adequate to quickly reduce viral burden thereby reducing the morbidity of the infection and, owing to reduced viral shedding, limiting infectivity. Schedule The course of therapy necessary to suppress the virus long enough for the host adaptive immune response to develop should be as short as possible and the dosing regimen should be simple and easy to follow to ensure compliance in a public health emergency. Risk of Resistance Emergence of a resistant virus post attack may allow the unchecked spread of disease through the population. The probability that a rapidly replicating virus circulating through a highly susceptible population during an epidemic would develop resistance is high. Resistance has developed to every widely used antiviral drug to date and has had a profound impact on the development of new antiviral agents [ 7 ]. The best approach to limiting viral breakthrough has been to develop potent drugs that target key replication proteins (enzymes) with a high genetic barrier to resistance. In addition, the resistant virus that ultimately emerges from such targeted drugs may be non-virulent, especially if mutations in the active site of a critical enzyme are required for the development of clinical resistance. Drug-Drug Interaction Antiviral therapy during a smallpox attack would be used for the treatment/prophylaxis of: (1) immunosuppressed patients who were exposed and are symptomatic with smallpox and, (2) those who received smallpox vaccination, developed complications from the vaccine and consequently need antiviral therapy. These groups of people will typically require significant medical management and be on multiple medications. The ideal smallpox antiviral will need to have minimal potential for drug interactions and will not require dosage adjustment in patients taking multiple concomitant medications. Formulation Characteristics The manufacturing process for the drug needs to be robust with the potential to accommodate surge capacity if necessary. The dose formulation should be easy to produce, and stable for long-term storage under a range of conditions. These properties represent the context in which we are developing CMX001 as a potential medical countermeasure for use in a smallpox attack. 3. CMX001 in vitro Efficacy and Resistance Testing CMX001 is active in vitro against a broad range of viruses from all five families of dsDNA viruses infecting humans, including multiple species of Orthopoxviridae . As shown in Table 1 , conjugation of the lipid to the phosphonate moiety of CDV to produce CMX001 results in significant decreases in apparent EC 50 values relative to those observed for CDV. In the case of the orthopoxviruses, the enhancement in activity (determined as the ratio of the EC 50 CDV/EC 50 CMX001) ranges from a high of 271-fold for variola virus to a low of 24-fold for ectromelia virus. The increased activity of CMX001 relative to CDV is attributable to the more efficient cellular uptake of CMX001 facilitated by the lipid chain [ 8 ]. CDV is transported into cells by a relatively inefficient method, fluid phase endocytosis [ 9 ]. CMX001 ( Figure 1a ) is formed by conjugating a lipid, 3-hexadecyloxy-1-propanol, to the phosphonate moiety of CDV. Once CMX001 is inside cells, CDV ( Figure 1b ) is liberated by phospholipase cleavage of the lipid ester linkage and activated by two successive phosphorylations, first to cidofovir monophosphate (CDV-P, Figure 1c ) and then to cidofovir diphosphate (CDV-PP, Figure 1d ) [ 10 ]. The CDV-PP acts as a competitive, alternative substrate inhibitor of the DNA directed DNA polymerases encoded by the herpesvirus, adenovirus and orthopoxvirus families of double-stranded DNA viruses (dsDNA) [ 11 – 14 ]. The broad spectrum activity of CMX001 against various species of orthopoxviruses was anticipated based on the mechanism of action of the drug, inhibition of the virally encoded polymerase by CDV-PP, and the high level of homology for this enzyme seen within the family. The amino acid sequences of the catalytic subunit of the polymerase have been aligned for cowpox virus (CPXV, strain Brighton Red), ECTV (strain Moscow), MPXV, (strain Zaire 1979-005), VACV (strains 3737 and Western Reserve), RPXV (strain Utrecht) and VARV (strains Bangladesh 1975 and India 7129). All of the subunits are 1005 residues in length, completely overlap, and have sequence identity ranging from 98.2% to 99.1%. Sequences were obtained from Poxvirus Bioinformatics Resource Center [ 15 ] and aligned with the NCBI Protein Blast program, BLASTP 2.2.24, [ 16 ]. This level of similarity in the target enzyme also supports the use of surrogates for VARV in the animal model studies required under the Animal Rule (21 CFR 314.600) to establish the efficacy of CMX001, or more specifically the efficacy of CDV-PP, the active antiviral formed from CMX001 ( Figure 1 ). In vitro experiments to generate orthopoxvirus strains resistant to CDV have been conducted and cross-resistance to CMX001 examined. Serial passage studies of camelpox, CPXV, MPXV and VACV viruses with CDV have all generated resistant strains [ 26 ]. The extent of viral resistance is reported to be on the same order as that observed for CDV-resistant forms of herpes simplex virus and CMV (10 to 27-fold). Resistant strains of VACV (strain WR) generated after 20 to 30 passages in the presence of increasing concentrations of CDV have been shown to be cross resistant to CMX001 [ 27 ]. Mutations conferring this resistance are located in the virally encoded DNA polymerase. Additionally, the development of resistance to CDV and CMX001 in VACV leads to a significant attenuation of virulence in mice [ 28 ], suggesting that any mutants that might become dominant from drug pressure during treatment of smallpox would be attenuated and less virulent. Twelve mutations in the polymerase domain of VACV have been associated with phenotypic resistance to CDV as shown in Table 2 . The specific roles of these mutations in CDV resistance have not been uniformly elucidated and some appear to be secondary; however, 11/12 of the sites involved are completely conserved among representative members of the orthopoxviruses suggesting the data obtained in VACV can be extrapolated to other members of this family, including VARV. This observation is in accord with the overall high level of sequence homology observed across the orthopoxviruses. 4. Establishing Activity under the Animal Efficacy Rule Because smallpox has been eradicated, the effectiveness of anti-VARV agents cannot be demonstrated in human clinical trials. In order to provide a development path for candidate smallpox therapeutics, the FDA promulgated the "Animal Efficacy Rule" in June of 2002 [ 31 ]. This rule establishes conditions under which animal models can be used to provide evidence of the effectiveness of an antiviral agent to treat a disease or condition for which clinical studies are impossible or unethical. As described in 21 CFR 314, subpart I, FDA will accept data from studies in animal models as evidence of efficacy when the following criteria are met: "(1) there is a reasonably well-understood pathophysiological mechanism of the toxicity of the substance and its prevention or substantial reduction by the product; (2) the effect is demonstrated in more than one animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans; (3) the animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity; (4) the data or information on the pharmacokinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans." Each of these points is discussed below in the context of the program to develop CMX001 under the Animal Efficacy Rule. 4.1. Pathophysiological Mechanisms Although our understanding of the pathogenesis of variola virus infection in humans is limited, it is clear that viral replication plays a key role in the lethality of the infection. Historically, the mortality rate from ordinary-type smallpox was approximately 30% (5–50%). Mortality correlated with the density of pock lesions on the skin, which almost certainly correlated with the intensity of the secondary viremia. Thus, an intervention that reduces viral replication should also reduce lesion count and increase survival. The antiviral mechanism by which CMX001 prevents or substantially reduces the toxicity of orthopoxviruses is understood. The drug arrests disease progression by blocking viral DNA replication and thereby reducing viral burden [ 13 , 30 , 32 ]. 4.2. Efficacy in Animal Models The optimal model for a human virus infection is an animal that can be naturally infected with the same virus; however, humans are the only natural host for VARV. Since the target enzyme of CMX001 is highly conserved, orthopoxviruses that naturally infect the model species are the next best choice. These natural infections have several hallmarks that may make them more desirable than laboratory contrived infections, including low virus dose infections that result in high mortality, relevant routes to induce severe disease in immunocompetent hosts, and virus-encoded host response modifier genes matched to host processes. Epidemiological data suggest that VARV is most often transmitted in large respiratory droplets making respiratory transmission the most relevant [ 2 ]. There is also considerable evidence suggesting that VARV infection of humans is initiated with a small dose of virus [ 33 ]. Thus for a model, a low inoculum delivered via the upper respiratory tract is the preferred route of infection. Unfortunately, no single animal model meets these criteria and fully reproduces the clinical and pathogenic course of VARV infection in humans. Since there is no single model of orthopoxvirus infection that completely replicates the clinical and pathophysiological course of human smallpox, CMX001 has been tested extensively in mouse, rabbit, and non-human primate models of orthopoxvirus infection. The results of these studies are summarized below. CMX001 in mouse models of orthopoxvirus infection The in vivo anti-orthopoxvirus activity of CMX001 has been extensively characterized in mice infected with ECTV, CPXV and VACV viruses. Ectromelia virus (mousepox) infection of mice has been used extensively as a model for orthopoxvirus pathogenesis and as a model for testing the efficacy of anti-orthopoxvirus therapeutics [ 34 ]. ECTV is a natural pathogen of mice and depending upon the mouse strain and route of inoculation, lethal infection can be initiated with a very small inoculum. Additionally, infection of mice with CPXV and VACV can be used to assess the efficacy of an antiviral against orthopoxviruses that can infect humans. The availability of an animal model(s) to help establish an effective dose for the treatment of vaccinia virus is critical in determining the potential utility of a drug in treating the side effects associated with a vaccinia derived, live attenuated virus smallpox vaccine. A large number of CMX001 regimens have been studied in ECTV-infected A/NCR mice. In this model, a 2.5 mg/kg dose of CMX001 given once daily for five days starting 4 h post-infection provides complete protection against a lethal intranasal challenge. When treatment is started as late as five days post-infection (3–4 days prior to the death of untreated controls), mice can be completely protected from mortality with a loading dose of 10 mg/kg followed by a maintenance dose of 2.5 mg/kg every other day for 14 days. A single 20 mg/kg oral dose of CMX001 given as late as four days post-infection provided 100% protection against lethal ECTV infection [ 35 , 36 ]. Against CPXV infection, oral administration of 10 mg/kg of CMX001 daily for five consecutive days was 100% protective in mice infected by either the aerosol inhalation or intranasal route when dosing was initiated 4 h post-infection [ 22 ]. A single oral dose of 12.5 mg/kg was 80% protective when given as early as five days before exposure, 100% effective when given one or three days before exposure, 100% effective when given one day post–exposure, and 87% protective when given as late as three days post-exposure [ 37 ]. CMX001 therapy showed significant protection (86%) against lethal VACV infection when initiated as late as 24 h post-infection (mean day of death for vehicle controls was 6.8) [ 37 ]. CMX001 in a rabbitpox model Rabbitpox virus is a subspecies of VACV that is naturally highly pathogenic in rabbits. Like other orthopoxvirus infections in their natural hosts, RPXV infection of rabbits shares many characteristics of smallpox. RPXV infection is readily transmitted between rabbits by the respiratory route and follows a pathophysiological progression similar to that of smallpox. Rabbits infected with RPXV undergo an incubation phase followed by the onset of fever, lesions, and ultimately respiratory distress and typically death [ 38 ]. Intradermal infection of rabbits allows for a controlled inoculum to be delivered while maintaining a course of disease nearly identical to that of the natural respiratory infection. Additionally, the lethal inoculum for intradermal infection is much lower than that required for intranasal infection, more closely mimicking the low inoculum required for human smallpox infection. Finally, intradermal infection results in a reliable occurrence of lesions, whereas with respiratory infection, rabbits sometimes die before rash development [ 39 ]. Ear lesions in the rabbitpox model, a result of systemic viral spread from the intradermal inoculation site on the thigh, typically appear beginning from three to five days post infection. As the desired trigger to initiate therapy for the treatment of smallpox is the onset of lesions, the reliable occurrence of lesions is a distinct advantage. CMX001 has been studied in New Zealand White rabbits infected intradermally with a lethal inoculum of RPXV [ 38 , 40 , 41 ]. Initial studies established the efficacy of CMX001 for pre-exposure and post-exposure prophylaxis. For example, 100% of animals administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated one day before challenge compared to 0% survival in animals administered placebo [ 38 ]. Delayed treatment experiments showed that CMX001 can be given late in the infection cycle, after appearance of clinical signs of disease (fever and lesions), and still prevent mortality. In a representative experiment, 100% of rabbits administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated four days after infection compared with 0% of placebo treated animals. Treatment initiated at five days post infection also provided a survival benefit (75% of CMX001-treated compared with 0% of placebo treated). In a series of randomized, blinded, placebo-controlled studies, administration of 20 mg/kg CMX001 initiated at the onset of lesions in the ears (mean 3.8 days post infection, range three to five days) provided a survival benefit over placebo-treated animals [ 40 ]. The results of these studies are summarized in Table 3 . Even a single dose of 20 mg/kg CMX001 given at the onset of lesions provided a statistically significant survival benefit compared to placebo [ 40 ]. Note that based on AUC 0–∞ , the 20 mg/kg dose in rabbits equates to a 0.67 mg/kg dose in humans (see Table 4 ). Doses in this range have been used to successfully treat fulminate disseminated dsDNA infections in patients. Monkeypox in Cynomolgus Monkeys Infection of cynomolgus monkeys with MPXV produces an exanthematous disease that can be used to evaluate the effect of a drug on skin lesions, viremia, and mortality. Because this model requires a large intravenous (i.v.) dose of virus (1–5 × 10 7 PFU of MPXV) to achieve a severe infection [ 42 – 45 ], the model may not reflect the pathophysiology of human disease, where the infectious dose is thought to be on the order of 100 PFU [ 46 ]. Animals manifest the first signs of illness within two to five days after infection and die within nine to 17 days (compared with six to 15 days and 16 to 22 days, respectively, in human smallpox) and have a near 100% mortality rate (as compared to 30% in humans). The distribution and progression of skin lesions in monkeys is similar to that in human smallpox. Although there may be a potential advantage to using a non-human primate as a model for a human disease, this advantage is offset by the very large intravenous inoculum required to induce severe disease. CMX001 was tested in the i.v. inoculation MPXV model at oral doses of 2.5, 5, and 10 mg/kg administered on days 1, 3, 6, 9, and 12 post–infection. Although these doses did not reduce mortality, an explanation was provided by pharmacokinetic analysis which showed that systemic exposure of cynomolgus monkeys to CMX001 following oral administration was very low compared to that seen in other species including mice, rabbits, and humans, with humans having by far the best systemic exposure (AUC) after oral administration (see Table 4 ). A subsequent study of MPXV in monkeys using intramuscular (i.m.) administration of CMX001 showed an equivocal effect of treatment, despite this route resulting in a higher systemic exposure to CMX001. This result was somewhat unexpected given that i.v. CDV had been shown to be effective in the model and that both CDV and CMX001 produce the same active antiviral metabolite, CDV-PP. However, comparison of the levels of CDV-PP after i.v. CDV treatment versus i.m. CMX001 treatment (in healthy monkeys) revealed that the concentration of CDV-PP was much higher in the peripheral blood mononuclear cells (PBMCs) of animals treated with i.v. CDV compared to those treated with i.m. CMX001 ( Figure 2 ). This result is consistent with intracellular conversion of CMX001 to CDV being inefficient in monkeys. Because of this metabolic difference between monkeys and humans, data obtained with CMX001 in monkeys cannot be used to model human exposure. To circumvent this issue, CDV treatment of monkeys will be used to model the exposure to the active antiviral metabolite, CDV-PP, which is common to both CDV and CMX001. This approach to using monkey model data to help establish a human dose is described under "Selection of an Effective Dose in Humans" below. 4.3. Selection of Endpoints With the exception of studies of VARV in monkeys where statistically powered mortality endpoints may not be feasible, the primary endpoint in animal efficacy studies is survival to 42 days post infection. This endpoint is clearly relevant to the desired benefit in humans. In VARV studies, due to the partial mortality in the model, the ethical concerns of using large numbers of monkeys to achieve statistical significance, and the practical constraints of handling large numbers of monkeys under biosafety level 4 conditions, the proposed primary endpoint is a reduction in the number of lesions. Historically, the mortality rate from ordinary-type smallpox was positively associated with the density of pock lesions, which almost certainly correlated with the intensity of the secondary viremia. Thus, an intervention that reduces viral replication should also reduce the number of lesions and increase survival. 4.4. Selection of an Effective Dose in Humans Straightforward use of different animal models of human smallpox for establishing the dose of a drug requires relatively consistent pharmacokinetic/pharmacodynamic profile of the drug across the animal model species. The pharmacokinetics (PK) of CMX001 have been determined in humans and in all animal species in which the drug has been used to study orthopoxvirus infection to date. Exposure to CMX001 after oral administration differs dramatically across species, with the lowest exposure occurring in monkeys and highest exposure occurring in humans (see Table 4 ). Even after achieving high plasma concentrations of CMX001 in monkeys by administration via non-oral routes, the conversion to the active moiety CDV-PP is low. The use of CDV as a surrogate for CMX001 in the monkey models avoids the differential PK and metabolism issues. As CDV-PP is the active antiviral metabolite that is common to both CMX001 and CDV, use of interspecies scaling through CDV-PP to extrapolate data from monkeys to humans is planned. In preliminary scaling studies, treatment with three doses of 20 mg/kg CMX001 has been shown to be effective in providing a survival benefit to rabbits infected with RPXV. In healthy rabbits, this treatment regimen resulted in an average CDV-PP concentration in PBMCs of 52.1 pg/10 6 cells when measured 24 hours after the third dose ( Figure 2 ). Similarly, treatment with three doses of 20 mg/kg CDV has also shown efficacy in providing a survival benefit to monkeys infected with MPXV [ 44 ]. In healthy monkeys, this treatment regimen resulted in average CDV-PP concentrations in PBMCs in the range of 52.9 to 75.6 pg/10 6 cells when measured from 12 hours after the first dose to 48 hours after the third dose ( Figure 2 ). Based upon these results, the target for an effective intracellular concentration of CDV-PP is in the range of approximately 50 pg/10 6 cells as measured in PBMCs. Reliable CDV-PP concentration data in PBMCs of humans treated with CMX001 is currently not available for comparison; however, in vitro studies with human PBMCs indicate that these CDV-PP levels will be achieved with doses of CMX001 currently being studied in clinical trials for treatment of other dsDNA virus infections. For example, a dose of 2 mg/kg CMX001 in humans produced a C max of approximately 0.6 μM (350 ng/mL) and an AUC of approximately 2650 h·ng/mL CMX001 in plasma (CMX001-102). Isolated human PBMCs incubated with 0.1 μM CMX001 for 24 hours (estimated AUC = 1348 h·ng/mL) produced a concentration of CDV-PP of approximately 79 pg/10 6 cells at 24 hours which is within the expected efficacious range as estimated from rabbits and monkeys. Furthermore, the long half-life of CDV-PP observed in human PBMCs in vitro indicates that infrequent dosing with CMX001 is sufficient to maintain an effective intracellular CDV-PP concentration. Additional studies measuring CDV-PP in the PBMCs of healthy and infected animals as well as in humans are planned. 4.1. Pathophysiological Mechanisms Although our understanding of the pathogenesis of variola virus infection in humans is limited, it is clear that viral replication plays a key role in the lethality of the infection. Historically, the mortality rate from ordinary-type smallpox was approximately 30% (5–50%). Mortality correlated with the density of pock lesions on the skin, which almost certainly correlated with the intensity of the secondary viremia. Thus, an intervention that reduces viral replication should also reduce lesion count and increase survival. The antiviral mechanism by which CMX001 prevents or substantially reduces the toxicity of orthopoxviruses is understood. The drug arrests disease progression by blocking viral DNA replication and thereby reducing viral burden [ 13 , 30 , 32 ]. 4.2. Efficacy in Animal Models The optimal model for a human virus infection is an animal that can be naturally infected with the same virus; however, humans are the only natural host for VARV. Since the target enzyme of CMX001 is highly conserved, orthopoxviruses that naturally infect the model species are the next best choice. These natural infections have several hallmarks that may make them more desirable than laboratory contrived infections, including low virus dose infections that result in high mortality, relevant routes to induce severe disease in immunocompetent hosts, and virus-encoded host response modifier genes matched to host processes. Epidemiological data suggest that VARV is most often transmitted in large respiratory droplets making respiratory transmission the most relevant [ 2 ]. There is also considerable evidence suggesting that VARV infection of humans is initiated with a small dose of virus [ 33 ]. Thus for a model, a low inoculum delivered via the upper respiratory tract is the preferred route of infection. Unfortunately, no single animal model meets these criteria and fully reproduces the clinical and pathogenic course of VARV infection in humans. Since there is no single model of orthopoxvirus infection that completely replicates the clinical and pathophysiological course of human smallpox, CMX001 has been tested extensively in mouse, rabbit, and non-human primate models of orthopoxvirus infection. The results of these studies are summarized below. CMX001 in mouse models of orthopoxvirus infection The in vivo anti-orthopoxvirus activity of CMX001 has been extensively characterized in mice infected with ECTV, CPXV and VACV viruses. Ectromelia virus (mousepox) infection of mice has been used extensively as a model for orthopoxvirus pathogenesis and as a model for testing the efficacy of anti-orthopoxvirus therapeutics [ 34 ]. ECTV is a natural pathogen of mice and depending upon the mouse strain and route of inoculation, lethal infection can be initiated with a very small inoculum. Additionally, infection of mice with CPXV and VACV can be used to assess the efficacy of an antiviral against orthopoxviruses that can infect humans. The availability of an animal model(s) to help establish an effective dose for the treatment of vaccinia virus is critical in determining the potential utility of a drug in treating the side effects associated with a vaccinia derived, live attenuated virus smallpox vaccine. A large number of CMX001 regimens have been studied in ECTV-infected A/NCR mice. In this model, a 2.5 mg/kg dose of CMX001 given once daily for five days starting 4 h post-infection provides complete protection against a lethal intranasal challenge. When treatment is started as late as five days post-infection (3–4 days prior to the death of untreated controls), mice can be completely protected from mortality with a loading dose of 10 mg/kg followed by a maintenance dose of 2.5 mg/kg every other day for 14 days. A single 20 mg/kg oral dose of CMX001 given as late as four days post-infection provided 100% protection against lethal ECTV infection [ 35 , 36 ]. Against CPXV infection, oral administration of 10 mg/kg of CMX001 daily for five consecutive days was 100% protective in mice infected by either the aerosol inhalation or intranasal route when dosing was initiated 4 h post-infection [ 22 ]. A single oral dose of 12.5 mg/kg was 80% protective when given as early as five days before exposure, 100% effective when given one or three days before exposure, 100% effective when given one day post–exposure, and 87% protective when given as late as three days post-exposure [ 37 ]. CMX001 therapy showed significant protection (86%) against lethal VACV infection when initiated as late as 24 h post-infection (mean day of death for vehicle controls was 6.8) [ 37 ]. CMX001 in a rabbitpox model Rabbitpox virus is a subspecies of VACV that is naturally highly pathogenic in rabbits. Like other orthopoxvirus infections in their natural hosts, RPXV infection of rabbits shares many characteristics of smallpox. RPXV infection is readily transmitted between rabbits by the respiratory route and follows a pathophysiological progression similar to that of smallpox. Rabbits infected with RPXV undergo an incubation phase followed by the onset of fever, lesions, and ultimately respiratory distress and typically death [ 38 ]. Intradermal infection of rabbits allows for a controlled inoculum to be delivered while maintaining a course of disease nearly identical to that of the natural respiratory infection. Additionally, the lethal inoculum for intradermal infection is much lower than that required for intranasal infection, more closely mimicking the low inoculum required for human smallpox infection. Finally, intradermal infection results in a reliable occurrence of lesions, whereas with respiratory infection, rabbits sometimes die before rash development [ 39 ]. Ear lesions in the rabbitpox model, a result of systemic viral spread from the intradermal inoculation site on the thigh, typically appear beginning from three to five days post infection. As the desired trigger to initiate therapy for the treatment of smallpox is the onset of lesions, the reliable occurrence of lesions is a distinct advantage. CMX001 has been studied in New Zealand White rabbits infected intradermally with a lethal inoculum of RPXV [ 38 , 40 , 41 ]. Initial studies established the efficacy of CMX001 for pre-exposure and post-exposure prophylaxis. For example, 100% of animals administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated one day before challenge compared to 0% survival in animals administered placebo [ 38 ]. Delayed treatment experiments showed that CMX001 can be given late in the infection cycle, after appearance of clinical signs of disease (fever and lesions), and still prevent mortality. In a representative experiment, 100% of rabbits administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated four days after infection compared with 0% of placebo treated animals. Treatment initiated at five days post infection also provided a survival benefit (75% of CMX001-treated compared with 0% of placebo treated). In a series of randomized, blinded, placebo-controlled studies, administration of 20 mg/kg CMX001 initiated at the onset of lesions in the ears (mean 3.8 days post infection, range three to five days) provided a survival benefit over placebo-treated animals [ 40 ]. The results of these studies are summarized in Table 3 . Even a single dose of 20 mg/kg CMX001 given at the onset of lesions provided a statistically significant survival benefit compared to placebo [ 40 ]. Note that based on AUC 0–∞ , the 20 mg/kg dose in rabbits equates to a 0.67 mg/kg dose in humans (see Table 4 ). Doses in this range have been used to successfully treat fulminate disseminated dsDNA infections in patients. Monkeypox in Cynomolgus Monkeys Infection of cynomolgus monkeys with MPXV produces an exanthematous disease that can be used to evaluate the effect of a drug on skin lesions, viremia, and mortality. Because this model requires a large intravenous (i.v.) dose of virus (1–5 × 10 7 PFU of MPXV) to achieve a severe infection [ 42 – 45 ], the model may not reflect the pathophysiology of human disease, where the infectious dose is thought to be on the order of 100 PFU [ 46 ]. Animals manifest the first signs of illness within two to five days after infection and die within nine to 17 days (compared with six to 15 days and 16 to 22 days, respectively, in human smallpox) and have a near 100% mortality rate (as compared to 30% in humans). The distribution and progression of skin lesions in monkeys is similar to that in human smallpox. Although there may be a potential advantage to using a non-human primate as a model for a human disease, this advantage is offset by the very large intravenous inoculum required to induce severe disease. CMX001 was tested in the i.v. inoculation MPXV model at oral doses of 2.5, 5, and 10 mg/kg administered on days 1, 3, 6, 9, and 12 post–infection. Although these doses did not reduce mortality, an explanation was provided by pharmacokinetic analysis which showed that systemic exposure of cynomolgus monkeys to CMX001 following oral administration was very low compared to that seen in other species including mice, rabbits, and humans, with humans having by far the best systemic exposure (AUC) after oral administration (see Table 4 ). A subsequent study of MPXV in monkeys using intramuscular (i.m.) administration of CMX001 showed an equivocal effect of treatment, despite this route resulting in a higher systemic exposure to CMX001. This result was somewhat unexpected given that i.v. CDV had been shown to be effective in the model and that both CDV and CMX001 produce the same active antiviral metabolite, CDV-PP. However, comparison of the levels of CDV-PP after i.v. CDV treatment versus i.m. CMX001 treatment (in healthy monkeys) revealed that the concentration of CDV-PP was much higher in the peripheral blood mononuclear cells (PBMCs) of animals treated with i.v. CDV compared to those treated with i.m. CMX001 ( Figure 2 ). This result is consistent with intracellular conversion of CMX001 to CDV being inefficient in monkeys. Because of this metabolic difference between monkeys and humans, data obtained with CMX001 in monkeys cannot be used to model human exposure. To circumvent this issue, CDV treatment of monkeys will be used to model the exposure to the active antiviral metabolite, CDV-PP, which is common to both CDV and CMX001. This approach to using monkey model data to help establish a human dose is described under "Selection of an Effective Dose in Humans" below. CMX001 in mouse models of orthopoxvirus infection The in vivo anti-orthopoxvirus activity of CMX001 has been extensively characterized in mice infected with ECTV, CPXV and VACV viruses. Ectromelia virus (mousepox) infection of mice has been used extensively as a model for orthopoxvirus pathogenesis and as a model for testing the efficacy of anti-orthopoxvirus therapeutics [ 34 ]. ECTV is a natural pathogen of mice and depending upon the mouse strain and route of inoculation, lethal infection can be initiated with a very small inoculum. Additionally, infection of mice with CPXV and VACV can be used to assess the efficacy of an antiviral against orthopoxviruses that can infect humans. The availability of an animal model(s) to help establish an effective dose for the treatment of vaccinia virus is critical in determining the potential utility of a drug in treating the side effects associated with a vaccinia derived, live attenuated virus smallpox vaccine. A large number of CMX001 regimens have been studied in ECTV-infected A/NCR mice. In this model, a 2.5 mg/kg dose of CMX001 given once daily for five days starting 4 h post-infection provides complete protection against a lethal intranasal challenge. When treatment is started as late as five days post-infection (3–4 days prior to the death of untreated controls), mice can be completely protected from mortality with a loading dose of 10 mg/kg followed by a maintenance dose of 2.5 mg/kg every other day for 14 days. A single 20 mg/kg oral dose of CMX001 given as late as four days post-infection provided 100% protection against lethal ECTV infection [ 35 , 36 ]. Against CPXV infection, oral administration of 10 mg/kg of CMX001 daily for five consecutive days was 100% protective in mice infected by either the aerosol inhalation or intranasal route when dosing was initiated 4 h post-infection [ 22 ]. A single oral dose of 12.5 mg/kg was 80% protective when given as early as five days before exposure, 100% effective when given one or three days before exposure, 100% effective when given one day post–exposure, and 87% protective when given as late as three days post-exposure [ 37 ]. CMX001 therapy showed significant protection (86%) against lethal VACV infection when initiated as late as 24 h post-infection (mean day of death for vehicle controls was 6.8) [ 37 ]. CMX001 in a rabbitpox model Rabbitpox virus is a subspecies of VACV that is naturally highly pathogenic in rabbits. Like other orthopoxvirus infections in their natural hosts, RPXV infection of rabbits shares many characteristics of smallpox. RPXV infection is readily transmitted between rabbits by the respiratory route and follows a pathophysiological progression similar to that of smallpox. Rabbits infected with RPXV undergo an incubation phase followed by the onset of fever, lesions, and ultimately respiratory distress and typically death [ 38 ]. Intradermal infection of rabbits allows for a controlled inoculum to be delivered while maintaining a course of disease nearly identical to that of the natural respiratory infection. Additionally, the lethal inoculum for intradermal infection is much lower than that required for intranasal infection, more closely mimicking the low inoculum required for human smallpox infection. Finally, intradermal infection results in a reliable occurrence of lesions, whereas with respiratory infection, rabbits sometimes die before rash development [ 39 ]. Ear lesions in the rabbitpox model, a result of systemic viral spread from the intradermal inoculation site on the thigh, typically appear beginning from three to five days post infection. As the desired trigger to initiate therapy for the treatment of smallpox is the onset of lesions, the reliable occurrence of lesions is a distinct advantage. CMX001 has been studied in New Zealand White rabbits infected intradermally with a lethal inoculum of RPXV [ 38 , 40 , 41 ]. Initial studies established the efficacy of CMX001 for pre-exposure and post-exposure prophylaxis. For example, 100% of animals administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated one day before challenge compared to 0% survival in animals administered placebo [ 38 ]. Delayed treatment experiments showed that CMX001 can be given late in the infection cycle, after appearance of clinical signs of disease (fever and lesions), and still prevent mortality. In a representative experiment, 100% of rabbits administered a dose of 10 mg CMX001/kg/day for five days survived when treatment was initiated four days after infection compared with 0% of placebo treated animals. Treatment initiated at five days post infection also provided a survival benefit (75% of CMX001-treated compared with 0% of placebo treated). In a series of randomized, blinded, placebo-controlled studies, administration of 20 mg/kg CMX001 initiated at the onset of lesions in the ears (mean 3.8 days post infection, range three to five days) provided a survival benefit over placebo-treated animals [ 40 ]. The results of these studies are summarized in Table 3 . Even a single dose of 20 mg/kg CMX001 given at the onset of lesions provided a statistically significant survival benefit compared to placebo [ 40 ]. Note that based on AUC 0–∞ , the 20 mg/kg dose in rabbits equates to a 0.67 mg/kg dose in humans (see Table 4 ). Doses in this range have been used to successfully treat fulminate disseminated dsDNA infections in patients. Monkeypox in Cynomolgus Monkeys Infection of cynomolgus monkeys with MPXV produces an exanthematous disease that can be used to evaluate the effect of a drug on skin lesions, viremia, and mortality. Because this model requires a large intravenous (i.v.) dose of virus (1–5 × 10 7 PFU of MPXV) to achieve a severe infection [ 42 – 45 ], the model may not reflect the pathophysiology of human disease, where the infectious dose is thought to be on the order of 100 PFU [ 46 ]. Animals manifest the first signs of illness within two to five days after infection and die within nine to 17 days (compared with six to 15 days and 16 to 22 days, respectively, in human smallpox) and have a near 100% mortality rate (as compared to 30% in humans). The distribution and progression of skin lesions in monkeys is similar to that in human smallpox. Although there may be a potential advantage to using a non-human primate as a model for a human disease, this advantage is offset by the very large intravenous inoculum required to induce severe disease. CMX001 was tested in the i.v. inoculation MPXV model at oral doses of 2.5, 5, and 10 mg/kg administered on days 1, 3, 6, 9, and 12 post–infection. Although these doses did not reduce mortality, an explanation was provided by pharmacokinetic analysis which showed that systemic exposure of cynomolgus monkeys to CMX001 following oral administration was very low compared to that seen in other species including mice, rabbits, and humans, with humans having by far the best systemic exposure (AUC) after oral administration (see Table 4 ). A subsequent study of MPXV in monkeys using intramuscular (i.m.) administration of CMX001 showed an equivocal effect of treatment, despite this route resulting in a higher systemic exposure to CMX001. This result was somewhat unexpected given that i.v. CDV had been shown to be effective in the model and that both CDV and CMX001 produce the same active antiviral metabolite, CDV-PP. However, comparison of the levels of CDV-PP after i.v. CDV treatment versus i.m. CMX001 treatment (in healthy monkeys) revealed that the concentration of CDV-PP was much higher in the peripheral blood mononuclear cells (PBMCs) of animals treated with i.v. CDV compared to those treated with i.m. CMX001 ( Figure 2 ). This result is consistent with intracellular conversion of CMX001 to CDV being inefficient in monkeys. Because of this metabolic difference between monkeys and humans, data obtained with CMX001 in monkeys cannot be used to model human exposure. To circumvent this issue, CDV treatment of monkeys will be used to model the exposure to the active antiviral metabolite, CDV-PP, which is common to both CDV and CMX001. This approach to using monkey model data to help establish a human dose is described under "Selection of an Effective Dose in Humans" below. 4.3. Selection of Endpoints With the exception of studies of VARV in monkeys where statistically powered mortality endpoints may not be feasible, the primary endpoint in animal efficacy studies is survival to 42 days post infection. This endpoint is clearly relevant to the desired benefit in humans. In VARV studies, due to the partial mortality in the model, the ethical concerns of using large numbers of monkeys to achieve statistical significance, and the practical constraints of handling large numbers of monkeys under biosafety level 4 conditions, the proposed primary endpoint is a reduction in the number of lesions. Historically, the mortality rate from ordinary-type smallpox was positively associated with the density of pock lesions, which almost certainly correlated with the intensity of the secondary viremia. Thus, an intervention that reduces viral replication should also reduce the number of lesions and increase survival. 4.4. Selection of an Effective Dose in Humans Straightforward use of different animal models of human smallpox for establishing the dose of a drug requires relatively consistent pharmacokinetic/pharmacodynamic profile of the drug across the animal model species. The pharmacokinetics (PK) of CMX001 have been determined in humans and in all animal species in which the drug has been used to study orthopoxvirus infection to date. Exposure to CMX001 after oral administration differs dramatically across species, with the lowest exposure occurring in monkeys and highest exposure occurring in humans (see Table 4 ). Even after achieving high plasma concentrations of CMX001 in monkeys by administration via non-oral routes, the conversion to the active moiety CDV-PP is low. The use of CDV as a surrogate for CMX001 in the monkey models avoids the differential PK and metabolism issues. As CDV-PP is the active antiviral metabolite that is common to both CMX001 and CDV, use of interspecies scaling through CDV-PP to extrapolate data from monkeys to humans is planned. In preliminary scaling studies, treatment with three doses of 20 mg/kg CMX001 has been shown to be effective in providing a survival benefit to rabbits infected with RPXV. In healthy rabbits, this treatment regimen resulted in an average CDV-PP concentration in PBMCs of 52.1 pg/10 6 cells when measured 24 hours after the third dose ( Figure 2 ). Similarly, treatment with three doses of 20 mg/kg CDV has also shown efficacy in providing a survival benefit to monkeys infected with MPXV [ 44 ]. In healthy monkeys, this treatment regimen resulted in average CDV-PP concentrations in PBMCs in the range of 52.9 to 75.6 pg/10 6 cells when measured from 12 hours after the first dose to 48 hours after the third dose ( Figure 2 ). Based upon these results, the target for an effective intracellular concentration of CDV-PP is in the range of approximately 50 pg/10 6 cells as measured in PBMCs. Reliable CDV-PP concentration data in PBMCs of humans treated with CMX001 is currently not available for comparison; however, in vitro studies with human PBMCs indicate that these CDV-PP levels will be achieved with doses of CMX001 currently being studied in clinical trials for treatment of other dsDNA virus infections. For example, a dose of 2 mg/kg CMX001 in humans produced a C max of approximately 0.6 μM (350 ng/mL) and an AUC of approximately 2650 h·ng/mL CMX001 in plasma (CMX001-102). Isolated human PBMCs incubated with 0.1 μM CMX001 for 24 hours (estimated AUC = 1348 h·ng/mL) produced a concentration of CDV-PP of approximately 79 pg/10 6 cells at 24 hours which is within the expected efficacious range as estimated from rabbits and monkeys. Furthermore, the long half-life of CDV-PP observed in human PBMCs in vitro indicates that infrequent dosing with CMX001 is sufficient to maintain an effective intracellular CDV-PP concentration. Additional studies measuring CDV-PP in the PBMCs of healthy and infected animals as well as in humans are planned. 5. Toxicology of CMX001 Twenty-six toxicology studies have been conducted to describe the toxicological profile and secondary pharmacology of CMX001 to support its clinical development. Most studies employed dose administration by the oral route; however, CMX001 has also been administered to animals topically and by the intravenous, subcutaneous and intramuscular routes. Thirteen week studies in rats and cynomolgus monkeys have been completed and a 39-week study in monkeys is ongoing. Dose administration was daily in studies up to 14 days duration and twice weekly in the 13 and 39 week studies because it appeared that a longer dosing interval would provide relief from the dose-limiting gastrointestinal (GI) toxicity allowing administration of higher doses. Topical administration resulted in quantifiable systemic exposure and parenteral administration was not well tolerated due to injection site reactions and poor hemocompatibility. The dose-limiting toxicity of CMX001 is gastrointestinal. In general, decreased food consumption and corresponding decreases in body weight were the first signs of CMX001-related GI toxicity. In rats given a single 100 mg/kg dose, these signs of toxicity appeared two to three days after administration followed by dehydration, anorexia and nonformed/liquid/absent feces beginning six to eight days after administration. Histopathology changes in the GI were present as early as 24 hours after a 100 mg/kg dose, most severe at six to seven days post-dose, and fully resolved by 14 days post-dose. The histological findings were described as enteritis or enteropathy generally centered in the ileum or jejunum. In monkeys, gastropathy was also observed. Similar findings were observed at lower doses in repeat dose studies with daily administration for up to 14 days; however, no GI findings were observed in rats and monkeys administered CMX001 twice weekly for up to 13 weeks at doses of up to 15 mg/kg suggesting an extended dosing interval allows the GI time to recover between administrations. CMX001 produced other toxicities that have previously been reported for cidofovir. CMX001 was positive in the chromosomal aberrations assay but negative in the Ames and in vivo micronucleus tests. Like cidofovir, there was a dose-related incidence of mammary adenocarcinomas in rats administered CMX001 however, no tumors were observed in a similar study in monkeys and no excess risk of carcinogenicity has been reported in humans administered cidofovir. Also like cidofovir, CMX001 had effects on reproduction and development including decreased fertility. Embryotoxicity, malformations (skeletal and visceral), and developmental effects including decreased body weight gain and delayed sexual maturation were observed at doses that caused maternal toxicity. In general, doses that did not produce these effects were identified and the dosing regimen required to produce the effects was either more frequent or longer than is anticipated for treatment of smallpox. Although CMX001 exposures (AUC) in humans were higher than exposures in animals at comparable doses, the toxicities observed in animals correlated with dose rather than with exposure (AUC) and are expected to be predictive of potential human toxicities despite differences in exposure. For example, no effect doses as well as doses that caused severe toxicity were the same in mice and monkeys even though exposures at a given dose were about 10-fold lower in monkeys compared with mice. To date, no dose-limiting toxicities have been observed in more than 300 patients administered CMX001 at doses that bracket the anticipated dose for smallpox given for durations of up to six months which exceeds the anticipated treatment course for smallpox. Exposures in humans were higher than those observed in animals given similar doses, however, the doses were below those that caused dose-limiting toxicity in animals and the correlation with dose rather than exposure appears to be confirmed by the absence of any dose-limiting toxicities in humans administered CMX001 to date. The most significant point of distinction between the toxicity of CMX001 and that of CDV, is nephrotoxicity, the dose-limiting toxicity of Vistide®. The clinical utility of cidofovir is limited by a risk of acute nephrotoxicity necessitating in-hospital administration by intravenous infusion preceded by hydration and renal protection using probenecid. Numerous endpoints were incorporated in the nonclinical development of CMX001 to monitor for potential effects on the kidney, including changes in serum chemistry (creatinine and BUN) and urinalysis values, changes in kidney weight, gross changes which included microscopic examination of the fresh cut (mid-line transverse section) surface of the kidney, and microscopic examination of fixed, stained sections by DACVP-certified veterinary pathologists. As judged by the absence of CMX001-related changes in any of these parameters, there was no evidence of cidofovir-like nephrotoxicity in any nonclinical study of CMX001. Hence, the dose-limiting toxicity of CMX001 appears to be gastrointestinal. In contrast to the nephrotoxicity produced by cidofovir, CMX001-related GI toxicity is easily monitored and rapidly reversible. 5.1. Species Differences and Similarities in CMX001 Pharmacokinetics and Metabolism The pharmacokinetics of CMX001 has been evaluated in single and multiple dose studies in mice, rats, rabbits and cynomolgus monkeys [ 47 , 48 ]. CMX001 was readily absorbed in all species after oral administration, with lower systemic plasma concentrations observed in monkeys relative to other species including human ( Table 4 ). The dose normalized exposure to CMX001 in humans (DN AUC 0–∞ ) is more than 150-fold higher than that observed in monkeys. This difference is attributed, in part, to a higher rate of oxidative hepatic metabolism of CMX001 in monkeys. The primary mode of elimination of CMX001 is via metabolism, based on lack of urinary elimination of CMX001 in animals and humans, as well as reasonably close predictions of in vivo clearance based on in vitro hepatocyte studies. After i.v. or oral administration of 14 C-CMX001 to monkeys or mice, less than 0.1% of the dose is excreted as unchanged parent drug in urine. After oral administration of CMX001 to humans, CMX001 was below detection in most urine samples. After in vitro incubation with cryopreserved suspended hepatocytes, 14 C-CMX001 was degraded more rapidly when incubated with cynomolgus or rhesus monkey hepatocytes than when incubated with rabbit or human hepatocyte incubations. Using intrinsic clearance obtained from this study to predict in vivo clearance based on the well-stirred model of hepatic clearance [ 49 ], a reasonably close prediction of cynomolgus monkey clearance is obtained (actual mean clearance values 22 and 25 mL/min/kg after i.v. administration of 1 and 4 mg/kg CMX001 to cynomolgus monkeys, respectively, compared to predicted 19 mL/min/kg), further supporting that metabolism by the liver is a major route of elimination for CMX001 [ 50 ]. The predicted versus actual clearance values for rabbits were 11 versus 16 mL/min/kg (i.v. administration of 4 mg/kg) and for humans were 6 versus 13 mL/min/kg (oral administration of 2 mg/kg). The qualitative in vitro and in vivo metabolite profile was similar across species, though the quantities of metabolites differed between species. After incubation of 14 C-CMX001 with mouse, rat, rabbit, monkey and human hepatocytes, the major metabolites determined by HPLC-MS-MS and radiochemical detection included CDV, CMX103 (3-(4-butanoic acid)propyl hydrogen ((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonate), CMX064 (3-hydroxypropyl hydrogen ((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonate); CMX108 (monohydroxylation on hexadecyl alkyl chain); and CMX108 glucuronide [ 50 ]. After administration of 14 C-CMX001 to cynomolgus monkeys, the major metabolite in plasma and excreta was identified as CMX064; CDV, CMX103 and four additional oxidative metabolites (lipid chain-shortened carboxylic acids) were also detected in plasma and excreta by HPLC-MS-MS. After administration of 14 C-CMX001 to mice, the major metabolite in plasma and excreta was CMX103, with lesser amounts of CDV and CMX064 also present. The concentrations of six metabolites (previously identified in animal ADME studies) were quantified in plasma samples following oral administration of CMX001 to humans (Clinical Study CMX001-103). The three major human metabolites were CMX103 (130% of CMX001 AUC), CDV (90% of CMX001 AUC) and CMX064 (50% of CMX001 AUC). The enzymes responsible for metabolism of CMX001 have not been fully elucidated. However, upon incubation of 14 C-CMX001 with human hepatocytes in the presence of the cytochrome P450 (CYP) inhibitor, 1-aminobenztriazole, the amount of CMX103 formed was reduced by approximately 50%, suggesting that the formation of CMX103 was partially mediated by CYP enzymes [ 51 ]. 5.2. OAT-mediated Secretion and Kidney Distribution CDV is actively secreted from blood into kidney proximal tubule cells by the organic anion transporters (OATs), leading to high concentrations in kidney proximal tubules and CDV-induced nephrotoxicity [ 52 ]. As shown in Figure 3 and in contrast to CDV, CMX001 uptake is not enhanced in vitro in cells expressing OAT1 compared to control cells that do not express this transporter (net uptake is approximately zero, after incubation with OAT1-expressing cells and non-OAT1-expressing control cells at a concentration of 5 μM of test drug for 5 min), nor is uptake reduced by the OAT inhibitor probenecid. These results indicate that CMX001 is not a substrate for OATs [ 53 ]. The distribution of radioactivity to the kidney seen in mice following administration of CMX001 is supportive of these in vitro findings. Peak concentrations of radioactivity in kidney cortex, the tissue which contains renal proximal tubules, was more than 30-fold lower after oral administration of 5 mg/kg 14 C-CMX001 compared to that observed after i.v. administration of an equivalent dose of 14 C-CDV. In addition, the tissue:plasma ratio in kidney cortex ranged from 4:1 at the time of peak concentration (4 h), and increased to 25:1 at 48 h after CMX001 administration compared to much higher ratios following i.v. cidofovir (21:1 at the time of peak concentration (0.5 h), with an increase to 400:1, 4 h after cidofovir administration). Thus, CMX001 has a low potential to cause CDV-induced nephrotoxicity because it is not itself a substrate for OATs [ 54 ]. 5.1. Species Differences and Similarities in CMX001 Pharmacokinetics and Metabolism The pharmacokinetics of CMX001 has been evaluated in single and multiple dose studies in mice, rats, rabbits and cynomolgus monkeys [ 47 , 48 ]. CMX001 was readily absorbed in all species after oral administration, with lower systemic plasma concentrations observed in monkeys relative to other species including human ( Table 4 ). The dose normalized exposure to CMX001 in humans (DN AUC 0–∞ ) is more than 150-fold higher than that observed in monkeys. This difference is attributed, in part, to a higher rate of oxidative hepatic metabolism of CMX001 in monkeys. The primary mode of elimination of CMX001 is via metabolism, based on lack of urinary elimination of CMX001 in animals and humans, as well as reasonably close predictions of in vivo clearance based on in vitro hepatocyte studies. After i.v. or oral administration of 14 C-CMX001 to monkeys or mice, less than 0.1% of the dose is excreted as unchanged parent drug in urine. After oral administration of CMX001 to humans, CMX001 was below detection in most urine samples. After in vitro incubation with cryopreserved suspended hepatocytes, 14 C-CMX001 was degraded more rapidly when incubated with cynomolgus or rhesus monkey hepatocytes than when incubated with rabbit or human hepatocyte incubations. Using intrinsic clearance obtained from this study to predict in vivo clearance based on the well-stirred model of hepatic clearance [ 49 ], a reasonably close prediction of cynomolgus monkey clearance is obtained (actual mean clearance values 22 and 25 mL/min/kg after i.v. administration of 1 and 4 mg/kg CMX001 to cynomolgus monkeys, respectively, compared to predicted 19 mL/min/kg), further supporting that metabolism by the liver is a major route of elimination for CMX001 [ 50 ]. The predicted versus actual clearance values for rabbits were 11 versus 16 mL/min/kg (i.v. administration of 4 mg/kg) and for humans were 6 versus 13 mL/min/kg (oral administration of 2 mg/kg). The qualitative in vitro and in vivo metabolite profile was similar across species, though the quantities of metabolites differed between species. After incubation of 14 C-CMX001 with mouse, rat, rabbit, monkey and human hepatocytes, the major metabolites determined by HPLC-MS-MS and radiochemical detection included CDV, CMX103 (3-(4-butanoic acid)propyl hydrogen ((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonate), CMX064 (3-hydroxypropyl hydrogen ((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonate); CMX108 (monohydroxylation on hexadecyl alkyl chain); and CMX108 glucuronide [ 50 ]. After administration of 14 C-CMX001 to cynomolgus monkeys, the major metabolite in plasma and excreta was identified as CMX064; CDV, CMX103 and four additional oxidative metabolites (lipid chain-shortened carboxylic acids) were also detected in plasma and excreta by HPLC-MS-MS. After administration of 14 C-CMX001 to mice, the major metabolite in plasma and excreta was CMX103, with lesser amounts of CDV and CMX064 also present. The concentrations of six metabolites (previously identified in animal ADME studies) were quantified in plasma samples following oral administration of CMX001 to humans (Clinical Study CMX001-103). The three major human metabolites were CMX103 (130% of CMX001 AUC), CDV (90% of CMX001 AUC) and CMX064 (50% of CMX001 AUC). The enzymes responsible for metabolism of CMX001 have not been fully elucidated. However, upon incubation of 14 C-CMX001 with human hepatocytes in the presence of the cytochrome P450 (CYP) inhibitor, 1-aminobenztriazole, the amount of CMX103 formed was reduced by approximately 50%, suggesting that the formation of CMX103 was partially mediated by CYP enzymes [ 51 ]. 5.2. OAT-mediated Secretion and Kidney Distribution CDV is actively secreted from blood into kidney proximal tubule cells by the organic anion transporters (OATs), leading to high concentrations in kidney proximal tubules and CDV-induced nephrotoxicity [ 52 ]. As shown in Figure 3 and in contrast to CDV, CMX001 uptake is not enhanced in vitro in cells expressing OAT1 compared to control cells that do not express this transporter (net uptake is approximately zero, after incubation with OAT1-expressing cells and non-OAT1-expressing control cells at a concentration of 5 μM of test drug for 5 min), nor is uptake reduced by the OAT inhibitor probenecid. These results indicate that CMX001 is not a substrate for OATs [ 53 ]. The distribution of radioactivity to the kidney seen in mice following administration of CMX001 is supportive of these in vitro findings. Peak concentrations of radioactivity in kidney cortex, the tissue which contains renal proximal tubules, was more than 30-fold lower after oral administration of 5 mg/kg 14 C-CMX001 compared to that observed after i.v. administration of an equivalent dose of 14 C-CDV. In addition, the tissue:plasma ratio in kidney cortex ranged from 4:1 at the time of peak concentration (4 h), and increased to 25:1 at 48 h after CMX001 administration compared to much higher ratios following i.v. cidofovir (21:1 at the time of peak concentration (0.5 h), with an increase to 400:1, 4 h after cidofovir administration). Thus, CMX001 has a low potential to cause CDV-induced nephrotoxicity because it is not itself a substrate for OATs [ 54 ]. 6. Clinical Experience with CMX001 CMX001 is currently in clinical development for the prophylaxis and treatment of multiple dsDNA viral infections including: (1) the treatment of smallpox under the "Animal Rule" using animal efficacy data and human safety data, (2) prophylaxis/preemption of cytomegalovirus disease in human stem cell transplant (HSCT) recipients, and (3) preemption treatment of adenovirus disease in pediatric HSCT recipients. CMV and AdV are common, typically mild and self-limited, infections in the healthy host, but cause life- and graft-threatening disease in transplant patients as well as severe opportunistic infections in other immunocompromised individuals. The demonstration of safety and efficacy of CMX001 in humans with these life-threatening dsDNA virus infections can provide critical supportive data for the registration package for the treatment of smallpox under the Animal Rule. In draft guidance for industry entitled "Smallpox (Variola) Infection: Developing Drugs for the Treatment or Prevention" published in November of 2007, FDA states that "because smallpox is no longer a naturally occurring disease, data from studies of a candidate in other human illnesses might play a more important role than usual in the evaluation of drug safety." In this same guidance FDA also states that human data are particularly compelling if they involve the study of "viral infections related to variola." Certainly in the case of CMX001, pharmacokinetic and pharmacodynamic parameters for doses that demonstrate clinical efficacy for the treatment of dsDNA viral infections can be compared directly to pharmacokinetic and pharmacodynamic parameters necessary for the prevention of mortality in animal models of human smallpox ( i.e. , animals infected with dsDNA viruses in the family Orthopoxviridae ). This comparison will validate that drug exposures necessary to treat smallpox can be achieved in humans and that these doses are efficacious in humans as evidenced by activity against similar dsDNA virus infections. CMX001 has been administered to healthy human volunteers in two completed Phase 1 clinical trials (designated CMX001-102 and CMX001-103). Two clinical trials are ongoing: a Phase 1b/2a trial (CMX001-104) in renal and hematopoietic stem cell transplants (HSCT) patients with BK virus viruria, and a Phase 2 trial (CMX001-201) in HSCT recipients to evaluate CMX001 for the prophylaxis and/or pre-emptive treatment of CMV infection in this population. In addition over 105 patients have been treated for various dsDNA virus infections under Emergency INDs or the ex-US equivalent. 6.1. CMX001-102 CMX001–102 was a Phase 1, dose-escalation, pharmacokinetic (PK), first time in humans (FTIH) study of the safety and tolerability of CMX001 in healthy human volunteers. A solution formulation of CMX001 was administered as a weight-based dose to volunteers in a total of nine single dose cohorts (.025, .05, 0.1, 0.2, 0.4, 0.6, 1, 1.5 and 2 mg/kg) and five multiple dose cohorts (0.1, 0.2, 0.4, 0.6 and 1.0 mg/kg). Subjects in the multiple dose cohorts received a total of three doses of CMX001, one dose every six days. Each cohort enrolled six subjects randomized 2:1 (active: placebo). Following the completion of each cohort, all safety data and subsequent dose escalations were reviewed and approved by a Data Safety Monitoring Board (DSMB) and the FDA. Safety analysis included clinical and laboratory assessments as well as wireless capsule endoscopy (WCE) which was conducted before and after dosing to look for macroscopic evidence of GI toxicity. There were no severe adverse events (SAEs), no adverse events (AEs) that prevented dose escalation, and no evidence of GI toxicity. A total of 30 treatment-emergent adverse events were reported by 16 subjects (30% of subjects who received at least one dose of CMX001 or placebo). Among the 36 subjects who received CMX001, nine (25%) subjects reported at least one adverse event and among the 18 subjects who received placebo, seven (39%) subjects reported at least one adverse event. No dose-related trend in the incidence or severity of AEs was observed. After a single dose under fasting conditions, CMX001 was readily absorbed with the time to maximum plasma concentration (T max ) ranging from two to three hours. Maximum plasma concentration (C max ) and systemic exposure (AUC) increased approximately in proportion to dose over the range of 0.025 to 2.0 mg/kg. The half-life of elimination (t 1/2 elim ) increased with increasing dose, ranging from 6.15 hours at 0.025 mg/kg to 32.7 hours at 1.5 mg/kg, presumably due to better definition of the elimination phase at higher doses. CMX001 is not eliminated in urine; however, CDV, a metabolite of CMX001 is eliminated in urine. No changes in PK parameters were observed after repeat administration. 6.2. CMX001-103 CMX001-103 was a Phase 1 comparative bioavailability study of CMX001 solution versus tablets, plus a comparison of PK parameters for CMX001 and CDV in subjects administered CMX001 after fasting overnight versus having eaten a high fat meal within 30 minutes of dosing. CMX001 was administered as a fixed dose to 24 volunteers in three single doses (40 mg solution, fasted; 40 mg tablet following a high fat breakfast; and 40 mg tablet fasted). Each dose was separated by a 14-day washout period. CMX001 was generally safe and well tolerated by healthy volunteers. The most frequently reported adverse events were headache (17%), increased blood CPK (17%), increased ALT (13%), nausea (8%), and oropharyngeal pain (8%). This study showed that CMX001 administered as a tablet in the fasted state produces similar C max and AUC values to those observed after CMX001 administration as solution in the fasted state, and that CMX001 administered as tablet in the fed state as compared with the fasted state demonstrates a significant food effect as evidenced by decreased C max , AUC ∞ and AUC last values. Peak plasma concentrations (C max ) and systemic exposure (AUC 0–∞ ) following administration in the fed state were reduced approximately 48% and 28%, respectively, compared with C max and AUC 0–∞ following administration to fasted subjects. In addition, the median time to peak plasma concentrations in fed subjects was approximately twice that for fasted subjects. Changing the dose formulation from solution to tablet may reduce exposure to CMX001 by a maximum of approximately 13%. 6.3. CMX001-104 CMX001-104, a study of the safety, tolerability, and preliminary antiviral activity of CMX001 in renal transplant and HSCT recipients with BK viruria, is nearing completion. A total of twenty-eight subjects were enrolled into this study. All received fixed doses of 10, 20, or 40 mg of CMX001 or placebo once or twice weekly for up to 28 days. No serious adverse events attributable to study drug ( i.e. , CMX001 or placebo) have been reported. 6.4. CMX001-201 CMX001-201, a multicenter, randomized, double-blind, placebo-controlled, dose-escalation study of the safety, tolerability, and ability of CMX001 to prevent or control post-transplant CMV infection in CMV seropositive HSCT recipients is underway. Up to 6 dose-escalation cohorts of 32 subjects per cohort are planned. An expansion phase of this study is also planned to enroll up to 200 patients at a dose assessed during the dose-escalation phase and selected based on preliminary safety and efficacy. The expansion phase will increase enrollment at the selected dose to obtain the statistical power needed to show a difference in efficacy between CMX001 and placebo in the prevention of CMV. Forty subjects were enrolled in cohort 1 and 39 subjects were enrolled in cohort 2. Subjects were randomized 3:1 to receive once weekly doses of 40 mg (cohort 1) or 100 mg (cohort 2) of CMX001 or matching placebo. Treatment with study drug was continued for up to 90 days (or 13 weeks) post-transplant. Enrollment into cohort 3, at a once weekly dose of 200 mg CMX001 or placebo is ongoing. 6.5. Emergency IND Experience Additional clinical experience with CMX001 has been gained through the emergency treatment of various dsDNA virus infections under Emergency IND (EIND) or ex-US equivalent [ 55 – 57 ]. More than 105 patients have received CMX001 through emergency single patient protocols. Patients from one month to 69 years of age have been treated, with the longest treatment duration in excess of six months. Results of the EIND experience have been reported for the use of CMX001 to treat recurrent CMV (a case series involving three transplant patients who had failed valganciclovir therapy), AdV infection (a case series of thirteen immunocompromised patients with a median age of 12 years, range 0.75 to 66, with disseminated AdV infection) and progressive vaccinia (a single case with complications from smallpox vaccination). For example, AdV viremia declined by almost 1000-fold (2.9 log 10 ) in patients who received eight weeks of CMX001 therapy [ 55 ]. In an analysis of pharmacokinetic data from 49 patients treated under EINDs with CMX001, higher clearance resulting in lower systemic exposure (AUC 0–∞ ) was observed in pediatric (age ≤ 18 years) patients relative to adults at doses of 2, 3 and 4 mg/kg. At a given dose, pediatric patients typically had 30% to 50% of the average adult exposure. In both pediatric and adult patients, C max and AUC 0–∞ generally increased greater than in proportion to dose. In a second analysis conducted separately for both pediatric and adult patients, systemic exposure to CMX001 was comparable between patients with normal renal function and those with moderate to severe renal impairment, in both the pediatric and adult populations, indicating that dose adjustment is not needed in patients with renal impairment. In this analysis, it was found that CMX001 exposure is not affected by hemodialysis [ 48 ]. 6.6. Additional Studies in Planning Stage Two additional clinical studies are under development. A randomized, double-blind, placebo-controlled study evaluating the safety and efficacy of pre-emptive treatment with CMX001 in children for the prevention of AdV disease following HSCT is planned. Also, a multicenter, open-label study of CMX001 treatment of serious diseases or conditions caused by dsDNA viruses is in the initial stages of enrollment. In the open-label study, up to 200 patients without viable alternatives, including other studies of CMX001, will be enrolled for the treatment of diseases caused by poxviruses (vaccinia virus and molluscum contagiosum), adenoviruses, herpesviruses (CMV, HSV, EBV, VZV, HHV6), polyomaviruses (BKV or JCV), and HPV. Treatment with CMX001 in this protocol may be continued twice weekly for up to six months, as needed, based on the clinical disease state of the patient. 6.1. CMX001-102 CMX001–102 was a Phase 1, dose-escalation, pharmacokinetic (PK), first time in humans (FTIH) study of the safety and tolerability of CMX001 in healthy human volunteers. A solution formulation of CMX001 was administered as a weight-based dose to volunteers in a total of nine single dose cohorts (.025, .05, 0.1, 0.2, 0.4, 0.6, 1, 1.5 and 2 mg/kg) and five multiple dose cohorts (0.1, 0.2, 0.4, 0.6 and 1.0 mg/kg). Subjects in the multiple dose cohorts received a total of three doses of CMX001, one dose every six days. Each cohort enrolled six subjects randomized 2:1 (active: placebo). Following the completion of each cohort, all safety data and subsequent dose escalations were reviewed and approved by a Data Safety Monitoring Board (DSMB) and the FDA. Safety analysis included clinical and laboratory assessments as well as wireless capsule endoscopy (WCE) which was conducted before and after dosing to look for macroscopic evidence of GI toxicity. There were no severe adverse events (SAEs), no adverse events (AEs) that prevented dose escalation, and no evidence of GI toxicity. A total of 30 treatment-emergent adverse events were reported by 16 subjects (30% of subjects who received at least one dose of CMX001 or placebo). Among the 36 subjects who received CMX001, nine (25%) subjects reported at least one adverse event and among the 18 subjects who received placebo, seven (39%) subjects reported at least one adverse event. No dose-related trend in the incidence or severity of AEs was observed. After a single dose under fasting conditions, CMX001 was readily absorbed with the time to maximum plasma concentration (T max ) ranging from two to three hours. Maximum plasma concentration (C max ) and systemic exposure (AUC) increased approximately in proportion to dose over the range of 0.025 to 2.0 mg/kg. The half-life of elimination (t 1/2 elim ) increased with increasing dose, ranging from 6.15 hours at 0.025 mg/kg to 32.7 hours at 1.5 mg/kg, presumably due to better definition of the elimination phase at higher doses. CMX001 is not eliminated in urine; however, CDV, a metabolite of CMX001 is eliminated in urine. No changes in PK parameters were observed after repeat administration. 6.2. CMX001-103 CMX001-103 was a Phase 1 comparative bioavailability study of CMX001 solution versus tablets, plus a comparison of PK parameters for CMX001 and CDV in subjects administered CMX001 after fasting overnight versus having eaten a high fat meal within 30 minutes of dosing. CMX001 was administered as a fixed dose to 24 volunteers in three single doses (40 mg solution, fasted; 40 mg tablet following a high fat breakfast; and 40 mg tablet fasted). Each dose was separated by a 14-day washout period. CMX001 was generally safe and well tolerated by healthy volunteers. The most frequently reported adverse events were headache (17%), increased blood CPK (17%), increased ALT (13%), nausea (8%), and oropharyngeal pain (8%). This study showed that CMX001 administered as a tablet in the fasted state produces similar C max and AUC values to those observed after CMX001 administration as solution in the fasted state, and that CMX001 administered as tablet in the fed state as compared with the fasted state demonstrates a significant food effect as evidenced by decreased C max , AUC ∞ and AUC last values. Peak plasma concentrations (C max ) and systemic exposure (AUC 0–∞ ) following administration in the fed state were reduced approximately 48% and 28%, respectively, compared with C max and AUC 0–∞ following administration to fasted subjects. In addition, the median time to peak plasma concentrations in fed subjects was approximately twice that for fasted subjects. Changing the dose formulation from solution to tablet may reduce exposure to CMX001 by a maximum of approximately 13%. 6.3. CMX001-104 CMX001-104, a study of the safety, tolerability, and preliminary antiviral activity of CMX001 in renal transplant and HSCT recipients with BK viruria, is nearing completion. A total of twenty-eight subjects were enrolled into this study. All received fixed doses of 10, 20, or 40 mg of CMX001 or placebo once or twice weekly for up to 28 days. No serious adverse events attributable to study drug ( i.e. , CMX001 or placebo) have been reported. 6.4. CMX001-201 CMX001-201, a multicenter, randomized, double-blind, placebo-controlled, dose-escalation study of the safety, tolerability, and ability of CMX001 to prevent or control post-transplant CMV infection in CMV seropositive HSCT recipients is underway. Up to 6 dose-escalation cohorts of 32 subjects per cohort are planned. An expansion phase of this study is also planned to enroll up to 200 patients at a dose assessed during the dose-escalation phase and selected based on preliminary safety and efficacy. The expansion phase will increase enrollment at the selected dose to obtain the statistical power needed to show a difference in efficacy between CMX001 and placebo in the prevention of CMV. Forty subjects were enrolled in cohort 1 and 39 subjects were enrolled in cohort 2. Subjects were randomized 3:1 to receive once weekly doses of 40 mg (cohort 1) or 100 mg (cohort 2) of CMX001 or matching placebo. Treatment with study drug was continued for up to 90 days (or 13 weeks) post-transplant. Enrollment into cohort 3, at a once weekly dose of 200 mg CMX001 or placebo is ongoing. 6.5. Emergency IND Experience Additional clinical experience with CMX001 has been gained through the emergency treatment of various dsDNA virus infections under Emergency IND (EIND) or ex-US equivalent [ 55 – 57 ]. More than 105 patients have received CMX001 through emergency single patient protocols. Patients from one month to 69 years of age have been treated, with the longest treatment duration in excess of six months. Results of the EIND experience have been reported for the use of CMX001 to treat recurrent CMV (a case series involving three transplant patients who had failed valganciclovir therapy), AdV infection (a case series of thirteen immunocompromised patients with a median age of 12 years, range 0.75 to 66, with disseminated AdV infection) and progressive vaccinia (a single case with complications from smallpox vaccination). For example, AdV viremia declined by almost 1000-fold (2.9 log 10 ) in patients who received eight weeks of CMX001 therapy [ 55 ]. In an analysis of pharmacokinetic data from 49 patients treated under EINDs with CMX001, higher clearance resulting in lower systemic exposure (AUC 0–∞ ) was observed in pediatric (age ≤ 18 years) patients relative to adults at doses of 2, 3 and 4 mg/kg. At a given dose, pediatric patients typically had 30% to 50% of the average adult exposure. In both pediatric and adult patients, C max and AUC 0–∞ generally increased greater than in proportion to dose. In a second analysis conducted separately for both pediatric and adult patients, systemic exposure to CMX001 was comparable between patients with normal renal function and those with moderate to severe renal impairment, in both the pediatric and adult populations, indicating that dose adjustment is not needed in patients with renal impairment. In this analysis, it was found that CMX001 exposure is not affected by hemodialysis [ 48 ]. 6.6. Additional Studies in Planning Stage Two additional clinical studies are under development. A randomized, double-blind, placebo-controlled study evaluating the safety and efficacy of pre-emptive treatment with CMX001 in children for the prevention of AdV disease following HSCT is planned. Also, a multicenter, open-label study of CMX001 treatment of serious diseases or conditions caused by dsDNA viruses is in the initial stages of enrollment. In the open-label study, up to 200 patients without viable alternatives, including other studies of CMX001, will be enrolled for the treatment of diseases caused by poxviruses (vaccinia virus and molluscum contagiosum), adenoviruses, herpesviruses (CMV, HSV, EBV, VZV, HHV6), polyomaviruses (BKV or JCV), and HPV. Treatment with CMX001 in this protocol may be continued twice weekly for up to six months, as needed, based on the clinical disease state of the patient. 7. Conclusions CMX001 has demonstrated in vitro and in vivo efficacy against orthopoxvirus infections. In the rabbitpox model it significantly improved survival even when administered after lesions were detected. Although the antiviral activity of CMX001 is not evaluable in monkeys due to species specific metabolism/anabolism issues which result in low intracellular levels of the active antiviral, CDV is an effective surrogate for CMX001 and scaling animal results to humans via CDV-PP should allow development to proceed using the variola model in monkeys combined with proof of efficacy against similar dsDNA virus infections in humans. Current data suggest the 2 mg/kg dose of CMX001 which has been used to treat dsDNA virus infections in patients should produce CDV-PP levels above those attained in rabbits dosed with levels of CMX001 which effectively prevented mortality after appearance of lesions. The reductions in adenovirus viremia reported in EIND patients following treatment with CMX001 provide further support for potential human efficacy from a non-orthopox dsDNA virus model that has important similarities to VARV, including disseminated, lytic infection of epithelial cells. The emerging efficacy, safety, pharmacokinetic and resistance profiles of CMX001 make it a promising antiviral for use in the treatment of smallpox.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789758/

Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira . Since genetic transformation remains technically limited for pathogenic Leptospira , a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo , tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens. Introduction Leptospirosis, caused by spirochete bacteria of the genus Leptospira , is a zoonotic disease of high public health impact [1] . Globally, nearly 900,000 people are infected annually through contact with contaminated water, infected tissue or urine of mammalian reservoir hosts [2] . Phylogenetic analyses have resolved the genus into 3 distinct lineages, which are the focus of a pan- Leptospira genome project supported by the NIAID Genome Sequencing Center: nine pathogenic species; five intermediate species (eg. L. fainei, L. licerasiae ); and six non-infectious saprophytic species (i.e. L. biflexa ) ( Fig. 1A ) [3] – [6] . The greatest burden of disease is caused by the pathogenic species, mainly affecting people living in poverty and with poor sanitation [1] , [2] , [7] . Epidemics of leptospirosis associated with floods, monsoons, or hurricanes have a high morbidity and mortality with case fatality rates ranging as high as 20–25% in hospitalized patients leading to refractory shock, jaundice, renal failure, and pulmonary hemorrhage [1] . 10.1371/journal.pntd.0002468.g001 Figure 1 Pathogenomic analysis of Leptospira interrogans serovar Lai strain 556021 to identify virulence related genes. ( A ) Schematic of phylogenetic relatedness of "Pathogenic" (P), "Intermediate" (I) and "Saprophytic" (S) members of the genus Leptospira . ( B ) Workflow to identify putative virulence-associated genes. Asterisk denotes a hypothetical position in which a SNV has been identified ( C ) Genomic Locations of SNPs and PF07598 paralogs in the reference genome of L. interrogans serovar Lai strain 56601. Each concentric circle represents genomic data and is numbered from the outermost to the innermost circle. The outermost circles represent the predicted CDS on the + and − strands, respectively, colored by functional role categories (see key). The following circle descriptions apply to chromosome I. The third circle notes the location of predicted prophage regions (olive) and the LPS region (slate). The fourth circle indicates those CDS found to have non-synonymous amino acid substitutions (black) as well as the location of CDS annotated as "transposase" in Genbank (salmon). The fifth circle represents the location of the 12 PF07598 family members (blue). The innermost circle denotes atypical regions (χ 2 value). For chromosome II, the outermost and innermost circles are the same as for chromosome I; however, the third circle notes the location of transposases (salmon), while the fourth circle indicates the location of the CDS found to have non-synonymous amino acid substitutions (black). Despite its severity and global importance, the molecular pathogenesis of leptospirosis remains poorly understood [8] . Leptospira penetrate mucosal epithelium and damaged integument then hematogenously disseminate to localize within multiple organs, including the liver and kidney, within 72 hours. Leptospiremia may continue for up to two weeks after onset of symptoms with blood bacterial concentrations reaching as high as 10 6 –10 7 organisms/mL in infected patients [9] , [10] . The only virulence factor genetically defined to date is the surface lipoprotein Loa22 [11] , but mechanisms by which it contributes to disease pathogenesis remain unknown. Other virulence-associated genes include heme oxygenase [12] , LPS [13] , clpB [14] , and flagellar components [15] , [16] . Although random transposon mutagenesis has been used to identify a few putative leptospiral virulence-related genes [17] [18] , further progress has been hindered by the lack of efficient gene-targeted mutagenesis techniques in pathogenic Leptospira [8] . We used a functional systems biology (pathogenomic) approach to identify candidate virulence genes, by genomic comparison of a culture-attenuated Leptospira interrogans serovar Lai strain 56601 (LD 50 >10 9 )( Fig. 1B ) with its virulent, isogenic parent (LD 50 <100) [19] . In vivo relevance of identified candidate genes was determined by quantification of expression of candidate genes on day 4 after hamster infection in blood, liver, and kidney compared to in vitro culture. Materials and Methods Ethics statement This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in AAALAC-approved facilities. The experimental animal work was approved by the Institutional Animal Care and Use Committee of the University of California San Diego under protocol S03128H. Bacterial strain maintenance and attenuation of L. interrogans serovar Lai strain 56601 All strains were maintained in vitro in Ellinghausen-McCullough-Johnson -Harris (EMJH) media using standard protocols and are available from BEI Resources. L. interrogans serovar Lai strain 56601 was obtained from Dr. David Haake (UCLA). Virulence was selected for by serial passage through hamsters so that the P1 strain used in the present study had an LD 50 of ∼10 organisms. L. interrogans serovar Lai strain 56601_P1 was attenuated by 18 bi-weekly subcultures in vitro . Virulence was assessed every five to ten subcultures using three-week-old male Golden Syrian Hamsters. Following a final subculture, genomic DNA was prepared from this attenuated strain (designated P19) on which next generation sequencing was carried out. Genome assembly of virulent P1 and attenuated P19 L. interrogans serovar Lai strain 56601 and non-synonymous SNV (nsSNV) detection We generated 4,379,515 and 5,340,095 unpaired shotgun reads from L. interrogans serovar Lai 56601_P1 and 56601_P19, respectively using next generation sequencing technology. All reads were 36 bases long. Both genomes were assembled using the comparative assembler AMOScmp. The AMOSCmp-shortReads-alignmentTrimmed pipeline that runs within AMOScmp, was used to look for exact matches of each read to the published L. interrogans serovar Lai 56601 genome of at least 20 bp, permitting a maximum consensus error rate of 0.06% (i.e. at most two mismatches in any read). This script runs a reference-based trimming of the 3′-end of the reads prior to assembly. We found that trimming of at most 4 bases from the 3′-end of the reads based on their matches to the reference produced better assemblies than un-trimmed reads. The P1 assembly used 3,919,609 reads, leaving 459,906 unassembled singletons, while the P19 assembly used 4,915,295 leaving 424,800 singleton reads. The 56601_P1 genome was assembled into 167 contigs with an average length of 28,124 kb and an N50 length of 105,604 kb and the P19 genome into 97 contigs, average length 48,417 and N50 of 190,406. We checked the quality of both assemblies using the amosvalidate pipeline, which runs within AMOScmp. This pipeline identifies misassembly features such as increased read depth and correlated SNVs (i.e. one or more reads with the same SNV, which is unlikely to be due to sequencing error), both indicative of collapsed repeats. We found that both assemblies were high quality with at most 5 potential misassembly features in longer contigs. These potential misassemblies were inspected manually using the Hawkeye viewer and reassembled if necessary using minimus, which employs a stricter assembly algorithm. The unfinished 56601_P1a and 56601_P19 genomes were aligned and SNVs identified using the MUMmer v3.22 software package. RT-qPCR in vivo gene expression analysis Three wk old Golden Syrian Hamsters were infected via intraperitoneal injection with 10 8 low passage L. interrogans serovar Lai strain 56601. 96 hours post infection total RNA was collected using TRIzol (Invitrogen) from blood, liver, and kidney tissue, as well as from a 96-hour EMJH culture of L. interrogans grown at 30°C. Total RNA was reverse transcribed using a QuantiTect reverse transcription kit (Qiagen). cDNA was amplified using a CFX96 thermal cycler (Bio-Rad) using PerfeCta SYBR Green FastMix (Quanta Biosciences). PCR was carried out at 95°C for 3 min, a touchdown gradient of 14 cycles of (94°C 10 s, 80°C 45 s) decreasing 1°C/cycle, followed by 40 cycles of (94°C 30 s, 65°C 45 s). Ct values were normalized to the leptospiral 16S rRNA gene and expression fold change calculated using the Pfaffl method [20] . Primer sequences are listed in Table S3 in Text S1 . Domain architecture analysis of LA_4008 and other related AGC proteins Domain architecture comparison of LA_4008 with orthologs of Myxococcus xanthus , Corallococcus coralloides , Stigmatella aurantiaca , and Mycobacterium tuberculosis using NCBI CD Search, SMART, and TPRPred. Protein homology analysis was carried out using BLAST using the following reference sequences: LA_4008 (NP_714188.1), MXAN_4545 (YP_632713.1), COCOR_04748 (YP_005370712.1), STAUR_4866 (YP_003954471.1), Rv0386 (CCP43116). The coverage for the query sequence, statistical significance (E-value), and maximum amino acid identify ("Max Ident") are indicated at right for each predicted primary sequence. Identified domains were then graphically represented using the DOG 1.0 program ( http://dog.biocuckoo.org ) Leptospira Concentrated Culture Supernatant (CCS) L. interrogans Lai 56601 or L. licerasiae Varillal were grown in EMJH media +10% heat inactivated rabbit serum at 37°C on a rotating shaker for 96 hr. Culture was centrifuged for 30 min at 10,000× g. Supernatant was decanted, filtered through a .22 µm syringe filter unit (Millipore), and concentrated 10× in an Amicon Ultra 10K MWCO centrifugal filter unit (Millipore). No leptospires were observed in the CCS after concentration using darkfield microscopy. CCS cAMP elevating activity CCS was incubated with monolayers of THP-1,a human monocyte/macrophage cell line. At 4, 6, and 20 hours cells were rinsed 3× in PBS and analyzed for cAMP (Direct cAMP EIA kit, Enzo Life Sciences). Secondly, CCS from L. interrogans Lai and L. licerasiae were incubated with THP-1 monolayers for 4 hours, and assayed for cAMP. CCS immunodepletion studies Rabbits were used to generate anti-peptide antisera against LA_4008 using a protein specific, sixteen amino acid fragment (SVEEDPLTREIDRKQK) conjugated to keyhole limpet hemocyanin as a carrier protein (Pacific Immunology, Ramona, CA). The IgG fractions from pre-immunization and production bleeds were purified using a Melon Gel IgG Purification kit (Thermo Scientific) and covalently linked to magnetic beads using a NanoLink BeadLink Kit (Solulink). Antibody linked beads were incubated with CCS overnight at 4°C on a rotating shaker. Beads were separated on a QuadroMACS separation unit (Miltenyi Biotec). Depleted CCS was applied to THP-1 monolayers and incubated for 4 hours. Cells were rinsed 3× in PBS and analyzed for total cAMP using the Direct cAMP EIA kit. Phylogenetic analysis of PF07598 paralogous protein family L. interrogans , L. borgpetersenii and B. bacilliformis full-length sequences were downloaded from the Uniprot databa se ( http://www.uniprot.com ) and aligned using MAFFT v7 ( http://mafft.cbrc.jp/alignment/software ) with default parameters. The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman frequency model [21] . Statistical support of the tree topology was obtained from 500 bootstrap replicates. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 271 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [22] . Pan-genomic analysis of attenuated genes and PF07598 orthologs The genomic data analyzed here are publically available and are from newly generated, unpublished Leptospira whole genome sequence data produced by the JCVI as part of the white paper " Leptospira Genomics and Human Health," sponsored by the NIAID-funded Genome Sequencing Centers. PanOCT [23] was run using default settings with the exception that a dynamically determined pairwise cutoff was implemented, not available in the current release, but available upon request. The following genomes, representing all 20 Leptospira spp. were used: L. alexanderi sv. Manha 3 str. L 60 T (Genbank:AHMT00000000), L. alstoni sv. Pingchang str. 80-412 (Genbank:AOHD00000000), L. biflexa sv. Patoc str. Patoc I Paris (Genbank:CP000786), L. borgpetersenii sv. Javanica str. UI 09931 (Genbank:AHNP00000000), L. broomii sv. undetermined str. 5399 T (Genbank:AHMO00000000), L. fainei sv. Hurstbridge str. BUT 6 T (Genbank:AKWZ00000000), L. inadai sv. Lyme str. 10 T (Genbank:AHMM00000000), L. interrogans sv. Copenhageni str. Fiocruz L1-130 (Genbank:AE016823), L. interrogans sv. Copenhageni str. M20 (Genbank:AOGV00000000), L. interrogans sv. Lai str. 56601 (Genbank:AE010300), L. kirschneri sv. Cynopteri str. 3522 C T (Genbank:AHMN00000000), L. kmetyi sv. undetermined str. Bejo-Iso9 T (Genbank:AHMP00000000), L. licerasiae sv. Varillal str. VAR 010 T (Genbank:AHOO00000000), L. meyeri sv. Hardjo str. Went 5 (Genbank:AKXE00000000), L. noguchii sv. Panama str. CZ 214 T (Genbank: AKWY00000000), L. santarosai sv. Shermani str. 1342K T (AOHB00000000), L. terpstrae sv. Hualin str. LT 11-33 T (Genbank: AOGW00000000), L. vanthielii sv. Holland str. WaZ Holland (Genbank:AOGY00000000), L. weilii sv. Ranarum str. ICF T (Genbank:AOHC00000000), L. wolbachii sv. Codice str. CDC (Genbank:AOGZ00000000), L. wolffii sv. undetermined str. Khorat-H2 T (Genbank:AKWX00000000), L. yanagawae sv. Saopaulo str. Sao Paulo T (Genbank:AOGX00000000). Statistics Data were analyzed using GraphPad Prism 5.0. Significance was assessed using one-way ANOVA followed by Tukey's HSD post hoc testing. P-values are reported as *** = p<0.001, ** = p<0.01, * = p<0.05. Ethics statement This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in AAALAC-approved facilities. The experimental animal work was approved by the Institutional Animal Care and Use Committee of the University of California San Diego under protocol S03128H. Bacterial strain maintenance and attenuation of L. interrogans serovar Lai strain 56601 All strains were maintained in vitro in Ellinghausen-McCullough-Johnson -Harris (EMJH) media using standard protocols and are available from BEI Resources. L. interrogans serovar Lai strain 56601 was obtained from Dr. David Haake (UCLA). Virulence was selected for by serial passage through hamsters so that the P1 strain used in the present study had an LD 50 of ∼10 organisms. L. interrogans serovar Lai strain 56601_P1 was attenuated by 18 bi-weekly subcultures in vitro . Virulence was assessed every five to ten subcultures using three-week-old male Golden Syrian Hamsters. Following a final subculture, genomic DNA was prepared from this attenuated strain (designated P19) on which next generation sequencing was carried out. Genome assembly of virulent P1 and attenuated P19 L. interrogans serovar Lai strain 56601 and non-synonymous SNV (nsSNV) detection We generated 4,379,515 and 5,340,095 unpaired shotgun reads from L. interrogans serovar Lai 56601_P1 and 56601_P19, respectively using next generation sequencing technology. All reads were 36 bases long. Both genomes were assembled using the comparative assembler AMOScmp. The AMOSCmp-shortReads-alignmentTrimmed pipeline that runs within AMOScmp, was used to look for exact matches of each read to the published L. interrogans serovar Lai 56601 genome of at least 20 bp, permitting a maximum consensus error rate of 0.06% (i.e. at most two mismatches in any read). This script runs a reference-based trimming of the 3′-end of the reads prior to assembly. We found that trimming of at most 4 bases from the 3′-end of the reads based on their matches to the reference produced better assemblies than un-trimmed reads. The P1 assembly used 3,919,609 reads, leaving 459,906 unassembled singletons, while the P19 assembly used 4,915,295 leaving 424,800 singleton reads. The 56601_P1 genome was assembled into 167 contigs with an average length of 28,124 kb and an N50 length of 105,604 kb and the P19 genome into 97 contigs, average length 48,417 and N50 of 190,406. We checked the quality of both assemblies using the amosvalidate pipeline, which runs within AMOScmp. This pipeline identifies misassembly features such as increased read depth and correlated SNVs (i.e. one or more reads with the same SNV, which is unlikely to be due to sequencing error), both indicative of collapsed repeats. We found that both assemblies were high quality with at most 5 potential misassembly features in longer contigs. These potential misassemblies were inspected manually using the Hawkeye viewer and reassembled if necessary using minimus, which employs a stricter assembly algorithm. The unfinished 56601_P1a and 56601_P19 genomes were aligned and SNVs identified using the MUMmer v3.22 software package. RT-qPCR in vivo gene expression analysis Three wk old Golden Syrian Hamsters were infected via intraperitoneal injection with 10 8 low passage L. interrogans serovar Lai strain 56601. 96 hours post infection total RNA was collected using TRIzol (Invitrogen) from blood, liver, and kidney tissue, as well as from a 96-hour EMJH culture of L. interrogans grown at 30°C. Total RNA was reverse transcribed using a QuantiTect reverse transcription kit (Qiagen). cDNA was amplified using a CFX96 thermal cycler (Bio-Rad) using PerfeCta SYBR Green FastMix (Quanta Biosciences). PCR was carried out at 95°C for 3 min, a touchdown gradient of 14 cycles of (94°C 10 s, 80°C 45 s) decreasing 1°C/cycle, followed by 40 cycles of (94°C 30 s, 65°C 45 s). Ct values were normalized to the leptospiral 16S rRNA gene and expression fold change calculated using the Pfaffl method [20] . Primer sequences are listed in Table S3 in Text S1 . Domain architecture analysis of LA_4008 and other related AGC proteins Domain architecture comparison of LA_4008 with orthologs of Myxococcus xanthus , Corallococcus coralloides , Stigmatella aurantiaca , and Mycobacterium tuberculosis using NCBI CD Search, SMART, and TPRPred. Protein homology analysis was carried out using BLAST using the following reference sequences: LA_4008 (NP_714188.1), MXAN_4545 (YP_632713.1), COCOR_04748 (YP_005370712.1), STAUR_4866 (YP_003954471.1), Rv0386 (CCP43116). The coverage for the query sequence, statistical significance (E-value), and maximum amino acid identify ("Max Ident") are indicated at right for each predicted primary sequence. Identified domains were then graphically represented using the DOG 1.0 program ( http://dog.biocuckoo.org ) Leptospira Concentrated Culture Supernatant (CCS) L. interrogans Lai 56601 or L. licerasiae Varillal were grown in EMJH media +10% heat inactivated rabbit serum at 37°C on a rotating shaker for 96 hr. Culture was centrifuged for 30 min at 10,000× g. Supernatant was decanted, filtered through a .22 µm syringe filter unit (Millipore), and concentrated 10× in an Amicon Ultra 10K MWCO centrifugal filter unit (Millipore). No leptospires were observed in the CCS after concentration using darkfield microscopy. CCS cAMP elevating activity CCS was incubated with monolayers of THP-1,a human monocyte/macrophage cell line. At 4, 6, and 20 hours cells were rinsed 3× in PBS and analyzed for cAMP (Direct cAMP EIA kit, Enzo Life Sciences). Secondly, CCS from L. interrogans Lai and L. licerasiae were incubated with THP-1 monolayers for 4 hours, and assayed for cAMP. CCS immunodepletion studies Rabbits were used to generate anti-peptide antisera against LA_4008 using a protein specific, sixteen amino acid fragment (SVEEDPLTREIDRKQK) conjugated to keyhole limpet hemocyanin as a carrier protein (Pacific Immunology, Ramona, CA). The IgG fractions from pre-immunization and production bleeds were purified using a Melon Gel IgG Purification kit (Thermo Scientific) and covalently linked to magnetic beads using a NanoLink BeadLink Kit (Solulink). Antibody linked beads were incubated with CCS overnight at 4°C on a rotating shaker. Beads were separated on a QuadroMACS separation unit (Miltenyi Biotec). Depleted CCS was applied to THP-1 monolayers and incubated for 4 hours. Cells were rinsed 3× in PBS and analyzed for total cAMP using the Direct cAMP EIA kit. Phylogenetic analysis of PF07598 paralogous protein family L. interrogans , L. borgpetersenii and B. bacilliformis full-length sequences were downloaded from the Uniprot databa se ( http://www.uniprot.com ) and aligned using MAFFT v7 ( http://mafft.cbrc.jp/alignment/software ) with default parameters. The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman frequency model [21] . Statistical support of the tree topology was obtained from 500 bootstrap replicates. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 271 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [22] . Pan-genomic analysis of attenuated genes and PF07598 orthologs The genomic data analyzed here are publically available and are from newly generated, unpublished Leptospira whole genome sequence data produced by the JCVI as part of the white paper " Leptospira Genomics and Human Health," sponsored by the NIAID-funded Genome Sequencing Centers. PanOCT [23] was run using default settings with the exception that a dynamically determined pairwise cutoff was implemented, not available in the current release, but available upon request. The following genomes, representing all 20 Leptospira spp. were used: L. alexanderi sv. Manha 3 str. L 60 T (Genbank:AHMT00000000), L. alstoni sv. Pingchang str. 80-412 (Genbank:AOHD00000000), L. biflexa sv. Patoc str. Patoc I Paris (Genbank:CP000786), L. borgpetersenii sv. Javanica str. UI 09931 (Genbank:AHNP00000000), L. broomii sv. undetermined str. 5399 T (Genbank:AHMO00000000), L. fainei sv. Hurstbridge str. BUT 6 T (Genbank:AKWZ00000000), L. inadai sv. Lyme str. 10 T (Genbank:AHMM00000000), L. interrogans sv. Copenhageni str. Fiocruz L1-130 (Genbank:AE016823), L. interrogans sv. Copenhageni str. M20 (Genbank:AOGV00000000), L. interrogans sv. Lai str. 56601 (Genbank:AE010300), L. kirschneri sv. Cynopteri str. 3522 C T (Genbank:AHMN00000000), L. kmetyi sv. undetermined str. Bejo-Iso9 T (Genbank:AHMP00000000), L. licerasiae sv. Varillal str. VAR 010 T (Genbank:AHOO00000000), L. meyeri sv. Hardjo str. Went 5 (Genbank:AKXE00000000), L. noguchii sv. Panama str. CZ 214 T (Genbank: AKWY00000000), L. santarosai sv. Shermani str. 1342K T (AOHB00000000), L. terpstrae sv. Hualin str. LT 11-33 T (Genbank: AOGW00000000), L. vanthielii sv. Holland str. WaZ Holland (Genbank:AOGY00000000), L. weilii sv. Ranarum str. ICF T (Genbank:AOHC00000000), L. wolbachii sv. Codice str. CDC (Genbank:AOGZ00000000), L. wolffii sv. undetermined str. Khorat-H2 T (Genbank:AKWX00000000), L. yanagawae sv. Saopaulo str. Sao Paulo T (Genbank:AOGX00000000). Statistics Data were analyzed using GraphPad Prism 5.0. Significance was assessed using one-way ANOVA followed by Tukey's HSD post hoc testing. P-values are reported as *** = p<0.001, ** = p<0.01, * = p<0.05. Results Pathogenomic identification of protein coding genes in Leptospira interrogans serovar Lai and patterns of tissue-specific up-regulation in vivo Comparison of the wild type and attenuated L. interrogans Lai 55601 genomes identified 41 non-synonymous single nucleotide variants (nsSNVs) in a total of 35 protein-coding genes (CDS; Table S1 in in Text S1 ). P19 sequence analysis revealed that all SNVs were homogeneous within the culture population; minority populations were not detected at the limit of detection of the Illumina sequencing platform (<4%). For the purposes of this study, therefore, the bacterial populations were considered clonal. Filtering to include CDS restricted to pathogenic Leptospira species identified 11 genes ( Fig. 2K ). These CDS are highly conserved among pathogenic Leptospira species ( Fig. 2K ). In vivo transcriptional analysis identified that of these 11 pathogen-specific genes, 10 were up-regulated in vivo during acute hamster infection ( Fig. 2 , normalized to the 16S rDNA gene, Fig. S1 in Text S1 ). Transcriptional up-regulation of CDS was as high as several thousand-fold, with a much higher dynamic range than found with in vitro conditions used in previously reported systems biology analyses (summarized in Table S2 in Text S1 ). 10.1371/journal.pntd.0002468.g002 Figure 2 In vivo transcriptional analysis of putative virulence-associated genes. In vivo relevance of the identified virulence-related genes, mRNA transcript levels of the genes identified by the pathogenomics approach was assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 d after hamster infection, compared to log phase in vitro cultured Leptospira . Leptospiral gene expression levels in infected tissue vs. EMJH were expressed logarithmically as the log 2 of the fold change between the two conditions ( A–J ). 16S rRNA transcript levels (previously validated [61] ) were used to normalize gene expression in tissues and under the different conditions (Fig. S1 in Text S1 ). Expression of 10/11 identified genes was detectable in vivo in all three tissues assayed; the exception was the hypothetical protein LA_0979. The remaining 10 genes were detected in all three tissues assayed. Expression varied between groups of animals, and interestingly, the highest levels of up-regulation were found in leptospires isolated from the blood of infected animals, with transcript levels also being up in bacteria from the liver. Virulence-associated genes were variably up-regulated in kidney. The data represented are the mean ± SEM of 3 independent experiments (n = 7 animals). ( K ) Leptospira species distribution of the 11 virulence-associated genes identified and associated single nucleotide variants found in coding sequences of the avirulent passage (P19) strain. Protein code is according to the annotated protein database; Accession is the GenBank code for the protein. Identification of a putative leptospiral protein with host cAMP elevating activity Of particular interest is LA_4008, a putative adenylate/guanylate cyclase (AGC) that lacks transmembrane helices typical of integral membrane cyclases involved in signal transduction, suggesting that this protein may be soluble. While another adenylate/guanylate cyclase was found in our screen in pathogens and intermediates (Table S1 in Text S1 ; LA_0027), this protein is predicted to be a housekeeping gene, a membrane- bound and intracellular, and not likely to be found in the extracellular milieu. Orthologs of LA_4008 are found only in pathogenic Leptospira and the intermediately pathogenic strain L. fainei , Fig. 2K . Other bacterial adenylate cyclases lacking transmembrane domains include the soluble cyclase class of toxins of the pathogens Mycobacterium tuberculosis, Bordetella pertussis, Bacillus anthracis, Yersinia pestis, and Pseudomonas aeruginosa which modulate host cellular responses to infection [24] . Sequence analysis by SMART, TPRPred, and NCBI conserved domain (CD) search revealed a unique domain architecture for LA_4008 consisting of two tandem N-terminal class III cyclase homology domains followed immediately by an AAA-ATPase domain, and finally a series of C-terminal tetratricopeptide (TPR) domains ( Fig. 3 ), that are known to mediate protein-protein interactions and have recently been recognized as components of bacterial virulence mechanisms [25] , [26] . LA_4008 also shares striking homology to a toxin (NCBI protein cluster PCLA_814229) shared by predatory species of the δ-proteobacterial order Myxococcales ( Fig. 3 ). The domain structure shared by this protein cluster is reminiscent of another pathogenesis-related adenylate cyclase, Rv0386 of Mycobacterium tuberculosis . This domain structure, with precedent in both pathogenic and environmental bacteria, has been experimentally shown to increase cyclic AMP levels in host macrophages and impair the innate immune response to infection ( Fig. 3 ), [27] , [28] . To test whether LA_4008 has the potential to elevate cyclic AMP in host cells, concentrated L. interrogans serovar Lai strain 56601 EMJH culture supernatant (CCS) was added to in vitro monolayer cultures of macrophage-like THP-1 cells and the cells were washed and lysed at various times over 20 hr, and intracellular cyclic AMP levels were assayed. All CCS preparations were microscopically confirmed to be absent of visible leptospires prior to all experiments. We observed that a L. interrogans -derived soluble factor from culture supernatant stimulated a transient rise in intracellular macrophage cAMP levels, peaking at four hours ( Fig. 4A ). Next, the cAMP elevating activity of CCS was compared between L. interrogans serovar Lai (which has LA_4008), and the intermediate L. licerasiae serovar Varillal ( Fig. 4B ) (which does not have an ortholog of LA_4008). The results of this experiment was consistent with the hypothesis that cAMP elevating activity may be related to pathogenic Leptospira species containing the LA_4008 AGC but not by intermediate Leptospira ( Fig. 1A ), and therefore is not a general feature of all infectious leptospires. To further test if LA_4008 is responsible for the elevated target cell cAMP, CCS was digested with proteinase K prior to addition to THP-1 cells (important because some bacterial LPS can also elevate cAMP levels in host cells [29] ) and, more critically, immune-depleted with a specific anti-LA_4008 antibody before adding the CCS to THP-1 cells. As a control, CCS was also immune-depleted with pre-immune serum of the host animal in which the anti-LA_4008 antiserum was generated. Both protease treatment and specific immunodepletion, but not non-specific depletion blocked CCS-mediated increases in intracellular cAMP levels in THP-1 cells ( Fig. 4C ), consistent with the hypothesis that LA_4008 from L. interrogans Lai is a cAMP-elevating factor in host cells. 10.1371/journal.pntd.0002468.g003 Figure 3 Ortholog sequence analysis of pathogenic Leptospira adenylate/guanylate cyclase compared to predatory environmental bacteria and the pathogen, Mycobacterium tuberculosis . Domain architecture comparison of LA_4008 with orthologs of Myxococcus xanthus , Corallococcus coralloides , Stigmatella aurantiaca , and Mycobacterium tuberculosis using NCBI CD Search, SMART, and TPRPred. Protein homology analysis was carried out using BLAST using the following reference sequences: LA_4008 (NP_714188.1), MXAN_4545 (YP_632713.1), COCOR_04748 (YP_005370712.1), STAUR_4866 (YP_003954471.1), Rv0386 (CCP43116). The coverage for the query sequence, statistical significance (E-value), and maximum amino acid identify ("Max Ident") are indicated at right for each predicted primary sequence. 10.1371/journal.pntd.0002468.g004 Figure 4 Confirmation of cAMP induction in target mammalian cells by LA_4008 activity in leptospiral culture supernatant. ( A ) THP-1 cell monolayers were treated with leptospire-free concentrated culture supernatant (CCS) from L. interrogans Lai or EMJH negative control. ( B ) THP-1 monolayers were treated with CCS from L. interrogans Lai or L. licerasiae Varillal, NT = not treated. ( C ) THP-1 cell monolayers were treated with CCS, CCS that was immunoprecipitated (IP) with specific anti-peptide antibody raised in rabbits and non-specific anti-LA 4008 antibody, and CCS that was digested with proteinase K. Values in all experiments are represented as the mean (n = 3) ± SD. Identification of a paralogous gene family shared by pathogenic Leptospira , Bartonella bacilliformis , and Bartonella australis with profound, tissue-specific up-regulation in vivo in an acute leptospirosis infection model in hamsters During our analysis of attenuation mutations we identified two members (LA_3490, LA_3388) of a newly discovered paralogous gene family that is shared between pathogenic Leptospira but conspicuously absent in the intermediate and saprophytic species. All full-length members of this family (PF07598/DUF1561) are predicted to have secretory signal peptides, although degenerate forms do occur. Past the signal peptide, Cys residues are invariant at twelve positions, and occur nowhere else, suggesting a conserved pattern of disulfide bond formation and implying extracellular function (Fig. S2 in Text S1 ). In a given genome, the most closely related paralogs are often tandem. Otherwise, gene neighborhood analysis provided no clue to protein function. Paralog counts in pathogenic Leptospira range from two in the leptospire L. santarosai to 12 in L. kirschneri serovar Cynopteri and L. interrogans ( Fig. 5A ). Interestingly the PF07598 gene family has also been recently described in the unrelated α-proteobacteria species Bartonella bacilliformis and Bartonella australis . B. bacilliformis has 15 paralogs in its genome with B. australis having nearly the same ( Fig. 5B ) [30] . In addition single gene copies were found in three animal-infecting ε -proteobacteria, Helicobacter hepaticus , H. mustelae , and H. cetorum . 10.1371/journal.pntd.0002468.g005 Figure 5 Phylogenetic and in vivo gene expression analysis of the PF07598 paralogous gene family shared by pathogenic Leptospira and Bartonella bacilliformis . ( A ) Distribution of the paralogous gene family shared by Leptospira and Bartonella bacilliformis in the genus Leptospira . P, pathogen; I, intermediate; S, saprophyte. ( B ) An unrooted phylogenetic tree was constructed of protein sequences from all identifiable homologs of the DUF1561 protein family found in GenBank and the PATRIC databases, which included predicted sequences from the following bacteria ( Helicobacter spp. and B. bacilliformis genome locus tags and protein sequences used for constructing the tree are listed in Table S4 in Text S1 ): L. interrogans Lai, L. borgpeterseni Hardjo; Helicobacter cetorum , H. hepaticus and H. mustelae ; and B. bacilliformis full-length sequences were aligned using MAFFT. Node labels represent support from 500 bootstrap replicates. Tree drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. Analyses were conducted in MEGA5. ( C–E ) In vivo relevance of the leptospiral paralogous gene family was assessed in the acute hamster infection model as described in Fig. 1 . Transcript levels of the genes were assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 days after hamster infection and compared to log phase in vitro cultured Leptospira . Leptospiral gene expression levels in infected tissue vs. EMJH medium alone were expressed logarithmically as the log 2 of the fold change between the two conditions. Solid bars indicate proteins containing predicted signal peptides that suggest extracellular presence, i.e. secretion or cell-surface, of the protein, consistent with bacterial interaction with the host. Data represented are the mean ± SEM of 3 independent experiments (n = 7 animals). There are great phylogenetic distances separating the genera that contain this gene family, but paralogs are restricted to select animal-infecting species within each lineage; suggesting that these proteins may be uniquely related to host adaptation. All 12 members of the leptospiral PF07598 gene family were analyzed for in vivo expression in hamsters acutely infected with virulent, wild type L. interrogans Lai 55601. All members of this gene family were up-regulated in blood and liver to varying degrees, with LA_3490 and LA_3388, both containing secretory signal peptide sequences, being most highly up-regulated (more than ∼1000-fold); all members of this gene family were up-regulated in the circulation and liver to varying degrees. In contrast, up-regulation of other members this gene family significantly varied among experimental animals in kidney ( Fig. 5C–E ). Other pathogenomically-identified putative virulence genes in Leptospira spp Other pathogenomically-associated virulence genes include the following: LA_1056 : This gene has two predicted transmembrane helices and shares a conserved PHA00965 domain with tail proteins found in Gram-positive bacteriophages. This protein shows similarity to phage tape measure proteins after repeated rounds PSI-BLAST. Recent studies involving the phage-encoded pblA in Streptococcus mitis have identified a sequence weakly reminiscent of a tape measure motif protein by PSI-BLAST as an adhesin-type molecule used for bacterial attachment to platelets [31] – [34] . LA_1765 : This protein has similarity to spvB, a protein from a group of plasmid-encoded virulence genes that mediate lethal infection in nontyphoid Salmonella strains [35] . LA_1533 : a flavin-dependent thymidylate synthase. This unusual and newly described class of enzyme is expressed by many clinically relevant pathogens, including Bacillus anthracis , Borellia burgdorferi , Campylobacter jejuni , Clostridium difficile , Helicobacter pylori , Mycobacterium tuberculosis , and Treponema pallidum during infection as part of an alternative thymidine synthesis pathway [36] – [38] . LA_0202 : a gene of unknown function previously reported to be transcriptionally up-regulated in virulent L. interrogans Lai 55601 when compared to another avirulent strain [39] . LA_1568 : a putative lipoprotein with β-propeller repeats that has not been previously studied. Lipoproteins are important mediators of spirochete virulence, with the L. interrogans genome encoding over one hundred lipoproteins [40] , the function and localization of many remain unclear. Pathogenomic identification of protein coding genes in Leptospira interrogans serovar Lai and patterns of tissue-specific up-regulation in vivo Comparison of the wild type and attenuated L. interrogans Lai 55601 genomes identified 41 non-synonymous single nucleotide variants (nsSNVs) in a total of 35 protein-coding genes (CDS; Table S1 in in Text S1 ). P19 sequence analysis revealed that all SNVs were homogeneous within the culture population; minority populations were not detected at the limit of detection of the Illumina sequencing platform (<4%). For the purposes of this study, therefore, the bacterial populations were considered clonal. Filtering to include CDS restricted to pathogenic Leptospira species identified 11 genes ( Fig. 2K ). These CDS are highly conserved among pathogenic Leptospira species ( Fig. 2K ). In vivo transcriptional analysis identified that of these 11 pathogen-specific genes, 10 were up-regulated in vivo during acute hamster infection ( Fig. 2 , normalized to the 16S rDNA gene, Fig. S1 in Text S1 ). Transcriptional up-regulation of CDS was as high as several thousand-fold, with a much higher dynamic range than found with in vitro conditions used in previously reported systems biology analyses (summarized in Table S2 in Text S1 ). 10.1371/journal.pntd.0002468.g002 Figure 2 In vivo transcriptional analysis of putative virulence-associated genes. In vivo relevance of the identified virulence-related genes, mRNA transcript levels of the genes identified by the pathogenomics approach was assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 d after hamster infection, compared to log phase in vitro cultured Leptospira . Leptospiral gene expression levels in infected tissue vs. EMJH were expressed logarithmically as the log 2 of the fold change between the two conditions ( A–J ). 16S rRNA transcript levels (previously validated [61] ) were used to normalize gene expression in tissues and under the different conditions (Fig. S1 in Text S1 ). Expression of 10/11 identified genes was detectable in vivo in all three tissues assayed; the exception was the hypothetical protein LA_0979. The remaining 10 genes were detected in all three tissues assayed. Expression varied between groups of animals, and interestingly, the highest levels of up-regulation were found in leptospires isolated from the blood of infected animals, with transcript levels also being up in bacteria from the liver. Virulence-associated genes were variably up-regulated in kidney. The data represented are the mean ± SEM of 3 independent experiments (n = 7 animals). ( K ) Leptospira species distribution of the 11 virulence-associated genes identified and associated single nucleotide variants found in coding sequences of the avirulent passage (P19) strain. Protein code is according to the annotated protein database; Accession is the GenBank code for the protein. Identification of a putative leptospiral protein with host cAMP elevating activity Of particular interest is LA_4008, a putative adenylate/guanylate cyclase (AGC) that lacks transmembrane helices typical of integral membrane cyclases involved in signal transduction, suggesting that this protein may be soluble. While another adenylate/guanylate cyclase was found in our screen in pathogens and intermediates (Table S1 in Text S1 ; LA_0027), this protein is predicted to be a housekeeping gene, a membrane- bound and intracellular, and not likely to be found in the extracellular milieu. Orthologs of LA_4008 are found only in pathogenic Leptospira and the intermediately pathogenic strain L. fainei , Fig. 2K . Other bacterial adenylate cyclases lacking transmembrane domains include the soluble cyclase class of toxins of the pathogens Mycobacterium tuberculosis, Bordetella pertussis, Bacillus anthracis, Yersinia pestis, and Pseudomonas aeruginosa which modulate host cellular responses to infection [24] . Sequence analysis by SMART, TPRPred, and NCBI conserved domain (CD) search revealed a unique domain architecture for LA_4008 consisting of two tandem N-terminal class III cyclase homology domains followed immediately by an AAA-ATPase domain, and finally a series of C-terminal tetratricopeptide (TPR) domains ( Fig. 3 ), that are known to mediate protein-protein interactions and have recently been recognized as components of bacterial virulence mechanisms [25] , [26] . LA_4008 also shares striking homology to a toxin (NCBI protein cluster PCLA_814229) shared by predatory species of the δ-proteobacterial order Myxococcales ( Fig. 3 ). The domain structure shared by this protein cluster is reminiscent of another pathogenesis-related adenylate cyclase, Rv0386 of Mycobacterium tuberculosis . This domain structure, with precedent in both pathogenic and environmental bacteria, has been experimentally shown to increase cyclic AMP levels in host macrophages and impair the innate immune response to infection ( Fig. 3 ), [27] , [28] . To test whether LA_4008 has the potential to elevate cyclic AMP in host cells, concentrated L. interrogans serovar Lai strain 56601 EMJH culture supernatant (CCS) was added to in vitro monolayer cultures of macrophage-like THP-1 cells and the cells were washed and lysed at various times over 20 hr, and intracellular cyclic AMP levels were assayed. All CCS preparations were microscopically confirmed to be absent of visible leptospires prior to all experiments. We observed that a L. interrogans -derived soluble factor from culture supernatant stimulated a transient rise in intracellular macrophage cAMP levels, peaking at four hours ( Fig. 4A ). Next, the cAMP elevating activity of CCS was compared between L. interrogans serovar Lai (which has LA_4008), and the intermediate L. licerasiae serovar Varillal ( Fig. 4B ) (which does not have an ortholog of LA_4008). The results of this experiment was consistent with the hypothesis that cAMP elevating activity may be related to pathogenic Leptospira species containing the LA_4008 AGC but not by intermediate Leptospira ( Fig. 1A ), and therefore is not a general feature of all infectious leptospires. To further test if LA_4008 is responsible for the elevated target cell cAMP, CCS was digested with proteinase K prior to addition to THP-1 cells (important because some bacterial LPS can also elevate cAMP levels in host cells [29] ) and, more critically, immune-depleted with a specific anti-LA_4008 antibody before adding the CCS to THP-1 cells. As a control, CCS was also immune-depleted with pre-immune serum of the host animal in which the anti-LA_4008 antiserum was generated. Both protease treatment and specific immunodepletion, but not non-specific depletion blocked CCS-mediated increases in intracellular cAMP levels in THP-1 cells ( Fig. 4C ), consistent with the hypothesis that LA_4008 from L. interrogans Lai is a cAMP-elevating factor in host cells. 10.1371/journal.pntd.0002468.g003 Figure 3 Ortholog sequence analysis of pathogenic Leptospira adenylate/guanylate cyclase compared to predatory environmental bacteria and the pathogen, Mycobacterium tuberculosis . Domain architecture comparison of LA_4008 with orthologs of Myxococcus xanthus , Corallococcus coralloides , Stigmatella aurantiaca , and Mycobacterium tuberculosis using NCBI CD Search, SMART, and TPRPred. Protein homology analysis was carried out using BLAST using the following reference sequences: LA_4008 (NP_714188.1), MXAN_4545 (YP_632713.1), COCOR_04748 (YP_005370712.1), STAUR_4866 (YP_003954471.1), Rv0386 (CCP43116). The coverage for the query sequence, statistical significance (E-value), and maximum amino acid identify ("Max Ident") are indicated at right for each predicted primary sequence. 10.1371/journal.pntd.0002468.g004 Figure 4 Confirmation of cAMP induction in target mammalian cells by LA_4008 activity in leptospiral culture supernatant. ( A ) THP-1 cell monolayers were treated with leptospire-free concentrated culture supernatant (CCS) from L. interrogans Lai or EMJH negative control. ( B ) THP-1 monolayers were treated with CCS from L. interrogans Lai or L. licerasiae Varillal, NT = not treated. ( C ) THP-1 cell monolayers were treated with CCS, CCS that was immunoprecipitated (IP) with specific anti-peptide antibody raised in rabbits and non-specific anti-LA 4008 antibody, and CCS that was digested with proteinase K. Values in all experiments are represented as the mean (n = 3) ± SD. Identification of a paralogous gene family shared by pathogenic Leptospira , Bartonella bacilliformis , and Bartonella australis with profound, tissue-specific up-regulation in vivo in an acute leptospirosis infection model in hamsters During our analysis of attenuation mutations we identified two members (LA_3490, LA_3388) of a newly discovered paralogous gene family that is shared between pathogenic Leptospira but conspicuously absent in the intermediate and saprophytic species. All full-length members of this family (PF07598/DUF1561) are predicted to have secretory signal peptides, although degenerate forms do occur. Past the signal peptide, Cys residues are invariant at twelve positions, and occur nowhere else, suggesting a conserved pattern of disulfide bond formation and implying extracellular function (Fig. S2 in Text S1 ). In a given genome, the most closely related paralogs are often tandem. Otherwise, gene neighborhood analysis provided no clue to protein function. Paralog counts in pathogenic Leptospira range from two in the leptospire L. santarosai to 12 in L. kirschneri serovar Cynopteri and L. interrogans ( Fig. 5A ). Interestingly the PF07598 gene family has also been recently described in the unrelated α-proteobacteria species Bartonella bacilliformis and Bartonella australis . B. bacilliformis has 15 paralogs in its genome with B. australis having nearly the same ( Fig. 5B ) [30] . In addition single gene copies were found in three animal-infecting ε -proteobacteria, Helicobacter hepaticus , H. mustelae , and H. cetorum . 10.1371/journal.pntd.0002468.g005 Figure 5 Phylogenetic and in vivo gene expression analysis of the PF07598 paralogous gene family shared by pathogenic Leptospira and Bartonella bacilliformis . ( A ) Distribution of the paralogous gene family shared by Leptospira and Bartonella bacilliformis in the genus Leptospira . P, pathogen; I, intermediate; S, saprophyte. ( B ) An unrooted phylogenetic tree was constructed of protein sequences from all identifiable homologs of the DUF1561 protein family found in GenBank and the PATRIC databases, which included predicted sequences from the following bacteria ( Helicobacter spp. and B. bacilliformis genome locus tags and protein sequences used for constructing the tree are listed in Table S4 in Text S1 ): L. interrogans Lai, L. borgpeterseni Hardjo; Helicobacter cetorum , H. hepaticus and H. mustelae ; and B. bacilliformis full-length sequences were aligned using MAFFT. Node labels represent support from 500 bootstrap replicates. Tree drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. Analyses were conducted in MEGA5. ( C–E ) In vivo relevance of the leptospiral paralogous gene family was assessed in the acute hamster infection model as described in Fig. 1 . Transcript levels of the genes were assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 days after hamster infection and compared to log phase in vitro cultured Leptospira . Leptospiral gene expression levels in infected tissue vs. EMJH medium alone were expressed logarithmically as the log 2 of the fold change between the two conditions. Solid bars indicate proteins containing predicted signal peptides that suggest extracellular presence, i.e. secretion or cell-surface, of the protein, consistent with bacterial interaction with the host. Data represented are the mean ± SEM of 3 independent experiments (n = 7 animals). There are great phylogenetic distances separating the genera that contain this gene family, but paralogs are restricted to select animal-infecting species within each lineage; suggesting that these proteins may be uniquely related to host adaptation. All 12 members of the leptospiral PF07598 gene family were analyzed for in vivo expression in hamsters acutely infected with virulent, wild type L. interrogans Lai 55601. All members of this gene family were up-regulated in blood and liver to varying degrees, with LA_3490 and LA_3388, both containing secretory signal peptide sequences, being most highly up-regulated (more than ∼1000-fold); all members of this gene family were up-regulated in the circulation and liver to varying degrees. In contrast, up-regulation of other members this gene family significantly varied among experimental animals in kidney ( Fig. 5C–E ). Other pathogenomically-identified putative virulence genes in Leptospira spp Other pathogenomically-associated virulence genes include the following: LA_1056 : This gene has two predicted transmembrane helices and shares a conserved PHA00965 domain with tail proteins found in Gram-positive bacteriophages. This protein shows similarity to phage tape measure proteins after repeated rounds PSI-BLAST. Recent studies involving the phage-encoded pblA in Streptococcus mitis have identified a sequence weakly reminiscent of a tape measure motif protein by PSI-BLAST as an adhesin-type molecule used for bacterial attachment to platelets [31] – [34] . LA_1765 : This protein has similarity to spvB, a protein from a group of plasmid-encoded virulence genes that mediate lethal infection in nontyphoid Salmonella strains [35] . LA_1533 : a flavin-dependent thymidylate synthase. This unusual and newly described class of enzyme is expressed by many clinically relevant pathogens, including Bacillus anthracis , Borellia burgdorferi , Campylobacter jejuni , Clostridium difficile , Helicobacter pylori , Mycobacterium tuberculosis , and Treponema pallidum during infection as part of an alternative thymidine synthesis pathway [36] – [38] . LA_0202 : a gene of unknown function previously reported to be transcriptionally up-regulated in virulent L. interrogans Lai 55601 when compared to another avirulent strain [39] . LA_1568 : a putative lipoprotein with β-propeller repeats that has not been previously studied. Lipoproteins are important mediators of spirochete virulence, with the L. interrogans genome encoding over one hundred lipoproteins [40] , the function and localization of many remain unclear. Discussion Here we describe the use of pathogenomics to identify novel potential virulence genes in the pathogenic spirochete Leptospira interrogans . Previous work to identify mechanisms of pathogenesis by gene knockouts and transposon mutagenesis has not yet yielded detailed mechanistic insights into the role of individual genes play in the pathogenesis of leptospirosis. It has long been known in the leptospirosis field that serial in vitro passage of pathogenic Leptospira yielded attenuated organisms; the converse, serial passage of liver homogenates of infected animals selects for virulence. A previous study explored the genomic and proteomic differences between a pathogenic L. interrogans serovar Lai strain 56601, and an avirulent strain IPAV [41] . These data must be carefully considered because the analyzed strains are not isogenic (the IPAV strain is of unknown provenance since details of its original isolation are unavailable) nor do they provide any in vivo relevance for identified genes, focusing instead on proteomic differences between strains during in vitro EMJH culture. Our current study, which employed whole genome sequence comparison of an attenuated strain with its isogenic pathogenic parent, yielded a small set of protein coding genes (CDS) with point mutations. While most of the 11 specific mutations found here cannot be quantitatively attributed to specific aspects of virulence or pathogenicity, our pathogenomic approach yielded the identification of a novel leptospiral AGC with cAMP elevating activity in host cells and a hitherto unstudied large gene family that is broadly up-regulated, in a tissue-specific manner, in vivo during an animal model of acute leptospirosis. The identification of a non-transmembrane bound AGC in pathogenic Leptospira is particularly important for two reasons. First, the primary structure implies a non-housekeeping function since the protein is not predicted to be membrane-associated, unlike the housekeeping AGC. Second, the host cell cAMP elevating activity of LA_4008 reported in this study is the first demonstrated evidence of a possible biological mechanism that could contribute to virulence for Leptospira . Although long established and accepted as a virulence mechanism in other pathogens, the evidence of elevation of host cAMP levels by L. interrogans suggests a previously unknown mechanism of pathogenesis and immune evasion for this bacterium, especially given recent evidence that pathogenic Leptospira may reside within macrophages in vivo [42] , [43] . Manipulation of intracellular cAMP levels in immune cells may be an important means of attenuating host responses to infection [44] , an enticing hypothesis given the up-regulation of this gene upon leptospiral entry into the bloodstream observed in this study. Many human pathogens exploit host cell cAMP signaling during infection, for example, the pore-forming toxin CyaA of the respiratory pathogen Bordetella pertussis penetrates host cells where it catalyzes the unregulated conversion of cellular ATP to cAMP, thereby impairing superoxide production, chemotaxis, cytokine production, and phagocytosis [45] – [47] . Similar effects are caused by the edema factor (EF) of Bacillus anthracis , the ExoY toxin of Pseudomonas aeruginosa , and the AGC toxin of Yersinia pestis [24] , [48] – [50] . Due to an unexpected loss of the cryogenically preserved stock cultures, we were unable to assess the cAMP elevating activity of the attenuated P19 strain. However, we would hypothesize that the attenuated SNV-containing variant could have either absolute elimination or quantitative reduction in cAMP elevating activity; this possibility will be addressed directly in ongoing experiments by quantifying the effect of recombinantly producing wild type and mutant LA_4008 on THP-1 and other target cells. We also believe that any observed reduction in activity would have been a quantitative not qualitative difference. Regardless, our findings demonstrate that LA_4008 contributes to a transitory increase in cAMP levels in host cells, and that further experiments are certainly needed to assess the functional consequences of cAMP intoxication in host immune cells during leptospirosis. To formally determine the role of LA_4008 in Leptopira pathogenesis is the subject of ongoing experiments, including determining whether this protein modulates mechanisms of evading host defenses. The identification of a paralogous protein family shared by pathogenic Leptospira spp., and two α-proteobacteria B. bacilliformis , and B. australis was particularly intriguing. The observation that this gene family expanded in pathogenic Leptospira and the two Bartonella spp. suggests that ancestors of these pathogens must have co-existed at some time and place in the past. Phylogenetic analysis suggests a common origin of this gene family, and revealed a greater divergence in the Bartonella members, indicated by greater branch length differences ( Fig. 5B ). Regardless of the source of the primary ortholog, the founding gene was presumably transferred after the branching of pathogenic Leptospira from the other clades of Leptospira , although it is also possible that gene loss occurred in intermediates or saprophytes evolved from pathogens. Although we cannot speculate on the molecular mechanism of gene transfer, it is interesting to consider the conditions that would have been conducive to such an event. L. interrogans is a globally distributed bacterium that can infect many vertebrate hosts as well as live in the environment; it is considered an extracellular parasite, although evidence is mounting that Leptospira [42] , [43] , [51] are able to persist within macrophages and transverse epithelial cells [52] . B bacilliformis and B. australis are facultative intracellular pathogens found only in a specific region of South America [53] and Australia respectively. The PF07598 family shared between pathogenic Leptospira might be shared by other Bartonella species that have yet to be sequenced or even identified, such as those recently found in Thailand [54] , [55] . The maintenance of multiple members of this paralogous gene family clearly must confer a selective advantage to these pathogens. We performed a meta-analysis of 6 previous studies [12] , [56] – [60] that explored transcriptional responses of L. interrogans during exposure to host-like physiological conditions (Table S2 in Text S1 ), and discovered that the expression levels of several of these genes occurs in response to multiple stimuli. This implies that L interrogans responds to signals from the host milieu that lead to the alteration of expression of these genes in a differential manner during its infection cycle. The identification of leptospiral AGC and PF07598 gene family orthologs in specific species of evolutionary distant alpha and delta-proteobacteria was an unexpected and exciting discovery. Given the broad host range of Leptospira as well as their environmental persistence, the horizontal gene transfers our findings imply emphasize how the soil context within the unique transmission cycle of Leptospira has likely shaped the evolution of pathogenic mechanisms for these bacteria. Our investigation was not without limitations. The attenuation experiment was done only once. While genes of pathogenetic interest were identified here, whether these mutations occurred stochastically or not remains to be determined. Accumulation of mutations during the attenuation process was not assessed so that step-wise accumulation of mutations could not be attributed to a level of virulence. Proteomic comparisons between ex-vivo -isolated and EMJH cultured leptospires were not performed, as our study only focused on gene transcriptional levels, which do not necessarily correlate with protein expression levels. It would be interesting to undertake such ex-vivo proteomic investigations in Leptospira ; especially given the vast transcriptional up-regulation of identified genes upon entry into host tissues. Further investigation remains to define the precise mechanisms of how the identified genes in our study relate to the virulence and pathogenesis of leptospirosis, as a majority of these genes have undiscovered functions. We show here that a systems biology-pathogenomic approach to infer virulence-related genes in Leptospira interrogans identified a notable set of hitherto unstudied genes with both pathogenetic and evolutionary significance, including a putative soluble adenylate/guanylate cyclase (AGC), and a paralogous gene family shared by pathogenic Leptospira and the distantly related pathogens B. bacilliformis , a human-specific pathogen geographically restricted to the Andes mountains of South America, and B. australis , a species currently known to only infect kangaroos. This pathogenomic approach is generalizable beyond prokaryotes and particularly relevant to novel virulence gene identification in any pathogen capable of in vitro attenuation. Given the recalcitrant nature of pathogenic leptospires to genetic manipulation, this approach represents an improved method to identify important virulence genes in pathogens whose pathogenesis remains poorly defined by current research strategies, and highlights the extraordinary insights into bacterial pathogenesis and evolutionary biology that large scale genomic sequencing can produce in the context of simple experimentation. These genes will hopefully spur much needed research into the pathogenesis of this neglected disease, but many may also represent rational choices for new vaccine studies. Supporting Information Text S1 Supporting information. Includes: Figure S1. Validation of 16S rDNA Gene to Normalize Leptospira In Vivo Gene Expression. Figure S2. Alignment of Bartonella bacilliformis and Leptospira interrogans serovar Lai anonymous paralog families. Table S1. Leptospira Species Distribution of Pathogenomically-Discovered Genes. Table S2. Differential Expression of Gene Family Members During Exposure of L. interrogans to Host-like Conditions. Table S3. Primers used for In-vivo RT-qPCR Analysis. Table S4. Genome locus tags and GenBank protein sequence accession numbers for Bartonella bacilliformis and Helicobacter spp. PF07598 family homologs used to construct Figure 5A . (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3669167/

Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis

Patients with inflammatory bowel diseases (IBD) harbour intestinal bacterial communities with altered composition compared with healthy counterparts; however, it is unknown whether changes in the microbiota are associated with genetic susceptibility of individuals for developing disease or instead reflect other changes in the intestinal environment related to the disease itself. Since deficiencies in the innate immune receptors Nod1 and Nod2 are linked to IBD, we tested the hypothesis that Nod-signaling alters intestinal immune profiles and subsequently alters bacterial community structure. We used qPCR to analyze expression patterns of selected immune mediators in the ileum and cecum of Nod-deficient mice compared with their Nod-sufficient littermates and assessed the relative abundance of major bacterial groups sampled from the ileum, cecum and colon. The Nod1-deficient ileum exhibited significantly lower expression of Nod2, Muc2, α- and β-defensins and keratinocyte-derived chemokine (KC), suggesting a weakened epithelial barrier compared with WT littermates; however, there were no significant differences in the relative abundance of targeted bacterial groups, indicating that Nod1-associated immune differences alone do not promote dysbiosis. Furthermore, Nod2-deficient mice did not display any changes in the expression of immune markers or bacterial communities. Shifts in bacterial communities that were observed in this study correlated with housing conditions and were independent of genotype. These findings emphasize the importance of using F2 littermate controls to minimize environmental sources of variation in microbial analyses, to establish baseline conditions for host-microbe homeostasis in Nod-deficient mice and to strengthen models for testing factors contributing to microbial dysbiosis associated with IBD.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064307/

Anthrax edema toxin has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in an isolated perfused rat heart model

While anthrax edema toxin produces pronounced tachycardia and lethal toxin depresses left ventricular (LV) ejection fraction in in vivo models, whether these changes reflect direct cardiac effects as opposed to indirect ones related to preload or afterload alterations is unclear. In the present study, the effects of edema toxin and lethal toxin were investigated in a constant pressure isolated perfused rat heart model. Compared with control hearts, edema toxin at doses comparable to or less than a dose that produced an 80% lethality rate (LD 80 ) in vivo in rats (200, 100, and 50 ng/ml) produced rapid increases in heart rate (HR), coronary flow (CF), LV developed pressure (LVDP), dP/d t max , and rate-pressure product (RPP) that were most pronounced and persisted with the lowest dose ( P ≤ 0.003). Edema toxin (50 ng/ml) increased effluent and myocardial cAMP levels ( P ≤ 0.002). Compared with dobutamine, edema toxin produced similar myocardial changes, but these occurred more slowly and persisted longer. Increases in HR, CF, and cAMP with edema toxin were inhibited by a monoclonal antibody blocking toxin uptake and by adefovir, which inhibits the toxin's intracellular adenyl cyclase activity ( P ≤ 0.05). Lethal toxin at an LD 80 dose (50 ng/ml) had no significant effect on heart function but a much higher dose (500 ng/ml) reduced all parameters ( P ≤ 0.05). In conclusion, edema toxin produced cAMP-mediated myocardial chronotropic, inotropic, and vasodilatory effects. Vasodilation systemically with edema toxin could contribute to shock during anthrax while masking potential inotropic effects. Although lethal toxin produced myocardial depression, this only occurred at high doses, and its relevance to in vivo findings is unclear.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355939/

Predicting support for non-pharmaceutical interventions during infectious outbreaks: a four region analysis

Non-pharmaceutical interventions (NPIs) are an important public health tool for responding to infectious disease outbreaks, including pandemics. However, little is known about the individual characteristics associated with support for NPIs, or whether they are consistent across regions. This study draws on survey data from four regions—Hong Kong, Singapore, Taiwan, and the United States—collected following the Severe Acute Respiratory Syndrome (SARS) outbreak of 2002–03, and employs regression techniques to estimate predictors of NPI support. It finds that characteristics associated with NPI support vary widely by region, possibly because of cultural variation and prior experience, and that minority groups tend to be less supportive of NPIs when arrest is the consequence of noncompliance. Prior experience of face-mask usage also results in increased support for future usage, as well as other NPIs. Policymakers should be attentive to local preferences and to the application of compulsory interventions. It is speculated here that some public health interventions may serve as 'gateway' exposures to future public health interventions.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149714/

Principles of Parasitism

Introduction Access the complete reference list online at http://www.expertconsult.com The relationship between two living organisms can be classified as parasitic, symbiotic, or commensal. 1 , 2 , 3 This same classification scheme can be used to describe relationships between microorganisms and more complex living organisms that act as hosts. The term parasite is used here in its broad sense to mean a microorganism interacting with another organism (either vertebrate or invertebrate) in the same ecologic niche. The following definitions are used in this chapter: Parasitism : Association between two different organisms wherein one benefits at the expense of the other. All infectious agents causing illness belong to this category. Commensalism : Association between two organisms in which one derives benefit from the other without causing it any harm. This intermediate category is not uniformly accepted. Often, upon detailed analysis, the relationship turns out to be either parasitic or symbiotic. 2 Symbiosis or mutualism : Both organisms benefit from the relationship. The type of relationship also depends on host factors. For example, bacteria normally inhabiting the bowel live in an apparent commensal or (by inhibiting potential pathogens) symbiotic relationship with humans. However, in cases of cirrhosis with consequent hepatic insufficiency, bacteria can become a dangerous source of ammonia that leads to hepatic encephalopathy. A commensal relationship can be transformed into a potentially harmful one. Microbial Factors Principles of Microbial Evolution and Classification The Earth is approximately 4.5 to 5 billion years old. There is good fossil evidence of microbial life approximately 3.5 billion years ago. Microbial life (stromatolites) was mostly photosynthetic, unicellular, and anaerobic. 1 , 4 Eukaryotes, bacteria, and archaea evolved from a still hypothetical universal common ancestor. 5 , 6 , 7 Eukaryotes then evolved into protozoans, metazoans, plants, and animals, as we know them today. Moreover, there is strong evidence that primitive eukaryotic cells established relationships with bacterial organisms that later evolved into cytoplasmic organelles, such as chloroplasts in plants and mitochondria in animals. 8 To put things into perspective, approximately five-sixths of the history of life on Earth has been exclusively microbial. Human beings appeared on the planet only 2 million years ago as very late newcomers to the biosphere. Life was initially anaerobic, but with the appearance of photosynthetic organisms and chloroplasts, oxygen was released into the atmosphere for the first time. 9 Radiation in the upper atmosphere created the ozone layer from molecular oxygen, which then shielded the Earth's surface from dangerous radiation. Nucleic acids were therefore protected from harmful mutations. Organisms had to evolve to survive in the presence of oxygen. A few of the ancient anaerobes were able to survive in the highly oxidant atmosphere, and they represent the anaerobes as we know them today. The phylogeny of living organisms is based on molecular approaches, particularly analysis of ribosomal RNA. 5 , 6 , 10 Because of the antiquity of the protein synthesis machinery, these molecules are excellent evolutionary clocks. For prokaryotes, the 16S subunit of ribosomes is the most useful for classification purposes. Viruses deserve special comment because of their molecular simplicity and at the same time their importance as human pathogens and as possible agents of hereditary changes and cancer. 11 , 12 A virus is a genetic element with either DNA or RNA coated by protein of viral origin and sometimes enveloped by lipid material of host origin. Some viruses have enzymes that are necessary for their replication. The only criterion that these organisms fulfill to be considered living organisms is that of reproduction. They are inert particles when outside of the host cell, and once they have access into a cell they become active and the cell is subverted to produce more viral particles. Sometimes the cell dies in the process, and sometimes the relationship is stable. Viral hosts include bacteria, protozoa, animals, and plants. The classification of viruses is based on different criteria than the ones used for other organisms. The major criteria are type of nucleic acid, presence or absence of an envelope, manner of replication, and morphologic characteristics. 12 , 13 Simpler forms of self-replicating organisms include virusoids and viroids. 14 , 15 The former are satellite RNAs that are found encapsidated in the proteins encoded by their helper virus (e.g., hepatitis caused by the hepatitis D virus delta agent in conjunction with hepatitis B virus). The viroids are mostly plant pathogens that consist of single-stranded circular RNA molecules. The concept of "infectious agent" was revolutionized by the discovery of proteinaceous infectious agents known as prions. These proteins are responsible for neurodegenerative diseases in animals and humans. The protein particles lack nucleic acids but still are able to reproduce and trigger conformational changes in host proteins, leading to cell death. 16 , 17 In contrast, protozoa are nucleated, single-cell organisms that, depending on the species, replicate by means as simple as binary fission (e.g., Trichomonas ) or as complex as involving multiple sexual and asexual stages in both animal and invertebrate hosts (e.g., plasmodia). Protozoa include amebae (e.g., Entamoeba histolytica ), flagellates (e.g., Giardia lamblia ), ciliates (e.g., Balantidium coli ), and sporozoa (e.g., Cryptosporidium ). Even more complex are helminths, which are multicellular metazoan organisms with highly developed internal organs, including alimentary and reproductive tracts. The helminths include nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). Many helminths have complex life cycles with multiple developmental stages both in the animal host and in intermediate invertebrate or vertebrate hosts. Because of their size, helminths, the macroparasites, are solely extracellular pathogens; because of their prolonged life cycles and generation times, their capacity for genetic alteration is diminished compared to smaller, simpler microbes (the microparasites). Development of Microbial Virulence Evolution of Virulence The traditional view assumes that natural selection would favor evolution toward a benign coexistence between host and parasite. 18 , 19 A modern view of evolution of virulence focuses on the tradeoff between the benefits that pathogens accrue through increased exploitation of hosts and the costs that result from any effects of disease that reduce transmission to susceptible hosts. 19 , 20 From this point of view, virulence could be the evolved as well as the primitive stage of the association between host and parasite, depending on the development of enhanced rather than reduced transmission. According to Levin 19 and Levin and Svanborg-Eden, 20 there are three alternative models to explain evolution of a microparasite's virulence: direct selection, coincidental evolution, and short-sighted within-host selection. The direct selection model states that there is a direct relationship between the parasite's virulence and its rate of infectious transmission. The best documented and often cited example is that of the dramatic changes in virulence that the myxoma virus underwent after being released into the wild in Australia to "control" the population of wild rabbits. In the beginning, rabbit mortality and viral transmission rates were high. As the population of rabbits was decimated, the virulence of the virus decreased and its rate of transmission actually increased. This outcome is explained by the longer survival and duration of the period of shedding of the virus. At the same time, more resistant rabbits increased in number due to the selection process. 21 According to the coincidental evolution model, the factors responsible for the virulence of a microparasite evolved for some purpose other than to provide the parasite with some advantage within a host or for its transmission to other hosts. Clostridial toxins are good examples in this category. There is no beneficial reason to kill a human host who became infected by Clostridium tetani spores from soil in order for the parasite to survive. They are mostly soil bacteria and do not need humans for their survival. 19 Short-sighted within-host evolution posits that the parasites responsible for the morbidity and mortality of the host are selected for as a consequence of within-host evolution since that produces a local advantage for their survival within the host. The host dies and the rate of transmission would decrease. This is an example of evolutionary myopia in which the long-term consequences of killing a host would not matter to the parasite. 22 , 23 Natural selection is a local phenomenon that happens at a given time and place and goes perfectly with this model. Bacteria such as Neisseria meningitidis that normally live attached to human pharyngeal epithelial cells sometimes invade the central nervous system (CNS) and kill the host. Their replication in the CNS is favored since competition is low and defenses are not as abundant as in the tonsillar areas. 19 It follows from the previous paragraphs that evolutionary theories addressing virulence have predicted that virulence of microbes evolves in response to changes in conditions or tradeoffs. One of the most commonly considered "tradeoffs" is between the benefit of a high within-host microbial density (allowing efficient transmission to the next host) and the cost of a reduced longevity of the infection in the host due to high parasite density that tends to kill the host. Other approaches have been proposed such as the role of how pathogenic mechanisms affect virulence evolution. According to this model, pathogenic mechanisms that manipulate host immunity or escape from the host immune response (longer survival in the host) dominate as causes of virulence compared to mechanisms that incrementally alter transmission (higher microbial density and increased mortality). 24 , 25 Furthermore, natural selection would act more strongly on mutations influencing clearance than on mutations influencing transmission. 26 Another cited model to explain dynamics of virulence evolution is the so-called "source–sink" model in which evolution of bacterial pathogens is evaluated from ecological points of view that microbes switch from permanent (source) and transient (sink) habitats. 27 The generation times of mammalian hosts are much longer than those of microorganisms. Therefore, genetic mutations in these hosts, on which natural selection acts, take longer to become part of a large population. Nevertheless, there is evidence that specific microorganisms can exert selective pressure on the gene pool of human hosts. The evidence is strongest for the potentially lethal infections caused by falciparum malaria. In regions of the world where falciparum malaria is endemic, including Africa, there is a high prevalence of genetic mutations that alter hemoglobin structure or synthesis, decreasing or abolishing falciparum malaria parasites' survival. 28 The selective pressure of malaria on human gene expression is not confined solely to affecting erythrocytes but also likely involves the immune system, cytokines, and other systems. 29 Other Modes of Altering Virulence and Pathogenicity Although the selective pressures of evolution generally exert changes over a multitude of centuries, there are other mechanisms that may more rapidly alter microbial pathogenicity, virulence, and drug susceptibility. The expression of mutated genes in microorganisms is heightened when there are greater numbers of organisms and their generation times are brief. Hence, altered gene expression in helminths will be slow to be expressed, whereas in microparasites genetic alterations will be likely to develop. For mycobacterial infections, large numbers of bacilli that persist for a long time facilitate the genetic emergence of drug resistance to a single agent, and this likelihood underlies the principle of using more than a single drug to treat tuberculosis. Even more rapidly dividing microparasites can develop genetic alterations, and this is especially true when the fidelity of genetic replication is poor. This is prominent in human immunodeficiency virus type 1 (HIV-1), whose reverse transcriptase lacks a 3′ exonuclease proofreading activity. 30 Alterations in cell tropism, pathogenicity, and drug sensitivity are frequent in HIV-1 infections. Again, several antiviral agents must be employed concomitantly to circumvent the highly frequent mutations that alter drug susceptibility in HIV-1 strains. In addition to their own genetic material, many classes of microparasites either contain or are capable of acquiring transferable genetic elements in the form of plasmids, transposons, or bacteriophages. These transferable genetic elements also provide a means for the spread of resistance to antibacterial drugs, an increasing problem in all regions of the world. 31 Causes of Acute or Chronic Infections in Individuals One obvious impact of an infectious disease is on the individual infected. Hence, in any region of the world independent of other infectious diseases or malnutrition, the acute infection will cause morbidity and potential mortality in the infected human host. Among otherwise healthy people, the immediate impact of the infection is the symptomatic acute illness. For some infections that have prolonged courses, their impact may also continue over many years. Chronic infections include most of those caused by helminthic parasites, which characteristically live for years; persisting mycobacterial infections; and retroviral infections (HIV-1, HIV-2, and human T-cell lymphotropic virus type 1). Finally, the sequelae of some infections can include the development of neoplasms. Examples include hepatocellular carcinomas associated with chronic hepatitis B and C viral infections, bladder tumors with urinary schistosomiasis, cholangiocarcinomas with biliary fluke infections, and gastric adenocarcinomas and lymphomas associated with Helicobacter pylori infections. Causes of Widespread Infections in Populations Infectious diseases may affect not only individuals but also large groups of people or entire populations due to epidemic or highly endemic transmission. Throughout human history, a few microorganisms have been responsible for great epidemics and massive numbers of dead or crippled people as a result of infections spreading locally or throughout the world. 32 , 33 , 34 , 35 Typhus has been associated almost always with situations that involve overcrowding, famine, war, natural disasters, and poverty. The outcomes of several European wars were affected by the morbidity and mortality inflicted by typhus or other diseases on the military. Typhus epidemics were common during the world wars of the twentieth century and in the concentration camps where the ecological conditions were ideal for such a disease to spread. 30 Today, typhus and other rickettsioses are still public health problems in some countries, but overall the disease was brought under control after its life cycle was described and antibiotics, insecticides, and public health measures became available. 34 Bubonic plague, caused by Yersinia pestis , is another disease that has shaped history, especially in Europe during the Middle Ages. 35 Millions of people were affected by pandemics that spread throughout the continent. Tuberculosis, smallpox, and measles had a tremendous effect on the native populations of the Americas after Columbus's voyages to the New World. It has been estimated that 90% of the population in Mexico was killed by these pathogens, which were novel to the native residents. Acquired immunodeficiency syndrome (AIDS) represents the modern pandemic that will continue to affect human history for at least decades. Other examples are cholera and influenza, which are capable of causing pandemics. 36 In addition to widespread diseases caused by epidemic spread of infections, some infectious diseases, because of their highly endemic prevalence in populations, continue to affect large segments of the world's population. These include enteric and respiratory infections, measles, malaria, tuberculosis and schistosomiasis. Furthermore, even the staggering mortality and morbidity of these tropical infectious diseases do not control populations but are associated with population overgrowth. This is true not only across the different countries of the world but also throughout the history of developed countries. Thus, the impact of these infections is not solely on the individual but, because of their highly endemic or epidemic occurrence, on populations. This has consequences on economic, political, and social functioning of entire societies. 37 Polyparasitism and Effects on Nutrition and Growth In an otherwise healthy and fully nourished person, a new infection is likely to be the only active infection in that person. In contrast, in regions where enteric and other infections are highly prevalent because of inadequate sanitation and poor socioeconomic conditions, adults and especially children may harbor several infections or be subject to repeated episodes of new enteric pathogens. Thus, the polyparasitism of multiple concurrent or recurrent infections adds a new dimension to the impact of acute infections, not often encountered in developed countries. Moreover, the subclinical impact of a number of tropical infectious diseases is beginning to become apparent. Increasing data suggest that even "asymptomatic" giardial, 38 cryptosporidial, 39 and enteroaggregative Escherichia coli 40 infections may be very important in predisposing to malnutrition, thus reflecting a clinically important impact, even in the absence of overt clinical disease such as diarrhea. Likewise, chronic intestinal helminth infections also have a major impact on nutrition in those with already marginal nutrition. Anthelminthic therapy in these children, who lack symptomatic infections, has led to increases in growth, exercise tolerance, and scholastic performance. 41 , 42 Principles of Microbial Evolution and Classification The Earth is approximately 4.5 to 5 billion years old. There is good fossil evidence of microbial life approximately 3.5 billion years ago. Microbial life (stromatolites) was mostly photosynthetic, unicellular, and anaerobic. 1 , 4 Eukaryotes, bacteria, and archaea evolved from a still hypothetical universal common ancestor. 5 , 6 , 7 Eukaryotes then evolved into protozoans, metazoans, plants, and animals, as we know them today. Moreover, there is strong evidence that primitive eukaryotic cells established relationships with bacterial organisms that later evolved into cytoplasmic organelles, such as chloroplasts in plants and mitochondria in animals. 8 To put things into perspective, approximately five-sixths of the history of life on Earth has been exclusively microbial. Human beings appeared on the planet only 2 million years ago as very late newcomers to the biosphere. Life was initially anaerobic, but with the appearance of photosynthetic organisms and chloroplasts, oxygen was released into the atmosphere for the first time. 9 Radiation in the upper atmosphere created the ozone layer from molecular oxygen, which then shielded the Earth's surface from dangerous radiation. Nucleic acids were therefore protected from harmful mutations. Organisms had to evolve to survive in the presence of oxygen. A few of the ancient anaerobes were able to survive in the highly oxidant atmosphere, and they represent the anaerobes as we know them today. The phylogeny of living organisms is based on molecular approaches, particularly analysis of ribosomal RNA. 5 , 6 , 10 Because of the antiquity of the protein synthesis machinery, these molecules are excellent evolutionary clocks. For prokaryotes, the 16S subunit of ribosomes is the most useful for classification purposes. Viruses deserve special comment because of their molecular simplicity and at the same time their importance as human pathogens and as possible agents of hereditary changes and cancer. 11 , 12 A virus is a genetic element with either DNA or RNA coated by protein of viral origin and sometimes enveloped by lipid material of host origin. Some viruses have enzymes that are necessary for their replication. The only criterion that these organisms fulfill to be considered living organisms is that of reproduction. They are inert particles when outside of the host cell, and once they have access into a cell they become active and the cell is subverted to produce more viral particles. Sometimes the cell dies in the process, and sometimes the relationship is stable. Viral hosts include bacteria, protozoa, animals, and plants. The classification of viruses is based on different criteria than the ones used for other organisms. The major criteria are type of nucleic acid, presence or absence of an envelope, manner of replication, and morphologic characteristics. 12 , 13 Simpler forms of self-replicating organisms include virusoids and viroids. 14 , 15 The former are satellite RNAs that are found encapsidated in the proteins encoded by their helper virus (e.g., hepatitis caused by the hepatitis D virus delta agent in conjunction with hepatitis B virus). The viroids are mostly plant pathogens that consist of single-stranded circular RNA molecules. The concept of "infectious agent" was revolutionized by the discovery of proteinaceous infectious agents known as prions. These proteins are responsible for neurodegenerative diseases in animals and humans. The protein particles lack nucleic acids but still are able to reproduce and trigger conformational changes in host proteins, leading to cell death. 16 , 17 In contrast, protozoa are nucleated, single-cell organisms that, depending on the species, replicate by means as simple as binary fission (e.g., Trichomonas ) or as complex as involving multiple sexual and asexual stages in both animal and invertebrate hosts (e.g., plasmodia). Protozoa include amebae (e.g., Entamoeba histolytica ), flagellates (e.g., Giardia lamblia ), ciliates (e.g., Balantidium coli ), and sporozoa (e.g., Cryptosporidium ). Even more complex are helminths, which are multicellular metazoan organisms with highly developed internal organs, including alimentary and reproductive tracts. The helminths include nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). Many helminths have complex life cycles with multiple developmental stages both in the animal host and in intermediate invertebrate or vertebrate hosts. Because of their size, helminths, the macroparasites, are solely extracellular pathogens; because of their prolonged life cycles and generation times, their capacity for genetic alteration is diminished compared to smaller, simpler microbes (the microparasites). Development of Microbial Virulence Evolution of Virulence The traditional view assumes that natural selection would favor evolution toward a benign coexistence between host and parasite. 18 , 19 A modern view of evolution of virulence focuses on the tradeoff between the benefits that pathogens accrue through increased exploitation of hosts and the costs that result from any effects of disease that reduce transmission to susceptible hosts. 19 , 20 From this point of view, virulence could be the evolved as well as the primitive stage of the association between host and parasite, depending on the development of enhanced rather than reduced transmission. According to Levin 19 and Levin and Svanborg-Eden, 20 there are three alternative models to explain evolution of a microparasite's virulence: direct selection, coincidental evolution, and short-sighted within-host selection. The direct selection model states that there is a direct relationship between the parasite's virulence and its rate of infectious transmission. The best documented and often cited example is that of the dramatic changes in virulence that the myxoma virus underwent after being released into the wild in Australia to "control" the population of wild rabbits. In the beginning, rabbit mortality and viral transmission rates were high. As the population of rabbits was decimated, the virulence of the virus decreased and its rate of transmission actually increased. This outcome is explained by the longer survival and duration of the period of shedding of the virus. At the same time, more resistant rabbits increased in number due to the selection process. 21 According to the coincidental evolution model, the factors responsible for the virulence of a microparasite evolved for some purpose other than to provide the parasite with some advantage within a host or for its transmission to other hosts. Clostridial toxins are good examples in this category. There is no beneficial reason to kill a human host who became infected by Clostridium tetani spores from soil in order for the parasite to survive. They are mostly soil bacteria and do not need humans for their survival. 19 Short-sighted within-host evolution posits that the parasites responsible for the morbidity and mortality of the host are selected for as a consequence of within-host evolution since that produces a local advantage for their survival within the host. The host dies and the rate of transmission would decrease. This is an example of evolutionary myopia in which the long-term consequences of killing a host would not matter to the parasite. 22 , 23 Natural selection is a local phenomenon that happens at a given time and place and goes perfectly with this model. Bacteria such as Neisseria meningitidis that normally live attached to human pharyngeal epithelial cells sometimes invade the central nervous system (CNS) and kill the host. Their replication in the CNS is favored since competition is low and defenses are not as abundant as in the tonsillar areas. 19 It follows from the previous paragraphs that evolutionary theories addressing virulence have predicted that virulence of microbes evolves in response to changes in conditions or tradeoffs. One of the most commonly considered "tradeoffs" is between the benefit of a high within-host microbial density (allowing efficient transmission to the next host) and the cost of a reduced longevity of the infection in the host due to high parasite density that tends to kill the host. Other approaches have been proposed such as the role of how pathogenic mechanisms affect virulence evolution. According to this model, pathogenic mechanisms that manipulate host immunity or escape from the host immune response (longer survival in the host) dominate as causes of virulence compared to mechanisms that incrementally alter transmission (higher microbial density and increased mortality). 24 , 25 Furthermore, natural selection would act more strongly on mutations influencing clearance than on mutations influencing transmission. 26 Another cited model to explain dynamics of virulence evolution is the so-called "source–sink" model in which evolution of bacterial pathogens is evaluated from ecological points of view that microbes switch from permanent (source) and transient (sink) habitats. 27 The generation times of mammalian hosts are much longer than those of microorganisms. Therefore, genetic mutations in these hosts, on which natural selection acts, take longer to become part of a large population. Nevertheless, there is evidence that specific microorganisms can exert selective pressure on the gene pool of human hosts. The evidence is strongest for the potentially lethal infections caused by falciparum malaria. In regions of the world where falciparum malaria is endemic, including Africa, there is a high prevalence of genetic mutations that alter hemoglobin structure or synthesis, decreasing or abolishing falciparum malaria parasites' survival. 28 The selective pressure of malaria on human gene expression is not confined solely to affecting erythrocytes but also likely involves the immune system, cytokines, and other systems. 29 Other Modes of Altering Virulence and Pathogenicity Although the selective pressures of evolution generally exert changes over a multitude of centuries, there are other mechanisms that may more rapidly alter microbial pathogenicity, virulence, and drug susceptibility. The expression of mutated genes in microorganisms is heightened when there are greater numbers of organisms and their generation times are brief. Hence, altered gene expression in helminths will be slow to be expressed, whereas in microparasites genetic alterations will be likely to develop. For mycobacterial infections, large numbers of bacilli that persist for a long time facilitate the genetic emergence of drug resistance to a single agent, and this likelihood underlies the principle of using more than a single drug to treat tuberculosis. Even more rapidly dividing microparasites can develop genetic alterations, and this is especially true when the fidelity of genetic replication is poor. This is prominent in human immunodeficiency virus type 1 (HIV-1), whose reverse transcriptase lacks a 3′ exonuclease proofreading activity. 30 Alterations in cell tropism, pathogenicity, and drug sensitivity are frequent in HIV-1 infections. Again, several antiviral agents must be employed concomitantly to circumvent the highly frequent mutations that alter drug susceptibility in HIV-1 strains. In addition to their own genetic material, many classes of microparasites either contain or are capable of acquiring transferable genetic elements in the form of plasmids, transposons, or bacteriophages. These transferable genetic elements also provide a means for the spread of resistance to antibacterial drugs, an increasing problem in all regions of the world. 31 Causes of Acute or Chronic Infections in Individuals One obvious impact of an infectious disease is on the individual infected. Hence, in any region of the world independent of other infectious diseases or malnutrition, the acute infection will cause morbidity and potential mortality in the infected human host. Among otherwise healthy people, the immediate impact of the infection is the symptomatic acute illness. For some infections that have prolonged courses, their impact may also continue over many years. Chronic infections include most of those caused by helminthic parasites, which characteristically live for years; persisting mycobacterial infections; and retroviral infections (HIV-1, HIV-2, and human T-cell lymphotropic virus type 1). Finally, the sequelae of some infections can include the development of neoplasms. Examples include hepatocellular carcinomas associated with chronic hepatitis B and C viral infections, bladder tumors with urinary schistosomiasis, cholangiocarcinomas with biliary fluke infections, and gastric adenocarcinomas and lymphomas associated with Helicobacter pylori infections. Causes of Widespread Infections in Populations Infectious diseases may affect not only individuals but also large groups of people or entire populations due to epidemic or highly endemic transmission. Throughout human history, a few microorganisms have been responsible for great epidemics and massive numbers of dead or crippled people as a result of infections spreading locally or throughout the world. 32 , 33 , 34 , 35 Typhus has been associated almost always with situations that involve overcrowding, famine, war, natural disasters, and poverty. The outcomes of several European wars were affected by the morbidity and mortality inflicted by typhus or other diseases on the military. Typhus epidemics were common during the world wars of the twentieth century and in the concentration camps where the ecological conditions were ideal for such a disease to spread. 30 Today, typhus and other rickettsioses are still public health problems in some countries, but overall the disease was brought under control after its life cycle was described and antibiotics, insecticides, and public health measures became available. 34 Bubonic plague, caused by Yersinia pestis , is another disease that has shaped history, especially in Europe during the Middle Ages. 35 Millions of people were affected by pandemics that spread throughout the continent. Tuberculosis, smallpox, and measles had a tremendous effect on the native populations of the Americas after Columbus's voyages to the New World. It has been estimated that 90% of the population in Mexico was killed by these pathogens, which were novel to the native residents. Acquired immunodeficiency syndrome (AIDS) represents the modern pandemic that will continue to affect human history for at least decades. Other examples are cholera and influenza, which are capable of causing pandemics. 36 In addition to widespread diseases caused by epidemic spread of infections, some infectious diseases, because of their highly endemic prevalence in populations, continue to affect large segments of the world's population. These include enteric and respiratory infections, measles, malaria, tuberculosis and schistosomiasis. Furthermore, even the staggering mortality and morbidity of these tropical infectious diseases do not control populations but are associated with population overgrowth. This is true not only across the different countries of the world but also throughout the history of developed countries. Thus, the impact of these infections is not solely on the individual but, because of their highly endemic or epidemic occurrence, on populations. This has consequences on economic, political, and social functioning of entire societies. 37 Polyparasitism and Effects on Nutrition and Growth In an otherwise healthy and fully nourished person, a new infection is likely to be the only active infection in that person. In contrast, in regions where enteric and other infections are highly prevalent because of inadequate sanitation and poor socioeconomic conditions, adults and especially children may harbor several infections or be subject to repeated episodes of new enteric pathogens. Thus, the polyparasitism of multiple concurrent or recurrent infections adds a new dimension to the impact of acute infections, not often encountered in developed countries. Moreover, the subclinical impact of a number of tropical infectious diseases is beginning to become apparent. Increasing data suggest that even "asymptomatic" giardial, 38 cryptosporidial, 39 and enteroaggregative Escherichia coli 40 infections may be very important in predisposing to malnutrition, thus reflecting a clinically important impact, even in the absence of overt clinical disease such as diarrhea. Likewise, chronic intestinal helminth infections also have a major impact on nutrition in those with already marginal nutrition. Anthelminthic therapy in these children, who lack symptomatic infections, has led to increases in growth, exercise tolerance, and scholastic performance. 41 , 42 Evolution of Virulence The traditional view assumes that natural selection would favor evolution toward a benign coexistence between host and parasite. 18 , 19 A modern view of evolution of virulence focuses on the tradeoff between the benefits that pathogens accrue through increased exploitation of hosts and the costs that result from any effects of disease that reduce transmission to susceptible hosts. 19 , 20 From this point of view, virulence could be the evolved as well as the primitive stage of the association between host and parasite, depending on the development of enhanced rather than reduced transmission. According to Levin 19 and Levin and Svanborg-Eden, 20 there are three alternative models to explain evolution of a microparasite's virulence: direct selection, coincidental evolution, and short-sighted within-host selection. The direct selection model states that there is a direct relationship between the parasite's virulence and its rate of infectious transmission. The best documented and often cited example is that of the dramatic changes in virulence that the myxoma virus underwent after being released into the wild in Australia to "control" the population of wild rabbits. In the beginning, rabbit mortality and viral transmission rates were high. As the population of rabbits was decimated, the virulence of the virus decreased and its rate of transmission actually increased. This outcome is explained by the longer survival and duration of the period of shedding of the virus. At the same time, more resistant rabbits increased in number due to the selection process. 21 According to the coincidental evolution model, the factors responsible for the virulence of a microparasite evolved for some purpose other than to provide the parasite with some advantage within a host or for its transmission to other hosts. Clostridial toxins are good examples in this category. There is no beneficial reason to kill a human host who became infected by Clostridium tetani spores from soil in order for the parasite to survive. They are mostly soil bacteria and do not need humans for their survival. 19 Short-sighted within-host evolution posits that the parasites responsible for the morbidity and mortality of the host are selected for as a consequence of within-host evolution since that produces a local advantage for their survival within the host. The host dies and the rate of transmission would decrease. This is an example of evolutionary myopia in which the long-term consequences of killing a host would not matter to the parasite. 22 , 23 Natural selection is a local phenomenon that happens at a given time and place and goes perfectly with this model. Bacteria such as Neisseria meningitidis that normally live attached to human pharyngeal epithelial cells sometimes invade the central nervous system (CNS) and kill the host. Their replication in the CNS is favored since competition is low and defenses are not as abundant as in the tonsillar areas. 19 It follows from the previous paragraphs that evolutionary theories addressing virulence have predicted that virulence of microbes evolves in response to changes in conditions or tradeoffs. One of the most commonly considered "tradeoffs" is between the benefit of a high within-host microbial density (allowing efficient transmission to the next host) and the cost of a reduced longevity of the infection in the host due to high parasite density that tends to kill the host. Other approaches have been proposed such as the role of how pathogenic mechanisms affect virulence evolution. According to this model, pathogenic mechanisms that manipulate host immunity or escape from the host immune response (longer survival in the host) dominate as causes of virulence compared to mechanisms that incrementally alter transmission (higher microbial density and increased mortality). 24 , 25 Furthermore, natural selection would act more strongly on mutations influencing clearance than on mutations influencing transmission. 26 Another cited model to explain dynamics of virulence evolution is the so-called "source–sink" model in which evolution of bacterial pathogens is evaluated from ecological points of view that microbes switch from permanent (source) and transient (sink) habitats. 27 The generation times of mammalian hosts are much longer than those of microorganisms. Therefore, genetic mutations in these hosts, on which natural selection acts, take longer to become part of a large population. Nevertheless, there is evidence that specific microorganisms can exert selective pressure on the gene pool of human hosts. The evidence is strongest for the potentially lethal infections caused by falciparum malaria. In regions of the world where falciparum malaria is endemic, including Africa, there is a high prevalence of genetic mutations that alter hemoglobin structure or synthesis, decreasing or abolishing falciparum malaria parasites' survival. 28 The selective pressure of malaria on human gene expression is not confined solely to affecting erythrocytes but also likely involves the immune system, cytokines, and other systems. 29 Other Modes of Altering Virulence and Pathogenicity Although the selective pressures of evolution generally exert changes over a multitude of centuries, there are other mechanisms that may more rapidly alter microbial pathogenicity, virulence, and drug susceptibility. The expression of mutated genes in microorganisms is heightened when there are greater numbers of organisms and their generation times are brief. Hence, altered gene expression in helminths will be slow to be expressed, whereas in microparasites genetic alterations will be likely to develop. For mycobacterial infections, large numbers of bacilli that persist for a long time facilitate the genetic emergence of drug resistance to a single agent, and this likelihood underlies the principle of using more than a single drug to treat tuberculosis. Even more rapidly dividing microparasites can develop genetic alterations, and this is especially true when the fidelity of genetic replication is poor. This is prominent in human immunodeficiency virus type 1 (HIV-1), whose reverse transcriptase lacks a 3′ exonuclease proofreading activity. 30 Alterations in cell tropism, pathogenicity, and drug sensitivity are frequent in HIV-1 infections. Again, several antiviral agents must be employed concomitantly to circumvent the highly frequent mutations that alter drug susceptibility in HIV-1 strains. In addition to their own genetic material, many classes of microparasites either contain or are capable of acquiring transferable genetic elements in the form of plasmids, transposons, or bacteriophages. These transferable genetic elements also provide a means for the spread of resistance to antibacterial drugs, an increasing problem in all regions of the world. 31 Causes of Acute or Chronic Infections in Individuals One obvious impact of an infectious disease is on the individual infected. Hence, in any region of the world independent of other infectious diseases or malnutrition, the acute infection will cause morbidity and potential mortality in the infected human host. Among otherwise healthy people, the immediate impact of the infection is the symptomatic acute illness. For some infections that have prolonged courses, their impact may also continue over many years. Chronic infections include most of those caused by helminthic parasites, which characteristically live for years; persisting mycobacterial infections; and retroviral infections (HIV-1, HIV-2, and human T-cell lymphotropic virus type 1). Finally, the sequelae of some infections can include the development of neoplasms. Examples include hepatocellular carcinomas associated with chronic hepatitis B and C viral infections, bladder tumors with urinary schistosomiasis, cholangiocarcinomas with biliary fluke infections, and gastric adenocarcinomas and lymphomas associated with Helicobacter pylori infections. Causes of Widespread Infections in Populations Infectious diseases may affect not only individuals but also large groups of people or entire populations due to epidemic or highly endemic transmission. Throughout human history, a few microorganisms have been responsible for great epidemics and massive numbers of dead or crippled people as a result of infections spreading locally or throughout the world. 32 , 33 , 34 , 35 Typhus has been associated almost always with situations that involve overcrowding, famine, war, natural disasters, and poverty. The outcomes of several European wars were affected by the morbidity and mortality inflicted by typhus or other diseases on the military. Typhus epidemics were common during the world wars of the twentieth century and in the concentration camps where the ecological conditions were ideal for such a disease to spread. 30 Today, typhus and other rickettsioses are still public health problems in some countries, but overall the disease was brought under control after its life cycle was described and antibiotics, insecticides, and public health measures became available. 34 Bubonic plague, caused by Yersinia pestis , is another disease that has shaped history, especially in Europe during the Middle Ages. 35 Millions of people were affected by pandemics that spread throughout the continent. Tuberculosis, smallpox, and measles had a tremendous effect on the native populations of the Americas after Columbus's voyages to the New World. It has been estimated that 90% of the population in Mexico was killed by these pathogens, which were novel to the native residents. Acquired immunodeficiency syndrome (AIDS) represents the modern pandemic that will continue to affect human history for at least decades. Other examples are cholera and influenza, which are capable of causing pandemics. 36 In addition to widespread diseases caused by epidemic spread of infections, some infectious diseases, because of their highly endemic prevalence in populations, continue to affect large segments of the world's population. These include enteric and respiratory infections, measles, malaria, tuberculosis and schistosomiasis. Furthermore, even the staggering mortality and morbidity of these tropical infectious diseases do not control populations but are associated with population overgrowth. This is true not only across the different countries of the world but also throughout the history of developed countries. Thus, the impact of these infections is not solely on the individual but, because of their highly endemic or epidemic occurrence, on populations. This has consequences on economic, political, and social functioning of entire societies. 37 Polyparasitism and Effects on Nutrition and Growth In an otherwise healthy and fully nourished person, a new infection is likely to be the only active infection in that person. In contrast, in regions where enteric and other infections are highly prevalent because of inadequate sanitation and poor socioeconomic conditions, adults and especially children may harbor several infections or be subject to repeated episodes of new enteric pathogens. Thus, the polyparasitism of multiple concurrent or recurrent infections adds a new dimension to the impact of acute infections, not often encountered in developed countries. Moreover, the subclinical impact of a number of tropical infectious diseases is beginning to become apparent. Increasing data suggest that even "asymptomatic" giardial, 38 cryptosporidial, 39 and enteroaggregative Escherichia coli 40 infections may be very important in predisposing to malnutrition, thus reflecting a clinically important impact, even in the absence of overt clinical disease such as diarrhea. Likewise, chronic intestinal helminth infections also have a major impact on nutrition in those with already marginal nutrition. Anthelminthic therapy in these children, who lack symptomatic infections, has led to increases in growth, exercise tolerance, and scholastic performance. 41 , 42 Microbial Interactions with Human Hosts Just as microorganisms have evolved over centuries or longer, mammalian hosts have evolved to contain and limit the deleterious consequences of infections with diverse microbes. The human immune system is composed of multiple elements, including those of innate immunity and those of adaptive immunity. Many of the elements of innate immunity are more primitive and found in invertebrate organisms, whereas the adaptive immune responses have evolved further in vertebrate hosts. Microorganisms that successfully infect human hosts must, at least in the short term, overcome elements of the host immune system, which then may react further to attempt to control these infections. The study of microbial pathogenesis has been revolutionized with the advent of comparative genomics and the multitude of genomic tools that have become available in the last two decades or so. The first bacterium whose genome was fully sequenced was that of a laboratory strain of Haemophilus influenzae in 1995, followed by Mycoplasma genitalium the same year. Since then over 250 bacterial genome sequences have become available. Discovery of virulence genes has therefore expanded rapidly and has benefited from other strategies such as powerful computational methods, genetic signatures, analysis of physical linkage to accessory genetic elements, and biochemical and genetic approaches that depend on comprehensive genome sequence information. 43 Furthermore, proteomics-based methods have also been used in combination with genome-sequence analysis to define new virulence factors. 43 For obligate intracellular pathogens, whose genetic manipulation is far more difficult than for other microbes, the use of genomic and post-genomic tools (genomic-microarray methods and proteomics) has been virtually the only path to discovery of virulence factors. In general, these tools have advanced the understanding of virulence factors in pathogenic microorganisms exponentially. Microorganisms that infect humans are exogenous to the host and must colonize or penetrate epithelial barriers to gain access to the host. Except for infections acquired during the intrauterine period, infectious agents must bridge host epithelial surfaces, the keratinized epithelium of the skin, or the mucosal epithelium of the respiratory, gastrointestinal, or genitourinary tracts. Ultimately, there are four types of microbial localization in the host ( Fig. 1.1 ). Some microbes will enter intracellular sites either within the cytoplasm or within vesicular or vacuolar compartments in cells. Other microbes remain extracellular, either at epithelial surfaces or within the host in the blood, lymph, or tissues. Figure 1.1 Microbial localization. Interactions at Epithelial Barrier Surfaces The barrier functions occurring at epithelial surfaces are part of the innate host defenses and are important in determining the outcome of interactions of potential pathogens with the host. Interactions at epithelial barriers involved in defense against external microbes include not only the physical properties of the epithelial surfaces but also the overlying mucous phase, the ciliated or other propulsive activities facilitating microbe clearance, and the normal microbial flora. Normal Flora Vertebrate warm-blooded organisms, such as humans, are an ideal site for the survival of many microbes and provide a rich source of organic material and a constant temperature and pH. Microbes coexist with us in and on our bodies, especially on epithelial surfaces where there is contact with the outside world, such as the bowel, upper respiratory tract, mouth, skin, and distal portions of the genitourinary tract. 1 , 2 , 43 Most of these microorganisms are highly adapted to live with us and do not cause any harm. The presence of the same type of microorganisms at a particular site in the absence of disease is called colonization. Normal colonizing microbial flora help to limit access by potentially pathogenic microorganisms. One condition predisposing to infection is the alteration of the normal epithelial flora, as occurs with antibiotic therapy, since this may allow for the proliferation of pathogenic organisms normally held in balance by the endogenous normal microbial flora. Examples include Candida vaginitis or the development of pseudomembranous colitis due to toxigenic Clostridium difficile , which may complicate antibiotic therapy. Adhesion to the Epithelium Microorganisms maintain themselves in or on their host by adhesion to cells or the extracellular matrix. Adhesins are encoded by chromosomal genes, plasmids, or phages. 44 They are usually divided into fimbrial and afimbrial adhesins. 45 Fimbrial adhesins are present in organisms such as Neisseria gonorrhoeae and are in part responsible for the attachment to genitourinary tract epithelium, preventing the bacteria from being washed out by the urine stream. 46 , 47 An example of an afimbrial adhesin is the filamentous hemagglutinin of Bordetella pertussis , which is responsible for the attachment of B. pertussis to epithelial cells in the respiratory tract. 48 Adhesins attach to receptors in the host. These receptors include proteins, glycolipids, and carbohydrates exposed on the surface of cells or in the extracellular matrix. 44 Integrins are one class of proteins present on eukaryotic cell surfaces that can serve as bacterial receptors. 44 Helicobacter pylori binds to Lewis blood group antigen present in the gastric epithelium. 49 Neisseria has a ligand that binds to CD66 molecules on epithelial cells. Some pathogens have even more evolved interactions with the host and activate signal transduction mechanisms in the host cell, which in turn upregulate other molecules that aid in the adhesion process. 2 , 44 Certain strains of enteropathogenic E. coli possess type III secretion or contact-mediated systems. 50 In such cases, the secretion and synthesis of virulence factors is modulated by contact with host surfaces. The systems are complex (more than 20 genes are involved) and have not been elucidated completely at the molecular level. 51 , 52 Penetration of the Epithelial Barriers Some microbes do not have the means to penetrate skin barriers and are only able to gain access through bites produced by arthropods (e.g., rickettsiae, arboviruses, plasmodia, and filariae). 53 , 54 In such cases, microbes may be introduced by direct inoculation (e.g., rickettsiae, arboviruses, and plasmodia) or may gain access by migrating through the puncture site (filariae). Other microbes (e.g., skin bacteria and fungi) depend on mechanical disruption of the skin (e.g., due to burns, trauma, or intravenous catheters) to invade deeper structures. 55 Still others invade when defenses on mucosal surfaces are lowered due to combined local or generalized immunosuppression and altered mucosal integrity (mucositis) due to chemotherapy or malnutrition (e.g., Candida spp. and anaerobic and other enteric bacteria in the bowel). Some microbes do not invade tissues at all and affect the host locally and systemically by liberating toxins at the site of colonization (e.g., diphtheria exotoxin). 44 For enteric pathogens, some, including poliovirus, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Campylobacter jejuni , Yersinia enterocolitica , and Yersinia pseudotuberculosis , gain access to the host across the intestinal epithelium by utilizing uptake in specialized epithelial M cells. 56 Internalization of some microorganisms is also achieved through other mechanisms, such as sequential "zipper-like" encircling of the organisms triggered by bacterial ligands and cellular receptors, as occurs in infections caused by Listeria monocytogenes . 44 The trigger mechanism of the bacteria induces massive rearrangements of cytoskeletal proteins such as actin, which results in membrane ruffles, as occur with shigellosis and salmonellosis. 44 In the genitourinary tract, invasion of some agents (e.g., HIV-1) is facilitated by mucosal erosions caused by other infectious agents. 57 Spread from the Portal of Entry Once the organisms gain access to the body after overcoming the first lines of defense, they either spread to other sites of the body or reproduce locally and often invade surrounding tissues. Local spread is facilitated by a number of factors, including collagenases, hyaluronidases, fibrinolysis, and other enzymes. They are produced by a wide range of organisms, and the role of these enzymes in invasion is, in some cases, controversial. 2 Lymphatic spread occurs in most cases once the organisms gain access to subepithelial tissues or serosal surfaces. Lymphatic vessels are distributed in most tissues of the body, with few exceptions such as the brain. Lymph is carried by lymphatic vessels to regional lymph nodes, where it circulates through the node and eventually returns to the systemic circulation through the thoracic duct and the great lymphatic vein. One to three liters of lymph is returned to the systemic circulation every day. Most pathogens are filtered in lymph nodes before reaching the systemic circulation, but some actually reproduce either in the endothelium of lymphatic vessels (e.g., Mycobacterium leprae ) 2 , 58 or in tissue macrophages present in the lymph nodes (e.g., Brucella spp.) or lymphocytes (HIV and herpesviruses, including Epstein–Barr virus). 59 Some organisms reach the systemic circulation after overwhelming the defenses in the lymph nodes (e.g., Bacillus anthracis and Y. pestis ). Microorganisms carried in the blood are transported either extracellularly (e.g., most of those causing bacteremia) or intracellularly. Intracellular pathogens are carried by red blood cells (e.g., Plasmodium, Babesia, Colorado tick fever virus, and Bartonella ), monocytes (e.g., measles virus, cytomegalovirus, and Toxoplasma ), or neutrophils (e.g., Anaplasma phagocytophilum , Ehrlichia ewingii , and some pyogenic bacteria). 2 , 60 Once in the blood, by initial lymphatic or hematogenous spread, the microorganisms have access to virtually any site in the body. However, some pathogens exhibit tropism for certain tissues. This tropism depends on multiple factors, including the anatomy of the microcirculation in a given tissue (fenestrated capillaries versus continuous endothelial lining), receptors present on certain endothelial cells, and the presence of mononuclear phagocytic cells in organs such as bone marrow, liver, and spleen. 2 Other less common routes of spread include peripheral nerves (e.g., rabies and varicella zoster virus), cerebrospinal fluid (after the organisms traverse the blood–brain barrier), and serosal cavities. Localization in the Host Microbes that have gained access to the host at or through epithelial barriers then, depending on the properties and size of the pathogens, have the capacity either to seek intracellular sites or remain extracellular (see Fig. 1.1 ). Mechanisms of host immune responses to the microorganisms vary depending on their sites of localization. Intracellular Localization Specific microorganisms use highly developed processes to gain access to and survive within host cells. The microorganisms may reside either in the cytoplasm or within vesicular or vacuolar compartments of targeted cells. Targeting and penetration of cells is governed by the interactions of microbial surface proteins that may engage host cell molecules that function as receptors for the microbial ligands. The entry of malarial parasites into erythrocytes is a good example, and the nature of the erythrocyte receptors used by different malarial parasite species governs which red blood cells are infected. Plasmodium vivax binds to the Duffy blood group antigens present on some people's red blood cell membranes. The expression of the Duffy blood group antigen is genetically determined, and this antigen is present mostly in whites and Asians and largely absent in blacks of sub-Saharan African ancestry. 61 , 62 , 63 , 64 This genetic absence of a receptor on red blood cells required for vivax malaria's survival explains why vivax malaria is rare in regions of Africa. Plasmodium vivax also exhibits a characteristic restriction in the age of erythrocytes it infects. Only young red blood cells and reticulocytes are susceptible to infection, even though the Duffy blood group antigen is present on red blood cells of all ages. The basis for this restriction to younger red blood cells also rests with receptor-mediated limitations. Plasmodium vivax parasites contain reticulocyte-binding proteins, which recognize and bind to reticulocyte-specific antigens on the red blood cell surface. 65 , 66 Thus, host cell receptor–microbial ligand interactions have an impact on the geographic range of infections based on host genetic differences in requisite receptor expression and on the specific cells that a microbe may enter. Another example of the intricacies of microbe–receptor interactions has been recognized with HIV-1. Although CD4 is the primary cellular receptor for HIV entry, binding to CD4 alone is not sufficient for entry of HIV-1 into cells. Cellular coreceptors that are members of the chemokine receptor family of seven-transmembrane G protein-coupled molecules are also important. T-cell tropic strains use the CXCR-4 chemokine receptor, and macrophage tropic HIV-1 strains use the CCR-3 and CCR-5 chemokine receptors as coreceptors in concert with CD4. The differences among strains of HIV-1 in their capacities to bind to different chemokine receptor–coreceptors may help explain differences in cell tropism and pathogenicity, the lack of infectability of nonprimate cells, and, for those with genetically altered coreceptors, the apparent resistance to HIV-1 infection of some individuals. 67 , 68 , 69 , 70 Typical of those etiologic agents that have an intracellular localization are viruses. The entry of these agents into cells is increasingly recognized to be dependent on their interactions with specific host cell proteins that act as their "receptors." For instance, host cell molecules that function as viral receptors include multiple isoforms of membrane cofactor protein (CD46), a complement regulatory protein, for measles; the integrin, intracellular adhesion molecule-1 (ICAM-1), for rhinovirus; erythrocyte P antigen for parvovirus B19; and the C3d complement receptor (CR2) for Epstein–Barr virus. 71 , 72 , 73 , 74 Microbes that exist principally within the cytoplasm are sequestered from many immune response mechanisms active on extracellular pathogens, including antibody and phagocytic cells. Viral intracellular proteins will be processed and displayed with class I major histocompatibility complex (MHC) proteins, which enable CD8 cytotoxic T cells to recognize and kill the virally infected cell. Other microbes are internalized within phagocytic cells, especially macrophages. Once internalized in host cells, organisms such as Salmonella , Mycobacterium , Chlamydia , and Legionella use an extraordinary assortment of mechanisms to prevent their phagocytic vacuole from fusing with the host cells' acidifying lysosomes. 75 , 76 , 77 For some parasites, the intracellular environment is an important determinant of parasitism. For example, Leishmania and Coxiella (unlike other pathogens) benefit from the acidic environment of the macrophage phagolysosome. Leishmania use the proton gradient across the lysosome to drive the energy-dependent uptake of two important substrates: glucose and proline. 78 Thus, Leishmania amastigotes actually survive in the macrophage phagolysosome because they benefit from its proton gradient and because they avoid activating the processes that normally kill ingested microorganisms. Leishmanial lipophosphoglycan inhibits the action of β-galactosidase, chelates calcium, inhibits protein kinase C and the oxidative burst, and may scavenge toxic oxygen metabolites. 79 Conversely, other intracellular pathogens such as Toxoplasma gondii survive within the macrophage by using an alternative pathway of entry that avoids fusion of the parasitophorous vacuole with lysosomes. 76 , 80 In contrast, dead or antibody-coated T. gondii enter via the Fc receptor and are routed to a different intracellular compartment, which fuses with the lysosome, and are then killed in the phagolysosome. 76 , 80 , 81 Other organisms, such as Shigella , Listeria , and Rickettsia , breach their vacuolar membrane to multiply freely in the cytoplasm and may also usurp host cellular actin to propel their further spread to neighboring cells, continuing to exploit their intracellular sanctuary. 82 , 83 , 84 Immune responses against microbes within macrophages rely heavily on class II MHC-mediated presentation of host antigenic peptides to T-helper 1 (Th1) types of CD4 + T cells, which then augment the microbicidal activities of the macrophages. Extracellular Localization Some types of microbes that remain extracellular typically reside at epithelial surfaces, including bacteria such as N. gonorrhoeae , H. pylori , Vibrio cholerae , and E. coli , and helminths such as adult Ascaris lumbricoides , hookworms, and Trichuris trichiura . Mucosal immune responses, including IgA and leukocytes, participate in host immune reactions to these pathogens. Other microbes that survive extracellularly are present within the blood, lymph, or tissues of the host, and these organisms include fungi, viruses, bacteria, protozoa, and notably the helminths. Multicellular helminths, due to their large size, remain forever extracellular and may be found in the blood (e.g., microfilariae), lymph (adult lymphatic filarial worms), tissues (migrating larvae and adult stages of some helminths), and cerebrospinal fluid. Host defense against extracellular pathogens uses antibodies, complement, phagocytic cells, and, for helminths, IgE, eosinophils, and mast cells. 85 Tissue Damage There are multiple mechanisms by which microbes inflict damage on host tissues. Direct Damage or Alteration of Host Cell Function Host cells can be killed directly by the infectious agent, as in some viral or bacterial infections that are highly cytopathic (e.g., yellow fever virus in hepatocytes and Salmonella in macrophages). 86 , 87 Some microorganisms multiply intracellularly until the cell bursts and dies (e.g., Rickettsia prowazekii ). 34 Some bacteria, viruses, and other parasites, such as Shigella , HIV-1, and Listeria , can induce apoptosis of host cells. 59 , 88 , 89 Apoptosis is triggered by different mechanisms, such as activation of the interleukin-converting enzyme pathway. 90 , 91 This form of programmed cell death is probably more widespread as a mechanism of cell death in infectious diseases than previously thought. Damage is sometimes caused by toxins secreted by bacterial cells (exotoxins). In this case, bacteria can either invade host tissues or colonize mucosal sites and then release toxins at the mucosal site that are absorbed systemically and cause distant damage. 92 Exotoxins can act through different pathways that damage the components of the cell membranes such as phospholipids 88 , 93 or affect signaling pathways (e.g., V. cholerae ). 44 , 94 Other exotoxins, such as streptolysins and listeriolysins, alter membrane permeability. Still others, such as exfoliatin (e.g., Staphylococcus aureus ) and elastase (e.g., Pseudomonas spp.), are capable of degrading extracellular elements. 2 Some toxins are translocated to the intracellular environment, where they affect multiple enzymatic systems. These toxins are classified according to their enzymatic activity, such as adenosine diphosphate (ADP) ribosyl transferase (e.g., diphtheria toxin, P. aeruginosa exotoxin A, and pertussis toxin), depurinase (e.g., Shiga toxin), adenylate cyclase (e.g., pertussis hemolysin and anthrax edema factor), and zinc protease (e.g., tetanus). 94 The end result ranges from blockade of protein synthesis and cell death or blockade of exocytosis (especially CNS neurotransmitters at the synaptic cleft) 95 , 96 to increases of cyclic adenosine monophosphate (AMP) or cyclic guanosine monophosphate (GMP) and changes in cell permeability. 94 Still other organisms, such as C. difficile , produce toxins that change basic cell signaling transducers such as Rho to alter cell function or affect their spread. Finally, organisms can interact with host cell or microbial transcriptional regulation of genes (such as iron-binding proteins for uropathogenic E. coli 92 , 97 ) or cytokine release (such as H. pylori or enteroaggregative E. coli 40 , 98 , 99 , 100 ) to enhance their survival or elicit pathogenic responses. The evolutionary advantages to a microbe of its remarkable array of traits that we call "virulence" hold many of the clues to their control, if we can but truly understand them. Endotoxins are a subset of lipopolysaccharides present in the outer membrane of Gram-negative bacteria that can trigger a wide variety of responses in the host, including massive cytokine release leading to hypotension and shock. 101 , 102 These deleterious effects occur with high-grade invasion of the blood by Gram-negative bacteria, including enteric Gram-negative bacteremias and meningococcemia. Indirect Damage Damage to the host may also develop as a consequence of immune reactions to the infectious agents. One scheme for classifying immunopathologic responses divides the reactions into four types based on the elements of the immune response involved. 103 Type I reactions involve elements of strong Th2 responses that lead to increased IgE, eosinophilia, and eosinophil and mast cell activation. Adverse reactions of this type include the development of urticaria (with several helminthic parasites), the occurrence of potentially life-threatening anaphylactic shock in IgE-mediated mast cell degranulation (e.g., triggered by systemic release of antigens from echinococcal cysts 104 ), and exuberant eosinophilic infiltration of tissues due to migrating helminth larvae (e.g., Löffler's pneumonia with the pulmonary migration of Ascaris larvae). Type II reactions are also dependent on elements of Th2 cell responses that lead to increased IgM and then IgG antibodies directed toward the infectious agents. These antibodies, if cross-reactive with host antigens, may lead to complement-mediated cytotoxicity or antibody-dependent cell-mediated cytotoxicity by natural killer cells, which have Fc receptors. An example of this type of immunopathologic response is the uncommon hemolytic anemia associated with Mycoplasma pneumoniae infection that is mediated by complement-induced hemolysis triggered by IgM (cold agglutinin) antibodies against erythrocyte I antigen. Type III reactions are caused by the deposition of immune complexes. When neither antibody nor antigen is present in excess of the other, the complexing of antibodies with soluble antigen results in the formation of immune complexes that may cause disease. This may develop acutely as antibody titers rise in the presence of microbial antigens, causing the syndrome of serum sickness. In addition, when soluble antigen is persistently abundant, sustained formation of immune complexes develops, leading to chronic immune complex-mediated tissue damage (especially glomerulonephritis), as found in subacute bacterial endocarditis, chronic hepatitis B antigenemia, and chronic Plasmodium malariae infections. 105 Type IV reactions include adverse reactions mediated by macrophages and cytotoxic T cells. Examples are damage caused by granulomas in leprosy, tuberculosis, tertiary syphilis, and fungal infections. Likewise, granulomas developing around schistosomal eggs, depending on their location, may cause ureteral obstruction or hepatic presinusoidal lesions. Other deleterious inflammatory reactions in this category are mediated by parasite-elicited host cytokines, such as the hepatic fibrosis elicited by schistosomal eggs. Interactions at Epithelial Barrier Surfaces The barrier functions occurring at epithelial surfaces are part of the innate host defenses and are important in determining the outcome of interactions of potential pathogens with the host. Interactions at epithelial barriers involved in defense against external microbes include not only the physical properties of the epithelial surfaces but also the overlying mucous phase, the ciliated or other propulsive activities facilitating microbe clearance, and the normal microbial flora. Normal Flora Vertebrate warm-blooded organisms, such as humans, are an ideal site for the survival of many microbes and provide a rich source of organic material and a constant temperature and pH. Microbes coexist with us in and on our bodies, especially on epithelial surfaces where there is contact with the outside world, such as the bowel, upper respiratory tract, mouth, skin, and distal portions of the genitourinary tract. 1 , 2 , 43 Most of these microorganisms are highly adapted to live with us and do not cause any harm. The presence of the same type of microorganisms at a particular site in the absence of disease is called colonization. Normal colonizing microbial flora help to limit access by potentially pathogenic microorganisms. One condition predisposing to infection is the alteration of the normal epithelial flora, as occurs with antibiotic therapy, since this may allow for the proliferation of pathogenic organisms normally held in balance by the endogenous normal microbial flora. Examples include Candida vaginitis or the development of pseudomembranous colitis due to toxigenic Clostridium difficile , which may complicate antibiotic therapy. Adhesion to the Epithelium Microorganisms maintain themselves in or on their host by adhesion to cells or the extracellular matrix. Adhesins are encoded by chromosomal genes, plasmids, or phages. 44 They are usually divided into fimbrial and afimbrial adhesins. 45 Fimbrial adhesins are present in organisms such as Neisseria gonorrhoeae and are in part responsible for the attachment to genitourinary tract epithelium, preventing the bacteria from being washed out by the urine stream. 46 , 47 An example of an afimbrial adhesin is the filamentous hemagglutinin of Bordetella pertussis , which is responsible for the attachment of B. pertussis to epithelial cells in the respiratory tract. 48 Adhesins attach to receptors in the host. These receptors include proteins, glycolipids, and carbohydrates exposed on the surface of cells or in the extracellular matrix. 44 Integrins are one class of proteins present on eukaryotic cell surfaces that can serve as bacterial receptors. 44 Helicobacter pylori binds to Lewis blood group antigen present in the gastric epithelium. 49 Neisseria has a ligand that binds to CD66 molecules on epithelial cells. Some pathogens have even more evolved interactions with the host and activate signal transduction mechanisms in the host cell, which in turn upregulate other molecules that aid in the adhesion process. 2 , 44 Certain strains of enteropathogenic E. coli possess type III secretion or contact-mediated systems. 50 In such cases, the secretion and synthesis of virulence factors is modulated by contact with host surfaces. The systems are complex (more than 20 genes are involved) and have not been elucidated completely at the molecular level. 51 , 52 Penetration of the Epithelial Barriers Some microbes do not have the means to penetrate skin barriers and are only able to gain access through bites produced by arthropods (e.g., rickettsiae, arboviruses, plasmodia, and filariae). 53 , 54 In such cases, microbes may be introduced by direct inoculation (e.g., rickettsiae, arboviruses, and plasmodia) or may gain access by migrating through the puncture site (filariae). Other microbes (e.g., skin bacteria and fungi) depend on mechanical disruption of the skin (e.g., due to burns, trauma, or intravenous catheters) to invade deeper structures. 55 Still others invade when defenses on mucosal surfaces are lowered due to combined local or generalized immunosuppression and altered mucosal integrity (mucositis) due to chemotherapy or malnutrition (e.g., Candida spp. and anaerobic and other enteric bacteria in the bowel). Some microbes do not invade tissues at all and affect the host locally and systemically by liberating toxins at the site of colonization (e.g., diphtheria exotoxin). 44 For enteric pathogens, some, including poliovirus, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Campylobacter jejuni , Yersinia enterocolitica , and Yersinia pseudotuberculosis , gain access to the host across the intestinal epithelium by utilizing uptake in specialized epithelial M cells. 56 Internalization of some microorganisms is also achieved through other mechanisms, such as sequential "zipper-like" encircling of the organisms triggered by bacterial ligands and cellular receptors, as occurs in infections caused by Listeria monocytogenes . 44 The trigger mechanism of the bacteria induces massive rearrangements of cytoskeletal proteins such as actin, which results in membrane ruffles, as occur with shigellosis and salmonellosis. 44 In the genitourinary tract, invasion of some agents (e.g., HIV-1) is facilitated by mucosal erosions caused by other infectious agents. 57 Normal Flora Vertebrate warm-blooded organisms, such as humans, are an ideal site for the survival of many microbes and provide a rich source of organic material and a constant temperature and pH. Microbes coexist with us in and on our bodies, especially on epithelial surfaces where there is contact with the outside world, such as the bowel, upper respiratory tract, mouth, skin, and distal portions of the genitourinary tract. 1 , 2 , 43 Most of these microorganisms are highly adapted to live with us and do not cause any harm. The presence of the same type of microorganisms at a particular site in the absence of disease is called colonization. Normal colonizing microbial flora help to limit access by potentially pathogenic microorganisms. One condition predisposing to infection is the alteration of the normal epithelial flora, as occurs with antibiotic therapy, since this may allow for the proliferation of pathogenic organisms normally held in balance by the endogenous normal microbial flora. Examples include Candida vaginitis or the development of pseudomembranous colitis due to toxigenic Clostridium difficile , which may complicate antibiotic therapy. Adhesion to the Epithelium Microorganisms maintain themselves in or on their host by adhesion to cells or the extracellular matrix. Adhesins are encoded by chromosomal genes, plasmids, or phages. 44 They are usually divided into fimbrial and afimbrial adhesins. 45 Fimbrial adhesins are present in organisms such as Neisseria gonorrhoeae and are in part responsible for the attachment to genitourinary tract epithelium, preventing the bacteria from being washed out by the urine stream. 46 , 47 An example of an afimbrial adhesin is the filamentous hemagglutinin of Bordetella pertussis , which is responsible for the attachment of B. pertussis to epithelial cells in the respiratory tract. 48 Adhesins attach to receptors in the host. These receptors include proteins, glycolipids, and carbohydrates exposed on the surface of cells or in the extracellular matrix. 44 Integrins are one class of proteins present on eukaryotic cell surfaces that can serve as bacterial receptors. 44 Helicobacter pylori binds to Lewis blood group antigen present in the gastric epithelium. 49 Neisseria has a ligand that binds to CD66 molecules on epithelial cells. Some pathogens have even more evolved interactions with the host and activate signal transduction mechanisms in the host cell, which in turn upregulate other molecules that aid in the adhesion process. 2 , 44 Certain strains of enteropathogenic E. coli possess type III secretion or contact-mediated systems. 50 In such cases, the secretion and synthesis of virulence factors is modulated by contact with host surfaces. The systems are complex (more than 20 genes are involved) and have not been elucidated completely at the molecular level. 51 , 52 Penetration of the Epithelial Barriers Some microbes do not have the means to penetrate skin barriers and are only able to gain access through bites produced by arthropods (e.g., rickettsiae, arboviruses, plasmodia, and filariae). 53 , 54 In such cases, microbes may be introduced by direct inoculation (e.g., rickettsiae, arboviruses, and plasmodia) or may gain access by migrating through the puncture site (filariae). Other microbes (e.g., skin bacteria and fungi) depend on mechanical disruption of the skin (e.g., due to burns, trauma, or intravenous catheters) to invade deeper structures. 55 Still others invade when defenses on mucosal surfaces are lowered due to combined local or generalized immunosuppression and altered mucosal integrity (mucositis) due to chemotherapy or malnutrition (e.g., Candida spp. and anaerobic and other enteric bacteria in the bowel). Some microbes do not invade tissues at all and affect the host locally and systemically by liberating toxins at the site of colonization (e.g., diphtheria exotoxin). 44 For enteric pathogens, some, including poliovirus, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Campylobacter jejuni , Yersinia enterocolitica , and Yersinia pseudotuberculosis , gain access to the host across the intestinal epithelium by utilizing uptake in specialized epithelial M cells. 56 Internalization of some microorganisms is also achieved through other mechanisms, such as sequential "zipper-like" encircling of the organisms triggered by bacterial ligands and cellular receptors, as occurs in infections caused by Listeria monocytogenes . 44 The trigger mechanism of the bacteria induces massive rearrangements of cytoskeletal proteins such as actin, which results in membrane ruffles, as occur with shigellosis and salmonellosis. 44 In the genitourinary tract, invasion of some agents (e.g., HIV-1) is facilitated by mucosal erosions caused by other infectious agents. 57 Spread from the Portal of Entry Once the organisms gain access to the body after overcoming the first lines of defense, they either spread to other sites of the body or reproduce locally and often invade surrounding tissues. Local spread is facilitated by a number of factors, including collagenases, hyaluronidases, fibrinolysis, and other enzymes. They are produced by a wide range of organisms, and the role of these enzymes in invasion is, in some cases, controversial. 2 Lymphatic spread occurs in most cases once the organisms gain access to subepithelial tissues or serosal surfaces. Lymphatic vessels are distributed in most tissues of the body, with few exceptions such as the brain. Lymph is carried by lymphatic vessels to regional lymph nodes, where it circulates through the node and eventually returns to the systemic circulation through the thoracic duct and the great lymphatic vein. One to three liters of lymph is returned to the systemic circulation every day. Most pathogens are filtered in lymph nodes before reaching the systemic circulation, but some actually reproduce either in the endothelium of lymphatic vessels (e.g., Mycobacterium leprae ) 2 , 58 or in tissue macrophages present in the lymph nodes (e.g., Brucella spp.) or lymphocytes (HIV and herpesviruses, including Epstein–Barr virus). 59 Some organisms reach the systemic circulation after overwhelming the defenses in the lymph nodes (e.g., Bacillus anthracis and Y. pestis ). Microorganisms carried in the blood are transported either extracellularly (e.g., most of those causing bacteremia) or intracellularly. Intracellular pathogens are carried by red blood cells (e.g., Plasmodium, Babesia, Colorado tick fever virus, and Bartonella ), monocytes (e.g., measles virus, cytomegalovirus, and Toxoplasma ), or neutrophils (e.g., Anaplasma phagocytophilum , Ehrlichia ewingii , and some pyogenic bacteria). 2 , 60 Once in the blood, by initial lymphatic or hematogenous spread, the microorganisms have access to virtually any site in the body. However, some pathogens exhibit tropism for certain tissues. This tropism depends on multiple factors, including the anatomy of the microcirculation in a given tissue (fenestrated capillaries versus continuous endothelial lining), receptors present on certain endothelial cells, and the presence of mononuclear phagocytic cells in organs such as bone marrow, liver, and spleen. 2 Other less common routes of spread include peripheral nerves (e.g., rabies and varicella zoster virus), cerebrospinal fluid (after the organisms traverse the blood–brain barrier), and serosal cavities. Localization in the Host Microbes that have gained access to the host at or through epithelial barriers then, depending on the properties and size of the pathogens, have the capacity either to seek intracellular sites or remain extracellular (see Fig. 1.1 ). Mechanisms of host immune responses to the microorganisms vary depending on their sites of localization. Intracellular Localization Specific microorganisms use highly developed processes to gain access to and survive within host cells. The microorganisms may reside either in the cytoplasm or within vesicular or vacuolar compartments of targeted cells. Targeting and penetration of cells is governed by the interactions of microbial surface proteins that may engage host cell molecules that function as receptors for the microbial ligands. The entry of malarial parasites into erythrocytes is a good example, and the nature of the erythrocyte receptors used by different malarial parasite species governs which red blood cells are infected. Plasmodium vivax binds to the Duffy blood group antigens present on some people's red blood cell membranes. The expression of the Duffy blood group antigen is genetically determined, and this antigen is present mostly in whites and Asians and largely absent in blacks of sub-Saharan African ancestry. 61 , 62 , 63 , 64 This genetic absence of a receptor on red blood cells required for vivax malaria's survival explains why vivax malaria is rare in regions of Africa. Plasmodium vivax also exhibits a characteristic restriction in the age of erythrocytes it infects. Only young red blood cells and reticulocytes are susceptible to infection, even though the Duffy blood group antigen is present on red blood cells of all ages. The basis for this restriction to younger red blood cells also rests with receptor-mediated limitations. Plasmodium vivax parasites contain reticulocyte-binding proteins, which recognize and bind to reticulocyte-specific antigens on the red blood cell surface. 65 , 66 Thus, host cell receptor–microbial ligand interactions have an impact on the geographic range of infections based on host genetic differences in requisite receptor expression and on the specific cells that a microbe may enter. Another example of the intricacies of microbe–receptor interactions has been recognized with HIV-1. Although CD4 is the primary cellular receptor for HIV entry, binding to CD4 alone is not sufficient for entry of HIV-1 into cells. Cellular coreceptors that are members of the chemokine receptor family of seven-transmembrane G protein-coupled molecules are also important. T-cell tropic strains use the CXCR-4 chemokine receptor, and macrophage tropic HIV-1 strains use the CCR-3 and CCR-5 chemokine receptors as coreceptors in concert with CD4. The differences among strains of HIV-1 in their capacities to bind to different chemokine receptor–coreceptors may help explain differences in cell tropism and pathogenicity, the lack of infectability of nonprimate cells, and, for those with genetically altered coreceptors, the apparent resistance to HIV-1 infection of some individuals. 67 , 68 , 69 , 70 Typical of those etiologic agents that have an intracellular localization are viruses. The entry of these agents into cells is increasingly recognized to be dependent on their interactions with specific host cell proteins that act as their "receptors." For instance, host cell molecules that function as viral receptors include multiple isoforms of membrane cofactor protein (CD46), a complement regulatory protein, for measles; the integrin, intracellular adhesion molecule-1 (ICAM-1), for rhinovirus; erythrocyte P antigen for parvovirus B19; and the C3d complement receptor (CR2) for Epstein–Barr virus. 71 , 72 , 73 , 74 Microbes that exist principally within the cytoplasm are sequestered from many immune response mechanisms active on extracellular pathogens, including antibody and phagocytic cells. Viral intracellular proteins will be processed and displayed with class I major histocompatibility complex (MHC) proteins, which enable CD8 cytotoxic T cells to recognize and kill the virally infected cell. Other microbes are internalized within phagocytic cells, especially macrophages. Once internalized in host cells, organisms such as Salmonella , Mycobacterium , Chlamydia , and Legionella use an extraordinary assortment of mechanisms to prevent their phagocytic vacuole from fusing with the host cells' acidifying lysosomes. 75 , 76 , 77 For some parasites, the intracellular environment is an important determinant of parasitism. For example, Leishmania and Coxiella (unlike other pathogens) benefit from the acidic environment of the macrophage phagolysosome. Leishmania use the proton gradient across the lysosome to drive the energy-dependent uptake of two important substrates: glucose and proline. 78 Thus, Leishmania amastigotes actually survive in the macrophage phagolysosome because they benefit from its proton gradient and because they avoid activating the processes that normally kill ingested microorganisms. Leishmanial lipophosphoglycan inhibits the action of β-galactosidase, chelates calcium, inhibits protein kinase C and the oxidative burst, and may scavenge toxic oxygen metabolites. 79 Conversely, other intracellular pathogens such as Toxoplasma gondii survive within the macrophage by using an alternative pathway of entry that avoids fusion of the parasitophorous vacuole with lysosomes. 76 , 80 In contrast, dead or antibody-coated T. gondii enter via the Fc receptor and are routed to a different intracellular compartment, which fuses with the lysosome, and are then killed in the phagolysosome. 76 , 80 , 81 Other organisms, such as Shigella , Listeria , and Rickettsia , breach their vacuolar membrane to multiply freely in the cytoplasm and may also usurp host cellular actin to propel their further spread to neighboring cells, continuing to exploit their intracellular sanctuary. 82 , 83 , 84 Immune responses against microbes within macrophages rely heavily on class II MHC-mediated presentation of host antigenic peptides to T-helper 1 (Th1) types of CD4 + T cells, which then augment the microbicidal activities of the macrophages. Extracellular Localization Some types of microbes that remain extracellular typically reside at epithelial surfaces, including bacteria such as N. gonorrhoeae , H. pylori , Vibrio cholerae , and E. coli , and helminths such as adult Ascaris lumbricoides , hookworms, and Trichuris trichiura . Mucosal immune responses, including IgA and leukocytes, participate in host immune reactions to these pathogens. Other microbes that survive extracellularly are present within the blood, lymph, or tissues of the host, and these organisms include fungi, viruses, bacteria, protozoa, and notably the helminths. Multicellular helminths, due to their large size, remain forever extracellular and may be found in the blood (e.g., microfilariae), lymph (adult lymphatic filarial worms), tissues (migrating larvae and adult stages of some helminths), and cerebrospinal fluid. Host defense against extracellular pathogens uses antibodies, complement, phagocytic cells, and, for helminths, IgE, eosinophils, and mast cells. 85 Intracellular Localization Specific microorganisms use highly developed processes to gain access to and survive within host cells. The microorganisms may reside either in the cytoplasm or within vesicular or vacuolar compartments of targeted cells. Targeting and penetration of cells is governed by the interactions of microbial surface proteins that may engage host cell molecules that function as receptors for the microbial ligands. The entry of malarial parasites into erythrocytes is a good example, and the nature of the erythrocyte receptors used by different malarial parasite species governs which red blood cells are infected. Plasmodium vivax binds to the Duffy blood group antigens present on some people's red blood cell membranes. The expression of the Duffy blood group antigen is genetically determined, and this antigen is present mostly in whites and Asians and largely absent in blacks of sub-Saharan African ancestry. 61 , 62 , 63 , 64 This genetic absence of a receptor on red blood cells required for vivax malaria's survival explains why vivax malaria is rare in regions of Africa. Plasmodium vivax also exhibits a characteristic restriction in the age of erythrocytes it infects. Only young red blood cells and reticulocytes are susceptible to infection, even though the Duffy blood group antigen is present on red blood cells of all ages. The basis for this restriction to younger red blood cells also rests with receptor-mediated limitations. Plasmodium vivax parasites contain reticulocyte-binding proteins, which recognize and bind to reticulocyte-specific antigens on the red blood cell surface. 65 , 66 Thus, host cell receptor–microbial ligand interactions have an impact on the geographic range of infections based on host genetic differences in requisite receptor expression and on the specific cells that a microbe may enter. Another example of the intricacies of microbe–receptor interactions has been recognized with HIV-1. Although CD4 is the primary cellular receptor for HIV entry, binding to CD4 alone is not sufficient for entry of HIV-1 into cells. Cellular coreceptors that are members of the chemokine receptor family of seven-transmembrane G protein-coupled molecules are also important. T-cell tropic strains use the CXCR-4 chemokine receptor, and macrophage tropic HIV-1 strains use the CCR-3 and CCR-5 chemokine receptors as coreceptors in concert with CD4. The differences among strains of HIV-1 in their capacities to bind to different chemokine receptor–coreceptors may help explain differences in cell tropism and pathogenicity, the lack of infectability of nonprimate cells, and, for those with genetically altered coreceptors, the apparent resistance to HIV-1 infection of some individuals. 67 , 68 , 69 , 70 Typical of those etiologic agents that have an intracellular localization are viruses. The entry of these agents into cells is increasingly recognized to be dependent on their interactions with specific host cell proteins that act as their "receptors." For instance, host cell molecules that function as viral receptors include multiple isoforms of membrane cofactor protein (CD46), a complement regulatory protein, for measles; the integrin, intracellular adhesion molecule-1 (ICAM-1), for rhinovirus; erythrocyte P antigen for parvovirus B19; and the C3d complement receptor (CR2) for Epstein–Barr virus. 71 , 72 , 73 , 74 Microbes that exist principally within the cytoplasm are sequestered from many immune response mechanisms active on extracellular pathogens, including antibody and phagocytic cells. Viral intracellular proteins will be processed and displayed with class I major histocompatibility complex (MHC) proteins, which enable CD8 cytotoxic T cells to recognize and kill the virally infected cell. Other microbes are internalized within phagocytic cells, especially macrophages. Once internalized in host cells, organisms such as Salmonella , Mycobacterium , Chlamydia , and Legionella use an extraordinary assortment of mechanisms to prevent their phagocytic vacuole from fusing with the host cells' acidifying lysosomes. 75 , 76 , 77 For some parasites, the intracellular environment is an important determinant of parasitism. For example, Leishmania and Coxiella (unlike other pathogens) benefit from the acidic environment of the macrophage phagolysosome. Leishmania use the proton gradient across the lysosome to drive the energy-dependent uptake of two important substrates: glucose and proline. 78 Thus, Leishmania amastigotes actually survive in the macrophage phagolysosome because they benefit from its proton gradient and because they avoid activating the processes that normally kill ingested microorganisms. Leishmanial lipophosphoglycan inhibits the action of β-galactosidase, chelates calcium, inhibits protein kinase C and the oxidative burst, and may scavenge toxic oxygen metabolites. 79 Conversely, other intracellular pathogens such as Toxoplasma gondii survive within the macrophage by using an alternative pathway of entry that avoids fusion of the parasitophorous vacuole with lysosomes. 76 , 80 In contrast, dead or antibody-coated T. gondii enter via the Fc receptor and are routed to a different intracellular compartment, which fuses with the lysosome, and are then killed in the phagolysosome. 76 , 80 , 81 Other organisms, such as Shigella , Listeria , and Rickettsia , breach their vacuolar membrane to multiply freely in the cytoplasm and may also usurp host cellular actin to propel their further spread to neighboring cells, continuing to exploit their intracellular sanctuary. 82 , 83 , 84 Immune responses against microbes within macrophages rely heavily on class II MHC-mediated presentation of host antigenic peptides to T-helper 1 (Th1) types of CD4 + T cells, which then augment the microbicidal activities of the macrophages. Extracellular Localization Some types of microbes that remain extracellular typically reside at epithelial surfaces, including bacteria such as N. gonorrhoeae , H. pylori , Vibrio cholerae , and E. coli , and helminths such as adult Ascaris lumbricoides , hookworms, and Trichuris trichiura . Mucosal immune responses, including IgA and leukocytes, participate in host immune reactions to these pathogens. Other microbes that survive extracellularly are present within the blood, lymph, or tissues of the host, and these organisms include fungi, viruses, bacteria, protozoa, and notably the helminths. Multicellular helminths, due to their large size, remain forever extracellular and may be found in the blood (e.g., microfilariae), lymph (adult lymphatic filarial worms), tissues (migrating larvae and adult stages of some helminths), and cerebrospinal fluid. Host defense against extracellular pathogens uses antibodies, complement, phagocytic cells, and, for helminths, IgE, eosinophils, and mast cells. 85 Tissue Damage There are multiple mechanisms by which microbes inflict damage on host tissues. Direct Damage or Alteration of Host Cell Function Host cells can be killed directly by the infectious agent, as in some viral or bacterial infections that are highly cytopathic (e.g., yellow fever virus in hepatocytes and Salmonella in macrophages). 86 , 87 Some microorganisms multiply intracellularly until the cell bursts and dies (e.g., Rickettsia prowazekii ). 34 Some bacteria, viruses, and other parasites, such as Shigella , HIV-1, and Listeria , can induce apoptosis of host cells. 59 , 88 , 89 Apoptosis is triggered by different mechanisms, such as activation of the interleukin-converting enzyme pathway. 90 , 91 This form of programmed cell death is probably more widespread as a mechanism of cell death in infectious diseases than previously thought. Damage is sometimes caused by toxins secreted by bacterial cells (exotoxins). In this case, bacteria can either invade host tissues or colonize mucosal sites and then release toxins at the mucosal site that are absorbed systemically and cause distant damage. 92 Exotoxins can act through different pathways that damage the components of the cell membranes such as phospholipids 88 , 93 or affect signaling pathways (e.g., V. cholerae ). 44 , 94 Other exotoxins, such as streptolysins and listeriolysins, alter membrane permeability. Still others, such as exfoliatin (e.g., Staphylococcus aureus ) and elastase (e.g., Pseudomonas spp.), are capable of degrading extracellular elements. 2 Some toxins are translocated to the intracellular environment, where they affect multiple enzymatic systems. These toxins are classified according to their enzymatic activity, such as adenosine diphosphate (ADP) ribosyl transferase (e.g., diphtheria toxin, P. aeruginosa exotoxin A, and pertussis toxin), depurinase (e.g., Shiga toxin), adenylate cyclase (e.g., pertussis hemolysin and anthrax edema factor), and zinc protease (e.g., tetanus). 94 The end result ranges from blockade of protein synthesis and cell death or blockade of exocytosis (especially CNS neurotransmitters at the synaptic cleft) 95 , 96 to increases of cyclic adenosine monophosphate (AMP) or cyclic guanosine monophosphate (GMP) and changes in cell permeability. 94 Still other organisms, such as C. difficile , produce toxins that change basic cell signaling transducers such as Rho to alter cell function or affect their spread. Finally, organisms can interact with host cell or microbial transcriptional regulation of genes (such as iron-binding proteins for uropathogenic E. coli 92 , 97 ) or cytokine release (such as H. pylori or enteroaggregative E. coli 40 , 98 , 99 , 100 ) to enhance their survival or elicit pathogenic responses. The evolutionary advantages to a microbe of its remarkable array of traits that we call "virulence" hold many of the clues to their control, if we can but truly understand them. Endotoxins are a subset of lipopolysaccharides present in the outer membrane of Gram-negative bacteria that can trigger a wide variety of responses in the host, including massive cytokine release leading to hypotension and shock. 101 , 102 These deleterious effects occur with high-grade invasion of the blood by Gram-negative bacteria, including enteric Gram-negative bacteremias and meningococcemia. Indirect Damage Damage to the host may also develop as a consequence of immune reactions to the infectious agents. One scheme for classifying immunopathologic responses divides the reactions into four types based on the elements of the immune response involved. 103 Type I reactions involve elements of strong Th2 responses that lead to increased IgE, eosinophilia, and eosinophil and mast cell activation. Adverse reactions of this type include the development of urticaria (with several helminthic parasites), the occurrence of potentially life-threatening anaphylactic shock in IgE-mediated mast cell degranulation (e.g., triggered by systemic release of antigens from echinococcal cysts 104 ), and exuberant eosinophilic infiltration of tissues due to migrating helminth larvae (e.g., Löffler's pneumonia with the pulmonary migration of Ascaris larvae). Type II reactions are also dependent on elements of Th2 cell responses that lead to increased IgM and then IgG antibodies directed toward the infectious agents. These antibodies, if cross-reactive with host antigens, may lead to complement-mediated cytotoxicity or antibody-dependent cell-mediated cytotoxicity by natural killer cells, which have Fc receptors. An example of this type of immunopathologic response is the uncommon hemolytic anemia associated with Mycoplasma pneumoniae infection that is mediated by complement-induced hemolysis triggered by IgM (cold agglutinin) antibodies against erythrocyte I antigen. Type III reactions are caused by the deposition of immune complexes. When neither antibody nor antigen is present in excess of the other, the complexing of antibodies with soluble antigen results in the formation of immune complexes that may cause disease. This may develop acutely as antibody titers rise in the presence of microbial antigens, causing the syndrome of serum sickness. In addition, when soluble antigen is persistently abundant, sustained formation of immune complexes develops, leading to chronic immune complex-mediated tissue damage (especially glomerulonephritis), as found in subacute bacterial endocarditis, chronic hepatitis B antigenemia, and chronic Plasmodium malariae infections. 105 Type IV reactions include adverse reactions mediated by macrophages and cytotoxic T cells. Examples are damage caused by granulomas in leprosy, tuberculosis, tertiary syphilis, and fungal infections. Likewise, granulomas developing around schistosomal eggs, depending on their location, may cause ureteral obstruction or hepatic presinusoidal lesions. Other deleterious inflammatory reactions in this category are mediated by parasite-elicited host cytokines, such as the hepatic fibrosis elicited by schistosomal eggs. Direct Damage or Alteration of Host Cell Function Host cells can be killed directly by the infectious agent, as in some viral or bacterial infections that are highly cytopathic (e.g., yellow fever virus in hepatocytes and Salmonella in macrophages). 86 , 87 Some microorganisms multiply intracellularly until the cell bursts and dies (e.g., Rickettsia prowazekii ). 34 Some bacteria, viruses, and other parasites, such as Shigella , HIV-1, and Listeria , can induce apoptosis of host cells. 59 , 88 , 89 Apoptosis is triggered by different mechanisms, such as activation of the interleukin-converting enzyme pathway. 90 , 91 This form of programmed cell death is probably more widespread as a mechanism of cell death in infectious diseases than previously thought. Damage is sometimes caused by toxins secreted by bacterial cells (exotoxins). In this case, bacteria can either invade host tissues or colonize mucosal sites and then release toxins at the mucosal site that are absorbed systemically and cause distant damage. 92 Exotoxins can act through different pathways that damage the components of the cell membranes such as phospholipids 88 , 93 or affect signaling pathways (e.g., V. cholerae ). 44 , 94 Other exotoxins, such as streptolysins and listeriolysins, alter membrane permeability. Still others, such as exfoliatin (e.g., Staphylococcus aureus ) and elastase (e.g., Pseudomonas spp.), are capable of degrading extracellular elements. 2 Some toxins are translocated to the intracellular environment, where they affect multiple enzymatic systems. These toxins are classified according to their enzymatic activity, such as adenosine diphosphate (ADP) ribosyl transferase (e.g., diphtheria toxin, P. aeruginosa exotoxin A, and pertussis toxin), depurinase (e.g., Shiga toxin), adenylate cyclase (e.g., pertussis hemolysin and anthrax edema factor), and zinc protease (e.g., tetanus). 94 The end result ranges from blockade of protein synthesis and cell death or blockade of exocytosis (especially CNS neurotransmitters at the synaptic cleft) 95 , 96 to increases of cyclic adenosine monophosphate (AMP) or cyclic guanosine monophosphate (GMP) and changes in cell permeability. 94 Still other organisms, such as C. difficile , produce toxins that change basic cell signaling transducers such as Rho to alter cell function or affect their spread. Finally, organisms can interact with host cell or microbial transcriptional regulation of genes (such as iron-binding proteins for uropathogenic E. coli 92 , 97 ) or cytokine release (such as H. pylori or enteroaggregative E. coli 40 , 98 , 99 , 100 ) to enhance their survival or elicit pathogenic responses. The evolutionary advantages to a microbe of its remarkable array of traits that we call "virulence" hold many of the clues to their control, if we can but truly understand them. Endotoxins are a subset of lipopolysaccharides present in the outer membrane of Gram-negative bacteria that can trigger a wide variety of responses in the host, including massive cytokine release leading to hypotension and shock. 101 , 102 These deleterious effects occur with high-grade invasion of the blood by Gram-negative bacteria, including enteric Gram-negative bacteremias and meningococcemia. Indirect Damage Damage to the host may also develop as a consequence of immune reactions to the infectious agents. One scheme for classifying immunopathologic responses divides the reactions into four types based on the elements of the immune response involved. 103 Type I reactions involve elements of strong Th2 responses that lead to increased IgE, eosinophilia, and eosinophil and mast cell activation. Adverse reactions of this type include the development of urticaria (with several helminthic parasites), the occurrence of potentially life-threatening anaphylactic shock in IgE-mediated mast cell degranulation (e.g., triggered by systemic release of antigens from echinococcal cysts 104 ), and exuberant eosinophilic infiltration of tissues due to migrating helminth larvae (e.g., Löffler's pneumonia with the pulmonary migration of Ascaris larvae). Type II reactions are also dependent on elements of Th2 cell responses that lead to increased IgM and then IgG antibodies directed toward the infectious agents. These antibodies, if cross-reactive with host antigens, may lead to complement-mediated cytotoxicity or antibody-dependent cell-mediated cytotoxicity by natural killer cells, which have Fc receptors. An example of this type of immunopathologic response is the uncommon hemolytic anemia associated with Mycoplasma pneumoniae infection that is mediated by complement-induced hemolysis triggered by IgM (cold agglutinin) antibodies against erythrocyte I antigen. Type III reactions are caused by the deposition of immune complexes. When neither antibody nor antigen is present in excess of the other, the complexing of antibodies with soluble antigen results in the formation of immune complexes that may cause disease. This may develop acutely as antibody titers rise in the presence of microbial antigens, causing the syndrome of serum sickness. In addition, when soluble antigen is persistently abundant, sustained formation of immune complexes develops, leading to chronic immune complex-mediated tissue damage (especially glomerulonephritis), as found in subacute bacterial endocarditis, chronic hepatitis B antigenemia, and chronic Plasmodium malariae infections. 105 Type IV reactions include adverse reactions mediated by macrophages and cytotoxic T cells. Examples are damage caused by granulomas in leprosy, tuberculosis, tertiary syphilis, and fungal infections. Likewise, granulomas developing around schistosomal eggs, depending on their location, may cause ureteral obstruction or hepatic presinusoidal lesions. Other deleterious inflammatory reactions in this category are mediated by parasite-elicited host cytokines, such as the hepatic fibrosis elicited by schistosomal eggs. Immune Interactions Immune Evasion The human immune system has evolved in concert with microbes and is very sophisticated, especially with regard to host defenses against microbes, but the system is not perfect. Interactions of the immune system with microbes are an ongoing affair. Microbes have a high mutation rate compared to human beings. Microbes have evolved a diversity of mechanisms that can enable microorganisms to subvert immediate immunologically mediated elimination ( Table 1.1 ). Persistence within the host is necessary for the propagation of some parasites. Table 1.1 Mechanisms of Immune Evasion Used by Pathogenic Microbes Type of Immune Response Immune Evasion Mechanisms Innate immunity Block natural killer cells Recognition avoidance Complement inactivation or blockage Avoidance of phagocytosis by macrophages and polymorphonuclear neutrophils Manipulation of cell surface Early induced response Modulation of inflammatory response Interference with signaling pathways Interference with RNAi Antimicrobial peptide degradation Modulation of endosomal trafficking Modulation of cytoskeleton Adaptive immunity Induce apoptosis Interfere with receptors and signaling Modulation of antigen presentation and processing Modulation of cell maturation Superantigens Protective immunity Antigenic variation Phase shift Escape mutants There are multiple mechanisms by which microbes can persist in the body and evade the immune system. Tolerance is defined as specific reduction in the response of the immune system to a given antigen. 106 , 107 In the case of transplacental infection, the fetus develops a certain degree of tolerance to antigens to which it is exposed. The immune system of fetuses is rather incompletely developed in utero , and microorganisms survive easily. Cytomegalovirus infects the fetus transplacentally and produces extensive damage to multiple tissues. After delivery, infants continue shedding virions for weeks to months because they are unable to destroy the virus. Other mechanisms include the production of superantigens that stimulate a large population of T cells, which then become deleted if the encounter occurs during early development. Exposure to massive amounts of antigen in the circulation can also lead to tolerance. 2 , 103 Immunosuppression is a well-demonstrated phenomenon that occurs during certain infections caused by viruses, bacteria, protozoa, and helminths. These infections usually involve the lymphoid tissues and macrophages and hamper the immune response. Intracellular pathogens that are able to spread from cell to cell without exposure to the extracellular compartment can avoid exposure to some elements of the immune system. In other cases, pathogens reside in sites relatively inaccessible to the immune system, such as glandular luminal spaces or kidney tubules. In many infections, antibodies are produced but do not effect microbial killing. Sometimes, antibody avidity is low, the epitopes against which the antibody is directed are not critical to the microorganism's survival, or the mechanism of immune elimination is not antibody dependent. 2 Other microorganisms have developed means of counteracting specific elements of immune responses, such as production of an IgA-degrading enzyme, IgAase, by certain strains of N. gonorrhoeae . 108 Some strains of amebae also produce proteases that destroy complement. 2 Reactivation of infections in old age due to waning immunity has been well demonstrated in cases of tuberculosis and varicella zoster virus, allowing transmission to new hosts. One well-studied mechanism of immune evasion is the capability of changing the antigenic structure by genetic mutation or by programmed sequential expression of genes encoding different surface antigens. 109 Antigenic drift and recombination between influenzaviruses affecting humans and animals are well documented. Borrelia recurrentis and Trypanosoma gambiense are also capable of changing their surface antigens after antibodies control the initial bloodstream infection. 110 , 111 The new antigens are not recognized by the antibodies, allowing relapse of the infection. Parasites in which sexual reproduction is possible benefit enormously. 112 Genetic variability introduced by crossing over during meiotic divisions is much greater than the variability introduced by asexual reproduction. As many as four crossovers on a single pair of chromosomes have been demonstrated in P. falciparum . 113 Microparasites also have multiple mechanisms by which they can evade the initial line of defense provided by phagocytes. These strategies include killing of the phagocyte (e.g., Streptococcus pyogenes and Entamoeba histolytica ), inhibition of chemotaxis (e.g., Clostridium perfringens ), decreased internalization of microbes by phagocytic cells (e.g., T. gondii ), inhibition of opsonins (e.g., Treponema pallidum ), inhibition of phagolysosome fusion (e.g., M. leprae and Mycobacterium tuberculosis ), and escape from the phagosome into the cytoplasm (e.g., Rickettsia spp., Trypanosoma cruzi , and Listeria ). 2 , 44 , 75 , 92 With cell-to-cell spread, microorganisms may be minimally exposed to complement, antibodies, or phagocytes in the extracellular or intravascular spaces. 77 , 78 Rickettsial infections spread from cell to cell throughout the infected foci in the endothelial layer of the microvasculature. 82 , 83 , 94 Macroparasites, the helminths, have evolved diverse mechanisms that enable them to survive in vivo . 85 Characteristically, helminths live for months to years in infected hosts within the lumen of the bowel, within tissues, or in the blood or lymphatic vessels. Many helminths are in intimate and recurring contact with all elements of the immune system. As a consequence of their size, helminthic worms do not use intracellular mechanisms to evade immune responses but have evolved a number of capabilities that permit their survival. For instance, interference with antigen processing has been well documented in animal models and patients infected with the filarial nematodes Brugia malayi and Onchocerca volvulus . These helminths produce a family of proteins called the cystatins that are capable of inhibiting proteases responsible for antigen degradation and subsequent presentation through MHC class II pathways in antigen-presenting cells. These proteins are also capable of modulating T-cell proliferation and elicit upregulation of interleukin-10 (IL-10) expression. Other modulators include helminthic derivatives of arachidonic acid such as lipoxin A4, which is capable of blocking production of IL-12 in dendritic cells. Helminthic prostaglandins can also inhibit IL-12 production by dendritic cells. Since helminths have very complex genomes (~21 000 protein-encoding genes in some of them), they are capable of producing a large variety of proteins. Some of them are cytokines and related proteins also capable of modulating the host immune response to their advantage. For example, B. malayi has been shown to express transforming growth factor (TGF)-β-like proteins capable of binding TGF-β human receptors. Other cytokines include macrophage-migration inhibition factors produced by several nematodes including B. malayi . Blockade of effector mechanisms has also been demonstrated in some helminth infections, including proteases that target effector molecules such as eotaxin. Neutrophil proteases can also be inhibited by serpins. Immune Evasion The human immune system has evolved in concert with microbes and is very sophisticated, especially with regard to host defenses against microbes, but the system is not perfect. Interactions of the immune system with microbes are an ongoing affair. Microbes have a high mutation rate compared to human beings. Microbes have evolved a diversity of mechanisms that can enable microorganisms to subvert immediate immunologically mediated elimination ( Table 1.1 ). Persistence within the host is necessary for the propagation of some parasites. Table 1.1 Mechanisms of Immune Evasion Used by Pathogenic Microbes Type of Immune Response Immune Evasion Mechanisms Innate immunity Block natural killer cells Recognition avoidance Complement inactivation or blockage Avoidance of phagocytosis by macrophages and polymorphonuclear neutrophils Manipulation of cell surface Early induced response Modulation of inflammatory response Interference with signaling pathways Interference with RNAi Antimicrobial peptide degradation Modulation of endosomal trafficking Modulation of cytoskeleton Adaptive immunity Induce apoptosis Interfere with receptors and signaling Modulation of antigen presentation and processing Modulation of cell maturation Superantigens Protective immunity Antigenic variation Phase shift Escape mutants There are multiple mechanisms by which microbes can persist in the body and evade the immune system. Tolerance is defined as specific reduction in the response of the immune system to a given antigen. 106 , 107 In the case of transplacental infection, the fetus develops a certain degree of tolerance to antigens to which it is exposed. The immune system of fetuses is rather incompletely developed in utero , and microorganisms survive easily. Cytomegalovirus infects the fetus transplacentally and produces extensive damage to multiple tissues. After delivery, infants continue shedding virions for weeks to months because they are unable to destroy the virus. Other mechanisms include the production of superantigens that stimulate a large population of T cells, which then become deleted if the encounter occurs during early development. Exposure to massive amounts of antigen in the circulation can also lead to tolerance. 2 , 103 Immunosuppression is a well-demonstrated phenomenon that occurs during certain infections caused by viruses, bacteria, protozoa, and helminths. These infections usually involve the lymphoid tissues and macrophages and hamper the immune response. Intracellular pathogens that are able to spread from cell to cell without exposure to the extracellular compartment can avoid exposure to some elements of the immune system. In other cases, pathogens reside in sites relatively inaccessible to the immune system, such as glandular luminal spaces or kidney tubules. In many infections, antibodies are produced but do not effect microbial killing. Sometimes, antibody avidity is low, the epitopes against which the antibody is directed are not critical to the microorganism's survival, or the mechanism of immune elimination is not antibody dependent. 2 Other microorganisms have developed means of counteracting specific elements of immune responses, such as production of an IgA-degrading enzyme, IgAase, by certain strains of N. gonorrhoeae . 108 Some strains of amebae also produce proteases that destroy complement. 2 Reactivation of infections in old age due to waning immunity has been well demonstrated in cases of tuberculosis and varicella zoster virus, allowing transmission to new hosts. One well-studied mechanism of immune evasion is the capability of changing the antigenic structure by genetic mutation or by programmed sequential expression of genes encoding different surface antigens. 109 Antigenic drift and recombination between influenzaviruses affecting humans and animals are well documented. Borrelia recurrentis and Trypanosoma gambiense are also capable of changing their surface antigens after antibodies control the initial bloodstream infection. 110 , 111 The new antigens are not recognized by the antibodies, allowing relapse of the infection. Parasites in which sexual reproduction is possible benefit enormously. 112 Genetic variability introduced by crossing over during meiotic divisions is much greater than the variability introduced by asexual reproduction. As many as four crossovers on a single pair of chromosomes have been demonstrated in P. falciparum . 113 Microparasites also have multiple mechanisms by which they can evade the initial line of defense provided by phagocytes. These strategies include killing of the phagocyte (e.g., Streptococcus pyogenes and Entamoeba histolytica ), inhibition of chemotaxis (e.g., Clostridium perfringens ), decreased internalization of microbes by phagocytic cells (e.g., T. gondii ), inhibition of opsonins (e.g., Treponema pallidum ), inhibition of phagolysosome fusion (e.g., M. leprae and Mycobacterium tuberculosis ), and escape from the phagosome into the cytoplasm (e.g., Rickettsia spp., Trypanosoma cruzi , and Listeria ). 2 , 44 , 75 , 92 With cell-to-cell spread, microorganisms may be minimally exposed to complement, antibodies, or phagocytes in the extracellular or intravascular spaces. 77 , 78 Rickettsial infections spread from cell to cell throughout the infected foci in the endothelial layer of the microvasculature. 82 , 83 , 94 Macroparasites, the helminths, have evolved diverse mechanisms that enable them to survive in vivo . 85 Characteristically, helminths live for months to years in infected hosts within the lumen of the bowel, within tissues, or in the blood or lymphatic vessels. Many helminths are in intimate and recurring contact with all elements of the immune system. As a consequence of their size, helminthic worms do not use intracellular mechanisms to evade immune responses but have evolved a number of capabilities that permit their survival. For instance, interference with antigen processing has been well documented in animal models and patients infected with the filarial nematodes Brugia malayi and Onchocerca volvulus . These helminths produce a family of proteins called the cystatins that are capable of inhibiting proteases responsible for antigen degradation and subsequent presentation through MHC class II pathways in antigen-presenting cells. These proteins are also capable of modulating T-cell proliferation and elicit upregulation of interleukin-10 (IL-10) expression. Other modulators include helminthic derivatives of arachidonic acid such as lipoxin A4, which is capable of blocking production of IL-12 in dendritic cells. Helminthic prostaglandins can also inhibit IL-12 production by dendritic cells. Since helminths have very complex genomes (~21 000 protein-encoding genes in some of them), they are capable of producing a large variety of proteins. Some of them are cytokines and related proteins also capable of modulating the host immune response to their advantage. For example, B. malayi has been shown to express transforming growth factor (TGF)-β-like proteins capable of binding TGF-β human receptors. Other cytokines include macrophage-migration inhibition factors produced by several nematodes including B. malayi . Blockade of effector mechanisms has also been demonstrated in some helminth infections, including proteases that target effector molecules such as eotaxin. Neutrophil proteases can also be inhibited by serpins. Emerging Infectious Diseases The concept of emerging infectious diseases is not new but has been the focus of attention due to the resurgence of old infectious diseases that were thought to be controlled and the recognition of new pathogens as humans increase their interaction with the biosphere. By definition, an emerging infectious disease is one that has newly appeared in the population or has existed but is rapidly increasing in incidence or geographic range. 114 Others define emerging infections as "new emerging or drug resistant infections whose incidence in humans has increased within the last two decades, or whose incidence threatens to increase in the near future." Emerging infections are classified by some as newly emerging, re-emerging/resurging, and deliberately emerging. Since 1967, after a widely publicized statement from the US Surgeon General declaring victory in the war against infectious diseases, more than 85 new pathogens have been described. These include viruses, bacteria, protozoans, and helminths. These pathogens can cause a wide diversity of syndromes including acute respiratory infections (influenza A H1N5, H1N1, SARS-CoV, Sin Nombre virus, human metapneumovirus), systemic diseases caused by viral hemorrhagic fever viruses (Lassa, Ebola, dengue), encephalitic syndromes (Nipah virus, West Nile virus), arthropod-borne agents ( Borrelia burgdorferi , Rickettsia spp., Ehrlichia spp., Anaplasma ), enteric pathogens ( Cryptosporidium , microsporidia), chronic viral diseases (HIV-1 and 2, human T-cell lymphotropic virus types 1–3, human herpesviruses 6–8), hepatotropic viruses (hepatitis C and E), and other infectious agents. The factors involved in the emergence or reemergence of infectious diseases are complex and include ecological changes (deforestation, reforestation, flooding, and climatic changes), changes in human demographics and behavior (sexual, cultural, and war), increased international travel, technological advances (organ transplantation and antibiotics), microbial evolution with the appearance of antibiotic-resistant or antigenically distinct strains, and deficiencies in surveillance and public health policy. 113 , 115 , 116 , 117 The classic triad of microbe, host, and environment is again exemplified. Tropical Infectious Diseases Globally, as assessed in terms of disability-adjusted life years (DALYs), which measure morbidity and mortality, 118 infectious diseases in 1990 accounted for 36.4% of total DALYs. Infectious disease DALYs were considerably in excess of those attributable to cancer (5.9%), heart disease (3.1%), cerebrovascular disease (3.2%), or chronic lung disease (3.5%). 119 However, these calculations admittedly miss the disproportionate impact of tropical infectious diseases on the still rapidly increasing populations living in impoverished, tropical areas, and they grossly underestimate the major developmental impact of common childhood enteric, helminthic, and other infections. 38 , 120 , 121 , 122 For those caring for individual patients with infectious diseases, appropriate diagnosis and treatment are important considerations for the individual. Even more important is the consideration of approaches that will lead to diminished acquisition of infectious diseases. For some infectious agents, immunization holds promise, as witnessed by the successful global eradication of smallpox and the potential eradication of poliomyelitis. Greater progress in the control of infectious diseases, however, rests with improvements related to socioeconomic conditions of the population at risk. In developed countries, tuberculosis was diminished well before the introduction of the first antimicrobial agents active against M. tuberculosis , and the reduction was attributable to improved socioeconomic conditions. For the major infectious diseases of the tropics, improvements in sanitation, living conditions, and general public health will be critical in helping control the impact of the diverse infectious agents that currently contribute to human morbidity and mortality. The impact of these infections is related not only to their effect on the health of the infected individual but also to their contribution to the morbidity associated with malnutrition and to their larger societal impact as an impediment to the full development of the political, economic, and social potential of entire populations.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113555/

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866897/

Biomedical Applications of Nisin

Nisin is a bacteriocin produced by a group of Gram-positive bacteria that belongs to Lactococcus and Streptococcus species. Nisin is classified as a Type A (I) lantibiotic that is synthesized from mRNA and the translated peptide contains several unusual amino acids due to post-translational modifications. Over the past few decades, nisin has been used widely as a food biopreservative. Since then, many natural and genetically modified variants of nisin have been identified and studied for their unique antimicrobial properties. Nisin is an FDA approved and GRAS (generally regarded as safe) peptide with recognized potential for clinical use. Over the past two decades the application of nisin has been extended to biomedical fields. Studies have reported that nisin can prevent the growth of drug-resistant bacterial strains, such as methicillin resistant Staphylococcus aureus , Streptococcus pneumoniae , Enterococci and Clostridium difficile . Nisin has now been shown to have antimicrobial activity against both Gram-positive and Gram-negative disease-associated pathogens. Nisin has been reported to have anti-biofilm properties and can work synergistically in combination with conventional therapeutic drugs. In addition, like host defense peptides, nisin may activate the adaptive immune response and have an immunomodulatory role. Increasing evidence indicates that nisin can influence the growth of tumors and exhibit selective cytotoxicity towards cancer cells. Collectively, the application of nisin has advanced beyond its role as a food biopreservative. Thus, this review will describe and compare studies on nisin and provide insight into its future biomedical applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161017/

An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

Background Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism. Results To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells. Conclusion The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action. Background Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism. Results To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells. Conclusion The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action. Background Normal somatic cells undergo a finite number of divisions before entering a state of irreversible growth arrest termed cellular senescence [ 1 ]. This is triggered in response to a variety of intrinsic and extrinsic stimuli including progressive telomere shortening or changes in telomeric structure at the ends of chromosomes or other forms of genotoxic stress such as oncogene activation, or DNA damage or oxidative stress, resulting in a DNA damage response and growth arrest via activation of the p53 tumour suppressor pathway [ 2 , 3 ]. Non-genotoxic stress induces senescence by a telomere independent mechanism involving activation of the p16-pRB pathway by up-regulation of p16 INK4a [ 3 , 4 ]. Cellular senescence acts as an important tumour suppressor mechanism. Overcoming senescence and acquiring a limitless replicative potential has been proposed to be one of the key events required for malignant transformation [ 5 ]. Senescence is thought to have evolved as an example of antagonistic pleiotrophy, whereby its beneficial traits in a reproductively active individual have deleterious effects later in life [ 6 , 7 ]. The underlying mechanisms that control cellular senescence, the signal transduction pathways involved and how the diverse signals that result in senescence are all integrated, remain poorly defined. Moreover the downstream effectors of the p53-p21 and p16-pRB pathways that result in the irreversible growth arrest and entry into senescence are unknown. The discovery of RNA interference as a mechanism to silence gene expression has revolutionized studies on mammalian gene expression and has permitted loss-of-function genome-wide genetic screens, to identify genes involved in a variety of cellular processes, to be performed [ 8 - 12 ]. A number of shRNA libraries have been developed for carrying out such genome-wide screens, one of which is the pSM2 Retroviral shRNAmir library [ 13 ] (Thermo Scientific Open Biosystems). This library has several unique features that make it very versatile and efficient for large-scale screens particularly the human microRNA-30 (miR30) adapted design which increases knockdown specificity and efficiency [ 14 ]. Here we present a RNA interference screen using the human pSM2 retroviral shRNAmir library, carried out in the conditionally immortal HMF3A human fibroblasts, to identify genes whose silencing bypasses senescence arrest induced by activation of the p53-p21 and p16-pRB pathways. The primary screen identified 112 known genes and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of the known targets with genes known to be up-regulated upon senescence by micro-array expression profiling, identified 4 common genes whose expression was reversed when senescence was bypassed upon inactivation of the p53-p21 and p16-pRB pathways. Results To directly identify the downstream effectors of the p53-p21 and p16-pRB pathways, we have utilized the conditionally immortal HMF3A human fibroblasts that were derived by immortalising adult human mammary fibroblasts with the catalytic subunit of human telomerase and a thermolabile U19tsA58 mutant of SV40 Large T antigen [ 15 ]. These cells are immortal if grown at 34°C but undergo a senescence arrest upon inactivation of the thermolabile U19tsA58 T antigen resulting in the activation of the p53-p21 and p16-pRB pathways [ 15 ]. They are stringently temperature sensitive but senescence can be readily bypassed by inactivation of the p53-p21 or the p16-pRB pathway [ 16 ]. To facilitate efficient transduction of these cells by retroviral infection, they were transduced with the full length murine ecotropic retroviral receptor and CL3 EcoR cells derived, that most closely mirror the parental cells [ 16 ]. The temperature dependent senescent arrest of CL3 EcoR cells and its bypass upon inactivation of p21 CIP1 by silencing or sequestration of the RB family of proteins by HPV16 E7 are shown in Figure 1 . Figure 1 Characteristics of CL3 EcoR cells . a : CL3 EcoR cells are immortal at 34°C but undergo a senescence arrest upon shift up 38°C. b : Senescence is bypassed upon silencing of p21 CIP1 using pRSp21F or sequestration of RB family of proteins by HPV16 E7. The pSM2 library version 1.3 comprising 15,148 constructs targetting 9,392 human cancer associated genes was amplified and each 96 well plate used to prepare a pool of plasmid DNA; each of the 100 pools contained between 150 to 200 different shRNAmir constructs with each gene being represented by 1 to 3 shRNAmirs. To ensure that CL3 EcoR cells were sufficiently sensitive to identify a single shRNAmir construct in a pool of 200 shRNAmir constructs, pRSp21F (a p21 CIP1 shRNA construct) [ 17 ] was mixed in a ratio of 1:200 with pRSLaminA/C and used to assay bypass of senescence in CL3 EcoR cells. The pRSp21F construct was used because the pSM2 library version 1.3 did not contain any silencing constructs for p21 CIP1 . Silencing of LaminA/C did not bypass senescence, very few growing colonies were obtained (Figure 2a ) whereas silencing of p21 CIP1 was very efficient and produced essentially a confluent monolayer of growing cells (Figure 2b ). The 1:200 p21 CIP1 /LaminA/C mix produced numerous distinct densely growing colonies (Figure 2c ) indicating that CL3 EcoR cells and the procedure were sufficiently sensitive to generate colonies in which senescence had been overcome. Figure 2 Sensitivity of the screen . CL3 EcoR cells were infected with retroviruses prepared from pRSLamin A/C (a) , pRSp21F (b) , or a 1/200 mix of pRSp21F/pRSLamin A/C (c) . After puromycin selection, cells were reseeded at 8.5 × 10 4 per 15 cm plate or 0.5 × 10 4 per well in 6-well plates and shifted to 38°C for 3 weeks. ShRNA interference screen The formula: ln [1-0.95]/ln [1-1/(Library Size)] recommended for genetic screens by the Nolan lab ( http://www.stanford.edu/group/nolan/screens/screens.html ), suggested that approximately 1000 independent infectious events would be sufficient for a 99% confidence that all shRNAs within a pool had been sampled. To ensure that the screen would be saturating, virus sufficient to yield 10,000 infectious events was utilised for each pool (shown in Additional File 1 ). The screen was performed in successive waves of 10 pools. To minimise variation and background reversion, CL3 EcoR cells were used at the same passage for every pool. Virus prepared from pRSp21F and pRSLamin A/C was used as positive and negative controls respectively to evaluate the level of background and ensure that the complementation assay worked for each round of the screen. Stably transduced cells were trypsinised and reseeded. Three weeks after reseeding, flasks were examined to identify growing colonies; a representative colony is shown in Figure 3 . Each colony was examined microscopically to ensure it comprised growing cells and the number of colonies obtained for each pool determined. The number of stably transduced cells, the number of flasks reseeded and the number of colonies obtained for each flask at 38°C are shown in Additional File 2 . The flasks which contained more densely growing/bigger colonies (indicated in red in Additional File 2 ) were trypsinised, replated and used for extracting genomic DNA when confluent. Figure 3 ShRNA screen . CL3 EcoR cells were transduced with ecotropic retroviruses prepared from each of the 100 pools of shRNAmirs, selected with puromycin, reseeded and shifted up to 38°C for 3 weeks. Densely growing colonies were considered to have bypassed senescence and potentially contain candidate shRNAmirs. 34 out of 100 pools yielded healthy growing colonies; pools 13, 78 and 82 particularly gave a higher number of colonies which were also larger. Pools 16, 18, 19, 21 and 80 also yielded colonies but they were smaller. For each pool that contained growing colonies, 1 to 4 flasks containing the highest number of growing colonies, were reseeded for extracting genomic DNA and resulted in a total of 81 sub-pools. Identification of shRNAmirs The shRNAmir proviral inserts were by amplified by two rounds of nested PCR using pSM2 specific primers, the amplified products TOPO cloned and plasmid DNA extracted from at least six colonies sequenced to identify all shRNAs present within each pool; for some pools, DNA from many more colonies was sequenced. The hair pin sequence was determined by searching for the miR-30 context and the miR-30 loop that are common to all inserts and frame the hair pin. The sequence of the hair pin was used to identify the target gene by searching the pSM2 data base or by BLASTN analysis of the NCBI human genome data base ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Sequences that could not be linked back to the list of hair pin sequences within the Open Biosystems collection or were not 100% homologous to a gene within Genbank were not pursued and are presented in Additional File 3 . The rescued shRNAmir hair pins identified 112 known genes and another 29 shRNAmirs targetting as yet unidentified loci. For each pool, the number of times that sequence was obtained, the corresponding insert reference, gene name and the number of shRNAmir constructs for that gene within the SM2 library are shown in Table 1 . The last column of the table indicates if the recovered insert was a match to a hair pin present in that particular pool ("match") or if it was present within another pool ("listed in pool X"). 14 hair pins were isolated from incorrect pools. Some inserts were detected several times in multiple pools. For example, the hair pin v2HS_119967 (corresponding to LOC155004) listed within pool 52 was also identified from pool 3, 4, 5, 9, 12, 30, 59, 60, 64, 71, 72, 74, 77, and 98 suggesting that there may have been some cross-contamination of the library perhaps during replica plating. However, this was not a problem since the aim of our screen was to identify shRNAmirs that are able to bypass senescence and does not depend upon the identity of the pool. Since this insert was isolated from so many pools, it could be that it is a strong positive or that it was highly represented within the library and likely to be a false positive. Table 1 Primary screen Pool Insert Gene name Gene symbol Freq Cons per Gene Library location 3 v2HS_63142 keratin associated protein 5-9 KRTAP5-9 3/9 1 match v2HS_119967 LOC155004 LOC155004 4/9 2 pool 52 v2HS_56766 acyl-CoA synthetase medium-chain family member 3 ACSM3 1/9 2 match v2HS_59294 glycoprotein hormone alpha 2 GPHA2 1/9 1 match v2HS_53974 PRO0255 protein PRO0255 5/8 1 match v2HS_63482 paired box gene 1 PAX1 2/8 1 match 4 v2HS_119967 LOC155004 LOC155004 2/10 2 pool 52 v2HS_66751 LOC344082 LOC344082 4/10 1 match v2HS_70011 serum amyloid A-like 1 SAAL1 2/10 2 match v2HS_98079 human solute carrier family 22 SLC22A3 1/10 2 pool 79 5 v2HS_108647 LOC349868 LOC349868 3/8 1 pool 79 v2HS_70473 polymerase (DNA directed), mu POLM 2/8 v2HS_71958 human olfactory receptor, family 5, subfamily P, member 3 OR5P3 3/8 1 match v2HS_119967 LOC155004 LOC155004 7/12 2 pool 52 v2HS_66652 protein phosphatase 3, catalytic subunit, β isoform PPP3CB 1/12 1 match v2HS_70473 polymerase (DNA directed), mu POLM 4/12 7 v2HS_112910 human cyclin-dependent kinase 8 CDK8 1/10 3 match v2HS_98079 solute carrier family 22, member3 SLC22A3 1/10 2 pool 79 v2HS_97017 sterile alpha motif containing 4a SAMD4A 1/10 v2HS_69776 cytochrome P450, family 4, subfamily Z, polypeptide CYP4Z2P 1/10 2 match 2 pseudogene 9 v2HS_108647 LOC349868 LOC349868 1/8 1 pool 79 v2HS_119967 LOC155004 LOC155004 1/8 2 pool 52 v2HS_62506 family with sequence similarity 181, member B FAM181B 1/8 1 match v2HS_55950 prostate stem cell antigen PSCA 2/13 1 match v2HS_119967 LOC155004 LOC155004 3/13 2 pool 52 v2HS_70312 TRK-fused gene TFG 4/13 3 match v2HS_71740 ataxin 10 ATXN10 1/13 1 match v2HS_98079 solute carrier family 22, member 3 SLC22A3 1/13 2 pool 79 v2HS_65121 sorting nexin 12 SNX12 1/13 1 match v2HS_64384 amyloid beta precursor protein binding family A member 2 APBA2 1/6 1 match v2HS_97017 sterile alpha motif containing 4A SAMD4A 1/6 12 v2HS_119967 LOC155004 LOC155004 5/11 2 pool 52 v2HS_58950 signal-regulatory protein beta 2 SIRPB2 3/11 2 match protein tyrosine phosphatase, non-receptor type 13 PTPN13 1/10 v2HS_119967 LOC155004 LOC155004 5/10 2 pool 52 13 v2HS_55731 phenylalanine-tRNA synthetase-like, beta subunit FARSLB 1/12 2 match v2HS_59258 dynein, light chain, roadblock-type 1 DYNLRB1 3/12 1 match v2HS_59891 TAO kinase 1 TAOK1 5/12 3 match v2HS_162164 iodotyrosine deiodinase IYD 1/12 cell cycle exit and neuronal differentiation 1 CEND1 1/12 transmembrane protein 168 TMEM168 2/5 v2HS_68714 abl-interactor 1 ABL1 3/5 3 match cell cycle exit and neuronal differentiation 1 CEND1 4/6 v2HS_58952 signal regulatory protein gamma SIRPG 1/6 2 pool 14 v2HS_59653 LOC342404 LOC342404 1/6 1 match v2HS_55731 phenylalanyl-tRNA synthetase like, beta subunit FARSLB 1/15 2 match v2HS_64320 Smith-Magenis syndrome chromosome region, candidate 7 SMCR7 1/15 1 match v2HS_71174 peroxisome proliferator-activated receptor, gamma PPARGC1A 3/15 1 match v2HS_71453 similar to IAP-associated factor VIAF1; phosducin-like 2/15 1 match 16 v2HS_68478 CD28 antigen CD28 8/12 2 pool 13 v2HS_63107 dopamine beta hydoxylase DBH 2/12 1 match v2HS_61750 LOC100129230 LOC100129230 7/13 3 match LOC9142 LOC9142 2/13 V2HS_62831 membrane bound O-acyltransferase domain containing 1 MBOAT1 3/13 2 match 18 v2HS_63989 STAR-related lipid transfer (START) domain containing 6 STARD6 1/14 2 match LOC730256 LOC730256 1/14 19 v2HS_58958 TMEM9 domain family, member B/C11orf15 TMEM9B 3/10 1 match v2HS_57051 RNA binding motif, single stranded interacting protein 1 RBMS1 5/10 1 match v2HS_106158 LOC345672 LOC345672 1/10 2 pool 82 v2HS_59560 chromosome 13 open reading frame 15 C13orf15 10/10 1 match 21 v2HS_55312 glucosamine-phosphate N-acetyltransferase 1 GNPNAT1 6/9 2 match v2HS_68437 LOC157503 LOC157503 2/9 3 match 30 v2HS_119967 LOC155004 LOC155004 6/14 2 pool 52 v2HS_34338 protein phosphatase 4, regulatory subunit 2 PPP4R2 7/14 3 match v2HS_119967 LOC155004 LOC155004 1/13 2 pool 52 v2HS_36467 solute carrier family 33 (acetyl-CoA transporter), member 1 SLC33A1 8/13 3 match v2HS_46793 ubiquitin-like modifier activating enzyme 3 UBA3 1/13 1 match 32 v2HS_42104 citrate lyase beta like (CLYBL), transcript variant 1 CLYBL 5/7 1 match LOC100129563 LOC 10012956 1/7 41 v2HS_48278 choline kinase-like CHKL 6/6 1 match 54 v2HS_112629 basic transcription factor 3, like 1 BTF3L1 1/5 1 match v2HS_121153 LOC221399 LOC221399 2/5 1 match v2HS_125538 LOC350103 LOC350103 1/5 1 match v2HS_129527 LOC351851 LOC351851 6/6 1 match v2HS_112629 basic transcription factor 3, like 1 BTF3L1 2/6 1 match 55 v2HS_117465 SH3 domain binding glutamic acid-rich protein like 2 SH3BGRL2 2/6 1 match v2HS_119051 LOC90841 LOC90841 1/6 1 match v2HS_116174 YTH domain containing 2 YTHDC2 1/7 2 pool 58 v2HS_119120 hypothetical protein FLJ20032 FLJ20032 6/7 2 match 56 V2HS_120429 six-twelve leukemia (STL), non-coding RNA STL 5/6 1 match v2HS_115231 rab23, member RAS oncogene family RAB23 4/6 4 match v2HS_117914 transketolase-like 2 TKTL2 2/6 1 match v2HS_117239 chromosome 9 open reading frame 58 C9orf58 3/6 1 match v2HS_120429 six-twelve leukemia (STL), non-coding RNA STL 1/6 1 match v2HS_119206 lon peptidase 2, peroxisomal LONP2 2/6 1 pool 146 58 v2HS_116174 YTH domain containing 2 YTHDC2 1/4 2 match v2HS_118722 layilin LAYN 2/4 2 match v2HS_112838 ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 3/7 4 match v2HS_112982 chromodomain helicase DNA binding protein 3 CHD3 1/7 3 match v2HS_116174 YTH domain containing 2 YTHDC2 1/7 2 match v2HS_112838 ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 1/12 4 match v2HS_118254 WD repeat and FYVE domain containing 2 WDFY2 1/12 1 match v2HS_122548 similar to ribosomal protein S3A RPS3A 4/12 1 pool 53 v2HS_120982 coiled coil domain containing 129 CCDC129 5/12 2 match 59 v2HS_111554 interleukin 2 IL2 8/10 2 match v2HS_115544 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 DDX47 2/10 4 match v2HS_116377 transmemembrane protein 135 TMEM135 1/9 2 match v2HS_117064 CD1E molecule CD1E 3/9 1 match v2HS_119967 LOC155004 LOC155004 4/9 2 pool 52 v2HS_120757 olfactory receptor, family 8, subfamily K, member 1 OR8K1 1/9 2 match v2HS_108647 LOC349868 LOC349868 1/8 1 pool 79 v2HS_115544 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 DDX47 3/8 4 match v2HS_116833 intermediate filament protein syncoilin SYNC 1/8 2 match 60 v2HS_121585 dual specificity phosphatase 3 DUSP3 10/11 1 match v2HS_117903 glutamine rich 2 QRICH2 4/11 1 match v2HS_119967 LOC155004 LOC155004 1/11 2 pool 52 v2HS_121585 dual specificity phosphatase 3 DUSP3 2/11 1 match v2HS_128131 LOC351061 LOC351061 2/11 1 match v2HS_98079 solute carrier family 22 member 3 SLC22A3 1/11 2 pool 79 v2HS_119967 LOC155004 LOC155004 8/10 2 pool 52 v2HS_121585 dual specificity phosphatase 3 DUSP3 2/10 1 match 64 v2HS_119967 LOC155004 LOC155004 10/11 2 pool 52 v2HS_121013 protease serine 54 PRSS54 3/12 1 match 66 v2HS_117675 latent transforming growth factor β binding protein 2/3 LTBP2/3 15/15 v2HS_118530 RNA exonuclease 1 homolog like 1 REXO1L1 5/13 3 match v2HS_117675 latent transforming growth factor β binding protein 2/3 LTBP2/3 8/13 v2HS_118530 REX1, RNA exonuclease 1 homolog-like 2 REXO1L1 1/11 3 match 71 v2HS_103818 LOC284804 LOC284804 2/16 1 pool 78 v2HS_119967 LOC155004 LOC155004 9/16 2 pool 52 v2HS_97981 LOC51152 LOC51152 5/16 2 pool 79 72 v2HS_119967 LOC155004 LOC155004 9/10 2 pool 52 74 v2HS_119967 LOC155004 LOC155004 11/12 2 pool 52 77 v2HS_119967 LOC155004 LOC155004 12/12 2 pool 52 78 v2HS_94763 paired related homeobox 1 PRRX1 1/8 1 match v2HS_99138 solute carrier family 25 SLC25A21 7/8 2 match v2HS_102207 transmembrane protein 63B TMEM63B 1/12 1 match v2HS_103818 LOC284804 LOC284804 4/12 1 match V2HS_95356 armadillo repeat containing, X-linked 2 ARMCX2 2/12 2 match v2HS_98650 mitochondrial ribosomal protein 63 MRP63 1/12 1 match v2HS_102155 similar to hypothetical protein KIAA0286 (HA6800) KIAA0286 1/8 1 match v2HS_108506 LOC349839 LOC349839 1/8 2 match v2HS_96236 zinc finger and BTB domain containing 1 ZBTB1 2/8 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 3/10 v2HS_96236 zinc finger and BTB domain containing 1 ZBTB1 1/10 1 match 79 v2HS_105974 LOC345597 LOC345597 3/11 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 3/11 v2HS_98079 solute carrier family 22, member 3 SLC22A3 4/11 2 match v2HS_108647 LOC349868 LOC349868 2/7 1 match v2HS_98079 solute carrier family 22, member 3 SLC22A3 5/7 2 match v2HS_101943 anthrax toxin receptor-like ANTXRL 1/11 3 match v2HS_105093 human solute carrier family 35, member F4 SLC35F4 2/11 1 match v2HS_106472 LOC345713 LOC345713 1/11 1 match v2HS_107395 LOC346589 LOC346589 1/11 1 match v2HS_91777 nuclear receptor co-repressor N-Cor 1 NCOR1 4/11 2 match Non-protein coding RNA 282 NCRNA00282 1/11 80 v2HS_102441 adenylate cyclase 1 ADCY1 5/11 1 match v2HS_130882 glutamate receptor, metabotropic 3 GRM3 1/11 3 pool 91 v2HS_97368 yippee-like 5 YPEL5 2/11 1 match v2HS_102441 adenylate cyclase 1 ADCY1 5/10 1 match 82 v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/11 v2HS_101845 human prickle-like 2 (Drosophila) PRICKLE2 3/11 1 match v2HS_109096 LOC349975 LOC349975 1/11 1 pool 84 v2HS_99423 coiled coil domain containing 70 CCDC70 1/11 1 match v2HS_109596 1/14 v2HS_108399 LOC349811 LOC349811 3/14 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/14 v2HS_93536 proteolipid protein 1 PLP1 5/14 1 match v2HS_97017 sterile alpha motif domain containing 4A SAMD4A 1/14 v2HS_106158 LOC345672 LOC345672 3/14 2 match v2HS_93615 p53 TP53 3/14 1 match v2HS_95112 RAS p21 protein activator 4 RASA4 1/14 1 match v2HS_99525 BCL2 like 12 BCL2L12 1/14 2 match v2HS_97017 sterile alpha motif domain containing 4A SAMD4A 4/14 v2HS_94640 aryl hydrocarbon receptor nuclear translocator-like ARNTL 3/10 2 match v2HS_100174 desmoglein 4 DSG4 2/10 2 pool 145 v2HS_96026 adnp homeobox 2 ADNP2 3/10 1 match v2HS_100819 Rho GTPase activating protein 20 ARHGAP20 2/10 1 match 84 v2HS_106409 LOC345700 LOC345700 1/11 1 match v2HS_95019 zinc finger protein 16 ZNF16 9/11 1 match v2HS_97152 ubiquitin-conjugating enzyme E2, J1 UBE2J1 1/11 1 match v2HS_95019 zinc finger protein 16 ZNF16 10/11 1 match 94 v2HS_141495 zinc finger protein 454 ZNF454 7/12 2 pool 96 95 v2HS_130457 radial spoke head 10 homolog B (Chlamydomonas) RSPH10B 6/7 3 match v2HS_131154 potassium inwardly-rectifying channel, subfamily J KCNJ2 1/7 3 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/3 96 chromosome 9 open reading frame 123 C9orf123 5/13 98 v2HS_141367 cDNA FLJ37626/LOC285500 FLJ37626 10/10 2 match v2HS_119967 LOC155004 LOC155004 2/11 2 pool 52 v2HS_133299 insulin-like growth factor binding protein 6 IGFBP6 3/11 2 match v2HS_135564 chromosome X open reading frame 57 CXORF57 5/11 1 match For each candidate the DNA pool from which it was isolated, the insert reference, target gene, frequency of isolation, as well as the number of shRNAmir constructs for that gene within the SM2 library are indicated. The last column shows if the recovered insert was a match to a hair pin in that particular pool ("match") or if it was from another pool ("pool X"). Pool 13, 78 and 82 that produced more colonies and colonies that were larger and healthier than others, identified the following genes: Pool 13: DYNLRB1, FARSLB, PPARGC1A, TAOK1, CEND1, ABL1, LOC342404, TMEM168, SIRPG, IYD and SMCR7; Pool 78: PRRX1, SLC25A21, ARMCX2, LOC284804, LOC349839, MRP63, TMEM63B, ZBTB1, KIA0286 and EIF4A1; and Pool 82: EIF4A1, CCDC70, PRICKLE2, PLP1, SAMD4A, RASA4, TP53, ARNTL, BCL2L12, ADNP2, DSG4, LOC345672, LOC349975, LOC349811 and ARHGAP20. Moreover, pool 82, one of the pools that gave the best senescence bypass, contained the only shRNAmir targetting p53, v2HS_93615 identified in the screen, thereby internally validating it. Overlap of the primary candidates of the shRNA screen with microarray data for genes up-regulated upon senescence in CL3 EcoR cells To prioritise the candidates identified from the primary screen for functional validation, they were compared to genes up-regulated upon senescence and whose expression was reversed when senescence was abrogated upon inactivation of the p53 and pRB pathways [[ 16 ]; microarray expression profiling data is available from Gene Expression Omnibus database accession number GSE24810]. This identified 4 common genes, ATXN10, LAYN, LTBP2/3 and TMEM9B. The microarray expression profiling data presented in Table 2 showed that they were all up-regulated upon senescence growth arrest: ATXN10 by 1.3 fold (p-value 1 × 10 -3 ), LAYN by 2 fold (p-value 2 × 10 -4 ), LTBP2/3 by 2.5 (p-value 9 × 10 -8 ), 1.7 (p-value 5 × 10 -9 ), 1.5 (p-value 8 × 10 -4 ) and 1.4 fold (p-value 5 × 10 -5 ) and TMEM9B by 1.4 (p-value 1 × 10 -7 ) and 1.3 fold (p-value 2 × 10 -4 ) respectively. The data in Table 2 further show that up-regulation of these candidates was reversed when senescence was bypassed upon inactivation of the p53-p21 and p16-pRB pathways by silencing p53 (pRS_p53) or p21 CIP1 (pRS_p21) or by sequestration of the RB family of proteins by HPV16 E7 or by expression of the dominant negative E2F-DB protein. Table 2 Microarray expression profiling data for common genes Probe Symbol GA p-value HS p-value pRS_p53 p-value pRS_p21 p-value E7 p-value E2F_DB p-value 208832_at ATXN10 0.36 1E-03 0.25 3E-02 -0.18 2E-01 -0.35 2E-03 0.02 9E-01 -0.23 4E-02 228080_at LAYN 1.04 2E-04 0.30 3E-01 -0.95 1E-03 -0.70 2E-02 -0.56 6E-02 -0.11 8E-01 219922_s_at LTBP2/3 1.32 9E-08 0.66 5E-03 -1.04 2E-05 -1.32 3E-07 -1.19 1E-06 -1.41 4E-08 223690_at LTBP2/3 0.75 5E-09 -0.14 3E-01 -0.52 1E-05 -0.91 2E-10 -1.27 1E-14 -1.22 3E-14 204682_at LTBP2/3 0.57 8E-04 -0.48 8E-03 -0.39 4E-02 -0.94 1E-06 -1.62 3E-12 -2.02 4E-15 227308_x_at LTBP2/3 0.46 5E-05 0.03 9E-01 -0.43 3E-04 -0.57 4E-06 -0.28 2E-02 -0.55 4E-06 218065_s_at TMEM9B 0.44 1E-07 -0.11 2E-01 -0.33 6E-05 -0.38 9E-06 -0.20 1E-02 -0.18 2E-02 222507_s_at TMEM9B 0.39 2E-04 -0.19 1E-01 -0.28 1E-02 -0.34 2E-03 -0.12 3E-01 -0.07 6E-01 Log 2 fold changes in gene expression in CL3 EcoR cells upon senescence arrest are indicated as GA and upon shift up of control temperature insensitive HMF3S cells, from 34°C to 38°C, as HS respectively. Also indicated are the data obtained for these genes from CL3 EcoR cells in which senescence had been bypassed by silencing of p53 (pRS_p53) or p21 CIP1 (pRS_p21) or by inactivation of pRB using HPV16 E7 (E7) or by expression of the dominant negative E2F-DB protein. The identification of TMEM9B was particularly remarkable because the microarray analysis has suggested that senescence growth arrest in CL3 EcoR cells is associated with activation of the NF-κB signalling pathway and TMEM9B has previously been shown to be able to activate NF-κB dependent reporter constructs [ 18 , 19 ]. To determine if silencing of TMEM9B would bypass senescence, 4 GIPZ lentiviral silencing constructs (v2LHS_247318, 58957, 58958 and 58959; Thermo Scientific Open Biosystems) targetting TMEM9B were obtained, pooled and introduced into CL3 EcoR cells after packaging as lentiviruses. The GIPZ lentiviral library contains the same hair pins as the retroviral library but is more stable and the constructs are packaged as lentiviruses rather than retroviruses. Lentiviral human GIPZ Lamin A/C shRNAmir (v2LHS_62719) was used as a negative control. Silencing of TMEM9B was clearly able to bypass senescence (Figure 4a ). Moreover each of the constructs was individually able to overcome senescence arrest with v2LHS_58957 being the most efficient [ 16 ]. Figure 4 Silencing targets identified from the primary screen . CL3 EcoR cells were infected in triplicate with lentiviruses prepared from the indicated GIPZ shRNAmir constructs targetting TMEM9B (a) , LTBP2/3 (b) , ATXN10 (c) and LAYN (d) . Cells resistant to 6 μg/ml puromycin were isolated, reseeded and assayed for bypass of senescence by culturing at 38°C for 3 weeks. Lentiviruses prepared from the GIPZ shRNAmir Lamin A/C (v2LHS_62719) construct were used as the negative control. For TMEM9B, the mix of constructs comprised v2LHS_247318, 58957, 58958 and 58959. To determine if ATXN10, LAYN and LTBP2/3 were also able to directly bypass senescence, lentivirus constructs from the GIPZ lentiviral shRNAmir library were used. The complementation assay for LTBP2/3, in Figure 4b , showed that silencing LTBP2/3 with the one available silencing construct (v2LHS_34089) clearly yielded healthy growing colonies. Silencing of ATXN10 was tested using 4 different silencing constructs v2LHS_71735, 71736, 71737 and 71740. All four constructs were able to overcome senescence and yielded more growing colonies than the Lamin A/C negative control (Figure 4c ). Silencing of LAYN was tested using 2 different silencing constructs v2LHS_265009 and 118722; both constructs were able to bypass growth arrest and produced growing colonies (Figure 4d ). Taken together our results showed that silencing of TMEM9B, ATXN10, LAYN and LTBP2/3 was able to bypass senescence in the conditionally immortal human fibroblasts. ShRNA interference screen The formula: ln [1-0.95]/ln [1-1/(Library Size)] recommended for genetic screens by the Nolan lab ( http://www.stanford.edu/group/nolan/screens/screens.html ), suggested that approximately 1000 independent infectious events would be sufficient for a 99% confidence that all shRNAs within a pool had been sampled. To ensure that the screen would be saturating, virus sufficient to yield 10,000 infectious events was utilised for each pool (shown in Additional File 1 ). The screen was performed in successive waves of 10 pools. To minimise variation and background reversion, CL3 EcoR cells were used at the same passage for every pool. Virus prepared from pRSp21F and pRSLamin A/C was used as positive and negative controls respectively to evaluate the level of background and ensure that the complementation assay worked for each round of the screen. Stably transduced cells were trypsinised and reseeded. Three weeks after reseeding, flasks were examined to identify growing colonies; a representative colony is shown in Figure 3 . Each colony was examined microscopically to ensure it comprised growing cells and the number of colonies obtained for each pool determined. The number of stably transduced cells, the number of flasks reseeded and the number of colonies obtained for each flask at 38°C are shown in Additional File 2 . The flasks which contained more densely growing/bigger colonies (indicated in red in Additional File 2 ) were trypsinised, replated and used for extracting genomic DNA when confluent. Figure 3 ShRNA screen . CL3 EcoR cells were transduced with ecotropic retroviruses prepared from each of the 100 pools of shRNAmirs, selected with puromycin, reseeded and shifted up to 38°C for 3 weeks. Densely growing colonies were considered to have bypassed senescence and potentially contain candidate shRNAmirs. 34 out of 100 pools yielded healthy growing colonies; pools 13, 78 and 82 particularly gave a higher number of colonies which were also larger. Pools 16, 18, 19, 21 and 80 also yielded colonies but they were smaller. For each pool that contained growing colonies, 1 to 4 flasks containing the highest number of growing colonies, were reseeded for extracting genomic DNA and resulted in a total of 81 sub-pools. Identification of shRNAmirs The shRNAmir proviral inserts were by amplified by two rounds of nested PCR using pSM2 specific primers, the amplified products TOPO cloned and plasmid DNA extracted from at least six colonies sequenced to identify all shRNAs present within each pool; for some pools, DNA from many more colonies was sequenced. The hair pin sequence was determined by searching for the miR-30 context and the miR-30 loop that are common to all inserts and frame the hair pin. The sequence of the hair pin was used to identify the target gene by searching the pSM2 data base or by BLASTN analysis of the NCBI human genome data base ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Sequences that could not be linked back to the list of hair pin sequences within the Open Biosystems collection or were not 100% homologous to a gene within Genbank were not pursued and are presented in Additional File 3 . The rescued shRNAmir hair pins identified 112 known genes and another 29 shRNAmirs targetting as yet unidentified loci. For each pool, the number of times that sequence was obtained, the corresponding insert reference, gene name and the number of shRNAmir constructs for that gene within the SM2 library are shown in Table 1 . The last column of the table indicates if the recovered insert was a match to a hair pin present in that particular pool ("match") or if it was present within another pool ("listed in pool X"). 14 hair pins were isolated from incorrect pools. Some inserts were detected several times in multiple pools. For example, the hair pin v2HS_119967 (corresponding to LOC155004) listed within pool 52 was also identified from pool 3, 4, 5, 9, 12, 30, 59, 60, 64, 71, 72, 74, 77, and 98 suggesting that there may have been some cross-contamination of the library perhaps during replica plating. However, this was not a problem since the aim of our screen was to identify shRNAmirs that are able to bypass senescence and does not depend upon the identity of the pool. Since this insert was isolated from so many pools, it could be that it is a strong positive or that it was highly represented within the library and likely to be a false positive. Table 1 Primary screen Pool Insert Gene name Gene symbol Freq Cons per Gene Library location 3 v2HS_63142 keratin associated protein 5-9 KRTAP5-9 3/9 1 match v2HS_119967 LOC155004 LOC155004 4/9 2 pool 52 v2HS_56766 acyl-CoA synthetase medium-chain family member 3 ACSM3 1/9 2 match v2HS_59294 glycoprotein hormone alpha 2 GPHA2 1/9 1 match v2HS_53974 PRO0255 protein PRO0255 5/8 1 match v2HS_63482 paired box gene 1 PAX1 2/8 1 match 4 v2HS_119967 LOC155004 LOC155004 2/10 2 pool 52 v2HS_66751 LOC344082 LOC344082 4/10 1 match v2HS_70011 serum amyloid A-like 1 SAAL1 2/10 2 match v2HS_98079 human solute carrier family 22 SLC22A3 1/10 2 pool 79 5 v2HS_108647 LOC349868 LOC349868 3/8 1 pool 79 v2HS_70473 polymerase (DNA directed), mu POLM 2/8 v2HS_71958 human olfactory receptor, family 5, subfamily P, member 3 OR5P3 3/8 1 match v2HS_119967 LOC155004 LOC155004 7/12 2 pool 52 v2HS_66652 protein phosphatase 3, catalytic subunit, β isoform PPP3CB 1/12 1 match v2HS_70473 polymerase (DNA directed), mu POLM 4/12 7 v2HS_112910 human cyclin-dependent kinase 8 CDK8 1/10 3 match v2HS_98079 solute carrier family 22, member3 SLC22A3 1/10 2 pool 79 v2HS_97017 sterile alpha motif containing 4a SAMD4A 1/10 v2HS_69776 cytochrome P450, family 4, subfamily Z, polypeptide CYP4Z2P 1/10 2 match 2 pseudogene 9 v2HS_108647 LOC349868 LOC349868 1/8 1 pool 79 v2HS_119967 LOC155004 LOC155004 1/8 2 pool 52 v2HS_62506 family with sequence similarity 181, member B FAM181B 1/8 1 match v2HS_55950 prostate stem cell antigen PSCA 2/13 1 match v2HS_119967 LOC155004 LOC155004 3/13 2 pool 52 v2HS_70312 TRK-fused gene TFG 4/13 3 match v2HS_71740 ataxin 10 ATXN10 1/13 1 match v2HS_98079 solute carrier family 22, member 3 SLC22A3 1/13 2 pool 79 v2HS_65121 sorting nexin 12 SNX12 1/13 1 match v2HS_64384 amyloid beta precursor protein binding family A member 2 APBA2 1/6 1 match v2HS_97017 sterile alpha motif containing 4A SAMD4A 1/6 12 v2HS_119967 LOC155004 LOC155004 5/11 2 pool 52 v2HS_58950 signal-regulatory protein beta 2 SIRPB2 3/11 2 match protein tyrosine phosphatase, non-receptor type 13 PTPN13 1/10 v2HS_119967 LOC155004 LOC155004 5/10 2 pool 52 13 v2HS_55731 phenylalanine-tRNA synthetase-like, beta subunit FARSLB 1/12 2 match v2HS_59258 dynein, light chain, roadblock-type 1 DYNLRB1 3/12 1 match v2HS_59891 TAO kinase 1 TAOK1 5/12 3 match v2HS_162164 iodotyrosine deiodinase IYD 1/12 cell cycle exit and neuronal differentiation 1 CEND1 1/12 transmembrane protein 168 TMEM168 2/5 v2HS_68714 abl-interactor 1 ABL1 3/5 3 match cell cycle exit and neuronal differentiation 1 CEND1 4/6 v2HS_58952 signal regulatory protein gamma SIRPG 1/6 2 pool 14 v2HS_59653 LOC342404 LOC342404 1/6 1 match v2HS_55731 phenylalanyl-tRNA synthetase like, beta subunit FARSLB 1/15 2 match v2HS_64320 Smith-Magenis syndrome chromosome region, candidate 7 SMCR7 1/15 1 match v2HS_71174 peroxisome proliferator-activated receptor, gamma PPARGC1A 3/15 1 match v2HS_71453 similar to IAP-associated factor VIAF1; phosducin-like 2/15 1 match 16 v2HS_68478 CD28 antigen CD28 8/12 2 pool 13 v2HS_63107 dopamine beta hydoxylase DBH 2/12 1 match v2HS_61750 LOC100129230 LOC100129230 7/13 3 match LOC9142 LOC9142 2/13 V2HS_62831 membrane bound O-acyltransferase domain containing 1 MBOAT1 3/13 2 match 18 v2HS_63989 STAR-related lipid transfer (START) domain containing 6 STARD6 1/14 2 match LOC730256 LOC730256 1/14 19 v2HS_58958 TMEM9 domain family, member B/C11orf15 TMEM9B 3/10 1 match v2HS_57051 RNA binding motif, single stranded interacting protein 1 RBMS1 5/10 1 match v2HS_106158 LOC345672 LOC345672 1/10 2 pool 82 v2HS_59560 chromosome 13 open reading frame 15 C13orf15 10/10 1 match 21 v2HS_55312 glucosamine-phosphate N-acetyltransferase 1 GNPNAT1 6/9 2 match v2HS_68437 LOC157503 LOC157503 2/9 3 match 30 v2HS_119967 LOC155004 LOC155004 6/14 2 pool 52 v2HS_34338 protein phosphatase 4, regulatory subunit 2 PPP4R2 7/14 3 match v2HS_119967 LOC155004 LOC155004 1/13 2 pool 52 v2HS_36467 solute carrier family 33 (acetyl-CoA transporter), member 1 SLC33A1 8/13 3 match v2HS_46793 ubiquitin-like modifier activating enzyme 3 UBA3 1/13 1 match 32 v2HS_42104 citrate lyase beta like (CLYBL), transcript variant 1 CLYBL 5/7 1 match LOC100129563 LOC 10012956 1/7 41 v2HS_48278 choline kinase-like CHKL 6/6 1 match 54 v2HS_112629 basic transcription factor 3, like 1 BTF3L1 1/5 1 match v2HS_121153 LOC221399 LOC221399 2/5 1 match v2HS_125538 LOC350103 LOC350103 1/5 1 match v2HS_129527 LOC351851 LOC351851 6/6 1 match v2HS_112629 basic transcription factor 3, like 1 BTF3L1 2/6 1 match 55 v2HS_117465 SH3 domain binding glutamic acid-rich protein like 2 SH3BGRL2 2/6 1 match v2HS_119051 LOC90841 LOC90841 1/6 1 match v2HS_116174 YTH domain containing 2 YTHDC2 1/7 2 pool 58 v2HS_119120 hypothetical protein FLJ20032 FLJ20032 6/7 2 match 56 V2HS_120429 six-twelve leukemia (STL), non-coding RNA STL 5/6 1 match v2HS_115231 rab23, member RAS oncogene family RAB23 4/6 4 match v2HS_117914 transketolase-like 2 TKTL2 2/6 1 match v2HS_117239 chromosome 9 open reading frame 58 C9orf58 3/6 1 match v2HS_120429 six-twelve leukemia (STL), non-coding RNA STL 1/6 1 match v2HS_119206 lon peptidase 2, peroxisomal LONP2 2/6 1 pool 146 58 v2HS_116174 YTH domain containing 2 YTHDC2 1/4 2 match v2HS_118722 layilin LAYN 2/4 2 match v2HS_112838 ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 3/7 4 match v2HS_112982 chromodomain helicase DNA binding protein 3 CHD3 1/7 3 match v2HS_116174 YTH domain containing 2 YTHDC2 1/7 2 match v2HS_112838 ectonucleoside triphosphate diphosphohydrolase 3 ENTPD3 1/12 4 match v2HS_118254 WD repeat and FYVE domain containing 2 WDFY2 1/12 1 match v2HS_122548 similar to ribosomal protein S3A RPS3A 4/12 1 pool 53 v2HS_120982 coiled coil domain containing 129 CCDC129 5/12 2 match 59 v2HS_111554 interleukin 2 IL2 8/10 2 match v2HS_115544 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 DDX47 2/10 4 match v2HS_116377 transmemembrane protein 135 TMEM135 1/9 2 match v2HS_117064 CD1E molecule CD1E 3/9 1 match v2HS_119967 LOC155004 LOC155004 4/9 2 pool 52 v2HS_120757 olfactory receptor, family 8, subfamily K, member 1 OR8K1 1/9 2 match v2HS_108647 LOC349868 LOC349868 1/8 1 pool 79 v2HS_115544 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 DDX47 3/8 4 match v2HS_116833 intermediate filament protein syncoilin SYNC 1/8 2 match 60 v2HS_121585 dual specificity phosphatase 3 DUSP3 10/11 1 match v2HS_117903 glutamine rich 2 QRICH2 4/11 1 match v2HS_119967 LOC155004 LOC155004 1/11 2 pool 52 v2HS_121585 dual specificity phosphatase 3 DUSP3 2/11 1 match v2HS_128131 LOC351061 LOC351061 2/11 1 match v2HS_98079 solute carrier family 22 member 3 SLC22A3 1/11 2 pool 79 v2HS_119967 LOC155004 LOC155004 8/10 2 pool 52 v2HS_121585 dual specificity phosphatase 3 DUSP3 2/10 1 match 64 v2HS_119967 LOC155004 LOC155004 10/11 2 pool 52 v2HS_121013 protease serine 54 PRSS54 3/12 1 match 66 v2HS_117675 latent transforming growth factor β binding protein 2/3 LTBP2/3 15/15 v2HS_118530 RNA exonuclease 1 homolog like 1 REXO1L1 5/13 3 match v2HS_117675 latent transforming growth factor β binding protein 2/3 LTBP2/3 8/13 v2HS_118530 REX1, RNA exonuclease 1 homolog-like 2 REXO1L1 1/11 3 match 71 v2HS_103818 LOC284804 LOC284804 2/16 1 pool 78 v2HS_119967 LOC155004 LOC155004 9/16 2 pool 52 v2HS_97981 LOC51152 LOC51152 5/16 2 pool 79 72 v2HS_119967 LOC155004 LOC155004 9/10 2 pool 52 74 v2HS_119967 LOC155004 LOC155004 11/12 2 pool 52 77 v2HS_119967 LOC155004 LOC155004 12/12 2 pool 52 78 v2HS_94763 paired related homeobox 1 PRRX1 1/8 1 match v2HS_99138 solute carrier family 25 SLC25A21 7/8 2 match v2HS_102207 transmembrane protein 63B TMEM63B 1/12 1 match v2HS_103818 LOC284804 LOC284804 4/12 1 match V2HS_95356 armadillo repeat containing, X-linked 2 ARMCX2 2/12 2 match v2HS_98650 mitochondrial ribosomal protein 63 MRP63 1/12 1 match v2HS_102155 similar to hypothetical protein KIAA0286 (HA6800) KIAA0286 1/8 1 match v2HS_108506 LOC349839 LOC349839 1/8 2 match v2HS_96236 zinc finger and BTB domain containing 1 ZBTB1 2/8 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 3/10 v2HS_96236 zinc finger and BTB domain containing 1 ZBTB1 1/10 1 match 79 v2HS_105974 LOC345597 LOC345597 3/11 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 3/11 v2HS_98079 solute carrier family 22, member 3 SLC22A3 4/11 2 match v2HS_108647 LOC349868 LOC349868 2/7 1 match v2HS_98079 solute carrier family 22, member 3 SLC22A3 5/7 2 match v2HS_101943 anthrax toxin receptor-like ANTXRL 1/11 3 match v2HS_105093 human solute carrier family 35, member F4 SLC35F4 2/11 1 match v2HS_106472 LOC345713 LOC345713 1/11 1 match v2HS_107395 LOC346589 LOC346589 1/11 1 match v2HS_91777 nuclear receptor co-repressor N-Cor 1 NCOR1 4/11 2 match Non-protein coding RNA 282 NCRNA00282 1/11 80 v2HS_102441 adenylate cyclase 1 ADCY1 5/11 1 match v2HS_130882 glutamate receptor, metabotropic 3 GRM3 1/11 3 pool 91 v2HS_97368 yippee-like 5 YPEL5 2/11 1 match v2HS_102441 adenylate cyclase 1 ADCY1 5/10 1 match 82 v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/11 v2HS_101845 human prickle-like 2 (Drosophila) PRICKLE2 3/11 1 match v2HS_109096 LOC349975 LOC349975 1/11 1 pool 84 v2HS_99423 coiled coil domain containing 70 CCDC70 1/11 1 match v2HS_109596 1/14 v2HS_108399 LOC349811 LOC349811 3/14 1 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/14 v2HS_93536 proteolipid protein 1 PLP1 5/14 1 match v2HS_97017 sterile alpha motif domain containing 4A SAMD4A 1/14 v2HS_106158 LOC345672 LOC345672 3/14 2 match v2HS_93615 p53 TP53 3/14 1 match v2HS_95112 RAS p21 protein activator 4 RASA4 1/14 1 match v2HS_99525 BCL2 like 12 BCL2L12 1/14 2 match v2HS_97017 sterile alpha motif domain containing 4A SAMD4A 4/14 v2HS_94640 aryl hydrocarbon receptor nuclear translocator-like ARNTL 3/10 2 match v2HS_100174 desmoglein 4 DSG4 2/10 2 pool 145 v2HS_96026 adnp homeobox 2 ADNP2 3/10 1 match v2HS_100819 Rho GTPase activating protein 20 ARHGAP20 2/10 1 match 84 v2HS_106409 LOC345700 LOC345700 1/11 1 match v2HS_95019 zinc finger protein 16 ZNF16 9/11 1 match v2HS_97152 ubiquitin-conjugating enzyme E2, J1 UBE2J1 1/11 1 match v2HS_95019 zinc finger protein 16 ZNF16 10/11 1 match 94 v2HS_141495 zinc finger protein 454 ZNF454 7/12 2 pool 96 95 v2HS_130457 radial spoke head 10 homolog B (Chlamydomonas) RSPH10B 6/7 3 match v2HS_131154 potassium inwardly-rectifying channel, subfamily J KCNJ2 1/7 3 match v2HS_184999 eukaryotic initiation factor 4A isoform 1 EIF4A1 1/3 96 chromosome 9 open reading frame 123 C9orf123 5/13 98 v2HS_141367 cDNA FLJ37626/LOC285500 FLJ37626 10/10 2 match v2HS_119967 LOC155004 LOC155004 2/11 2 pool 52 v2HS_133299 insulin-like growth factor binding protein 6 IGFBP6 3/11 2 match v2HS_135564 chromosome X open reading frame 57 CXORF57 5/11 1 match For each candidate the DNA pool from which it was isolated, the insert reference, target gene, frequency of isolation, as well as the number of shRNAmir constructs for that gene within the SM2 library are indicated. The last column shows if the recovered insert was a match to a hair pin in that particular pool ("match") or if it was from another pool ("pool X"). Pool 13, 78 and 82 that produced more colonies and colonies that were larger and healthier than others, identified the following genes: Pool 13: DYNLRB1, FARSLB, PPARGC1A, TAOK1, CEND1, ABL1, LOC342404, TMEM168, SIRPG, IYD and SMCR7; Pool 78: PRRX1, SLC25A21, ARMCX2, LOC284804, LOC349839, MRP63, TMEM63B, ZBTB1, KIA0286 and EIF4A1; and Pool 82: EIF4A1, CCDC70, PRICKLE2, PLP1, SAMD4A, RASA4, TP53, ARNTL, BCL2L12, ADNP2, DSG4, LOC345672, LOC349975, LOC349811 and ARHGAP20. Moreover, pool 82, one of the pools that gave the best senescence bypass, contained the only shRNAmir targetting p53, v2HS_93615 identified in the screen, thereby internally validating it. Overlap of the primary candidates of the shRNA screen with microarray data for genes up-regulated upon senescence in CL3 EcoR cells To prioritise the candidates identified from the primary screen for functional validation, they were compared to genes up-regulated upon senescence and whose expression was reversed when senescence was abrogated upon inactivation of the p53 and pRB pathways [[ 16 ]; microarray expression profiling data is available from Gene Expression Omnibus database accession number GSE24810]. This identified 4 common genes, ATXN10, LAYN, LTBP2/3 and TMEM9B. The microarray expression profiling data presented in Table 2 showed that they were all up-regulated upon senescence growth arrest: ATXN10 by 1.3 fold (p-value 1 × 10 -3 ), LAYN by 2 fold (p-value 2 × 10 -4 ), LTBP2/3 by 2.5 (p-value 9 × 10 -8 ), 1.7 (p-value 5 × 10 -9 ), 1.5 (p-value 8 × 10 -4 ) and 1.4 fold (p-value 5 × 10 -5 ) and TMEM9B by 1.4 (p-value 1 × 10 -7 ) and 1.3 fold (p-value 2 × 10 -4 ) respectively. The data in Table 2 further show that up-regulation of these candidates was reversed when senescence was bypassed upon inactivation of the p53-p21 and p16-pRB pathways by silencing p53 (pRS_p53) or p21 CIP1 (pRS_p21) or by sequestration of the RB family of proteins by HPV16 E7 or by expression of the dominant negative E2F-DB protein. Table 2 Microarray expression profiling data for common genes Probe Symbol GA p-value HS p-value pRS_p53 p-value pRS_p21 p-value E7 p-value E2F_DB p-value 208832_at ATXN10 0.36 1E-03 0.25 3E-02 -0.18 2E-01 -0.35 2E-03 0.02 9E-01 -0.23 4E-02 228080_at LAYN 1.04 2E-04 0.30 3E-01 -0.95 1E-03 -0.70 2E-02 -0.56 6E-02 -0.11 8E-01 219922_s_at LTBP2/3 1.32 9E-08 0.66 5E-03 -1.04 2E-05 -1.32 3E-07 -1.19 1E-06 -1.41 4E-08 223690_at LTBP2/3 0.75 5E-09 -0.14 3E-01 -0.52 1E-05 -0.91 2E-10 -1.27 1E-14 -1.22 3E-14 204682_at LTBP2/3 0.57 8E-04 -0.48 8E-03 -0.39 4E-02 -0.94 1E-06 -1.62 3E-12 -2.02 4E-15 227308_x_at LTBP2/3 0.46 5E-05 0.03 9E-01 -0.43 3E-04 -0.57 4E-06 -0.28 2E-02 -0.55 4E-06 218065_s_at TMEM9B 0.44 1E-07 -0.11 2E-01 -0.33 6E-05 -0.38 9E-06 -0.20 1E-02 -0.18 2E-02 222507_s_at TMEM9B 0.39 2E-04 -0.19 1E-01 -0.28 1E-02 -0.34 2E-03 -0.12 3E-01 -0.07 6E-01 Log 2 fold changes in gene expression in CL3 EcoR cells upon senescence arrest are indicated as GA and upon shift up of control temperature insensitive HMF3S cells, from 34°C to 38°C, as HS respectively. Also indicated are the data obtained for these genes from CL3 EcoR cells in which senescence had been bypassed by silencing of p53 (pRS_p53) or p21 CIP1 (pRS_p21) or by inactivation of pRB using HPV16 E7 (E7) or by expression of the dominant negative E2F-DB protein. The identification of TMEM9B was particularly remarkable because the microarray analysis has suggested that senescence growth arrest in CL3 EcoR cells is associated with activation of the NF-κB signalling pathway and TMEM9B has previously been shown to be able to activate NF-κB dependent reporter constructs [ 18 , 19 ]. To determine if silencing of TMEM9B would bypass senescence, 4 GIPZ lentiviral silencing constructs (v2LHS_247318, 58957, 58958 and 58959; Thermo Scientific Open Biosystems) targetting TMEM9B were obtained, pooled and introduced into CL3 EcoR cells after packaging as lentiviruses. The GIPZ lentiviral library contains the same hair pins as the retroviral library but is more stable and the constructs are packaged as lentiviruses rather than retroviruses. Lentiviral human GIPZ Lamin A/C shRNAmir (v2LHS_62719) was used as a negative control. Silencing of TMEM9B was clearly able to bypass senescence (Figure 4a ). Moreover each of the constructs was individually able to overcome senescence arrest with v2LHS_58957 being the most efficient [ 16 ]. Figure 4 Silencing targets identified from the primary screen . CL3 EcoR cells were infected in triplicate with lentiviruses prepared from the indicated GIPZ shRNAmir constructs targetting TMEM9B (a) , LTBP2/3 (b) , ATXN10 (c) and LAYN (d) . Cells resistant to 6 μg/ml puromycin were isolated, reseeded and assayed for bypass of senescence by culturing at 38°C for 3 weeks. Lentiviruses prepared from the GIPZ shRNAmir Lamin A/C (v2LHS_62719) construct were used as the negative control. For TMEM9B, the mix of constructs comprised v2LHS_247318, 58957, 58958 and 58959. To determine if ATXN10, LAYN and LTBP2/3 were also able to directly bypass senescence, lentivirus constructs from the GIPZ lentiviral shRNAmir library were used. The complementation assay for LTBP2/3, in Figure 4b , showed that silencing LTBP2/3 with the one available silencing construct (v2LHS_34089) clearly yielded healthy growing colonies. Silencing of ATXN10 was tested using 4 different silencing constructs v2LHS_71735, 71736, 71737 and 71740. All four constructs were able to overcome senescence and yielded more growing colonies than the Lamin A/C negative control (Figure 4c ). Silencing of LAYN was tested using 2 different silencing constructs v2LHS_265009 and 118722; both constructs were able to bypass growth arrest and produced growing colonies (Figure 4d ). Taken together our results showed that silencing of TMEM9B, ATXN10, LAYN and LTBP2/3 was able to bypass senescence in the conditionally immortal human fibroblasts. Discussion To directly identify the downstream effectors of the p53-p21 and p16-pRB pathways crucial for mediating entry into senescence, we carried out a loss-of-function RNA interference screen in the conditionally immortal HMF3A human fibroblasts. This identified 112 known genes including p53 and another 29 shRNAmirs targetting unidentified loci. Comparison of these known targets with genes up-regulated upon senescence in these cells identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes using lentiviral shRNAmirs bypasses senescence in the HMF3A cells. Although none of these genes has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which we have found to have a causal role in promoting senescence. The 112 known targets identified by the shRNA screen comprise a wide variety of genes but most importantly one of them was the only p53 shRNAmir (v2HS_93615 from pool 82) present within this library, thereby internally validating the screen. Moreover all of the primary targets were identified from single shRNAmirs even though we have subsequently shown that other shRNAmirs corresponding to these targets present within the library are able to bypass senescence. It is not clear why other shRNAmirs were not isolated in the screen; however this is exactly what has been observed previously by others such as Westbrook and colleagues [ 20 ]. Nevertheless it remains to be demonstrated which of the targets identified by the primary screen are able to bypass senescence when assayed individually. TMEM9B was one of the 4 genes in common between the shRNA screen and genes known to be up-regulated upon senescence in HMF3A cells [ 16 ]. Moreover expression of TMEM9B was down-regulated when senescence was bypassed upon abrogation of the p53-p21 or p16-pRB pathways. TMEM9B is a glycosylated protein that localises to lysosome membranes and partially to early endosomes. It has been shown to be a component of TNF signalling and a module shared between the interleukin-1 and Toll-like receptor pathways. It is also essential for TNF activation of both NF-κB and MAPK pathways by acting downstream of RIP1 and upstream of the MAPK and IκB kinases at the level of the TAK1 complex [ 19 ]. TMEM9B was also identified in a large scale study to identify genes activating NF-κB and MAPK signalling pathways [ 18 ]. These results are all consistent with our finding that in the conditionally immortal HMF3A cells, senescence growth arrest is associated with an activation of NF-κB signalling and suppression of this pathway bypasses senescence [ 16 ]. The latent TGFβ-binding protein 2/3 (LTBP2/3) hair pin sequence was identified from pool 66. Up-regulation of LTBP2/3 expression upon growth arrest was reversed when senescence was overcome. LTBPs are secreted proteins initially identified through their binding to TGFβ and may be involved in their assembly, secretion and targetting [ 21 ]. LTBP2/3 in particular has been found to play an essential role in the secretion and targetting of TGFβ1 [ 22 ]. Since silencing of LTBP2/3 can bypass senescence in HMF3A cells, it suggests that LTBP2/3 may be linked with the control of cell growth and be playing a role in suppressing tumour progression perhaps through regulation of TGFβ. This is in accordance with the identification of TGFβ as a senescence-inducing factor in the human lung A549 adenocarcinoma cells [ 23 ]. It is also in accordance with several other reports suggesting that TGFβ1 is capable of inducing cellular senescence. For instance, stimulation of human diploid fibroblasts with TGFβ1 triggers the appearance of senescence associated-β-galactosidase activity and increases steady state mRNA levels of senescence associated genes including APO J, fibronectin, and M22 [ 24 - 26 ]; both APO J (clusterin) and fibronectin are up-regulated in CL3 cells upon senescence arrest and this is reversed when senescence is bypassed [ 16 ]. Ataxin (ATXN) 10 was slightly up-regulated upon senescence arrest which was reversed upon silencing of p53 and p21 CIP1 or ectopic expression of the dominant negative E2F-DB protein. Spinocerebellar ataxia type 10 (SCA10) is a dominantly inherited disorder characterized by ataxia, seizures and anticipation caused by an intronic ATTCT pentanucleotide repeat expansion. The product of SCA10 encodes the novel protein, ATXN10, previously known as E46L, which is widely expressed in the brain and belongs to the family of armadillo repeat proteins. Although clinical features of the disease are well characterized, very little is known about ATXN10. ATXN10 knock down by RNAi has recently been shown to cause increased apoptosis in primary cerebellar cultures, implicated in SCA10 pathogenesis [ 27 , 28 ]. This is in contrast to our finding that silencing of ATXN10 in HMF3A cells by four different shRNAmirs did not cause apoptosis but promoted growth and permitted a bypass of senescence. Layilin (LAYN) identified from pool 58 was 2 fold up-regulated upon senescence arrest, which was reversed upon abrogation of the growth arrest by inactivation of either the p53-p21 or the p16-pRB pathways. Moreover two different LAYN shRNAmirs were found to directly bypass senescence in HMF3A cells. Layilin is a widely expressed integral membrane hyaluronan receptor, originally identified as a binding partner of talin located in membrane ruffles. Talin is responsible, along with its adaptor proteins, for maintaining the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Recently layilin has been suggested to play a crucial role in lymphatic metastasis of lung carcinoma A549 cells [ 29 ]. In addition to the genes described above, a number of other interesting genes particularly TAOK1, RAS4A and ARMCX2 were identified. TAOK1 is a micro-tubule affinity-regulating kinase required for both chromosome congression and checkpoint-induced anaphase delay [ 30 ]. It is known to activate the p38MAPK pathway through the specific phosphorylation of MKK3. This is a complex pathway responsive to stress stimuli and involved in cell differentiation and apoptosis and has been shown to have an important causative role in senescence [ 31 ]. RAS4A encodes a member of the GAP1 family of GTPase-activating proteins that have been identified to suppress the Ras/mitogen-activated protein kinase pathway in response to an elevation of Ca 2+ ions. Stimuli that increase intracellular Ca 2+ levels result in the translocation of this protein to the plasma membrane, where it activates Ras GTPase activity resulting in Ras being converted from the active GTP-bound state to the inactive GDP-bound state and suppression of downstream signalling [ 32 ]. ARMCX2 encodes a member of the ALEX family of proteins and may play a role in tumour suppression. This protein contains a potential N-terminal transmembrane domain and a single Armadillo repeat; armadillo repeat containing proteins are involved in development, maintenance of tissue integrity and suppressing carcinomas [ 33 ]. Conclusions The RNA interference screen has identified 112 known candidate proteins including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Although none of them except p53 had previously been linked to senescence or known to be downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways, directly silencing four of these candidates, TMEM9B, ATXN10, LAYN and LTBP2/3 bypassed senescence in CL3 EcoR cells. It remains to be determined whether direct silencing of any of the other primary candidates will also bypass senescence. Any genes that can bypass senescence upon silencing are novel starting points for identifying the signalling networks that act downstream of p53 and pRB to induce cellular senescence. The genes/proteins identified in the screen are also potential tumour suppressors, and a mechanistic dissection of their mode of action and role in cancer will undoubtedly provide new avenues for further research. Methods Cell Culture CL3 EcoR cells were maintained at 34°C ± 0.5°C [ 15 ]. Temperature shift experiments were performed at 38°C ± 0.5°C. Phoenix ecotropic and HEK293T cells were obtained from the ATCC and maintained at 37°C. Cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% (v/v) heat inactivated foetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. All media and components were obtained from Invitrogen. Viral packaging and infection Lentiviruses were prepared according to Besnier et al. [ 34 ]. Ecotropic viruses were prepared by transfecting 10 μg of retroviral plasmid DNA into phoenix ecotropic cells by FuGENE 6 Transfection reagent (Roche), according to the manufacturer's instructions. 24 hrs post-transfection, media was changed and fresh medium added. 48 hrs post-transfection, retroviral supernatant was harvested, filtered through a 0.45 μm filter and either used immediately or frozen at -80°C. A second harvest was prepared by adding fresh media to the plates and harvesting the virus supernatant the next day. Cells were infected with virus supernatants for 24 hrs at 34°C. Four days post-infection, antibiotic selection was added (2 μg/ml puromycin for pRS and pSM2 retroviruses or 6 μg/ml puromycin for pGIPZ shRNAmir lentiviruses; Invitrogen). Selection of cells infected with human GIPZ lentiviral shRNAmir constructs in puromycin at 6 μg/ml, enriches for cells with higher levels of shRNAmir expression. For the senescence bypass assay, the stably transduced cells were plated at 5 × 10 4 cells in T-75 flasks or at 1.6 × 10 4 cells in T-25 flasks and incubated overnight at 34°C. Next day the medium was changed and the cells shifted to 38°C for 3 weeks. Flasks which contained more densely growing or bigger colonies were trypsinised, replated and used for extracting genomic DNA when confluent. Cell Culture CL3 EcoR cells were maintained at 34°C ± 0.5°C [ 15 ]. Temperature shift experiments were performed at 38°C ± 0.5°C. Phoenix ecotropic and HEK293T cells were obtained from the ATCC and maintained at 37°C. Cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% (v/v) heat inactivated foetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. All media and components were obtained from Invitrogen. Viral packaging and infection Lentiviruses were prepared according to Besnier et al. [ 34 ]. Ecotropic viruses were prepared by transfecting 10 μg of retroviral plasmid DNA into phoenix ecotropic cells by FuGENE 6 Transfection reagent (Roche), according to the manufacturer's instructions. 24 hrs post-transfection, media was changed and fresh medium added. 48 hrs post-transfection, retroviral supernatant was harvested, filtered through a 0.45 μm filter and either used immediately or frozen at -80°C. A second harvest was prepared by adding fresh media to the plates and harvesting the virus supernatant the next day. Cells were infected with virus supernatants for 24 hrs at 34°C. Four days post-infection, antibiotic selection was added (2 μg/ml puromycin for pRS and pSM2 retroviruses or 6 μg/ml puromycin for pGIPZ shRNAmir lentiviruses; Invitrogen). Selection of cells infected with human GIPZ lentiviral shRNAmir constructs in puromycin at 6 μg/ml, enriches for cells with higher levels of shRNAmir expression. For the senescence bypass assay, the stably transduced cells were plated at 5 × 10 4 cells in T-75 flasks or at 1.6 × 10 4 cells in T-25 flasks and incubated overnight at 34°C. Next day the medium was changed and the cells shifted to 38°C for 3 weeks. Flasks which contained more densely growing or bigger colonies were trypsinised, replated and used for extracting genomic DNA when confluent. Competing interests The authors declare that they have no competing interests. Authors' contributions ER and LM-carried out the screen. ER and PSJ rescued the inserts. CJL and AA amplified and provided the shRNAmir library. PSJ wrote the manuscript. All authors have read and approved the final manuscript. Supplementary Material Additional file 1 Volume of virus supernatants used for the screen . The table shows the volume of virus supernatants used for each of the pools. Click here for file Additional file 2 Primary screen . This table shows the pool number, the number of cells obtained after puromycin selection, the number of T-75 and T-25cm2 flasks reseeded and the number of growing colonies observed after 3 weeks at 38°C. Numbers indicated in red correspond to flasks that were reseeded for extracting genomic DNA. Click here for file Additional file 3 Unidentified inserts . This table shows the hair pin sequences that were not 100% homologous to a gene by BLASTN analysis of the NCBI human genome data base ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) or could not be linked back to a hair pin sequence within the Open Biosystems SM2 library; the number of times the insert was isolated is also indicated. Click here for file Acknowledgements We are indebted to Catherine King (the UCL shRNA library core facility) for providing constructs, Gary Adamson for DNA sequencing and to Ray Young for graphics. PSJ gratefully acknowledges financial support from the Wellcome Trust (078305) and an equipment grant from the Brain Research Trust.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807221/

Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome

The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1β through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515070/

Additions to the knowledge of Nevada carabid beetles ( Coleoptera : Carabidae ) and a preliminary list of carabids from the Great Basin National Park

Abstract Background Additions to the list of Carabidae known for Nevada, USA and carabid beetles found in the Great Basin National Park, NV are reported with notes on ecology and identification resources. New information For 79 species of carabids, we present 57 new state records, two state records previously reported in online resources, one confirmation of a previous questionable record for the state, and report 22 records for the Great Basin National Park that includes three new state records. Background Additions to the list of Carabidae known for Nevada, USA and carabid beetles found in the Great Basin National Park, NV are reported with notes on ecology and identification resources. New information For 79 species of carabids, we present 57 new state records, two state records previously reported in online resources, one confirmation of a previous questionable record for the state, and report 22 records for the Great Basin National Park that includes three new state records. Introduction Carabids of Nevada Carabidae is family of beetles that have a cosmopolitan distribution and are often very common in the United States. Surprisingly, the number of published records of carabid species from Nevada is staggeringly low. In the recent catalog of North American carabid beetles ( Bousquet 2012 ) the total number of species known for Nevada placed the state as 49th (242 species), only surpassing Delaware (206 species). For comparison, among the 50 US states, Delaware is ranked 49th for total land area, with only 644,718 ha (2,489 sq mi.), while Nevada is ranked 7th with 28,635,092 ha (110,560.71 sq mi.). Compared to adjacent states (California, Oregon, Arizona, Idaho, and Utah) Nevada had only about a third to three quarters the number of species reported. These discrepancies suggested to us that the Nevada fauna was significantly underreported. To better document Nevada's biodiversity, our study aimed to expand the checklist of the carabid fauna in Nevada by identifying specimens from several western North American Entomological collections and undertaking collecting trips to Nevada. The Ecologial Setting in Nevada The state of Nevada is composed of five ecoregions ( U.S. Geological Survey, Department of the Interior, U.S.A 2006 , Nevada Fish & Wildlife Office 2014 ). Three cover the majority of the state: the Northern, Central, and Mojave Basins and Ranges, while two ecoregions, Sierra Nevada Range and Arizona-Mexico Plateau, comprise a relatively small portion of Nevada. Within these ecoregions Nevada's seasonally hot, dry climate produces a plethora of different ecosystems that support mainly shrubs and grasses at low and medium elevations and conifer trees at higher elevations; however, pockets of isolated environments allow endemic species of plants to thrive and provide habitat for a variety of carabid beetles. The largest ecoregion in Nevada is the Central Basin and Range. This ecoregion is composed mainly of north-south oriented mountain ranges alternating with dry shrub and grass covered basins. At mid elevations, the mountains are characterised with scattered forests of woodland and mountain brush. Due to the Sierra Nevada Mountains, Nevada's western region is under a rain shadow and as a result the dry-tolerant piñon-juniper woodlands are common as are stands of ponderosa, lodgepole and white pine. At high elevations (above 2700 m), seasonal moisture supports the growth of high elevation conifers species and aspen groves, with sagebrush and mountain-mahogany in the understory. The precipitation at this elevation also provides perennial streams and ponds with water. In the Pleistocene much of central Nevada was under what was one of the largest lakes in North America, Lake Lahontan. All that remains of the lake now are playas and a few remnants lakes. The Central Basin and Range is now high desert plain with the exception of small, often seasonal wetlands, typically alkali flats or saline lakes that are replenished by seasonal rains and runoff. In the west and central part of the region there are characteristic rolling valleys with mountian ranges that have alluvial fan outwashes flowing into the lower basins. Diversity in this region is rather low due to its aridity. With a few exceptions, there are almost no continuous woodlands. The highest elevations in this region, those that extend above treeline, are densely covered with mountain big sagebrush, western serviceberry, snowberry, and low sagebrush, but very few trees. The easternmost part of the Central Basin and Range ecoregion consists of sagebrush valleys, woodlands, mountains, and saline basins. Much of the soil in this region is shallow due to a combination of heavy summer rains and the limestone and dolomite bedrock. As a result, the sagebrush valleys consist of grasses and brush capable of tolerating shallow soils. The woodland zones consist of mainly piñon and juniper forests. Historically, these forests only occurred at higher elevations due to annual natural fires, but fire suppression has allowed the forests to move further down towards the sagebrush valleys. In the more mountainous areas, for example Great Basin National Park (GBNP), various conifers (white fir, Douglas fir, Engelmann spruce, bristlecone pines) dominate. Although high enough to form an alpine zone, climate conditions do not favour the retention of water as most of it rapidly flows out via springs and streams. The GBNP includes a range of the typical floral zones for the region with sagebrush dominated lower elevations, rising through juniper, piñon pine, ponderosa pine, and grassy mountain meadows. Above treeline the habitat is rocky with few plants. Situated on Wheeler Peak, which rises to nearly 4000 m elevation, is a small alpine glacier, the remnant of a more extensive glacier from the last glacial maximum (LGM). Erosional deposition from the glacier's meltwater is evident in much of the of the most-visited parts of the park. The Northern Basin and Range, which is shared with Oregon and Idaho, is a cooler, more mesic, and less mountainous than the Central Basin and Range. This ecoregion consists of high lava plateaus to the northeast, high lava plains to the northwest and semi-arid uplands scattered across northern Nevada. The high lava plateau receives more precipitation and experiences colder winters resulting in cool season grasses such as bluebunch wheatgrass and Idaho fescue. The high lava plains, a large sagebrush steppe, are very similar to the high lava plateau in climate and vegetation, but have many ephemeral pools that are home to a diverse fauna and flora. In the far south of Nevada lies the Mojave Basin and Range, a region extending from California to Arizona and southwestern Utah. The warmer, drier climate and milder winters favours a flora dominated by creosote bush at lower elevations. This desert region is far from uniform and is comprised of three subregions: the Amargosa Desert, creosote-bush dominated basins, and arid footslopes. The Amargosa desert is in the rain shadow of the Spring Mountains and is an internally-drained basin with the greatest temperature extremes. Most of the flora is dry adapted creosote bush and bursage. Further east, creosote bush-dominated basins are scattered between the Mojave Desert mountain ranges, grasslands, blackbrush plains, Joshua trees woodlands, and cactus dominated areas. The Spring Mountains prominently rise above the basin habitats with Mount Charleston reaching over 3600 m elevation. Along the elevational gradient various scrub and bush zones lead to juniper, piñon pine, mountain mahogany, culminating in montane conifers. The Sierra Nevada ecoregion is a mountainous region that is mainly in California but a small part of this ecoregion extends into Nevada along its western border, notably including the foothills of the Carson Range. At mid-elevations, the dry forest is composed of a mix of conifers, including California white fir, incense cedar, Jeffrey pine, with an understory of sagebrush, antelope bitterbrush, and manzanita. At elevations between 7500-9500 ft, heavy snowfall and summer droughts favours vegetation adapted to season extremes like red fir, white pine and mountain hemlock. Though a relatively small area, this extension of the Sierra Nevada fauna is significant as it is not found elsewhere in the state of Nevada. The Arizona-Mexico Plateau ( Ruhlman et al. 2010 ) is the ecoregion with the smallest area among the five in Nevada. It is represented by an area of the Virgin Mountains at the Nevada and Arizona border, which is the western limit of the region that extends east into New Mexico. The region has semiarid grassland and desert-scrub, piñon pine uplands, and ponderosa pine at the highest elevations. Carabids of Nevada Carabidae is family of beetles that have a cosmopolitan distribution and are often very common in the United States. Surprisingly, the number of published records of carabid species from Nevada is staggeringly low. In the recent catalog of North American carabid beetles ( Bousquet 2012 ) the total number of species known for Nevada placed the state as 49th (242 species), only surpassing Delaware (206 species). For comparison, among the 50 US states, Delaware is ranked 49th for total land area, with only 644,718 ha (2,489 sq mi.), while Nevada is ranked 7th with 28,635,092 ha (110,560.71 sq mi.). Compared to adjacent states (California, Oregon, Arizona, Idaho, and Utah) Nevada had only about a third to three quarters the number of species reported. These discrepancies suggested to us that the Nevada fauna was significantly underreported. To better document Nevada's biodiversity, our study aimed to expand the checklist of the carabid fauna in Nevada by identifying specimens from several western North American Entomological collections and undertaking collecting trips to Nevada. The Ecologial Setting in Nevada The state of Nevada is composed of five ecoregions ( U.S. Geological Survey, Department of the Interior, U.S.A 2006 , Nevada Fish & Wildlife Office 2014 ). Three cover the majority of the state: the Northern, Central, and Mojave Basins and Ranges, while two ecoregions, Sierra Nevada Range and Arizona-Mexico Plateau, comprise a relatively small portion of Nevada. Within these ecoregions Nevada's seasonally hot, dry climate produces a plethora of different ecosystems that support mainly shrubs and grasses at low and medium elevations and conifer trees at higher elevations; however, pockets of isolated environments allow endemic species of plants to thrive and provide habitat for a variety of carabid beetles. The largest ecoregion in Nevada is the Central Basin and Range. This ecoregion is composed mainly of north-south oriented mountain ranges alternating with dry shrub and grass covered basins. At mid elevations, the mountains are characterised with scattered forests of woodland and mountain brush. Due to the Sierra Nevada Mountains, Nevada's western region is under a rain shadow and as a result the dry-tolerant piñon-juniper woodlands are common as are stands of ponderosa, lodgepole and white pine. At high elevations (above 2700 m), seasonal moisture supports the growth of high elevation conifers species and aspen groves, with sagebrush and mountain-mahogany in the understory. The precipitation at this elevation also provides perennial streams and ponds with water. In the Pleistocene much of central Nevada was under what was one of the largest lakes in North America, Lake Lahontan. All that remains of the lake now are playas and a few remnants lakes. The Central Basin and Range is now high desert plain with the exception of small, often seasonal wetlands, typically alkali flats or saline lakes that are replenished by seasonal rains and runoff. In the west and central part of the region there are characteristic rolling valleys with mountian ranges that have alluvial fan outwashes flowing into the lower basins. Diversity in this region is rather low due to its aridity. With a few exceptions, there are almost no continuous woodlands. The highest elevations in this region, those that extend above treeline, are densely covered with mountain big sagebrush, western serviceberry, snowberry, and low sagebrush, but very few trees. The easternmost part of the Central Basin and Range ecoregion consists of sagebrush valleys, woodlands, mountains, and saline basins. Much of the soil in this region is shallow due to a combination of heavy summer rains and the limestone and dolomite bedrock. As a result, the sagebrush valleys consist of grasses and brush capable of tolerating shallow soils. The woodland zones consist of mainly piñon and juniper forests. Historically, these forests only occurred at higher elevations due to annual natural fires, but fire suppression has allowed the forests to move further down towards the sagebrush valleys. In the more mountainous areas, for example Great Basin National Park (GBNP), various conifers (white fir, Douglas fir, Engelmann spruce, bristlecone pines) dominate. Although high enough to form an alpine zone, climate conditions do not favour the retention of water as most of it rapidly flows out via springs and streams. The GBNP includes a range of the typical floral zones for the region with sagebrush dominated lower elevations, rising through juniper, piñon pine, ponderosa pine, and grassy mountain meadows. Above treeline the habitat is rocky with few plants. Situated on Wheeler Peak, which rises to nearly 4000 m elevation, is a small alpine glacier, the remnant of a more extensive glacier from the last glacial maximum (LGM). Erosional deposition from the glacier's meltwater is evident in much of the of the most-visited parts of the park. The Northern Basin and Range, which is shared with Oregon and Idaho, is a cooler, more mesic, and less mountainous than the Central Basin and Range. This ecoregion consists of high lava plateaus to the northeast, high lava plains to the northwest and semi-arid uplands scattered across northern Nevada. The high lava plateau receives more precipitation and experiences colder winters resulting in cool season grasses such as bluebunch wheatgrass and Idaho fescue. The high lava plains, a large sagebrush steppe, are very similar to the high lava plateau in climate and vegetation, but have many ephemeral pools that are home to a diverse fauna and flora. In the far south of Nevada lies the Mojave Basin and Range, a region extending from California to Arizona and southwestern Utah. The warmer, drier climate and milder winters favours a flora dominated by creosote bush at lower elevations. This desert region is far from uniform and is comprised of three subregions: the Amargosa Desert, creosote-bush dominated basins, and arid footslopes. The Amargosa desert is in the rain shadow of the Spring Mountains and is an internally-drained basin with the greatest temperature extremes. Most of the flora is dry adapted creosote bush and bursage. Further east, creosote bush-dominated basins are scattered between the Mojave Desert mountain ranges, grasslands, blackbrush plains, Joshua trees woodlands, and cactus dominated areas. The Spring Mountains prominently rise above the basin habitats with Mount Charleston reaching over 3600 m elevation. Along the elevational gradient various scrub and bush zones lead to juniper, piñon pine, mountain mahogany, culminating in montane conifers. The Sierra Nevada ecoregion is a mountainous region that is mainly in California but a small part of this ecoregion extends into Nevada along its western border, notably including the foothills of the Carson Range. At mid-elevations, the dry forest is composed of a mix of conifers, including California white fir, incense cedar, Jeffrey pine, with an understory of sagebrush, antelope bitterbrush, and manzanita. At elevations between 7500-9500 ft, heavy snowfall and summer droughts favours vegetation adapted to season extremes like red fir, white pine and mountain hemlock. Though a relatively small area, this extension of the Sierra Nevada fauna is significant as it is not found elsewhere in the state of Nevada. The Arizona-Mexico Plateau ( Ruhlman et al. 2010 ) is the ecoregion with the smallest area among the five in Nevada. It is represented by an area of the Virgin Mountains at the Nevada and Arizona border, which is the western limit of the region that extends east into New Mexico. The region has semiarid grassland and desert-scrub, piñon pine uplands, and ponderosa pine at the highest elevations. Materials and Methods Specimen sources and identification methods Specimens examined were from the following institutions: Cornell University Insect Collection (CUIC), Ithaca, NY; Essig Museum of Entomology (EMEC), University of California, Berkeley; Monte L. Bean Life Science Museum, Arthropod Collection, Brigham Young University (BYUC); California Academy of Sciences, San Francisco, CA (CAS) including the former University of Nevada, Las Vegas (UNLV) holdings now at CAS; the teaching collection held at the Great Basin National Park (GBNP); the Oregon State University Arthropod Collection, Corvallis, OR (OSAC); and the collection of Peter W. Messer, Wisconsin (PWMC). Additional records were sought from the Symbiota Collections of Arthropods Network (SCAN) at http://symbiota4.acis.ufl.edu/scan/portal and Bug Guide at http://bugguide.net/ . We collected additional specimens in the GBNP from 10-15 June 2015 and collections were made in other regions of Nevada by K. Will in June of 2009, 2012, 2013, and May 2016. Collecting methods used included ramp traps, pitfall traps, UV light traps, leaf litter sifting, or hand collected during the day or at night using headlamps. The primary identification references used were Lindroth's Ground Beetles of Canada and Alaska, parts 1-6 ( Lindroth 1961 , Lindroth 1963 , Lindroth 1966 , Lindroth 1968 , Lindroth 1969a , Lindroth 1969b ); American Beetles, chapter 6 ( Ball and Bousquet 2000 ) and published genus level treatment published since Lindroth's work, as appropriate. EMEC and CAS were the primary comparative reference collections. Except as noted, taxonomic names follow Bousquet (2012) . Specimens in EMEC and CAS that are identified by the revisor of a group, e.g. G. Noonan for various Harpalini or F. Heike for Zabrini , were accepted as authoritatively identified. Several authoritative records are in litteris from various sources as noted with specimen records. All other determinations were done by KWW. Specimen sources and identification methods Specimens examined were from the following institutions: Cornell University Insect Collection (CUIC), Ithaca, NY; Essig Museum of Entomology (EMEC), University of California, Berkeley; Monte L. Bean Life Science Museum, Arthropod Collection, Brigham Young University (BYUC); California Academy of Sciences, San Francisco, CA (CAS) including the former University of Nevada, Las Vegas (UNLV) holdings now at CAS; the teaching collection held at the Great Basin National Park (GBNP); the Oregon State University Arthropod Collection, Corvallis, OR (OSAC); and the collection of Peter W. Messer, Wisconsin (PWMC). Additional records were sought from the Symbiota Collections of Arthropods Network (SCAN) at http://symbiota4.acis.ufl.edu/scan/portal and Bug Guide at http://bugguide.net/ . We collected additional specimens in the GBNP from 10-15 June 2015 and collections were made in other regions of Nevada by K. Will in June of 2009, 2012, 2013, and May 2016. Collecting methods used included ramp traps, pitfall traps, UV light traps, leaf litter sifting, or hand collected during the day or at night using headlamps. The primary identification references used were Lindroth's Ground Beetles of Canada and Alaska, parts 1-6 ( Lindroth 1961 , Lindroth 1963 , Lindroth 1966 , Lindroth 1968 , Lindroth 1969a , Lindroth 1969b ); American Beetles, chapter 6 ( Ball and Bousquet 2000 ) and published genus level treatment published since Lindroth's work, as appropriate. EMEC and CAS were the primary comparative reference collections. Except as noted, taxonomic names follow Bousquet (2012) . Specimens in EMEC and CAS that are identified by the revisor of a group, e.g. G. Noonan for various Harpalini or F. Heike for Zabrini , were accepted as authoritatively identified. Several authoritative records are in litteris from various sources as noted with specimen records. All other determinations were done by KWW. Results We identified nearly 900 specimens from localities across the state of Nevada. This includes 79 species of carabids that represent 57 new state records, two state records previously reported online, one confirmation of a previous questionable record for the state, and report 22 records for the Great Basin National Park (GBNP) that includes three of the new state records. Nearly half, 26 of 57, species newly reported for Nevada are shared with three or more adjacent states. Specimen occurance data For all new state records and GBNP records see Suppl. material 1 for the complete specimen occurance data. Full data are also published in GBIF DOI: http://doi.org/10.15468/dl.stpda0 . Also under each species entry the species are linked to a query of the Essig Museum database that pulls all records for that species used in this study and allows for downloading and mapping of those records. Species Accounts Notiophilini Notiophilus nitens LeConte, 1857. New state record. The single specimen of N. nitens from the Lamoille Creek Power Plant Picnic area (Elko County) represents the first report of this tribe from Nevada. This is a northwestern species from the bordering states of Oregon and Idaho. Species of notiophilines are day-active predators. Notiophilus nitens is known to be found in open grasslands in hilly country and is tolerant of dry conditions, and possibly xerophilus. The Nevada record represents the known southern limit of the species. Lindroth (1961) provides a key to species and ecological information. Loricerini Loricera pilicornis pilicornis (Fabricius, 1775). Report of online record. A Holarctic distributed species that can be very common seasonally, near water or on wet, organic-rich mud. It is already reported from all the states surrounding Nevada and so was expected to be found. The single specimen represented as an image posted to BugGuide is all that is known to us. http://bugguide.net/node/view/898466 "South Fork Lake, Elko County, Nevada, USA, June 17, 2004" Image submitted by M. Romero, identification by P. Messer. Accessed 30 June 2016. Keys to species and ecological information were published by Ball and Erwin (1969) and Lindroth (1961) . Carabini Calosoma (Camegonia) prominens LeConte, 1853. New state record. This southwestern species is common in the bordering states of Arizona and California. It is notibly common at lights in Arizona. The single specimen from the Toiyabe Mountains (Nye County) is the northernmost record. These beetles are predaceous, nocturnally active, and powerful fliers. Gidaspow (1959) provides a key to species. Carabus (Tanaocarabus) taedatus agassii LeConte, 1850. GBNP record. We found beetles of this widespread, fairly common species under rocks during the day and walking at night in open, rocky coniferous forest sites from 2000-3000m. A key to species and ecological information was provided by Lindroth (1961). A discussion and reference regarding the various subspecies is provided by Bousquet (2012) . Cicindelini Tiger beetles have been well treated, including taxa from Nevada, by Pearson et al. (2006) . The numerous subspecies and variants, and the different genus-level concepts used in collections we studied make identification challenging. We did note what appear to be specimens of Cicindela willistoni pseudosenilis Horn, 1900, which would represent a new record for Nevada. However, it seems most likely that these specimens are an intergrade or variant of Cicindela willistoni echo Casey, 1897, which is a subspecies found in Nevada. It is likely that a focused effort might turn up new species or new records, but this was beyond the scope of our study. Clivinini Clivina (Clivina) punctulata LeConte, 1852. New state record. This species is found throughout California. They are found along ponds and riparian habitats and probably nocturnal and fossorial, as is typical for the genus. The two specimens from the Humboldt River, above Rye Patch Reservoir (Pershing County) are the easternmost records. Bousquet (1997) provides a key to species. Schizogenius (Schizogenius) depressus LeConte, 1852. New state record. This species is known from all the states surrounding Nevada and so is an expected part of the fauna. They are found among the gravel at the edge of streams in fairly open habitat. Whitehead (1972) provided a key to species and ecological notes. Scaritini Scarites (Scarites) subterraneus Fabricius, 1775. New state record. This is a widespread species known from the neighboring states of California and Arizona. It is a eurytopic species that is tolerant of disturbance and frequently found in agricultural settings. The 11 specimens we studied are all from in or near Las Vegas and probably its occurrence in the state is due to human transport. Bousquet and Skelley (2010) provide a key to species. Patrobini Diplous (Platidius) aterrimus (Dejean, 1828). New state record. A decidedly northern species known from the neighboring states of Oregon, Idaho, and Utah that is found on the gravel shores of fast running streams and rivers. The four specimens we examined include three from the east side of the Sierra Nevada Mountains and one from the Spring Mountains. The Spring Mts. record is a significant southern range extension, but that range is known for other isolated populations such as the recently described Nebria ( Catonebria ) baumanni Kavanaugh, 2015. Lindroth (1961) provides key to species and ecological information. Diplous (Platidius) filicornis (Casey, 1918). New state record. Known from the adjacent states of Oregon and northern California that is found along fast, cold flowing streams. Records from Washoe Co. are from the east side of the Sierra, which is likely the eastern limit of its distribution. Key to species and ecology provided by Lindroth (1961) . Patrobus fossifrons (Eschscholtz, 1823). New state record. A decidedly western beetle, previously known from California, Oregon, Idaho, and Utah. A very hygrophilus species found on lake shores and along slow moving water under debris, e.g. dead catatail ( Typha sp). Lindroth (1961) provides key to species and ecological information. Trechini Trechus (Trechus) tenuiscapus Lindroth, 1961. New state record and GBNP record. Fig. 1 A species found in the northwest with records known for the adjacent states of Oregon and Idaho. Specimens from our sampling in the GBNP were found in open conifer forest above 2300 m. Beetles were found during the day in leaf litter and active at night. This is a significant southern expansion of the known range for this species. Bembidiini Species of Bembidion can often be identified using the keys provided by Lindroth (1963) . However, many species are difficult to identify and a considerable amount of confusion still surrounds many names proposed by T.L. Casey for western species. Confident identification of the specimens that are the basis for the records presented here was only possible with assistance and/or direct identifications provided by D.R. Maddison and J. Sproul (Oregon State University), who are presently revising the genus. Lindroth (1963) also gives ecological information for most species. Bembidion (Bembidion) mutatum Gemminger & Harold, 1868. New state record. A species with a transcontinental distribution in the north known from the neighboring states of Idaho and Utah. Recorded from Douglas County. Bembidion (Eupetedromus) incrematum LeConte, 1860. New state record. A Holarctic species, known from the adjacent states of California, Idaho, and Oregon. Specimens collected on the muddy shore of a man-made reservoir in Lander County. Bembidion (Furcacampa) fuchsii Blaisdell, 1902. New state record. A northwestern species found in the adjacent states of California, Oregon, and Idaho. Found in wet meadow with light cover of Populus and Salix in the Ruby Mountains, Elko County. Bembidion (Furcacampa) versicolor (LeConte, 1847). New state record. A widespread species that can be very abundant in a variety of wetland habitats. Previously known from the adjacent states of Idaho and Oregon. Bembidion (Hirmoplataphus) concolor (Kirby, 1837). GBNP record. A transcontinental North American species found near water. While Lindroth (1963) notes that they are found on bare gravel or coarse sand near running water or on lake shores, all specimens collected in GBNP were under rocks and debris near the water in a seasonal lake at about 3000 m elevation in rocky, clay soil. Bembidion (Hirmoplataphus) quadrulum LeConte, 1861. New state record. A western species, known from all states bordering Nevada and so is an expected species in the fauna. Collected in Elko Co., along Lamoille Creek above 2100 m elevation. Bembidion (Liocosmius) horni Hayward, 1897. New state record. Found across southern California, Arizona, and Utah. Bembidion (Notaphus) approximatum (LeConte, 1852). Confirmation of state record. Reported from the adjacent states of California and Oregon. Specimens previously attributed to this species were apparently misidentifications ( Bousquet 2012 , Lindroth 1963 ). Here we report on 33 confirmed specimens from Nye, Elko, and Lincoln Counties. Bembidion (Notaphus) graphicum Casey, 1918. New state record. A fairly widespread species previously known from the adjacent states of Arizona, Oregon, and Utah. Bembidion (Notaphus) intermedium (Kirby, 1837). New state record. A transcontinental, primarily northern species not reported from any states adjacent to Nevada. There are records from Montana, Wyoming, and Colorado. Found in sandy habitat along the Humboldt River. Bembidion (Notaphus) nubiculosum Chaudoir, 1868. New state record. Found in the southwestern US, known from the adjacent states of Arizona and California. Bembidion (Notaphus) obtusangulum LeConte, 1863. New state record. A midwest to western US species distributed generally in the north and in its southern limited extended along the Rocky and Sierra Mountains. Reported from the adjacent states of California, Oregon, Idaho, and Utah. Bembidion (Notaphus) umbratum (LeConte, 1847). New state record. In collections, specimens identified as B. umbratum may be B. variolosum (Motschulsky, 1859), which is maintained as a junior synonym by Bousquet (2012) following Lindroth (1963) . These are distinct species (D.R. Maddison in litt.) with confirmed specimens of both species known from Nevada. Bembidion umbratum is found in the adjacent states of California, Oregon, and Idaho. Bembidion (Peryphodes) ephippigerum (LeConte, 1852). New state record. Previously only reported from California. The single specimen from Washoe Co. is a slight expansion eastward of the species as it was known from several eastern California counties in the Sierras. Bembidion (Peryphus) nevadense Ulke, 1875. GBNP record. A western North American montane species. Specimens from GBNP were collected near small and medium size flowing water and, most abundantly, under rocks and debris near the water in a seasonal lake at about 3000 m elevation in rocky, clay soil. Bembidion (Peryphus) striola (LeConte, 1852). New state record. Reported previously from California and Oregon primarily in coastal counties. Bembidion (Plataphus) laxatum Casey, 1918. New state record. GBNP record. Reported previously from California and Washington, with an unconfirmed record in British Columbia. Six specimens collected above 3000 m elevation, on wet, rocky, open soil around the shore of Teresa Lake, Great Basin National Park, White Pine County. Bembidion (Testediolum) nebraskense LeConte, 1863. GBNP record. A fairly common western North American species found in the park at around 2100m elevation along Snake Creek near a few pools of water. Bembidion (Testediolum) obscuripenne Blaisdell, 1902. New state record. A western montane species, previously known from the adjacent states of California and Oregon. Found in Lander County along a small stream in open habitat. Bembidion (Trepanedoris) acutifrons LeConte, 1879. New state record. A western species, known from the adjacent states of Oregon and Utah. The single specimen we examined was collected near the Humboldt River in Eureka Co., but its exact habitat is not known. Bembidion (Trepanedoris) anguliferum (LeConte, 1852). New state record. Previously only reported from California. Elaphropus (Barytachys) anthrax (LeConte, 1852). New state record. The species was previously reported from the adjacent states of California, Oregon, and Idaho. Hayward (1900) provides a key to species. Elaphropus (Barytachys) conjugens (Notman, 1919). New state record. This species was known only from southern Arizona and the Nye and Clark County records are a significant extension northward. Hayward (1900) provided a key to species (as Tachys trechiformis Hayward, 1900). Elaphropus (Barytachys) dolosus (LeConte, 1848). New state record. A widely distributed species previously known from the adjacent states of California and Arizona found on the sandy banks of rivers and lakes. Lindroth (1963) provides a key to species and ecological information. Polyderis rufotestacea (Hayward, 1900). New state record. A western species, known from almost all states adjacent to Nevada; Arizona, California, Oregon, and Idaho. Hayward (1900) provides a key to species. Psydrini Psydrus piceus LeConte, 1846. GBNP record. Distributed transcontinentally in the north but restricted to the west in the southern part of its range. The single specimen collected in the GBNP was found under the bark on a log of an unidentified conifer. These beetles are always found under bark in deadwood and they are regularly collected in the Californian Sierras under the bark of dead Pinus and Pseudotsuga . The pygidial gland secretions emitted when they are disturbed are extremely pungent. Lindroth (1961) provided a key to species. Brachinini Brachinus (Neobrachinus) elongatulus Chaudoir, 1876. New state record. Often found to be extremely common along the shores of lakes and streams. Known previously from the adjacent states of Arizona, California, and Oregon. Flight wing is full and they are frequently attracted to lights. Erwin (1970) provides a key to species and ecological information. Brachinus (Neobrachinus) phaeocerus Chaudoir, 1868. Report of online record. A southern species, known from as far west as the adjacent state of Arizona. This record pushes the distribution northward. The single specimen represented as an image posted to BugGuide is all that is known to us. http://bugguide.net/node/view/725859 "Las Vegas in the Red Rock Canyon National Conservation Area, Clark County, Nevada, USA April 23, 2012" image contributed by D. Lund in November 2012. Identification by T.L. Erwin. Accessed 23 June 2016. Erwin (1970) provides a key to species and ecological information. Brachinus (Neobrachinus) quadripennis Dejean, 1825. New state record. A very widespread species known from California, Oregon, Idaho, and Utah. Its occurrence in Nevada is expected. Erwin (1970) provides a key to species and ecological information. Chlaeniini Chlaenius (Chlaeniellus) obsoletus LeConte, 1851. New state record. A southwestern species previously reported from California and Arizona. Sometimes found along medium sized streams. Bell (1960) provides a key to species. Chlaenius (Chlaeniellus) pennsylvanicus blanditus Casey, 1920. New state record. The nominate subspecies is transcontinental in the north and so is found in the adjacent states of Oregon and Idaho, while C. pennsylvanicus blanditus is from Arizona and Utah. This subspecies is more of a green color and stouter in form but otherwise is little different than the nominate. Like most Chlaenius species, C. pennsylvanicus blanditus is hygrophilus and found near water, probably slow-flowing or standing water. Key to species and subspecies is provided by Bell (1960) . Galeritini Galerita (Progaleritina) lecontei lecontei Dejean, 1831. New state record. The northern subspecies of a southern species, ( G. lecontei bicoloripes Reichardt is found in central Mexico) known from the adjacent states of Arizona and California. These beetles often come to lights and are nocturnally active in various types of mesic woodlands. Lindroth (1969a) provides a key to species. Harpalini Bradycellus (Liocellus) nitidus (Dejean, 1829). New state record. Known from all the states surrounding Nevada except for Idaho and so is an expected part of the Nevada fauna. Beetles are found along seasonal streams under rocks and in gravel, but nothing is known about their life history. Fall (1905) and Casey (1924) provide keys to species and descriptions, however, the subgenus Liocellus is in need of revision. Lindroth (1968) includes this species in his key as well. Bradycellus (Stenocellus) rivalis LeConte, 1858. New state record. This is a fairly common species in southern California and is also reported from Arizona. Sometimes it appears abundantly at lights in California. Its habits are unknown, but probably like other Bradycellus they are hygrophilus. Lindroth (1968) includes this species in his key. Notiobia (Anisotarsus) terminata (Say, 1823). New state record. A very widespread species, but only reported from Arizona among the states adjacent to Nevada. The single specimen we studied was from an urban area in the southern part of the Las Vegas region. As these beetles are known to thrive in disturbed habitats, this record may be due to human transport and it is unknown if an endemic or naturalized population occurs in Nevada. Lindroth (1968) has a key to species and ecological notes. Selenophorus ( Selenophorus ) famulus Casey, 1914. New state record. This species is known from southern California and Arizona. A record for a single female specimen from Mesquite, NV, Clark County was sent to us by P. Messer (in litt.). The specimen is deposited in his collection. No published keys cover this or many species of Selenophorus and a revision of the group is much needed. If this species is typical for the genus in its ecology, then it is an arid habitat species, found in sandy soils with sparse vegetation. Stenolophus (Agonoderus) comma (Fabricius, 1775). New state record. An extraordinarily widespread species, often extremely abundant and attracted to lights. It is known from every state adjacent to Nevada and so it is only surprising that it had not been recorded from Nevada previously. Lindroth (1968) provides a key to species and ecological notes. Stenolophus (Stenolophus) fuliginosus Dejean, 1829. New state record. A widespread species in the middle latitudes of North America previously reported from California, Oregon, and Idaho. Beetles are found on the shore of slow or still waters among Carex and Typha . The key by Lindroth (1968) covers this species. Stenolophus (Stenolophus) ochropezus (Say, 1823). New state record. Arguably one of the most common and widespread species of carabid beetle in North America. Reported from the adjacent states of Arizona, California, and Utah. Common on wet mud along the shoreline of still waters and in other wet areas with vegetation cover. Lindroth (1968) provides a key to species and ecological notes. Bradycellus (Stenocellus) congener (LeConte, 1847). GBNP record. A common, transcontinentally distributed species. The key by Lindroth (1968) covers this species. Discoderus amoenus LeConte, 1863. GBNP record. This species is found from Wyoming to southern California, and being xerophilic, is relatively common in Nevada. The genus is in need of revision. Casey (1914) covers many species, but the work is incomplete. Harpalus (Opadius) fraternus LeConte, 1852. GBNP record. A fairly common species found in sites across the park. Collected in pitfall traps, headlamp searching at night and under woody debris during the day. All specimens collected in GBNP and others studied (EMEC) from Nevada are from stands of aspen or aspen mixed with either willow or fir. This differs from Lindroth's description of the ecology of the species further north, as being "[i]n dry, open country with scarce vegetation, often on sandy soil" ( Lindroth 1968 ). Harpalus (Harpalobius) fuscipalpis Sturm, 1818. GBNP record. A Holarctic species. One specimen was collected near the Baker Creek Campground in rocky, lightly vegetated habitat at night. Key to species and ecology provided by Lindroth (1968) as Harpalellus basilaris Kirby. Harpalus (Harpalus) ellipsis LeConte, 1847. New State record. Known previously from adjacent states of Arizona, Utah, Idaho, and Oregon. Key to species by Lindroth (1968) and Noonan (1991) . This species can only be confidently separated from Harpalus obnixus Casey, a species previously reported from Nevada, by examination of the male genitalia. Harpalus opacipennis (Haldeman 1843). GBNP record. A widespread North American species. Found throughout the park at 2000-2400 m elevation by pit fall traping and night searching in grassy open habitat or areas of light, low brush. Key to species provided by Lindroth (1968) who also notes this species is common on sand and gravel soils in areas of sparse vegitation. Platynini Agonum (Agonum) placidum (Say, 1823). New state record and GBNP record. Fig. 2 A widespread species known from all the states surrounding Nevada and so an expected part of the fauna. We collected specimens in the GBNP while headlamp searching at night, in sites at around 2000 m elevation, along streams, looking under rocks. A specimen is also known from the park at over 3000 m elevation near Stella Lake. Other records for A. placidum are on the east slope of the Sierras. Liebherr (1994) provides a key to species and Lindroth (1966) includes ecological notes. Agonum (Europhilus) gratiosum (Mannerheim, 1853). New state record. This species has a northern Holarctic distribution and has been found in the neighboring states of California, Oregon, and Utah. These beetles are found in fairly open habitats with moist soils, but not necessarily close to water. However, specimens from Ruby Lake (Elko Co.) were found near the lake under dead Typha . Liebherr (1994) provides a key to species and Lindroth (1966) , Lindroth (1969a) includes ecological notes. Agonum (Platynomicrus) nigriceps LeConte, 1846. New state record. A Holarctic, decidedly northern species only reported from Idaho of the adjacent states. This is a very hygrophilous species found among and on the plants growing in the water. Beetles can be readly collected by treading the vegetation into the water. The eight specimens we examined from the Humboldt River area in Pershing County seem a modest southward extension of this northern species. The single specimen from Las Vegas Valley, in Clark County is a remarkable southern record. Liebherr (1994) provides a key to species and includes ecological notes. Rhadine sp. GBNP record. A single specimen of Rhadine was collected in the Baker Creek Campground latrine. Significant effort was made to find additional specimens in the area, but none were found. The taxonomy of the genus is in need of revision. In addition to the single specimen we sampled during the study, numerous specimens were seen in collections, but they cannot be confidently identified. The genus includes both cave and surface dwelling species ( Gómez et al. 2016 ). Pterostichini Pterostichus (Pseudomaseus) luctuosus (Dejean, 1828). New state record. A widespread and relatively commonly collected species in the north and middle latitudes of North America, especially in the east. Of the states surrounding Nevada, it is only reported from Idaho. It is a decidedly hygrophilus and can be found near water and in very wet, marsh areas. The habitat where the Nevada specimens were collected, Ruby Lake (Elko Co.), is very typical for them. Lindroth (1966) provides a key to species and ecological notes. Pterostichus (Hypherpes) protractus LeConte, 1860. GBNP record. One of the most widespread species of the subgenus, ranging from northern New Mexico to Alberta, Canada, west to California. Found throughout Nevada in coniferous forests from near treeline to 2000 m (and probably lower) elevation. At lower elevations confined to riparian habitat under woody debris and in thick leaf litter. These nocturnal predatory beetles were the most common species found during our sampling at GBNP. They were significantly abundant in areas with ponderosa pine and fir. Lindroth (1966) provides a key to species, however, the key does not cover the numerous southern species in the subgenus, which is in need of revision. Pterostichus (Bothriopterus) lustrans LeConte, 1851. GBNP record. A western North American species found in numerous locations across Nevada. In the GBNP specimens were collected from multiple sites in open vegetation along the riparian corridor, though not in tight association with the stream. Lindroth (1966) provides a key to species. Sphodrini Synuchus dubius (LeConte, 1854). GBNP record. A central southwest species reported from the bordering states of Arizona and Utah. Specimens examined are from three general localities in the eastern part of the state. We collected specimens from several locations in the Great Basin National Park by searching the forest leaf litter at night in areas above 2000 m. These are flightless beetles and other localities for this species are at similar elevations, suggesting significantly disjunct populations. Lindroth (1966) provides a key to species. Laemostenus (Laemostenus) complanatus (Dejean, 1828). New state record. A globally subcosmopolitan species that is an adventive and synanthropic. Known from the adjacent states of California and Oregon, especially in near-coastal regions in urban areas such as watered lawns and parks. The single specimen we studied is only labeled Clark Co., but if there is an established population it would probably be in human maintained habitat in the Las Vegas area. Lindroth (1966) provides a key to species. Calathus (Acalathus) advena (LeConte, 1846). GBNP record. A widespread forest, leaf litter species that occurs in the northeastern part of Nevada. In the conifer forest habitat of the GBNP we found this to be the most commonly encountered species. Frequently found with Pterostichus protractus . Lindroth (1966) and Ball and Nègre (1972) provide keys to species and ecological notes. Zabrini Amara (Amarocelia) farcta LeConte, 1855. GBNP record A western North American species found in various open habitats near water. All specimens collected in GBNP were under rocks and debris near the water in a seasonal lake at about 3000 m elevation. Lindroth (1968) provides a key that includes this species. Amara (Bradytus) lindrothi Hieke, 1990. GBNP record. Known from localities widely distributed across western North America. GBNP is near its southern limit. Specimens were collected in open conifer forest between 3000 and 3200 m elevation, on wet ground, under rocks and debris. Identification is possible using the description and illustrations provided by Hieke (1990) . Amara (Celia) idahoana (Casey, 1924). New state record. Found in the west, in states north of Nevada, including the adjacent states of Idaho and Oregon. Records from Nevada extend the range significantly southward. However, while the record from near Weeks (Lyon Co.) is in an irrigated area near the Carson River, the record from Nye County is in a desert habitat and may be a mislabeled specimen. Lindroth (1968) provides a key to species and ecological notes. Amara (Celia) sinuosa (Casey, 1918). New state record. A northern species, known from the adjacent states of Idaho and Utah. The single specimen we examined was collected in 1936 and labeled Governors Canyon, Nevada. We have been unable to find a match for that exact locality. Lindroth (1968) provides a key to species and notes that the species is found "[o]n open, dry, sandy or gravelly ground." Amara (Curtonotus) carinata (LeConte, 1847). New state record. A widespread species found in all states adjacent to Nevada and so an expected part of the carabid fauna. Beetles are found in grasslands, frequently near alkaline habitats. Lindroth (1968) provides provides a key to species and ecological notes. Amara (Paracelia) quenseli quenseli (Schönherr, 1806). GBNP record. A Holarctic species that is frequently common. Specimens were collected across the GBNP, during the day and at night in open, grassy habitat. Lindroth (1968) provides a key that includes this species. Lebiini Apristus pugetanus Casey, 1920. New state record. A western North American species, already known from all the states surrounding Nevada and so it is an expected member of the fauna. The beetles are found in gravel and sand along rivers, creek and springs, although they are often found some distance from the water. They are frequently day active. Lindroth (1968) provides a key to species. Axinopalpus biplagiatus (Dejean, 1825). New state record. A widespread species found in all states adjacent to Nevada and so an expected part of the carabid fauna. Little is known about the ecology and habits of this species but it has been collected in a variety of open habitats and of the ones we studied from Nevada, one was taken from Joshua Trees. Lindroth (1968) discusses this species, but there are more species in the region and there is no revision of the group. Comparison with types and original descriptions must be used for identification. Axinopalpus fusciceps LeConte, 1851. New state record. A western species, found in the middle latitudes of North America, south into Guatemala. Know from scattered records in the adjacent states of Arizona, California, and Idaho. Nothing is known of this species habits and there is no key to species for the genus. For identification recourse to original descriptions is needed. Cymindis (Tarulus) arizonensis Schaeffer, 1910. New state record. This species is known from southwestern California and Arizona. The record from Clark Co. is a slight expansion of its range. Lindroth (1968) provides a key to species and discussion of the taxonomic difficulties related to this species. Lebia (Lebia) perita Casey, 1920. New state record. A western species, known previously from the adjacent states of California, Oregon, and Idaho. One specimen from Spencer Hot Spring (Landers County) was taken on whiteflower rabbitbrush ( Chrysothamnus albidus ). This marks the easternmost record for the species. Madge (1967) provides a key to species and distributional data. Zuphiini Pseudaptinus (Thalpius) rufulus (LeConte, 1851). New state record. A western North American species, known previously from Arizona, California, and Oregon. Little is known about the habits of this species. A key to species of the subgenus Thalpius was published by Messer (2011) . Pseudomorphini Pseudomorpha castanea Casey, 1909. GBNP record. This species was previosuly reported from California, Oregon, and Utah. As far as known, pseudomorphines are myrmecophiles and ovoviviparous ( Liebherr and Kavanaugh 1985 ). Notman (1925) includes a key to species, however, many undescribed species exist (T.L. Erwin, in litt.) and the genus is in need of revision. Specimen occurance data For all new state records and GBNP records see Suppl. material 1 for the complete specimen occurance data. Full data are also published in GBIF DOI: http://doi.org/10.15468/dl.stpda0 . Also under each species entry the species are linked to a query of the Essig Museum database that pulls all records for that species used in this study and allows for downloading and mapping of those records. Species Accounts Notiophilini Notiophilus nitens LeConte, 1857. New state record. The single specimen of N. nitens from the Lamoille Creek Power Plant Picnic area (Elko County) represents the first report of this tribe from Nevada. This is a northwestern species from the bordering states of Oregon and Idaho. Species of notiophilines are day-active predators. Notiophilus nitens is known to be found in open grasslands in hilly country and is tolerant of dry conditions, and possibly xerophilus. The Nevada record represents the known southern limit of the species. Lindroth (1961) provides a key to species and ecological information. Loricerini Loricera pilicornis pilicornis (Fabricius, 1775). Report of online record. A Holarctic distributed species that can be very common seasonally, near water or on wet, organic-rich mud. It is already reported from all the states surrounding Nevada and so was expected to be found. The single specimen represented as an image posted to BugGuide is all that is known to us. http://bugguide.net/node/view/898466 "South Fork Lake, Elko County, Nevada, USA, June 17, 2004" Image submitted by M. Romero, identification by P. Messer. Accessed 30 June 2016. Keys to species and ecological information were published by Ball and Erwin (1969) and Lindroth (1961) . Carabini Calosoma (Camegonia) prominens LeConte, 1853. New state record. This southwestern species is common in the bordering states of Arizona and California. It is notibly common at lights in Arizona. The single specimen from the Toiyabe Mountains (Nye County) is the northernmost record. These beetles are predaceous, nocturnally active, and powerful fliers. Gidaspow (1959) provides a key to species. Carabus (Tanaocarabus) taedatus agassii LeConte, 1850. GBNP record. We found beetles of this widespread, fairly common species under rocks during the day and walking at night in open, rocky coniferous forest sites from 2000-3000m. A key to species and ecological information was provided by Lindroth (1961). A discussion and reference regarding the various subspecies is provided by Bousquet (2012) . Cicindelini Tiger beetles have been well treated, including taxa from Nevada, by Pearson et al. (2006) . The numerous subspecies and variants, and the different genus-level concepts used in collections we studied make identification challenging. We did note what appear to be specimens of Cicindela willistoni pseudosenilis Horn, 1900, which would represent a new record for Nevada. However, it seems most likely that these specimens are an intergrade or variant of Cicindela willistoni echo Casey, 1897, which is a subspecies found in Nevada. It is likely that a focused effort might turn up new species or new records, but this was beyond the scope of our study. Clivinini Clivina (Clivina) punctulata LeConte, 1852. New state record. This species is found throughout California. They are found along ponds and riparian habitats and probably nocturnal and fossorial, as is typical for the genus. The two specimens from the Humboldt River, above Rye Patch Reservoir (Pershing County) are the easternmost records. Bousquet (1997) provides a key to species. Schizogenius (Schizogenius) depressus LeConte, 1852. New state record. This species is known from all the states surrounding Nevada and so is an expected part of the fauna. They are found among the gravel at the edge of streams in fairly open habitat. Whitehead (1972) provided a key to species and ecological notes. Scaritini Scarites (Scarites) subterraneus Fabricius, 1775. New state record. This is a widespread species known from the neighboring states of California and Arizona. It is a eurytopic species that is tolerant of disturbance and frequently found in agricultural settings. The 11 specimens we studied are all from in or near Las Vegas and probably its occurrence in the state is due to human transport. Bousquet and Skelley (2010) provide a key to species. Patrobini Diplous (Platidius) aterrimus (Dejean, 1828). New state record. A decidedly northern species known from the neighboring states of Oregon, Idaho, and Utah that is found on the gravel shores of fast running streams and rivers. The four specimens we examined include three from the east side of the Sierra Nevada Mountains and one from the Spring Mountains. The Spring Mts. record is a significant southern range extension, but that range is known for other isolated populations such as the recently described Nebria ( Catonebria ) baumanni Kavanaugh, 2015. Lindroth (1961) provides key to species and ecological information. Diplous (Platidius) filicornis (Casey, 1918). New state record. Known from the adjacent states of Oregon and northern California that is found along fast, cold flowing streams. Records from Washoe Co. are from the east side of the Sierra, which is likely the eastern limit of its distribution. Key to species and ecology provided by Lindroth (1961) . Patrobus fossifrons (Eschscholtz, 1823). New state record. A decidedly western beetle, previously known from California, Oregon, Idaho, and Utah. A very hygrophilus species found on lake shores and along slow moving water under debris, e.g. dead catatail ( Typha sp). Lindroth (1961) provides key to species and ecological information. Trechini Trechus (Trechus) tenuiscapus Lindroth, 1961. New state record and GBNP record. Fig. 1 A species found in the northwest with records known for the adjacent states of Oregon and Idaho. Specimens from our sampling in the GBNP were found in open conifer forest above 2300 m. Beetles were found during the day in leaf litter and active at night. This is a significant southern expansion of the known range for this species. Bembidiini Species of Bembidion can often be identified using the keys provided by Lindroth (1963) . However, many species are difficult to identify and a considerable amount of confusion still surrounds many names proposed by T.L. Casey for western species. Confident identification of the specimens that are the basis for the records presented here was only possible with assistance and/or direct identifications provided by D.R. Maddison and J. Sproul (Oregon State University), who are presently revising the genus. Lindroth (1963) also gives ecological information for most species. Bembidion (Bembidion) mutatum Gemminger & Harold, 1868. New state record. A species with a transcontinental distribution in the north known from the neighboring states of Idaho and Utah. Recorded from Douglas County. Bembidion (Eupetedromus) incrematum LeConte, 1860. New state record. A Holarctic species, known from the adjacent states of California, Idaho, and Oregon. Specimens collected on the muddy shore of a man-made reservoir in Lander County. Bembidion (Furcacampa) fuchsii Blaisdell, 1902. New state record. A northwestern species found in the adjacent states of California, Oregon, and Idaho. Found in wet meadow with light cover of Populus and Salix in the Ruby Mountains, Elko County. Bembidion (Furcacampa) versicolor (LeConte, 1847). New state record. A widespread species that can be very abundant in a variety of wetland habitats. Previously known from the adjacent states of Idaho and Oregon. Bembidion (Hirmoplataphus) concolor (Kirby, 1837). GBNP record. A transcontinental North American species found near water. While Lindroth (1963) notes that they are found on bare gravel or coarse sand near running water or on lake shores, all specimens collected in GBNP were under rocks and debris near the water in a seasonal lake at about 3000 m elevation in rocky, clay soil. Bembidion (Hirmoplataphus) quadrulum LeConte, 1861. New state record. A western species, known from all states bordering Nevada and so is an expected species in the fauna. Collected in Elko Co., along Lamoille Creek above 2100 m elevation. Bembidion (Liocosmius) horni Hayward, 1897. New state record. Found across southern California, Arizona, and Utah. Bembidion (Notaphus) approximatum (LeConte, 1852). Confirmation of state record. Reported from the adjacent states of California and Oregon. Specimens previously attributed to this species were apparently misidentifications ( Bousquet 2012 , Lindroth 1963 ). Here we report on 33 confirmed specimens from Nye, Elko, and Lincoln Counties. Bembidion (Notaphus) graphicum Casey, 1918. New state record. A fairly widespread species previously known from the adjacent states of Arizona, Oregon, and Utah. Bembidion (Notaphus) intermedium (Kirby, 1837). New state record. A transcontinental, primarily northern species not reported from any states adjacent to Nevada. There are records from Montana, Wyoming, and Colorado. Found in sandy habitat along the Humboldt River. Bembidion (Notaphus) nubiculosum Chaudoir, 1868. New state record. Found in the southwestern US, known from the adjacent states of Arizona and California. Bembidion (Notaphus) obtusangulum LeConte, 1863. New state record. A midwest to western US species distributed generally in the north and in its southern limited extended along the Rocky and Sierra Mountains. Reported from the adjacent states of California, Oregon, Idaho, and Utah. Bembidion (Notaphus) umbratum (LeConte, 1847). New state record. In collections, specimens identified as B. umbratum may be B. variolosum (Motschulsky, 1859), which is maintained as a junior synonym by Bousquet (2012) following Lindroth (1963) . These are distinct species (D.R. Maddison in litt.) with confirmed specimens of both species known from Nevada. Bembidion umbratum is found in the adjacent states of California, Oregon, and Idaho. Bembidion (Peryphodes) ephippigerum (LeConte, 1852). New state record. Previously only reported from California. The single specimen from Washoe Co. is a slight expansion eastward of the species as it was known from several eastern California counties in the Sierras. Bembidion (Peryphus) nevadense Ulke, 1875. GBNP record. A western North American montane species. Specimens from GBNP were collected near small and medium size flowing water and, most abundantly, under rocks and debris near the water in a seasonal lake at about 3000 m elevation in rocky, clay soil. Bembidion (Peryphus) striola (LeConte, 1852). New state record. Reported previously from California and Oregon primarily in coastal counties. Bembidion (Plataphus) laxatum Casey, 1918. New state record. GBNP record. Reported previously from California and Washington, with an unconfirmed record in British Columbia. Six specimens collected above 3000 m elevation, on wet, rocky, open soil around the shore of Teresa Lake, Great Basin National Park, White Pine County. Bembidion (Testediolum) nebraskense LeConte, 1863. GBNP record. A fairly common western North American species found in the park at around 2100m elevation along Snake Creek near a few pools of water. Bembidion (Testediolum) obscuripenne Blaisdell, 1902. New state record. A western montane species, previously known from the adjacent states of California and Oregon. Found in Lander County along a small stream in open habitat. Bembidion (Trepanedoris) acutifrons LeConte, 1879. New state record. A western species, known from the adjacent states of Oregon and Utah. The single specimen we examined was collected near the Humboldt River in Eureka Co., but its exact habitat is not known. Bembidion (Trepanedoris) anguliferum (LeConte, 1852). New state record. Previously only reported from California. Elaphropus (Barytachys) anthrax (LeConte, 1852). New state record. The species was previously reported from the adjacent states of California, Oregon, and Idaho. Hayward (1900) provides a key to species. Elaphropus (Barytachys) conjugens (Notman, 1919). New state record. This species was known only from southern Arizona and the Nye and Clark County records are a significant extension northward. Hayward (1900) provided a key to species (as Tachys trechiformis Hayward, 1900). Elaphropus (Barytachys) dolosus (LeConte, 1848). New state record. A widely distributed species previously known from the adjacent states of California and Arizona found on the sandy banks of rivers and lakes. Lindroth (1963) provides a key to species and ecological information. Polyderis rufotestacea (Hayward, 1900). New state record. A western species, known from almost all states adjacent to Nevada; Arizona, California, Oregon, and Idaho. Hayward (1900) provides a key to species. Psydrini Psydrus piceus LeConte, 1846. GBNP record. Distributed transcontinentally in the north but restricted to the west in the southern part of its range. The single specimen collected in the GBNP was found under the bark on a log of an unidentified conifer. These beetles are always found under bark in deadwood and they are regularly collected in the Californian Sierras under the bark of dead Pinus and Pseudotsuga . The pygidial gland secretions emitted when they are disturbed are extremely pungent. Lindroth (1961) provided a key to species. Brachinini Brachinus (Neobrachinus) elongatulus Chaudoir, 1876. New state record. Often found to be extremely common along the shores of lakes and streams. Known previously from the adjacent states of Arizona, California, and Oregon. Flight wing is full and they are frequently attracted to lights. Erwin (1970) provides a key to species and ecological information. Brachinus (Neobrachinus) phaeocerus Chaudoir, 1868. Report of online record. A southern species, known from as far west as the adjacent state of Arizona. This record pushes the distribution northward. The single specimen represented as an image posted to BugGuide is all that is known to us. http://bugguide.net/node/view/725859 "Las Vegas in the Red Rock Canyon National Conservation Area, Clark County, Nevada, USA April 23, 2012" image contributed by D. Lund in November 2012. Identification by T.L. Erwin. Accessed 23 June 2016. Erwin (1970) provides a key to species and ecological information. Brachinus (Neobrachinus) quadripennis Dejean, 1825. New state record. A very widespread species known from California, Oregon, Idaho, and Utah. Its occurrence in Nevada is expected. Erwin (1970) provides a key to species and ecological information. Chlaeniini Chlaenius (Chlaeniellus) obsoletus LeConte, 1851. New state record. A southwestern species previously reported from California and Arizona. Sometimes found along medium sized streams. Bell (1960) provides a key to species. Chlaenius (Chlaeniellus) pennsylvanicus blanditus Casey, 1920. New state record. The nominate subspecies is transcontinental in the north and so is found in the adjacent states of Oregon and Idaho, while C. pennsylvanicus blanditus is from Arizona and Utah. This subspecies is more of a green color and stouter in form but otherwise is little different than the nominate. Like most Chlaenius species, C. pennsylvanicus blanditus is hygrophilus and found near water, probably slow-flowing or standing water. Key to species and subspecies is provided by Bell (1960) . Galeritini Galerita (Progaleritina) lecontei lecontei Dejean, 1831. New state record. The northern subspecies of a southern species, ( G. lecontei bicoloripes Reichardt is found in central Mexico) known from the adjacent states of Arizona and California. These beetles often come to lights and are nocturnally active in various types of mesic woodlands. Lindroth (1969a) provides a key to species. Harpalini Bradycellus (Liocellus) nitidus (Dejean, 1829). New state record. Known from all the states surrounding Nevada except for Idaho and so is an expected part of the Nevada fauna. Beetles are found along seasonal streams under rocks and in gravel, but nothing is known about their life history. Fall (1905) and Casey (1924) provide keys to species and descriptions, however, the subgenus Liocellus is in need of revision. Lindroth (1968) includes this species in his key as well. Bradycellus (Stenocellus) rivalis LeConte, 1858. New state record. This is a fairly common species in southern California and is also reported from Arizona. Sometimes it appears abundantly at lights in California. Its habits are unknown, but probably like other Bradycellus they are hygrophilus. Lindroth (1968) includes this species in his key. Notiobia (Anisotarsus) terminata (Say, 1823). New state record. A very widespread species, but only reported from Arizona among the states adjacent to Nevada. The single specimen we studied was from an urban area in the southern part of the Las Vegas region. As these beetles are known to thrive in disturbed habitats, this record may be due to human transport and it is unknown if an endemic or naturalized population occurs in Nevada. Lindroth (1968) has a key to species and ecological notes. Selenophorus ( Selenophorus ) famulus Casey, 1914. New state record. This species is known from southern California and Arizona. A record for a single female specimen from Mesquite, NV, Clark County was sent to us by P. Messer (in litt.). The specimen is deposited in his collection. No published keys cover this or many species of Selenophorus and a revision of the group is much needed. If this species is typical for the genus in its ecology, then it is an arid habitat species, found in sandy soils with sparse vegetation. Stenolophus (Agonoderus) comma (Fabricius, 1775). New state record. An extraordinarily widespread species, often extremely abundant and attracted to lights. It is known from every state adjacent to Nevada and so it is only surprising that it had not been recorded from Nevada previously. Lindroth (1968) provides a key to species and ecological notes. Stenolophus (Stenolophus) fuliginosus Dejean, 1829. New state record. A widespread species in the middle latitudes of North America previously reported from California, Oregon, and Idaho. Beetles are found on the shore of slow or still waters among Carex and Typha . The key by Lindroth (1968) covers this species. Stenolophus (Stenolophus) ochropezus (Say, 1823). New state record. Arguably one of the most common and widespread species of carabid beetle in North America. Reported from the adjacent states of Arizona, California, and Utah. Common on wet mud along the shoreline of still waters and in other wet areas with vegetation cover. Lindroth (1968) provides a key to species and ecological notes. Bradycellus (Stenocellus) congener (LeConte, 1847). GBNP record. A common, transcontinentally distributed species. The key by Lindroth (1968) covers this species. Discoderus amoenus LeConte, 1863. GBNP record. This species is found from Wyoming to southern California, and being xerophilic, is relatively common in Nevada. The genus is in need of revision. Casey (1914) covers many species, but the work is incomplete. Harpalus (Opadius) fraternus LeConte, 1852. GBNP record. A fairly common species found in sites across the park. Collected in pitfall traps, headlamp searching at night and under woody debris during the day. All specimens collected in GBNP and others studied (EMEC) from Nevada are from stands of aspen or aspen mixed with either willow or fir. This differs from Lindroth's description of the ecology of the species further north, as being "[i]n dry, open country with scarce vegetation, often on sandy soil" ( Lindroth 1968 ). Harpalus (Harpalobius) fuscipalpis Sturm, 1818. GBNP record. A Holarctic species. One specimen was collected near the Baker Creek Campground in rocky, lightly vegetated habitat at night. Key to species and ecology provided by Lindroth (1968) as Harpalellus basilaris Kirby. Harpalus (Harpalus) ellipsis LeConte, 1847. New State record. Known previously from adjacent states of Arizona, Utah, Idaho, and Oregon. Key to species by Lindroth (1968) and Noonan (1991) . This species can only be confidently separated from Harpalus obnixus Casey, a species previously reported from Nevada, by examination of the male genitalia. Harpalus opacipennis (Haldeman 1843). GBNP record. A widespread North American species. Found throughout the park at 2000-2400 m elevation by pit fall traping and night searching in grassy open habitat or areas of light, low brush. Key to species provided by Lindroth (1968) who also notes this species is common on sand and gravel soils in areas of sparse vegitation. Platynini Agonum (Agonum) placidum (Say, 1823). New state record and GBNP record. Fig. 2 A widespread species known from all the states surrounding Nevada and so an expected part of the fauna. We collected specimens in the GBNP while headlamp searching at night, in sites at around 2000 m elevation, along streams, looking under rocks. A specimen is also known from the park at over 3000 m elevation near Stella Lake. Other records for A. placidum are on the east slope of the Sierras. Liebherr (1994) provides a key to species and Lindroth (1966) includes ecological notes. Agonum (Europhilus) gratiosum (Mannerheim, 1853). New state record. This species has a northern Holarctic distribution and has been found in the neighboring states of California, Oregon, and Utah. These beetles are found in fairly open habitats with moist soils, but not necessarily close to water. However, specimens from Ruby Lake (Elko Co.) were found near the lake under dead Typha . Liebherr (1994) provides a key to species and Lindroth (1966) , Lindroth (1969a) includes ecological notes. Agonum (Platynomicrus) nigriceps LeConte, 1846. New state record. A Holarctic, decidedly northern species only reported from Idaho of the adjacent states. This is a very hygrophilous species found among and on the plants growing in the water. Beetles can be readly collected by treading the vegetation into the water. The eight specimens we examined from the Humboldt River area in Pershing County seem a modest southward extension of this northern species. The single specimen from Las Vegas Valley, in Clark County is a remarkable southern record. Liebherr (1994) provides a key to species and includes ecological notes. Rhadine sp. GBNP record. A single specimen of Rhadine was collected in the Baker Creek Campground latrine. Significant effort was made to find additional specimens in the area, but none were found. The taxonomy of the genus is in need of revision. In addition to the single specimen we sampled during the study, numerous specimens were seen in collections, but they cannot be confidently identified. The genus includes both cave and surface dwelling species ( Gómez et al. 2016 ). Pterostichini Pterostichus (Pseudomaseus) luctuosus (Dejean, 1828). New state record. A widespread and relatively commonly collected species in the north and middle latitudes of North America, especially in the east. Of the states surrounding Nevada, it is only reported from Idaho. It is a decidedly hygrophilus and can be found near water and in very wet, marsh areas. The habitat where the Nevada specimens were collected, Ruby Lake (Elko Co.), is very typical for them. Lindroth (1966) provides a key to species and ecological notes. Pterostichus (Hypherpes) protractus LeConte, 1860. GBNP record. One of the most widespread species of the subgenus, ranging from northern New Mexico to Alberta, Canada, west to California. Found throughout Nevada in coniferous forests from near treeline to 2000 m (and probably lower) elevation. At lower elevations confined to riparian habitat under woody debris and in thick leaf litter. These nocturnal predatory beetles were the most common species found during our sampling at GBNP. They were significantly abundant in areas with ponderosa pine and fir. Lindroth (1966) provides a key to species, however, the key does not cover the numerous southern species in the subgenus, which is in need of revision. Pterostichus (Bothriopterus) lustrans LeConte, 1851. GBNP record. A western North American species found in numerous locations across Nevada. In the GBNP specimens were collected from multiple sites in open vegetation along the riparian corridor, though not in tight association with the stream. Lindroth (1966) provides a key to species. Sphodrini Synuchus dubius (LeConte, 1854). GBNP record. A central southwest species reported from the bordering states of Arizona and Utah. Specimens examined are from three general localities in the eastern part of the state. We collected specimens from several locations in the Great Basin National Park by searching the forest leaf litter at night in areas above 2000 m. These are flightless beetles and other localities for this species are at similar elevations, suggesting significantly disjunct populations. Lindroth (1966) provides a key to species. Laemostenus (Laemostenus) complanatus (Dejean, 1828). New state record. A globally subcosmopolitan species that is an adventive and synanthropic. Known from the adjacent states of California and Oregon, especially in near-coastal regions in urban areas such as watered lawns and parks. The single specimen we studied is only labeled Clark Co., but if there is an established population it would probably be in human maintained habitat in the Las Vegas area. Lindroth (1966) provides a key to species. Calathus (Acalathus) advena (LeConte, 1846). GBNP record. A widespread forest, leaf litter species that occurs in the northeastern part of Nevada. In the conifer forest habitat of the GBNP we found this to be the most commonly encountered species. Frequently found with Pterostichus protractus . Lindroth (1966) and Ball and Nègre (1972) provide keys to species and ecological notes. Zabrini Amara (Amarocelia) farcta LeConte, 1855. GBNP record A western North American species found in various open habitats near water. All specimens collected in GBNP were under rocks and debris near the water in a seasonal lake at about 3000 m elevation. Lindroth (1968) provides a key that includes this species. Amara (Bradytus) lindrothi Hieke, 1990. GBNP record. Known from localities widely distributed across western North America. GBNP is near its southern limit. Specimens were collected in open conifer forest between 3000 and 3200 m elevation, on wet ground, under rocks and debris. Identification is possible using the description and illustrations provided by Hieke (1990) . Amara (Celia) idahoana (Casey, 1924). New state record. Found in the west, in states north of Nevada, including the adjacent states of Idaho and Oregon. Records from Nevada extend the range significantly southward. However, while the record from near Weeks (Lyon Co.) is in an irrigated area near the Carson River, the record from Nye County is in a desert habitat and may be a mislabeled specimen. Lindroth (1968) provides a key to species and ecological notes. Amara (Celia) sinuosa (Casey, 1918). New state record. A northern species, known from the adjacent states of Idaho and Utah. The single specimen we examined was collected in 1936 and labeled Governors Canyon, Nevada. We have been unable to find a match for that exact locality. Lindroth (1968) provides a key to species and notes that the species is found "[o]n open, dry, sandy or gravelly ground." Amara (Curtonotus) carinata (LeConte, 1847). New state record. A widespread species found in all states adjacent to Nevada and so an expected part of the carabid fauna. Beetles are found in grasslands, frequently near alkaline habitats. Lindroth (1968) provides provides a key to species and ecological notes. Amara (Paracelia) quenseli quenseli (Schönherr, 1806). GBNP record. A Holarctic species that is frequently common. Specimens were collected across the GBNP, during the day and at night in open, grassy habitat. Lindroth (1968) provides a key that includes this species. Lebiini Apristus pugetanus Casey, 1920. New state record. A western North American species, already known from all the states surrounding Nevada and so it is an expected member of the fauna. The beetles are found in gravel and sand along rivers, creek and springs, although they are often found some distance from the water. They are frequently day active. Lindroth (1968) provides a key to species. Axinopalpus biplagiatus (Dejean, 1825). New state record. A widespread species found in all states adjacent to Nevada and so an expected part of the carabid fauna. Little is known about the ecology and habits of this species but it has been collected in a variety of open habitats and of the ones we studied from Nevada, one was taken from Joshua Trees. Lindroth (1968) discusses this species, but there are more species in the region and there is no revision of the group. Comparison with types and original descriptions must be used for identification. Axinopalpus fusciceps LeConte, 1851. New state record. A western species, found in the middle latitudes of North America, south into Guatemala. Know from scattered records in the adjacent states of Arizona, California, and Idaho. Nothing is known of this species habits and there is no key to species for the genus. For identification recourse to original descriptions is needed. Cymindis (Tarulus) arizonensis Schaeffer, 1910. New state record. This species is known from southwestern California and Arizona. The record from Clark Co. is a slight expansion of its range. Lindroth (1968) provides a key to species and discussion of the taxonomic difficulties related to this species. Lebia (Lebia) perita Casey, 1920. New state record. A western species, known previously from the adjacent states of California, Oregon, and Idaho. One specimen from Spencer Hot Spring (Landers County) was taken on whiteflower rabbitbrush ( Chrysothamnus albidus ). This marks the easternmost record for the species. Madge (1967) provides a key to species and distributional data. Zuphiini Pseudaptinus (Thalpius) rufulus (LeConte, 1851). New state record. A western North American species, known previously from Arizona, California, and Oregon. Little is known about the habits of this species. A key to species of the subgenus Thalpius was published by Messer (2011) . Pseudomorphini Pseudomorpha castanea Casey, 1909. GBNP record. This species was previosuly reported from California, Oregon, and Utah. As far as known, pseudomorphines are myrmecophiles and ovoviviparous ( Liebherr and Kavanaugh 1985 ). Notman (1925) includes a key to species, however, many undescribed species exist (T.L. Erwin, in litt.) and the genus is in need of revision. Discussion We identified nearly 900 specimens from localities across the state of Nevada (Fig. 3 ). Nearly half, 26 of 57, species newly reported for Nevada are shared with three or more adjacent states. Only Bembidion ( Notaphus ) intermedium is a trans-state extralimital for Nevada. There is no strong pattern in the regional similarity of the new records, i.e., about half of the species have relatively northern distributions that include Oregon, Idaho and northern California and remaining species are either more southern or widespread. Qualitatively, the current list of species of carabids found in Nevada appears to reflect the habitats that are available, with many species that are known to be riparian and seasonal wetland associates or obligate open habitat grassland species. The only non-native species found during our study was Laemostenus ( Laemostenus ) complanatus and it is not clear that this species has established. It is strongly associated with human development in other areas where it has been introduced (e.g., California) and has not shown a tendency to move into undisturbed habitats. The 57 species we report here grows the list of carabid species for Nevada to 299, much closer to its ecologically comparable neighbors Idaho (338 species) and Utah (323 species) (numbers from Bousquet (2012) ). It is expected that Oregon (478 species), with its exceptionally diverse coastal fauna and California (646 species), with its much larger area and multiple bioregions, will have many more species than Nevada. In fact, it is likely that all of these western states have many dozens, perhaps hundreds of species yet to be reported. For California, estimates of the true fauna is over 800 species (K. Will and D.H. Kavanaugh, unpubl.). Conclusions While ongoing monographic research and targeted collecting efforts for surveys, bioblitzes, and ecological monitoring ( Kao et al. 2012 ) are critical to collecting baseline data on faunal distributions, our museum and university collections hold many decades of data on carabids that have yet to be exposed. Our study drew on previously collected material spanning more than 100 years (1913- present). Recent efforts to digitize insect and spider collections ( Baker 2011 , Hill et al. 2012 , iDigBio 2017 ) and online communities for sharing images (e.g. BugGuide 2017 ) are greatly accelerating the process of discovery and exposure of more complete species distributions. We anticipate a surge in the number of species known for Nevada and other states in western USA in the near future because of this. Supplementary Material Supplementary material 1 Occurence data for new state records and GBNP records for carabids of Nevada Data type: occurences Brief description: All specimen records recorded in the EssigDB for species that are new state records and GBNP records for carabids of Nevada File: oo_121705.csv Will, Madan, Hsu

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135788/

Targeting Metallo-proteins by Fragment-Based Lead Discovery

It has been estimated that nearly one third of functional proteins contain a metal ion. These constitute a wide variety of possible drug targets including metalloproteinases, dehydrogenases, oxidoreductases, hydrolases, deacetylases or many others in which the metal ion is either of catalytic or structural nature. Despite the predominant role of a metal ion in so many classes of drug targets, current high-throughput screening techniques do not usually produce viable hits against these proteins, likely due to the lack of proper metal binding pharmacophores in the current screening libraries. Herein we describe a novel fragment based drug discovery approach using a metal targeting fragment library that is based on a variety of distinct classes of metal-binding groups designed to reliably anchor the fragments at the target's metal ions. We show that the approach can effectively identify novel, potent and selective agents that can be readily developed into metalloprotein-targeted therapeutics.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10706207/

Mmp2 Deficiency Leads to Defective Parturition and High Dystocia Rates in Mice

Parturition is the final and essential step for mammalian reproduction. While the uterus is quiescent during pregnancy, fundamental changes arise in the myometrial contractility, inducing fetal expulsion. Extracellular matrix (ECM) remodeling is fundamental for these events. The gelatinases subgroup of matrix metalloproteinases (MMPs), MMP2 and MMP9, participate in uterine ECM remodeling throughout pregnancy and parturition. However, their loss-of-function effect is unknown. Here, we determined the result of eliminating Mmp2 and/or Mmp9 on parturition in vivo, using single- and double-knockout (dKO) mice. The dystocia rates were measured in each genotype, and uterine tissue was collected from nulliparous synchronized females at the ages of 2, 4, 9 and 12 months. Very high percentages of dystocia (40–55%) were found in the Mmp2 −/− and dKO females, contrary to the Mmp9 −/− and wild-type females. The histological analysis of the uterus and cervix revealed that Mmp2 −/− tissues undergo marked structural alterations, including highly enlarged myometrial, endometrial and luminal cavity. Increased collagen deposition was also demonstrated, suggesting a mechanism of extensive fibrosis in the Mmp2 −/− myometrium, which may result in dystocia. Overall, this study describes a new role for MMP2 in myometrium remodeling during mammalian parturition process, highlighting a novel cause for dystocia due to a loss in MMP2 activity in the uterine tissue. 1. Introduction Parturition is the final and most critical step for successful mammalian reproduction. During pregnancy, the uterus undergoes a period of quiescence, which is fundamental for fetal growth and development. However, at some point, dramatic changes occur in myometrial contractility, resulting in the efficient expulsion of the fetus. These events are necessary for the survival of both the fetus and the mother. However, their underlying mechanisms are only partially defined. Additionally, since preterm births account for ~11.5% of all live births in the US and are the main cause of perinatal mortality and morbidity worldwide [ 1 , 2 ], elucidating the molecular pathways that control parturition will contribute to combating preterm births or, on the other hand, to optimizing protocols for medically induced labor [ 3 ]. In all mammals, the uterine wall consists of three major elements: (1) the endometrium; (2) the myometrium, which consists of an inner circular layer and an outer longitudinal layer of oriented smooth muscle; and (3) the perimetrium outer layer. Notably, the endometrium, which contains multiple secreting glands, undergoes extensive remodeling throughout the estrus cycle [ 4 ]. Moreover, the myometrium layer of the uterus undergoes a transition from a quiescence state during gestation to contractility mode during labor, a mandatory shift for executing natural parturition. By secreting and/or responding to progesterone and estrogen signaling, the myometrium plays an essential role in regulating its activities, which also involves profound tissue remodeling, which, in turn, regulates the myometrium's activities. However, while research from past decades has provided critical insights into the parturition process, the molecular mechanisms involved in its initiation remain largely unknown [ 1 , 2 , 5 , 6 ]. Matrix metalloproteinases (MMPs) are a large family of enzymes known for their ability to degrade different components and proteins in the extracellular matrix (ECM). Therefore, they were found to be involved in many physiological processes, such as in ovulation and embryo implantation [ 7 , 8 , 9 , 10 ]. For example, Novaro et al., showed that in rats, at the time of implantation, the blastocyst starts to produce nitric oxide, which leads to increased levels of MMP2, which is necessary for the tissue-remodeling process that takes place in the implantation sites [ 7 ]. Such MMPs were also found to participate in pathological conditions, including cancer-cell metastasis, multiple sclerosis and Alzheimer's disease [ 11 , 12 , 13 , 14 ]. The gelatinase subfamily of MMPs has only two members, MMP2 and MMP9. These secreted enzymes share a similar protein structure, comparable biological activity in different biological systems and shared substrates in vitro, such as gelatin [ 15 , 16 , 17 , 18 , 19 ]. In addition, we previously found that during development, both MMP2 and MMP9 are required for executing the migration of the unique population of neural crest cells (NCCs) in both chick and mouse embryos, and each MMP compensates for the loss of the other [ 20 , 21 , 22 ]. On the other hand, in our recent study on skeleton development, we found that each gelatinase has an individual and unique role; while MMP2 regulates intramembranous ossification affecting the development of the skull and the cortices, MMP9 controls the endochondral ossification affecting the longitudinal growth of the skeleton, as well as the overall body length [ 23 ]. Hence, their redundant or individual activities are highly context-dependent. Interestingly, several MMPs, including MMP1, MMP2, MMP3, MMP7, MMP9, MMP10 and MMP11, and TIMPs such as TIMP1, TIMP2 and TIMP3, were previously found to be expressed in the uterus in various species [ 24 ]. Furthermore, their balanced function was shown to be essential for normal uterine tissue remodeling throughout the estrus cycle, as well as during pregnancy, parturition and postpartum uterine involution in both rodents and humans [ 24 , 25 ]. For example, Engelen et al., showed that in cows, there is an increase in MMP2 levels, right before calving, which is associated with collagen degradation that allows cervix ripening [ 26 ]. Furthermore, Vadillo-Ortega et al. described how in humans, MMP9 activity increases in amniochorion with the onset of labor [ 27 ]. These studies suggested that MMPs and TIMPs participate in the dynamic regulation of uterus remodeling. However, the comparable or individual roles of each MMP have not been explored. Here, we set out to determine whether the gelatinases, MMP2 and MMP9, have a combined or individual role in uterine function during parturition process, using single- and double-knockout (dKO) mice for both. 2. Results 2.1. Mmp2 Loss Results in Defective Parturition Process and Dystocia Dystocia is defined as a difficult and prolonged process of delivery with a negative outcome. When breeding the different MMP2/MMP9-null colonies, we frequently observed pregnant females with dystocia. Hence, we decided to methodologically characterize the occurrence of dystocia in the following genotypes: WT, Mmp2 +/− , Mmp2 −/− , Mmp9 +/− , Mmp9 −/− , Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− and Mmp2 −/− Mmp9 −/− (dKO). Dystocia was diagnosed in both timed and untimed pregnancies; in the timed pregnancies, dystocia was determined when a pregnancy reached gestational day (GD) 22.5 according to vaginal plug observation, but did not result in normal parturition, as reflected by the females that which still had one or more (dead) fetuses in their uterus. Other timed pregnancies that resulted in vaginal bleeding at GD18.5–GD21.5 without any live newborns during the following 24 h, but with dead fetuses remaining in the uterus, were also diagnosed as dystocia. Additionally, untimed pregnancies in which the females had reached the end of pregnancy without any apparent parturition or newborns in the cage, but with dead fetuses remaining in the uterus, were also considered as dystocia ( Figure 1 A–C). In all cases, the females were euthanized. The dystocia percentages were calculated for the females under and over 6 months (M) of age and were measured for each female's-genotype as the number of pregnancies ending in dystocia versus the total number of pregnancies for which the female had the same genotype. Markedly, when the females were older than 6 M, 54.5% of all the Mmp2 −/− pregnancies (12/22) resulted in dystocia ( Figure 1 D). In contrast, no cases of dystocia were found in the WT females (0/31), and 15.6% of the Mmp9 −/− pregnancies (5/32) resulted in dystocia ( Figure 1 D). Additionally, both heterozygous Mmp2 +/− and Mmp9 +/− showed a dystocia rate of 8.3% (2/24 and 1/12, respectively) in the >6 M-old females ( Figure 1 D). Moreover, measuring the dystocia frequency among females with double mutations in the Mmp2 and Mmp9 genes, such as the mixed genotypes Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− or double-full-knockout ( Mmp2 −/− Mmp9 −/− , dKO), revealed varied results, such as 10.3% (4/39), 42.9% (9/21), 0% (0/11) and 40% (6/15), respectively ( Figure 1 D). Notably, in the females younger than 6 M, we found much lower rates of dystocia in general, but the pattern of higher dystocia occurrence in the Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO, compared to either the WT or the other Mmp9 mixed genotypes, was similar ( Figure S1A ). This ruled out the possibility of these high percentages being age-related. Intriguingly, these data show that the loss of one or two alleles of Mmp9 to the Mmp2 -KO background (i.e., Mmp2 −/− Mmp9 +/− and dKO) leads to a somewhat reversed phenotype of a decreased dystocia incidence (40-42.9%) in relation to the rate in the Mmp2 −/− females (54.5%) ( Figure 1 D). Overall, this analysis suggests that the occurrence of dystocia is largely linked to Mmp2 loss, as well as indicating that the additional loss of the other gelatinase, Mmp9 , reduces this phenotype to a certain extent. Next, we evaluated whether the failure to undergo normal parturition is linked to in utero fetal death. The viability of the fetuses and litter size were measured at GD18.5 and P0, revealing that regardless of the genotype, the entire litter was alive at GD18.5, with a similar number of fetuses at P0 (5.18–6.97) ( Figure 1 E). Notably, when dKO pairs were mated, a smaller litter size was found compared to when either WT/ Mmp2 −/− or Mmp9 −/− parents were mated, but this was not statistically significant ( p = 0.0508, Figure 1 E). Furthermore, the measurement of the genotype distribution of the WT, Mmp2 +/− and Mmp2 −/− in the neonates generated from the mating of the Mmp2 +/− females and males demonstrated the following averaged ratios of all the examined litters, as expected according to the Mendelian inheritance: 26.6%, 47.7% and 25.7% for WT, Mmp2 +/− and Mmp2 −/− , respectively ( Figure 1 F). Moreover, when the Mmp2 +/− Mmp9 +/− pairs were mated, and the relative distribution of each genotype was measured at P0, the percentage of the dKO offspring was not lower than expected ( Figure S1B ). Together, these results rule out any association between fetal death and dystocia in the Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO genotypes. 2.2. Mmp2 −/− Nulliparous Females Present Relatively Short Uterine Horns After unraveling the connection between Mmp2 loss and dystocia occurrence, we next set out to determine the effect of Mmp2 loss on the uterus morphology. Nulliparous females were used to examine this effect of Mmp2 loss on the naïve uterine tissue. In mice, the estrus cycle is short (4–5 days) and comprises four precise phases: proestrus, estrus, metestrus and diestrus. As natural morphological changes are known to occur in the uterine wall during the estrus cycle, it is crucial to synchronize the estrus cycle between females, in order to ascertain that morphological differences that may appear in the different genotypes are not estrus-phase related [ 6 , 28 ]. Hence, the females were synchronized and uteri were collected from 4–12 females from the WT, Mmp2 −/− , Mmp9 −/− and dKO genotypes at three ages: 8 w, 4 M and 8–9.5 M. The decision to examine the females at different ages was based on the observation that dystocia incidents mostly occur among older females ( Figure 1 D and Figure S1A ). The length of the uterus horn was measured and found to be somewhat shorter in the Mmp2 −/− samples compared to the WT at all the examined ages ( Figure 2 ); however, this difference was not statistically significant, possibly due to the small sample size ( p = 0.0935, p = 0.1991 and p = 0.33 for WT vs. Mmp2 −/− at 8 w, 4 M and 8–9.5 M, respectively). Additionally, the H&E staining of the sections taken from the different uteri at 12 M demonstrated that some Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO samples were markedly abnormal, with extensive luminal areas and secretary glands, compared to the WTs ( Figure 2 P and Figure S2 ). These tissues also demonstrated a very thin myometrial layer and almost no endometrium stroma compared to the WTs ( Figure 2 P). Even though these tissues were collected from unsynchronized females, their abnormalities are most likely not contributed from differences in the estrus phase, since normal uterine tissues in any estrus stage do not present such severe abnormalities [ 29 , 30 , 31 ]. 2.3. Mmp2 −/− Uterus Demonstrates Enlarged Myometrium, Endometrium and Lumen To further determine whether the pathophysiology of the dystocia is associated with the abnormal anatomy of the uterus only at older stages, a histological analysis was performed on the uteri harvested at the ages of 8 w, 4 M and 8–9.5 M ( Figure 3 ), similar to those analyzed in Figure 2 A–O. We found that at 8 w and 4 M, the uterine tissues of the Mmp2 −/− , Mmp9 −/− and dKO presented a normal appearance compared to the WT, as determined by the overall tissue size and the relative area measured by each layer of the uterine wall ( Figure 3 A–H). However, at 4 M, the Mmp2 −/− tissue started to display a dissimilar structure, with an increased number of secreting glands in the endometrial sub-tissue, compared to other genotypes ( Figure 3 E–H). However, at the ages of 8–9.5 M, a much more significant enlargement of the entire tissue area was found in the Mmp2 −/− compared to the other genotypes ( Figure 3 I–L). Furthermore, specific area measurements of each layer, such as the myometrium, endometrium and lumen, demonstrated that while at 8 w and 4 M, no differences were found between the different genotypes, at 8–9.5 M, the area of all three tissue layers was significantly enlarged in the Mmp2 −/− compared to the WT ( Figure 3 U–X, compared to M–P and Q–T). Notably, concomitantly with the relatively low proportion of the dystocia occurrence in the Mmp9 −/− females (8.3%, Figure 1 ), the histology of these uteri did not present major differences compared to the WTs in terms of the total tissue area, or the myometrium, endometrium and lumen areas. Furthermore, the histological characteristics of the dKO ( Mmp2 −/− Mmp9 −/− ) uteri did not demonstrate a severer phenotype than that of the Mmp2 −/− at any age ( Figure 3 ). Additionally, the specific measurements of the dKO histology demonstrated that while the total area and the myometrium, endometrium and lumen areas of the Mmp2 −/− differed from those of the WT significantly ( Figure 3 U–X), the same parameters in the dKO did not differ from either the Mmp2 −/− nor the WT. This result, which was in agreement with the less prominent percentage of dystocia observed in the dKO (40%) females compared to the Mmp2 −/− (54.5%) ( Figure 1 D), strongly suggests that the additional loss of Mmp9 against the background of Mmp2 loss (i.e., in dKO and Mmp2 −/− , respectively) activates a compensatory mechanism that partially resolves the phenotype. 2.4. Mmp2 −/− Nulliparous Uterus at 8 M Demonstrates Signs of Myometrial Fibrosis Collagen accumulation in associated with fibrosis in several tissues, including the liver, kidney and uterus [ 32 , 33 , 34 , 35 , 36 ]. For example, Xu et al. showed enhanced collagen expression and fibrosis in the mouse uterus, which are associated with Notch1 signaling [ 35 ]. Furthermore, one of the main substrates known to be degraded by MMP2 is collagen [ 37 , 38 ] and, previously, it was demonstrated that, in cows, there is an increase in MMP2 levels 5 days before calving, which is accompanied with denatured collagen, suggesting that this step enables cervix ripening [ 26 ]. Hence, we next asked whether the observed pathophysiology in the Mmp2 -nulls is coupled with abnormal collagen deposition in the uterine wall, which may be associated with perturbed uterine-wall morphology, as shown in Figure 3 . Masson's trichrome staining for collagen fibrils was performed on transverse sections from the WT and Mmp2 −/− nulliparous uteri at 8 M, demonstrating increased collagen fibrils in the Mmp2 −/− tissue, as shown by the enhanced staining (blue), mostly in the circular myometrium, compared to the WT ( Figure 4 A,B; blue, arrows). Additionally, the Masson trichrome staining on the cervix samples also showed increased levels of collagen in the Mmp2 −/− compared to the WT ( Figure S3A,B ). This result suggests a possible mechanism in which the disrupted uterine functionality during parturition in Mmp2 -KO females is the insufficient remodeling of the uterine ECM, which results in fibrotic myometrium. This fibrosis can be the cause of inadequate contractions of the uterus during parturition, which, in turn, may lead to dystocia. The fact that the other genotypes showed much less accumulation of collagen in the uterine tissue is in agreement with the lower dystocia rates and the relatively normal uterine histology ( Figure S3C–F ), further highlighting the dominant role of MMP2, rather than MMP9, in this process. 2.5. Morphometric Analysis of the Pelvic Bone in Mmp2 −/− Females Cephalopelvic disproportion (CPD) is a leading cause of dystocia in women, which occurs due to a mismatch between the fetal head size and size of the maternal pelvis, resulting in mechanical "failure to progress" in labor; if untreated, the consequence is obstructed labor, which can endanger the lives of both mother and fetus [ 39 ]. Since we previously revealed that Mmp2 has a role in intramembranous ossification and its loss leads to wider skulls as early as in P0 [ 23 ], we next set out to determine whether the pelvic structure is also affected by Mmp2 loss. Using a µCT scanner, the pelvic bones from the WT and Mmp2 −/− females at 8 M of age (n = 5 and 4, respectively) were analyzed. Measuring the pelvic bone length and width revealed a significantly shorter pelvic bone in the Mmp2 −/− females compared to the WT ( Figure 5 A–C). The pelvic width also demonstrated a great tendency towards being smaller ( p value = 0.0567), possibly due to the small sample size. Notably, in addition to a smaller pelvis, obstruction can also be associated with fetal overgrowth [ 40 ]. However, when measuring the body weights of the P0 newborns, we did not reveal a major weight difference between the Mmp2 −/− and WT mice, indicating that the dystocia phenomenon does not result from overweight fetuses ( Figure S4A ). Consequently, the Mmp2 −/− females of different ages (4 w, 8 w, 6 M) also did not reveal any significant weight differences compared to the WT ( Figure S4B–D ), suggesting that maternal overweight/underweight is also not associated with the occurrence of dystocia upon Mmp2 -KO. Altogether, these results raise the possibility of a mechanical mechanism that is involved in the high rates of dystocia in Mmp2 −/− females, which is driven by the combination of a relatively small pelvic bone with a wider skull in fetuses. 2.1. Mmp2 Loss Results in Defective Parturition Process and Dystocia Dystocia is defined as a difficult and prolonged process of delivery with a negative outcome. When breeding the different MMP2/MMP9-null colonies, we frequently observed pregnant females with dystocia. Hence, we decided to methodologically characterize the occurrence of dystocia in the following genotypes: WT, Mmp2 +/− , Mmp2 −/− , Mmp9 +/− , Mmp9 −/− , Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− and Mmp2 −/− Mmp9 −/− (dKO). Dystocia was diagnosed in both timed and untimed pregnancies; in the timed pregnancies, dystocia was determined when a pregnancy reached gestational day (GD) 22.5 according to vaginal plug observation, but did not result in normal parturition, as reflected by the females that which still had one or more (dead) fetuses in their uterus. Other timed pregnancies that resulted in vaginal bleeding at GD18.5–GD21.5 without any live newborns during the following 24 h, but with dead fetuses remaining in the uterus, were also diagnosed as dystocia. Additionally, untimed pregnancies in which the females had reached the end of pregnancy without any apparent parturition or newborns in the cage, but with dead fetuses remaining in the uterus, were also considered as dystocia ( Figure 1 A–C). In all cases, the females were euthanized. The dystocia percentages were calculated for the females under and over 6 months (M) of age and were measured for each female's-genotype as the number of pregnancies ending in dystocia versus the total number of pregnancies for which the female had the same genotype. Markedly, when the females were older than 6 M, 54.5% of all the Mmp2 −/− pregnancies (12/22) resulted in dystocia ( Figure 1 D). In contrast, no cases of dystocia were found in the WT females (0/31), and 15.6% of the Mmp9 −/− pregnancies (5/32) resulted in dystocia ( Figure 1 D). Additionally, both heterozygous Mmp2 +/− and Mmp9 +/− showed a dystocia rate of 8.3% (2/24 and 1/12, respectively) in the >6 M-old females ( Figure 1 D). Moreover, measuring the dystocia frequency among females with double mutations in the Mmp2 and Mmp9 genes, such as the mixed genotypes Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− or double-full-knockout ( Mmp2 −/− Mmp9 −/− , dKO), revealed varied results, such as 10.3% (4/39), 42.9% (9/21), 0% (0/11) and 40% (6/15), respectively ( Figure 1 D). Notably, in the females younger than 6 M, we found much lower rates of dystocia in general, but the pattern of higher dystocia occurrence in the Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO, compared to either the WT or the other Mmp9 mixed genotypes, was similar ( Figure S1A ). This ruled out the possibility of these high percentages being age-related. Intriguingly, these data show that the loss of one or two alleles of Mmp9 to the Mmp2 -KO background (i.e., Mmp2 −/− Mmp9 +/− and dKO) leads to a somewhat reversed phenotype of a decreased dystocia incidence (40-42.9%) in relation to the rate in the Mmp2 −/− females (54.5%) ( Figure 1 D). Overall, this analysis suggests that the occurrence of dystocia is largely linked to Mmp2 loss, as well as indicating that the additional loss of the other gelatinase, Mmp9 , reduces this phenotype to a certain extent. Next, we evaluated whether the failure to undergo normal parturition is linked to in utero fetal death. The viability of the fetuses and litter size were measured at GD18.5 and P0, revealing that regardless of the genotype, the entire litter was alive at GD18.5, with a similar number of fetuses at P0 (5.18–6.97) ( Figure 1 E). Notably, when dKO pairs were mated, a smaller litter size was found compared to when either WT/ Mmp2 −/− or Mmp9 −/− parents were mated, but this was not statistically significant ( p = 0.0508, Figure 1 E). Furthermore, the measurement of the genotype distribution of the WT, Mmp2 +/− and Mmp2 −/− in the neonates generated from the mating of the Mmp2 +/− females and males demonstrated the following averaged ratios of all the examined litters, as expected according to the Mendelian inheritance: 26.6%, 47.7% and 25.7% for WT, Mmp2 +/− and Mmp2 −/− , respectively ( Figure 1 F). Moreover, when the Mmp2 +/− Mmp9 +/− pairs were mated, and the relative distribution of each genotype was measured at P0, the percentage of the dKO offspring was not lower than expected ( Figure S1B ). Together, these results rule out any association between fetal death and dystocia in the Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO genotypes. 2.2. Mmp2 −/− Nulliparous Females Present Relatively Short Uterine Horns After unraveling the connection between Mmp2 loss and dystocia occurrence, we next set out to determine the effect of Mmp2 loss on the uterus morphology. Nulliparous females were used to examine this effect of Mmp2 loss on the naïve uterine tissue. In mice, the estrus cycle is short (4–5 days) and comprises four precise phases: proestrus, estrus, metestrus and diestrus. As natural morphological changes are known to occur in the uterine wall during the estrus cycle, it is crucial to synchronize the estrus cycle between females, in order to ascertain that morphological differences that may appear in the different genotypes are not estrus-phase related [ 6 , 28 ]. Hence, the females were synchronized and uteri were collected from 4–12 females from the WT, Mmp2 −/− , Mmp9 −/− and dKO genotypes at three ages: 8 w, 4 M and 8–9.5 M. The decision to examine the females at different ages was based on the observation that dystocia incidents mostly occur among older females ( Figure 1 D and Figure S1A ). The length of the uterus horn was measured and found to be somewhat shorter in the Mmp2 −/− samples compared to the WT at all the examined ages ( Figure 2 ); however, this difference was not statistically significant, possibly due to the small sample size ( p = 0.0935, p = 0.1991 and p = 0.33 for WT vs. Mmp2 −/− at 8 w, 4 M and 8–9.5 M, respectively). Additionally, the H&E staining of the sections taken from the different uteri at 12 M demonstrated that some Mmp2 −/− , Mmp2 −/− Mmp9 +/− and dKO samples were markedly abnormal, with extensive luminal areas and secretary glands, compared to the WTs ( Figure 2 P and Figure S2 ). These tissues also demonstrated a very thin myometrial layer and almost no endometrium stroma compared to the WTs ( Figure 2 P). Even though these tissues were collected from unsynchronized females, their abnormalities are most likely not contributed from differences in the estrus phase, since normal uterine tissues in any estrus stage do not present such severe abnormalities [ 29 , 30 , 31 ]. 2.3. Mmp2 −/− Uterus Demonstrates Enlarged Myometrium, Endometrium and Lumen To further determine whether the pathophysiology of the dystocia is associated with the abnormal anatomy of the uterus only at older stages, a histological analysis was performed on the uteri harvested at the ages of 8 w, 4 M and 8–9.5 M ( Figure 3 ), similar to those analyzed in Figure 2 A–O. We found that at 8 w and 4 M, the uterine tissues of the Mmp2 −/− , Mmp9 −/− and dKO presented a normal appearance compared to the WT, as determined by the overall tissue size and the relative area measured by each layer of the uterine wall ( Figure 3 A–H). However, at 4 M, the Mmp2 −/− tissue started to display a dissimilar structure, with an increased number of secreting glands in the endometrial sub-tissue, compared to other genotypes ( Figure 3 E–H). However, at the ages of 8–9.5 M, a much more significant enlargement of the entire tissue area was found in the Mmp2 −/− compared to the other genotypes ( Figure 3 I–L). Furthermore, specific area measurements of each layer, such as the myometrium, endometrium and lumen, demonstrated that while at 8 w and 4 M, no differences were found between the different genotypes, at 8–9.5 M, the area of all three tissue layers was significantly enlarged in the Mmp2 −/− compared to the WT ( Figure 3 U–X, compared to M–P and Q–T). Notably, concomitantly with the relatively low proportion of the dystocia occurrence in the Mmp9 −/− females (8.3%, Figure 1 ), the histology of these uteri did not present major differences compared to the WTs in terms of the total tissue area, or the myometrium, endometrium and lumen areas. Furthermore, the histological characteristics of the dKO ( Mmp2 −/− Mmp9 −/− ) uteri did not demonstrate a severer phenotype than that of the Mmp2 −/− at any age ( Figure 3 ). Additionally, the specific measurements of the dKO histology demonstrated that while the total area and the myometrium, endometrium and lumen areas of the Mmp2 −/− differed from those of the WT significantly ( Figure 3 U–X), the same parameters in the dKO did not differ from either the Mmp2 −/− nor the WT. This result, which was in agreement with the less prominent percentage of dystocia observed in the dKO (40%) females compared to the Mmp2 −/− (54.5%) ( Figure 1 D), strongly suggests that the additional loss of Mmp9 against the background of Mmp2 loss (i.e., in dKO and Mmp2 −/− , respectively) activates a compensatory mechanism that partially resolves the phenotype. 2.4. Mmp2 −/− Nulliparous Uterus at 8 M Demonstrates Signs of Myometrial Fibrosis Collagen accumulation in associated with fibrosis in several tissues, including the liver, kidney and uterus [ 32 , 33 , 34 , 35 , 36 ]. For example, Xu et al. showed enhanced collagen expression and fibrosis in the mouse uterus, which are associated with Notch1 signaling [ 35 ]. Furthermore, one of the main substrates known to be degraded by MMP2 is collagen [ 37 , 38 ] and, previously, it was demonstrated that, in cows, there is an increase in MMP2 levels 5 days before calving, which is accompanied with denatured collagen, suggesting that this step enables cervix ripening [ 26 ]. Hence, we next asked whether the observed pathophysiology in the Mmp2 -nulls is coupled with abnormal collagen deposition in the uterine wall, which may be associated with perturbed uterine-wall morphology, as shown in Figure 3 . Masson's trichrome staining for collagen fibrils was performed on transverse sections from the WT and Mmp2 −/− nulliparous uteri at 8 M, demonstrating increased collagen fibrils in the Mmp2 −/− tissue, as shown by the enhanced staining (blue), mostly in the circular myometrium, compared to the WT ( Figure 4 A,B; blue, arrows). Additionally, the Masson trichrome staining on the cervix samples also showed increased levels of collagen in the Mmp2 −/− compared to the WT ( Figure S3A,B ). This result suggests a possible mechanism in which the disrupted uterine functionality during parturition in Mmp2 -KO females is the insufficient remodeling of the uterine ECM, which results in fibrotic myometrium. This fibrosis can be the cause of inadequate contractions of the uterus during parturition, which, in turn, may lead to dystocia. The fact that the other genotypes showed much less accumulation of collagen in the uterine tissue is in agreement with the lower dystocia rates and the relatively normal uterine histology ( Figure S3C–F ), further highlighting the dominant role of MMP2, rather than MMP9, in this process. 2.5. Morphometric Analysis of the Pelvic Bone in Mmp2 −/− Females Cephalopelvic disproportion (CPD) is a leading cause of dystocia in women, which occurs due to a mismatch between the fetal head size and size of the maternal pelvis, resulting in mechanical "failure to progress" in labor; if untreated, the consequence is obstructed labor, which can endanger the lives of both mother and fetus [ 39 ]. Since we previously revealed that Mmp2 has a role in intramembranous ossification and its loss leads to wider skulls as early as in P0 [ 23 ], we next set out to determine whether the pelvic structure is also affected by Mmp2 loss. Using a µCT scanner, the pelvic bones from the WT and Mmp2 −/− females at 8 M of age (n = 5 and 4, respectively) were analyzed. Measuring the pelvic bone length and width revealed a significantly shorter pelvic bone in the Mmp2 −/− females compared to the WT ( Figure 5 A–C). The pelvic width also demonstrated a great tendency towards being smaller ( p value = 0.0567), possibly due to the small sample size. Notably, in addition to a smaller pelvis, obstruction can also be associated with fetal overgrowth [ 40 ]. However, when measuring the body weights of the P0 newborns, we did not reveal a major weight difference between the Mmp2 −/− and WT mice, indicating that the dystocia phenomenon does not result from overweight fetuses ( Figure S4A ). Consequently, the Mmp2 −/− females of different ages (4 w, 8 w, 6 M) also did not reveal any significant weight differences compared to the WT ( Figure S4B–D ), suggesting that maternal overweight/underweight is also not associated with the occurrence of dystocia upon Mmp2 -KO. Altogether, these results raise the possibility of a mechanical mechanism that is involved in the high rates of dystocia in Mmp2 −/− females, which is driven by the combination of a relatively small pelvic bone with a wider skull in fetuses. 3. Discussion In this study, we examined the effect of Mmp2 knockout on nulliparous uterine development and its function during parturition process in female mice. We showed that while the KO of the other gelatinase, Mmp9 , was found to affect parturition only partially, Mmp2 loss led to a severe defective parturition process with very high rates of dystocia. We demonstrated that 54.5% of all the Mmp2 −/− pregnancies in the females older than 6 M resulted in dystocia, in which the females presented with at least one dead fetus still in the uterus. The dystocia rate in the WT females was found to be 0% and in the Mmp9 −/− females, it was less pronounced (15.6%). Surprisingly, the total dystocia proportion decreased in the Mmp2 −/− Mmp9 +/− and dKO females to 42.9% and 40%, respectively. Furthermore, the histological analysis of the nulliparous uteri of all four genotypes at several ages revealed a significantly enlarged tissue in the Mmp2 −/− , rather than in the Mmp9 −/− , dKO or WT, with increased myometrium, endometrium and lumen areas. Importantly, a greater accumulation of collagen fibrils was found in the Mmp2 −/− uterine tissue compared to the WT. As MMP2 is largely known for the proteolytic activity through which it degrades collagen (among other ECM molecules) [ 38 , 41 , 42 ], it is reasonable to speculate that when this activity is lost, collagen will accumulate in the uterine ECM, leading to myometrial fibrosis. Our data support this possibility and suggest that the increased fibrosis in nulliparous Mmp2 −/− uteri may be one of the causes of insufficient contractions during parturition, ending with dystocia and in utero fetal death. Moreover, normal levels of collagen fibrils were also demonstrated in the Mmp9 −/− and dKO uteri ( Figure S3 ), which is in alignment with our observation of lower rates of dystocia in these genotypes. Nevertheless, the dystocia rates that were found in the Mmp9 −/− (15.6%) are still considered high and non-negligible compared to the WT (0%). This implies that increased collagen deposition, which leads to fibrosis, is most likely not the only mechanism involved. Furthermore, while, during pregnancy, the cervix must be firm to prevent premature fetus expulsion, when reaching term, cervical ripening is initiated by a cascade of events that includes ECM remodeling, making the cervix more compliant with parturition [ 3 , 43 , 44 ]. Based on the higher accumulation of collagen fibrils, both in the uterine myometrium and in the cervixes of Mmp2 −/− females, it is possible that, in addition to the uteri, the cervixes of these females also fail to properly remodel the ECM, resulting in cervical fibrosis. Our suggested role for MMP2 in uterus-ECM remodeling is further supported by previous studies on mice which were knocked out for Anthrax toxin receptor 2 ( Antxr2 ) and presented defective parturition and dystocia [ 45 , 46 ]. It was suggested that this resulted from fibrosis in the uterus and cervix due to the aberrant deposition of collagens and fibronectin, together with disrupted myometrial cell layers. Notably, a decrease in the active form of MMP2 was found in the Antxr2 −/− uteri, leading to the hypothesis that ANTXR2 activates MMP2 by regulating MMP14 activity in the uterus. Furthermore, a different study found in Geranylgeranyl pyrophosphate synthase ( Ggps1) -KO mice that 75% of all pregnancies ended with dystocia [ 47 ]. The isolation of uterine muscle strips from nonpregnant mice revealed that the spontaneous contraction rate and amplitude in the Ggps1 -KO mice was largely decreased. Additionally, the activation of the Rho/Rho-associated protein kinase (Rock) pathway, which is associated with smooth-muscle contraction, was significantly decreased in the myometria of Ggps1 -nulls. Thus, the authors concluded that Ggps1 deletion disrupts the RhoA/Rock2 pathway, causing uterine contraction and parturition problems. Interestingly, Rho was previously found to regulate MMP expression in various cell types [ 48 , 49 , 50 ], raising the possibility that the Rho/Rock2 pathway is also involved in governing parturition processes in Mmp2 −/− females. Moreover, it is well known that both MMP2 and MMP9 participate in the remodeling of the mammary gland during pregnancy, lactation and involution [ 51 , 52 ]. Additionally, it was found that Mmp2 −/− mice show differences in mammary-gland structure and impaired lactation [ 53 ]. Therefore, it will be of great interest to investigate in the future whether the loss of Mmp2 also perturbs tissue remodeling in other gestation-related organs, such as the mammary gland and ovaries. Dystocia in women is often associated with cephalopelvic disproportion (CPD), which arises when the fetal head size does not align with the size of the maternal pelvis [ 39 ]. This mismatch leads to a mechanical "failure to progress" during labor. If left untreated, this can result in obstructed labor, posing a significant risk to the lives of both the mother and the fetus [ 39 ]. Therefore, an additional possible mechanism for the dystocia in Mmp2 −/− mice may be related to skeletal pathology [ 23 ]. Indeed, we recently uncovered that during skeleton development, MMP2 participates in intramembranous ossification and its loss results in shorter but wider skulls in neonates [ 23 ]. Since the pelvic bones are also composed of irregular and flat bones, which develop through intramembranous ossification [ 54 , 55 , 56 ], we raised the hypothesis that the pelvic bones of the Mmp2 −/− females may have been impaired. The measurement of the lengths and widths of the pelvic bones demonstrated a clear trend towards smaller pelvic bones in Mmp2 −/− females compared to the WT, leading to thinner spaces in the birth canals. Based on this result, we cannot rule out that, similarly to the skull, the pelvic bones of the Mmp2 −/− mice developed abnormally. Thus, the impairment of the pelvis in Mmp2 −/− pregnant females, along with the wider skulls we observed previously in the Mmp2 −/− fetuses, can lead to CPD and consequent dystocia, as previously reported in women [ 39 ]. Moreover, the fact that some, but not all, of the P0 Mmp2 −/− newborns had significantly wider skulls [ 23 ], can explain why some fetuses are expelled from the uterus while others become stuck and die. Interestingly, past analyses of the different phenotypes of various Mmp -knockout mice, such as Mmp1 , Mmp2 , Mmp3 , Mmp7 , Mmp9 , Mmp11 , Mmp12 and Mmp14 , along with Timp1, Timp2 and Timp3 Kos, did not report an impact on the reproductive axis [ 57 , 58 , 59 ]. However, subsequent analyses revealed effects on reproduction, such as lower pregnancy rates in Timp1-null females compared to WTs (52% vs. 78%), as well as significantly fewer pups per litter [ 25 , 60 ]. Since it was found, in fibrosarcoma cells in vitro, that TIMP is able to activate proMMP2 by binding its hemopexin domain [ 59 ], further studies are needed to explore whether MMP2 activity is impaired in Timp1 -null uteri. A confounding factor when aiming to disclose MMPs' action in any physiological or pathological condition is that the removal of one MMP often results in compensation by other MMP sub-members. For example, the deletion of Mmp7 results in a 10–12-fold increase in Mmp3 and Mmp10 during uterine involution [ 61 ]. Furthermore, our previous studies highlighted that the loss of one gelatinase ( Mmp2 or Mmp9 ) in embryonic NCCs is compensated by the other, leading to the normal migration of NCCs [ 21 ]. Concomitantly, our recent RNAseq analysis on bone tissue revealed that the loss of both gelatinases leads to an increase in the expression of other MMPs, such as Mmp13 , Mmp14 and Mmp15 [ 23 ]. However, this was not evident in the single KOs (each displaying a different bone phenotype), indicating that while the activity of both gelatinases is not redundant in the different bones, only in the absence of both MMP2 and MMP9, the transcription levels of other MMPs become elevated. Based on our findings, it is also possible that in the reproductive system, the genetic loss of one MMP may be compensated by other MMPs, preventing the occurrence of a specific fertility-related phenotype in animal models. Thus, it can be suggested that the different results observed in the Mmp2 −/− and Mmp9 −/− females occur through a unilateral compensation mechanism between the two gelatinases, through which Mmp2 can compensate, to some degree, for the loss of Mmp9 , but not vice versa; hence, the remaining Mmp9 alleles in Mmp2 −/− cannot rescue the defective parturition phenotype. Moreover, as MMPs are secreted as pro-proteins, which must be cleaved in order to become active, the compensation by other MMPs may relate to their enzymatic activation/inhibition, in addition to their expression levels. Therefore, a multiomics analysis [ 62 , 63 ] is required to compare between the transcriptome, proteome and degradome profiles of the uterine tissues in the different genotypes to elucidate the full spectrum of MMPs and other substrates involved in uterine-wall remodeling in the presence or absence of MMP2 and MMP9. 4. Conclusions, Strengths and Limitations This study demonstrates that more than 54% of Mmp2 −/− pregnancies in females over the age of 6 M end with severe dystocia. This percentage is reduced to 42.9% and 40% with the additional loss of one or both alleles of the other gelatinase, Mmp9 , respectively, i.e., in Mmp2 −/− Mmp9 +/− and dKO. The histological analysis revealed increased and abnormal uterine-tissue sizes and suggested a mechanism through which Mmp2 loss leads to collagen accumulation and fibrosis in the myometrium, preventing it from contracting efficiently. Additionally, a smaller pelvis was demonstrated in the Mmp2 −/− compared to the WT females, suggesting the contribution of a biomechanical factor to the high rates of dystocia in Mmp2 −/− females. The main strength of this study is that this was the first time Mmp2 /9 loss was studied in the context of physiological parturition processes in mice. The extremely high rates of dystocia found in the Mmp2 -nulls strongly suggest that this gelatinase is a fundamental regulator of uterine functionality during parturition. The causal relationship between the severe dystocia and the abnormal structure of the uterine wall in the Mmp2 -nulls shown in this study calls for a further in-depth analysis of the action of gelatinases during normal and abnormal labor, and of the compensatory mechanisms that may be present or missing in the absence of one or both gelatinases. However, while mice are the gold standard for biomedical research, the conclusions from our genetically modified mouse models cannot be directly applied to obstructive or pre-term births in women. Hence, further studies are required to illuminate the roles and mechanisms of action of gelatinases in dystocia etiology and prevention. 5. Materials and Methods 5.1. Animals We used Mmp2 −/− [ 64 , 65 ], Mmp9 −/− [ 57 ], dKO ( Mmp2 −/− Mmp9 −/− ) [ 23 ] and wild-type (WT) C57BL/6J female mice. The WT and Mmp9 −/− mice were purchased from Harlan laboratories (Rehovot, Israel), while Mmp2 −/− mice were provided by Martignetti Lab (Mount Sinai School of Medicine, NY, USA). The dKO mice, as well as additional genotypes ( Mmp2 +/− , Mmp9 +/− , Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− ), were generated by us [ 21 , 23 ]. Mice were maintained at the Hebrew University Specific Pathogen Free animal facility according to animal-care regulations. All procedures were approved by the Hebrew University Animal Care Committee (license 21-16781-3). Genotyping was performed as previously described [ 21 ]. 5.2. Dystocia Assessment Mating in the study was conducted as part of breeding-colony maintenance for other studies [ 21 , 23 ]. Females of different genotypes were mated with males of different genotypes. Dystocia percentages were measured for each female's genotype as the number of pregnancies ending in dystocia versus the total number of pregnancies for the same genotype. Dystocia was diagnosed in three different situations: (1) (most common) when a pregnancy reached GD22.5 according to vaginal plug observation, but did not result in normal parturition, as reflected by females that still had one or more (dead) fetuses in their uterus; (2) vaginal bleeding at GD18.5–GD21.5 with remaining of dead fetuses in uterus, which could not be expelled from the uterus even on the next day; (3) when GD was not determined by vaginal plug, but it was clear upon visual observation that females had reached term without apparent parturition, or newborns in the cage but with dead fetuses remaining in uterus and deterioration in females' physical condition (hunched posture). Notably, in all of the above cases, females presented with reduced muscle tone in their abdomen, which was observed by elevation of the uterus towards the mouse's chest (as shown in Figure 1 A). 5.3. Genotype Distribution and Litter-Size Measurements Males and females from Mmp2 +/− genotype (n = 4) were mated at the ages of 4–8 months overnight (~16 h), and then females were monitored for vaginal plug, which was determined as gestational day (GD) 0.5. Eighteen days later, at GD18.5, females were euthanized, and genotyping was performed on a piece of tail from their fetuses. The relative distribution of each possible genotype (WT, Mmp2 +/− and Mmp2 −/− ) was calculated and compared to the expected ratio according to Mendel's law for genetic distribution. For litter-size measurements, 37 pairs of WT females and males were mated, and the number of neonates was counted at postnatal day (P) 0. The same procedure was conducted for 11 pairs of Mmp2 −/− , 11 pairs of Mmp9 −/− and 11 pairs of dKO females and males. 5.4. Collection of Uterine Tissues Nulliparous females were euthanized at the ages of 8 weeks (w), 4 months (M) and 8–9.5 M (n = 4–12) to collect their uterine tissues. Prior to that, females were treated with Depo-Provera (Pfizer Inc., New York City, NY, USA), which contains medroxyprogesterone acetate (MPA), for synchronization into diestrus stage. Diestrus stage was chosen according to our previous studies and experience [ 66 ]. The MPA (3 mg per mouse) was subcutaneously injected once on day 1 and a second time on day 5. Synchronized diestrus stage was reached 7 days after first Depo-Provera administration [ 67 ]. When indicated, uteri at 12 M of age were collected without estrus-cycle synchronization. After uterine collection, their gross morphology was assessed, followed by histological analysis. 5.5. Histology Uterine tissue was fixed with 4% paraformaldehyde overnight at 4 °C, dehydrated with increased concentrations of ethanol, cleared with xylene and embedded in paraffin. Transverse sections (5 µm) were prepared with microtome (Leica, Germany). For staining, slides were washed twice in xylene for paraffin removal and rehydrated using decreasing ethanol concentrations. The H&E staining and Masson's trichrome staining were conducted as described previously [ 68 , 69 , 70 ]. Sections were imaged by light microscopy (Axio Imager M1 with AxioCam MRm camera, Zeiss, Germany) or scanned at ×20 magnification, using a Panoramic Flash III 250 scanner (Dhistech3, The Technion, Haifa, Israel). 5.6. µCT Scanning and Morphometric Analysis Pelvic bones from WT and Mmp2 −/− females (n = 5 and 4, respectively) were dissected at the ages of 7–11 months. Scanning was performed using a Skyscan 1272 X-ray computed microtomography device. Images were obtained by 100 kV X-ray tube voltages, using a 0.25 mm aluminum filter, at 4000 ms of exposure time, at the highest special resolution of 15 µm. For each specimen, a series of 900 projection images were obtained with a rotation step of 0.5°, averaging two frames, for a total rotation of 180°. Flat-field correction was performed at the beginning of every scan for a specific zone and image format. A stack of 2D X-ray shadow projections was reconstructed to obtain images using NRecon software (version number 1.1.18, 1272 control software; Skyscan, Bruker, Belgium). Reconstructed scans were volume-rendered (Amira software v.6.4, FEI, Hillsboro, OR, USA) to visualize the 3D morphologies of the selected samples [ 71 ], to allow measurements of the pelvis' lengths and widths. 5.7. Quantification and Statistical Analysis All data are expressed as the mean ± SEM (standard error). The significance of differences between groups was determined using JMP 15.0 Statistical Discovery Software (SAS Institute 2000) and GraphPad Prism 9.0 using one-way ANOVA followed by Tukey–Kramer HSD test and t -test. Differences were considered significant at p ≤ 0.05. 5.1. Animals We used Mmp2 −/− [ 64 , 65 ], Mmp9 −/− [ 57 ], dKO ( Mmp2 −/− Mmp9 −/− ) [ 23 ] and wild-type (WT) C57BL/6J female mice. The WT and Mmp9 −/− mice were purchased from Harlan laboratories (Rehovot, Israel), while Mmp2 −/− mice were provided by Martignetti Lab (Mount Sinai School of Medicine, NY, USA). The dKO mice, as well as additional genotypes ( Mmp2 +/− , Mmp9 +/− , Mmp2 +/− Mmp9 +/− , Mmp2 −/− Mmp9 +/− , Mmp2 +/− Mmp9 −/− ), were generated by us [ 21 , 23 ]. Mice were maintained at the Hebrew University Specific Pathogen Free animal facility according to animal-care regulations. All procedures were approved by the Hebrew University Animal Care Committee (license 21-16781-3). Genotyping was performed as previously described [ 21 ]. 5.2. Dystocia Assessment Mating in the study was conducted as part of breeding-colony maintenance for other studies [ 21 , 23 ]. Females of different genotypes were mated with males of different genotypes. Dystocia percentages were measured for each female's genotype as the number of pregnancies ending in dystocia versus the total number of pregnancies for the same genotype. Dystocia was diagnosed in three different situations: (1) (most common) when a pregnancy reached GD22.5 according to vaginal plug observation, but did not result in normal parturition, as reflected by females that still had one or more (dead) fetuses in their uterus; (2) vaginal bleeding at GD18.5–GD21.5 with remaining of dead fetuses in uterus, which could not be expelled from the uterus even on the next day; (3) when GD was not determined by vaginal plug, but it was clear upon visual observation that females had reached term without apparent parturition, or newborns in the cage but with dead fetuses remaining in uterus and deterioration in females' physical condition (hunched posture). Notably, in all of the above cases, females presented with reduced muscle tone in their abdomen, which was observed by elevation of the uterus towards the mouse's chest (as shown in Figure 1 A). 5.3. Genotype Distribution and Litter-Size Measurements Males and females from Mmp2 +/− genotype (n = 4) were mated at the ages of 4–8 months overnight (~16 h), and then females were monitored for vaginal plug, which was determined as gestational day (GD) 0.5. Eighteen days later, at GD18.5, females were euthanized, and genotyping was performed on a piece of tail from their fetuses. The relative distribution of each possible genotype (WT, Mmp2 +/− and Mmp2 −/− ) was calculated and compared to the expected ratio according to Mendel's law for genetic distribution. For litter-size measurements, 37 pairs of WT females and males were mated, and the number of neonates was counted at postnatal day (P) 0. The same procedure was conducted for 11 pairs of Mmp2 −/− , 11 pairs of Mmp9 −/− and 11 pairs of dKO females and males. 5.4. Collection of Uterine Tissues Nulliparous females were euthanized at the ages of 8 weeks (w), 4 months (M) and 8–9.5 M (n = 4–12) to collect their uterine tissues. Prior to that, females were treated with Depo-Provera (Pfizer Inc., New York City, NY, USA), which contains medroxyprogesterone acetate (MPA), for synchronization into diestrus stage. Diestrus stage was chosen according to our previous studies and experience [ 66 ]. The MPA (3 mg per mouse) was subcutaneously injected once on day 1 and a second time on day 5. Synchronized diestrus stage was reached 7 days after first Depo-Provera administration [ 67 ]. When indicated, uteri at 12 M of age were collected without estrus-cycle synchronization. After uterine collection, their gross morphology was assessed, followed by histological analysis. 5.5. Histology Uterine tissue was fixed with 4% paraformaldehyde overnight at 4 °C, dehydrated with increased concentrations of ethanol, cleared with xylene and embedded in paraffin. Transverse sections (5 µm) were prepared with microtome (Leica, Germany). For staining, slides were washed twice in xylene for paraffin removal and rehydrated using decreasing ethanol concentrations. The H&E staining and Masson's trichrome staining were conducted as described previously [ 68 , 69 , 70 ]. Sections were imaged by light microscopy (Axio Imager M1 with AxioCam MRm camera, Zeiss, Germany) or scanned at ×20 magnification, using a Panoramic Flash III 250 scanner (Dhistech3, The Technion, Haifa, Israel). 5.6. µCT Scanning and Morphometric Analysis Pelvic bones from WT and Mmp2 −/− females (n = 5 and 4, respectively) were dissected at the ages of 7–11 months. Scanning was performed using a Skyscan 1272 X-ray computed microtomography device. Images were obtained by 100 kV X-ray tube voltages, using a 0.25 mm aluminum filter, at 4000 ms of exposure time, at the highest special resolution of 15 µm. For each specimen, a series of 900 projection images were obtained with a rotation step of 0.5°, averaging two frames, for a total rotation of 180°. Flat-field correction was performed at the beginning of every scan for a specific zone and image format. A stack of 2D X-ray shadow projections was reconstructed to obtain images using NRecon software (version number 1.1.18, 1272 control software; Skyscan, Bruker, Belgium). Reconstructed scans were volume-rendered (Amira software v.6.4, FEI, Hillsboro, OR, USA) to visualize the 3D morphologies of the selected samples [ 71 ], to allow measurements of the pelvis' lengths and widths. 5.7. Quantification and Statistical Analysis All data are expressed as the mean ± SEM (standard error). The significance of differences between groups was determined using JMP 15.0 Statistical Discovery Software (SAS Institute 2000) and GraphPad Prism 9.0 using one-way ANOVA followed by Tukey–Kramer HSD test and t -test. Differences were considered significant at p ≤ 0.05.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173440/

POLYMERASE CHAIN REACTION

Introduction Polymerase chain reaction (PCR) is a technology for exponential amplification of a fragment of DNA. (The PCR is covered by patents owned by Hoffman-La Roche. A license is required to use the PCR process.) The limit of its sensitivity is a single molecule, making PCR a superb qualitative tool for the specific detection of rare DNA sequences. Under proper conditions, the yield of amplified DNA is proportional to the initial number of target molecules, rendering it a quantitative analytical tool as well. Since its original description in 1985, PCR has evolved into an assemblage of varied methodologies almost universally used in basic biological research, biotechnology, clinical research, clinical diagnostics, forensics, food technology, environmental testing, archaeology and anthropology, and other fields. Even though other nucleic acid amplification technologies have been described, PCR remains by far the most widely used. Biochemical Basis of PCR PCR involves the enzymatic synthesis of millions of copies of a specific DNA segment. The exponential amplification of a very small amount of template DNA is achieved using a heat-stable DNA polymerase and an automated heat block that is capable of rapid changes of temperature. The template DNA molecule is initially denatured to two single strands by heating to high temperature (typically 90–95°C) in the first stage of the PCR cycle ( Figure 1A ). Two small oligonucleotides that are complementary to sequences on opposite strands of the template molecule and that flank the DNA segment to be amplified are used as primers for the DNA polymerase. The second stage of the PCR cycle consists of cooling the reaction, which permits the annealing of the single-stranded oligonucleotide primers to the denatured template molecule ( Figure 1B ). The third stage of the reaction is the extension of the new strand from the annealed primer in a 5′ to 3′ direction by the heat-stable polymerase ( Figure 1C ). This is performed at the optimum temperature for the polymerase (68–72°C). The most commonly used polymerase is the enzyme isolated from Thermus aquaticus ( Taq DNA polymerase). After the first cycle, each template molecule has been amplified to two molecules. These in turn are denatured in the next cycle and amplified to produce four molecules. The four molecules are amplified to eight in the third cycle, and so on. Each successive cycle effectively doubles the amount of DNA product. The three-stage cycle of denaturation, annealing, and primer extension is repeated 25–40 times in a typical PCR procedure. The product of such a reaction is a large quantity (10 −9 to 10 −8 mol l −1 in a 10–100 μl volume) of a double-stranded DNA molecule whose length is determined by the distance between the primer sites on the original template molecule. Typically, PCR products or amplicons are 100–3000 bp in length, although much longer (up to ∼50000 bp) amplifications are possible under specific conditions. PCR products are visualized by electrophoresis followed by staining with fluorescent dyes or by hybridization to labeled oligonucleotide probes. Figure 1 One cycle of a PCR. (A) The template is shown as a double-stranded DNA molecule, which is denatured to two single strands. (B) The short oligonucleotide primers anneal to the complementary regions of the single strands. (C) The polymerase generates a new strand from each single-stranded molecule, creating two double-stranded molecules. Technical Aspects of PCR One of the advantages of PCR technology is the speed and simplicity with which the technique can be performed. A PCR reaction can be set up in a short time, and multiple samples can be handled easily. Components of the amplification reaction (template DNA, DNA polymerase, oligonucleotide primers, buffers) are added to the amplification vessel. The most common containers for PCR are microcentrifuge tubes (0.5–0.2 ml) with thin walls to facilitate rapid heat transfer or multiwell plates (96- or 384-well plates being the most common). Some commercially available systems utilize specially designed vessels such as capillary tubes. The reaction tubes are then placed into an automated programmable thermal cycler for controlled heating and cooling for denaturation, annealing, and extension reactions. Following amplification, some type of DNA sequence analysis is typically performed, although technology for real-time monitoring of amplification obviates the need even to open the PCR tube after cycling is complete. Template DNA One of the advantages of PCR is that DNA from relatively clean samples need not be highly purified to be used as a template for amplification. It is possible to simply heat samples to near boiling to release DNA, then to add the resulting lysate to the PCR reaction tube. Such simplicity is the hallmark of PCR. With some types of biological samples, inhibitors of PCR such as heme or polyanionic mucopolysaccharides must be removed by DNA purification. Virtually any form of DNA can be used as a template in a PCR reaction. Plasmids, cosmids, phagemids, lambda phage, M13 phage, genomic DNA, and many other sources of DNA have all been used successfully. A PCR can be performed directly on colonies or plaques. Although it is possible to amplify a single DNA molecule, a typical PCR reaction uses 10 5 –10 6 copies of the target DNA as template. In practice, this means adding ∼1 μg of eukaryotic genomic DNA, 10 ng of yeast DNA, or 1 ng of bacterial DNA. The concentration of DNA is not critical for most applications, however, and a PCR reaction will work with a wide range of template concentrations. Often the concentration of template DNA is unknown and does not need to be determined. If necessary, a series of control reactions, each containing a different amount of template, can be performed. Biochemical Components DNA polymerase A wide range of DNA polymerases is available for PCR. A cloned heat-stable DNA polymerase from Thermus aquaticus ( Taq DNA polymerase) is the most commonly used. Other enzymes such as DNA polymerase from Pyrococcus furiosus ( Pfu DNA polymerase) or Thermoccocus litoralis (Vent™ DNA polymerase, New England Biolabs) have been reported to have a lower error rate than Taq polymerase and may be advantageous for some applications. Typically, 0.5–2.5 units of enzyme are used per 50 μl reaction. Oligonucleotide primers Oligonucleotide primers are usually used at a concentration of 1 μmol l −1 in PCR reactions (50 pmol per 50 μl reaction). Concentrations between 0.1 and 1 μmol l −1 can be used. Higher concentrations may increase nonspecific annealing of primers and thus to nonspecific amplification products. PCR primers are normally between 18 and 30 nucleotides in length and should preferably have a guanine+cytosine (G+C) content of ∼50%. The temperature at which half the DNA molecules will be double-stranded, T m , in degree celsius, for a primer is estimated by the rule-of-thumb equation: 2×(number of As and Ts)+4×number of Gs and Cs) (A=adenine, T=thymine). However, more accurate calculation of oligonucleotide T m requires consideration of the effects of ionic strength and neighboring bases in the DNA strand (nearest neighbor T m calculation methods). Software is available for performing these calculations. The T m values for the two primers in a reaction should be similar, and the annealing temperature used is normally ∼5°C below the T m . As annealing temperature approaches T m , more specific amplifications are achieved, but overall yields may decrease. Despite careful calculations, empirical testing of annealing temperatures is essential for a well-optimized PCR assay. Complementarity at the 3′ ends of primer pairs should be avoided as this promotes the formation of primer oligomers (primer dimers). Such artifactual products are themselves templates for PCR amplification and compete with the desired products for deoxynucleotide triphosphates (dNTPs), polymerase, and primers. This leads to a decreased yield of the desired product and the presence of nonspecific products that may complicate analysis. A primer oligomer forms when two or more primers anneal to each other and are extended during PCR. The double-stranded product acts as a template during subsequent rounds of amplification and competes with the desired product. In general, low annealing temperatures, high enzyme concentrations, and high primer concentrations increase the probability of primer oligomerization. However, it is stringency during the initial cycle of PCR that is most critical for primer oligomer formation, as well as for nonspecific PCR products in general. Various techniques for maintaining high stringency up until the point when primers and enzyme are mixed together have been described (generally called Hot Start). Hot Start methods involve mixing of enzyme and primers only after reactions have reached a stringent temperature. A simple technique for Hot Start is manual addition of a small volume (typically 5–10 μl) of reaction buffer containing the enzyme to the other components maintained in the thermal cycler at ∼80°C, then continuing with standard PCR amplification. Another method involves sealing a portion of the amplification reaction under wax, then adding the remaining reactants above the wax barrier. Enzymatic methods to accomplish the same goal include the use of a modified DNA polymerase that is blocked with a thermolabile group or the use of uracil DNA glycosylase (uracil- N -glycosylase, UNG), deoxyuridine triphosphate (dUTP), and a brief initial incubation at 50°C. Typically, dUTP is substituted on an equimolar basis for deoxythymidine triphosphate (dTTP) (200 μmol l −1 ), but higher concentrations of dUTP (125–300 μmol l −1 ) may be beneficial in some assays. Uracil DNA glycosylase is used at 1–2 Units per reaction. This method ensures that any nonspecific product or primer oligomer that is generated during the initial low-stringency conditions will contain uracil (U) and therefore be susceptible to cleavage by UNG. Uracil is excised during the 50°C incubation, and strand breakage occurs at these abasic sites during the initial denaturation step. In addition to inactivating any contaminating amplicons, this method reduces nonspecific products and primer oligomer. Because residual UNG can degrade the U-containing amplicons, PCR products must be analyzed immediately or stored at 4°C for short periods of time or frozen for longer times. Alternately, UNG can be removed by extraction with organic solvents or digested with proteases. The 3′ end of a PCR primer should be perfectly complementary to the template DNA. Failure of the 3′ end to hybridize results in inefficient amplification. Internal sequences are less critical. Provided a suitable annealing temperature is used, degenerate primers (primers that consist of a pool of different but closely related oligonucleotides) may be used to ensure that primers will anneal to highly variable sequences or sequences that are only partially known. These primers are designed from amino acid sequences or from a comparison of similar genes from other organisms. Sequences at the 5′ end of the primer are least critical. Providing the hybridizing portion of the primer is long enough to ensure annealing to the template DNA, nonhomologous 5′ extensions (regions that do not correspond to the sequence of the template) can be used to introduce restriction enzyme sites into PCR products to be cloned or to add other sequences such as phage RNA polymerase promoters. Multiplex PCR enables the detection of multiple gene sequences within the same reaction. Because several sets of PCR primers must function in the same reaction without interference, primer design and optimization of reaction conditions are critical. Primers are often labeled with detectable groups to facilitate post-PCR analysis. Radioactive isotopes, haptens such as biotin, or fluorescent dyes are the most widely used. Labels attached to the 5′ end of a primer have little or no effect on amplification. Many heat-stable polymerases used for PCR exhibit a template-independent activity that adds deoxynucleosides (predominantly deoxyadenosine) to the 3′ ends of all double-stranded molecules in the reaction. These A-overhangs must be filled in prior to blunt-end cloning of PCR products. Alternatively, special vectors are available that have single 3′ T-overhangs at the cloning site ready for insertion of the PCR product. Choosing a polymerase without this activity or adjusting reaction conditions to either minimize or maximize the extent of A-overhangs may produce sharper peaks in high-resolution electrophoretic analysis. Deoxynucleoside triphosphates dNTPs are typically used at a concentration of ∼200 μmol l −1 each dNTP in a PCR reaction. Excessively high concentrations promote nonspecific product formation. Modified dNTPs are sometimes used to label PCR products with radioactive or fluorescent markers or with haptens such as biotin, fluorescein, or digoxygenin. The modified dNTP is typically used at a concentration (0.1–1 μmol l −1 ) much lower than the unmodified dNTP (∼200 μmol l −1 ). Probes can be easily generated using PCR amplification with a labeled nucleotide, followed by removal of the unincorporated label. Buffer components Several reagents containing buffer ions, monovalent salts, and divalent cations required for polymerase activity, have been described for use in PCR amplification. The most widely used buffer consists of 10 mmol l −1 Tris–HCl (pH 8.3 at room temperature) and 50 mmol l −1 KCl. At the extension temperature (72°C), the pH of this Tris buffer falls to 7.2, near the optimum for Taq DNA polymerase. Sulfate-containing buffers such as 20 mmol l −1 Tris–SO 4 (pH 8.5–9.0 at room temperature) and 20 mmol l −1 (NH 4 ) 2 SO 4 are also widely used. Monovalent cations (K + or NH 4 + ) are included to adjust ionic strength. DNA polymerases require divalent cations for activity, and PCR reactions contain MgCl 2 (1–5 mmol l −1 ). In general, higher MgCl 2 concentrations promote nonspecific primer annealing and nonspecific product amplification. Reaction buffers usually include a low percentage (0.1–0.5%) of nonionic detergent such as Igepal CA-630, Tween 20, or Triton X-100 to minimize adsorption of polymerase to the surfaces of the PCR tube. Proteins (gelatin or bovine serum albumin) are sometimes included at similar concentrations as nonionic detergents for the same reason. Other components that have been reported to increase PCR product yields or specificity on specific templates include betaine, dimethylsulfoxide, formamide, and glycerol. Thermal Cycling Equipment The PCR process involves thermal cycling between denaturation, annealing, and extension temperatures ( Figure 1 ). Each incubation step is a segment, and a complete round of denaturation, annealing, and extension is a cycle. First, samples are heated to a temperature adequate to melt the double-stranded DNA template and any secondary structure in the primers. Denaturation is most commonly performed at 94°C, although temperatures of 97–99°C may be required for templates with high GC content. After the first several cycles, denaturation temperature can be lowered to 90–92°C to reduce loss of polymerase activity through heat denaturation during the course of PCR. Next, samples are cooled to an annealing temperature at which the primers will hybridize to their target sequences. The choice of annealing temperature depends on primer T m , but is usually in the range of 50–60°C. Finally, the samples are heated to the extension temperature (68–72°C). If primers are designed to permit efficient annealing at 60–68°C, the annealing and extension steps can be combined into one step. Typical PCR amplification is performed over 20–30 cycles, although 40 or more cycles may be used with template DNA at very low initial concentration or with otherwise marginal amplifications involving, for example, degenerate primers or poor-quality template DNA. Although it is possible to perform 20 or even 40 cycles of PCR manually using sandbaths, waterbaths, or heat blocks and physically moving the samples from one to the other at timed intervals, the tedium involved makes this approach impractical. Many different types of machines are commercially available to perform thermal cycling. Some cyclers use robotic arms to transfer a rack of PCR samples from one heat block to another. Other machines use resistive heating elements, compressors, water circulators, fan-forced air, high-intensity lamps, Peltier elements, or combinations of these heating and cooling devices. Microprocessors enable the user to program the instrument for time and temperature of each segment, and the number of cycles to be performed. Thermal cyclers that can monitor progress of the amplification reaction while it is taking place are called real-time thermal cyclers. Real-time instruments are designed to monitor fluorescence from labels whose emission intensity is proportional to the amount of amplified DNA. Flexibility varies greatly among the available machines, but all instruments can measure fluorescence at least once during the annealing segment of every cycle. Intercalating dyes such as ethidium bromide or SYBR Green that are selective for double-stranded DNA provide the simplest method. Probes that hybridize adjacent to one another on one strand of the amplicon can be designed to have a fluorescence energy transfer donor and acceptor that will be close enough for energy transfer if and only if hybridization occurs. Emission from the hybridized donor- and acceptor-labeled probes is monitored during each cycle. A third approach is to use the 5′ nuclease activity of Taq DNA polymerase to cleave a probe labeled with a fluorophore and a quencher. When Taq DNA polymerase (and some, but not all, other heat-stable polymerases) encounters the probe, it cuts it, dissociating the fluorophore and quencher. As with energy transfer methods, amplification is detectable as an increase in fluorescence. In addition to monitoring fluorescence, real-time thermal cyclers provide data analysis for quantitation of initial template concentration. Real-time PCR has become the standard for quantitative PCR. Post-PCR Analysis Unless real-time thermal cyclers are used, some form of manipulation or analysis of PCR products must be performed. In preparative applications, amplicons are inserted into a suitable vector and propagated by standard molecular biological methods. In analytical applications, some form of DNA size or sequence analysis is performed. In many cases, high-performance liquid chromatography or electrophoresis on agarose or polyacrylamide gels to confirm that the amplicon is the expected size is sufficient. In clinical testing, hybridization of an oligonucleotide probe complementary to a sequence between the PCR primers is more commonly used to confirm specificity of the amplified DNA. Probe hybridization can be combined with DNA size analysis by electrophoresis or can be performed using methods analogous to immunoassays. Template DNA One of the advantages of PCR is that DNA from relatively clean samples need not be highly purified to be used as a template for amplification. It is possible to simply heat samples to near boiling to release DNA, then to add the resulting lysate to the PCR reaction tube. Such simplicity is the hallmark of PCR. With some types of biological samples, inhibitors of PCR such as heme or polyanionic mucopolysaccharides must be removed by DNA purification. Virtually any form of DNA can be used as a template in a PCR reaction. Plasmids, cosmids, phagemids, lambda phage, M13 phage, genomic DNA, and many other sources of DNA have all been used successfully. A PCR can be performed directly on colonies or plaques. Although it is possible to amplify a single DNA molecule, a typical PCR reaction uses 10 5 –10 6 copies of the target DNA as template. In practice, this means adding ∼1 μg of eukaryotic genomic DNA, 10 ng of yeast DNA, or 1 ng of bacterial DNA. The concentration of DNA is not critical for most applications, however, and a PCR reaction will work with a wide range of template concentrations. Often the concentration of template DNA is unknown and does not need to be determined. If necessary, a series of control reactions, each containing a different amount of template, can be performed. Biochemical Components DNA polymerase A wide range of DNA polymerases is available for PCR. A cloned heat-stable DNA polymerase from Thermus aquaticus ( Taq DNA polymerase) is the most commonly used. Other enzymes such as DNA polymerase from Pyrococcus furiosus ( Pfu DNA polymerase) or Thermoccocus litoralis (Vent™ DNA polymerase, New England Biolabs) have been reported to have a lower error rate than Taq polymerase and may be advantageous for some applications. Typically, 0.5–2.5 units of enzyme are used per 50 μl reaction. Oligonucleotide primers Oligonucleotide primers are usually used at a concentration of 1 μmol l −1 in PCR reactions (50 pmol per 50 μl reaction). Concentrations between 0.1 and 1 μmol l −1 can be used. Higher concentrations may increase nonspecific annealing of primers and thus to nonspecific amplification products. PCR primers are normally between 18 and 30 nucleotides in length and should preferably have a guanine+cytosine (G+C) content of ∼50%. The temperature at which half the DNA molecules will be double-stranded, T m , in degree celsius, for a primer is estimated by the rule-of-thumb equation: 2×(number of As and Ts)+4×number of Gs and Cs) (A=adenine, T=thymine). However, more accurate calculation of oligonucleotide T m requires consideration of the effects of ionic strength and neighboring bases in the DNA strand (nearest neighbor T m calculation methods). Software is available for performing these calculations. The T m values for the two primers in a reaction should be similar, and the annealing temperature used is normally ∼5°C below the T m . As annealing temperature approaches T m , more specific amplifications are achieved, but overall yields may decrease. Despite careful calculations, empirical testing of annealing temperatures is essential for a well-optimized PCR assay. Complementarity at the 3′ ends of primer pairs should be avoided as this promotes the formation of primer oligomers (primer dimers). Such artifactual products are themselves templates for PCR amplification and compete with the desired products for deoxynucleotide triphosphates (dNTPs), polymerase, and primers. This leads to a decreased yield of the desired product and the presence of nonspecific products that may complicate analysis. A primer oligomer forms when two or more primers anneal to each other and are extended during PCR. The double-stranded product acts as a template during subsequent rounds of amplification and competes with the desired product. In general, low annealing temperatures, high enzyme concentrations, and high primer concentrations increase the probability of primer oligomerization. However, it is stringency during the initial cycle of PCR that is most critical for primer oligomer formation, as well as for nonspecific PCR products in general. Various techniques for maintaining high stringency up until the point when primers and enzyme are mixed together have been described (generally called Hot Start). Hot Start methods involve mixing of enzyme and primers only after reactions have reached a stringent temperature. A simple technique for Hot Start is manual addition of a small volume (typically 5–10 μl) of reaction buffer containing the enzyme to the other components maintained in the thermal cycler at ∼80°C, then continuing with standard PCR amplification. Another method involves sealing a portion of the amplification reaction under wax, then adding the remaining reactants above the wax barrier. Enzymatic methods to accomplish the same goal include the use of a modified DNA polymerase that is blocked with a thermolabile group or the use of uracil DNA glycosylase (uracil- N -glycosylase, UNG), deoxyuridine triphosphate (dUTP), and a brief initial incubation at 50°C. Typically, dUTP is substituted on an equimolar basis for deoxythymidine triphosphate (dTTP) (200 μmol l −1 ), but higher concentrations of dUTP (125–300 μmol l −1 ) may be beneficial in some assays. Uracil DNA glycosylase is used at 1–2 Units per reaction. This method ensures that any nonspecific product or primer oligomer that is generated during the initial low-stringency conditions will contain uracil (U) and therefore be susceptible to cleavage by UNG. Uracil is excised during the 50°C incubation, and strand breakage occurs at these abasic sites during the initial denaturation step. In addition to inactivating any contaminating amplicons, this method reduces nonspecific products and primer oligomer. Because residual UNG can degrade the U-containing amplicons, PCR products must be analyzed immediately or stored at 4°C for short periods of time or frozen for longer times. Alternately, UNG can be removed by extraction with organic solvents or digested with proteases. The 3′ end of a PCR primer should be perfectly complementary to the template DNA. Failure of the 3′ end to hybridize results in inefficient amplification. Internal sequences are less critical. Provided a suitable annealing temperature is used, degenerate primers (primers that consist of a pool of different but closely related oligonucleotides) may be used to ensure that primers will anneal to highly variable sequences or sequences that are only partially known. These primers are designed from amino acid sequences or from a comparison of similar genes from other organisms. Sequences at the 5′ end of the primer are least critical. Providing the hybridizing portion of the primer is long enough to ensure annealing to the template DNA, nonhomologous 5′ extensions (regions that do not correspond to the sequence of the template) can be used to introduce restriction enzyme sites into PCR products to be cloned or to add other sequences such as phage RNA polymerase promoters. Multiplex PCR enables the detection of multiple gene sequences within the same reaction. Because several sets of PCR primers must function in the same reaction without interference, primer design and optimization of reaction conditions are critical. Primers are often labeled with detectable groups to facilitate post-PCR analysis. Radioactive isotopes, haptens such as biotin, or fluorescent dyes are the most widely used. Labels attached to the 5′ end of a primer have little or no effect on amplification. Many heat-stable polymerases used for PCR exhibit a template-independent activity that adds deoxynucleosides (predominantly deoxyadenosine) to the 3′ ends of all double-stranded molecules in the reaction. These A-overhangs must be filled in prior to blunt-end cloning of PCR products. Alternatively, special vectors are available that have single 3′ T-overhangs at the cloning site ready for insertion of the PCR product. Choosing a polymerase without this activity or adjusting reaction conditions to either minimize or maximize the extent of A-overhangs may produce sharper peaks in high-resolution electrophoretic analysis. Deoxynucleoside triphosphates dNTPs are typically used at a concentration of ∼200 μmol l −1 each dNTP in a PCR reaction. Excessively high concentrations promote nonspecific product formation. Modified dNTPs are sometimes used to label PCR products with radioactive or fluorescent markers or with haptens such as biotin, fluorescein, or digoxygenin. The modified dNTP is typically used at a concentration (0.1–1 μmol l −1 ) much lower than the unmodified dNTP (∼200 μmol l −1 ). Probes can be easily generated using PCR amplification with a labeled nucleotide, followed by removal of the unincorporated label. Buffer components Several reagents containing buffer ions, monovalent salts, and divalent cations required for polymerase activity, have been described for use in PCR amplification. The most widely used buffer consists of 10 mmol l −1 Tris–HCl (pH 8.3 at room temperature) and 50 mmol l −1 KCl. At the extension temperature (72°C), the pH of this Tris buffer falls to 7.2, near the optimum for Taq DNA polymerase. Sulfate-containing buffers such as 20 mmol l −1 Tris–SO 4 (pH 8.5–9.0 at room temperature) and 20 mmol l −1 (NH 4 ) 2 SO 4 are also widely used. Monovalent cations (K + or NH 4 + ) are included to adjust ionic strength. DNA polymerases require divalent cations for activity, and PCR reactions contain MgCl 2 (1–5 mmol l −1 ). In general, higher MgCl 2 concentrations promote nonspecific primer annealing and nonspecific product amplification. Reaction buffers usually include a low percentage (0.1–0.5%) of nonionic detergent such as Igepal CA-630, Tween 20, or Triton X-100 to minimize adsorption of polymerase to the surfaces of the PCR tube. Proteins (gelatin or bovine serum albumin) are sometimes included at similar concentrations as nonionic detergents for the same reason. Other components that have been reported to increase PCR product yields or specificity on specific templates include betaine, dimethylsulfoxide, formamide, and glycerol. DNA polymerase A wide range of DNA polymerases is available for PCR. A cloned heat-stable DNA polymerase from Thermus aquaticus ( Taq DNA polymerase) is the most commonly used. Other enzymes such as DNA polymerase from Pyrococcus furiosus ( Pfu DNA polymerase) or Thermoccocus litoralis (Vent™ DNA polymerase, New England Biolabs) have been reported to have a lower error rate than Taq polymerase and may be advantageous for some applications. Typically, 0.5–2.5 units of enzyme are used per 50 μl reaction. Oligonucleotide primers Oligonucleotide primers are usually used at a concentration of 1 μmol l −1 in PCR reactions (50 pmol per 50 μl reaction). Concentrations between 0.1 and 1 μmol l −1 can be used. Higher concentrations may increase nonspecific annealing of primers and thus to nonspecific amplification products. PCR primers are normally between 18 and 30 nucleotides in length and should preferably have a guanine+cytosine (G+C) content of ∼50%. The temperature at which half the DNA molecules will be double-stranded, T m , in degree celsius, for a primer is estimated by the rule-of-thumb equation: 2×(number of As and Ts)+4×number of Gs and Cs) (A=adenine, T=thymine). However, more accurate calculation of oligonucleotide T m requires consideration of the effects of ionic strength and neighboring bases in the DNA strand (nearest neighbor T m calculation methods). Software is available for performing these calculations. The T m values for the two primers in a reaction should be similar, and the annealing temperature used is normally ∼5°C below the T m . As annealing temperature approaches T m , more specific amplifications are achieved, but overall yields may decrease. Despite careful calculations, empirical testing of annealing temperatures is essential for a well-optimized PCR assay. Complementarity at the 3′ ends of primer pairs should be avoided as this promotes the formation of primer oligomers (primer dimers). Such artifactual products are themselves templates for PCR amplification and compete with the desired products for deoxynucleotide triphosphates (dNTPs), polymerase, and primers. This leads to a decreased yield of the desired product and the presence of nonspecific products that may complicate analysis. A primer oligomer forms when two or more primers anneal to each other and are extended during PCR. The double-stranded product acts as a template during subsequent rounds of amplification and competes with the desired product. In general, low annealing temperatures, high enzyme concentrations, and high primer concentrations increase the probability of primer oligomerization. However, it is stringency during the initial cycle of PCR that is most critical for primer oligomer formation, as well as for nonspecific PCR products in general. Various techniques for maintaining high stringency up until the point when primers and enzyme are mixed together have been described (generally called Hot Start). Hot Start methods involve mixing of enzyme and primers only after reactions have reached a stringent temperature. A simple technique for Hot Start is manual addition of a small volume (typically 5–10 μl) of reaction buffer containing the enzyme to the other components maintained in the thermal cycler at ∼80°C, then continuing with standard PCR amplification. Another method involves sealing a portion of the amplification reaction under wax, then adding the remaining reactants above the wax barrier. Enzymatic methods to accomplish the same goal include the use of a modified DNA polymerase that is blocked with a thermolabile group or the use of uracil DNA glycosylase (uracil- N -glycosylase, UNG), deoxyuridine triphosphate (dUTP), and a brief initial incubation at 50°C. Typically, dUTP is substituted on an equimolar basis for deoxythymidine triphosphate (dTTP) (200 μmol l −1 ), but higher concentrations of dUTP (125–300 μmol l −1 ) may be beneficial in some assays. Uracil DNA glycosylase is used at 1–2 Units per reaction. This method ensures that any nonspecific product or primer oligomer that is generated during the initial low-stringency conditions will contain uracil (U) and therefore be susceptible to cleavage by UNG. Uracil is excised during the 50°C incubation, and strand breakage occurs at these abasic sites during the initial denaturation step. In addition to inactivating any contaminating amplicons, this method reduces nonspecific products and primer oligomer. Because residual UNG can degrade the U-containing amplicons, PCR products must be analyzed immediately or stored at 4°C for short periods of time or frozen for longer times. Alternately, UNG can be removed by extraction with organic solvents or digested with proteases. The 3′ end of a PCR primer should be perfectly complementary to the template DNA. Failure of the 3′ end to hybridize results in inefficient amplification. Internal sequences are less critical. Provided a suitable annealing temperature is used, degenerate primers (primers that consist of a pool of different but closely related oligonucleotides) may be used to ensure that primers will anneal to highly variable sequences or sequences that are only partially known. These primers are designed from amino acid sequences or from a comparison of similar genes from other organisms. Sequences at the 5′ end of the primer are least critical. Providing the hybridizing portion of the primer is long enough to ensure annealing to the template DNA, nonhomologous 5′ extensions (regions that do not correspond to the sequence of the template) can be used to introduce restriction enzyme sites into PCR products to be cloned or to add other sequences such as phage RNA polymerase promoters. Multiplex PCR enables the detection of multiple gene sequences within the same reaction. Because several sets of PCR primers must function in the same reaction without interference, primer design and optimization of reaction conditions are critical. Primers are often labeled with detectable groups to facilitate post-PCR analysis. Radioactive isotopes, haptens such as biotin, or fluorescent dyes are the most widely used. Labels attached to the 5′ end of a primer have little or no effect on amplification. Many heat-stable polymerases used for PCR exhibit a template-independent activity that adds deoxynucleosides (predominantly deoxyadenosine) to the 3′ ends of all double-stranded molecules in the reaction. These A-overhangs must be filled in prior to blunt-end cloning of PCR products. Alternatively, special vectors are available that have single 3′ T-overhangs at the cloning site ready for insertion of the PCR product. Choosing a polymerase without this activity or adjusting reaction conditions to either minimize or maximize the extent of A-overhangs may produce sharper peaks in high-resolution electrophoretic analysis. Deoxynucleoside triphosphates dNTPs are typically used at a concentration of ∼200 μmol l −1 each dNTP in a PCR reaction. Excessively high concentrations promote nonspecific product formation. Modified dNTPs are sometimes used to label PCR products with radioactive or fluorescent markers or with haptens such as biotin, fluorescein, or digoxygenin. The modified dNTP is typically used at a concentration (0.1–1 μmol l −1 ) much lower than the unmodified dNTP (∼200 μmol l −1 ). Probes can be easily generated using PCR amplification with a labeled nucleotide, followed by removal of the unincorporated label. Buffer components Several reagents containing buffer ions, monovalent salts, and divalent cations required for polymerase activity, have been described for use in PCR amplification. The most widely used buffer consists of 10 mmol l −1 Tris–HCl (pH 8.3 at room temperature) and 50 mmol l −1 KCl. At the extension temperature (72°C), the pH of this Tris buffer falls to 7.2, near the optimum for Taq DNA polymerase. Sulfate-containing buffers such as 20 mmol l −1 Tris–SO 4 (pH 8.5–9.0 at room temperature) and 20 mmol l −1 (NH 4 ) 2 SO 4 are also widely used. Monovalent cations (K + or NH 4 + ) are included to adjust ionic strength. DNA polymerases require divalent cations for activity, and PCR reactions contain MgCl 2 (1–5 mmol l −1 ). In general, higher MgCl 2 concentrations promote nonspecific primer annealing and nonspecific product amplification. Reaction buffers usually include a low percentage (0.1–0.5%) of nonionic detergent such as Igepal CA-630, Tween 20, or Triton X-100 to minimize adsorption of polymerase to the surfaces of the PCR tube. Proteins (gelatin or bovine serum albumin) are sometimes included at similar concentrations as nonionic detergents for the same reason. Other components that have been reported to increase PCR product yields or specificity on specific templates include betaine, dimethylsulfoxide, formamide, and glycerol. Thermal Cycling Equipment The PCR process involves thermal cycling between denaturation, annealing, and extension temperatures ( Figure 1 ). Each incubation step is a segment, and a complete round of denaturation, annealing, and extension is a cycle. First, samples are heated to a temperature adequate to melt the double-stranded DNA template and any secondary structure in the primers. Denaturation is most commonly performed at 94°C, although temperatures of 97–99°C may be required for templates with high GC content. After the first several cycles, denaturation temperature can be lowered to 90–92°C to reduce loss of polymerase activity through heat denaturation during the course of PCR. Next, samples are cooled to an annealing temperature at which the primers will hybridize to their target sequences. The choice of annealing temperature depends on primer T m , but is usually in the range of 50–60°C. Finally, the samples are heated to the extension temperature (68–72°C). If primers are designed to permit efficient annealing at 60–68°C, the annealing and extension steps can be combined into one step. Typical PCR amplification is performed over 20–30 cycles, although 40 or more cycles may be used with template DNA at very low initial concentration or with otherwise marginal amplifications involving, for example, degenerate primers or poor-quality template DNA. Although it is possible to perform 20 or even 40 cycles of PCR manually using sandbaths, waterbaths, or heat blocks and physically moving the samples from one to the other at timed intervals, the tedium involved makes this approach impractical. Many different types of machines are commercially available to perform thermal cycling. Some cyclers use robotic arms to transfer a rack of PCR samples from one heat block to another. Other machines use resistive heating elements, compressors, water circulators, fan-forced air, high-intensity lamps, Peltier elements, or combinations of these heating and cooling devices. Microprocessors enable the user to program the instrument for time and temperature of each segment, and the number of cycles to be performed. Thermal cyclers that can monitor progress of the amplification reaction while it is taking place are called real-time thermal cyclers. Real-time instruments are designed to monitor fluorescence from labels whose emission intensity is proportional to the amount of amplified DNA. Flexibility varies greatly among the available machines, but all instruments can measure fluorescence at least once during the annealing segment of every cycle. Intercalating dyes such as ethidium bromide or SYBR Green that are selective for double-stranded DNA provide the simplest method. Probes that hybridize adjacent to one another on one strand of the amplicon can be designed to have a fluorescence energy transfer donor and acceptor that will be close enough for energy transfer if and only if hybridization occurs. Emission from the hybridized donor- and acceptor-labeled probes is monitored during each cycle. A third approach is to use the 5′ nuclease activity of Taq DNA polymerase to cleave a probe labeled with a fluorophore and a quencher. When Taq DNA polymerase (and some, but not all, other heat-stable polymerases) encounters the probe, it cuts it, dissociating the fluorophore and quencher. As with energy transfer methods, amplification is detectable as an increase in fluorescence. In addition to monitoring fluorescence, real-time thermal cyclers provide data analysis for quantitation of initial template concentration. Real-time PCR has become the standard for quantitative PCR. Post-PCR Analysis Unless real-time thermal cyclers are used, some form of manipulation or analysis of PCR products must be performed. In preparative applications, amplicons are inserted into a suitable vector and propagated by standard molecular biological methods. In analytical applications, some form of DNA size or sequence analysis is performed. In many cases, high-performance liquid chromatography or electrophoresis on agarose or polyacrylamide gels to confirm that the amplicon is the expected size is sufficient. In clinical testing, hybridization of an oligonucleotide probe complementary to a sequence between the PCR primers is more commonly used to confirm specificity of the amplified DNA. Probe hybridization can be combined with DNA size analysis by electrophoresis or can be performed using methods analogous to immunoassays. Considerations Fidelity For preparative applications and the analysis of single-base changes, fidelity of the DNA polymerase is important. The fidelity of the DNA polymerase in a PCR reaction determines the similarity between the sequence of the PCR product and the original template. Taq DNA polymerase sometimes incorporates an incorrect base in the growing DNA strand, and this error is propagated during subsequent rounds of cycling. The higher the fidelity, the more closely the sequence of the PCR product reflects the sequence of the original DNA template. A range of DNA polymerases is commercially available, and certain enzymes have demonstrably higher fidelity. In addition to intrinsic differences among the various heat-stable polymerases, fidelity is influenced by a number of factors. It is therefore possible to increase or decrease the number of mismatches between the product and initial template. Conditions of low fidelity may be chosen to introduce random point mutations into a PCR product. More general applications benefit from accurate amplification. Fidelity is improved by keeping polymerase, dNTP, and MgCl 2 concentrations as low as practical, using the same concentrations of each of the four dNTPs, maintaining an annealing temperature near the T m of the primers, and programming a short extension segment and as few cycles as practical. Cross-Contamination Because PCR primers are incorporated into each molecule of PCR product, amplicons are themselves suitable templates for amplification. Re-amplification of previously amplified DNA is a major problem for PCR. To reduce the likelihood of contamination of reagents, separate laboratory work areas should be designated for reagent preparation, sample preparation, reaction assembly and thermal cycling, and post-PCR analysis. Depending on the analytical rigor required and the resources available, well-isolated rooms for each step of the process will be beneficial. In the research laboratory, however, it is more common to rely on separate work areas and equipment for pre- and postamplification work and gowning of laboratory personnel in cleanroom garments. Laboratory equipment, especially pipettors and shared equipment, and laboratory personnel are the most common sources of PCR product contamination. Clinical laboratories or other analytical laboratories now employ physical separation of work areas and equipment in addition to a biochemical method using the enzyme uracil DNA glycosylase (UNG) to prevent amplicon contamination. If PCR reactions are performed with dUTP rather than dTTP, amplicons will be susceptible to digestion by UNG. This method has been called PCR sterilization. The need to avoid contamination with amplified DNA constitutes an incentive to use real-time PCR for qualitative as well as quantitative assays. With real-time PCR, tubes containing amplified DNA need never be opened. No matter what precautions are taken, negative controls containing no DNA template must always be included with each batch of PCR reactions. If low-copy detection is the assay goal, then multiple negative controls are required to adequately test for amplicon contamination. Reverse Transcription PCR PCR can be used to detect and quantify RNA if RNA is first reverse-transcribed to complementary DNA (cDNA). Reverse transcriptases from Maloney murine leukemia virus (MMLV-RT) or avian myeloblastoma virus (AMV-RT) are commonly used. Either the buffer optimal for the reverse transcription (RT) or the PCR buffer is suitable in most cases, and reverse transcription is typically performed at 37–42°C for 15–60 min. Because these enzymes are heat-labile, they are inactivated during the first denaturation segment of PCR. Heat-stable reverse transcriptases enable reverse transcription at higher temperatures to denature RNA secondary structure. In the presence of Mn 2+ , some DNA-dependent DNA polymerases such as the enzyme from Thermus thermophilus ( Tth DNA polymerase) have activity on RNA templates. Using MnCl 2 for reverse transcription can enable cDNA synthesis with the same enzyme used for PCR. Unless a balance between MnCl 2 and MgCl 2 is carefully identified, Mn 2+ must be removed or chelated, and Mg 2+ must be added prior to PCR. RT-PCR can be performed within intact cells ( in situ PCR) to identify cells expressing particular genes or to assess the presence of disease-related genes. Thermal cyclers are available to automate the process on microscope slides. Fidelity For preparative applications and the analysis of single-base changes, fidelity of the DNA polymerase is important. The fidelity of the DNA polymerase in a PCR reaction determines the similarity between the sequence of the PCR product and the original template. Taq DNA polymerase sometimes incorporates an incorrect base in the growing DNA strand, and this error is propagated during subsequent rounds of cycling. The higher the fidelity, the more closely the sequence of the PCR product reflects the sequence of the original DNA template. A range of DNA polymerases is commercially available, and certain enzymes have demonstrably higher fidelity. In addition to intrinsic differences among the various heat-stable polymerases, fidelity is influenced by a number of factors. It is therefore possible to increase or decrease the number of mismatches between the product and initial template. Conditions of low fidelity may be chosen to introduce random point mutations into a PCR product. More general applications benefit from accurate amplification. Fidelity is improved by keeping polymerase, dNTP, and MgCl 2 concentrations as low as practical, using the same concentrations of each of the four dNTPs, maintaining an annealing temperature near the T m of the primers, and programming a short extension segment and as few cycles as practical. Cross-Contamination Because PCR primers are incorporated into each molecule of PCR product, amplicons are themselves suitable templates for amplification. Re-amplification of previously amplified DNA is a major problem for PCR. To reduce the likelihood of contamination of reagents, separate laboratory work areas should be designated for reagent preparation, sample preparation, reaction assembly and thermal cycling, and post-PCR analysis. Depending on the analytical rigor required and the resources available, well-isolated rooms for each step of the process will be beneficial. In the research laboratory, however, it is more common to rely on separate work areas and equipment for pre- and postamplification work and gowning of laboratory personnel in cleanroom garments. Laboratory equipment, especially pipettors and shared equipment, and laboratory personnel are the most common sources of PCR product contamination. Clinical laboratories or other analytical laboratories now employ physical separation of work areas and equipment in addition to a biochemical method using the enzyme uracil DNA glycosylase (UNG) to prevent amplicon contamination. If PCR reactions are performed with dUTP rather than dTTP, amplicons will be susceptible to digestion by UNG. This method has been called PCR sterilization. The need to avoid contamination with amplified DNA constitutes an incentive to use real-time PCR for qualitative as well as quantitative assays. With real-time PCR, tubes containing amplified DNA need never be opened. No matter what precautions are taken, negative controls containing no DNA template must always be included with each batch of PCR reactions. If low-copy detection is the assay goal, then multiple negative controls are required to adequately test for amplicon contamination. Reverse Transcription PCR PCR can be used to detect and quantify RNA if RNA is first reverse-transcribed to complementary DNA (cDNA). Reverse transcriptases from Maloney murine leukemia virus (MMLV-RT) or avian myeloblastoma virus (AMV-RT) are commonly used. Either the buffer optimal for the reverse transcription (RT) or the PCR buffer is suitable in most cases, and reverse transcription is typically performed at 37–42°C for 15–60 min. Because these enzymes are heat-labile, they are inactivated during the first denaturation segment of PCR. Heat-stable reverse transcriptases enable reverse transcription at higher temperatures to denature RNA secondary structure. In the presence of Mn 2+ , some DNA-dependent DNA polymerases such as the enzyme from Thermus thermophilus ( Tth DNA polymerase) have activity on RNA templates. Using MnCl 2 for reverse transcription can enable cDNA synthesis with the same enzyme used for PCR. Unless a balance between MnCl 2 and MgCl 2 is carefully identified, Mn 2+ must be removed or chelated, and Mg 2+ must be added prior to PCR. RT-PCR can be performed within intact cells ( in situ PCR) to identify cells expressing particular genes or to assess the presence of disease-related genes. Thermal cyclers are available to automate the process on microscope slides. Applications Clinical Diagnostics The main clinical application of PCR technology is in the diagnosis of disease, and in many cases the speed and simplicity of PCRs have revolutionized clinical diagnosis. Bacteria that are difficult or impossible to culture on artificial media can now be detected by PCR. The causative organism of syphilis, Treponema pallidum , can be detected by a PCR assay in which a gene encoding a specific membrane protein is amplified. PCR is being employed to investigate the pathogenicity of Mycoplasma genitalium . As few as four organisms can be detected by this method. PCR-based detection assays have also been developed for Mycobacterium tuberculosis , an organism that takes up to several weeks to identify by conventional means owing to its slow growth on artificial media. M. tuberculosis , the bacterium that causes tuberculosis, infects about one-third of the world's population and is most prevalent in developing countries. Recently, this organism has re-emerged as a significant pathogen in the developed world, especially among the poor and homeless, and antibiotic-resistant strains are developing. PCR tests have become essential for rapid diagnosis and epidemiology of diseases such as tuberculosis. Amplification of viral genes forms the basis of PCR-based detection systems for viral pathogens. The human immunodeficiency virus (HIV), which is responsible for AIDS, is currently detected primarily by immunological means. However, as with most viral infections, it takes some time following infection for antibodies to develop, and there is therefore a period when a patient infected with the virus would appear negative by conventional tests. A PCR assay can be used to detect the presence of the virus in this case. Proviral DNA that has integrated into the genomes of leucocytes can be detected, or the viral RNA genome can be detected in plasma by RT-PCR. Identification of HIV by PCR methods has played an important role in AIDS diagnostics and research, as well as improved safety testing in blood banks. PCR assays have also been used in epidemiological studies and in the identification of other retroviruses such as human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2). PCR procedures have also been developed to detect human papillomaviruses. This diverse group of viruses can cause a number of diseases. A PCR assay can distinguish the relatively harmless strains from those that cause serious diseases such as cervical cancer. Current laboratory methods for the diagnosis of human enteroviruses, which cause infection in children, are slow and lack sensitivity. Detection is hindered by the low level of viruses present in clinical specimens. PCR technology, however, provides a rapid and accurate diagnostic test. PCR assays have also been used to detect coronaviruses, cytomegalovirus, human herpesvirus type 6, adenovirus, Epstein–Barr virus, rotaviruses, human parvovirus, and herpes simplex viruses, among many others. In addition to detection, PCR assays can be designed to differentiate between different strains or serotypes of a particular virus. Parasitic infections cause important human diseases such as leishmaniasis and malaria. About 40% of the world's population is at risk from malaria, and drug-resistant strains have emerged and are spreading rapidly. As well as being helpful in diagnosis of disease, a PCR assay can distinguish between drug-resistant and drug-sensitive strains, thus enabling effective treatment to be prescribed. Many PCR diagnostic kits are now commercially available. Hoffman-La Roche has introduced kits for Chlamydia , M. tuberculosis , HIV, hepatitis C virus, and others. It is likely that an even wider range of PCR diagnostic kits will become available, and PCR testing will continue to have a huge impact in the clinical diagnostic laboratory. Despite the simplicity and rapidity of PCR from the perspective of the research laboratory, it is viewed as a complex test in the clinical laboratory. The expenses of highly trained personnel and commercial PCR kits coupled with concerns about false positive test results due to contamination with previously amplified DNA have so far confined this promising technology to clinical research and specialty testing labs. Eventually, improved technology and assay automation will enable PCR testing to be as common in diagnostic laboratories as immunoassays. Molecular Genetics In addition to being an invaluable tool for the detection and diagnosis of infectious diseases, PCR tests have also proved useful in the diagnosis and analysis of genetic diseases. Many genetic disorders are caused by specific mutations, and the fragment of DNA involved can be amplified by PCR. DNA sequencing, high-resolution size analysis on sequencing gels, single-strand conformation polymorphism analysis, Southern blotting and probe hybridization, allele-specific oligonucleotide probes, and other techniques are used to test for the presence of mutations. This approach has been used to detect mutations causing diseases such as sickle cell anemia, beta-thalassemia, cystic fibrosis, and many others. Genetic diseases caused by trinucleotide repeat expansion can be tested using PCR followed by high-resolution electrophoretic analysis on sequencing gels. A commercial test for fragile X syndrome employs novel PCR reagents and thermal cycling conditions to enable amplification of (CGG) repeats up to 800 repeat units in length in the same reaction containing a normal allele (usually 29–31 repeat units). In general, PCR should be considered a versatile template onto which can be superimposed known DNA sequences for any disorder, whether genetic or infectious in nature, to rapidly configure a high-performance test. PCR is routinely used in the analysis of human leucocyte antigen (HLA) polymorphisms (HLA typing) to assess donor compatibility prior to bone marrow or organ transplantation. The use of PCR in transfusion medicine is also being investigated, and the basis of a system for ABO blood typing using PCR has been described. Prenatal Diagnostics Prenatal diagnosis of genetic disease is normally carried out on a sample of amniotic fluid obtained by amniocentesis or by chorionic villus sampling (CVS). Conventional diagnostic tests for diseases such as cystic fibrosis can take as long as 2 weeks. Results of a PCR test can be available in 1 day. If the option to terminate a pregnancy is taken, this time savings can be of enormous benefit. The availability of PCR technology creates an incentive for the development of noninvasive prenatal sampling techniques. Because amniocentesis and CVS carry a certain amount of risk, less invasive sampling procedures are desirable. A small number of cells of fetal origin circulate in the maternal blood. Methods to purify these cells have been described. Mutations and polymorphisms on the Y chromosome already can be tested using maternal blood samples without the need to purify fetal cells. It seems likely that in the future PCR tests will be used for prenatal diagnosis of a whole range of genetic diseases from a sample of maternal blood. PCR testing is currently available for preimplantation genetic diagnosis for in vitro fertilization. One cell is removed from an eight-cell zygote for PCR testing. Basic Research PCR has become an indispensable tool in the molecular biology laboratory. Many traditional methods and protocols have now been replaced by PCR-based techniques. One of the most common techniques employed by molecular biologists involves the cloning of DNA fragments into plasmid vectors. The plasmids are maintained in bacterial strains. To determine which bacterial colony contains the plasmid of interest, transformant colonies can be analyzed by PCR using primers designed from the vector (plasmid) DNA, bracketing the insert. In this way, colonies can be screened to find one containing a plasmid with an insert of a particular size, obviating the need to screen transformants by plasmid DNA preparation followed by restriction enzyme digestion. PCR can also be used to screen libraries for clones containing a particular DNA fragment or gene. This strategy dispenses with the filter hybridization step and can be both quicker and more sensitive than traditional methods. PCR is also invaluable in mutagenesis, radioactive, and nonradioactive labeling of DNA fragments, and many other applications. In addition, the technology developed for PCR amplification has been applied to the extremely important techniques of DNA sequencing and gene expression analysis. PCR is being used in phylogenetic and evolutionary studies. PCR amplification and screening of ribosomal RNA (rRNA) genes and mitochondrial DNA have been used to estimate relationships between species and subspecies. Besides studies on the relationships of contemporary species, PCR allows studies on extinct species because it can be performed on material in museum collections. Amplification and sequencing of mitochondrial DNA are also used in evolutionary studies. Forensics PCR has become an essential tool in forensic science. Many methods that exploit differences in DNA to identify individuals or to distinguish between individuals require large amounts of DNA (>100 ng). However, PCR permits the amplification of minute amounts of DNA from biological evidence found at the scene of a crime and has greatly facilitated the identification of individuals. Now, sufficient DNA for analysis can be obtained from very small amounts of biological material, such as a single hair or a small blood stain. Other Applications Monitoring of foods for pathogens is increasingly performed using PCR analysis. Direct detection of microbial DNA or RNA can enable detection of contamination before visible signs appear. Assays for enterotoxigenic E. coli ( E. coli 0157:H7), Listeria monocytogenes , Salmonella , and other pathogens are increasingly a component of food safety testing. Food products and ingredients can be tested for the presence of genetically modified organisms using PCR primers specific for the inserted gene sequence. Environmental samples can be tested for coliform and other bacteria or for biowarfare agents such as smallpox or anthrax. Because of its exquisite sensitivity, PCR amplification seems assured to find even more applications across research and analytical disciplines wherever there is a need to detect small amounts of genetic material. Clinical Diagnostics The main clinical application of PCR technology is in the diagnosis of disease, and in many cases the speed and simplicity of PCRs have revolutionized clinical diagnosis. Bacteria that are difficult or impossible to culture on artificial media can now be detected by PCR. The causative organism of syphilis, Treponema pallidum , can be detected by a PCR assay in which a gene encoding a specific membrane protein is amplified. PCR is being employed to investigate the pathogenicity of Mycoplasma genitalium . As few as four organisms can be detected by this method. PCR-based detection assays have also been developed for Mycobacterium tuberculosis , an organism that takes up to several weeks to identify by conventional means owing to its slow growth on artificial media. M. tuberculosis , the bacterium that causes tuberculosis, infects about one-third of the world's population and is most prevalent in developing countries. Recently, this organism has re-emerged as a significant pathogen in the developed world, especially among the poor and homeless, and antibiotic-resistant strains are developing. PCR tests have become essential for rapid diagnosis and epidemiology of diseases such as tuberculosis. Amplification of viral genes forms the basis of PCR-based detection systems for viral pathogens. The human immunodeficiency virus (HIV), which is responsible for AIDS, is currently detected primarily by immunological means. However, as with most viral infections, it takes some time following infection for antibodies to develop, and there is therefore a period when a patient infected with the virus would appear negative by conventional tests. A PCR assay can be used to detect the presence of the virus in this case. Proviral DNA that has integrated into the genomes of leucocytes can be detected, or the viral RNA genome can be detected in plasma by RT-PCR. Identification of HIV by PCR methods has played an important role in AIDS diagnostics and research, as well as improved safety testing in blood banks. PCR assays have also been used in epidemiological studies and in the identification of other retroviruses such as human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2). PCR procedures have also been developed to detect human papillomaviruses. This diverse group of viruses can cause a number of diseases. A PCR assay can distinguish the relatively harmless strains from those that cause serious diseases such as cervical cancer. Current laboratory methods for the diagnosis of human enteroviruses, which cause infection in children, are slow and lack sensitivity. Detection is hindered by the low level of viruses present in clinical specimens. PCR technology, however, provides a rapid and accurate diagnostic test. PCR assays have also been used to detect coronaviruses, cytomegalovirus, human herpesvirus type 6, adenovirus, Epstein–Barr virus, rotaviruses, human parvovirus, and herpes simplex viruses, among many others. In addition to detection, PCR assays can be designed to differentiate between different strains or serotypes of a particular virus. Parasitic infections cause important human diseases such as leishmaniasis and malaria. About 40% of the world's population is at risk from malaria, and drug-resistant strains have emerged and are spreading rapidly. As well as being helpful in diagnosis of disease, a PCR assay can distinguish between drug-resistant and drug-sensitive strains, thus enabling effective treatment to be prescribed. Many PCR diagnostic kits are now commercially available. Hoffman-La Roche has introduced kits for Chlamydia , M. tuberculosis , HIV, hepatitis C virus, and others. It is likely that an even wider range of PCR diagnostic kits will become available, and PCR testing will continue to have a huge impact in the clinical diagnostic laboratory. Despite the simplicity and rapidity of PCR from the perspective of the research laboratory, it is viewed as a complex test in the clinical laboratory. The expenses of highly trained personnel and commercial PCR kits coupled with concerns about false positive test results due to contamination with previously amplified DNA have so far confined this promising technology to clinical research and specialty testing labs. Eventually, improved technology and assay automation will enable PCR testing to be as common in diagnostic laboratories as immunoassays. Molecular Genetics In addition to being an invaluable tool for the detection and diagnosis of infectious diseases, PCR tests have also proved useful in the diagnosis and analysis of genetic diseases. Many genetic disorders are caused by specific mutations, and the fragment of DNA involved can be amplified by PCR. DNA sequencing, high-resolution size analysis on sequencing gels, single-strand conformation polymorphism analysis, Southern blotting and probe hybridization, allele-specific oligonucleotide probes, and other techniques are used to test for the presence of mutations. This approach has been used to detect mutations causing diseases such as sickle cell anemia, beta-thalassemia, cystic fibrosis, and many others. Genetic diseases caused by trinucleotide repeat expansion can be tested using PCR followed by high-resolution electrophoretic analysis on sequencing gels. A commercial test for fragile X syndrome employs novel PCR reagents and thermal cycling conditions to enable amplification of (CGG) repeats up to 800 repeat units in length in the same reaction containing a normal allele (usually 29–31 repeat units). In general, PCR should be considered a versatile template onto which can be superimposed known DNA sequences for any disorder, whether genetic or infectious in nature, to rapidly configure a high-performance test. PCR is routinely used in the analysis of human leucocyte antigen (HLA) polymorphisms (HLA typing) to assess donor compatibility prior to bone marrow or organ transplantation. The use of PCR in transfusion medicine is also being investigated, and the basis of a system for ABO blood typing using PCR has been described. Prenatal Diagnostics Prenatal diagnosis of genetic disease is normally carried out on a sample of amniotic fluid obtained by amniocentesis or by chorionic villus sampling (CVS). Conventional diagnostic tests for diseases such as cystic fibrosis can take as long as 2 weeks. Results of a PCR test can be available in 1 day. If the option to terminate a pregnancy is taken, this time savings can be of enormous benefit. The availability of PCR technology creates an incentive for the development of noninvasive prenatal sampling techniques. Because amniocentesis and CVS carry a certain amount of risk, less invasive sampling procedures are desirable. A small number of cells of fetal origin circulate in the maternal blood. Methods to purify these cells have been described. Mutations and polymorphisms on the Y chromosome already can be tested using maternal blood samples without the need to purify fetal cells. It seems likely that in the future PCR tests will be used for prenatal diagnosis of a whole range of genetic diseases from a sample of maternal blood. PCR testing is currently available for preimplantation genetic diagnosis for in vitro fertilization. One cell is removed from an eight-cell zygote for PCR testing. Basic Research PCR has become an indispensable tool in the molecular biology laboratory. Many traditional methods and protocols have now been replaced by PCR-based techniques. One of the most common techniques employed by molecular biologists involves the cloning of DNA fragments into plasmid vectors. The plasmids are maintained in bacterial strains. To determine which bacterial colony contains the plasmid of interest, transformant colonies can be analyzed by PCR using primers designed from the vector (plasmid) DNA, bracketing the insert. In this way, colonies can be screened to find one containing a plasmid with an insert of a particular size, obviating the need to screen transformants by plasmid DNA preparation followed by restriction enzyme digestion. PCR can also be used to screen libraries for clones containing a particular DNA fragment or gene. This strategy dispenses with the filter hybridization step and can be both quicker and more sensitive than traditional methods. PCR is also invaluable in mutagenesis, radioactive, and nonradioactive labeling of DNA fragments, and many other applications. In addition, the technology developed for PCR amplification has been applied to the extremely important techniques of DNA sequencing and gene expression analysis. PCR is being used in phylogenetic and evolutionary studies. PCR amplification and screening of ribosomal RNA (rRNA) genes and mitochondrial DNA have been used to estimate relationships between species and subspecies. Besides studies on the relationships of contemporary species, PCR allows studies on extinct species because it can be performed on material in museum collections. Amplification and sequencing of mitochondrial DNA are also used in evolutionary studies. Forensics PCR has become an essential tool in forensic science. Many methods that exploit differences in DNA to identify individuals or to distinguish between individuals require large amounts of DNA (>100 ng). However, PCR permits the amplification of minute amounts of DNA from biological evidence found at the scene of a crime and has greatly facilitated the identification of individuals. Now, sufficient DNA for analysis can be obtained from very small amounts of biological material, such as a single hair or a small blood stain. Other Applications Monitoring of foods for pathogens is increasingly performed using PCR analysis. Direct detection of microbial DNA or RNA can enable detection of contamination before visible signs appear. Assays for enterotoxigenic E. coli ( E. coli 0157:H7), Listeria monocytogenes , Salmonella , and other pathogens are increasingly a component of food safety testing. Food products and ingredients can be tested for the presence of genetically modified organisms using PCR primers specific for the inserted gene sequence. Environmental samples can be tested for coliform and other bacteria or for biowarfare agents such as smallpox or anthrax. Because of its exquisite sensitivity, PCR amplification seems assured to find even more applications across research and analytical disciplines wherever there is a need to detect small amounts of genetic material. See also : AMPLIFICATION REACTIONS; DNA SEQUENCING; ELECTROPHORESIS | Nucleic Acids; FORENSIC SCIENCES | DNA Profiling; NUCLEIC ACIDS | Chromatographic and Electrophoretic Methods; NUCLEIC ACIDS | Immunoassays.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237246/

A case of ulceroglandular tularemia presenting with lymphadenopathy and an ulcer on a linear morphoea lesion surrounded by erysipelas

Tularemia is a zoonosis caused by the infection of Francisella tularensis (a gram-negative aerobic bacterium). Transmission to other animals or humans usually occurs through insect or tick bites, direct contact with a contaminated environment (mud or water), infected animals – mainly lagomorphs – or by ingesting undercooked meat or inhaling contaminated dust (hay or soil). This paper discusses the case of a 32-year-old man, who came to our Emergency Room presenting with persistent fever, inguinal lymphadenopathy, and an ulcer on his left lower limb on a linear morphoea lesion that had been there for some time. The lesion was surrounded by erysipelas. After hospitalization and tests, the patient was diagnosed with ulceroglandular tularemia. Antibiotic treatment with doxycycline resolved the clinical picture, but not the morphoea lesion. Introduction Francisella tularensis ( F. tularensis ) are transmitted to other animals or humans through insect or tick bites, direct contact with a contaminated environment, infected animals, or by ingesting undercooked meat or inhaling contaminated dust. 1 , 2 Ulceroglandular tularemia, one of the six main clinical forms of this disease, is the most common and clinically recognizable. Symptoms include fever, inflammation of the infected area, and enlarged lymph nodes. 9 Treatment include gentamicin intramuscular or intravenous for moderate to severe infections and doxycycline or ciprofloxacin for milder infections. 13 Case report Medical history A 32-year-old man presenting with morphoea (circumscribed scleroderma) on his left lower limb had been regularly treated with topical steroids by his dermatologist. The patient also presented with recurrent ulcers on the lower limbs, which had been evaluated on several occasions at a hospital in Zurich and attributed to microtraumas. However, the patient did not report any trauma during history-taking. He was not taking medications and did not have any known allergies. He did report to have regular contact with his cat and dog but did not remember to have ever been bitten. He did not remember any tick bites either, and he had not had the opportunity to travel abroad over the previous 8 weeks. He is a carpenter. The patient came to the Emergency Room of our Regional Hospital presenting with left groin pain and a body temperature of up to 39°C with shivers. Blood tests showed leukocytosis (14 g/L) with normal C-reactive protein (CRP). An inguinal ultrasound examination showed subcutaneous edema and numerous benign reactive enlarged lymph nodes ( Figure 1 ). A chest X-ray did not reveal any suspicious alterations. The images of the pelvic X-ray were compatible with trochanteric insertional tendinopathy. Therefore, the patient was diagnosed with acute pubalgia in insertional tendinopathy and discharged with anti-inflammatory treatment. However, the next day, the patient returned to the Emergency Room as the groin pain had gotten worse. Status The patient's vitals were characterized by high body temperature (39.2°C), hypotension (106/46 mmHg), tachycardia (116 bpm), and normal saturation. The lower left limb had a 12×6 mm superficial pretibial ulcer with a fibrin bed on the (healed) linear morphoea lesion we knew about, and erythema with ill-defined borders ( Figure 2 ). The patient also presented with erythema on the thigh, which extended to the lower limb ( Figure 3 ). We suspected left leg erysipelas due to secondary infection of the underlying morphoea lesion. The patient was admitted to the Internal Medicine Department, where he was given an intravenous antibiotic treatment with a high-dose (2.2 g) formulation of amoxicillin/clavulanic acid three times a day. Results During the following days the fever persisted, accompanied by the worsening of the localized edema and an increase in inflammatory parameters, with CRP peaking at 329 mg/L on day 5 and leukocytosis at 15×10 9 /L on day 7. The erythrocyte sedimentation rate was 110 mm/h. Tests also showed a positive antistreptolysin titer (508 U/L). The antibiotics were switched to carbapenems (imipenem/cilastatin 500 mg/500 mg four times a day), which led to a slight improvement, although the fever persisted. The patient was then transferred to the Department of Dermatology for specialist care. During his stay, four pairs of blood cultures were collected at febrile peaks, but no microorganisms developed from them. The perimalleolar ulcer smears and the HIV serological tests were also negative. The lower extremity venous duplex examination excluded any deep venous thrombosis, while the soft tissue ultrasound of the inguinal region showed reactive subcutaneous lymphadenopathy surrounded by edematous inflammatory tissue with no evidence of abscess. The ankle X-ray showed no findings indicative of osteomyelitis. At that point, our colleagues at the Department of Dermatology asked the Department of Infectious Diseases for a consultation. Serological tests for F. tularensis and Bartonella henselae and Bartonella quintana were carried out. Differential diagnosis indicates something intermediate between an infection caused by Francisella , infection by Bartonella (because of the enlarged lymph node), and/or streptococcal infection (positive anti-streptolysin titer) starting from the traumatic pretibial ulcer. Our laboratory uses an immunofluorescent assay (IFA) for B. henselae and B. quintana (IFA-IgM and IFA-IgG) and traditional ELISA enzyme immunoassay for F. tularensis , giving results for both IgM ang IgG. At the same time, a doxycycline treatment was administered in addition to the imipenem/cilastatin combination. Diagnosis and treatment The serological test for B. quintana and B. henselae was negative ( B. quintana IgM: < 1/20; B. quintana IgG: < 1/64; B. henselae IgM: < 1/20; B. henselae IgG: < 1/64) while the serological test for F. tularensis was positive (IgM: 33.4 U/mL; IgG: 3 U/ml). Therefore, the patient was diagnosed with ulceroglandular tularemia with leg erysipelas secondary to the underlying morphoea lesion. The carbapenem treatment was suspended, so the patient was now treated only with doxycycline. Dalibour water compresses with cream and a silver sulfadiazine pack were applied locally. A class III corticosteroid was applied to the perilesional erythematous skin. Finally, a compression bandage was applied to the patient's left leg. Evolution From a clinical point of view, the patient showed a rapid remission of symptoms. The fever disappeared even after the first dose of antibiotic, and the lymphadenopathy regressed gradually. Upon discharge, CRP levels had decreased to 25 mg/L without leukocytosis. The patient continued the antibiotic treatment for 3 weeks. The Cantonal Medical Service has requested an environmental investigation to identify the source of infection, which is still unknown. Medical history A 32-year-old man presenting with morphoea (circumscribed scleroderma) on his left lower limb had been regularly treated with topical steroids by his dermatologist. The patient also presented with recurrent ulcers on the lower limbs, which had been evaluated on several occasions at a hospital in Zurich and attributed to microtraumas. However, the patient did not report any trauma during history-taking. He was not taking medications and did not have any known allergies. He did report to have regular contact with his cat and dog but did not remember to have ever been bitten. He did not remember any tick bites either, and he had not had the opportunity to travel abroad over the previous 8 weeks. He is a carpenter. The patient came to the Emergency Room of our Regional Hospital presenting with left groin pain and a body temperature of up to 39°C with shivers. Blood tests showed leukocytosis (14 g/L) with normal C-reactive protein (CRP). An inguinal ultrasound examination showed subcutaneous edema and numerous benign reactive enlarged lymph nodes ( Figure 1 ). A chest X-ray did not reveal any suspicious alterations. The images of the pelvic X-ray were compatible with trochanteric insertional tendinopathy. Therefore, the patient was diagnosed with acute pubalgia in insertional tendinopathy and discharged with anti-inflammatory treatment. However, the next day, the patient returned to the Emergency Room as the groin pain had gotten worse. Status The patient's vitals were characterized by high body temperature (39.2°C), hypotension (106/46 mmHg), tachycardia (116 bpm), and normal saturation. The lower left limb had a 12×6 mm superficial pretibial ulcer with a fibrin bed on the (healed) linear morphoea lesion we knew about, and erythema with ill-defined borders ( Figure 2 ). The patient also presented with erythema on the thigh, which extended to the lower limb ( Figure 3 ). We suspected left leg erysipelas due to secondary infection of the underlying morphoea lesion. The patient was admitted to the Internal Medicine Department, where he was given an intravenous antibiotic treatment with a high-dose (2.2 g) formulation of amoxicillin/clavulanic acid three times a day. Results During the following days the fever persisted, accompanied by the worsening of the localized edema and an increase in inflammatory parameters, with CRP peaking at 329 mg/L on day 5 and leukocytosis at 15×10 9 /L on day 7. The erythrocyte sedimentation rate was 110 mm/h. Tests also showed a positive antistreptolysin titer (508 U/L). The antibiotics were switched to carbapenems (imipenem/cilastatin 500 mg/500 mg four times a day), which led to a slight improvement, although the fever persisted. The patient was then transferred to the Department of Dermatology for specialist care. During his stay, four pairs of blood cultures were collected at febrile peaks, but no microorganisms developed from them. The perimalleolar ulcer smears and the HIV serological tests were also negative. The lower extremity venous duplex examination excluded any deep venous thrombosis, while the soft tissue ultrasound of the inguinal region showed reactive subcutaneous lymphadenopathy surrounded by edematous inflammatory tissue with no evidence of abscess. The ankle X-ray showed no findings indicative of osteomyelitis. At that point, our colleagues at the Department of Dermatology asked the Department of Infectious Diseases for a consultation. Serological tests for F. tularensis and Bartonella henselae and Bartonella quintana were carried out. Differential diagnosis indicates something intermediate between an infection caused by Francisella , infection by Bartonella (because of the enlarged lymph node), and/or streptococcal infection (positive anti-streptolysin titer) starting from the traumatic pretibial ulcer. Our laboratory uses an immunofluorescent assay (IFA) for B. henselae and B. quintana (IFA-IgM and IFA-IgG) and traditional ELISA enzyme immunoassay for F. tularensis , giving results for both IgM ang IgG. At the same time, a doxycycline treatment was administered in addition to the imipenem/cilastatin combination. Diagnosis and treatment The serological test for B. quintana and B. henselae was negative ( B. quintana IgM: < 1/20; B. quintana IgG: < 1/64; B. henselae IgM: < 1/20; B. henselae IgG: < 1/64) while the serological test for F. tularensis was positive (IgM: 33.4 U/mL; IgG: 3 U/ml). Therefore, the patient was diagnosed with ulceroglandular tularemia with leg erysipelas secondary to the underlying morphoea lesion. The carbapenem treatment was suspended, so the patient was now treated only with doxycycline. Dalibour water compresses with cream and a silver sulfadiazine pack were applied locally. A class III corticosteroid was applied to the perilesional erythematous skin. Finally, a compression bandage was applied to the patient's left leg. Evolution From a clinical point of view, the patient showed a rapid remission of symptoms. The fever disappeared even after the first dose of antibiotic, and the lymphadenopathy regressed gradually. Upon discharge, CRP levels had decreased to 25 mg/L without leukocytosis. The patient continued the antibiotic treatment for 3 weeks. The Cantonal Medical Service has requested an environmental investigation to identify the source of infection, which is still unknown. Discussion Tularemia is a zoonosis caused by F. tularensis , a gram-negative aerobic bacterium. Subspecies tularensis is the most virulent strain, whereas subspecies holarctica causes most of the infections in humans and animals. This microorganism mainly infects lagomorphs, ie, rabbits, hares, rodents, beavers, muskrats, squirrels, hamsters, and voles. F. tularensis can persist for several weeks in animal carcasses, mud, and water. Laboratory personnel, farmers, veterinarians, hunters, landscapers, and butchers can come in contact with infection sources at work. Contact with infected animals, cats, birds, insects that bite or sting (especially ticks), and contaminated food and water also pose a threat. This infection is usually transmitted to other animals or humans through insect or tick bites, direct contact with a contaminated environment or infected animals (animal hunting, skinning, or slaughtering), or by ingesting undercooked meat or inhaling contaminated dust (eg, hay or soil). Person-to-person transmission has never been reported. 1 , 2 Epidemiology Many cases of tularemia have been reported in North America, Japan, Russia, Central Asia, Nordic countries, and the Balkans. Although epidemics occurred also in Russia and Eastern Europe, Nordic countries and the USA remain endemic countries for this disease. The annual incidence of tularemia in the USA is 0.5–5 cases per million people. In the period between 2001–2010, the incidence of tularemia in Kosovo was 5.2 cases per 100,000 people per year, the highest in Europe. 3 , 4 Interestingly, tularemia has not been reported in the UK, Iceland, Africa, South America, and Antarctica. Until 2014 Australia has reported only two cases of tularemia related to F. tularensis . 5 , 6 Over the past few years, climate change, wars, natural disasters, human travels, and animal migration have been the main causes of the increasing number of tularemia cases worldwide. Global warming has caused an increase in the average temperature throughout the year, which has led to a significant change in the cycle of tularemia. A study carried out in Sweden – in which a climate scenario was created with a 2°C increase in the annual average temperature for the time period 2010–2100 – has shown an increase in outbreaks of F. tularensis . 7 In Switzerland (with a population of 8,482,200 at the end of 2017), 8 where case reporting is mandatory, the average incidence is seven cases per year. Over the past 10 years, the highest number of cases was reported in 2017 with 66 cases. Pathogenesis The incubation period is 3–5 days (more rarely 1–21 days). The microorganism proliferates in the inoculation site and, after exiting infected cells through lysis, it causes an acute inflammatory reaction that results in tissue necrosis. It then spreads to the regional lymph nodes and throughout the body via the lymphohematogenous route. Clinical signs The course of the disease varies depending on modes of transmission, affected organs, and pathogenic subspecies. It usually manifests with fever, progressive inflammation of the infected area, and enlarged lymph nodes. Untreated tularemia infections are fatal in 5%–15% of cases. 1 , 2 There are six main clinical forms of tularemia based on the inoculation site and virulence of the infectious agent. These are described in the following. Ulceroglandular tularemia is the most common and clinically recognizable form. Usually, the patient has had some recent contact with animals or has been exposed to vectors (insects, especially ticks). Clinical signs include fever, an erythematous papule with a central eschar where inoculation occurred (eg, tick bite), and frequent regional, cervical, or occipital firm, elastic adenopathies. Glandular tularemia manifests with painful regional lymphadenopathy involving one or more lymph nodes, without any identifiable skin lesion. This form is transmitted in the same way as the ulceroglandular form but does not have the characteristic skin lesion at the inoculation site. Absent or blunted fever may contribute to diagnostic delays. The differential diagnosis for both glandular and ulceroglandular tularemia includes cat-scratch disease, neoplasms, syphilis, mycobacterial infection, lymphogranuloma venereum, streptococcal or staphylococcal lymphadenitis, toxoplasmosis, Herpes simplex, fungal infection, anthrax, and the plague. 9 , 10 Oropharyngeal tularemia presents with a sore throat (with or without ulceration) and marked cervical lymphadenopathy, which can be unilateral. Pneumonic tularemia may occur as a primary infection after inhalation of infected aerosols or as secondary infection following hematogenous spread of organisms in other forms of the disease. 11 Infected pleural fluid is exudative, negative on Gram stain, and usually contains more than 1,000 leukocytes/mm 3 . 12 Oculoglandular tularemia is characterized by purulent conjunctivitis, palpebral ulcers, and periocular lymphadenopathy. 13 Typhoidal tularemia manifests without regional lymphadenopathy or other distinctive clinical signs. It is often associated with underlying chronic systemic diseases or even sepsis. 10 , 14 Epidemiology Many cases of tularemia have been reported in North America, Japan, Russia, Central Asia, Nordic countries, and the Balkans. Although epidemics occurred also in Russia and Eastern Europe, Nordic countries and the USA remain endemic countries for this disease. The annual incidence of tularemia in the USA is 0.5–5 cases per million people. In the period between 2001–2010, the incidence of tularemia in Kosovo was 5.2 cases per 100,000 people per year, the highest in Europe. 3 , 4 Interestingly, tularemia has not been reported in the UK, Iceland, Africa, South America, and Antarctica. Until 2014 Australia has reported only two cases of tularemia related to F. tularensis . 5 , 6 Over the past few years, climate change, wars, natural disasters, human travels, and animal migration have been the main causes of the increasing number of tularemia cases worldwide. Global warming has caused an increase in the average temperature throughout the year, which has led to a significant change in the cycle of tularemia. A study carried out in Sweden – in which a climate scenario was created with a 2°C increase in the annual average temperature for the time period 2010–2100 – has shown an increase in outbreaks of F. tularensis . 7 In Switzerland (with a population of 8,482,200 at the end of 2017), 8 where case reporting is mandatory, the average incidence is seven cases per year. Over the past 10 years, the highest number of cases was reported in 2017 with 66 cases. Pathogenesis The incubation period is 3–5 days (more rarely 1–21 days). The microorganism proliferates in the inoculation site and, after exiting infected cells through lysis, it causes an acute inflammatory reaction that results in tissue necrosis. It then spreads to the regional lymph nodes and throughout the body via the lymphohematogenous route. Clinical signs The course of the disease varies depending on modes of transmission, affected organs, and pathogenic subspecies. It usually manifests with fever, progressive inflammation of the infected area, and enlarged lymph nodes. Untreated tularemia infections are fatal in 5%–15% of cases. 1 , 2 There are six main clinical forms of tularemia based on the inoculation site and virulence of the infectious agent. These are described in the following. Ulceroglandular tularemia is the most common and clinically recognizable form. Usually, the patient has had some recent contact with animals or has been exposed to vectors (insects, especially ticks). Clinical signs include fever, an erythematous papule with a central eschar where inoculation occurred (eg, tick bite), and frequent regional, cervical, or occipital firm, elastic adenopathies. Glandular tularemia manifests with painful regional lymphadenopathy involving one or more lymph nodes, without any identifiable skin lesion. This form is transmitted in the same way as the ulceroglandular form but does not have the characteristic skin lesion at the inoculation site. Absent or blunted fever may contribute to diagnostic delays. The differential diagnosis for both glandular and ulceroglandular tularemia includes cat-scratch disease, neoplasms, syphilis, mycobacterial infection, lymphogranuloma venereum, streptococcal or staphylococcal lymphadenitis, toxoplasmosis, Herpes simplex, fungal infection, anthrax, and the plague. 9 , 10 Oropharyngeal tularemia presents with a sore throat (with or without ulceration) and marked cervical lymphadenopathy, which can be unilateral. Pneumonic tularemia may occur as a primary infection after inhalation of infected aerosols or as secondary infection following hematogenous spread of organisms in other forms of the disease. 11 Infected pleural fluid is exudative, negative on Gram stain, and usually contains more than 1,000 leukocytes/mm 3 . 12 Oculoglandular tularemia is characterized by purulent conjunctivitis, palpebral ulcers, and periocular lymphadenopathy. 13 Typhoidal tularemia manifests without regional lymphadenopathy or other distinctive clinical signs. It is often associated with underlying chronic systemic diseases or even sepsis. 10 , 14 Diagnosis In clinical practice, the diagnosis of tularemia remains based on serological methods. 1 However, a recent study to evaluate the performance of the serological tests available, ie, ELISA (Serion ELISA) and immunochromatographic test (ICT) compared to that of the microscopic agglutination test (MAT) and indirect immunofluorescence assay (IFA), highlighted the limits of the serological methods. The traditional IgG and IgM ELISA tests for F. tularensis are two commercial kits that allow specific detection of IgM or IgG antibodies. ICT and MAT tests detect both anti- Francisella IgM and IgG, while for IFA detection it is necessary to use two separate tests, one for IgM antibodies (IFA-IgM) and one for IgG (IFA-IgG). The results of the study showed how ELISA performed better than MAT and IFA in detecting F. tularensis antibodies in serum samples taken from patients with tularemia during the first 2 weeks after the onset of symptoms. However, early detection with low F. tularensis antibody titers can be associated with a higher risk of false-positives due to cross-reacting antibodies, especially between Francisella , Brucella , Yersinia enterocolitica species, and mimivirus capsid antigens. The residual antibody titers may persist for years, leading to false-positives. ELISA tests allow for the early detection of specific antibodies but have lower specificity than MAT and IFA and need to be confirmed by these. On the other hand, the ELISA tests available on the market for the detection and titration of F. tularensis IgM or IgG antibodies are easier to automate and standardize. 15 IgM for F. tularensis usually appear after 2–3 weeks from beginning of symptoms, and may persist together with IgG for longer than 10 years after infection. A definitive serological diagnosis should be supported by seroconversion or a 4-fold titer increase, or confirmed by a positive specific PCR of biopsy specimen. 12 In our report, unfortunately, a convalescent serum of our patient was not available, neither was there a serum sample before symptoms started, therefore diagnosis of tularemia relied on a positive IgM in the presence of a compatible acute clinical picture and a immediate effectiveness of the specific treatment with doxycycline. As for the ICT test, this study revealed a much lower specificity than MAT, IFA, and ELISA tests. Its use in hospital laboratories should be discouraged; however, it remains a valuable tool for field studies, as it allows for quick and easy screening of people and animals with potential F. tularensis infection. In the case we are describing, the Infectious Diseases Department did not suggest lesion biopsy because F. tularensis is rarely detected in clinical samples by culture or PCR. 15 Treatment The antibiotic treatment must be administered in all suspected or confirmed cases, although there have been cases of spontaneous regression without a specific treatment. 9 Aminoglycosides (gentamicin) are the drugs of choice for severe infections. Tetracyclines (doxycycline) – also widely used for the prophylaxis and treatment of tularemia – are associated with a higher relapse rate. Recent experience has demonstrated the excellent efficacy of ciprofloxacin, which is preferred for oral use. The treatment period is 10–14 days with aminoglycosides and ciprofloxacin and 21 days with tetracyclines. 13 Suppurative lymph nodes require surgical drainage; pleural effusion requires thoracentesis to rule out empyema. 9 , 10 Prevention People exposed to the risk of infection can undergo antibiotic prophylaxis under medical supervision to prevent the onset of the disease. 1 There is no safe, authorized vaccine available on the market. A potential candidate vaccine (LVS – Live Vaccine Strain) does not meet the criteria for general use. The first live attenuated vaccine candidate was made from F. tularensis subsp. holarctica strains in the former Soviet Union and, after serial passage in the USA, it led to the preparation of LVS. Although it demonstrated the ability to induce protective immunity against a low-dose aerosol challenge of the Schu S4 strain, the Food and Drug Administration did not approve LVS as a human vaccine due to its undefined attenuation mechanism, insufficient protection against respiratory infection by type A strains of Francisella , phenotypic instability, and partial virulence after aerosol vaccination. Therefore, although live attenuated vaccines can be promising, for safety reasons, current trends in prophylaxis are focusing on vaccines based on bacterial subunits rather than on live attenuated strains. Subunit vaccines are considered safer due to their composition, which consists of synthetic or isolated microbial antigens. However, since Francisella is an intracellular pathogen, a Francisella subunit vaccine must induce a cell-mediated response, and the identification of specific T-cell epitopes is not a trivial matter. Computational whole-genome analysis, which detects immunogenic Francisella peptides associated with major histocompatibility complex class I (MHCI), constitutes one of the most promising approaches. Alternatively, a protein array based approach could identify major histocompatibility complex class II (MHCII) epitopes of various serological targets. These new approaches are expected to provide new peptide epitopes for the development of effective subunit vaccines. 16 Essentials for medical practice F. tularensis mainly infects lagomorphs, but can persist for several weeks in animal carcasses, mud, and water. The infection is maintained through the life cycles between ticks and rodents. Tularemia manifests 3–5 days after incubation, depending on how it was transmitted. Symptoms include fever, inflammation of the infected area, and enlarged lymph nodes. Of the six main clinical forms of this disease, ulceroglandular tularemia is the most common and clinically recognizable. Treatment must be administered in all suspected or confirmed cases. Moderate to severe infections are usually treated with 5 mg/kg of gentamicin a day administered intramuscularly or intravenously every 8 hours for 10–14 days. Milder infections are treated with 100 mg of doxycycline administered orally twice a day for 21 days or with 500–750 mg of ciprofloxacin administered orally twice a day for 10–14 days. Treatment The antibiotic treatment must be administered in all suspected or confirmed cases, although there have been cases of spontaneous regression without a specific treatment. 9 Aminoglycosides (gentamicin) are the drugs of choice for severe infections. Tetracyclines (doxycycline) – also widely used for the prophylaxis and treatment of tularemia – are associated with a higher relapse rate. Recent experience has demonstrated the excellent efficacy of ciprofloxacin, which is preferred for oral use. The treatment period is 10–14 days with aminoglycosides and ciprofloxacin and 21 days with tetracyclines. 13 Suppurative lymph nodes require surgical drainage; pleural effusion requires thoracentesis to rule out empyema. 9 , 10 Prevention People exposed to the risk of infection can undergo antibiotic prophylaxis under medical supervision to prevent the onset of the disease. 1 There is no safe, authorized vaccine available on the market. A potential candidate vaccine (LVS – Live Vaccine Strain) does not meet the criteria for general use. The first live attenuated vaccine candidate was made from F. tularensis subsp. holarctica strains in the former Soviet Union and, after serial passage in the USA, it led to the preparation of LVS. Although it demonstrated the ability to induce protective immunity against a low-dose aerosol challenge of the Schu S4 strain, the Food and Drug Administration did not approve LVS as a human vaccine due to its undefined attenuation mechanism, insufficient protection against respiratory infection by type A strains of Francisella , phenotypic instability, and partial virulence after aerosol vaccination. Therefore, although live attenuated vaccines can be promising, for safety reasons, current trends in prophylaxis are focusing on vaccines based on bacterial subunits rather than on live attenuated strains. Subunit vaccines are considered safer due to their composition, which consists of synthetic or isolated microbial antigens. However, since Francisella is an intracellular pathogen, a Francisella subunit vaccine must induce a cell-mediated response, and the identification of specific T-cell epitopes is not a trivial matter. Computational whole-genome analysis, which detects immunogenic Francisella peptides associated with major histocompatibility complex class I (MHCI), constitutes one of the most promising approaches. Alternatively, a protein array based approach could identify major histocompatibility complex class II (MHCII) epitopes of various serological targets. These new approaches are expected to provide new peptide epitopes for the development of effective subunit vaccines. 16 Essentials for medical practice F. tularensis mainly infects lagomorphs, but can persist for several weeks in animal carcasses, mud, and water. The infection is maintained through the life cycles between ticks and rodents. Tularemia manifests 3–5 days after incubation, depending on how it was transmitted. Symptoms include fever, inflammation of the infected area, and enlarged lymph nodes. Of the six main clinical forms of this disease, ulceroglandular tularemia is the most common and clinically recognizable. Treatment must be administered in all suspected or confirmed cases. Moderate to severe infections are usually treated with 5 mg/kg of gentamicin a day administered intramuscularly or intravenously every 8 hours for 10–14 days. Milder infections are treated with 100 mg of doxycycline administered orally twice a day for 21 days or with 500–750 mg of ciprofloxacin administered orally twice a day for 10–14 days.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582765/

A Two-stage Multiplex Method for Quantitative Analysis of Botulinum Neurotoxin type A, B, E, and F by MALDI-TOF Mass Spectrometry

In this publication we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g. 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 hr), so that toxin levels lower than 1 mouse LD 50 or 55 attomole/mL could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, /B, /E, or /F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and to save valuable clinical samples.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10472150/

Composition and functional diversity of bacterial communities during swine carcass decomposition

Objective This study investigated the changes in bacterial communities within decomposing swine microcosms, comparing soil with or without intact microbial communities, and under aerobic and anaerobic conditions. Methods The experimental microcosms consisted of four conditions: UA, unsterilized soil–aerobic condition; SA, sterilized soil–aerobic condition; UAn, unsterilized soil–anaerobic condition; and San, sterilized soil–anaerobic condition. The microcosms were prepared by mixing 112.5 g of soil and 37.5 g of ground carcass, which were then placed in sterile containers. The carcass-soil mixture was sampled at day 0, 5, 10, 30, and 60 of decomposition, and the bacterial communities that formed during carcass decomposition were assessed using Illumina MiSeq sequencing of the 16S rRNA gene. Results A total of 1,687 amplicon sequence variants representing 22 phyla and 805 genera were identified in the microcosms. The Chao1 and Shannon diversity indices varied in between microcosms at each period (p<0.05). Metagenomic analysis showed variation in the taxa composition across the burial microcosms during decomposition, with Firmicutes being the dominant phylum, followed by Proteobacteria. At the genus level, Bacillus and Clostridium were the main genera within Firmicutes. Functional prediction revealed that the most abundant Kyoto encyclopedia of genes and genomes metabolic functions were carbohydrate and amino acid metabolisms. Conclusion This study demonstrated a higher bacteria diversity in UA and UAn microcosms than in SA and SAn microcosms. In addition, the taxonomic composition of the microbial community also exhibited changes, highlighting the impact of soil sterilization and oxygen on carcass decomposition. Furthermore, this study provided insights into the microbial communities associated with decomposing swine carcasses in microcosm. Objective This study investigated the changes in bacterial communities within decomposing swine microcosms, comparing soil with or without intact microbial communities, and under aerobic and anaerobic conditions. Methods The experimental microcosms consisted of four conditions: UA, unsterilized soil–aerobic condition; SA, sterilized soil–aerobic condition; UAn, unsterilized soil–anaerobic condition; and San, sterilized soil–anaerobic condition. The microcosms were prepared by mixing 112.5 g of soil and 37.5 g of ground carcass, which were then placed in sterile containers. The carcass-soil mixture was sampled at day 0, 5, 10, 30, and 60 of decomposition, and the bacterial communities that formed during carcass decomposition were assessed using Illumina MiSeq sequencing of the 16S rRNA gene. Results A total of 1,687 amplicon sequence variants representing 22 phyla and 805 genera were identified in the microcosms. The Chao1 and Shannon diversity indices varied in between microcosms at each period (p<0.05). Metagenomic analysis showed variation in the taxa composition across the burial microcosms during decomposition, with Firmicutes being the dominant phylum, followed by Proteobacteria. At the genus level, Bacillus and Clostridium were the main genera within Firmicutes. Functional prediction revealed that the most abundant Kyoto encyclopedia of genes and genomes metabolic functions were carbohydrate and amino acid metabolisms. Conclusion This study demonstrated a higher bacteria diversity in UA and UAn microcosms than in SA and SAn microcosms. In addition, the taxonomic composition of the microbial community also exhibited changes, highlighting the impact of soil sterilization and oxygen on carcass decomposition. Furthermore, this study provided insights into the microbial communities associated with decomposing swine carcasses in microcosm. INTRODUCTION Outbreaks of contagious animal diseases such as foot-and-mouth disease and African swine fever can lead to significant losses in the livestock industry [ 1 ]. Burial is a commonly used method for disposing of both daily and disease-related animal mortalities [ 2 ]. However, the metabolites from decomposing carcasses may have adverse effects on the environment, such as soil and groundwater pollution, as well as posing a risk to human and animal health [ 3 ]. The decomposition of carcasses can cause dynamic changes in the bacterial communities in the soil, which can adversely affect the environment and lead to possible disease outbreak. During a disease outbreak, proper disposal of animals associated with infectious pathogens should be implemented to minimize the risk of disease spreading. The disinfection of burial pits for contagious disease-related animal mortalities is usually performed to reduce the spread of disease or reduce the contamination in the environment [ 4 ]. Some methods for soil sterilization are through the use of chemicals or heat which can be effective in killing off the microorganisms in the soil [ 5 ]. Owing to the heat generated during sterilization, there are decreases in microbial biomass and enzyme activity, resulting in the inactivation of enzymes released by soil microorganisms. The decomposition of buried carcasses mostly relies on the capacity of microbes to generate extracellular proteolytic enzymes, which aid in the breakdown of complex organic matter polymers into smaller oligomeric and monomeric molecules [ 6 ]. The rate of carcass decomposition is significantly influenced by microbial activity both within, on, and around the carcasses, as it contributes to the maintenance of soil quality through its involvement in organic matter dynamics, nutrient cycling, and decomposition [ 7 ]. In addition, various biotic and abiotic factors can influence the carcass and can cause an adverse effect in the soil microbiome [ 8 ]. Aside from the sterilization of soil, the availability of oxygen can affect decomposition and contribute to the changes in the microbial community during carcass decomposition. Various studies have shown that decomposition typically occurs at a faster rate under aerobic conditions [ 9 ], while others report faster decomposition under anaerobic conditions [ 10 ]. The microbial communities involved in the decomposition of animal carcasses may vary depending on whether the animals were buried or left to decompose naturally in the environment; thus, different aerobic and anaerobic bacteria may be involved in the decomposition of animal carcasses. Several studies have been conducted to characterize the microbial community composition in decomposing carcasses. However, limited research has been conducted on the microbial community structure of swine carcasses in soil, with or without indigenous microbial communities, during aerobic or anaerobic decomposition. These factors may contribute to the changes in the microbial community composition in decomposing carcasses. Therefore, it is necessary to investigate the changes in the bacterial community in animal burial soil. Thus, the present study focused on the investigation of the changes in the composition and functional diversity of bacterial communities of decomposing swine carcasses in a burial microcosm under the influence of various conditions: carcasses buried in either i) unsterilized soil (soil with an intact microbial community) or ii) soil that was sterilized and was incubated either aerobically or anaerobically. MATERIALS AND METHODS Ethical statement This study was conducted in accordance with the guidelines and regulations set by the Institutional Animal Care and Use Committee (Approval number: SCNU IACUC-2019-7) of Sunchon National University (Suncheon, Korea). All experimental protocols were approved by the aforementioned governing body. Soil preparation Soil (10 kg) was collected from an agricultural field at the experimental farm of the Sunchon National University. The raw soil comprised sandy loam soil and had a pH (1:5H 2 O) of 5.84 and a moisture content of 23.70%. The soil was sieved (2 mm) and split into two portions. One portion of the collected soil was sterilized, while the other was kept unsterilized. The sterilized soil was prepared by autoclaving the soil (121°C at 15 psi for 30 minutes) thrice over 4 days to eliminate microbes, fungi, and their spores [ 5 ]. Pre-processing of carcass A young domestic pig ( Sus scrofa L.), weighing 10.0±2.0 kg, was purchased commercially and used in the study. The swine was sacrificed, and carcasses were preprocessed prior to decomposition. The carcass was carefully separated from the bones, and the blood and internal organs were collected. Following bone removal, the carcass, skin, blood, and internal organs were homogenized in a mixer and used for the laboratory microcosm to simulate the decomposition process. Preparation of microcosm for carcass decomposition A laboratory microcosm was prepared for the decomposition of the swine carcass. The burial microcosms used two types of soil (soil with intact microbes (unsterilized soil)); ii. soil that was sterilized and two incubation conditions (aerobic and anaerobic conditions). The experimental microcosms were: UA, unsterilized soil – aerobic condition; SA, sterilized soil – aerobic condition; UAn, unsterilized soil – anaerobic condition; and San, sterilized soil – anaerobic condition ( Supplementary Figure S1 ). Soil (112.5 g) and homogenized carcass (37.5 g) were mixed thoroughly and distributed into sterile containers (dimensions: 109 mm×152 mm×58 mm). The soil/carcass ratio depicts the heavy burial conditions of an estimated 550 pig carcasses in a 100 m 2 burial area [ 11 ]. The anaerobic condition was achieved by sealing the container and placing it in a 5% CO 2 incubator, while for aerobic conditions, the lid of the container was pierced to allow air to pass through the hole before placing it in the incubator. All experimental setups were conducted in triplicate and incubated at 25°C for a total of 60 days. Sample collection Approximately 10 g of the carcass-soil mixture samples were collected from the microcosms at the initial placement time and after 5, 10, 30, and 60 days of decomposition. The samples were placed in sterile conical tubes and kept at −80°C until used. Library construction and amplicon sequencing The carcass-soil samples were sent to Macrogen Inc. (Seoul, Korea) for metagenomic sequencing analysis. The bacterial communities were characterized by analyzing the V3–V4 region of the 16S rRNA gene according to the 16S Metagenomics Library Prep Guide (15044223 Rev. B) [ 12 ]. Paired-end sequencing was performed on a MiSeq platform (Illumina, San Diego, CA, USA) using v3 reagents at Macrogen Inc. (Korea). Bioinformatics and data analyses Following sequencing, the raw data was classified by sample using an index sequence, and paired-end FASTQ files were generated for each sample. Subsequently, the raw sequences were demultiplexed, and barcodes and adaptors sequence were removed using the Cutadapt v3.2 program [ 13 ]. Sequence reads were clustered into amplicon sequence variants (ASVs) according to the standard pipeline workflow of Divisive Amplicon Denoising Algorithm 2 (DADA2) v1.18.0 [ 14 ]. For the paired-end reads, forward and reverse reads were truncated at 250 bp and 200 bp, respectively, and sequences with expected errors of ≥2 were excluded. The QIIME v1.9 program was used for the comparative analysis of the microbial community [ 15 ]. Each of the DNA sequences was annotated to the species level using BLAST+ (v.2.9.0) against the Reference Database (NCBI 16S Microbial DB) [ 16 ]. Data analysis and visualization were conducted using the MicrobiomeAnalyst web-based tool [ 17 , 18 ]. The processed sequence data were imported into MicrobiomeAnalyst and filtered for low count and low variance using the default settings. This resulted in the removal of 631 low abundance features based on prevalence and 39 low variance features based on inter-quantile range). Subsequently, data normalization was performed using 'total sum scaling' as the scaling method. Shannon's diversity index and Chao1 richness were calculated and used to compare the alpha diversity in the microcosms. Statistical analysis was performed using the general linear model procedure of the Statistical Analysis System (SAS) program version 9.4 (SAS Institute Inc., Cary, NC, USA). Two-way analysis of variance was used to examine the effects of the presence or absence of soil microbes, oxygen availability, and their interaction in the alpha diversity indices. Tukey's honestly significant difference post hoc test was used to compare significant differences between burial microcosms at each time point, with a significance level set at p<0.05. Beta diversity at the genus level was assessed based on the Bray-Curtis distance method, and the results were visualized using principal coordinate analysis (PCoA). Bacterial abundance profiles at the phylum, genus, and species levels were represented using stacked bar graphs. Venn diagram of unique and core bacterial genera was drawn using jvenn [ 19 ] to highlight the similarities and shared sequences between the different microcosms. Analysis of the core microbiome was carried out at the genus level using the MicrobiomeAnalyst with sample prevalence at 20% and a relative abundance cutoff of 0.01%. Prediction of functional profile of bacterial communities in decomposing swine carcass MicrobiomeAnalyst was employed to predict the functional profiles of bacterial communities associated with decomposing swine carcasses based on 16S rRNA gene sequencing data [ 17 , 18 ]. The ASV table and metadata table were uploaded to the Marker Data Profiling Module. Functional profiles were determined by analyzing the ASVs using Tax4Fun in MicrobiomeAnalyst. The resulting Kyoto encyclopedia of genes and genomes (KEGG) Orthology (KO) table [ 20 ] was then imported to the Shotgun Data Profiling Module, and diversity and association analyses were investigated. Ethical statement This study was conducted in accordance with the guidelines and regulations set by the Institutional Animal Care and Use Committee (Approval number: SCNU IACUC-2019-7) of Sunchon National University (Suncheon, Korea). All experimental protocols were approved by the aforementioned governing body. Soil preparation Soil (10 kg) was collected from an agricultural field at the experimental farm of the Sunchon National University. The raw soil comprised sandy loam soil and had a pH (1:5H 2 O) of 5.84 and a moisture content of 23.70%. The soil was sieved (2 mm) and split into two portions. One portion of the collected soil was sterilized, while the other was kept unsterilized. The sterilized soil was prepared by autoclaving the soil (121°C at 15 psi for 30 minutes) thrice over 4 days to eliminate microbes, fungi, and their spores [ 5 ]. Pre-processing of carcass A young domestic pig ( Sus scrofa L.), weighing 10.0±2.0 kg, was purchased commercially and used in the study. The swine was sacrificed, and carcasses were preprocessed prior to decomposition. The carcass was carefully separated from the bones, and the blood and internal organs were collected. Following bone removal, the carcass, skin, blood, and internal organs were homogenized in a mixer and used for the laboratory microcosm to simulate the decomposition process. Preparation of microcosm for carcass decomposition A laboratory microcosm was prepared for the decomposition of the swine carcass. The burial microcosms used two types of soil (soil with intact microbes (unsterilized soil)); ii. soil that was sterilized and two incubation conditions (aerobic and anaerobic conditions). The experimental microcosms were: UA, unsterilized soil – aerobic condition; SA, sterilized soil – aerobic condition; UAn, unsterilized soil – anaerobic condition; and San, sterilized soil – anaerobic condition ( Supplementary Figure S1 ). Soil (112.5 g) and homogenized carcass (37.5 g) were mixed thoroughly and distributed into sterile containers (dimensions: 109 mm×152 mm×58 mm). The soil/carcass ratio depicts the heavy burial conditions of an estimated 550 pig carcasses in a 100 m 2 burial area [ 11 ]. The anaerobic condition was achieved by sealing the container and placing it in a 5% CO 2 incubator, while for aerobic conditions, the lid of the container was pierced to allow air to pass through the hole before placing it in the incubator. All experimental setups were conducted in triplicate and incubated at 25°C for a total of 60 days. Sample collection Approximately 10 g of the carcass-soil mixture samples were collected from the microcosms at the initial placement time and after 5, 10, 30, and 60 days of decomposition. The samples were placed in sterile conical tubes and kept at −80°C until used. Library construction and amplicon sequencing The carcass-soil samples were sent to Macrogen Inc. (Seoul, Korea) for metagenomic sequencing analysis. The bacterial communities were characterized by analyzing the V3–V4 region of the 16S rRNA gene according to the 16S Metagenomics Library Prep Guide (15044223 Rev. B) [ 12 ]. Paired-end sequencing was performed on a MiSeq platform (Illumina, San Diego, CA, USA) using v3 reagents at Macrogen Inc. (Korea). Bioinformatics and data analyses Following sequencing, the raw data was classified by sample using an index sequence, and paired-end FASTQ files were generated for each sample. Subsequently, the raw sequences were demultiplexed, and barcodes and adaptors sequence were removed using the Cutadapt v3.2 program [ 13 ]. Sequence reads were clustered into amplicon sequence variants (ASVs) according to the standard pipeline workflow of Divisive Amplicon Denoising Algorithm 2 (DADA2) v1.18.0 [ 14 ]. For the paired-end reads, forward and reverse reads were truncated at 250 bp and 200 bp, respectively, and sequences with expected errors of ≥2 were excluded. The QIIME v1.9 program was used for the comparative analysis of the microbial community [ 15 ]. Each of the DNA sequences was annotated to the species level using BLAST+ (v.2.9.0) against the Reference Database (NCBI 16S Microbial DB) [ 16 ]. Data analysis and visualization were conducted using the MicrobiomeAnalyst web-based tool [ 17 , 18 ]. The processed sequence data were imported into MicrobiomeAnalyst and filtered for low count and low variance using the default settings. This resulted in the removal of 631 low abundance features based on prevalence and 39 low variance features based on inter-quantile range). Subsequently, data normalization was performed using 'total sum scaling' as the scaling method. Shannon's diversity index and Chao1 richness were calculated and used to compare the alpha diversity in the microcosms. Statistical analysis was performed using the general linear model procedure of the Statistical Analysis System (SAS) program version 9.4 (SAS Institute Inc., Cary, NC, USA). Two-way analysis of variance was used to examine the effects of the presence or absence of soil microbes, oxygen availability, and their interaction in the alpha diversity indices. Tukey's honestly significant difference post hoc test was used to compare significant differences between burial microcosms at each time point, with a significance level set at p<0.05. Beta diversity at the genus level was assessed based on the Bray-Curtis distance method, and the results were visualized using principal coordinate analysis (PCoA). Bacterial abundance profiles at the phylum, genus, and species levels were represented using stacked bar graphs. Venn diagram of unique and core bacterial genera was drawn using jvenn [ 19 ] to highlight the similarities and shared sequences between the different microcosms. Analysis of the core microbiome was carried out at the genus level using the MicrobiomeAnalyst with sample prevalence at 20% and a relative abundance cutoff of 0.01%. Prediction of functional profile of bacterial communities in decomposing swine carcass MicrobiomeAnalyst was employed to predict the functional profiles of bacterial communities associated with decomposing swine carcasses based on 16S rRNA gene sequencing data [ 17 , 18 ]. The ASV table and metadata table were uploaded to the Marker Data Profiling Module. Functional profiles were determined by analyzing the ASVs using Tax4Fun in MicrobiomeAnalyst. The resulting Kyoto encyclopedia of genes and genomes (KEGG) Orthology (KO) table [ 20 ] was then imported to the Shotgun Data Profiling Module, and diversity and association analyses were investigated. RESULTS Bacterial species richness and diversity The alpha diversity represented by Chao1 and Shannon's diversity indices for the different microcosms is shown in Figure 1 . Chao1 showed significant differences among burial microcosms (p<0.05) in all time points ( Figure 1a ). Chao1 was significantly higher (p<0.05) in UA and UAn microcosms than in SA and SAn microcosms during the initial placement, then decreased throughout the decomposition period. At the end of the study period, lowest Chao1 were observed in UA, SA, and SAn microcosms. The Shannon's diversity index showed significant differences among the microcosms in all periods (p<0.05) ( Figure 1b ). At day 0, Shannon index in UA and UAn were significantly higher compared to SA and SAn microcosms. At days 5, 10, 30, and 60, Shannon index was significantly different between treatments (p<0.05). Moreover, the Shannon's diversity index in the UA and UAn microcosms were higher than those in SA and SAn microcosms until day 30. At day 60, Shannon's diversity index were higher in UAn and SAn than in UA and SA microcosms (p<0.05). Comparisons of the microcosms at different time points using PCoA revealed that the samples at day 0 showed a well-differentiated bacterial profile. Moreover, axis 1 and axis 2 explained 52% and 26.9% of the variance, respectively. The first two principal components (Axis1+Axis2) accounted for 78.9% of the total variation ( Figure 2 ). Microcosm samples on days 5 and 10 were grouped together and at different distances from the other time points. In addition, at the beginning of decomposition (day 0) and after the late stages (days 30 and 60), samples were found dispersed in axes, indicating a differentiated bacterial community based on 16S rRNA amplicon sequencing. Core microbiome In total, 1,687 ASVs were detected in all soil microcosm samples, of which 22 phyla, 805 genera, and 1,687 species were identified across all samples. The Venn diagram represents the shared bacterial species within all microcosms, as well as the unique species within the different samples ( Figure 3a ). Results showed that 101, 22, 92, and 27 genera were uniquely present in UA, SA, UAn, and SAn, respectively ( Supplementary Table S1 ). We found 7 unique bacterial genera ( Alistipes , Natranaerovigra , Acinetobacter , Ralstonia , Achromobacter , Novosphingobium , and Comomonas ) between UA and SA ( Supplementary Table S2 ). In addition, 7 unique bacterial genera ( Geothermomicrobium , Ramlibacter , Jeotgalibacillus , Allisonella , Rothia , Thermobacillus , and N eglecta ) were identified between UAn and SAn. Meanwhile, 208 and 10 unique genera were found between UA and UAn microcosms and SA and SAn microcosms, respectively. Moreover, we found 214 genera were shared among all four microcosms ( Supplementary Table Table S3 ). Although 805 genera were identified in the analyzed samples, 14 genera, Clostridium , Bacillus , Lactobacillus , Pseudescherichia , Enterococcus , Paraclostridium , Eubacterium , Pediococcus , Anaerosalibacter , Rummeliibacillus , Schnuerera , T errisporobacter , Corynebacterium , and Neobacillus , constituted the core microbiome of the analyzed samples from the swine burial microcosms ( Figure 3b ). Bacterial community composition during decomposition The taxonomic composition of each microcosm was analyzed and compared at the phylum, genus, and species levels. The dominant taxa varied among the microcosms at different time points. Evaluation of the bacterial ASVs revealed that Firmicutes was the major phylum identified in all the microcosms, followed by Proteobacteria and Actinobacteria ( Figure 4 ). Classification of the identified bacterial ASVs revealed that during the initial day (day 0), Firmicutes was the most dominant phylum in the burial microcosm, which represented 57.62%, 90.68%, 57.43%, and 90.30% of the relative abundances for UA, SA, UAn, and SAn microcosms, respectively. Actinobacteria was the next most abundant phylum in the UA (17.92%) and UAn (17.84%) microcosms. On day 5, the relative abundance of Firmicutes increased in UA (79.98%) and UAn (75.39%), whereas the abundance was reduced in SA (53.66%) and SAn (68%). Proteobacteria increased in abundance in all samples, particularly in SA (45.89%), and SAn (31.52%). After 10 days, the relative abundance of Proteobacteria was reduced in all samples, more particularly in SA and SAn, where an apparent reduction in abundance was observed. Moreover, Firmicutes increased in abundance in all samples and became the most abundant phylum at this point. On subsequent days (days 30 and 60), a clear domination of the members of the phylum Firmicutes, which represented 96% to 99% of the population in all samples was observed, whereas the abundance of other bacterial phyla decreased. As shown in Figure 5 , Bacillus , Clostridium , Enterococcus , and Lactobacillus were among the major bacterial genera in the microcosms. The abundances of these bacterial genera shifted throughout the decomposition period. On day 0, Lactobacillus and Clostridium were the most dominant genera in the samples; however, a higher abundance was observed in microcosms SA and SAn than what was seen in UA and UAn. After 5 days, Clostridium , Pseudescherichia , Enterococcus , Paraclostridium , and Pediococcus were among the genera that increased in abundance. Moreover, the abundance of Pseudescherichia sharply increased in all the samples, particularly in the SA and SAn. In addition, Clostridium and Paraclostridium increased in abundance in the UA and UAn microcosms. In contrast, Enterococcus abundance increased in all microcosms, particularly in SA, UAn, and SAn. On day 10, Clostridium increased in all samples and was identified as the most dominant genus. A higher abundance of Clostridium was observed in the UAn and SAn microcosms. Meanwhile, the relative abundances of Pseudescherichia , Paraclostridium , and Enterococcus were reduced in all samples, whereas the abundance of Rummeliibacillus increased, particularly in the microcosms SA and SAn. On day 30, members of Bacillus dominated UAn, SA, and SAn at 65.84%, 50.75%, and 69.24%, respectively. Consequently, a sharp decline in the abundance of Clostridium was observed in UAn, SA, and SAn, whereas the abundance in UA was maintained. In addition, Anaerosalibacter was detected in UA at an abundance of 12.73% which was higher than that in other burial microcosms. On day 60, members of the Bacillus continued to be dominant in all samples. Moreover, a higher abundance of Bacillus was observed in microcosms UA and SA than in UAn and SAn. The major bacterial species in each burial microcosm are shown in Figure 6 . The composition of bacterial species varied in each burial microcosm and at each time point. On the initial day, Lactobacillus ultunensis , L. johnsonii , and Clostridium saudiense were the most abundant species in all samples. However, higher abundances of these species were found in the SA and SAn microcosms compared to the UA and UAn microcosms. By day 5, Pseudescherichia vulneris , Clostridium sporogenes , Enterococcus faecalis , Paraclostridium benzoelyticium , and Pediococcus pentosaceus were among the dominant genera observed in the samples. Specifically, P. vulneris was more abundant in SA and SAn, while C. sporogenes was more abundant in UA, UAn, and SAn, and E. faecalis was abundant in SA, UAn, and SAn. In addition, the relative abundance of P. pentosaceus increased in all the samples. We also identified P. benzoelyticium in UA and UAn microcosms. By day 10, C. sporogenes decreased in abundance in UA but increased in SA, UAn, and SAn. In particular, the abundance in UAn and SAn was high, with a relative abundance of 20.06% and 35.28%, respectively. Meanwhile, the relative abundances of P. vulneris , P. benzoelyticium , E. faecalis , and P. pentosaceus was reduced. On day 30, a clear dominance of Bacillus paralicheniformis was noted in the microcosms of SA, UAn, and SAn. Anaerosalibacter bizertensis was detected in the UA microcosm with an abundance of 12.73%. By day 60, the abundance of B. paralicheniformis increased in all samples and was the most dominant genus at this point, reaching a relative abundance of approximately 59.99% to 82.98% at the end of the experiment. Prediction of the functional profile of bacterial communities associated with decomposing swine carcass The probable functions of the decomposition microbiome were inspected by Tax4Fun in the MicrobiomeAnalyst tool. Diversity analysis revealed 5,418 KEGG orthologs, 1,230 KEGG pathways, and 11 KEGG metabolic functions in decomposing swine carcasses. The most abundant KEGG metabolic functions were carbohydrate and amino acid metabolisms ( Figure 7a ). There were 22 predicted clusters of orthologous groups of proteins (COG) in all microcosms and the most abundant COG were amino acid transport and metabolism, followed by inorganic ion transport and metabolism, and carbohydrate transport and metabolism ( Figure 7b ). Bacterial species richness and diversity The alpha diversity represented by Chao1 and Shannon's diversity indices for the different microcosms is shown in Figure 1 . Chao1 showed significant differences among burial microcosms (p<0.05) in all time points ( Figure 1a ). Chao1 was significantly higher (p<0.05) in UA and UAn microcosms than in SA and SAn microcosms during the initial placement, then decreased throughout the decomposition period. At the end of the study period, lowest Chao1 were observed in UA, SA, and SAn microcosms. The Shannon's diversity index showed significant differences among the microcosms in all periods (p<0.05) ( Figure 1b ). At day 0, Shannon index in UA and UAn were significantly higher compared to SA and SAn microcosms. At days 5, 10, 30, and 60, Shannon index was significantly different between treatments (p<0.05). Moreover, the Shannon's diversity index in the UA and UAn microcosms were higher than those in SA and SAn microcosms until day 30. At day 60, Shannon's diversity index were higher in UAn and SAn than in UA and SA microcosms (p<0.05). Comparisons of the microcosms at different time points using PCoA revealed that the samples at day 0 showed a well-differentiated bacterial profile. Moreover, axis 1 and axis 2 explained 52% and 26.9% of the variance, respectively. The first two principal components (Axis1+Axis2) accounted for 78.9% of the total variation ( Figure 2 ). Microcosm samples on days 5 and 10 were grouped together and at different distances from the other time points. In addition, at the beginning of decomposition (day 0) and after the late stages (days 30 and 60), samples were found dispersed in axes, indicating a differentiated bacterial community based on 16S rRNA amplicon sequencing. Core microbiome In total, 1,687 ASVs were detected in all soil microcosm samples, of which 22 phyla, 805 genera, and 1,687 species were identified across all samples. The Venn diagram represents the shared bacterial species within all microcosms, as well as the unique species within the different samples ( Figure 3a ). Results showed that 101, 22, 92, and 27 genera were uniquely present in UA, SA, UAn, and SAn, respectively ( Supplementary Table S1 ). We found 7 unique bacterial genera ( Alistipes , Natranaerovigra , Acinetobacter , Ralstonia , Achromobacter , Novosphingobium , and Comomonas ) between UA and SA ( Supplementary Table S2 ). In addition, 7 unique bacterial genera ( Geothermomicrobium , Ramlibacter , Jeotgalibacillus , Allisonella , Rothia , Thermobacillus , and N eglecta ) were identified between UAn and SAn. Meanwhile, 208 and 10 unique genera were found between UA and UAn microcosms and SA and SAn microcosms, respectively. Moreover, we found 214 genera were shared among all four microcosms ( Supplementary Table Table S3 ). Although 805 genera were identified in the analyzed samples, 14 genera, Clostridium , Bacillus , Lactobacillus , Pseudescherichia , Enterococcus , Paraclostridium , Eubacterium , Pediococcus , Anaerosalibacter , Rummeliibacillus , Schnuerera , T errisporobacter , Corynebacterium , and Neobacillus , constituted the core microbiome of the analyzed samples from the swine burial microcosms ( Figure 3b ). Bacterial community composition during decomposition The taxonomic composition of each microcosm was analyzed and compared at the phylum, genus, and species levels. The dominant taxa varied among the microcosms at different time points. Evaluation of the bacterial ASVs revealed that Firmicutes was the major phylum identified in all the microcosms, followed by Proteobacteria and Actinobacteria ( Figure 4 ). Classification of the identified bacterial ASVs revealed that during the initial day (day 0), Firmicutes was the most dominant phylum in the burial microcosm, which represented 57.62%, 90.68%, 57.43%, and 90.30% of the relative abundances for UA, SA, UAn, and SAn microcosms, respectively. Actinobacteria was the next most abundant phylum in the UA (17.92%) and UAn (17.84%) microcosms. On day 5, the relative abundance of Firmicutes increased in UA (79.98%) and UAn (75.39%), whereas the abundance was reduced in SA (53.66%) and SAn (68%). Proteobacteria increased in abundance in all samples, particularly in SA (45.89%), and SAn (31.52%). After 10 days, the relative abundance of Proteobacteria was reduced in all samples, more particularly in SA and SAn, where an apparent reduction in abundance was observed. Moreover, Firmicutes increased in abundance in all samples and became the most abundant phylum at this point. On subsequent days (days 30 and 60), a clear domination of the members of the phylum Firmicutes, which represented 96% to 99% of the population in all samples was observed, whereas the abundance of other bacterial phyla decreased. As shown in Figure 5 , Bacillus , Clostridium , Enterococcus , and Lactobacillus were among the major bacterial genera in the microcosms. The abundances of these bacterial genera shifted throughout the decomposition period. On day 0, Lactobacillus and Clostridium were the most dominant genera in the samples; however, a higher abundance was observed in microcosms SA and SAn than what was seen in UA and UAn. After 5 days, Clostridium , Pseudescherichia , Enterococcus , Paraclostridium , and Pediococcus were among the genera that increased in abundance. Moreover, the abundance of Pseudescherichia sharply increased in all the samples, particularly in the SA and SAn. In addition, Clostridium and Paraclostridium increased in abundance in the UA and UAn microcosms. In contrast, Enterococcus abundance increased in all microcosms, particularly in SA, UAn, and SAn. On day 10, Clostridium increased in all samples and was identified as the most dominant genus. A higher abundance of Clostridium was observed in the UAn and SAn microcosms. Meanwhile, the relative abundances of Pseudescherichia , Paraclostridium , and Enterococcus were reduced in all samples, whereas the abundance of Rummeliibacillus increased, particularly in the microcosms SA and SAn. On day 30, members of Bacillus dominated UAn, SA, and SAn at 65.84%, 50.75%, and 69.24%, respectively. Consequently, a sharp decline in the abundance of Clostridium was observed in UAn, SA, and SAn, whereas the abundance in UA was maintained. In addition, Anaerosalibacter was detected in UA at an abundance of 12.73% which was higher than that in other burial microcosms. On day 60, members of the Bacillus continued to be dominant in all samples. Moreover, a higher abundance of Bacillus was observed in microcosms UA and SA than in UAn and SAn. The major bacterial species in each burial microcosm are shown in Figure 6 . The composition of bacterial species varied in each burial microcosm and at each time point. On the initial day, Lactobacillus ultunensis , L. johnsonii , and Clostridium saudiense were the most abundant species in all samples. However, higher abundances of these species were found in the SA and SAn microcosms compared to the UA and UAn microcosms. By day 5, Pseudescherichia vulneris , Clostridium sporogenes , Enterococcus faecalis , Paraclostridium benzoelyticium , and Pediococcus pentosaceus were among the dominant genera observed in the samples. Specifically, P. vulneris was more abundant in SA and SAn, while C. sporogenes was more abundant in UA, UAn, and SAn, and E. faecalis was abundant in SA, UAn, and SAn. In addition, the relative abundance of P. pentosaceus increased in all the samples. We also identified P. benzoelyticium in UA and UAn microcosms. By day 10, C. sporogenes decreased in abundance in UA but increased in SA, UAn, and SAn. In particular, the abundance in UAn and SAn was high, with a relative abundance of 20.06% and 35.28%, respectively. Meanwhile, the relative abundances of P. vulneris , P. benzoelyticium , E. faecalis , and P. pentosaceus was reduced. On day 30, a clear dominance of Bacillus paralicheniformis was noted in the microcosms of SA, UAn, and SAn. Anaerosalibacter bizertensis was detected in the UA microcosm with an abundance of 12.73%. By day 60, the abundance of B. paralicheniformis increased in all samples and was the most dominant genus at this point, reaching a relative abundance of approximately 59.99% to 82.98% at the end of the experiment. Prediction of the functional profile of bacterial communities associated with decomposing swine carcass The probable functions of the decomposition microbiome were inspected by Tax4Fun in the MicrobiomeAnalyst tool. Diversity analysis revealed 5,418 KEGG orthologs, 1,230 KEGG pathways, and 11 KEGG metabolic functions in decomposing swine carcasses. The most abundant KEGG metabolic functions were carbohydrate and amino acid metabolisms ( Figure 7a ). There were 22 predicted clusters of orthologous groups of proteins (COG) in all microcosms and the most abundant COG were amino acid transport and metabolism, followed by inorganic ion transport and metabolism, and carbohydrate transport and metabolism ( Figure 7b ). DISCUSSION Microbes play a crucial role in decomposition, as they produce degradative enzymes and can utilize a diverse range of carrion substrates, including internal tissues, organs, skin, hair, and even bone. Therefore, identifying the decomposition ecology in swine microcosms is crucial to strengthen the current knowledge of the microbiology of decomposing carcasses. Previous research on decomposing swine and mice has revealed that bacterial communities undergo changes in major phyla over time, which align with specific visual indicators of body decomposition [ 21 , 22 ]. The variances in the microbial composition observed in our study could potentially be attributed to changes in dominant phyla. Lauber et al [ 5 ] revealed that the presence of soil microbial communities has a substantial impact on accelerating the rates of carrion decomposition. Our findings showed that Chao1 index was comparable between UA and UAn microcosms and in SA and SAn microcosms, particularly at day 0 to 10. At days 30 and 60, Chao1 showed variations between different microcosms, with UAn being the highest. Moreover, Shannon's diversity index showed that variations in bacterial composition was observed among the different microcosms in all periods. These results suggest that the removal of the indigenous microbes in the soil and the oxygen availability during decomposition influenced the changes in the bacterial composition. Moreover, the changes in the bacterial communities suggest that various bacterial species may have played a role during decomposition. Firmicutes, Proteobacteria, and Actinobacteria were the predominant bacterial phyla in the microcosms. This is consistent with other studies that have reported similar findings regardless of the type of carcass [ 5 , 23 ]. Interestingly, Firmicutes were found to increase in abundance in all microcosms as the decomposition process advanced over time, particularly at days 30 and 60 of decomposition. Several studies also reported the replacement of Proteobacteria by Firmicutes as the dominant phylum during the later stages of decay in swine models [ 3 , 23 ]. Firmicutes are known to be actively involved in the degradation of large macromolecules such as proteins, complex fats, and polycarbohydrates into their constituent building blocks [ 24 ]. Additionally, members of Firmicutes are facultative anaerobes or anaerobes, which can thrive in environments with limited oxygen availability and can compete over other bacteria that are less adapted to low oxygen environments. Furthermore, Firmicutes are often among the first groups of bacteria to colonize and initiate the decomposition process in organic matter. Their ability to quickly establish a presence and initiate degradation is advantageous in resource-rich environments such as carcasses, where there is an abundant supply of organic matter. Thus, Firmicutes tend to be more abundant during decomposition processes. The taxa belonging to Proteobacteria are often linked to meat spoilage and have been detected on the skin of slaughtered animals [ 23 ]. Additionally, Proteobacteria are commonly found in soil and play a significant role in the decomposition of fats and carbohydrates [ 25 ]. The genera Clostridium , Bacillus , and Lactobacillus were the most prevalent core microbes identified from all the swine burial microcosms in the study. The detection of the core microbiota from the microcosms suggests that these bacteria are associated with carcass decomposition. These genera were predominantly present in decomposing carcasses [ 7 , 25 ]. This study indicated notable variations in the genera during decomposition. During the initial day, Lactobacillus was found to be more abundant in SA and SAn microcosms than in UA and UAn microcosms. The dominance of these bacteria was due to being part of the gut microflora of the animal [ 26 ]. Lactobacillus spp. are known to be involved in the breakdown of lipids and complex carbohydrates in animal carcasses [ 27 ]. On day 5 of decomposition, the abundance of Enterococcus increased, whereas that of Lactobacillus decreased in all microcosms. Similarly, Li et al [ 28 ] reported that during the early stage of decomposition, gas accumulation caused bloating and rupture of the carcass, leading to a shift from internal to external conditions. This shift resulted in a decrease in anaerobe bacteria like Lactobacillus , while the facultative anaerobe Enterococcus took advantage of the changed conditions and thrived. In addition, Iancu et al [ 29 ] also reported an increase in the abundance of E. faecalis , whereas Hauther et al [ 30 ] reported a decrease in the abundance of members of Lactobacillus . E. faecalis is commonly found in human and animal gastrointestinal tracts and can ferment glucose and catabolize carbohydrates, diamino acids, and glycerol [ 29 ]. Our findings showed a notable increase in the abundance of Bacillus towards the end of the incubation period in all microcosms. Bacillus spp. are microorganisms associated with adipocyte decomposition and capable of denitrification [ 25 ]. Furthermore, they are known to produce a wide range of non-peptide and peptide antimicrobial compounds that effectively inhibit the growth of other bacteria [ 31 ]. The increased abundance of Bacillus towards the later stage of decomposition may be attributed to the synergistic or antagonistic interactions between Bacillus and other bacteria, which led to the alteration of the microbial community structure. A high abundance of B. paralicheniformis was identified in all microcosms, particularly in microcosms under aerobic conditions, suggesting that this bacterium may positively be associated with swine carcass decomposition. The bacterial community in the microcosms was dominated by Clostridium , particularly in UA, UAn, and SAn microcosms. Clostridium spp. are part of the normal gut microflora and are anaerobic organisms, but several species may survive in the presence of a small amount of oxygen [ 32 ]; therefore, these factors were likely the reason for this high abundance in these microcosms. Members of Clostridium spp. are known to play a crucial role in biomass breakdown, as they synthesize a wide variety of extracellular enzymes that aid in the degradation of various compounds, such as carbohydrates, lipids, amino acids, alcohols, and purines [ 33 ]. Additionally, several studies have highlighted the significant role of Clostridium spp. in carcass decomposition, as they can make up to 20% of the postmortem microbiome and possess proteolytic ability, fast growth rate, and anaerobic capabilities, making them well-suited for decomposing carcasses [ 34 ]. Similarly, our findings revealed an abundance of approximately 20% for this genus. Among these species, C. saudiense and C. sporogenes were identified in the microcosms. The abundance of C. sporogenes was substantially higher on days 5, 10, and 60 in the SAn and UAn microcosms, whereas C. saudiense was more abundant on day 0 and decreased in abundance as decomposition progressed in the SA and SAn microcosms. It has been reported that C. sporogenes was one of the most abundant species during decomposition [ 28 ]. The microbiota associated with carcass decomposition demonstrated diverse functional pathways. This diversity reflects the potential roles of microbes as decomposers. We detected expected increases in the expression of genes related to carbohydrate and amino acid metabolism. The up-regulation of carbohydrate and amino acid metabolism suggests that nutritional utilization plays a crucial role in determining which species become dominant [ 35 ]. Furthermore, Firmicutes have been reported to ferment amino acids and peptides into propionate and butyrate, which can contribute to the production of odor. Moreover, the up-regulation of carbohydrate metabolism is associated with increases in concentrations of hydrogen, carbon dioxide, hydrogen sulfide, and methane during decomposition [ 36 ]. On day 5, we observed an upregulation in the expression of genes related to carbohydrate metabolism and amino acid metabolism, which subsequently decreased on day 10. These findings indicate that the degradation of amino acids and carbohydrates within the carcasses decreased, likely due to the release of nutrient-rich fluids into the surrounding environment [ 37 ]. Several studies have focused on the quantification and identification of bacterial species associated with decomposition. Identifying the changes in bacterial community is significant for further understanding the decomposition microbiome. It is also important to note that laboratory microcosm experiments are just one tool for investigating the complex processes of decomposition in soil, and their results may not always be directly applicable to natural settings. Nonetheless, such experiments can provide valuable insights into the underlying changes in the microbial community during decomposition and help inform our understanding of the ecological and environmental impacts of animal carcass disposal. Overall, our findings provide microbiome information on carcasses decomposed in soil with or without microbes under different conditions of oxygen availability. The results of the present study are beneficial for estimating the microbes associated with the decomposition of swine carcasses. However, quantitative differences must be expected as each carcass has its unique microbiome composition. CONCLUSION This study evaluated the changes in the bacterial communities of different microcosms of decomposing swine carcasses over a 60 day period. Our findings demonstrated the composition of the bacterial communities was significantly influenced by factors such as soil sterilization and oxygen availability. In particular, Chao1 and Shannon diversity indices were significantly higher in UA and UAn compared to SA and SAn at the start of the experiment; however, diversity indices decreased over time. Variations in the bacterial taxonomic composition between UA and UAn to SA and SAn microcosms were observed throughout the decomposition period, suggesting that the removal of indigenous microbes in the soil and oxygen availability during decomposition influenced the shifts in the microbial composition. In addition, we detected predicted functional genes associated with the decomposition of carcasses. These findings provide valuable insights into the underlying changes in the diversity, structure, and composition of bacterial communities in swine carcasses decomposed under different conditions in vitro . Despite these findings, the association and functional role of these bacterial species on carcass decomposition is limited; thus, further research is necessary in this field. SUPPLEMENTARY MATERIAL Supplementary file is available from: https://doi.org/10.5713/ab.23.0140 Supplementary Figure S1 . Illustration of the incubation boxes for aerobic and anaerobic conditions. Supplementary Table S1 . Taxonomic classification of bacterial genera present only in UA, SA, UAn, and SAn samples Supplementary Table S2 . Taxonomic classification of shared bacterial genera present between UA, SA, UAn, and SAn samples Supplementary Table S3 . Taxonomic classification of shared bacterial genera present in UA, SA, UAn, and SAn samples

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271185/

Lung Infections

A review of pulmonary infections of all types with diagnostic and morphological features. Diagnostic Tools and Strategies The histories of pathology and microbiology are intertwined. 6 Pathologists should add their diagnostic techniques to those of microbiology for the best diagnostic yield ( Table 7.2 ). 7 Unfortunately the diagnostic work-up and reporting of findings in anatomic pathology and microbiology typically run along nonintersecting paths, often without one group knowing (or acknowledging) the findings of the other. An interdisciplinary approach based on mutual understanding and communication is the ideal scenario for clinical management. 8 Our concept of an integrated morphologic and microbiologic approach is presented schematically in Fig. 7.1 and with greater detail for specific situations in which bacterial ( Fig. 7.2 ), mycobacterial ( Fig. 7.3 ), fungal ( Fig. 7.4 ), or viral ( Fig. 7.5 ) pathogens are suspected. Specific species diagnoses are typically not possible from most pathology specimens, and attempts at pure morphologic diagnosis can be misleading. Pathologic findings should always be correlated with microbiologic findings. Accordingly, foresight is required on the part of the intraoperative pathologist in obtaining and properly handling tissues for culture. 9 The pathologic report should correlate the relevant microbiologic findings. Table 7.2 Diagnostic Tools of the Microbiologist Table 7.2 Activity Objective Pre-/intra-/postoperative consultation Information exchange and strategies Direct visualization (smears and imprints) Rapid detection Culture Identification of genus and species; susceptibility studies Antigen detection Rapid identification Serologic testing Specific antibody response Molecular techniques Sensitive and specific detection/identification Report Traditional versus interpretive format Figure 7.1 Schematic for the work-up of a respiratory specimen for suspected infection. BAL , Bronchoalveolar lavage; FNA , fine-needle aspiration; FS , frozen section; HC , histochemistry; H&E , hematoxylin and eosin (stain); IHC , immunohistochemistry studies; ISH , in situ hybridization; PCR , polymerase chain reaction (assay). Figure 7.1 Figure 7.2 Integrated morphologic and microbiologic approach to laboratory diagnosis of bacterial infection. BAL , Bronchoalveolar lavage; BAP , blood agar plate; BCYE , buffered charcoal yeast extract; CBAP , chocolate blood agar plate; MAC , MacConkey agar; RT , room temperature. Figure 7.2 Figure 7.3 Integrated morphologic and microbiologic approach to the laboratory diagnosis of mycobacterial infection. Bac-Tec , BD BACTEC Instrumented Mycobaterial Growth Systems; BAL , bronchoalveolar lavage; ESP , ESP Culture System; HPLC , high-performance liquid chromatography; MB/Bact Alert , Biomerieux Bact/alert 3D; PCR , polymerase chain reaction (assay); TMA , transcription-mediated amplification. Figure 7.3 Figure 7.4 Integrated morphologic and microbiologic approach to laboratory diagnosis of fungal infection. BAL , Bronchoalveolar lavage; BHI , brain-heart infusion; DFA , direct immunofluorescence assay; RT , room temperature; SDA , Sabouraud dextrose agar. Figure 7.4 Figure 7.5 Integrated morphologic and microbiologic approach to laboratory diagnosis of viral infection. BAL , Bronchoalveolar lavage; EIA , enzyme immunoassay; IF , immunofluorescence; PCR , polymerase chain reaction assay. Figure 7.5 Knowledge of the Clinical Setting Identification of the patient's risk factors and immune status is important because these parameters typically influence the spectrum of histopathologic changes, the types of etiologic agents, and number of organisms. 10 , 11 , 12 , 13 , 14 , 15 The degree of immunosuppression can influence the burden of organisms making the etiologic organisms more difficult to demonstrate histopathologically. For example, organisms are less often found in lung tissues from patients with normal or near-normal immunity. In this setting, cultures, serologic studies, and epidemiologic data must be relied on to provide the diagnosis. 16 Contrast the tedious search for rare acid-fast organisms in reactivation tuberculosis granulomas with Mycobacterium avium infection in acquired immunodeficiency syndrome (AIDS) patients. In the AIDS patient, M. avium infection typically manifests poorly formed granulomas or simply histiocytic infiltrates, with an overabundance of organisms identified by tissue acid-fast stains. Similarly, Pneumocystis organisms may be easily identified in patients with AIDS, but when immunosuppression is less severe (such as that produced by corticosteroids therapy for arthritis), the organism is rare. The relationship between the level of immunity, burden of organisms, and patterns of disease is illustrated for cryptococcosis in Fig. 7.6 . Figure 7.6 Cryptococcosis: Correlation of pathologic patterns with immunity level and organism burden. With cryptococcal pneumonia in patients with normal or near-normal immunity, granuloma formation with few organisms is characteristic. In immunocompromised patients, typical findings include histiocytic infiltrates or mucoid pneumonia with little or no inflammatory reaction and many organisms. Figure 7.6 (Data from Mark EJ. Case records of the Massachusetts General Hospital. N Engl J Med . 2002;347:518–524.) In the immunocompromised patient, there is always a broader differential diagnosis. 17 In addition to infection, other disorders come into consideration, such as pulmonary involvement by preexisting disease, drug-induced and treatment-related injury, noninfectious interstitial pneumonias, malignancy, and new pulmonary diseases unrelated to the patient's immunocompromised state, such as aspiration, heart failure, and pulmonary embolism. When immunosuppression is intentional, as in transplant recipients, unique additional challenges come into play, such as transplant rejection, graft-versus-host disease, and Epstein–Barr virus (EBV)–associated lymphoproliferative disorders. Immunosuppressed persons are at risk for multiple simultaneous infections, so when one organism is found, a careful search for others is always warranted ( Fig. 7.7 ). Figure 7.7 Co-infection with dual pulmonary pathogens. (A) Spherule of Coccidioides (S) and Mycobacterium avium complex acid-fast bacilli (Ziehl–Neelsen/Hematoxylin and eosin stains). (B) Toxoplasma pseudocysts (T) and cytomegalovirus-infected alveolar lining cell (arrow) . (C) Clusters of Pneumocystis cysts (P) in the midst of H. capsulatum yeast cells (h) (Grocott methenamine silver stain). Figure 7.7 A number of well-characterized genetic disorders of immunity and cellular function are known to predispose affected persons to lung infection. 18 , 19 , 20 , 21 Cystic fibrosis bears special recognition in this context because it is associated with reproducible patterns of lung disease and susceptibility to a wide spectrum of infectious organisms. This genetic disease of autosomal recessive inheritance involves mutation of the CFTR gene, which affects the ability of epithelial cells to effectively transport chloride and water across cell membranes. As a result, many organs, including the lungs, develop excessively viscous mucous secretions that cannot be cleared from the airways effectively. In the lung, retention of such secretions leads to progressive and widespread bronchiectasis with airway obstruction, which in turn paves the way for recurrent infection ( Fig. 7.8 ). Bacterial organisms commonly isolated include Pseudomonasaeruginosa (both mucoid and nonmucoid strains), Haemophilus influenzae , Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Burkholderia cepacia complex, Stenotrophomonas maltophilia , and Achromobacter xylosoxidans . 22 Polymicrobial infections are not uncommon, and some of these pathogens, especially certain subspecies within the B. cepacia complex, are linked to an adverse prognosis. 23 Cystic fibrosis is also a risk factor for nontuberculous mycobacterial infection and allergic bronchopulmonary fungal disease, and the condition is potentially exacerbated by superimposed viral infections. 24 , 25 , 26 , 27 Figure 7.8 Changes of cystic fibrosis in the lung. (A) Explant from a 13-year-old patient. (B) Advanced disease at autopsy. Figure 7.8 Pattern Recognition Knowledge of the radiologic pattern of infectious lung disease in a given patient often helps to narrow the scope of the differential diagnosis. 28 , 29 Patterns of lung infection seen on high-resolution computed tomography (HRCT) are typically dominated by increased attenuation (opacity). Such opacities may occur as one or more localized densities (nodule, mass, or infiltrate), as ground-glass opacities (attenuation that allows underlying lung structures to be visible), or consolidation (attenuation that overshadows underlying structures). 30 Review of the chest imaging studies and the pace of the disease (acute, subacute, and chronic) can be very helpful in arriving at a clinically relevant diagnosis. ( Fig. 7.9 ). Fortunately the recognized histopathologic patterns of lung infection are fairly limited (airway disease, acute lung injury, cellular infiltrates, alveolar filling, and nodules), and these typically correlate with a particular group of organisms ( Table 7.3 ). Figure 7.9 Miliary pattern of tuberculosis. (A) Chest film, closeup view of miliary infiltrate. (B) Gross cut surface of pulmonary parenchyma with miliary nodules. (C) Histopathologic features of miliary necrotizing granulomas. Figure 7.9 Table 7.3 Histopathologic Patterns and Most Agents of Pulmonary Infection Table 7.3 Pattern Most Common Agent(s) Airway disease — Bronchitis/bronchiolitis Virus; bacteria; Mycoplasma Bronchiectasis Bacteria; mycobacteria Acute exudative pneumonia — Purulent (neutrophilic) Bacteria Lobular (bronchopneumonia Bacteria Confluent (lobar pneumonia) Bacteria With granules Agents of botryomycosis ( Staphylococcus aureus ), actinomycosis ( Actinomyces israelii ) Eosinophilic Parasites Foamy alveolar cast Pneumocystis Acute diffuse/localized alveolar damage Virus; polymicrobial Chronic pneumonia — Fibroinflammatory Bacteria Organizing diffuse/localized alveolar damage Virus Eosinophilic Parasite Histiocytic Mycobacteria Interstitial pneumonia — Perivascular lymphoid Virus; atypical agents Eosinophilic Parasite Granulomatous Mycobacteria Nodules — Large — Necrotizing Fungi; mycobacteria Granulomatous Fungi; mycobacteria Fibrocaseous Fungi; mycobacteria Calcified Fungi; mycobacteria Miliary — Necrotizing Viral; mycobacteria; fungi Granulomatous Fungi Cavities and cysts Fungi; mycobacteria Intravascular/infarct Fungi Spindle cell pseudotumor Mycobacteria Minimal ("id") reaction Polymicrobial Useful Tissue Stains in Lung Infection Some pathologists have an aversion to the use of special stains for identifying organisms in tissue sections based on less than optimal specificity and sensitivity and the technical difficulty of performing some of these (especially silver impregnation methods, such as the Dieterle, Steiner, and Warthin-Starry stains). Nevertheless, several tissue section staining techniques are quite useful in detecting bacteria, mycobacteria, and fungi in tissue sections. A list of these is presented in Box 7.2 . These stains should be applied as part of an algorithmic strategy for acute lung injury, especially in the immunocompromised patient. 31 For example, when bacteria are being sought, some pathologists would prefer to begin with the tissue Gram stain (e.g., Brown and Hopps, Brown and Brenn; Figs. 7.10 and 7.11 ), but silver impregnation techniques (e.g., Warthin-Starry) are actually more sensitive and a good starting point for approaching a suspected bacterial infection. By coating the bacteria with metallic silver, the bacterial silhouettes are enhanced ( Fig. 7.12 ) and become more visible. 31 Other stains (e.g., Giemsa) will sometimes detect bacteria that do not stain well with more conventional stains ( Fig. 7.13 ). The Grocott methenamine silver (GMS) stain ( Fig. 7.14 ) is the best stain for most fungi in tissue; it also stains Actinomycetes , Nocardia , Pneumocystis (cysts), free-living soil amebae, algal cells, the spores of certain microsporidia, and the cytoplasmic inclusions of cytomegalovirus (CMV). 7 Box 7.2 Useful Tissue Stains in Lung Infection Gram stain Brown and Brenn Brown and Hopps Silver stains Warthin-Starry Steiner Dieterle Fungal stains Grocott methenamine silver (GMS) Periodic acid–Schiff reagent (PAS) Mycobacterial stains Ziehl–Neelsen (heat) Kinyon (cold) Auramine O (fluorochrome) Fite-Faraco (peanut or mineral oil) Other tissue stains Giemsa; Diff-Quik Mucicarmine Modified trichrome (Weber) Fontana–Masson Chemofluorescent (optical brighteners) Immunofluorescent antibodies Immunohistochemical Alt-text: Box 7.2 Figure 7.10 Gram-negative bacilli (Escherichia coli) in alveolar exudate (Brown and Hopps stain). Figure 7.10 Figure 7.11 (A) Gram-positive cocci in clusters: Staphylococcus aureus . (B) Gram-positive cocci in pairs/chains: 1: Streptococcus pneumoniae . 2: Streptococcus pyogenes . (C) Gram-negative diplococci: Neisseria meningitidis , Neisseria gonorrhoeae , Moraxella catarrhalis. *(D) Short gram-positive bacilli/coccobacilli: Corynebacterium jeikeium , Listeria monocytogenes . (E) Filamentous gram-positive bacilli: Nocardia spp., Actinomyces spp., Rhodococcus equi , Bartonella henselae . †(F) Gram-negative coccobacilli: Haemophilus influenzae , Acinetobacter baumannii . (G) Large gram-negative bacilli: Klebsiella pneumoniae , Escherichia coli , Serratia marcescens , Salmonella typhi , Yersinia pestis , Proteus mirabilis , Proteus vulgaris , Enterobacter spp., Salmonella spp., Yersinia enterocolitica . (H) Faintly staining gram-negative bacilli: Legionella spp, Francisella tularensis , Brucella spp, Bordetella spp. (I) Long slender gram-negative bacilli: Pseudomonas aeruginosa , Burkholderia pseudomallei , Burkholderia cepacia . Note: *Technically Moraxella catarrhalis has been placed in a bacillary genus, although this organism does have coccal morphology and responds as a coccus in the penicillin test. †Sometimes filamentous. Figure 7.11 (Bacterial Gram stain montage courtesy Drs. A.E. McCullough, S. Stewart, and L. Burdeaux, Mayo Clinic Hospital Microbiology Laboratory, Scottsdale, Arizona; From Tomashefksi JF, et al. Dail & Hammer's Pulmonary Pathology , 3rd ed. 2008:246. with permission of Springer.) Figure 7.12 Black (silver-coated) bacilli (Legionella pneumophila) in alveolar exudate (Dieterle stain). Figure 7.12 Figure 7.13 Bacillary organisms in alveolar exudates (Giemsa stain). Figure 7.13 Figure 7.14 Angioinvasive Aspergillus species (Grocott methenamine silver stain). Figure 7.14 (Courtesy Dr. Francis Chandler, Augusta, Georgia.) Most mycobacteria stain well with the Ziehl–Neelsen procedure ( Fig. 7.15 ), but the auramine-rhodamine fluorescent procedure is superior in terms of sensitivity ( Fig. 7.16 ). Nocardia organisms, Legionella micdadei , and Rhodococcus equi are weakly or partially acid-fast, and the use of modified acid-fast stains such the Fite-Faraco technique is more satisfactory for the identification of these organisms. Some mycobacterial species, such as M. avium complex (MAC), are also periodic acid–Schiff reagent (PAS)-positive, GMS-positive, and weakly gram-positive. Figure 7.15 Acid-fast bacilli: Mycobacterium tuberculosis (Ziehl–Neelsen stain). Figure 7.15 Figure 7.16 Fluorescent bacillary organisms: Mycobacterium tuberculosis . (A) Tissue section with two bacilli. Note beaded character in closeup view (inset) . (Auramine-rhodamine stain.) (B) Low-power view. Figure 7.16 Finally, for the identification of most protozoa and helminths, as well as viral inclusions, a good-quality hematoxylin and eosin (H&E)–stained section suffices; in fact, a well-prepared H&E section alone is diagnostic for many infectious diseases. This stain can often detect and even distinguish between bacterial cocci and bacilli when the burden of organisms is high ( Fig. 7.17 ). Figure 7.17 Streptococci in necrotizing pneumonia. Figure 7.17 Immunologic and Molecular Techniques The application of ancillary studies—such as immunohistochemistry, in situ hybridization ( Fig. 7.18 ), 32 or nucleic acid amplification technology—can provide a specific etiologic diagnosis in certain cases. These techniques have the best chance of diagnosing infections caused by fastidious species that are difficult or impossible to culture from fresh samples; they are also useful in situations where only formalin-fixed, paraffin-embedded tissues are available. Immunohistochemical reagents for microbiologic detection are becoming increasingly available and provide added power to determining specific diagnoses on formalin-fixed paraffin-embedded tissue ( Fig. 7.19 ). 33 Although these techniques provide the diagnostic equivalence of culture confirmation, they are not without limitations and diagnostic pitfalls. The polymerase chain reaction (PCR) method first introduced in the 1980s has undergone a number of modifications. Non-PCR DNA amplification methods and methods based not on the amplification of the DNA target per se but on amplification of the signal or probe have also been introduced. 34 Among the more recently available technologies is the rapid-cycle real-time PCR assay, representing an especially powerful advance in that it is significantly more sensitive than culture. The adaptation of various amplification methods to real-time and multiplex formats enables laboratories to detect a wide range of respiratory pathogens. Furthermore the transition from traditional and analyte-specific methods to more global technologies such as PCR arrays, liquid bead arrays, microarrays, and high-throughput DNA sequencing is under way. Over time, these methods will find a place in laboratories of all sizes and dramatically impact the speed and accuracy of microbiologic testing practice for all types of microorganisms. 35 , 36 , 37 , 38 , 39 Figure 7.18 Blastomyces dermatitidis . In situ hybridization. Figure 7.18 (Courtesy Ricardo Lloyd, MD, Rochester, Minnesota.) Figure 7.19 Herpes simplex virus necrotizing pneumonitis (immunohistochemical stain). Figure 7.19 Limiting Factors in Diagnosis Needless to say, the diagnostic tools employed by both pathologists and microbiologists have their limitations in terms of sensitivity and specificity. 7 Some common tools are listed in Box 7.3 . Culture alone cannot distinguish contamination from colonization, or in the case of viruses, asymptomatic shedding from true infection. Molecular tests may require specialized, often costly equipment and are susceptible to false-positive and false-negative results. 37 If a surgical biopsy is available, correlation of the histopathologic features can help assign an etiologic role to an agent recovered in culture or help establish if the microbiologically discovered organism has caused any microscopically visible lesion. The host inflammatory pattern and morphologic features of an organism can be characteristic for certain types of infections, but often the organism's morphology alone is not sufficient for a diagnosis at the genus or species level. Furthermore, the classic histopathologic findings for a given infection may be incomplete or lacking, making specific morphologic diagnosis possible for relatively few organisms. For example, the etiologic diagnosis is straightforward when large spherules with endospores characteristic of Coccidioides species are present, when the small budding yeasts of Histoplasma capsulatum are seen, or when yeasts with the large mucoid capsules of Cryptococcus neoformans are identified. However, atypical forms of these organisms can be confusing. 40 Similarly, hyphal morphology is helpful when it is characteristic of a specific genus or group, but the many look-alikes ( Fig. 7.20 ) require separation by searching for subtle differences under high magnification (or oil immersion) or by relying on special techniques and culture. 41 Box 7.3 Limitations of Diagnostic Tools Morphology Histopathologic examination: Inflammatory changes nonspecific, atypical, or absent; organisms not visualized or nonspecific morphology (e.g., " Aspergillus -like"); unexpected or unfamiliar site Special stains, immunohistochemical/molecular techniques: Sensitivity and specificity issues; misinterpretation (e.g., aberrant forms, artifacts, nonmicrobial mimics); limited reagents, false-negative and false-positive results Cytopathologic analysis: Limitations similar to those with histopathologic examination Microbiology Direct visualization: Sensitivity and specificity Culture/identification: Normal flora versus pathogens; colonization or asymptomatic shedding versus invasion; difficult, dangerous, or slow to grow; treated; fixed, contaminated tissue; too small or nonrepresentative sample Serologic studies: Single sample; no early response or lack of response; nondiagnostic for highly prevalent/persistent microbe; cross reaction; acute versus chronic; false-positive result on IgM tests Alt-text: Box 7.3 Figure 7.20 Coccidioides immitis demonstrating biphasic features versus those of other organisms. Culture grew C. immitis and Fusarium species. (A) Spherules and mycelia; (B) mycelia; (C) ruptured spherules with endospores (Grocott methenamine silver stain). Figure 7.20 Certain viruses may have characteristic inclusions in tissue, but there are notable pitfalls. For example, the eosinophilic intranuclear inclusions of adenovirus may resemble the early inclusions in herpes simplex virus (HSV) or CMV, especially when the typical smudged cellular forms of adenovirus are absent. Also, simulators of viral cytopathic effect (CPE), such as macronucleoli, optically clear nuclei, and intranuclear cytoplasmic invaginations, can occur in a number of conditions and need to be recognized ( Fig. 7.21 ). Figure 7.21 Macronucleolus mimicking a viral inclusion in an alveolar lining cell. Figure 7.21 Pseudomicrobe artifacts also have been recognized on routine and special stains for the identification of bacteria and fungi. Such potential artifacts include fragmented reticulin fibers, pigments, calcium deposits, Hamazaki-Wesenberg (yellow-brown) yeast-like bodies ( Fig. 7.22 ), pollen grains, and even lymphoglandular bodies. 42 For all of these reasons, the pathologist must maintain a high threshold for diagnosing organisms on morphologic grounds. If any question remains, it is best to repeat special stains liberally on deeper levels or in different tissue blocks. Figure 7.22 Yellow-brown Hamazaki-Wesenberg bodies (A) Hematoxylin and eosin; (B) Grocott methenamine silver stain. Figure 7.22 Role of Cytopathologic Examination in Diagnosis of Lung Infection A wide variety of infectious diseases of the lung—including bacterial, mycobacterial, fungal, viral, and parasitic—can be diagnosed through exfoliative or fine-needle aspiration cytologic techniques. 43 , 44 , 45 , 46 Fine-needle aspiration is an especially powerful tool compared with the exfoliative cytology study of respiratory secretions: sputum, bronchial washings/brushings, and bronchoalveolar lavage (BAL) fluid. The usefulness of exfoliative cytology examination is often limited owing to the difficulty of distinguishing colonizing/contaminant organisms in the airways from true pathogens. Nonetheless both diagnostic techniques are complementary and have been used in recent years to evaluate pneumonias and pulmonary nodules in both immunocompetent and immunocompromised patients. Mass-like infiltrates are often the target of aspiration biopsy needles when suspicion or exclusion of an infectious process ranks high in the differential diagnosis. Besides the morphologic features of the microorganism, important cytologic clues to the diagnosis include the accompanying cellular response and the presence and character of any necrotic debris, as outlined in Table 7.4 . Although nonspecific, such features can suggest certain possibilities to the cytopathologist and assist the microbiology laboratory in triaging the specimen. 47 To this end, the presence of a cytopathologist, microscope, and staining setup during the aspiration process can be useful. The cytopathologist can correlate the clinical setting, radiologic features, and clues from the gross character of the aspirate (color, consistency, odor, and so on), thereby assisting in narrowing the diagnostic possibilities and avoiding false-positive and false-negative diagnoses. 48 Also, immediate evaluation of smears by rapid stain procedures allows the cytopathologist to either make or suggest a specific diagnosis, as with the preparation and evaluation of a frozen section during intraoperative consultation. Smears can be prepared for special stains, needle rinses can be performed for culture and other ancillary studies, and additional aspirations may be encouraged for these purposes. 49 Special stains for bacteria, mycobacteria, and fungi should be used whenever the character of the aspirate and the clinical setting (e.g., compromised immune status) indicate that such studies may be useful. Table 7.4 Fine-Needle Aspiration Patterns of Pulmonary Infectious Diseases Table 7.4 Pattern Possible Etiologic Agent(s) Acute purulent inflammation/ abscess Bacteria, fungi Granuloma pattern (epithelioid cells with or without necrosis): Caseous/necrotizing; suppurative epithelial mixed Mycobacteria, bacteria, parasites, fungi Foamy alveolar cast pattern Pneumocystis jirovecii Histiocytic Mycobacteria, bacteria, fungi Chronic inflammation (lymphocyte and plasma cell) Virus, other, agent not otherwise specified Null ("id") reaction Virus, any, other Some interventionists prefer to provide only a needle core biopsy in lieu of an aspirate for a variety of reasons. These two techniques can be viewed as complementary; whereas needle core biopsies work well for neoplasms and many granulomas, the aspirate is often superior for diagnosing many types of infections, especially bacterial abscesses. Sometimes a rapid and specific etiologic diagnosis is possible at the bedside, based on the microscopic features of the organism itself. However, when the organism is not readily apparent or its features are inconclusive, the microbiology laboratory can be invaluable for its role in isolation and identification. 49 BAL, typically performed in the evaluation of infection in an immunocompromised host, provides a standard panel of microbiology results, which should always be correlated with the cytology findings. 50 , 51 Summary The successful treatment of pulmonary infections depends on accurate identification of the pathogen involved. In turn, this requires collecting the best specimens, transporting them to the anatomic and microbiology sections of the laboratory under optimal conditions, and processing them with techniques appropriate for the spectrum of possible etiologic disorders. An interdisciplinary approach enhances this process. It is best that pathologists, clinicians, and microbiologists communicate frequently and recognize the strengths and weaknesses of their respective disciplines. Joint strategies can be developed for the approach to certain types of suspected infections, helping to foster the development of laboratory foresight in surgical colleagues and medical consultants. It is good practice to look up the microbiologic and culture results in interpreting the biopsy. Communication and consideration of the histologic and microbiologic methods of diagnosis should be symbiotic. An example of such collaboration is presented in Box 7.4 . Box 7.4 Work-up of Pulmonary Infections Pre/Intraoperative Consultation Inquiry regarding History Risk factors; immune status Radiographic pattern Advise regarding How and what to collect What cultures and tests to order Devices, media, and containers for obtaining and transporting specimens Fixatives for morphologic study Written Protocol Handling tissue for cultures Special stains and ancillary tests Logistics Requisition—designed to communicate Morphologic Examination Inflammatory pattern Persistence and repeat studies Oil immersion studies if necessary Strict criteria for positive Consider multiple pathogens Report Presumptive versus definitive diagnosis; correlate with results of culture, other studies Comment Clinicopathologic-microbiologic correlation Differential diagnosis Ancillary tests Suggestions for further work-up Alt-text: Box 7.4 Knowledge of the Clinical Setting Identification of the patient's risk factors and immune status is important because these parameters typically influence the spectrum of histopathologic changes, the types of etiologic agents, and number of organisms. 10 , 11 , 12 , 13 , 14 , 15 The degree of immunosuppression can influence the burden of organisms making the etiologic organisms more difficult to demonstrate histopathologically. For example, organisms are less often found in lung tissues from patients with normal or near-normal immunity. In this setting, cultures, serologic studies, and epidemiologic data must be relied on to provide the diagnosis. 16 Contrast the tedious search for rare acid-fast organisms in reactivation tuberculosis granulomas with Mycobacterium avium infection in acquired immunodeficiency syndrome (AIDS) patients. In the AIDS patient, M. avium infection typically manifests poorly formed granulomas or simply histiocytic infiltrates, with an overabundance of organisms identified by tissue acid-fast stains. Similarly, Pneumocystis organisms may be easily identified in patients with AIDS, but when immunosuppression is less severe (such as that produced by corticosteroids therapy for arthritis), the organism is rare. The relationship between the level of immunity, burden of organisms, and patterns of disease is illustrated for cryptococcosis in Fig. 7.6 . Figure 7.6 Cryptococcosis: Correlation of pathologic patterns with immunity level and organism burden. With cryptococcal pneumonia in patients with normal or near-normal immunity, granuloma formation with few organisms is characteristic. In immunocompromised patients, typical findings include histiocytic infiltrates or mucoid pneumonia with little or no inflammatory reaction and many organisms. Figure 7.6 (Data from Mark EJ. Case records of the Massachusetts General Hospital. N Engl J Med . 2002;347:518–524.) In the immunocompromised patient, there is always a broader differential diagnosis. 17 In addition to infection, other disorders come into consideration, such as pulmonary involvement by preexisting disease, drug-induced and treatment-related injury, noninfectious interstitial pneumonias, malignancy, and new pulmonary diseases unrelated to the patient's immunocompromised state, such as aspiration, heart failure, and pulmonary embolism. When immunosuppression is intentional, as in transplant recipients, unique additional challenges come into play, such as transplant rejection, graft-versus-host disease, and Epstein–Barr virus (EBV)–associated lymphoproliferative disorders. Immunosuppressed persons are at risk for multiple simultaneous infections, so when one organism is found, a careful search for others is always warranted ( Fig. 7.7 ). Figure 7.7 Co-infection with dual pulmonary pathogens. (A) Spherule of Coccidioides (S) and Mycobacterium avium complex acid-fast bacilli (Ziehl–Neelsen/Hematoxylin and eosin stains). (B) Toxoplasma pseudocysts (T) and cytomegalovirus-infected alveolar lining cell (arrow) . (C) Clusters of Pneumocystis cysts (P) in the midst of H. capsulatum yeast cells (h) (Grocott methenamine silver stain). Figure 7.7 A number of well-characterized genetic disorders of immunity and cellular function are known to predispose affected persons to lung infection. 18 , 19 , 20 , 21 Cystic fibrosis bears special recognition in this context because it is associated with reproducible patterns of lung disease and susceptibility to a wide spectrum of infectious organisms. This genetic disease of autosomal recessive inheritance involves mutation of the CFTR gene, which affects the ability of epithelial cells to effectively transport chloride and water across cell membranes. As a result, many organs, including the lungs, develop excessively viscous mucous secretions that cannot be cleared from the airways effectively. In the lung, retention of such secretions leads to progressive and widespread bronchiectasis with airway obstruction, which in turn paves the way for recurrent infection ( Fig. 7.8 ). Bacterial organisms commonly isolated include Pseudomonasaeruginosa (both mucoid and nonmucoid strains), Haemophilus influenzae , Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Burkholderia cepacia complex, Stenotrophomonas maltophilia , and Achromobacter xylosoxidans . 22 Polymicrobial infections are not uncommon, and some of these pathogens, especially certain subspecies within the B. cepacia complex, are linked to an adverse prognosis. 23 Cystic fibrosis is also a risk factor for nontuberculous mycobacterial infection and allergic bronchopulmonary fungal disease, and the condition is potentially exacerbated by superimposed viral infections. 24 , 25 , 26 , 27 Figure 7.8 Changes of cystic fibrosis in the lung. (A) Explant from a 13-year-old patient. (B) Advanced disease at autopsy. Figure 7.8 Pattern Recognition Knowledge of the radiologic pattern of infectious lung disease in a given patient often helps to narrow the scope of the differential diagnosis. 28 , 29 Patterns of lung infection seen on high-resolution computed tomography (HRCT) are typically dominated by increased attenuation (opacity). Such opacities may occur as one or more localized densities (nodule, mass, or infiltrate), as ground-glass opacities (attenuation that allows underlying lung structures to be visible), or consolidation (attenuation that overshadows underlying structures). 30 Review of the chest imaging studies and the pace of the disease (acute, subacute, and chronic) can be very helpful in arriving at a clinically relevant diagnosis. ( Fig. 7.9 ). Fortunately the recognized histopathologic patterns of lung infection are fairly limited (airway disease, acute lung injury, cellular infiltrates, alveolar filling, and nodules), and these typically correlate with a particular group of organisms ( Table 7.3 ). Figure 7.9 Miliary pattern of tuberculosis. (A) Chest film, closeup view of miliary infiltrate. (B) Gross cut surface of pulmonary parenchyma with miliary nodules. (C) Histopathologic features of miliary necrotizing granulomas. Figure 7.9 Table 7.3 Histopathologic Patterns and Most Agents of Pulmonary Infection Table 7.3 Pattern Most Common Agent(s) Airway disease — Bronchitis/bronchiolitis Virus; bacteria; Mycoplasma Bronchiectasis Bacteria; mycobacteria Acute exudative pneumonia — Purulent (neutrophilic) Bacteria Lobular (bronchopneumonia Bacteria Confluent (lobar pneumonia) Bacteria With granules Agents of botryomycosis ( Staphylococcus aureus ), actinomycosis ( Actinomyces israelii ) Eosinophilic Parasites Foamy alveolar cast Pneumocystis Acute diffuse/localized alveolar damage Virus; polymicrobial Chronic pneumonia — Fibroinflammatory Bacteria Organizing diffuse/localized alveolar damage Virus Eosinophilic Parasite Histiocytic Mycobacteria Interstitial pneumonia — Perivascular lymphoid Virus; atypical agents Eosinophilic Parasite Granulomatous Mycobacteria Nodules — Large — Necrotizing Fungi; mycobacteria Granulomatous Fungi; mycobacteria Fibrocaseous Fungi; mycobacteria Calcified Fungi; mycobacteria Miliary — Necrotizing Viral; mycobacteria; fungi Granulomatous Fungi Cavities and cysts Fungi; mycobacteria Intravascular/infarct Fungi Spindle cell pseudotumor Mycobacteria Minimal ("id") reaction Polymicrobial Useful Tissue Stains in Lung Infection Some pathologists have an aversion to the use of special stains for identifying organisms in tissue sections based on less than optimal specificity and sensitivity and the technical difficulty of performing some of these (especially silver impregnation methods, such as the Dieterle, Steiner, and Warthin-Starry stains). Nevertheless, several tissue section staining techniques are quite useful in detecting bacteria, mycobacteria, and fungi in tissue sections. A list of these is presented in Box 7.2 . These stains should be applied as part of an algorithmic strategy for acute lung injury, especially in the immunocompromised patient. 31 For example, when bacteria are being sought, some pathologists would prefer to begin with the tissue Gram stain (e.g., Brown and Hopps, Brown and Brenn; Figs. 7.10 and 7.11 ), but silver impregnation techniques (e.g., Warthin-Starry) are actually more sensitive and a good starting point for approaching a suspected bacterial infection. By coating the bacteria with metallic silver, the bacterial silhouettes are enhanced ( Fig. 7.12 ) and become more visible. 31 Other stains (e.g., Giemsa) will sometimes detect bacteria that do not stain well with more conventional stains ( Fig. 7.13 ). The Grocott methenamine silver (GMS) stain ( Fig. 7.14 ) is the best stain for most fungi in tissue; it also stains Actinomycetes , Nocardia , Pneumocystis (cysts), free-living soil amebae, algal cells, the spores of certain microsporidia, and the cytoplasmic inclusions of cytomegalovirus (CMV). 7 Box 7.2 Useful Tissue Stains in Lung Infection Gram stain Brown and Brenn Brown and Hopps Silver stains Warthin-Starry Steiner Dieterle Fungal stains Grocott methenamine silver (GMS) Periodic acid–Schiff reagent (PAS) Mycobacterial stains Ziehl–Neelsen (heat) Kinyon (cold) Auramine O (fluorochrome) Fite-Faraco (peanut or mineral oil) Other tissue stains Giemsa; Diff-Quik Mucicarmine Modified trichrome (Weber) Fontana–Masson Chemofluorescent (optical brighteners) Immunofluorescent antibodies Immunohistochemical Alt-text: Box 7.2 Figure 7.10 Gram-negative bacilli (Escherichia coli) in alveolar exudate (Brown and Hopps stain). Figure 7.10 Figure 7.11 (A) Gram-positive cocci in clusters: Staphylococcus aureus . (B) Gram-positive cocci in pairs/chains: 1: Streptococcus pneumoniae . 2: Streptococcus pyogenes . (C) Gram-negative diplococci: Neisseria meningitidis , Neisseria gonorrhoeae , Moraxella catarrhalis. *(D) Short gram-positive bacilli/coccobacilli: Corynebacterium jeikeium , Listeria monocytogenes . (E) Filamentous gram-positive bacilli: Nocardia spp., Actinomyces spp., Rhodococcus equi , Bartonella henselae . †(F) Gram-negative coccobacilli: Haemophilus influenzae , Acinetobacter baumannii . (G) Large gram-negative bacilli: Klebsiella pneumoniae , Escherichia coli , Serratia marcescens , Salmonella typhi , Yersinia pestis , Proteus mirabilis , Proteus vulgaris , Enterobacter spp., Salmonella spp., Yersinia enterocolitica . (H) Faintly staining gram-negative bacilli: Legionella spp, Francisella tularensis , Brucella spp, Bordetella spp. (I) Long slender gram-negative bacilli: Pseudomonas aeruginosa , Burkholderia pseudomallei , Burkholderia cepacia . Note: *Technically Moraxella catarrhalis has been placed in a bacillary genus, although this organism does have coccal morphology and responds as a coccus in the penicillin test. †Sometimes filamentous. Figure 7.11 (Bacterial Gram stain montage courtesy Drs. A.E. McCullough, S. Stewart, and L. Burdeaux, Mayo Clinic Hospital Microbiology Laboratory, Scottsdale, Arizona; From Tomashefksi JF, et al. Dail & Hammer's Pulmonary Pathology , 3rd ed. 2008:246. with permission of Springer.) Figure 7.12 Black (silver-coated) bacilli (Legionella pneumophila) in alveolar exudate (Dieterle stain). Figure 7.12 Figure 7.13 Bacillary organisms in alveolar exudates (Giemsa stain). Figure 7.13 Figure 7.14 Angioinvasive Aspergillus species (Grocott methenamine silver stain). Figure 7.14 (Courtesy Dr. Francis Chandler, Augusta, Georgia.) Most mycobacteria stain well with the Ziehl–Neelsen procedure ( Fig. 7.15 ), but the auramine-rhodamine fluorescent procedure is superior in terms of sensitivity ( Fig. 7.16 ). Nocardia organisms, Legionella micdadei , and Rhodococcus equi are weakly or partially acid-fast, and the use of modified acid-fast stains such the Fite-Faraco technique is more satisfactory for the identification of these organisms. Some mycobacterial species, such as M. avium complex (MAC), are also periodic acid–Schiff reagent (PAS)-positive, GMS-positive, and weakly gram-positive. Figure 7.15 Acid-fast bacilli: Mycobacterium tuberculosis (Ziehl–Neelsen stain). Figure 7.15 Figure 7.16 Fluorescent bacillary organisms: Mycobacterium tuberculosis . (A) Tissue section with two bacilli. Note beaded character in closeup view (inset) . (Auramine-rhodamine stain.) (B) Low-power view. Figure 7.16 Finally, for the identification of most protozoa and helminths, as well as viral inclusions, a good-quality hematoxylin and eosin (H&E)–stained section suffices; in fact, a well-prepared H&E section alone is diagnostic for many infectious diseases. This stain can often detect and even distinguish between bacterial cocci and bacilli when the burden of organisms is high ( Fig. 7.17 ). Figure 7.17 Streptococci in necrotizing pneumonia. Figure 7.17 Immunologic and Molecular Techniques The application of ancillary studies—such as immunohistochemistry, in situ hybridization ( Fig. 7.18 ), 32 or nucleic acid amplification technology—can provide a specific etiologic diagnosis in certain cases. These techniques have the best chance of diagnosing infections caused by fastidious species that are difficult or impossible to culture from fresh samples; they are also useful in situations where only formalin-fixed, paraffin-embedded tissues are available. Immunohistochemical reagents for microbiologic detection are becoming increasingly available and provide added power to determining specific diagnoses on formalin-fixed paraffin-embedded tissue ( Fig. 7.19 ). 33 Although these techniques provide the diagnostic equivalence of culture confirmation, they are not without limitations and diagnostic pitfalls. The polymerase chain reaction (PCR) method first introduced in the 1980s has undergone a number of modifications. Non-PCR DNA amplification methods and methods based not on the amplification of the DNA target per se but on amplification of the signal or probe have also been introduced. 34 Among the more recently available technologies is the rapid-cycle real-time PCR assay, representing an especially powerful advance in that it is significantly more sensitive than culture. The adaptation of various amplification methods to real-time and multiplex formats enables laboratories to detect a wide range of respiratory pathogens. Furthermore the transition from traditional and analyte-specific methods to more global technologies such as PCR arrays, liquid bead arrays, microarrays, and high-throughput DNA sequencing is under way. Over time, these methods will find a place in laboratories of all sizes and dramatically impact the speed and accuracy of microbiologic testing practice for all types of microorganisms. 35 , 36 , 37 , 38 , 39 Figure 7.18 Blastomyces dermatitidis . In situ hybridization. Figure 7.18 (Courtesy Ricardo Lloyd, MD, Rochester, Minnesota.) Figure 7.19 Herpes simplex virus necrotizing pneumonitis (immunohistochemical stain). Figure 7.19 Limiting Factors in Diagnosis Needless to say, the diagnostic tools employed by both pathologists and microbiologists have their limitations in terms of sensitivity and specificity. 7 Some common tools are listed in Box 7.3 . Culture alone cannot distinguish contamination from colonization, or in the case of viruses, asymptomatic shedding from true infection. Molecular tests may require specialized, often costly equipment and are susceptible to false-positive and false-negative results. 37 If a surgical biopsy is available, correlation of the histopathologic features can help assign an etiologic role to an agent recovered in culture or help establish if the microbiologically discovered organism has caused any microscopically visible lesion. The host inflammatory pattern and morphologic features of an organism can be characteristic for certain types of infections, but often the organism's morphology alone is not sufficient for a diagnosis at the genus or species level. Furthermore, the classic histopathologic findings for a given infection may be incomplete or lacking, making specific morphologic diagnosis possible for relatively few organisms. For example, the etiologic diagnosis is straightforward when large spherules with endospores characteristic of Coccidioides species are present, when the small budding yeasts of Histoplasma capsulatum are seen, or when yeasts with the large mucoid capsules of Cryptococcus neoformans are identified. However, atypical forms of these organisms can be confusing. 40 Similarly, hyphal morphology is helpful when it is characteristic of a specific genus or group, but the many look-alikes ( Fig. 7.20 ) require separation by searching for subtle differences under high magnification (or oil immersion) or by relying on special techniques and culture. 41 Box 7.3 Limitations of Diagnostic Tools Morphology Histopathologic examination: Inflammatory changes nonspecific, atypical, or absent; organisms not visualized or nonspecific morphology (e.g., " Aspergillus -like"); unexpected or unfamiliar site Special stains, immunohistochemical/molecular techniques: Sensitivity and specificity issues; misinterpretation (e.g., aberrant forms, artifacts, nonmicrobial mimics); limited reagents, false-negative and false-positive results Cytopathologic analysis: Limitations similar to those with histopathologic examination Microbiology Direct visualization: Sensitivity and specificity Culture/identification: Normal flora versus pathogens; colonization or asymptomatic shedding versus invasion; difficult, dangerous, or slow to grow; treated; fixed, contaminated tissue; too small or nonrepresentative sample Serologic studies: Single sample; no early response or lack of response; nondiagnostic for highly prevalent/persistent microbe; cross reaction; acute versus chronic; false-positive result on IgM tests Alt-text: Box 7.3 Figure 7.20 Coccidioides immitis demonstrating biphasic features versus those of other organisms. Culture grew C. immitis and Fusarium species. (A) Spherules and mycelia; (B) mycelia; (C) ruptured spherules with endospores (Grocott methenamine silver stain). Figure 7.20 Certain viruses may have characteristic inclusions in tissue, but there are notable pitfalls. For example, the eosinophilic intranuclear inclusions of adenovirus may resemble the early inclusions in herpes simplex virus (HSV) or CMV, especially when the typical smudged cellular forms of adenovirus are absent. Also, simulators of viral cytopathic effect (CPE), such as macronucleoli, optically clear nuclei, and intranuclear cytoplasmic invaginations, can occur in a number of conditions and need to be recognized ( Fig. 7.21 ). Figure 7.21 Macronucleolus mimicking a viral inclusion in an alveolar lining cell. Figure 7.21 Pseudomicrobe artifacts also have been recognized on routine and special stains for the identification of bacteria and fungi. Such potential artifacts include fragmented reticulin fibers, pigments, calcium deposits, Hamazaki-Wesenberg (yellow-brown) yeast-like bodies ( Fig. 7.22 ), pollen grains, and even lymphoglandular bodies. 42 For all of these reasons, the pathologist must maintain a high threshold for diagnosing organisms on morphologic grounds. If any question remains, it is best to repeat special stains liberally on deeper levels or in different tissue blocks. Figure 7.22 Yellow-brown Hamazaki-Wesenberg bodies (A) Hematoxylin and eosin; (B) Grocott methenamine silver stain. Figure 7.22 Morphology Histopathologic examination: Inflammatory changes nonspecific, atypical, or absent; organisms not visualized or nonspecific morphology (e.g., " Aspergillus -like"); unexpected or unfamiliar site Special stains, immunohistochemical/molecular techniques: Sensitivity and specificity issues; misinterpretation (e.g., aberrant forms, artifacts, nonmicrobial mimics); limited reagents, false-negative and false-positive results Cytopathologic analysis: Limitations similar to those with histopathologic examination Microbiology Direct visualization: Sensitivity and specificity Culture/identification: Normal flora versus pathogens; colonization or asymptomatic shedding versus invasion; difficult, dangerous, or slow to grow; treated; fixed, contaminated tissue; too small or nonrepresentative sample Serologic studies: Single sample; no early response or lack of response; nondiagnostic for highly prevalent/persistent microbe; cross reaction; acute versus chronic; false-positive result on IgM tests Role of Cytopathologic Examination in Diagnosis of Lung Infection A wide variety of infectious diseases of the lung—including bacterial, mycobacterial, fungal, viral, and parasitic—can be diagnosed through exfoliative or fine-needle aspiration cytologic techniques. 43 , 44 , 45 , 46 Fine-needle aspiration is an especially powerful tool compared with the exfoliative cytology study of respiratory secretions: sputum, bronchial washings/brushings, and bronchoalveolar lavage (BAL) fluid. The usefulness of exfoliative cytology examination is often limited owing to the difficulty of distinguishing colonizing/contaminant organisms in the airways from true pathogens. Nonetheless both diagnostic techniques are complementary and have been used in recent years to evaluate pneumonias and pulmonary nodules in both immunocompetent and immunocompromised patients. Mass-like infiltrates are often the target of aspiration biopsy needles when suspicion or exclusion of an infectious process ranks high in the differential diagnosis. Besides the morphologic features of the microorganism, important cytologic clues to the diagnosis include the accompanying cellular response and the presence and character of any necrotic debris, as outlined in Table 7.4 . Although nonspecific, such features can suggest certain possibilities to the cytopathologist and assist the microbiology laboratory in triaging the specimen. 47 To this end, the presence of a cytopathologist, microscope, and staining setup during the aspiration process can be useful. The cytopathologist can correlate the clinical setting, radiologic features, and clues from the gross character of the aspirate (color, consistency, odor, and so on), thereby assisting in narrowing the diagnostic possibilities and avoiding false-positive and false-negative diagnoses. 48 Also, immediate evaluation of smears by rapid stain procedures allows the cytopathologist to either make or suggest a specific diagnosis, as with the preparation and evaluation of a frozen section during intraoperative consultation. Smears can be prepared for special stains, needle rinses can be performed for culture and other ancillary studies, and additional aspirations may be encouraged for these purposes. 49 Special stains for bacteria, mycobacteria, and fungi should be used whenever the character of the aspirate and the clinical setting (e.g., compromised immune status) indicate that such studies may be useful. Table 7.4 Fine-Needle Aspiration Patterns of Pulmonary Infectious Diseases Table 7.4 Pattern Possible Etiologic Agent(s) Acute purulent inflammation/ abscess Bacteria, fungi Granuloma pattern (epithelioid cells with or without necrosis): Caseous/necrotizing; suppurative epithelial mixed Mycobacteria, bacteria, parasites, fungi Foamy alveolar cast pattern Pneumocystis jirovecii Histiocytic Mycobacteria, bacteria, fungi Chronic inflammation (lymphocyte and plasma cell) Virus, other, agent not otherwise specified Null ("id") reaction Virus, any, other Some interventionists prefer to provide only a needle core biopsy in lieu of an aspirate for a variety of reasons. These two techniques can be viewed as complementary; whereas needle core biopsies work well for neoplasms and many granulomas, the aspirate is often superior for diagnosing many types of infections, especially bacterial abscesses. Sometimes a rapid and specific etiologic diagnosis is possible at the bedside, based on the microscopic features of the organism itself. However, when the organism is not readily apparent or its features are inconclusive, the microbiology laboratory can be invaluable for its role in isolation and identification. 49 BAL, typically performed in the evaluation of infection in an immunocompromised host, provides a standard panel of microbiology results, which should always be correlated with the cytology findings. 50 , 51 Summary The successful treatment of pulmonary infections depends on accurate identification of the pathogen involved. In turn, this requires collecting the best specimens, transporting them to the anatomic and microbiology sections of the laboratory under optimal conditions, and processing them with techniques appropriate for the spectrum of possible etiologic disorders. An interdisciplinary approach enhances this process. It is best that pathologists, clinicians, and microbiologists communicate frequently and recognize the strengths and weaknesses of their respective disciplines. Joint strategies can be developed for the approach to certain types of suspected infections, helping to foster the development of laboratory foresight in surgical colleagues and medical consultants. It is good practice to look up the microbiologic and culture results in interpreting the biopsy. Communication and consideration of the histologic and microbiologic methods of diagnosis should be symbiotic. An example of such collaboration is presented in Box 7.4 . Box 7.4 Work-up of Pulmonary Infections Pre/Intraoperative Consultation Inquiry regarding History Risk factors; immune status Radiographic pattern Advise regarding How and what to collect What cultures and tests to order Devices, media, and containers for obtaining and transporting specimens Fixatives for morphologic study Written Protocol Handling tissue for cultures Special stains and ancillary tests Logistics Requisition—designed to communicate Morphologic Examination Inflammatory pattern Persistence and repeat studies Oil immersion studies if necessary Strict criteria for positive Consider multiple pathogens Report Presumptive versus definitive diagnosis; correlate with results of culture, other studies Comment Clinicopathologic-microbiologic correlation Differential diagnosis Ancillary tests Suggestions for further work-up Alt-text: Box 7.4 Pre/Intraoperative Consultation Inquiry regarding History Risk factors; immune status Radiographic pattern Advise regarding How and what to collect What cultures and tests to order Devices, media, and containers for obtaining and transporting specimens Fixatives for morphologic study Written Protocol Handling tissue for cultures Special stains and ancillary tests Logistics Requisition—designed to communicate Morphologic Examination Inflammatory pattern Persistence and repeat studies Oil immersion studies if necessary Strict criteria for positive Consider multiple pathogens Report Presumptive versus definitive diagnosis; correlate with results of culture, other studies Comment Clinicopathologic-microbiologic correlation Differential diagnosis Ancillary tests Suggestions for further work-up Bacterial Pneumonias The surgical pathologist rarely receives biopsy specimens from patients with community-acquired or nosocomial pneumonias. Most of these infections are suspected clinically by symptoms and physical and radiologic findings; some are confirmed immediately by Gram stains (or later by culture) performed on respiratory secretions in the microbiology laboratory. Serologic studies sometimes prove to be diagnostic. Even when conventional microbiologic approaches are applied, however, approximately 50% of bacterial pneumonias remain undiagnosed. 52 , 53 , 54 Patients with mild disease are often not tested and treated empirically with antibiotic regimens following established guidelines. By contrast, patients with severe disease, whether immunocompromised or not, often become candidates for invasive procedures. Etiologic Agents Bacterial pneumonia may be classified according to various parameters including pathogenesis, epidemiology, anatomic pattern, clinical course, and organism type ( Box 7.5 ). 55 Using bacterial type as a starting point allows the pathologist to correlate anatomic and histopathologic patterns of lung injury with categories of etiologic agents. Box 7.5 Classification of Bacterial Pneumonia Pathogenesis Primary Exogenous Endogenous Secondary Epidemiology Community-acquired Nosocomial Anatomic Type Lobular Lobar Clinical Course Acute Chronic Bacterial Type Pyogenic species Atypical agents Granule/filamentous group Alt-text: Box 7.5 The pyogenic bacteria most commonly associated with community-acquired pneumonias include S. pneumoniae , H. influenzae , and Moraxella catarrhalis . 54 Other pathogens such as Legionella species, Chlamydia pneumoniae , and Mycoplasma pneumoniae (often referred to as the atypical group ) are clinically important, but controversy exists with regard to the relative frequency of these organisms as etiologic agents. Although community-acquired pneumonia is considered to be fundamentally different in children and in adults, severe or complicated pneumonias in both of these age groups are of similar etiology. 56 The enteric gram-negative bacilli cause relatively few community-acquired pneumonias, whereas they account for most of the nosocomial pneumonias, along with Pseudomonas species, Acinetobacter species, S. aureus , and anaerobes. 57 , 58 Most nosocomial pneumonias result from aspiration of these bacterial species that colonize the oropharynx of hospitalized patients, and such pneumonias can be polymicrobial. Any of the bacterial organisms listed (including mixtures with fungi and viruses) can cause pneumonia in immunocompromised patients. 14 , 59 Ventilator-associated pneumonia is a special subset of nosocomial pneumonia and an important cause of morbidity and mortality in the intensive care unit. 60 , 61 , 62 The bacterial etiology in this setting is quite diverse and dependent on such factors as patient characteristics, underlying lung disease, and geographical location. 63 Most recently, an increase in skin and soft tissue staphylococcal infections due to methicillin-resistant strains has led to the recognition of these organisms as an important cause of both community-acquired and nosocomial pneumonia with attendant morbidity and mortality. 64 In rare nosocomial pneumonias, a number of unusual organisms, such as Salmonella , Rhodococcus , and Leptospira species, may be the etiologic agent. 65 , 66 The atypical pneumonia agents do not commonly produce lobar consolidation. Although this potentially implicates a wide variety of bacterial, viral, and protozoal pathogens, a selective list by convention includes M. pneumoniae , Legionella species, and C. pneumoniae as the three dominant nonzoonotic pathogens, and Coxiella burnetii (the agent of Q fever), Chlamydia psittaci (causing psittacosis in people), and Francisella tularensis (causing tularemia) as the three more common zoonotic pathogens. 67 , 68 The filamentous/granule group refers to those bacteria that form long, thin, branching filaments in tissues, such as Actinomyces (anaerobic actinomycetes) or Nocardia (aerobic actinomycetes). 69 Botryomycosis is caused by nonfilamentous bacteria, especially S. aureus , or gram-negative bacilli, such as P. aeruginosa and Escherichia coli , which form organized aggregates referred to as grains or granules . 70 Histopathology Bacterial lung injury patterns will vary in accordance with the virulence of the organism and the host response. These patterns are further modulated by therapeutic or immunologic factors. Although some of the patterns presented in Box 7.6 are characteristic, none are diagnostic. Overlap and mixed patterns occur. Box 7.6 Histopathologic Patterns in Bacterial Lung Injury Bronchitis/bronchiolitis Acute exudative pneumonia Lobular (bronchopneumonia) Confluent (lobar pneumonia) With granules Fibroinflammatory and/or organizing pneumonia Interstitial pneumonia Nodular/necrotizing lesions Miliary lesions Abscess Alt-text: Box 7.6 Acute Exudative Pneumonia Acute exudative pneumonia is most often caused by pyogenic bacteria, such as streptococci, which typically produce a neutrophil-rich intra-alveolar exudate (i.e., alveolar filling) with variable amounts of fibrin and red cells. Pathologists recognize this constellation of findings as acute lobular pneumonia ( Fig. 7.23 ), which usually correlates with patchy segmental infiltrates on the chest film (consolidation pattern on HRCT). 29 , 71 , 72 , 73 Figure 7.23 Alveoli filled with fibrinopurulent exudate with variable hemorrhage. Figure 7.23 With increasing organism virulence and disease severity, lobular exudates may become confluent (i.e., lobar pneumonia). In milder cases, the disease may be limited to the airways (bronchitis/bronchiolitis) with a mixed cellular infiltrate of mononuclear cells and neutrophils ( Fig. 7.24 ). One very common manifestation of such airway-limited infection has been designated as acute exacerbation of chronic obstructive pulmonary disease (COPD). A majority of these exacerbations are caused by particular bacteria (specifically H. influenzae , S. pneumoniae , and M. catarrhalis ) with approximately one third resulting from viral airway infections, typically resulting from rhinovirus, respiratory syncytial virus (RSV), and human metapneumovirus. 74 Figure 7.24 Bronchiolitis with intraluminal exudate. Figure 7.24 Nodular/Necrotizing Lesions Nodular inflammatory infiltrates with or without necrotizing features ( Fig. 7.25 ) are characteristic of infection by certain species, such as R. equi ( Fig. 7.26 ). 75 Necrotizing pneumonias may also be produced by pyogenic bacteria such as S. aureus , Streptococcus pyogenes , and the gram-negative bacilli— Klebsiella , Acinetobacter , Pseudomonas , and Burkholderia species. Figure 7.25 Nodular histiocytic infiltrate in rhodococcal pneumonia. Figure 7.25 Figure 7.26 Rhodococcus equi bacilli in macrophage. Figure 7.26 Miliary Lesions A subset of the nodular histopathologic pattern, miliary infection ( Fig. 7.27 ), strongly implies pneumonia secondary to the hematogenous spread of bacteria (septicemia). This pattern of infection can be seen with other organisms, such as Nocardia and the anaerobic Actinomycetes. In these settings, histopathologic examination may show a combination of both nodular disease and alveolar filling. Figure 7.27 Necrotizing pneumonia, miliary pattern. Figure 7.27 Aspiration Pneumonia and Lung Abscess There are multiple scenarios for aspiration pneumonia, including cases caused by chemical pneumonitis (so-called Mendelson syndrome), airway obstruction, exogenous lipoid pneumonia, chronic interstitial fibrosis, diffuse bronchiolar disease, bacterial pneumonia, and lung abscess. 76 , 77 Aspiration pneumonia refers specifically to the aspiration of bacteria in oropharyngeal secretions, with the bacterial species depending on whether the aspiration event occurs in the community or hospital setting. Recognition of food particles (so-called pulses) is important in diagnosis. These may or may not be invested by giant cells but are usually found in purulent exudate or granulomatous foci. In the organizing phase of the pneumonia, food particles may be found within polyps of organizing pneumonia in the alveolar ducts and alveoli. Lobular pneumonia, lipoid pneumonia, organizing pneumonia, and bronchiolitis, alone or in combination, may also be seen. 73 , 78 The pathogens in lung abscess ( Fig. 7.28 ) usually encompass a polymicrobial mixture of aerobic and anaerobic bacteria, 79 and formation of such abscesses most often is secondary to aspiration ( Fig. 7.29 ). Infections due to Actinomyces species ( Fig. 7.30 ) and Nocardia species may also manifest this pattern, as can those infections caused by certain pyogenic bacteria, such as S. aureus and the other organisms listed previously for necrotizing pneumonias. Granulomatous inflammation with foreign bodies may be present if aspiration is the cause ( Fig. 7.31 ). Figure 7.28 Lung abscess showing gross evidence of chronicity with fibrosis in surrounding parenchyma. Figure 7.28 Figure 7.29 (A) and (B) Lung abscess with polymicrobial bacterial population (Gram stain). Figure 7.29 Figure 7.30 Lung abscess with sulfur granule of actinomycosis in purulent exudate. Figure 7.30 Figure 7.31 Aspiration pneumonia. Giant cells surround vegetable matter (FB) in purulent exudates, organizing pneumonia (OP) , bronchiolitis (BR) , artery (A) . Figure 7.31 Chronic Bacterial Pneumonias Chronic bacterial infections ( Fig. 7.32 ) that are slow to resolve as a result of inappropriate initial therapy, involvement with certain microbial species, a noninfectious comorbid process, or an inadequate host response can produce a nonspecific fibroinflammatory pattern, with lymphoplasmacytic infiltrates, macrophages, or organization with polyps of immature fibroblasts in alveolar ducts and alveolar spaces. 80 , 81 , 82 , 83 If not resorbed, polyps of airspace organization may become polyps of intra-alveolar fibrosis, which sometimes ossify (dendriform ossification). Such scarring in chronic pneumonia is often associated with localized interlobular septal and pleural thickening ( Fig. 7.33 ), producing a jigsaw puzzle pattern of scarring best seen at scanning magnification. Figure 7.32 Chronic pneumonia. (A) Lymphoplasmacytic infiltrate. (B) Fascicles of fibroblasts in alveolar ducts and spaces. Figure 7.32 Figure 7.33 Chronic pneumonia with thickened interlobular septum. Figure 7.33 Diffuse alveolar damage is the histopathologic correlate of the acute respiratory distress syndrome (ARDS), and today lung infection is the leading cause of diffuse alveolar damage and ARDS in the United States. 84 Diffuse alveolar damage may coexist with any of the necroinflammatory patterns described earlier. The initial exudative phase of this ARDS is accompanied by hyaline membranes ( Fig. 7.34 ), the later organizing phase by airspace and interstitial fibroplasia. In clinical practice, diffuse alveolar damage accompanied by tissue necrosis is nearly always a manifestation of lung infection. Figure 7.34 Bacterial pneumonia with hyaline membranes (HM) at periphery. Figure 7.34 The atypical pneumonias include the well-described cases due to Legionella species and the less well-described cases caused by other organisms comprising the atypical group. Legionella infection typically results in an intensely neutrophilic acute fibrinopurulent lobular pneumonia ( Fig. 7.35A ). 3 , 5 , 71 Legionella bacilli can be identified in silver impregnation-stained sections ( Fig. 7.35B ) or recovered in culture, but newer diagnostic methods, such as real-time PCR and in situ hybridization ( Fig. 7.36 ), can also be applied when standard approaches fail. 85 The histopathologic patterns associated with the other members of the atypical group (i.e., Chlamydia , Mycoplasma ) are not well characterized, mainly because investigation of these pneumonias rarely includes biopsy. The few well-documented cases of Mycoplasma , Chlamydia , and Coxiella infections resemble viral bronchitis or bronchiolitis, with mixed inflammatory infiltrates in airway walls and in the adjacent interstitium ( Fig. 7.37 ). 86 , 87 Relative sparing of the peribronchiolar alveolar spaces has been described, although patchy organized fibrinous exudates are seen in some cases and complications may superimpose additional findings. Figure 7.35 (A) Legionnaire's disease with intraalveolar necroinflammatory exudates (N) and hemorrhage. (B) Enhanced silhouette of Legionella bacilli (LB) in alveolar exudate with silver impregnation (Dieterle stain). Figure 7.35 Figure 7.36 Legionnaire's disease. Detection of organisms by in situ DNA hybridization. Figure 7.36 (Courtesy R.V. Lloyd, MD, Rochester, Minnesota.) Figure 7.37 Mycoplasma pneumonia. Bronchiolitis with patchy infiltrates in peribronchial interstitium. Figure 7.37 The grains and granules formed by the Actinomycetes and bacteria of botryomycosis may have a uniform tinctorial hue on routine H&E–stained sections, but sometimes these bacterial aggregations display a distinctive body with a hematoxylinophilic core and an outer investment of eosinophilic material; formation of this array is referred to as the Splendore-Hoeppli phenomenon ( Fig. 7.38 ). Actinomycetes species tend to form similar-appearing granules, and both they and the bacteria of botryomycosis are typically found in the midst of purulent exudates. 69 , 88 , 89 , 90 Nocardia species may aggregate in colonies simulating granules, but with a much looser texture ( Fig. 7.39 ) and more monochromatic tinctorial properties. 91 Rarely, these colonies may be identical in appearance to the grains or granules of botryomycosis or actinomycosis in H&E sections. Figure 7.38 Botryomycosis granule with hematoxylinophilic core and eosinophilic investment known as the Splendore-Hoeppli effect . Figure 7.38 Figure 7.39 Loose-textured aggregate of Nocardia filamentous bacteria surrounded by neutrophils. Figure 7.39 Bacterial Agents of Bioterrorism The potential for use of microbial pathogens as agents of bioterrorism requires that clinicians be alert to this possibility when community-acquired pneumonias are found to be caused by these agents. In turn, pathologists must become familiar with the histopathologic features these agents can produce. 92 Respiratory disease caused by the inhalation of Bacillus anthracis , Yersinia pestis , and F. tularensis is especially pertinent in this context and is discussed next. 93 Bacillus anthracis In 1877, Robert Koch's conclusive demonstration that B. anthracis was the etiologic agent of anthrax revolutionized medicine by linking microbial cause and effect. 6 Inhalational anthrax causes a severe hemorrhagic mediastinitis. 94 , 95 , 96 , 97 , 98 This pathologic process in combination with the toxemia ( B. anthracis produces an exotoxin with three potent components—protective antigen, lethal factor, and edema factor) from the ensuing massive bacteremia severely compromises pulmonary function, leading to death in 40% or more of the cases. Pleural effusion may be present, but pneumonia generally is minor and secondary. In those patients in whom pulmonary parenchymal changes are found, the alveolar spaces contain a serosanguineous fluid with minimal fibrin deposits and some mononuclear cells but few if any neutrophils. 97 Large gram-positive bacilli (some may appear partially gram-negative) without spores pervade the alveolar septal vessels, with a few in the alveolar spaces. This distribution suggests hematogenous rather than airway acquisition. Hemorrhagic mediastinitis in a previously healthy adult is essentially pathognomonic for inhalational anthrax. The lymph node parenchyma generally is teeming with intact and fragmented gram-positive bacilli, which can be identified as B. anthracis by immunohistochemical studies. 96 , 97 Cultures of blood and pleural fluid, if available, are likely to yield the earliest positive diagnostic results. 98 Sputum studies are much less useful in this regard. Specific guidelines for pathology and microbiology specimens for anthrax diagnosis (as well as other potential agents of bioterrorism) are current and available on the Centers for Disease Control and Prevention (CDC) website. 99 Yersinia pestis Primary pneumonic plague follows inhalation of Y. pestis bacilli in a potential bioterrorism scenario. 100 , 101 The infection begins as bronchiolitis and alveolitis that progress to a lobular and eventual lobar consolidation. 102 The histopathologic features evolve over time, beginning with a serosanguineous intraalveolar fluid accumulation with variable fibrin deposits ( Fig. 7.40 ), progressing through a fibrinopurulent phase, and culminating in a necrotizing lesion. 103 The presence of myriad bacilli in the intraalveolar exudates with significantly fewer organisms in the interstitium (a characteristic of primary pneumonia) is one of several pulmonary and extrapulmonary features used to distinguish primary from secondary pneumonic plague. 104 These bacilli may be obvious in H&E-stained sections ( Fig. 7.41 ) but generally are better visualized with Giemsa rather than Gram stain. Immunohistochemical staining provides a rapid and specific diagnosis. 102 In contrast to inhalational anthrax, sputum Gram stain and culture are useful tests that are likely to yield a positive result at clinical presentation. Also, because sepsis is an integral component of the pneumonia, it is important to collect blood culture specimens. 105 Figure 7.40 Plague pneumonia, early phase. Edema, fibrin, and sparse inflammatory cells are evident. Figure 7.40 Figure 7.41 Yersinia pestis bacilli in alveolar space. Figure 7.41 Francisella tularensis Inhalation of F. tularensis bacilli following a bioterrorism aerosol release is generally expected to result in a slowly progressing pneumonia with a lower case-fatality rate than with either inhalational anthrax or plague. 104 , 106 Initially a hemorrhagic and ulcerative bronchiolitis is followed by a fibrinous lobular pneumonia with many macrophages but relatively few neutrophils ( Fig. 7.42 ). Necrosis then supervenes and evolves into a granulomatous reaction. The small, gram-negative coccobacillary organisms are difficult to identify in a tissue Gram stain, and the use of silvering techniques (e.g., Steiner, Dieterle, Warthin-Starry) is required to enhance their silhouette. 107 Specific fluorescent antibody testing for formalin-fixed tissue and immunohistochemical studies are available through public health laboratories. In the microbiology laboratory, Gram stain and culture of respiratory secretions are useful for diagnosis, but blood cultures are often negative. Antigen detection and molecular techniques, such as PCR amplification, can also identify F. tularensis . Serologic tests are available but probably would not provide timely information in an outbreak situation. 104 Figure 7.42 Tularemia. Fibrinous lobular pneumonia phase. Figure 7.42 Cytopathology The stereotypic cellular response to pyogenic bacteria is acute inflammation, characterized by variable numbers of neutrophils. Bacteria may be visualized in various stained preparations made from respiratory tract secretions and washings using the Papanicolaou and Diff-Quik methods. 45 The clinical significance of bacteria in such specimens may be limited owing to potential contamination by oral flora and the problem of distinguishing colonization from infection. However, when the upper respiratory tract can be bypassed by means of either transtracheal or transthoracic needle aspiration, the presence of bacteria becomes much more significant, especially when sheets of neutrophils or necroinflammatory debris are present ( Fig. 7.43A ), as would be the case with a typical lobar or lobular consolidation, lung abscess, or other complex pneumonia. 53 , 90 , 108 , 109 In this context, transthoracic needle aspiration can establish the etiologic diagnosis of community-acquired and nosocomial pneumonias in both children and adults when coupled with modern microbiologic methods. 49 , 58 , 110 , 111 Proponents consider it an underused technique the potential benefits of which, in experienced hands, outweigh the modest associated risks. Figure 7.43 (A) Purulent exudate of nodular pulmonary infiltrate in fine-needle aspirate (alcohol-fixed). (B) Streptococci ( viridans group) in cytoplasm of neutrophil seen in fine-needle aspirate (Diff-Quik preparation). Figure 7.43 Many types of bacilli and cocci can be seen within and around neutrophils on Diff-Quik–stained smears ( Fig. 7.43B ). A smear can also be prepared for Gram stain and the aspirate needle rinsed in nonbacteriostatic sterile saline or nutrient broths for culture. The size (length and width) and shape of organisms and the Gram reaction allow rough categorization of organisms into groups, such as enteric-type bacilli, pseudomonads, fusiform anaerobic-type bacilli, tiny coccobacillary types suggestive of the Haemophilus–Bacteroides group ( Fig. 7.44 ), or gram-positive cocci. 112 Branching filamentous forms suggest Actinomycetes or Nocardia species ( Fig. 7.45 ), with the latter distinguished by being partially acid-fast. 113 , 114 Figure 7.44 (A) Fusiform bacteria ( Fusobacterium organisms) in cytoplasm of neutrophil in fine-needle aspirate (Gram stain). (B) Coccobacilli (Haemophilus influenzae) in cytoplasm of leukocyte in fine-needle aspirate (Gram stain). Figure 7.44 Figure 7.45 Nocardia. Loose, feathery cluster of bacilli in purulent exudate seen in a fine-needle aspirate: alcohol-fixed, Hematoxylin and eosin stain (HE) ; Gram stain (Gram) ; Grocott methenamine silver stain (GMS) ; Ziehl–Neelsen stain (ZN) . Figure 7.45 Although most aspirated cavitary lung lesions with the abscess pattern are the result of bacterial infection, considerations in the differential diagnosis include necrotic neoplasm (particularly squamous cell carcinoma), granulomatosis with polyangiitis, and nonbacterial infections associated with suppurative granulomas such as those due to fungi and mycobacteria. Microbiology Microbiology techniques in current use for the laboratory diagnosis of bacterial pneumonia are summarized in Box 7.7 . 39 , 115 , 116 , 117 The traditional morphologic and functional approach to microbiologic diagnosis is gradually shifting to molecular methods, and diagnostic arrays of common respiratory pathogens are marketed by several vendors; they are adjusted for laboratory size, for individual random access testing, or for test batching in larger laboratories. Box 7.7 Laboratory Diagnosis of Bacterial Pneumonia Direct detection of organisms Gram stain; other stains of respiratory secretions and fluids Direct fluorescent antibody stain Histopathologic/cytopathologic examination Immunohistochemistry Antigen detection (with Legionella pneumophila [LP1] and Streptococcus pneumoniae ) Culture Conventional media for usual pyogenic bacteria Special media for fastidious or atypical agents Serologic testing Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.7 The work-up of respiratory secretions such as sputum in the microbiology laboratory may or may not be indicated based on the clinical and immunologic status of the patient. The value of microbiologic work-up for community-acquired pneumonias has been questioned for some time, and evolving guidelines from two specialty societies—the American Thoracic Society and the Infectious Disease Society of America—have lately coalesced. 118 , 119 , 120 , 121 Despite microbiologic testing, in a retrospective review of 2259 patients with radiographic evidence of pneumonia hospitalized from January 2010–June 2012 in selected US communities, no pathogen was detected in the majority of patients. 122 When a carefully collected specimen reveals one or two predominant bacterial morphotypes on a well-prepared Gram stain ( Fig. 7.46 ), especially in the presence of neutrophils and few or no squamous cells, a presumptive diagnosis can be offered and correlated with whatever grows on culture plates. 123 , 124 , 125 A mixed bacterial population is usually considered nondiagnostic, especially in the absence of inflammation or the presence of many benign oral squamous cells. Pneumonia in the hospitalized or immunocompromised patient requires an aggressive strategy to collect a good sputum sample for Gram stain and culture. If this attempt is unsatisfactory or the findings are nondiagnostic, then the use of invasive techniques beginning with fiberoptic bronchoscopy and BAL with protected catheters should be considered. 60 , 62 , 126 Anaerobic pulmonary infections, typically in the form of a lung abscess, can also be approached in this way or with transthoracic needle aspiration. 79 Figure 7.46 Sputum Gram stain. (A) Gram-positive diplococci (Streptococcus pneumoniae) with neutrophils, but no squamous cells. (B) Gram-positive diplococci (S. pneumoniae) and gram-negative coccobacilli (Haemophilus influenzae) . Figure 7.46 Gram staining of tissue sections from bronchoscopic or surgical biopsy specimens is notoriously insensitive and nonspecific. As with sputum, the presence of a predominant bacterial morphotype in a distinctive necroinflammatory background carries diagnostic weight, especially when correlated with available clinical and laboratory data. Because histology laboratories do not generally observe the same level of caution in reagent preparation and storage as microbiology laboratories, it is worth remembering that tissue sections are prone to false-positive results from in vitro bacterial contamination. In those cases where bacteria are visible on H&E-stained sections, the Gram stain can be helpful in confirming a presumptive etiology. For example, pairs and chains of gram-positive cocci in a necroinflammatory background suggest a streptococcal pneumonia, whereas numerous slender gram-negative bacilli investing and infiltrating blood vessels are characteristic of Pseudomonas pneumonia ( Fig. 7.47 ). Other types of gram-negative pneumonias ( Fig. 7.48 ) can also be confirmed with well-prepared Gram stains. 81 In the case of an abscess, a mixture of gram-positive cocci and gram-negative bacilli in tissue (illustrated earlier in Fig. 7.29 ) is a useful finding that is helpful in supporting a diagnosis of an anaerobic infection. Figure 7.47 (A) Pseudomonas aeruginosa bacilli investing interstitial vessels (Brown and Hopps stain). (B) The slender gram-negative bacilli are nicely demonstrated on Gram stain. Figure 7.47 Figure 7.48 Burkholderia cepacia bacilli (Brown and Hopps stain). Figure 7.48 When organisms are sparse, other stains such as Giemsa or silver impregnation may highlight the organisms in the exudates ( Fig. 7.49 ). The Gram stain is also useful for evaluating infections with granules and allows differentiation of the agents of botryomycosis (the gram-positive cocci or gram-negative bacilli) from the filamentous Actinomyces organisms ( Fig. 7.50 ). Figure 7.49 Bacterial tetrads in alveolar exudate (Giemsa stain). Figure 7.49 Figure 7.50 Botryomycosis. Cluster of gram-positive cocci (Staphylococcus aureus) invested by gram-negative–staining Splendore-Hoeppli material (Brown and Brenn stain). Figure 7.50 (Courtesy Dr. Francis Chandler, Augusta, Georgia.) Staining with methenamine silver is the best procedure for detecting Nocardia organisms. The modified Ziehl–Neelsen stain allows for differentiation of Nocardia (positive) from the anaerobic Actinomyces (negative). 114 Commercially available immunohistochemical reagents exist for relatively few bacterial species. Immunohistochemistry testing for the potential bioterrorist agents discussed in this chapter is available through the CDC in Atlanta, Georgia. It is expected that commercial reagents will become increasingly available for the common etiologic agents in the near future. 33 Culture media that will allow recovery of common bacterial species causing pneumonia from various types of respiratory samples (secretions, washings, brushings, aspirates, and tissues) include sheep blood agar, chocolate agar, and McConkey agar. These media also will support growth of B. anthracis and Y. pestis . Buffered charcoal yeast extract (BCYE) agar is the primary medium for Legionella species. Because Legionella organisms survive poorly in respiratory secretions, rapid transport and immediate plating is essential for recovery. BCYE is also a good all-purpose medium for growing other fastidious species, including F. tularensis . However, F. tularensis grows best in cysteine-enriched media. 127 In addition to respiratory samples, blood can be obtained for cultures in patients sick enough to lead to suspicion of bacteremia; pleural fluid culture can be used when effusions are present. Positive cultures of these normally sterile fluids circumvent the interpretive problems associated with bacterial growth in sputum samples. The Actinomycetes are best isolated from invasive specimens such as needle aspirates and transbronchial and lung biopsy specimens. The laboratory should be alerted to search for these agents because special consideration must be given to culture setup and incubation conditions. 89 The Actinomycetes responsible for actinomycosis require anaerobic media and atmosphere as well as prolonged incubation. Nocardia , an aerobic Actinomycete, grows well on most nonselective media but requires extended incubation. Determination of colonial morphology, Gram and acid-fast stains, and a few biochemical tests generally suffice to identify these organisms at the genus level. However, genotype rather than phenotype characteristics are required to identify newly emergent species. 128 In general, the laboratory diagnosis of pneumonia caused by most of the atypical agents is difficult because systems are not routinely available or are costly, cumbersome, or unsafe. For the atypical agents ( Mycoplasma , Chlamydia , and Coxiella species), serologic testing has been the method of choice for diagnosis. 67 , 129 Classic cold agglutinin and complement fixation tests for these agents have largely been replaced by enzyme immunoassay and microimmunofluorescence testing. 87 , 130 , 131 Serologic methods are also useful for the diagnosis of tularemia because of the difficulty in culturing the fastidious bacterium. Legionella pneumonia is a common form of severe pneumonia that is not readily diagnosed for a number of reasons, including the organism's fastidiousness. 132 In the microbiology laboratory, the direct fluorescent antibody test and culture on buffered BCYE agar have been the mainstays of diagnosis. Culture is considered the diagnostic gold standard but is only 60% sensitive. Serologic testing is available for most of the Legionella pneumophila serotypes, which account for 90% of the pneumonia cases; however, the need to collect paired sera weeks apart limits its usefulness in the acutely ill patient. Antigen detection in urine has become commercially available for both L. pneumophila and S. pneumoniae ; because the need to collect acute and convalescent sera is obviated, it has become a frequently used diagnostic test. 132 , 133 Its advantage lies in its potential to effect early treatment decisions through rapid diagnosis. Its disadvantage lies in the fact that it identifies only patients infected with L. pneumophila serogroup 1 (LP1), the most prevalent species and serotype, but none of the non-LP1 serotypes or cases due to other Legionella species. 134 , 135 , 136 The use of molecular diagnostic tools (in situ hybridization and nucleic acid amplification by PCR or other methods) to detect these agents has been reported. 85 , 136 , 137 PCR nested assays are replacing more and more of the above classical methods with sensitive, specific, and rapid diagnostic technique. Multiplex assay, to detect multiple agents in a single reaction, would seem to be an ideal pursuit for the laboratory diagnosis of the most common community-acquired pneumonias, including those due to the atypical pneumonia agents. 36 , 138 , 139 , 140 , 141 Differential Diagnosis The key morphologic and microbiologic features of the bacterial pneumonias are summarized in Table 7.5 . The presence of purulent exudates or significant numbers of neutrophils in biopsy or cytologic samples should always trigger a search for bacterial infection. Because lung biopsies are usually performed late in the clinical course after procedures have been performed and bacterial infections have been excluded and/or treated with antibiotics, neutrophilic exudates may not signify bacterial infection unless accompanied by necrosis, as in an abscess. Instead, consideration should be given to one of several noninfectious acute inflammatory diseases, with an immunologic basis, that can mimic bacterial infection. Some of these include granulomatosis with polyangiitis, Goodpasture syndrome, systemic lupus erythematosus, and microscopic polyangiitis, all conditions that can produce acute inflammation predominantly involving alveolar septal blood vessels ("capillaritis"). On occasion, capillaritis can result in airspace accumulation of neutrophils, further raising concern for bronchopneumonia. Centrally necrotic or cavitary neoplasms of various types may mimic abscesses grossly and microscopically, and exceptionally well-differentiated adenocarcinomas containing glands filled with detritus may mimic inflammatory and bacterial diseases. Suppurative granulomas can have a bacterial, mycobacterial, or fungal etiology. Even the miliary necroinflammatory lesion typical of bacterial infection can be produced by viruses, some fungi, and even protozoa (e.g., Toxoplasmagondii ). Aspiration, common in hospitalized patients, may manifest with an acute bronchopneumonia/bronchiolitis type of pattern, and it should also be in the differential diagnosis as a contributing or etiologic factor when considering most pneumonias; microscopic identification of foreign material is a definitive diagnostic clue, frequently gained only from histopathologic examination. 78 Table 7.5 Bacterial Pneumonias: Summary of Pathologic Findings Table 7.5 Assessment Component Findings Pyogenic Bacteria Surgical pathology Acute purulent inflammation with/without necrosis; organization; diffuse alveolar damage may be present Cytopathology Acute inflammation with/without visible bacteria on Diff-Quik–stained smear Microbiology Gram stain reactivity and morphology (visual detection requires heavy bacterial burden: 10 6 organisms/g of tissue); culture-sterile lung tissue on standard nonselective and selective media (blood, chocolate and MacConkey agars); anaerobic broth and agars for abscesses; urinary antigen for Streptococcus pneumoniae Atypical Pneumonia Agents Surgical pathology Legionella pneumonia: fibrinopurulent with bacilli visible in silver-stained (Dieterle; Warthin-Starry) sections DAD often present Chlamydia and Mycoplasma infection: polymorphous bronchiolar and interstitial infiltrate Cytopathology Acute inflammation with bacilli stained with silver or by immunofluorescence ( Legionella pneumonia) Microbiology DFA for L. pneumophila serotypes; culture on selective (BCYE) agar for Legionella ; urinary antigen for Legionella ; serologic testing and/or PCR assay for Mycoplasma and Chlamydia Filamentous Granule Group Surgical pathology Granules or loose filamentous aggregates in purulent exudate with abscess formation and poorly formed granuloma in some cases Cytopathology Filamentous tangles or aggregates or granules with neutrophils and/or necroinflammatory background Microbiology Gram-positive branching filaments: Nocardia (aerobic actinomycete) and Actinomyces (anaerobic actinomycete); Nocardia partially acid-fast and GMS-positive Gram-positive cocci or gram-negative bacilli (botryomycosis); culture on standard nonselective media and selective (BCYE) media; anaerobic culture broths and media for Actinomyces BCYE , Buffered charcoal yeast extract; DAD , diffuse alveolar damage; DFA , direct fluorescence assay; GMS , Grocott methenamine silver. Etiologic Agents Bacterial pneumonia may be classified according to various parameters including pathogenesis, epidemiology, anatomic pattern, clinical course, and organism type ( Box 7.5 ). 55 Using bacterial type as a starting point allows the pathologist to correlate anatomic and histopathologic patterns of lung injury with categories of etiologic agents. Box 7.5 Classification of Bacterial Pneumonia Pathogenesis Primary Exogenous Endogenous Secondary Epidemiology Community-acquired Nosocomial Anatomic Type Lobular Lobar Clinical Course Acute Chronic Bacterial Type Pyogenic species Atypical agents Granule/filamentous group Alt-text: Box 7.5 The pyogenic bacteria most commonly associated with community-acquired pneumonias include S. pneumoniae , H. influenzae , and Moraxella catarrhalis . 54 Other pathogens such as Legionella species, Chlamydia pneumoniae , and Mycoplasma pneumoniae (often referred to as the atypical group ) are clinically important, but controversy exists with regard to the relative frequency of these organisms as etiologic agents. Although community-acquired pneumonia is considered to be fundamentally different in children and in adults, severe or complicated pneumonias in both of these age groups are of similar etiology. 56 The enteric gram-negative bacilli cause relatively few community-acquired pneumonias, whereas they account for most of the nosocomial pneumonias, along with Pseudomonas species, Acinetobacter species, S. aureus , and anaerobes. 57 , 58 Most nosocomial pneumonias result from aspiration of these bacterial species that colonize the oropharynx of hospitalized patients, and such pneumonias can be polymicrobial. Any of the bacterial organisms listed (including mixtures with fungi and viruses) can cause pneumonia in immunocompromised patients. 14 , 59 Ventilator-associated pneumonia is a special subset of nosocomial pneumonia and an important cause of morbidity and mortality in the intensive care unit. 60 , 61 , 62 The bacterial etiology in this setting is quite diverse and dependent on such factors as patient characteristics, underlying lung disease, and geographical location. 63 Most recently, an increase in skin and soft tissue staphylococcal infections due to methicillin-resistant strains has led to the recognition of these organisms as an important cause of both community-acquired and nosocomial pneumonia with attendant morbidity and mortality. 64 In rare nosocomial pneumonias, a number of unusual organisms, such as Salmonella , Rhodococcus , and Leptospira species, may be the etiologic agent. 65 , 66 The atypical pneumonia agents do not commonly produce lobar consolidation. Although this potentially implicates a wide variety of bacterial, viral, and protozoal pathogens, a selective list by convention includes M. pneumoniae , Legionella species, and C. pneumoniae as the three dominant nonzoonotic pathogens, and Coxiella burnetii (the agent of Q fever), Chlamydia psittaci (causing psittacosis in people), and Francisella tularensis (causing tularemia) as the three more common zoonotic pathogens. 67 , 68 The filamentous/granule group refers to those bacteria that form long, thin, branching filaments in tissues, such as Actinomyces (anaerobic actinomycetes) or Nocardia (aerobic actinomycetes). 69 Botryomycosis is caused by nonfilamentous bacteria, especially S. aureus , or gram-negative bacilli, such as P. aeruginosa and Escherichia coli , which form organized aggregates referred to as grains or granules . 70 Pathogenesis Primary Exogenous Endogenous Secondary Epidemiology Community-acquired Nosocomial Anatomic Type Lobular Lobar Clinical Course Acute Chronic Bacterial Type Pyogenic species Atypical agents Granule/filamentous group Histopathology Bacterial lung injury patterns will vary in accordance with the virulence of the organism and the host response. These patterns are further modulated by therapeutic or immunologic factors. Although some of the patterns presented in Box 7.6 are characteristic, none are diagnostic. Overlap and mixed patterns occur. Box 7.6 Histopathologic Patterns in Bacterial Lung Injury Bronchitis/bronchiolitis Acute exudative pneumonia Lobular (bronchopneumonia) Confluent (lobar pneumonia) With granules Fibroinflammatory and/or organizing pneumonia Interstitial pneumonia Nodular/necrotizing lesions Miliary lesions Abscess Alt-text: Box 7.6 Acute Exudative Pneumonia Acute exudative pneumonia is most often caused by pyogenic bacteria, such as streptococci, which typically produce a neutrophil-rich intra-alveolar exudate (i.e., alveolar filling) with variable amounts of fibrin and red cells. Pathologists recognize this constellation of findings as acute lobular pneumonia ( Fig. 7.23 ), which usually correlates with patchy segmental infiltrates on the chest film (consolidation pattern on HRCT). 29 , 71 , 72 , 73 Figure 7.23 Alveoli filled with fibrinopurulent exudate with variable hemorrhage. Figure 7.23 With increasing organism virulence and disease severity, lobular exudates may become confluent (i.e., lobar pneumonia). In milder cases, the disease may be limited to the airways (bronchitis/bronchiolitis) with a mixed cellular infiltrate of mononuclear cells and neutrophils ( Fig. 7.24 ). One very common manifestation of such airway-limited infection has been designated as acute exacerbation of chronic obstructive pulmonary disease (COPD). A majority of these exacerbations are caused by particular bacteria (specifically H. influenzae , S. pneumoniae , and M. catarrhalis ) with approximately one third resulting from viral airway infections, typically resulting from rhinovirus, respiratory syncytial virus (RSV), and human metapneumovirus. 74 Figure 7.24 Bronchiolitis with intraluminal exudate. Figure 7.24 Nodular/Necrotizing Lesions Nodular inflammatory infiltrates with or without necrotizing features ( Fig. 7.25 ) are characteristic of infection by certain species, such as R. equi ( Fig. 7.26 ). 75 Necrotizing pneumonias may also be produced by pyogenic bacteria such as S. aureus , Streptococcus pyogenes , and the gram-negative bacilli— Klebsiella , Acinetobacter , Pseudomonas , and Burkholderia species. Figure 7.25 Nodular histiocytic infiltrate in rhodococcal pneumonia. Figure 7.25 Figure 7.26 Rhodococcus equi bacilli in macrophage. Figure 7.26 Miliary Lesions A subset of the nodular histopathologic pattern, miliary infection ( Fig. 7.27 ), strongly implies pneumonia secondary to the hematogenous spread of bacteria (septicemia). This pattern of infection can be seen with other organisms, such as Nocardia and the anaerobic Actinomycetes. In these settings, histopathologic examination may show a combination of both nodular disease and alveolar filling. Figure 7.27 Necrotizing pneumonia, miliary pattern. Figure 7.27 Aspiration Pneumonia and Lung Abscess There are multiple scenarios for aspiration pneumonia, including cases caused by chemical pneumonitis (so-called Mendelson syndrome), airway obstruction, exogenous lipoid pneumonia, chronic interstitial fibrosis, diffuse bronchiolar disease, bacterial pneumonia, and lung abscess. 76 , 77 Aspiration pneumonia refers specifically to the aspiration of bacteria in oropharyngeal secretions, with the bacterial species depending on whether the aspiration event occurs in the community or hospital setting. Recognition of food particles (so-called pulses) is important in diagnosis. These may or may not be invested by giant cells but are usually found in purulent exudate or granulomatous foci. In the organizing phase of the pneumonia, food particles may be found within polyps of organizing pneumonia in the alveolar ducts and alveoli. Lobular pneumonia, lipoid pneumonia, organizing pneumonia, and bronchiolitis, alone or in combination, may also be seen. 73 , 78 The pathogens in lung abscess ( Fig. 7.28 ) usually encompass a polymicrobial mixture of aerobic and anaerobic bacteria, 79 and formation of such abscesses most often is secondary to aspiration ( Fig. 7.29 ). Infections due to Actinomyces species ( Fig. 7.30 ) and Nocardia species may also manifest this pattern, as can those infections caused by certain pyogenic bacteria, such as S. aureus and the other organisms listed previously for necrotizing pneumonias. Granulomatous inflammation with foreign bodies may be present if aspiration is the cause ( Fig. 7.31 ). Figure 7.28 Lung abscess showing gross evidence of chronicity with fibrosis in surrounding parenchyma. Figure 7.28 Figure 7.29 (A) and (B) Lung abscess with polymicrobial bacterial population (Gram stain). Figure 7.29 Figure 7.30 Lung abscess with sulfur granule of actinomycosis in purulent exudate. Figure 7.30 Figure 7.31 Aspiration pneumonia. Giant cells surround vegetable matter (FB) in purulent exudates, organizing pneumonia (OP) , bronchiolitis (BR) , artery (A) . Figure 7.31 Chronic Bacterial Pneumonias Chronic bacterial infections ( Fig. 7.32 ) that are slow to resolve as a result of inappropriate initial therapy, involvement with certain microbial species, a noninfectious comorbid process, or an inadequate host response can produce a nonspecific fibroinflammatory pattern, with lymphoplasmacytic infiltrates, macrophages, or organization with polyps of immature fibroblasts in alveolar ducts and alveolar spaces. 80 , 81 , 82 , 83 If not resorbed, polyps of airspace organization may become polyps of intra-alveolar fibrosis, which sometimes ossify (dendriform ossification). Such scarring in chronic pneumonia is often associated with localized interlobular septal and pleural thickening ( Fig. 7.33 ), producing a jigsaw puzzle pattern of scarring best seen at scanning magnification. Figure 7.32 Chronic pneumonia. (A) Lymphoplasmacytic infiltrate. (B) Fascicles of fibroblasts in alveolar ducts and spaces. Figure 7.32 Figure 7.33 Chronic pneumonia with thickened interlobular septum. Figure 7.33 Diffuse alveolar damage is the histopathologic correlate of the acute respiratory distress syndrome (ARDS), and today lung infection is the leading cause of diffuse alveolar damage and ARDS in the United States. 84 Diffuse alveolar damage may coexist with any of the necroinflammatory patterns described earlier. The initial exudative phase of this ARDS is accompanied by hyaline membranes ( Fig. 7.34 ), the later organizing phase by airspace and interstitial fibroplasia. In clinical practice, diffuse alveolar damage accompanied by tissue necrosis is nearly always a manifestation of lung infection. Figure 7.34 Bacterial pneumonia with hyaline membranes (HM) at periphery. Figure 7.34 The atypical pneumonias include the well-described cases due to Legionella species and the less well-described cases caused by other organisms comprising the atypical group. Legionella infection typically results in an intensely neutrophilic acute fibrinopurulent lobular pneumonia ( Fig. 7.35A ). 3 , 5 , 71 Legionella bacilli can be identified in silver impregnation-stained sections ( Fig. 7.35B ) or recovered in culture, but newer diagnostic methods, such as real-time PCR and in situ hybridization ( Fig. 7.36 ), can also be applied when standard approaches fail. 85 The histopathologic patterns associated with the other members of the atypical group (i.e., Chlamydia , Mycoplasma ) are not well characterized, mainly because investigation of these pneumonias rarely includes biopsy. The few well-documented cases of Mycoplasma , Chlamydia , and Coxiella infections resemble viral bronchitis or bronchiolitis, with mixed inflammatory infiltrates in airway walls and in the adjacent interstitium ( Fig. 7.37 ). 86 , 87 Relative sparing of the peribronchiolar alveolar spaces has been described, although patchy organized fibrinous exudates are seen in some cases and complications may superimpose additional findings. Figure 7.35 (A) Legionnaire's disease with intraalveolar necroinflammatory exudates (N) and hemorrhage. (B) Enhanced silhouette of Legionella bacilli (LB) in alveolar exudate with silver impregnation (Dieterle stain). Figure 7.35 Figure 7.36 Legionnaire's disease. Detection of organisms by in situ DNA hybridization. Figure 7.36 (Courtesy R.V. Lloyd, MD, Rochester, Minnesota.) Figure 7.37 Mycoplasma pneumonia. Bronchiolitis with patchy infiltrates in peribronchial interstitium. Figure 7.37 The grains and granules formed by the Actinomycetes and bacteria of botryomycosis may have a uniform tinctorial hue on routine H&E–stained sections, but sometimes these bacterial aggregations display a distinctive body with a hematoxylinophilic core and an outer investment of eosinophilic material; formation of this array is referred to as the Splendore-Hoeppli phenomenon ( Fig. 7.38 ). Actinomycetes species tend to form similar-appearing granules, and both they and the bacteria of botryomycosis are typically found in the midst of purulent exudates. 69 , 88 , 89 , 90 Nocardia species may aggregate in colonies simulating granules, but with a much looser texture ( Fig. 7.39 ) and more monochromatic tinctorial properties. 91 Rarely, these colonies may be identical in appearance to the grains or granules of botryomycosis or actinomycosis in H&E sections. Figure 7.38 Botryomycosis granule with hematoxylinophilic core and eosinophilic investment known as the Splendore-Hoeppli effect . Figure 7.38 Figure 7.39 Loose-textured aggregate of Nocardia filamentous bacteria surrounded by neutrophils. Figure 7.39 Acute Exudative Pneumonia Acute exudative pneumonia is most often caused by pyogenic bacteria, such as streptococci, which typically produce a neutrophil-rich intra-alveolar exudate (i.e., alveolar filling) with variable amounts of fibrin and red cells. Pathologists recognize this constellation of findings as acute lobular pneumonia ( Fig. 7.23 ), which usually correlates with patchy segmental infiltrates on the chest film (consolidation pattern on HRCT). 29 , 71 , 72 , 73 Figure 7.23 Alveoli filled with fibrinopurulent exudate with variable hemorrhage. Figure 7.23 With increasing organism virulence and disease severity, lobular exudates may become confluent (i.e., lobar pneumonia). In milder cases, the disease may be limited to the airways (bronchitis/bronchiolitis) with a mixed cellular infiltrate of mononuclear cells and neutrophils ( Fig. 7.24 ). One very common manifestation of such airway-limited infection has been designated as acute exacerbation of chronic obstructive pulmonary disease (COPD). A majority of these exacerbations are caused by particular bacteria (specifically H. influenzae , S. pneumoniae , and M. catarrhalis ) with approximately one third resulting from viral airway infections, typically resulting from rhinovirus, respiratory syncytial virus (RSV), and human metapneumovirus. 74 Figure 7.24 Bronchiolitis with intraluminal exudate. Figure 7.24 Nodular/Necrotizing Lesions Nodular inflammatory infiltrates with or without necrotizing features ( Fig. 7.25 ) are characteristic of infection by certain species, such as R. equi ( Fig. 7.26 ). 75 Necrotizing pneumonias may also be produced by pyogenic bacteria such as S. aureus , Streptococcus pyogenes , and the gram-negative bacilli— Klebsiella , Acinetobacter , Pseudomonas , and Burkholderia species. Figure 7.25 Nodular histiocytic infiltrate in rhodococcal pneumonia. Figure 7.25 Figure 7.26 Rhodococcus equi bacilli in macrophage. Figure 7.26 Miliary Lesions A subset of the nodular histopathologic pattern, miliary infection ( Fig. 7.27 ), strongly implies pneumonia secondary to the hematogenous spread of bacteria (septicemia). This pattern of infection can be seen with other organisms, such as Nocardia and the anaerobic Actinomycetes. In these settings, histopathologic examination may show a combination of both nodular disease and alveolar filling. Figure 7.27 Necrotizing pneumonia, miliary pattern. Figure 7.27 Aspiration Pneumonia and Lung Abscess There are multiple scenarios for aspiration pneumonia, including cases caused by chemical pneumonitis (so-called Mendelson syndrome), airway obstruction, exogenous lipoid pneumonia, chronic interstitial fibrosis, diffuse bronchiolar disease, bacterial pneumonia, and lung abscess. 76 , 77 Aspiration pneumonia refers specifically to the aspiration of bacteria in oropharyngeal secretions, with the bacterial species depending on whether the aspiration event occurs in the community or hospital setting. Recognition of food particles (so-called pulses) is important in diagnosis. These may or may not be invested by giant cells but are usually found in purulent exudate or granulomatous foci. In the organizing phase of the pneumonia, food particles may be found within polyps of organizing pneumonia in the alveolar ducts and alveoli. Lobular pneumonia, lipoid pneumonia, organizing pneumonia, and bronchiolitis, alone or in combination, may also be seen. 73 , 78 The pathogens in lung abscess ( Fig. 7.28 ) usually encompass a polymicrobial mixture of aerobic and anaerobic bacteria, 79 and formation of such abscesses most often is secondary to aspiration ( Fig. 7.29 ). Infections due to Actinomyces species ( Fig. 7.30 ) and Nocardia species may also manifest this pattern, as can those infections caused by certain pyogenic bacteria, such as S. aureus and the other organisms listed previously for necrotizing pneumonias. Granulomatous inflammation with foreign bodies may be present if aspiration is the cause ( Fig. 7.31 ). Figure 7.28 Lung abscess showing gross evidence of chronicity with fibrosis in surrounding parenchyma. Figure 7.28 Figure 7.29 (A) and (B) Lung abscess with polymicrobial bacterial population (Gram stain). Figure 7.29 Figure 7.30 Lung abscess with sulfur granule of actinomycosis in purulent exudate. Figure 7.30 Figure 7.31 Aspiration pneumonia. Giant cells surround vegetable matter (FB) in purulent exudates, organizing pneumonia (OP) , bronchiolitis (BR) , artery (A) . Figure 7.31 Chronic Bacterial Pneumonias Chronic bacterial infections ( Fig. 7.32 ) that are slow to resolve as a result of inappropriate initial therapy, involvement with certain microbial species, a noninfectious comorbid process, or an inadequate host response can produce a nonspecific fibroinflammatory pattern, with lymphoplasmacytic infiltrates, macrophages, or organization with polyps of immature fibroblasts in alveolar ducts and alveolar spaces. 80 , 81 , 82 , 83 If not resorbed, polyps of airspace organization may become polyps of intra-alveolar fibrosis, which sometimes ossify (dendriform ossification). Such scarring in chronic pneumonia is often associated with localized interlobular septal and pleural thickening ( Fig. 7.33 ), producing a jigsaw puzzle pattern of scarring best seen at scanning magnification. Figure 7.32 Chronic pneumonia. (A) Lymphoplasmacytic infiltrate. (B) Fascicles of fibroblasts in alveolar ducts and spaces. Figure 7.32 Figure 7.33 Chronic pneumonia with thickened interlobular septum. Figure 7.33 Diffuse alveolar damage is the histopathologic correlate of the acute respiratory distress syndrome (ARDS), and today lung infection is the leading cause of diffuse alveolar damage and ARDS in the United States. 84 Diffuse alveolar damage may coexist with any of the necroinflammatory patterns described earlier. The initial exudative phase of this ARDS is accompanied by hyaline membranes ( Fig. 7.34 ), the later organizing phase by airspace and interstitial fibroplasia. In clinical practice, diffuse alveolar damage accompanied by tissue necrosis is nearly always a manifestation of lung infection. Figure 7.34 Bacterial pneumonia with hyaline membranes (HM) at periphery. Figure 7.34 The atypical pneumonias include the well-described cases due to Legionella species and the less well-described cases caused by other organisms comprising the atypical group. Legionella infection typically results in an intensely neutrophilic acute fibrinopurulent lobular pneumonia ( Fig. 7.35A ). 3 , 5 , 71 Legionella bacilli can be identified in silver impregnation-stained sections ( Fig. 7.35B ) or recovered in culture, but newer diagnostic methods, such as real-time PCR and in situ hybridization ( Fig. 7.36 ), can also be applied when standard approaches fail. 85 The histopathologic patterns associated with the other members of the atypical group (i.e., Chlamydia , Mycoplasma ) are not well characterized, mainly because investigation of these pneumonias rarely includes biopsy. The few well-documented cases of Mycoplasma , Chlamydia , and Coxiella infections resemble viral bronchitis or bronchiolitis, with mixed inflammatory infiltrates in airway walls and in the adjacent interstitium ( Fig. 7.37 ). 86 , 87 Relative sparing of the peribronchiolar alveolar spaces has been described, although patchy organized fibrinous exudates are seen in some cases and complications may superimpose additional findings. Figure 7.35 (A) Legionnaire's disease with intraalveolar necroinflammatory exudates (N) and hemorrhage. (B) Enhanced silhouette of Legionella bacilli (LB) in alveolar exudate with silver impregnation (Dieterle stain). Figure 7.35 Figure 7.36 Legionnaire's disease. Detection of organisms by in situ DNA hybridization. Figure 7.36 (Courtesy R.V. Lloyd, MD, Rochester, Minnesota.) Figure 7.37 Mycoplasma pneumonia. Bronchiolitis with patchy infiltrates in peribronchial interstitium. Figure 7.37 The grains and granules formed by the Actinomycetes and bacteria of botryomycosis may have a uniform tinctorial hue on routine H&E–stained sections, but sometimes these bacterial aggregations display a distinctive body with a hematoxylinophilic core and an outer investment of eosinophilic material; formation of this array is referred to as the Splendore-Hoeppli phenomenon ( Fig. 7.38 ). Actinomycetes species tend to form similar-appearing granules, and both they and the bacteria of botryomycosis are typically found in the midst of purulent exudates. 69 , 88 , 89 , 90 Nocardia species may aggregate in colonies simulating granules, but with a much looser texture ( Fig. 7.39 ) and more monochromatic tinctorial properties. 91 Rarely, these colonies may be identical in appearance to the grains or granules of botryomycosis or actinomycosis in H&E sections. Figure 7.38 Botryomycosis granule with hematoxylinophilic core and eosinophilic investment known as the Splendore-Hoeppli effect . Figure 7.38 Figure 7.39 Loose-textured aggregate of Nocardia filamentous bacteria surrounded by neutrophils. Figure 7.39 Bacterial Agents of Bioterrorism The potential for use of microbial pathogens as agents of bioterrorism requires that clinicians be alert to this possibility when community-acquired pneumonias are found to be caused by these agents. In turn, pathologists must become familiar with the histopathologic features these agents can produce. 92 Respiratory disease caused by the inhalation of Bacillus anthracis , Yersinia pestis , and F. tularensis is especially pertinent in this context and is discussed next. 93 Bacillus anthracis In 1877, Robert Koch's conclusive demonstration that B. anthracis was the etiologic agent of anthrax revolutionized medicine by linking microbial cause and effect. 6 Inhalational anthrax causes a severe hemorrhagic mediastinitis. 94 , 95 , 96 , 97 , 98 This pathologic process in combination with the toxemia ( B. anthracis produces an exotoxin with three potent components—protective antigen, lethal factor, and edema factor) from the ensuing massive bacteremia severely compromises pulmonary function, leading to death in 40% or more of the cases. Pleural effusion may be present, but pneumonia generally is minor and secondary. In those patients in whom pulmonary parenchymal changes are found, the alveolar spaces contain a serosanguineous fluid with minimal fibrin deposits and some mononuclear cells but few if any neutrophils. 97 Large gram-positive bacilli (some may appear partially gram-negative) without spores pervade the alveolar septal vessels, with a few in the alveolar spaces. This distribution suggests hematogenous rather than airway acquisition. Hemorrhagic mediastinitis in a previously healthy adult is essentially pathognomonic for inhalational anthrax. The lymph node parenchyma generally is teeming with intact and fragmented gram-positive bacilli, which can be identified as B. anthracis by immunohistochemical studies. 96 , 97 Cultures of blood and pleural fluid, if available, are likely to yield the earliest positive diagnostic results. 98 Sputum studies are much less useful in this regard. Specific guidelines for pathology and microbiology specimens for anthrax diagnosis (as well as other potential agents of bioterrorism) are current and available on the Centers for Disease Control and Prevention (CDC) website. 99 Yersinia pestis Primary pneumonic plague follows inhalation of Y. pestis bacilli in a potential bioterrorism scenario. 100 , 101 The infection begins as bronchiolitis and alveolitis that progress to a lobular and eventual lobar consolidation. 102 The histopathologic features evolve over time, beginning with a serosanguineous intraalveolar fluid accumulation with variable fibrin deposits ( Fig. 7.40 ), progressing through a fibrinopurulent phase, and culminating in a necrotizing lesion. 103 The presence of myriad bacilli in the intraalveolar exudates with significantly fewer organisms in the interstitium (a characteristic of primary pneumonia) is one of several pulmonary and extrapulmonary features used to distinguish primary from secondary pneumonic plague. 104 These bacilli may be obvious in H&E-stained sections ( Fig. 7.41 ) but generally are better visualized with Giemsa rather than Gram stain. Immunohistochemical staining provides a rapid and specific diagnosis. 102 In contrast to inhalational anthrax, sputum Gram stain and culture are useful tests that are likely to yield a positive result at clinical presentation. Also, because sepsis is an integral component of the pneumonia, it is important to collect blood culture specimens. 105 Figure 7.40 Plague pneumonia, early phase. Edema, fibrin, and sparse inflammatory cells are evident. Figure 7.40 Figure 7.41 Yersinia pestis bacilli in alveolar space. Figure 7.41 Francisella tularensis Inhalation of F. tularensis bacilli following a bioterrorism aerosol release is generally expected to result in a slowly progressing pneumonia with a lower case-fatality rate than with either inhalational anthrax or plague. 104 , 106 Initially a hemorrhagic and ulcerative bronchiolitis is followed by a fibrinous lobular pneumonia with many macrophages but relatively few neutrophils ( Fig. 7.42 ). Necrosis then supervenes and evolves into a granulomatous reaction. The small, gram-negative coccobacillary organisms are difficult to identify in a tissue Gram stain, and the use of silvering techniques (e.g., Steiner, Dieterle, Warthin-Starry) is required to enhance their silhouette. 107 Specific fluorescent antibody testing for formalin-fixed tissue and immunohistochemical studies are available through public health laboratories. In the microbiology laboratory, Gram stain and culture of respiratory secretions are useful for diagnosis, but blood cultures are often negative. Antigen detection and molecular techniques, such as PCR amplification, can also identify F. tularensis . Serologic tests are available but probably would not provide timely information in an outbreak situation. 104 Figure 7.42 Tularemia. Fibrinous lobular pneumonia phase. Figure 7.42 Bacillus anthracis In 1877, Robert Koch's conclusive demonstration that B. anthracis was the etiologic agent of anthrax revolutionized medicine by linking microbial cause and effect. 6 Inhalational anthrax causes a severe hemorrhagic mediastinitis. 94 , 95 , 96 , 97 , 98 This pathologic process in combination with the toxemia ( B. anthracis produces an exotoxin with three potent components—protective antigen, lethal factor, and edema factor) from the ensuing massive bacteremia severely compromises pulmonary function, leading to death in 40% or more of the cases. Pleural effusion may be present, but pneumonia generally is minor and secondary. In those patients in whom pulmonary parenchymal changes are found, the alveolar spaces contain a serosanguineous fluid with minimal fibrin deposits and some mononuclear cells but few if any neutrophils. 97 Large gram-positive bacilli (some may appear partially gram-negative) without spores pervade the alveolar septal vessels, with a few in the alveolar spaces. This distribution suggests hematogenous rather than airway acquisition. Hemorrhagic mediastinitis in a previously healthy adult is essentially pathognomonic for inhalational anthrax. The lymph node parenchyma generally is teeming with intact and fragmented gram-positive bacilli, which can be identified as B. anthracis by immunohistochemical studies. 96 , 97 Cultures of blood and pleural fluid, if available, are likely to yield the earliest positive diagnostic results. 98 Sputum studies are much less useful in this regard. Specific guidelines for pathology and microbiology specimens for anthrax diagnosis (as well as other potential agents of bioterrorism) are current and available on the Centers for Disease Control and Prevention (CDC) website. 99 Yersinia pestis Primary pneumonic plague follows inhalation of Y. pestis bacilli in a potential bioterrorism scenario. 100 , 101 The infection begins as bronchiolitis and alveolitis that progress to a lobular and eventual lobar consolidation. 102 The histopathologic features evolve over time, beginning with a serosanguineous intraalveolar fluid accumulation with variable fibrin deposits ( Fig. 7.40 ), progressing through a fibrinopurulent phase, and culminating in a necrotizing lesion. 103 The presence of myriad bacilli in the intraalveolar exudates with significantly fewer organisms in the interstitium (a characteristic of primary pneumonia) is one of several pulmonary and extrapulmonary features used to distinguish primary from secondary pneumonic plague. 104 These bacilli may be obvious in H&E-stained sections ( Fig. 7.41 ) but generally are better visualized with Giemsa rather than Gram stain. Immunohistochemical staining provides a rapid and specific diagnosis. 102 In contrast to inhalational anthrax, sputum Gram stain and culture are useful tests that are likely to yield a positive result at clinical presentation. Also, because sepsis is an integral component of the pneumonia, it is important to collect blood culture specimens. 105 Figure 7.40 Plague pneumonia, early phase. Edema, fibrin, and sparse inflammatory cells are evident. Figure 7.40 Figure 7.41 Yersinia pestis bacilli in alveolar space. Figure 7.41 Francisella tularensis Inhalation of F. tularensis bacilli following a bioterrorism aerosol release is generally expected to result in a slowly progressing pneumonia with a lower case-fatality rate than with either inhalational anthrax or plague. 104 , 106 Initially a hemorrhagic and ulcerative bronchiolitis is followed by a fibrinous lobular pneumonia with many macrophages but relatively few neutrophils ( Fig. 7.42 ). Necrosis then supervenes and evolves into a granulomatous reaction. The small, gram-negative coccobacillary organisms are difficult to identify in a tissue Gram stain, and the use of silvering techniques (e.g., Steiner, Dieterle, Warthin-Starry) is required to enhance their silhouette. 107 Specific fluorescent antibody testing for formalin-fixed tissue and immunohistochemical studies are available through public health laboratories. In the microbiology laboratory, Gram stain and culture of respiratory secretions are useful for diagnosis, but blood cultures are often negative. Antigen detection and molecular techniques, such as PCR amplification, can also identify F. tularensis . Serologic tests are available but probably would not provide timely information in an outbreak situation. 104 Figure 7.42 Tularemia. Fibrinous lobular pneumonia phase. Figure 7.42 Cytopathology The stereotypic cellular response to pyogenic bacteria is acute inflammation, characterized by variable numbers of neutrophils. Bacteria may be visualized in various stained preparations made from respiratory tract secretions and washings using the Papanicolaou and Diff-Quik methods. 45 The clinical significance of bacteria in such specimens may be limited owing to potential contamination by oral flora and the problem of distinguishing colonization from infection. However, when the upper respiratory tract can be bypassed by means of either transtracheal or transthoracic needle aspiration, the presence of bacteria becomes much more significant, especially when sheets of neutrophils or necroinflammatory debris are present ( Fig. 7.43A ), as would be the case with a typical lobar or lobular consolidation, lung abscess, or other complex pneumonia. 53 , 90 , 108 , 109 In this context, transthoracic needle aspiration can establish the etiologic diagnosis of community-acquired and nosocomial pneumonias in both children and adults when coupled with modern microbiologic methods. 49 , 58 , 110 , 111 Proponents consider it an underused technique the potential benefits of which, in experienced hands, outweigh the modest associated risks. Figure 7.43 (A) Purulent exudate of nodular pulmonary infiltrate in fine-needle aspirate (alcohol-fixed). (B) Streptococci ( viridans group) in cytoplasm of neutrophil seen in fine-needle aspirate (Diff-Quik preparation). Figure 7.43 Many types of bacilli and cocci can be seen within and around neutrophils on Diff-Quik–stained smears ( Fig. 7.43B ). A smear can also be prepared for Gram stain and the aspirate needle rinsed in nonbacteriostatic sterile saline or nutrient broths for culture. The size (length and width) and shape of organisms and the Gram reaction allow rough categorization of organisms into groups, such as enteric-type bacilli, pseudomonads, fusiform anaerobic-type bacilli, tiny coccobacillary types suggestive of the Haemophilus–Bacteroides group ( Fig. 7.44 ), or gram-positive cocci. 112 Branching filamentous forms suggest Actinomycetes or Nocardia species ( Fig. 7.45 ), with the latter distinguished by being partially acid-fast. 113 , 114 Figure 7.44 (A) Fusiform bacteria ( Fusobacterium organisms) in cytoplasm of neutrophil in fine-needle aspirate (Gram stain). (B) Coccobacilli (Haemophilus influenzae) in cytoplasm of leukocyte in fine-needle aspirate (Gram stain). Figure 7.44 Figure 7.45 Nocardia. Loose, feathery cluster of bacilli in purulent exudate seen in a fine-needle aspirate: alcohol-fixed, Hematoxylin and eosin stain (HE) ; Gram stain (Gram) ; Grocott methenamine silver stain (GMS) ; Ziehl–Neelsen stain (ZN) . Figure 7.45 Although most aspirated cavitary lung lesions with the abscess pattern are the result of bacterial infection, considerations in the differential diagnosis include necrotic neoplasm (particularly squamous cell carcinoma), granulomatosis with polyangiitis, and nonbacterial infections associated with suppurative granulomas such as those due to fungi and mycobacteria. Microbiology Microbiology techniques in current use for the laboratory diagnosis of bacterial pneumonia are summarized in Box 7.7 . 39 , 115 , 116 , 117 The traditional morphologic and functional approach to microbiologic diagnosis is gradually shifting to molecular methods, and diagnostic arrays of common respiratory pathogens are marketed by several vendors; they are adjusted for laboratory size, for individual random access testing, or for test batching in larger laboratories. Box 7.7 Laboratory Diagnosis of Bacterial Pneumonia Direct detection of organisms Gram stain; other stains of respiratory secretions and fluids Direct fluorescent antibody stain Histopathologic/cytopathologic examination Immunohistochemistry Antigen detection (with Legionella pneumophila [LP1] and Streptococcus pneumoniae ) Culture Conventional media for usual pyogenic bacteria Special media for fastidious or atypical agents Serologic testing Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.7 The work-up of respiratory secretions such as sputum in the microbiology laboratory may or may not be indicated based on the clinical and immunologic status of the patient. The value of microbiologic work-up for community-acquired pneumonias has been questioned for some time, and evolving guidelines from two specialty societies—the American Thoracic Society and the Infectious Disease Society of America—have lately coalesced. 118 , 119 , 120 , 121 Despite microbiologic testing, in a retrospective review of 2259 patients with radiographic evidence of pneumonia hospitalized from January 2010–June 2012 in selected US communities, no pathogen was detected in the majority of patients. 122 When a carefully collected specimen reveals one or two predominant bacterial morphotypes on a well-prepared Gram stain ( Fig. 7.46 ), especially in the presence of neutrophils and few or no squamous cells, a presumptive diagnosis can be offered and correlated with whatever grows on culture plates. 123 , 124 , 125 A mixed bacterial population is usually considered nondiagnostic, especially in the absence of inflammation or the presence of many benign oral squamous cells. Pneumonia in the hospitalized or immunocompromised patient requires an aggressive strategy to collect a good sputum sample for Gram stain and culture. If this attempt is unsatisfactory or the findings are nondiagnostic, then the use of invasive techniques beginning with fiberoptic bronchoscopy and BAL with protected catheters should be considered. 60 , 62 , 126 Anaerobic pulmonary infections, typically in the form of a lung abscess, can also be approached in this way or with transthoracic needle aspiration. 79 Figure 7.46 Sputum Gram stain. (A) Gram-positive diplococci (Streptococcus pneumoniae) with neutrophils, but no squamous cells. (B) Gram-positive diplococci (S. pneumoniae) and gram-negative coccobacilli (Haemophilus influenzae) . Figure 7.46 Gram staining of tissue sections from bronchoscopic or surgical biopsy specimens is notoriously insensitive and nonspecific. As with sputum, the presence of a predominant bacterial morphotype in a distinctive necroinflammatory background carries diagnostic weight, especially when correlated with available clinical and laboratory data. Because histology laboratories do not generally observe the same level of caution in reagent preparation and storage as microbiology laboratories, it is worth remembering that tissue sections are prone to false-positive results from in vitro bacterial contamination. In those cases where bacteria are visible on H&E-stained sections, the Gram stain can be helpful in confirming a presumptive etiology. For example, pairs and chains of gram-positive cocci in a necroinflammatory background suggest a streptococcal pneumonia, whereas numerous slender gram-negative bacilli investing and infiltrating blood vessels are characteristic of Pseudomonas pneumonia ( Fig. 7.47 ). Other types of gram-negative pneumonias ( Fig. 7.48 ) can also be confirmed with well-prepared Gram stains. 81 In the case of an abscess, a mixture of gram-positive cocci and gram-negative bacilli in tissue (illustrated earlier in Fig. 7.29 ) is a useful finding that is helpful in supporting a diagnosis of an anaerobic infection. Figure 7.47 (A) Pseudomonas aeruginosa bacilli investing interstitial vessels (Brown and Hopps stain). (B) The slender gram-negative bacilli are nicely demonstrated on Gram stain. Figure 7.47 Figure 7.48 Burkholderia cepacia bacilli (Brown and Hopps stain). Figure 7.48 When organisms are sparse, other stains such as Giemsa or silver impregnation may highlight the organisms in the exudates ( Fig. 7.49 ). The Gram stain is also useful for evaluating infections with granules and allows differentiation of the agents of botryomycosis (the gram-positive cocci or gram-negative bacilli) from the filamentous Actinomyces organisms ( Fig. 7.50 ). Figure 7.49 Bacterial tetrads in alveolar exudate (Giemsa stain). Figure 7.49 Figure 7.50 Botryomycosis. Cluster of gram-positive cocci (Staphylococcus aureus) invested by gram-negative–staining Splendore-Hoeppli material (Brown and Brenn stain). Figure 7.50 (Courtesy Dr. Francis Chandler, Augusta, Georgia.) Staining with methenamine silver is the best procedure for detecting Nocardia organisms. The modified Ziehl–Neelsen stain allows for differentiation of Nocardia (positive) from the anaerobic Actinomyces (negative). 114 Commercially available immunohistochemical reagents exist for relatively few bacterial species. Immunohistochemistry testing for the potential bioterrorist agents discussed in this chapter is available through the CDC in Atlanta, Georgia. It is expected that commercial reagents will become increasingly available for the common etiologic agents in the near future. 33 Culture media that will allow recovery of common bacterial species causing pneumonia from various types of respiratory samples (secretions, washings, brushings, aspirates, and tissues) include sheep blood agar, chocolate agar, and McConkey agar. These media also will support growth of B. anthracis and Y. pestis . Buffered charcoal yeast extract (BCYE) agar is the primary medium for Legionella species. Because Legionella organisms survive poorly in respiratory secretions, rapid transport and immediate plating is essential for recovery. BCYE is also a good all-purpose medium for growing other fastidious species, including F. tularensis . However, F. tularensis grows best in cysteine-enriched media. 127 In addition to respiratory samples, blood can be obtained for cultures in patients sick enough to lead to suspicion of bacteremia; pleural fluid culture can be used when effusions are present. Positive cultures of these normally sterile fluids circumvent the interpretive problems associated with bacterial growth in sputum samples. The Actinomycetes are best isolated from invasive specimens such as needle aspirates and transbronchial and lung biopsy specimens. The laboratory should be alerted to search for these agents because special consideration must be given to culture setup and incubation conditions. 89 The Actinomycetes responsible for actinomycosis require anaerobic media and atmosphere as well as prolonged incubation. Nocardia , an aerobic Actinomycete, grows well on most nonselective media but requires extended incubation. Determination of colonial morphology, Gram and acid-fast stains, and a few biochemical tests generally suffice to identify these organisms at the genus level. However, genotype rather than phenotype characteristics are required to identify newly emergent species. 128 In general, the laboratory diagnosis of pneumonia caused by most of the atypical agents is difficult because systems are not routinely available or are costly, cumbersome, or unsafe. For the atypical agents ( Mycoplasma , Chlamydia , and Coxiella species), serologic testing has been the method of choice for diagnosis. 67 , 129 Classic cold agglutinin and complement fixation tests for these agents have largely been replaced by enzyme immunoassay and microimmunofluorescence testing. 87 , 130 , 131 Serologic methods are also useful for the diagnosis of tularemia because of the difficulty in culturing the fastidious bacterium. Legionella pneumonia is a common form of severe pneumonia that is not readily diagnosed for a number of reasons, including the organism's fastidiousness. 132 In the microbiology laboratory, the direct fluorescent antibody test and culture on buffered BCYE agar have been the mainstays of diagnosis. Culture is considered the diagnostic gold standard but is only 60% sensitive. Serologic testing is available for most of the Legionella pneumophila serotypes, which account for 90% of the pneumonia cases; however, the need to collect paired sera weeks apart limits its usefulness in the acutely ill patient. Antigen detection in urine has become commercially available for both L. pneumophila and S. pneumoniae ; because the need to collect acute and convalescent sera is obviated, it has become a frequently used diagnostic test. 132 , 133 Its advantage lies in its potential to effect early treatment decisions through rapid diagnosis. Its disadvantage lies in the fact that it identifies only patients infected with L. pneumophila serogroup 1 (LP1), the most prevalent species and serotype, but none of the non-LP1 serotypes or cases due to other Legionella species. 134 , 135 , 136 The use of molecular diagnostic tools (in situ hybridization and nucleic acid amplification by PCR or other methods) to detect these agents has been reported. 85 , 136 , 137 PCR nested assays are replacing more and more of the above classical methods with sensitive, specific, and rapid diagnostic technique. Multiplex assay, to detect multiple agents in a single reaction, would seem to be an ideal pursuit for the laboratory diagnosis of the most common community-acquired pneumonias, including those due to the atypical pneumonia agents. 36 , 138 , 139 , 140 , 141 Differential Diagnosis The key morphologic and microbiologic features of the bacterial pneumonias are summarized in Table 7.5 . The presence of purulent exudates or significant numbers of neutrophils in biopsy or cytologic samples should always trigger a search for bacterial infection. Because lung biopsies are usually performed late in the clinical course after procedures have been performed and bacterial infections have been excluded and/or treated with antibiotics, neutrophilic exudates may not signify bacterial infection unless accompanied by necrosis, as in an abscess. Instead, consideration should be given to one of several noninfectious acute inflammatory diseases, with an immunologic basis, that can mimic bacterial infection. Some of these include granulomatosis with polyangiitis, Goodpasture syndrome, systemic lupus erythematosus, and microscopic polyangiitis, all conditions that can produce acute inflammation predominantly involving alveolar septal blood vessels ("capillaritis"). On occasion, capillaritis can result in airspace accumulation of neutrophils, further raising concern for bronchopneumonia. Centrally necrotic or cavitary neoplasms of various types may mimic abscesses grossly and microscopically, and exceptionally well-differentiated adenocarcinomas containing glands filled with detritus may mimic inflammatory and bacterial diseases. Suppurative granulomas can have a bacterial, mycobacterial, or fungal etiology. Even the miliary necroinflammatory lesion typical of bacterial infection can be produced by viruses, some fungi, and even protozoa (e.g., Toxoplasmagondii ). Aspiration, common in hospitalized patients, may manifest with an acute bronchopneumonia/bronchiolitis type of pattern, and it should also be in the differential diagnosis as a contributing or etiologic factor when considering most pneumonias; microscopic identification of foreign material is a definitive diagnostic clue, frequently gained only from histopathologic examination. 78 Table 7.5 Bacterial Pneumonias: Summary of Pathologic Findings Table 7.5 Assessment Component Findings Pyogenic Bacteria Surgical pathology Acute purulent inflammation with/without necrosis; organization; diffuse alveolar damage may be present Cytopathology Acute inflammation with/without visible bacteria on Diff-Quik–stained smear Microbiology Gram stain reactivity and morphology (visual detection requires heavy bacterial burden: 10 6 organisms/g of tissue); culture-sterile lung tissue on standard nonselective and selective media (blood, chocolate and MacConkey agars); anaerobic broth and agars for abscesses; urinary antigen for Streptococcus pneumoniae Atypical Pneumonia Agents Surgical pathology Legionella pneumonia: fibrinopurulent with bacilli visible in silver-stained (Dieterle; Warthin-Starry) sections DAD often present Chlamydia and Mycoplasma infection: polymorphous bronchiolar and interstitial infiltrate Cytopathology Acute inflammation with bacilli stained with silver or by immunofluorescence ( Legionella pneumonia) Microbiology DFA for L. pneumophila serotypes; culture on selective (BCYE) agar for Legionella ; urinary antigen for Legionella ; serologic testing and/or PCR assay for Mycoplasma and Chlamydia Filamentous Granule Group Surgical pathology Granules or loose filamentous aggregates in purulent exudate with abscess formation and poorly formed granuloma in some cases Cytopathology Filamentous tangles or aggregates or granules with neutrophils and/or necroinflammatory background Microbiology Gram-positive branching filaments: Nocardia (aerobic actinomycete) and Actinomyces (anaerobic actinomycete); Nocardia partially acid-fast and GMS-positive Gram-positive cocci or gram-negative bacilli (botryomycosis); culture on standard nonselective media and selective (BCYE) media; anaerobic culture broths and media for Actinomyces BCYE , Buffered charcoal yeast extract; DAD , diffuse alveolar damage; DFA , direct fluorescence assay; GMS , Grocott methenamine silver. Mycobacterial Infections The surgical pathologist tends to encounter mycobacterial infections in lung biopsies when standard clinical diagnostic approaches to pulmonary infiltrates are unsuccessful and the lesions persist or progress. Tuberculosis is but one of several different types of lung infection that can manifest clinically as community-acquired pneumonia, resulting in delay until an invasive procedure such as transbronchial biopsy, transthoracic needle biopsy, or surgical lung biopsy is performed, often as a "last resort" effort. 142 , 143 In recent years, delays in the diagnosis of mycobacterial infection have markedly decreased, thanks in part to recommendations from the CDC for improving laboratory turnaround time and to the response of the diagnostics industry with better methods and technology. Because direct acid-fast smears of respiratory specimens yield negative findings in at least half of the cases, 144 and because many mycobacterial species are fastidious and slow-growing, the biopsy results may be the first suggestion of a mycobacterial infection. The biopsy findings can also define the organism's relationship to a histopathologic lesion and host response—important information in evaluating the significance of a culture result. Although an isolate of M. tuberculosis is always taken seriously, obtaining a single isolate of a nontuberculous mycobacterium from the respiratory tract does not necessarily implicate the organism as the cause of disease. 145 Etiologic Agents The mycobacterial species can be categorized in two clinically relevant groups: Mycobacterium tuberculosis complex (MTC) and the nontuberculous mycobacteria (NTM). MTC includes the subspecies Mycobacterium tuberculosis , Mycobacterium bovis , Mycobacterium africanum , and Mycobacterium microti . The last three species produce tuberculosis in some areas of the world, but in the United States the prevalence of such disease is very low. Mycobacterium tuberculosis M. tuberculosis is the most virulent mycobacterial species and an unequivocal pathogen that is responsible for numerous deaths worldwide. This organism is the etiologic agent of tuberculosis in its various forms, which are listed in Box 7.8 . Box 7.8 Classification of Tuberculosis Primary tuberculosis Exogenous first infection Exogenous reinfection Progressive primary tuberculosis Postprimary tuberculosis Endogenous reactivation Exogenous infection in BCG-vaccinated persons Exogenous superinfection BCG, Bacille Calmette-Guérin. Alt-text: Box 7.8 Data from Allen E. Tuberculosis and other mycobacterial infections of the lung. In: Churg AM, Thurlbeck WM, eds. Pathology of the Lung, 2nd ed. New York: Thieme; 1995:233, Table 13.1. Primary tuberculosis occurs in patients without previous exposure or with the loss of acquired immunity. Progressive primary tuberculosis occurs in patients with inadequate acquired immunity—that is, impaired cellular immunity. Postprimary tuberculosis, also referred to as secondary or reinfection-reactivation tuberculosis , occurs in patients with previous immunity to the organism and accounts for most clinical cases of tuberculosis. 146 , 147 Many clinical experts consider that most cases of active tuberculosis in adults with normal immunity arise from reactivation of latent infection (postprimary tuberculosis), whereas reinfection with a new strain derived from the environment (primary or postprimary tuberculosis) can occur in the immunocompromised patient. More recently, DNA fingerprinting methods (genotyping) have challenged this dogma by showing that exogenous reinfection accounts for a significant percentage of cases in some areas of the world. 148 Miliary tuberculosis and extrapulmonary disease can occur with any of these forms. 146 , 149 Primary tuberculosis is usually a mild illness that is often not recognized. The bacillemia that occurs during its development can seed extrapulmonary organs and set the stage for subsequent reactivation. Approximately 5% of patients pass through latency to postprimary disease within 2 years of primary infection, and another 5% do so later in their lives. 150 Nontuberculous Mycobacteria Recognized NTM species, many of which were identified during the past decade, number more than 125. 151 , 152 However, relatively few cause pulmonary disease. 145 , 153 , 154 , 155 These organisms are acquired from the environment, where they are ubiquitous. In contrast with M. tuberculosis , the NTM are not spread from person to person. In most instances, patients in whom NTM infection develops have chronic lung disease and other risk factors, such as AIDS, alcoholism, or diabetes. Reports of NTM infections in nonimmunocompromised patients are increasing. 16 , 156 MAC and then Mycobacterium kansasii are the most frequent isolates in all settings. Among a growing number of species causing lung disease are Mycobacterium abscessus , Mycobacteriumfortuitum , Mycobacterium szulgai , Mycobacterium simiae , Mycobacterium xenopi , Mycobacterium malmoense , Mycobacterium celatum , Mycobacterium asiaticum , and Mycobacterium shimodii . These species manifest marked geographic variability with respect to prevalence and severity. Of note, however, since 1985, more MAC isolates than M. tuberculosis have been reported in the United States. 145 Histopathology The histopathologic patterns produced by mycobacteria are listed in Box 7.9 . The radiologic, gross, and microscopic patterns of mycobacterial disease reflect the virulence of the various mycobacterial species as well as the patient's prior exposure and immune status. 157 , 158 , 159 , 160 Box 7.9 Histopathologic Patterns in Mycobacterial Lung Injury Large nodules with or without cavities Well-formed granuloma Poorly formed granuloma Suppurative granuloma Histiocytic aggregates Miliary nodules Calcified nodules Granulomatous interstitial pneumonitis Bronchitis/bronchiectasis Spindle cell pseudotumors Alt-text: Box 7.9 Primary Tuberculosis Mycobacterium tuberculosis occurs typically in the best-aerated lung regions (anterior segments of the upper lobes, lingua and middle lobe, or basal segments of the lower lobes). 158 The disease passes through progressive phases of exudation, recruitment of macrophages and T lymphocytes, and granuloma formation followed by repair with granulation tissue, fibrosis, and mineralization. 147 , 161 Macrophage-laden bacilli also travel to the hilar lymph nodes, where the phases are repeated. This combination of events produces the classic Ghon complex, consisting of a peripheral 1- to 2-cm lung nodule ( Fig. 7.51 ) and an enlarged, sometimes calcified hilar lymph node. In both locations, the histopathologic hallmark is a necrotizing granuloma ( Fig. 7.52 ) composed of epithelioid cells with variable numbers of Langhans giant cells, a peripheral investment of lymphocytes, and a central zone of caseation necrosis, a form of necrosis attributed to apoptosis. 146 , 162 A spectrum of lesions may be seen, from the tuberculoid "hard" granuloma without necrosis and rare organisms to the multibacillary necrotic lesion with scant epithelioid cells. 163 In a minority of patients the lesions enlarge and progress as a result of increased necrosis or liquifaction. Figure 7.51 Tuberculoma removed from right upper lobe. Figure 7.51 Figure 7.52 (A) Tuberculoid granuloma with central zone of caseation necrosis surrounded by epithelioid cells, giant cells, and outer investment of lymphocytes. (B) Palisade of epithelioid histiocytes in giant cells at edge of necrotic zone. Figure 7.52 The complications of tuberculosis are listed in Box 7.10 and illustrated in Fig. 7.53 . Other complications may include extension into blood vessels with miliary ( Fig. 7.54 ) or systemic dissemination, lymphatic drainage into the pleura with granulomatous pleuritis and effusions, involvement of bronchi by bronchocentric granulomatous lesions ( Fig. 7.55 ), or tuberculous bronchopneumonia. Granulomas may also encroach upon blood vessels, mimicking a "granulomatous" vasculitis. The hemophagocytic syndrome, which has been implicated in a variety of bacterial, viral, and parasitic infections, has also been associated with tuberculosis. 164 Box 7.10 Complications of Tuberculosis Miliary tuberculosis Granulomatous pleuritis and effusions Tuberculous bronchopneumonia Extrapulmonary dissemination to Meninges Kidney Bone Other Alt-text: Box 7.10 Figure 7.53 Complications of tuberculosis. Invasion of arteries (a) with miliary spread; bronchi (br) with tuberculous bronchopneumonia; lymphatics (l) with granulomatous pleuritis and effusions. Invasion of septal (s) veins (v) leads to extrapulmonary dissemination. Figure 7.53 Figure 7.54 Miliary tuberculosis. (A) Miliary pattern. (B) Epithelioid granulomas with necrotic central zones. Figure 7.54 Figure 7.55 Bronchocentric granuloma in mycobacterial infection. Only a small focus of residual bronchial epithelium (b) remains. Figure 7.55 Postprimary Tuberculosis Postprimary tuberculosis, the most common form in adults, typically involves the apices of the upper lobes, producing granulomatous lesions with greater caseation, often with cavities and variable degrees of fibrosis and retraction of the parenchyma. 149 , 160 Fibrosis and bronchiectasis occur with the healing of cavities and is the major cause of pulmonary disability in this disease. 165 Recent studies have proposed that postprimary disease begins as a form of lipoid pneumonia, with bacilli-laden foamy alveolar macrophages and bronchiolar obstruction progressing to caseating cavitary disease and microvascular occlusion due to delayed-type hypersensitivity. 166 Extension to other lobes or hilar/mediastinal lymph nodes and miliary spread through the lungs and to extrapulmonary sites can occur. Other presentation patterns include acute and organizing diffuse alveolar damage with advanced or miliary disease, acute tuberculous bronchopneumonia, and the solitary pulmonary nodule (tuberculoma). A proximal endobronchial form may mimic a neoplasm, noteworthy for its necrosis and large numbers of bacilli. 167 Because characteristic granulomatous morphology may not be visible around the necrotic material, stains for mycobacteria should be considered for all necrotic endobronchial samples. Tuberculous Pleurisy Tuberculosis is a rare cause of chronic pleural effusion. Pleural biopsy may be included when clinical suspicion for tuberculosis is high, both to improve recovery of the organisms and to visualize granulomas. The presence of pleural caseating granulomas can be considered nearly diagnostic of tuberculous pleural effusion and a powerful indication for treatment 168 ; lack of true caseation in the granulomas expands the differential diagnosis to include sarcoid, fungal infection, and rheumatoid disease. Nontuberculous Mycobacterial Infections NTM infections may be similar to those due to M. tuberculosis , but certain differences have been noted. For example, the NTM pathogens do not cause the same sequence of primary or postprimary disease, and systemic dissemination does not occur except in the immunocompromised patient. M. kansasii is more virulent than MAC, and the infection-associated histopathologic pattern is more like that produced by M. tuberculosis. 169 Infections due to MAC and other common pulmonary NTM pathogens generally manifest as one of five clinicopathologic entities: solitary pulmonary nodule, chronic progressive pulmonary disease, disseminated disease, chronic bronchiolitis with bronchiectasis, and hypersensitivity-like pneumonitis. 147 , 170 Solitary pulmonary nodules generally exhibit granulomas resembling those caused by M. tuberculosis . Chronic progressive disease also resembles tuberculosis, with upper lobe thin-walled cavities and granulomatous inflammation with or without caseous necrosis ( Fig. 7.56 ). Multiple confluent granulomas in fibrosis can mimic sarcoidosis. Organisms are usually sparse and more difficult to find in the immunocompetent patient. This presentation most often is seen in patients with underlying chronic lung disease, such as COPD, bronchiectasis, cystic fibrosis, pneumoconiosis, reflux disease, or preexisting cavitary lung disease of any cause (including old tuberculous cavities). Figure 7.56 Nonnecrotizing granuloma in infection due to Mycobacterium avium complex (MAC). Figure 7.56 Disseminated disease is typically associated with the immunocompromise produced by human immunodeficiency virus (HIV) infection, in which the disease tends to target the gastrointestinal tract (the likely portal of entry) and pulmonary and reticuloendothelial disease signifies dissemination. 171 In this setting, NTM bacilli (predominantly MAC) proliferate characteristically to high levels in poorly formed granulomas or in sheets and clusters of plump, finely vacuolated macrophages ("pseudo-Gaucher" cells) containing abundant phagocytosed intracytoplasmic bacilli ( Fig. 7.57 ). Figure 7.57 (A) Clusters of macrophages in Mycobacterium avium complex (MAC) infection in a patient with AIDS. (B) Myriad acid-fast bacilli (MAC) in histiocytic infiltrate (Ziehl–Neelsen stain). Figure 7.57 A distinctive form of NTM disease occurs as the "Lady Windermere syndrome." In the classic clinical scenario, an elderly, nonsmoking, immunocompetent woman of particular habits, demeanor, and body type presents with multiple pulmonary nodules, preferentially involving the middle lobe and lingula. The airway-centric granulomas and bronchiectasis can be subtle or pronounced ( Fig. 7.58 ); these findings have been recognized as one of the patterns of middle lobe syndrome. 172 NTM bacilli can also colonize bronchiectatic lung from any cause, with resultant granulomatous inflammation predominantly affecting the airway walls—presumably as a result of localized decreased mucociliary clearance. Figure 7.58 Middle lobe syndrome. (A) Bronchiectasis with peribronchial granulomas containing Mycobacterium avium complex. (B) Airway mucosa with granuloma. Figure 7.58 Hypersensitivity-like pulmonary disease has been associated with contaminated water in hot tubs ("hot tub lung") and other environmental sources such as humidifiers and air conditioners. 16 Biopsy reveals a miliary bronchiolocentric and interstitial granulomatous pattern, similar to that produced by hypersensitivity pneumonitis ( Fig. 7.59 ). A similar infection-colonization-hypersensitivity syndrome has been described in workers exposed to metalworking fluid aerosols. 173 The clinical, radiologic, and pathologic findings are similar to disease associated with hot tub use and other water sources except that a distinctive rapid-growing NTM species, M. immunogenum , has been recovered almost exclusively. Organisms are difficult to find in these cases but can sometimes be recovered in culture or with molecular techniques. Whether this entity represents an infection, a colonization, a hypersensitivity reaction, or a hybrid condition remains unresolved at this time. Figure 7.59 Hot tub lung. (A) Nonnecrotizing granuloma. (B) Computed tomographic image with features resembling those of hypersensitivity pneumonitis. Figure 7.59 A rare morphologic manifestation of mycobacterial infection is the so-called spindle cell inflammatory pseudotumor ( Fig. 7.60 ), which may occur in lung, skin, lymph nodes, and a number of other sites in immunocompromised patients. 174 The etiologic agents usually are NTM (MAC and M. kansasii ), but M. tuberculosis has also been identified in some cases. Another uncommon variant is proximal endobronchial disease, discussed earlier in the spectrum of postprimary tuberculosis. Most cases are due to M. avium complex and manifest as polypoid lesions in immunocompromised HIV-infected patients, but this lesion may also be seen in immunocompetent persons. 175 Figure 7.60 Spindle cell pseudotumor. (A) The fascicles of fibroblasts with scattered lymphocytes. (B) Myriad acid-fast bacilli (Ziehl–Neelsen stain). Figure 7.60 Certain species of rapidly growing mycobacteria (RGMs) are capable of producing pulmonary disease, albeit infrequently. 145 , 176 , 177 M. abscessus is the third most frequently recovered NTM respiratory pathogen in the United States, after M. avium complex and M. kansasii . M. abscessus produces chronic lung infection that has a striking clinical and pathologic similarity to M. avium complex infection, including the propensity to involve the lungs of patients with bronchiectasis. The RGMs have also been thought to colonize lipoid pneumonia 178 ; however, it is more likely that the pathogenesis of the lung injury pattern caused by the RGMs is similar to that seen in skin and soft tissue cases, in which various combinations of suppurative foci, poorly formed or necrotizing granulomas, scattered multinucleated giant cells, and vacuoles are typical (termed pseudocysts ). 179 These combined features may mimic lipoid pneumonia and constitute an important clue to the presence of RGM infection. Cytopathology Fine-needle aspiration biopsy has been successfully used to diagnose both tuberculous pulmonary lesions and nontuberculous mycobacterial infections. 180 , 181 The finding of finely granular amorphous necrotic debris associated with aggregates of epithelioid histiocytes (with or without multinucleate giant cells; Fig. 7.61 ) is suggestive of a mycobacterial or fungal infection. 182 In this setting, necrotic cancers must be excluded by a thorough search for atypical cells. Epithelioid granulomas manifest a similar cellular pattern, but the granular necrotic debris is absent. Another pattern that may be seen, particularly in specimens from the immunocompromised patient, is a pure histiocytic or macrophage reaction with few or no epithelioid or multinucleate giant cells or necrotic debris. Numerous bacilli may be present in the distended cytoplasm of histiocytes and in the extracellular background. In air-dried (Diff-Quik) and alcohol-fixed (H&E- or Papanicolaou-stained) smears, the bacilli may be recognized as negative images ( Fig. 7.62 ) Figure 7.61 Necrotizing granuloma in Mycobacterium kansasii infection. Sheets of epithelioid cells in a background of granular necroinflammatory debris are evident in this fine-needle aspirate (Diff-Quik preparation). Figure 7.61 Figure 7.62 Pseudo-Gaucher histiocytes filled with myriad mycobacteria are seen as negative images in this fine-needle aspirate (Diff-Quik preparation). Figure 7.62 The use of fine-needle aspirate to target and harvest potential microbiologically positive diagnostic specimens is an important technique, especially in underdeveloped countries where bronchoscopy may not be available. Fine-needle aspiration, especially of affected lymph nodes, combined with an automated rapid PCR diagnostic platform where available (such as Xpert MTB/RIF, Cepheid, Sunnyvale, California) is a public health opportunity for faster diagnosis and containment of disease in areas with a large incidence. 181 , 183 Microbiology The traditional as well as newer molecular approaches to the laboratory diagnosis of mycobacterial lung infection are outlined in Box 7.11 . The mycobacterium is a slender, slightly curved bacillus 4 µm in length, often with a beaded appearance; the length, curvature, and beading are sometimes accentuated in M. kansasii . 184 In tissue sections or on smears, the Ziehl–Neelsen acid-fast stain or auramine-rhodamine fluorescent stains are most often recommended for best visualization; practice among pathologists in the use of acid-fast stains, quality control, and their perception of the value of such stains varies considerably. 185 Organisms are most often found within the area of granulomatous reaction at the immediate periphery of the necrotic zone of the granulomas or the cellular reactive process in the lining of cavities. Sections from several tissue blocks may be required to find organisms. Bacilli are rarely found in the absence of necrosis except in smears from immunocompromised patients, in which they are visible and abundant within pseudo-Gaucher cells on H&E-stained sections, or as ghosted intracellular outlines with Giemsa-type stains. Dead bacilli lose their acid-fast character but may sometimes be identified with the GMS stain. The NTM, especially the RGM, may be more sensitive to acid alcohol decolorization and may not stain well or at all with the auramine-rhodamine method. 145 There are several recent series of confirmed identification using anti-MPT64 for immunostaining of the M. tuberculosis complex in pathology and cytology specimens. 186 , 187 , 188 Differentiation of mycobacterial species in Ziehl–Neelsen–positive, formalin-fixed sections has also been achieved by in situ hybridization techniques with specific nucleic acid probes. 189 , 190 , 191 PCR amplification plus identification is likely to be the most sensitive technique in those cases in which the lesion is suspected to harbor mycobacteria but yields a negative result on acid-fast staining. 192 This technique may also be useful in cases in which the characteristic granulomatous pattern of inflammation is lacking or mycobacteria have been identified in acid-fast–stained sections but culture results remain negative or cultures were not performed. 193 , 194 Box 7.11 Laboratory Diagnosis of Mycobacterial Lung Infection Direct detection of organisms Ziehl–Neelsen; Kinyon acid-fast stains Auramine O fluorescent stain Histopathologic/cytopathologic examination Immunohistochemical studies Culture Conventional solid and broth media Radiometric liquid media system Nonradiometric (fluorescent; colorimetric) liquid media systems Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.11 Conventional wisdom states that culture is more sensitive than direct examination; however, the literature clearly documents cases where acid-fast stains on tissue biopsies succeeded when cultures of tissue failed—an outcome that speaks to the virtue of perseverance in the face of compelling histopathologic findings. 195 Furthermore, tissue culture is prone to sampling error unless more than one site is sampled. 196 Specimens may also be smear-positive and culture-negative in patients whose disease has been treated. When only a rare bacillus is found, strict criteria must be maintained and artifactual pseudo acid-fast bacilli excluded. As a general rule, a cutoff value of three organisms for a positive result seems prudent. False-positive smears can also result from contamination with local tap water, which may harbor mycobacteria. Traditional solid media (Lowenstein–Jensen, Petragani, and Middlebrook agars) have given way to liquid media (radiometric and nonradiometric) as the first-line systems. Liquid media have demonstrated increased recovery of mycobacteria and decreased time to detection. They also facilitate rapid and accurate susceptibility testing. 144 , 197 Some of these liquid systems are manual with visual inspection, whereas others are fully automated and continuously monitored. Most laboratories back up liquid systems with conventional media because no system, at this time, is capable of identifying all isolates. Commercially available DNA probes that hybridize to the mycobacterial RNA have largely replaced traditional biochemical testing, and these methods have significantly shortened the time to identification of M. tuberculosis and selected NTM. 198 For identification of the less frequently isolated species of NTM, for which probes are not available, it usually is necessary to send specimens to reference or state laboratories, where identification is accomplished by either biochemical testing, cell wall analysis using chromatographic techniques, or genotypic sequencing. 151 The rapid differentiation of M. tuberculosis from NTM species is clinically very important because the latter are much less infectious. In this context, molecular techniques have decreased the time to detection and identification of mycobacteria to less than 3 weeks in most instances. Direct nucleic acid amplification testing of clinical specimens using commercially available PCR or transcription-mediated amplification (TMA) methods can reduce detection and identification times to less than 8 hours. 196 Immunochromatographic techniques based on the detection of secreted mycobacterial proteins have the potential to reduce these times even further. 199 Although nucleic acid amplification is faster, its overall accuracy is higher than that of smears but less than that of culture. 198 In fact, no single test at this time has sufficient sensitivity and specificity to stand alone; therefore the use of a combination of available techniques, depending on the clinical and economic setting, may be the best overall strategy. 200 , 201 Interpretation of a culture isolate can sometimes be difficult. The presence of M. tuberculosis is always significant. M. kansasii is an important pathogen, and its isolation is usually also significant, although it may represent colonization. The significance of other NTM isolates is variable depending on whether there is clinical and radiologic evidence of disease. It is in this setting that histopathologic examination plays an important role. M. avium complex can be isolated from the respiratory tract of otherwise healthy adults as well as HIV-infected patients with no clinical or radiologic evidence of disease. The American Thoracic Society has proposed diagnostic criteria requiring that certain clinical, radiologic, and laboratory parameters be met in order to prove pathogenicity. 145 A synopsis of the key morphologic and microbiologic attributes of mycobacterial lung infections is presented in Table 7.6 . Mycobacteria produce a wide spectrum of inflammatory patterns, both granulomatous and nongranulomatous. Although the potential differential diagnostic listing is long, in practical terms major considerations are fungal infections, sarcoidosis, granulomatosis with polyangiitis, and bacterial infections that produce suppurative granulomas, such as those due to Nocardia , Actinomyces , Brucella , and Francisella species. Generally, the use of special stains and cultures will resolve most diagnostic dilemmas. Granulomatosis with polyangiitis can usually be excluded based on the lack of the characteristic tinctorial properties of the necrosis in the granulomas and absence of vasculitis or capillaritis. When necrosis is absent or sparse in a mycobacterial infection, sarcoidosis can be difficult to exclude. Radiologic evidence of bilateral hilar adenopathy and other systemic findings of sarcoidosis often resolve the issue. Table 7.6 Mycobacterial Pneumonias: Summary of Pathologic Findings Table 7.6 Assessment Component Findings Mycobacterial Tuberculosis Surgical pathology Necrotizing (tuberculoid) granulomas Cytopathology Epithelioid cells and necroinflammatory debris; acid-fast bacilli detected with Ziehl–Neelsen or auramine O stains of cell block sections, more sensitive than smears Microbiology Acid-fast bacilli detected with Ziehl–Neelsen, Kinyon stains, or fluorescent bacilli with auramine O stain; culture on Lowenstein-Jensen and Middlebrook selective and nonselective agar and/or liquid media systems, DNA probes, or NAA for identification Nontuberculous Mycobacteria (MOTT) Surgical pathology Granulomas generally with less necrosis; often epithelioid only; unusual patterns (e.g., pseudo-Gaucher and spindle cell proliferation in immunocompromised patients) Cytopathology Epithelioid cells; pseudo-Gaucher or spindle cells with little or no necrosis; negative images in Diff-Quik, confirmed as acid-fast bacilli with Ziehl–Neelsen organisms sparse, except in immunocompromised patient Microbiology As for Mycobacterium tuberculosis MOTT , Mycobacteria other than M. tuberculosis ; NAA , nucleic acid amplification. Etiologic Agents The mycobacterial species can be categorized in two clinically relevant groups: Mycobacterium tuberculosis complex (MTC) and the nontuberculous mycobacteria (NTM). MTC includes the subspecies Mycobacterium tuberculosis , Mycobacterium bovis , Mycobacterium africanum , and Mycobacterium microti . The last three species produce tuberculosis in some areas of the world, but in the United States the prevalence of such disease is very low. Mycobacterium tuberculosis M. tuberculosis is the most virulent mycobacterial species and an unequivocal pathogen that is responsible for numerous deaths worldwide. This organism is the etiologic agent of tuberculosis in its various forms, which are listed in Box 7.8 . Box 7.8 Classification of Tuberculosis Primary tuberculosis Exogenous first infection Exogenous reinfection Progressive primary tuberculosis Postprimary tuberculosis Endogenous reactivation Exogenous infection in BCG-vaccinated persons Exogenous superinfection BCG, Bacille Calmette-Guérin. Alt-text: Box 7.8 Data from Allen E. Tuberculosis and other mycobacterial infections of the lung. In: Churg AM, Thurlbeck WM, eds. Pathology of the Lung, 2nd ed. New York: Thieme; 1995:233, Table 13.1. Primary tuberculosis occurs in patients without previous exposure or with the loss of acquired immunity. Progressive primary tuberculosis occurs in patients with inadequate acquired immunity—that is, impaired cellular immunity. Postprimary tuberculosis, also referred to as secondary or reinfection-reactivation tuberculosis , occurs in patients with previous immunity to the organism and accounts for most clinical cases of tuberculosis. 146 , 147 Many clinical experts consider that most cases of active tuberculosis in adults with normal immunity arise from reactivation of latent infection (postprimary tuberculosis), whereas reinfection with a new strain derived from the environment (primary or postprimary tuberculosis) can occur in the immunocompromised patient. More recently, DNA fingerprinting methods (genotyping) have challenged this dogma by showing that exogenous reinfection accounts for a significant percentage of cases in some areas of the world. 148 Miliary tuberculosis and extrapulmonary disease can occur with any of these forms. 146 , 149 Primary tuberculosis is usually a mild illness that is often not recognized. The bacillemia that occurs during its development can seed extrapulmonary organs and set the stage for subsequent reactivation. Approximately 5% of patients pass through latency to postprimary disease within 2 years of primary infection, and another 5% do so later in their lives. 150 Nontuberculous Mycobacteria Recognized NTM species, many of which were identified during the past decade, number more than 125. 151 , 152 However, relatively few cause pulmonary disease. 145 , 153 , 154 , 155 These organisms are acquired from the environment, where they are ubiquitous. In contrast with M. tuberculosis , the NTM are not spread from person to person. In most instances, patients in whom NTM infection develops have chronic lung disease and other risk factors, such as AIDS, alcoholism, or diabetes. Reports of NTM infections in nonimmunocompromised patients are increasing. 16 , 156 MAC and then Mycobacterium kansasii are the most frequent isolates in all settings. Among a growing number of species causing lung disease are Mycobacterium abscessus , Mycobacteriumfortuitum , Mycobacterium szulgai , Mycobacterium simiae , Mycobacterium xenopi , Mycobacterium malmoense , Mycobacterium celatum , Mycobacterium asiaticum , and Mycobacterium shimodii . These species manifest marked geographic variability with respect to prevalence and severity. Of note, however, since 1985, more MAC isolates than M. tuberculosis have been reported in the United States. 145 Mycobacterium tuberculosis M. tuberculosis is the most virulent mycobacterial species and an unequivocal pathogen that is responsible for numerous deaths worldwide. This organism is the etiologic agent of tuberculosis in its various forms, which are listed in Box 7.8 . Box 7.8 Classification of Tuberculosis Primary tuberculosis Exogenous first infection Exogenous reinfection Progressive primary tuberculosis Postprimary tuberculosis Endogenous reactivation Exogenous infection in BCG-vaccinated persons Exogenous superinfection BCG, Bacille Calmette-Guérin. Alt-text: Box 7.8 Data from Allen E. Tuberculosis and other mycobacterial infections of the lung. In: Churg AM, Thurlbeck WM, eds. Pathology of the Lung, 2nd ed. New York: Thieme; 1995:233, Table 13.1. Primary tuberculosis occurs in patients without previous exposure or with the loss of acquired immunity. Progressive primary tuberculosis occurs in patients with inadequate acquired immunity—that is, impaired cellular immunity. Postprimary tuberculosis, also referred to as secondary or reinfection-reactivation tuberculosis , occurs in patients with previous immunity to the organism and accounts for most clinical cases of tuberculosis. 146 , 147 Many clinical experts consider that most cases of active tuberculosis in adults with normal immunity arise from reactivation of latent infection (postprimary tuberculosis), whereas reinfection with a new strain derived from the environment (primary or postprimary tuberculosis) can occur in the immunocompromised patient. More recently, DNA fingerprinting methods (genotyping) have challenged this dogma by showing that exogenous reinfection accounts for a significant percentage of cases in some areas of the world. 148 Miliary tuberculosis and extrapulmonary disease can occur with any of these forms. 146 , 149 Primary tuberculosis is usually a mild illness that is often not recognized. The bacillemia that occurs during its development can seed extrapulmonary organs and set the stage for subsequent reactivation. Approximately 5% of patients pass through latency to postprimary disease within 2 years of primary infection, and another 5% do so later in their lives. 150 Nontuberculous Mycobacteria Recognized NTM species, many of which were identified during the past decade, number more than 125. 151 , 152 However, relatively few cause pulmonary disease. 145 , 153 , 154 , 155 These organisms are acquired from the environment, where they are ubiquitous. In contrast with M. tuberculosis , the NTM are not spread from person to person. In most instances, patients in whom NTM infection develops have chronic lung disease and other risk factors, such as AIDS, alcoholism, or diabetes. Reports of NTM infections in nonimmunocompromised patients are increasing. 16 , 156 MAC and then Mycobacterium kansasii are the most frequent isolates in all settings. Among a growing number of species causing lung disease are Mycobacterium abscessus , Mycobacteriumfortuitum , Mycobacterium szulgai , Mycobacterium simiae , Mycobacterium xenopi , Mycobacterium malmoense , Mycobacterium celatum , Mycobacterium asiaticum , and Mycobacterium shimodii . These species manifest marked geographic variability with respect to prevalence and severity. Of note, however, since 1985, more MAC isolates than M. tuberculosis have been reported in the United States. 145 Histopathology The histopathologic patterns produced by mycobacteria are listed in Box 7.9 . The radiologic, gross, and microscopic patterns of mycobacterial disease reflect the virulence of the various mycobacterial species as well as the patient's prior exposure and immune status. 157 , 158 , 159 , 160 Box 7.9 Histopathologic Patterns in Mycobacterial Lung Injury Large nodules with or without cavities Well-formed granuloma Poorly formed granuloma Suppurative granuloma Histiocytic aggregates Miliary nodules Calcified nodules Granulomatous interstitial pneumonitis Bronchitis/bronchiectasis Spindle cell pseudotumors Alt-text: Box 7.9 Primary Tuberculosis Mycobacterium tuberculosis occurs typically in the best-aerated lung regions (anterior segments of the upper lobes, lingua and middle lobe, or basal segments of the lower lobes). 158 The disease passes through progressive phases of exudation, recruitment of macrophages and T lymphocytes, and granuloma formation followed by repair with granulation tissue, fibrosis, and mineralization. 147 , 161 Macrophage-laden bacilli also travel to the hilar lymph nodes, where the phases are repeated. This combination of events produces the classic Ghon complex, consisting of a peripheral 1- to 2-cm lung nodule ( Fig. 7.51 ) and an enlarged, sometimes calcified hilar lymph node. In both locations, the histopathologic hallmark is a necrotizing granuloma ( Fig. 7.52 ) composed of epithelioid cells with variable numbers of Langhans giant cells, a peripheral investment of lymphocytes, and a central zone of caseation necrosis, a form of necrosis attributed to apoptosis. 146 , 162 A spectrum of lesions may be seen, from the tuberculoid "hard" granuloma without necrosis and rare organisms to the multibacillary necrotic lesion with scant epithelioid cells. 163 In a minority of patients the lesions enlarge and progress as a result of increased necrosis or liquifaction. Figure 7.51 Tuberculoma removed from right upper lobe. Figure 7.51 Figure 7.52 (A) Tuberculoid granuloma with central zone of caseation necrosis surrounded by epithelioid cells, giant cells, and outer investment of lymphocytes. (B) Palisade of epithelioid histiocytes in giant cells at edge of necrotic zone. Figure 7.52 The complications of tuberculosis are listed in Box 7.10 and illustrated in Fig. 7.53 . Other complications may include extension into blood vessels with miliary ( Fig. 7.54 ) or systemic dissemination, lymphatic drainage into the pleura with granulomatous pleuritis and effusions, involvement of bronchi by bronchocentric granulomatous lesions ( Fig. 7.55 ), or tuberculous bronchopneumonia. Granulomas may also encroach upon blood vessels, mimicking a "granulomatous" vasculitis. The hemophagocytic syndrome, which has been implicated in a variety of bacterial, viral, and parasitic infections, has also been associated with tuberculosis. 164 Box 7.10 Complications of Tuberculosis Miliary tuberculosis Granulomatous pleuritis and effusions Tuberculous bronchopneumonia Extrapulmonary dissemination to Meninges Kidney Bone Other Alt-text: Box 7.10 Figure 7.53 Complications of tuberculosis. Invasion of arteries (a) with miliary spread; bronchi (br) with tuberculous bronchopneumonia; lymphatics (l) with granulomatous pleuritis and effusions. Invasion of septal (s) veins (v) leads to extrapulmonary dissemination. Figure 7.53 Figure 7.54 Miliary tuberculosis. (A) Miliary pattern. (B) Epithelioid granulomas with necrotic central zones. Figure 7.54 Figure 7.55 Bronchocentric granuloma in mycobacterial infection. Only a small focus of residual bronchial epithelium (b) remains. Figure 7.55 Postprimary Tuberculosis Postprimary tuberculosis, the most common form in adults, typically involves the apices of the upper lobes, producing granulomatous lesions with greater caseation, often with cavities and variable degrees of fibrosis and retraction of the parenchyma. 149 , 160 Fibrosis and bronchiectasis occur with the healing of cavities and is the major cause of pulmonary disability in this disease. 165 Recent studies have proposed that postprimary disease begins as a form of lipoid pneumonia, with bacilli-laden foamy alveolar macrophages and bronchiolar obstruction progressing to caseating cavitary disease and microvascular occlusion due to delayed-type hypersensitivity. 166 Extension to other lobes or hilar/mediastinal lymph nodes and miliary spread through the lungs and to extrapulmonary sites can occur. Other presentation patterns include acute and organizing diffuse alveolar damage with advanced or miliary disease, acute tuberculous bronchopneumonia, and the solitary pulmonary nodule (tuberculoma). A proximal endobronchial form may mimic a neoplasm, noteworthy for its necrosis and large numbers of bacilli. 167 Because characteristic granulomatous morphology may not be visible around the necrotic material, stains for mycobacteria should be considered for all necrotic endobronchial samples. Tuberculous Pleurisy Tuberculosis is a rare cause of chronic pleural effusion. Pleural biopsy may be included when clinical suspicion for tuberculosis is high, both to improve recovery of the organisms and to visualize granulomas. The presence of pleural caseating granulomas can be considered nearly diagnostic of tuberculous pleural effusion and a powerful indication for treatment 168 ; lack of true caseation in the granulomas expands the differential diagnosis to include sarcoid, fungal infection, and rheumatoid disease. Nontuberculous Mycobacterial Infections NTM infections may be similar to those due to M. tuberculosis , but certain differences have been noted. For example, the NTM pathogens do not cause the same sequence of primary or postprimary disease, and systemic dissemination does not occur except in the immunocompromised patient. M. kansasii is more virulent than MAC, and the infection-associated histopathologic pattern is more like that produced by M. tuberculosis. 169 Infections due to MAC and other common pulmonary NTM pathogens generally manifest as one of five clinicopathologic entities: solitary pulmonary nodule, chronic progressive pulmonary disease, disseminated disease, chronic bronchiolitis with bronchiectasis, and hypersensitivity-like pneumonitis. 147 , 170 Solitary pulmonary nodules generally exhibit granulomas resembling those caused by M. tuberculosis . Chronic progressive disease also resembles tuberculosis, with upper lobe thin-walled cavities and granulomatous inflammation with or without caseous necrosis ( Fig. 7.56 ). Multiple confluent granulomas in fibrosis can mimic sarcoidosis. Organisms are usually sparse and more difficult to find in the immunocompetent patient. This presentation most often is seen in patients with underlying chronic lung disease, such as COPD, bronchiectasis, cystic fibrosis, pneumoconiosis, reflux disease, or preexisting cavitary lung disease of any cause (including old tuberculous cavities). Figure 7.56 Nonnecrotizing granuloma in infection due to Mycobacterium avium complex (MAC). Figure 7.56 Disseminated disease is typically associated with the immunocompromise produced by human immunodeficiency virus (HIV) infection, in which the disease tends to target the gastrointestinal tract (the likely portal of entry) and pulmonary and reticuloendothelial disease signifies dissemination. 171 In this setting, NTM bacilli (predominantly MAC) proliferate characteristically to high levels in poorly formed granulomas or in sheets and clusters of plump, finely vacuolated macrophages ("pseudo-Gaucher" cells) containing abundant phagocytosed intracytoplasmic bacilli ( Fig. 7.57 ). Figure 7.57 (A) Clusters of macrophages in Mycobacterium avium complex (MAC) infection in a patient with AIDS. (B) Myriad acid-fast bacilli (MAC) in histiocytic infiltrate (Ziehl–Neelsen stain). Figure 7.57 A distinctive form of NTM disease occurs as the "Lady Windermere syndrome." In the classic clinical scenario, an elderly, nonsmoking, immunocompetent woman of particular habits, demeanor, and body type presents with multiple pulmonary nodules, preferentially involving the middle lobe and lingula. The airway-centric granulomas and bronchiectasis can be subtle or pronounced ( Fig. 7.58 ); these findings have been recognized as one of the patterns of middle lobe syndrome. 172 NTM bacilli can also colonize bronchiectatic lung from any cause, with resultant granulomatous inflammation predominantly affecting the airway walls—presumably as a result of localized decreased mucociliary clearance. Figure 7.58 Middle lobe syndrome. (A) Bronchiectasis with peribronchial granulomas containing Mycobacterium avium complex. (B) Airway mucosa with granuloma. Figure 7.58 Hypersensitivity-like pulmonary disease has been associated with contaminated water in hot tubs ("hot tub lung") and other environmental sources such as humidifiers and air conditioners. 16 Biopsy reveals a miliary bronchiolocentric and interstitial granulomatous pattern, similar to that produced by hypersensitivity pneumonitis ( Fig. 7.59 ). A similar infection-colonization-hypersensitivity syndrome has been described in workers exposed to metalworking fluid aerosols. 173 The clinical, radiologic, and pathologic findings are similar to disease associated with hot tub use and other water sources except that a distinctive rapid-growing NTM species, M. immunogenum , has been recovered almost exclusively. Organisms are difficult to find in these cases but can sometimes be recovered in culture or with molecular techniques. Whether this entity represents an infection, a colonization, a hypersensitivity reaction, or a hybrid condition remains unresolved at this time. Figure 7.59 Hot tub lung. (A) Nonnecrotizing granuloma. (B) Computed tomographic image with features resembling those of hypersensitivity pneumonitis. Figure 7.59 A rare morphologic manifestation of mycobacterial infection is the so-called spindle cell inflammatory pseudotumor ( Fig. 7.60 ), which may occur in lung, skin, lymph nodes, and a number of other sites in immunocompromised patients. 174 The etiologic agents usually are NTM (MAC and M. kansasii ), but M. tuberculosis has also been identified in some cases. Another uncommon variant is proximal endobronchial disease, discussed earlier in the spectrum of postprimary tuberculosis. Most cases are due to M. avium complex and manifest as polypoid lesions in immunocompromised HIV-infected patients, but this lesion may also be seen in immunocompetent persons. 175 Figure 7.60 Spindle cell pseudotumor. (A) The fascicles of fibroblasts with scattered lymphocytes. (B) Myriad acid-fast bacilli (Ziehl–Neelsen stain). Figure 7.60 Certain species of rapidly growing mycobacteria (RGMs) are capable of producing pulmonary disease, albeit infrequently. 145 , 176 , 177 M. abscessus is the third most frequently recovered NTM respiratory pathogen in the United States, after M. avium complex and M. kansasii . M. abscessus produces chronic lung infection that has a striking clinical and pathologic similarity to M. avium complex infection, including the propensity to involve the lungs of patients with bronchiectasis. The RGMs have also been thought to colonize lipoid pneumonia 178 ; however, it is more likely that the pathogenesis of the lung injury pattern caused by the RGMs is similar to that seen in skin and soft tissue cases, in which various combinations of suppurative foci, poorly formed or necrotizing granulomas, scattered multinucleated giant cells, and vacuoles are typical (termed pseudocysts ). 179 These combined features may mimic lipoid pneumonia and constitute an important clue to the presence of RGM infection. Primary Tuberculosis Mycobacterium tuberculosis occurs typically in the best-aerated lung regions (anterior segments of the upper lobes, lingua and middle lobe, or basal segments of the lower lobes). 158 The disease passes through progressive phases of exudation, recruitment of macrophages and T lymphocytes, and granuloma formation followed by repair with granulation tissue, fibrosis, and mineralization. 147 , 161 Macrophage-laden bacilli also travel to the hilar lymph nodes, where the phases are repeated. This combination of events produces the classic Ghon complex, consisting of a peripheral 1- to 2-cm lung nodule ( Fig. 7.51 ) and an enlarged, sometimes calcified hilar lymph node. In both locations, the histopathologic hallmark is a necrotizing granuloma ( Fig. 7.52 ) composed of epithelioid cells with variable numbers of Langhans giant cells, a peripheral investment of lymphocytes, and a central zone of caseation necrosis, a form of necrosis attributed to apoptosis. 146 , 162 A spectrum of lesions may be seen, from the tuberculoid "hard" granuloma without necrosis and rare organisms to the multibacillary necrotic lesion with scant epithelioid cells. 163 In a minority of patients the lesions enlarge and progress as a result of increased necrosis or liquifaction. Figure 7.51 Tuberculoma removed from right upper lobe. Figure 7.51 Figure 7.52 (A) Tuberculoid granuloma with central zone of caseation necrosis surrounded by epithelioid cells, giant cells, and outer investment of lymphocytes. (B) Palisade of epithelioid histiocytes in giant cells at edge of necrotic zone. Figure 7.52 The complications of tuberculosis are listed in Box 7.10 and illustrated in Fig. 7.53 . Other complications may include extension into blood vessels with miliary ( Fig. 7.54 ) or systemic dissemination, lymphatic drainage into the pleura with granulomatous pleuritis and effusions, involvement of bronchi by bronchocentric granulomatous lesions ( Fig. 7.55 ), or tuberculous bronchopneumonia. Granulomas may also encroach upon blood vessels, mimicking a "granulomatous" vasculitis. The hemophagocytic syndrome, which has been implicated in a variety of bacterial, viral, and parasitic infections, has also been associated with tuberculosis. 164 Box 7.10 Complications of Tuberculosis Miliary tuberculosis Granulomatous pleuritis and effusions Tuberculous bronchopneumonia Extrapulmonary dissemination to Meninges Kidney Bone Other Alt-text: Box 7.10 Figure 7.53 Complications of tuberculosis. Invasion of arteries (a) with miliary spread; bronchi (br) with tuberculous bronchopneumonia; lymphatics (l) with granulomatous pleuritis and effusions. Invasion of septal (s) veins (v) leads to extrapulmonary dissemination. Figure 7.53 Figure 7.54 Miliary tuberculosis. (A) Miliary pattern. (B) Epithelioid granulomas with necrotic central zones. Figure 7.54 Figure 7.55 Bronchocentric granuloma in mycobacterial infection. Only a small focus of residual bronchial epithelium (b) remains. Figure 7.55 Postprimary Tuberculosis Postprimary tuberculosis, the most common form in adults, typically involves the apices of the upper lobes, producing granulomatous lesions with greater caseation, often with cavities and variable degrees of fibrosis and retraction of the parenchyma. 149 , 160 Fibrosis and bronchiectasis occur with the healing of cavities and is the major cause of pulmonary disability in this disease. 165 Recent studies have proposed that postprimary disease begins as a form of lipoid pneumonia, with bacilli-laden foamy alveolar macrophages and bronchiolar obstruction progressing to caseating cavitary disease and microvascular occlusion due to delayed-type hypersensitivity. 166 Extension to other lobes or hilar/mediastinal lymph nodes and miliary spread through the lungs and to extrapulmonary sites can occur. Other presentation patterns include acute and organizing diffuse alveolar damage with advanced or miliary disease, acute tuberculous bronchopneumonia, and the solitary pulmonary nodule (tuberculoma). A proximal endobronchial form may mimic a neoplasm, noteworthy for its necrosis and large numbers of bacilli. 167 Because characteristic granulomatous morphology may not be visible around the necrotic material, stains for mycobacteria should be considered for all necrotic endobronchial samples. Tuberculous Pleurisy Tuberculosis is a rare cause of chronic pleural effusion. Pleural biopsy may be included when clinical suspicion for tuberculosis is high, both to improve recovery of the organisms and to visualize granulomas. The presence of pleural caseating granulomas can be considered nearly diagnostic of tuberculous pleural effusion and a powerful indication for treatment 168 ; lack of true caseation in the granulomas expands the differential diagnosis to include sarcoid, fungal infection, and rheumatoid disease. Nontuberculous Mycobacterial Infections NTM infections may be similar to those due to M. tuberculosis , but certain differences have been noted. For example, the NTM pathogens do not cause the same sequence of primary or postprimary disease, and systemic dissemination does not occur except in the immunocompromised patient. M. kansasii is more virulent than MAC, and the infection-associated histopathologic pattern is more like that produced by M. tuberculosis. 169 Infections due to MAC and other common pulmonary NTM pathogens generally manifest as one of five clinicopathologic entities: solitary pulmonary nodule, chronic progressive pulmonary disease, disseminated disease, chronic bronchiolitis with bronchiectasis, and hypersensitivity-like pneumonitis. 147 , 170 Solitary pulmonary nodules generally exhibit granulomas resembling those caused by M. tuberculosis . Chronic progressive disease also resembles tuberculosis, with upper lobe thin-walled cavities and granulomatous inflammation with or without caseous necrosis ( Fig. 7.56 ). Multiple confluent granulomas in fibrosis can mimic sarcoidosis. Organisms are usually sparse and more difficult to find in the immunocompetent patient. This presentation most often is seen in patients with underlying chronic lung disease, such as COPD, bronchiectasis, cystic fibrosis, pneumoconiosis, reflux disease, or preexisting cavitary lung disease of any cause (including old tuberculous cavities). Figure 7.56 Nonnecrotizing granuloma in infection due to Mycobacterium avium complex (MAC). Figure 7.56 Disseminated disease is typically associated with the immunocompromise produced by human immunodeficiency virus (HIV) infection, in which the disease tends to target the gastrointestinal tract (the likely portal of entry) and pulmonary and reticuloendothelial disease signifies dissemination. 171 In this setting, NTM bacilli (predominantly MAC) proliferate characteristically to high levels in poorly formed granulomas or in sheets and clusters of plump, finely vacuolated macrophages ("pseudo-Gaucher" cells) containing abundant phagocytosed intracytoplasmic bacilli ( Fig. 7.57 ). Figure 7.57 (A) Clusters of macrophages in Mycobacterium avium complex (MAC) infection in a patient with AIDS. (B) Myriad acid-fast bacilli (MAC) in histiocytic infiltrate (Ziehl–Neelsen stain). Figure 7.57 A distinctive form of NTM disease occurs as the "Lady Windermere syndrome." In the classic clinical scenario, an elderly, nonsmoking, immunocompetent woman of particular habits, demeanor, and body type presents with multiple pulmonary nodules, preferentially involving the middle lobe and lingula. The airway-centric granulomas and bronchiectasis can be subtle or pronounced ( Fig. 7.58 ); these findings have been recognized as one of the patterns of middle lobe syndrome. 172 NTM bacilli can also colonize bronchiectatic lung from any cause, with resultant granulomatous inflammation predominantly affecting the airway walls—presumably as a result of localized decreased mucociliary clearance. Figure 7.58 Middle lobe syndrome. (A) Bronchiectasis with peribronchial granulomas containing Mycobacterium avium complex. (B) Airway mucosa with granuloma. Figure 7.58 Hypersensitivity-like pulmonary disease has been associated with contaminated water in hot tubs ("hot tub lung") and other environmental sources such as humidifiers and air conditioners. 16 Biopsy reveals a miliary bronchiolocentric and interstitial granulomatous pattern, similar to that produced by hypersensitivity pneumonitis ( Fig. 7.59 ). A similar infection-colonization-hypersensitivity syndrome has been described in workers exposed to metalworking fluid aerosols. 173 The clinical, radiologic, and pathologic findings are similar to disease associated with hot tub use and other water sources except that a distinctive rapid-growing NTM species, M. immunogenum , has been recovered almost exclusively. Organisms are difficult to find in these cases but can sometimes be recovered in culture or with molecular techniques. Whether this entity represents an infection, a colonization, a hypersensitivity reaction, or a hybrid condition remains unresolved at this time. Figure 7.59 Hot tub lung. (A) Nonnecrotizing granuloma. (B) Computed tomographic image with features resembling those of hypersensitivity pneumonitis. Figure 7.59 A rare morphologic manifestation of mycobacterial infection is the so-called spindle cell inflammatory pseudotumor ( Fig. 7.60 ), which may occur in lung, skin, lymph nodes, and a number of other sites in immunocompromised patients. 174 The etiologic agents usually are NTM (MAC and M. kansasii ), but M. tuberculosis has also been identified in some cases. Another uncommon variant is proximal endobronchial disease, discussed earlier in the spectrum of postprimary tuberculosis. Most cases are due to M. avium complex and manifest as polypoid lesions in immunocompromised HIV-infected patients, but this lesion may also be seen in immunocompetent persons. 175 Figure 7.60 Spindle cell pseudotumor. (A) The fascicles of fibroblasts with scattered lymphocytes. (B) Myriad acid-fast bacilli (Ziehl–Neelsen stain). Figure 7.60 Certain species of rapidly growing mycobacteria (RGMs) are capable of producing pulmonary disease, albeit infrequently. 145 , 176 , 177 M. abscessus is the third most frequently recovered NTM respiratory pathogen in the United States, after M. avium complex and M. kansasii . M. abscessus produces chronic lung infection that has a striking clinical and pathologic similarity to M. avium complex infection, including the propensity to involve the lungs of patients with bronchiectasis. The RGMs have also been thought to colonize lipoid pneumonia 178 ; however, it is more likely that the pathogenesis of the lung injury pattern caused by the RGMs is similar to that seen in skin and soft tissue cases, in which various combinations of suppurative foci, poorly formed or necrotizing granulomas, scattered multinucleated giant cells, and vacuoles are typical (termed pseudocysts ). 179 These combined features may mimic lipoid pneumonia and constitute an important clue to the presence of RGM infection. Cytopathology Fine-needle aspiration biopsy has been successfully used to diagnose both tuberculous pulmonary lesions and nontuberculous mycobacterial infections. 180 , 181 The finding of finely granular amorphous necrotic debris associated with aggregates of epithelioid histiocytes (with or without multinucleate giant cells; Fig. 7.61 ) is suggestive of a mycobacterial or fungal infection. 182 In this setting, necrotic cancers must be excluded by a thorough search for atypical cells. Epithelioid granulomas manifest a similar cellular pattern, but the granular necrotic debris is absent. Another pattern that may be seen, particularly in specimens from the immunocompromised patient, is a pure histiocytic or macrophage reaction with few or no epithelioid or multinucleate giant cells or necrotic debris. Numerous bacilli may be present in the distended cytoplasm of histiocytes and in the extracellular background. In air-dried (Diff-Quik) and alcohol-fixed (H&E- or Papanicolaou-stained) smears, the bacilli may be recognized as negative images ( Fig. 7.62 ) Figure 7.61 Necrotizing granuloma in Mycobacterium kansasii infection. Sheets of epithelioid cells in a background of granular necroinflammatory debris are evident in this fine-needle aspirate (Diff-Quik preparation). Figure 7.61 Figure 7.62 Pseudo-Gaucher histiocytes filled with myriad mycobacteria are seen as negative images in this fine-needle aspirate (Diff-Quik preparation). Figure 7.62 The use of fine-needle aspirate to target and harvest potential microbiologically positive diagnostic specimens is an important technique, especially in underdeveloped countries where bronchoscopy may not be available. Fine-needle aspiration, especially of affected lymph nodes, combined with an automated rapid PCR diagnostic platform where available (such as Xpert MTB/RIF, Cepheid, Sunnyvale, California) is a public health opportunity for faster diagnosis and containment of disease in areas with a large incidence. 181 , 183 Microbiology The traditional as well as newer molecular approaches to the laboratory diagnosis of mycobacterial lung infection are outlined in Box 7.11 . The mycobacterium is a slender, slightly curved bacillus 4 µm in length, often with a beaded appearance; the length, curvature, and beading are sometimes accentuated in M. kansasii . 184 In tissue sections or on smears, the Ziehl–Neelsen acid-fast stain or auramine-rhodamine fluorescent stains are most often recommended for best visualization; practice among pathologists in the use of acid-fast stains, quality control, and their perception of the value of such stains varies considerably. 185 Organisms are most often found within the area of granulomatous reaction at the immediate periphery of the necrotic zone of the granulomas or the cellular reactive process in the lining of cavities. Sections from several tissue blocks may be required to find organisms. Bacilli are rarely found in the absence of necrosis except in smears from immunocompromised patients, in which they are visible and abundant within pseudo-Gaucher cells on H&E-stained sections, or as ghosted intracellular outlines with Giemsa-type stains. Dead bacilli lose their acid-fast character but may sometimes be identified with the GMS stain. The NTM, especially the RGM, may be more sensitive to acid alcohol decolorization and may not stain well or at all with the auramine-rhodamine method. 145 There are several recent series of confirmed identification using anti-MPT64 for immunostaining of the M. tuberculosis complex in pathology and cytology specimens. 186 , 187 , 188 Differentiation of mycobacterial species in Ziehl–Neelsen–positive, formalin-fixed sections has also been achieved by in situ hybridization techniques with specific nucleic acid probes. 189 , 190 , 191 PCR amplification plus identification is likely to be the most sensitive technique in those cases in which the lesion is suspected to harbor mycobacteria but yields a negative result on acid-fast staining. 192 This technique may also be useful in cases in which the characteristic granulomatous pattern of inflammation is lacking or mycobacteria have been identified in acid-fast–stained sections but culture results remain negative or cultures were not performed. 193 , 194 Box 7.11 Laboratory Diagnosis of Mycobacterial Lung Infection Direct detection of organisms Ziehl–Neelsen; Kinyon acid-fast stains Auramine O fluorescent stain Histopathologic/cytopathologic examination Immunohistochemical studies Culture Conventional solid and broth media Radiometric liquid media system Nonradiometric (fluorescent; colorimetric) liquid media systems Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.11 Conventional wisdom states that culture is more sensitive than direct examination; however, the literature clearly documents cases where acid-fast stains on tissue biopsies succeeded when cultures of tissue failed—an outcome that speaks to the virtue of perseverance in the face of compelling histopathologic findings. 195 Furthermore, tissue culture is prone to sampling error unless more than one site is sampled. 196 Specimens may also be smear-positive and culture-negative in patients whose disease has been treated. When only a rare bacillus is found, strict criteria must be maintained and artifactual pseudo acid-fast bacilli excluded. As a general rule, a cutoff value of three organisms for a positive result seems prudent. False-positive smears can also result from contamination with local tap water, which may harbor mycobacteria. Traditional solid media (Lowenstein–Jensen, Petragani, and Middlebrook agars) have given way to liquid media (radiometric and nonradiometric) as the first-line systems. Liquid media have demonstrated increased recovery of mycobacteria and decreased time to detection. They also facilitate rapid and accurate susceptibility testing. 144 , 197 Some of these liquid systems are manual with visual inspection, whereas others are fully automated and continuously monitored. Most laboratories back up liquid systems with conventional media because no system, at this time, is capable of identifying all isolates. Commercially available DNA probes that hybridize to the mycobacterial RNA have largely replaced traditional biochemical testing, and these methods have significantly shortened the time to identification of M. tuberculosis and selected NTM. 198 For identification of the less frequently isolated species of NTM, for which probes are not available, it usually is necessary to send specimens to reference or state laboratories, where identification is accomplished by either biochemical testing, cell wall analysis using chromatographic techniques, or genotypic sequencing. 151 The rapid differentiation of M. tuberculosis from NTM species is clinically very important because the latter are much less infectious. In this context, molecular techniques have decreased the time to detection and identification of mycobacteria to less than 3 weeks in most instances. Direct nucleic acid amplification testing of clinical specimens using commercially available PCR or transcription-mediated amplification (TMA) methods can reduce detection and identification times to less than 8 hours. 196 Immunochromatographic techniques based on the detection of secreted mycobacterial proteins have the potential to reduce these times even further. 199 Although nucleic acid amplification is faster, its overall accuracy is higher than that of smears but less than that of culture. 198 In fact, no single test at this time has sufficient sensitivity and specificity to stand alone; therefore the use of a combination of available techniques, depending on the clinical and economic setting, may be the best overall strategy. 200 , 201 Interpretation of a culture isolate can sometimes be difficult. The presence of M. tuberculosis is always significant. M. kansasii is an important pathogen, and its isolation is usually also significant, although it may represent colonization. The significance of other NTM isolates is variable depending on whether there is clinical and radiologic evidence of disease. It is in this setting that histopathologic examination plays an important role. M. avium complex can be isolated from the respiratory tract of otherwise healthy adults as well as HIV-infected patients with no clinical or radiologic evidence of disease. The American Thoracic Society has proposed diagnostic criteria requiring that certain clinical, radiologic, and laboratory parameters be met in order to prove pathogenicity. 145 A synopsis of the key morphologic and microbiologic attributes of mycobacterial lung infections is presented in Table 7.6 . Mycobacteria produce a wide spectrum of inflammatory patterns, both granulomatous and nongranulomatous. Although the potential differential diagnostic listing is long, in practical terms major considerations are fungal infections, sarcoidosis, granulomatosis with polyangiitis, and bacterial infections that produce suppurative granulomas, such as those due to Nocardia , Actinomyces , Brucella , and Francisella species. Generally, the use of special stains and cultures will resolve most diagnostic dilemmas. Granulomatosis with polyangiitis can usually be excluded based on the lack of the characteristic tinctorial properties of the necrosis in the granulomas and absence of vasculitis or capillaritis. When necrosis is absent or sparse in a mycobacterial infection, sarcoidosis can be difficult to exclude. Radiologic evidence of bilateral hilar adenopathy and other systemic findings of sarcoidosis often resolve the issue. Table 7.6 Mycobacterial Pneumonias: Summary of Pathologic Findings Table 7.6 Assessment Component Findings Mycobacterial Tuberculosis Surgical pathology Necrotizing (tuberculoid) granulomas Cytopathology Epithelioid cells and necroinflammatory debris; acid-fast bacilli detected with Ziehl–Neelsen or auramine O stains of cell block sections, more sensitive than smears Microbiology Acid-fast bacilli detected with Ziehl–Neelsen, Kinyon stains, or fluorescent bacilli with auramine O stain; culture on Lowenstein-Jensen and Middlebrook selective and nonselective agar and/or liquid media systems, DNA probes, or NAA for identification Nontuberculous Mycobacteria (MOTT) Surgical pathology Granulomas generally with less necrosis; often epithelioid only; unusual patterns (e.g., pseudo-Gaucher and spindle cell proliferation in immunocompromised patients) Cytopathology Epithelioid cells; pseudo-Gaucher or spindle cells with little or no necrosis; negative images in Diff-Quik, confirmed as acid-fast bacilli with Ziehl–Neelsen organisms sparse, except in immunocompromised patient Microbiology As for Mycobacterium tuberculosis MOTT , Mycobacteria other than M. tuberculosis ; NAA , nucleic acid amplification. Fungal Pneumonias The pathologist examining fungi can provide at least a provisional diagnosis at the group or genus level and make a judgment about fungal invasion or the presence of fungi as a pathogen, saprophyte, or allergen. One effective diagnostic strategy available is the rapid identification of fungi in frozen sections, routine sections, or cytologic samples. 45 , 202 , 203 This approach is especially important when opportunistic infection is being considered in the immunocompromised patient. Prudent practice requires caution in morphologic diagnosis alone; integration of microbiologic data and histopathologic findings is required. Etiologic Agents Nearly 70,000 fungi are known, and approximately 100 have been recovered from respiratory infections. 204 A small number are implicated as pathogenic on a consistent basis; these are listed in Box 7.12 . Box 7.12 Common Fungal Pathogens in the Lung Dimorphic fungi (mycelia at 25–30°C; yeast at 37°C) Blastomyces dermatitidis Coccidioides immitis Histoplasma capsulatum Paracoccidioides braziliensis Sporothrix schenckii Penicillium marneffei Yeasts Cryptococcus neoformans Candida spp. Hyaline (nonpigmented) molds Aspergillus spp. Zygomycetes organisms Phaeoid (pigmented; dematiaceous) molds Bipolaris spp. , Alternaria, Curvularia Pseudoallescheria boydii/Scedosporium apiospermum Miscellaneous pathogens Pneumocystis jirovecii Alt-text: Box 7.12 Histopathology Like mycobacterial species, fungal pathogens typically produce one or more nodular lesions in the normal host ( Fig. 7.63 ); these may become cavitary as the lesions evolve ( Fig. 7.64 ). Inflammatory histopathologic patterns that suggest the presence of a fungal infection are summarized in Box 7.13 . As is the case for other etiologic agents, there are no absolutely diagnostic patterns. Overlap is common and atypical reactions occur, ranging from overwhelming diffuse alveolar damage, little or no reaction, or sheets of organisms in the immunocompromised patient. Proximal endobronchial disease mimicking a neoplasm has also been described for various fungal species. 205 Detection of the etiologic agent in tissue by microscopic examination, ancillary tests, or culture confers specificity and significance to the listed patterns. Large spherules with endospores characteristic of Coccidioides immitis or yeast with large mucoid capsules of C. neoformans can be diagnostic. However, atypical forms of these organisms can be misleading and challenging. For example, in aerated cavities or in the setting of bronchopleural fistula, Coccidioides species may produce branching septate and moniliform hyphae or immature morula-like spherules mimicking other fungi (e.g., hyaline molds and Blastomyces dermatitidis ). 40 Similarly, C. neoformans , H. capsulatum , and S. schenckii have been reported to produce hyphae or pseudohyphae in tissue, whereas acapsular C. neoformans may mimic other yeasts or Pneumocystis organisms. 206 Figure 7.63 Coccidioides granuloma. Figure 7.63 Figure 7.64 Cavitary aspergilloma. Figure 7.64 Box 7.13 Histopathologic Patterns in Fungal Lung Injury Large nodules Nonnecrotizing granulomas Necrotizing granulomas Suppurative granulomas Poorly formed granulomas Cavitary lesions Miliary nodules Acute bronchopneumonia Airway disease Intravascular changes/infarct Diffuse alveolar damage, acute and organizing Foamy alveolar casts Alt-text: Box 7.13 Mycelial morphology is helpful when it is characteristic of a specific genus or group. For example, broad, sparsely septate, nonparallel, twisted or irregular in diameter, thin-walled mycelia with variable wide-angle branching characterize Zygomycetes. Progressively proliferating, regularly septate, 45-degree angle, dichotomously branching mycelia with parallel walls are typical of Aspergillus species ( Fig. 7.65 ). In the case of Aspergillus , an important point is that only the presence of a fruiting body (conidiophore with sterigmata and conidia) permits diagnosis at the genus level, and there are many Aspergillus look-alikes in tissue, such as Fusarium , Paecilomyces , Acremonium , Bipolaris , Pseudallescheria boydii , and its asexual anamorph Scedosporium apiospermum . 206 Sometimes careful examination of tissue with special stains under high magnification or oil immersion will reveal clues, such as in situ sporulation, allowing a more definitive diagnosis. 41 However, these clues are often subtle, and it is important to defer to culture whenever possible. 207 Typical morphologic injury patterns and related etiologic agents are detailed next. Figure 7.65 Aspergillus species. (A) Septate mycelia with 45-degree angle branching (Grocott methenamine silver stain). (B) Fruiting body (conidiophore with sterigmata and conidia) (Grocott methenamine silver stain). Figure 7.65 Blastomycosis Blastomycosis, the chronic granulomatous and suppurative infection produced by B. dermatitidis , is essentially a North American disease, concentrated in the Ohio and the Mississippi River valleys. The prevalence of infection is particularly high in the state of Mississippi. Blastomycosis is the third most common endemic mycosis in North America, following histoplasmosis and coccidioidomycosis. It may occur in patients with normal immunity as well as those immunocompromised by diseases or medical therapy. 208 , 209 , 210 The isolated nodular manifestation can simulate lung cancer radiologically. 211 The disease almost always begins in the lungs, although skin and bone are other common sites of involvement. In the lung, pathologic manifestations include focal or diffuse infiltrates, rare lobar consolidation, miliary nodules, solitary nodules, and acute or organizing diffuse alveolar damage ( Box 7.14 ). 208 , 211 , 212 , 213 Necrotizing granulomas are characteristic and often of the suppurative type ( Fig. 7.66A ), but nonnecrotizing granulomas may be found as well. Box 7.14 Histopathologic Patterns in Pulmonary Blastomycosis Acute pneumonia Lobular Lobar Diffuse alveolar damage Miliary nodule Solitary nodule Alt-text: Box 7.14 Figure 7.66 Blastomycosis. (A) The suppurative granuloma is characteristic. (B) Double-contour-wall yeast with broad-based budding. Figure 7.66 The broad-based budding yeast forms of Blastomyces are refractile and have double-contoured walls. Multinucleate yeast cells are typically 8 to 15 µm in diameter, with some forms measuring up to 30 µm ( Fig. 7.66B ). These large forms can mimic small Coccidioides spherules, 214 whereas smaller forms ("microforms") can mimic C. neoformans . 213 Coccidioidomycosis Endemic in the Lower Sonoran life zone of the southwestern United States, the soil fungus C. immitis and the more recently recognized, morphologically identical, and genomically similar species Coccidioides posadasii 215 may be encountered outside the endemic area as a result of fomite transmission of arthroconidia (e.g., Asian textile workers handling imported Arizona cotton) or in travelers who have returned from an endemic area. Most primary pulmonary infections are asymptomatic. The exceptionally wide spectrum of pulmonary pathology in patients with clinically evident disease is outlined in Box 7.15 . The true prevalence of the disease is significantly underestimated in endemic regions of the Southwest, where it is thought to account for nearly 30% of community-acquired pneumonias in some metropolitan areas. 216 , 217 , 218 , 219 Granulomas are characteristic and may occur with or without necrosis. Intact spherules induce fibrocaseous granulomas ( Fig. 7.67A ), whereas ruptured spherules may incite suppurative and bronchocentric granulomatosis (BCG)-like reactions ( Fig. 7.67B ). 216 Box 7.15 Histopathologic Patterns in Coccidioidal Respiratory Tract Disease Airway disease Pharyngeal granuloma Laryngeal granuloma Tracheobronchial granuloma Pulmonary parenchymal disease Acute pneumonia Eosinophilic pneumonia Chronic progressive infection Fibrocavitary lesions Bronchopleural fistula; empyema Solitary pulmonary nodule Disseminated disease Miliary Extrapulmonary Alt-text: Box 7.15 Figure 7.67 Coccidioidomycosis. (A) Fibrocaseous granuloma. (B) Bronchocentric granulomatosis–like granuloma. (C) Coccidioides immitis . Both small (arrow) and large spherules with and without endospores can be seen. (D) Biphasic pattern with mycelia and spore-like swellings (Grocott methenamine silver stain). Figure 7.67 The large mature spherule (up to 40 to 60 µm in diameter) has a thick refractile wall lined by or filled with endospores; it constitutes the key diagnostic finding ( Fig. 7.67C ). This finding allows the distinction of coccidioidomycosis from other fungal infections such as blastomycosis and histoplasmosis, which are associated with similar histopathologic reaction patterns. In aerated cavities or in the setting of bronchopleural fistula, mycelia resembling various hyaline molds may be seen with or without a variety of mature and immature spherules ( Figs. 7.20 and 7.67D ). Coccidioides spherule look-alikes include large-variant B. dermatitidis, adiaspiromycosis, pollen grains, and pulses (legume seeds). Histoplasmosis Histoplasmosis, the most common pulmonary fungal infection worldwide, is endemic in the Ohio and the Mississippi River valleys of North America and is the most common endemic mycosis in AIDS. 220 The clinical forms of H. capsulatum infection 55 , 203 , 221 , 222 are presented in Box 7.16 . The histopathologic correlates include a spectrum ranging from an exudative to a granulomatous process influenced by such factors as the fungal burden and the immune status of the patient. In patients with normal defenses, the characteristic histopathology is dominated by well-formed necrotizing and nonnecrotizing granulomas occurring as solitary lesions indistinguishable from other granulomatous infections. Other presentations include miliary nodules ( Fig. 7.68 ), cavitary lesions, and laminated fibrous solitary nodules ( Fig. 7.69 ) that may be partially calcified (sometimes referred to as residual granulomas ). In patients with impaired immunity, striking macrophage response with numerous intracellular yeasts is a characteristic pattern ( Fig. 7.70A ). The exudative lesion resembles acute lobular pneumonia with fibrinopurulent exudates. 223 Box 7.16 Clinical Forms of Pulmonary Histoplasmosis Benign, self-limited Acute Acute respiratory distress syndrome Acute self-limited, upper lobe (in smokers with emphysema) Chronic Asymptomatic pulmonary nodule, with or without calcification ("histoplasmoma") Progressive (chronic cavitary) pulmonary Progressive disseminated Mediastinal Lymphadenopathy Middle lobe syndrome Fibrosis Alt-text: Box 7.16 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.6. Figure 7.68 Histoplasmosis. Miliary nodule with central zone of necrosis invested by epithelioid histiocytes, multinucleate giant cells, and outer collarette of lymphocytes. Figure 7.68 Figure 7.69 Histoplasmoma. Characteristic gross appearance of the persistent granulomatous nodule. Note the fibrous wall (arrow) surrounding caseous necrosis (n) . Figure 7.69 Figure 7.70 Histoplasmosis in an immunocompromised patient. (A) Numerous Histoplasma capsulatum yeast cells in macrophages. (B) Clusters of H. capsulatum yeast cells in macrophages. Note the narrow-based budding (arrow) (Grocott methenamine silver stain). Figure 7.70 H. capsulatum organisms are yeasts (2 to 5 µm), with narrow-based unequal budding ( Fig. 7.71 ; Fig. 7.70B ). They may be seen on H&E-stained sections and, when numerous, appear as small refractile ovoid structures within macrophages. Yeasts typically occur in clusters but may be rare in old granulomas. A search for budding organisms in these situations may prove futile. Sometimes, yeasts may have dark-staining foci resembling Pneumocystis organisms. Also, some yeast cells may be surrounded by a clear space and may be mistaken for Cryptococcus . 55 Other look-alikes include Candida species, P. marneffei , capsule-deficient cryptococci, intracellular B. dermatitidis , and Hamazaki-Wesenberg bodies. Figure 7.71 Tinctorial and morphologic attributes of H. capsulatum in stained clinical specimens. (A) Diff-Quik: BAL fluid smear showing extracellular yeast forms. (B) Giemsa stain: tissue touch preparation demonstrating numerous yeast within a histiocyte and in the surrounding space. (C) GMS stain: abundant black-colored yeast cells in a tissue touch preparation. Both extracellular and intrahistiocytic forms are seen. (D) Gram stain: red-colored yeast cells in a blood culture smear. (E) Hematoxylin and eosin stain: liver biopsy containing numerous intracellular and extracellular yeast forms. Note the presence of colorless halos surrounding the yeast. (F) Mucicarmine stain: yeast forms are barely visible without the aid of increased contrast. (G) Periodic acid–Schiff stain: magenta-colored yeast cells are evident scattered throughout the epidermis of a skin biopsy specimen. (H) Wright-Giemsa: yeast forms evident within a monocyte in a peripheral blood smear. Scale bar, 10 mm; original magnification, ×1000. Figure 7.71 (From Wheat LJ, Azar MM, Bahr NC, et al. Histoplasmosis. Inf Dis Clin NA . 2016;30:216.) Paracoccidioidomycosis (South American Blastomycosis) Seven clinical forms occur, but they rarely cause lung infections in North America. In areas of high endemicity, such as Brazil, the several forms can mimic malignancy or sarcoidosis. 224 , 225 The histopathology resembles that of other mycoses and can be exudative or granulomatous. Paracoccidioides braziliensis appears as a large spherical yeast (10 to 60 µm) with multiple buds attached by narrow necks (a "steering wheel" or "ship's wheel" appearance). 226 When budding is sparse, look-alikes include H. capsulatum with small intracellular forms, B. dermatitidis and capsule-deficient cryptococci for medium-sized forms, and C. immitis or C. posadasii for large forms. Sporotrichosis Infection by Sporothrix schenckii is usually confined to the skin, subcutis, and lymphatic pathways, but the organism can disseminate to the lungs. Rarely, S. schenckii is a primary pulmonary pathogen. 227 The organism can produce cavitary disease in the form of a single lesion. Infection may be bilateral and apical, progressive and destructive, or it may be identified clinically as a solitary pulmonary nodule. Microscopically, caseous and suppurative granulomas ( Fig. 7.72A ) occur with variable numbers of round to oval, small (2 to 3 µm), narrow budding yeast ( Fig. 7.72B ) or cigar-shaped forms. 228 Nonnecrotizing granulomas also occur. Asteroid bodies are an important clue, especially when organisms are sparse, as is often the case. Look-alikes include H. capsulatum , acapsular cryptococci, Candida organisms, and Hamazaki-Wesenberg bodies. Figure 7.72 Sporotrichosis. (A) Cavitary granuloma manifesting as a solitary pulmonary nodule. (B) A rare, oval, narrow budding yeast (Grocott methenamine silver stain). Figure 7.72 Penicilliosis Southeast Asia is the endemic setting of the unique dimorphic fungus Penicillium marneffei . The disease it produces is not seen in North America except in travelers, especially immunocompromised persons. It is a common opportunistic infections in AIDS patients in Southeast Asia and a significant clue to the presence of AIDS in that area. 229 The respiratory tract is the portal of entry, with pulmonary infiltrates and disseminated disease, especially involving the skin. Microscopically, alveolar macrophages stuffed with spherical to oval yeast-like cells (2.5 to 5 µm) are seen, each with a single transverse septum; short hyphal forms and elongated, curved "sausage" forms may be formed in necrotic and cavitary lesions. 230 , 231 The septum distinguishes it from H. capsulatum , its look-alike. 229 , 232 Cryptococcosis C. neoformans is a ubiquitous, facultative intracellular yeast. Pulmonary cryptococcosis occurs worldwide but has a particularly high incidence in the United States. 233 The pathogenicity and histopathologic features of lung infection depends largely on the patient's immune status, as illustrated earlier in Fig. 7.7 and summarized in Box 7.17 . In the normal host, a substantial proportion of cryptococcal infections are asymptomatic, others are symptomatic, with infiltrates or nodules. Immunocompromised patients are almost invariably symptomatic and often develop disseminated disease with a predilection for the brain and meninges. Pulmonary injury patterns include single or multiple large nodules, segmental or diffuse infiltrates, cavitary lesions, and miliary nodules. Normal hosts most often develop nodules comprising fibrocaseous granulomas ( Fig. 7.73A ), or granulomatous pneumonia ( Fig. 7.73B ). Immunocompromised patients are more likely to have histiocytic ( Fig. 7.73C ) or mucoid infiltrates without inflammation ( Fig. 7.73D ). Box 7.17 Histopathologic Patterns in Cryptococcal Lung Disease In order of associated decrease in immune function: Fibrocaseous granuloma Granulomatous pneumonia Histiocytic pneumonia Mucoid pneumonia Intracapillary cryptococcosis Alt-text: Box 7.17 Reprinted with permission from Mark EJ. Case records of the Massachusetts General Hospital. N Engl J Med. 2002;347:518–524. Figure 7.73 Cryptococcosis. (A) Solitary pulmonary nodule with small satellite granulomas. (B) Granulomatous pneumonia with clusters of pale staining yeast in clear spaces surrounded by histiocytes and multinucleated giant cells. (C) Histiocytic pneumonia. (D) Mucoid pneumonia with no inflammatory cell reaction. Figure 7.73 The cryptococcal organisms are round yeast forms ranging in diameter from 2 to 15 µm, with an average size of 4 to 7 µm. Cryptococcal yeasts are visible on H&E-stained sections as pale gray to light blue structures, frequently with attached smaller buds. They often occur in clusters and can sometimes be found within giant cells. 203 The mucicarmine stain highlights the capsule ( Fig. 7.74A ); but with capsule-deficient forms ( Fig. 7.74B ) the pleomorphic appearance can be confused with that of other yeast forms (e.g., H. capsulatum , B. dermatitidis , S. schenckii ) and sometimes Pneumocystis . Figure 7.74 (A) Intravascular cryptococcus. Yeast cells with stained capsules (mucicarmine stain). (B) Capsule-deficient cryptococcus (Grocott methenamine silver stain). Figure 7.74 The lungs of patients with the most severe immunodeficiency may show myriad yeasts in alveolar septal capillaries ( Fig. 7.74A ), with little if any intraalveolar reaction 234 ; this form of the disease may also be associated with mucoid pneumonia. 235 The mucoid pneumonia ( Fig. 7.75A ) of cryptococcal infection can be confirmed with mucin stains such as Alcian blue ( Fig. 7.75B ). Another microscopic pattern recently described in HIV-infected patients is the inflammatory spindle cell pseudotumor, a lesion much more commonly associated with mycobacterial infection. 236 Figure 7.75 Cryptococcal mucoid pneumonia. (A) Myriad blue-gray yeast cells in mucoid matrix. (B) Alcian blue mucin stain accentuates the mucoid matrix. Figure 7.75 Candidiasis Candida organisms are yeasts that can produce pseudohyphae and are the most common invasive fungal pathogens in humans. Secondary Candida pneumonia is relatively common, but primary Candida pneumonia is rare in other than immunocompromised patients in the intensive care unit. 55 In general, Candida albicans is the most frequently isolated of the more than 100 known species, which include a few rare and emerging human pathogens. Candida glabrata and Candida tropicalis , together with C. albicans , account for 95% of bloodstream infections, the principal route for the acquisition of Candida pneumonia. 237 A non–blood-borne route to pneumonia results from aspiration of organisms from a heavily colonized or infected oropharynx. When the infection is blood-borne, miliary nodules with a necroinflammatory center and a hemorrhagic rim reflect an intravascular distribution of fungi. In the case of aspiration, the organisms may be found in the airways associated with an alveolar filling pattern of bronchopneumonia ( Fig. 7.76A ) 238 or, much less commonly, a bronchocentric granulomatosis pattern. Figure 7.76 (A) Candida bronchopneumonia. (B) Candida yeast cells—blastoconidia (Grocott methenamine silver stain). Figure 7.76 In tissue sections, oval budding yeast-like cells (blastoconidia) 2 to 6 µm in diameter may appear with pseudohyphae that constrict at points of budding, creating the impression of bulging rather than parallel walls ( Fig. 7.76B ). The pseudohyphae branch at acute angles and can overlap in width with the true hyphae of Aspergillus , from which they must be distinguished. Among the medically important species, Candida glabrata (formerly Torulopsis glabrata ) and Candida parapsilosis produce only yeast cells in tissue, in contrast with most other Candida species, which produce both yeast and pseudohyphae. 203 Other look-alikes include H. capsulatum , Trichosporon beigelii , and Malassezia furfur , depending on whether pseudohyphae or yeast forms alone are present. They can be distinguished from Histoplasma by their extracellular location and Gram stain positivity. T. beigelii tends to be somewhat larger and more pleomorphic. Malassezia is clinically associated with parenteral nutrition, Intralipid, and indwelling catheters. Pulmonary lesions include pneumonia, mycotic thromboemboli, infarcts, and vasculitis. M. furfur may be found in small arteries, where the organisms appear as small 2- to 5-µm yeast-like cells. They form distinctive unipolar broad-based buds but no pseudohyphae. 55 Aspergillosis Aspergillus species and other hyaline and dematiaceous molds have emerged as significant causes of morbidity and death in the immunocompromised host. Worldwide, species of Aspergillus are the most common invasive molds. They are the second most common fungal pathogens after Candida species but, in contrast with Candida , are more commonly isolated from the lung. Several species are recognized, but Aspergillus fumigatus is the one most often seen in the clinical laboratory and most often isolated from the lungs of immunocompromised patients. 239 Respiratory aspergillosis can be classified into a colonizing or saprophytic form (intrabronchial and preexisting cavity fungus ball, Fig. 7.77A ); hypersensitivity forms (allergic bronchopulmonary aspergillosis, including mucoid impaction of bronchi and hypersensitivity pneumonitis; Fig. 7.77B ); and invasive disease (minimally invasive–chronic necrotizing or angioinvasive–disseminated, Box 7.18 ). 55 , 240 , 241 , 242 , 243 Invasive disease ( Fig. 7.78 ) tends to occur in immunocompromised patients, including those with prolonged neutropenia, transplant recipients (especially hematopoietic stem cell and lung transplants), advanced AIDS, and the inherited immune deficiency disorder referred to as chronic granulomatous disease of childhood . The clinicopathologic features of invasive disease reflect these host-associated risk factors. 244 In patients with neutropenia, a characteristic angioinvasive pattern occurs, with intravascular spread resulting in hemorrhagic infarcts ( Fig. 7.79 ). In the nonneutropenic patient, the necroinflammatory pattern tends to lack this angioinvasive feature. 245 Some cases defy categorization (e.g., bronchocentric and miliary patterns; Fig. 7.80 ) or may be hybrids of infection and hypersensitivity. 246 Figure 7.77 (A) Aspergillosis fungus ball. (B) Allergic bronchopulmonary aspergillosis. Intraluminal allergic mucin with laminated clusters of eosinophils can be seen in inspissated basophilic mucin with scattered Charcot–Leyden crystals. Figure 7.77 Box 7.18 Histopathologic Patterns in Pulmonary Aspergillosis Colonization Fungus ball Hypersensitivity reaction Allergic bronchopulmonary aspergillosis Eosinophilic pneumonia Mucoid impaction Bronchocentric granulomatosis Hypersensitivity pneumonitis Invasive Acute invasive aspergillosis Necrotizing pseudomembranous tracheobronchitis Chronic necrotizing pneumonia Bronchopleural fistula Empyema Alt-text: Box 7.18 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.10. Figure 7.78 Resected lung specimen from an immunocompromised patient with necrotizing Aspergillus pneumonia. Figure 7.78 Figure 7.79 Invasive aspergillosis. (A) Hemorrhagic infarct. (B) 45-degree angle branching septate hyphae. Figure 7.79 Figure 7.80 Bronchocentric aspergillosis. (A) Bronchiole expanded and filled with purulent exudate. (B) Miliary aspergillosis. Colony of organisms with hyaline membranes evident at periphery of the image (lower right) . Figure 7.80 Microscopically, septate hyphae, dichotomously branched at a 45-degree angle, have uniform, consistent width (3 to 6 µm) without constrictions at points of septation . When numerous, as in some angioinvasive lesions and fungus balls, these features can be readily appreciated in H&E-stained sections. Fruiting heads of Aspergillus (shown earlier in Fig. 7.65 ) are sometimes formed in cavities. Oxalate crystals, visible in plane-polarized light ( Fig. 7.81 ), are an important clue to Aspergillus infection when hyphae cannot be identified. Figure 7.81 (A) Pale yellow oxalate crystal sheaths in necroinflammatory debris. (B) Birefringent oxalates seen under polarized light. Figure 7.81 Look-alikes include various hyaline molds such as Zygomycetes and Candida species as well as Pseudallescheria boydii . 247 Another look-alike is Fusarium species. Fusariosis is an emerging mycosis in the immunocompromised host, and Fusarium is the second most common opportunistic pathogen after Aspergillus species in immunosuppressed patients with hematologic malignancies. 248 The clinical and pathologic features in the lung and at sites of dissemination mimic those of aspergillosis, and the mycelia are essentially indistinguishable. Isolation in culture, immunohistochemistry, or molecular techniques, such as in situ hybridization or PCR amplification, is required for definitive diagnosis. Other previously uncommon but newly emerging hyaline molds that may be difficult to distinguish from Aspergillus in tissue are Paecilomyces , Acremonium , Scedosporium , and Basidiobolus . 237 , 249 , 250 Zygomycosis The taxonomic organization of the fungal phylum Zygomycota includes the class Zygomycetes, which is subdivided into two orders: Mucorales and Entomophthorales. These orders contain the agents of human zygomycosis. 251 The order Mucorales includes the genera Absidia , Apophysomyces , Rhizopus , Rhizomucor, and Mucor , from which the often taxonomically incorrect term mucormycosis is derived. In fact, most infections are due to Rhizopus and Absidia species. 252 The zygomycete species share clinical and pathologic features with invasive Aspergillus species, being angiotropic and capable of inducing hemorrhagic infarcts with sparse inflammation. Clinical syndromes produced by these fungi include rhinocerebral, pulmonary, cutaneous, and gastrointestinal infections with a predilection for neonates. 253 Hematopoietic malignancies and diabetes mellitus with acidosis underlie most cases of pulmonary infection in children and adults. 254 , 255 Box 7.19 lists a broad spectrum of pulmonary diseases that includes solitary or multiple and bilateral nodular lesions, segmental or lobar consolidation, cavitary lesions, fistulas, and infarcts ( Figs. 7.82 and 7.83 ); direct extension into mediastinal, thoracic soft tissue, chest wall, and diaphragm; chronic tracheal and endobronchial infection; and fungus balls similar to those seen with aspergilloma. 256 An endobronchial syndrome with a propensity for blood vessel erosion has also been described, sometimes resulting in fatal hemoptysis. 257 Box 7.19 Histopathologic Patterns in Pulmonary Zygomycosis Acute lobular or lobar pneumonia Nodules Cavities Endobronchial mass Fistulas Infarcts Thoracic soft tissue/mediastinum Fungus ball Alt-text: Box 7.19 Figure 7.82 Resected lung specimen from patient with necrotizing pneumonia caused by zygomycosis. Figure 7.82 Figure 7.83 Zygomycosis. (A) Nodular infarct. (B) Intravascular organisms (arrows). Vessel at right arrow is shown at high magnification (inset) (Grocott methenamine silver stain). Figure 7.83 Hyphae are broad (6 to 25 µm), thin-walled, and pauciseptate ( Fig. 7.84A ). They display variation in width, with twisted, nonparallel contours and random wide-angle branching nearing 90 degrees. 203 They also have a tendency to fragment more commonly than Aspergillus organisms, which tend to retain their elongated sweeping profiles. Additional features include variability in tinctorial staining in H&E sections, ranging from basophilia to eosinophilia. In frozen sections, hyphae may show weak staining, and they often have a bubbly or vacuolated appearance. 256 In addition to being angiotropic, they are neurotropic. 258 In lesions exposed to air, the hyphae may form ovoid or spherical thick-walled chlamydoconidia, within or at the terminal ends ( Fig. 7.84B ). 259 Look-alikes at the lower-width range include Aspergillus and other Aspergillus -like hyaline molds. The pseudohyphae of Candida species can sometimes be similar. Figure 7.84 Zygomycosis. (A) Twisted pauciseptate, broad mycelia characteristic of Zygomycetes (Grocott methenamine silver stain). (B) Endobronchial zygomycosis with chlamydospores. Figure 7.84 Phaeohyphomycosis A few genera of dematiaceous molds produce infections resembling those of Aspergillus , including allergic bronchopulmonary disease ( Fig. 7.85A ) and bronchocentric granulomatosis patterns. 260 , 261 The more than 80 genera and species of these saprophytes, which occur naturally in wood, soil, and decaying matter, include Bipolaris , Exserohilum , Xylohypha , Alternaria , and Curvularia , among others. 203 The unique appearance of these fungi is due to their cell wall melanin content. In the allergic mucin or other deposits of necroinflammatory debris, the phaeoid (dark brown- to black-pigmented) hyphae (2 to 6 µm in diameter) are generally sparse but can resemble Aspergillus and other hyaline molds, especially when lightly pigmented or nonpigmented. Typically only small mycelial fragments are seen, which may be mistaken for artifacts, sometimes with terminal swellings resembling chlamydoconidia ( Fig. 7.85B ). The dematiaceous agents of subcutaneous forms of chromoblastomycosis appear as pigmented muriform cells in granulomas, and they do not form mycelia. Chromoblastomycosis is rarely encountered in the lung. Another Aspergillus look-alike is P. boydii , an organism that is sometimes grouped with the dematiaceous fungi. P. boydii usually exhibits a more ragged, disorganized, and densely clustered pattern of mycelia. Clinically, localized disease may be cured by excision alone; systemic disease is often refractory to treatment. 262 Figure 7.85 Allergic bronchopulmonary fungal disease. (A) Ectatic bronchus with thick eosinophilic basement membrane and intraluminal necroinflammatory debris. (B) Mycelial fragments of Bipolaris organisms (Grocott methenamine silver stain). Figure 7.85 Pneumocystosis The face of Pneumocystis pneumonia continues to change. Once considered to be a protozoan, this organism is now classified as a fungus, and the species infecting humans has been renamed Pneumocystis jirovecii (formerly Pneumocystis carinii ). 263 Once a disease of malnourished or leukemic children, today Pneumocystis infection is identified most commonly in patients with defective immunity, especially AIDS, or those on immunosuppressive therapies for hematopoietic malignancies, organ transplants, and collagen vascular diseases. With the success of contemporary therapy for AIDS, the pathologist is now more likely to encounter the disease in the latter group of patients in whom it is apt to be more subtle. 264 The classic pattern during the HIV epidemic was the foamy alveolar cast ( Fig. 7.86 ) with moderate to numerous organisms, type II pneumocyte hyperplasia, and a scant to moderate interstitial lymphoplasmacytic infiltrate. 265 , 266 Figure 7.86 Pneumocystis pneumonia. (A) Lymphoplasmacytic interstitial infiltrate and intraalveolar foamy alveolar cast. (B) Numerous yeast-like cells of Pneumocystis jirovecii of various shapes (Grocott methenamine silver stain). Figure 7.86 In recent years a number of atypical and unusual patterns have been described that are worth recognizing. 55 , 267 , 268 These are listed in Box 7.20 . P. jirovecii infection can mimic any lung injury pattern, ranging from acute diffuse alveolar damage with hyaline membranes ( Fig. 7.87 ) and minimal or no foamy exudates to an organizing phase with sparse organisms. There is also a spectrum of granulomatous infection, both nonnecrotizing and necrotizing, that may overlap morphologically with mycobacterial or other fungal infections, particularly histoplasmosis ( Fig. 7.88 ). Cavitary disease, solitary pulmonary nodules that may be relatively fibrotic, cysts, and dystrophic calcification are also described. 268 , 269 , 270 Box 7.20 Histopathologic Patterns in Pulmonary Pneumocystis Infection Foamy alveolar cast Diffuse alveolar damage "Id" reaction (minimal-change reaction) Granulomas Miliary disease Vascular invasion/vasculitis/infarct Lymphoid interstitial pneumonia Cavities and cysts Subpleural blebs and bullae Microcalcification Alt-text: Box 7.20 Figure 7.87 Pneumocystis pneumonia. (A) Diffuse alveolar damage pattern with hyaline membranes. (B) Cysts in hyaline membrane (Grocott methenamine silver stain). Figure 7.87 Figure 7.88 Pneumocystis pneumonia. (A) Miliary granuloma with central necrosis. (B) Sparse organisms in granuloma (Grocott methenamine silver stain). Figure 7.88 Microscopically, the three life stages of the organism are still referred to by protozoan terminology as sporozoites, trophozoites, and cysts. The cyst is the most common form seen by pathologists. On silver stains the cyst is seen as an oval (4 to 7 µm) yeast-like cell that may be collapsed, helmet-shaped, or variably crescentic. The intracystic dot or paired–comma structures are important keys to distinguishing P. jirovecii cysts from look-alikes such as Histoplasma , the capsule-deficient form of Cryptococcus , Candida species, and even overstained red blood cells. Sporozoites and trophozoites are seen to best advantage in touch imprints and cytologic preparations of respiratory samples. Cytopathology Many of the fungal pathogens involving the respiratory tract can be detected by cytologic techniques in sputum samples, bronchial washings and brushings, BAL fluid samples, and needle aspirates. 46 The aspirates and other samples can also be submitted for culture and ancillary studies. 271 The four most common yeast forms— C. neoformans , C. immitis or C. posadasii , H. capsulatum , and B. dermatitidis —must be distinguished from each other, and P. jirovecii can also enter the differential diagnosis. 45 Morphologic features of these organisms are often better visualized in cytologic preparations than in tissue sections, usually permitting a rapid and definitive diagnosis on smears prepared using routine stains (Papanicolaou, Diff-Quik, and H&E). More specific fungal stains (GMS, Gridley, and Fontana–Masson) can often be held in reserve. Amorphous granular debris and epithelioid cells characterize many necrotizing granulomas. Typically a background of neutrophils is seen when suppurative granulomas are aspirated. Histoplasma infections may manifest an epithelioid or phagocytic cell population. Cryptococcal infections can be similar or may be associated with little or no accompanying inflammation in the immunocompromised patient. Cytology of Common Yeast Forms Morphologic features of some of the more common yeast forms that the pathologist may encounter in cytologic material are presented in Table 7.7 . C. neoformans organisms are seen as are single budding yeast forms with a narrow, pinched-off base, approximately 4 to 7 µm in diameter but ranging in size from 2 to 15 µm. In needle aspirates, the mucoid capsule investing the yeast imparts a "spare tire" appearance ( Fig. 7.89 ). Figure 7.89 Cryptococcus neoformans . In this fine-needle aspirate, clusters of yeast cells resembling "spare tires" are invested by capsule in a sparse inflammatory background (alcohol-fixed). Figure 7.89 B. dermatitidis organisms are refractile, double-contoured yeast forms and range in diameter from 8 to 15 µm with broad-based budding ( Fig. 7.90 ). An internal amorphous mass can be appreciated in some stained preparations. Smaller or larger yeast cells can be mistaken for C. neoformans or C. immitis , respectively. Figure 7.90 Blastomyces dermatitidis. (A) Necroinflammatory infiltrate with refractile yeast forms. (B) Periodic acid–Schiff staining highlights the double-contoured yeast with broad-based budding (see inset for greater detail). Figure 7.90 C. immitis/C. posadasii spherules exhibit a variety of sizes and shapes, ranging from large spherules packed with endospores ( Fig. 7.91A ) to empty, collapsed spheres and small immature spherules. 272 The latter may overlap with Blastomyces and other yeasts. Mycelial forms of Coccidioides species, with arthrospores, may be found in aspirates of cavitary nodules exposed to air ( Fig. 7.91B ). Figure 7.91 Coccidioides species. (A) Negative-staining spherule in suppurative inflammatory background in a fine-needle aspirate (alcohol-fixed). (B) Ruptured spherules and mycelia with arthrospores in granular necrotic background in another fine-needle aspirate (alcohol-fixed). Figure 7.91 H. capsulatum yeast cells are small (2 to 5 µm) and stain poorly in routine smears, but the presence of this pathogen can be suspected on the basis of the dot-like refractile appearance of these cells in the cytoplasm of macrophages. In Diff-Quik–stained smears, the characteristic purple, polarized yeast forms ( Fig. 7.92 ) are discernible, and they are outlined entirely in GMS-stained smears. Figure 7.92 Histoplasma capsulatum. Clusters of purple polarized yeast cells are readily seen in this fine-needle aspirate (Diff-Quik preparation). Figure 7.92 P. jirovecii is most commonly identified in exfoliative samples and aspirates by the presence of the foamy alveolar cast, which varies from eosinophilic to basophilic and is highly characteristic ( Fig. 7.93A ). These organisms rarely occur singly. The GMS stain outlines the characteristic cysts ( Fig. 7.93B ). Figure 7.93 Pneumocystis jirovecii. (A) Foamy alveolar cast in bronchial washing (ThinPrep Papanicolaou stain). (B) Cysts with intracystic dot in bronchial washing (ThinPrep, Grocott methenamine silver stain). Figure 7.93 Table 7.7 Morphologic Features of Selective Yeast Forms Table 7.7 Feature Small Intermediate Large Candida Pneumocystis Histoplasma Cryptococcus Blastomyces Coccidioides Size (µm) 3–4 5–8 2–5 5–15 8–20 20–200 Shape Oval Pleomorphic Oval Pleomorphic Round Round Budding None None Narrow-based Narrow-based Broad-based None Wall thickness Thin Thin Thin Thin Thick Thick Hyphae/pseudohyphae Common; characteristic Absent Rare Rare Rare Occasional Other features Single and chains Intracystic body Trophozoite forms Intracellular Refractile Mucicarmine + capsule Acapsular forms Double-contour wall Endospores, immature spherules Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:87. Cytology of Common Mycelial Forms The cytopathologist's most frequent challenge is the interpretation of mycelial forms in exfoliated material, especially the distinction between Aspergillus look-alikes—Zygomycetes and Candida hyphae. The morphologic features of some of the more common agents are compared in Table 7.8 . Candida species are readily seen and easily diagnosed when both yeasts and pseudohyphae are present. However, interpretation of their significance is difficult in all except transthoracic needle aspirates, where the presence of any mycelial structure, particularly in the setting of mass-like and cavitary infiltrates, provides strong morphologic evidence of infection. Aspergillus species are characterized by septate mycelia that branch at angles approaching 45 degrees ( Fig. 7.94 ). Aspergillus hyphae lack constrictions at points of septation. However, Aspergillus organisms cannot be differentiated from one of their mimics by morphology alone unless accompanied by a fruiting body. A rapid in situ hybridization technique specific for Aspergillus species can be performed on pulmonary cytocentrifuge preparations, as well as on tissue. 273 Figure 7.94 Aspergillus species. (A) Twisted, sparsely septate mycelia are difficult to differentiate from mimics, including Zygomycetes, in this fine-needle aspirate (Diff-Quik preparation). (B) Characteristic mycelia in a bronchial washing (Papanicolaou stain). Figure 7.94 Zygomycete mycelia are distinguished from Aspergillus and Candida forms by their often broader width and their pleomorphic, twisted ribbon–like, pauciseptate features. Of note, however, in aspirates of aspergilloma, the mycelia may also have a twisted appearance. Table 7.8 Morphologic Features of Selected Fungal Mycelia Table 7.8 Feature Aspergillus Bipolaris Zygomycetes Pseudallescheria Boydii Fusarium Width (µm) 3–6 2–6 5–20 2–5 3–8 Contour Parallel Parallel Irregular Parallel Parallel Branching Dichotomous Haphazard Wide angle Haphazard 90-degree angle Branch orientation Parallel Random Random Random Random Septation Frequent Frequent Infrequent Frequent Frequent Phaeoid (Brown) No Yes No Usually not No Angioinvasive Yes No Yes Yes Yes Other features Fruiting body; oxalate crystals sometimes Chlamydoconidia sometimes One of many dematiaceous genera Rarely chlamydoconidia Aspergillus "lookalikes" Aspergillus "lookalikes" Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:204. A potential pitfall in the evaluation of cytopathologic specimens in fungal infections (both exfoliative samples and needle aspirates) is the confounding presence of atypical reactive squamous cells and type II pneumocytes, which can mimic the cytologic atypia of malignant neoplasms. 48 Furthermore, the pathologist interpreting lung biopsy findings, especially with transbronchial specimens, should always attempt to correlate such findings with samples that may have been collected for cytologic or microbiologic study. This is especially advisable because etiologic agents that escape detection in tissue, such as Pneumocystis , Aspergillus , and CMV, may be found in washings or lavage fluid. 274 Microbiology Complementary laboratory methods are often required for the diagnosis of fungal infection; these are listed in Box 7.21 . 204 Under the microscope, many fungi are readily apparent in H&E-stained sections, where they appear colorless (negative staining) or phaeoid (naturally pigmented). The GMS stain is the best histologic stain for demonstrating fungi when they are sparse or not visible on H&E sections. However, some fungi, notably the Zygomycetes, may stain poorly with GMS. The GMS preparation can be counterstained with H&E, allowing coevaluation of the host inflammatory response. The Fontana–Masson stain has been used to detect melanin in C. neoformans and phaeoid fungi, but many Aspergillus species and some Zygomycetes will also stain with this reagent. 259 , 275 The PAS stain can be useful in select circumstances, and histochemical stains for mucin (Alcian blue or mucicarmine) are useful for C. neoformans infections. The PAS and mucin preparations can also be counterstained with GMS or Fontana–Masson to simultaneously highlight cell walls and capsules of cryptococci. It is important to recognize that not everything that stains with the silver methods is a fungus, and care must be taken to distinguish organisms from pseudomicrobes, such as overstained red cells, white blood cell nuclei, reticulin and elastic fibers, calcium deposits, and even Hamazaki-Wesenberg bodies. 42 Box 7.21 Laboratory Diagnosis of Fungal Pneumonia Direct detection of organisms Chemofluorescence stains Direct fluorescent antibody stain Histopathologic/cytopathologic examination Immunohistochemical studies Antigen detection (in suspected histoplasmosis and cryptococcosis) Culture Emmons modified Sabouraud agar Brain-heart infusion agar Special and selective media Serologic testing Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.21 In the microbiology laboratory, the age-old technique of direct light microscopic visualization of fluids, exudates, and tissue homogenates treated with potassium hydroxide (the KOH wet prep) is being replaced by chemofluorescent cotton-brightening agents (such as calcofluor white and fungiqual). Fluorescence microscopy with these reagents can detect a wide variety of fungi in wet mounts as well as frozen sections and paraffin-embedded tissue. 276 , 277 Laboratory techniques for the identification of fungi (gross colonial and microscopic morphologic analysis after isolation on fungal media, followed by biochemical testing) are the principal means to a species-specific etiologic diagnosis. For deep tissues, including the lung and other sterile sites, the Emmons modification of Sabouraud glucose agar with chloramphenicol is recommended by many mycologists. 278 Additional use of enriched media such as brain-heart infusion agar can improve recovery of C. neoformans , B. dermatitidis , and H. capsulatum . Selective media containing cycloheximide are not recommended for normally sterile sites because they are potentially inhibitory for yeasts, such as Cryptococcus and Candida species, and molds, such as Aspergillus and the Zygomycetes. The interpretation of a positive fungal culture must be made in the clinical context. In the absence of proof of tissue invasion or compelling ancillary data, the interpretation of laboratory results requires considerable judgment. Many fungi are ubiquitous in the environment, and most fungal isolates from nonsterile respiratory samples do not represent disease unless there are also significant risk factors such as HIV infection, organ transplantation, or immunocompromising drug therapy. 279 For most of the dimorphic fungi, in vitro hyphae-to-yeast conversion studies have given way to commercially available nucleic acid probes for rapid specific identification. Procurement of tissue for culture before formalin fixation is important whenever fungal infections are suspected. The tissue sample should be kept moist using sterile, nonbacteriostatic saline or Ringer's solution. Specimens are minced but not ground before plating. The value of bringing multiple, often complementary laboratory methods to bear on inconclusive morphologic findings cannot be overemphasized. In this context, while culture has been considered the most reliable method for definitive diagnosis and histopathology often the fastest, the greatest yield results from combining histopathology with traditional culture and one or more of the newer molecular methods. 280 , 281 Culture may fail to yield an isolate even in the face of positive microscopic findings. In fact, the yield from tissue specimens, needle aspirates, BAL fluid samples, and bronchial washings is quite low for molds and other fungi for reasons that are not entirely clear. 49 , 282 Immunofluorescence testing using specific monoclonal antibodies can achieve a rapid and specific diagnosis in selected infections, especially when tissue has not been submitted for culture. Antibodies directed against the antigens of Aspergillus species and selected other fungi have been described, but most are not yet commercially available. For the problematic case, the mycology section of the CDC can provide assistance. Immunohistochemical identification of fungi can be accomplished fairly easily for those species for which reagents are commercially available. 33 , 283 , 284 Molecular techniques, including in situ hybridization and amplification technologies such as PCR, are other powerful tools that can provide rapid, accurate diagnosis for yeasts and molds that may be present in small numbers or manifest overlapping histologic features. 277 , 285 , 286 , 287 A few laboratories (including the CDC) are performing such assays. Use of quantitative real-time PCR assays on blood, body fluids, and other samples holds promise for relatively rapid definitive diagnosis when routine methods of isolation and identification fail in critical situations. 288 , 289 Serologic tests can support a morphologic diagnosis when positive titers are present, but effective serodiagnosis of systemic fungal infections is not available for most fungi. 290 Unfortunately an antibody response does not necessarily correlate with invasive disease, and an antibody response may be lacking for various reasons. False-positive results due to cross reactions and false-negative results due to a variety of reasons plague many of these assays. Some of the most accurate serologic tests (with high sensitivity and specificity) for fungal infections are those for histoplasmosis and coccidioidomycosis, yet tests for both have limitations that must be recognized in interpreting results. 291 , 292 The detection of macromolecular antigens shed into various body fluids requires a relatively large microbial burden, which tends to limit sensitivity for most fungal infections except histoplasmosis and crytococcosis. 280 For these two fungi, useful antigen detection techniques are available using serum, urine, cerebrospinal fluid, and BAL fluid. They are especially sensitive in patients with defective immunity. 271 , 292 In patients with pneumonia and normal immunity, however, these tests may be positive in lavage fluid but negative in urine unless the disease has disseminated. Other assays designed to detect antigens or metabolites of invasive fungi include those for 1,3β- d -glucan, a cell wall component of several fungi such as Aspergillus , Candida , Fusarium , and others, and for galactomannan, a polysaccharide antigen in the cell wall of Aspergillus ; these assays have shown fair sensitivity and specificity. 203 , 293 , 294 Differential Diagnosis A synopsis of the key morphologic and mycologic features of the fungal pneumonias is presented in Table 7.9 . 295 When H&E and GMS stains fail to detect fungal elements, the use of ancillary procedures may provide the specific diagnosis. Sometimes, if tissue or other patient specimens have been submitted for culture, the answer may lie in the mycology section of the microbiology laboratory, as many species begin to grow in a matter of days. When fungi are not readily identified by any of these techniques or strategies, other granulomatous infections should be considered, especially mycobacterial, uncommon bacterial (e.g., tularemia, brucellosis), and parasitic infections. Noninfectious necrotizing and nonnecrotizing granulomatous disorders also enter the differential diagnosis. These include granulomatosis with polyangiitis, idiopathic bronchocentric granulomatosis, aspiration, sarcoidosis, rheumatoid nodules, pyoderma gangrenosum–like lung lesions in patients with inflammatory bowel disease, and Churg-Strauss syndrome. 224 Table 7.9 Fungal Pneumonias: Summary of Pathologic Findings Table 7.9 Assessment Component Findings Blastomycosis Surgical pathology Suppurative granuloma most characteristic; also, tuberculoid (necrotizing) types; round, thick-walled (double-contour) yeast with broad-based budding Cytopathology Neutrophils and epithelioid cells with characteristic refractile yeast cell with double-contoured wall and broad-based budding Microbiology Characteristic yeast seen on wet mount, KOH- and calcofluor-stained smear; culture-sterile lung tissue on nonselective fungal media (e.g., Emmons modified Sabouraud) and enriched media (e.g., brain-heart infusion); add selective media for bronchial/transbronchial samples; colonies produce oval conidia on terminal ends of conidiophore at right angle to mycelium; confirm with DNA probe; serologic studies not useful Coccidioidomycosis Surgical pathology Fibrocaseous granuloma; large intact and/or ruptured spherules, full or partially or completely empty of endospores; mycelial forms in aerated cavities and fistula Cytopathology Necroinflammatory debris with epithelioid histiocytes; intact, viable, colorless spherules with variable number of endospores and/or ruptured degenerating forms with stained wall; range in size from large mature to small immature types Microbiology Characteristic mature spherules in wet mount, KOH- and calcofluor-stained smear; culture of sterile lung tissue on nonselective fungal media yields mycelia with characteristic arthroconidia; confirm with DNA probe; serologic diagnosis with tests for IgG and IgM antibodies by immunodiffusion, EIA; complement fixation for titers Histoplasmosis Surgical pathology Macrophage reaction and/or granulomas, based on immunity, including miliary and solitary pulmonary, variably hyalinized nodule; small, thin-walled, oval yeasts with narrow-based buds, often refractile Cytopathology Macrophage and epithelioid cells with characteristic yeast cell, often intracellular, stained purple with Diff-Quik, black with GMS Microbiology Rarely detected by direct examination of most clinical specimens; culture-sterile lung tissue on nonselective and enriched fungal media produces tuberculate macroconidia; confirm with DNA probe; antigen detection by EIA available for BAL fluid, CSF, serum, and urine Paracoccidioidomycosis Surgical pathology Exudative or granulomatous lesion with large, globose yeast cell with multiple buds Cytopathology Suppurative or granulomatous reaction with characteristic yeast cell Microbiology Direct detection in wet mount, KOH- and calcofluor-stained smear; culture-sterile lung tissue on standard nonselective fungal media; serologic testing by immunodiffusion, EIA; complement fixation for titer Sporotrichosis Surgical pathology Necrotizing granuloma, often cavitary with small, usually round, sometimes cigar-shaped yeast with sparse, narrow buds Cytopathology Suppurative or necrotizing granuloma pattern; yeast cells generally sparse or absent Microbiology Rarely detected by direct examination of most clinical specimens; culture of sterile lung tissue on nonselective fungal media yields thin, hyphae-bearing conidia in a rosette pattern; converts to a yeast phase at 37°C on blood agar; no serologic tests Penicilliosis Surgical pathology Alveolar macrophages stuffed with yeast cells resemble Histoplasma species, but with septum reflecting binary fission, not budding reproduction Cytopathology Macrophage with intracellular characteristic yeast forms Microbiology Culture of sterile lung tissue on nonselective fungal media yields a mold with a red pigment evident as culture ages; erect conidiophores, sometimes branched with metulae bearing one or several phialides with long, loose chains of oval conidia; new urinary antigen test Cryptococcosis Surgical pathology Granulomas, histiocytic infiltrate or mucoid pneumonia, based on immunity with pale, round, budding pleomorphic yeast cells, often in clusters; mucoid capsules usually present; acapsular types sometimes seen Cytopathology Yeast cell with mucoid capsular halo resembles "spare tire"; combination of mucicarmine and GMS or Fontana–Masson outlines capsule and cell wall; background of epithelioid cells or necroinflammatory debris may be sparse or absent Microbiology Oval to lemon-shaped calcofluor-positive yeast cell with capsule in India ink–stained touch imprint; culture on nonselective fungal media yields mucoid yeast-type colonies; no pseudohyphae; germ tube–negative; dark brown pigment on birdseed (niger) agar; confirm with biochemical tests; antigen detection test (latex agglutination or EIA) on serum, BAL fluid, CSF, and needle aspirates Candidiasis Surgical pathology Miliary necroinflammatory lesions or bronchopneumonia with small, oval, budding yeasts with or without pseudohyphae; C. glabrata yeast only Cytopathology Yeasts and/or pseudohyphae in a necroinflammatory background Microbiology Budding yeasts and pseudohyphae in wet mounts, KOH- and calcofluor-stained smears; cultures on selective and nonselective fungal media yield creamy tan to white yeast-type colonies; identification by germ tube production, carbohydrate assimilation, and cornmeal agar morphology Aspergillosis Surgical pathology Various forms include saprophytic (fungus ball), allergic (ABPA and mucoid impaction), hypersensitivity pneumonitis, and invasive disease ranging in severity from minimal chronic necrotizing to extensive pneumonia; angiotrophic with necrotizing infarcts; also hybrid forms of disease; septate, dichotomous, 45-degree angle mycelia; oxalate crystals; presence of fruiting body is genus-specific Cytopathology Tangled clusters of septate mycelia in a necroinflammatory background; may appear sparsely septate and twisted, mimicking Zygomycetes Microbiology Positive staining of mycelia with calcofluor and GMS; culture of sterile lung tissue on nonselective fungal media produces mold-type colonies in a range of colors; species differentiation by conidial and conidiophore morphology Zygomycosis Surgical pathology Nodular lesions, lobar consolidations, cavitary lesions, fungus balls, and airway infections commonly necrotizing and ischemic secondary to angioinvasion; broad pauciseptate mycelia with 90-degree angle, branching, often with twisted-ribbon morphology Cytopathology Pauciseptate mycelia, often with twisted-ribbon morphology in a necroinflammatory background Microbiology Positive staining of mycelia with calcofluor and GMS; rapidly growing cottony colonies are grown on most nonselective fungal media, but "controlled baiting" with bread sometimes necessary; identification based on presence and locations of rhizoids, shape of sporangia, presence of columellae, and shape of sporangiospores Phaeohyphomycosis Surgical pathology Allergic bronchopulmonary fungal disease similar to aspergillosis Cytopathology Similar to ABPA pattern—"allergic mucin" with eosinophils, Charcot–Leiden crystals in inspissated mucus; fungal mycelial fragments sparse or absent Microbiology Dematiaceous (phaeoid) dark brown to black colonies on nonselective fungal media; identified by shape and cross walls of multicell, pigmented conidia Pneumocystosis Surgical pathology Pneumonia with foamy alveolar cast is classic; other patterns include diffuse alveolar damage, granulomatous lesions, and minimal changes; variable numbers of cysts noted in GMS-stained sections Cytopathology Foamy alveolar cast with characteristic cysts outlined by GMS Microbiology Causative organism: formerly Pneumocystis carinii , classified as a fungus and renamed Pneumocystis jirovecii ; cannot be cultured; Detection is with fluorescent monoclonal antibody assay or GMS-stained smears ABPA, Allergic bronchopulmonary aspergillosis; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; GMS, Grocott methenamine silver; IgG, IgM, immunoglobulins G and M; KOH, potassium hydroxide. Etiologic Agents Nearly 70,000 fungi are known, and approximately 100 have been recovered from respiratory infections. 204 A small number are implicated as pathogenic on a consistent basis; these are listed in Box 7.12 . Box 7.12 Common Fungal Pathogens in the Lung Dimorphic fungi (mycelia at 25–30°C; yeast at 37°C) Blastomyces dermatitidis Coccidioides immitis Histoplasma capsulatum Paracoccidioides braziliensis Sporothrix schenckii Penicillium marneffei Yeasts Cryptococcus neoformans Candida spp. Hyaline (nonpigmented) molds Aspergillus spp. Zygomycetes organisms Phaeoid (pigmented; dematiaceous) molds Bipolaris spp. , Alternaria, Curvularia Pseudoallescheria boydii/Scedosporium apiospermum Miscellaneous pathogens Pneumocystis jirovecii Alt-text: Box 7.12 Histopathology Like mycobacterial species, fungal pathogens typically produce one or more nodular lesions in the normal host ( Fig. 7.63 ); these may become cavitary as the lesions evolve ( Fig. 7.64 ). Inflammatory histopathologic patterns that suggest the presence of a fungal infection are summarized in Box 7.13 . As is the case for other etiologic agents, there are no absolutely diagnostic patterns. Overlap is common and atypical reactions occur, ranging from overwhelming diffuse alveolar damage, little or no reaction, or sheets of organisms in the immunocompromised patient. Proximal endobronchial disease mimicking a neoplasm has also been described for various fungal species. 205 Detection of the etiologic agent in tissue by microscopic examination, ancillary tests, or culture confers specificity and significance to the listed patterns. Large spherules with endospores characteristic of Coccidioides immitis or yeast with large mucoid capsules of C. neoformans can be diagnostic. However, atypical forms of these organisms can be misleading and challenging. For example, in aerated cavities or in the setting of bronchopleural fistula, Coccidioides species may produce branching septate and moniliform hyphae or immature morula-like spherules mimicking other fungi (e.g., hyaline molds and Blastomyces dermatitidis ). 40 Similarly, C. neoformans , H. capsulatum , and S. schenckii have been reported to produce hyphae or pseudohyphae in tissue, whereas acapsular C. neoformans may mimic other yeasts or Pneumocystis organisms. 206 Figure 7.63 Coccidioides granuloma. Figure 7.63 Figure 7.64 Cavitary aspergilloma. Figure 7.64 Box 7.13 Histopathologic Patterns in Fungal Lung Injury Large nodules Nonnecrotizing granulomas Necrotizing granulomas Suppurative granulomas Poorly formed granulomas Cavitary lesions Miliary nodules Acute bronchopneumonia Airway disease Intravascular changes/infarct Diffuse alveolar damage, acute and organizing Foamy alveolar casts Alt-text: Box 7.13 Mycelial morphology is helpful when it is characteristic of a specific genus or group. For example, broad, sparsely septate, nonparallel, twisted or irregular in diameter, thin-walled mycelia with variable wide-angle branching characterize Zygomycetes. Progressively proliferating, regularly septate, 45-degree angle, dichotomously branching mycelia with parallel walls are typical of Aspergillus species ( Fig. 7.65 ). In the case of Aspergillus , an important point is that only the presence of a fruiting body (conidiophore with sterigmata and conidia) permits diagnosis at the genus level, and there are many Aspergillus look-alikes in tissue, such as Fusarium , Paecilomyces , Acremonium , Bipolaris , Pseudallescheria boydii , and its asexual anamorph Scedosporium apiospermum . 206 Sometimes careful examination of tissue with special stains under high magnification or oil immersion will reveal clues, such as in situ sporulation, allowing a more definitive diagnosis. 41 However, these clues are often subtle, and it is important to defer to culture whenever possible. 207 Typical morphologic injury patterns and related etiologic agents are detailed next. Figure 7.65 Aspergillus species. (A) Septate mycelia with 45-degree angle branching (Grocott methenamine silver stain). (B) Fruiting body (conidiophore with sterigmata and conidia) (Grocott methenamine silver stain). Figure 7.65 Blastomycosis Blastomycosis, the chronic granulomatous and suppurative infection produced by B. dermatitidis , is essentially a North American disease, concentrated in the Ohio and the Mississippi River valleys. The prevalence of infection is particularly high in the state of Mississippi. Blastomycosis is the third most common endemic mycosis in North America, following histoplasmosis and coccidioidomycosis. It may occur in patients with normal immunity as well as those immunocompromised by diseases or medical therapy. 208 , 209 , 210 The isolated nodular manifestation can simulate lung cancer radiologically. 211 The disease almost always begins in the lungs, although skin and bone are other common sites of involvement. In the lung, pathologic manifestations include focal or diffuse infiltrates, rare lobar consolidation, miliary nodules, solitary nodules, and acute or organizing diffuse alveolar damage ( Box 7.14 ). 208 , 211 , 212 , 213 Necrotizing granulomas are characteristic and often of the suppurative type ( Fig. 7.66A ), but nonnecrotizing granulomas may be found as well. Box 7.14 Histopathologic Patterns in Pulmonary Blastomycosis Acute pneumonia Lobular Lobar Diffuse alveolar damage Miliary nodule Solitary nodule Alt-text: Box 7.14 Figure 7.66 Blastomycosis. (A) The suppurative granuloma is characteristic. (B) Double-contour-wall yeast with broad-based budding. Figure 7.66 The broad-based budding yeast forms of Blastomyces are refractile and have double-contoured walls. Multinucleate yeast cells are typically 8 to 15 µm in diameter, with some forms measuring up to 30 µm ( Fig. 7.66B ). These large forms can mimic small Coccidioides spherules, 214 whereas smaller forms ("microforms") can mimic C. neoformans . 213 Coccidioidomycosis Endemic in the Lower Sonoran life zone of the southwestern United States, the soil fungus C. immitis and the more recently recognized, morphologically identical, and genomically similar species Coccidioides posadasii 215 may be encountered outside the endemic area as a result of fomite transmission of arthroconidia (e.g., Asian textile workers handling imported Arizona cotton) or in travelers who have returned from an endemic area. Most primary pulmonary infections are asymptomatic. The exceptionally wide spectrum of pulmonary pathology in patients with clinically evident disease is outlined in Box 7.15 . The true prevalence of the disease is significantly underestimated in endemic regions of the Southwest, where it is thought to account for nearly 30% of community-acquired pneumonias in some metropolitan areas. 216 , 217 , 218 , 219 Granulomas are characteristic and may occur with or without necrosis. Intact spherules induce fibrocaseous granulomas ( Fig. 7.67A ), whereas ruptured spherules may incite suppurative and bronchocentric granulomatosis (BCG)-like reactions ( Fig. 7.67B ). 216 Box 7.15 Histopathologic Patterns in Coccidioidal Respiratory Tract Disease Airway disease Pharyngeal granuloma Laryngeal granuloma Tracheobronchial granuloma Pulmonary parenchymal disease Acute pneumonia Eosinophilic pneumonia Chronic progressive infection Fibrocavitary lesions Bronchopleural fistula; empyema Solitary pulmonary nodule Disseminated disease Miliary Extrapulmonary Alt-text: Box 7.15 Figure 7.67 Coccidioidomycosis. (A) Fibrocaseous granuloma. (B) Bronchocentric granulomatosis–like granuloma. (C) Coccidioides immitis . Both small (arrow) and large spherules with and without endospores can be seen. (D) Biphasic pattern with mycelia and spore-like swellings (Grocott methenamine silver stain). Figure 7.67 The large mature spherule (up to 40 to 60 µm in diameter) has a thick refractile wall lined by or filled with endospores; it constitutes the key diagnostic finding ( Fig. 7.67C ). This finding allows the distinction of coccidioidomycosis from other fungal infections such as blastomycosis and histoplasmosis, which are associated with similar histopathologic reaction patterns. In aerated cavities or in the setting of bronchopleural fistula, mycelia resembling various hyaline molds may be seen with or without a variety of mature and immature spherules ( Figs. 7.20 and 7.67D ). Coccidioides spherule look-alikes include large-variant B. dermatitidis, adiaspiromycosis, pollen grains, and pulses (legume seeds). Histoplasmosis Histoplasmosis, the most common pulmonary fungal infection worldwide, is endemic in the Ohio and the Mississippi River valleys of North America and is the most common endemic mycosis in AIDS. 220 The clinical forms of H. capsulatum infection 55 , 203 , 221 , 222 are presented in Box 7.16 . The histopathologic correlates include a spectrum ranging from an exudative to a granulomatous process influenced by such factors as the fungal burden and the immune status of the patient. In patients with normal defenses, the characteristic histopathology is dominated by well-formed necrotizing and nonnecrotizing granulomas occurring as solitary lesions indistinguishable from other granulomatous infections. Other presentations include miliary nodules ( Fig. 7.68 ), cavitary lesions, and laminated fibrous solitary nodules ( Fig. 7.69 ) that may be partially calcified (sometimes referred to as residual granulomas ). In patients with impaired immunity, striking macrophage response with numerous intracellular yeasts is a characteristic pattern ( Fig. 7.70A ). The exudative lesion resembles acute lobular pneumonia with fibrinopurulent exudates. 223 Box 7.16 Clinical Forms of Pulmonary Histoplasmosis Benign, self-limited Acute Acute respiratory distress syndrome Acute self-limited, upper lobe (in smokers with emphysema) Chronic Asymptomatic pulmonary nodule, with or without calcification ("histoplasmoma") Progressive (chronic cavitary) pulmonary Progressive disseminated Mediastinal Lymphadenopathy Middle lobe syndrome Fibrosis Alt-text: Box 7.16 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.6. Figure 7.68 Histoplasmosis. Miliary nodule with central zone of necrosis invested by epithelioid histiocytes, multinucleate giant cells, and outer collarette of lymphocytes. Figure 7.68 Figure 7.69 Histoplasmoma. Characteristic gross appearance of the persistent granulomatous nodule. Note the fibrous wall (arrow) surrounding caseous necrosis (n) . Figure 7.69 Figure 7.70 Histoplasmosis in an immunocompromised patient. (A) Numerous Histoplasma capsulatum yeast cells in macrophages. (B) Clusters of H. capsulatum yeast cells in macrophages. Note the narrow-based budding (arrow) (Grocott methenamine silver stain). Figure 7.70 H. capsulatum organisms are yeasts (2 to 5 µm), with narrow-based unequal budding ( Fig. 7.71 ; Fig. 7.70B ). They may be seen on H&E-stained sections and, when numerous, appear as small refractile ovoid structures within macrophages. Yeasts typically occur in clusters but may be rare in old granulomas. A search for budding organisms in these situations may prove futile. Sometimes, yeasts may have dark-staining foci resembling Pneumocystis organisms. Also, some yeast cells may be surrounded by a clear space and may be mistaken for Cryptococcus . 55 Other look-alikes include Candida species, P. marneffei , capsule-deficient cryptococci, intracellular B. dermatitidis , and Hamazaki-Wesenberg bodies. Figure 7.71 Tinctorial and morphologic attributes of H. capsulatum in stained clinical specimens. (A) Diff-Quik: BAL fluid smear showing extracellular yeast forms. (B) Giemsa stain: tissue touch preparation demonstrating numerous yeast within a histiocyte and in the surrounding space. (C) GMS stain: abundant black-colored yeast cells in a tissue touch preparation. Both extracellular and intrahistiocytic forms are seen. (D) Gram stain: red-colored yeast cells in a blood culture smear. (E) Hematoxylin and eosin stain: liver biopsy containing numerous intracellular and extracellular yeast forms. Note the presence of colorless halos surrounding the yeast. (F) Mucicarmine stain: yeast forms are barely visible without the aid of increased contrast. (G) Periodic acid–Schiff stain: magenta-colored yeast cells are evident scattered throughout the epidermis of a skin biopsy specimen. (H) Wright-Giemsa: yeast forms evident within a monocyte in a peripheral blood smear. Scale bar, 10 mm; original magnification, ×1000. Figure 7.71 (From Wheat LJ, Azar MM, Bahr NC, et al. Histoplasmosis. Inf Dis Clin NA . 2016;30:216.) Paracoccidioidomycosis (South American Blastomycosis) Seven clinical forms occur, but they rarely cause lung infections in North America. In areas of high endemicity, such as Brazil, the several forms can mimic malignancy or sarcoidosis. 224 , 225 The histopathology resembles that of other mycoses and can be exudative or granulomatous. Paracoccidioides braziliensis appears as a large spherical yeast (10 to 60 µm) with multiple buds attached by narrow necks (a "steering wheel" or "ship's wheel" appearance). 226 When budding is sparse, look-alikes include H. capsulatum with small intracellular forms, B. dermatitidis and capsule-deficient cryptococci for medium-sized forms, and C. immitis or C. posadasii for large forms. Sporotrichosis Infection by Sporothrix schenckii is usually confined to the skin, subcutis, and lymphatic pathways, but the organism can disseminate to the lungs. Rarely, S. schenckii is a primary pulmonary pathogen. 227 The organism can produce cavitary disease in the form of a single lesion. Infection may be bilateral and apical, progressive and destructive, or it may be identified clinically as a solitary pulmonary nodule. Microscopically, caseous and suppurative granulomas ( Fig. 7.72A ) occur with variable numbers of round to oval, small (2 to 3 µm), narrow budding yeast ( Fig. 7.72B ) or cigar-shaped forms. 228 Nonnecrotizing granulomas also occur. Asteroid bodies are an important clue, especially when organisms are sparse, as is often the case. Look-alikes include H. capsulatum , acapsular cryptococci, Candida organisms, and Hamazaki-Wesenberg bodies. Figure 7.72 Sporotrichosis. (A) Cavitary granuloma manifesting as a solitary pulmonary nodule. (B) A rare, oval, narrow budding yeast (Grocott methenamine silver stain). Figure 7.72 Penicilliosis Southeast Asia is the endemic setting of the unique dimorphic fungus Penicillium marneffei . The disease it produces is not seen in North America except in travelers, especially immunocompromised persons. It is a common opportunistic infections in AIDS patients in Southeast Asia and a significant clue to the presence of AIDS in that area. 229 The respiratory tract is the portal of entry, with pulmonary infiltrates and disseminated disease, especially involving the skin. Microscopically, alveolar macrophages stuffed with spherical to oval yeast-like cells (2.5 to 5 µm) are seen, each with a single transverse septum; short hyphal forms and elongated, curved "sausage" forms may be formed in necrotic and cavitary lesions. 230 , 231 The septum distinguishes it from H. capsulatum , its look-alike. 229 , 232 Cryptococcosis C. neoformans is a ubiquitous, facultative intracellular yeast. Pulmonary cryptococcosis occurs worldwide but has a particularly high incidence in the United States. 233 The pathogenicity and histopathologic features of lung infection depends largely on the patient's immune status, as illustrated earlier in Fig. 7.7 and summarized in Box 7.17 . In the normal host, a substantial proportion of cryptococcal infections are asymptomatic, others are symptomatic, with infiltrates or nodules. Immunocompromised patients are almost invariably symptomatic and often develop disseminated disease with a predilection for the brain and meninges. Pulmonary injury patterns include single or multiple large nodules, segmental or diffuse infiltrates, cavitary lesions, and miliary nodules. Normal hosts most often develop nodules comprising fibrocaseous granulomas ( Fig. 7.73A ), or granulomatous pneumonia ( Fig. 7.73B ). Immunocompromised patients are more likely to have histiocytic ( Fig. 7.73C ) or mucoid infiltrates without inflammation ( Fig. 7.73D ). Box 7.17 Histopathologic Patterns in Cryptococcal Lung Disease In order of associated decrease in immune function: Fibrocaseous granuloma Granulomatous pneumonia Histiocytic pneumonia Mucoid pneumonia Intracapillary cryptococcosis Alt-text: Box 7.17 Reprinted with permission from Mark EJ. Case records of the Massachusetts General Hospital. N Engl J Med. 2002;347:518–524. Figure 7.73 Cryptococcosis. (A) Solitary pulmonary nodule with small satellite granulomas. (B) Granulomatous pneumonia with clusters of pale staining yeast in clear spaces surrounded by histiocytes and multinucleated giant cells. (C) Histiocytic pneumonia. (D) Mucoid pneumonia with no inflammatory cell reaction. Figure 7.73 The cryptococcal organisms are round yeast forms ranging in diameter from 2 to 15 µm, with an average size of 4 to 7 µm. Cryptococcal yeasts are visible on H&E-stained sections as pale gray to light blue structures, frequently with attached smaller buds. They often occur in clusters and can sometimes be found within giant cells. 203 The mucicarmine stain highlights the capsule ( Fig. 7.74A ); but with capsule-deficient forms ( Fig. 7.74B ) the pleomorphic appearance can be confused with that of other yeast forms (e.g., H. capsulatum , B. dermatitidis , S. schenckii ) and sometimes Pneumocystis . Figure 7.74 (A) Intravascular cryptococcus. Yeast cells with stained capsules (mucicarmine stain). (B) Capsule-deficient cryptococcus (Grocott methenamine silver stain). Figure 7.74 The lungs of patients with the most severe immunodeficiency may show myriad yeasts in alveolar septal capillaries ( Fig. 7.74A ), with little if any intraalveolar reaction 234 ; this form of the disease may also be associated with mucoid pneumonia. 235 The mucoid pneumonia ( Fig. 7.75A ) of cryptococcal infection can be confirmed with mucin stains such as Alcian blue ( Fig. 7.75B ). Another microscopic pattern recently described in HIV-infected patients is the inflammatory spindle cell pseudotumor, a lesion much more commonly associated with mycobacterial infection. 236 Figure 7.75 Cryptococcal mucoid pneumonia. (A) Myriad blue-gray yeast cells in mucoid matrix. (B) Alcian blue mucin stain accentuates the mucoid matrix. Figure 7.75 Candidiasis Candida organisms are yeasts that can produce pseudohyphae and are the most common invasive fungal pathogens in humans. Secondary Candida pneumonia is relatively common, but primary Candida pneumonia is rare in other than immunocompromised patients in the intensive care unit. 55 In general, Candida albicans is the most frequently isolated of the more than 100 known species, which include a few rare and emerging human pathogens. Candida glabrata and Candida tropicalis , together with C. albicans , account for 95% of bloodstream infections, the principal route for the acquisition of Candida pneumonia. 237 A non–blood-borne route to pneumonia results from aspiration of organisms from a heavily colonized or infected oropharynx. When the infection is blood-borne, miliary nodules with a necroinflammatory center and a hemorrhagic rim reflect an intravascular distribution of fungi. In the case of aspiration, the organisms may be found in the airways associated with an alveolar filling pattern of bronchopneumonia ( Fig. 7.76A ) 238 or, much less commonly, a bronchocentric granulomatosis pattern. Figure 7.76 (A) Candida bronchopneumonia. (B) Candida yeast cells—blastoconidia (Grocott methenamine silver stain). Figure 7.76 In tissue sections, oval budding yeast-like cells (blastoconidia) 2 to 6 µm in diameter may appear with pseudohyphae that constrict at points of budding, creating the impression of bulging rather than parallel walls ( Fig. 7.76B ). The pseudohyphae branch at acute angles and can overlap in width with the true hyphae of Aspergillus , from which they must be distinguished. Among the medically important species, Candida glabrata (formerly Torulopsis glabrata ) and Candida parapsilosis produce only yeast cells in tissue, in contrast with most other Candida species, which produce both yeast and pseudohyphae. 203 Other look-alikes include H. capsulatum , Trichosporon beigelii , and Malassezia furfur , depending on whether pseudohyphae or yeast forms alone are present. They can be distinguished from Histoplasma by their extracellular location and Gram stain positivity. T. beigelii tends to be somewhat larger and more pleomorphic. Malassezia is clinically associated with parenteral nutrition, Intralipid, and indwelling catheters. Pulmonary lesions include pneumonia, mycotic thromboemboli, infarcts, and vasculitis. M. furfur may be found in small arteries, where the organisms appear as small 2- to 5-µm yeast-like cells. They form distinctive unipolar broad-based buds but no pseudohyphae. 55 Aspergillosis Aspergillus species and other hyaline and dematiaceous molds have emerged as significant causes of morbidity and death in the immunocompromised host. Worldwide, species of Aspergillus are the most common invasive molds. They are the second most common fungal pathogens after Candida species but, in contrast with Candida , are more commonly isolated from the lung. Several species are recognized, but Aspergillus fumigatus is the one most often seen in the clinical laboratory and most often isolated from the lungs of immunocompromised patients. 239 Respiratory aspergillosis can be classified into a colonizing or saprophytic form (intrabronchial and preexisting cavity fungus ball, Fig. 7.77A ); hypersensitivity forms (allergic bronchopulmonary aspergillosis, including mucoid impaction of bronchi and hypersensitivity pneumonitis; Fig. 7.77B ); and invasive disease (minimally invasive–chronic necrotizing or angioinvasive–disseminated, Box 7.18 ). 55 , 240 , 241 , 242 , 243 Invasive disease ( Fig. 7.78 ) tends to occur in immunocompromised patients, including those with prolonged neutropenia, transplant recipients (especially hematopoietic stem cell and lung transplants), advanced AIDS, and the inherited immune deficiency disorder referred to as chronic granulomatous disease of childhood . The clinicopathologic features of invasive disease reflect these host-associated risk factors. 244 In patients with neutropenia, a characteristic angioinvasive pattern occurs, with intravascular spread resulting in hemorrhagic infarcts ( Fig. 7.79 ). In the nonneutropenic patient, the necroinflammatory pattern tends to lack this angioinvasive feature. 245 Some cases defy categorization (e.g., bronchocentric and miliary patterns; Fig. 7.80 ) or may be hybrids of infection and hypersensitivity. 246 Figure 7.77 (A) Aspergillosis fungus ball. (B) Allergic bronchopulmonary aspergillosis. Intraluminal allergic mucin with laminated clusters of eosinophils can be seen in inspissated basophilic mucin with scattered Charcot–Leyden crystals. Figure 7.77 Box 7.18 Histopathologic Patterns in Pulmonary Aspergillosis Colonization Fungus ball Hypersensitivity reaction Allergic bronchopulmonary aspergillosis Eosinophilic pneumonia Mucoid impaction Bronchocentric granulomatosis Hypersensitivity pneumonitis Invasive Acute invasive aspergillosis Necrotizing pseudomembranous tracheobronchitis Chronic necrotizing pneumonia Bronchopleural fistula Empyema Alt-text: Box 7.18 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.10. Figure 7.78 Resected lung specimen from an immunocompromised patient with necrotizing Aspergillus pneumonia. Figure 7.78 Figure 7.79 Invasive aspergillosis. (A) Hemorrhagic infarct. (B) 45-degree angle branching septate hyphae. Figure 7.79 Figure 7.80 Bronchocentric aspergillosis. (A) Bronchiole expanded and filled with purulent exudate. (B) Miliary aspergillosis. Colony of organisms with hyaline membranes evident at periphery of the image (lower right) . Figure 7.80 Microscopically, septate hyphae, dichotomously branched at a 45-degree angle, have uniform, consistent width (3 to 6 µm) without constrictions at points of septation . When numerous, as in some angioinvasive lesions and fungus balls, these features can be readily appreciated in H&E-stained sections. Fruiting heads of Aspergillus (shown earlier in Fig. 7.65 ) are sometimes formed in cavities. Oxalate crystals, visible in plane-polarized light ( Fig. 7.81 ), are an important clue to Aspergillus infection when hyphae cannot be identified. Figure 7.81 (A) Pale yellow oxalate crystal sheaths in necroinflammatory debris. (B) Birefringent oxalates seen under polarized light. Figure 7.81 Look-alikes include various hyaline molds such as Zygomycetes and Candida species as well as Pseudallescheria boydii . 247 Another look-alike is Fusarium species. Fusariosis is an emerging mycosis in the immunocompromised host, and Fusarium is the second most common opportunistic pathogen after Aspergillus species in immunosuppressed patients with hematologic malignancies. 248 The clinical and pathologic features in the lung and at sites of dissemination mimic those of aspergillosis, and the mycelia are essentially indistinguishable. Isolation in culture, immunohistochemistry, or molecular techniques, such as in situ hybridization or PCR amplification, is required for definitive diagnosis. Other previously uncommon but newly emerging hyaline molds that may be difficult to distinguish from Aspergillus in tissue are Paecilomyces , Acremonium , Scedosporium , and Basidiobolus . 237 , 249 , 250 Zygomycosis The taxonomic organization of the fungal phylum Zygomycota includes the class Zygomycetes, which is subdivided into two orders: Mucorales and Entomophthorales. These orders contain the agents of human zygomycosis. 251 The order Mucorales includes the genera Absidia , Apophysomyces , Rhizopus , Rhizomucor, and Mucor , from which the often taxonomically incorrect term mucormycosis is derived. In fact, most infections are due to Rhizopus and Absidia species. 252 The zygomycete species share clinical and pathologic features with invasive Aspergillus species, being angiotropic and capable of inducing hemorrhagic infarcts with sparse inflammation. Clinical syndromes produced by these fungi include rhinocerebral, pulmonary, cutaneous, and gastrointestinal infections with a predilection for neonates. 253 Hematopoietic malignancies and diabetes mellitus with acidosis underlie most cases of pulmonary infection in children and adults. 254 , 255 Box 7.19 lists a broad spectrum of pulmonary diseases that includes solitary or multiple and bilateral nodular lesions, segmental or lobar consolidation, cavitary lesions, fistulas, and infarcts ( Figs. 7.82 and 7.83 ); direct extension into mediastinal, thoracic soft tissue, chest wall, and diaphragm; chronic tracheal and endobronchial infection; and fungus balls similar to those seen with aspergilloma. 256 An endobronchial syndrome with a propensity for blood vessel erosion has also been described, sometimes resulting in fatal hemoptysis. 257 Box 7.19 Histopathologic Patterns in Pulmonary Zygomycosis Acute lobular or lobar pneumonia Nodules Cavities Endobronchial mass Fistulas Infarcts Thoracic soft tissue/mediastinum Fungus ball Alt-text: Box 7.19 Figure 7.82 Resected lung specimen from patient with necrotizing pneumonia caused by zygomycosis. Figure 7.82 Figure 7.83 Zygomycosis. (A) Nodular infarct. (B) Intravascular organisms (arrows). Vessel at right arrow is shown at high magnification (inset) (Grocott methenamine silver stain). Figure 7.83 Hyphae are broad (6 to 25 µm), thin-walled, and pauciseptate ( Fig. 7.84A ). They display variation in width, with twisted, nonparallel contours and random wide-angle branching nearing 90 degrees. 203 They also have a tendency to fragment more commonly than Aspergillus organisms, which tend to retain their elongated sweeping profiles. Additional features include variability in tinctorial staining in H&E sections, ranging from basophilia to eosinophilia. In frozen sections, hyphae may show weak staining, and they often have a bubbly or vacuolated appearance. 256 In addition to being angiotropic, they are neurotropic. 258 In lesions exposed to air, the hyphae may form ovoid or spherical thick-walled chlamydoconidia, within or at the terminal ends ( Fig. 7.84B ). 259 Look-alikes at the lower-width range include Aspergillus and other Aspergillus -like hyaline molds. The pseudohyphae of Candida species can sometimes be similar. Figure 7.84 Zygomycosis. (A) Twisted pauciseptate, broad mycelia characteristic of Zygomycetes (Grocott methenamine silver stain). (B) Endobronchial zygomycosis with chlamydospores. Figure 7.84 Phaeohyphomycosis A few genera of dematiaceous molds produce infections resembling those of Aspergillus , including allergic bronchopulmonary disease ( Fig. 7.85A ) and bronchocentric granulomatosis patterns. 260 , 261 The more than 80 genera and species of these saprophytes, which occur naturally in wood, soil, and decaying matter, include Bipolaris , Exserohilum , Xylohypha , Alternaria , and Curvularia , among others. 203 The unique appearance of these fungi is due to their cell wall melanin content. In the allergic mucin or other deposits of necroinflammatory debris, the phaeoid (dark brown- to black-pigmented) hyphae (2 to 6 µm in diameter) are generally sparse but can resemble Aspergillus and other hyaline molds, especially when lightly pigmented or nonpigmented. Typically only small mycelial fragments are seen, which may be mistaken for artifacts, sometimes with terminal swellings resembling chlamydoconidia ( Fig. 7.85B ). The dematiaceous agents of subcutaneous forms of chromoblastomycosis appear as pigmented muriform cells in granulomas, and they do not form mycelia. Chromoblastomycosis is rarely encountered in the lung. Another Aspergillus look-alike is P. boydii , an organism that is sometimes grouped with the dematiaceous fungi. P. boydii usually exhibits a more ragged, disorganized, and densely clustered pattern of mycelia. Clinically, localized disease may be cured by excision alone; systemic disease is often refractory to treatment. 262 Figure 7.85 Allergic bronchopulmonary fungal disease. (A) Ectatic bronchus with thick eosinophilic basement membrane and intraluminal necroinflammatory debris. (B) Mycelial fragments of Bipolaris organisms (Grocott methenamine silver stain). Figure 7.85 Pneumocystosis The face of Pneumocystis pneumonia continues to change. Once considered to be a protozoan, this organism is now classified as a fungus, and the species infecting humans has been renamed Pneumocystis jirovecii (formerly Pneumocystis carinii ). 263 Once a disease of malnourished or leukemic children, today Pneumocystis infection is identified most commonly in patients with defective immunity, especially AIDS, or those on immunosuppressive therapies for hematopoietic malignancies, organ transplants, and collagen vascular diseases. With the success of contemporary therapy for AIDS, the pathologist is now more likely to encounter the disease in the latter group of patients in whom it is apt to be more subtle. 264 The classic pattern during the HIV epidemic was the foamy alveolar cast ( Fig. 7.86 ) with moderate to numerous organisms, type II pneumocyte hyperplasia, and a scant to moderate interstitial lymphoplasmacytic infiltrate. 265 , 266 Figure 7.86 Pneumocystis pneumonia. (A) Lymphoplasmacytic interstitial infiltrate and intraalveolar foamy alveolar cast. (B) Numerous yeast-like cells of Pneumocystis jirovecii of various shapes (Grocott methenamine silver stain). Figure 7.86 In recent years a number of atypical and unusual patterns have been described that are worth recognizing. 55 , 267 , 268 These are listed in Box 7.20 . P. jirovecii infection can mimic any lung injury pattern, ranging from acute diffuse alveolar damage with hyaline membranes ( Fig. 7.87 ) and minimal or no foamy exudates to an organizing phase with sparse organisms. There is also a spectrum of granulomatous infection, both nonnecrotizing and necrotizing, that may overlap morphologically with mycobacterial or other fungal infections, particularly histoplasmosis ( Fig. 7.88 ). Cavitary disease, solitary pulmonary nodules that may be relatively fibrotic, cysts, and dystrophic calcification are also described. 268 , 269 , 270 Box 7.20 Histopathologic Patterns in Pulmonary Pneumocystis Infection Foamy alveolar cast Diffuse alveolar damage "Id" reaction (minimal-change reaction) Granulomas Miliary disease Vascular invasion/vasculitis/infarct Lymphoid interstitial pneumonia Cavities and cysts Subpleural blebs and bullae Microcalcification Alt-text: Box 7.20 Figure 7.87 Pneumocystis pneumonia. (A) Diffuse alveolar damage pattern with hyaline membranes. (B) Cysts in hyaline membrane (Grocott methenamine silver stain). Figure 7.87 Figure 7.88 Pneumocystis pneumonia. (A) Miliary granuloma with central necrosis. (B) Sparse organisms in granuloma (Grocott methenamine silver stain). Figure 7.88 Microscopically, the three life stages of the organism are still referred to by protozoan terminology as sporozoites, trophozoites, and cysts. The cyst is the most common form seen by pathologists. On silver stains the cyst is seen as an oval (4 to 7 µm) yeast-like cell that may be collapsed, helmet-shaped, or variably crescentic. The intracystic dot or paired–comma structures are important keys to distinguishing P. jirovecii cysts from look-alikes such as Histoplasma , the capsule-deficient form of Cryptococcus , Candida species, and even overstained red blood cells. Sporozoites and trophozoites are seen to best advantage in touch imprints and cytologic preparations of respiratory samples. Blastomycosis Blastomycosis, the chronic granulomatous and suppurative infection produced by B. dermatitidis , is essentially a North American disease, concentrated in the Ohio and the Mississippi River valleys. The prevalence of infection is particularly high in the state of Mississippi. Blastomycosis is the third most common endemic mycosis in North America, following histoplasmosis and coccidioidomycosis. It may occur in patients with normal immunity as well as those immunocompromised by diseases or medical therapy. 208 , 209 , 210 The isolated nodular manifestation can simulate lung cancer radiologically. 211 The disease almost always begins in the lungs, although skin and bone are other common sites of involvement. In the lung, pathologic manifestations include focal or diffuse infiltrates, rare lobar consolidation, miliary nodules, solitary nodules, and acute or organizing diffuse alveolar damage ( Box 7.14 ). 208 , 211 , 212 , 213 Necrotizing granulomas are characteristic and often of the suppurative type ( Fig. 7.66A ), but nonnecrotizing granulomas may be found as well. Box 7.14 Histopathologic Patterns in Pulmonary Blastomycosis Acute pneumonia Lobular Lobar Diffuse alveolar damage Miliary nodule Solitary nodule Alt-text: Box 7.14 Figure 7.66 Blastomycosis. (A) The suppurative granuloma is characteristic. (B) Double-contour-wall yeast with broad-based budding. Figure 7.66 The broad-based budding yeast forms of Blastomyces are refractile and have double-contoured walls. Multinucleate yeast cells are typically 8 to 15 µm in diameter, with some forms measuring up to 30 µm ( Fig. 7.66B ). These large forms can mimic small Coccidioides spherules, 214 whereas smaller forms ("microforms") can mimic C. neoformans . 213 Coccidioidomycosis Endemic in the Lower Sonoran life zone of the southwestern United States, the soil fungus C. immitis and the more recently recognized, morphologically identical, and genomically similar species Coccidioides posadasii 215 may be encountered outside the endemic area as a result of fomite transmission of arthroconidia (e.g., Asian textile workers handling imported Arizona cotton) or in travelers who have returned from an endemic area. Most primary pulmonary infections are asymptomatic. The exceptionally wide spectrum of pulmonary pathology in patients with clinically evident disease is outlined in Box 7.15 . The true prevalence of the disease is significantly underestimated in endemic regions of the Southwest, where it is thought to account for nearly 30% of community-acquired pneumonias in some metropolitan areas. 216 , 217 , 218 , 219 Granulomas are characteristic and may occur with or without necrosis. Intact spherules induce fibrocaseous granulomas ( Fig. 7.67A ), whereas ruptured spherules may incite suppurative and bronchocentric granulomatosis (BCG)-like reactions ( Fig. 7.67B ). 216 Box 7.15 Histopathologic Patterns in Coccidioidal Respiratory Tract Disease Airway disease Pharyngeal granuloma Laryngeal granuloma Tracheobronchial granuloma Pulmonary parenchymal disease Acute pneumonia Eosinophilic pneumonia Chronic progressive infection Fibrocavitary lesions Bronchopleural fistula; empyema Solitary pulmonary nodule Disseminated disease Miliary Extrapulmonary Alt-text: Box 7.15 Figure 7.67 Coccidioidomycosis. (A) Fibrocaseous granuloma. (B) Bronchocentric granulomatosis–like granuloma. (C) Coccidioides immitis . Both small (arrow) and large spherules with and without endospores can be seen. (D) Biphasic pattern with mycelia and spore-like swellings (Grocott methenamine silver stain). Figure 7.67 The large mature spherule (up to 40 to 60 µm in diameter) has a thick refractile wall lined by or filled with endospores; it constitutes the key diagnostic finding ( Fig. 7.67C ). This finding allows the distinction of coccidioidomycosis from other fungal infections such as blastomycosis and histoplasmosis, which are associated with similar histopathologic reaction patterns. In aerated cavities or in the setting of bronchopleural fistula, mycelia resembling various hyaline molds may be seen with or without a variety of mature and immature spherules ( Figs. 7.20 and 7.67D ). Coccidioides spherule look-alikes include large-variant B. dermatitidis, adiaspiromycosis, pollen grains, and pulses (legume seeds). Histoplasmosis Histoplasmosis, the most common pulmonary fungal infection worldwide, is endemic in the Ohio and the Mississippi River valleys of North America and is the most common endemic mycosis in AIDS. 220 The clinical forms of H. capsulatum infection 55 , 203 , 221 , 222 are presented in Box 7.16 . The histopathologic correlates include a spectrum ranging from an exudative to a granulomatous process influenced by such factors as the fungal burden and the immune status of the patient. In patients with normal defenses, the characteristic histopathology is dominated by well-formed necrotizing and nonnecrotizing granulomas occurring as solitary lesions indistinguishable from other granulomatous infections. Other presentations include miliary nodules ( Fig. 7.68 ), cavitary lesions, and laminated fibrous solitary nodules ( Fig. 7.69 ) that may be partially calcified (sometimes referred to as residual granulomas ). In patients with impaired immunity, striking macrophage response with numerous intracellular yeasts is a characteristic pattern ( Fig. 7.70A ). The exudative lesion resembles acute lobular pneumonia with fibrinopurulent exudates. 223 Box 7.16 Clinical Forms of Pulmonary Histoplasmosis Benign, self-limited Acute Acute respiratory distress syndrome Acute self-limited, upper lobe (in smokers with emphysema) Chronic Asymptomatic pulmonary nodule, with or without calcification ("histoplasmoma") Progressive (chronic cavitary) pulmonary Progressive disseminated Mediastinal Lymphadenopathy Middle lobe syndrome Fibrosis Alt-text: Box 7.16 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.6. Figure 7.68 Histoplasmosis. Miliary nodule with central zone of necrosis invested by epithelioid histiocytes, multinucleate giant cells, and outer collarette of lymphocytes. Figure 7.68 Figure 7.69 Histoplasmoma. Characteristic gross appearance of the persistent granulomatous nodule. Note the fibrous wall (arrow) surrounding caseous necrosis (n) . Figure 7.69 Figure 7.70 Histoplasmosis in an immunocompromised patient. (A) Numerous Histoplasma capsulatum yeast cells in macrophages. (B) Clusters of H. capsulatum yeast cells in macrophages. Note the narrow-based budding (arrow) (Grocott methenamine silver stain). Figure 7.70 H. capsulatum organisms are yeasts (2 to 5 µm), with narrow-based unequal budding ( Fig. 7.71 ; Fig. 7.70B ). They may be seen on H&E-stained sections and, when numerous, appear as small refractile ovoid structures within macrophages. Yeasts typically occur in clusters but may be rare in old granulomas. A search for budding organisms in these situations may prove futile. Sometimes, yeasts may have dark-staining foci resembling Pneumocystis organisms. Also, some yeast cells may be surrounded by a clear space and may be mistaken for Cryptococcus . 55 Other look-alikes include Candida species, P. marneffei , capsule-deficient cryptococci, intracellular B. dermatitidis , and Hamazaki-Wesenberg bodies. Figure 7.71 Tinctorial and morphologic attributes of H. capsulatum in stained clinical specimens. (A) Diff-Quik: BAL fluid smear showing extracellular yeast forms. (B) Giemsa stain: tissue touch preparation demonstrating numerous yeast within a histiocyte and in the surrounding space. (C) GMS stain: abundant black-colored yeast cells in a tissue touch preparation. Both extracellular and intrahistiocytic forms are seen. (D) Gram stain: red-colored yeast cells in a blood culture smear. (E) Hematoxylin and eosin stain: liver biopsy containing numerous intracellular and extracellular yeast forms. Note the presence of colorless halos surrounding the yeast. (F) Mucicarmine stain: yeast forms are barely visible without the aid of increased contrast. (G) Periodic acid–Schiff stain: magenta-colored yeast cells are evident scattered throughout the epidermis of a skin biopsy specimen. (H) Wright-Giemsa: yeast forms evident within a monocyte in a peripheral blood smear. Scale bar, 10 mm; original magnification, ×1000. Figure 7.71 (From Wheat LJ, Azar MM, Bahr NC, et al. Histoplasmosis. Inf Dis Clin NA . 2016;30:216.) Paracoccidioidomycosis (South American Blastomycosis) Seven clinical forms occur, but they rarely cause lung infections in North America. In areas of high endemicity, such as Brazil, the several forms can mimic malignancy or sarcoidosis. 224 , 225 The histopathology resembles that of other mycoses and can be exudative or granulomatous. Paracoccidioides braziliensis appears as a large spherical yeast (10 to 60 µm) with multiple buds attached by narrow necks (a "steering wheel" or "ship's wheel" appearance). 226 When budding is sparse, look-alikes include H. capsulatum with small intracellular forms, B. dermatitidis and capsule-deficient cryptococci for medium-sized forms, and C. immitis or C. posadasii for large forms. Sporotrichosis Infection by Sporothrix schenckii is usually confined to the skin, subcutis, and lymphatic pathways, but the organism can disseminate to the lungs. Rarely, S. schenckii is a primary pulmonary pathogen. 227 The organism can produce cavitary disease in the form of a single lesion. Infection may be bilateral and apical, progressive and destructive, or it may be identified clinically as a solitary pulmonary nodule. Microscopically, caseous and suppurative granulomas ( Fig. 7.72A ) occur with variable numbers of round to oval, small (2 to 3 µm), narrow budding yeast ( Fig. 7.72B ) or cigar-shaped forms. 228 Nonnecrotizing granulomas also occur. Asteroid bodies are an important clue, especially when organisms are sparse, as is often the case. Look-alikes include H. capsulatum , acapsular cryptococci, Candida organisms, and Hamazaki-Wesenberg bodies. Figure 7.72 Sporotrichosis. (A) Cavitary granuloma manifesting as a solitary pulmonary nodule. (B) A rare, oval, narrow budding yeast (Grocott methenamine silver stain). Figure 7.72 Penicilliosis Southeast Asia is the endemic setting of the unique dimorphic fungus Penicillium marneffei . The disease it produces is not seen in North America except in travelers, especially immunocompromised persons. It is a common opportunistic infections in AIDS patients in Southeast Asia and a significant clue to the presence of AIDS in that area. 229 The respiratory tract is the portal of entry, with pulmonary infiltrates and disseminated disease, especially involving the skin. Microscopically, alveolar macrophages stuffed with spherical to oval yeast-like cells (2.5 to 5 µm) are seen, each with a single transverse septum; short hyphal forms and elongated, curved "sausage" forms may be formed in necrotic and cavitary lesions. 230 , 231 The septum distinguishes it from H. capsulatum , its look-alike. 229 , 232 Cryptococcosis C. neoformans is a ubiquitous, facultative intracellular yeast. Pulmonary cryptococcosis occurs worldwide but has a particularly high incidence in the United States. 233 The pathogenicity and histopathologic features of lung infection depends largely on the patient's immune status, as illustrated earlier in Fig. 7.7 and summarized in Box 7.17 . In the normal host, a substantial proportion of cryptococcal infections are asymptomatic, others are symptomatic, with infiltrates or nodules. Immunocompromised patients are almost invariably symptomatic and often develop disseminated disease with a predilection for the brain and meninges. Pulmonary injury patterns include single or multiple large nodules, segmental or diffuse infiltrates, cavitary lesions, and miliary nodules. Normal hosts most often develop nodules comprising fibrocaseous granulomas ( Fig. 7.73A ), or granulomatous pneumonia ( Fig. 7.73B ). Immunocompromised patients are more likely to have histiocytic ( Fig. 7.73C ) or mucoid infiltrates without inflammation ( Fig. 7.73D ). Box 7.17 Histopathologic Patterns in Cryptococcal Lung Disease In order of associated decrease in immune function: Fibrocaseous granuloma Granulomatous pneumonia Histiocytic pneumonia Mucoid pneumonia Intracapillary cryptococcosis Alt-text: Box 7.17 Reprinted with permission from Mark EJ. Case records of the Massachusetts General Hospital. N Engl J Med. 2002;347:518–524. Figure 7.73 Cryptococcosis. (A) Solitary pulmonary nodule with small satellite granulomas. (B) Granulomatous pneumonia with clusters of pale staining yeast in clear spaces surrounded by histiocytes and multinucleated giant cells. (C) Histiocytic pneumonia. (D) Mucoid pneumonia with no inflammatory cell reaction. Figure 7.73 The cryptococcal organisms are round yeast forms ranging in diameter from 2 to 15 µm, with an average size of 4 to 7 µm. Cryptococcal yeasts are visible on H&E-stained sections as pale gray to light blue structures, frequently with attached smaller buds. They often occur in clusters and can sometimes be found within giant cells. 203 The mucicarmine stain highlights the capsule ( Fig. 7.74A ); but with capsule-deficient forms ( Fig. 7.74B ) the pleomorphic appearance can be confused with that of other yeast forms (e.g., H. capsulatum , B. dermatitidis , S. schenckii ) and sometimes Pneumocystis . Figure 7.74 (A) Intravascular cryptococcus. Yeast cells with stained capsules (mucicarmine stain). (B) Capsule-deficient cryptococcus (Grocott methenamine silver stain). Figure 7.74 The lungs of patients with the most severe immunodeficiency may show myriad yeasts in alveolar septal capillaries ( Fig. 7.74A ), with little if any intraalveolar reaction 234 ; this form of the disease may also be associated with mucoid pneumonia. 235 The mucoid pneumonia ( Fig. 7.75A ) of cryptococcal infection can be confirmed with mucin stains such as Alcian blue ( Fig. 7.75B ). Another microscopic pattern recently described in HIV-infected patients is the inflammatory spindle cell pseudotumor, a lesion much more commonly associated with mycobacterial infection. 236 Figure 7.75 Cryptococcal mucoid pneumonia. (A) Myriad blue-gray yeast cells in mucoid matrix. (B) Alcian blue mucin stain accentuates the mucoid matrix. Figure 7.75 Candidiasis Candida organisms are yeasts that can produce pseudohyphae and are the most common invasive fungal pathogens in humans. Secondary Candida pneumonia is relatively common, but primary Candida pneumonia is rare in other than immunocompromised patients in the intensive care unit. 55 In general, Candida albicans is the most frequently isolated of the more than 100 known species, which include a few rare and emerging human pathogens. Candida glabrata and Candida tropicalis , together with C. albicans , account for 95% of bloodstream infections, the principal route for the acquisition of Candida pneumonia. 237 A non–blood-borne route to pneumonia results from aspiration of organisms from a heavily colonized or infected oropharynx. When the infection is blood-borne, miliary nodules with a necroinflammatory center and a hemorrhagic rim reflect an intravascular distribution of fungi. In the case of aspiration, the organisms may be found in the airways associated with an alveolar filling pattern of bronchopneumonia ( Fig. 7.76A ) 238 or, much less commonly, a bronchocentric granulomatosis pattern. Figure 7.76 (A) Candida bronchopneumonia. (B) Candida yeast cells—blastoconidia (Grocott methenamine silver stain). Figure 7.76 In tissue sections, oval budding yeast-like cells (blastoconidia) 2 to 6 µm in diameter may appear with pseudohyphae that constrict at points of budding, creating the impression of bulging rather than parallel walls ( Fig. 7.76B ). The pseudohyphae branch at acute angles and can overlap in width with the true hyphae of Aspergillus , from which they must be distinguished. Among the medically important species, Candida glabrata (formerly Torulopsis glabrata ) and Candida parapsilosis produce only yeast cells in tissue, in contrast with most other Candida species, which produce both yeast and pseudohyphae. 203 Other look-alikes include H. capsulatum , Trichosporon beigelii , and Malassezia furfur , depending on whether pseudohyphae or yeast forms alone are present. They can be distinguished from Histoplasma by their extracellular location and Gram stain positivity. T. beigelii tends to be somewhat larger and more pleomorphic. Malassezia is clinically associated with parenteral nutrition, Intralipid, and indwelling catheters. Pulmonary lesions include pneumonia, mycotic thromboemboli, infarcts, and vasculitis. M. furfur may be found in small arteries, where the organisms appear as small 2- to 5-µm yeast-like cells. They form distinctive unipolar broad-based buds but no pseudohyphae. 55 Aspergillosis Aspergillus species and other hyaline and dematiaceous molds have emerged as significant causes of morbidity and death in the immunocompromised host. Worldwide, species of Aspergillus are the most common invasive molds. They are the second most common fungal pathogens after Candida species but, in contrast with Candida , are more commonly isolated from the lung. Several species are recognized, but Aspergillus fumigatus is the one most often seen in the clinical laboratory and most often isolated from the lungs of immunocompromised patients. 239 Respiratory aspergillosis can be classified into a colonizing or saprophytic form (intrabronchial and preexisting cavity fungus ball, Fig. 7.77A ); hypersensitivity forms (allergic bronchopulmonary aspergillosis, including mucoid impaction of bronchi and hypersensitivity pneumonitis; Fig. 7.77B ); and invasive disease (minimally invasive–chronic necrotizing or angioinvasive–disseminated, Box 7.18 ). 55 , 240 , 241 , 242 , 243 Invasive disease ( Fig. 7.78 ) tends to occur in immunocompromised patients, including those with prolonged neutropenia, transplant recipients (especially hematopoietic stem cell and lung transplants), advanced AIDS, and the inherited immune deficiency disorder referred to as chronic granulomatous disease of childhood . The clinicopathologic features of invasive disease reflect these host-associated risk factors. 244 In patients with neutropenia, a characteristic angioinvasive pattern occurs, with intravascular spread resulting in hemorrhagic infarcts ( Fig. 7.79 ). In the nonneutropenic patient, the necroinflammatory pattern tends to lack this angioinvasive feature. 245 Some cases defy categorization (e.g., bronchocentric and miliary patterns; Fig. 7.80 ) or may be hybrids of infection and hypersensitivity. 246 Figure 7.77 (A) Aspergillosis fungus ball. (B) Allergic bronchopulmonary aspergillosis. Intraluminal allergic mucin with laminated clusters of eosinophils can be seen in inspissated basophilic mucin with scattered Charcot–Leyden crystals. Figure 7.77 Box 7.18 Histopathologic Patterns in Pulmonary Aspergillosis Colonization Fungus ball Hypersensitivity reaction Allergic bronchopulmonary aspergillosis Eosinophilic pneumonia Mucoid impaction Bronchocentric granulomatosis Hypersensitivity pneumonitis Invasive Acute invasive aspergillosis Necrotizing pseudomembranous tracheobronchitis Chronic necrotizing pneumonia Bronchopleural fistula Empyema Alt-text: Box 7.18 Reprinted with permission from Travis WD, Colby TV, Koss MN, et al. Lung infections. In: King D, ed. Atlas of Non-Tumor Pathology , Fascicle 2. Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology; 2002:539–728, Table 12.10. Figure 7.78 Resected lung specimen from an immunocompromised patient with necrotizing Aspergillus pneumonia. Figure 7.78 Figure 7.79 Invasive aspergillosis. (A) Hemorrhagic infarct. (B) 45-degree angle branching septate hyphae. Figure 7.79 Figure 7.80 Bronchocentric aspergillosis. (A) Bronchiole expanded and filled with purulent exudate. (B) Miliary aspergillosis. Colony of organisms with hyaline membranes evident at periphery of the image (lower right) . Figure 7.80 Microscopically, septate hyphae, dichotomously branched at a 45-degree angle, have uniform, consistent width (3 to 6 µm) without constrictions at points of septation . When numerous, as in some angioinvasive lesions and fungus balls, these features can be readily appreciated in H&E-stained sections. Fruiting heads of Aspergillus (shown earlier in Fig. 7.65 ) are sometimes formed in cavities. Oxalate crystals, visible in plane-polarized light ( Fig. 7.81 ), are an important clue to Aspergillus infection when hyphae cannot be identified. Figure 7.81 (A) Pale yellow oxalate crystal sheaths in necroinflammatory debris. (B) Birefringent oxalates seen under polarized light. Figure 7.81 Look-alikes include various hyaline molds such as Zygomycetes and Candida species as well as Pseudallescheria boydii . 247 Another look-alike is Fusarium species. Fusariosis is an emerging mycosis in the immunocompromised host, and Fusarium is the second most common opportunistic pathogen after Aspergillus species in immunosuppressed patients with hematologic malignancies. 248 The clinical and pathologic features in the lung and at sites of dissemination mimic those of aspergillosis, and the mycelia are essentially indistinguishable. Isolation in culture, immunohistochemistry, or molecular techniques, such as in situ hybridization or PCR amplification, is required for definitive diagnosis. Other previously uncommon but newly emerging hyaline molds that may be difficult to distinguish from Aspergillus in tissue are Paecilomyces , Acremonium , Scedosporium , and Basidiobolus . 237 , 249 , 250 Zygomycosis The taxonomic organization of the fungal phylum Zygomycota includes the class Zygomycetes, which is subdivided into two orders: Mucorales and Entomophthorales. These orders contain the agents of human zygomycosis. 251 The order Mucorales includes the genera Absidia , Apophysomyces , Rhizopus , Rhizomucor, and Mucor , from which the often taxonomically incorrect term mucormycosis is derived. In fact, most infections are due to Rhizopus and Absidia species. 252 The zygomycete species share clinical and pathologic features with invasive Aspergillus species, being angiotropic and capable of inducing hemorrhagic infarcts with sparse inflammation. Clinical syndromes produced by these fungi include rhinocerebral, pulmonary, cutaneous, and gastrointestinal infections with a predilection for neonates. 253 Hematopoietic malignancies and diabetes mellitus with acidosis underlie most cases of pulmonary infection in children and adults. 254 , 255 Box 7.19 lists a broad spectrum of pulmonary diseases that includes solitary or multiple and bilateral nodular lesions, segmental or lobar consolidation, cavitary lesions, fistulas, and infarcts ( Figs. 7.82 and 7.83 ); direct extension into mediastinal, thoracic soft tissue, chest wall, and diaphragm; chronic tracheal and endobronchial infection; and fungus balls similar to those seen with aspergilloma. 256 An endobronchial syndrome with a propensity for blood vessel erosion has also been described, sometimes resulting in fatal hemoptysis. 257 Box 7.19 Histopathologic Patterns in Pulmonary Zygomycosis Acute lobular or lobar pneumonia Nodules Cavities Endobronchial mass Fistulas Infarcts Thoracic soft tissue/mediastinum Fungus ball Alt-text: Box 7.19 Figure 7.82 Resected lung specimen from patient with necrotizing pneumonia caused by zygomycosis. Figure 7.82 Figure 7.83 Zygomycosis. (A) Nodular infarct. (B) Intravascular organisms (arrows). Vessel at right arrow is shown at high magnification (inset) (Grocott methenamine silver stain). Figure 7.83 Hyphae are broad (6 to 25 µm), thin-walled, and pauciseptate ( Fig. 7.84A ). They display variation in width, with twisted, nonparallel contours and random wide-angle branching nearing 90 degrees. 203 They also have a tendency to fragment more commonly than Aspergillus organisms, which tend to retain their elongated sweeping profiles. Additional features include variability in tinctorial staining in H&E sections, ranging from basophilia to eosinophilia. In frozen sections, hyphae may show weak staining, and they often have a bubbly or vacuolated appearance. 256 In addition to being angiotropic, they are neurotropic. 258 In lesions exposed to air, the hyphae may form ovoid or spherical thick-walled chlamydoconidia, within or at the terminal ends ( Fig. 7.84B ). 259 Look-alikes at the lower-width range include Aspergillus and other Aspergillus -like hyaline molds. The pseudohyphae of Candida species can sometimes be similar. Figure 7.84 Zygomycosis. (A) Twisted pauciseptate, broad mycelia characteristic of Zygomycetes (Grocott methenamine silver stain). (B) Endobronchial zygomycosis with chlamydospores. Figure 7.84 Phaeohyphomycosis A few genera of dematiaceous molds produce infections resembling those of Aspergillus , including allergic bronchopulmonary disease ( Fig. 7.85A ) and bronchocentric granulomatosis patterns. 260 , 261 The more than 80 genera and species of these saprophytes, which occur naturally in wood, soil, and decaying matter, include Bipolaris , Exserohilum , Xylohypha , Alternaria , and Curvularia , among others. 203 The unique appearance of these fungi is due to their cell wall melanin content. In the allergic mucin or other deposits of necroinflammatory debris, the phaeoid (dark brown- to black-pigmented) hyphae (2 to 6 µm in diameter) are generally sparse but can resemble Aspergillus and other hyaline molds, especially when lightly pigmented or nonpigmented. Typically only small mycelial fragments are seen, which may be mistaken for artifacts, sometimes with terminal swellings resembling chlamydoconidia ( Fig. 7.85B ). The dematiaceous agents of subcutaneous forms of chromoblastomycosis appear as pigmented muriform cells in granulomas, and they do not form mycelia. Chromoblastomycosis is rarely encountered in the lung. Another Aspergillus look-alike is P. boydii , an organism that is sometimes grouped with the dematiaceous fungi. P. boydii usually exhibits a more ragged, disorganized, and densely clustered pattern of mycelia. Clinically, localized disease may be cured by excision alone; systemic disease is often refractory to treatment. 262 Figure 7.85 Allergic bronchopulmonary fungal disease. (A) Ectatic bronchus with thick eosinophilic basement membrane and intraluminal necroinflammatory debris. (B) Mycelial fragments of Bipolaris organisms (Grocott methenamine silver stain). Figure 7.85 Pneumocystosis The face of Pneumocystis pneumonia continues to change. Once considered to be a protozoan, this organism is now classified as a fungus, and the species infecting humans has been renamed Pneumocystis jirovecii (formerly Pneumocystis carinii ). 263 Once a disease of malnourished or leukemic children, today Pneumocystis infection is identified most commonly in patients with defective immunity, especially AIDS, or those on immunosuppressive therapies for hematopoietic malignancies, organ transplants, and collagen vascular diseases. With the success of contemporary therapy for AIDS, the pathologist is now more likely to encounter the disease in the latter group of patients in whom it is apt to be more subtle. 264 The classic pattern during the HIV epidemic was the foamy alveolar cast ( Fig. 7.86 ) with moderate to numerous organisms, type II pneumocyte hyperplasia, and a scant to moderate interstitial lymphoplasmacytic infiltrate. 265 , 266 Figure 7.86 Pneumocystis pneumonia. (A) Lymphoplasmacytic interstitial infiltrate and intraalveolar foamy alveolar cast. (B) Numerous yeast-like cells of Pneumocystis jirovecii of various shapes (Grocott methenamine silver stain). Figure 7.86 In recent years a number of atypical and unusual patterns have been described that are worth recognizing. 55 , 267 , 268 These are listed in Box 7.20 . P. jirovecii infection can mimic any lung injury pattern, ranging from acute diffuse alveolar damage with hyaline membranes ( Fig. 7.87 ) and minimal or no foamy exudates to an organizing phase with sparse organisms. There is also a spectrum of granulomatous infection, both nonnecrotizing and necrotizing, that may overlap morphologically with mycobacterial or other fungal infections, particularly histoplasmosis ( Fig. 7.88 ). Cavitary disease, solitary pulmonary nodules that may be relatively fibrotic, cysts, and dystrophic calcification are also described. 268 , 269 , 270 Box 7.20 Histopathologic Patterns in Pulmonary Pneumocystis Infection Foamy alveolar cast Diffuse alveolar damage "Id" reaction (minimal-change reaction) Granulomas Miliary disease Vascular invasion/vasculitis/infarct Lymphoid interstitial pneumonia Cavities and cysts Subpleural blebs and bullae Microcalcification Alt-text: Box 7.20 Figure 7.87 Pneumocystis pneumonia. (A) Diffuse alveolar damage pattern with hyaline membranes. (B) Cysts in hyaline membrane (Grocott methenamine silver stain). Figure 7.87 Figure 7.88 Pneumocystis pneumonia. (A) Miliary granuloma with central necrosis. (B) Sparse organisms in granuloma (Grocott methenamine silver stain). Figure 7.88 Microscopically, the three life stages of the organism are still referred to by protozoan terminology as sporozoites, trophozoites, and cysts. The cyst is the most common form seen by pathologists. On silver stains the cyst is seen as an oval (4 to 7 µm) yeast-like cell that may be collapsed, helmet-shaped, or variably crescentic. The intracystic dot or paired–comma structures are important keys to distinguishing P. jirovecii cysts from look-alikes such as Histoplasma , the capsule-deficient form of Cryptococcus , Candida species, and even overstained red blood cells. Sporozoites and trophozoites are seen to best advantage in touch imprints and cytologic preparations of respiratory samples. Cytopathology Many of the fungal pathogens involving the respiratory tract can be detected by cytologic techniques in sputum samples, bronchial washings and brushings, BAL fluid samples, and needle aspirates. 46 The aspirates and other samples can also be submitted for culture and ancillary studies. 271 The four most common yeast forms— C. neoformans , C. immitis or C. posadasii , H. capsulatum , and B. dermatitidis —must be distinguished from each other, and P. jirovecii can also enter the differential diagnosis. 45 Morphologic features of these organisms are often better visualized in cytologic preparations than in tissue sections, usually permitting a rapid and definitive diagnosis on smears prepared using routine stains (Papanicolaou, Diff-Quik, and H&E). More specific fungal stains (GMS, Gridley, and Fontana–Masson) can often be held in reserve. Amorphous granular debris and epithelioid cells characterize many necrotizing granulomas. Typically a background of neutrophils is seen when suppurative granulomas are aspirated. Histoplasma infections may manifest an epithelioid or phagocytic cell population. Cryptococcal infections can be similar or may be associated with little or no accompanying inflammation in the immunocompromised patient. Cytology of Common Yeast Forms Morphologic features of some of the more common yeast forms that the pathologist may encounter in cytologic material are presented in Table 7.7 . C. neoformans organisms are seen as are single budding yeast forms with a narrow, pinched-off base, approximately 4 to 7 µm in diameter but ranging in size from 2 to 15 µm. In needle aspirates, the mucoid capsule investing the yeast imparts a "spare tire" appearance ( Fig. 7.89 ). Figure 7.89 Cryptococcus neoformans . In this fine-needle aspirate, clusters of yeast cells resembling "spare tires" are invested by capsule in a sparse inflammatory background (alcohol-fixed). Figure 7.89 B. dermatitidis organisms are refractile, double-contoured yeast forms and range in diameter from 8 to 15 µm with broad-based budding ( Fig. 7.90 ). An internal amorphous mass can be appreciated in some stained preparations. Smaller or larger yeast cells can be mistaken for C. neoformans or C. immitis , respectively. Figure 7.90 Blastomyces dermatitidis. (A) Necroinflammatory infiltrate with refractile yeast forms. (B) Periodic acid–Schiff staining highlights the double-contoured yeast with broad-based budding (see inset for greater detail). Figure 7.90 C. immitis/C. posadasii spherules exhibit a variety of sizes and shapes, ranging from large spherules packed with endospores ( Fig. 7.91A ) to empty, collapsed spheres and small immature spherules. 272 The latter may overlap with Blastomyces and other yeasts. Mycelial forms of Coccidioides species, with arthrospores, may be found in aspirates of cavitary nodules exposed to air ( Fig. 7.91B ). Figure 7.91 Coccidioides species. (A) Negative-staining spherule in suppurative inflammatory background in a fine-needle aspirate (alcohol-fixed). (B) Ruptured spherules and mycelia with arthrospores in granular necrotic background in another fine-needle aspirate (alcohol-fixed). Figure 7.91 H. capsulatum yeast cells are small (2 to 5 µm) and stain poorly in routine smears, but the presence of this pathogen can be suspected on the basis of the dot-like refractile appearance of these cells in the cytoplasm of macrophages. In Diff-Quik–stained smears, the characteristic purple, polarized yeast forms ( Fig. 7.92 ) are discernible, and they are outlined entirely in GMS-stained smears. Figure 7.92 Histoplasma capsulatum. Clusters of purple polarized yeast cells are readily seen in this fine-needle aspirate (Diff-Quik preparation). Figure 7.92 P. jirovecii is most commonly identified in exfoliative samples and aspirates by the presence of the foamy alveolar cast, which varies from eosinophilic to basophilic and is highly characteristic ( Fig. 7.93A ). These organisms rarely occur singly. The GMS stain outlines the characteristic cysts ( Fig. 7.93B ). Figure 7.93 Pneumocystis jirovecii. (A) Foamy alveolar cast in bronchial washing (ThinPrep Papanicolaou stain). (B) Cysts with intracystic dot in bronchial washing (ThinPrep, Grocott methenamine silver stain). Figure 7.93 Table 7.7 Morphologic Features of Selective Yeast Forms Table 7.7 Feature Small Intermediate Large Candida Pneumocystis Histoplasma Cryptococcus Blastomyces Coccidioides Size (µm) 3–4 5–8 2–5 5–15 8–20 20–200 Shape Oval Pleomorphic Oval Pleomorphic Round Round Budding None None Narrow-based Narrow-based Broad-based None Wall thickness Thin Thin Thin Thin Thick Thick Hyphae/pseudohyphae Common; characteristic Absent Rare Rare Rare Occasional Other features Single and chains Intracystic body Trophozoite forms Intracellular Refractile Mucicarmine + capsule Acapsular forms Double-contour wall Endospores, immature spherules Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:87. Cytology of Common Mycelial Forms The cytopathologist's most frequent challenge is the interpretation of mycelial forms in exfoliated material, especially the distinction between Aspergillus look-alikes—Zygomycetes and Candida hyphae. The morphologic features of some of the more common agents are compared in Table 7.8 . Candida species are readily seen and easily diagnosed when both yeasts and pseudohyphae are present. However, interpretation of their significance is difficult in all except transthoracic needle aspirates, where the presence of any mycelial structure, particularly in the setting of mass-like and cavitary infiltrates, provides strong morphologic evidence of infection. Aspergillus species are characterized by septate mycelia that branch at angles approaching 45 degrees ( Fig. 7.94 ). Aspergillus hyphae lack constrictions at points of septation. However, Aspergillus organisms cannot be differentiated from one of their mimics by morphology alone unless accompanied by a fruiting body. A rapid in situ hybridization technique specific for Aspergillus species can be performed on pulmonary cytocentrifuge preparations, as well as on tissue. 273 Figure 7.94 Aspergillus species. (A) Twisted, sparsely septate mycelia are difficult to differentiate from mimics, including Zygomycetes, in this fine-needle aspirate (Diff-Quik preparation). (B) Characteristic mycelia in a bronchial washing (Papanicolaou stain). Figure 7.94 Zygomycete mycelia are distinguished from Aspergillus and Candida forms by their often broader width and their pleomorphic, twisted ribbon–like, pauciseptate features. Of note, however, in aspirates of aspergilloma, the mycelia may also have a twisted appearance. Table 7.8 Morphologic Features of Selected Fungal Mycelia Table 7.8 Feature Aspergillus Bipolaris Zygomycetes Pseudallescheria Boydii Fusarium Width (µm) 3–6 2–6 5–20 2–5 3–8 Contour Parallel Parallel Irregular Parallel Parallel Branching Dichotomous Haphazard Wide angle Haphazard 90-degree angle Branch orientation Parallel Random Random Random Random Septation Frequent Frequent Infrequent Frequent Frequent Phaeoid (Brown) No Yes No Usually not No Angioinvasive Yes No Yes Yes Yes Other features Fruiting body; oxalate crystals sometimes Chlamydoconidia sometimes One of many dematiaceous genera Rarely chlamydoconidia Aspergillus "lookalikes" Aspergillus "lookalikes" Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:204. A potential pitfall in the evaluation of cytopathologic specimens in fungal infections (both exfoliative samples and needle aspirates) is the confounding presence of atypical reactive squamous cells and type II pneumocytes, which can mimic the cytologic atypia of malignant neoplasms. 48 Furthermore, the pathologist interpreting lung biopsy findings, especially with transbronchial specimens, should always attempt to correlate such findings with samples that may have been collected for cytologic or microbiologic study. This is especially advisable because etiologic agents that escape detection in tissue, such as Pneumocystis , Aspergillus , and CMV, may be found in washings or lavage fluid. 274 Cytology of Common Yeast Forms Morphologic features of some of the more common yeast forms that the pathologist may encounter in cytologic material are presented in Table 7.7 . C. neoformans organisms are seen as are single budding yeast forms with a narrow, pinched-off base, approximately 4 to 7 µm in diameter but ranging in size from 2 to 15 µm. In needle aspirates, the mucoid capsule investing the yeast imparts a "spare tire" appearance ( Fig. 7.89 ). Figure 7.89 Cryptococcus neoformans . In this fine-needle aspirate, clusters of yeast cells resembling "spare tires" are invested by capsule in a sparse inflammatory background (alcohol-fixed). Figure 7.89 B. dermatitidis organisms are refractile, double-contoured yeast forms and range in diameter from 8 to 15 µm with broad-based budding ( Fig. 7.90 ). An internal amorphous mass can be appreciated in some stained preparations. Smaller or larger yeast cells can be mistaken for C. neoformans or C. immitis , respectively. Figure 7.90 Blastomyces dermatitidis. (A) Necroinflammatory infiltrate with refractile yeast forms. (B) Periodic acid–Schiff staining highlights the double-contoured yeast with broad-based budding (see inset for greater detail). Figure 7.90 C. immitis/C. posadasii spherules exhibit a variety of sizes and shapes, ranging from large spherules packed with endospores ( Fig. 7.91A ) to empty, collapsed spheres and small immature spherules. 272 The latter may overlap with Blastomyces and other yeasts. Mycelial forms of Coccidioides species, with arthrospores, may be found in aspirates of cavitary nodules exposed to air ( Fig. 7.91B ). Figure 7.91 Coccidioides species. (A) Negative-staining spherule in suppurative inflammatory background in a fine-needle aspirate (alcohol-fixed). (B) Ruptured spherules and mycelia with arthrospores in granular necrotic background in another fine-needle aspirate (alcohol-fixed). Figure 7.91 H. capsulatum yeast cells are small (2 to 5 µm) and stain poorly in routine smears, but the presence of this pathogen can be suspected on the basis of the dot-like refractile appearance of these cells in the cytoplasm of macrophages. In Diff-Quik–stained smears, the characteristic purple, polarized yeast forms ( Fig. 7.92 ) are discernible, and they are outlined entirely in GMS-stained smears. Figure 7.92 Histoplasma capsulatum. Clusters of purple polarized yeast cells are readily seen in this fine-needle aspirate (Diff-Quik preparation). Figure 7.92 P. jirovecii is most commonly identified in exfoliative samples and aspirates by the presence of the foamy alveolar cast, which varies from eosinophilic to basophilic and is highly characteristic ( Fig. 7.93A ). These organisms rarely occur singly. The GMS stain outlines the characteristic cysts ( Fig. 7.93B ). Figure 7.93 Pneumocystis jirovecii. (A) Foamy alveolar cast in bronchial washing (ThinPrep Papanicolaou stain). (B) Cysts with intracystic dot in bronchial washing (ThinPrep, Grocott methenamine silver stain). Figure 7.93 Table 7.7 Morphologic Features of Selective Yeast Forms Table 7.7 Feature Small Intermediate Large Candida Pneumocystis Histoplasma Cryptococcus Blastomyces Coccidioides Size (µm) 3–4 5–8 2–5 5–15 8–20 20–200 Shape Oval Pleomorphic Oval Pleomorphic Round Round Budding None None Narrow-based Narrow-based Broad-based None Wall thickness Thin Thin Thin Thin Thick Thick Hyphae/pseudohyphae Common; characteristic Absent Rare Rare Rare Occasional Other features Single and chains Intracystic body Trophozoite forms Intracellular Refractile Mucicarmine + capsule Acapsular forms Double-contour wall Endospores, immature spherules Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:87. Cytology of Common Mycelial Forms The cytopathologist's most frequent challenge is the interpretation of mycelial forms in exfoliated material, especially the distinction between Aspergillus look-alikes—Zygomycetes and Candida hyphae. The morphologic features of some of the more common agents are compared in Table 7.8 . Candida species are readily seen and easily diagnosed when both yeasts and pseudohyphae are present. However, interpretation of their significance is difficult in all except transthoracic needle aspirates, where the presence of any mycelial structure, particularly in the setting of mass-like and cavitary infiltrates, provides strong morphologic evidence of infection. Aspergillus species are characterized by septate mycelia that branch at angles approaching 45 degrees ( Fig. 7.94 ). Aspergillus hyphae lack constrictions at points of septation. However, Aspergillus organisms cannot be differentiated from one of their mimics by morphology alone unless accompanied by a fruiting body. A rapid in situ hybridization technique specific for Aspergillus species can be performed on pulmonary cytocentrifuge preparations, as well as on tissue. 273 Figure 7.94 Aspergillus species. (A) Twisted, sparsely septate mycelia are difficult to differentiate from mimics, including Zygomycetes, in this fine-needle aspirate (Diff-Quik preparation). (B) Characteristic mycelia in a bronchial washing (Papanicolaou stain). Figure 7.94 Zygomycete mycelia are distinguished from Aspergillus and Candida forms by their often broader width and their pleomorphic, twisted ribbon–like, pauciseptate features. Of note, however, in aspirates of aspergilloma, the mycelia may also have a twisted appearance. Table 7.8 Morphologic Features of Selected Fungal Mycelia Table 7.8 Feature Aspergillus Bipolaris Zygomycetes Pseudallescheria Boydii Fusarium Width (µm) 3–6 2–6 5–20 2–5 3–8 Contour Parallel Parallel Irregular Parallel Parallel Branching Dichotomous Haphazard Wide angle Haphazard 90-degree angle Branch orientation Parallel Random Random Random Random Septation Frequent Frequent Infrequent Frequent Frequent Phaeoid (Brown) No Yes No Usually not No Angioinvasive Yes No Yes Yes Yes Other features Fruiting body; oxalate crystals sometimes Chlamydoconidia sometimes One of many dematiaceous genera Rarely chlamydoconidia Aspergillus "lookalikes" Aspergillus "lookalikes" Modified from Chandler FW, Watts JC. Pathologic Diagnosis of Fungal Infections. Chicago: ASCP Press; 1987:204. A potential pitfall in the evaluation of cytopathologic specimens in fungal infections (both exfoliative samples and needle aspirates) is the confounding presence of atypical reactive squamous cells and type II pneumocytes, which can mimic the cytologic atypia of malignant neoplasms. 48 Furthermore, the pathologist interpreting lung biopsy findings, especially with transbronchial specimens, should always attempt to correlate such findings with samples that may have been collected for cytologic or microbiologic study. This is especially advisable because etiologic agents that escape detection in tissue, such as Pneumocystis , Aspergillus , and CMV, may be found in washings or lavage fluid. 274 Microbiology Complementary laboratory methods are often required for the diagnosis of fungal infection; these are listed in Box 7.21 . 204 Under the microscope, many fungi are readily apparent in H&E-stained sections, where they appear colorless (negative staining) or phaeoid (naturally pigmented). The GMS stain is the best histologic stain for demonstrating fungi when they are sparse or not visible on H&E sections. However, some fungi, notably the Zygomycetes, may stain poorly with GMS. The GMS preparation can be counterstained with H&E, allowing coevaluation of the host inflammatory response. The Fontana–Masson stain has been used to detect melanin in C. neoformans and phaeoid fungi, but many Aspergillus species and some Zygomycetes will also stain with this reagent. 259 , 275 The PAS stain can be useful in select circumstances, and histochemical stains for mucin (Alcian blue or mucicarmine) are useful for C. neoformans infections. The PAS and mucin preparations can also be counterstained with GMS or Fontana–Masson to simultaneously highlight cell walls and capsules of cryptococci. It is important to recognize that not everything that stains with the silver methods is a fungus, and care must be taken to distinguish organisms from pseudomicrobes, such as overstained red cells, white blood cell nuclei, reticulin and elastic fibers, calcium deposits, and even Hamazaki-Wesenberg bodies. 42 Box 7.21 Laboratory Diagnosis of Fungal Pneumonia Direct detection of organisms Chemofluorescence stains Direct fluorescent antibody stain Histopathologic/cytopathologic examination Immunohistochemical studies Antigen detection (in suspected histoplasmosis and cryptococcosis) Culture Emmons modified Sabouraud agar Brain-heart infusion agar Special and selective media Serologic testing Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.21 In the microbiology laboratory, the age-old technique of direct light microscopic visualization of fluids, exudates, and tissue homogenates treated with potassium hydroxide (the KOH wet prep) is being replaced by chemofluorescent cotton-brightening agents (such as calcofluor white and fungiqual). Fluorescence microscopy with these reagents can detect a wide variety of fungi in wet mounts as well as frozen sections and paraffin-embedded tissue. 276 , 277 Laboratory techniques for the identification of fungi (gross colonial and microscopic morphologic analysis after isolation on fungal media, followed by biochemical testing) are the principal means to a species-specific etiologic diagnosis. For deep tissues, including the lung and other sterile sites, the Emmons modification of Sabouraud glucose agar with chloramphenicol is recommended by many mycologists. 278 Additional use of enriched media such as brain-heart infusion agar can improve recovery of C. neoformans , B. dermatitidis , and H. capsulatum . Selective media containing cycloheximide are not recommended for normally sterile sites because they are potentially inhibitory for yeasts, such as Cryptococcus and Candida species, and molds, such as Aspergillus and the Zygomycetes. The interpretation of a positive fungal culture must be made in the clinical context. In the absence of proof of tissue invasion or compelling ancillary data, the interpretation of laboratory results requires considerable judgment. Many fungi are ubiquitous in the environment, and most fungal isolates from nonsterile respiratory samples do not represent disease unless there are also significant risk factors such as HIV infection, organ transplantation, or immunocompromising drug therapy. 279 For most of the dimorphic fungi, in vitro hyphae-to-yeast conversion studies have given way to commercially available nucleic acid probes for rapid specific identification. Procurement of tissue for culture before formalin fixation is important whenever fungal infections are suspected. The tissue sample should be kept moist using sterile, nonbacteriostatic saline or Ringer's solution. Specimens are minced but not ground before plating. The value of bringing multiple, often complementary laboratory methods to bear on inconclusive morphologic findings cannot be overemphasized. In this context, while culture has been considered the most reliable method for definitive diagnosis and histopathology often the fastest, the greatest yield results from combining histopathology with traditional culture and one or more of the newer molecular methods. 280 , 281 Culture may fail to yield an isolate even in the face of positive microscopic findings. In fact, the yield from tissue specimens, needle aspirates, BAL fluid samples, and bronchial washings is quite low for molds and other fungi for reasons that are not entirely clear. 49 , 282 Immunofluorescence testing using specific monoclonal antibodies can achieve a rapid and specific diagnosis in selected infections, especially when tissue has not been submitted for culture. Antibodies directed against the antigens of Aspergillus species and selected other fungi have been described, but most are not yet commercially available. For the problematic case, the mycology section of the CDC can provide assistance. Immunohistochemical identification of fungi can be accomplished fairly easily for those species for which reagents are commercially available. 33 , 283 , 284 Molecular techniques, including in situ hybridization and amplification technologies such as PCR, are other powerful tools that can provide rapid, accurate diagnosis for yeasts and molds that may be present in small numbers or manifest overlapping histologic features. 277 , 285 , 286 , 287 A few laboratories (including the CDC) are performing such assays. Use of quantitative real-time PCR assays on blood, body fluids, and other samples holds promise for relatively rapid definitive diagnosis when routine methods of isolation and identification fail in critical situations. 288 , 289 Serologic tests can support a morphologic diagnosis when positive titers are present, but effective serodiagnosis of systemic fungal infections is not available for most fungi. 290 Unfortunately an antibody response does not necessarily correlate with invasive disease, and an antibody response may be lacking for various reasons. False-positive results due to cross reactions and false-negative results due to a variety of reasons plague many of these assays. Some of the most accurate serologic tests (with high sensitivity and specificity) for fungal infections are those for histoplasmosis and coccidioidomycosis, yet tests for both have limitations that must be recognized in interpreting results. 291 , 292 The detection of macromolecular antigens shed into various body fluids requires a relatively large microbial burden, which tends to limit sensitivity for most fungal infections except histoplasmosis and crytococcosis. 280 For these two fungi, useful antigen detection techniques are available using serum, urine, cerebrospinal fluid, and BAL fluid. They are especially sensitive in patients with defective immunity. 271 , 292 In patients with pneumonia and normal immunity, however, these tests may be positive in lavage fluid but negative in urine unless the disease has disseminated. Other assays designed to detect antigens or metabolites of invasive fungi include those for 1,3β- d -glucan, a cell wall component of several fungi such as Aspergillus , Candida , Fusarium , and others, and for galactomannan, a polysaccharide antigen in the cell wall of Aspergillus ; these assays have shown fair sensitivity and specificity. 203 , 293 , 294 Differential Diagnosis A synopsis of the key morphologic and mycologic features of the fungal pneumonias is presented in Table 7.9 . 295 When H&E and GMS stains fail to detect fungal elements, the use of ancillary procedures may provide the specific diagnosis. Sometimes, if tissue or other patient specimens have been submitted for culture, the answer may lie in the mycology section of the microbiology laboratory, as many species begin to grow in a matter of days. When fungi are not readily identified by any of these techniques or strategies, other granulomatous infections should be considered, especially mycobacterial, uncommon bacterial (e.g., tularemia, brucellosis), and parasitic infections. Noninfectious necrotizing and nonnecrotizing granulomatous disorders also enter the differential diagnosis. These include granulomatosis with polyangiitis, idiopathic bronchocentric granulomatosis, aspiration, sarcoidosis, rheumatoid nodules, pyoderma gangrenosum–like lung lesions in patients with inflammatory bowel disease, and Churg-Strauss syndrome. 224 Table 7.9 Fungal Pneumonias: Summary of Pathologic Findings Table 7.9 Assessment Component Findings Blastomycosis Surgical pathology Suppurative granuloma most characteristic; also, tuberculoid (necrotizing) types; round, thick-walled (double-contour) yeast with broad-based budding Cytopathology Neutrophils and epithelioid cells with characteristic refractile yeast cell with double-contoured wall and broad-based budding Microbiology Characteristic yeast seen on wet mount, KOH- and calcofluor-stained smear; culture-sterile lung tissue on nonselective fungal media (e.g., Emmons modified Sabouraud) and enriched media (e.g., brain-heart infusion); add selective media for bronchial/transbronchial samples; colonies produce oval conidia on terminal ends of conidiophore at right angle to mycelium; confirm with DNA probe; serologic studies not useful Coccidioidomycosis Surgical pathology Fibrocaseous granuloma; large intact and/or ruptured spherules, full or partially or completely empty of endospores; mycelial forms in aerated cavities and fistula Cytopathology Necroinflammatory debris with epithelioid histiocytes; intact, viable, colorless spherules with variable number of endospores and/or ruptured degenerating forms with stained wall; range in size from large mature to small immature types Microbiology Characteristic mature spherules in wet mount, KOH- and calcofluor-stained smear; culture of sterile lung tissue on nonselective fungal media yields mycelia with characteristic arthroconidia; confirm with DNA probe; serologic diagnosis with tests for IgG and IgM antibodies by immunodiffusion, EIA; complement fixation for titers Histoplasmosis Surgical pathology Macrophage reaction and/or granulomas, based on immunity, including miliary and solitary pulmonary, variably hyalinized nodule; small, thin-walled, oval yeasts with narrow-based buds, often refractile Cytopathology Macrophage and epithelioid cells with characteristic yeast cell, often intracellular, stained purple with Diff-Quik, black with GMS Microbiology Rarely detected by direct examination of most clinical specimens; culture-sterile lung tissue on nonselective and enriched fungal media produces tuberculate macroconidia; confirm with DNA probe; antigen detection by EIA available for BAL fluid, CSF, serum, and urine Paracoccidioidomycosis Surgical pathology Exudative or granulomatous lesion with large, globose yeast cell with multiple buds Cytopathology Suppurative or granulomatous reaction with characteristic yeast cell Microbiology Direct detection in wet mount, KOH- and calcofluor-stained smear; culture-sterile lung tissue on standard nonselective fungal media; serologic testing by immunodiffusion, EIA; complement fixation for titer Sporotrichosis Surgical pathology Necrotizing granuloma, often cavitary with small, usually round, sometimes cigar-shaped yeast with sparse, narrow buds Cytopathology Suppurative or necrotizing granuloma pattern; yeast cells generally sparse or absent Microbiology Rarely detected by direct examination of most clinical specimens; culture of sterile lung tissue on nonselective fungal media yields thin, hyphae-bearing conidia in a rosette pattern; converts to a yeast phase at 37°C on blood agar; no serologic tests Penicilliosis Surgical pathology Alveolar macrophages stuffed with yeast cells resemble Histoplasma species, but with septum reflecting binary fission, not budding reproduction Cytopathology Macrophage with intracellular characteristic yeast forms Microbiology Culture of sterile lung tissue on nonselective fungal media yields a mold with a red pigment evident as culture ages; erect conidiophores, sometimes branched with metulae bearing one or several phialides with long, loose chains of oval conidia; new urinary antigen test Cryptococcosis Surgical pathology Granulomas, histiocytic infiltrate or mucoid pneumonia, based on immunity with pale, round, budding pleomorphic yeast cells, often in clusters; mucoid capsules usually present; acapsular types sometimes seen Cytopathology Yeast cell with mucoid capsular halo resembles "spare tire"; combination of mucicarmine and GMS or Fontana–Masson outlines capsule and cell wall; background of epithelioid cells or necroinflammatory debris may be sparse or absent Microbiology Oval to lemon-shaped calcofluor-positive yeast cell with capsule in India ink–stained touch imprint; culture on nonselective fungal media yields mucoid yeast-type colonies; no pseudohyphae; germ tube–negative; dark brown pigment on birdseed (niger) agar; confirm with biochemical tests; antigen detection test (latex agglutination or EIA) on serum, BAL fluid, CSF, and needle aspirates Candidiasis Surgical pathology Miliary necroinflammatory lesions or bronchopneumonia with small, oval, budding yeasts with or without pseudohyphae; C. glabrata yeast only Cytopathology Yeasts and/or pseudohyphae in a necroinflammatory background Microbiology Budding yeasts and pseudohyphae in wet mounts, KOH- and calcofluor-stained smears; cultures on selective and nonselective fungal media yield creamy tan to white yeast-type colonies; identification by germ tube production, carbohydrate assimilation, and cornmeal agar morphology Aspergillosis Surgical pathology Various forms include saprophytic (fungus ball), allergic (ABPA and mucoid impaction), hypersensitivity pneumonitis, and invasive disease ranging in severity from minimal chronic necrotizing to extensive pneumonia; angiotrophic with necrotizing infarcts; also hybrid forms of disease; septate, dichotomous, 45-degree angle mycelia; oxalate crystals; presence of fruiting body is genus-specific Cytopathology Tangled clusters of septate mycelia in a necroinflammatory background; may appear sparsely septate and twisted, mimicking Zygomycetes Microbiology Positive staining of mycelia with calcofluor and GMS; culture of sterile lung tissue on nonselective fungal media produces mold-type colonies in a range of colors; species differentiation by conidial and conidiophore morphology Zygomycosis Surgical pathology Nodular lesions, lobar consolidations, cavitary lesions, fungus balls, and airway infections commonly necrotizing and ischemic secondary to angioinvasion; broad pauciseptate mycelia with 90-degree angle, branching, often with twisted-ribbon morphology Cytopathology Pauciseptate mycelia, often with twisted-ribbon morphology in a necroinflammatory background Microbiology Positive staining of mycelia with calcofluor and GMS; rapidly growing cottony colonies are grown on most nonselective fungal media, but "controlled baiting" with bread sometimes necessary; identification based on presence and locations of rhizoids, shape of sporangia, presence of columellae, and shape of sporangiospores Phaeohyphomycosis Surgical pathology Allergic bronchopulmonary fungal disease similar to aspergillosis Cytopathology Similar to ABPA pattern—"allergic mucin" with eosinophils, Charcot–Leiden crystals in inspissated mucus; fungal mycelial fragments sparse or absent Microbiology Dematiaceous (phaeoid) dark brown to black colonies on nonselective fungal media; identified by shape and cross walls of multicell, pigmented conidia Pneumocystosis Surgical pathology Pneumonia with foamy alveolar cast is classic; other patterns include diffuse alveolar damage, granulomatous lesions, and minimal changes; variable numbers of cysts noted in GMS-stained sections Cytopathology Foamy alveolar cast with characteristic cysts outlined by GMS Microbiology Causative organism: formerly Pneumocystis carinii , classified as a fungus and renamed Pneumocystis jirovecii ; cannot be cultured; Detection is with fluorescent monoclonal antibody assay or GMS-stained smears ABPA, Allergic bronchopulmonary aspergillosis; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; GMS, Grocott methenamine silver; IgG, IgM, immunoglobulins G and M; KOH, potassium hydroxide. Viral Pneumonia Viruses cause more infections than all other types of microorganisms combined and involve the respiratory tract more commonly than other organ systems. 296 Fortunately, the lung diseases produced by viruses are usually mild and self-limited. Nevertheless, viruses cause major public health illnesses and account for many of the new and emerging diseases in current headlines. At times viruses are also capable of producing serious and life-threatening infections that come to the attention of pathologists in both immunocompromised patients and young, healthy persons. 297 The viruses that commonly infect the lung are listed in Table 7.10 . 298 Table 7.10 Viral Pathogens of the Lung Table 7.10 RNA Viruses DNA Viruses Influenza virus Adenovirus Parainfluenza virus Herpes simplex virus Respiratory syncytial virus Varicella-zoster virus Measles virus Cytomegalovirus Hantavirus Epstein–Barr virus Etiologic Agents The conventional respiratory viruses—influenza virus, parainfluenza virus, RSV, and adenovirus—cause outbreaks of respiratory illness in the general population each year. In infants, the elderly, and in patients with chronic diseases, these pathogens can cause serious pneumonias. Pneumonia in immunocompromised persons is usually attributed to the herpesviruses (HSV and CMV). Less appreciated is that the conventional respiratory viruses are also frequent causes of respiratory illness in these patients and that such infections result in high rates of morbidity and mortality. 299 Newly recognized respiratory viruses 300 , 301 include H5N1, a highly pathogenic strain of influenza. First detected in 1997 in Hong Kong, it has since spread to Europe, the Middle East, and Africa. Another unique, triple-reassortment swine-origin influenza virus A, H1N1 (S-OIV), emerged in 2009 as the cause of outbreaks sustained by person-to-person transmission in multiple countries. It was characterized by respiratory illness of variable severity ranging from self-limited disease resembling seasonal flu to severe illness requiring hospitalization and occasionally eventuating in death from respiratory failure. 302 An acute cardiopulmonary syndrome in the southwestern United States was etiologically linked to a new hantavirus referred to as Sin Nombre ("without a name"). The severe acute respiratory syndrome (SARS) epidemic, which began in southern China and was carried by travelers to 33 other countries and 5 continents, was caused by SARS-CoV, a newly recognized coronavirus. Four other coronaviruses linked to respiratory illnesses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) have since been reported. 303 Human metapneumovirus, a paramyxovirus closely related to RSV clinically and pathologically, has become recognized as one of the leading causes of respiratory illness in children and can also cause illness in adults and immunocompromised patients. 304 Human bocavirus (HBoV) has been isolated in several countries from children with wheezing. 305 Other viruses such as the picornavirus group (rhinovirus and enterovirus) can cause pneumonia, as can polyomavirus (BK virus). 306 Parvovirus B19, an Erythrovirus, has long been known to cause disease, primarily in maternal–fetal and pediatric patients. Recently an autoimmune-type pneumonitis associated with serologic evidence of parvovirus B19 has also been described. 308 The evolution of diagnostic laboratory methods and large-scale molecular screening suggests that more viruses will be linked to respiratory tract disease in the future. Histopathology The respiratory tract viruses have a tendency to target specific regions of the tracheobronchial tree and lungs, producing characteristic clinical syndromes. However, sufficient overlap clinically, radiologically, and pathologically often limits a strict interpretation of findings for a definitive diagnosis. The information in Box 7.22 can sometimes be useful in narrowing the search for a specific etiologic agent. The microscopic findings in most pulmonary viral infections include the direct effect of the virus as well as the host's inflammatory response. The clinical outcome depends upon the virulence of the organism and the nature of the host response, be it diffuse alveolar damage, diffuse or patchy bronchiolitis and interstitial pneumonitis, giant cell reactions, or even minimal change. 309 Box 7.22 Histopathologic Patterns in Viral Lung Injury Diffuse alveolar damage Bronchitis and bronchiolitis Diffuse interstitial pneumonia Perivascular lymphoid infiltrates Miliary small nodules Airspace organization— Bronchiolitis obliterans –organizing pneumonia (BOOP) pattern Calcified nodules Alt-text: Box 7.22 The histopathologic diagnosis of viral infection is impossible without identification of the characteristic CPE. The term cytopathic effect has traditionally been used by virologists to describe cellular changes in unstained cell culture monolayers seen by light microscopy, 310 , 311 but it can be applied to all virus-associated nuclear and cytoplasmic alterations seen on H&E-stained slides or highlighted by immunohistochemical staining, molecular in situ–based methodology, or ultrastructural localization. 312 , 313 Diffuse alveolar damage, often with bronchiolitis, is the most typical pattern of viral lung injury. As noted earlier, however, diffuse alveolar damage also occurs in bacterial, mycobacterial, and fungal pneumonias, so a careful search for specific viral CPE becomes important in this setting. For the surgical pathologist, CPE manifests mainly as the viral inclusion present in the nucleus or cytoplasm of an infected cell. Viral inclusions confer diagnostic specificity to the pathologic pattern of injury in which they are found, and for the common respiratory tract viruses, the features are presented in Table 7.11 . Finally, it is worth mentioning that most clinically significant viral pneumonias that have CPE also show necrosis somewhere in the biopsy. Table 7.11 Cytopathic Effects in Pulmonary Infections With Selected Viruses Table 7.11 Virus Presence of Inclusions Inclusion Characteristics Intranuclear Intracytoplasmic Herpes simplex virus; varicella-zoster virus + − Early ground-glass appearance; later eosinophilic (Cowdry A type) multinucleate cells Adenovirus + − Early eosinophilic (Cowdry A); later basophilic, smudged nucleus Cytomegalovirus + + Cytomegaly with large "owl eye" amphophilic (Cowdry A) nuclear and multiple smaller basophilic (GMS-positive), cytoplasmic type Respiratory syncytial virus − + Eosinophilic smooth, small, often indistinct; multinucleate syncytia in some cases Measles virus + + Eosinophilic nuclear (Cowdry A) in multinucleate cells; cytoplasmic type—eosinophilic, pleomorphic Parainfluenza virus − + Rarely observed, pleomorphic, eosinophilic; multinucleate syncytia rarely Influenza virus − − No inclusions or other distinctive cytopathic effects Influenza Virus Influenza viruses are the most pathogenic of the respiratory viruses and predispose patients most commonly to secondary bacterial pneumonia. These viruses also account for the greatest public health burden. Annually they cause epidemic outbreaks of respiratory disease that are often associated with considerable morbidity; periodically, they produce pandemics with high mortality rates. These viruses target the ciliated epithelium of the tracheobronchial tree, producing necrotizing bronchitis and bronchiolitis and a spectrum of changes that vary depending on the stage of the disease (early vs. late), outcome (fatal vs. nonfatal), and the presence or absence of secondary bacterial pneumonia. Uncomplicated influenza pneumonia is rarely biopsied today. Based on historical data from bronchoscopic biopsies performed in the 1950s and early 1960s, the histopathologic findings in nonfatal uncomplicated influenza are those of active tracheobronchitis. 314 Necrosis and desquamation of the epithelial cells to the basement membrane is associated with a relatively scant lymphocytic infiltrate; however, in more severe cases, the virus and its attendant inflammatory response spread more distally into the respiratory bronchioles and alveoli, with hemorrhage, edema, fibrinous exudate with hyaline membranes, and patchy interstitial cellular infiltrates ( Fig. 7.95 ). This constellation of findings comprises the lesion of characterization . 315 In contemporary pathologic terms this would correspond to diffuse alveolar damage and, clinically, a primary viral pneumonia. Depending on the clinical course and time of lung biopsy (or autopsy) within the first 2 weeks of illness, the process may be in the acute and/or organizing phase. 316 , 317 Later, the airway epithelial damage may pave the way for secondary bacterial pneumonia, which accounts for much of the morbidity and mortality of influenza and may obscure the features of primary viral pneumonia. 318 Figure 7.95 Influenza virus. (A) Bronchiolitis with intraluminal necroinflammatory debris. (B) Acute diffuse alveolar damage pattern with hyaline membranes. Figure 7.95 From 2003 through 2008, 391 human cases of highly pathogenic avian influenza involving the H5N1 strain were recorded with 247 deaths. 319 The histopathologic changes observed in the few autopsied cases fall within the spectrum of findings described during the pandemics of 1918, 1957, and 1968 and in fatal cases of interpandemic (seasonal) influenza. 317 A characteristic feature of the H5N1 and 1918 cases is the high mortality rate, especially among previously healthy older children and young adults. Excessively high levels of cytokines and chemokines are thought to play an important role in the pathogenesis of the acute lung injury pattern seen in these fatal cases of influenza. 320 Because these viruses produce no characteristic cellular inclusions, etiologic diagnosis is not possible by morphology alone and requires antigen detection by immunofluorescence, immunohistochemistry, in situ hybridization, or culture. 321 The influenza virus genome steadily shifts over the passage of time; infections occur in those without previous immunity to each new strain. Rapid characterization of each viral variant is a continuous challenge, with numerous virologic methods concentrating on rapid point-of-care methods needed for effective prophylaxis. 322 Parainfluenza Virus Parainfluenza virus comprises four serotypes (I to IV) that typically target the upper respiratory tract, classically in the form of croup. 323 Some cases involve distal airways, as in infections due to RSV and influenza virus, but are milder, with less morbidity and requiring fewer hospitalizations. A few documented cases have been described with a diffuse alveolar damage pattern or an interstitial pneumonitis with giant cells, the latter resembling those of measles and RSV infection. The giant cells of parainfluenza tend to be larger and have more intracytoplasmic inclusions. 55 Parainfluenza virus is a potential opportunist in immunocompromised patients, especially children with congenital immunodeficiency disorders, 324 in whom fatal pneumonitis with disseminated disease may occur. 325 Respiratory Syncytial Virus RSV causes more significant respiratory infections in early childhood than those attributable to either influenza viruses or parainfluenza viruses. 326 , 327 The annual outbreaks of bronchiolitis and pneumonia in infants are especially severe during the first year of life and in those of low birth weight or with cardiopulmonary disease. 323 Considered primarily a childhood virus, RSV has more recently been recognized as the etiologic agent of pneumonia in community-dwelling and high-risk adults with chronic lung disease requiring hospitalization. 324 , 328 , 329 Also, RSV is often an unsuspected opportunistic pathogen in immunocompromised patients. 299 , 330 RSV targets the epithelium of the distal airway, producing bronchiolitis with disorganization of the epithelium and epithelial cell sloughing ( Fig. 7.96A ). 307 In fatal cases, airway obstruction due to sloughed cell detritus, mucus, and fibrin is compounded by airway lymphoid hyperplasia. 331 Diffuse alveolar damage may be seen in immunocompromised patients. Giant cells (syncytia), similar to the cytopathic changes seen in cell culture, may be present in alveolar ducts and airspaces around areas of bronchiolitis ( Fig. 7.96B ). Eosinophilic inclusions in cytoplasm may be seen in tissues and cytology specimens from immunosuppressed patients, but these are difficult to confirm as diagnostic of RSV without immunohistochemistry. Figure 7.96 Respiratory syncytial virus. (A) Bronchiolitis with intraluminal sloughing. (B) Bronchiolitis with giant cell syncytia. Figure 7.96 Human Metapneumovirus Human metapneumovirus, a newly recognized paramyxovirus, is a leading cause of respiratory tract disease in infants, with annual epidemics occurring during the winter and early spring months. 304 , 332 The virus also causes disease in immunocompromised patients 333 and likely explains some lower respiratory tract infections in the elderly. The clinical spectrum of croup, bronchiolitis, and pneumonia is similar to that for infections due to other paramyxoviruses, such as RSV and parainfluenza virus. The pathologic features are not well characterized because few well-documented cases have included biopsy in the evaluation. However, histopathologic assessment of lung tissue in severe cases has revealed acute and organizing diffuse alveolar damage as well as smudge cell formation. 334 , 335 The definitive identification of the virus can be established in tissue culture, but monoclonal antibody reagents and molecular techniques (real-time PCR assay) are the current diagnostic methods of choice. Measles Virus The measles virus causes a highly communicable childhood viral exanthem worldwide that, unlike varicella (chickenpox), leads to complications that are common and serious. 336 Measles pneumonia accounts for the vast majority of measles-related deaths, and most of these are a consequence of secondary pneumonia (bacterial or viral) or attributable to an aberrant immune response. Despite vaccination, measles is still a global pathogen and has resurfaced due to variation in vaccination rates, even in the United States. 337 , 338 Primary viral pneumonia occurs but is uncommon, even in immunocompromised hosts. Microscopically, bronchial and bronchiolar epithelial degeneration and reactive hyperplasia with squamous metaplasia is typically accompanied by peribronchial inflammation. Diffuse alveolar damage may occur, and quantitative immunohistochemical studies have revealed severe immune dysfunction with loss of key effector cells and their cytokines. 339 Characteristic giant cells show distinctive intranuclear eosinophilic inclusions surrounded by halos ( Fig. 7.97 ). This is the classic measles injury pattern 307 and is referred to as Hecht giant cell pneumonia . Minute intracytoplasmic eosinophilic inclusions precede the development of the intranuclear inclusions and are often difficult to identify. Pneumonia with giant cells should always suggest measles, but similar changes can be seen in RSV and parainfluenza pneumonias, and not all cases of measles pneumonia have these giant cells. 307 Hard metal pneumoconiosis (giant cell interstitial pneumonia) is in the differential diagnosis, but the overall appearance of hard metal disease is one of a chronic disease with some fibrosis and few if any acute changes. In the absence of giant cells, the cellular interstitial pneumonia must be differentiated from those caused by other viruses and atypical pneumonia agents as well as from nonspecific interstitial pneumonia. Figure 7.97 Measles virus pneumonia with characteristic eosinophilic intranuclear inclusions in giant cell. Figure 7.97 Hantavirus The recently identified hantavirus produces a rapidly evolving cardiopulmonary syndrome with a high mortality rate. This disorder first came to public attention as an emerging infection following an outbreak in the southwestern United States in 1993; it was causally linked to a previously unrecognized hantavirus. All members of this genus are zoonotic and are found in rodents around the world. The specific type responsible for the cardiopulmonary syndrome, Sin Nombre , is present in rodent feces and is acquired from the environment through inhalation. It produces florid pulmonary edema with pleural effusions, variable fibrin deposits, and focal wispy hyaline membranes ( Fig. 7.98A ). 340 Immunoblast-like cells are present in vascular spaces and in the peripheral blood ( Fig. 7.98B ). Morphologic diagnosis is presumptive because hantaviral antigen in endothelial cells, detected by immunohistochemistry, is required for definitive diagnosis. 341 In the appropriate clinical setting, clues to the diagnosis can sometimes be found in a constellation of morphologic findings on a peripheral blood smear, and confirmation can be achieved serologically by the detection of hantavirus-specific immunoglobulin M (IgM) antibodies or by the detection of hantavirus RNA by PCR assay in peripheral blood leukocytes. 342 , 343 , 344 Figure 7.98 Hantavirus. (A) Pulmonary edema with fibrin deposits. (B) Immunoblast-like cells in alveolar capillaries at arrows. Figure 7.98 Coronaviruses Coronaviruses are ubiquitous RNA viruses known to cause disease in many animals. At least five different coronaviruses are known to infect humans, and these cluster into two antigenic groups. 303 They, along with the rhinoviruses, are responsible for a majority of common colds. Coinfections with other respiratory viruses occur in infants and children presenting with more severe respiratory disease. In certain epidemiologic situations, they can cause pneumonia in children, frail elderly individuals, and immunocompromised adults. 345 , 346 In November 2002, the appearance of an atypical pneumonia in China, subsequently labeled SARS, became an alarming global health problem in the period of a few months. 347 The disease was linked (Koch's postulates were fulfilled) by means of tissue culture isolation, electron microscopy, and molecular analysis to an emergent novel coronavirus, proposed as the Urbani strain of SARS-associated coronavirus. 348 Clinically the disease ranges from a nonhypoxemic febrile respiratory disease (with minimal symptoms in some patients) to one of severe pulmonary dysfunction, manifesting as ARDS and eventuating in death for approximately 5% of the patients affected. 349 In the reported cases, the chest x-ray appearance on presentation was either normal or the chest film showed unilateral, predominantly peripheral areas of consolidation that progressed to bilateral, patchy consolidation, the degree and extent of which correlated with the development of respiratory failure. In patients who presented with a normal x-ray appearance, CT scans often revealed bilateral ground-glass consolidation resembling that in bronchiolitis obliterans with organizing pneumonia (cryptogenic organizing pneumonia). Lymphopenia and elevated LDH were helpful clues, but the clinical, radiologic, and laboratory features, although characteristic, were not distinguishable from those in patients with pneumonia caused by other viruses and bacteria and various atypical agents. Histopathologic findings in lung biopsy and autopsy tissues included acute lung injury (diffuse alveolar damage) in various stages of organization. 350 , 351 Lung biopsy specimens in milder cases showed relatively scant intraalveolar fibrin deposits with some congestion and edema ( Fig. 7.99 ). However, the spectrum of findings included acute fibrinous pneumonia, hyaline membrane formation, interstitial lymphocytic infiltrates, desquamation of alveolar pneumocytes, and areas undergoing organization of the acute-phase injury. 352 In some patients, multinucleate syncytial cells reminiscent of the CPE seen in influenza virus, RSV, and measles virus infections were noted. Viral inclusions were not identified, and initial immunohistochemical studies failed to reveal viral antigen. Subsequent investigations detected virus in epithelial cells (predominantly type II pneumocytes) and alveolar macrophages using immunohistochemical staining, in situ hybridization, RT-PCR methods, and electron microscopy. A unique coronavirus ( Fig. 7.100 ) was finally implicated as the etiologic agent. 352 , 353 Comparative histopathologic studies in fatal cases of SARS and H5N1 avian influenza reveal similarities and differences. 354 Both infections feature acute and organizing diffuse alveolar damage, but SARS appears to be more frequently associated with subacute injury with intraalveolar organization, whereas H5N1 virus causes a more fulminant diffuse alveolar damage pattern with patchy interstitial inflammation and paucicellular fibrosis. Figure 7.99 Coronavirus pneumonia: Severe acute respiratory syndrome. (A) and (B) Acute fibrinous lung injury is evident. Figure 7.99 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Figure 7.100 Coronavirus-infected cell can be seen in this electron photomicrograph. Figure 7.100 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Adenovirus Adenovirus comprises several genera, with multiple serotypes that cause infections of the upper and lower respiratory tract, conjunctiva, and gut. Respiratory tract infections are most common and account for approximately 5% to 10% of pediatric pneumonias. These can be especially severe in neonates, children, and immunocompromised persons. 307 , 355 In the lung, adenovirus infection produces two patterns of lung injury: diffuse alveolar damage with or without necrotizing bronchiolitis and pneumonitis with "dirty" or karyorrhectic necrosis 356 ( Fig. 7.101 ). These patterns may coexist in some cases, and the pneumonia may be accompanied by hemorrhage secondary to adenovirus-induced endothelial cell damage. 357 Two types of adenoviral CPE may be seen. Initially an eosinophilic (Cowdry A) intranuclear inclusion occurs surrounded by a halo with marginated chromatin, similar to HSV ( Fig. 7.102A ). This later enlarges and becomes amphophilic and then more basophilic, obliterating the nuclear membrane and producing the characteristic smudge cell ( Fig. 7.102B ). 307 Figure 7.101 Adenoviral pneumonia. (A) Necrosis (N) and diffuse alveolar damage (hm) . (B) Necrotizing bronchiolitis. hm, hyaline membrane. Figure 7.101 Figure 7.102 Adenovirus. (A) Cowdry A intranuclear inclusions. (B) Smudged cell. Figure 7.102 Herpes Simplex Viruses HSVs types I and II have had traditional assigned roles as etiologic agents of mucocutaneous disease of the head and neck (type I) and genitalia (type II). Considerable crossover has been documented, however, with both types isolated from patients with disease at either site. Tracheobronchitis and pneumonia due to these viruses are rare in healthy adults with intact immune systems. They occur primarily in patients with underlying pulmonary disease and in association with inhalational and intubational trauma. They also occur in neonates and in patients who are immunosuppressed or compromised by various chronic diseases. Characteristic lesions include tracheobronchitis ( Fig. 7.103A ) with ulcers and hemorrhagic diffuse alveolar damage. Necrosis in a miliary small (or rarely large) nodular pattern is a helpful clue and the best location to identify CPE ( Fig. 7.103B ). 73 Like adenovirus, HSV also has two types of CPE: Initially a ground-glass amphophilic intranuclear inclusion, Cowdry B , appears with marginated chromatin; later a single eosinophilic Cowdry A inclusion ( Fig. 7.104 ) surrounded by a halo, similar to that seen with adenovirus, develops. The Cowdry A inclusion is considered noninfectious, as it is devoid of nucleic acid protein and is thought to represent the nuclear "scar" of HSV infection. 307 In the absence of smudge cells, HSV and adenoviral infections can look identical. Fortunately, immunohistochemistry or in situ hybridization can often resolve this differential diagnosis. Figure 7.103 Herpes simplex virus pneumonia. (A) Tracheobronchitis. Note cells with ground-glass inclusion. (B) Miliary nodular pattern of hemorrhagic necrosis. Figure 7.103 Figure 7.104 Herpes simplex virus pneumonia. Note two types of nuclear cytopathic effects: Cowdry A ground-glass type (short arrow) and Cowdry B eosinophilic inclusion (arrowhead) . Compare with cytomegalovirus intranuclear and cytoplasmic inclusion at long arrow. Figure 7.104 Varicella-Zoster Virus Varicella-zoster virus (VZV) infection produces considerable morbidity in the newborn, the adult, and the immunocompromised host, both in its primary form (varicella) and in its reactivated form (zoster). Varicella pneumonia is rarely observed in otherwise healthy children but is a major complication of adult varicella, occurring in approximately 10% to 15% of adults with VZV. In affected adults without underlying diseases and normal immunity, the course is generally mild and self-limited. Nevertheless, fatality rates of up to 10% have been reported. 307 By contrast, high mortality rates (25% to 45%) have been noted among some cohorts of immunosuppressed patients. Microscopically, small miliary nodules of necrosis are seen, associated with interstitial pneumonitis, edema, fibrin deposits, or patchy hyaline membranes ( Fig. 7.105A ). HSV-like intranuclear inclusions are present but may be sparse and difficult to identify. A miliary pattern of calcified nodules ( Fig. 7.105B ) may be present in the healed phase. 358 Figure 7.105 Varicella pneumonia. (A) A hemorrhagic miliary nodule. (B) Late phase with calcified nodules. Figure 7.105 Cytomegalovirus CMV infections are acquired throughout life. This virus can cause considerable morbidity and even death in the neonate, but infection is generally asymptomatic in older healthy children and adults. As in the case of other herpesviruses, primary infection is followed by latency, which persists until immune deficiency or immunosuppressive therapy causes it to reactivate and disseminate. CMV has therefore become one of the most common opportunists in patients with AIDS and those who receive organ transplants. In these settings, CMV can produce a variety of patterns, including one with minimal changes where only scattered alveolar lining cells with typical viropathic changes are seen. The CPE of CMV produces cytomegalic cells with large, round to oval, smooth "owl eye" eosinophilic to basophilic intranuclear inclusions surrounded by a clear halo ( Fig. 7.106A ). Figure 7.106 Cytomegalovirus (CMV) pneumonia. (A) Multiple characteristic intranuclear and intracytoplasmic inclusions in alveolar lining cells. Note Grocott methenamine silver–positive staining of inclusions (inset) . (B) Miliary nodule pattern of CMV pneumonia. Figure 7.106 (A, Courtesy Dr. Francis Chandler, Augusta, Georgia.) Later, multiple eosinophilic cytoplasmic inclusions develop that may be positive on staining with PAS and GMS ( Fig. 7.106A , inset). The more numerous the cytomegalic cells, the greater the clinical significance. In some cases, atypical inclusions may be seen in cells that are not significantly enlarged, and the nuclei may contain dark-staining homogeneous inclusions that may lack a clear halo. Despite their atypical appearance, these inclusions will usually be highlighted with immunohistochemical stains. 359 Another typical pattern that suggests viral infection is the presence of small miliary nodules with a central hemorrhage surrounded by necrotic alveolar walls ( Fig. 7.106B ). 73 Interstitial pneumonitis is the least common pattern of CMV infection. Ulcers may be seen in the trachea and bronchi, but they occur less often than in herpetic infections. In CMV pneumonias, it is advisable to look for other pathogens, typically P. jirovecii ( Fig. 7.107 ); but bacteria, fungi, protozoa, and other viruses are all possible coinfecting organisms. 360 Figure 7.107 Cytomegalovirus-infected alveolar lining cells associated with the foamy alveolar casts of Pneumocystis jirovecii . Figure 7.107 Epstein–Barr Virus EBV infections are usually acquired in childhood and are generally asymptomatic. The pathologist most often encounters this virus in the lung in the context of pulmonary lymphomas or in other EBV-associated lymphoproliferative disorders that can occur in transplant recipients and other immunocompromised patients. However, the most common symptomatic primary EBV infection is infectious mononucleosis. Most of these patients recover uneventfully, but a few develop one or more complications. Pneumonitis is one of them, albeit rare and not well characterized. The few reports describing pathology indicate a nonspecific lymphocytic interstitial pneumonitis, which may be bronchiolocentric ( Fig. 7.108 ). 361 , 362 CPE is absent, and although serologic studies can be supportive of a clinicopathologic diagnosis, etiologic proof of EBV infection requires the demonstration of the virus in lymphoid cells by in situ hybridization for EBV-encoded RNA-1 (EBER-1). Figure 7.108 Epstein–Barr virus pneumonitis. (A) Nonspecific cellular interstitial pneumonitis. (B) Patchy interstitial infiltrate. Figure 7.108 Cytopathology The cytologic features of viral infections in the respiratory tract are most likely to be found in exfoliative specimens, such as bronchial washings and BAL fluid samples, rather than needle aspirates, although viral diagnosis has been achieved with this technique. 363 , 364 This is because viral infections are less likely to produce radiologic mass–like infiltrates, which are the most common targets of needle biopsy procedures. Herpes simplex virus, CMV ( Fig. 7.109 ), and adenovirus are the most commonly identified viral pathogens in respiratory cytologic specimens, but varicella virus, parainfluenza virus, RSV, human metapneumovirus, and measles virus have also been detected. Figure 7.109 Cytomegalovirus pneumonitis with characteristic cytopathic effect. (A) Fine-needle aspirate. (B) Bronchoalveolar lavage specimen. Figure 7.109 Characteristic CPE produced by these viruses is often better appreciated in cytologic smears than in tissue sections, which may, in fact, yield a negative result. Therefore review of any cytology sample taken at the time of biopsy can be valuable. Other, less specific changes may be found. These include ciliocytophoria (free cilia complexes with terminal bars) and cytologic atypia mimicking cancer. 46 Microbiology Diagnostic virology is the newest of the microbiology and infectious disease specialties to have benefited from the technologic revolution in laboratory medicine. Rapid and accurate diagnosis can often be achieved today using practical, convenient laboratory methods that employ reliable, commercially available mammalian cells, media, and reagent systems. 297 , 365 , 366 This has allowed many rural and small urban hospital laboratories to provide timely viral diagnostic services not possible a short time ago. It is predicted that self-contained, rapid-cycle real-time PCR methods will one day account for the majority of viral assays in laboratories of all sizes. As a result, the pathologist who suspects a viral infection will increasingly have a variety of tools to obtain an etiologic diagnosis when morphologic manifestations are suggestive of viral infection. The basic approaches to viral diagnosis in the laboratory are listed in Box 7.23 . In questionable cases, confirmation by immunohistochemical studies ( Fig. 7.110A ), in situ hybridization ( Fig. 7.110B ), or electron microscopy may be helpful. 32 , 367 Many of the traditional methods of viral detection, detailed later, are being augmented by respiratory panel assays based on the detection of nucleic acid and compiled around common respiratory viral and bacterial pathogens. 368 , 369 Box 7.23 Laboratory Diagnosis of Viral Pneumonia Direct detection of organisms Histopathologic/cytopathologic examination for cytopathic effect (CPE) Immunohistochemical studies Electron microscopy Antigen detection Direct fluorescent antibody test Enzyme immunoassay Culture Conventional roller tube technique Shell vial technique Serologic studies Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.23 Figure 7.110 (A) Respiratory syncytial virus cytoplasmic inclusions detected by immunohistochemical staining. (B) Cytomegalovirus-infected cell with cytoplasmic inclusions detected by in situ hybridization. Figure 7.110 (Courtesy R.V. Lloyd, MD, Rochester, Minnesota.) The diagnosis of viral respiratory infections can also be based on antigen detection and culture ( Fig. 7.111 ). Direct antigen detection in clinical specimens collected by nasopharyngeal swabs, nasal washings, and aspirates or BAL fluid (but not sputum samples or, with rare exception, throat swabs) is performed using monoclonal antibodies by either immunofluorescence microscopy or enzyme immunoassay. By using a single reagent containing the monoclonal antibodies against several viruses and dual fluorochromes, the common respiratory viruses can be rapidly screened by direct immunofluorescence testing. Positive specimens can then be tested with individual reagents to determine the specific etiologic agent, while negative specimens can be submitted for culture. 370 Enzyme immunoassay includes methods that offer speed and convenience at the point of care. However, they are less sensitive than standard virologic methods, which must still be used to test negative specimens. Direct detection can also be accomplished in cellular samples, including tissue, by in situ hybridization or amplification techniques such as PCR. For RNA viruses, PCR amplification uses a reverse transcriptase (RT) step. PCR methodology has recently evolved into multiplex formats, and novel systems have been introduced that combine multiplex PCR chemistry with electron microarray (DNA chip) technology or fluid microsphere-based systems, permitting the simultaneous detection of a wide array of respiratory viruses and other pathogens. 371 , 372 , 373 , 374 , 375 , 376 Figure 7.111 Respiratory syncytial virus (RSV) infection. (A) RSV cytopathic effect in tissue culture. (B) RSV antigen in nasopharyngeal swab specimen detected by direct immunofluorescence microscopy. Figure 7.111 These systems have the potential to more rapidly and accurately diagnose acute infections and may also allow the study of complex coinfections and the active monitoring of outbreaks of influenza and other viral illnesses. 377 Panels composed of common respiratory bacterial and viral pathogens are available; these are based on nucleic acid detection by nested PCR and come by several brand names. Such panels typically encompass many of the respiratory viruses detailed previously with specimens obtained through sampling with a nasopharyngeal swab. 378 , 379 Traditional viral cultures in tubes with various types of cell monolayers are currently performed with greater sensitivity and turnaround time using the shell vial technique. This technique uses centrifugation of clinical specimen suspensions onto coverslipped cell monolayers followed by brief incubation (1 to 2 days) and antigen detection. 365 It is important, therefore, to preserve a portion of tissue from a bronchial or transbronchial biopsy or thoracotomy specimen in viral transport medium, especially with an immunocompromised patient, who may not have had BAL fluid submitted for culture. Shell vials, although faster than the traditional tube culture method, are still a slow method based on viral growth and are being replaced by direct nucleic acid detection. Viral serologic testing has commonly been used for diagnosis but may be the least sensitive approach. A positive serodiagnosis is typically based on a fourfold rise in titer between acute and convalescent sera and therefore cannot be achieved by this means in the acutely ill patient; antigen detection or culture of respiratory tract specimens is much preferred. However, a serologic strategy, utilizing a panel of antigens in an immunofluorescence or enzyme immunoassay format on a single specimen, is useful in suspected EBV infections. 380 A case also can be made for the benefit of CMV serologic testing for assessing the antibody status of organ donors and recipients for predicting the risk of posttransplantation CMV disease. When tissue is not available or findings are inconclusive, tests for the detection of actual disease in these transplant recipients include the p65 antigenemia assay on peripheral blood leukocytes and amplification or quantitation of CMV DNA in various peripheral blood compartments (plasma, whole blood, and leukocytes). 381 These assays may eventually replace culture of BAL fluid for surveillance of CMV infection in such patients. 382 The detection of virus in respiratory secretions (including BAL fluid), urine, or blood establishes the presence of virus but does not necessarily implicate it as the etiologic agent of a pneumonia. Quantitation of viral load by real-time PCR amplification, however, can be useful in this regard by linking high viral load with infection. 383 Differential Diagnosis A synopsis of the key morphologic and microbiologic features of the viral pneumonias is presented in Table 7.12 . In the absence of CPE, diffuse alveolar damage and other patterns of lung injury are not diagnostic of viral infection. Diffuse alveolar damage is a nonspecific response to many types of infection, including bacterial, mycobacterial, fungal, and protozoal, all of which must be considered in the differential diagnosis. In addition, other noninfectious causes include reactions to drugs, radiation, toxic inhalants, and shock of any type. Occasionally, CPE may not be diagnostic; for example, the early inclusions of adenovirus, HSV, and CMV may be quite similar. In most cases, immunohistochemistry or molecular techniques can resolve the diagnostic dilemma. Mimics of CPE that must be ruled out include macronuclei in both reactive processes and occult neoplastic infiltrates and intranuclear cytoplasmic invaginations, which can occur in a variety of cells. Cytoplasmic viral inclusions can also be simulated by aggregated altered protein and particulate matter. Table 7.12 Viral Pneumonias: Summary of Pathologic Findings Table 7.12 Assessment Component Findings Influenza Virus Surgical pathology Diffuse alveolar damage, bronchitis, and bronchiolitis; secondary acute purulent pneumonia; antigen detection by immunofluorescence, immunohistochemical, or in situ hybridization studies Cytopathology Nonspecific changes may include presence of reactive-type pneumocytes; ciliocytophoria Microbiology Antigen detection by DFA or EIA; culture on primary monkey kidney cells: noncytopathic; detection by hemadsorption Respiratory Syncytial Virus Surgical pathology Bronchiolitis with lumen detritus; may be associated with syncytial giant cells; diffuse alveolar damage in immunocompromised patients; confirm with immunohistochemistry Cytopathology Giant cell syncytia characteristic but often not seen; eosinophilic inclusions may be seen in bronchial epithelial cells of immunocompromised patients; rarely in those of normal hosts; rarely diagnosed by cytology alone Microbiology Antigen detection by DFA and EIA usually more sensitive than culture; cultures on continuous epithelial cell lines (Hep-2) and primary monkey kidney yield characteristic syncytial CPEs Measles Virus Surgical pathology Bronchitis, bronchiolitis, diffuse alveolar damage with giant cells containing Cowdry A inclusions and small cytoplasmic inclusions Cytopathology Eosinophilic intranuclear and cytoplasmic inclusions; rarely diagnosed by cytology Microbiology Antigen detection by DFA and EIA; culture on primary monkey kidney produces spindle cell or multinucleate CPE; serologic testing (for measles-specific IgM) available Hantavirus Surgical pathology Pulmonary edema pattern with variable fibrin deposits; immunoblast-like cells in vascular spaces; confirm by immunohistochemistry Cytopathology Noncytopathic Microbiology Serology: Hantavirus-specific IgM or detection of specific RNA by PCR assay in peripheral blood leukocytes Adenovirus Surgical pathology Diffuse alveolar damage with or without necrotizing bronchiolitis and/or pneumonitis with necrosis and karyorrhexis Cytopathology Early Cowdry A intranuclear inclusions, later smudge cell; reactive and reparative-type atypia in background Microbiology Antigen detection by EIA and DFA; culture on continuous epithelial cell lines produces characteristic grape-like clustered cytopathic effect Herpesvirus Surgical pathology Tracheobronchitis; diffuse alveolar damage; miliary necroinflammatory lesions Cytopathology Ground-glass (Cowdry B) intranuclear inclusions; later Cowdry A inclusions in multinucleated cells, often with a "seeds in a pomegranate" appearance on Pap-, H&E-, and Diff-Quik–stained smears Background reactive and reparative atypia Microbiology Antigen detection by immunofluorescence; culture on diploid fibroblasts produces characteristic cytopathic effect, sometimes within 24 hours;serologic testing less useful Varicella-Zoster Virus Surgical pathology Miliary necroinflammatory lesions; calcified nodules in healed phase Cytopathology Intranuclear Cowdry A inclusions sparse and less welldefined than with herpes simplex Microbiology Antigen detection by immunofluorescence; culture on human embryonic lung or Vero cells produces CPE more slowly than for herpesviruses (3–7 days); serologic testing available Cytomegalovirus Surgical pathology Minimal changes with scattered cytomegalic cells; miliary necroinflammatory lesions; interstitial pneumonitis Cytopathology Large "owl eye" Cowdry A inclusions with halo; cytoplasmic inclusions stained with GMS Microbiology Culture on human diploid fibroblasts produces characteristic CPE slowly in traditional tube cultures but more rapidly with use of shell vial technique p65 antigenemia assay; PCR assay; selective application of serology useful Epstein–Barr Virus Surgical pathology Polymorphous lymphoid interstitial pneumonitis; confirm by in situ hybridization Cytopathology Noncytopathic Microbiology No routine culture; diagnosis by serologic testing using panel of antibodies (EA; IgG and IgM VCA; EBNA) CPE , Cytopathic effect; DFA , direct immunofluorescence antibody (test); EA , early antigen; EBNA , Epstein–Barr virus–determined nuclear antigen; EIA , enzyme immunoassay; GMS , Grocott methenamine silver; H&E , hematoxylin and eosin; IgG, IgM , immunoglobulins G and M; Pap , Papanicolaou; PCR , polymerase chain reaction; VCA , viral capsid antigen. Etiologic Agents The conventional respiratory viruses—influenza virus, parainfluenza virus, RSV, and adenovirus—cause outbreaks of respiratory illness in the general population each year. In infants, the elderly, and in patients with chronic diseases, these pathogens can cause serious pneumonias. Pneumonia in immunocompromised persons is usually attributed to the herpesviruses (HSV and CMV). Less appreciated is that the conventional respiratory viruses are also frequent causes of respiratory illness in these patients and that such infections result in high rates of morbidity and mortality. 299 Newly recognized respiratory viruses 300 , 301 include H5N1, a highly pathogenic strain of influenza. First detected in 1997 in Hong Kong, it has since spread to Europe, the Middle East, and Africa. Another unique, triple-reassortment swine-origin influenza virus A, H1N1 (S-OIV), emerged in 2009 as the cause of outbreaks sustained by person-to-person transmission in multiple countries. It was characterized by respiratory illness of variable severity ranging from self-limited disease resembling seasonal flu to severe illness requiring hospitalization and occasionally eventuating in death from respiratory failure. 302 An acute cardiopulmonary syndrome in the southwestern United States was etiologically linked to a new hantavirus referred to as Sin Nombre ("without a name"). The severe acute respiratory syndrome (SARS) epidemic, which began in southern China and was carried by travelers to 33 other countries and 5 continents, was caused by SARS-CoV, a newly recognized coronavirus. Four other coronaviruses linked to respiratory illnesses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) have since been reported. 303 Human metapneumovirus, a paramyxovirus closely related to RSV clinically and pathologically, has become recognized as one of the leading causes of respiratory illness in children and can also cause illness in adults and immunocompromised patients. 304 Human bocavirus (HBoV) has been isolated in several countries from children with wheezing. 305 Other viruses such as the picornavirus group (rhinovirus and enterovirus) can cause pneumonia, as can polyomavirus (BK virus). 306 Parvovirus B19, an Erythrovirus, has long been known to cause disease, primarily in maternal–fetal and pediatric patients. Recently an autoimmune-type pneumonitis associated with serologic evidence of parvovirus B19 has also been described. 308 The evolution of diagnostic laboratory methods and large-scale molecular screening suggests that more viruses will be linked to respiratory tract disease in the future. Histopathology The respiratory tract viruses have a tendency to target specific regions of the tracheobronchial tree and lungs, producing characteristic clinical syndromes. However, sufficient overlap clinically, radiologically, and pathologically often limits a strict interpretation of findings for a definitive diagnosis. The information in Box 7.22 can sometimes be useful in narrowing the search for a specific etiologic agent. The microscopic findings in most pulmonary viral infections include the direct effect of the virus as well as the host's inflammatory response. The clinical outcome depends upon the virulence of the organism and the nature of the host response, be it diffuse alveolar damage, diffuse or patchy bronchiolitis and interstitial pneumonitis, giant cell reactions, or even minimal change. 309 Box 7.22 Histopathologic Patterns in Viral Lung Injury Diffuse alveolar damage Bronchitis and bronchiolitis Diffuse interstitial pneumonia Perivascular lymphoid infiltrates Miliary small nodules Airspace organization— Bronchiolitis obliterans –organizing pneumonia (BOOP) pattern Calcified nodules Alt-text: Box 7.22 The histopathologic diagnosis of viral infection is impossible without identification of the characteristic CPE. The term cytopathic effect has traditionally been used by virologists to describe cellular changes in unstained cell culture monolayers seen by light microscopy, 310 , 311 but it can be applied to all virus-associated nuclear and cytoplasmic alterations seen on H&E-stained slides or highlighted by immunohistochemical staining, molecular in situ–based methodology, or ultrastructural localization. 312 , 313 Diffuse alveolar damage, often with bronchiolitis, is the most typical pattern of viral lung injury. As noted earlier, however, diffuse alveolar damage also occurs in bacterial, mycobacterial, and fungal pneumonias, so a careful search for specific viral CPE becomes important in this setting. For the surgical pathologist, CPE manifests mainly as the viral inclusion present in the nucleus or cytoplasm of an infected cell. Viral inclusions confer diagnostic specificity to the pathologic pattern of injury in which they are found, and for the common respiratory tract viruses, the features are presented in Table 7.11 . Finally, it is worth mentioning that most clinically significant viral pneumonias that have CPE also show necrosis somewhere in the biopsy. Table 7.11 Cytopathic Effects in Pulmonary Infections With Selected Viruses Table 7.11 Virus Presence of Inclusions Inclusion Characteristics Intranuclear Intracytoplasmic Herpes simplex virus; varicella-zoster virus + − Early ground-glass appearance; later eosinophilic (Cowdry A type) multinucleate cells Adenovirus + − Early eosinophilic (Cowdry A); later basophilic, smudged nucleus Cytomegalovirus + + Cytomegaly with large "owl eye" amphophilic (Cowdry A) nuclear and multiple smaller basophilic (GMS-positive), cytoplasmic type Respiratory syncytial virus − + Eosinophilic smooth, small, often indistinct; multinucleate syncytia in some cases Measles virus + + Eosinophilic nuclear (Cowdry A) in multinucleate cells; cytoplasmic type—eosinophilic, pleomorphic Parainfluenza virus − + Rarely observed, pleomorphic, eosinophilic; multinucleate syncytia rarely Influenza virus − − No inclusions or other distinctive cytopathic effects Influenza Virus Influenza viruses are the most pathogenic of the respiratory viruses and predispose patients most commonly to secondary bacterial pneumonia. These viruses also account for the greatest public health burden. Annually they cause epidemic outbreaks of respiratory disease that are often associated with considerable morbidity; periodically, they produce pandemics with high mortality rates. These viruses target the ciliated epithelium of the tracheobronchial tree, producing necrotizing bronchitis and bronchiolitis and a spectrum of changes that vary depending on the stage of the disease (early vs. late), outcome (fatal vs. nonfatal), and the presence or absence of secondary bacterial pneumonia. Uncomplicated influenza pneumonia is rarely biopsied today. Based on historical data from bronchoscopic biopsies performed in the 1950s and early 1960s, the histopathologic findings in nonfatal uncomplicated influenza are those of active tracheobronchitis. 314 Necrosis and desquamation of the epithelial cells to the basement membrane is associated with a relatively scant lymphocytic infiltrate; however, in more severe cases, the virus and its attendant inflammatory response spread more distally into the respiratory bronchioles and alveoli, with hemorrhage, edema, fibrinous exudate with hyaline membranes, and patchy interstitial cellular infiltrates ( Fig. 7.95 ). This constellation of findings comprises the lesion of characterization . 315 In contemporary pathologic terms this would correspond to diffuse alveolar damage and, clinically, a primary viral pneumonia. Depending on the clinical course and time of lung biopsy (or autopsy) within the first 2 weeks of illness, the process may be in the acute and/or organizing phase. 316 , 317 Later, the airway epithelial damage may pave the way for secondary bacterial pneumonia, which accounts for much of the morbidity and mortality of influenza and may obscure the features of primary viral pneumonia. 318 Figure 7.95 Influenza virus. (A) Bronchiolitis with intraluminal necroinflammatory debris. (B) Acute diffuse alveolar damage pattern with hyaline membranes. Figure 7.95 From 2003 through 2008, 391 human cases of highly pathogenic avian influenza involving the H5N1 strain were recorded with 247 deaths. 319 The histopathologic changes observed in the few autopsied cases fall within the spectrum of findings described during the pandemics of 1918, 1957, and 1968 and in fatal cases of interpandemic (seasonal) influenza. 317 A characteristic feature of the H5N1 and 1918 cases is the high mortality rate, especially among previously healthy older children and young adults. Excessively high levels of cytokines and chemokines are thought to play an important role in the pathogenesis of the acute lung injury pattern seen in these fatal cases of influenza. 320 Because these viruses produce no characteristic cellular inclusions, etiologic diagnosis is not possible by morphology alone and requires antigen detection by immunofluorescence, immunohistochemistry, in situ hybridization, or culture. 321 The influenza virus genome steadily shifts over the passage of time; infections occur in those without previous immunity to each new strain. Rapid characterization of each viral variant is a continuous challenge, with numerous virologic methods concentrating on rapid point-of-care methods needed for effective prophylaxis. 322 Parainfluenza Virus Parainfluenza virus comprises four serotypes (I to IV) that typically target the upper respiratory tract, classically in the form of croup. 323 Some cases involve distal airways, as in infections due to RSV and influenza virus, but are milder, with less morbidity and requiring fewer hospitalizations. A few documented cases have been described with a diffuse alveolar damage pattern or an interstitial pneumonitis with giant cells, the latter resembling those of measles and RSV infection. The giant cells of parainfluenza tend to be larger and have more intracytoplasmic inclusions. 55 Parainfluenza virus is a potential opportunist in immunocompromised patients, especially children with congenital immunodeficiency disorders, 324 in whom fatal pneumonitis with disseminated disease may occur. 325 Respiratory Syncytial Virus RSV causes more significant respiratory infections in early childhood than those attributable to either influenza viruses or parainfluenza viruses. 326 , 327 The annual outbreaks of bronchiolitis and pneumonia in infants are especially severe during the first year of life and in those of low birth weight or with cardiopulmonary disease. 323 Considered primarily a childhood virus, RSV has more recently been recognized as the etiologic agent of pneumonia in community-dwelling and high-risk adults with chronic lung disease requiring hospitalization. 324 , 328 , 329 Also, RSV is often an unsuspected opportunistic pathogen in immunocompromised patients. 299 , 330 RSV targets the epithelium of the distal airway, producing bronchiolitis with disorganization of the epithelium and epithelial cell sloughing ( Fig. 7.96A ). 307 In fatal cases, airway obstruction due to sloughed cell detritus, mucus, and fibrin is compounded by airway lymphoid hyperplasia. 331 Diffuse alveolar damage may be seen in immunocompromised patients. Giant cells (syncytia), similar to the cytopathic changes seen in cell culture, may be present in alveolar ducts and airspaces around areas of bronchiolitis ( Fig. 7.96B ). Eosinophilic inclusions in cytoplasm may be seen in tissues and cytology specimens from immunosuppressed patients, but these are difficult to confirm as diagnostic of RSV without immunohistochemistry. Figure 7.96 Respiratory syncytial virus. (A) Bronchiolitis with intraluminal sloughing. (B) Bronchiolitis with giant cell syncytia. Figure 7.96 Human Metapneumovirus Human metapneumovirus, a newly recognized paramyxovirus, is a leading cause of respiratory tract disease in infants, with annual epidemics occurring during the winter and early spring months. 304 , 332 The virus also causes disease in immunocompromised patients 333 and likely explains some lower respiratory tract infections in the elderly. The clinical spectrum of croup, bronchiolitis, and pneumonia is similar to that for infections due to other paramyxoviruses, such as RSV and parainfluenza virus. The pathologic features are not well characterized because few well-documented cases have included biopsy in the evaluation. However, histopathologic assessment of lung tissue in severe cases has revealed acute and organizing diffuse alveolar damage as well as smudge cell formation. 334 , 335 The definitive identification of the virus can be established in tissue culture, but monoclonal antibody reagents and molecular techniques (real-time PCR assay) are the current diagnostic methods of choice. Measles Virus The measles virus causes a highly communicable childhood viral exanthem worldwide that, unlike varicella (chickenpox), leads to complications that are common and serious. 336 Measles pneumonia accounts for the vast majority of measles-related deaths, and most of these are a consequence of secondary pneumonia (bacterial or viral) or attributable to an aberrant immune response. Despite vaccination, measles is still a global pathogen and has resurfaced due to variation in vaccination rates, even in the United States. 337 , 338 Primary viral pneumonia occurs but is uncommon, even in immunocompromised hosts. Microscopically, bronchial and bronchiolar epithelial degeneration and reactive hyperplasia with squamous metaplasia is typically accompanied by peribronchial inflammation. Diffuse alveolar damage may occur, and quantitative immunohistochemical studies have revealed severe immune dysfunction with loss of key effector cells and their cytokines. 339 Characteristic giant cells show distinctive intranuclear eosinophilic inclusions surrounded by halos ( Fig. 7.97 ). This is the classic measles injury pattern 307 and is referred to as Hecht giant cell pneumonia . Minute intracytoplasmic eosinophilic inclusions precede the development of the intranuclear inclusions and are often difficult to identify. Pneumonia with giant cells should always suggest measles, but similar changes can be seen in RSV and parainfluenza pneumonias, and not all cases of measles pneumonia have these giant cells. 307 Hard metal pneumoconiosis (giant cell interstitial pneumonia) is in the differential diagnosis, but the overall appearance of hard metal disease is one of a chronic disease with some fibrosis and few if any acute changes. In the absence of giant cells, the cellular interstitial pneumonia must be differentiated from those caused by other viruses and atypical pneumonia agents as well as from nonspecific interstitial pneumonia. Figure 7.97 Measles virus pneumonia with characteristic eosinophilic intranuclear inclusions in giant cell. Figure 7.97 Hantavirus The recently identified hantavirus produces a rapidly evolving cardiopulmonary syndrome with a high mortality rate. This disorder first came to public attention as an emerging infection following an outbreak in the southwestern United States in 1993; it was causally linked to a previously unrecognized hantavirus. All members of this genus are zoonotic and are found in rodents around the world. The specific type responsible for the cardiopulmonary syndrome, Sin Nombre , is present in rodent feces and is acquired from the environment through inhalation. It produces florid pulmonary edema with pleural effusions, variable fibrin deposits, and focal wispy hyaline membranes ( Fig. 7.98A ). 340 Immunoblast-like cells are present in vascular spaces and in the peripheral blood ( Fig. 7.98B ). Morphologic diagnosis is presumptive because hantaviral antigen in endothelial cells, detected by immunohistochemistry, is required for definitive diagnosis. 341 In the appropriate clinical setting, clues to the diagnosis can sometimes be found in a constellation of morphologic findings on a peripheral blood smear, and confirmation can be achieved serologically by the detection of hantavirus-specific immunoglobulin M (IgM) antibodies or by the detection of hantavirus RNA by PCR assay in peripheral blood leukocytes. 342 , 343 , 344 Figure 7.98 Hantavirus. (A) Pulmonary edema with fibrin deposits. (B) Immunoblast-like cells in alveolar capillaries at arrows. Figure 7.98 Coronaviruses Coronaviruses are ubiquitous RNA viruses known to cause disease in many animals. At least five different coronaviruses are known to infect humans, and these cluster into two antigenic groups. 303 They, along with the rhinoviruses, are responsible for a majority of common colds. Coinfections with other respiratory viruses occur in infants and children presenting with more severe respiratory disease. In certain epidemiologic situations, they can cause pneumonia in children, frail elderly individuals, and immunocompromised adults. 345 , 346 In November 2002, the appearance of an atypical pneumonia in China, subsequently labeled SARS, became an alarming global health problem in the period of a few months. 347 The disease was linked (Koch's postulates were fulfilled) by means of tissue culture isolation, electron microscopy, and molecular analysis to an emergent novel coronavirus, proposed as the Urbani strain of SARS-associated coronavirus. 348 Clinically the disease ranges from a nonhypoxemic febrile respiratory disease (with minimal symptoms in some patients) to one of severe pulmonary dysfunction, manifesting as ARDS and eventuating in death for approximately 5% of the patients affected. 349 In the reported cases, the chest x-ray appearance on presentation was either normal or the chest film showed unilateral, predominantly peripheral areas of consolidation that progressed to bilateral, patchy consolidation, the degree and extent of which correlated with the development of respiratory failure. In patients who presented with a normal x-ray appearance, CT scans often revealed bilateral ground-glass consolidation resembling that in bronchiolitis obliterans with organizing pneumonia (cryptogenic organizing pneumonia). Lymphopenia and elevated LDH were helpful clues, but the clinical, radiologic, and laboratory features, although characteristic, were not distinguishable from those in patients with pneumonia caused by other viruses and bacteria and various atypical agents. Histopathologic findings in lung biopsy and autopsy tissues included acute lung injury (diffuse alveolar damage) in various stages of organization. 350 , 351 Lung biopsy specimens in milder cases showed relatively scant intraalveolar fibrin deposits with some congestion and edema ( Fig. 7.99 ). However, the spectrum of findings included acute fibrinous pneumonia, hyaline membrane formation, interstitial lymphocytic infiltrates, desquamation of alveolar pneumocytes, and areas undergoing organization of the acute-phase injury. 352 In some patients, multinucleate syncytial cells reminiscent of the CPE seen in influenza virus, RSV, and measles virus infections were noted. Viral inclusions were not identified, and initial immunohistochemical studies failed to reveal viral antigen. Subsequent investigations detected virus in epithelial cells (predominantly type II pneumocytes) and alveolar macrophages using immunohistochemical staining, in situ hybridization, RT-PCR methods, and electron microscopy. A unique coronavirus ( Fig. 7.100 ) was finally implicated as the etiologic agent. 352 , 353 Comparative histopathologic studies in fatal cases of SARS and H5N1 avian influenza reveal similarities and differences. 354 Both infections feature acute and organizing diffuse alveolar damage, but SARS appears to be more frequently associated with subacute injury with intraalveolar organization, whereas H5N1 virus causes a more fulminant diffuse alveolar damage pattern with patchy interstitial inflammation and paucicellular fibrosis. Figure 7.99 Coronavirus pneumonia: Severe acute respiratory syndrome. (A) and (B) Acute fibrinous lung injury is evident. Figure 7.99 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Figure 7.100 Coronavirus-infected cell can be seen in this electron photomicrograph. Figure 7.100 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Adenovirus Adenovirus comprises several genera, with multiple serotypes that cause infections of the upper and lower respiratory tract, conjunctiva, and gut. Respiratory tract infections are most common and account for approximately 5% to 10% of pediatric pneumonias. These can be especially severe in neonates, children, and immunocompromised persons. 307 , 355 In the lung, adenovirus infection produces two patterns of lung injury: diffuse alveolar damage with or without necrotizing bronchiolitis and pneumonitis with "dirty" or karyorrhectic necrosis 356 ( Fig. 7.101 ). These patterns may coexist in some cases, and the pneumonia may be accompanied by hemorrhage secondary to adenovirus-induced endothelial cell damage. 357 Two types of adenoviral CPE may be seen. Initially an eosinophilic (Cowdry A) intranuclear inclusion occurs surrounded by a halo with marginated chromatin, similar to HSV ( Fig. 7.102A ). This later enlarges and becomes amphophilic and then more basophilic, obliterating the nuclear membrane and producing the characteristic smudge cell ( Fig. 7.102B ). 307 Figure 7.101 Adenoviral pneumonia. (A) Necrosis (N) and diffuse alveolar damage (hm) . (B) Necrotizing bronchiolitis. hm, hyaline membrane. Figure 7.101 Figure 7.102 Adenovirus. (A) Cowdry A intranuclear inclusions. (B) Smudged cell. Figure 7.102 Herpes Simplex Viruses HSVs types I and II have had traditional assigned roles as etiologic agents of mucocutaneous disease of the head and neck (type I) and genitalia (type II). Considerable crossover has been documented, however, with both types isolated from patients with disease at either site. Tracheobronchitis and pneumonia due to these viruses are rare in healthy adults with intact immune systems. They occur primarily in patients with underlying pulmonary disease and in association with inhalational and intubational trauma. They also occur in neonates and in patients who are immunosuppressed or compromised by various chronic diseases. Characteristic lesions include tracheobronchitis ( Fig. 7.103A ) with ulcers and hemorrhagic diffuse alveolar damage. Necrosis in a miliary small (or rarely large) nodular pattern is a helpful clue and the best location to identify CPE ( Fig. 7.103B ). 73 Like adenovirus, HSV also has two types of CPE: Initially a ground-glass amphophilic intranuclear inclusion, Cowdry B , appears with marginated chromatin; later a single eosinophilic Cowdry A inclusion ( Fig. 7.104 ) surrounded by a halo, similar to that seen with adenovirus, develops. The Cowdry A inclusion is considered noninfectious, as it is devoid of nucleic acid protein and is thought to represent the nuclear "scar" of HSV infection. 307 In the absence of smudge cells, HSV and adenoviral infections can look identical. Fortunately, immunohistochemistry or in situ hybridization can often resolve this differential diagnosis. Figure 7.103 Herpes simplex virus pneumonia. (A) Tracheobronchitis. Note cells with ground-glass inclusion. (B) Miliary nodular pattern of hemorrhagic necrosis. Figure 7.103 Figure 7.104 Herpes simplex virus pneumonia. Note two types of nuclear cytopathic effects: Cowdry A ground-glass type (short arrow) and Cowdry B eosinophilic inclusion (arrowhead) . Compare with cytomegalovirus intranuclear and cytoplasmic inclusion at long arrow. Figure 7.104 Varicella-Zoster Virus Varicella-zoster virus (VZV) infection produces considerable morbidity in the newborn, the adult, and the immunocompromised host, both in its primary form (varicella) and in its reactivated form (zoster). Varicella pneumonia is rarely observed in otherwise healthy children but is a major complication of adult varicella, occurring in approximately 10% to 15% of adults with VZV. In affected adults without underlying diseases and normal immunity, the course is generally mild and self-limited. Nevertheless, fatality rates of up to 10% have been reported. 307 By contrast, high mortality rates (25% to 45%) have been noted among some cohorts of immunosuppressed patients. Microscopically, small miliary nodules of necrosis are seen, associated with interstitial pneumonitis, edema, fibrin deposits, or patchy hyaline membranes ( Fig. 7.105A ). HSV-like intranuclear inclusions are present but may be sparse and difficult to identify. A miliary pattern of calcified nodules ( Fig. 7.105B ) may be present in the healed phase. 358 Figure 7.105 Varicella pneumonia. (A) A hemorrhagic miliary nodule. (B) Late phase with calcified nodules. Figure 7.105 Cytomegalovirus CMV infections are acquired throughout life. This virus can cause considerable morbidity and even death in the neonate, but infection is generally asymptomatic in older healthy children and adults. As in the case of other herpesviruses, primary infection is followed by latency, which persists until immune deficiency or immunosuppressive therapy causes it to reactivate and disseminate. CMV has therefore become one of the most common opportunists in patients with AIDS and those who receive organ transplants. In these settings, CMV can produce a variety of patterns, including one with minimal changes where only scattered alveolar lining cells with typical viropathic changes are seen. The CPE of CMV produces cytomegalic cells with large, round to oval, smooth "owl eye" eosinophilic to basophilic intranuclear inclusions surrounded by a clear halo ( Fig. 7.106A ). Figure 7.106 Cytomegalovirus (CMV) pneumonia. (A) Multiple characteristic intranuclear and intracytoplasmic inclusions in alveolar lining cells. Note Grocott methenamine silver–positive staining of inclusions (inset) . (B) Miliary nodule pattern of CMV pneumonia. Figure 7.106 (A, Courtesy Dr. Francis Chandler, Augusta, Georgia.) Later, multiple eosinophilic cytoplasmic inclusions develop that may be positive on staining with PAS and GMS ( Fig. 7.106A , inset). The more numerous the cytomegalic cells, the greater the clinical significance. In some cases, atypical inclusions may be seen in cells that are not significantly enlarged, and the nuclei may contain dark-staining homogeneous inclusions that may lack a clear halo. Despite their atypical appearance, these inclusions will usually be highlighted with immunohistochemical stains. 359 Another typical pattern that suggests viral infection is the presence of small miliary nodules with a central hemorrhage surrounded by necrotic alveolar walls ( Fig. 7.106B ). 73 Interstitial pneumonitis is the least common pattern of CMV infection. Ulcers may be seen in the trachea and bronchi, but they occur less often than in herpetic infections. In CMV pneumonias, it is advisable to look for other pathogens, typically P. jirovecii ( Fig. 7.107 ); but bacteria, fungi, protozoa, and other viruses are all possible coinfecting organisms. 360 Figure 7.107 Cytomegalovirus-infected alveolar lining cells associated with the foamy alveolar casts of Pneumocystis jirovecii . Figure 7.107 Epstein–Barr Virus EBV infections are usually acquired in childhood and are generally asymptomatic. The pathologist most often encounters this virus in the lung in the context of pulmonary lymphomas or in other EBV-associated lymphoproliferative disorders that can occur in transplant recipients and other immunocompromised patients. However, the most common symptomatic primary EBV infection is infectious mononucleosis. Most of these patients recover uneventfully, but a few develop one or more complications. Pneumonitis is one of them, albeit rare and not well characterized. The few reports describing pathology indicate a nonspecific lymphocytic interstitial pneumonitis, which may be bronchiolocentric ( Fig. 7.108 ). 361 , 362 CPE is absent, and although serologic studies can be supportive of a clinicopathologic diagnosis, etiologic proof of EBV infection requires the demonstration of the virus in lymphoid cells by in situ hybridization for EBV-encoded RNA-1 (EBER-1). Figure 7.108 Epstein–Barr virus pneumonitis. (A) Nonspecific cellular interstitial pneumonitis. (B) Patchy interstitial infiltrate. Figure 7.108 Influenza Virus Influenza viruses are the most pathogenic of the respiratory viruses and predispose patients most commonly to secondary bacterial pneumonia. These viruses also account for the greatest public health burden. Annually they cause epidemic outbreaks of respiratory disease that are often associated with considerable morbidity; periodically, they produce pandemics with high mortality rates. These viruses target the ciliated epithelium of the tracheobronchial tree, producing necrotizing bronchitis and bronchiolitis and a spectrum of changes that vary depending on the stage of the disease (early vs. late), outcome (fatal vs. nonfatal), and the presence or absence of secondary bacterial pneumonia. Uncomplicated influenza pneumonia is rarely biopsied today. Based on historical data from bronchoscopic biopsies performed in the 1950s and early 1960s, the histopathologic findings in nonfatal uncomplicated influenza are those of active tracheobronchitis. 314 Necrosis and desquamation of the epithelial cells to the basement membrane is associated with a relatively scant lymphocytic infiltrate; however, in more severe cases, the virus and its attendant inflammatory response spread more distally into the respiratory bronchioles and alveoli, with hemorrhage, edema, fibrinous exudate with hyaline membranes, and patchy interstitial cellular infiltrates ( Fig. 7.95 ). This constellation of findings comprises the lesion of characterization . 315 In contemporary pathologic terms this would correspond to diffuse alveolar damage and, clinically, a primary viral pneumonia. Depending on the clinical course and time of lung biopsy (or autopsy) within the first 2 weeks of illness, the process may be in the acute and/or organizing phase. 316 , 317 Later, the airway epithelial damage may pave the way for secondary bacterial pneumonia, which accounts for much of the morbidity and mortality of influenza and may obscure the features of primary viral pneumonia. 318 Figure 7.95 Influenza virus. (A) Bronchiolitis with intraluminal necroinflammatory debris. (B) Acute diffuse alveolar damage pattern with hyaline membranes. Figure 7.95 From 2003 through 2008, 391 human cases of highly pathogenic avian influenza involving the H5N1 strain were recorded with 247 deaths. 319 The histopathologic changes observed in the few autopsied cases fall within the spectrum of findings described during the pandemics of 1918, 1957, and 1968 and in fatal cases of interpandemic (seasonal) influenza. 317 A characteristic feature of the H5N1 and 1918 cases is the high mortality rate, especially among previously healthy older children and young adults. Excessively high levels of cytokines and chemokines are thought to play an important role in the pathogenesis of the acute lung injury pattern seen in these fatal cases of influenza. 320 Because these viruses produce no characteristic cellular inclusions, etiologic diagnosis is not possible by morphology alone and requires antigen detection by immunofluorescence, immunohistochemistry, in situ hybridization, or culture. 321 The influenza virus genome steadily shifts over the passage of time; infections occur in those without previous immunity to each new strain. Rapid characterization of each viral variant is a continuous challenge, with numerous virologic methods concentrating on rapid point-of-care methods needed for effective prophylaxis. 322 Parainfluenza Virus Parainfluenza virus comprises four serotypes (I to IV) that typically target the upper respiratory tract, classically in the form of croup. 323 Some cases involve distal airways, as in infections due to RSV and influenza virus, but are milder, with less morbidity and requiring fewer hospitalizations. A few documented cases have been described with a diffuse alveolar damage pattern or an interstitial pneumonitis with giant cells, the latter resembling those of measles and RSV infection. The giant cells of parainfluenza tend to be larger and have more intracytoplasmic inclusions. 55 Parainfluenza virus is a potential opportunist in immunocompromised patients, especially children with congenital immunodeficiency disorders, 324 in whom fatal pneumonitis with disseminated disease may occur. 325 Respiratory Syncytial Virus RSV causes more significant respiratory infections in early childhood than those attributable to either influenza viruses or parainfluenza viruses. 326 , 327 The annual outbreaks of bronchiolitis and pneumonia in infants are especially severe during the first year of life and in those of low birth weight or with cardiopulmonary disease. 323 Considered primarily a childhood virus, RSV has more recently been recognized as the etiologic agent of pneumonia in community-dwelling and high-risk adults with chronic lung disease requiring hospitalization. 324 , 328 , 329 Also, RSV is often an unsuspected opportunistic pathogen in immunocompromised patients. 299 , 330 RSV targets the epithelium of the distal airway, producing bronchiolitis with disorganization of the epithelium and epithelial cell sloughing ( Fig. 7.96A ). 307 In fatal cases, airway obstruction due to sloughed cell detritus, mucus, and fibrin is compounded by airway lymphoid hyperplasia. 331 Diffuse alveolar damage may be seen in immunocompromised patients. Giant cells (syncytia), similar to the cytopathic changes seen in cell culture, may be present in alveolar ducts and airspaces around areas of bronchiolitis ( Fig. 7.96B ). Eosinophilic inclusions in cytoplasm may be seen in tissues and cytology specimens from immunosuppressed patients, but these are difficult to confirm as diagnostic of RSV without immunohistochemistry. Figure 7.96 Respiratory syncytial virus. (A) Bronchiolitis with intraluminal sloughing. (B) Bronchiolitis with giant cell syncytia. Figure 7.96 Human Metapneumovirus Human metapneumovirus, a newly recognized paramyxovirus, is a leading cause of respiratory tract disease in infants, with annual epidemics occurring during the winter and early spring months. 304 , 332 The virus also causes disease in immunocompromised patients 333 and likely explains some lower respiratory tract infections in the elderly. The clinical spectrum of croup, bronchiolitis, and pneumonia is similar to that for infections due to other paramyxoviruses, such as RSV and parainfluenza virus. The pathologic features are not well characterized because few well-documented cases have included biopsy in the evaluation. However, histopathologic assessment of lung tissue in severe cases has revealed acute and organizing diffuse alveolar damage as well as smudge cell formation. 334 , 335 The definitive identification of the virus can be established in tissue culture, but monoclonal antibody reagents and molecular techniques (real-time PCR assay) are the current diagnostic methods of choice. Measles Virus The measles virus causes a highly communicable childhood viral exanthem worldwide that, unlike varicella (chickenpox), leads to complications that are common and serious. 336 Measles pneumonia accounts for the vast majority of measles-related deaths, and most of these are a consequence of secondary pneumonia (bacterial or viral) or attributable to an aberrant immune response. Despite vaccination, measles is still a global pathogen and has resurfaced due to variation in vaccination rates, even in the United States. 337 , 338 Primary viral pneumonia occurs but is uncommon, even in immunocompromised hosts. Microscopically, bronchial and bronchiolar epithelial degeneration and reactive hyperplasia with squamous metaplasia is typically accompanied by peribronchial inflammation. Diffuse alveolar damage may occur, and quantitative immunohistochemical studies have revealed severe immune dysfunction with loss of key effector cells and their cytokines. 339 Characteristic giant cells show distinctive intranuclear eosinophilic inclusions surrounded by halos ( Fig. 7.97 ). This is the classic measles injury pattern 307 and is referred to as Hecht giant cell pneumonia . Minute intracytoplasmic eosinophilic inclusions precede the development of the intranuclear inclusions and are often difficult to identify. Pneumonia with giant cells should always suggest measles, but similar changes can be seen in RSV and parainfluenza pneumonias, and not all cases of measles pneumonia have these giant cells. 307 Hard metal pneumoconiosis (giant cell interstitial pneumonia) is in the differential diagnosis, but the overall appearance of hard metal disease is one of a chronic disease with some fibrosis and few if any acute changes. In the absence of giant cells, the cellular interstitial pneumonia must be differentiated from those caused by other viruses and atypical pneumonia agents as well as from nonspecific interstitial pneumonia. Figure 7.97 Measles virus pneumonia with characteristic eosinophilic intranuclear inclusions in giant cell. Figure 7.97 Hantavirus The recently identified hantavirus produces a rapidly evolving cardiopulmonary syndrome with a high mortality rate. This disorder first came to public attention as an emerging infection following an outbreak in the southwestern United States in 1993; it was causally linked to a previously unrecognized hantavirus. All members of this genus are zoonotic and are found in rodents around the world. The specific type responsible for the cardiopulmonary syndrome, Sin Nombre , is present in rodent feces and is acquired from the environment through inhalation. It produces florid pulmonary edema with pleural effusions, variable fibrin deposits, and focal wispy hyaline membranes ( Fig. 7.98A ). 340 Immunoblast-like cells are present in vascular spaces and in the peripheral blood ( Fig. 7.98B ). Morphologic diagnosis is presumptive because hantaviral antigen in endothelial cells, detected by immunohistochemistry, is required for definitive diagnosis. 341 In the appropriate clinical setting, clues to the diagnosis can sometimes be found in a constellation of morphologic findings on a peripheral blood smear, and confirmation can be achieved serologically by the detection of hantavirus-specific immunoglobulin M (IgM) antibodies or by the detection of hantavirus RNA by PCR assay in peripheral blood leukocytes. 342 , 343 , 344 Figure 7.98 Hantavirus. (A) Pulmonary edema with fibrin deposits. (B) Immunoblast-like cells in alveolar capillaries at arrows. Figure 7.98 Coronaviruses Coronaviruses are ubiquitous RNA viruses known to cause disease in many animals. At least five different coronaviruses are known to infect humans, and these cluster into two antigenic groups. 303 They, along with the rhinoviruses, are responsible for a majority of common colds. Coinfections with other respiratory viruses occur in infants and children presenting with more severe respiratory disease. In certain epidemiologic situations, they can cause pneumonia in children, frail elderly individuals, and immunocompromised adults. 345 , 346 In November 2002, the appearance of an atypical pneumonia in China, subsequently labeled SARS, became an alarming global health problem in the period of a few months. 347 The disease was linked (Koch's postulates were fulfilled) by means of tissue culture isolation, electron microscopy, and molecular analysis to an emergent novel coronavirus, proposed as the Urbani strain of SARS-associated coronavirus. 348 Clinically the disease ranges from a nonhypoxemic febrile respiratory disease (with minimal symptoms in some patients) to one of severe pulmonary dysfunction, manifesting as ARDS and eventuating in death for approximately 5% of the patients affected. 349 In the reported cases, the chest x-ray appearance on presentation was either normal or the chest film showed unilateral, predominantly peripheral areas of consolidation that progressed to bilateral, patchy consolidation, the degree and extent of which correlated with the development of respiratory failure. In patients who presented with a normal x-ray appearance, CT scans often revealed bilateral ground-glass consolidation resembling that in bronchiolitis obliterans with organizing pneumonia (cryptogenic organizing pneumonia). Lymphopenia and elevated LDH were helpful clues, but the clinical, radiologic, and laboratory features, although characteristic, were not distinguishable from those in patients with pneumonia caused by other viruses and bacteria and various atypical agents. Histopathologic findings in lung biopsy and autopsy tissues included acute lung injury (diffuse alveolar damage) in various stages of organization. 350 , 351 Lung biopsy specimens in milder cases showed relatively scant intraalveolar fibrin deposits with some congestion and edema ( Fig. 7.99 ). However, the spectrum of findings included acute fibrinous pneumonia, hyaline membrane formation, interstitial lymphocytic infiltrates, desquamation of alveolar pneumocytes, and areas undergoing organization of the acute-phase injury. 352 In some patients, multinucleate syncytial cells reminiscent of the CPE seen in influenza virus, RSV, and measles virus infections were noted. Viral inclusions were not identified, and initial immunohistochemical studies failed to reveal viral antigen. Subsequent investigations detected virus in epithelial cells (predominantly type II pneumocytes) and alveolar macrophages using immunohistochemical staining, in situ hybridization, RT-PCR methods, and electron microscopy. A unique coronavirus ( Fig. 7.100 ) was finally implicated as the etiologic agent. 352 , 353 Comparative histopathologic studies in fatal cases of SARS and H5N1 avian influenza reveal similarities and differences. 354 Both infections feature acute and organizing diffuse alveolar damage, but SARS appears to be more frequently associated with subacute injury with intraalveolar organization, whereas H5N1 virus causes a more fulminant diffuse alveolar damage pattern with patchy interstitial inflammation and paucicellular fibrosis. Figure 7.99 Coronavirus pneumonia: Severe acute respiratory syndrome. (A) and (B) Acute fibrinous lung injury is evident. Figure 7.99 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Figure 7.100 Coronavirus-infected cell can be seen in this electron photomicrograph. Figure 7.100 (Courtesy Dr. Oi-Yee Cheung, Queen Elizabeth Hospital, Hong Kong, China.) Adenovirus Adenovirus comprises several genera, with multiple serotypes that cause infections of the upper and lower respiratory tract, conjunctiva, and gut. Respiratory tract infections are most common and account for approximately 5% to 10% of pediatric pneumonias. These can be especially severe in neonates, children, and immunocompromised persons. 307 , 355 In the lung, adenovirus infection produces two patterns of lung injury: diffuse alveolar damage with or without necrotizing bronchiolitis and pneumonitis with "dirty" or karyorrhectic necrosis 356 ( Fig. 7.101 ). These patterns may coexist in some cases, and the pneumonia may be accompanied by hemorrhage secondary to adenovirus-induced endothelial cell damage. 357 Two types of adenoviral CPE may be seen. Initially an eosinophilic (Cowdry A) intranuclear inclusion occurs surrounded by a halo with marginated chromatin, similar to HSV ( Fig. 7.102A ). This later enlarges and becomes amphophilic and then more basophilic, obliterating the nuclear membrane and producing the characteristic smudge cell ( Fig. 7.102B ). 307 Figure 7.101 Adenoviral pneumonia. (A) Necrosis (N) and diffuse alveolar damage (hm) . (B) Necrotizing bronchiolitis. hm, hyaline membrane. Figure 7.101 Figure 7.102 Adenovirus. (A) Cowdry A intranuclear inclusions. (B) Smudged cell. Figure 7.102 Herpes Simplex Viruses HSVs types I and II have had traditional assigned roles as etiologic agents of mucocutaneous disease of the head and neck (type I) and genitalia (type II). Considerable crossover has been documented, however, with both types isolated from patients with disease at either site. Tracheobronchitis and pneumonia due to these viruses are rare in healthy adults with intact immune systems. They occur primarily in patients with underlying pulmonary disease and in association with inhalational and intubational trauma. They also occur in neonates and in patients who are immunosuppressed or compromised by various chronic diseases. Characteristic lesions include tracheobronchitis ( Fig. 7.103A ) with ulcers and hemorrhagic diffuse alveolar damage. Necrosis in a miliary small (or rarely large) nodular pattern is a helpful clue and the best location to identify CPE ( Fig. 7.103B ). 73 Like adenovirus, HSV also has two types of CPE: Initially a ground-glass amphophilic intranuclear inclusion, Cowdry B , appears with marginated chromatin; later a single eosinophilic Cowdry A inclusion ( Fig. 7.104 ) surrounded by a halo, similar to that seen with adenovirus, develops. The Cowdry A inclusion is considered noninfectious, as it is devoid of nucleic acid protein and is thought to represent the nuclear "scar" of HSV infection. 307 In the absence of smudge cells, HSV and adenoviral infections can look identical. Fortunately, immunohistochemistry or in situ hybridization can often resolve this differential diagnosis. Figure 7.103 Herpes simplex virus pneumonia. (A) Tracheobronchitis. Note cells with ground-glass inclusion. (B) Miliary nodular pattern of hemorrhagic necrosis. Figure 7.103 Figure 7.104 Herpes simplex virus pneumonia. Note two types of nuclear cytopathic effects: Cowdry A ground-glass type (short arrow) and Cowdry B eosinophilic inclusion (arrowhead) . Compare with cytomegalovirus intranuclear and cytoplasmic inclusion at long arrow. Figure 7.104 Varicella-Zoster Virus Varicella-zoster virus (VZV) infection produces considerable morbidity in the newborn, the adult, and the immunocompromised host, both in its primary form (varicella) and in its reactivated form (zoster). Varicella pneumonia is rarely observed in otherwise healthy children but is a major complication of adult varicella, occurring in approximately 10% to 15% of adults with VZV. In affected adults without underlying diseases and normal immunity, the course is generally mild and self-limited. Nevertheless, fatality rates of up to 10% have been reported. 307 By contrast, high mortality rates (25% to 45%) have been noted among some cohorts of immunosuppressed patients. Microscopically, small miliary nodules of necrosis are seen, associated with interstitial pneumonitis, edema, fibrin deposits, or patchy hyaline membranes ( Fig. 7.105A ). HSV-like intranuclear inclusions are present but may be sparse and difficult to identify. A miliary pattern of calcified nodules ( Fig. 7.105B ) may be present in the healed phase. 358 Figure 7.105 Varicella pneumonia. (A) A hemorrhagic miliary nodule. (B) Late phase with calcified nodules. Figure 7.105 Cytomegalovirus CMV infections are acquired throughout life. This virus can cause considerable morbidity and even death in the neonate, but infection is generally asymptomatic in older healthy children and adults. As in the case of other herpesviruses, primary infection is followed by latency, which persists until immune deficiency or immunosuppressive therapy causes it to reactivate and disseminate. CMV has therefore become one of the most common opportunists in patients with AIDS and those who receive organ transplants. In these settings, CMV can produce a variety of patterns, including one with minimal changes where only scattered alveolar lining cells with typical viropathic changes are seen. The CPE of CMV produces cytomegalic cells with large, round to oval, smooth "owl eye" eosinophilic to basophilic intranuclear inclusions surrounded by a clear halo ( Fig. 7.106A ). Figure 7.106 Cytomegalovirus (CMV) pneumonia. (A) Multiple characteristic intranuclear and intracytoplasmic inclusions in alveolar lining cells. Note Grocott methenamine silver–positive staining of inclusions (inset) . (B) Miliary nodule pattern of CMV pneumonia. Figure 7.106 (A, Courtesy Dr. Francis Chandler, Augusta, Georgia.) Later, multiple eosinophilic cytoplasmic inclusions develop that may be positive on staining with PAS and GMS ( Fig. 7.106A , inset). The more numerous the cytomegalic cells, the greater the clinical significance. In some cases, atypical inclusions may be seen in cells that are not significantly enlarged, and the nuclei may contain dark-staining homogeneous inclusions that may lack a clear halo. Despite their atypical appearance, these inclusions will usually be highlighted with immunohistochemical stains. 359 Another typical pattern that suggests viral infection is the presence of small miliary nodules with a central hemorrhage surrounded by necrotic alveolar walls ( Fig. 7.106B ). 73 Interstitial pneumonitis is the least common pattern of CMV infection. Ulcers may be seen in the trachea and bronchi, but they occur less often than in herpetic infections. In CMV pneumonias, it is advisable to look for other pathogens, typically P. jirovecii ( Fig. 7.107 ); but bacteria, fungi, protozoa, and other viruses are all possible coinfecting organisms. 360 Figure 7.107 Cytomegalovirus-infected alveolar lining cells associated with the foamy alveolar casts of Pneumocystis jirovecii . Figure 7.107 Epstein–Barr Virus EBV infections are usually acquired in childhood and are generally asymptomatic. The pathologist most often encounters this virus in the lung in the context of pulmonary lymphomas or in other EBV-associated lymphoproliferative disorders that can occur in transplant recipients and other immunocompromised patients. However, the most common symptomatic primary EBV infection is infectious mononucleosis. Most of these patients recover uneventfully, but a few develop one or more complications. Pneumonitis is one of them, albeit rare and not well characterized. The few reports describing pathology indicate a nonspecific lymphocytic interstitial pneumonitis, which may be bronchiolocentric ( Fig. 7.108 ). 361 , 362 CPE is absent, and although serologic studies can be supportive of a clinicopathologic diagnosis, etiologic proof of EBV infection requires the demonstration of the virus in lymphoid cells by in situ hybridization for EBV-encoded RNA-1 (EBER-1). Figure 7.108 Epstein–Barr virus pneumonitis. (A) Nonspecific cellular interstitial pneumonitis. (B) Patchy interstitial infiltrate. Figure 7.108 Cytopathology The cytologic features of viral infections in the respiratory tract are most likely to be found in exfoliative specimens, such as bronchial washings and BAL fluid samples, rather than needle aspirates, although viral diagnosis has been achieved with this technique. 363 , 364 This is because viral infections are less likely to produce radiologic mass–like infiltrates, which are the most common targets of needle biopsy procedures. Herpes simplex virus, CMV ( Fig. 7.109 ), and adenovirus are the most commonly identified viral pathogens in respiratory cytologic specimens, but varicella virus, parainfluenza virus, RSV, human metapneumovirus, and measles virus have also been detected. Figure 7.109 Cytomegalovirus pneumonitis with characteristic cytopathic effect. (A) Fine-needle aspirate. (B) Bronchoalveolar lavage specimen. Figure 7.109 Characteristic CPE produced by these viruses is often better appreciated in cytologic smears than in tissue sections, which may, in fact, yield a negative result. Therefore review of any cytology sample taken at the time of biopsy can be valuable. Other, less specific changes may be found. These include ciliocytophoria (free cilia complexes with terminal bars) and cytologic atypia mimicking cancer. 46 Microbiology Diagnostic virology is the newest of the microbiology and infectious disease specialties to have benefited from the technologic revolution in laboratory medicine. Rapid and accurate diagnosis can often be achieved today using practical, convenient laboratory methods that employ reliable, commercially available mammalian cells, media, and reagent systems. 297 , 365 , 366 This has allowed many rural and small urban hospital laboratories to provide timely viral diagnostic services not possible a short time ago. It is predicted that self-contained, rapid-cycle real-time PCR methods will one day account for the majority of viral assays in laboratories of all sizes. As a result, the pathologist who suspects a viral infection will increasingly have a variety of tools to obtain an etiologic diagnosis when morphologic manifestations are suggestive of viral infection. The basic approaches to viral diagnosis in the laboratory are listed in Box 7.23 . In questionable cases, confirmation by immunohistochemical studies ( Fig. 7.110A ), in situ hybridization ( Fig. 7.110B ), or electron microscopy may be helpful. 32 , 367 Many of the traditional methods of viral detection, detailed later, are being augmented by respiratory panel assays based on the detection of nucleic acid and compiled around common respiratory viral and bacterial pathogens. 368 , 369 Box 7.23 Laboratory Diagnosis of Viral Pneumonia Direct detection of organisms Histopathologic/cytopathologic examination for cytopathic effect (CPE) Immunohistochemical studies Electron microscopy Antigen detection Direct fluorescent antibody test Enzyme immunoassay Culture Conventional roller tube technique Shell vial technique Serologic studies Molecular methods In situ hybridization DNA amplification Alt-text: Box 7.23 Figure 7.110 (A) Respiratory syncytial virus cytoplasmic inclusions detected by immunohistochemical staining. (B) Cytomegalovirus-infected cell with cytoplasmic inclusions detected by in situ hybridization. Figure 7.110 (Courtesy R.V. Lloyd, MD, Rochester, Minnesota.) The diagnosis of viral respiratory infections can also be based on antigen detection and culture ( Fig. 7.111 ). Direct antigen detection in clinical specimens collected by nasopharyngeal swabs, nasal washings, and aspirates or BAL fluid (but not sputum samples or, with rare exception, throat swabs) is performed using monoclonal antibodies by either immunofluorescence microscopy or enzyme immunoassay. By using a single reagent containing the monoclonal antibodies against several viruses and dual fluorochromes, the common respiratory viruses can be rapidly screened by direct immunofluorescence testing. Positive specimens can then be tested with individual reagents to determine the specific etiologic agent, while negative specimens can be submitted for culture. 370 Enzyme immunoassay includes methods that offer speed and convenience at the point of care. However, they are less sensitive than standard virologic methods, which must still be used to test negative specimens. Direct detection can also be accomplished in cellular samples, including tissue, by in situ hybridization or amplification techniques such as PCR. For RNA viruses, PCR amplification uses a reverse transcriptase (RT) step. PCR methodology has recently evolved into multiplex formats, and novel systems have been introduced that combine multiplex PCR chemistry with electron microarray (DNA chip) technology or fluid microsphere-based systems, permitting the simultaneous detection of a wide array of respiratory viruses and other pathogens. 371 , 372 , 373 , 374 , 375 , 376 Figure 7.111 Respiratory syncytial virus (RSV) infection. (A) RSV cytopathic effect in tissue culture. (B) RSV antigen in nasopharyngeal swab specimen detected by direct immunofluorescence microscopy. Figure 7.111 These systems have the potential to more rapidly and accurately diagnose acute infections and may also allow the study of complex coinfections and the active monitoring of outbreaks of influenza and other viral illnesses. 377 Panels composed of common respiratory bacterial and viral pathogens are available; these are based on nucleic acid detection by nested PCR and come by several brand names. Such panels typically encompass many of the respiratory viruses detailed previously with specimens obtained through sampling with a nasopharyngeal swab. 378 , 379 Traditional viral cultures in tubes with various types of cell monolayers are currently performed with greater sensitivity and turnaround time using the shell vial technique. This technique uses centrifugation of clinical specimen suspensions onto coverslipped cell monolayers followed by brief incubation (1 to 2 days) and antigen detection. 365 It is important, therefore, to preserve a portion of tissue from a bronchial or transbronchial biopsy or thoracotomy specimen in viral transport medium, especially with an immunocompromised patient, who may not have had BAL fluid submitted for culture. Shell vials, although faster than the traditional tube culture method, are still a slow method based on viral growth and are being replaced by direct nucleic acid detection. Viral serologic testing has commonly been used for diagnosis but may be the least sensitive approach. A positive serodiagnosis is typically based on a fourfold rise in titer between acute and convalescent sera and therefore cannot be achieved by this means in the acutely ill patient; antigen detection or culture of respiratory tract specimens is much preferred. However, a serologic strategy, utilizing a panel of antigens in an immunofluorescence or enzyme immunoassay format on a single specimen, is useful in suspected EBV infections. 380 A case also can be made for the benefit of CMV serologic testing for assessing the antibody status of organ donors and recipients for predicting the risk of posttransplantation CMV disease. When tissue is not available or findings are inconclusive, tests for the detection of actual disease in these transplant recipients include the p65 antigenemia assay on peripheral blood leukocytes and amplification or quantitation of CMV DNA in various peripheral blood compartments (plasma, whole blood, and leukocytes). 381 These assays may eventually replace culture of BAL fluid for surveillance of CMV infection in such patients. 382 The detection of virus in respiratory secretions (including BAL fluid), urine, or blood establishes the presence of virus but does not necessarily implicate it as the etiologic agent of a pneumonia. Quantitation of viral load by real-time PCR amplification, however, can be useful in this regard by linking high viral load with infection. 383 Differential Diagnosis A synopsis of the key morphologic and microbiologic features of the viral pneumonias is presented in Table 7.12 . In the absence of CPE, diffuse alveolar damage and other patterns of lung injury are not diagnostic of viral infection. Diffuse alveolar damage is a nonspecific response to many types of infection, including bacterial, mycobacterial, fungal, and protozoal, all of which must be considered in the differential diagnosis. In addition, other noninfectious causes include reactions to drugs, radiation, toxic inhalants, and shock of any type. Occasionally, CPE may not be diagnostic; for example, the early inclusions of adenovirus, HSV, and CMV may be quite similar. In most cases, immunohistochemistry or molecular techniques can resolve the diagnostic dilemma. Mimics of CPE that must be ruled out include macronuclei in both reactive processes and occult neoplastic infiltrates and intranuclear cytoplasmic invaginations, which can occur in a variety of cells. Cytoplasmic viral inclusions can also be simulated by aggregated altered protein and particulate matter. Table 7.12 Viral Pneumonias: Summary of Pathologic Findings Table 7.12 Assessment Component Findings Influenza Virus Surgical pathology Diffuse alveolar damage, bronchitis, and bronchiolitis; secondary acute purulent pneumonia; antigen detection by immunofluorescence, immunohistochemical, or in situ hybridization studies Cytopathology Nonspecific changes may include presence of reactive-type pneumocytes; ciliocytophoria Microbiology Antigen detection by DFA or EIA; culture on primary monkey kidney cells: noncytopathic; detection by hemadsorption Respiratory Syncytial Virus Surgical pathology Bronchiolitis with lumen detritus; may be associated with syncytial giant cells; diffuse alveolar damage in immunocompromised patients; confirm with immunohistochemistry Cytopathology Giant cell syncytia characteristic but often not seen; eosinophilic inclusions may be seen in bronchial epithelial cells of immunocompromised patients; rarely in those of normal hosts; rarely diagnosed by cytology alone Microbiology Antigen detection by DFA and EIA usually more sensitive than culture; cultures on continuous epithelial cell lines (Hep-2) and primary monkey kidney yield characteristic syncytial CPEs Measles Virus Surgical pathology Bronchitis, bronchiolitis, diffuse alveolar damage with giant cells containing Cowdry A inclusions and small cytoplasmic inclusions Cytopathology Eosinophilic intranuclear and cytoplasmic inclusions; rarely diagnosed by cytology Microbiology Antigen detection by DFA and EIA; culture on primary monkey kidney produces spindle cell or multinucleate CPE; serologic testing (for measles-specific IgM) available Hantavirus Surgical pathology Pulmonary edema pattern with variable fibrin deposits; immunoblast-like cells in vascular spaces; confirm by immunohistochemistry Cytopathology Noncytopathic Microbiology Serology: Hantavirus-specific IgM or detection of specific RNA by PCR assay in peripheral blood leukocytes Adenovirus Surgical pathology Diffuse alveolar damage with or without necrotizing bronchiolitis and/or pneumonitis with necrosis and karyorrhexis Cytopathology Early Cowdry A intranuclear inclusions, later smudge cell; reactive and reparative-type atypia in background Microbiology Antigen detection by EIA and DFA; culture on continuous epithelial cell lines produces characteristic grape-like clustered cytopathic effect Herpesvirus Surgical pathology Tracheobronchitis; diffuse alveolar damage; miliary necroinflammatory lesions Cytopathology Ground-glass (Cowdry B) intranuclear inclusions; later Cowdry A inclusions in multinucleated cells, often with a "seeds in a pomegranate" appearance on Pap-, H&E-, and Diff-Quik–stained smears Background reactive and reparative atypia Microbiology Antigen detection by immunofluorescence; culture on diploid fibroblasts produces characteristic cytopathic effect, sometimes within 24 hours;serologic testing less useful Varicella-Zoster Virus Surgical pathology Miliary necroinflammatory lesions; calcified nodules in healed phase Cytopathology Intranuclear Cowdry A inclusions sparse and less welldefined than with herpes simplex Microbiology Antigen detection by immunofluorescence; culture on human embryonic lung or Vero cells produces CPE more slowly than for herpesviruses (3–7 days); serologic testing available Cytomegalovirus Surgical pathology Minimal changes with scattered cytomegalic cells; miliary necroinflammatory lesions; interstitial pneumonitis Cytopathology Large "owl eye" Cowdry A inclusions with halo; cytoplasmic inclusions stained with GMS Microbiology Culture on human diploid fibroblasts produces characteristic CPE slowly in traditional tube cultures but more rapidly with use of shell vial technique p65 antigenemia assay; PCR assay; selective application of serology useful Epstein–Barr Virus Surgical pathology Polymorphous lymphoid interstitial pneumonitis; confirm by in situ hybridization Cytopathology Noncytopathic Microbiology No routine culture; diagnosis by serologic testing using panel of antibodies (EA; IgG and IgM VCA; EBNA) CPE , Cytopathic effect; DFA , direct immunofluorescence antibody (test); EA , early antigen; EBNA , Epstein–Barr virus–determined nuclear antigen; EIA , enzyme immunoassay; GMS , Grocott methenamine silver; H&E , hematoxylin and eosin; IgG, IgM , immunoglobulins G and M; Pap , Papanicolaou; PCR , polymerase chain reaction; VCA , viral capsid antigen. Parasitic Infections Approximately 300 species of helminth worms and 70 species of protozoa have been acquired by humans during our short history on Earth. 384 Most of these are rare, but approximately 90 are relatively common and some have been found in the lung. 385 , 386 , 387 , 388 , 389 With travel to endemic areas and emergence (or reemergence) of parasitic pathogens in immunocompromised patients, pathologists will see these organisms 36 as exotic pulmonary conditions. Etiologic Agents Several parasite species migrate through the lungs as part of their normal life cycle, but few preferentially infect the human lung. 390 Most are aberrant pulmonary localizations in the human host, where they become lost in transit or are part of a secondary disseminated infection from another organ system, often in the setting of compromised immunity. The listing of etiologic agents in Box 7.24 is selective, based on the more common pathogens known to be associated with pulmonary involvement. Box 7.24 Some Common Parasitic Lung Pathogens Protozoa Toxoplasma gondii Entamoeba histolytica Cryptosporidia Microsporidia Metazoa (Helminths) Nematodes Dirofilaria immitis Strongyloides stercoralis Cestodes Echinococcus spp. Trematodes Paragonimus spp. Schistosoma spp. Alt-text: Box 7.24 Histopathology When parasites in the form of adult worms, larvae, or eggs invade or become deposited in lung tissue, they usually provoke an intense inflammatory reaction with neutrophils, eosinophils, and various mononuclear cells. One or more of the patterns listed in Box 7.25 may be identified. When the predominant site of involvement is the bronchial mucosa, a bronchitis and bronchiolitis pattern is observed; when they become impacted in pulmonary arteries, a nodular angiocentric pattern is observed, although it may be overshadowed by thrombosis and infarction. Some parasites invade the alveolar parenchyma, resulting in a pattern of miliary small nodules or pneumonitis. Naturally none of these patterns are consistently present and combinations of patterns may be seen. In some cases, an acute Loeffler-like eosinophilic pneumonia may reflect an allergic reaction to the transient passage of larvae through the pulmonary vasculature. Box 7.25 Histopathologic Patterns in Parasitic Lung Injury Eosinophilic pneumonia Large nodule(s) Miliary small nodules Bronchitis and bronchiolitis Abscess, cavities, and cysts Intravascular reaction Alt-text: Box 7.25 The various patterns, although nondiagnostic, can be suggestive of a parasitic infection, particularly when they incorporate a heavy eosinophilic infiltrate or granulomatous component. Eosinophilic lung disease, with or without blood eosinophilia, has a diverse etiology but is particularly characteristic of parasitic infection, especially in the tropics. 385 In the United States, other infections, such as coccidioidomycosis, must be considered, in addition to the many noninfectious causes of pulmonary eosinophilia. The challenge for the pathologist is the identification of a parasite, distinguishing it from artifact or foreign body, and classifying it as precisely as possible based on its size and unique morphologic features. Once the presence of suggestive morphologic features has been confirmed, the patient's travel or avocation history can help to further narrow the scope of the differential diagnosis. Of interest, a common "parasite" encountered in clinical practice is not a parasite at all but aspirated vegetable material simulating the complex structure of an organism. 391 Toxoplasmosis T. gondii is an obligate intracellular protozoan and a common opportunist in patients with AIDS, the disease underlying most cases of toxoplasmosis seen in recent years. The brain and retina are most commonly involved in these patients, but pulmonary lesions may also be present in cases of disseminated disease. These often take the form of miliary small nodules with fibrinous exudates, which may progress to a confluent fibrinopurulent pneumonia. 392 Free forms (crescent-shaped tachyzoites) and cysts may be identified ( Fig. 7.112 ). Pseudocysts packed with tachyzoites can be distinguished from true cysts with bradyzoites by staining of the latter with PAS and GMS. 393 Serology is the main method of diagnosis in the acute phase, and serology with concomitant radiologic findings in appropriate settings in immunocompromised hosts usually obviates the need for direct demonstration of the organisms. Either PCR on the specimen or immunohistochemistry can be used to demonstrate the organisms. 394 Figure 7.112 Toxoplasmosis. (A) Tachyzoites. (B) Pseudocysts packed with tachyzoites. Figure 7.112 Amebiasis Amebic dysentery becomes invasive in a small percentage of patients. When the trophozoites leave the gut, they most commonly travel to the liver. From the liver, either by direct extension or rarely by hematogenous spread, the lungs may become involved. In this scenario, abscesses composed of liquefactive debris—with few neutrophils, distinguishable from bacterial abscess where neutrophils are dominant—may be seen, most often in the right lower lobe adjacent to the liver. 395 , 396 Trophozoites can be best seen at the margin of viable tissue ( Fig. 7.113 ). They resemble histiocytes but are usually larger, with a lower nucleocytoplasmic ratio. A tiny central karyosome within a round nucleus having vesicular chromatin is characteristic. 397 , 398 Bronchial fistula formation and empyema can occur as complications; amebas may be found in sputum and pleural fluid, respectively, in these situations. For free-living amebic species (those of the genera Acanthamoeba , Balamuthia , Naegleria ), the central nervous system is the principal focus of infection. However disseminated disease including lung infection ( Fig. 7.114 ) may occur in certain epidemiologic situations, especially those involving compromised immune status, or in lung transplants. 399 , 400 , 401 Figure 7.113 Amoebic trophozoite in lung tissue (arrows) . Note delicate marginal nuclear chromatin with small central karyosome and small red blood cell in cytoplasm. Figure 7.113 (Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Figure 7.114 Free-living ameba in lung tissue from an immunocompromised patient. (A) Necroinflammatory nodule. (B) Encysted form, black arrow and left upper inset ; trophozoite, white arrow and right lower inset . Figure 7.114 Cryptosporidiosis Ten species of the intracellular coccidian protozoa are currently recognized, but one of them, Cryptosporidium parvum , causes most human infections. 402 Clinically, infection due to this organism may have three major manifestations: asymptomatic shedding, acute watery diarrhea that lasts for approximately 2 weeks, and persistent diarrhea that lasts several weeks. Patients with AIDS have a wider spectrum of disease severity and duration that includes a fulminant cholera-like illness. 402 These patients are most likely to manifest extraintestinal disease. In the lung, the organism targets the epithelium of the airways, just as it does the surface epithelium of the gut and biliary tract. 403 In H&E sections, cryptosporidia appear as small (4 to 6 µm in diameter) round to oval protrusions from the cell surface. Electron microscopy reveals that they are intracellular but extracytoplasmic. In addition to H&E, they stain with Giemsa, PAS, GMS, and acid-fast stains. A mild to moderate chronic inflammatory cell infiltrate is usually present in the submucosa. Pulmonary cryptosporidiosis is largely a case report event, most reports being from earlier phases of the AIDS epidemic 404 —a surprise in reviewing acid-fast stains for more common organisms. 405 Newer reports suggest that respiratory cryptosporidiosis may occur in immunocompetent children with cryptosporidial diarrhea and cough. 406 , 407 Microsporidiosis The microsporidia are obligate intracellular spore-forming protozoa. More than 140 genera and 1200 species are recognized, but only 7 genera and a few species have been confirmed as human pathogens. 408 They are opportunists that have recently emerged in severely immunocompromised patients, AIDS patients, and transplant recipients, with case reports of pulmonary infections in the immunocompromised population. 409 , 410 , 411 Clinically they primarily cause chronic diarrhea and cholangitis. In the lung, they cause bronchitis or bronchiolitis (or both), usually in patients who also have intestinal infections or disease at other sites, especially the biliary tract. 412 The predominant pathologic changes are in the airways, which show a mixed inflammatory cell infiltrate of mononuclear and polymorphonuclear leukocytes. 413 The organisms are found within vacuoles in the apical portion of epithelial cells lining the airways. They appear as very small (1 to 1.5 µm in diameter) basophilic dots whose recognition depends on organism load. However, even when heavy, the findings can be subtle. Also, as with cryptosporidiosis, their presence is often overlooked or obscured by coexistent pneumonias. Special stains—such as modified trichrome, Warthin-Starry–type silver, and Gram stains—are more sensitive and specific, especially when used in combination. 414 Leishmaniasis Leishmaniasis ( Leishmania donovani infection) is transmitted to humans by several species of the Phlebotomus sand fly. 415 Pulmonary leishmaniasis has been reported in HIV-infected patients and transplant recipients. 385 , 416 , 417 The organisms ( L. donovani amastigotes) can be found in the alveoli and alveolar septa and may be recovered in BAL fluid from these patients. 418 They also can be found in bronchoscopic biopsies ( Fig. 7.115 ). Serologic testing for leishmaniasis has been suggested as part of the pretransplantation work-up in endemic areas. 419 A rapid PCR-amplified diagnostic method has been described. 420 Figure 7.115 Leishmania donovani in bronchoscopic biopsy specimens obtained from a North African immigrant to Sicily. (A) Lower-power view of cellular infiltrate. (B) High-power view of dot-like organisms. Figure 7.115 (Courtesy Dr. Francesca Guddo, Palermo, Italy.) Dirofilariasis The zoonosis caused by Dirofilaria immitis , a parasite of dogs and other mammals, is transmitted by mosquitos and black flies to humans. 421 , 422 , 423 Larvae injected by these insect vectors migrate from the subcutis into veins and travel to the heart, where they die before maturing into adult worms. They are then washed into the lungs by the pulmonary arterial blood flow, where they form the nidus of a thrombus. Formation of an infarct follows, typically manifesting as an asymptomatic solitary pulmonary nodule ("coin lesion") in the lung periphery ( Fig. 7.116 ) that may be visualized on a positron emission tomography (PET) scan. 424 , 425 , 426 Microscopically the nodule resembles a typical infarct with a core of coagulation necrosis but also containing degenerated worm fragments in the remnant of an arteriole ( Figs. 7.117 and 7.118 ). A peripheral investment of chronic granulation tissue forms an interface with the alveolated parenchyma. "Step" sections and trichrome stains may be needed when H&E sections do not show the parasite. 427 Figure 7.116 Dirofilarial nodule, gross specimen. Figure 7.116 Figure 7.117 Dirofilarial nodule, with worm remnants in organizing thrombosed vessel. Figure 7.117 Figure 7.118 Dirofilariasis. (A) Intact worm cross section ×260. (B) Showing body cavity layers ×360. Surrounding necrosis in both figures. Figure 7.118 (From Abhisek, B, Reilly P, Perez A, et al. Human pulmonary dirofilariasis presenting as a solitary pulmonary nodule: a case report and brief review of literature. Resp Med Case Rep . 2013;10:40–42.) Strongyloidiasis Strongyloides is a parasite most often found in patients or travelers in the tropics, but endemic foci are present in the southeastern United States. Rhabditiform larvae of the nematode Strongyloides stercoralis , after hatching from ingested eggs, 428 invade the small intestinal mucosa. At this site occult infection may remain asymptomatic for years. Dissemination typically follows debilitation brought on by immunocompromising diseases and therapies. When this occurs, filariform larvae leave the gut and travel through the pulmonary vasculature. When they penetrate alveoli ( Fig. 7.119 ), they provoke hemorrhage and inflammation. 429 , 430 , 431 Loeffler syndrome, eosinophilic pneumonia, and abscesses may develop. When migration is interrupted, filariform larvae may metamorphose in situ to adult worms, which can produce eggs and rhabditiform larvae. Larvae identified in the sputum indicate hyperinfection. 432 Disseminated strongyloidiasis is but one example of an infection that may become manifest, particularly in immunocompromised patients, years after emigration from or travel to an endemic area harboring pathogens that are considered unusual or exotic by pathologists in the United States. Figure 7.119 Filariform larva of Strongyloides stercoralis penetrating into alveolar space with associated inflammation. Figure 7.119 Echinococcosis Echinococcosis is a zoonosis that occurs wherever sheep, dogs or other canids, and humans live in close contact. Ingested eggs of the tapeworm Echinococcus hatch in the gut, releasing oncospheres, which then invade the mucosa, enter the circulation, and travel to various sites, where they develop into hydatid cysts. 433 , 434 In the lung, unilocular slow-growing cysts are produced by Echinococcus granulosus . 435 Echinococcus multilocularis proliferates by budding, producing an alveolar pattern of microvesicles. 398 The cyst of E. granulosus has a trilayered membrane ( Fig. 7.120A ) with an outer fibrous, middle-laminated hyaline, and inner germinal layer that gives rise to brood capsules containing infective protoscolices with hooklets and suckers ( Fig. 7.120B ). The layers usually become separated in tissue, with the outer fibrous layer containing chronic inflammatory cells that form an interface with the alveolated parenchyma. Cysts that rupture into bronchi may be expectorated as debris with protoscolices or portions of the cyst wall. Abscesses and granulomas may also form in the lung, pleura, and chest wall. 436 Figure 7.120 Echinococcus granulosus. (A) Cyst with trilayered membrane. (B) Brood capsules. Figure 7.120 Paragonimiasis The parasite Paragonimus targets the lung and is acquired by the ingestion of freshwater crabs or crayfish infected with the metacercarial larvae of Paragonimus species. 437 Most cases worldwide are due to P. westermani , but several other species exist in Asia, Africa, and South and Latin America. In the United States, infections due to P. kellicotti have been reported. 390 The disease manifestations are related to the migratory route and the inflammatory response these hermaphroditic flukes stimulate as they enter lung parenchyma and travel to sites near larger bronchioles or bronchi. Typically an area of eosinophil-rich inflammatory reaction surrounds them, and this reactive process may evolve to form a fibrous pseudocyst or capsule containing worms, exudate, and debris ( Fig. 7.121A ). Cysts rupturing into bronchioles may result in eggs, blood, and inflammatory cells being coughed up in the sputum. Alternatively, eggs may become embedded in parenchyma, producing nodular granulomatous lesions ( Fig. 7.121B ) that progress to scars. 438 The eggs are yellowish, ovoid, and operculated, measuring 75 to 110 µm by 45 to 60 µm. The opercula unfortunately are not easily seen in tissue; however, the eggs are birefringent under polarized light, which helps to distinguish them from nonbirefringent schistosome eggs ( Fig. 7.122 ). 390 Figure 7.121 (A) Paragonimus westermani with yellowish refractile eggs in eosinophil-rich exudates. (B) Distorted egg of Paragonimus kellicotti in granuloma. Figure 7.121 Figure 7.122 Paragonimiasis. (A) Granulomatous reaction to egg. (B) Single egg in polarized light. (C) Chronic eosinophilic pneumonia with many eggs. (D) Giant cell reaction; pigment in eggs. Figure 7.122 (Courtesy A.E. McCullough, MD.) Schistosomiasis The public health burden of schistosomiasis is enormous. This parasitic infection affects 200 million people in 74 countries while continuing to expand its geographical range. 439 , 440 The life cycle and disease manifestations of the three major Schistosoma species— Schistosoma mansoni , Schistosoma haematobium , and Schistosoma japonicum —involve eggs, snail intermediate hosts, and free-swimming cercaria, which penetrate the skin of susceptible animals and people and develop into adult worms. The male and female worms eventually come to reside in various human venous plexuses, depending on the species, where egg deposition occurs. Pulmonary schistosomiasis comprises both acute and chronic forms. The acute disease, referred to as Katayama syndrome , manifests with fever, chills, weight loss, gastrointestinal symptoms, myalgia, and urticaria in patients with no previous exposure to the parasite. Acute larval pneumonitis and a Loeffler-like eosinophilic pneumonia may be seen in this setting. 439 , 441 Chronic pulmonary disease is almost always secondary to severe hepatic involvement with portal hypertension. In this setting, the eggs of S. mansoni , and rarely S. japonicum or S. haematobium , may be shunted through portosystemic collateral veins to the lungs. The eggs lodge in arterioles, provoking a characteristic granulomatous endarteritis with pulmonary symptoms and radiologic infiltrates. 442 , 443 When the endarteritis is accompanied by angiomatoid changes, the lesion is considered pathognomonic for pulmonary schistosomiasis. 390 Eggs typically are surrounded by epithelioid cells and collagen ( Fig. 7.123 ). Most schistosome eggs do not exhibit birefringence and are larger than Paragonimus eggs, with which they share a superficial resemblance. Adult schistosomes may rarely be found in pulmonary blood vessels. Worldwide, given the burden of disease in Africa and Asia, chronic disease is associated for unclear reasons with pulmonary hypertension. 444 Figure 7.123 (A) Schistosome eggs in lung parenchyma. (B) Eggs of Schistosoma japonicum. Figure 7.123 (A and B, Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Visceral Larva Migrans The common parasites that cause visceral larva migrans are the dog tapeworm, Toxocara canis , and the less common cat tapeworm, Toxocara cati . When embryonated eggs are ingested by an intermediate host, typically a child with a history of pica, they hatch into infective larvae in the intestine. Subsequently, the larvae penetrate the intestinal wall, gain access to the circulation, and are carried to many organs, including the lungs. This is the end point, for their growth is arrested by a granulomatous reaction and they never mature into adult worms. The granulomatous reaction usually has a conspicuous eosinophilic component, and larvae may be seen. 445 Cytopathology The cytologic literature contains many reports of the successful identification of parasites in pulmonary specimens recovered by exfoliative (sputum, bronchial washing or brushing, BAL fluid, pleural fluid) and needle aspiration techniques. Some of these are listed in Box 7.26 . 418 , 424 , 425 , 426 , 436 , 446 , 447 , 448 , 449 , 450 , 451 , 452 , 453 , 454 , 455 , 456 , 457 Commonly cited in textbooks and reviews is the finding of Strongyloides stercoralis larvae in expectorated sputum or bronchial washings of patients with hyperinfections ( Fig. 7.124 ). Also common are reports of Echinococcus protoscolices and hooklets in needle aspirates from patients with pleuropulmonary disease. 45 , 46 Use of large-bore and cutting needle biopsies has traditionally been contraindicated in the setting of suspected Echinococcus infections; reports of success with fine-needle aspiration without untoward reactions suggest that this technique is a relatively safe procedure in which the benefits outweigh the risks. 448 Box 7.26 Parasites Reported in Respiratory Cytology Specimens Toxoplasma Amebae Trichomonas Cryptosporidia Microsporidia Leishmania Paragonimus Echinococcus Strongyloides Schistosoma Dirofilaria Microfilariae Alt-text: Box 7.26 Figure 7.124 Strongyloides stercoralis larvae in bronchial washing. (A) Larval fragments in cell block. (B) ThinPrep smear. Figure 7.124 Cytologic analysis is a sensitive and often preferred method to diagnose cryptosporidiosis, microsporidiosis, and other respiratory tract infections in the immunocompromised patient because it has the advantage of being less invasive. Specimens such as bronchial washings and BAL fluids can be prepared by high-speed centrifugation followed by standard smear preparation, cytocentrifugation, or ThinPrep technology. A battery of special stains—including Gram, modified trichrome, Giemsa, GMS, acid-fast, chemofluorescent, and immunofluorescent, depending on reagent availability—can then be applied to detect cryptosporidial oocysts, microsporidial spores, or other etiologic agents. The morphologic features of many of the aforementioned organisms are usually better defined in cytologic preparations than in tissue biopsy specimens provided that obscuring background debris is limited and that the cytopreparation technique and staining have been well performed. Pseudoparasites such as vegetable matter, textile fibers, pollens, red cell "ghosts," and other extraneous material must be recognized and excluded. Thus, as for all of the various categories of microorganisms cited in this chapter, cytopathologic examination adds synergy to surgical pathologic and microbiologic methods. Microbiology The laboratory diagnosis of parasitic disease depends on the collection of appropriate specimens, which, in turn, requires appropriate clinical evaluation. For example, just as stool examination is the most efficient means of diagnosing most intestinal protozoa and helminths, respiratory specimens (e.g., sputum samples, bronchial washings, BAL fluid samples, touch imprints of lung biopsy tissue) can provide a specific etiologic diagnosis when pulmonary infections are suspected. 458 As in the case of cytologic samples, these specimens often reveal the characteristic microanatomic features of parasite larvae and eggs that usually cannot be readily seen when they are embedded in tissue. Moreover, the identification of organisms in respiratory specimens is diagnostic of pulmonary infection, whereas the presence of the organism in the feces of a patient suspected to have pulmonary disease provides only presumptive evidence. Serodiagnosis with immunologic and molecular methods can be useful when parasites are located deep within tissue, such as the lung, and not easily accessible to biopsy or cytologic sampling. 396 The effectiveness of serodiagnosis of parasitic diseases has been hampered by tests with low sensitivity and specificity, mainly as a result of the complex composition of parasitic antigens and the occurrence of frequent cross reactions. 458 In recent years, however, significant refinements in antigenic preparations and improvements in technology have resulted in assays with greater predictive value. The newer tests are based on enzyme immunoassay and immunoblot methodology. Many test kits are commercially available, and diagnostic services are available from the CDC and other reference laboratories. 459 With protozoal infections, serologic testing is especially useful for the diagnosis of toxoplasmosis. Several commercial kits are available for detection of IgG and IgM antibodies; however, false-negative results are possible in immunocompromised patients, and positive results must be interpreted with caution, especially when the index of clinical suspicion is low. 460 Real-time PCR analysis has been used for the diagnosis of toxoplasmosis in the immunocompromised patient. 461 , 462 Antibody determinations also have value in cases of pulmonary and other tissue-invasive forms of amebiasis as compared with antigen detection methods, which are more useful for noninvasive amebic intestinal diseases. However, the best diagnostic approach to invasive disease may be the use of serologic testing, antigen detection, and PCR methods in various combinations. 463 For the identification of cryptosporidia, the new immunofluorescence tests and enzyme immunoassays that have been developed for intestinal infections may have application in respiratory infections. Similar tests are not available for the microsporidia, and diagnosis of infection with these organisms continues to rely on direct staining techniques. For the helminths, serodiagnosis is possible for Echinococcus , Paragonimus , Strongyloides , and Schistosoma species using enzyme immunoassay methods, which have fair sensitivity and specificity. 385 , 459 The available tests for Dirofilaria suffer from poor sensitivity and specificity and are not clinically useful at this time. Differential Diagnosis The key morphologic and microbiologic features of selected parasitic lung infections are summarized in Table 7.13 . In the absence of eggs, larvae, worms, or trophozoites, the various inflammatory patterns must be distinguished from those of other infections and various noninfectious processes due to toxins, drugs, and such entities as asthma, allergic bronchopulmonary aspergillosis, and pulmonary vasculitis syndromes including Churg-Strauss and hypereosinophilic syndromes. 464 Acute and chronic forms of eosinophilic pneumonia, as previously emphasized, have a varied etiology that includes parasitic infections. 465 False-positive morphologic diagnosis of a parasitic infection may be based on the presence of objects resembling parasites, 391 , 466 such as lentils in aspiration pneumonia, pollen grains, or Liesegang rings. These ring-like structures can simulate various types of nematodes. 467 Careful attention to the microanatomy of an apparent foreign body and comparison with parasites illustrated in atlases can often resolve such diagnostic dilemmas. Some cases, however, may require referral to pathologists with specialized training and experience in parasitic diseases. Table 7.13 Parasitic Pneumonias: Summary of Pathologic Findings Table 7.13 Assessment Component Findings Toxoplasmosis Surgical pathology Miliary small necroinflammatory nodules with fibrin; fibrinous pneumonia Cytopathology Crescent-shaped tachyzoites, pseudocysts, and true cysts Microbiology Serologic diagnosis by IFA or EIA; identification of tachyzoites or pseudocyst in tissue Amebiasis Surgical pathology Lung abscess Cytopathology Trophozoite in necroinflammatory debris resembles histiocytes; confirm with immunohistochemistry Microbiology Identification of trophozoite characteristics; serologic methods positive in most cases of extraintestinal disease; DNA probes Cryptosporidiosis Surgical pathology Bronchitis and/or bronchiolitis with cryptosporidia seen on H&E sections as small, round protrusions along the epithelial surface of the mucosa Cytopathology Red oocysts in smears prepared from bronchial washes and BAL fluid stained with modified acid-fast stains Microbiology Findings on direct examination of specimens similar to those on cytologic examination; immunofluorescence and enzyme immunoassays developed for intestinal infection Microsporidiosis Surgical pathology Bronchitis and/or bronchiolitis; small basophilic dots in vacuoles may be visible in H&E-stained sections when burden of organism is heavy; highlighted with Gram and modified trichrome stains; toluidine blue stain on plastic sections; electron microscopy Cytopathology Characteristic pink capsule-shaped spores with dark band in modified trichrome-stained preparations of BAL fluid Giemsa, Gram, and chemofluorescence stains also useful Microbiology Findings on direct examination of fluids similar to those on cytologic examination; culture in research setting by special arrangement; molecular methods Dirofilariasis Surgical pathology Solitary pulmonary nodule with infarct pattern and worm fragments Cytopathology Intact or fragmented worm in necroinflammatory debris Microbiology Identification of characteristic roundworm in tissues; serologic studies not useful Strongyloides Infection Surgical pathology Eosinophilic pneumonia, abscess, Loeffler syndrome with filariform larvae Cytopathology Filariform larvae in sputum indicate hyperinfection Microbiology Primary diagnostic stage in stool is rhabitiform larvae; filariform larvae may be seen in sputum and lung tissue; eggs resemble hookworm eggs but are rarely seen Echinococcus Infection Surgical pathology Trilayered cyst with brood capsules containing protoscolices; fibrous wall forms interface with lung parenchyma; sometimes abscess and granulomas Cytopathology Protoscolices with sucker and hooklets or detached hooklets in granular background debris Microbiology Identification of hooklets and protoscolices in needle aspirates, pleural fluid, and sputum; serologic testing available Paragonimiasis Surgical pathology Eosinophilic pneumonia; fibrous pseudocysts containing worms and necroinflammatory debris; egg granulomas Cytopathology Yellow ovoid birefringent eggs with flattened operculum Microbiology Identification of characteristic egg in sputum or tissue; serologic testing available Schistosomiasis Surgical pathology Granulomatous endarteritis; eggs in epithelioid granulomas Cytopathology Characteristic nonbirefringent, nonoperculated eggs; presence and position of spine determines species Microbiology Embryonated eggs may be present in feces or urine; not sputum;serologic testing available BAL , Bronchoalveolar fluid; EIA , enzyme immunoassay; H&E , hematoxylin and eosin; IFA , immunofluorescence assay. Etiologic Agents Several parasite species migrate through the lungs as part of their normal life cycle, but few preferentially infect the human lung. 390 Most are aberrant pulmonary localizations in the human host, where they become lost in transit or are part of a secondary disseminated infection from another organ system, often in the setting of compromised immunity. The listing of etiologic agents in Box 7.24 is selective, based on the more common pathogens known to be associated with pulmonary involvement. Box 7.24 Some Common Parasitic Lung Pathogens Protozoa Toxoplasma gondii Entamoeba histolytica Cryptosporidia Microsporidia Metazoa (Helminths) Nematodes Dirofilaria immitis Strongyloides stercoralis Cestodes Echinococcus spp. Trematodes Paragonimus spp. Schistosoma spp. Alt-text: Box 7.24 Protozoa Toxoplasma gondii Entamoeba histolytica Cryptosporidia Microsporidia Metazoa (Helminths) Nematodes Dirofilaria immitis Strongyloides stercoralis Cestodes Echinococcus spp. Trematodes Paragonimus spp. Schistosoma spp. Histopathology When parasites in the form of adult worms, larvae, or eggs invade or become deposited in lung tissue, they usually provoke an intense inflammatory reaction with neutrophils, eosinophils, and various mononuclear cells. One or more of the patterns listed in Box 7.25 may be identified. When the predominant site of involvement is the bronchial mucosa, a bronchitis and bronchiolitis pattern is observed; when they become impacted in pulmonary arteries, a nodular angiocentric pattern is observed, although it may be overshadowed by thrombosis and infarction. Some parasites invade the alveolar parenchyma, resulting in a pattern of miliary small nodules or pneumonitis. Naturally none of these patterns are consistently present and combinations of patterns may be seen. In some cases, an acute Loeffler-like eosinophilic pneumonia may reflect an allergic reaction to the transient passage of larvae through the pulmonary vasculature. Box 7.25 Histopathologic Patterns in Parasitic Lung Injury Eosinophilic pneumonia Large nodule(s) Miliary small nodules Bronchitis and bronchiolitis Abscess, cavities, and cysts Intravascular reaction Alt-text: Box 7.25 The various patterns, although nondiagnostic, can be suggestive of a parasitic infection, particularly when they incorporate a heavy eosinophilic infiltrate or granulomatous component. Eosinophilic lung disease, with or without blood eosinophilia, has a diverse etiology but is particularly characteristic of parasitic infection, especially in the tropics. 385 In the United States, other infections, such as coccidioidomycosis, must be considered, in addition to the many noninfectious causes of pulmonary eosinophilia. The challenge for the pathologist is the identification of a parasite, distinguishing it from artifact or foreign body, and classifying it as precisely as possible based on its size and unique morphologic features. Once the presence of suggestive morphologic features has been confirmed, the patient's travel or avocation history can help to further narrow the scope of the differential diagnosis. Of interest, a common "parasite" encountered in clinical practice is not a parasite at all but aspirated vegetable material simulating the complex structure of an organism. 391 Toxoplasmosis T. gondii is an obligate intracellular protozoan and a common opportunist in patients with AIDS, the disease underlying most cases of toxoplasmosis seen in recent years. The brain and retina are most commonly involved in these patients, but pulmonary lesions may also be present in cases of disseminated disease. These often take the form of miliary small nodules with fibrinous exudates, which may progress to a confluent fibrinopurulent pneumonia. 392 Free forms (crescent-shaped tachyzoites) and cysts may be identified ( Fig. 7.112 ). Pseudocysts packed with tachyzoites can be distinguished from true cysts with bradyzoites by staining of the latter with PAS and GMS. 393 Serology is the main method of diagnosis in the acute phase, and serology with concomitant radiologic findings in appropriate settings in immunocompromised hosts usually obviates the need for direct demonstration of the organisms. Either PCR on the specimen or immunohistochemistry can be used to demonstrate the organisms. 394 Figure 7.112 Toxoplasmosis. (A) Tachyzoites. (B) Pseudocysts packed with tachyzoites. Figure 7.112 Amebiasis Amebic dysentery becomes invasive in a small percentage of patients. When the trophozoites leave the gut, they most commonly travel to the liver. From the liver, either by direct extension or rarely by hematogenous spread, the lungs may become involved. In this scenario, abscesses composed of liquefactive debris—with few neutrophils, distinguishable from bacterial abscess where neutrophils are dominant—may be seen, most often in the right lower lobe adjacent to the liver. 395 , 396 Trophozoites can be best seen at the margin of viable tissue ( Fig. 7.113 ). They resemble histiocytes but are usually larger, with a lower nucleocytoplasmic ratio. A tiny central karyosome within a round nucleus having vesicular chromatin is characteristic. 397 , 398 Bronchial fistula formation and empyema can occur as complications; amebas may be found in sputum and pleural fluid, respectively, in these situations. For free-living amebic species (those of the genera Acanthamoeba , Balamuthia , Naegleria ), the central nervous system is the principal focus of infection. However disseminated disease including lung infection ( Fig. 7.114 ) may occur in certain epidemiologic situations, especially those involving compromised immune status, or in lung transplants. 399 , 400 , 401 Figure 7.113 Amoebic trophozoite in lung tissue (arrows) . Note delicate marginal nuclear chromatin with small central karyosome and small red blood cell in cytoplasm. Figure 7.113 (Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Figure 7.114 Free-living ameba in lung tissue from an immunocompromised patient. (A) Necroinflammatory nodule. (B) Encysted form, black arrow and left upper inset ; trophozoite, white arrow and right lower inset . Figure 7.114 Cryptosporidiosis Ten species of the intracellular coccidian protozoa are currently recognized, but one of them, Cryptosporidium parvum , causes most human infections. 402 Clinically, infection due to this organism may have three major manifestations: asymptomatic shedding, acute watery diarrhea that lasts for approximately 2 weeks, and persistent diarrhea that lasts several weeks. Patients with AIDS have a wider spectrum of disease severity and duration that includes a fulminant cholera-like illness. 402 These patients are most likely to manifest extraintestinal disease. In the lung, the organism targets the epithelium of the airways, just as it does the surface epithelium of the gut and biliary tract. 403 In H&E sections, cryptosporidia appear as small (4 to 6 µm in diameter) round to oval protrusions from the cell surface. Electron microscopy reveals that they are intracellular but extracytoplasmic. In addition to H&E, they stain with Giemsa, PAS, GMS, and acid-fast stains. A mild to moderate chronic inflammatory cell infiltrate is usually present in the submucosa. Pulmonary cryptosporidiosis is largely a case report event, most reports being from earlier phases of the AIDS epidemic 404 —a surprise in reviewing acid-fast stains for more common organisms. 405 Newer reports suggest that respiratory cryptosporidiosis may occur in immunocompetent children with cryptosporidial diarrhea and cough. 406 , 407 Microsporidiosis The microsporidia are obligate intracellular spore-forming protozoa. More than 140 genera and 1200 species are recognized, but only 7 genera and a few species have been confirmed as human pathogens. 408 They are opportunists that have recently emerged in severely immunocompromised patients, AIDS patients, and transplant recipients, with case reports of pulmonary infections in the immunocompromised population. 409 , 410 , 411 Clinically they primarily cause chronic diarrhea and cholangitis. In the lung, they cause bronchitis or bronchiolitis (or both), usually in patients who also have intestinal infections or disease at other sites, especially the biliary tract. 412 The predominant pathologic changes are in the airways, which show a mixed inflammatory cell infiltrate of mononuclear and polymorphonuclear leukocytes. 413 The organisms are found within vacuoles in the apical portion of epithelial cells lining the airways. They appear as very small (1 to 1.5 µm in diameter) basophilic dots whose recognition depends on organism load. However, even when heavy, the findings can be subtle. Also, as with cryptosporidiosis, their presence is often overlooked or obscured by coexistent pneumonias. Special stains—such as modified trichrome, Warthin-Starry–type silver, and Gram stains—are more sensitive and specific, especially when used in combination. 414 Leishmaniasis Leishmaniasis ( Leishmania donovani infection) is transmitted to humans by several species of the Phlebotomus sand fly. 415 Pulmonary leishmaniasis has been reported in HIV-infected patients and transplant recipients. 385 , 416 , 417 The organisms ( L. donovani amastigotes) can be found in the alveoli and alveolar septa and may be recovered in BAL fluid from these patients. 418 They also can be found in bronchoscopic biopsies ( Fig. 7.115 ). Serologic testing for leishmaniasis has been suggested as part of the pretransplantation work-up in endemic areas. 419 A rapid PCR-amplified diagnostic method has been described. 420 Figure 7.115 Leishmania donovani in bronchoscopic biopsy specimens obtained from a North African immigrant to Sicily. (A) Lower-power view of cellular infiltrate. (B) High-power view of dot-like organisms. Figure 7.115 (Courtesy Dr. Francesca Guddo, Palermo, Italy.) Dirofilariasis The zoonosis caused by Dirofilaria immitis , a parasite of dogs and other mammals, is transmitted by mosquitos and black flies to humans. 421 , 422 , 423 Larvae injected by these insect vectors migrate from the subcutis into veins and travel to the heart, where they die before maturing into adult worms. They are then washed into the lungs by the pulmonary arterial blood flow, where they form the nidus of a thrombus. Formation of an infarct follows, typically manifesting as an asymptomatic solitary pulmonary nodule ("coin lesion") in the lung periphery ( Fig. 7.116 ) that may be visualized on a positron emission tomography (PET) scan. 424 , 425 , 426 Microscopically the nodule resembles a typical infarct with a core of coagulation necrosis but also containing degenerated worm fragments in the remnant of an arteriole ( Figs. 7.117 and 7.118 ). A peripheral investment of chronic granulation tissue forms an interface with the alveolated parenchyma. "Step" sections and trichrome stains may be needed when H&E sections do not show the parasite. 427 Figure 7.116 Dirofilarial nodule, gross specimen. Figure 7.116 Figure 7.117 Dirofilarial nodule, with worm remnants in organizing thrombosed vessel. Figure 7.117 Figure 7.118 Dirofilariasis. (A) Intact worm cross section ×260. (B) Showing body cavity layers ×360. Surrounding necrosis in both figures. Figure 7.118 (From Abhisek, B, Reilly P, Perez A, et al. Human pulmonary dirofilariasis presenting as a solitary pulmonary nodule: a case report and brief review of literature. Resp Med Case Rep . 2013;10:40–42.) Strongyloidiasis Strongyloides is a parasite most often found in patients or travelers in the tropics, but endemic foci are present in the southeastern United States. Rhabditiform larvae of the nematode Strongyloides stercoralis , after hatching from ingested eggs, 428 invade the small intestinal mucosa. At this site occult infection may remain asymptomatic for years. Dissemination typically follows debilitation brought on by immunocompromising diseases and therapies. When this occurs, filariform larvae leave the gut and travel through the pulmonary vasculature. When they penetrate alveoli ( Fig. 7.119 ), they provoke hemorrhage and inflammation. 429 , 430 , 431 Loeffler syndrome, eosinophilic pneumonia, and abscesses may develop. When migration is interrupted, filariform larvae may metamorphose in situ to adult worms, which can produce eggs and rhabditiform larvae. Larvae identified in the sputum indicate hyperinfection. 432 Disseminated strongyloidiasis is but one example of an infection that may become manifest, particularly in immunocompromised patients, years after emigration from or travel to an endemic area harboring pathogens that are considered unusual or exotic by pathologists in the United States. Figure 7.119 Filariform larva of Strongyloides stercoralis penetrating into alveolar space with associated inflammation. Figure 7.119 Echinococcosis Echinococcosis is a zoonosis that occurs wherever sheep, dogs or other canids, and humans live in close contact. Ingested eggs of the tapeworm Echinococcus hatch in the gut, releasing oncospheres, which then invade the mucosa, enter the circulation, and travel to various sites, where they develop into hydatid cysts. 433 , 434 In the lung, unilocular slow-growing cysts are produced by Echinococcus granulosus . 435 Echinococcus multilocularis proliferates by budding, producing an alveolar pattern of microvesicles. 398 The cyst of E. granulosus has a trilayered membrane ( Fig. 7.120A ) with an outer fibrous, middle-laminated hyaline, and inner germinal layer that gives rise to brood capsules containing infective protoscolices with hooklets and suckers ( Fig. 7.120B ). The layers usually become separated in tissue, with the outer fibrous layer containing chronic inflammatory cells that form an interface with the alveolated parenchyma. Cysts that rupture into bronchi may be expectorated as debris with protoscolices or portions of the cyst wall. Abscesses and granulomas may also form in the lung, pleura, and chest wall. 436 Figure 7.120 Echinococcus granulosus. (A) Cyst with trilayered membrane. (B) Brood capsules. Figure 7.120 Paragonimiasis The parasite Paragonimus targets the lung and is acquired by the ingestion of freshwater crabs or crayfish infected with the metacercarial larvae of Paragonimus species. 437 Most cases worldwide are due to P. westermani , but several other species exist in Asia, Africa, and South and Latin America. In the United States, infections due to P. kellicotti have been reported. 390 The disease manifestations are related to the migratory route and the inflammatory response these hermaphroditic flukes stimulate as they enter lung parenchyma and travel to sites near larger bronchioles or bronchi. Typically an area of eosinophil-rich inflammatory reaction surrounds them, and this reactive process may evolve to form a fibrous pseudocyst or capsule containing worms, exudate, and debris ( Fig. 7.121A ). Cysts rupturing into bronchioles may result in eggs, blood, and inflammatory cells being coughed up in the sputum. Alternatively, eggs may become embedded in parenchyma, producing nodular granulomatous lesions ( Fig. 7.121B ) that progress to scars. 438 The eggs are yellowish, ovoid, and operculated, measuring 75 to 110 µm by 45 to 60 µm. The opercula unfortunately are not easily seen in tissue; however, the eggs are birefringent under polarized light, which helps to distinguish them from nonbirefringent schistosome eggs ( Fig. 7.122 ). 390 Figure 7.121 (A) Paragonimus westermani with yellowish refractile eggs in eosinophil-rich exudates. (B) Distorted egg of Paragonimus kellicotti in granuloma. Figure 7.121 Figure 7.122 Paragonimiasis. (A) Granulomatous reaction to egg. (B) Single egg in polarized light. (C) Chronic eosinophilic pneumonia with many eggs. (D) Giant cell reaction; pigment in eggs. Figure 7.122 (Courtesy A.E. McCullough, MD.) Schistosomiasis The public health burden of schistosomiasis is enormous. This parasitic infection affects 200 million people in 74 countries while continuing to expand its geographical range. 439 , 440 The life cycle and disease manifestations of the three major Schistosoma species— Schistosoma mansoni , Schistosoma haematobium , and Schistosoma japonicum —involve eggs, snail intermediate hosts, and free-swimming cercaria, which penetrate the skin of susceptible animals and people and develop into adult worms. The male and female worms eventually come to reside in various human venous plexuses, depending on the species, where egg deposition occurs. Pulmonary schistosomiasis comprises both acute and chronic forms. The acute disease, referred to as Katayama syndrome , manifests with fever, chills, weight loss, gastrointestinal symptoms, myalgia, and urticaria in patients with no previous exposure to the parasite. Acute larval pneumonitis and a Loeffler-like eosinophilic pneumonia may be seen in this setting. 439 , 441 Chronic pulmonary disease is almost always secondary to severe hepatic involvement with portal hypertension. In this setting, the eggs of S. mansoni , and rarely S. japonicum or S. haematobium , may be shunted through portosystemic collateral veins to the lungs. The eggs lodge in arterioles, provoking a characteristic granulomatous endarteritis with pulmonary symptoms and radiologic infiltrates. 442 , 443 When the endarteritis is accompanied by angiomatoid changes, the lesion is considered pathognomonic for pulmonary schistosomiasis. 390 Eggs typically are surrounded by epithelioid cells and collagen ( Fig. 7.123 ). Most schistosome eggs do not exhibit birefringence and are larger than Paragonimus eggs, with which they share a superficial resemblance. Adult schistosomes may rarely be found in pulmonary blood vessels. Worldwide, given the burden of disease in Africa and Asia, chronic disease is associated for unclear reasons with pulmonary hypertension. 444 Figure 7.123 (A) Schistosome eggs in lung parenchyma. (B) Eggs of Schistosoma japonicum. Figure 7.123 (A and B, Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Visceral Larva Migrans The common parasites that cause visceral larva migrans are the dog tapeworm, Toxocara canis , and the less common cat tapeworm, Toxocara cati . When embryonated eggs are ingested by an intermediate host, typically a child with a history of pica, they hatch into infective larvae in the intestine. Subsequently, the larvae penetrate the intestinal wall, gain access to the circulation, and are carried to many organs, including the lungs. This is the end point, for their growth is arrested by a granulomatous reaction and they never mature into adult worms. The granulomatous reaction usually has a conspicuous eosinophilic component, and larvae may be seen. 445 Toxoplasmosis T. gondii is an obligate intracellular protozoan and a common opportunist in patients with AIDS, the disease underlying most cases of toxoplasmosis seen in recent years. The brain and retina are most commonly involved in these patients, but pulmonary lesions may also be present in cases of disseminated disease. These often take the form of miliary small nodules with fibrinous exudates, which may progress to a confluent fibrinopurulent pneumonia. 392 Free forms (crescent-shaped tachyzoites) and cysts may be identified ( Fig. 7.112 ). Pseudocysts packed with tachyzoites can be distinguished from true cysts with bradyzoites by staining of the latter with PAS and GMS. 393 Serology is the main method of diagnosis in the acute phase, and serology with concomitant radiologic findings in appropriate settings in immunocompromised hosts usually obviates the need for direct demonstration of the organisms. Either PCR on the specimen or immunohistochemistry can be used to demonstrate the organisms. 394 Figure 7.112 Toxoplasmosis. (A) Tachyzoites. (B) Pseudocysts packed with tachyzoites. Figure 7.112 Amebiasis Amebic dysentery becomes invasive in a small percentage of patients. When the trophozoites leave the gut, they most commonly travel to the liver. From the liver, either by direct extension or rarely by hematogenous spread, the lungs may become involved. In this scenario, abscesses composed of liquefactive debris—with few neutrophils, distinguishable from bacterial abscess where neutrophils are dominant—may be seen, most often in the right lower lobe adjacent to the liver. 395 , 396 Trophozoites can be best seen at the margin of viable tissue ( Fig. 7.113 ). They resemble histiocytes but are usually larger, with a lower nucleocytoplasmic ratio. A tiny central karyosome within a round nucleus having vesicular chromatin is characteristic. 397 , 398 Bronchial fistula formation and empyema can occur as complications; amebas may be found in sputum and pleural fluid, respectively, in these situations. For free-living amebic species (those of the genera Acanthamoeba , Balamuthia , Naegleria ), the central nervous system is the principal focus of infection. However disseminated disease including lung infection ( Fig. 7.114 ) may occur in certain epidemiologic situations, especially those involving compromised immune status, or in lung transplants. 399 , 400 , 401 Figure 7.113 Amoebic trophozoite in lung tissue (arrows) . Note delicate marginal nuclear chromatin with small central karyosome and small red blood cell in cytoplasm. Figure 7.113 (Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Figure 7.114 Free-living ameba in lung tissue from an immunocompromised patient. (A) Necroinflammatory nodule. (B) Encysted form, black arrow and left upper inset ; trophozoite, white arrow and right lower inset . Figure 7.114 Cryptosporidiosis Ten species of the intracellular coccidian protozoa are currently recognized, but one of them, Cryptosporidium parvum , causes most human infections. 402 Clinically, infection due to this organism may have three major manifestations: asymptomatic shedding, acute watery diarrhea that lasts for approximately 2 weeks, and persistent diarrhea that lasts several weeks. Patients with AIDS have a wider spectrum of disease severity and duration that includes a fulminant cholera-like illness. 402 These patients are most likely to manifest extraintestinal disease. In the lung, the organism targets the epithelium of the airways, just as it does the surface epithelium of the gut and biliary tract. 403 In H&E sections, cryptosporidia appear as small (4 to 6 µm in diameter) round to oval protrusions from the cell surface. Electron microscopy reveals that they are intracellular but extracytoplasmic. In addition to H&E, they stain with Giemsa, PAS, GMS, and acid-fast stains. A mild to moderate chronic inflammatory cell infiltrate is usually present in the submucosa. Pulmonary cryptosporidiosis is largely a case report event, most reports being from earlier phases of the AIDS epidemic 404 —a surprise in reviewing acid-fast stains for more common organisms. 405 Newer reports suggest that respiratory cryptosporidiosis may occur in immunocompetent children with cryptosporidial diarrhea and cough. 406 , 407 Microsporidiosis The microsporidia are obligate intracellular spore-forming protozoa. More than 140 genera and 1200 species are recognized, but only 7 genera and a few species have been confirmed as human pathogens. 408 They are opportunists that have recently emerged in severely immunocompromised patients, AIDS patients, and transplant recipients, with case reports of pulmonary infections in the immunocompromised population. 409 , 410 , 411 Clinically they primarily cause chronic diarrhea and cholangitis. In the lung, they cause bronchitis or bronchiolitis (or both), usually in patients who also have intestinal infections or disease at other sites, especially the biliary tract. 412 The predominant pathologic changes are in the airways, which show a mixed inflammatory cell infiltrate of mononuclear and polymorphonuclear leukocytes. 413 The organisms are found within vacuoles in the apical portion of epithelial cells lining the airways. They appear as very small (1 to 1.5 µm in diameter) basophilic dots whose recognition depends on organism load. However, even when heavy, the findings can be subtle. Also, as with cryptosporidiosis, their presence is often overlooked or obscured by coexistent pneumonias. Special stains—such as modified trichrome, Warthin-Starry–type silver, and Gram stains—are more sensitive and specific, especially when used in combination. 414 Leishmaniasis Leishmaniasis ( Leishmania donovani infection) is transmitted to humans by several species of the Phlebotomus sand fly. 415 Pulmonary leishmaniasis has been reported in HIV-infected patients and transplant recipients. 385 , 416 , 417 The organisms ( L. donovani amastigotes) can be found in the alveoli and alveolar septa and may be recovered in BAL fluid from these patients. 418 They also can be found in bronchoscopic biopsies ( Fig. 7.115 ). Serologic testing for leishmaniasis has been suggested as part of the pretransplantation work-up in endemic areas. 419 A rapid PCR-amplified diagnostic method has been described. 420 Figure 7.115 Leishmania donovani in bronchoscopic biopsy specimens obtained from a North African immigrant to Sicily. (A) Lower-power view of cellular infiltrate. (B) High-power view of dot-like organisms. Figure 7.115 (Courtesy Dr. Francesca Guddo, Palermo, Italy.) Dirofilariasis The zoonosis caused by Dirofilaria immitis , a parasite of dogs and other mammals, is transmitted by mosquitos and black flies to humans. 421 , 422 , 423 Larvae injected by these insect vectors migrate from the subcutis into veins and travel to the heart, where they die before maturing into adult worms. They are then washed into the lungs by the pulmonary arterial blood flow, where they form the nidus of a thrombus. Formation of an infarct follows, typically manifesting as an asymptomatic solitary pulmonary nodule ("coin lesion") in the lung periphery ( Fig. 7.116 ) that may be visualized on a positron emission tomography (PET) scan. 424 , 425 , 426 Microscopically the nodule resembles a typical infarct with a core of coagulation necrosis but also containing degenerated worm fragments in the remnant of an arteriole ( Figs. 7.117 and 7.118 ). A peripheral investment of chronic granulation tissue forms an interface with the alveolated parenchyma. "Step" sections and trichrome stains may be needed when H&E sections do not show the parasite. 427 Figure 7.116 Dirofilarial nodule, gross specimen. Figure 7.116 Figure 7.117 Dirofilarial nodule, with worm remnants in organizing thrombosed vessel. Figure 7.117 Figure 7.118 Dirofilariasis. (A) Intact worm cross section ×260. (B) Showing body cavity layers ×360. Surrounding necrosis in both figures. Figure 7.118 (From Abhisek, B, Reilly P, Perez A, et al. Human pulmonary dirofilariasis presenting as a solitary pulmonary nodule: a case report and brief review of literature. Resp Med Case Rep . 2013;10:40–42.) Strongyloidiasis Strongyloides is a parasite most often found in patients or travelers in the tropics, but endemic foci are present in the southeastern United States. Rhabditiform larvae of the nematode Strongyloides stercoralis , after hatching from ingested eggs, 428 invade the small intestinal mucosa. At this site occult infection may remain asymptomatic for years. Dissemination typically follows debilitation brought on by immunocompromising diseases and therapies. When this occurs, filariform larvae leave the gut and travel through the pulmonary vasculature. When they penetrate alveoli ( Fig. 7.119 ), they provoke hemorrhage and inflammation. 429 , 430 , 431 Loeffler syndrome, eosinophilic pneumonia, and abscesses may develop. When migration is interrupted, filariform larvae may metamorphose in situ to adult worms, which can produce eggs and rhabditiform larvae. Larvae identified in the sputum indicate hyperinfection. 432 Disseminated strongyloidiasis is but one example of an infection that may become manifest, particularly in immunocompromised patients, years after emigration from or travel to an endemic area harboring pathogens that are considered unusual or exotic by pathologists in the United States. Figure 7.119 Filariform larva of Strongyloides stercoralis penetrating into alveolar space with associated inflammation. Figure 7.119 Echinococcosis Echinococcosis is a zoonosis that occurs wherever sheep, dogs or other canids, and humans live in close contact. Ingested eggs of the tapeworm Echinococcus hatch in the gut, releasing oncospheres, which then invade the mucosa, enter the circulation, and travel to various sites, where they develop into hydatid cysts. 433 , 434 In the lung, unilocular slow-growing cysts are produced by Echinococcus granulosus . 435 Echinococcus multilocularis proliferates by budding, producing an alveolar pattern of microvesicles. 398 The cyst of E. granulosus has a trilayered membrane ( Fig. 7.120A ) with an outer fibrous, middle-laminated hyaline, and inner germinal layer that gives rise to brood capsules containing infective protoscolices with hooklets and suckers ( Fig. 7.120B ). The layers usually become separated in tissue, with the outer fibrous layer containing chronic inflammatory cells that form an interface with the alveolated parenchyma. Cysts that rupture into bronchi may be expectorated as debris with protoscolices or portions of the cyst wall. Abscesses and granulomas may also form in the lung, pleura, and chest wall. 436 Figure 7.120 Echinococcus granulosus. (A) Cyst with trilayered membrane. (B) Brood capsules. Figure 7.120 Paragonimiasis The parasite Paragonimus targets the lung and is acquired by the ingestion of freshwater crabs or crayfish infected with the metacercarial larvae of Paragonimus species. 437 Most cases worldwide are due to P. westermani , but several other species exist in Asia, Africa, and South and Latin America. In the United States, infections due to P. kellicotti have been reported. 390 The disease manifestations are related to the migratory route and the inflammatory response these hermaphroditic flukes stimulate as they enter lung parenchyma and travel to sites near larger bronchioles or bronchi. Typically an area of eosinophil-rich inflammatory reaction surrounds them, and this reactive process may evolve to form a fibrous pseudocyst or capsule containing worms, exudate, and debris ( Fig. 7.121A ). Cysts rupturing into bronchioles may result in eggs, blood, and inflammatory cells being coughed up in the sputum. Alternatively, eggs may become embedded in parenchyma, producing nodular granulomatous lesions ( Fig. 7.121B ) that progress to scars. 438 The eggs are yellowish, ovoid, and operculated, measuring 75 to 110 µm by 45 to 60 µm. The opercula unfortunately are not easily seen in tissue; however, the eggs are birefringent under polarized light, which helps to distinguish them from nonbirefringent schistosome eggs ( Fig. 7.122 ). 390 Figure 7.121 (A) Paragonimus westermani with yellowish refractile eggs in eosinophil-rich exudates. (B) Distorted egg of Paragonimus kellicotti in granuloma. Figure 7.121 Figure 7.122 Paragonimiasis. (A) Granulomatous reaction to egg. (B) Single egg in polarized light. (C) Chronic eosinophilic pneumonia with many eggs. (D) Giant cell reaction; pigment in eggs. Figure 7.122 (Courtesy A.E. McCullough, MD.) Schistosomiasis The public health burden of schistosomiasis is enormous. This parasitic infection affects 200 million people in 74 countries while continuing to expand its geographical range. 439 , 440 The life cycle and disease manifestations of the three major Schistosoma species— Schistosoma mansoni , Schistosoma haematobium , and Schistosoma japonicum —involve eggs, snail intermediate hosts, and free-swimming cercaria, which penetrate the skin of susceptible animals and people and develop into adult worms. The male and female worms eventually come to reside in various human venous plexuses, depending on the species, where egg deposition occurs. Pulmonary schistosomiasis comprises both acute and chronic forms. The acute disease, referred to as Katayama syndrome , manifests with fever, chills, weight loss, gastrointestinal symptoms, myalgia, and urticaria in patients with no previous exposure to the parasite. Acute larval pneumonitis and a Loeffler-like eosinophilic pneumonia may be seen in this setting. 439 , 441 Chronic pulmonary disease is almost always secondary to severe hepatic involvement with portal hypertension. In this setting, the eggs of S. mansoni , and rarely S. japonicum or S. haematobium , may be shunted through portosystemic collateral veins to the lungs. The eggs lodge in arterioles, provoking a characteristic granulomatous endarteritis with pulmonary symptoms and radiologic infiltrates. 442 , 443 When the endarteritis is accompanied by angiomatoid changes, the lesion is considered pathognomonic for pulmonary schistosomiasis. 390 Eggs typically are surrounded by epithelioid cells and collagen ( Fig. 7.123 ). Most schistosome eggs do not exhibit birefringence and are larger than Paragonimus eggs, with which they share a superficial resemblance. Adult schistosomes may rarely be found in pulmonary blood vessels. Worldwide, given the burden of disease in Africa and Asia, chronic disease is associated for unclear reasons with pulmonary hypertension. 444 Figure 7.123 (A) Schistosome eggs in lung parenchyma. (B) Eggs of Schistosoma japonicum. Figure 7.123 (A and B, Courtesy Ronald Neafi, Armed Forces Institute of Pathology, Washington, DC.) Visceral Larva Migrans The common parasites that cause visceral larva migrans are the dog tapeworm, Toxocara canis , and the less common cat tapeworm, Toxocara cati . When embryonated eggs are ingested by an intermediate host, typically a child with a history of pica, they hatch into infective larvae in the intestine. Subsequently, the larvae penetrate the intestinal wall, gain access to the circulation, and are carried to many organs, including the lungs. This is the end point, for their growth is arrested by a granulomatous reaction and they never mature into adult worms. The granulomatous reaction usually has a conspicuous eosinophilic component, and larvae may be seen. 445 Cytopathology The cytologic literature contains many reports of the successful identification of parasites in pulmonary specimens recovered by exfoliative (sputum, bronchial washing or brushing, BAL fluid, pleural fluid) and needle aspiration techniques. Some of these are listed in Box 7.26 . 418 , 424 , 425 , 426 , 436 , 446 , 447 , 448 , 449 , 450 , 451 , 452 , 453 , 454 , 455 , 456 , 457 Commonly cited in textbooks and reviews is the finding of Strongyloides stercoralis larvae in expectorated sputum or bronchial washings of patients with hyperinfections ( Fig. 7.124 ). Also common are reports of Echinococcus protoscolices and hooklets in needle aspirates from patients with pleuropulmonary disease. 45 , 46 Use of large-bore and cutting needle biopsies has traditionally been contraindicated in the setting of suspected Echinococcus infections; reports of success with fine-needle aspiration without untoward reactions suggest that this technique is a relatively safe procedure in which the benefits outweigh the risks. 448 Box 7.26 Parasites Reported in Respiratory Cytology Specimens Toxoplasma Amebae Trichomonas Cryptosporidia Microsporidia Leishmania Paragonimus Echinococcus Strongyloides Schistosoma Dirofilaria Microfilariae Alt-text: Box 7.26 Figure 7.124 Strongyloides stercoralis larvae in bronchial washing. (A) Larval fragments in cell block. (B) ThinPrep smear. Figure 7.124 Cytologic analysis is a sensitive and often preferred method to diagnose cryptosporidiosis, microsporidiosis, and other respiratory tract infections in the immunocompromised patient because it has the advantage of being less invasive. Specimens such as bronchial washings and BAL fluids can be prepared by high-speed centrifugation followed by standard smear preparation, cytocentrifugation, or ThinPrep technology. A battery of special stains—including Gram, modified trichrome, Giemsa, GMS, acid-fast, chemofluorescent, and immunofluorescent, depending on reagent availability—can then be applied to detect cryptosporidial oocysts, microsporidial spores, or other etiologic agents. The morphologic features of many of the aforementioned organisms are usually better defined in cytologic preparations than in tissue biopsy specimens provided that obscuring background debris is limited and that the cytopreparation technique and staining have been well performed. Pseudoparasites such as vegetable matter, textile fibers, pollens, red cell "ghosts," and other extraneous material must be recognized and excluded. Thus, as for all of the various categories of microorganisms cited in this chapter, cytopathologic examination adds synergy to surgical pathologic and microbiologic methods. Microbiology The laboratory diagnosis of parasitic disease depends on the collection of appropriate specimens, which, in turn, requires appropriate clinical evaluation. For example, just as stool examination is the most efficient means of diagnosing most intestinal protozoa and helminths, respiratory specimens (e.g., sputum samples, bronchial washings, BAL fluid samples, touch imprints of lung biopsy tissue) can provide a specific etiologic diagnosis when pulmonary infections are suspected. 458 As in the case of cytologic samples, these specimens often reveal the characteristic microanatomic features of parasite larvae and eggs that usually cannot be readily seen when they are embedded in tissue. Moreover, the identification of organisms in respiratory specimens is diagnostic of pulmonary infection, whereas the presence of the organism in the feces of a patient suspected to have pulmonary disease provides only presumptive evidence. Serodiagnosis with immunologic and molecular methods can be useful when parasites are located deep within tissue, such as the lung, and not easily accessible to biopsy or cytologic sampling. 396 The effectiveness of serodiagnosis of parasitic diseases has been hampered by tests with low sensitivity and specificity, mainly as a result of the complex composition of parasitic antigens and the occurrence of frequent cross reactions. 458 In recent years, however, significant refinements in antigenic preparations and improvements in technology have resulted in assays with greater predictive value. The newer tests are based on enzyme immunoassay and immunoblot methodology. Many test kits are commercially available, and diagnostic services are available from the CDC and other reference laboratories. 459 With protozoal infections, serologic testing is especially useful for the diagnosis of toxoplasmosis. Several commercial kits are available for detection of IgG and IgM antibodies; however, false-negative results are possible in immunocompromised patients, and positive results must be interpreted with caution, especially when the index of clinical suspicion is low. 460 Real-time PCR analysis has been used for the diagnosis of toxoplasmosis in the immunocompromised patient. 461 , 462 Antibody determinations also have value in cases of pulmonary and other tissue-invasive forms of amebiasis as compared with antigen detection methods, which are more useful for noninvasive amebic intestinal diseases. However, the best diagnostic approach to invasive disease may be the use of serologic testing, antigen detection, and PCR methods in various combinations. 463 For the identification of cryptosporidia, the new immunofluorescence tests and enzyme immunoassays that have been developed for intestinal infections may have application in respiratory infections. Similar tests are not available for the microsporidia, and diagnosis of infection with these organisms continues to rely on direct staining techniques. For the helminths, serodiagnosis is possible for Echinococcus , Paragonimus , Strongyloides , and Schistosoma species using enzyme immunoassay methods, which have fair sensitivity and specificity. 385 , 459 The available tests for Dirofilaria suffer from poor sensitivity and specificity and are not clinically useful at this time. Differential Diagnosis The key morphologic and microbiologic features of selected parasitic lung infections are summarized in Table 7.13 . In the absence of eggs, larvae, worms, or trophozoites, the various inflammatory patterns must be distinguished from those of other infections and various noninfectious processes due to toxins, drugs, and such entities as asthma, allergic bronchopulmonary aspergillosis, and pulmonary vasculitis syndromes including Churg-Strauss and hypereosinophilic syndromes. 464 Acute and chronic forms of eosinophilic pneumonia, as previously emphasized, have a varied etiology that includes parasitic infections. 465 False-positive morphologic diagnosis of a parasitic infection may be based on the presence of objects resembling parasites, 391 , 466 such as lentils in aspiration pneumonia, pollen grains, or Liesegang rings. These ring-like structures can simulate various types of nematodes. 467 Careful attention to the microanatomy of an apparent foreign body and comparison with parasites illustrated in atlases can often resolve such diagnostic dilemmas. Some cases, however, may require referral to pathologists with specialized training and experience in parasitic diseases. Table 7.13 Parasitic Pneumonias: Summary of Pathologic Findings Table 7.13 Assessment Component Findings Toxoplasmosis Surgical pathology Miliary small necroinflammatory nodules with fibrin; fibrinous pneumonia Cytopathology Crescent-shaped tachyzoites, pseudocysts, and true cysts Microbiology Serologic diagnosis by IFA or EIA; identification of tachyzoites or pseudocyst in tissue Amebiasis Surgical pathology Lung abscess Cytopathology Trophozoite in necroinflammatory debris resembles histiocytes; confirm with immunohistochemistry Microbiology Identification of trophozoite characteristics; serologic methods positive in most cases of extraintestinal disease; DNA probes Cryptosporidiosis Surgical pathology Bronchitis and/or bronchiolitis with cryptosporidia seen on H&E sections as small, round protrusions along the epithelial surface of the mucosa Cytopathology Red oocysts in smears prepared from bronchial washes and BAL fluid stained with modified acid-fast stains Microbiology Findings on direct examination of specimens similar to those on cytologic examination; immunofluorescence and enzyme immunoassays developed for intestinal infection Microsporidiosis Surgical pathology Bronchitis and/or bronchiolitis; small basophilic dots in vacuoles may be visible in H&E-stained sections when burden of organism is heavy; highlighted with Gram and modified trichrome stains; toluidine blue stain on plastic sections; electron microscopy Cytopathology Characteristic pink capsule-shaped spores with dark band in modified trichrome-stained preparations of BAL fluid Giemsa, Gram, and chemofluorescence stains also useful Microbiology Findings on direct examination of fluids similar to those on cytologic examination; culture in research setting by special arrangement; molecular methods Dirofilariasis Surgical pathology Solitary pulmonary nodule with infarct pattern and worm fragments Cytopathology Intact or fragmented worm in necroinflammatory debris Microbiology Identification of characteristic roundworm in tissues; serologic studies not useful Strongyloides Infection Surgical pathology Eosinophilic pneumonia, abscess, Loeffler syndrome with filariform larvae Cytopathology Filariform larvae in sputum indicate hyperinfection Microbiology Primary diagnostic stage in stool is rhabitiform larvae; filariform larvae may be seen in sputum and lung tissue; eggs resemble hookworm eggs but are rarely seen Echinococcus Infection Surgical pathology Trilayered cyst with brood capsules containing protoscolices; fibrous wall forms interface with lung parenchyma; sometimes abscess and granulomas Cytopathology Protoscolices with sucker and hooklets or detached hooklets in granular background debris Microbiology Identification of hooklets and protoscolices in needle aspirates, pleural fluid, and sputum; serologic testing available Paragonimiasis Surgical pathology Eosinophilic pneumonia; fibrous pseudocysts containing worms and necroinflammatory debris; egg granulomas Cytopathology Yellow ovoid birefringent eggs with flattened operculum Microbiology Identification of characteristic egg in sputum or tissue; serologic testing available Schistosomiasis Surgical pathology Granulomatous endarteritis; eggs in epithelioid granulomas Cytopathology Characteristic nonbirefringent, nonoperculated eggs; presence and position of spine determines species Microbiology Embryonated eggs may be present in feces or urine; not sputum;serologic testing available BAL , Bronchoalveolar fluid; EIA , enzyme immunoassay; H&E , hematoxylin and eosin; IFA , immunofluorescence assay. 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A peripheral lung nodule/granuloma and calcified hilar lymph node B. A feature of postprimary/reactivation tuberculosis C. A feature of primary tuberculosis D. a and b only E. a and c only ANSWER: E 5. Regarding the nontuberculous mycobacteria, all of the following are correct EXCEPT: A. They produce histopathologic lesions similar to Mycobacterium tuberculosis . B. They are not acquired person to person. C. They can produce histiocytic infiltrates and spindle cell lesions. D. They can cause disseminated disease in the immunocompromised. E. Mycobacterium bovis and Mycobacterium africanum are two of more than 100 species of nontuberculous mycobacteria. ANSWER: E 6. Mycobacterium abscessus is: A. Part of the Mycobacterium tuberculosis complex B. The leading rapid-growing mycobacterium recovered from the lung C. The leading slow-growing mycobacterium recovered from the lung D. The etiologic agent most associated with middle lobe syndrome E. c and d only ANSWER: B 7. All of the following statements regarding coccidioidomycosis are correct EXCEPT: A. It is caused by inhalation of arthrospores in alkaline soil of the Sonoran life zone. B. It is caused by a biphasic fungus that forms yeasts in tissue and hyphae only in laboratory media. C. Serology offers a sensitive method for laboratory diagnosis. D. It can present as community-acquired pneumonia. E. It can be associated with blood eosinophilia and eosinophilic pneumonia. ANSWER: B 8. Clinical forms of histoplasmosis include: A. Asymptomatic infection B. Solitary pulmonary nodule C. Cavitary granuloma D. Fibrosing mediastinitis E. All of the above ANSWER: E 9. Aspergillus fungal microscopic look-alikes in tissue include: A. Most Zygomycetes species B. Most Fusarium species C. Bipolaris spicifera D. All of the above E. a and b only ANSWER: D 10. Yellowish, oval, birefringent eggs in an eosinophil-rich exudate are characteristic of which of the following parasites? A. Paragonimus B. Schistosomes C. Strongyloides D. Ascaris E. Echinococcus ANSWER: A 11. Which of the following statements concerning nontuberculous mycobacterial infection is/are TRUE? A. It follows the same sequence of primary and postprimary disease as Mycobacterium tuberculosis. B. It manifests three distinct clinicopathologic entities. C. It is treated aggressively, often with the addition of cytotoxic agents. D. Disseminated disease mainly affects human immunodeficiency virus (HIV)–infected individuals. E. All of the above. ANSWER: D 12. True or false: Blastomycosis is endemic to the Pacific Northwest region of the United States. A. True B. False ANSWER: B 13. True or false: Histoplasmosis is the most common pulmonary fungal infection worldwide. A. True B. False ANSWER: A 14. True or false: In children under the age of 1 year, respiratory syncytial virus occurs more frequently than influenza or parainfluenza viral infection. A. True B. False ANSWER: A 15. What is this? Unlabelled image A. Giemsa stain of Candida pneumonia B. Trichrome stain of Aspergillus infection C. Von Kossa stain of malakoplakia D. Periodic acid–Schiff stain of Pneumocystis E. None of the above ANSWER: E 16. What is this? Unlabelled image A. Osteomyelitis with bacterial stain B. Mycobacterial granuloma with rhodamine-auramine C. Malakoplakia with silver impregnation technique D. Fibrinoid eosinophilia with Strongyloides worms under fluorescence E. None of the above ANSWER: B 17. What is this? Unlabelled image A. Pneumocystis B. Hantavirus C. Blastomyces D. Coccidioides E. None of the above ANSWER: D 18. What are these brown structures? Unlabelled image A. So-called brown bodies of blastomycosis B. Hamazaki-Wesenberg bodies C. Fungal yeast forms of Candida D. Sideroplanum spores E. None of the above ANSWER: B 19. What is this? Unlabelled image A. Aspirated vegetable material B. Migratory parasite C. Sulfur granule of Actinomyces D. Eosinophilic pneumonia body E. None of the above ANSWER: C 20. What are these? Unlabelled image A. Aspirated vegetable material B. Migratory parasites C. Mucor hyphae D. Aspergillus hyphae E. None of the above ANSWER: D 21. Which statement regarding stains for bacterial pneumonias is false? A. The agent of anthrax pneumonia is a large gram-positive rod that may be seen in alveolar septal vessels. B. It is difficult to demonstrate the organism of tularemia with a stain. C. Nocardia species are partially acid-fast. D. Legionella species can be seen with silver-based stains. E. Rhodococcus is an animal pathogen that rarely cause pneumonia in humans; it is best demonstrated in a GMS stain. ANSWER: E 22. With respect to Aspergillus species, which is a true statement? A. Aspergillus species are nonpigmented. B. Aspergillus hyphae branch at a right angles. C. Aspergillus species are positive in a Gram stain. D. Galactomannan antigen can be used as an adjunct to the diagnosis of Aspergillus in a BAL specimen. E. Aspergillus hyphae always taper to a thin point. ANSWER: D 23. You identified yeast cells in a lung biopsy. The yeast has a thick cell wall and in one section there is a yeast cell with wide-based budding. Which statement is most likely to be true about this patient? A. The patient is from Mississippi. B. He has a diagnostic serology test. C. The organism will be recoverable from a culture in about a week. D. He has a cavitary lung lesion on his chest x-ray. E. The biopsy shows compact granulomas following alveolar septae. ANSWER: A 24. Your patient has had a slightly enlarging nodule in the base of the right lower lobe for a year. He says he was sick after an adventure vacation to Arizona last year, where he went spelunking. Serologies for coccidioidomycosis are negative. A wedge biopsy was performed to the remove the nodule and, on frozen section, you saw a large spherule with internal endospores in a thick-walled necrotic granuloma. Which of the following pieces of information can you offer the surgeon? A. The diagnosis of the nodule is coccidioidomycosis. B. The nodule is probably due to coccidioidomycosis, but you are qualifying your diagnosis since the serology was negative. C. The nodule is due to sporotrichosis. D. The etiology of the nodule is unknown, since the granuloma could have commensal organisms in it and you feel you should wait for culture results. E. This is a nodule of prior histoplasmosis. ANSWER: A 25. You have identified a granuloma that you think is probably from histoplasmosis in the biopsy you are examining. It might help to confirm the diagnosis if you: A. Examine the medical record for results of beta-D-glucan in the serum B. Examine the medical record for results of serum or urine antigen testing C. Cut deeper sections and do GMS on multiple levels D. All of the above E. b and c ANSWER: E 26. A bone marrow transplant patient has hemoptysis. You find some ribbon-like hyphae in a hemorrhagic portion of her lung biopsy. Which of these features is most likely to help you differentiate whether this fungus is Aspergillus or Mucor? A. The morphology of the hyphal branching B. The negative Gram stain C. The serum galactomannan result D. The way the organism is invading vessels E. Waiting for the culture ANSWER: C 27. Consider Paracoccidioides infection. Which is untrue? A. It is common in Brazil. B. It is most commonly symptomatic after inhalation infection. C. It has narrow-based budding. D. It is more common in men. E. It will have multiple buds in yeast cells. ANSWER: B 28. The cytopathologic effect of cytomegalovirus is characterized by: A. Cellular enlargement B. Red-purple inclusions in the nucleus C. Red-purple granules in the cytoplasm D. Lack of correlation with viral load in blood E. All of the above ANSWER: E 29. Pulmonary dirofilariasis is most typically characterized by: A. Eosinophilia in the majority of patients B. Degenerating parasites in a histiocyte-rimmed necrotic single pulmonary nodule C. Diagnosis by excisional lung biopsy D. b and c E. All of the above ANSWER: D 30. Which organism is incorrectly described? A. Actinomyces, a filamentous bacterium, is positive in Gram and GMS stains. B. Botryomycosis involves a gram-positive collection of rods and cocci. C. Legionella is a gram-negative organism that will not grow on standard media owing to a requirement for cysteine. D. Cryptococcus is a yeast with a mucicarmine-positive capsule. E. Candida is a gram-positive fungus only rarely associated with giant cells or granulomas. ANSWER: B Case 1 History A 40-year-old white male with history of allograft liver transplantation presents after several weeks of dyspnea. Ground-glass infiltrate in chest x-ray. Open lung biopsy performed. Pathologic Findings Diffuse alveolar damage ( eSlide 7.1A ) and characteristic inclusions ( eSlide 7.1B ). Diagnosis Cytomegalovirus pneumonitis. History A 40-year-old white male with history of allograft liver transplantation presents after several weeks of dyspnea. Ground-glass infiltrate in chest x-ray. Open lung biopsy performed. Pathologic Findings Diffuse alveolar damage ( eSlide 7.1A ) and characteristic inclusions ( eSlide 7.1B ). Diagnosis Cytomegalovirus pneumonitis. Case 2 History A 42-year-old white female on steroids and immune-modifying drugs for rheumatoid arthritis presents with dyspnea and bilateral diffuse lung infiltrates. A right lower lobe wedge biopsy is performed. Pathologic Findings Fluffy alveolar infiltrate, somewhat fibrinous appearing ( eSlide 7.2A ) with some suggestion of internal structure at high power ( eSlide 7.2B ). GMS stain demonstrates organisms diagnostic of Pneumocystis in exudates ( eSlide 7.2C ). Diagnosis Pneumocystis pneumonia. History A 42-year-old white female on steroids and immune-modifying drugs for rheumatoid arthritis presents with dyspnea and bilateral diffuse lung infiltrates. A right lower lobe wedge biopsy is performed. Pathologic Findings Fluffy alveolar infiltrate, somewhat fibrinous appearing ( eSlide 7.2A ) with some suggestion of internal structure at high power ( eSlide 7.2B ). GMS stain demonstrates organisms diagnostic of Pneumocystis in exudates ( eSlide 7.2C ). Diagnosis Pneumocystis pneumonia. Case 3 History An asymptomatic 81-year-old Japanese white female who had a normal chest x-ray a year earlier is found, on repeat chest x-ray, to have a solitary pulmonary nodule 1.5 cm in diameter in the right middle lobe. The nodule is removed by wedge excision. Pathologic Findings Discrete nodule with slightly organized rim and diffuse central necrosis ( eSlide 7.3A ). Areas in the nodule show necrotic residual organisms in cross section ( eSlide 7.3B ) surrounded by bland necrosis. A higher-power view suggests a layered body wall with a central lumen ( eSlide 7.3C ). Diagnosis Most consistent with dirofilarial granuloma. History An asymptomatic 81-year-old Japanese white female who had a normal chest x-ray a year earlier is found, on repeat chest x-ray, to have a solitary pulmonary nodule 1.5 cm in diameter in the right middle lobe. The nodule is removed by wedge excision. Pathologic Findings Discrete nodule with slightly organized rim and diffuse central necrosis ( eSlide 7.3A ). Areas in the nodule show necrotic residual organisms in cross section ( eSlide 7.3B ) surrounded by bland necrosis. A higher-power view suggests a layered body wall with a central lumen ( eSlide 7.3C ). Diagnosis Most consistent with dirofilarial granuloma. Case 4 History Three months after heart transplantation a 60-year-old male is admitted with atrial fibrillation, left-sided pleuritic pain, and a 2-week history of nonproductive cough. Chest CT shows two mass-like lesions, one abutting the mediastinum and one on the anterior chest wall, ranging from 2 to 5 cm in maximum diameter. There is no significant adenopathy. BAL without biopsy is performed; nondiagnostic culture results show an Aspergillus antigen index on BAL fluid of >3.75 (normal 3.75 (normal <0.5). CT guided needle biopsy of anterior mass is then performed. Pathologic Findings Acute and organizing pneumonia ( eSlide 7.4A ) with hemosiderin and neutrophilic exudate ( eSlide 7.4B ). GMS fungal stain reveals small bits of fragmented hyphae dispersed through the biopsy ( eSlide 7.4C ). These vary in width and are too fragmented to be definitively identified morphologically ( eSlide 7.4D ), although they are consistent with Aspergillus species. The greatly elevated Aspergillus antigen from the recent BAL fluid is highly suggestive of Aspergillus infection. Initial calcofluor fungal smear in the microbiology lab made from a needle core biopsy was negative. The culture of the tissue obtained at the lung biopsy subsequently grew Aspergillus fumigatus. Diagnosis Acute and organizing pneumonia from Aspergillus fumigatus. Case 5 History A 32-year-old HIV-positive black male with AIDS, recently treated Pneumocystis jirovecii pneumonia, returns for a follow-up chest x-ray. He has a persistent left upper lobe infiltrate, low-grade fever, and general malaise. Open lung biopsy of the lesion is performed. Pathologic Findings Solid infiltrate, slightly nodular with pleural adhesion ( eSlide 7.5A ), both spindle cell and round cell infiltrate with background lymphocytic infiltrate ( eSlide 7.5B ). Acid-fast stain shows numerous acid-fast organisms throughout the most cellular portions of the mass ( eSlide 7.5C ). Culture of tissue obtained at open lung biopsy grew Mycobacterium avium . Diagnosis M. avium pseudotumor. History A 32-year-old HIV-positive black male with AIDS, recently treated Pneumocystis jirovecii pneumonia, returns for a follow-up chest x-ray. He has a persistent left upper lobe infiltrate, low-grade fever, and general malaise. Open lung biopsy of the lesion is performed. Pathologic Findings Solid infiltrate, slightly nodular with pleural adhesion ( eSlide 7.5A ), both spindle cell and round cell infiltrate with background lymphocytic infiltrate ( eSlide 7.5B ). Acid-fast stain shows numerous acid-fast organisms throughout the most cellular portions of the mass ( eSlide 7.5C ). Culture of tissue obtained at open lung biopsy grew Mycobacterium avium . Diagnosis M. avium pseudotumor.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749544/

Deceptology in cancer and vaccine sciences: Seeds of immune destruction‐mini electric shocks in mitochondria: Neuroplasticity‐electrobiology of response profiles and increased induced diseases in four generations – A hypothesis

Abstract From Rockefeller's support of patent medicine to Gates' patent vaccines, medical establishment invested a great deal in intellectual ignorance. Through the control over medical education and research it has created a public illusion to prop up corporate profit and encouraged the lust for money and power. An overview of data on cancer and vaccine sciences, the status of Americans' health, a survey of repeated failed projects, economic toxicity, and heavy drug consumption or addiction among young and old provide compelling evidence that in the twentieth century nearly all classic disease categories (congenital, inheritance, neonatal, or induced) shifted to increase induced diseases. Examples of this deceptology in ignoring or minimizing, and mocking fundamental discoveries and theories in cancer and vaccine sciences are attacks on research showing that (a), effective immunity is responsible for defending and killing pathogens and defective cancerous cells, correcting and repairing genetic mutations; (b) viruses cause cancer; and (c), abnormal gene mutations are often the consequences of (and secondary to) disturbances in effective immunity. The outcomes of cancer reductionist approaches to therapies reveal failure rates of 90% (+/‐5) for solid tumors; loss of over 50 million lives and waste of $30‐50 trillions on too many worthless, out‐of‐focus, and irresponsible projects. Current emphasis on vaccination of public with pathogen‐specific vaccines and ingredients seems new terms for drugging young and old. Cumulative exposures to low level carcinogens and environmental hazards or high energy electronic devices (EMF; 5G) are additional triggers to vaccine toxicities (antigen‐mitochondrial overload) or "seeds of immune destruction" that create mini electrical shocks (molecular sinks holes) in highly synchronized and regulated immune network that retard time‐energy‐dependent biorhythms in organs resulting in causes, exacerbations or consequences of mild, moderate or severe immune disorders. Four generations of drug‐dependent Americans strongly suggest that medical establishment has practiced decades of intellectual deception through its claims on "war on cancer"; that cancer is 100, 200, or 1000 diseases; identification of "individual" genetic mutations to cure diseases; "vaccines are safe". Such immoral and unethical practices, along with intellectual harassment and bullying, censoring or silencing of independent and competent professionals ("Intellectual Me Too") present grave concerns, far greater compared with the sexual harassment of 'Me Too' movement that was recently spearheaded by NIH. The principal driving forces behind conducting deceptive and illogical medical/cancer and vaccine projects seem to be; (a) huge return of investment and corporate profit for selling drugs and vaccines; (b) maintenance of abusive power over public health; (c) global control of population growth via increased induction of diseases, infertility, decline in life‐span, and death. An overview of accidental discoveries that we established and extended since 1980s, on models of acute and chronic ocular inflammatory diseases, provides series of the first evidence for a direct link between inflammation and multistep immune dysfunction in tumorigenesis and angiogenesis. Results are relevant to demonstrate that current emphasis on vaccinating the unborn, newborn, or infant would induce immediate or long‐term immune disorders (eg, low birth weight, preterm birth, fatigue, autism, epilepsy/seizures, BBB leakage, autoimmune, neurodegenerative or digestive diseases, obesity, diabetes, cardiovascular problems, or cancers). Vaccination of the unborn is likely to disturb trophoblast‐embryo‐fetus‐placenta biology and orderly growth of embryo‐fetus, alter epithelial‐mesenchymal transition or constituent‐inducible receptors, damage mitochondria, and diverse function of histamine‐histidine pathways. Significant increased in childhood illnesses are likely due to toxicities of vaccine and incipient (eg, metals [Al, Hg], detergents, fetal tissue, DNA/RNA) that retard bioenergetics of mitochondria, alter polarization‐depolarization balance of tumoricidal (Yin) and tumorigenic (Yang) properties of immunity. Captivated by complex electobiology of immunity, this multidisciplinary perspective is an attempt to initiate identifying bases for increased induction of immune disorders in three to four generations in America. We hypothesize that (a) gene‐environment‐immune biorhythms parallel neuronal function (brain neuroplasticity) with super‐packages of inducible (adaptive or horizontal) electronic signals and (b) autonomic sympathetic and parasympathetic circuitry that shape immunity (Yin‐Yang) cannot be explained by limited genomics (innate, perpendicular) that conventionally explain certain inherited diseases (eg, sickle cell anemia, progeria). Future studies should focus on deep learning of complex electrobiology of immunity that requires differential bioenergetics from mitochondria and cytoplasm. Approaches to limit or control excessive activation of gene‐environment‐immunity are keys to assess accurate disease risk formulations, prevent inducible diseases, and develop universal safe vaccines that promote health, the most basic human right. 1 INTRODUCTION A great deal of intelligence can be invested in ignorance when the need for illusion is deep! Saul Bellow We have remained in the dark long enough to talk about the light . Sohrab Sepehri In the 20th century, the patent medical establishment, led by a coalition of Governments‐Big Pharma and Venture‐Capitalists‐'Philanthropists' (disease investors) invested a great deal on intellectual ignorance in medical education and research projects because the need for a public illusion that would generate corporate profit seemed deep and dark. The illusion fed on medical projects initiated by the twisted and profitable cholesterol story and extended to other fabricated and expensive projects on cancer and vaccine sciences that repeatedly failed. Significant increased in the sick population in America (young and old), heavy consumption and addiction to drugs and the current emphasis to vaccinate the public with pathogen‐specific vaccines demonstrate that motives of the medical establishment in conducting too many intellectually fraudulent projects are diametrically opposing the mission to improve public health by preventing diseases or saving lives [reviewed in refs. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. To better appreciate the extent of this intellectual deception, fraud, and chaos in conducting scientific/clinical projects that were practiced by the medical establishment against public health, the author chose the word of "deceptology." Deceptology in medical/cancer and vaccine sciences refers to a combination of adjectives such as deception, prank, magic, scam, fraud, cons, and propaganda; fear‐mongering, bias, nepotism, and heavy bribes in conducting projects. Tremendous financial gain and absolute power to control public health were accomplished by endless intellectual deception (scientific/medical Ponzi schemes) in marketing failed projects. As detailed below, novel ideas and discoveries are routinely hijacked, fragmented, and used as a front to collect funding from taxpayers. Decisions on expensive and out‐of‐focus projects are associated with ignoring medical morals or ethics and conflict of interest, while practicing heavy intellectual bullying, harassing, and silencing of independent and competent professionals 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . The "medical establishment" refers to a highly organized and powerful group, a hierarchy with military‐like structure that functions like an elite tribe and operates globally. The medical establishment is an intimate and co‐dependent partnership between decision makers in governments (eg, NIH‐FDA‐CDC‐DHHS, WHO) * , Big Pharma, venture capitalists 'philanthropists' (disease investors), medical/cancer research and treatment centers, organizations (eg, AMA, AACR, ASCO, ACS, APS), insurance companies, major food industry players and the main stream media outlets that collectively control the marketing of foods, drugs, and vaccines. Globally, the estimated total number of members in medical establishment including the world's largest lobbying and support groups, handlers/cronies (institutes' directors, department chairs, and staff) is 4–6 millions. In this power structure, the role of policymakers, with/without scientific/medical backgrounds who are largely influenced (including monetarily) by lobbying groups cannot be ignored. This power structure channels funds and sponsors legislations on behalf of the medical establishment (eg, recent actions to mandate vaccination " Give Kids a Shot "; 'Moonshot' initiative [2016] that increased funding for NCI/NIH by $1.6 B for HPV vaccines and cancer research; or Cancer Act by President Nixon [1970] that increased funding for cancer research by 1.6 B to resolve cancer problem in 8 years!!). 1 , 7 , 8 , 12 , 18 †' ‡ Details on financial structures and cycles of collaborations between governmental agencies, pharmaceutical companies, and medical education programs on the promotion of drugs are provided in an informative book by Marcia Angell, MD (past Editor‐In‐Chief of New England Journal of Medicine ). 2 It describes how drug companies operate within this system [" Buying Influence‐How the Industry Makes Sure It Gets Its Way…Bribing Doctors—or Nurturing Consultants… "] by selling drugs that scientists/physicians advocate on behalf of industry; and by charging taxpayers twice, once for supporting research of government‐academia and again paying for prescription drugs at prices that are set by manufacturers. In another instructive book, John Geyman, MD 3 describes the market‐driven healthcare that changed medical practices; that physicians frequently ignore the principles of the medical profession (patient welfare, patient autonomy, social justice, medical ethics, and moral values of healthcare) and doing no harm. Geyman demonstrates that physicians routinely misdiagnose or over‐diagnose diseases and prescribe drugs that are promoted by Big Pharma, while downplaying conflicts of interest and ignoring medical ethics by accepting fees for consultations and honoraria to lecture on behalf of drug companies who are also organizers of continuing medical education programs. 3 In a series of informative books, Harris Coulter, MD, describes the history and philosophical approaches to medicine from the time of Hippocrates to the 20th century and the conflicts between the two systems of empiricism and rationalism, 15 , 16 or as we have described the differences between integrated and reductionist approaches to the medical sciences. 1 , 5 , 8 , 10 , 11 , 12 , 20 In another comprehensive publication, by an independent vaccine and health journalist, Jeremy Hammond describes the FDA vaccine approval process " The Government Is the Vaccine Industry ". Hammond details how the perception that the government is serving on behalf of the public is far from the truth. 4 Suzanne Humphries, MD and Roman Bystriany 17 in an eye‐opening book describe the history behind polio vaccines and induction of diseases despite protests against vaccination since 1919. The quotation by Eustace Mullins that public vaccination is " Murder by Injection" (reviewed in ref. 17 ) has scientific merits as we describe below the toxicity of current drugs and vaccines. In another report, 18 Brandon Turbeville describes that The American Legislative Exchange Council (ALEC), a "non‐profitable organization" is a " driving force behind the current drive for mandatory vaccine bills … a source of enormous profits for drug manufacturers …". Details on operation of ALEC, along with names of companies, contributors, beneficiaries within and outside government, and the agenda for vaccine mandates are provided in this report. 18 This multidimensional perspective/hypothesis is a brief overview of identifying a century of intellectual deception, chaos, and fraud that the medical establishment practiced in conducting failed cancer and vaccine sciences. Ignoring the truth about the motives behind too many ill‐conceived, reckless, and failed projects that led to the loss of millions of precious lives and creation of three to four generations of sick and drug‐dependent people and the huge economic burden to the society are no longer tolerable, acceptable, or sustainable. The first steps to switching the disease‐care mentality of the medical establishment are to present sufficient biological evidence to reveal the the hidden agenda behind this massive intellectual deception in conducting medical/cancer and vaccine sciences. The hidden agenda seeks to deny health to the public by chipping away and destroying the body's natural immunity in an effort to regulate population growth, maintain an ill, easily controlled worker population, and effectuate wealth transfer from the public to the establishment. Future scientific directions should include cleaning up the massive misinformation (scientific noise and frauds) on cancer and vaccine sciences, while focusing on designs of cost‐effective systematic and logical studies to promote immunity, assess accurate risk formulations, develop safe/universal vaccines with the goal to improve health and prevent diseases for a healthier and more productive society. 2 FROM ROCKEFELLER MEDICINE TO GATES VACCINES: REDUCTIONIST AND FRAUD APPROACHES TO CANCER AND VACCINE SCIENCES. INDUCTION OF DISEASES IN YOUNG AND OLD FOR DRUG SALE In 1900s, the Rockefeller and other "philanthropists" (who should be identified as "disease investors") supported medical school education programs with an eye toward influencing drug development and sale. The major drug business initiated with an intellectually twisted story on cholesterol‐lowering drugs that continue to generate billions of dollars for the industry. The side‐effects of such drugs made millions of older individuals sick and brought additional profits for drug manufacturers and food industry. 1 , 6 , 7 , 8 § In a recent documentary, Aseem Malhotra, MD, noted it was " time for a full public parliamentary inquiry into the controversial drug and to fully expose the great cholesterol and statin con ." 6 ** The ill‐designed studies that promoted and educated physicians for use of cholesterol inhibitors became bases for a shared Nobel prize by Brown and Goldstein and development of statins and their derivatives. 6 ††A century ago, the leading causes of death were pneumonia/influenza, tuberculosis, and diarrhea followed by heart disease and stroke. In all likelihood, at the turn of past century, heart disease and stroke were also the consequences of serious infections that shortened life expectancy, particularly in poor neighborhoods, and in the absence of antibiotics and better hygiene. Available statistics show that in 1900s, cancer occurred occasionally, as a genetic disorder (inherited disease category) at the rate of 5%. 1 , 5 , 8 Eight decades ago the National Institutes of Health (NIH) or "the hidden crown jewel of corruption in the government" 8 were established and received funding from taxpayers; and in collaborations with other governmental health agencies and centers within DHHS, had the "mission" to improve public health, prevent and treat diseases, and save lives. 1 , 8 , 12 , 13 However, despite improved hygiene and development of antibiotics and modern diagnostic technologies, the health status of Americans became significantly lower compared with the previous two to four generations at the same age and lowest compared with other developed nations. Since 1955s, after public was introduced to virus‐contaminated polio vaccines, cancer incident and mortality and numerous other diseases sharply increased, particularly in America. In 2013, the American Association for Cancer Research (AACR, among the largest cancer organizations and lobbying group for establishment) announced that one‐third of women (33%) and half of men (50%) develop cancer in their life‐time. 1 , 5 , 8 , 12 , 13 , 24 Major associated factors in the increased induction of diseases, shorten life expectancy or death in America are combinations of consumption of too many drugs, reductionist approaches to cancer research and therapy, as well as toxicities of vaccines that target the unborn, newborn, infant, or individuals (young and old) who are immune‐compromised (see below). Decision makers in medicine, major food and drug industry, or agricultural and electronic companies constantly design, advertise, and encourage public to use and be exposed to low level carcinogens (eg, glyphosate/ herbicides, pesticides, food additives and preservatives, artificial sweeteners, GMOs, chemical, biological and environmental hazards, or high energy electronic gadgets [4/5G devices]) that cumulatively weaken and interfere with the amazing electrobiology/ biorhythms that shape immunity and causing induction of mild, moderate or severe immune disorders. 1 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 Objections, debates, questions, or suggested solutions on cancer or vaccine projects and clinical trials by independent and competent scientists are ignored or perceived as "dangerous" by decision makers 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 17 , 22 (Khatami, NCI/NIH scientific and legal documents, since 1998). In brief, the reductionist approach to cancer sciences is the real "dangerous" intellectual deception that made solving cancer a profitable myth‐making machine for the medical establishment. Recently, we presented evidence that cancer is an induced disease of the 20th century, created by the medical establishment by allowing baby boomers to consume virus‐contaminated polio vaccines since 1955s. 1 , 5 We also presented evidence that, unlike popularized notions that cancer is 100, 200, or 1000 diseases, cancer is only one disease; the severely disrupted loss of highly regulated biorhythms of effective immunity, provided through tumoricidal (Yin) and tumorigenic (Yang) arms (autonomic sympathetic and parasympathetic) of acute inflammation. 5 In this multidisciplinary perspective, we further provide evidence that nearly all clinically and pathologically established disease categories (neonatal, hereditary, congenital, and induced) that occasionally occurred at the rates of 1‐5% in the past century, have been shifted to increase the population of induced diseases in young and old. To achieve maximum disease status, particularly in America medical establishment employed combined methods of (a) heavy advertisements for the consumption of numerous drugs; (b) frequent vaccination of young and old with pathogen‐specific vaccines; and (c) cumulative exposures of public to environmental hazards. Major methods that establishment continue to employ on utilizing reductionist approaches to cancer and vaccine projects that created tremendous misunderstanding, misinformation, debates, and controversies and resulted in increased diseases in young and old are listed below: 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 §§ ' *** ' †††Definitions of inflammation/immunity, whether inflammation is protective in preventing cancer or it causes cancer; Identifying too many genetic mutations to develop and sell drugs (eg, monoclonal antibodies, inhibitors of growth factors); Claims of "targeted" therapy, "personalized" or "precision" medicine, or immunotherapy; Claims that "vaccines are safe," with little serious safety and efficacy tests. Vaccine manufacturers have no liability or responsibility toward vaccine injuries; Incentives and royalties that scientists/physicians receive for advocating pathogen‐specific vaccines (eg, flu, HPV, meningitis, shingles, Hep a, b, c, MMR, EBOLA, ZIKA) or the "upcoming coronavirus vaccines"; as well as efforts to minimize voices of concern about vaccines safety; Heavy propaganda on the consumption of too many drugs for minor or major health conditions (eg, headache, muscle pain, allergies, depression, mood swings, cholesterol, indigestion, colitis, gastritis, sleep disorders, or cancers) With regard to vaccines, Maurice Hilleman who developed several vaccines at Merck, in an interesting interview stated that " vaccines have to be considered the bargain basement technology for the twentieth century ." ‡‡‡ One of the most dangerous plans of the establishment is the heavy propaganda campaign to vaccinate the unborn, newborn, infant, toddler, and teenagers with a total of 72 doses of 16 different pathogen‐specific vaccines by the time they are 18 years old. As detailed below, the presence of active or inactive specific pathogens and adjuvants in current vaccines are hypothesized as causes, aggregations‐exacerbations, and consequences of significant increased in immune disorders in young and old in the twentieth century, 1 , 5 , 8 , 20 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 §§§ (Figure 1 ). FIGURE 1 Schematic representation that current pathogen‐specific vaccines and incipient given to unborn (fetus), newborn or infant (age 0 to 24 mons), toddler or children (2‐16 years) are causes, exacerbations (aggregations) or consequences of development of a wide range of immune disorders that are often features of age‐associated chronic illnesses. Vaccination of unborn is depicted to lead to SIDS, preterm birth or low weight at birth, and associated underdeveloped immunity as bases for increased mortality and induction of childhood diseases. It depicts that current vaccines and metal‐containing ingredients are seeds of immune destruction, particularly affecting mitochondria and oxido‐redox potentials and immune‐metabolic‐hormonal‐neuronal activities. The scheme also represents that current vaccines and incipient shift/increase the classic categories of disease (inheritance, congenital, neonatal, and induced) to induced diseases. See text 3 IGNORING, WHILE ABUSING FUNDAMENTAL DISCOVERIES AND THEORIES OF IMMUNITY: CAUSES AND CONSEQUENCES OF DISEASES OR CANCER For evil to flourish, all that is needed is for good people to do nothing . Edmund Burke. The ability of inflammatory cells to destroy cancerous cells was first observed by Ilya Metchnikoff in the 19th century when the microscope was invented. Metchnikoff's report on phagocytic properties of inflammatory cells was the basis to study innate immunity. Paul Ehrlich also established the concept of antigen‐antibody complementarities and basis to study adaptive immunity. In 1908, the Nobel prize was shared between Ehrlich and Metchnikoff for their pioneering work in immunology and host defense mechanisms (reviewed in refs. 1 , 8 ). 3.1 Initiators of cancer (tumor) growth and theory of immune surveillance In 1910‐1911, the important and careful studies of Peyton Rous led to the discovery that viruses induce cancer. The main factor that was transmissible in chicken leukemia, lymphoma, sarcoma, and other neoplasms was a filterable virus (reviewed in refs. 1 , 8 , 20 ). Rous' visionary work demonstrated the cumulative effects of the "initiators" in carcinogenesis. The integrated and generalized description of "initiators" in carcinogenesis that Rous defined was later extended and supported by the important theory of immune surveillance of Burnet in 1957 48 and by our accidental discoveries (1980s) on direct evidence for cumulative effects of immune disruptors (antigens) in the initiation of multistep tumorigenesis and angiogenesis. Our earlier discoveries led to definitions of the Yin‐Yang‐like interplay of inflammation/immunity in the maintenance of health or induction and progression of nearly all acute and chronic diseases including site‐specific cancers (see below) 1 , 5 , 8 , 20 , 24 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 (Figure 2 ). FIGURE 2 Schematic representation that inflammation and aging are co‐risk factors in developmental phases of immune dysfunction in multistep tumorigenesis and angiogenesis. The left panel depicts initial stages of our 'accidental' discoveries on inflammation‐induced identifiable immune dysfunction in ocular tissue responses during (a) acute phase responses or self‐terminating inflammation (reversible); (b) intermediate phase, down‐regulation phenomenon accompanied with mild tissue atrophy and neovascularization (potentially reversible); and (c) chronic phase, induction of massive lymphoid hyperplasia and tumorigenesis and angiogenesis (irreversible?). The right panel represents chronic inflammation and continued stages of tissue growth (d,e), advancing to cancer malignancies and angiogenesis in site‐specific tissue. The complex scheme demonstrates that majorities of translational medicine and clinical trials are conducted in identification of endless damaged molecules at advanced stages of carcinogenesis for drug development and therapy (red arrows in phase e, 'cancer tsunami'). Modified from Exp Opin Biol Ther; Informa Healthcare, 2011. 47 All Rights reserved The immune surveillance theory of Burnet was based on a decade of extensive analyses and integration of data from several scientific disciplines such as developmental biology, embryology, immunology, pathophysiology, and oncology of his era. Unlike the current reductionist approaches to cancer and vaccine sciences, Burnet realized that scientists like himself " believe that at every stage in scientific development it is necessary to provide the best available generalizations as a guide to effective work, both in the application of knowledge to human needs and in the planning of future research…." . Burnet explained that" …Cancer is a negative condition‐ a manifestation of the breakdown in one or more aspects of the positive control that welds the cells of the body into a single functioning unit‐the organism as a whole…The failure in cancer is due not to any weakness of the organism but to a change in the character of the cells rendering them in one way or another insusceptible to the normal control. This statement is self‐evident when we consider the phenomena of metastasis and experimental transplantation… . " 48 Despite the fundamental knowledge that viruses cause cancer, in 1955's/1960's, the public was allowed to consume virus‐contaminated polio vaccines. This intellectually criminal act by decision makers sharply increased cancer incidence, mortality, and morbidity of baby boomers and the subsequent generations (see below). It should be noted that prior to vaccination of public with virus‐contaminated polio vaccines, decision makers minimized, downplayed, ignored, and harassed a highly competent and concerned microbiologist (Bernice Eddy) at NIH who discovered that the polio vaccines had live and filterable viruses (eg, Simian virus, SV‐40) and predicted that contaminated polio vaccines could cause cancer epidemic. 1 , 5 , 8 , 17 The loss of too many lives and numerous polio vaccine injuries resulted in lawsuits against NIH and DHHS and resignation of directors of NIH and DHHS, constituting little more than a slap on the hand!. 1 Despite this record, the power of the medical establishment over exclusive decision‐making and over public health in general increased to its current scary level. In the last few decades induction of several infective respiratory diseases such as Swine flu, SARS or MERS, Zika, and the current pandemic on coronavirus (Covid‐ 19) that resulted in global lockdown, the crash of the economy, scare tactics and heavy publicity, debates, controversies for masking, treating and marketing vaccines on a global scale that are parroted by major media have created serious scientific/medical and legal concerns about future of public health internationally 81 , 83 , 86 , 87 (manuscript in preparation). **** ' ††††The basis for the emergence of increased risk of pathogenic and retrovirus infections (eg, flu, HIV) or SARS, MERS, and coronavirus that created an urgent need for developing pathogen‐specific vaccines initiated questions on the potential presence of live pathogens (similar to SV‐40) in the media that vaccines were prepared and consumed by the public in the past three decades 1 , 20 (manuscript in preparation) ‡‡‡‡ 3.2 Ignoring while abusing evidence for link between inflammation and tumorigenesis and angiogenesis It is dangerous to be right, when the government is wrong . Voltaire Epidemiological reports on circumstantial evidence for an association between sites of prior injuries/chronic irritation or inflammation and the increased risk of cancer have been documented for a century. 1 , 5 , 8 , 10 , 41 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 In few such articles, professionals noted major gaps on direct evidence for a link between inflammation and induction of carcinogenesis and angiogenesis. Biological gaps were also noted on evidence for identifiable stages of immune alterations toward tumorigenesis or cancer. The cancer establishment has continued ignoring these important biological gaps on the initiation events that lead to immune dysfunction toward tumorigenesis and angiogenesis. Since 1998, analyses of data on our original studies that were conducted on experimental models of acute and chronic ocular inflammatory diseases, unexpectedly demonstrated a series of evidence that satisfied at least two of the major knowledge gaps on the role of inflammation in cancer immunobiology 1 , 5 , 8 , 20 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 (Figure 2 ): Evidence on direct association between inflammation and induction of tumorigenesis and angiogenesis; Time course kinetics of inflammation‐induced at least three identifiable phases of immune dysfunction toward multistep tumorigenesis; In 2014, further analyses of original data also revealed the first evidence on sequential interactions and synergies between activated host and recruiting immune and non‐immune cells in the direction of tumor growth. These data also incorporate the missing evidence on immune disruptor‐induced initial events in altering immune responses. 42 3.3 Author's accidental discoveries: Initiation events in tumorigenesis and angiogenesis‐ Direct evidence for a link between inflammation in multistep immune dysfunction. Intellectual challenges at NCI/NIH since 1998 (Intellectual Me Too!) Since 1998 at NCI/NIH, Khatami followed the logical, careful, and integrated approaches of Burnet that led him to the theory of immune surveillance. Analyses of original data that the author's team established at the University of Pennsylvania on experimental models of ocular acute and chronic inflammatory diseases in 1980s, led her to a series of reports that were suggestive of the first and only series of data on direct association between inflammation (initiation events) and multistep tumorigenesis and angiogenesis. 1 , 5 , 8 , 20 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 50 Further analyses and integration of data in the fields of immunology‐inflammation, cancer, and developmental biology, aging, biomarkers, molecular diagnosis and therapeutics led to recent definitions of inflammation for maintenance of health or induction of diseases. A number of concepts and comprehensive proposals were submitted in an effort to promote the role of inflammation in cancer research for early molecular diagnosis, design of clinical trials, use of patients' biospecimen, and potential agents (eg, SH‐containing agents, captopril, Sulindac, aspirin) for cancer chemoprevention. Khatami also developed a working project, standardizing cancer biomarkers criteria for developing effective databases for oncology research, using an inflammatory mediator (M‐CSF), as a prototype to tailor and test the sensitivity and specificity of M‐CSF, in comparison with conventional mediators (NCI‐Invention, Fed. Reg, 2005). 1 , 5 , 8 , 10 , 20 , 41 , 42 , 52 Decision makers and their handlers at NCI/NIH, severely opposed, ignored, denied, minimized, and rejected the submitted concepts and comprehensive proposals on the important role of inflammation in cancer research and therapy. However, in the last two decades, it seems that Khatami's challenging efforts awakened the entire cancer community around the world. Members of the establishment fragmented the submitted ideas and used them as a front for collecting more funding from the cancer‐stricken public. Significant increased funded projects focus on isolated numerous cellular and molecular aspects of inflammation‐immunity for cancer research and therapy; using site‐specific tumor models, expensive specific technologies, and related networks. However, the reductionist approaches on the topic of inflammation created further confusion and ongoing debates on what inflammation does, whether it prevents cancer or it causes cancer, 1 , 5 , 8 , 20 , 51 (NCI/NIH scientific and legal documents, since 1998). It is noteworthy that current literature in the field is flooded with hundreds of thousands of articles on the structures and substructures of numerous pathogens, their roles in experimental models of diseases; or numerous identified genetic mutations in cancer molecular tsunami to use specific expensive technologies for research, diagnosis or treatment and pathogen‐specific vaccine technologies. However, peculiarly, except for our accidental discoveries that were established in 1980s, very little is known about how stimuli (immune disruptors, pathogens) systematically would induce initiation processes in altering immune response dynamics toward time‐dependent multistage disease development. 3.1 Initiators of cancer (tumor) growth and theory of immune surveillance In 1910‐1911, the important and careful studies of Peyton Rous led to the discovery that viruses induce cancer. The main factor that was transmissible in chicken leukemia, lymphoma, sarcoma, and other neoplasms was a filterable virus (reviewed in refs. 1 , 8 , 20 ). Rous' visionary work demonstrated the cumulative effects of the "initiators" in carcinogenesis. The integrated and generalized description of "initiators" in carcinogenesis that Rous defined was later extended and supported by the important theory of immune surveillance of Burnet in 1957 48 and by our accidental discoveries (1980s) on direct evidence for cumulative effects of immune disruptors (antigens) in the initiation of multistep tumorigenesis and angiogenesis. Our earlier discoveries led to definitions of the Yin‐Yang‐like interplay of inflammation/immunity in the maintenance of health or induction and progression of nearly all acute and chronic diseases including site‐specific cancers (see below) 1 , 5 , 8 , 20 , 24 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 (Figure 2 ). FIGURE 2 Schematic representation that inflammation and aging are co‐risk factors in developmental phases of immune dysfunction in multistep tumorigenesis and angiogenesis. The left panel depicts initial stages of our 'accidental' discoveries on inflammation‐induced identifiable immune dysfunction in ocular tissue responses during (a) acute phase responses or self‐terminating inflammation (reversible); (b) intermediate phase, down‐regulation phenomenon accompanied with mild tissue atrophy and neovascularization (potentially reversible); and (c) chronic phase, induction of massive lymphoid hyperplasia and tumorigenesis and angiogenesis (irreversible?). The right panel represents chronic inflammation and continued stages of tissue growth (d,e), advancing to cancer malignancies and angiogenesis in site‐specific tissue. The complex scheme demonstrates that majorities of translational medicine and clinical trials are conducted in identification of endless damaged molecules at advanced stages of carcinogenesis for drug development and therapy (red arrows in phase e, 'cancer tsunami'). Modified from Exp Opin Biol Ther; Informa Healthcare, 2011. 47 All Rights reserved The immune surveillance theory of Burnet was based on a decade of extensive analyses and integration of data from several scientific disciplines such as developmental biology, embryology, immunology, pathophysiology, and oncology of his era. Unlike the current reductionist approaches to cancer and vaccine sciences, Burnet realized that scientists like himself " believe that at every stage in scientific development it is necessary to provide the best available generalizations as a guide to effective work, both in the application of knowledge to human needs and in the planning of future research…." . Burnet explained that" …Cancer is a negative condition‐ a manifestation of the breakdown in one or more aspects of the positive control that welds the cells of the body into a single functioning unit‐the organism as a whole…The failure in cancer is due not to any weakness of the organism but to a change in the character of the cells rendering them in one way or another insusceptible to the normal control. This statement is self‐evident when we consider the phenomena of metastasis and experimental transplantation… . " 48 Despite the fundamental knowledge that viruses cause cancer, in 1955's/1960's, the public was allowed to consume virus‐contaminated polio vaccines. This intellectually criminal act by decision makers sharply increased cancer incidence, mortality, and morbidity of baby boomers and the subsequent generations (see below). It should be noted that prior to vaccination of public with virus‐contaminated polio vaccines, decision makers minimized, downplayed, ignored, and harassed a highly competent and concerned microbiologist (Bernice Eddy) at NIH who discovered that the polio vaccines had live and filterable viruses (eg, Simian virus, SV‐40) and predicted that contaminated polio vaccines could cause cancer epidemic. 1 , 5 , 8 , 17 The loss of too many lives and numerous polio vaccine injuries resulted in lawsuits against NIH and DHHS and resignation of directors of NIH and DHHS, constituting little more than a slap on the hand!. 1 Despite this record, the power of the medical establishment over exclusive decision‐making and over public health in general increased to its current scary level. In the last few decades induction of several infective respiratory diseases such as Swine flu, SARS or MERS, Zika, and the current pandemic on coronavirus (Covid‐ 19) that resulted in global lockdown, the crash of the economy, scare tactics and heavy publicity, debates, controversies for masking, treating and marketing vaccines on a global scale that are parroted by major media have created serious scientific/medical and legal concerns about future of public health internationally 81 , 83 , 86 , 87 (manuscript in preparation). **** ' ††††The basis for the emergence of increased risk of pathogenic and retrovirus infections (eg, flu, HIV) or SARS, MERS, and coronavirus that created an urgent need for developing pathogen‐specific vaccines initiated questions on the potential presence of live pathogens (similar to SV‐40) in the media that vaccines were prepared and consumed by the public in the past three decades 1 , 20 (manuscript in preparation) ‡‡‡‡ 3.2 Ignoring while abusing evidence for link between inflammation and tumorigenesis and angiogenesis It is dangerous to be right, when the government is wrong . Voltaire Epidemiological reports on circumstantial evidence for an association between sites of prior injuries/chronic irritation or inflammation and the increased risk of cancer have been documented for a century. 1 , 5 , 8 , 10 , 41 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 In few such articles, professionals noted major gaps on direct evidence for a link between inflammation and induction of carcinogenesis and angiogenesis. Biological gaps were also noted on evidence for identifiable stages of immune alterations toward tumorigenesis or cancer. The cancer establishment has continued ignoring these important biological gaps on the initiation events that lead to immune dysfunction toward tumorigenesis and angiogenesis. Since 1998, analyses of data on our original studies that were conducted on experimental models of acute and chronic ocular inflammatory diseases, unexpectedly demonstrated a series of evidence that satisfied at least two of the major knowledge gaps on the role of inflammation in cancer immunobiology 1 , 5 , 8 , 20 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 (Figure 2 ): Evidence on direct association between inflammation and induction of tumorigenesis and angiogenesis; Time course kinetics of inflammation‐induced at least three identifiable phases of immune dysfunction toward multistep tumorigenesis; In 2014, further analyses of original data also revealed the first evidence on sequential interactions and synergies between activated host and recruiting immune and non‐immune cells in the direction of tumor growth. These data also incorporate the missing evidence on immune disruptor‐induced initial events in altering immune responses. 42 3.3 Author's accidental discoveries: Initiation events in tumorigenesis and angiogenesis‐ Direct evidence for a link between inflammation in multistep immune dysfunction. Intellectual challenges at NCI/NIH since 1998 (Intellectual Me Too!) Since 1998 at NCI/NIH, Khatami followed the logical, careful, and integrated approaches of Burnet that led him to the theory of immune surveillance. Analyses of original data that the author's team established at the University of Pennsylvania on experimental models of ocular acute and chronic inflammatory diseases in 1980s, led her to a series of reports that were suggestive of the first and only series of data on direct association between inflammation (initiation events) and multistep tumorigenesis and angiogenesis. 1 , 5 , 8 , 20 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 50 Further analyses and integration of data in the fields of immunology‐inflammation, cancer, and developmental biology, aging, biomarkers, molecular diagnosis and therapeutics led to recent definitions of inflammation for maintenance of health or induction of diseases. A number of concepts and comprehensive proposals were submitted in an effort to promote the role of inflammation in cancer research for early molecular diagnosis, design of clinical trials, use of patients' biospecimen, and potential agents (eg, SH‐containing agents, captopril, Sulindac, aspirin) for cancer chemoprevention. Khatami also developed a working project, standardizing cancer biomarkers criteria for developing effective databases for oncology research, using an inflammatory mediator (M‐CSF), as a prototype to tailor and test the sensitivity and specificity of M‐CSF, in comparison with conventional mediators (NCI‐Invention, Fed. Reg, 2005). 1 , 5 , 8 , 10 , 20 , 41 , 42 , 52 Decision makers and their handlers at NCI/NIH, severely opposed, ignored, denied, minimized, and rejected the submitted concepts and comprehensive proposals on the important role of inflammation in cancer research and therapy. However, in the last two decades, it seems that Khatami's challenging efforts awakened the entire cancer community around the world. Members of the establishment fragmented the submitted ideas and used them as a front for collecting more funding from the cancer‐stricken public. Significant increased funded projects focus on isolated numerous cellular and molecular aspects of inflammation‐immunity for cancer research and therapy; using site‐specific tumor models, expensive specific technologies, and related networks. However, the reductionist approaches on the topic of inflammation created further confusion and ongoing debates on what inflammation does, whether it prevents cancer or it causes cancer, 1 , 5 , 8 , 20 , 51 (NCI/NIH scientific and legal documents, since 1998). It is noteworthy that current literature in the field is flooded with hundreds of thousands of articles on the structures and substructures of numerous pathogens, their roles in experimental models of diseases; or numerous identified genetic mutations in cancer molecular tsunami to use specific expensive technologies for research, diagnosis or treatment and pathogen‐specific vaccine technologies. However, peculiarly, except for our accidental discoveries that were established in 1980s, very little is known about how stimuli (immune disruptors, pathogens) systematically would induce initiation processes in altering immune response dynamics toward time‐dependent multistage disease development. 4 INTELLECTUAL IGNORANCE THAT ABNORMAL GENE MUTATIONS IN CARCINOGENESIS ARE CONSEQUENCES OF SEVERELY DISTURBED IMMUNITY. IDENTIFYING ENDLESS MUTATIONS IN RESEARCH AND DRUG DEVELOPMENT THAT FAILED PATIENTS The truth is incontrovertible; malice may attack it, ignorance may deride it, but in the end, there it is . Winston Churchill Advances in technologies for identification and sequencing of genetic mutations and over‐ or under‐expression of gene products provide evidence that individual patients have different average rates of evolving mutations during examination and growth patterns of cancer mass. For example, patients with lung cancer demonstrate 200‐300 mutations per tumor, while patients with esophageal or colon cancer present 50‐100 DNA mutations per tumor. 1 , 5 , 8 , 11 , 20 , 49 The Cancer Genome Atlas (an NCI‐funded project) identified thousands of gene mutations in too many site‐specific cancers. Over 30,000 gene mutations are reported in breast cancer tissue alone. 1 , 11 , 54 The mutations that are identified in cancer molecular tsunami for the purpose of drug development and claims of "targeted" therapy, "precision," or "personalized" medicine, or recently fashionable 'immunotherapy' have repeatedly failed patients. Abnormal or excessive activation or deactivation of genomic pathways (eg, chromosomal, DNA/ RNA, hypo‐, hyper‐methylation and epigenomic modifications) and associated expression and co‐expression of tumoricidal or tumorigenic mediators, receptors and decoy molecules, enzymes/proteins/growth factors (eg, TNFRs, ILs, IRAK‐M, SODs, mTOR, FGF, MMPs, cMyc) are the results of overstimulation (exhaustion) of molecular components of the synchronized immune response dynamics; loss of electrobiology and skewed balance between tumoricidal (apoptosis, Yin or degeneration) and tumorigenic (wound healing, Yang, or regeneration) properties of immunity. The evolving mutational patterns and the number of mutations at specific cancer sites (cancer molecular tsunami) make such expensive projects, intellectually deceptive, worthless, and irresponsible and they are "scientific/medical Ponzi schemes." 1 , 5 , 8 , 10 , 11 , 49 , 50 , 51 Decision makers of such ill‐conceived and dangerous therapeutic projects who often use a combination of chemotherapy with whole or partial body radiation, totally disregard the important molecular compensatory mechanisms of immune responses toward inhibitors of apoptotic factors (eg, monoclonal antibodies, growth factors‐kinases inhibitors) against specific factors, enzymes or receptor molecules. 1 , 5 , 8 , 20 Other failed clinical trials include the use of hormone replacement therapy; finasteride (synthetic 4‐azasteroid) to inhibit type II‐5‐α‐reductase that converts androgen testosterone to 5‐α‐dihydrotestosterone; PSA measurement for prostate cancer therapy and diagnosis. 1 , 5 , 8 , 10 , 11 , 12 §§§§ Such therapies in an already immune‐compromised patient often induce immune tsunami (cytokine storm) in tissues/organs and lead to relapse, fatigue, cachexia, sarcopenia, thromboembolism, multiple organ failures (MOFs), and death. 1 , 8 , 10 , 11 , 20 The quality of blood (eg, fresh/young, frozen or old and storage procedures, using preservatives and duration of blood storage) that are employed for transfusion or iron supplementation in therapy‐induced anemia is also among factors that are often ignored or not reported in the literature. There are considerable differences in the outcomes of such procedures (eg, iron toxicities, "storage lessions," infections, complications with bleeding and thrombocytopenia, compatibility, age, and immune status of donor or recipient), 55 , 56 (manuscript in preparation). 5 AUTHOR'S SUMMARY OF ACCIDENTAL DISCOVERIES‐MULTISTEP IMMUNE DYSFUNCTION: SETTING STAGE FOR VACCINE‐INDUCED INJURIES It is better to deserve a prize and not have it, than to have a prize and not deserve it . Mark Twain Summary results of a series of our original studies and recent analyses of data are relevant when toxicities of vaccines and adjuvants are discussed. Guinea pig eyes were repeatedly stimulated and challenged with topical administration of FLOA (immune disruptor, antigen) with or without infective agents (A. Summ, parasite and extracts), adjuvants (pertussis), tumor‐promoting agents (TPAs) for up to 30 months. Major clinical, histopathological, and immunological findings and observations are summarized below 1 , 5 , 8 , 20 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 50 , 51 , 52 : Acute/Immediate, Self‐Terminating Responses (within 2 weeks of tissue sensitization/stimulation with antigen/stimulus): clinical strong or weak reactions associated with varying degrees of tissue edema, tearing, scratching and vascular hyperpermeability, induction of IgE antibody synthesis, release of histamine and prostaglandin (PGF‐1α) as first and secondary mediators; Intermediate Phase (Down‐Regulation Phenomenon) (within couple of mons of repeated stimulation of CALTs): clinical desensitization, heavy infiltration of eosinophils into epithelium, goblet/mucus secreting cells and ocular secretion; Chronic Phase (within 30 mons of tissue stimulation): induction of tumor‐like lesions, extensive angiogenesis, massive hyperplasia of lymphoid tissues (upper and lower bulbar conjunctiva, activation of macrophages, impaired boundary of lymphoid tissue and infiltration of different size lymphocytes into epithelial tissues, follicular formation, epithelial thickening and/or thinning (necrosis or growth often noted in the same tissue sections), increased MCs degranulation ('leaky' MCs), histiocytes (DCs) and lymphatic channel activations; Newborn Sensitivity toward Antigen Challenge : Preliminary observations demonstrated that newborn guinea pig eyes, born from sensitized parents responded to 1 st or 2 nd challenge by antigen, suggesting predisposition of fetus/unborn tissues, involving B/plasma cells and MCs sensitization/activation, through parental sensitization. These observations suggest that parental sensitization induced genetic mutations and increased (induced) allergies (inheritance, congenital or neonatal?) in unborn and newborn animals; Local and Distal Tissue Sensitization : Animals with strong ocular responses often presented wheezing‐like reactions, suggesting sensitization and activation of lung mast cells (preliminary observations); Mixture of Antigen with TPAs : Mixing antigen with tumor promoting agents (TPAs, phorbol esters) shifted induction of tumorigenesis‐ hyperplasia to earlier time‐frames (within 6 months) suggesting activation of kinases (preliminary observations) ***** . Extension and confirmation of these accidental discoveries and observations are among projects that perhaps deserve taxpayers' investment. Outcomes are expected to provide insightful understanding of short‐ or long‐term effects of oxidative stress, or toxicities of drugs and vaccines during pregnancy and after birth in induction of diverse immune disorders 1 , 5 , 20 , 58 (Figures 1 and 2 ) (see below). 5.1 Recent Definitions of Effective Immunity: Yin and Yang of Acute Inflammation: Vaccine‐Induced Injuries or Destruction of Natural Biorhythms Definitions of acute and chronic inflammation (Yin‐Yang) was first reported in 2008 43 and further extended since then. 1 , 5 , 8 , 20 , 24 , 42 , 43 , 44 , 47 , 50 , 51 Effective immunity was defined as the balance between two highly regulated and biologically opposing arms, termed the Yin (pro‐inflammatory, apoptosis, tumoricidal, degeneration) and the Yang (post‐inflammatory, growth promoting, wound healing, tumorigenic, regeneration) of self‐terminating properties of acute inflammation with dual or biphasic roles. Protective mechanisms of acute inflammation involve amazingly precise electro‐molecular (bioelectrical) signal transduction communications that are synchronized (time‐dependent electro chemical control switches with circadian behaviors) or autonomic sympathetic and parasympathetic activities between immune and non‐immune systems, involving innate and adoptive immune cells and vascular‐metabolic‐neuronal‐hormonal/endocrine‐lipids/adipocytes; or cell mediated and humoral immunity (CMI, HI). Self‐terminating properties of acute inflammation requires differential energy expenditure from mitochondrial oxidative phosphorylation (burst of energy, ATP hydrolysis) during Yin events; and low energy (ATP hydrolysis) from glycolysis (glucose metabolism) during Yang events. Dual processes in Yin‐Yang events would allow mitochondrial recovery and biosynthesis of TCA intermediates. Depending on the nature and potency of stimuli and susceptibility or type of host tissue (eg, immune‐responsive or immune‐privileged), Yin events involve generation of precise quantities of danger molecules, pro‐inflammatory cytokines/chemokines, oxidants and enzymes [eg, TLRs, vasoactive agents (histamine), NO, TNFα, PGs, neurotoxins, ILs, ROS, caspases/oxidases] from activated cells for the purpose of destroying internal or external foreign entities and injured host tissues. Immediately following Yin events, phenotypes of activated immune and non‐immune system with dual properties provide specific signals to express mediators with reducing or anti‐inflammatory properties for resolving inflammation and repairing, reconstructing or remodeling the injured host tissue. Yang pathways include expression of decoy receptor molecules, antioxidants, growth factors, hormones, enzymes and cytokines (eg, ILdRs, superoxide dismutases (SODs), kinases, IRAKM, TNFdRs, INFs, FGF, VEGF). 1 , 5 , 8 , 20 , 24 , 43 , 44 , 51 , 52 In general, the molecular/cellular components that make‐up the highly synchronized and controlled signal transduction mechanisms of effective immunity (CMI or HI), play dual roles during an inflammatory condition. For example, stimuli would induce activation of macrophages (MΦs, M1/tumoricidal and M2/tumorigenic phenotypes) or other antigen presenting cells (APCs). M1 phenotypes induce appropriate and often simultaneous electrochemical signals to express receptors/danger molecules, surface proinflammatory mediators, and activate T and B cells, vasculature, mucus‐secreting cells, as well as activation of metabolic and neuronal pathways. Major outcomes of stimuli‐induced activation of cells are burst of energy (ATP hydrolysis) from mitochondria (oxidative phosphorylation) accompanied by release of ROS and numerous other tumoricidal (Yin) mediators, to destroy unwanted agents and infected/injured host tissue. Following destruction of stimuli, simultaneously M2 and their counterparts in T (eg,Treg) and B cells, vasculature and metabolic‐neuronal systems, signal for shutdown/resting status of mitochondria (allowing regeneration of TCA cycle), and expression of tumorigenic (post‐inflammatory) mediators (Yang) to terminate and resolve inflammation, and to repair host tissue. Oxidative stress or unresolved (subclinical) inflammation or continuous stimulation of tissues was proposed, as a common denominator in initiation and progression of nearly all chronic diseases (eg, asthma, emphysema, hypertension, gastritis, colitis, thyroiditis, prostitis, atherosclerosis, multiple sclerosis, ALS, lupus, Alzheimer's, Parkinson's, obesity, adult‐ onset diabetes and cardiovascular complications, stroke and site‐specific cancers) that are often features of age‐associated illnesses. 1 , 5 , 8 , 20 , 42 , 43 , 44 , 49 , 51 , 52 , 53 Author's original concept on definitions of Yin and Yang properties of effective immunity seems to serve larger applications, than originally proposed, for understanding of the complex biphasic and synchronized activities of system biology (autonomic sympathetic and parasympathetic neuronal system) for maintenance of health or initiation of diseases. 1 , 5 , 20 Analyses and integration of a wide range of data on infections, drugs and vaccine‐related topics support a hypothesis that frequent infections, irritations and vaccination with pathogen‐specific vaccines and incipient, cause overstimulation (exhaustion) of mitochondria that would adversely influence the electrobiology of immune response profiles (Yin‐Yang) and pose diverse health consequences in young and old, particularly in the unborn, newborn, infant or immune‐compromised individuals (see below) 1 , 5 , 8 , 20 , 23 , 24 , 43 , 44 , 47 , 49 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 (Figures 1 , 2 , 3 ). FIGURE 3 Schematic representation of toxicities of vaccines altering fetus growth and development and bases for immediate or long‐term immune disorders, at different stages of life. The complex scheme represents that current pathogen‐specific vaccines (MMR, Hep, Flu, dTab, HPV, meningitis) and adjuvants [Al, Hg, detergents, solvents growth factors (PS80, Silica A, fetal serum)] would alter immune electromagnetic response profiles in tissues and damage differential bioenergetics of mitochondrial oxidative phosphorylation, leading to diverse immune disorders. It also depicts that vaccination of unborn/newborn and infants lead to shifted disease categories (congenital, hereditary, neonatal or induced) to increase the induced diseases (black box). See text 5.2 Development of mitochondria and effective immunity [Yin‐Yang] after birth: Thermodynamic laws of open access system in growth and development: A hypothesis Recently, we theorized that mitochondria and Yin arm of immunity are fully developed/functional after birth; when the newborn is exposed to atmospheric oxygen pressure and for completion of organ development; during which infant becomes independent from mother's immunity (within 2 years after birth). After birth and throughout adulthood and aging, the human body requires an effective immunity to combat diseases. The multi‐cellular complex signal communications of effective immunity require differential energy‐demand processes for tear (degeneration, Yin) and wear (regeneration, Yang) to effectively defend and maintain individual health (power within) against all elements that are perceived threatening body's survival (power without) 1 , 5 , 20 , 24 (manuscript in preparation). In a working model in an attempt to explain the complex electrobioloy and differential roles of mitochondria in autonomic neuronal sympathetic‐parasympathetic or on‐off signal switches of effective immunity, the author theorized that the law of thermodynamics of open systems would apply, in varying degrees, to the growth patterns of human biology; from orderly growth and development of fetus, to adulthood, aging and disease processes. 5 , 20 During fetus growth, except for those events that are required for organogenesis and angiogenesis, occurring under low/limited oxygen pressure of protective environment of placenta, Yin arm of immunity and mitochondria are not fully developed and not required. Otherwise, oxidative stress and expression of apoptotic factors could result in fetus abortion, preterm birth, low growth rate (low weight at birth), retardation or defects in fetus organ development or childhood cancers. 5 , 20 As detailed below, a potential factor in reported increased in childhood diseases or cancers or SIDs are presence of oxidative stress‐ (eg, vaccination of pregnant woman) and expression of exaggerated wound healing or apoptotic factors that are likely to alter/skew expression of constituent vs. induced receptors and adversely affect growth of newborn, immediately after birth or later on during adulthood or aging process 1 , 5 , 20 (manuscript in preparation)] (Figures 1 and 3 ). Immunologically, one may argue that exposure of unborn, newborn and infant to even 'safe vaccines' or other biologics (stimuli) could retard‐impair and threaten proper development of mitochondria and tissues/organs (eg, lung, kidneys, liver, brain, reproductive system) causing immediate‐short‐, or long‐term health consequences. As shown below, pathogen‐specific vaccines (eg, polio, swine flu, hepatitis a, b, c, MMRs, meningitis, shingles, anthrax, pertussis, HPV, SARS, Ebola, Zika, or 'covid‐19') and incipient weaken/destroy the complex electrobiology of immunity and Not Promote It !. 1 , 5 , 8 , 11 , 17 , 20 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 5.3 Low energy consuming arm of immunity from glycolysis (Yang): Vaccination during pregnancy disturb delicate biology of trophoblast‐placenta and orderly fetus growth. Hypothesis Orderly growth of fetus or disorderly growth of cancer masses requires low energy consumption from glycolysis (Warburg effect); where growth processes occur under low oxygen tension. The orderly growth of fetal mass was suggested to be peculiarly comparable to the disorderly growth of cancer cells having undeveloped or dysfunctional mitochondria, respectively. 1 , 5 In brief, after birth the dual capacity of effective immunity (Yin‐tumoricidal v. Yang‐tumorigenic) and mitochondrial function are required for differential energy consumption and time‐dependent electrochemical signals (synchronized on/off switches) for maintenance of health. Protection of complex electrobiology of immunity (immune neuroplasticity) that parallels neuronal behaviors (autonomic sympathetic and parasympathetic) is the most important aspect of human health and well being. 5.1 Recent Definitions of Effective Immunity: Yin and Yang of Acute Inflammation: Vaccine‐Induced Injuries or Destruction of Natural Biorhythms Definitions of acute and chronic inflammation (Yin‐Yang) was first reported in 2008 43 and further extended since then. 1 , 5 , 8 , 20 , 24 , 42 , 43 , 44 , 47 , 50 , 51 Effective immunity was defined as the balance between two highly regulated and biologically opposing arms, termed the Yin (pro‐inflammatory, apoptosis, tumoricidal, degeneration) and the Yang (post‐inflammatory, growth promoting, wound healing, tumorigenic, regeneration) of self‐terminating properties of acute inflammation with dual or biphasic roles. Protective mechanisms of acute inflammation involve amazingly precise electro‐molecular (bioelectrical) signal transduction communications that are synchronized (time‐dependent electro chemical control switches with circadian behaviors) or autonomic sympathetic and parasympathetic activities between immune and non‐immune systems, involving innate and adoptive immune cells and vascular‐metabolic‐neuronal‐hormonal/endocrine‐lipids/adipocytes; or cell mediated and humoral immunity (CMI, HI). Self‐terminating properties of acute inflammation requires differential energy expenditure from mitochondrial oxidative phosphorylation (burst of energy, ATP hydrolysis) during Yin events; and low energy (ATP hydrolysis) from glycolysis (glucose metabolism) during Yang events. Dual processes in Yin‐Yang events would allow mitochondrial recovery and biosynthesis of TCA intermediates. Depending on the nature and potency of stimuli and susceptibility or type of host tissue (eg, immune‐responsive or immune‐privileged), Yin events involve generation of precise quantities of danger molecules, pro‐inflammatory cytokines/chemokines, oxidants and enzymes [eg, TLRs, vasoactive agents (histamine), NO, TNFα, PGs, neurotoxins, ILs, ROS, caspases/oxidases] from activated cells for the purpose of destroying internal or external foreign entities and injured host tissues. Immediately following Yin events, phenotypes of activated immune and non‐immune system with dual properties provide specific signals to express mediators with reducing or anti‐inflammatory properties for resolving inflammation and repairing, reconstructing or remodeling the injured host tissue. Yang pathways include expression of decoy receptor molecules, antioxidants, growth factors, hormones, enzymes and cytokines (eg, ILdRs, superoxide dismutases (SODs), kinases, IRAKM, TNFdRs, INFs, FGF, VEGF). 1 , 5 , 8 , 20 , 24 , 43 , 44 , 51 , 52 In general, the molecular/cellular components that make‐up the highly synchronized and controlled signal transduction mechanisms of effective immunity (CMI or HI), play dual roles during an inflammatory condition. For example, stimuli would induce activation of macrophages (MΦs, M1/tumoricidal and M2/tumorigenic phenotypes) or other antigen presenting cells (APCs). M1 phenotypes induce appropriate and often simultaneous electrochemical signals to express receptors/danger molecules, surface proinflammatory mediators, and activate T and B cells, vasculature, mucus‐secreting cells, as well as activation of metabolic and neuronal pathways. Major outcomes of stimuli‐induced activation of cells are burst of energy (ATP hydrolysis) from mitochondria (oxidative phosphorylation) accompanied by release of ROS and numerous other tumoricidal (Yin) mediators, to destroy unwanted agents and infected/injured host tissue. Following destruction of stimuli, simultaneously M2 and their counterparts in T (eg,Treg) and B cells, vasculature and metabolic‐neuronal systems, signal for shutdown/resting status of mitochondria (allowing regeneration of TCA cycle), and expression of tumorigenic (post‐inflammatory) mediators (Yang) to terminate and resolve inflammation, and to repair host tissue. Oxidative stress or unresolved (subclinical) inflammation or continuous stimulation of tissues was proposed, as a common denominator in initiation and progression of nearly all chronic diseases (eg, asthma, emphysema, hypertension, gastritis, colitis, thyroiditis, prostitis, atherosclerosis, multiple sclerosis, ALS, lupus, Alzheimer's, Parkinson's, obesity, adult‐ onset diabetes and cardiovascular complications, stroke and site‐specific cancers) that are often features of age‐associated illnesses. 1 , 5 , 8 , 20 , 42 , 43 , 44 , 49 , 51 , 52 , 53 Author's original concept on definitions of Yin and Yang properties of effective immunity seems to serve larger applications, than originally proposed, for understanding of the complex biphasic and synchronized activities of system biology (autonomic sympathetic and parasympathetic neuronal system) for maintenance of health or initiation of diseases. 1 , 5 , 20 Analyses and integration of a wide range of data on infections, drugs and vaccine‐related topics support a hypothesis that frequent infections, irritations and vaccination with pathogen‐specific vaccines and incipient, cause overstimulation (exhaustion) of mitochondria that would adversely influence the electrobiology of immune response profiles (Yin‐Yang) and pose diverse health consequences in young and old, particularly in the unborn, newborn, infant or immune‐compromised individuals (see below) 1 , 5 , 8 , 20 , 23 , 24 , 43 , 44 , 47 , 49 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 (Figures 1 , 2 , 3 ). FIGURE 3 Schematic representation of toxicities of vaccines altering fetus growth and development and bases for immediate or long‐term immune disorders, at different stages of life. The complex scheme represents that current pathogen‐specific vaccines (MMR, Hep, Flu, dTab, HPV, meningitis) and adjuvants [Al, Hg, detergents, solvents growth factors (PS80, Silica A, fetal serum)] would alter immune electromagnetic response profiles in tissues and damage differential bioenergetics of mitochondrial oxidative phosphorylation, leading to diverse immune disorders. It also depicts that vaccination of unborn/newborn and infants lead to shifted disease categories (congenital, hereditary, neonatal or induced) to increase the induced diseases (black box). See text 5.2 Development of mitochondria and effective immunity [Yin‐Yang] after birth: Thermodynamic laws of open access system in growth and development: A hypothesis Recently, we theorized that mitochondria and Yin arm of immunity are fully developed/functional after birth; when the newborn is exposed to atmospheric oxygen pressure and for completion of organ development; during which infant becomes independent from mother's immunity (within 2 years after birth). After birth and throughout adulthood and aging, the human body requires an effective immunity to combat diseases. The multi‐cellular complex signal communications of effective immunity require differential energy‐demand processes for tear (degeneration, Yin) and wear (regeneration, Yang) to effectively defend and maintain individual health (power within) against all elements that are perceived threatening body's survival (power without) 1 , 5 , 20 , 24 (manuscript in preparation). In a working model in an attempt to explain the complex electrobioloy and differential roles of mitochondria in autonomic neuronal sympathetic‐parasympathetic or on‐off signal switches of effective immunity, the author theorized that the law of thermodynamics of open systems would apply, in varying degrees, to the growth patterns of human biology; from orderly growth and development of fetus, to adulthood, aging and disease processes. 5 , 20 During fetus growth, except for those events that are required for organogenesis and angiogenesis, occurring under low/limited oxygen pressure of protective environment of placenta, Yin arm of immunity and mitochondria are not fully developed and not required. Otherwise, oxidative stress and expression of apoptotic factors could result in fetus abortion, preterm birth, low growth rate (low weight at birth), retardation or defects in fetus organ development or childhood cancers. 5 , 20 As detailed below, a potential factor in reported increased in childhood diseases or cancers or SIDs are presence of oxidative stress‐ (eg, vaccination of pregnant woman) and expression of exaggerated wound healing or apoptotic factors that are likely to alter/skew expression of constituent vs. induced receptors and adversely affect growth of newborn, immediately after birth or later on during adulthood or aging process 1 , 5 , 20 (manuscript in preparation)] (Figures 1 and 3 ). Immunologically, one may argue that exposure of unborn, newborn and infant to even 'safe vaccines' or other biologics (stimuli) could retard‐impair and threaten proper development of mitochondria and tissues/organs (eg, lung, kidneys, liver, brain, reproductive system) causing immediate‐short‐, or long‐term health consequences. As shown below, pathogen‐specific vaccines (eg, polio, swine flu, hepatitis a, b, c, MMRs, meningitis, shingles, anthrax, pertussis, HPV, SARS, Ebola, Zika, or 'covid‐19') and incipient weaken/destroy the complex electrobiology of immunity and Not Promote It !. 1 , 5 , 8 , 11 , 17 , 20 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 5.3 Low energy consuming arm of immunity from glycolysis (Yang): Vaccination during pregnancy disturb delicate biology of trophoblast‐placenta and orderly fetus growth. Hypothesis Orderly growth of fetus or disorderly growth of cancer masses requires low energy consumption from glycolysis (Warburg effect); where growth processes occur under low oxygen tension. The orderly growth of fetal mass was suggested to be peculiarly comparable to the disorderly growth of cancer cells having undeveloped or dysfunctional mitochondria, respectively. 1 , 5 In brief, after birth the dual capacity of effective immunity (Yin‐tumoricidal v. Yang‐tumorigenic) and mitochondrial function are required for differential energy consumption and time‐dependent electrochemical signals (synchronized on/off switches) for maintenance of health. Protection of complex electrobiology of immunity (immune neuroplasticity) that parallels neuronal behaviors (autonomic sympathetic and parasympathetic) is the most important aspect of human health and well being. 6 VACCINE SCIENCES: CURRENT IMMUNOLOGICAL SAFETY CONCERNS Be a yardstick of quality. Some people aren't used to an environment where excellence is expected . Steve Jobs The concept of vaccination, or rather immunization, for protecting, promoting and defending individual health against viruses, bacteria or parasites developed in the eighteenth century, well before the important theory of immune surveillance was developed, and before better hygiene or antibiotics improved public health and reduced many preventable infectious diseases and increased longevity. The concept of protecting public health by immunization also existed before Rockefeller patent medicine and Gates patent vaccines invested a great deal in medical education to influence promotion of drug sale and to vaccinate the public with pathogen‐specific vaccines in toxic media and associated debates and controversies. 1 , 2 , 4 , 5 , 12 , 13 , 17 , 18 , 20 , 21 , 22 , 23 , 24 , 28 , 30 , 31 , 32 , 33 , 36 , 37 , 38 , 39 , 40 , 59 , 60 , 61 , 62 , 63 , 66 , 67 , 68 , 70 , 73 , 77 , 83 , 93 , 94 , 96 , 97 , 110 , 111 , 116 , 128 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 146 , 147 , 148 , 149 , 150 , 156 , 158 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 168 , 169 , 170 , 171 , 172 , 174 , 175 , 181 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 205 , 206 , 212 Seven/eight decades ago, vaccines were considered relatively safe and effective in promoting/boosting immunity and preventing diseases when healthy children (2 years or older) were vaccinated with few dead/inactivated pathogens (eg, measles, mumps, diphtheria, smallpox) that were prepared in saline solutions. The overall review of data on epidemiological studies and/or comparison of vaccinated and unvaccinated children at different settings around the globe, despite variations in methods and procedures, suggest that natural exposures to infective agents (eg, measles and mumps) are associated with lower rates of mortality from chronic diseases such as atherosclerotic and cardiovascular diseases 31 , 174 †††††. In general, outcomes of an acute inflammation (eg, responses to infective agents) are lymphocyte‐derived clonal expansion, increased synthesis of pathogen‐(or allergen) specific antibodies and memory cells. 1 , 5 , 8 , 12 , 20 , 24 , 25 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 Synthesis of antibodies (eg, IgGs, IgE, IgA, IgM) and memory cells (B/plasma or T cells) are needed for priming the immune system (boosting immunity). Upon next exposure to similarly structured infective agents, the host's primed immune system unleashes appropriate and precise quantities of required neutralizing antibodies and pro‐, and anti‐inflammatory mediators [eg, TLRs, vasoactive agents (histamine), cytokines, oxidants, enzymes, neurotoxins or growth factors and antioxidants] to destroy pathogens and injured/infected tissues and also to repair and remodel infected host tissues (see above). Therefore, occasional exposures of healthy children to infective agents are expected to boost natural immunity and prevent many diseases throughout life. 1 , 5 , 8 , 20 Even occasional exposure of healthy adults to potent pathogens (eg, meningococcal, coronavirus) is likely to protect the body from cardiovascular and respiratory diseases or cancer, if the victims successfully survive pathogen‐induced cytokine storms (immune tsunami or exaggerated immune responses) that are expressed against such pathogens, 1 , 5 , 20 , 24 (unpublished observations). In brief, it takes approximately 2 years for newborn‐infant to be immunologically independent from mother's immunity. Newborn's exposure to oxygen pressure and parallel completions of mitochondria and organ development and Yin (tumoricidal) arm of immunity provide the growing baby the required natural protection (defending capacity, power within) against external and internal foreign elements for maintenance of health. 1 , 5 We also proposed that after birth, the Yin arm of effective immunity is required for metabolism of essential branched amino acids (eg, val, leu, isoleu) for protein biosynthesis and structural integrities of tissues and mitochondrial function. 1 , 5 , 20 7 VACCINE TOXICITIES: RETARDATION OF MITOCHONDRIA AND IMMUNE RESPONSE PROFILES. SEEDS OF IMMUNE DESTRUCTION AND INDUCTION OF MILD, MODERATE OR SEVERE IMMUNE DISORDERS IN UNBORN, NEWBORN, INFANT, CHILDREN AND ADULTS As noted above, the current emphasis to frequently vaccinate public with pathogen‐specific vaccines that are prepared in toxic incipient/media are likely the major factors in causes, exacerbation and consequences of impaired (retarded) mitochondrial function and Yin (tumoricidal) capacities of effective immunity in reported increased disease status in young and old in America. Stimuli (vaccines)‐induced oxidative stress and suppression of immunity are likely the major risk factors in significant increase in allergies and other immune disorders (eg, asthma, autism, tics and Tourette's syndrome, hot flashes, fatigue, epilepsy, vasculitis, urticaria, pancreatitis, obesity, type I or II diabetes and cardiovascular complications, anemia, stroke, encephalitis and other neurodegenerative and autoimmune diseases or cancers) and impaired (lowered) fertility rates in younger generations in America 1 , 5 , 8 , 12 , 13 , 14 , 17 , 19 , 20 , 21 , 22 , 24 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 36 , 37 , 38 , 39 , 40 , 49 , 50 , 51 , 52 , 53 , 57 , 116 , 117 , 176 , 192 , 220 (Figures 3 and 4 ). FIGURE 4 Schematic representation of toxicities of vaccines and incipient in altering mitochondrial function and diverse activities of histidine‐histamine pathways toward increased induction of diseases. Vaccine incipient/excipient (eg, metals, growth factors, DNA/RNA, and fetal tissues) are depicted to alter immune‐neuronal response dynamics, influencing genomic, mitochondrial, metabolic, and physiological functions of gastric secretion, energy levels (ATP/ADP), cell cycle and brain activities as well as vasculature, tissue growth or necrosis. The scheme depicts that 140–200 genes are involved in histamine‐histidine metabolic‐neuro‐immune pathways. Altered tissue bioenergetics is depicted to cause induction of mild, moderate, or severe immune disorders (black box). The scheme also represents that current vaccines shift the incidence of all classically known diseases (congenital, hereditary, neonatal, or induced) to increase the level of induced diseases. See text 7.1 Debates and controversies on reported safety of vaccines and incipient Analyses of data on vaccines and the impact on health of the unborn/neonatal, newborn and infant include review of documents on regulatory governmental agencies (eg, FDA, CDC, WHO), Public Health Informatics Network, American Medical Informatics Association, National Animal Health Management Emergency Management System and USDA, information on funding support from government, industry and 'philanthropists' to study and promote vaccines, manufacturers' inserts on vaccines contraindications, reports on spontaneous electronic adverse events on cancer drugs and vaccines, websites and blogs as well as, congressional debates, legal and financial incentives to professionals for promoting and publicizing vaccines, experts depositions in vaccine injury courts and awards to vaccine injured individuals 1 , 5 , 18 , 64 , 77 , 94 , 136 , 137 , 140 , 141 , 142 , 143 , 144 , 169 , 172 , 175 , 176 , 177 , 178 , 179 ‡‡‡‡‡ ' §§§§§ , ****** ' ††††††' ‡‡‡‡‡‡ . Unfortunately, governmental guidelines, particularly on cancer or vaccine‐related statistics, safety procedures, vaccine effectiveness or reported injuries are provided on behalf of medical establishment with little independent evaluation, considerations or validation of biological sciences, medical ethics and conflicts of interest, or safety considerations. The official guidelines by governmental agencies are skewed, biased and often laced with deception and cover ups on disease causes (eg, SIDS, vaccine injuries, drug toxicities, clinical trials on exclusion/inclusion criteria). Often official information is not worthy of serious considerations once the scientific pros and cons are weighed, particularly regarding information on root causes of cancer epidemics, over‐, under‐diagnosis of diseases, toxicities of drugs, biologics or vaccines safety 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 17 , 20 , 21 , 22 , 23 , 50 , 51 , 52 , 64 , 69 , 71 , 93 , 136 , 137 , 140 , 141 , 157 , 164 , 177 , 192 §§§§§§ , ******* , †††††††, ‡‡‡‡‡‡‡ , §§§§§§§ . Often, credible scientific information that is prepared by scientists who work in the government or academia are ignored, dismissed or abused when members of the establishment decide to publicize drugs or vaccines of their choice. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 11 , 12 , 13 , 17 , 18 , 34 , 36 , 37 , 38 , 39 , 64 , 69 , 71 , 77 , 94 , 143 , 149 , 169 , 172 , 176 , 178 , 180 , 181 , 182 , 183 , 184 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 In general, diversities and extent of immune disorders [acute inflammatory responses, delayed hypersensitivity reactions, mild (subclinical inflammation, oxidative stress), moderate or severe diseases] that occur immediately after vaccination or within few days‐weeks‐ months or years later, depend on several interdependent factors as outlined below (Figures 1 , 2 , 3 , 4 ): Age of vaccinated individual; Health status of vaccinated individual; Dosage, frequency and rout of vaccination; Period between subsequent vaccination; Composition of media/adjuvants (incipient) in vaccines; Nature (quality/composition, potency) of pathogenic particles in vaccines; Quality control (placebo) status of vaccines, procedures and standard tests for safety before public consumption; Quality of clinical trials (eg, inclusion or exclusion criteria, crossover trials and safety recommendations and approval of vaccines); Time course on reported vaccine injuries and follow up on inclusion of VAERS; Flu shot ingredients, given to pregnant women could disturb the intricate biological networks of trophoblast‐embryo‐fetus‐placenta that are required for orderly growth of fetus. Under incomplete mitochondrial development and hypoxic conditions of placenta, organogenesis and angiogenesis of embryo‐fetus require proper architectural organization and functioning of trophoblast epithelium for providing and consuming appropriate and sufficient growth factors/hormones, nutrients, enzymes, trace elements (metals) and respective constituent or induced receptor molecules. Exposure of the embryo‐fetus to flu shot and incipient, could pose serious threats to survival and health of both mother and fetus including altered nutrients in organogenesis, transition of myoblasts to myotube development, ratios of constituent/induced receptors, as potential contributors in growth retardation or growth promotion, fetus abortion or impaired health of newborn, infant, toddler, that also influence immunity during adulthood and aging process 1 , 5 , 8 , 20 , 24 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 129 , 192 (manuscript in preparation)] (Figures 1 , 3 and 4 ). Examples of deception that are frequently applied to the safety of drugs or vaccines are controversies on exclusion criteria or crossover practices in conducting clinical trials for obtaining approval of drugs or vaccines. 11 , 113 , 139 , 140 , 141 , 187 , 189 , 190 , 191 , 196 , 197 In the majority of clinical trials, decision makers select and recruit healthy individuals for testing safety of drugs or vaccines. Any individual with minor or major illnesses are excluded. This allows manufacturers to show maximum benefits and minimum harms of tested biologics. However, even under such selective criteria, healthy participants often experience various degrees of side‐effects that may or may not be acknowledged or documented during the marketing of such drugs. Vaccine approval voting is conducted through FDA (eg, VRBPAC) or CDC (eg, ACIP) committees, whose members are often industry, government employers or grantees (principal investigators) who receive funding to study and patent drugs/vaccines and to collaborate with manufacturers for large scale development and for marketing to the general public, healthy or not. 2 , 8 , 18 , 70 An example of vaccine propaganda is found in the marketing of HPV vaccines (Gardasil or Cervarix) that were approved to target the young generation, claiming to prevent cervical cancer. Segments of papilloma virus (types 6,11,18) and recombinant DNA technologies are used in media/incipient that has combination of Al, PS80, SIO2, Saponin. 8 , 12 , 14 , 19 , 24 , 110 , 131 , 132 , 133 , 134 , 137 , 138 , 139 , 143 Review of related data suggest that HPV vaccines and adjuvants are associated with mild or severe adverse reactions (VAERS), including autoimmune diseases, fibromyalgia, tachycardia, ovarian failure, fatigue, without any benefits in preventing incidences of cervical and related cancers 8 , 12 , 14 , 66 , 70 , 71 , 108 , 110 , 136 , 137 , 138 , 139 , 143 , ††††††††' ‡‡‡‡‡‡‡‡ . In a comprehensive review 14 Giannotta and Giannotta described the mechanisms of adverse effects of vaccines (eg, HPV) and incipient in autonomic neuronal system and development of autism spectrum disorder (ASD), fatigue and vaccine‐induced altered behaviors of immune cells (microglial and astrocytes) in the brain and associated loss of BBB. 7.2 Vaccines incipient (ingredient, adjuvant, excipient): Mini electric shocks and molecular sink holes in mitochondria with loss of biorhythms and induction of diseases Inactivated (or live) pathogens in vaccines, on their own, are immunogens (stimuli or immune disruptors) and could over‐stimulate immunity. According to manufacturer's inserts, governmental or published scientific data, majority of pathogen‐specific vaccines are prepared in media containing combinations of metals, chemicals‐biological agents such as aluminum (Al, as hydroxide or phosphate salts), mercury (Hg, thimerosal), detergents, solvents or preservatives [eg, CTAB, polymyxin, neomycin, saponin, formaldehyde, silica and derivatives, solutes (sorbitol, polysorbate 80 or 20, Tween 20), glyphosate‐herbicide, octylphenol ethoxylate or octoxynol‐10 (Triton X‐100)], genetically engineered DNA/RNA, yeast extracts, fetal tissues and organ parts or fragments 12 , 14 , 24 , 38 , 67 , 73 , 74 , 77 , 94 , 96 , 128 , 130 , 131 , 132 , 141 , 144 , 148 , 152 , 153 , 156 , 165 , 167 , 168 §§§§§§§§ ' ********* . Majority of these vaccine ingredients are not natural agents and do not participate in biochemical pathways in human physiology. These ingredients are additional foreign agents that overwhelm the immune system (see below). The ingredients that are perceived as 'natural' [eg, fetal serum, clumps of tissue/organ or DNA particles, proteins‐peptides (eg, ovalbumin, egg proteins, serum albumin, hydrolyzed porcine gelatin), amino acids (arginine, glutamate, or L‐histidine)] could disturb physiological activities and immune responses, particularly affecting the growing embryo‐fetus (unborn/neonate), newborn and infant whose organ systems, gut microbiome composition and immunity are not fully developed, or individuals who are immune compromised 1 , 4 , 5 , 8 , 12 , 14 , 17 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 50 , 58 , 61 , 62 , 63 , 64 , 66 , 67 , 68 , 70 , 71 , 141 , 144 , 148 , 152 , 153 , 156 , 165 , 167 , 168 (Figures 3 , 4 , 5 ). FIGURE 5 Schematic representation of toxicities of metals and other ingredients in pathogen‐specific vaccines in induction of mild, moderate, or severe immune disorders. Metals such as aluminum (Al) or mercury (Hg) are depicted to compete with essential trace elements and alter tissue biological activities (see blue box and center divided circle), influencing mitochondrial bioenergetics metabolism and immune response profiles in tissues. The scheme depicts that vaccines and ingredients differentially influence tissues that are immune‐privileged (brain, CNS, reproductive organs) or immune‐responsive (epithelial, endothelial, mucus, fibroblast) in the genesis of wide ranges of immune disorders (tissue necrosis or growth). See text 7.3 Toxicities of metals in vaccines: Mini electric shocks in mitochondria altering electrobiology of vascular‐metabolic‐neuronal‐hormonal pathways Aluminum (Al and its salts) or mercury (Hg, thimerosal) possess inert properties in nature, with cationic capabilities (eg, Al +3 , Hg +2 ) to interact with charged molecules and act as electronic magnets. Biologically, presence of these metals in injected vaccines could compete, scavenge (chelate) or act as cationic sinks and damage the function of essential metals and trace elements (Fe +2 , Fe +3 , Cu +1 , Cu +2 , Zn +2 , Ca +2 , Mg +2 , Se +2 ) that are required for a wide range of cellular functions. In general, presence of Al or Hg in vaccines is likely to interfere with required proton pumping and maintenance of differential electronic charges across cellular components. Among numerous cellular functions that are likely influenced by the presence of Al or Hg are transport and function of intra‐, extra‐cellular charged proteins, amino acids and cationic‐anionic trace elements across membranes [eg, ATP/ADP/AMP, Na + /K + exchanger, Na + /H + exchanger, water channels, active/passive transport of solutes, osmolytes or nutrients (eg, myo‐inositol, pyridoxal phosphate, ascorbic acid)]. Overview and integration of fragmented data on vaccine‐related topics, vaccine injuries and inflammatory/immune disorders suggest that presence of non‐functional metals in vaccines create mini‐electronic shocks or 'molecular sink holes' and induce biological defects in mitochondria, membranes and chromosoms, damaging the regulations of biological activities in tissues including alterations of gut microbiome profiles and neuronal behaviors (Figures 1 , 3 – 5 ) 1 , 5 , 14 , 36 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 (manuscript in preparation)] †††††††††, ‡‡‡‡‡‡‡‡‡ . For example, copper (Cu 1+ or Cu 2+ ) is an essential trace element (cation) and plays crucial roles as a cofactor in mitochondrial cytochrome C oxidase and numerous other biological activities including neuronal function, wound healing, biosynthesis of collagen and vasculature. Normally, copper ions are bound to carrier molecules and distributed via carrier proteins ('copper chaperones') for protecting/preventing tissue damage. 36 , 72 , 84 , 85 , 91 , 92 , 153 , 183 Excess amount of copper (free) could be detrimental to respiratory chain reactions and generation of toxic hydroxyl radicals (HO 0 ) causing oxidative damage not only to mitochondria, but also other extracellular‐intracellular proteins or nucleic acids and lipids (Fenton reaction) and tissue oxido‐redox potentials. 1 , 5 , 14 , 20 , 29 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 53 , 55 , 72 , 73 , 78 , 84 , 85 , 88 , 89 , 90 , 91 , 92 , 155 , 182 , 183 Furthermore, copper and Zn +2 (another trace element and antioxidant) are involved in detoxifying mitochondrial ROS and superoxide dismutase1 (SOD1) activities, regulation of Cu‐mediated production of O 2 /ROS. Related studies suggest that trace elements (Cu, zinc, Fe), influence regulations of transport of triglycerides in gastrointestinal tract and are important in the function of red blood cells and endoplasmic reticulum activities. While mechanisms of toxicities or interactions between Al or Hg and trace elements on biological pathways are not well understood, it is highly likely that tissues are sensitive to such metals in vaccines, particularly affecting tissue bioenergetics (Figures 3 , 4 , 5 ). The presence of Al or detergents in vaccines could induce retardation/overload of mitochondria, causing elevated levels of Cu 2+ in tissues (eg, liver) that would lead to vaccine‐induced respiratory or neuronal illnesses (eg, Wilson and other mitochondrial diseases). 1 , 29 , 36 , 39 , 40 , 48 , 49 , 53 , 54 , 55 , 56 , 57 , 74 , 75 , 114 , 151 , 163 , 183 Injected vaccines containing Al or Hg, at various stages of pregnancy, could retard fetus growth and development involving important generation, utilization, or recycling of glutathione‐related pathways (GSH: GSSH and NAD + : NADH) and further altering oxido‐redox potentials and incomplete mitochondria and organ development, growth impairment, immune and mitochondrial diseases after birth. 1 , 5 , 90 , 102 , 114 , 117 , 118 , 151 , 152 , 153 , 154 , 155 , 163 , 182 In a detailed retrospective epidemiological study, using automated Vaccine Safety Datalink (VSD), Young et al 36 reported that vaccines containing Hg (thimerosal) were associated with neurological developmental disorders (eg, anaphylaxis, autism, ASD, tics, attention deficit disorder, and emotional disturbances) in newborn (7 and 13 months old) perhaps, due to mitochondrial dysfunction. Evaluation of data on reported vaccine injuries (eg, fatigue, hypotonia, neuropathological episodes of epilepsy, Rett syndrome or encephalomyopathy, or cancers) suggest a range of electrochemical signal defects in the function of B/plasma cells, receptor/surface molecules that could retard and alter, among other pathways, memory B cells complexes, expression of immunoglobulins, mutations in mitochondrial complexes (I, II, III, or IV), related genetic/epigenetic modifications as contributing factors in impairing pathways of oxidative phosphorylation (mitophagy), autophagy and altered endoplasmic reticulum. 1 , 5 , 14 , 90 , 102 , 114 , 115 , 116 , 117 , 118 , 151 , 152 , 153 , 154 , 155 , 163 , 166 , 182 , 183 7.4 Presence of l ‐histidine in vaccines: histamine‐histidine interface‐exchange Histidine is a natural and essential amino acid (nutrient) and structural backbone of a variety of important proteins and enzymes with diverse biological functions. With its unique imidazole side chain, histidine plays critical roles in immune response dynamics, associated with renal, neuronal, ocular and gastrointestinal biological activities. There are up to 200 genes that mediate activities of histdine‐containing proteins, such as histidine metabolic enzymes (eg, histidine decarboxylase [HDC], amino oxidase [AO]), carrier proteins, and chelating agents of trace elements (eg, Zn). Analyses of data on PTH1 or PHT1 and histidine‐histamine homeostasis and histamine receptors in neuronal tissues (eg, brain) suggest that presence of l ‐His, together with Al or Hg, in vaccines alter histidine‐histamine ratio and neuropeptide regulation, particularly affecting the developing brain of fetus or newborn. 5 , 19 , 20 , 36 , 38 , 41 , 67 , 79 , 95 , 96 , 105 , 122 , 123 , 124 , 125 , 126 , 127 , 150 , 176 , 177 , 186 , 188 , 194 In addition to its role in neuronal tissues histamine (catecholamine, an alkali, a potent vasoactive agent) acts as a key element and the most versatile biogenic amine, having diverse and antagonistic properties in mammalian physiology. Histamine is synthesized by enzyme histidine decarboxylase (HDC), an enzyme present in all tissues. Histamine and its receptor molecules (HRs 1–4 and subfamilies) play diverse roles in human development, acute and chronic inflammation, acid‐base balance, digestion, mucosal activities, vascular function and permeability, neuronal activities as well as, growth of cancer mass. 1 , 5 , 20 , 95 , 124 , 126 , 127 , 150 , 199 , 200 , 201 , 202 , 203 Results of our original studies on inflammation‐induced multistep tumorigenesis and angiogenesis led to recent hypotheses that low level release of histamine (independent from IgE‐fcR degranulation of MCs ["leaky" or exhausted MCs]) is a factor in induction of tissue/cell growth that alter immune responses, including induction of autoimmune and neurodegenerative diseases or tumorigenesis and angiogenesis. 1 , 5 , 8 , 20 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 52 Summary of diverse biological activities that are affected by abnormal levels (deficiencies or excesses) of histidine‐histamine in tissues, including altered nutritional intake, genetic mutations in production or metabolism of histidine or histamine are provided below 1 , 5 , 20 , 41 , 42 , 43 , 44 , 46 , 79 , 95 , 105 , 106 , 109 , 124 , 126 , 127 , 128 , 148 , 152 , 172 , 176 , 177 , 200 , 201 , 202 , 203 (Figures 4 and 5 ): Early embryonic‐fetus growth, organogenesis‐angiogenesis; Vasculature, innate immune cells (eg,MCs) and neuronal activities after birth; DNA transcription involving Zn‐imidazole at active sites of enzymes (eg, carbonic anhydrase‐CA); Hymolytic and redox reactions; Adenosyl methionine and ATP binding site of actin; Hydroxylation of galactosylceramide and maintenance of myelin sheath structure; Thyrotropin‐releasing hormone; Serine esterase activities of trypsin, chymotrypsin; acetylcholinesterase and blood clotting and complement cascades; Food or PH‐induced gastrin release of histamine from enterochromamaffin‐like cell (ECL) and activation of HDC; Induction of tissue growth, tumorigenesis, angiogenesis, and cancer; Among major histidine‐histamine‐associated diseases are histinemia, kidney disease, anemia, and cancers. Hitinemia is an inherited autosomal‐recessive metabolic disorder where lack/impaired histidase activity cause an increased level of histidine and its metabolites in blood and urine and decreased uronic acid in skin and blood or elevated levels of histaminase (diaminase) activities and related metabolites and neurotransmitters such as L‐dopamine and calcitonine. 1 , 5 , 20 , 101 , 103 , 124 , 185 , 197 , 198 , 199 , 200 , 201 Rate of histinemia was shown comparable with another inherited metabolic disorder, phenylketonuria. Histinemia is associated with defects in mild neurological disorders and slow‐down of speech. Chronic kidney disease, in contrast, is associated with low histidine levels and impaired metabolites such as histamine. Furthermore, low plasma levels of histidine are associated with a higher level of histamine (perhaps increased allergies), oxidative stress and retardation of mitochondrial energy, that also affect glomerular capillaries and filtration ability of kidneys and vascular/arterial endothelium associated with pruritus. Abnormal activation of stomach digestive enzymes/hormones (gastrin) and hypergastrinemia along with altered/increased mucosal histamine production or HDC have been suggested in hyperplasia of enterochromaffin‐like cells (ECLCs or ECL). Histidine is involved in erythropoiesis, hemoglobin biosynthesis and protection of RBC in circulation and the damaging effects of ROS. 1 , 14 , 17 , 20 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 95 , 105 , 106 , 109 , 124 , 126 , 127 , 128 , 129 , 148 , 152 , 172 , 176 , 177 , 202 , 203 , 222 Anemia is also associated with histidine deficiency and oxidative stress. The presence of Al in vaccines is likely to impair histidine metabolism, by interfering with iron‐requiring proteins including transferrin, biosynthesis of erythropoiesis and RBC or complement activation cascades and contribute to vasculature lesions (eg, vasculitis) and anemia 1 , 5 , 20 , 47 , 72 , 75 , 82 , 222 (manuscript in preparation) (Figure 4 ). As noted above, our preliminary studies that newborn guinea pigs, born from sensitized animals, manifested strong ocular reactions (MCs activation) upon first or second challenge with antigen, suggested parental‐fetus sensitization of MCs/plasma cells and lymphoid organs, and/or premature biosynthesis of immunoglobulins (eg IgE) that influence fetus genetic predisposition and epigenetic modifications presented as diverse altered immune responses. 1 , 5 , 20 Production of lactate from glycolysis and presence of essential amino acids (Ala) and histamine are characteristics of egg embryonic growth or human placenta in the transformation of myoblast to myotube and contractile myofibroblasts during organogenesis and angiogenesis. 1 , 20 , 219 Data on vaccine‐related topics and increased allergies, autism, autoimmune and neurodevelopmental disorders in children indirectly support our reported observations and recent hypotheses on the role of histamine in immune disorders or cancers. 1 , 5 , 8 , 10 , 12 , 13 , 14 , 20 , 23 , 24 , 36 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 , 60 , 95 , 141 , 185 , 197 , 198 , 199 , 200 , 201 It is further suggested that current vaccines could induce vascular lesions by damaging endothelial cells, MMPs, heparin sulfate enzymes, GFs, and related oxidative damage in BBB, neuronal tissues, and RBC. Impaired vascular activities and altered ratios of pro‐and anti‐angiogenic factors are likely to alter vasculature function (eg, toning, permeability and hyper‐permeability) under inflammatory conditions and significantly contribute to the genesis and progression of nearly all diseases. 1 , 5 , 20 Considering that vasculature is the "tree of life", we suggest vaccine injections alter important and diverse biological functions of these tissues as listed below 1 , 5 , 8 , 20 (Figures 1 , 2 , 3 , 4 , 5 ): Vasculogenesis is the earliest events in fetus growth and development; Delivery of nutrient and oxygen to the tissues, and removal of gases and waste products from the tissues; keeping in mind that mechanisms of vasculature interactions are somewhat different in immune‐privileged and immune‐responsive tissues; Major participant (gatekeeper) and facilitator in inflammatory responses during cellular proliferation, differentiation, and infiltration of inflammatory cells into infected/injured target tissue; contributing to both apoptosis (Yin) and wound healing (Yang) processes, under acute and chronic inflammatory conditions or carcinogenesis; In summary, vaccine‐related oxidative stress could lay a foundation to cause, exacerbate, and be a consequence of a wide range of mild, moderate, or severe immune disorders. While, diverse contraindications of vaccines have been observed even in healthy subjects in clinical trials (exclusion criteria!) and identified in manufacturers' inserts, heavy publicity to vaccinate the general public overlooks the health problems when industry abuses or ignores such information and targets the general public, healthy or not!, particularly because industry has little/no liability for testing the safety of vaccines 4 , 13 , 17 , 19 , 24 , 59 , 60 , 64 , 66 , 93 , 145 , 157 , 186 , 189 , 190 , 191 , 193 , 194 §§§§§§§§§ 7.1 Debates and controversies on reported safety of vaccines and incipient Analyses of data on vaccines and the impact on health of the unborn/neonatal, newborn and infant include review of documents on regulatory governmental agencies (eg, FDA, CDC, WHO), Public Health Informatics Network, American Medical Informatics Association, National Animal Health Management Emergency Management System and USDA, information on funding support from government, industry and 'philanthropists' to study and promote vaccines, manufacturers' inserts on vaccines contraindications, reports on spontaneous electronic adverse events on cancer drugs and vaccines, websites and blogs as well as, congressional debates, legal and financial incentives to professionals for promoting and publicizing vaccines, experts depositions in vaccine injury courts and awards to vaccine injured individuals 1 , 5 , 18 , 64 , 77 , 94 , 136 , 137 , 140 , 141 , 142 , 143 , 144 , 169 , 172 , 175 , 176 , 177 , 178 , 179 ‡‡‡‡‡ ' §§§§§ , ****** ' ††††††' ‡‡‡‡‡‡ . Unfortunately, governmental guidelines, particularly on cancer or vaccine‐related statistics, safety procedures, vaccine effectiveness or reported injuries are provided on behalf of medical establishment with little independent evaluation, considerations or validation of biological sciences, medical ethics and conflicts of interest, or safety considerations. The official guidelines by governmental agencies are skewed, biased and often laced with deception and cover ups on disease causes (eg, SIDS, vaccine injuries, drug toxicities, clinical trials on exclusion/inclusion criteria). Often official information is not worthy of serious considerations once the scientific pros and cons are weighed, particularly regarding information on root causes of cancer epidemics, over‐, under‐diagnosis of diseases, toxicities of drugs, biologics or vaccines safety 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 17 , 20 , 21 , 22 , 23 , 50 , 51 , 52 , 64 , 69 , 71 , 93 , 136 , 137 , 140 , 141 , 157 , 164 , 177 , 192 §§§§§§ , ******* , †††††††, ‡‡‡‡‡‡‡ , §§§§§§§ . Often, credible scientific information that is prepared by scientists who work in the government or academia are ignored, dismissed or abused when members of the establishment decide to publicize drugs or vaccines of their choice. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 11 , 12 , 13 , 17 , 18 , 34 , 36 , 37 , 38 , 39 , 64 , 69 , 71 , 77 , 94 , 143 , 149 , 169 , 172 , 176 , 178 , 180 , 181 , 182 , 183 , 184 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 In general, diversities and extent of immune disorders [acute inflammatory responses, delayed hypersensitivity reactions, mild (subclinical inflammation, oxidative stress), moderate or severe diseases] that occur immediately after vaccination or within few days‐weeks‐ months or years later, depend on several interdependent factors as outlined below (Figures 1 , 2 , 3 , 4 ): Age of vaccinated individual; Health status of vaccinated individual; Dosage, frequency and rout of vaccination; Period between subsequent vaccination; Composition of media/adjuvants (incipient) in vaccines; Nature (quality/composition, potency) of pathogenic particles in vaccines; Quality control (placebo) status of vaccines, procedures and standard tests for safety before public consumption; Quality of clinical trials (eg, inclusion or exclusion criteria, crossover trials and safety recommendations and approval of vaccines); Time course on reported vaccine injuries and follow up on inclusion of VAERS; Flu shot ingredients, given to pregnant women could disturb the intricate biological networks of trophoblast‐embryo‐fetus‐placenta that are required for orderly growth of fetus. Under incomplete mitochondrial development and hypoxic conditions of placenta, organogenesis and angiogenesis of embryo‐fetus require proper architectural organization and functioning of trophoblast epithelium for providing and consuming appropriate and sufficient growth factors/hormones, nutrients, enzymes, trace elements (metals) and respective constituent or induced receptor molecules. Exposure of the embryo‐fetus to flu shot and incipient, could pose serious threats to survival and health of both mother and fetus including altered nutrients in organogenesis, transition of myoblasts to myotube development, ratios of constituent/induced receptors, as potential contributors in growth retardation or growth promotion, fetus abortion or impaired health of newborn, infant, toddler, that also influence immunity during adulthood and aging process 1 , 5 , 8 , 20 , 24 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 129 , 192 (manuscript in preparation)] (Figures 1 , 3 and 4 ). Examples of deception that are frequently applied to the safety of drugs or vaccines are controversies on exclusion criteria or crossover practices in conducting clinical trials for obtaining approval of drugs or vaccines. 11 , 113 , 139 , 140 , 141 , 187 , 189 , 190 , 191 , 196 , 197 In the majority of clinical trials, decision makers select and recruit healthy individuals for testing safety of drugs or vaccines. Any individual with minor or major illnesses are excluded. This allows manufacturers to show maximum benefits and minimum harms of tested biologics. However, even under such selective criteria, healthy participants often experience various degrees of side‐effects that may or may not be acknowledged or documented during the marketing of such drugs. Vaccine approval voting is conducted through FDA (eg, VRBPAC) or CDC (eg, ACIP) committees, whose members are often industry, government employers or grantees (principal investigators) who receive funding to study and patent drugs/vaccines and to collaborate with manufacturers for large scale development and for marketing to the general public, healthy or not. 2 , 8 , 18 , 70 An example of vaccine propaganda is found in the marketing of HPV vaccines (Gardasil or Cervarix) that were approved to target the young generation, claiming to prevent cervical cancer. Segments of papilloma virus (types 6,11,18) and recombinant DNA technologies are used in media/incipient that has combination of Al, PS80, SIO2, Saponin. 8 , 12 , 14 , 19 , 24 , 110 , 131 , 132 , 133 , 134 , 137 , 138 , 139 , 143 Review of related data suggest that HPV vaccines and adjuvants are associated with mild or severe adverse reactions (VAERS), including autoimmune diseases, fibromyalgia, tachycardia, ovarian failure, fatigue, without any benefits in preventing incidences of cervical and related cancers 8 , 12 , 14 , 66 , 70 , 71 , 108 , 110 , 136 , 137 , 138 , 139 , 143 , ††††††††' ‡‡‡‡‡‡‡‡ . In a comprehensive review 14 Giannotta and Giannotta described the mechanisms of adverse effects of vaccines (eg, HPV) and incipient in autonomic neuronal system and development of autism spectrum disorder (ASD), fatigue and vaccine‐induced altered behaviors of immune cells (microglial and astrocytes) in the brain and associated loss of BBB. 7.2 Vaccines incipient (ingredient, adjuvant, excipient): Mini electric shocks and molecular sink holes in mitochondria with loss of biorhythms and induction of diseases Inactivated (or live) pathogens in vaccines, on their own, are immunogens (stimuli or immune disruptors) and could over‐stimulate immunity. According to manufacturer's inserts, governmental or published scientific data, majority of pathogen‐specific vaccines are prepared in media containing combinations of metals, chemicals‐biological agents such as aluminum (Al, as hydroxide or phosphate salts), mercury (Hg, thimerosal), detergents, solvents or preservatives [eg, CTAB, polymyxin, neomycin, saponin, formaldehyde, silica and derivatives, solutes (sorbitol, polysorbate 80 or 20, Tween 20), glyphosate‐herbicide, octylphenol ethoxylate or octoxynol‐10 (Triton X‐100)], genetically engineered DNA/RNA, yeast extracts, fetal tissues and organ parts or fragments 12 , 14 , 24 , 38 , 67 , 73 , 74 , 77 , 94 , 96 , 128 , 130 , 131 , 132 , 141 , 144 , 148 , 152 , 153 , 156 , 165 , 167 , 168 §§§§§§§§ ' ********* . Majority of these vaccine ingredients are not natural agents and do not participate in biochemical pathways in human physiology. These ingredients are additional foreign agents that overwhelm the immune system (see below). The ingredients that are perceived as 'natural' [eg, fetal serum, clumps of tissue/organ or DNA particles, proteins‐peptides (eg, ovalbumin, egg proteins, serum albumin, hydrolyzed porcine gelatin), amino acids (arginine, glutamate, or L‐histidine)] could disturb physiological activities and immune responses, particularly affecting the growing embryo‐fetus (unborn/neonate), newborn and infant whose organ systems, gut microbiome composition and immunity are not fully developed, or individuals who are immune compromised 1 , 4 , 5 , 8 , 12 , 14 , 17 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 50 , 58 , 61 , 62 , 63 , 64 , 66 , 67 , 68 , 70 , 71 , 141 , 144 , 148 , 152 , 153 , 156 , 165 , 167 , 168 (Figures 3 , 4 , 5 ). FIGURE 5 Schematic representation of toxicities of metals and other ingredients in pathogen‐specific vaccines in induction of mild, moderate, or severe immune disorders. Metals such as aluminum (Al) or mercury (Hg) are depicted to compete with essential trace elements and alter tissue biological activities (see blue box and center divided circle), influencing mitochondrial bioenergetics metabolism and immune response profiles in tissues. The scheme depicts that vaccines and ingredients differentially influence tissues that are immune‐privileged (brain, CNS, reproductive organs) or immune‐responsive (epithelial, endothelial, mucus, fibroblast) in the genesis of wide ranges of immune disorders (tissue necrosis or growth). See text 7.3 Toxicities of metals in vaccines: Mini electric shocks in mitochondria altering electrobiology of vascular‐metabolic‐neuronal‐hormonal pathways Aluminum (Al and its salts) or mercury (Hg, thimerosal) possess inert properties in nature, with cationic capabilities (eg, Al +3 , Hg +2 ) to interact with charged molecules and act as electronic magnets. Biologically, presence of these metals in injected vaccines could compete, scavenge (chelate) or act as cationic sinks and damage the function of essential metals and trace elements (Fe +2 , Fe +3 , Cu +1 , Cu +2 , Zn +2 , Ca +2 , Mg +2 , Se +2 ) that are required for a wide range of cellular functions. In general, presence of Al or Hg in vaccines is likely to interfere with required proton pumping and maintenance of differential electronic charges across cellular components. Among numerous cellular functions that are likely influenced by the presence of Al or Hg are transport and function of intra‐, extra‐cellular charged proteins, amino acids and cationic‐anionic trace elements across membranes [eg, ATP/ADP/AMP, Na + /K + exchanger, Na + /H + exchanger, water channels, active/passive transport of solutes, osmolytes or nutrients (eg, myo‐inositol, pyridoxal phosphate, ascorbic acid)]. Overview and integration of fragmented data on vaccine‐related topics, vaccine injuries and inflammatory/immune disorders suggest that presence of non‐functional metals in vaccines create mini‐electronic shocks or 'molecular sink holes' and induce biological defects in mitochondria, membranes and chromosoms, damaging the regulations of biological activities in tissues including alterations of gut microbiome profiles and neuronal behaviors (Figures 1 , 3 – 5 ) 1 , 5 , 14 , 36 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 (manuscript in preparation)] †††††††††, ‡‡‡‡‡‡‡‡‡ . For example, copper (Cu 1+ or Cu 2+ ) is an essential trace element (cation) and plays crucial roles as a cofactor in mitochondrial cytochrome C oxidase and numerous other biological activities including neuronal function, wound healing, biosynthesis of collagen and vasculature. Normally, copper ions are bound to carrier molecules and distributed via carrier proteins ('copper chaperones') for protecting/preventing tissue damage. 36 , 72 , 84 , 85 , 91 , 92 , 153 , 183 Excess amount of copper (free) could be detrimental to respiratory chain reactions and generation of toxic hydroxyl radicals (HO 0 ) causing oxidative damage not only to mitochondria, but also other extracellular‐intracellular proteins or nucleic acids and lipids (Fenton reaction) and tissue oxido‐redox potentials. 1 , 5 , 14 , 20 , 29 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 53 , 55 , 72 , 73 , 78 , 84 , 85 , 88 , 89 , 90 , 91 , 92 , 155 , 182 , 183 Furthermore, copper and Zn +2 (another trace element and antioxidant) are involved in detoxifying mitochondrial ROS and superoxide dismutase1 (SOD1) activities, regulation of Cu‐mediated production of O 2 /ROS. Related studies suggest that trace elements (Cu, zinc, Fe), influence regulations of transport of triglycerides in gastrointestinal tract and are important in the function of red blood cells and endoplasmic reticulum activities. While mechanisms of toxicities or interactions between Al or Hg and trace elements on biological pathways are not well understood, it is highly likely that tissues are sensitive to such metals in vaccines, particularly affecting tissue bioenergetics (Figures 3 , 4 , 5 ). The presence of Al or detergents in vaccines could induce retardation/overload of mitochondria, causing elevated levels of Cu 2+ in tissues (eg, liver) that would lead to vaccine‐induced respiratory or neuronal illnesses (eg, Wilson and other mitochondrial diseases). 1 , 29 , 36 , 39 , 40 , 48 , 49 , 53 , 54 , 55 , 56 , 57 , 74 , 75 , 114 , 151 , 163 , 183 Injected vaccines containing Al or Hg, at various stages of pregnancy, could retard fetus growth and development involving important generation, utilization, or recycling of glutathione‐related pathways (GSH: GSSH and NAD + : NADH) and further altering oxido‐redox potentials and incomplete mitochondria and organ development, growth impairment, immune and mitochondrial diseases after birth. 1 , 5 , 90 , 102 , 114 , 117 , 118 , 151 , 152 , 153 , 154 , 155 , 163 , 182 In a detailed retrospective epidemiological study, using automated Vaccine Safety Datalink (VSD), Young et al 36 reported that vaccines containing Hg (thimerosal) were associated with neurological developmental disorders (eg, anaphylaxis, autism, ASD, tics, attention deficit disorder, and emotional disturbances) in newborn (7 and 13 months old) perhaps, due to mitochondrial dysfunction. Evaluation of data on reported vaccine injuries (eg, fatigue, hypotonia, neuropathological episodes of epilepsy, Rett syndrome or encephalomyopathy, or cancers) suggest a range of electrochemical signal defects in the function of B/plasma cells, receptor/surface molecules that could retard and alter, among other pathways, memory B cells complexes, expression of immunoglobulins, mutations in mitochondrial complexes (I, II, III, or IV), related genetic/epigenetic modifications as contributing factors in impairing pathways of oxidative phosphorylation (mitophagy), autophagy and altered endoplasmic reticulum. 1 , 5 , 14 , 90 , 102 , 114 , 115 , 116 , 117 , 118 , 151 , 152 , 153 , 154 , 155 , 163 , 166 , 182 , 183 7.4 Presence of l ‐histidine in vaccines: histamine‐histidine interface‐exchange Histidine is a natural and essential amino acid (nutrient) and structural backbone of a variety of important proteins and enzymes with diverse biological functions. With its unique imidazole side chain, histidine plays critical roles in immune response dynamics, associated with renal, neuronal, ocular and gastrointestinal biological activities. There are up to 200 genes that mediate activities of histdine‐containing proteins, such as histidine metabolic enzymes (eg, histidine decarboxylase [HDC], amino oxidase [AO]), carrier proteins, and chelating agents of trace elements (eg, Zn). Analyses of data on PTH1 or PHT1 and histidine‐histamine homeostasis and histamine receptors in neuronal tissues (eg, brain) suggest that presence of l ‐His, together with Al or Hg, in vaccines alter histidine‐histamine ratio and neuropeptide regulation, particularly affecting the developing brain of fetus or newborn. 5 , 19 , 20 , 36 , 38 , 41 , 67 , 79 , 95 , 96 , 105 , 122 , 123 , 124 , 125 , 126 , 127 , 150 , 176 , 177 , 186 , 188 , 194 In addition to its role in neuronal tissues histamine (catecholamine, an alkali, a potent vasoactive agent) acts as a key element and the most versatile biogenic amine, having diverse and antagonistic properties in mammalian physiology. Histamine is synthesized by enzyme histidine decarboxylase (HDC), an enzyme present in all tissues. Histamine and its receptor molecules (HRs 1–4 and subfamilies) play diverse roles in human development, acute and chronic inflammation, acid‐base balance, digestion, mucosal activities, vascular function and permeability, neuronal activities as well as, growth of cancer mass. 1 , 5 , 20 , 95 , 124 , 126 , 127 , 150 , 199 , 200 , 201 , 202 , 203 Results of our original studies on inflammation‐induced multistep tumorigenesis and angiogenesis led to recent hypotheses that low level release of histamine (independent from IgE‐fcR degranulation of MCs ["leaky" or exhausted MCs]) is a factor in induction of tissue/cell growth that alter immune responses, including induction of autoimmune and neurodegenerative diseases or tumorigenesis and angiogenesis. 1 , 5 , 8 , 20 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 52 Summary of diverse biological activities that are affected by abnormal levels (deficiencies or excesses) of histidine‐histamine in tissues, including altered nutritional intake, genetic mutations in production or metabolism of histidine or histamine are provided below 1 , 5 , 20 , 41 , 42 , 43 , 44 , 46 , 79 , 95 , 105 , 106 , 109 , 124 , 126 , 127 , 128 , 148 , 152 , 172 , 176 , 177 , 200 , 201 , 202 , 203 (Figures 4 and 5 ): Early embryonic‐fetus growth, organogenesis‐angiogenesis; Vasculature, innate immune cells (eg,MCs) and neuronal activities after birth; DNA transcription involving Zn‐imidazole at active sites of enzymes (eg, carbonic anhydrase‐CA); Hymolytic and redox reactions; Adenosyl methionine and ATP binding site of actin; Hydroxylation of galactosylceramide and maintenance of myelin sheath structure; Thyrotropin‐releasing hormone; Serine esterase activities of trypsin, chymotrypsin; acetylcholinesterase and blood clotting and complement cascades; Food or PH‐induced gastrin release of histamine from enterochromamaffin‐like cell (ECL) and activation of HDC; Induction of tissue growth, tumorigenesis, angiogenesis, and cancer; Among major histidine‐histamine‐associated diseases are histinemia, kidney disease, anemia, and cancers. Hitinemia is an inherited autosomal‐recessive metabolic disorder where lack/impaired histidase activity cause an increased level of histidine and its metabolites in blood and urine and decreased uronic acid in skin and blood or elevated levels of histaminase (diaminase) activities and related metabolites and neurotransmitters such as L‐dopamine and calcitonine. 1 , 5 , 20 , 101 , 103 , 124 , 185 , 197 , 198 , 199 , 200 , 201 Rate of histinemia was shown comparable with another inherited metabolic disorder, phenylketonuria. Histinemia is associated with defects in mild neurological disorders and slow‐down of speech. Chronic kidney disease, in contrast, is associated with low histidine levels and impaired metabolites such as histamine. Furthermore, low plasma levels of histidine are associated with a higher level of histamine (perhaps increased allergies), oxidative stress and retardation of mitochondrial energy, that also affect glomerular capillaries and filtration ability of kidneys and vascular/arterial endothelium associated with pruritus. Abnormal activation of stomach digestive enzymes/hormones (gastrin) and hypergastrinemia along with altered/increased mucosal histamine production or HDC have been suggested in hyperplasia of enterochromaffin‐like cells (ECLCs or ECL). Histidine is involved in erythropoiesis, hemoglobin biosynthesis and protection of RBC in circulation and the damaging effects of ROS. 1 , 14 , 17 , 20 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 95 , 105 , 106 , 109 , 124 , 126 , 127 , 128 , 129 , 148 , 152 , 172 , 176 , 177 , 202 , 203 , 222 Anemia is also associated with histidine deficiency and oxidative stress. The presence of Al in vaccines is likely to impair histidine metabolism, by interfering with iron‐requiring proteins including transferrin, biosynthesis of erythropoiesis and RBC or complement activation cascades and contribute to vasculature lesions (eg, vasculitis) and anemia 1 , 5 , 20 , 47 , 72 , 75 , 82 , 222 (manuscript in preparation) (Figure 4 ). As noted above, our preliminary studies that newborn guinea pigs, born from sensitized animals, manifested strong ocular reactions (MCs activation) upon first or second challenge with antigen, suggested parental‐fetus sensitization of MCs/plasma cells and lymphoid organs, and/or premature biosynthesis of immunoglobulins (eg IgE) that influence fetus genetic predisposition and epigenetic modifications presented as diverse altered immune responses. 1 , 5 , 20 Production of lactate from glycolysis and presence of essential amino acids (Ala) and histamine are characteristics of egg embryonic growth or human placenta in the transformation of myoblast to myotube and contractile myofibroblasts during organogenesis and angiogenesis. 1 , 20 , 219 Data on vaccine‐related topics and increased allergies, autism, autoimmune and neurodevelopmental disorders in children indirectly support our reported observations and recent hypotheses on the role of histamine in immune disorders or cancers. 1 , 5 , 8 , 10 , 12 , 13 , 14 , 20 , 23 , 24 , 36 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 49 , 50 , 51 , 52 , 60 , 95 , 141 , 185 , 197 , 198 , 199 , 200 , 201 It is further suggested that current vaccines could induce vascular lesions by damaging endothelial cells, MMPs, heparin sulfate enzymes, GFs, and related oxidative damage in BBB, neuronal tissues, and RBC. Impaired vascular activities and altered ratios of pro‐and anti‐angiogenic factors are likely to alter vasculature function (eg, toning, permeability and hyper‐permeability) under inflammatory conditions and significantly contribute to the genesis and progression of nearly all diseases. 1 , 5 , 20 Considering that vasculature is the "tree of life", we suggest vaccine injections alter important and diverse biological functions of these tissues as listed below 1 , 5 , 8 , 20 (Figures 1 , 2 , 3 , 4 , 5 ): Vasculogenesis is the earliest events in fetus growth and development; Delivery of nutrient and oxygen to the tissues, and removal of gases and waste products from the tissues; keeping in mind that mechanisms of vasculature interactions are somewhat different in immune‐privileged and immune‐responsive tissues; Major participant (gatekeeper) and facilitator in inflammatory responses during cellular proliferation, differentiation, and infiltration of inflammatory cells into infected/injured target tissue; contributing to both apoptosis (Yin) and wound healing (Yang) processes, under acute and chronic inflammatory conditions or carcinogenesis; In summary, vaccine‐related oxidative stress could lay a foundation to cause, exacerbate, and be a consequence of a wide range of mild, moderate, or severe immune disorders. While, diverse contraindications of vaccines have been observed even in healthy subjects in clinical trials (exclusion criteria!) and identified in manufacturers' inserts, heavy publicity to vaccinate the general public overlooks the health problems when industry abuses or ignores such information and targets the general public, healthy or not!, particularly because industry has little/no liability for testing the safety of vaccines 4 , 13 , 17 , 19 , 24 , 59 , 60 , 64 , 66 , 93 , 145 , 157 , 186 , 189 , 190 , 191 , 193 , 194 §§§§§§§§§ 8 ECONOMIC BURDEN OF MEDICINE: WEALTH TRANSFER FROM TAXPAYERS TO ESTABLISHMENT: PUBLIC HEALTH PURCHASED! "If you would be a real seeker after truth, it is necessary that at least once in your life you doubt, as far as possible, all things" , René Descartes Since the rise of the purchasing power of Rockefeller influenced medical education and research, it seems the era of caring for public health and safety or real 'standard of care' has been gradually replaced by the philosophy of how to abuse power (intellectually, politically, and financially) by chipping away natural immunity and health through the abuse of drugs and pushing of vaccines. The strong partnership between governments, industry, and venture capitalists‐'philanthropist' has significantly weakened the conflict of interest compliance and medical ethics in conducting taxpayers‐supported projects. The drug industry has taken increasing control over the major media and of public policymaking in Congress, as well as eliminating vaccine liabilities, and changing consent forms in hospitals for patients for receiving care. 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 17 , 18 , 64 , 167 , 171 , 181 , 187 , 189 , 196 , 205 , 206 , 207 According to a financial analyses of healthcare 'The Saker Blog', the entire medical system (together with the insurance industry) has been ultimately controlled " by one giant oligarch… Its annual value was $3.7 trillion, amounting to 17.9% of GDP (2018). That is nearly double the average of developed Western countries…" However, " The enormous expense does not buy Americans any better health than the Europeans get for half the price; in fact the health outcomes are far inferior in the US. In life expectancy, the US has fallen down to 33rd place, even overtaken by Cuba…Exorbitant prices on drugs, medical treatment and health insurance are crushing consumers …" ********** As noted above, members of scientific boards and councils or review committees (eg, NCI‐BSA or NCAB, FDA, CDC review or approval groups for voting on drugs) and staff within DHHS or policymakers often have direct or indirect financial ties with drug industry‐government‐venture capitalist "philanthropists" complex, and act as rubber stamps for approving and conducting the repeatedly failed projects that are pushed by the establishment. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 17 , 18 , 21 , 142 , 171 , 180 , 181 , 186 , 187 , 189 , 192 , 193 , 194 , 196 , 202 , 203 , 204 , 207 Independent and competent professionals are becoming seriously concerned that over the last seven decades, chronic diseases that are often features of age‐associated immune disorders including cancers are manifested in children and younger generations who require hospitalization and the consumption of drugs. Table 1 shows the rising cost of vaccines, from 2000 to 2014, to vaccinate one child. TABLE 1 Cost to immunize one child in the public sector has risen by over 500% since 2000 2000 2002 2004 2006 2008 2010 2012 2013 2014 DTaP $46.25 $59.65 $62.05 $63.98 $63.25 $66.25 $75.00 $76.90 $76.90 Polio $31.00 $34.64 $40.40 $43.28 $45.92 $46.96 $48.96 $49.68 $49.84 MMR $30.16 $31.22 $32.38 $34.56 $36.52 $37.27 $38.66 $39.52 $39.82 Hib $21.96 $28.44 $33.60 $31.74 $33.78 $34.53 $35.91 $27.99 $28.08 Hep B $27.18 $28.11 $27.45 $27.65 $28.50 $30.75 $32.19 $32.79 $33.00 Varicella $37.14 $40.87 $47.02 $113.80 b $123.00 $134.16 $144.98 $150.72 $156.68 PCV $88.50 a $183.96 $203.00 $230.36 $265.76 $367.00 $408.12 $428.48 $449.76 Flu – – $30.00 $69.18 $205.36 d $175.67 $186.44 $217.39 $280.16 Tdap – – – $30.75 c $30.75 $28.54 $29.59 $24.63 $30.25 MCV‐4 – – – $68.00 $76.35 $79.75 $164.24 $138.72 $164.24 Hep A – – – $24.31 $24.50 $26.50 $29.50 $30.50 $32.30 Rotavirus – – – $156.00 $171.60 $167.50 $182.04 $184.30 $190.40 HPV – – – – $301.77 e $288.24 $335.89 $321.47 $363.09 TOTAL f $282.19 $406.89 $475.90 $893.61 $1407.06 $1483.12 $1620.15 $1711.52 $1894.52 a In 2000, the PCV cost to fully vaccinate one child was for half the calendar year. The CDC contract was not in place until July 1, 2000. b In 2006, ACIP recommended two doses of varicella. c Tdap replaced Td as the adolescent booster recommended by ACIP in June 2005, to provide protection against pertussis. The cost of Td has not been included in previous years due to the absence of a CDC contract. d In 2008, ACIP recommended annual influenza vaccination for all children up to age 18. Two doses are needed the first year of vaccination and 1 dose is needed annually thereafter, for a total of 20 doses. e Beginning in 2007 the total represents the cost to fully vaccinate a female including the HPV vaccine. The HPV vaccine is also recommended for males as of late 2011. f The cost of recommended vaccines is significantly higher when combination vaccines are factored in to the total cost. This table shows only the lower cost of single vaccines. TOTAL represents the cost to vaccinate one child with vaccines universally recommended from birth through 18 years of age using federal contract prices. Source: Centers for Disease Control and Prevention. Association of State and Territorial Health Officials (ASTHO), March 2015. John Wiley & Sons, Ltd. 9 WORTHINESS OF CURRENT SCIENTIFIC PUBLICATIONS ON CANCER AND VACCINE SCIENCES: WHEN GOVERNMENTS‐POLITICIANS‐DISEASE INVESTORS (PHILANTHROPISTS) DECIDE ON PUBLIC HEALTH SCIENCE! You can't depend on your eyes when your imagination is out of focus . Mark Twain There are well over 25 million basic and clinical articles, books, and documents on cancer and vaccine topics. Despite the high cost of cancer sciences and therapeutics, using sophisticated, advanced, and specific technologies, the worthiness, caliber, and effectiveness of published studies, authored by those who occupy high positions and have accumulated scientific recognitions (eg, Nobel prizes), in "high impact" or "peer‐reviewed" journals are in decline. Too many expensive projects that are designed on reductionist approaches to cancer and vaccine sciences have failed the public. Numerous publications have been accepted or rejected because of political views of decision makers. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 158 , 167 , 171 , 223 ‡‡‡‡‡‡‡‡‡‡ ' §§§§§§§§§§ Comparing the worthiness of publications in the past seven decades with those fundamental discoveries that stood the test of time, one can easily conclude that in the 18th or 19th century, the work of dedicated scientists who searched for the scientific truth to solve health‐biological problems were accomplished with limited resources and in the absence of highly modern technologies. As noted above, in the 20th century, it appears that the majority of pioneering studies are intellectually ignored or rejected for motives that diametrically oppose solving medical problems and improving public health and well‐being. The public is losing trust in conventional and "peer‐reviewed" publications that are supported and promoted by members of the establishment in governments, academia, and industries. Published data by independent and competent scientists are often downplayed, attacked, censored, or rejected by reviewers if the results do not fit the motives of the medical establishment. Professionals with financial ties to government and industry often label views of independent and competent professionals as a 'threat' to public health! 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 15 , 16 , 17 , 22 , 34 , 71 , 142 , 145 , 167 , 196 , 205 , 206 , 207 (unpublished observations). Often independent, highly competent, and concerned professionals who are frustrated with the diseased condition of the money‐driven mentality of the medical system discuss or publish their views on different sites on Internet. In recent years, even quality independent scientific blogs are being controlled and censored under fabricated reasons (unpublished data). 8 , 223 10 FUTURE TRENDS OF MEDICAL SYSTEM FOR PUBLIC HEALTH: EXPANDING FACILITIES FOR CHRONIC DISEASE CARE AND PUSHING DRUGS OR VACCINES TO YOUNG AND OLD! CORPORATE PROFIT OVER PUBLIC HEALTH AND HUMANITY If people let government decide which food they eat and medicines they take, their bodies will soon be in as sorry a state as are the souls of those who live under tyranny . Thomas Jefferson Evidence for extension of establishment's power in forming a 'global supreme leadership in medicine' to minister and control public health comes from the recently intensified partnership between governments, Big Pharma, and venture capitalists"philanthropists." On September 11, 2019, DHHS announced that it was sponsoring a health center [" Awards More than $50 Million to Establish New Health Center Sites "]. The announcement states that " This new funding will increase access to health care for more than 400,000 new patients,… " *********** . At about the same time‐frame, to match taxpayers' investment in 'healthcare' in expanding hospitals, vaccine manufacturers also announced hiring more staff for vaccine development †††††††††††. The Bill and Melinda Gates Foundation is also 'investing' millions in vaccinating children, in under‐developed countries and for corona virus vaccines globally ‡‡‡‡‡‡‡‡‡‡‡ ! All of this sounds "great" except that vaccine injury courts have awarded billions of dollars to a small percentage of vaccine‐injured individuals who became aware of the relationship between vaccines and their illnesses or those who could legally afford to report their vaccine injuries. Available statistics on injury claims that are filed in the Federal Vaccine Injury Compensation Program (VICP) and other governmental and private organization programs show that public vaccinations with polio, smallpox, and Swine flu vaccines killed millions and left many more millions seriously injured, hospitalized, disabled and drug‐ dependent. At least 22 000 were killed after smallpox vaccination alone. 1 , 2 , 3 , 4 , 5 , 6 , 14 , 17 §§§§§§§§§§§ , ************ , ††††††††††††, ************* 11 TWENTIETH CENTURY MEDICINE: SHIFTING DISEASE CATEGORIES TO INCREASE INDUCED DISEASES IN YOUNG AND OLD: WEALTH TRANSFER FROM the PUBLIC TO CORPORATE AMERICA! It's easier to fool people, than to convince them they have been fooled . Mark Twain A closer look at the current disease status of three to four generations in America, demonstrates that the major disease categories that are clinically, pathologically, and symptomatically identified as congenital, hereditary, neonatal and induced diseases have been shifted to increase the population toward induced diseases (Figures 1 , 3 – 5 ). Integration of the scattered data on epidemiological, environmental, clinical and basic research on developmental biology, inflammatory diseases, cancer and vaccine sciences, treatment options are outlined below 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 17 , 24 , 50 , 71 , 82 , 86 , 93 , 119 , 120 , 128 , 136 , 150 , 151 , 152 , 153 , 155 , 156 , 176 , 177 , 182 , 183 , 184 , 185 , 203 , 204 , 205 , 206 , 207 , 209 , 210 , 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 , 220 †††††††††††††, ‡‡‡‡‡‡‡‡‡‡‡‡‡ , §§§§§§§§§§§§§ , ************** : Significant increase in the incidence of allergies, asthma, anaphylactic, anemia, emphysema, autoimmune and neuronal dysfunction, obesity, hypertension, diabetes and cardiovascular complications, stroke, gastritis, colitis, fibromyalgia, thyroiditis, lupus, Alzheimer's, Parkinson's, and site‐specific cancers in young and older adults. These disease incidences that require hospitalization and consumption of various drugs are at the level of near epidemic proportions. Asthma and allergies among children mandate that schools provide drugs, inhalers, and extremely expensive Epipens for asthmatic children; Increased incidence of autism spectrum disorder (ASD) is an example of a range of immune‐neuronal‐metabolic illnesses that sharply rose among young and growing population in the last few decades. In 1950s and 1960s, the estimated rates of reported autism were 1/10,000 in America. In 1990s this rate rose to 1/ 5000. In 2014, the CDC estimated that 1/45 children were autistic (NHIS data), a 30% increase from 1 of 150 in 2002 (just in 12 years!). In 2018, it was estimated that the rate of autism will be four times higher in boys compared with girls. Therefore, about one in five boys will suffer from ASD, not including the "unexplained" SIDS or other illnesses that are not reported as relevant. With the current trend, it is estimated that one in two boys (50%) could become autistic and manifest multiple physical and intellectual disabilities between 2025–2032. Increased population of individuals who suffer from neurodegenerative and autoimmune diseases and require long‐term consumption of drugs further complicate overall well being, productivity and hopefulness that youth will be able to contribute to the society; 12 TOWARD UNIVERSAL AND SAFE VACCINE DEVELOPMENT: TIMELY CHALLENGES TO IMPROVE NATURAL IMMUNITY AND PUBLIC HEALTH Intellectually, with the fundamental knowledge that effective immunity is responsible for defending and protecting the body against internal or external foreign (non‐self) elements (eg, infectious agents, pollutants, non‐functional proteins, mutated genes, defective or cancerous cells), it is hard to accept that medical decision makers could be so incompetent not to realize that research priorities should have been directed to a systematic understanding of the highly regulated multi‐layer complex bioelectrical signals that make up an effective immunity. By abusing fundamental information on the role of immunity, the medical system continues to conduct projects that chip away at immunity, even before completion of the immune system in the unborn or newborn or infant. Pathogen‐specific vaccines and adjuvants seem to be "safer" new terms for drugging young and old populations and claiming that 'vaccines promote immunity'! Developing universal and safe vaccines that boost immunity and induce effective neutralizing antibodies and memory cells against a wide range of pathogenic structures [eg, envelope glycosylated proteins or glycoproteins (hemagglutinine‐rich structures), mannosyl‐rich proteins, glycolipids and related enzymes and receptor molecules] are yet to be seriously considered. Recent attempts toward 'universal' flu vaccines, while provide useful additional information and insights into shared or special structural and sub‐structural characteristics or compatibility among a few pathogens (eg, HIV, influenza) and the relation to host receptor glycoproteins and induction of immune responses (eg, IgGs) they are far from being considered as universal vaccines or safer treatment options 1 , 32 , 86 , 96 , 136 , 210 , 211 , 212 (manuscript in preparation). Such efforts for making a vaccine against pathogenic components of one infective family structure (eg, flu, SARS, or covid‐19) should not be claimed as 'universal'. Outcomes of projects that are claimed as 'universal' vaccines are likely to present infinite response possibilities in vivo within the host site‐specific tissues. Depending on evolving pathogenic components (eg, molecular envelop glycoproteins, receptor or surface molecules) or host exposure tissue sites, as well as the health status or the age of individual in responding to pathogenic components, or how vaccines are developed (eg, composition of the incipient or engineered viral modifications [DNA or RNA]) could induce additional complicated and unknown electrochemical immune responses. 1 , 5 , 8 , 12 , 14 , 19 , 20 , 22 , 32 , 62 , 106 , 129 , 162 , 172 , 177 , 185 , 194 13 DEFINITIONS OF HEALTH AND PHILANTHROPY IN TWENTIETH CENTURY: POWER TO PURCHASE PUBLIC HEALTH! In 2011, Chris Hedges, a respected and independent journalist described the current health situation ' Industry's lobbyist and industry money wrote the healthcare bill ' ††††††††††††††. Unfortunately, the originalities of thinkers of the 18 th or 19 th century, who searched for the scientific truth suffered a great deal in the 20 th century. They were replaced by weakened ethical and moral standing and acceptance of egregious conflicts of interest tolerated and even encouraged by the medical might of the establishment, with its world's largest lobbying group who control healthcare, promote and mandate vaccines, and peddle drugs. Extensive debates, and controversies that were created by isolationists in cancer biology and treatment options as well as the emphasis on vaccines over immune system support resulted in conducting too many out‐of‐focus and expensive projects that repeatedly failed the public. Searching the truth in medical sciences, pioneers such as Pasteur, Rous, Metchnikoff, Ehrlich or Burnet employed highly intellectual logics by integrating and presenting credible discoveries of their era that stood the test of time. With limited resources and in the absence of modern technologies, these true scientists viewed infections as foundations of inflammatory chronic diseases. In the twentieth century, these logical, common sense and fundamental studies have been drastically altered, abused, and replaced by reductionist and chaotic tactics by profit‐power‐seeker mentalities of decision makers who disregard medical morality "to do no harm" in conducting medical sciences. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 17 , 18 The medical system seems to have lost its soul to the power of blood (dark) money in the 20th century. Rockefeller Patent Medicine and Gates' Patent Vaccines altered definitions of health and philanthropy. Health definitions changed from the absence of disease to management of the disease by drugs/vaccines. Definitions of philanthropy have changed from doing good deeds to benefit society and humanity, to investing in diseases to benefit the investors. Disease investors and venture capitalists ('philanthropists') are giving more to their tax‐deductible foundations with the goal to collect a lot more! The overall outcomes of 'philanthropists' involvement in collaboration with Big Pharma and governments on public health projects, particularly cancer and vaccines may be summarized as: Shifted/increased in induced diseases in young and old; Reduced and disregarded medical morality, ethics and conflicts of interest in conducting projects; Prevented independent validation of research and clinical projects by competent professionals; Abused power to control a sick and drug‐dependent society for population growth control and the maintenance of a complacent work force; Transfer of wealth from the public to the disease investors ('philanthropists') and collaborators; It is time to remind true 'philanthropists' to go back to do good deeds to benefit society. Supporting and improving hygiene, agriculture, clean drinking water, nutritional programs, and infrastructure in the third world or poor countries (eg, Samoa, Congo) where real human crisis/tragedies are happening would be considered doing good deed for humanity. Vaccine‐deficiency is not what the developed or undeveloped nations are suffering from! Immunologically, investing in vaccination (even if vaccines were 'safe') will not reduce diseases or death. Vaccines further complicate the disease status of poor people who suffer from malnutrition and lack of hygiene. The fear‐mongering tactics based on the threat of infectious diseases (eg, measles, Ebola, HPV, meningitis, flu, coronavirus) in developed or developing countries are an over‐exaggeration and cover‐up for pushing and selling drugs (vaccines) and controlling population growth. Malnutrition or bad nutrition (overeating of junk and unhealthy foods) are important factors in manifestations of diseases in developing‐poor nations or developed nations. 1 , 3 , 5 , 7 , 8 , 172 , 173 , 174 ‡‡‡‡‡‡‡‡‡‡‡‡‡‡ ' §§§§§§§§§§§§§§ Despite the fact that America invests the highest amount in advanced technologies and medical research and healthcare, the health status of Americans ranks last among other developed nations. The rates of reproduction and longevity are declining in America. Peculiarly, when the disease status in developed or under‐developed countries is compared, the overconsumption and overabundance of certain unhealthy foods, antibiotics, and drugs/vaccines, the overall health of Americans seems comparable with the levels of malnutrition and lack of hygiene in poor nations! 14 FUTURE DIRECTIONS: UNDERSTANDING AND PROMOTING ELECTROBIOLOGY OF EFFECTIVE IMMUNITY FOR MAINTENANCE OF HEALTH The autonomic sympathetic and parasympathetic behaviors and circuitry that shape human immunity (adaptive, horizontal) cannot be explained by limited genomics (innate, perpendicular) that conventionally described certain inherited diseases (eg, sickle cell anemia, progeria). 221 , 222 Even these genomic diseases are potentially immune/inflammation‐based conditions that irreversibly affected parental/ancestral chromosomal/genetic structures and functions. Future studies should focus on a deep and systematic understanding of the complex electrobiology of immunity with time‐dependent and differential bioenergetics involving mitochondria and cytoplasm under a wide range of environmental and inflammatory conditions. Approaches to limit or control excessive activation of gene‐environment‐immunity are keys to assessing accurate risk formulations, preventing inducible diseases, developing universal safe vaccines, and promoting health, the most basic human right. 1 , 5 , 20 15 CONCLUDING REMARKS: HUMAN BODY IS NOT DRUG‐DEFICIENT, IT IS NOT VACCINE‐DEFICIENT! 'There could be no greater a heinous crime, than the premeditated withholding of truth from the masses, to the point of their injury or death .(?) In the last seven decades, the guardians of public health, instead of promoting health and preventing diseases, successfully managed to chip away the naturally synchronized and complex molecular dynamics of effective immunity and increased and shifted the population of induced diseases in young and old. Frequent use of drugs and pathogen‐specific vaccines are seeds of immune destruction that induce electrochemical sinkholes (mini electronic shocks) in time‐dependent circadian (biorhythms, the Yin‐Yang balance) of signal transduction mechanisms that primarily paralyze (exhaust) mitochondrial function, damage proton pumping and lead to initiation and progression of mild, moderate or severe diseases and death in young and old. Heavy propaganda and demands to over‐vaccinate young and old populations, including the current fear‐mongering on the covid‐19 situation and lockdown (Medical Marshal Law) with the goal to vaccinate the public globally make us wonder whether " Governments love pandemics… for the same reason they love war.…" *************** or this lockdown is a political ideology that experiments how to strip public freedom and dignity " replace freedom with the terrifying dreams of intellectuals …." †††††††††††††††Evidence was presented that (a) human body is not drug‐deficient or vaccine‐deficient; (b) current pathogen‐specific vaccines weaken immunity, not promote it; (c) current vaccines are new terms for drugging young and old; (d) safe, effective and universal vaccines that promote natural immunity and prevent diseases are yet to be seriously considered by the medical establishment. Due to serious harms that continue to erode the public health, concerned independent scientific/medical experts, ethicists, attorneys, media, and policymakers are urged to take a closer look at intellectual crimes that the medical establishment has practiced against public health and to initiate appropriate actions before all hopes for a functional healthy society are lost. Policymakers, elected officials, and professionals should return to the common sense that our Forefathers valued and strive to serve the public for a ' more perfect Union' . Our universe can offer a lot more untapped resources to afford a larger human populations without the need to destroy and cause the extinction of human beings by inducing infertility and diseases to control population growth and shorten life span, as well as to continue to spawn a compliant and docile, drug‐dependent work force who can be eliminated at the push of syringe should they become expandable to their elite masters. It is a horrifying thought to choose between the less of the two evils for humanity; killing and destroying weaker nations by man‐made weapons under fabricated reasons for creating wars, or destroying public health and controlling the population by weaponizing cancer or over‐vaccinating the public under intellectual deceptions and claims of "war on cancer" or "vaccines are safe." The real outcomes of either choice seem the same, transfer of wealth to warmongers, loss of precious lives and lack of respect and hope to save humanity. These are challenging times for correcting the disease status that was created by Rockefeller medicine and continued by Gates vaccine. It requires a serious change of heart for policymakers and professionals, as well as a public awareness of the issues to help purge the deceptology in the cancer and vaccine sciences and hold the perpetrators of this deception responsible for fraudulent projects in the medical sciences. After all ' Of one Essence is the human race, Thusly has Creation put the Base; One Limb impacted is sufficient, For all Others to feel the Mace' Saadi Shirazi. CONFLICT OF INTEREST No conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10024721/

Secreted protease PRSS35 suppresses hepatocellular carcinoma by disabling CXCL2-mediated neutrophil extracellular traps

Hepatocytes function largely through the secretion of proteins that regulate cell proliferation, metabolism, and intercellular communications. During the progression of hepatocellular carcinoma (HCC), the hepatocyte secretome changes dynamically as both a consequence and a causative factor in tumorigenesis, although the full scope of secreted protein function in this process remains unclear. Here, we show that the secreted pseudo serine protease PRSS35 functions as a tumor suppressor in HCC. Mechanistically, we demonstrate that active PRSS35 is processed via cleavage by proprotein convertases. Active PRSS35 then suppresses protein levels of CXCL2 through targeted cleavage of tandem lysine (KK) recognition motif. Consequently, CXCL2 degradation attenuates neutrophil recruitment to tumors and formation of neutrophil extracellular traps, ultimately suppressing HCC progression. These findings expand our understanding of the hepatocyte secretome's role in cancer development while providing a basis for the clinical translation of PRRS35 as a therapeutic target or diagnostic biomarker. Introduction During the development of hepatocellular carcinoma (HCC), the expression of numerous proteins is altered in hepatocytes or HCC cells to facilitate tumor cell survival and proliferation 1 , 2 . As a major component of the liver proteome, the secretome of hepatocytes or HCC cells can also contribute determining factors in cancer progression 3 , 4 . In recent years, several secreted proteins have emerged as diagnostic markers or treatment targets for various types of cancers, which are still undergoing validation in clinical trials 5 . However, considerable gaps persist in our understanding of the scope and mechanisms of secreted proteins that participate in HCC pathogenesis, which could greatly advance the development of markers for early diagnosis and effective therapeutic strategies for this deadly disease. Several proteins have been established as tumor suppressors, and the loss of their function can directly regulate the proliferation and functions of different cancer cells 6 , 7 . With the development of cancer immunity, tumor suppressors also regulate leukocytes, including immune cells in the tumor microenvironment (TME), to mediate strong, indirect suppression of tumor progression 8 , 9 . As a major sub-type of leukocyte, neutrophils have been discovered as prevalent members of the TME, where they exert dual functions. On one hand, neutrophils mediate antitumor responses through the direct killing of tumor cells and by participating in cellular networks that mediate antitumor resistance. On the other hand, neutrophils also activate an inflammatory response that can promote tumor growth by driving angiogenesis, extracellular matrix remodeling, metastasis, and immunosuppression. Whether neutrophils function in a tumor-suppressive or tumor-promoting capacity depends on factors in the TME as well as their own diversity and plasticity 10 – 12 . While neutrophils affect tumor progression through multiple mechanisms, evidence is also currently emerging for a role of neutrophil extracellular traps (NETs), a product released by neutrophils 13 . NETs are large extracellular web-like structures consisting of chromatin DNA filaments coated with granule proteins, that may prevent or limit infection 14 , 15 by trapping and immobilizing bacteria 16 , fungi 17 , viruses 18 and parasites 19 so that they can be eliminated by other secreted anti-microbial compounds. The dysregulation of NETs formation or activity has been shown to drive the development of cancer and other immune-related diseases 13 . Nevertheless, our understanding of NETs involvement in cancer development is still in the infant stage. As a major proportion of the non-cellular components in the tumor microenvironment, secreted proteins serve as a means of intercellular communication between host leukocytes and tumor cells, some of which can regulate neutrophil behavior in the TME. Here, by secretome analysis of hepatocyte and liver cancer cell lines, we identify a secreted tumor suppressor, PRSS35, that inhibits HCC progression through cleavage of the chemokine CXCL2, which mediates pro-tumor neutrophil function. The findings described here expand the scope of our understanding of the role of interplay between secreted proteins and neutrophils in HCC development, and suggest a potential diagnostic marker and therapeutic target for this disease. Results PRSS35 protein abundance is decreased in the HCC secretome To identify potential factors related to the development and progression of HCC, we employed label-free proteomic analysis of the HCC secretome and compared the protein profiles of human PLC liver cancer cells with that of human THLE3 hepatocytes secreted in conditioned medium. We found that many proteins showed differential abundance in the conditioned medium used for PLC cell culture compared to that of THLE3 cells (Supplementary Fig. 1a , left panel), among which 236 proteins were designated as secreted proteins in the UniProt database 20 . Analysis of these 236 secreted proteins revealed that PRSS35 was the most significantly downregulated protein in the PLC secretome (Fig. 1a , Supplementary Fig. 1a , right panel and Supplementary Data 2 ). Western blot (WB) analysis using an antibody that recognizes the N-terminus of PRSS35 indicated that both intracellular and extracellular PRSS35 protein levels were markedly reduced in PLC, HepG2, and Hep3B liver cancer cells, relative to its accumulation in THLE3 cells (Fig. 1b ). Interestingly, the molecular weight of PRSS35 enriched in culture medium (SF-PRSS35) was far lower than that of full-length PRSS35 (FL-PRSS35) isolated from cell lysates (Fig. 1b ). Overexpression of PRSS35 in PLC, HepG2, and Hep3B liver cancer cells further led to the identification of a short form (SF-PRSS35) in the culture medium (Supplementary Fig. 1b ), suggesting that this short form was specifically secreted. Further WB analysis using antibodies targeted to different peptide regions of PRSS35 (i.e., designated N-PRSS35, M-PRSS35, and C-PRSS35 based on their respective antigen sequences, Supplementary Fig. 1c ) revealed that multiple short forms of PRSS35 protein were enriched in the culture medium of PLC cells overexpressing PRSS35 (Fig. 1c ). Mass spectrometry (MS) also confirmed that the shorter bands detected by SDS-PAGE indeed included several PRSS35 peptides (Fig. 1d and Supplementary Data 3 ). Collectively, these results identified PRSS35 as a secreted protein with significantly lower abundance in HCC cells, potentially due to cleavage into multiple fragments. Fig. 1 PRSS35 is a secreted protein that decreased in HCC patients. a Proteomic analysis and comparison of THLE3 and PLC secretome (without non-classical secreted proteins) presented as a volcano plot. The red transverse dashed line indicates adjusted P- value of 0.05. The left black longitudinal dashed line indicates a fold-change (FC) of 0.5 and the right black longitudinal dashed line indicates a FC of 2.0. Orange dots: significantly increased proteins in THLE3 secretome ( P  2.0). Green dots: significantly decreased proteins in THLE3 ( P  2.0). Green dots: significantly decreased proteins in THLE3 ( P 8. All data analysis was conducted using MaxQuant (version 1.5.8.5) with Andromeda search engine. The reviewed human proteins documented with key word "secreted" in Uniprot database was downloaded as target human secreted protein database, the reviewed human proteins in Uniprot database was downloaded as target human protein database. First search peptide mass tolerance was 20 p.p.m. and main search peptide mass tolerance was 4.5p.p.m. MS/MS match tolerance was set to 0.5 Da. For the peptide identification via peptide spectrum matching the FDR was controlled with a standard target-decoy approach. 1% peptide FDR was applied at PSM level. Enzyme specificity with trypsin was used, and maximum missing cleavages was set as two. The fixed modification was set as Carbamidomethyl (C) and variable modifications were oxidation (M), acetylation (Protein N-term). False discovery rate (FDR) thresholds for protein and peptide were set at 0.01. SILAC-labeled secretome profiling Collected secreted proteins and quantified using Bradford Protein Assay Kit (Sangon biology). For quantitative analysis, 200 µg proteins of both samples were combined prior to subsequent procedures. Guanidine hydrochloride were added to the lysate to a final concentration of 4 M for secreted proteins denature. Then the proteins were reduced with 5 mM dithiothreitol (DTT) at 37 °C for 45 min and alkylated with 15 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. The alkylation was quenched by DTT at the final concentration of 20 mM at room temperature. Then 100 µg protein mixture were transferred to the 3kD molecular weight cut-off filter, spun down and washed twice with 100 mM HEPES buffer (pH 8.0). Then the proteins were resuspended in 100 μl 50 mM HEPES (pH 8.0) in the filter and digested by trypsin at a protein to enzyme ratio of 50:1 (w/w) (overnight, 37 °C). Then the digested peptides were filtered with 10kD molecular weight cut-off filter, desalted, and fractionated into four fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). Peptide samples were vacuum dried for HPLC–MS/MS analysis. For the HPLC-MS/MS analysis, the peptides were analyzed on an QE-plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an EASY-nLC 1200 nano liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were dissolved in mobile phase buffer A (0.1% formic acid (FA) in water) and loaded onto Precolumn (Nano Trap 100 µm i.d. × 20 mm, Acclaim PepMap100 C18, 5 µm, 100 à ) and Analytical column (PepMap C18 2 μm 50 μm × 150 mm NV FS 1200 bar) by an autosampler. Then the peptides were separated and eluted with a linear gradient of 3%–8% of mobile phase buffer B (0.1% FA in 80% acetonitrile) for 3 min, 8–28% buffer B for 70 min, 28–45% buffer B for 35 min, 45–90% buffer B for 2 min, and 90% buffer B for 10 min at the flow rate of 300nL/min on HPLC system. The eluted peptides were ionized under high voltage (2.2 KV) and detected by QE-plus using nano-spray ion source (NSI). For full MS scan, peptides with 350–1700 m / z were detected at the resolution 70,000. The automatic gain control (AGC) target was set to 3 × 10 6 , and maximum ion injection time was set to 50 ms. The ions with intensity above 2 × 10 4 were subjected to fragmentation via high energy collision induced dissociation (HCD) with 27% normalized collision energy (NCE). The fragmented ions were analyzed in Orbitrap. The AGC target in the ion trap was set to 1 × 10 5 , and the isolation window was 1.6 m / z . The scan range fixed first mass at 110.0 m /z. The microscan was 1. The dynamic exclusion was set as 20 s. The charge exclusion were unassigned and charge states with 1, 7, 8, and >8. For the SILAC-labeled proteome profiling, the raw data were searched with Thermo Scientific Proteome Discoverer 2.2 with Seaquest HT (v1.17) search engine. The proteins documented with key word "secreted" in uniprot database was downloaded as target Human secreted protein database. SILAC 2plex was set as quantification method. Enzyme specificity with trypsin was used, and maximum missing cleavages was set as two. The mass error tolerance for precursor ions was 20 ppm and fragment ions was 0.5 Da. The fixed modification was set as Carbamidomethyl (C) and variable modifications were oxidation (M), acetylation (Protein N-term). False discovery rate (FDR) thresholds for protein and peptide were set at 0.01. High-throughput protease screen HTPS was performed as previously described 38 . In brief, we harvested PLC, HepG2, Hep3B and 293T cells, respectively. For lysis to achieve native lysate, we used HNN buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF, pH 7.8) supplemented with 0.5% NP-40 and protease inhibitor cocktail. Afterwards, the lysate was centrifuged at 14,000 × g for 15 min to remove any non-soluble material and the buffer was exchanged for 20 mM ammonium bicarbonate pH 7.8 using a filter device with molecular weight cut-off of 3kD. Afterward, native cell lysate standardized in 20 mM Ammonium bicarbonate pH 7.8 was added at a final 50 µg of total protein per tube and mixed with the purified PRSS35 or PRSS35-domain1 at 1/50 [E]/[S] ratio. The samples were incubated at 37 °C for 12 h and collected by a 15 min centrifugation at 1200 × g in a low-binding tube. The collection step was repeated by adding 100 µl of MS-grade water. The fractions were transferred to low-binding tubes (Eppendorf) and concentrated on the speed-vacuum to complete dryness. The samples were stored at −80 °C until analysis. Before analysis, the samples were resuspended in 20 µl of MS-grade water with 0.1% formic acid and the peptide concentration was determined with Nanodrop UV spectrometer. The sample concentration was adjusted to 1 µg/µl with water containing 0.1% formic acid. For the HPLC–MS/MS analysis, the peptides were analyzed on an QE-plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an EASY-nLC 1200 nano liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were dissolved in mobile phase buffer A (0.1% formic acid (FA) in water) and loaded onto Precolumn (Nano Trap 100 µm i.d. × 20 mm, Acclaim PepMap100 C18, 5 µm, 100 à ) and Analytical column (PepMap C18 2 μm 50 μm × 150 mm NV FS 1200 bar) by an autosampler. Then the peptides were separated and eluted with a linear gradient of 3–8% of mobile phase buffer B (0.1% FA in 80% acetonitrile) for 3 min, 8–28% buffer B for 70 min, 28–45% buffer B for 35 min, 45–90% buffer B for 2 min, and 90% buffer B for 10 min at the flow rate of 300nL/min on HPLC system. The eluted peptides were ionized under high voltage (2.2 KV) and detected by QE-plus using nano-spray ion source (NSI). For full MS scan, peptides with 350–1700 m / z were detected at the resolution 70,000. The automatic gain control (AGC) target was set to 1 × 10 6 , and maximum ion injection time was set to 50 ms. The ions with intensity above 2 × 10 4 were subjected to fragmentation via high energy collision induced dissociation (HCD) with 27% normalized collision energy (NCE). The fragmented ions were analyzed in Orbitrap. The AGC target in the ion trap was set to 1 × 10 5 , and the isolation window was 1.6 m / z . The scan range fixed first mass at 110.0 m / z . The microscan was 1. The dynamic exclusion was set as 20 s. The charge exclusion were unassigned and charge states with 1, 7, 8, and >8. The raw data was searched with MaxQuant (version 1.5.8.5) with Andromeda search engine using the reviewed human UniProt database. We set the digestion mode to unspecific and the maximal peptide length to 40AA as described elsewhere 37 , 38 . Acetylation (N-termini) and oxidation (M) were set as variable modifications. First search peptide mass tolerance was 20 p.p.m. and main search peptide mass tolerance was 4.5 p.p.m. MS/MS match tolerance was set to 0.5 Da. For the peptide identification via peptide spectrum matching the FDR was controlled with a standard target-decoy approach. 1% peptide FDR was applied at PSM level and only peptide hits with a PEP score ≤0.05 and a score >40 were retained for further analysis. The final list of proteins was the union of proteins identified in the respective samples and we included only proteins with a global protein PEP ≤ 0.01 into the final database. Potential contaminants were excluded from the subsequent data analysis. The protease cleavage window was used for the protease specificity determination using the iceLogo tool 39 . The reference set used for the calculation of the chance of the amino acid occurrence ( P value) at a certain position was the human reference proteome. Mice Mice were housed at the animal facility of University of Science and Technology of China. Four-week-old male C57BL/6J and ICR mice were purchased from SLAC ANIMAL COMPANY. Four-week-old male Balb/c-nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. PRSS35 −/− mice (C57BL/6J) were generated using CRISPR genome editing (target sequence: 5′-AACGAGGGTACCGCTGCAGC-3′ and 5′-ACTCGGAACAGCAGCGTAAA-3′) and were obtained from the animal facility of the University of Science and Technology of China. Animal studies All animals were housed at a suitable temperature (22–24 °C) and humidity (40–70%) under a 12/12-h light/dark cycle with unrestricted access to food and water for the duration of the experiment. All animal studies were conducted with approval from the Animal Research Ethics Committee of the University of Science and Technology of China. For syngeneic mouse tumor models, each group of Hepa1-6 cells (4 × 10 6 ) was injected subcutaneously into four-week-old male mice (C57BL/6J; SLAC ANIMAL COMPANY). Tumor volumes were calculated using the following formula: width (mm) × depth (mm) × length (mm) × 0.52. For xenograft model, each group of HepG2 cells (4 × 10 6 ) was injected subcutaneously into four-week-old male nude mice (BALB/c-nude mice; Beijing Vital River Laboratory Animal Technology Co., Ltd). The tumor burden was less than the maximum diameter (15 mm) approved by the Animal Research Ethics Committee of the University of Science and Technology of China. For YAP-5SA-induced-HCC models, plasmid DNA suspended in sterile Ringer's solution in a volume equal to 10% of the body weight was injected in 5–7 s via the tail vein of four-week-old male C57BL/6J or ICR (SLAC ANIMAL COMPANY) mice. The amount of injected DNA was 50 µg of per transposon plasmids together with 10 µg of PB transposase plasmids. At the end of animal studies, all mice were euthanized by inhaling carbon dioxide. Flow cytometry For mouse livers and tumors, samples were cut into pieces and treated with collagenase IV and DNase I for one hour. The cell suspension was passed through a 70 µm cell strainer (Corning). The single-cell suspension was centrifuged at 300 × g for 5 min at 4 °C. The cell pellet was then resuspended in FACS buffer (1% BSA in PBS) and blocked with CD16/32 for 20 min, followed by incubating with the indicated antibodies for 30 min on ice. The signal was detected by using a BD Fortessa with BD FACSDiva Software (V8.03) and was analyzed using Flowjo v10 software. FACS sequential gating strategies for FACS experiments in Fig. 4c , Supplementary Fig 3j, m , Supplementary Fig 4c, h were presented in Supplementary Fig 4o . Clinical human tissue specimen The 7 paired HCC lesions and the adjacent noncancerous clinical tissue samples were collected from HCC patients. The normal blood samples were collected from heathy physical examination persons. And the blood samples with HCC were collected from HCC patients. All the patients and physical examination persons are from the general surgery department, The first affiliated hospital of University of Science and Technology of China. The Supplementary Table 2 documented the clinicopathological characteristics of the 158 HCC patients used for the IHC assay (Supplementary Fig 1e ). And the Supplementary Table 5 documented the basic characteristics of the HCC patients and normal subjects whose sera were used for the ELISA assay (Fig. 1f ). We used two different batches of clinical patient specimens for the IHC analysis and the ELISA assay. To use these clinical materials for research purposes, prior patients' written informed consents and approval from the Institutional Research Ethics Committee of the first affiliated hospital of University of Science and Technology of China were obtained. Detection of serum MPO–DNA We detected serum MPO–DNA using a previously described capture ELISA method with slight modifications. 96-well microtiter plates were coated with 5 μg/ml anti-MPO monoclonal antibody (Proteintech, 22225-1-AP) as the capturing antibody overnight at 4 °C. After blocking in 1% BSA, patient serum together with peroxidase-labeled anti-DNA monoclonal antibody was added (component No.2 of the Cell Death Detection ELISA kit, Roche, 11774425001), incubated at room temperature for 2 h and then washed with PBS three times. The peroxidase substrate (Roche, 11774425001) was added. After incubation at 37 °C for 40 min, the optical density was measured at 405 nm using a microplate reader. Neutrophil isolation To isolate neutrophils from peripheral blood of mice, whole blood was collected via cardiac puncture (1 ml per animal) and suspended in HBSS (2 ml per animal) with 15 mM EDTA. After centrifugation (400 × g , 10 min, 4 °C), white cells were resuspended in 2 ml HBSS with 2 mM EDTA. Then, the cells were centrifuged (1500 × g , 30 min, room temperature) in a three-layer Percoll gradient (78%, 69%, and 52%) without braking. Neutrophils enriched in the interface of 69% and 78% layers were confirmed to be of > 95% purity by flow cytometric analysis (Supplementary Fig. 3g ). Neutrophil migration assays 5 ×10 5 freshly isolated mice neutrophils in RPMI 1640 were added to the upper chamber of transwell device (Corning, 3402), and a 1:1 mixture of RPMI 1640 and indicated cancer cell conditional media was added to the lower chamber as the chemoattractant. The migrated neutrophils in the lower chamber were counted after three hours. PRSS35 in vitro substrate assay To test for the cleavage of β-casein, PRSS35, PRSS35-domain1, PRSS35-domain2, or PRSS35-domain3 (0.025 mM final concentration) and β-casein (25 μM final concentration) were both added to a tube, respectively. Reactions were incubated for 12 h at 37 °C followed detection by WB. To test for the cleavage of the substrates Dabcyl-KKKK-Edans, Dabcyl-LKKE-Edans and Dabcyl-RINKKIEK-Edans, a final concentration of 25 μM of each substrate was used. PRSS35-domain1 (0.025 mM final concentration) was added to a 96-well black, clear-bottom plate in 99 μl PBS. Substrates were dissolved in DMSO at 100× the indicated concentration (25 μM final concentration) and 1 μl was added to each well. Reactions were incubated for 12 h at 37 °C while being read on a CLARIOstar plate reader per hour (Molecular Devices) using an excitation wavelength of 340 nm and an emission wavelength of 490 nm. To test for the cleavage of the substrate CXCL2, a final concentration of 25 μM of purified human CXCL2 was used. PRSS35-domain1 (0.5 mM final concentration) and human CXCL2 were both added to a tube. Reactions were incubated for 48 h at 37 °C followed detection by WB. Statistics and reproducibility The data are presented as either mean ± s.d. or mean ± s.e.m. as stated. Statistical analyses were performed using either Prism8 (GraphPad Software) or SPSS software Statistics 22 (IBM Corp.). Data distribution was assumed to be normal, but this was not formally tested. Two-tailed unpaired Student's t -test and one-, two-analysis of variance (ANOVA) were used to calculate P -values. The Tukey method was used to adjust multiple comparisons. Kaplan–Meier curves were used to depict survival function from lifetime data for human patients using the log-rank test. The relationship between expression of PRSS35 and clinicopathological characteristics was analyzed by chi-square test. P 8. All data analysis was conducted using MaxQuant (version 1.5.8.5) with Andromeda search engine. The reviewed human proteins documented with key word "secreted" in Uniprot database was downloaded as target human secreted protein database, the reviewed human proteins in Uniprot database was downloaded as target human protein database. First search peptide mass tolerance was 20 p.p.m. and main search peptide mass tolerance was 4.5p.p.m. MS/MS match tolerance was set to 0.5 Da. For the peptide identification via peptide spectrum matching the FDR was controlled with a standard target-decoy approach. 1% peptide FDR was applied at PSM level. Enzyme specificity with trypsin was used, and maximum missing cleavages was set as two. The fixed modification was set as Carbamidomethyl (C) and variable modifications were oxidation (M), acetylation (Protein N-term). False discovery rate (FDR) thresholds for protein and peptide were set at 0.01. SILAC-labeled secretome profiling Collected secreted proteins and quantified using Bradford Protein Assay Kit (Sangon biology). For quantitative analysis, 200 µg proteins of both samples were combined prior to subsequent procedures. Guanidine hydrochloride were added to the lysate to a final concentration of 4 M for secreted proteins denature. Then the proteins were reduced with 5 mM dithiothreitol (DTT) at 37 °C for 45 min and alkylated with 15 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. The alkylation was quenched by DTT at the final concentration of 20 mM at room temperature. Then 100 µg protein mixture were transferred to the 3kD molecular weight cut-off filter, spun down and washed twice with 100 mM HEPES buffer (pH 8.0). Then the proteins were resuspended in 100 μl 50 mM HEPES (pH 8.0) in the filter and digested by trypsin at a protein to enzyme ratio of 50:1 (w/w) (overnight, 37 °C). Then the digested peptides were filtered with 10kD molecular weight cut-off filter, desalted, and fractionated into four fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). Peptide samples were vacuum dried for HPLC–MS/MS analysis. For the HPLC-MS/MS analysis, the peptides were analyzed on an QE-plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an EASY-nLC 1200 nano liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were dissolved in mobile phase buffer A (0.1% formic acid (FA) in water) and loaded onto Precolumn (Nano Trap 100 µm i.d. × 20 mm, Acclaim PepMap100 C18, 5 µm, 100 à ) and Analytical column (PepMap C18 2 μm 50 μm × 150 mm NV FS 1200 bar) by an autosampler. Then the peptides were separated and eluted with a linear gradient of 3%–8% of mobile phase buffer B (0.1% FA in 80% acetonitrile) for 3 min, 8–28% buffer B for 70 min, 28–45% buffer B for 35 min, 45–90% buffer B for 2 min, and 90% buffer B for 10 min at the flow rate of 300nL/min on HPLC system. The eluted peptides were ionized under high voltage (2.2 KV) and detected by QE-plus using nano-spray ion source (NSI). For full MS scan, peptides with 350–1700 m / z were detected at the resolution 70,000. The automatic gain control (AGC) target was set to 3 × 10 6 , and maximum ion injection time was set to 50 ms. The ions with intensity above 2 × 10 4 were subjected to fragmentation via high energy collision induced dissociation (HCD) with 27% normalized collision energy (NCE). The fragmented ions were analyzed in Orbitrap. The AGC target in the ion trap was set to 1 × 10 5 , and the isolation window was 1.6 m / z . The scan range fixed first mass at 110.0 m /z. The microscan was 1. The dynamic exclusion was set as 20 s. The charge exclusion were unassigned and charge states with 1, 7, 8, and >8. For the SILAC-labeled proteome profiling, the raw data were searched with Thermo Scientific Proteome Discoverer 2.2 with Seaquest HT (v1.17) search engine. The proteins documented with key word "secreted" in uniprot database was downloaded as target Human secreted protein database. SILAC 2plex was set as quantification method. Enzyme specificity with trypsin was used, and maximum missing cleavages was set as two. The mass error tolerance for precursor ions was 20 ppm and fragment ions was 0.5 Da. The fixed modification was set as Carbamidomethyl (C) and variable modifications were oxidation (M), acetylation (Protein N-term). False discovery rate (FDR) thresholds for protein and peptide were set at 0.01. High-throughput protease screen HTPS was performed as previously described 38 . In brief, we harvested PLC, HepG2, Hep3B and 293T cells, respectively. For lysis to achieve native lysate, we used HNN buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF, pH 7.8) supplemented with 0.5% NP-40 and protease inhibitor cocktail. Afterwards, the lysate was centrifuged at 14,000 × g for 15 min to remove any non-soluble material and the buffer was exchanged for 20 mM ammonium bicarbonate pH 7.8 using a filter device with molecular weight cut-off of 3kD. Afterward, native cell lysate standardized in 20 mM Ammonium bicarbonate pH 7.8 was added at a final 50 µg of total protein per tube and mixed with the purified PRSS35 or PRSS35-domain1 at 1/50 [E]/[S] ratio. The samples were incubated at 37 °C for 12 h and collected by a 15 min centrifugation at 1200 × g in a low-binding tube. The collection step was repeated by adding 100 µl of MS-grade water. The fractions were transferred to low-binding tubes (Eppendorf) and concentrated on the speed-vacuum to complete dryness. The samples were stored at −80 °C until analysis. Before analysis, the samples were resuspended in 20 µl of MS-grade water with 0.1% formic acid and the peptide concentration was determined with Nanodrop UV spectrometer. The sample concentration was adjusted to 1 µg/µl with water containing 0.1% formic acid. For the HPLC–MS/MS analysis, the peptides were analyzed on an QE-plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an EASY-nLC 1200 nano liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were dissolved in mobile phase buffer A (0.1% formic acid (FA) in water) and loaded onto Precolumn (Nano Trap 100 µm i.d. × 20 mm, Acclaim PepMap100 C18, 5 µm, 100 à ) and Analytical column (PepMap C18 2 μm 50 μm × 150 mm NV FS 1200 bar) by an autosampler. Then the peptides were separated and eluted with a linear gradient of 3–8% of mobile phase buffer B (0.1% FA in 80% acetonitrile) for 3 min, 8–28% buffer B for 70 min, 28–45% buffer B for 35 min, 45–90% buffer B for 2 min, and 90% buffer B for 10 min at the flow rate of 300nL/min on HPLC system. The eluted peptides were ionized under high voltage (2.2 KV) and detected by QE-plus using nano-spray ion source (NSI). For full MS scan, peptides with 350–1700 m / z were detected at the resolution 70,000. The automatic gain control (AGC) target was set to 1 × 10 6 , and maximum ion injection time was set to 50 ms. The ions with intensity above 2 × 10 4 were subjected to fragmentation via high energy collision induced dissociation (HCD) with 27% normalized collision energy (NCE). The fragmented ions were analyzed in Orbitrap. The AGC target in the ion trap was set to 1 × 10 5 , and the isolation window was 1.6 m / z . The scan range fixed first mass at 110.0 m / z . The microscan was 1. The dynamic exclusion was set as 20 s. The charge exclusion were unassigned and charge states with 1, 7, 8, and >8. The raw data was searched with MaxQuant (version 1.5.8.5) with Andromeda search engine using the reviewed human UniProt database. We set the digestion mode to unspecific and the maximal peptide length to 40AA as described elsewhere 37 , 38 . Acetylation (N-termini) and oxidation (M) were set as variable modifications. First search peptide mass tolerance was 20 p.p.m. and main search peptide mass tolerance was 4.5 p.p.m. MS/MS match tolerance was set to 0.5 Da. For the peptide identification via peptide spectrum matching the FDR was controlled with a standard target-decoy approach. 1% peptide FDR was applied at PSM level and only peptide hits with a PEP score ≤0.05 and a score >40 were retained for further analysis. The final list of proteins was the union of proteins identified in the respective samples and we included only proteins with a global protein PEP ≤ 0.01 into the final database. Potential contaminants were excluded from the subsequent data analysis. The protease cleavage window was used for the protease specificity determination using the iceLogo tool 39 . The reference set used for the calculation of the chance of the amino acid occurrence ( P value) at a certain position was the human reference proteome. Mice Mice were housed at the animal facility of University of Science and Technology of China. Four-week-old male C57BL/6J and ICR mice were purchased from SLAC ANIMAL COMPANY. Four-week-old male Balb/c-nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. PRSS35 −/− mice (C57BL/6J) were generated using CRISPR genome editing (target sequence: 5′-AACGAGGGTACCGCTGCAGC-3′ and 5′-ACTCGGAACAGCAGCGTAAA-3′) and were obtained from the animal facility of the University of Science and Technology of China. Animal studies All animals were housed at a suitable temperature (22–24 °C) and humidity (40–70%) under a 12/12-h light/dark cycle with unrestricted access to food and water for the duration of the experiment. All animal studies were conducted with approval from the Animal Research Ethics Committee of the University of Science and Technology of China. For syngeneic mouse tumor models, each group of Hepa1-6 cells (4 × 10 6 ) was injected subcutaneously into four-week-old male mice (C57BL/6J; SLAC ANIMAL COMPANY). Tumor volumes were calculated using the following formula: width (mm) × depth (mm) × length (mm) × 0.52. For xenograft model, each group of HepG2 cells (4 × 10 6 ) was injected subcutaneously into four-week-old male nude mice (BALB/c-nude mice; Beijing Vital River Laboratory Animal Technology Co., Ltd). The tumor burden was less than the maximum diameter (15 mm) approved by the Animal Research Ethics Committee of the University of Science and Technology of China. For YAP-5SA-induced-HCC models, plasmid DNA suspended in sterile Ringer's solution in a volume equal to 10% of the body weight was injected in 5–7 s via the tail vein of four-week-old male C57BL/6J or ICR (SLAC ANIMAL COMPANY) mice. The amount of injected DNA was 50 µg of per transposon plasmids together with 10 µg of PB transposase plasmids. At the end of animal studies, all mice were euthanized by inhaling carbon dioxide. Flow cytometry For mouse livers and tumors, samples were cut into pieces and treated with collagenase IV and DNase I for one hour. The cell suspension was passed through a 70 µm cell strainer (Corning). The single-cell suspension was centrifuged at 300 × g for 5 min at 4 °C. The cell pellet was then resuspended in FACS buffer (1% BSA in PBS) and blocked with CD16/32 for 20 min, followed by incubating with the indicated antibodies for 30 min on ice. The signal was detected by using a BD Fortessa with BD FACSDiva Software (V8.03) and was analyzed using Flowjo v10 software. FACS sequential gating strategies for FACS experiments in Fig. 4c , Supplementary Fig 3j, m , Supplementary Fig 4c, h were presented in Supplementary Fig 4o . Clinical human tissue specimen The 7 paired HCC lesions and the adjacent noncancerous clinical tissue samples were collected from HCC patients. The normal blood samples were collected from heathy physical examination persons. And the blood samples with HCC were collected from HCC patients. All the patients and physical examination persons are from the general surgery department, The first affiliated hospital of University of Science and Technology of China. The Supplementary Table 2 documented the clinicopathological characteristics of the 158 HCC patients used for the IHC assay (Supplementary Fig 1e ). And the Supplementary Table 5 documented the basic characteristics of the HCC patients and normal subjects whose sera were used for the ELISA assay (Fig. 1f ). We used two different batches of clinical patient specimens for the IHC analysis and the ELISA assay. To use these clinical materials for research purposes, prior patients' written informed consents and approval from the Institutional Research Ethics Committee of the first affiliated hospital of University of Science and Technology of China were obtained. Detection of serum MPO–DNA We detected serum MPO–DNA using a previously described capture ELISA method with slight modifications. 96-well microtiter plates were coated with 5 μg/ml anti-MPO monoclonal antibody (Proteintech, 22225-1-AP) as the capturing antibody overnight at 4 °C. After blocking in 1% BSA, patient serum together with peroxidase-labeled anti-DNA monoclonal antibody was added (component No.2 of the Cell Death Detection ELISA kit, Roche, 11774425001), incubated at room temperature for 2 h and then washed with PBS three times. The peroxidase substrate (Roche, 11774425001) was added. After incubation at 37 °C for 40 min, the optical density was measured at 405 nm using a microplate reader. Neutrophil isolation To isolate neutrophils from peripheral blood of mice, whole blood was collected via cardiac puncture (1 ml per animal) and suspended in HBSS (2 ml per animal) with 15 mM EDTA. After centrifugation (400 × g , 10 min, 4 °C), white cells were resuspended in 2 ml HBSS with 2 mM EDTA. Then, the cells were centrifuged (1500 × g , 30 min, room temperature) in a three-layer Percoll gradient (78%, 69%, and 52%) without braking. Neutrophils enriched in the interface of 69% and 78% layers were confirmed to be of > 95% purity by flow cytometric analysis (Supplementary Fig. 3g ). Neutrophil migration assays 5 ×10 5 freshly isolated mice neutrophils in RPMI 1640 were added to the upper chamber of transwell device (Corning, 3402), and a 1:1 mixture of RPMI 1640 and indicated cancer cell conditional media was added to the lower chamber as the chemoattractant. The migrated neutrophils in the lower chamber were counted after three hours. PRSS35 in vitro substrate assay To test for the cleavage of β-casein, PRSS35, PRSS35-domain1, PRSS35-domain2, or PRSS35-domain3 (0.025 mM final concentration) and β-casein (25 μM final concentration) were both added to a tube, respectively. Reactions were incubated for 12 h at 37 °C followed detection by WB. To test for the cleavage of the substrates Dabcyl-KKKK-Edans, Dabcyl-LKKE-Edans and Dabcyl-RINKKIEK-Edans, a final concentration of 25 μM of each substrate was used. PRSS35-domain1 (0.025 mM final concentration) was added to a 96-well black, clear-bottom plate in 99 μl PBS. Substrates were dissolved in DMSO at 100× the indicated concentration (25 μM final concentration) and 1 μl was added to each well. Reactions were incubated for 12 h at 37 °C while being read on a CLARIOstar plate reader per hour (Molecular Devices) using an excitation wavelength of 340 nm and an emission wavelength of 490 nm. To test for the cleavage of the substrate CXCL2, a final concentration of 25 μM of purified human CXCL2 was used. PRSS35-domain1 (0.5 mM final concentration) and human CXCL2 were both added to a tube. Reactions were incubated for 48 h at 37 °C followed detection by WB. Statistics and reproducibility The data are presented as either mean ± s.d. or mean ± s.e.m. as stated. Statistical analyses were performed using either Prism8 (GraphPad Software) or SPSS software Statistics 22 (IBM Corp.). Data distribution was assumed to be normal, but this was not formally tested. Two-tailed unpaired Student's t -test and one-, two-analysis of variance (ANOVA) were used to calculate P -values. The Tukey method was used to adjust multiple comparisons. Kaplan–Meier curves were used to depict survival function from lifetime data for human patients using the log-rank test. The relationship between expression of PRSS35 and clinicopathological characteristics was analyzed by chi-square test. P < 0.05 was considered significant. The experiments were not randomized, except that mice were randomly grouped before different treatments. Data collection and analysis were not performed blind to the conditions of the experiments, except for IHC score analysis. Each experiment was repeated at least three times independently. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Supplementary information Supplementary Information Peer Review File Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Reporting Summary Supplementary Information Peer Review File Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Reporting Summary Source data Source Data Supplementary information The online version contains supplementary material available at 10.1038/s41467-023-37227-z. Peer review information Nature Communications thanks Michel Salzet, Alvaro Teijeira, Allan Tsung and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243045/

Does Covera-19 know ‘when to hold ‘em or ‘when to fold ‘em? A translational thought experiment

The function of proteins depends on their structure. The structural integrity of proteins is dynamic and depends on interacting nearby neighboring moieties that influence their properties and induce folding and structural changes. The conformational changes induced by these nearby neighbors in the micro-environmental milieu at that moment are guided by chemical or electrical bonding attractions. There are few literature references that describe the potential for environmental milieu changes to disfavor SARS-CoV-2 attachment to a receptor for survival outside of a host. There are many studies on the effects of pH (acid and base balance) supporting its importance for protein structure and function, but few focus on pH role in extracellular or intracellular protein or actionable requirements of Covera-19. 'Fold 'em or Hold 'em' is seen by the various functions and effects of furin as it seeks an acidic milieu for action or compatible amino acid sequences which is currently aided by its histidine component and the structural changes of proteins as they enter or exit the host. Questions throughout the text are posed to focus on current thoughts as reviewing applicable COVID-19 translational research science in order to understand the complexities of Covid-19. The pH needs of COVID-19 players and its journey through the human host and environment as well as some efficacious readily available repurposed drugs and out-of-the box and easily available treatments are reviewed. Introduction Interest is redirected to the receptor surroundings and intracellular organelle environment by focus on furin and pH as two basic pillars of this translational thought experiment to demonstrate the importance for understanding some of the important science underlying COVID-19's journey in humans. Using the current viral pandemic as a life saving model for therapeutic intervention by vaccines only and not other than 'vaccine' methods places ideational restrictions and impedes new treatment potentials. Several other treatment modalities for COVID-19 are introduced and supported by elegant studies and proven science. Viral activity, surface glycoproteins and interacting partners are affected by pH changes. Heat of ~104F [ 1 ] and washing/wiping surfaces with liquids/water and at a basic [alkaline] pH are important as are removable and replaceable paper or plastic covers on surfaces changed after every use. The literature has exploded with hundreds of studies and reports on the various symptoms, demographics, outcomes and massive numbers of new facts regarding the current novel human pathologic agent SARS-CoV-2. The names 2019-nCoV or SARS-CoV-2 (the virus) and COVID-19, Covera-19 or Covera (the illness) are simplified herein to "CV19". The current appropriate priorities (washing, masking, social distancing, testing, tracking and isolation) will reduce new infections, if strictly enforced, available or doable. The psychological societ al costs and results of population-wide fears and anxieties induced by unscientific actions at all levels are not addressed in this paper. The complexities of CV19 illness in the context of prior comorbid conditions, the many locations of receptor sites and mutations, based on the early New York hospitalization experience [ 2 ], are demonstrated and discussed using the early New York City model described below. A study of the presenting symptoms of CV19 from 5700 hospitalized patients in the New York City area from March 1, 2020 to April 1, 2020 revealed ~ 60% males and ~ 40% females. The most common comorbidities were hypertension [~ 57%], obesity [~ 42%], and diabetes [~ 34%]. The presenting symptoms were fever [~ 31%] and a respiratory rate greater than 24 breaths per minute in ~ 17%, with ~ 28% of those receiving supplemental oxygen. Co-morbidities include cancer, cardiovascular disease [arrhythmias, ischemia, coronary artery disease (CAD), and congestive heart failure (CHF)], hypertension, asthma, COPD/emphysema, obstructive sleep apnea, obesity and older age. Few patients [6.1%] had no comorbidities immune suppression, aids/HIV, history of organ transplant, renal disease, liver disease, smoking (~ 16%). All patients tested positive by the second test for CV19. Of the patients admitted to the intensive care unit (ICU) for intensive care and ventilation support [~ 20% of 5700, or 1151 ICU admissions], ~ 25% or 282 ventilated patients died [ 2 ]. The requirement for ventilation represents ~ 2.28% of the total studied patient population. The mortality rate may continue to decrease as treatment improves and the number of tested asymptomatic CV19-negative or asymptomatic CV19-positive patients increases, assuming that the whole population is tested. Testing and measuring 'hotspots' measures 'hotspots' only and not whole population numbers may not be statistically valid, and are easily manipulated. The CV19 morbidity and mortality numbers cannot be accurate until the whole population is tested regardless of symptoms so a true population basis can be established for true statistical population comparison. CV19 continues to prove that it is a potential serious threat to many populations, with some requiring rapid intervention and comprehensive care. Children are not as 'immune' to CV19 infection as initially thought. A recent JAMA article reported the clinical characteristics of many children with a pediatric inflammatory syndrome temporarily associated with CV19 [ 3 ], now called multisystem inflammatory syndrome in children (MIS-C), which can affect the heart, lungs, kidneys, brain, skin and eyes [ 4 ]. Is there a prolonged post-infection survivor syndrome in some patients? The T-lymphocytes are active but may potentially not be as effective as needed? A pandemic phylogenetic analysis of 84 distinct CV19 genomes in New York revealed entry paths from the US, Europe and the New York area [ 5 ]. The mortality statistics exhibit an elevated rate since many deaths are counted as due to CV19 if they are connected in any way. Deaths counted as 'due to CV19' actually may occur due to overwhelming sepsis and/or pneumonia. In reality, 'due to CV19' is a contributing factor that can be listed on its own line on the death certificate beneath the potential actual cause of death. Ventilator-treated CV19 patients who die have profound hypoxemia and may die of a myocardial infarction from insufficient oxygen or perhaps an arrhythmia. The immediate cause of death is myocardial infarction due to hypoxemia due to CV19 contributory effects (pneumonia or inflammation etc). Other practices are built into various semantics, as death certificate signers decide on death data certification. This is also found in other infectious diseases, such as influenza or HIV for example, which also have pneumonia and inflammation as co-morbidities, but the cause of death is listed commonly as Influenza and HIV/AIDS. Obviously this common practice is not strictly accurate, but is the current practice. Support for this reality has finally arrived from the National Center for Health Statistics, which states that only 6% of CV19-specific deaths are truthfully countable [ 6 , 7 ]. Death 'from' CV19 is different from death 'with' or 'due to (addition of)' CV19. These separate lines are self-explanatory on the death certificate. CV19 must accomplish the following in order to sustain itself and to multiply: Find a host > attach to and bind its receptor with the S1 spike protein > enter and travel through the receptor > initiate cell membrane fusion > create a pore and pass through to inside the host cell > enter an endosome > shed and leave the endosome to find the Host DNA and begin and finish the replication cycle > re-enter an exosome and travel to the host membranes > and 'exit' the host cell. The exit and leaving the exosome to 'exit' are not well established. Each of these steps has its own requirements and complexities including energy sources. The mouth/oropharynx and nose as entrances for CV19-laden air and 'droplets' provide a direct path to the oropharynx, trachea, and lungs but also provide receptor sites along the way [ 8 – 10 ]. Two important angiotensin receptor blocker (ARB) proteins (ACE2[angiotensin converting enzyme 2] and TMPRSS2 [transmembrane protease serine 2]) are strongly expressed in nasal passage goblet secretory cells, type II pneumocytes of the alveoli in the lungs, and absorptive enterocytes of the intestine-ileum and lower bowel [ 9 , 10 ]. Masking and distancing are important in personal prevention of both oral and nasal CV19 entry by potential aerosol and 'droplet' contagion. Masking does double duty—it prevents spread from infected patients with or without symptoms via CV19 aerosols or 'droplets' into the nasal passages and mouths/oropharynx of uninfected victims, and protects uninfected patients wearing masks. Surface contamination sources are a separate problem. Ziegler et al. [ 9 ] noted other entry points and receptor site locations for CV19: the conjunctivae of the eyes, the epidermal surfaces of blood vessels, and the tissue targets of viral spread via blood vessels to target organs. Is an open wound or absent skin [i.e., burns] an opening for CV19? Burns and wounds expose blood vessels with receptor sites. The target organs include the eyes, lungs, heart, all types of blood vessels, brain (neural cortex and brain stem, spinal fluid), nose, liver, kidneys and intestines (ileal sites or lower bowel) [ 10 ]. The conjunctival entry point in the eyes presents special problems in covering and protection—perhaps wearing glasses may help, but that is questionable. In later CV19 illnesses, this entry may lead to conjunctivitis and be potentially restricted by tear production in response to irritation from CV19 presence. The conjunctivae also have goblet cells with a potential role in CV19 systemic spread. What is the role of tears or ocular fluids in combatting CV19 entry? Are the epidermal surfaces of blood vessels in the conjunctivae facilitating systemic CV19 entry? The pH of the conjunctival goblet cells and tears/ocular fluids was measured at pH 6.3/6.5 to 7.23/7.6 [ 11 , 12 ], which may be advantageous to CV19 and its preference for acidic site activity. It is reported that borate may be a pH buffer in the conjunctivae and ocular fluids [ 11 ]. The conjunctivae as entry sites have been overshadowed by the emphasis on oral and nasal entry portals, hence the conjunctival CV19 entry site has received less attention and study. Loss of eyesight or ocular damage has not been mentioned in the CV19 literature. Does the higher pH range in the conjunctivae denature CV19 'S' protein so it cannot be cleaved by furin for spike S1 subunit-receptor attachment? What is the furin level in the conjunctival goblet cells compared to oral and nasal passage sites? The answer may lie in the environmental milieu of the conjunctivae, which is bathed by tears and other ocular fluids. The conjunctival CV19 entry portal may support using a plastic facial mask that also covers the eyes as a superior infection barrier. The facial plastic mask may also limit PPE exposure from CV19 positive patients. The gastrointestinal tract may be a 'pass-through' site for shed viral shells and proteins [ 10 ], but can also be an entry site from any source or from self-infection by asymptomatic positive individuals. The widespread systemic ACE2 and ARB (angiotensin converting enzyme 2 and angiotensin 2 receptor blocker sites) receptor distribution and viral entry may lead to multi-organ illness or potential multi-organ collapse and death. CV19's intracellular journey and actions Protein structure dictates function. Local environmental chemically or electrically induced conformational and structural changes [denaturation] may enhance or prevent agonist and host receptor proteins from interacting. Does ARB/ACE2 display a feedback loop in the CV19 entry process? After CV19 enters the cell via the ACE2/ARB receptor, uncoating of both CV19 and host membranes during the fusion process occurs, and the single-stranded RNA genome enters into the host cell cytoplasm via endosomal transport. CV19 then initiates its reverse RNA reproduction cycle leading to eventual 'exocytosis', as its newly formed virions exit from inside newly formed exosomes to seek a new host cell's ACE2/ARB receptors and repeat the process. The host is re-infected in a continuously expanding infection, both intracellularly and systemically. Are RNAs are made early from the first genes of the host DNA genome, and are multiple copies made? What is the fidelity of the subsequent mRNAs templated from the host DNA? Could these multiple copies be other sources of mutations if strict copying fidelity is not observed [ 13 ]? Where on the CV19 RNA and in what time-frame are the promoter and terminator genes active, if present at all? Where and when, early or late, on the CV19 RNA is this specific reverse transcriptase gene found? Is an enzyme involved in the production and initiation of this particular RNA reverse transcriptase? What will comparison with HIV reverse transcriptase models show? Could the built-in 'degeneracy' in the triplet genetic code (a different triplet code for the same amino acid in the mammalian genome) also be a potential adaptive evolutionary mechanism in CV19, as it uses only its RNA? It is generally accepted that, like poliovirus, when CV19 uses its reverse transcriptase to make its own RNA, protein synthesis ceases in the host. In support of this, Thoms et al. recently showed that "Nsp1 (nonstructural protein 1) from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of host messenger RNA (mRNA) translation both in vitro and in cells." [ 14 ]. Ban supports this by suggesting that Nsp1 suppresses host innate immune functions and interferes with mRNA binding, as the C-terminal domain of SARS-CoV-2 Nsp1 binds to the mRNA entry channel [ 15 ]. The high infection and transmissibility rate of CV19 was found to be due to another host protein called 'neuropilin-1', "which is recognized and bound by CV19's spike protein and also facilitates CV19 cell entry and infectivity [ 16 ]." This finding seeks urgent study and comparison with newer emerging variants or other mutant entry proteins. Additional intermediate conformational structures of proteins were addressed earlier by Bai and Englander [ 17 ], who stated that " All possible protein folding intermediates exist in equilibrium with the native protein at naïve as well as non-naïve conditions, with occupation determined by their free energy level." Principles of protein structure and folding are illustrated by Walls [ 18 ], Wrobel [ 19 ], and Wrapp [ 20 ]. PROTEIN GAME PLAYERS: 'Hold 'em or fold 'em or fold 'em and hold 'em?' Wrapp et al. [ 20 ], Watanabe et al. [ 21 ] and Cai et al. [ 22 ] report that the 'Spike ('S')' glycoprotein mediates cell entry and cell fusion. The 'S' protein is described as a "trimeric class 1 fusion protein composed of two subunits: one responsible for receptor binding (S1 subunit) and a membrane fusion subunit on the S2 subunit. The 'S' protein undergoes a 'hinge-like' conformational change to expose the receptor-binding domain. The spike surface is dominated by host-derived glycans, with each trimer displaying 66 N-linked glycosylation sites" [ 21 ]. The 'S1' spike subunit binds to an amino terminal, and the 'S2' spike subunit binds to a carboxyl site after cleavage by furin [ 23 ]. Importantly, furin cleaves the 'S' protein of CV19 into two proteins: S1 and S2. As above, S1 is responsible for receptor site attachment, and S2, with its fusion peptide site, is responsible for fusion of the CV19 and host cell membranes to allow the CV19 genome to enter the host cell [ 22 ]. These actions may also indicate potential intrareceptor site mechanisms as CV19 transits through the receptor site. All of the receptor interactions and intracellular activities require an energy source. Once the membranes of both the host and CV19 are fused, the S2 fusion peptide forms a 'fusion pore' allowing the CV19 genome to enter the host cell for endosomal 'entry' and transportation [ 24 ]. Another potential CV19 entry-enabling protein was recently identified by Liu et al. [ 25 ]: heparan sulfate (SO4), which is closely related to heparin. Liu et al. also demonstrated that heparan SO4 removal inhibits CV19 attachment to the ACE2/ARB receptor site. Heparan SO4 is also known to be and described as an 'adhesin'. Heparan SO4 provides an attachment site for CV19, plausibly holding it in the host receptor site area, and CV19 may not attach to the ACE2/ARB receptor site until it has adhered to the heparan SO4. Heparan SO4 attachment may be an early time-dependent or controlled event, and in this role it may enhance 'adhesion' of CV19 to the ARB/ACE2 receptor for subsequent actions to proceed. This 'adherence and delivery' action by heparan SO4 could be a critical early step in CV19 attachment to host cells because it 'guarantees' delivery of CV19 to the host cell receptor. Could heparan SO4 be blocked as a treatment, and the process halted at this early stage as well with prolonging time adherent dependency? Does heparanSO4 participate in a sequential process with other components? Heparan SO4 is a negatively charged polysaccharide that resided on the cell membrane. In the CV19 events sequence, it is derived from the host and onto the CV19 exterior and is 'held' in the host receptor site region. As Liu described removal or inactivation of heparan SO4 inhibits CV19 attachment to the ACE2/ARB receptor site [ 25 ]. Since heparan SO4 is a very acidic moiety, could it be inactivated by creating a basic environment and receptor site environment by pH adjustment? This may disallow an early receptor site CV19 attachment and CV19 cell entry? Hence creating an external basic environment may be an early potential CV19 entry denial into the host cell. This proposed model requires study and confirmation. CV19 is also bound by heparin, which leads to the question 'does it uses heparin as a transport vehicle for systemic spread and infection, as HIV does with its platelet 'taxi' [ 26 ]'? Use of heparin as a transport medium might help to explain the common coagulation problems of patients with CV19 infection because of heparin's potential unavailability for normal blood anticoagulation and the widespread CV19 distribution. Yu et al. showed activation of an alternative complement pathway that blocks CV19 spike proteins [ 27 ]. Bouhaddou et al. [ 28 ] discussed the complicated phosphorylation processes during CV19 invasion. The increase in CV19 proteins and decrease in host proteins during various phases of phosphorylation in the energy production cycle showed that this decrease in host protein caused the demise of the affected host cell by inhibiting host mRNA translation and mitotic kinases [ 28 ]. Furin "Furin is a pro-protein convertase that cleaves the protein and amino acid chain region called 'RXR/K/XR' of precursor proteins and transforms the pro-proteins into biologically active proteins and peptides" [ 29 ]. Furin participates in many nuclear, intracellular, membrane and endosomal actions. Furin has important roles as a proteolytic cleaver of capsular polyprotein precursors prior to viral RNA assembly of newly made CV19 in the acidic environment of the trans-Golgi network. Furin is a multi-functionally important protease that 'senses' its needed acidic pH environment necessary for function. The structure of furin has both an amino group and a carboxylic acid group but a net neutral charge, which plausibly allows 'zwitterionic' or dipolar-like behavior and functions. It is known that for furin action in various organelle sites requires an acidic pH [ 30 ]. The TMPRSS2 serine host protease in the receptor site of CV19 is acidic and hence a welcome partner for furin action. How 'acidic' or how 'weakly basic' a furin action site must be for function is not known. Other viral models may also provide acidic environments for furin activity and may be models for study and potential understanding of CV19. Furin is a common denominator in many cellular functions and acts as a ubiquitous cellular protein 'concertmaster'. Could furin be artificially modified and targeted to achieve desired outcomes? Pelleccia's laboratory [ 31 ] identified furin as a companion protease to TMPRSS-2 in enhancing CV19 transit to the ACE2/ARB receptor for host cell entry. Furin affects spike glycoprotein structure cleavage in other viral evolutionary processes [ 19 , 32 ] and has a long history as a protein 'cleaver'. A four-amino-acid insertion between the edges of the S1 and S2 Spike protein subunits is 'sensed' by furin and allows furin-mediated 'S' protein cleavage [ 33 ]. Roebrook et al. found that furin requires a negatively charged four-amino-acid motif (or low-basic four-amino-acid sequence?) in the substrate-binding region to cleave that site [ 34 ]. This action enables the 'open pre-attachment conformation' necessary for the CV19 spike 'S1' protein subunit to attach to the ACE2 receptor site. What gene directs insertion of these four amino acids for recognition by furin cleavage, and can it be modified? The ubiquitous furin is present in the ARB/ACE2 receptor site before the 'S' protein arrives to be cleaved. The furin 'S' protein cleavage site was also described by Coutard et al., who noted its absence in other SARS-like coronaviruses (CoVs) [ 33 ]. The absence of the 'S' protein furin cleavage site in other SARS-like CoV viruses is an important difference. The presence of a four-amino-acid cleavage site attractive to furin could indicate CV19 mutations or an unusual evolutionary event. The S1 subunit has a basic N-terminus that binds to ACE2/ARB, while the S2 subunit has an acidic C-terminus that interacts with TMPRSS2 and furin after attachment for passage through the receptor site and contains the fusion peptide. These oppositely charged terminals may enhance furin's 'zwitterion'-like or 'di-polar' behavior and give it a greater capability to affect other protein sites. Given that CV19 mutates frequently for improved survival and function, any or all of the descriptions found to date and described here may change as CV19 mutates. Cai et al. [ 22 ] and Roebrok et al. [ 34 ] support that the 'S' spike protein has both a 'prefusion' and a 'postfusion' state and that it undergoes the conformational change needed for successful host entry enabled by furin cleavage. They stated that the prefusion trimer structure has "three receptor-binding domains adjacent to the fusion peptide and that the postfusion structure has strategically placed N-linked glycans suggesting 'viral protection' against host immune responses and harsh external conditions" [ 22 ]. Wrobel et al. supported these observations [ 19 ]: "the human CV19 pathogen presents a more stable pre-cleavage form and an approximate 1,000-fold tighter binding of SARS-CoV-2 to human receptor than in bats." They further state that " these observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 and [but] facilitates the adoption of the open conformation change that is required for 'S1' to bind to the ACE2 receptor." Furin is implicated in mutations, tumor growth, viral and bacterial infections, protein cleavage and change into biologically active moieties. Some approaches have been suggested to stop furin activity. A furin inhibitor, a 2,5-dideoxystreptamine derivative, was designed to form a complex with furin [ 35 ] but needs further evaluation. Jean et al. [ 36 ] introduced alpha1-antitrypsin Portland as a 'bioengineered' serpin molecule selective for furin inhibition. They stated that the formation of a chemical moiety complexed with furin revealed activity inhibition of 100% complete after 2 min of exposure and described it as a "suicide substrate inhibitor'. Additional studies and clinical evaluations of furin inhibitors are expeditiously needed. Cheng et al. described several other furin inhibitors that may be useful after more supportive studies [ 37 ]. The use of furin blockers has not received enough attention given the critical function and presence of furin for most cell needs for protein cleavage, in the replication process and at the cell membrane receptor site. Other entities will likely continue to be identified concerning furin function as our knowledge and experience with CV19 or other viral agents grows [ 38 ]. Controlling and eradicating furin activity appears to be one of the primary keys to controlling CV19 and potentially other emerging viral pathogens. This action speaks to the fact that the singular target 'vaccine' focus has sidelined this and other treatment potentials. A requirement for furin action, as for all cellular functions, is an energy source since biosystemic function is based on kinetic models instead of thermodynamic models. Mutation plays Early in the CV19 outbreak, it became apparent that CV19 has the capability to evolve, as initially demonstrated by its movement from animal to human hosts [ 24 ]. This ability to mutate and adapt is still evolving. The current intermediate protein forms of CV19 that emerge or mutate into different structures have increased potential for infection and improved host entry [ 25 ]. Intermediate forms may be found as new mutations emerge and thus give rise to a dynamic structural milieu. The 'older' forms will be replaced in time by the newer mutants. The ACE2/ARB receptor accommodates the current newly mutated form of CV19, which may signal a small or single. CV19 has genome mutations at position 23,403, which are 'adaptations' to different geographical areas and their populations. This phenomenon was identified again recently [ 39 ] and indicates geographical mutations and changes toward increased infectivity. The 'D614G' (now named G614) mutation changes the virus spike protein that attaches to the CV19 receptor site. This effect partially demonstrates that an agonist can be modified for a better stoichiometric fit. Watanabe et al. [ 21 ] and Cai et al. [ 22 ] showed structural CV19 spike 'S' protein conformational changes but no receptor site changes. The earliest mutation version, G614, found in the CV19 spike 'S' protein still 'fits' to the ACE2/ARB receptor protein but with stronger ability to fuse with host membranes and an at least 3–10 times greater infection capability, causing a mutation for improved survival but not a large structural change while maintaining attachment ability to the ACE2/ARB receptor protein, as confirmed by Choe and Fazan [ 26 ]. As mentioned above, G614 seeks comparison with the current 'new' variant to confirm its true appearance timeline. Infectivity appears to peak before symptom onset at ~ 4 to 7 days after exposure [ 23 ]. He et al. [ 40 ] estimated that 44% of infected individuals were infected during the 'pre-symptomatic' (asymptomatic) stage within household clusters and settings. These infections are primarily from close contact, absent masking and via aerosols, 'droplets' or surfaces. The compilation of prior studies by Korber [ 39 ] also identified the G614 mutation and supports the earlier mutation model by Choe and Fazan [ 26 ], which shows that the 'G614' mutation is almost completely dominant in most countries and potentially now is being 're-discovered'. They also noted that 'G614' has "slightly changed the spike shape and protein structure to enhance and ease the viral membrane and host cell membrane fusion, which also supports a potential stronger infection rate of 3 to 10 times as before and that CV19 accumulates about two changes a month in its genome [ 26 ]". These data may all be or become 'moving targets'. In a larger sense, together with asymptomatic patients, non-masking and non-social distancing these mutations offer an explanation for the early and current rapid infection wave spreading across the globe, with a stronger affinity and infectious potential. The mutational ability of CV19 was further addressed and found to be more complex by Berrio et al., who importantly noted genome mutations independent of protein function impact [ 41 ]. Van Dorp et al's report from the University College of London characterized "patterns of diversity of the SARS-CoV-2 virus genome" by identifying ~ 198 recurrent genetic mutations [ 42 ], which may help to explain how the virus adapts to its various environments, survival needs and human hosts by use of this large potential mutational repertoire. Are there epigenetic signals and activators for the mutations that may be identified? Regarding the known potential ~ 198 recurrent mutations [ 42 ], do they enable or guide CV19 and its ubiquitous player furin to a specific genome domain for mutational induction once inside the host environment in order to be more successful? It may help to think of this mutational induction as "functional genetic flow" enabled by 'evolutionary pressure' which may be guided by epigenetic need to adapt to changing requirements? The current mutations [or 'variants'] may fit into the mutational potentials described above. Baum et al. suggested developing an antibody cocktail to prevent CV19 from developing rapid mutational escape in response to the use of individual antibodies [ 43 ]. Mutational escape may make single target neutralizing antibody vaccines less effective. Hansen and Baum et al. reported on potential humanized neutralizing antibody cocktails for anti-SARS-CoV-2 use. This cocktail aims to decrease the potential, raised by Baum et al. [ 43 ], for the emergence of CV19 escape mutants from the use of single antibody vaccines [ 44 ]. The notion of single antibody vaccine failure due to a more rapid mutation potential is most concerning, and the above studies [ 42 – 44 ] must be solidly confirmed expeditiously and watched for close and appropriate response since this appears to be a looming event. The various cocktail antibodies work separately but synergistically. They [ 43 , 44 ] also stated that this combination cocktail of survivor antibodies may overcome the presence of mutant forms that are present and have escaped treatment. Duffy questions why the CV19 mutation rates are so high [ 45 ]. The Medical Letter lists an early summary of CV19 therapy [ 46 ]. Konno et al. recently discovered a new interferon (IFN) antagonist which may positively impact therapy after confirmation and efficacy studies [ 47 ]. Impaired IFN responses are associated with CV19 disease. What are the furin levels at this early stage in CV19 invasion? A comparison of entry genomes of the current mutations could indicate whether changed genomes are found as a 'gene in situ tweak' mutation to enable CV19. Could the pathologic process, once under way, induce other and more efficient mutations? Are there are more mutations early in the infection cycle, with fewer over time? Perhaps the 'zwitterionic' furin's ability to 'sense' an acidic intracellular organelle site, adapt to its environment and maintain activity after mutation plays a role? Current CV19 genome testing is used as a method to determine the geographical spread of CV19. The papers by Watanabe et al. [ 21 ], Cai et al. [ 22 ] and Wrapp et al. [ 20 ] support the question 'hold' em or fold' em' and support that CV19 does both, depending on receptor site and intracellular actions, pH and CV19 mutational survival needs. Repurposed therapy and modifying the agonist and receptor The pharmacology and biochemistry of new or repurposed drugs potentially efficacious in interfering with extra- and intracellular CV19 actions is a broad area. It is helpful to consider and select methods that disturb the flow of energy in the TCA/Krebs cycle and methods that could interfere with CV19 attempts at entry, membrane fusion for entry and exit, replication and protein reproduction. RNA mechanisms, furin cleavage, endo- and exosome activity at entry and exit are also important potential considerations for interfering with CV19. Every action and function within a cell requires a source of energy, and CV19 is no different. The Medical Letter on Drugs and Therapeutics presents a large list of potential medications for CV19. Some potential therapeutic modalities will be discussed here. The Medical Letter does not address mutations and their therapy [ 46 ]. Earlier support of the protease TMPRSS-2 'activation' of the CV19 Spike protein was offered by Glowacka et al. [ 48 ], and followed by a report from Kawase et al. [ 49 ] who used serine and cysteine protease inhibitors to prevent SARS-CoV entry [ 49 ]. Zhou et al. also suggested using protease inhibitors targeting CoV and select filovirus entry [ 50 ]. Hoffman et al. [ 51 ] then showed that the 'S' protein action is facilitated by TMPRSS-2, which is localized to the ACE2/ARB CV19 receptor domain. This activity also enables CV19 attachment and entrance to the cell via the ACE2/ARB receptor. Hoffman et al. [ 51 ] also reported that the 'priming' of spike agonist protein by TMPRSS-2 was inhibited by an FDA-approved orphan drug serine protease inhibitor, camostat mesylate, which is currently used for pancreatitis and esophagitis in Japan but could be available worldwide. Importantly, Hoffman et al. found that alveolar lung cells were not invaded by CV19 if camostat mesylate was used [ 51 ]. This is a critically important finding and therapy that has the potential to stop CV19 in its attacks on pulmonary tissues by blocking TMPRSS-2 'priming' of the CV19 'S' spike protein. Note that TMPRSS-2 has a serine as a part of its structure. The FDA site on camostat mesylate describes its actions as follows [ 52 ]: "The mesylate salt form of camostat, an orally bioavailable, synthetic serine protease inhibitor, with (has) anti-inflammatory, antifibrotic, and potential antiviral activities. Upon oral administration, camostat and its metabolite 4-(4-guanidinebenzoyloxy) phenyl acetic acid (FOY 251) inhibit the activities of a variety of proteases, including trypsin, kallikrein, thrombin and plasmin, and C1r- and C1 esterases. Although the mechanism of action of camostat is not fully understood, trypsinogen activation in the pancreas is known to be a trigger in the development of pancreatitis. Camostat blocks the activation of trypsinogen to trypsin and the inflammatory cascade that follows. Camostat may also suppress the expression of the cytokines interleukin-1 (IL1b), Interleukin-6 [IL6] (known to be highly present in the lungs during CV19), tumor necrosis factor–alpha (TNF-a), and transforming growth factor-beta (TGF-beta), along with alpha-smooth muscle actin (alpha-SMA). These cytokines belong to the anti-inflammatory Th2 cellular immune response but are likely overwhelmed by the strong inflammatory opposing response. In addition, camostat inhibits the activity of TMPRSS2, the host cell serine protease that mediates viral cell entry for influenza virus and CoV, thereby inhibiting viral infection and follow-on replication [ 52 ]. Uno described camostat mesylate and its use, dosing and efficacy [ 53 ]. A current large-scale study of camostat is ongoing by the University of Aarhus in Denmark, with an endpoint in March 2021 [ 54 ]. Interleukin-6 [IL-6] blockade therapy has been used to reduce the macrophage inflammatory response, with some success [ 55 ]. As noted above, camostat may also block IL-6 and potentially support a stronger Th1-type cellular immune response. The drug remdesivir produced a statistical improvement in nonspecific clinical status compared with that for standard care with 'undefined clinical importance' [ 56 , 57 ] but has been approved for use. However, remdesivir may work better in combination with an IL-6 blocker or other antiviral drugs. Tortorici et al. suggested that strong human antibodies protect against CV19 [ 58 ], while Konno et al. reported on an interferon [IFN] antagonist [ 47 ]. Stauffer et al. mention using dexamethasone early [ 59 ]. Jurgeit et al. [ 60 ] report that the old anthelmintic drug niclosamide inhibits adenosine triphosphate (ATP) production by uncoupling oxidative phosphorylation and that it blocks endosomal acidification. Acidification of endosomes and acidic environments as discussed in this paper is a necessary environment for furin to be active in cellular endosomes, in the ARB receptor site, the fusion process of CV19 and host membranes, as well as Golgi body protein assembly. Niclosamide may become very useful once tried in human clinical scenarios. Niclosamide importantly also neutralizes endosomal and Golgi acidic environments [ 60 ]. Furin is a major actor in the acidic environments of the ARB receptor site, Golgi bodies and CV19 exit endosomal milieu. Further studies are expeditiously needed on niclosamide in the context of CV19 and other viral infections to support its potential CV19 and furin limiting actions by changing the environmental, intracellular, endosomal pH and TCA/Krebs cycle pathways. Niclosamide is available for repurposing and has been used safely as an anthelmintic for over 40 years in humans. Once shown to be dually useful as an oxidative phosphorylation inhibitor and organelle and endosomal pH changer in human targets or for CV19 therapy it may become a valuable re-purposed medication in treating CV19 or as a part of a medication cocktail as used in other illnesses. If found to do the same in human studies, then it can be considered safe for use since it has been used safely for over 40 years. It is plausibly desirable and useful for human CV19 or any current or potential emergent mutant or anti-viral therapy. Forty years of safe human use of niclosamide is sufficient to demonstrate recovery of its suppressive TCA/Krebs cycle activity and follow-on continued human health. There are many repurposing drugs and identification efforts described in the literature, and it is beyond the scope of this paper cover them all. They may be useful adjunct therapy while awaiting vaccine roll-outs as back-up therapy, and efficacy expectations and results are studied. Each activity in the cellular milieu requires a source of energy. This energy is derived from the ttricarboxylic acid cycle (Krebs cycle), as it shuttles various moieties through losses and additions of electrons and produces adenosine tri-phosphate (ATP), which eventually becomes cyclic adenosine monophosphate (cAMP) with serial loss of energy-laden phosphate (PO4) bonds at various sites in the TCA/Krebs cycle. cAMP then becomes the energy source for many cellular activities, including CV19 and furin actions. The relationships between phosphodiesterase (PDE) and cAMP are very complex and counterintuitive but deserve much more study for potential future understanding. If PDE is blocked, then cAMP is able to function. An increase in PDE could possibly prevent an energy source from enabling CV19 activity by blocking cAMP. Niclosamide [see above] also uncouples oxidative phosphorylation, and with its added endosomal acidic pH neutralization capability, as said, it could become a desirable drug in CV19 treatment. Aside from energy production interruption, furin also cannot act in acidic endosomal or other action sites without an energy source. Viral entry was also targeted as a strategy for broad-spectrum antivirals [ 61 ]. The well-described use of dexamethasone (6 mg daily) has shown promising results in combatting the inflammatory response in the lungs as an immune suppressive medication [ 59 , 62 ]. This is a welcome short-term effect for inflammation suppression, but long-term Decadron or other corticosteroid use may suppress the immune system further toward potentially undesirable consequences. The use of different corticoid steroids showed an undefined lowering of 28-day mortality [ 63 ]. Immune plays There are currently many ongoing studies seeking an effective vaccine in clinical trials, with some declared efficacious, safe and being administered. Many studies and efforts have been gathered under the general term vaccine but are not a vaccine in the traditional sense. The word vaccine brings comfort. It is concerning that every solution to counter CV19 are single target efforts, and that CV19 in the face of efforts to destroy or incapacitate it may be able to change its mode of attack or molecular biology as it has the capability to mutate depending on geography and adversity. Other supporting concerns are production, distribution and cost to patients. The effect of CV19 on the immune system appears to be a Th1-to-Th2 cellular immunity shift that allows infections, inflammation from macrophages and other problems to emerge. Th1 produces IFN gamma (IFN-G), increasing inflammation early, which is desirable in some cases of infection and illness, and Th2, whose cytokines include IL6, IL10, IP0 and macrophage inflammatory protein alpha, attract other Th2-acting reprogrammed macrophages and increases inflammation [ 64 ]. Via a very complicated pathway, monocyte-derived inflammatory macrophages add to the cytokine storm and blood coagulation pathway activation seen in late stages of CV19. The cytokine Interleukin-6 [IL6] is also present in many cases, and IL6 blockade has been used with some success in many patients with macrophage-induced inflammatory syndrome [ 54 ]. The drug tocilizumab has been found to reduce the mortality risk of some ventilator patients by 45%, allowing them to be extubated or out of the hospital within a month. The researchers cautioned that dexamethasone therapy is undesirable at the same time with tocilizumab [ 65 ]. Tocilizumab is also reported to reduce cytokine release syndrome and IL6 action, reducing mortality [ 66 , 67 ]. The role of monocyte-derived macrophages requires additional evaluation, including regarding the timing of signaling actions in order to discover their multifaceted function. Macrophages may play different roles in different tissues and environments. An excellent description of the complex macrophage action in the inflammatory setting and cytokine storms is found in the papers by Merad and Martin [ 64 ] and Schulert and Grom [ 55 ]. Macrophage-influenced 'immune dysfunction' or 'overreaction' is also commonly seen in some severe trauma patients with total system and tissue collapse from cytokine over-reaction [ 68 ]. A pertinent and helpful discussion of macrophage activity was found in the paper by Rydzynski-Moderbacher [ 69 ]. They pose that the macrophage response is more important than the antibody response. Macrophages derived from bone marrow monocytes circulate until they 'find' an intruder to arm themselves against and assist B cells in making antibodies and killer T cells in seeking CV19-infected cells for elimination. Other killer T-cells may also be sourced from survivor blood and transfused. Naïve T cells in the bone marrow of infected patients are a reservoir of useful blood cells, including other naïve white blood cells. The T-cells in the bone marrow could be harvested, multiplied to billions in number and given to a patient autologously via any available access routes. This procedure restores a 'naïve and robust' immune system that would act as a new Th1 infection-fighting immune system [ 70 ]. There are at least four ways to achieve a 'younger and newer' naïve immune system: by birth and inheritance, from cord blood, by early withdrawal and storage for need or use later [ 71 ], and by collecting a naïve and robust immune system from the bone marrow. If not drawn from cord blood at birth or stored later in life when healthy [ 71 ] and no immune suppression present, then the bone marrow site with only the white blood cells and macrophages harvested(not the stem cells) is always available. The white blood cells drawn from the above sources would give one a healthier and younger immune system to face any immune-suppressive situation or need. The bone marrow is also a resuscitation or IV infusion site for medications. The bone marrow reservoir of life-saving naïve white blood cells or red blood cells is always ready and available for extraction and use for health and/or life-saving needs. Intraosseous access for fluid and blood products transfusions is a common standard of care if intravenous access cannot be established. This therapeutic modality was presented as a poster and short discussion at the 2019 American Association for Advancement of Science (AAAS) meeting [ 70 ]. Mentioned by Charron [ 71 ] earlier and then Griffin [ 70 ] is that each of us has a new and renewable immune system in our bone marrow. This is a mixture with stem cells and macrophages, and the macrophages and or the stem cells may be harvested as needed. An immune system readily available for use may be plausibly considered as an available personal 'vaccine'. Absent from many reports older patients to CV19, as a co-morbidity is the fact that the immune system is also less functional as the patient ages. A recent timely article poignantly suggested that an aging immune system may allow exacerbation of CV19 illness and symptoms despite already presenting other known comorbidities [ 72 ]. Life-threatening illnesses, including pandemics, require investigating out-of-the-box and courageous visionary solutions based on sound science and studies. Viral infections stimulate production of various types of interferons [IFNs] that induce an antiviral state [ 73 ], and some successes using IFNs to treat CV19 infections have been reported. Ziegler et al. [ 9 ] also reported the upregulation of ARB/ACE2 via an IFN-stimulated gene as a host viral infection defense. IFN alpha may induce response refractoriness, which requires close attention and possible changing or halting of IFN therapy. IFN alpha has been used for over 50 years for treating HIV and hepatitis B and C safely. Grajales-Reyes and Colonna's excellent discussion of IFN responses offers explanation and understanding of IFN roles in viral pneumonias [ 73 ]. The role of IFNs is complex and may be useful for CV19 treatment once completely studied, including safety, IFN responses and dose timing issues. Studies of antibody signatures are emerging and relate to different outcomes. In a 22-patient cohort with the same Immunoglobin-G [IgG] levels, those who survived had spike-specific humoral antibody responses, while those who died had nucleocapsid-specific antibody elevations [ 74 ]. This finding was supported in an elegant study by Peng et al. [ 75 ], who reported that CV19-specific CD4+ and CD8+ T cell responses were found in most convalescent patients, while a significantly greater T- cell response was noted in those patients with severe illness. They concluded that "Differential subsets of CV19-specific T-cells can be associated with (specific) clinical outcomes." Matthew et al. reported that there are three immunotype profiles in CV19 patients: 1 . immunotype 1-associated disease severity with a "robust activated CD4 T-cell response, a paucity of circulating follicular helper cells (B cells? [GG]), activated CD8 T-cells, hyperactivated or exhausted CD8 cells and plasmablasts (PBs); 2. immunotype 2 – characterized by less CD4+ cell activation, Tbet effector CD4 and CD8 T-cells, and proliferating memory B cells and not associated with disease severity; and 3. immunotype 3-correlated negatively with disease severity and lacked obvious T and B cell responses." [ 76 ]. A recent report indicates 'ultrapotent' antibodies identified from recovered patients' sera that stop CV19 attachment to host cells while disrupting the infection machinery [ 58 ]. A systems biological assessment of immunity was presented by Arunachalam et al. [ 77 ], who noted that "The increase in pro-inflammatory mediators in the plasma, including IL6, TNFRNFS14, EN-RAGE, and OSM, coupled with suppressed innate immune responses in blood monocytes and dendritic cells (DCs) suggest a sepsis-like clinical condition." In this context, it has been previously suggested that pro-inflammatory cytokines and bacterial products in the plasma may play pathogenic roles in sepsis, and the combination of these factors could be important in determining patient survival. Notably, the plasma of severe and ICU patients had significantly elevated levels of bacterial DNA, as measured by PCR quantitation of the bacterial 16S ribosomal RNA gene product. This finding was "correlated with bacterial DNA and the plasma levels of inflammatory mediators" [ 77 ]. The well-known infection and inflammation theory with danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) is supported by these findings. This finding [ 77 ] opens pathways for additional therapeutic methods and is a welcome step toward unravelling the complex inflammatory molecular actions in CV19. CV19 patients with cytokine storms have fewer memory B cells, which are needed to develop a durable immune response. There appears to be a TNF alpha-mediated feedback loop that seems to shut down the germinal centers that produce memory B cells for the long-term anti-CV19 response [ 78 ]. Some immune responses are present but not as robust as those with B cells from germinal centers. The immune response to CV19 may be similar but not identical to the influenza immune response. The IFN-G action and role bear repeating as IFN-G is related to the effect of catecholamines, specifically adrenergics. In a highly charged anxiety situation, a CV19 critically ill patient on a ventilator who has adrenergic stimulation (i.e., adrenalin, epinephrine) blocks the action of IFN-G by inducing a shift to a Th2 cellular response that is not beneficial at this point. The use of a beta blocker (B1 and B2 receptors, not B1 only) could restore the function of IFN-G, as was elegantly described by Prass et al. in brain injuries with ischemic stroke [ 79 ]. The 'droplet' supremacy A 'droplet' is a 'droplet', or is it something else? It is generally accepted that CV19 travels from person to person on a droplet and that both are carried by the force of a sneeze or cough or some waft through air or as an aerosol. Webster defines 'droplet' as "a tiny drop, or a blob, driblet, drip, drop or a glob." A 'drop' is further defined as "the quantity of fluid that falls in one spherical mass" and 'a dose of medicine measured by drops or the smallest practical unit of liquid measure". Wallensky further adds that the milieu includes an aerosol as a part of the 'droplet cloud', which is anything less than five microns in size, with droplets ranging from five to ten microns in size [ 80 ]. These definitions lead to a dilemma of defining what it is that CV19 actually travels on! Has anyone ever seen or proven that a CV19 is on within a 'droplet'? Is there truly a structure that can be identified as the vehicle CV19 travels on/with or in? Can we modify the 'droplet' or a structure that CV19 travels on, with or amid so that CV 19 cannot escape, if it is indeed in, on or amid 'droplets'? Is it truly carried 'by' or 'with' droplets or simply by the air flow from a cough, sneeze, breath or 'by the wind' as an aerosol or in 'droplets? 'Amid' droplets may be an appropriate description since CV19 is likely amid a blob or a glob of moisture and other 'stuff' in the breath/wind or sputum of the CV19 carrier. A recent paper addressed the issue of detection and quantification of CV19 by 'droplet' digital PCR from nasal swabs but failed to differentiate the actual 'droplet' status of CV19. However, additional useful data derived from the study could be used as a better and improved CV19 positive or negative test [ 81 ]. The pH of the 'droplet' cloud was not studied, and it may be useful to do so. At the 'droplet' stage of its accidental journey and search for an acceptable environment and receptor site, CV19 likely does not mutate. If the pH can be brought to an unsurvivable value for CV19 in the 'droplet' or air cloud or aerosol stage (i.e., to basic pH), then that may plausibly become a critically important timing and opportunistic strategy to stop host receptor site entry. All fluids and viral component proteins or amino acids within the 'air' also have an optimal and functional pH. Can the 'air' carrying CV19 be made slightly alkaline to decrease CV19 viability? Perhaps the masks worn by all can be impregnated by a basic environment-inducing agent to help incapacitate CV19 on contact? Studies of how long a CV19 particle 'floats' around in the 'air' are difficult to find. Environmental microenvironment management CV19's ability to enter the host cell is a purely accidental event. The stoichiometric matching structures and the acidic pH of receptor proteins are not accidental, but CV19 has a better hand to play than the potential host! How or if they affect each other stoichiometrically while attaching and passing through will dictate whether the proteins involved will hold 'em or fold 'em. It has been reported that environmental UV-C light is effective for killing other viruses and kills CV19 on N95 masks [ 82 ]. If UV-C light kills CV19 on mask surfaces, why not on other surfaces? Far UV-C lights are already in use in hospitals to sterilize surgical supplies and instruments. The UV frequencies used (~ 200-225 nm) are safe and cause no damage. These UV lights could be easily installed in restaurants, buses, schools, cafeterias, theaters, and outdoors, as portable heaters are now in outdoor diners; the uses and potential small sterile areas are endless. The efficacy of UV-C lights depends on a simple distance vs effect relationship, and may not be a practical solution for CV19 eradication. It seeks expeditious study, experimentation and. This simple tool may offer a safe, useful and inexpensive expeditious solution to manage a part of the CV19 pandemic problem when coupled with surface covering, washing and potential eye protection. Potential effects of radiation/ultrasound or other signal energy sources could be theoretically useful if the globular CV19 structures are all the same and if the spikes are all symmetrically positioned on each virus with predictable signal-dampening effects or potential resonance effects. There are studies of the CV19 viral coat structure, but none have evaluated simple spike location, separation or symmetricality. This information may be useful for eventual studies of outer CV19 coat destruction, disrupting its structural strength, integrity, and stability, and potential theoretical response to ultrasound or other energy-based resonance matched signal sources. UV light at resonant frequencies of ~ 222 nm seems to be efficacious in killing CV19 [ 82 ] on small areas and surfaces. Why is this particular frequency effective? Are there other frequencies that may be helpful to apply as above in order to kill CV19? How does this UV light and frequency kill CV19? Hand held UV-lights are used to clean surfaces and kill viruses and surgical supplies. The proteins ('S', TMPRSS2, and furin) and their constituent amino acids or appendages in the CV19 spike and ACE2/ARB receptor are also prime study targets to determine their properties and potential for nonnative denaturation, environmental manipulation and pH changes. The mouth/oral cavity and pharynx along with nasal passages or conjunctivae are the main entry portals for droplets and aerosols and can likely tolerate a larger change in pH to make the environment unfavorable for CV19. The nasal passages and oral cavity appear important as environmental modification targets because of the ACE2/ARB receptor sites found there. Is CV19 in the mouth/oropharynx still in, on or with its droplet on the way to the trachea and lungs? Are nasal passage studies are underway to investigate prevention of CV19 entry into nasal goblet cells using nasal sprays, nanobodies and old repurposed disinfecting solution applications [ 83 ]? Does CV19 travel to other organs via blood vessel distribution or by catching a ride on a protein 'taxi', such as heparin? For example, HIV hitches a ride on platelets [ 26 ]. There may be many viral properties similar to those of HIV or other viruses. External pH of CV19 environment as found in the host before or during acceptor site attachment and transit may be changed to an unacceptable pH for CV19 survival, within host tolerability. pH The vascular receptor environment and most other pH-dependent sites for physiological and drug effects cannot tolerate a large swing in pH, with the exception of the duodenum, with a large pH swing of ~ 5.6–8.0. The normal blood/serum and overall tissue/body pH is ~ 7.35–7.45, which is tightly controlled by respiratory, renal and chemical/phosphate buffering systems. Furthe r studies may demonstrate a more general method of modifying the conformational milieu surrounding the receptor site by changing the local environmental pH within safe host physiologic parameters to induce denaturation. pH is defined as "a value to express acidity and alkalinity" based on the concentration of H+ ions by means of a logarithmic function defined by S.P.L Sorensen in 1909 as he wrote in his book "Ueber die Messung und die Bedeutung der Wasserstoff ionen konzentration bei enzymatischen Prozessen." This may be translated into English as "On the measurement and the meaning of the acid ion concentration in enzymatic processes."[Springer Verlag, 1909] It is important to understand that when the pH of a solution is decreased by one unit from neutral 7.0 to 6.9, the H+ concentration has increased tenfold. Small pH changes have tremendous consequences on protein structures and functions. Many current CV19 studies overlook environmental or pH effects on stoichiometric actions involving ligand or receptor site proteins or their intracellular, intranuclear and endosomal environments. While it is true that viruses do not have a pH per se, their glycoproteins and carbohydrate appendages do have optimal and minimal pH functional ranges. pH is of primary importance to proteins, receptors, and intracellular or nuclear micro-environmental functions. Thus pH manipulation may be a feasible method to affect protein structure changes, folding, denaturing and function. Serine, for example, as a part of the CV19 spike glycoprotein responsible for attachment to the ACE2/ARB receptor after being 'activated' by TMPRSS2 from the ACE2/ARB receptor, is acidic and could be amenable to a small environmental or pH manipulation toward the basic range and potentially inactivated as noted above in the heparan SO4 discussion. Both the 'S' protein, TMPRSS2 and furin have serine as a part of their structures. pH environmental changes cause protein ionization, which may induce morphological changes and an inability to function. The body has many sites of varying pH that can accommodate receptor actions from many different drugs, chemical moieties, proteins, enzymes or microbes, including viruses. Denaturing by inhospitable pH causes protein biological and chemical activity to be lost or changed. All structural degrees beyond the primary structure are changed when a protein or nucleic acid is denatured. It is well known that extreme pH changes affect some peptide bonds, while serine and threonine are destroyed by an alkaline/basic environment. This phenomenon may provide an opportunity for study of a potentially important useful therapy. It is posited that environmental factors in the ACE2/ARB and ligand receptor surroundings affect protein structure and folding and that pH may be a likely factor. Protein folding or unfolding can be initiated by altering the pH. Will CV19 hold 'em or fold 'em in the event of a pH change sufficient to make a change in the structure and function, as described by Zhang [ 83 ]? It is highly plausible that a change to basic pH or less acidic pH may inhibit furin activity. Endosomal and Golgi acidity may also potentially be blocked with niclosamide, which is available on the US market as a repurposed drug. As a rule, nothing is transported across a membrane in its ionized state. Changes in pH affect the amino acids and ionizable groups and residues of proteins. Once ionized by pH changes, folding of the protein can occur, dictating structural and functional changes [ 84 ]. Ionization and electrical properties are important in all stoichiometric receptor functions. In the case of CV19, the priming action of the 'S' protein changes it to an acceptable form by induced folding in a supportive pH acidic environment. It does not adopt its needed 'induced fit' until cleavage by furin. While we know that within the receptor site, the pH is acidic, we do not know any other features within the receptor site. For example, does the receptor channel have Na + or Ca++ gates that depend on other factors? What would be the effect of a pH change in and inside the receptor site region? The receptor site and the proteins of this active and mutating virus are likely sensitive to pH changes and manipulation, inducing denaturation. Denaturing [folding] a protein may include losing a hydrogen bond, a disulfide bond, a connector salt bridge, or a nonpolar covalent bond and cause loss of function. The need to tolerate small pH fluctuations to carry out their functions is generally characteristic of most cells. Talley and Alexov [ 85 ] address the issue of whether a protein or 'action' molecule can tolerate small alterations in pH and structure for function. They conclude by saying that "biological macromolecules have a necessity to tolerate small pH fluctuations", "that such a scenario could be achieved if the pH-optimum of activity is similar to the characteristic pH of the subcellular compartment because the activity does not change much around the pH optimum", and "at the same time, keeping activity unchanged upon pH fluctuations will require the 3D structure of the corresponding protein to maintain its structural integrity". The notion of variable and differential pH in different cellular components noted in this paper and its application to CV19 players is supported by Elferich et al. [ 86 ] as they explain pro-peptide 'convertases' and how they regulate activation of 'their protease domains by sensing the organellar pH within the secretory pathway". They support one of the basic notions posed in this paper by highlighting the relationship of furin and its relationship to organellar pH. Elferich et al. [ 86 ] related evolutionary analysis to the proportion of histidine residues within pro-peptide proteases, specifically furin. They state that furin "activates in the TGN (Tri-golgi network) at a pH of 6.5 (acidic)." They reason that the acid-base balancing is slower than the histidine hydrogen exchange at basic pH values, noting the importance of the chemical reaction speed of different entities. They concluded by stating that "they demonstrated the pKa of the conserved histidine in pro-protein 'convertase' is acid shifted with furin and consistent with its lower pH of activation" [ 86 ]. An earlier paper by Feliciangeli et al. [ 87 ] identified a pH sensor in the furin pro-peptide that regulates enzyme activation and showed that furin activation occurs in the endoplasmic reticulum, and that furin has a conserved histidine that acts as a pH sensor. In support, Williamson et al. [ 30 ] further concluded that "Different spatial distributions of histidine residues modulate the activation pH of pro-protein convertases", such as furin. Noted is also that furin may seek a specific amino acid sequence for cleavage supporting Coutard's et al. report [ 33 ]. Talley and Alexov [ 85 ] state that "….only activity is biologically important, that macromolecules can tolerate small pH fluctuations that are inevitable with cellular function", and that "our findings rationalize the efforts of correlating the pH of maximum stability and the characteristic pH of subcellular compartments since only the pH of activity is subject to evolutionary pressure". More specifically, one of the central assertions of this paper is that the pH of the extracellular environment, receptor site and intracellular compartments appears critically important in enabling CV19. Williamson et al. [ 88 ] reveal the pH-dependent activation of furin via a complicated combination of structural, mathematical and molecular dynamic simulations that suggest that "His-69 from the furin pro-domain serving as the pH sensor close to the TGN triggers movement of a loop region in the pro-peptide that modulates access to the cleavage site and thus allows for the tight pH regulation of furin activation". Williamson et al's [ 88 ] work establishes a model for further study, and potential furin control via environmental and pH manipulation, as espoused in this paper. Acidic transmembrane glycoproteins have attached carbohydrate appendages whose response to minimal pH environmental changes may induce structural folding changes. One source states that the optimal pH for renal ACE2/ARB receptor activity is ~ 5.5–7.5 [ 89 ]. Does CV19 adapt to the various tissue environments via a small but still ACE2/ARB-compatible stoichiometric fit adjustment? The normal pH range in the oral cavity is large, ~ 6.8–7.5, from acidic to alkaline. The nasal cavity mucosa is 5.5–6.5 and increases to 7.2–8.3 with rhinitis. The fact that rhinitis caused by CV19 may also increase nasal pH has not been addressed and may also be a potential pH change CV19 therapeutic target since furin prefers an acidic environment for activity. Hull addressed the issue of changing nasal pH with nasal medicine application in patients afflicted with the common cold [ 90 ], with some success. It is plausible that CV19 may be destroyed or blocked by nasal receptor environmental pH adjustment [ 91 ]. England et al. found nasal pH to be a reliable parameter [ 92 ]. The notion of pH manipulation as a plausible therapy has been introduced for both acidic pre-entry, acidic intracellular Golgi body and endosomal pH manipulation [ 61 ]. It is apparent that furin favors an acidic environment for cleavage activity [ 86 ]. Manipulating the pH of select organelles and external environment appears a plausible method to stop furin which seeks acidic environments for activity. Potentially early heparanSO4 action could be interfered with a basic environment. An earlier report of pH dependent SARS coronavirus entry was mediated by the spike glycoprotein may have been a predictor of current entry events [ 93 ]. Connecting thoughts: hold 'em or fold 'em? The above discussions explore spike protein characteristics and chemistry, receptor site protein action, heparan SO4 & heparin involvement, pH effects on pertinent proteins, furin characteristics and activities, mutation effects, immune system action, select drug repurposing, energy needs and the role of PDE[phosphodiesterase], translational interpretations open to modification by newer data, an apparent Th1-to-Th2 cellular immune shift, potential use of far UV-C light, 'droplet' transmission, and niclosamide action on the TCA cycle and pH manipulation. The focus on environmental therapy and CV19 treatment is highlighted with some specific therapeutic recommendations. Extra and intracellular environmental management may be possible even if and when the approaching agonist has the capability to mutate during current activity [as CV19 does] since each protein, including the mutated protein, has its optimal pH range, including and regardless of the current momentary structure. The optimum environmental factor modulating extracellular pH needed for protein function to proceed must be determined, sought and adjusted within the parameters of host and medication tolerance. Both extra or intracellular pH and host nuclear pH changes and their effect on furin await to be studied expeditiously at each step of CV19's life cycle, from 'droplets' and air relationships to host entry, viral replication, exosome acquisition and exit from the host. Furin is a common denominator in many of these events. It must be learned: 1.How or if furin can be blocked or its functions modified by manipulating the pH, from its creation in the endoplasmic reticulum [ 87 ] or as it acts in organelles; 2. How to blunt furin pH sensing; 3. How to use furin blockers [ 94 ]; 4. How to use pH manipulation to stop CV19 and restore health. Extracellular receptor proteins may be more easily influenced by environmental or pH adjustment since they are presumably more stable or easily affected proteins. An agonist/ligand may change/mutate and still 'fit' a nonmutated receptor. The current batch of highly visible vaccines being developed has efficacy according to reports. Since CV19 has the potential to mutate on demand, will they still be effective, or is a new vaccine needed for each mutant? Environmental or pH adjustments for CV19 and ACE2/ARB protein changes have the potential to become a more universally effective therapeutic tool regardless of the current mutational structure or differing microbial or chemical agonists/ligands. As said the main steps in the infection pathway are random attachment, entry, fusion, replication, and endocytic and exocytic membrane site activity, with each step having its own requirements. There is still much to be learned about these steps, although enough may be known to begin clinical studies. This translational thought experiment is offered as a possible explanation and exploratory therapeutic beginning. This translational thought experiment presents a potential coalescence of molecular environmental manipulation of pH in the context of 'hold 'em or fold 'em' and furin role interactions. Wu-Dunn and Spear support the environmental effect notions and focus in this paper [ 61 ] in an elegant and wide-ranging paper. Jurgeit et al. proposed the use of niclosamide beyond its current limited application [ 60 ]. As mentioned above, Niclosamide has been safely used for over 40 years in humans as an anthelmintic and has desirable and potentially efficacious actions in human illnesses and intracellular or intra-nuclear effects. Niclosamide uncouples oxidative phosphorylation and hence potentially denies a critical energy source for cellular, microbiological or viral and CV19 action [ 60 , 61 ]. Niclosamide also blocks endosomal acidification. Could it also block receptor site acidification to potentially halt CV19 entry? These are known basic requirements for furin action, its production and activity [ 87 ]. Niclosamide is available as a drug in the US market and has widespread effects on viral infections that could be useful as one of several drugs in a cocktail (remdesivir, niclosamide, camostat mesylate, tocilizumab, other furin inhibitors or PDE manipulators, etc.) or potentially alone. The discussed repurposed drugs meet the needs to stop CV19: halt host receptor site entry, interfere with the energy needs of CV19 to make proteins and exit (and the well-known increase in PDE inhibitory action on cAMP), as well as change the pH environment to further prevent furin sensing via histidine. The repurposed drug cocktail or combinations suggested in this paper and the cocktails suggested by Hansen [ 43 ] and Hansen and Baum [ 44 ] may allow the potential resuscitation of single-target vaccine failures due to CV19 potential in situ changes or adaptations and mutations. The predictions of multiple new mutations requiring new 'vaccine' development and multi-valent vaccine development herein are emerging now and seek expeditious attention lest we lose the progress made up to the present. The way forward, as are additions to 'vaccines,' is with multivalent vaccines combined with medicine or drug cocktails on CV19 as done with influenza and HIV therapies. This translational thought experiment presents notions that speak to attachment action by proteins, functions of proteins (adhesion, attachment, receptor site function, fusion of cell membranes), protein production in acidic environments, cleavage by furin, sensing by furin, furin's need for an acidic environment for action, insertion of specific and CV19-unique amino acid series for furin sensing in CV19, the role of histidine as a pH sensor, variability of histidine chemical reaction speed, proposed models of the roles of heparan-SO4, its removal and pH change for receptor site entry denial, mutations, and repurposed medications already available and shown to be safe. Various combination cocktails may be necessary for potential treatment of resistant mutations. Two major notions, furin control or destruction and the role pH management were advanced. Much of the uncertainty, the unknown nature and fear of CV19 could be ameliorated if we can control whether CV19 holds 'em or folds 'em. pH control may offer some predictability of CV19 action and behavior. It must be pointed out that a nationwide or global-wide vaccination campaign, with many requiring the scheduling of two separate vaccinations are labor and time intensive, and place a heavy burden upon most states, countries, patients, support labor forces as well as equipment needs and costs when compared to using re-purposed medications or medication cocktails. These may plausibly work as well upon study and trial, and are less labor intensive and costly. The nations unable to obtain or manufacture vaccines may be able to gain faster protection for their populations by using alternatives to vaccination upon validation of the alternative therapies and environmental notions offered here. While waiting for 'vaccine' availability issues to be solved the alternative potential solutions offered here may be a productive pathway while seeking and awaiting vaccines. The emerging mutations add urgency to seek alternatives to vaccines. The current and future pandemics deserve all potential discovery and therapeutic ideas to be taken seriously and thoroughly evaluated, regardless of how 'Swiftian' they are or whether they have ever been considered before in this newer demanding context or not. Energy-dependent cellular activities potentially affecting host TCA/Krebs cycle functions will likely recover rapidly once CV19 is destroyed as they likely have for over 40 years with the use of niclosamide. To the above suggested cocktails, one can add a 'newer and younger' immune system from autologous white blood cell infusion by either prior storage or bone-marrow sources for potential expedited return to good health [ 70 , 71 ]. Both reports reflect the essence and basis of translational thought experiments [ 70 , 71 ]. The translational thought experiments posed and suggested herein may stimulate further ideation and study. Timely support for the hand that CV19 plays [Hold'em or Fold'em?] arrived from Goethe University in Frankfurt, where it was noted that 'they have observed the RNA folding structures of the SARS-CoV-2 genome with which the virus controls the infection process" [ 95 ]. Enabling loop completion supporting reports include autophagy studies that point to endolysomal deacidification which may impede furin, [ 96 ], the association of autophagy with uncontrolled inflammation and delayed or absence of types I and III Interferons and increased cytokine production defects [ 97 ], and the plausible inhibition endosomal uncoating 'thus preventing endosomal actions in entry and exit by CV19 (author's comment)'. Autophagy control is thought to be via changes in activation phases of autophagy related genes [ 98 ]. These comments are merely superficial indicators of an extremely complex genetic interplay that is beginning to be unraveled. Niclosamide is also confirmed by Gassen et al. [ 98 ] as a potential antiviral agent as suggested in this paper before it began its prolonged editorial and review journey. The role of cystidine peptidase control is thought to impede cell entry and replication [ 99 ]. Pislar et al. [ 99 ] note that cathepsin inhibitors 'dual' inhibitory action on viral and host by lessening the positive immune response may support the needed multi-pronged therapeutic approach as suggested earlier in this paper by Baum et al. [ 43 ] and Hansen et al. [ 44 ] who also suggest a multi-pronged therapeutic approach to counter mutation escape by viral variants, In addressing the activity at and of the furin cleavage site, Xing et al. [ 100 ] note that many mutations are active at that site, and may lessen the importance of the cleavage site. Their interesting studies seek more supporting data to unravel a complex finding. Xia et al. [ 101 ] discuss the role of trypsin in the furin cleavage site and its mutation or change from a 'RRAR' configuration into a 'SSAR' configuration which appears equally effective in cleavage and viral entry, adding more confusion to a most complex and vexing situation. Pardhan et al. [ 102 ] review and support that the most significant ocular symptoms by patients with CV19 infection are 'sore eyes'. However, one source reports that there was loss of vision by a patient infected by CV19 [ 103 ]. Conclusion Translational thought experiments add value to science since without them science would not progress. Translational experimental ideation, based on and referenced with sound and proven science, deserves acknowledgment and recognition as a necessary equal partner in scientific or medical discovery and progress. To do so will encourage huge scientific progress and pave the way to the future. Will the question between Darwin's natural selection, where only those organisms best adapted to their environment survive, and Lamarck's notion of adaptive force, where organisms can alter themselves to meet the needs of their environments be answered? The roles of furin and pH appear to be in the midst of the chaotic environment that allows CV19 to merge Darwin's science with Lamarck's contributions. Regardless of the pH of the moment, whether external, receptor site, internal cell site or organelle, pH becomes a major determinant of protein or chemical structure [folding] and function. If histidine sensing disappears or amino acid sequences change by mutation or evolution, CV19 and its minion furin can still be managed within host tolerance of pH management. Furin works best in an acidic environment. The newest emerging 'mutant' has an optimal pH for function, and population penetrance may be determined by pH influence on the players and their momentary managed environmental pH. CV19 will change and mutate, but the role and importance of pH remains unchanged. pH management allows CV19 to hold'em or to fold'em and plausibly grants loop completion of this translational thought experiment and potential global application of non-vaccine therapy. Both Darwin and Lamarck are correct and imply separate but equal historical scientific truths.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109446/

New records of bee flies (Diptera, Bombyliidae) from Cuatro Ciénegas, Coahuila, Mexico

Abstract Forty one new records of species of Bombyliidae are reported for Coahuila in northeastern Mexico. Nine of these species are reported for the first time for the country. The specimens were collected in the Cuatro Ciénegas Basin and Sierra La Madera mountains during 2007–2013. The modified distributions of species are discussed. The gaps in the distribution of many species suggest an undersampling of this group of insects in the north of Mexico. Citation Ávalos-Hernández O, Kits J, Trujano-Ortega M, García-Vázquez UO, Cano-Santana Z (2014) New records of bee flies (Diptera, Bombyliidae) from Cuatro Ciénegas, Coahuila, Mexico. ZooKeys 422: 49–85. doi: 10.3897/zookeys.422.7598 Introduction The bee flies ( Bombyliidae ) belong to the superfamily Asiloidea and are the eighth most diverse family within Diptera with 5382 described species ( Pape et al. 2011 ). All species of Bombyliidae are parasitoids, hyperparasitoids or predators of immature stages of Coleoptera , Hymenoptera , Lepidoptera , Orthoptera , Neuroptera , and Diptera ( Yeates and Greathead 1997 , Boesi et al. 2009 ). Unlike most other taxa, bee flies are most abundant and diverse in arid and semiarid portions of the world ( Hull 1973 , Evenhuis 1989 ). In the immature stages these insects function as a natural control for populations of other insects and as adults are efficient pollinators ( Motten et al. 1981 , Kearns 2001 ). Some faunistic studies have been completed including Bombyliidae in Mexico ( Rodríguez-Ortuño 1989 , Ávalos-Hernández 2007 ), but the northern region of the country is poorly known for this family. Although Evenhuis and Greathead (1999) list 15 species of Bombyliidae for Coahuila, species richness in this state is probably higher as suggested by the richness of surrounding Mexican states with similar or even smaller size and similar ecosystems (e.g., Nuevo León, 37 species; Durango, 41 species) and of Texas (171 species), the nearest USA state. Cuatro Ciénegas Basin in the northeast of Coahuila is especially interesting because of its geological history and the presence of water ponds and gypsum dunes, which create a different environment from the surrounding areas. The basin was a shallow sea from the Pangea breakup until the Eocene, 40 Ma, when the Sierra Madre Oriental in the east of Mexico rose isolating the Basin from the Atlantic Ocean ( Souza et al. 2012 ). The physiology of Cuatro Ciénegas bacteria is similar to that of marine species, with which they are closely related ( Souza et al. 2006 ). According to Moreno-Letelier et al. (2012) this evidence indicates that some water was kept trapped in the Basin when the ocean retreated giving the basin unique characteristics. These characteristics produced a high number endemism for vertebrates and prokaryotes in Cuatro Ciénegas ( Souza et al. 2006 , 2012 ). The present study is the first known long-term systematic sampling of Diptera in Cuatro Ciénegas. The objective of this project is to complete the list of species of Bombyliidae in the basin and surrounding mountains. In this paper, 41 new species-level records for Coahuila from Cuatro Ciénegas are presented, including nine new records for Mexico. The modified distributions of the species are discussed. Methods Beeflies were collected at nine sites from Cuatro Ciénegas Basin and Sierra La Madera within the Municipality of Cuatrociénegas ( Figure 1 ). Abbreviations for study sites ( Table 1 ) are used throughout. Samplings were performed during 2007-2013, using aerial net and a Malaise trap. The Malaise trap had white polyester netting, was square in configuration, 210 cm tall and 120 cm wide and the collecting head located at the top. Trap was set from 9:00 to 17:00 when weather conditions allowed it. To avoid damage to the specimens no killing agent was used, insects were extracted at the end of the day. Specimens were pinned and labeled. Generic identification was carried out under a stereomicroscope according to the keys by Hall (1981b) and Kits et al. (2008) . Species were identified by the first and second authors with specialized keys for each genus and comparison with museum specimens, keys used for identification of each genera are specified below. Taxonomic classification and distribution data are based on Evenhuis and Greathead (1999) and host data are based on Hull (1973) , if not indicated otherwise. Distribution gaps are suggested as disjunct distribution patterns or the result of under sampling by comparing the location of records in Mexico with those in the southern states of the USA. All specimens are deposited in the Colección Nacional de Insectos (Instituto de Biología, Universidad Nacional Autónoma de México; CNIN-IBUNAM). Figure 1. Field work sites. Cuatro Ciénegas basin is located in Coahuila at northeast of Mexico. Sierra La Madera is located at northwest of the basin. Table 1. Field work sites in Cuatro Ciénegas. Vegetation according to Pinkava (1979) . Site (Code) Location Altitude (m) Vegetation 1 Churince (CHU) 26°50'30"N, 102°08'10"W 770 Gypsum dunes; sedges and marshes; mezquital, halophile 2 Rancho Orozco (ROR) 26°52'18"N, 102°05'17"W 740 Sedges and marshes; mezquital; halophile 3 Rancho Pozas Azules (RPA) 26°49'39"N, 102°01'24"W 710 Sedges and marshes; mezquital; halophile 4 Ejido Antiguos Mineros (EAM) 26°46'58"N, 102°00'20"W 725 Sedges and marshes; mezquital; halophile 5 El Cañón (ECA) 27°00'34"N, 102°04'42"W 780 Mezquital; desert scrub 6 Ejido El Oso (EEO) 27°03'08"N, 102°13'35"W 1085 Desert scrub; chaparral 7 Rancho El Espejo (REE) 27°13'19"N, 102°30'19"W 1425 Desert scrub; chaparral 8 Rancho El Chupadero (REC) 27°10'07"N, 102°34'26"W 1790 Desert scrub; chaparral; Pine-Oak forest 9 Rancho La Casita (RLC) 27°06'45"N, 102°23'40"W 1630 Desert scrub; chaparral; Pine-Oak forest Results A total of 41 new species-level records are presented for the state of Coahuila. Nine of these 41 species are recorded for the first time in Mexico, being their most southern records. Of the 15 species previously listed for Coahuila, two were collected during this study: Heterostylum robustum (Osten Sacken, 1877) (Material collected: CHU: Apr (1 M), Sep (1 M); EAM: Mar (2 M), Sep (1 F), Jun (2 F), Jul (1 F), Oct (1 F); ROR: Apr (1 F, 3 M), May (1 F, 2 M), Jul (3 F), Sep (1 F); RPA: Apr (1 F, 1 M), Jul (2 F, 2 M), Sep (1 M), Oct (2 M)); and Anastoechus melanohalteralis Tucker, 1907 (Material collected: EAM: Oct (1 M); ECA: Oct (1 F, 1M); ROR: Oct (7 F, 6 M); RPA: Sep (1 M)). New records of the species included in this paper are from 17 genera for which modern revisions are available. Six taxa of Hemipenthes (3), Lordotus (1), Paravilla (1) and Rhynchanthrax (1) could not be identified accurately, being probably undescribed species. Identification of species in another 10 genera found in the study (e.g. Villa , Chrysanthrax , and Exoprosopa ) is difficult and unreliable. The number of morphospecies and specimens collected of these genera are presented in Table 2 . Six species of Tmemophlebia (1), Geron (1), Exoprosopa (3) and Villa (1) previously listed for Coahuila were probably collected but specimens of these genera are still being identified. Taxonomic work will continue, updates of the species list and descriptions of the new taxa will be published in subsequent papers. Table 2. Updated list of genera and species of Bombyliidae in Coahuila (* species not collected in this study, but recorded previously in Coahuila; ** species most likely collected in this study, but identification not yet certain). Subfamily, genus and species name New record Unidentifiable material PHTHRIINAE Neacreotrichus Cockerell * Neacreotrichus consors (Osten Sacken, 1887) Poecilognathus Jaennicke Coahuila 1 morphospecies, 3 specimens Relictiphthiria Evenhuis * Relictiphthiria psi (Cresson, 1919) Tmemophlebia Evenhuis 1 morphospecies, 21 specimens ** Tmemophlebia coquilletti (Johnson, 1902) TOXOPHORINAE Geron Meigen 2 morphospecies, 194 specimens ** Geron holosericeus Walker, 1849 Systropus Wiedemann Coahuila 1 morphospecies, 5 specimens Toxophora Meigen Coahuila Toxophora maxima Coquillett, 1886 Coahuila Toxophora virgata Osten Sacken, 1877 Coahuila BOMBYLIINAE Anastoechus Osten Sacken Anastoechus melanohalteralis Tucker, 1907 Bombylius Linnaeus Bombylius (Bombylius) frommerorum Hall & Evenhuis, 1980 Coahuila * Bombylius (Bombylius) sylphae Evenhuis, 1984 * Bombylius (Parabombylius) aleophilus (Hall & Evenhuis, 1981) * Bombylius (Parabombylius) coahuilensis (Hall & Evenhuis, 1981) * Bombylius (Parabombylius) paradoxus (Hall & Evenhuis, 1981) * Bombylius (Parabombylius) syndesmus (Coquillett, 1894) Conophorus Meigen Coahuila 1 morphospecies, 3 specimens Heterostylum Macquart Heterostylum croceum Painter, 1930 Mexico Heterostylum robustum (Osten Sacken, 1877) Lordotus Loew Coahuila 1 morphospecies, 38 specimens Lordotus diplasus Hall, 1954 Coahuila Lordotus divisus Cresson, 1919 Coahuila Lordotus perplexus Johnson & Johnson, 1959 Coahuila Triploechus Edwards Coahuila Triploechus novus (Williston, 1893) Coahuila LOMATIINAE Ogcodocera Macquart Coahuila Ogcodocera analis Williston, 1901 Coahuila TOMOMYZINAE Paracosmus Osten Sacken Coahuila Paracosmus (Paracosmus) morrisoni Osten Sacken, 1887 Coahuila ANTHRACINAE Anthrax Scopoli Coahuila Anthrax atriplex Marston, 1970 Coahuila Anthrax cybele (Coquillett, 1894) Mexico Anthrax georgicus Macquart, 1834 Coahuila Anthrax irroratus Say, 1823 Coahuila Anthrax oedipus Fabricius, 1805 Coahuila Anthrax pauper (Loew, 1869) Mexico Anthrax seriepunctatus (Osten Sacken, 1886b) Coahuila Aphoebantus Loew Coahuila 4 morphospecies, 236 specimens Chrysanthrax Osten Sacken Coahuila 6 morphospecies, 240 specimens Dipalta Osten Sacken Coahuila Dipalta serpentina (Osten Sacken, 1877) Coahuila Exoprosopa Macquart 9 morphospecies, 395 specimens ** Exoprosopa aztec Painter & Painter, 1969 ** Exoprosopa butleri Johnson & Johnson, 1958 ** Exoprosopa dorcadion Osten Sacken, 1877 Hemipenthes Loew Coahuila 3 morphospecies, 146 specimens Hemipenthes jaennickeana (Osten Sacken, 1886a) Coahuila Hemipenthes lepidota (Osten Sacken, 1886b) Coahuila Hemipenthes scylla (Osten Sacken, 1887) Coahuila Hemipenthes sinuosa (Wiedemann, 1821) Coahuila Lepidanthrax Osten Sacken Coahuila Lepidanthrax arizonensis Hall, 1976 Mexico Lepidanthrax disiunctus (Wiedemann, 1830) Coahuila Lepidanthrax hesperus Hall, 1976 Coahuila Lepidanthrax hyposcelus Hall, 1976 Coahuila Lepidanthrax proboscideus (Loew, 1869) Coahuila Ligyra Newman Coahuila 1 morphospecies, 2 specimens Neodiplocampta Curran Coahuila Neodiplocampta (Neodiplocampta) miranda Hull & Martin, 1974 Coahuila Paravilla Painter Coahuila 1 morphospecies, 48 specimens Paravilla edititoides (Painter, 1933) Coahuila Paravilla flavipilosa (Cole, 1923) Coahuila Paravilla parvula Hall, 1981a Coahuila Paravilla separata (Walker, 1852) Mexico Poecilanthrax Osten Sacken Coahuila Poecilanthrax effrenus (Coquillett, 1887) Coahuila Poecilanthrax fasciatus Johnson & Johnson, 1957 Mexico Poecilanthrax hyalinipennis Painter & Hall, 1960 Mexico Poecilanthrax poecilogaster (Osten Sacken, 1886b) Coahuila Rhynchanthrax Painter Coahuila 1 morphospecies, 70 specimens Rhynchanthrax capreus (Coquillett, 1887) Mexico Rhynachantrax texanus (Painter, 1933) Coahuila Thyridanthrax Osten Sacken Coahuila Thyridanthrax pallidus (Coquillett, 1887) Mexico Thyridanthrax selene (Osten Sacken, 1886b) Coahuila Villa Lioy 9 morphospecies, 115 specimens ** Villa fumicosta Painter & Painter, 1962 Xenox Evenhuis Coahuila Xenox xylocopae (Marston, 1970) Coahuila A total of 28 genera were found during this study, of which 21 are new records for the state. Two genera previously listed for Coahuila ( Neacreotrichus and Relictiphthiria ) were not found in Cuatro Ciénegas area. With the new records presented here, the list of bee fly species in Coahuila increases to 56 ( Table 2 ). Subfamily Toxophorinae Taxon classification Animalia Diptera Bombyliidae Genus Toxophora Meigen Remarks. Toxophora is distributed worldwide, being more diverse in the Afrotropical and Palearctic regions. Mexico's fauna includes three Neotropical species and five Nearctic species. All Nearctic species of Mexico were distributed in the western half of the country. These two new records represent the first of this genus in Coahuila and the most eastern distribution of the Nearctic species in the country. The New World species of this genus were keyed using Cunha et al. (2011) . Taxon classification Animalia Diptera Bombyliidae Toxophora maxima Coquillett, 1886 Figure 2a, b Material examined. CHU: Jul (1 M); EEO: Jul (2 F, 2 M), Oct (1 F, 3 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila); USA (Arizona, California, Idaho, Kansas, New Mexico, Oklahoma, Oregon, Texas). Comments. In Mexico Toxophora maxima was only known from Baja California Peninsula and now Coahuila. This apparent gap in its distribution is probably due to undersampling. Sampling of the intermediate zones is necessary to know if these populations form a continuous unit as they do in the southern states of USA. Figure 2. Toxophora . Toxophora maxima , male (CNIN 1115) a dorsal view b lateral view; Toxophora virgata , male (CNIN 1109) c lateral view d posterior view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Toxophora virgata Osten Sacken, 1877 Figure 2c, d Material examined. EAM: Jun (1 F, 1 M), Jul (1 F); CHU: Aug (1 M), Oct (1 M); EEO: Jul (1 M), Oct (1 F, 1 M); RLC: Jun (1 M); ROR: Apr (1 F, 2 M); RPA: Oct (1 F). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora); USA (Arizona, California, Colorado, Georgia, Idaho, Nevada, New Mexico, Oklahoma, Texas, Utah). Known hosts. Odynerus sp. ( Vespidae ); Stenodynerus toltecus Saussure ( Vespidae ). Comments. This species is present in the all southwestern states of the USA and northwest of Mexico. This is the first record in the northeast of Mexico. The species is probably also present in Chihuahua, between Sonora and Coahuila. Subfamily Bombyliinae Taxon classification Animalia Diptera Bombyliidae Genus Bombylius Linnaeus Remarks. With 278 described species, Bombylius is the second most diverse genus of Bombyliidae . It has a worldwide distribution being especially diverse in the Palearctic and Nearctic regions. One endemic species is present in Coahuila: Bombylius (Parabombylius) coahuilensis (Hall & Evenhuis, 1981). Four other species are reported for the state: Bombylius sylphae Evenhuis, 1984, Bombylius aleophilus (Hall & Evenhuis, 1981), Bombylius paradoxus (Hall & Evenhuis, 1981), Bombylius syndesmus (Coquillett, 1894). A review with identification keys for Nearctic species is presented in Hall and Evenhuis (1980) , later Evenhuis (1984) revised and present keys for the comanche group of America. Taxon classification Animalia Diptera Bombyliidae Bombylius ( Bombylius ) frommerorum Hall & Evenhuis, 1980 Figure 3 Material examined. EEO: Aug (1 M), Oct (1 F). Known Nearctic records. Mexico (Chihuahua, Coahuila); USA (Arizona, New Mexico, Texas). Comments. This species is restricted to the southwest of the USA and north of Mexico. Figure 3. Bombylius (Bombylius) frommerorum , female (CNIN 772) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Heterostylum Macquart Remarks. The genus is only present in Nearctic and Neotropical regions. Although not as diverse as other genera (only 12 species), specimens from some species are abundant in the field. Heterostylum robustum was previously known from Coahuila and was collected during this study. This species is distributed from Canada to central Mexico. There are two revisions for this genus that contains identification keys, one by Hall and Evenhuis (1980) and the more recent by Cunha et al. (2007) . Taxon classification Animalia Diptera Bombyliidae Heterostylum croceum Painter, 1930 Figure 4 Material examined. REE: Apr (1 F). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Missouri, New Mexico, Texas). Comments. Heterostylum croceum is recorded for the first time in Mexico; previously known from the southern-central United States. Hall and Evenhuis (1980) suggest that Heterostylum croceum may be related to Heterostylum engelhardti Painter, 1930 or even be a subspecies of that taxon, Heterostylum croceum is the eastern form and Heterostylum engelhardti the western form (Arizona, California, Texas, Utah) although both species are present in Texas. Cunha et al. (2007) comment that Heterostylum engelhardti can be distinguished by the presence of white to very pale yellow hair and brown-tipped hairs on the abdomen compared with the darker yellow hairs in Heterostylum croceum . Figure 4. Heterostylum croceum , female (CNIN 858) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Lordotus Loew Remarks. Most of the 29 species in this exclusively Nearctic genus are distributed in the southwest of the USA and north of Mexico, although eight species are present in the northwest of the USA ( Lordotus apicula Coquillet, 1887; Lordotus bipartitus Painter, 1940; Lordotus diversus Coquillett, 1891; Lordotus gibbus Loew, 1863; Lordotus miscellus Coquillett, 1887; Lordotus pulchrissimus Williston, 1893; Lordotus striatus Painter, 1940; Lordotus zona Coquillett, 1887). The three species present in Coahuila are also found in California; their distribution probably includes all northern states of Mexico. Hall (1954) and Hall and Evenhuis (1982) present reviews of the genus and keys to the species. Taxon classification Animalia Diptera Bombyliidae Lordotus diplasus Hall, 1954 Figure 5a, b Material examined. CHU: Sep (2 M); RLC: Sep (2 M); RPA: Sep (1 F). Known Nearctic records. Mexico (Coahuila, Zacatecas); USA (Arizona, California, New Mexico). Figure 5. Lordotus . Lordotus diplasus , a female (CNIN 774) dorsal view b male (CNIN 861) dorsal view c Lordotus divisus , male (CNIN 777) dorsal view; Lordotus perplexus , female (CNIN 801) d dorsal view e lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lordotus divisus Cresson, 1919 Figure 5c Material examined. ECA: Mar (1 M), Apr (2 M); EEO: Apr (16 M); REE: Apr (4 M); ROR: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Baja California); USA (Arizona, California, Nevada, New Mexico, Texas). Taxon classification Animalia Diptera Bombyliidae Lordotus perplexus Johnson & Johnson, 1959 Figure 5d, e Material examined. CHU: Apr (1 H), ECA: Apr (1 H); EEO: Apr (4 F); REE: Apr (7 F); ROR: Apr (1 F). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Lordotus perplexus has the most southern distribution in the genus, reaching Sinaloa on the Pacific coast. Taxon classification Animalia Diptera Bombyliidae Genus Triploechus Edwards Remarks. Four species of Triploechus are present in Nearctic region: Triploechus luridus Hall, 1975; Triploechus novus (Williston, 1893); Triploechus sackeni (Bigot, 1892); Triploechus stagei Hall, 1975. Of these Triploechus stagei is endemic to Mexico and Triploechus novus has the widest distribution of this genus, being present in the south of the USA and center of Mexico. Hall and Evenhuis (1981) present a revision and key for species for this genus. Taxon classification Animalia Diptera Bombyliidae Triploechus novus (Williston, 1893) Figure 6 Material examined. CHU: Apr (7 F, 6 M); REE: Apr (1 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Durango, San Luis Potosí, Sonora); USA (Arizona, California, Nevada, New Mexico, Texas). Comments. This is a widespread and apparently common species. All specimens were collected in April so it may have a short flight season. Figure 6. Triploechus novus , female (CNIN 1237) dorsal view. Scale bar: 3 mm. Subfamily Lomatiinae Taxon classification Animalia Diptera Bombyliidae Genus Ogcodocera Macquart Remarks. The only two species in this genus have been collected from the neotropical part of Mexico to north of the USA and Canada. Ogcodocera leucoprocta (Wiedemann, 1828), not sampled during this study, is present in the whole Nearctic region from Canada to south of Mexico. Taxon classification Animalia Diptera Bombyliidae Ogcodocera analis Williston, 1901 Figure 7 Material examined. EEO: Aug (2 M), Oct (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos); USA (Arizona, Texas). Comments. This record is the first of this species in the north of Mexico, but it has been previously collected in the south of Mexico and in the south of USA, and thus is probably distributed across the whole country. Unlike Ogcodocera leucoprocta , Ogcodocera analis has its most northern distribution in Arizona and Texas. Figure 7. Ogcodocera analis , male (CNIN 146) dorsal view. Scale bar: 3 mm. Subfamily Tomomyzinae Taxon classification Animalia Diptera Bombyliidae Genus Paracosmus Osten Sacken Remarks. All five extant species of Paracosmus have Nearctic distributions, and all are present in California. Two of these species have been collected in the northwest of Mexico ( Paracosmus (Actherosia) rubicundus Melander, 1950 and Paracosmus (Paracosmus) morrisoni Osten Sacken, 1887). Taxon classification Animalia Diptera Bombyliidae Paracosmus ( Paracosmus ) morrisoni Osten Sacken, 1887 Figure 8 Material examined. EAM: Apr (1 F, 1 M); CHU: Apr (2 M), Jul (1 F), Aug (2 F); ECA: Apr (1 M); EEO: May (1 F); REE: Apr (1 F); ROR: Apr (2 M), May (1 F, 3 M); RPA: Apr (1 F). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Paracosmus (Paracosmus) morrisoni has the widest distribution within this genus, but in Mexico had previously only been recorded in Sonora. This record represent the most eastern distribution for the genus in the country. Figure 8. Paracosmus (Paracosmus) morrisoni , male (CNIN 832) a dorsal view b lateral view. All scale bars: 3 mm. Subfamily Anthracinae Taxon classification Animalia Diptera Bombyliidae Genus Anthrax Scopoli Remarks. This is a diverse genus with 248 species worldwide. Two old but complete revisions of the genus, including distribution maps and keys, were made by Marston (1963 , 1970 ). Thanks to these Anthrax species can be easily identified. Some Anthrax species are widely distributed occupying two biogeographic regions. From the seven Anthrax species collected in this study in Coahuila, just Anthrax cybele (Coquillett, 1894) has a restricted distribution. The other six species are widespread across the Nearctic region. Two species of Anthrax are reported for the first time for Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax atriplex Marston, 1970 Figure 9a Material examined. EAM Apr (1 F); ROR: Oct (2 M); RPA: Aug (1 M); Sep (1 M); Oct (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Durango, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oregon, Texas, Utah). Known host. Megachile gentilis Cresson ( Megachilidae ). Comments. This species may be present in all the north of Mexico, including Chihuahua, Nuevo León and possibly Sinaloa. Figure 9. Anthrax part I. a Anthrax atriplex , male (CNIN 1098) dorsal view b Anthrax cybele , male (CNIN 1087) dorsal view c Anthrax georgicus , female (CNIN 1071) dorsal view d Anthrax irroratus , male (CNIN 1027) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax cybele (Coquillett, 1894) Figure 9b Material examined. ECA: Apr (2 F); EEO: Apr (1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California). Comments. This is a rare species flying in April. Its distribution is disjunct so far, present in the southwest of the USA and northeast of Mexico. It is probably also found in New Mexico and Texas in the USA and Sonora and Chihuahua in Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax georgicus Macquart, 1834 Figure 9c Material examined. EAM: Mar (1 F), Apr (1 M), Jun (1 F, 2 M), Jul (2 F), Sep (2 M); ROR: Apr (1 F), Sep (1 M); RPA: Mar (1 M), Apr (1 F, 1 M), Jul (2 F, 1 M), Sep (6 F, 3 M), Oct (6 F, 3 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Ontario, Quebec, Saskatchewan); Mexico (Coahuila, Guerrero, Michoacán de Ocampo, Morelos, Nuevo León, Puebla, Sonora, Veracruz); USA (Arizona, Arkansas, California, Colorado, Connecticut, Delaware, District of Columbia, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Tennessee, Texas, Utah, Vermont, Virginia, Washington, West Virginia, Wisconsin, Wyoming). Comments. The range of Anthrax georgicus includes all North America and Central America (Nicaragua, Costa Rica) covering a wide diversity of habitats and environmental conditions. Common in the rainy season and present in the dry season (March), this species is probably present in most if not all states of Mexico, but has only been collected in eight of them. Taxon classification Animalia Diptera Bombyliidae Anthrax irroratus Say, 1823 Figure 9d Material examined. EAM: Apr (1 M), Aug (2 M), Oct (1 M); ECA: Apr (1 F), May (1 F, 1 M); EEO: Apr (2 F), Jul (4 F), Aug (1 M); REC: Apr (3 F, 10 M), Aug (1 M); REE: Aug (1 M); RLC: Jul (6 F, 10 M); ROR: Feb (1 M), Aug (5 M), Sep (1 M); RPA: Apr (1 M), Aug (2 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Nova Scotia, Ontario, Quebec, Saskatchewan); Mexico (Baja California, Baja California Sur, Coahuila, Colima, Guerrero, Michoacán, Morelos, Nayarit, Puebla, San Luis Potosí, Sinaloa, Sonora, Veracruz, Zacatecas); USA (Alaska, Arizona, Arkansas, California, Colorado, Connecticut, Idaho, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, Oregon, Pennsylvania, Tennessee, Texas, Utah, Virginia, West Virginia, Wyoming). Known hosts. Megachile gentilis Cresson ( Megachilidae ); Megachile mendica Cresson ( Megachilidae ); Dianthidium heterulkei fraternum Timberlake ( Megachilidae ); Aschmendiella bucconis denticulata Cresson ( Megachilidae ); Hylaeus asininus Cockrell and Casad ( Colletidae ). Scott and Strickler (1992) also reared Anthrax irroratus from Megachile relativa Cresson ( Megachilidae ) and Megachile inermis Provancher ( Megachilidae ). Comments. Anthrax irroratus , like Anthrax georgicus (above), is present in all of North America and reaches Central America and Caribbean islands (Honduras, Puerto Rico). More abundant than its congener, this species has been collected in 15 states in Mexico (including Oaxaca of the Neotropical region not listed above) and all regions of the USA. Anthrax irroratus should be collected in any systematic, long term Bombyliidae sample in Mexico and the USA. Taxon classification Animalia Diptera Bombyliidae Anthrax oedipus Fabricius, 1805 Figure 10a Material examined. ECA: Apr (1 F, 1 M), Jul (1 F); EEO: Apr (2 F, 1 M), May (1 F, 4 M), Jul (1 M); REC: Apr (1 F); REE: Apr (2 M); RLC: Jul (2 F, 4 M), Sep (1 F); RPA: Apr (1 M), Aug (1 M). Known Nearctic records. Mexico (Baja California, Coahuila, Nayarit, Morelos, Sinaloa, Sonora); USA (Nevada, Texas). Comments. Apparently closely related to Anthrax irroratus , Anthrax oedipus has a narrow distribution in the Nearctic region but is widely distributed in all South America. In the USA it has been collected only in two southern states, while it occurs in most of the northern states of Mexico and one central state (Morelos); it may be present in most areas from Texas to Argentina. Figure 10. Anthrax part II. a Anthrax oedipus , female (CNIN 1055) dorsal view b Anthrax pauper , female (CNIN 1085) dorsal view c Anthrax seriepunctatus , female (CNIN 1089) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax pauper (Loew, 1869) Figure 10b Material examined. CHU: Apr (1 F, 1 M). Known Nearctic records. Canada (Ontario); Mexico (Coahuila); USA (Alabama, Colorado, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Nebraska, New Jersey, New Mexico, New York, Oklahoma, Pennsylvania, Texas, Utah, Vermont, Virginia, Wisconsin). Comments. With just two specimens collected, Anthrax pauper appears to be a rare species in this region. This population is the most southern recorded of this species, mostly present in the center and east of the USA. Presumably adapted to colder climates, it is no coincidence that it was collected in the most elevated site sampled. Taxon classification Animalia Diptera Bombyliidae Anthrax seriepunctatus (Osten Sacken, 1886b) Figure 10c Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Aug (1 F), Sep (1 F); ECA: Jun (1 M); REE: Aug (1 F); RLC: Jun (1 F), Jul (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora, Puebla); USA (Arizona, Nevada, New Mexico, Texas). Comments. This species is recorded mostly from the south of the USA and north of Mexico, but its presence in Puebla in central Mexico suggests a wider distribution within the country, at least in all northern states. Taxon classification Animalia Diptera Bombyliidae Genus Dipalta Osten Sacken Remarks. Dipalta is a small genus with just two species. Dipalta banksi Johnson, 1921 is only present in eastern Canada and USA, while Dipalta serpentina is distributed from Central America to the northern USA. Taxon classification Animalia Diptera Bombyliidae Dipalta serpentina (Osten Sacken, 1877) Figure 11 Material examined. REC: Aug (1 M); RLC: Jul (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Hidalgo, México, Morelos, Puebla, San Luis Potosí, Sinaloa); USA (Arizona, Arkansas, California, Colorado, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kansas, Kentucky, Maine, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Mexico, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Utah, Washington, Wisconsin, Wyoming). Known host. Myrmeleon immaculatus De Geer ( Myrmeleontidae ). Comments. This species is probably present in all of Mexico, but this is the only record in the north of Mexico. Figure 11. Dipalta serpentina , male (CNIN 215) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Hemipenthes Loew Remarks. Hemipenthes is equally diverse in the Nearctic (29 species), Neotropical (26 species) and Palearctic (37 species) regions, with just six species in the Oriental region and one in the Afrotropical region. Four species of this genus were collected in Coahuila. All of these have broad distributions but apparently from poor sampling because records are not continuous, especially in Mexico. Ávalos-Hernández (2009) recently published a revision of Hemipenthes , with a key for Nearctic species. Taxon classification Animalia Diptera Bombyliidae Hemipenthes jaennickeana (Osten Sacken, 1886a) Figure 12a Material examined. REC: Apr (18 F), Aug (4 F); REE: Feb (3 F); RLC: Mar (7 F), Jul (23 F, 3 M), Sep (3 F). Known Nearctic records. Mexico (Coahuila, Morelos, Sonora); USA (Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, Oregon, Texas, Utah). Comments. Present mainly in the Pacific coast states of the USA and Mexico, from Oregon as far as Morelos in the center of Mexico. This record is the most eastern record in Mexico. Figure 12. Hemipenthes . a Hemipenthes jaennickeana , female (CNIN 1137) dorsal view b Hemipenthes lepidota , female (CNIN 200) dorsal view c Hemipenthes scylla , male (CNIN 725) dorsal view d Hemipenthes sinuosa , female (CNIN 1134) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Hemipenthes lepidota (Osten Sacken, 1886b) Figure 12b Material examined. EAM: Apr (1 M), Aug (1 F); CHU: Apr (1 F, 3 M), Aug (1 M); EEO: Jul (1 F, 4 M), Aug (1 F); REC: (1 M); REE: Apr (11 F, 2 M), Aug (1 F); RLC: Jun (1 F), Jul (3 F); RPA: Apr (1 F), Sep (4 F), Oct (2 F). Known Nearctic records. Canada (Alberta); Mexico (Baja California, Baja California Sur, Coahuila, Chihuahua, Guerrero, Morelos, Puebla, San Luis Potosí, Sonora, Tamaulipas); USA (Arizona, California, Colorado, Idaho, Louisiana, Nevada). Comments. This species is abundant in the rainy season in most of the Nearctic region but has not been collected in many states of Mexico or the USA where it probably is present. Taxon classification Animalia Diptera Bombyliidae Hemipenthes scylla (Osten Sacken, 1887) Figure 12c Material examined. REC: Apr (23 M), Aug (7 M); REE: Feb (5 M), Apr (2 M); RLC: Mar (8 M), Jul (8 M), Sep (9 M). Known Nearctic records. Mexico (Coahuila, Morelos, Guanajuato, Sonora); USA (Arizona, Texas). Comments. Males of this species are abundant all year long but females are unknown. There is no explanation for this lack of females in the collections. Extreme sexual dimorphism and misidentification of females can be dismiss, since there is no Hemipenthes species from which only females are known. One possible explanation is that females life span is too short and therefor encounter probabilities are low. Distribution is discontinuous with populations present in central and northern Mexico and the southern USA; it is unknown whether this species is present in between these areas. Taxon classification Animalia Diptera Bombyliidae Hemipenthes sinuosa (Wiedemann, 1821) Figure 12d Material examined. REC: Apr (3 F); REE: (Feb (1 F), Apr (2 F); RLC: Jul (1 F, 1 M), Sep (1 M); RPA: Sep (2 F). Known Nearctic records. Mexico (Coahuila, Morelos); USA (Alabama, Arizona, Arkansas, Connecticut, Delaware, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maryland, Massachusetts, Minnesota, Mississippi, Missouri, Nebraska, New Jersey, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Vermont, Virginia, West Virginia, Wisconsin). Known host. Neodiprion sertifer Geoff. ( Diprionidae ). Comments. Hemipenthes sinuosa is only known from Morelos in the center of Mexico and Coahuila in the northeast, but can be found almost in all of the USA. It is clearly undersampled in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Lepidanthrax Osten Sacken Remarks. Forty seven of the 52 species of Lepidanthrax are from the Nearctic region. Hall (1976) published a revision of this genus including keys for species. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax arizonensis Hall, 1976 Figure 13a Material examined. EEO: Mar (1 F); Oct (2 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona). Comments. Lepidanthrax arizonensis has a restricted distribution, being present only in Arizona and Coahuila, but probably is also present in Chihuahua, Texas and New Mexico. Figure 13. Lepidanthrax . a Lepidanthrax arizonensis , female (CNIN 1352) dorsal view b Lepidanthrax disiunctus female (CNIN 334) dorsal view c Lepidanthrax hesperus , male (CNIN 1339) dorsal view d Lepidanthrax hyposcelus , male (CNIN 369) dorsal view e Lepidanthrax proboscideus , male (CNIN 357) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax disiunctus (Wiedemann, 1830) Figure 13b Material examined. REC: Aug (2 F, 1 M). Known Nearctic records. Mexico (Coahuila, Distrito Federal, Guerrero, Veracruz); USA (Arizona). Comments. The distribution of Lepidanthrax disiunctus has its northern extreme in Arizona and its southern extreme in Oaxaca, in the southeast of Mexico. It seems this species is rarely collected, but widely distributed. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hesperus Hall, 1976 Figure 13c Material examined. EAM: Apr (2 M); CHU: Apr (2 F, 5 M); ROR: Apr (1 F, 3 M), May (1 F, 1 M); RPA: Apr (1 F, 14 M). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, New Mexico, Texas). Comments. This record is the first in northeastern Mexico. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hyposcelus Hall, 1976 Figure 13d Material examined. RLC: Sep (4 F, 15 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos, Puebla). Comments. Lepidanthrax hyposcelus is endemic to Mexico, previously only known from the southwest of the country; this record extends its distribution to the northeast of the country. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax proboscideus (Loew, 1869) Figure 13e Material examined. ECA: Sep (1 F, 2 M); EEO: Apr (1 F), Aug (1 F, 1 M), Oct (4 F, 15 M); ROR: Sep (2 M); RPA: Sep (2 M), Oct (1 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila, Durango, Guerrero, Morelos, Sonora); USA (Arizona, California, Nevada, New Mexico, Utah). Comments. Lepidanthrax proboscideus , Lepidanthrax fuscipennis Hall, 1976 and Lepidanthrax disiunctus are the only species of this genus distributed in both the Nearctic and Neotropical regions. Of these Lepidanthrax proboscideus extends as far as El Salvador, the most southern distribution for a Nearctic species of this genus. This is the first record of this species in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Neodiplocampta Curran Remarks. Neodiplocampta is a small American genus, more diverse in the Neotropical than the Nearctic region. Hull and Martin (1974) described seven of the 16 species and published a key for all species of the genus. Taxon classification Animalia Diptera Bombyliidae Neodiplocampta ( Neodiplocampta ) miranda Hull & Martin, 1974 Figure 14 Material examined. CHU: Aug (1 F); EEO: Aug (1 F, 1 M); ROR: Jul (1 F); Oct (1 M); RPA: Aug (1 F, 2 M). Known Nearctic records. Mexico (Coahuila, Guerrero, San Luis Potosi, Sinaloa, Sonora); USA (Arizona, California, Florida, Texas). Comments. Neodiplocampta (Neodiplocampta) miranda and Neodiplocampta (Agitonia) sepia Hull, 1966 are the only two species distributed in both biogeographic regions (Nearctic and Neotropical). This species is distributed from the south of the USA to Nicaragua, but has not been collected in most Mexican states. This lack of records is possibly due its low abundance. Figure 14. Neodiplocampta (Neodiplocampta) miranda , female (CNIN 225) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Paravilla Painter Remarks. Fifty five of the 58 species of the genus are Nearctic. All species of Paravilla collected in Coahuila were exclusively collected in the summer months from April to July. Hall (1981a) reviewed this genus and presented a key for species and description of new species. Taxon classification Animalia Diptera Bombyliidae Paravilla edititoides (Painter, 1933) Figure 15a Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Jul (2 F, 1 M); ECA: Apr (1 F, 1 M), Jul (1 M); EEO: Apr (1 F, 10 M), May (1 F, 2 M), Jul (9 M); REE: Apr (1 M); RLC: Jun (2 F, 5 M), Jul (1 F); ROR: Jul (1 F, 3 M); RPA: Oct (1 M). Known Nearctic records. Canada (Saskatchewan); Mexico (Chihuahua, Coahuila, Durango, Jalisco, México, Zacatecas); USA (Arizona, Colorado, Idaho, Kansas, Montana, Nebraska, New Mexico, Oklahoma, Utah, Texas, Wyoming). Comments. This species is very abundant and present in most of North America, from Canada as far as Jalisco in central Mexico. Figure 15. Paravilla . a Paravilla edititoides , male (CNIN 1272) dorsal view b Paravilla flavipilosa , male (CNIN 1125) dorsal view c Paravilla parvula , female (CNIN 884) dorsal view d Paravilla separata , female (CNIN 898) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Paravilla flavipilosa (Cole, 1923) Figure 15b Material examined. CHU: Apr (1 M); Jul (1 M); ECA: Apr (1 M); EEO: Apr (7 M), May (11 M); ROR: Apr (2 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Nuevo León); USA (Arizona, California, Colorado, Texas). Comments. Paravilla flavipilosa is abundant and restricted to the south of the USA and north of Mexico. Taxon classification Animalia Diptera Bombyliidae Paravilla parvula Hall, 1981a Figure 15c Material examined. EAM: Apr (1 F); CHU: Apr (1 M); RPA: Apr (7 F, 13 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Durango, Guanajuato, Hidalgo, Jalisco, México, Michoacán, Nuevo León, San Luis Potosí, Sonora, Zacatecas), USA (Arizona, New Mexico, Texas, Utah). Comments. Paravilla parvula is relatively well collected in northern and central Mexico. Its distribution also includes the south of the USA but no farther north than Utah. Taxon classification Animalia Diptera Bombyliidae Paravilla separata (Walker, 1852) Figure 15d Material examined. CHU: Apr (1 F); EEO: Apr (3 F); REE: Apr (5 F, 3 M); RPA: Apr (1 M). Known Nearctic records. Canada (Ontario, Manitoba); Mexico (Coahuila); USA (Alabama, Florida, Georgia, Iowa, Kansas, Michigan, Minnesota, Mississippi, Nebraska, Ohio, South Dakota, Wisconsin). Comments. Paravilla separata is present mainly in the eastern half of the USA, and southeastern Canada. This record in Coahuila represents the southern extreme of the distribution of this species, and is the first in Mexico. It may also be present in Tamaulipas and Nuevo León but doubtfully in the northwest of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Poecilanthrax Osten Sacken Remarks. Four species from this mainly Nearctic genus are recorded in Coahuila for the first time. Painter and Hall (1960) published a review of Poecilanthrax with a key and images of the species. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax effrenus (Coquillett, 1887) Figure 16a Material examined. EAM: Apr (1 F, 1 M), Jun (1 F, 1 M), Sep (1 F); CHU: Jun (1 F); ROR: May (10 F, 5 M), Jul (2 F, 3 M), Aug (1 M); RPA: Jun (4 F, 1 M), Jul (6 F, 6 M), Aug (1 F, 1 M), Sep (4 F), Oct (2 F, 1 M). Known Nearctic records. Mexico (Baja California Sur, Chihuahua, Coahuila, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oklahoma, Texas). Comments. This record fills a gap in Poecilanthrax effrenus distribution between northwest and northeast populations of Mexico. This species is probably present in Baja California and Nuevo León, but has not yet been recorded. Figure 16. Poecilanthrax . a Poecilanthrax effrenus , female (CNIN 1380) dorsal view b Poecilanthrax fasciatus , male (CNIN 218) dorsal view c Poecilanthrax hyalinipennis , female (CNIN 1365) dorsal view d Poecilanthrax poecilogaster , male (CNIN 1356) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax fasciatus Johnson & Johnson, 1957 Figure 16b Material examined. EAM: Sep (1 M); CHU: Oct (1 M); ROR: Oct (1 M); RPA: Oct (1 M). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Texas). Known host. Chorizagrotis auxiliaris Grote ( Noctuidae ). Comments. Poecilanthrax fasciatus is collected in Mexico for the first time, and this extends the southern limit of this species distribution. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax hyalinipennis Painter & Hall, 1960 Figure 16c Material examined. EAM: Mar (3 M), Oct (1 F); CHU: Oct (1 M); ROR: Oct (4 M); RPA: Sep (1 F), Oct (2 F, 6 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Utah). Comments. This record extends the distribution of Poecilanthrax hyalinipennis into the northwest of Mexico. Considering its distribution in the USA, this species may also be present in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax poecilogaster (Osten Sacken, 1886b) Figure 16d Material examined. REE: Apr (2 M). Known Nearctic records. Canada (Alberta, Manitoba, Ontario, Saskatchewan); Mexico (Coahuila, Morelos, Nuevo León, Sonora); USA (Arizona, California, Colorado, Idaho, Nevada, New Mexico, Oregon, Utah). Comments. Most of the records in the USA and Mexico of this rarely collected but widespread species are from Pacific Coast states, although, there are records from Nuevo Leon and Coahuila in northeast Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Rhynchanthrax Painter Remarks. Of the seven species of this exclusively Nearctic genus, six are present in Mexico, with Rhynchanthrax maria (Williston, 1901) and Rhynchanthrax nigrofimbriatus (Williston, 1901) being endemic to this country. Only Rhynchanthrax parvicornis (Loew, 1869) has not been collected in Mexico, but it is distributed across the southern USA and may also occur in the north of Mexico. Taxon classification Animalia Diptera Bombyliidae Rhynchanthrax capreus (Coquillett, 1887) Figure 17a Material examined. CHU: Apr (1 M), Aug (13 F, 6 M), Jul (12 F, 6 M), Sep (1 F, 3 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Colorado, Nebraska, Nevada, New Mexico, Utah). Comments. This is the first record of this species in Mexico. Rhynchanthrax capreus is the only species occurring in the northwest of the USA, while the other species in the genus are present mainly in the south and east of the country. This species may also be present in the northwest of Mexico (Baja California, Sonora, Chihuahua). Figure 17. Rhynchanthrax . a Rhynchanthrax capreus , female (CNIN 940) dorsal view b Rhynachantrax texanus , male (CNIN 263) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Rhynachantrax texanus (Painter, 1933) Figure 17b Material examined. EEO: May (1 M); RLC: Jun (1 F, 11 M), Jul (3 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Kansas, New Mexico, Texas). Comments. This is the most eastern record in Mexico for this species. In the USA it is distributed in the southern-center of the country, but in Mexico it has been collected in Sonora so it probably also occurs in Arizona. Taxon classification Animalia Diptera Bombyliidae Genus Thyridanthrax Osten Sacken Remarks. Thyridanthrax has twice as many species in the Palearctic region as in the Nearctic and Neotropical regions combined. All 12 species in North America are present in the USA with five also in Mexico. These are the first records of this genus in Coahuila. The distribution of Thyridanthrax selene (Osten Sacken, 1886b) and Thyridanthrax pallidus (Coquillett, 1887) are very similar, being present in all of the southern USA and probably also in all of northern Mexico, although they have been only collected in Sonora and Coahuila to date. Both species are rare and were collected only in April. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax pallidus (Coquillett, 1887) Figure 18b Material examined. REE: Apr (4 F, 1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Texas, Utah). Comments. This represents the first record of this species in Mexico. Figure 18. Thyridanthrax . a Thyridanthrax selene , male (CNIN 182) dorsal view b Thyridanthrax pallidus , female (CNIN 1162) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax selene (Osten Sacken, 1886b) Figure 18a Material examined. EAM: Apr (1 M); REE: Apr (2 F, 2 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Texas). Comments. This is the most eastern record in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Xenox Evenhuis Remarks. Of the five species that constitute this genus, four are present in Mexico. Taxon classification Animalia Diptera Bombyliidae Xenox xylocopae (Marston, 1970) Figure 19 Material examined. ECA: Sep (1 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Sonora), USA (Arizona, New Mexico, Texas). Known host. Xylocopa micheneri micheneri (Hurd) ( Apidae ) as reported by Minckley (1989) . Comments. Xenox xylocopae appears to be restricted to the northeast of Mexico and south of the USA. Three of the other species also have restricted and separate distributions: Xenox delila Loew, 1869 is present in the northwest of Mexico and California; Xenox nigritus (Schaeffer, 1768) occurs from the northeast of Mexico (Veracruz and Tamaulipas without overlap with Xenox xylocopae ) to South America; and Xenox tigrinus (De Geer, 1776) is present in the eastern USA and southern Ontario. Only Xenox habrosus (Marston, 1970) has a distribution overlapping with the other four species, being present in all of Mexico and the southwest of the USA. Figure 19. Xenox xylocopae , male (CNIN 1165) dorsal view. Scale bar: 3 mm. Subfamily Toxophorinae Taxon classification Animalia Diptera Bombyliidae Genus Toxophora Meigen Remarks. Toxophora is distributed worldwide, being more diverse in the Afrotropical and Palearctic regions. Mexico's fauna includes three Neotropical species and five Nearctic species. All Nearctic species of Mexico were distributed in the western half of the country. These two new records represent the first of this genus in Coahuila and the most eastern distribution of the Nearctic species in the country. The New World species of this genus were keyed using Cunha et al. (2011) . Taxon classification Animalia Diptera Bombyliidae Toxophora maxima Coquillett, 1886 Figure 2a, b Material examined. CHU: Jul (1 M); EEO: Jul (2 F, 2 M), Oct (1 F, 3 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila); USA (Arizona, California, Idaho, Kansas, New Mexico, Oklahoma, Oregon, Texas). Comments. In Mexico Toxophora maxima was only known from Baja California Peninsula and now Coahuila. This apparent gap in its distribution is probably due to undersampling. Sampling of the intermediate zones is necessary to know if these populations form a continuous unit as they do in the southern states of USA. Figure 2. Toxophora . Toxophora maxima , male (CNIN 1115) a dorsal view b lateral view; Toxophora virgata , male (CNIN 1109) c lateral view d posterior view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Toxophora virgata Osten Sacken, 1877 Figure 2c, d Material examined. EAM: Jun (1 F, 1 M), Jul (1 F); CHU: Aug (1 M), Oct (1 M); EEO: Jul (1 M), Oct (1 F, 1 M); RLC: Jun (1 M); ROR: Apr (1 F, 2 M); RPA: Oct (1 F). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora); USA (Arizona, California, Colorado, Georgia, Idaho, Nevada, New Mexico, Oklahoma, Texas, Utah). Known hosts. Odynerus sp. ( Vespidae ); Stenodynerus toltecus Saussure ( Vespidae ). Comments. This species is present in the all southwestern states of the USA and northwest of Mexico. This is the first record in the northeast of Mexico. The species is probably also present in Chihuahua, between Sonora and Coahuila. Taxon classification Animalia Diptera Bombyliidae Genus Toxophora Meigen Remarks. Toxophora is distributed worldwide, being more diverse in the Afrotropical and Palearctic regions. Mexico's fauna includes three Neotropical species and five Nearctic species. All Nearctic species of Mexico were distributed in the western half of the country. These two new records represent the first of this genus in Coahuila and the most eastern distribution of the Nearctic species in the country. The New World species of this genus were keyed using Cunha et al. (2011) . Remarks. Toxophora is distributed worldwide, being more diverse in the Afrotropical and Palearctic regions. Mexico's fauna includes three Neotropical species and five Nearctic species. All Nearctic species of Mexico were distributed in the western half of the country. These two new records represent the first of this genus in Coahuila and the most eastern distribution of the Nearctic species in the country. The New World species of this genus were keyed using Cunha et al. (2011) . Taxon classification Animalia Diptera Bombyliidae Toxophora maxima Coquillett, 1886 Figure 2a, b Material examined. CHU: Jul (1 M); EEO: Jul (2 F, 2 M), Oct (1 F, 3 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila); USA (Arizona, California, Idaho, Kansas, New Mexico, Oklahoma, Oregon, Texas). Comments. In Mexico Toxophora maxima was only known from Baja California Peninsula and now Coahuila. This apparent gap in its distribution is probably due to undersampling. Sampling of the intermediate zones is necessary to know if these populations form a continuous unit as they do in the southern states of USA. Figure 2. Toxophora . Toxophora maxima , male (CNIN 1115) a dorsal view b lateral view; Toxophora virgata , male (CNIN 1109) c lateral view d posterior view. All scale bars: 3 mm. Material examined. CHU: Jul (1 M); EEO: Jul (2 F, 2 M), Oct (1 F, 3 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila); USA (Arizona, California, Idaho, Kansas, New Mexico, Oklahoma, Oregon, Texas). Comments. In Mexico Toxophora maxima was only known from Baja California Peninsula and now Coahuila. This apparent gap in its distribution is probably due to undersampling. Sampling of the intermediate zones is necessary to know if these populations form a continuous unit as they do in the southern states of USA. Figure 2. Toxophora . Toxophora maxima , male (CNIN 1115) a dorsal view b lateral view; Toxophora virgata , male (CNIN 1109) c lateral view d posterior view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Toxophora virgata Osten Sacken, 1877 Figure 2c, d Material examined. EAM: Jun (1 F, 1 M), Jul (1 F); CHU: Aug (1 M), Oct (1 M); EEO: Jul (1 M), Oct (1 F, 1 M); RLC: Jun (1 M); ROR: Apr (1 F, 2 M); RPA: Oct (1 F). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora); USA (Arizona, California, Colorado, Georgia, Idaho, Nevada, New Mexico, Oklahoma, Texas, Utah). Known hosts. Odynerus sp. ( Vespidae ); Stenodynerus toltecus Saussure ( Vespidae ). Comments. This species is present in the all southwestern states of the USA and northwest of Mexico. This is the first record in the northeast of Mexico. The species is probably also present in Chihuahua, between Sonora and Coahuila. Material examined. EAM: Jun (1 F, 1 M), Jul (1 F); CHU: Aug (1 M), Oct (1 M); EEO: Jul (1 M), Oct (1 F, 1 M); RLC: Jun (1 M); ROR: Apr (1 F, 2 M); RPA: Oct (1 F). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora); USA (Arizona, California, Colorado, Georgia, Idaho, Nevada, New Mexico, Oklahoma, Texas, Utah). Known hosts. Odynerus sp. ( Vespidae ); Stenodynerus toltecus Saussure ( Vespidae ). Comments. This species is present in the all southwestern states of the USA and northwest of Mexico. This is the first record in the northeast of Mexico. The species is probably also present in Chihuahua, between Sonora and Coahuila. Subfamily Bombyliinae Taxon classification Animalia Diptera Bombyliidae Genus Bombylius Linnaeus Remarks. With 278 described species, Bombylius is the second most diverse genus of Bombyliidae . It has a worldwide distribution being especially diverse in the Palearctic and Nearctic regions. One endemic species is present in Coahuila: Bombylius (Parabombylius) coahuilensis (Hall & Evenhuis, 1981). Four other species are reported for the state: Bombylius sylphae Evenhuis, 1984, Bombylius aleophilus (Hall & Evenhuis, 1981), Bombylius paradoxus (Hall & Evenhuis, 1981), Bombylius syndesmus (Coquillett, 1894). A review with identification keys for Nearctic species is presented in Hall and Evenhuis (1980) , later Evenhuis (1984) revised and present keys for the comanche group of America. Taxon classification Animalia Diptera Bombyliidae Bombylius ( Bombylius ) frommerorum Hall & Evenhuis, 1980 Figure 3 Material examined. EEO: Aug (1 M), Oct (1 F). Known Nearctic records. Mexico (Chihuahua, Coahuila); USA (Arizona, New Mexico, Texas). Comments. This species is restricted to the southwest of the USA and north of Mexico. Figure 3. Bombylius (Bombylius) frommerorum , female (CNIN 772) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Heterostylum Macquart Remarks. The genus is only present in Nearctic and Neotropical regions. Although not as diverse as other genera (only 12 species), specimens from some species are abundant in the field. Heterostylum robustum was previously known from Coahuila and was collected during this study. This species is distributed from Canada to central Mexico. There are two revisions for this genus that contains identification keys, one by Hall and Evenhuis (1980) and the more recent by Cunha et al. (2007) . Taxon classification Animalia Diptera Bombyliidae Heterostylum croceum Painter, 1930 Figure 4 Material examined. REE: Apr (1 F). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Missouri, New Mexico, Texas). Comments. Heterostylum croceum is recorded for the first time in Mexico; previously known from the southern-central United States. Hall and Evenhuis (1980) suggest that Heterostylum croceum may be related to Heterostylum engelhardti Painter, 1930 or even be a subspecies of that taxon, Heterostylum croceum is the eastern form and Heterostylum engelhardti the western form (Arizona, California, Texas, Utah) although both species are present in Texas. Cunha et al. (2007) comment that Heterostylum engelhardti can be distinguished by the presence of white to very pale yellow hair and brown-tipped hairs on the abdomen compared with the darker yellow hairs in Heterostylum croceum . Figure 4. Heterostylum croceum , female (CNIN 858) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Lordotus Loew Remarks. Most of the 29 species in this exclusively Nearctic genus are distributed in the southwest of the USA and north of Mexico, although eight species are present in the northwest of the USA ( Lordotus apicula Coquillet, 1887; Lordotus bipartitus Painter, 1940; Lordotus diversus Coquillett, 1891; Lordotus gibbus Loew, 1863; Lordotus miscellus Coquillett, 1887; Lordotus pulchrissimus Williston, 1893; Lordotus striatus Painter, 1940; Lordotus zona Coquillett, 1887). The three species present in Coahuila are also found in California; their distribution probably includes all northern states of Mexico. Hall (1954) and Hall and Evenhuis (1982) present reviews of the genus and keys to the species. Taxon classification Animalia Diptera Bombyliidae Lordotus diplasus Hall, 1954 Figure 5a, b Material examined. CHU: Sep (2 M); RLC: Sep (2 M); RPA: Sep (1 F). Known Nearctic records. Mexico (Coahuila, Zacatecas); USA (Arizona, California, New Mexico). Figure 5. Lordotus . Lordotus diplasus , a female (CNIN 774) dorsal view b male (CNIN 861) dorsal view c Lordotus divisus , male (CNIN 777) dorsal view; Lordotus perplexus , female (CNIN 801) d dorsal view e lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lordotus divisus Cresson, 1919 Figure 5c Material examined. ECA: Mar (1 M), Apr (2 M); EEO: Apr (16 M); REE: Apr (4 M); ROR: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Baja California); USA (Arizona, California, Nevada, New Mexico, Texas). Taxon classification Animalia Diptera Bombyliidae Lordotus perplexus Johnson & Johnson, 1959 Figure 5d, e Material examined. CHU: Apr (1 H), ECA: Apr (1 H); EEO: Apr (4 F); REE: Apr (7 F); ROR: Apr (1 F). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Lordotus perplexus has the most southern distribution in the genus, reaching Sinaloa on the Pacific coast. Taxon classification Animalia Diptera Bombyliidae Genus Triploechus Edwards Remarks. Four species of Triploechus are present in Nearctic region: Triploechus luridus Hall, 1975; Triploechus novus (Williston, 1893); Triploechus sackeni (Bigot, 1892); Triploechus stagei Hall, 1975. Of these Triploechus stagei is endemic to Mexico and Triploechus novus has the widest distribution of this genus, being present in the south of the USA and center of Mexico. Hall and Evenhuis (1981) present a revision and key for species for this genus. Taxon classification Animalia Diptera Bombyliidae Triploechus novus (Williston, 1893) Figure 6 Material examined. CHU: Apr (7 F, 6 M); REE: Apr (1 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Durango, San Luis Potosí, Sonora); USA (Arizona, California, Nevada, New Mexico, Texas). Comments. This is a widespread and apparently common species. All specimens were collected in April so it may have a short flight season. Figure 6. Triploechus novus , female (CNIN 1237) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Bombylius Linnaeus Remarks. With 278 described species, Bombylius is the second most diverse genus of Bombyliidae . It has a worldwide distribution being especially diverse in the Palearctic and Nearctic regions. One endemic species is present in Coahuila: Bombylius (Parabombylius) coahuilensis (Hall & Evenhuis, 1981). Four other species are reported for the state: Bombylius sylphae Evenhuis, 1984, Bombylius aleophilus (Hall & Evenhuis, 1981), Bombylius paradoxus (Hall & Evenhuis, 1981), Bombylius syndesmus (Coquillett, 1894). A review with identification keys for Nearctic species is presented in Hall and Evenhuis (1980) , later Evenhuis (1984) revised and present keys for the comanche group of America. Remarks. With 278 described species, Bombylius is the second most diverse genus of Bombyliidae . It has a worldwide distribution being especially diverse in the Palearctic and Nearctic regions. One endemic species is present in Coahuila: Bombylius (Parabombylius) coahuilensis (Hall & Evenhuis, 1981). Four other species are reported for the state: Bombylius sylphae Evenhuis, 1984, Bombylius aleophilus (Hall & Evenhuis, 1981), Bombylius paradoxus (Hall & Evenhuis, 1981), Bombylius syndesmus (Coquillett, 1894). A review with identification keys for Nearctic species is presented in Hall and Evenhuis (1980) , later Evenhuis (1984) revised and present keys for the comanche group of America. Taxon classification Animalia Diptera Bombyliidae Bombylius ( Bombylius ) frommerorum Hall & Evenhuis, 1980 Figure 3 Material examined. EEO: Aug (1 M), Oct (1 F). Known Nearctic records. Mexico (Chihuahua, Coahuila); USA (Arizona, New Mexico, Texas). Comments. This species is restricted to the southwest of the USA and north of Mexico. Figure 3. Bombylius (Bombylius) frommerorum , female (CNIN 772) a dorsal view b lateral view. All scale bars: 3 mm. Material examined. EEO: Aug (1 M), Oct (1 F). Known Nearctic records. Mexico (Chihuahua, Coahuila); USA (Arizona, New Mexico, Texas). Comments. This species is restricted to the southwest of the USA and north of Mexico. Figure 3. Bombylius (Bombylius) frommerorum , female (CNIN 772) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Heterostylum Macquart Remarks. The genus is only present in Nearctic and Neotropical regions. Although not as diverse as other genera (only 12 species), specimens from some species are abundant in the field. Heterostylum robustum was previously known from Coahuila and was collected during this study. This species is distributed from Canada to central Mexico. There are two revisions for this genus that contains identification keys, one by Hall and Evenhuis (1980) and the more recent by Cunha et al. (2007) . Remarks. The genus is only present in Nearctic and Neotropical regions. Although not as diverse as other genera (only 12 species), specimens from some species are abundant in the field. Heterostylum robustum was previously known from Coahuila and was collected during this study. This species is distributed from Canada to central Mexico. There are two revisions for this genus that contains identification keys, one by Hall and Evenhuis (1980) and the more recent by Cunha et al. (2007) . Taxon classification Animalia Diptera Bombyliidae Heterostylum croceum Painter, 1930 Figure 4 Material examined. REE: Apr (1 F). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Missouri, New Mexico, Texas). Comments. Heterostylum croceum is recorded for the first time in Mexico; previously known from the southern-central United States. Hall and Evenhuis (1980) suggest that Heterostylum croceum may be related to Heterostylum engelhardti Painter, 1930 or even be a subspecies of that taxon, Heterostylum croceum is the eastern form and Heterostylum engelhardti the western form (Arizona, California, Texas, Utah) although both species are present in Texas. Cunha et al. (2007) comment that Heterostylum engelhardti can be distinguished by the presence of white to very pale yellow hair and brown-tipped hairs on the abdomen compared with the darker yellow hairs in Heterostylum croceum . Figure 4. Heterostylum croceum , female (CNIN 858) a dorsal view b lateral view. All scale bars: 3 mm. Material examined. REE: Apr (1 F). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Missouri, New Mexico, Texas). Comments. Heterostylum croceum is recorded for the first time in Mexico; previously known from the southern-central United States. Hall and Evenhuis (1980) suggest that Heterostylum croceum may be related to Heterostylum engelhardti Painter, 1930 or even be a subspecies of that taxon, Heterostylum croceum is the eastern form and Heterostylum engelhardti the western form (Arizona, California, Texas, Utah) although both species are present in Texas. Cunha et al. (2007) comment that Heterostylum engelhardti can be distinguished by the presence of white to very pale yellow hair and brown-tipped hairs on the abdomen compared with the darker yellow hairs in Heterostylum croceum . Figure 4. Heterostylum croceum , female (CNIN 858) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Lordotus Loew Remarks. Most of the 29 species in this exclusively Nearctic genus are distributed in the southwest of the USA and north of Mexico, although eight species are present in the northwest of the USA ( Lordotus apicula Coquillet, 1887; Lordotus bipartitus Painter, 1940; Lordotus diversus Coquillett, 1891; Lordotus gibbus Loew, 1863; Lordotus miscellus Coquillett, 1887; Lordotus pulchrissimus Williston, 1893; Lordotus striatus Painter, 1940; Lordotus zona Coquillett, 1887). The three species present in Coahuila are also found in California; their distribution probably includes all northern states of Mexico. Hall (1954) and Hall and Evenhuis (1982) present reviews of the genus and keys to the species. Remarks. Most of the 29 species in this exclusively Nearctic genus are distributed in the southwest of the USA and north of Mexico, although eight species are present in the northwest of the USA ( Lordotus apicula Coquillet, 1887; Lordotus bipartitus Painter, 1940; Lordotus diversus Coquillett, 1891; Lordotus gibbus Loew, 1863; Lordotus miscellus Coquillett, 1887; Lordotus pulchrissimus Williston, 1893; Lordotus striatus Painter, 1940; Lordotus zona Coquillett, 1887). The three species present in Coahuila are also found in California; their distribution probably includes all northern states of Mexico. Hall (1954) and Hall and Evenhuis (1982) present reviews of the genus and keys to the species. Taxon classification Animalia Diptera Bombyliidae Lordotus diplasus Hall, 1954 Figure 5a, b Material examined. CHU: Sep (2 M); RLC: Sep (2 M); RPA: Sep (1 F). Known Nearctic records. Mexico (Coahuila, Zacatecas); USA (Arizona, California, New Mexico). Figure 5. Lordotus . Lordotus diplasus , a female (CNIN 774) dorsal view b male (CNIN 861) dorsal view c Lordotus divisus , male (CNIN 777) dorsal view; Lordotus perplexus , female (CNIN 801) d dorsal view e lateral view. All scale bars: 3 mm. Material examined. CHU: Sep (2 M); RLC: Sep (2 M); RPA: Sep (1 F). Known Nearctic records. Mexico (Coahuila, Zacatecas); USA (Arizona, California, New Mexico). Figure 5. Lordotus . Lordotus diplasus , a female (CNIN 774) dorsal view b male (CNIN 861) dorsal view c Lordotus divisus , male (CNIN 777) dorsal view; Lordotus perplexus , female (CNIN 801) d dorsal view e lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lordotus divisus Cresson, 1919 Figure 5c Material examined. ECA: Mar (1 M), Apr (2 M); EEO: Apr (16 M); REE: Apr (4 M); ROR: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Baja California); USA (Arizona, California, Nevada, New Mexico, Texas). Material examined. ECA: Mar (1 M), Apr (2 M); EEO: Apr (16 M); REE: Apr (4 M); ROR: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Baja California); USA (Arizona, California, Nevada, New Mexico, Texas). Taxon classification Animalia Diptera Bombyliidae Lordotus perplexus Johnson & Johnson, 1959 Figure 5d, e Material examined. CHU: Apr (1 H), ECA: Apr (1 H); EEO: Apr (4 F); REE: Apr (7 F); ROR: Apr (1 F). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Lordotus perplexus has the most southern distribution in the genus, reaching Sinaloa on the Pacific coast. Material examined. CHU: Apr (1 H), ECA: Apr (1 H); EEO: Apr (4 F); REE: Apr (7 F); ROR: Apr (1 F). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Lordotus perplexus has the most southern distribution in the genus, reaching Sinaloa on the Pacific coast. Taxon classification Animalia Diptera Bombyliidae Genus Triploechus Edwards Remarks. Four species of Triploechus are present in Nearctic region: Triploechus luridus Hall, 1975; Triploechus novus (Williston, 1893); Triploechus sackeni (Bigot, 1892); Triploechus stagei Hall, 1975. Of these Triploechus stagei is endemic to Mexico and Triploechus novus has the widest distribution of this genus, being present in the south of the USA and center of Mexico. Hall and Evenhuis (1981) present a revision and key for species for this genus. Remarks. Four species of Triploechus are present in Nearctic region: Triploechus luridus Hall, 1975; Triploechus novus (Williston, 1893); Triploechus sackeni (Bigot, 1892); Triploechus stagei Hall, 1975. Of these Triploechus stagei is endemic to Mexico and Triploechus novus has the widest distribution of this genus, being present in the south of the USA and center of Mexico. Hall and Evenhuis (1981) present a revision and key for species for this genus. Taxon classification Animalia Diptera Bombyliidae Triploechus novus (Williston, 1893) Figure 6 Material examined. CHU: Apr (7 F, 6 M); REE: Apr (1 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Durango, San Luis Potosí, Sonora); USA (Arizona, California, Nevada, New Mexico, Texas). Comments. This is a widespread and apparently common species. All specimens were collected in April so it may have a short flight season. Figure 6. Triploechus novus , female (CNIN 1237) dorsal view. Scale bar: 3 mm. Material examined. CHU: Apr (7 F, 6 M); REE: Apr (1 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Coahuila, Durango, San Luis Potosí, Sonora); USA (Arizona, California, Nevada, New Mexico, Texas). Comments. This is a widespread and apparently common species. All specimens were collected in April so it may have a short flight season. Figure 6. Triploechus novus , female (CNIN 1237) dorsal view. Scale bar: 3 mm. Subfamily Lomatiinae Taxon classification Animalia Diptera Bombyliidae Genus Ogcodocera Macquart Remarks. The only two species in this genus have been collected from the neotropical part of Mexico to north of the USA and Canada. Ogcodocera leucoprocta (Wiedemann, 1828), not sampled during this study, is present in the whole Nearctic region from Canada to south of Mexico. Taxon classification Animalia Diptera Bombyliidae Ogcodocera analis Williston, 1901 Figure 7 Material examined. EEO: Aug (2 M), Oct (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos); USA (Arizona, Texas). Comments. This record is the first of this species in the north of Mexico, but it has been previously collected in the south of Mexico and in the south of USA, and thus is probably distributed across the whole country. Unlike Ogcodocera leucoprocta , Ogcodocera analis has its most northern distribution in Arizona and Texas. Figure 7. Ogcodocera analis , male (CNIN 146) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Ogcodocera Macquart Remarks. The only two species in this genus have been collected from the neotropical part of Mexico to north of the USA and Canada. Ogcodocera leucoprocta (Wiedemann, 1828), not sampled during this study, is present in the whole Nearctic region from Canada to south of Mexico. Remarks. The only two species in this genus have been collected from the neotropical part of Mexico to north of the USA and Canada. Ogcodocera leucoprocta (Wiedemann, 1828), not sampled during this study, is present in the whole Nearctic region from Canada to south of Mexico. Taxon classification Animalia Diptera Bombyliidae Ogcodocera analis Williston, 1901 Figure 7 Material examined. EEO: Aug (2 M), Oct (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos); USA (Arizona, Texas). Comments. This record is the first of this species in the north of Mexico, but it has been previously collected in the south of Mexico and in the south of USA, and thus is probably distributed across the whole country. Unlike Ogcodocera leucoprocta , Ogcodocera analis has its most northern distribution in Arizona and Texas. Figure 7. Ogcodocera analis , male (CNIN 146) dorsal view. Scale bar: 3 mm. Material examined. EEO: Aug (2 M), Oct (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos); USA (Arizona, Texas). Comments. This record is the first of this species in the north of Mexico, but it has been previously collected in the south of Mexico and in the south of USA, and thus is probably distributed across the whole country. Unlike Ogcodocera leucoprocta , Ogcodocera analis has its most northern distribution in Arizona and Texas. Figure 7. Ogcodocera analis , male (CNIN 146) dorsal view. Scale bar: 3 mm. Subfamily Tomomyzinae Taxon classification Animalia Diptera Bombyliidae Genus Paracosmus Osten Sacken Remarks. All five extant species of Paracosmus have Nearctic distributions, and all are present in California. Two of these species have been collected in the northwest of Mexico ( Paracosmus (Actherosia) rubicundus Melander, 1950 and Paracosmus (Paracosmus) morrisoni Osten Sacken, 1887). Taxon classification Animalia Diptera Bombyliidae Paracosmus ( Paracosmus ) morrisoni Osten Sacken, 1887 Figure 8 Material examined. EAM: Apr (1 F, 1 M); CHU: Apr (2 M), Jul (1 F), Aug (2 F); ECA: Apr (1 M); EEO: May (1 F); REE: Apr (1 F); ROR: Apr (2 M), May (1 F, 3 M); RPA: Apr (1 F). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Paracosmus (Paracosmus) morrisoni has the widest distribution within this genus, but in Mexico had previously only been recorded in Sonora. This record represent the most eastern distribution for the genus in the country. Figure 8. Paracosmus (Paracosmus) morrisoni , male (CNIN 832) a dorsal view b lateral view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Paracosmus Osten Sacken Remarks. All five extant species of Paracosmus have Nearctic distributions, and all are present in California. Two of these species have been collected in the northwest of Mexico ( Paracosmus (Actherosia) rubicundus Melander, 1950 and Paracosmus (Paracosmus) morrisoni Osten Sacken, 1887). Remarks. All five extant species of Paracosmus have Nearctic distributions, and all are present in California. Two of these species have been collected in the northwest of Mexico ( Paracosmus (Actherosia) rubicundus Melander, 1950 and Paracosmus (Paracosmus) morrisoni Osten Sacken, 1887). Taxon classification Animalia Diptera Bombyliidae Paracosmus ( Paracosmus ) morrisoni Osten Sacken, 1887 Figure 8 Material examined. EAM: Apr (1 F, 1 M); CHU: Apr (2 M), Jul (1 F), Aug (2 F); ECA: Apr (1 M); EEO: May (1 F); REE: Apr (1 F); ROR: Apr (2 M), May (1 F, 3 M); RPA: Apr (1 F). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Paracosmus (Paracosmus) morrisoni has the widest distribution within this genus, but in Mexico had previously only been recorded in Sonora. This record represent the most eastern distribution for the genus in the country. Figure 8. Paracosmus (Paracosmus) morrisoni , male (CNIN 832) a dorsal view b lateral view. All scale bars: 3 mm. Material examined. EAM: Apr (1 F, 1 M); CHU: Apr (2 M), Jul (1 F), Aug (2 F); ECA: Apr (1 M); EEO: May (1 F); REE: Apr (1 F); ROR: Apr (2 M), May (1 F, 3 M); RPA: Apr (1 F). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Nevada, Texas). Comments. Paracosmus (Paracosmus) morrisoni has the widest distribution within this genus, but in Mexico had previously only been recorded in Sonora. This record represent the most eastern distribution for the genus in the country. Figure 8. Paracosmus (Paracosmus) morrisoni , male (CNIN 832) a dorsal view b lateral view. All scale bars: 3 mm. Subfamily Anthracinae Taxon classification Animalia Diptera Bombyliidae Genus Anthrax Scopoli Remarks. This is a diverse genus with 248 species worldwide. Two old but complete revisions of the genus, including distribution maps and keys, were made by Marston (1963 , 1970 ). Thanks to these Anthrax species can be easily identified. Some Anthrax species are widely distributed occupying two biogeographic regions. From the seven Anthrax species collected in this study in Coahuila, just Anthrax cybele (Coquillett, 1894) has a restricted distribution. The other six species are widespread across the Nearctic region. Two species of Anthrax are reported for the first time for Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax atriplex Marston, 1970 Figure 9a Material examined. EAM Apr (1 F); ROR: Oct (2 M); RPA: Aug (1 M); Sep (1 M); Oct (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Durango, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oregon, Texas, Utah). Known host. Megachile gentilis Cresson ( Megachilidae ). Comments. This species may be present in all the north of Mexico, including Chihuahua, Nuevo León and possibly Sinaloa. Figure 9. Anthrax part I. a Anthrax atriplex , male (CNIN 1098) dorsal view b Anthrax cybele , male (CNIN 1087) dorsal view c Anthrax georgicus , female (CNIN 1071) dorsal view d Anthrax irroratus , male (CNIN 1027) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax cybele (Coquillett, 1894) Figure 9b Material examined. ECA: Apr (2 F); EEO: Apr (1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California). Comments. This is a rare species flying in April. Its distribution is disjunct so far, present in the southwest of the USA and northeast of Mexico. It is probably also found in New Mexico and Texas in the USA and Sonora and Chihuahua in Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax georgicus Macquart, 1834 Figure 9c Material examined. EAM: Mar (1 F), Apr (1 M), Jun (1 F, 2 M), Jul (2 F), Sep (2 M); ROR: Apr (1 F), Sep (1 M); RPA: Mar (1 M), Apr (1 F, 1 M), Jul (2 F, 1 M), Sep (6 F, 3 M), Oct (6 F, 3 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Ontario, Quebec, Saskatchewan); Mexico (Coahuila, Guerrero, Michoacán de Ocampo, Morelos, Nuevo León, Puebla, Sonora, Veracruz); USA (Arizona, Arkansas, California, Colorado, Connecticut, Delaware, District of Columbia, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Tennessee, Texas, Utah, Vermont, Virginia, Washington, West Virginia, Wisconsin, Wyoming). Comments. The range of Anthrax georgicus includes all North America and Central America (Nicaragua, Costa Rica) covering a wide diversity of habitats and environmental conditions. Common in the rainy season and present in the dry season (March), this species is probably present in most if not all states of Mexico, but has only been collected in eight of them. Taxon classification Animalia Diptera Bombyliidae Anthrax irroratus Say, 1823 Figure 9d Material examined. EAM: Apr (1 M), Aug (2 M), Oct (1 M); ECA: Apr (1 F), May (1 F, 1 M); EEO: Apr (2 F), Jul (4 F), Aug (1 M); REC: Apr (3 F, 10 M), Aug (1 M); REE: Aug (1 M); RLC: Jul (6 F, 10 M); ROR: Feb (1 M), Aug (5 M), Sep (1 M); RPA: Apr (1 M), Aug (2 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Nova Scotia, Ontario, Quebec, Saskatchewan); Mexico (Baja California, Baja California Sur, Coahuila, Colima, Guerrero, Michoacán, Morelos, Nayarit, Puebla, San Luis Potosí, Sinaloa, Sonora, Veracruz, Zacatecas); USA (Alaska, Arizona, Arkansas, California, Colorado, Connecticut, Idaho, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, Oregon, Pennsylvania, Tennessee, Texas, Utah, Virginia, West Virginia, Wyoming). Known hosts. Megachile gentilis Cresson ( Megachilidae ); Megachile mendica Cresson ( Megachilidae ); Dianthidium heterulkei fraternum Timberlake ( Megachilidae ); Aschmendiella bucconis denticulata Cresson ( Megachilidae ); Hylaeus asininus Cockrell and Casad ( Colletidae ). Scott and Strickler (1992) also reared Anthrax irroratus from Megachile relativa Cresson ( Megachilidae ) and Megachile inermis Provancher ( Megachilidae ). Comments. Anthrax irroratus , like Anthrax georgicus (above), is present in all of North America and reaches Central America and Caribbean islands (Honduras, Puerto Rico). More abundant than its congener, this species has been collected in 15 states in Mexico (including Oaxaca of the Neotropical region not listed above) and all regions of the USA. Anthrax irroratus should be collected in any systematic, long term Bombyliidae sample in Mexico and the USA. Taxon classification Animalia Diptera Bombyliidae Anthrax oedipus Fabricius, 1805 Figure 10a Material examined. ECA: Apr (1 F, 1 M), Jul (1 F); EEO: Apr (2 F, 1 M), May (1 F, 4 M), Jul (1 M); REC: Apr (1 F); REE: Apr (2 M); RLC: Jul (2 F, 4 M), Sep (1 F); RPA: Apr (1 M), Aug (1 M). Known Nearctic records. Mexico (Baja California, Coahuila, Nayarit, Morelos, Sinaloa, Sonora); USA (Nevada, Texas). Comments. Apparently closely related to Anthrax irroratus , Anthrax oedipus has a narrow distribution in the Nearctic region but is widely distributed in all South America. In the USA it has been collected only in two southern states, while it occurs in most of the northern states of Mexico and one central state (Morelos); it may be present in most areas from Texas to Argentina. Figure 10. Anthrax part II. a Anthrax oedipus , female (CNIN 1055) dorsal view b Anthrax pauper , female (CNIN 1085) dorsal view c Anthrax seriepunctatus , female (CNIN 1089) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax pauper (Loew, 1869) Figure 10b Material examined. CHU: Apr (1 F, 1 M). Known Nearctic records. Canada (Ontario); Mexico (Coahuila); USA (Alabama, Colorado, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Nebraska, New Jersey, New Mexico, New York, Oklahoma, Pennsylvania, Texas, Utah, Vermont, Virginia, Wisconsin). Comments. With just two specimens collected, Anthrax pauper appears to be a rare species in this region. This population is the most southern recorded of this species, mostly present in the center and east of the USA. Presumably adapted to colder climates, it is no coincidence that it was collected in the most elevated site sampled. Taxon classification Animalia Diptera Bombyliidae Anthrax seriepunctatus (Osten Sacken, 1886b) Figure 10c Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Aug (1 F), Sep (1 F); ECA: Jun (1 M); REE: Aug (1 F); RLC: Jun (1 F), Jul (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora, Puebla); USA (Arizona, Nevada, New Mexico, Texas). Comments. This species is recorded mostly from the south of the USA and north of Mexico, but its presence in Puebla in central Mexico suggests a wider distribution within the country, at least in all northern states. Taxon classification Animalia Diptera Bombyliidae Genus Dipalta Osten Sacken Remarks. Dipalta is a small genus with just two species. Dipalta banksi Johnson, 1921 is only present in eastern Canada and USA, while Dipalta serpentina is distributed from Central America to the northern USA. Taxon classification Animalia Diptera Bombyliidae Dipalta serpentina (Osten Sacken, 1877) Figure 11 Material examined. REC: Aug (1 M); RLC: Jul (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Hidalgo, México, Morelos, Puebla, San Luis Potosí, Sinaloa); USA (Arizona, Arkansas, California, Colorado, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kansas, Kentucky, Maine, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Mexico, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Utah, Washington, Wisconsin, Wyoming). Known host. Myrmeleon immaculatus De Geer ( Myrmeleontidae ). Comments. This species is probably present in all of Mexico, but this is the only record in the north of Mexico. Figure 11. Dipalta serpentina , male (CNIN 215) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Hemipenthes Loew Remarks. Hemipenthes is equally diverse in the Nearctic (29 species), Neotropical (26 species) and Palearctic (37 species) regions, with just six species in the Oriental region and one in the Afrotropical region. Four species of this genus were collected in Coahuila. All of these have broad distributions but apparently from poor sampling because records are not continuous, especially in Mexico. Ávalos-Hernández (2009) recently published a revision of Hemipenthes , with a key for Nearctic species. Taxon classification Animalia Diptera Bombyliidae Hemipenthes jaennickeana (Osten Sacken, 1886a) Figure 12a Material examined. REC: Apr (18 F), Aug (4 F); REE: Feb (3 F); RLC: Mar (7 F), Jul (23 F, 3 M), Sep (3 F). Known Nearctic records. Mexico (Coahuila, Morelos, Sonora); USA (Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, Oregon, Texas, Utah). Comments. Present mainly in the Pacific coast states of the USA and Mexico, from Oregon as far as Morelos in the center of Mexico. This record is the most eastern record in Mexico. Figure 12. Hemipenthes . a Hemipenthes jaennickeana , female (CNIN 1137) dorsal view b Hemipenthes lepidota , female (CNIN 200) dorsal view c Hemipenthes scylla , male (CNIN 725) dorsal view d Hemipenthes sinuosa , female (CNIN 1134) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Hemipenthes lepidota (Osten Sacken, 1886b) Figure 12b Material examined. EAM: Apr (1 M), Aug (1 F); CHU: Apr (1 F, 3 M), Aug (1 M); EEO: Jul (1 F, 4 M), Aug (1 F); REC: (1 M); REE: Apr (11 F, 2 M), Aug (1 F); RLC: Jun (1 F), Jul (3 F); RPA: Apr (1 F), Sep (4 F), Oct (2 F). Known Nearctic records. Canada (Alberta); Mexico (Baja California, Baja California Sur, Coahuila, Chihuahua, Guerrero, Morelos, Puebla, San Luis Potosí, Sonora, Tamaulipas); USA (Arizona, California, Colorado, Idaho, Louisiana, Nevada). Comments. This species is abundant in the rainy season in most of the Nearctic region but has not been collected in many states of Mexico or the USA where it probably is present. Taxon classification Animalia Diptera Bombyliidae Hemipenthes scylla (Osten Sacken, 1887) Figure 12c Material examined. REC: Apr (23 M), Aug (7 M); REE: Feb (5 M), Apr (2 M); RLC: Mar (8 M), Jul (8 M), Sep (9 M). Known Nearctic records. Mexico (Coahuila, Morelos, Guanajuato, Sonora); USA (Arizona, Texas). Comments. Males of this species are abundant all year long but females are unknown. There is no explanation for this lack of females in the collections. Extreme sexual dimorphism and misidentification of females can be dismiss, since there is no Hemipenthes species from which only females are known. One possible explanation is that females life span is too short and therefor encounter probabilities are low. Distribution is discontinuous with populations present in central and northern Mexico and the southern USA; it is unknown whether this species is present in between these areas. Taxon classification Animalia Diptera Bombyliidae Hemipenthes sinuosa (Wiedemann, 1821) Figure 12d Material examined. REC: Apr (3 F); REE: (Feb (1 F), Apr (2 F); RLC: Jul (1 F, 1 M), Sep (1 M); RPA: Sep (2 F). Known Nearctic records. Mexico (Coahuila, Morelos); USA (Alabama, Arizona, Arkansas, Connecticut, Delaware, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maryland, Massachusetts, Minnesota, Mississippi, Missouri, Nebraska, New Jersey, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Vermont, Virginia, West Virginia, Wisconsin). Known host. Neodiprion sertifer Geoff. ( Diprionidae ). Comments. Hemipenthes sinuosa is only known from Morelos in the center of Mexico and Coahuila in the northeast, but can be found almost in all of the USA. It is clearly undersampled in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Lepidanthrax Osten Sacken Remarks. Forty seven of the 52 species of Lepidanthrax are from the Nearctic region. Hall (1976) published a revision of this genus including keys for species. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax arizonensis Hall, 1976 Figure 13a Material examined. EEO: Mar (1 F); Oct (2 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona). Comments. Lepidanthrax arizonensis has a restricted distribution, being present only in Arizona and Coahuila, but probably is also present in Chihuahua, Texas and New Mexico. Figure 13. Lepidanthrax . a Lepidanthrax arizonensis , female (CNIN 1352) dorsal view b Lepidanthrax disiunctus female (CNIN 334) dorsal view c Lepidanthrax hesperus , male (CNIN 1339) dorsal view d Lepidanthrax hyposcelus , male (CNIN 369) dorsal view e Lepidanthrax proboscideus , male (CNIN 357) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax disiunctus (Wiedemann, 1830) Figure 13b Material examined. REC: Aug (2 F, 1 M). Known Nearctic records. Mexico (Coahuila, Distrito Federal, Guerrero, Veracruz); USA (Arizona). Comments. The distribution of Lepidanthrax disiunctus has its northern extreme in Arizona and its southern extreme in Oaxaca, in the southeast of Mexico. It seems this species is rarely collected, but widely distributed. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hesperus Hall, 1976 Figure 13c Material examined. EAM: Apr (2 M); CHU: Apr (2 F, 5 M); ROR: Apr (1 F, 3 M), May (1 F, 1 M); RPA: Apr (1 F, 14 M). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, New Mexico, Texas). Comments. This record is the first in northeastern Mexico. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hyposcelus Hall, 1976 Figure 13d Material examined. RLC: Sep (4 F, 15 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos, Puebla). Comments. Lepidanthrax hyposcelus is endemic to Mexico, previously only known from the southwest of the country; this record extends its distribution to the northeast of the country. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax proboscideus (Loew, 1869) Figure 13e Material examined. ECA: Sep (1 F, 2 M); EEO: Apr (1 F), Aug (1 F, 1 M), Oct (4 F, 15 M); ROR: Sep (2 M); RPA: Sep (2 M), Oct (1 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila, Durango, Guerrero, Morelos, Sonora); USA (Arizona, California, Nevada, New Mexico, Utah). Comments. Lepidanthrax proboscideus , Lepidanthrax fuscipennis Hall, 1976 and Lepidanthrax disiunctus are the only species of this genus distributed in both the Nearctic and Neotropical regions. Of these Lepidanthrax proboscideus extends as far as El Salvador, the most southern distribution for a Nearctic species of this genus. This is the first record of this species in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Neodiplocampta Curran Remarks. Neodiplocampta is a small American genus, more diverse in the Neotropical than the Nearctic region. Hull and Martin (1974) described seven of the 16 species and published a key for all species of the genus. Taxon classification Animalia Diptera Bombyliidae Neodiplocampta ( Neodiplocampta ) miranda Hull & Martin, 1974 Figure 14 Material examined. CHU: Aug (1 F); EEO: Aug (1 F, 1 M); ROR: Jul (1 F); Oct (1 M); RPA: Aug (1 F, 2 M). Known Nearctic records. Mexico (Coahuila, Guerrero, San Luis Potosi, Sinaloa, Sonora); USA (Arizona, California, Florida, Texas). Comments. Neodiplocampta (Neodiplocampta) miranda and Neodiplocampta (Agitonia) sepia Hull, 1966 are the only two species distributed in both biogeographic regions (Nearctic and Neotropical). This species is distributed from the south of the USA to Nicaragua, but has not been collected in most Mexican states. This lack of records is possibly due its low abundance. Figure 14. Neodiplocampta (Neodiplocampta) miranda , female (CNIN 225) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Paravilla Painter Remarks. Fifty five of the 58 species of the genus are Nearctic. All species of Paravilla collected in Coahuila were exclusively collected in the summer months from April to July. Hall (1981a) reviewed this genus and presented a key for species and description of new species. Taxon classification Animalia Diptera Bombyliidae Paravilla edititoides (Painter, 1933) Figure 15a Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Jul (2 F, 1 M); ECA: Apr (1 F, 1 M), Jul (1 M); EEO: Apr (1 F, 10 M), May (1 F, 2 M), Jul (9 M); REE: Apr (1 M); RLC: Jun (2 F, 5 M), Jul (1 F); ROR: Jul (1 F, 3 M); RPA: Oct (1 M). Known Nearctic records. Canada (Saskatchewan); Mexico (Chihuahua, Coahuila, Durango, Jalisco, México, Zacatecas); USA (Arizona, Colorado, Idaho, Kansas, Montana, Nebraska, New Mexico, Oklahoma, Utah, Texas, Wyoming). Comments. This species is very abundant and present in most of North America, from Canada as far as Jalisco in central Mexico. Figure 15. Paravilla . a Paravilla edititoides , male (CNIN 1272) dorsal view b Paravilla flavipilosa , male (CNIN 1125) dorsal view c Paravilla parvula , female (CNIN 884) dorsal view d Paravilla separata , female (CNIN 898) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Paravilla flavipilosa (Cole, 1923) Figure 15b Material examined. CHU: Apr (1 M); Jul (1 M); ECA: Apr (1 M); EEO: Apr (7 M), May (11 M); ROR: Apr (2 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Nuevo León); USA (Arizona, California, Colorado, Texas). Comments. Paravilla flavipilosa is abundant and restricted to the south of the USA and north of Mexico. Taxon classification Animalia Diptera Bombyliidae Paravilla parvula Hall, 1981a Figure 15c Material examined. EAM: Apr (1 F); CHU: Apr (1 M); RPA: Apr (7 F, 13 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Durango, Guanajuato, Hidalgo, Jalisco, México, Michoacán, Nuevo León, San Luis Potosí, Sonora, Zacatecas), USA (Arizona, New Mexico, Texas, Utah). Comments. Paravilla parvula is relatively well collected in northern and central Mexico. Its distribution also includes the south of the USA but no farther north than Utah. Taxon classification Animalia Diptera Bombyliidae Paravilla separata (Walker, 1852) Figure 15d Material examined. CHU: Apr (1 F); EEO: Apr (3 F); REE: Apr (5 F, 3 M); RPA: Apr (1 M). Known Nearctic records. Canada (Ontario, Manitoba); Mexico (Coahuila); USA (Alabama, Florida, Georgia, Iowa, Kansas, Michigan, Minnesota, Mississippi, Nebraska, Ohio, South Dakota, Wisconsin). Comments. Paravilla separata is present mainly in the eastern half of the USA, and southeastern Canada. This record in Coahuila represents the southern extreme of the distribution of this species, and is the first in Mexico. It may also be present in Tamaulipas and Nuevo León but doubtfully in the northwest of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Poecilanthrax Osten Sacken Remarks. Four species from this mainly Nearctic genus are recorded in Coahuila for the first time. Painter and Hall (1960) published a review of Poecilanthrax with a key and images of the species. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax effrenus (Coquillett, 1887) Figure 16a Material examined. EAM: Apr (1 F, 1 M), Jun (1 F, 1 M), Sep (1 F); CHU: Jun (1 F); ROR: May (10 F, 5 M), Jul (2 F, 3 M), Aug (1 M); RPA: Jun (4 F, 1 M), Jul (6 F, 6 M), Aug (1 F, 1 M), Sep (4 F), Oct (2 F, 1 M). Known Nearctic records. Mexico (Baja California Sur, Chihuahua, Coahuila, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oklahoma, Texas). Comments. This record fills a gap in Poecilanthrax effrenus distribution between northwest and northeast populations of Mexico. This species is probably present in Baja California and Nuevo León, but has not yet been recorded. Figure 16. Poecilanthrax . a Poecilanthrax effrenus , female (CNIN 1380) dorsal view b Poecilanthrax fasciatus , male (CNIN 218) dorsal view c Poecilanthrax hyalinipennis , female (CNIN 1365) dorsal view d Poecilanthrax poecilogaster , male (CNIN 1356) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax fasciatus Johnson & Johnson, 1957 Figure 16b Material examined. EAM: Sep (1 M); CHU: Oct (1 M); ROR: Oct (1 M); RPA: Oct (1 M). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Texas). Known host. Chorizagrotis auxiliaris Grote ( Noctuidae ). Comments. Poecilanthrax fasciatus is collected in Mexico for the first time, and this extends the southern limit of this species distribution. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax hyalinipennis Painter & Hall, 1960 Figure 16c Material examined. EAM: Mar (3 M), Oct (1 F); CHU: Oct (1 M); ROR: Oct (4 M); RPA: Sep (1 F), Oct (2 F, 6 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Utah). Comments. This record extends the distribution of Poecilanthrax hyalinipennis into the northwest of Mexico. Considering its distribution in the USA, this species may also be present in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax poecilogaster (Osten Sacken, 1886b) Figure 16d Material examined. REE: Apr (2 M). Known Nearctic records. Canada (Alberta, Manitoba, Ontario, Saskatchewan); Mexico (Coahuila, Morelos, Nuevo León, Sonora); USA (Arizona, California, Colorado, Idaho, Nevada, New Mexico, Oregon, Utah). Comments. Most of the records in the USA and Mexico of this rarely collected but widespread species are from Pacific Coast states, although, there are records from Nuevo Leon and Coahuila in northeast Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Rhynchanthrax Painter Remarks. Of the seven species of this exclusively Nearctic genus, six are present in Mexico, with Rhynchanthrax maria (Williston, 1901) and Rhynchanthrax nigrofimbriatus (Williston, 1901) being endemic to this country. Only Rhynchanthrax parvicornis (Loew, 1869) has not been collected in Mexico, but it is distributed across the southern USA and may also occur in the north of Mexico. Taxon classification Animalia Diptera Bombyliidae Rhynchanthrax capreus (Coquillett, 1887) Figure 17a Material examined. CHU: Apr (1 M), Aug (13 F, 6 M), Jul (12 F, 6 M), Sep (1 F, 3 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Colorado, Nebraska, Nevada, New Mexico, Utah). Comments. This is the first record of this species in Mexico. Rhynchanthrax capreus is the only species occurring in the northwest of the USA, while the other species in the genus are present mainly in the south and east of the country. This species may also be present in the northwest of Mexico (Baja California, Sonora, Chihuahua). Figure 17. Rhynchanthrax . a Rhynchanthrax capreus , female (CNIN 940) dorsal view b Rhynachantrax texanus , male (CNIN 263) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Rhynachantrax texanus (Painter, 1933) Figure 17b Material examined. EEO: May (1 M); RLC: Jun (1 F, 11 M), Jul (3 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Kansas, New Mexico, Texas). Comments. This is the most eastern record in Mexico for this species. In the USA it is distributed in the southern-center of the country, but in Mexico it has been collected in Sonora so it probably also occurs in Arizona. Taxon classification Animalia Diptera Bombyliidae Genus Thyridanthrax Osten Sacken Remarks. Thyridanthrax has twice as many species in the Palearctic region as in the Nearctic and Neotropical regions combined. All 12 species in North America are present in the USA with five also in Mexico. These are the first records of this genus in Coahuila. The distribution of Thyridanthrax selene (Osten Sacken, 1886b) and Thyridanthrax pallidus (Coquillett, 1887) are very similar, being present in all of the southern USA and probably also in all of northern Mexico, although they have been only collected in Sonora and Coahuila to date. Both species are rare and were collected only in April. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax pallidus (Coquillett, 1887) Figure 18b Material examined. REE: Apr (4 F, 1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Texas, Utah). Comments. This represents the first record of this species in Mexico. Figure 18. Thyridanthrax . a Thyridanthrax selene , male (CNIN 182) dorsal view b Thyridanthrax pallidus , female (CNIN 1162) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax selene (Osten Sacken, 1886b) Figure 18a Material examined. EAM: Apr (1 M); REE: Apr (2 F, 2 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Texas). Comments. This is the most eastern record in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Xenox Evenhuis Remarks. Of the five species that constitute this genus, four are present in Mexico. Taxon classification Animalia Diptera Bombyliidae Xenox xylocopae (Marston, 1970) Figure 19 Material examined. ECA: Sep (1 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Sonora), USA (Arizona, New Mexico, Texas). Known host. Xylocopa micheneri micheneri (Hurd) ( Apidae ) as reported by Minckley (1989) . Comments. Xenox xylocopae appears to be restricted to the northeast of Mexico and south of the USA. Three of the other species also have restricted and separate distributions: Xenox delila Loew, 1869 is present in the northwest of Mexico and California; Xenox nigritus (Schaeffer, 1768) occurs from the northeast of Mexico (Veracruz and Tamaulipas without overlap with Xenox xylocopae ) to South America; and Xenox tigrinus (De Geer, 1776) is present in the eastern USA and southern Ontario. Only Xenox habrosus (Marston, 1970) has a distribution overlapping with the other four species, being present in all of Mexico and the southwest of the USA. Figure 19. Xenox xylocopae , male (CNIN 1165) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Anthrax Scopoli Remarks. This is a diverse genus with 248 species worldwide. Two old but complete revisions of the genus, including distribution maps and keys, were made by Marston (1963 , 1970 ). Thanks to these Anthrax species can be easily identified. Some Anthrax species are widely distributed occupying two biogeographic regions. From the seven Anthrax species collected in this study in Coahuila, just Anthrax cybele (Coquillett, 1894) has a restricted distribution. The other six species are widespread across the Nearctic region. Two species of Anthrax are reported for the first time for Mexico. Remarks. This is a diverse genus with 248 species worldwide. Two old but complete revisions of the genus, including distribution maps and keys, were made by Marston (1963 , 1970 ). Thanks to these Anthrax species can be easily identified. Some Anthrax species are widely distributed occupying two biogeographic regions. From the seven Anthrax species collected in this study in Coahuila, just Anthrax cybele (Coquillett, 1894) has a restricted distribution. The other six species are widespread across the Nearctic region. Two species of Anthrax are reported for the first time for Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax atriplex Marston, 1970 Figure 9a Material examined. EAM Apr (1 F); ROR: Oct (2 M); RPA: Aug (1 M); Sep (1 M); Oct (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Durango, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oregon, Texas, Utah). Known host. Megachile gentilis Cresson ( Megachilidae ). Comments. This species may be present in all the north of Mexico, including Chihuahua, Nuevo León and possibly Sinaloa. Figure 9. Anthrax part I. a Anthrax atriplex , male (CNIN 1098) dorsal view b Anthrax cybele , male (CNIN 1087) dorsal view c Anthrax georgicus , female (CNIN 1071) dorsal view d Anthrax irroratus , male (CNIN 1027) dorsal view. All scale bars: 3 mm. Material examined. EAM Apr (1 F); ROR: Oct (2 M); RPA: Aug (1 M); Sep (1 M); Oct (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Durango, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oregon, Texas, Utah). Known host. Megachile gentilis Cresson ( Megachilidae ). Comments. This species may be present in all the north of Mexico, including Chihuahua, Nuevo León and possibly Sinaloa. Figure 9. Anthrax part I. a Anthrax atriplex , male (CNIN 1098) dorsal view b Anthrax cybele , male (CNIN 1087) dorsal view c Anthrax georgicus , female (CNIN 1071) dorsal view d Anthrax irroratus , male (CNIN 1027) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax cybele (Coquillett, 1894) Figure 9b Material examined. ECA: Apr (2 F); EEO: Apr (1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California). Comments. This is a rare species flying in April. Its distribution is disjunct so far, present in the southwest of the USA and northeast of Mexico. It is probably also found in New Mexico and Texas in the USA and Sonora and Chihuahua in Mexico. Material examined. ECA: Apr (2 F); EEO: Apr (1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California). Comments. This is a rare species flying in April. Its distribution is disjunct so far, present in the southwest of the USA and northeast of Mexico. It is probably also found in New Mexico and Texas in the USA and Sonora and Chihuahua in Mexico. Taxon classification Animalia Diptera Bombyliidae Anthrax georgicus Macquart, 1834 Figure 9c Material examined. EAM: Mar (1 F), Apr (1 M), Jun (1 F, 2 M), Jul (2 F), Sep (2 M); ROR: Apr (1 F), Sep (1 M); RPA: Mar (1 M), Apr (1 F, 1 M), Jul (2 F, 1 M), Sep (6 F, 3 M), Oct (6 F, 3 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Ontario, Quebec, Saskatchewan); Mexico (Coahuila, Guerrero, Michoacán de Ocampo, Morelos, Nuevo León, Puebla, Sonora, Veracruz); USA (Arizona, Arkansas, California, Colorado, Connecticut, Delaware, District of Columbia, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Tennessee, Texas, Utah, Vermont, Virginia, Washington, West Virginia, Wisconsin, Wyoming). Comments. The range of Anthrax georgicus includes all North America and Central America (Nicaragua, Costa Rica) covering a wide diversity of habitats and environmental conditions. Common in the rainy season and present in the dry season (March), this species is probably present in most if not all states of Mexico, but has only been collected in eight of them. Material examined. EAM: Mar (1 F), Apr (1 M), Jun (1 F, 2 M), Jul (2 F), Sep (2 M); ROR: Apr (1 F), Sep (1 M); RPA: Mar (1 M), Apr (1 F, 1 M), Jul (2 F, 1 M), Sep (6 F, 3 M), Oct (6 F, 3 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Ontario, Quebec, Saskatchewan); Mexico (Coahuila, Guerrero, Michoacán de Ocampo, Morelos, Nuevo León, Puebla, Sonora, Veracruz); USA (Arizona, Arkansas, California, Colorado, Connecticut, Delaware, District of Columbia, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Tennessee, Texas, Utah, Vermont, Virginia, Washington, West Virginia, Wisconsin, Wyoming). Comments. The range of Anthrax georgicus includes all North America and Central America (Nicaragua, Costa Rica) covering a wide diversity of habitats and environmental conditions. Common in the rainy season and present in the dry season (March), this species is probably present in most if not all states of Mexico, but has only been collected in eight of them. Taxon classification Animalia Diptera Bombyliidae Anthrax irroratus Say, 1823 Figure 9d Material examined. EAM: Apr (1 M), Aug (2 M), Oct (1 M); ECA: Apr (1 F), May (1 F, 1 M); EEO: Apr (2 F), Jul (4 F), Aug (1 M); REC: Apr (3 F, 10 M), Aug (1 M); REE: Aug (1 M); RLC: Jul (6 F, 10 M); ROR: Feb (1 M), Aug (5 M), Sep (1 M); RPA: Apr (1 M), Aug (2 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Nova Scotia, Ontario, Quebec, Saskatchewan); Mexico (Baja California, Baja California Sur, Coahuila, Colima, Guerrero, Michoacán, Morelos, Nayarit, Puebla, San Luis Potosí, Sinaloa, Sonora, Veracruz, Zacatecas); USA (Alaska, Arizona, Arkansas, California, Colorado, Connecticut, Idaho, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, Oregon, Pennsylvania, Tennessee, Texas, Utah, Virginia, West Virginia, Wyoming). Known hosts. Megachile gentilis Cresson ( Megachilidae ); Megachile mendica Cresson ( Megachilidae ); Dianthidium heterulkei fraternum Timberlake ( Megachilidae ); Aschmendiella bucconis denticulata Cresson ( Megachilidae ); Hylaeus asininus Cockrell and Casad ( Colletidae ). Scott and Strickler (1992) also reared Anthrax irroratus from Megachile relativa Cresson ( Megachilidae ) and Megachile inermis Provancher ( Megachilidae ). Comments. Anthrax irroratus , like Anthrax georgicus (above), is present in all of North America and reaches Central America and Caribbean islands (Honduras, Puerto Rico). More abundant than its congener, this species has been collected in 15 states in Mexico (including Oaxaca of the Neotropical region not listed above) and all regions of the USA. Anthrax irroratus should be collected in any systematic, long term Bombyliidae sample in Mexico and the USA. Material examined. EAM: Apr (1 M), Aug (2 M), Oct (1 M); ECA: Apr (1 F), May (1 F, 1 M); EEO: Apr (2 F), Jul (4 F), Aug (1 M); REC: Apr (3 F, 10 M), Aug (1 M); REE: Aug (1 M); RLC: Jul (6 F, 10 M); ROR: Feb (1 M), Aug (5 M), Sep (1 M); RPA: Apr (1 M), Aug (2 M). Known Nearctic records. Canada (Alberta, British Columbia, Manitoba, Northwest Territory, Nova Scotia, Ontario, Quebec, Saskatchewan); Mexico (Baja California, Baja California Sur, Coahuila, Colima, Guerrero, Michoacán, Morelos, Nayarit, Puebla, San Luis Potosí, Sinaloa, Sonora, Veracruz, Zacatecas); USA (Alaska, Arizona, Arkansas, California, Colorado, Connecticut, Idaho, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Missouri, Montana, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, New York, Oregon, Pennsylvania, Tennessee, Texas, Utah, Virginia, West Virginia, Wyoming). Known hosts. Megachile gentilis Cresson ( Megachilidae ); Megachile mendica Cresson ( Megachilidae ); Dianthidium heterulkei fraternum Timberlake ( Megachilidae ); Aschmendiella bucconis denticulata Cresson ( Megachilidae ); Hylaeus asininus Cockrell and Casad ( Colletidae ). Scott and Strickler (1992) also reared Anthrax irroratus from Megachile relativa Cresson ( Megachilidae ) and Megachile inermis Provancher ( Megachilidae ). Comments. Anthrax irroratus , like Anthrax georgicus (above), is present in all of North America and reaches Central America and Caribbean islands (Honduras, Puerto Rico). More abundant than its congener, this species has been collected in 15 states in Mexico (including Oaxaca of the Neotropical region not listed above) and all regions of the USA. Anthrax irroratus should be collected in any systematic, long term Bombyliidae sample in Mexico and the USA. Taxon classification Animalia Diptera Bombyliidae Anthrax oedipus Fabricius, 1805 Figure 10a Material examined. ECA: Apr (1 F, 1 M), Jul (1 F); EEO: Apr (2 F, 1 M), May (1 F, 4 M), Jul (1 M); REC: Apr (1 F); REE: Apr (2 M); RLC: Jul (2 F, 4 M), Sep (1 F); RPA: Apr (1 M), Aug (1 M). Known Nearctic records. Mexico (Baja California, Coahuila, Nayarit, Morelos, Sinaloa, Sonora); USA (Nevada, Texas). Comments. Apparently closely related to Anthrax irroratus , Anthrax oedipus has a narrow distribution in the Nearctic region but is widely distributed in all South America. In the USA it has been collected only in two southern states, while it occurs in most of the northern states of Mexico and one central state (Morelos); it may be present in most areas from Texas to Argentina. Figure 10. Anthrax part II. a Anthrax oedipus , female (CNIN 1055) dorsal view b Anthrax pauper , female (CNIN 1085) dorsal view c Anthrax seriepunctatus , female (CNIN 1089) dorsal view. All scale bars: 3 mm. Material examined. ECA: Apr (1 F, 1 M), Jul (1 F); EEO: Apr (2 F, 1 M), May (1 F, 4 M), Jul (1 M); REC: Apr (1 F); REE: Apr (2 M); RLC: Jul (2 F, 4 M), Sep (1 F); RPA: Apr (1 M), Aug (1 M). Known Nearctic records. Mexico (Baja California, Coahuila, Nayarit, Morelos, Sinaloa, Sonora); USA (Nevada, Texas). Comments. Apparently closely related to Anthrax irroratus , Anthrax oedipus has a narrow distribution in the Nearctic region but is widely distributed in all South America. In the USA it has been collected only in two southern states, while it occurs in most of the northern states of Mexico and one central state (Morelos); it may be present in most areas from Texas to Argentina. Figure 10. Anthrax part II. a Anthrax oedipus , female (CNIN 1055) dorsal view b Anthrax pauper , female (CNIN 1085) dorsal view c Anthrax seriepunctatus , female (CNIN 1089) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Anthrax pauper (Loew, 1869) Figure 10b Material examined. CHU: Apr (1 F, 1 M). Known Nearctic records. Canada (Ontario); Mexico (Coahuila); USA (Alabama, Colorado, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Nebraska, New Jersey, New Mexico, New York, Oklahoma, Pennsylvania, Texas, Utah, Vermont, Virginia, Wisconsin). Comments. With just two specimens collected, Anthrax pauper appears to be a rare species in this region. This population is the most southern recorded of this species, mostly present in the center and east of the USA. Presumably adapted to colder climates, it is no coincidence that it was collected in the most elevated site sampled. Material examined. CHU: Apr (1 F, 1 M). Known Nearctic records. Canada (Ontario); Mexico (Coahuila); USA (Alabama, Colorado, Illinois, Indiana, Kansas, Maryland, Massachusetts, Michigan, Nebraska, New Jersey, New Mexico, New York, Oklahoma, Pennsylvania, Texas, Utah, Vermont, Virginia, Wisconsin). Comments. With just two specimens collected, Anthrax pauper appears to be a rare species in this region. This population is the most southern recorded of this species, mostly present in the center and east of the USA. Presumably adapted to colder climates, it is no coincidence that it was collected in the most elevated site sampled. Taxon classification Animalia Diptera Bombyliidae Anthrax seriepunctatus (Osten Sacken, 1886b) Figure 10c Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Aug (1 F), Sep (1 F); ECA: Jun (1 M); REE: Aug (1 F); RLC: Jun (1 F), Jul (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora, Puebla); USA (Arizona, Nevada, New Mexico, Texas). Comments. This species is recorded mostly from the south of the USA and north of Mexico, but its presence in Puebla in central Mexico suggests a wider distribution within the country, at least in all northern states. Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Aug (1 F), Sep (1 F); ECA: Jun (1 M); REE: Aug (1 F); RLC: Jun (1 F), Jul (1 F, 2 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Sonora, Puebla); USA (Arizona, Nevada, New Mexico, Texas). Comments. This species is recorded mostly from the south of the USA and north of Mexico, but its presence in Puebla in central Mexico suggests a wider distribution within the country, at least in all northern states. Taxon classification Animalia Diptera Bombyliidae Genus Dipalta Osten Sacken Remarks. Dipalta is a small genus with just two species. Dipalta banksi Johnson, 1921 is only present in eastern Canada and USA, while Dipalta serpentina is distributed from Central America to the northern USA. Remarks. Dipalta is a small genus with just two species. Dipalta banksi Johnson, 1921 is only present in eastern Canada and USA, while Dipalta serpentina is distributed from Central America to the northern USA. Taxon classification Animalia Diptera Bombyliidae Dipalta serpentina (Osten Sacken, 1877) Figure 11 Material examined. REC: Aug (1 M); RLC: Jul (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Hidalgo, México, Morelos, Puebla, San Luis Potosí, Sinaloa); USA (Arizona, Arkansas, California, Colorado, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kansas, Kentucky, Maine, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Mexico, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Utah, Washington, Wisconsin, Wyoming). Known host. Myrmeleon immaculatus De Geer ( Myrmeleontidae ). Comments. This species is probably present in all of Mexico, but this is the only record in the north of Mexico. Figure 11. Dipalta serpentina , male (CNIN 215) dorsal view. Scale bar: 3 mm. Material examined. REC: Aug (1 M); RLC: Jul (1 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Hidalgo, México, Morelos, Puebla, San Luis Potosí, Sinaloa); USA (Arizona, Arkansas, California, Colorado, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kansas, Kentucky, Maine, Maryland, Massachusetts, Michigan, Minnesota, Missouri, Montana, Nebraska, Nevada, New Mexico, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Utah, Washington, Wisconsin, Wyoming). Known host. Myrmeleon immaculatus De Geer ( Myrmeleontidae ). Comments. This species is probably present in all of Mexico, but this is the only record in the north of Mexico. Figure 11. Dipalta serpentina , male (CNIN 215) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Hemipenthes Loew Remarks. Hemipenthes is equally diverse in the Nearctic (29 species), Neotropical (26 species) and Palearctic (37 species) regions, with just six species in the Oriental region and one in the Afrotropical region. Four species of this genus were collected in Coahuila. All of these have broad distributions but apparently from poor sampling because records are not continuous, especially in Mexico. Ávalos-Hernández (2009) recently published a revision of Hemipenthes , with a key for Nearctic species. Remarks. Hemipenthes is equally diverse in the Nearctic (29 species), Neotropical (26 species) and Palearctic (37 species) regions, with just six species in the Oriental region and one in the Afrotropical region. Four species of this genus were collected in Coahuila. All of these have broad distributions but apparently from poor sampling because records are not continuous, especially in Mexico. Ávalos-Hernández (2009) recently published a revision of Hemipenthes , with a key for Nearctic species. Taxon classification Animalia Diptera Bombyliidae Hemipenthes jaennickeana (Osten Sacken, 1886a) Figure 12a Material examined. REC: Apr (18 F), Aug (4 F); REE: Feb (3 F); RLC: Mar (7 F), Jul (23 F, 3 M), Sep (3 F). Known Nearctic records. Mexico (Coahuila, Morelos, Sonora); USA (Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, Oregon, Texas, Utah). Comments. Present mainly in the Pacific coast states of the USA and Mexico, from Oregon as far as Morelos in the center of Mexico. This record is the most eastern record in Mexico. Figure 12. Hemipenthes . a Hemipenthes jaennickeana , female (CNIN 1137) dorsal view b Hemipenthes lepidota , female (CNIN 200) dorsal view c Hemipenthes scylla , male (CNIN 725) dorsal view d Hemipenthes sinuosa , female (CNIN 1134) dorsal view. All scale bars: 3 mm. Material examined. REC: Apr (18 F), Aug (4 F); REE: Feb (3 F); RLC: Mar (7 F), Jul (23 F, 3 M), Sep (3 F). Known Nearctic records. Mexico (Coahuila, Morelos, Sonora); USA (Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, Oregon, Texas, Utah). Comments. Present mainly in the Pacific coast states of the USA and Mexico, from Oregon as far as Morelos in the center of Mexico. This record is the most eastern record in Mexico. Figure 12. Hemipenthes . a Hemipenthes jaennickeana , female (CNIN 1137) dorsal view b Hemipenthes lepidota , female (CNIN 200) dorsal view c Hemipenthes scylla , male (CNIN 725) dorsal view d Hemipenthes sinuosa , female (CNIN 1134) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Hemipenthes lepidota (Osten Sacken, 1886b) Figure 12b Material examined. EAM: Apr (1 M), Aug (1 F); CHU: Apr (1 F, 3 M), Aug (1 M); EEO: Jul (1 F, 4 M), Aug (1 F); REC: (1 M); REE: Apr (11 F, 2 M), Aug (1 F); RLC: Jun (1 F), Jul (3 F); RPA: Apr (1 F), Sep (4 F), Oct (2 F). Known Nearctic records. Canada (Alberta); Mexico (Baja California, Baja California Sur, Coahuila, Chihuahua, Guerrero, Morelos, Puebla, San Luis Potosí, Sonora, Tamaulipas); USA (Arizona, California, Colorado, Idaho, Louisiana, Nevada). Comments. This species is abundant in the rainy season in most of the Nearctic region but has not been collected in many states of Mexico or the USA where it probably is present. Material examined. EAM: Apr (1 M), Aug (1 F); CHU: Apr (1 F, 3 M), Aug (1 M); EEO: Jul (1 F, 4 M), Aug (1 F); REC: (1 M); REE: Apr (11 F, 2 M), Aug (1 F); RLC: Jun (1 F), Jul (3 F); RPA: Apr (1 F), Sep (4 F), Oct (2 F). Known Nearctic records. Canada (Alberta); Mexico (Baja California, Baja California Sur, Coahuila, Chihuahua, Guerrero, Morelos, Puebla, San Luis Potosí, Sonora, Tamaulipas); USA (Arizona, California, Colorado, Idaho, Louisiana, Nevada). Comments. This species is abundant in the rainy season in most of the Nearctic region but has not been collected in many states of Mexico or the USA where it probably is present. Taxon classification Animalia Diptera Bombyliidae Hemipenthes scylla (Osten Sacken, 1887) Figure 12c Material examined. REC: Apr (23 M), Aug (7 M); REE: Feb (5 M), Apr (2 M); RLC: Mar (8 M), Jul (8 M), Sep (9 M). Known Nearctic records. Mexico (Coahuila, Morelos, Guanajuato, Sonora); USA (Arizona, Texas). Comments. Males of this species are abundant all year long but females are unknown. There is no explanation for this lack of females in the collections. Extreme sexual dimorphism and misidentification of females can be dismiss, since there is no Hemipenthes species from which only females are known. One possible explanation is that females life span is too short and therefor encounter probabilities are low. Distribution is discontinuous with populations present in central and northern Mexico and the southern USA; it is unknown whether this species is present in between these areas. Material examined. REC: Apr (23 M), Aug (7 M); REE: Feb (5 M), Apr (2 M); RLC: Mar (8 M), Jul (8 M), Sep (9 M). Known Nearctic records. Mexico (Coahuila, Morelos, Guanajuato, Sonora); USA (Arizona, Texas). Comments. Males of this species are abundant all year long but females are unknown. There is no explanation for this lack of females in the collections. Extreme sexual dimorphism and misidentification of females can be dismiss, since there is no Hemipenthes species from which only females are known. One possible explanation is that females life span is too short and therefor encounter probabilities are low. Distribution is discontinuous with populations present in central and northern Mexico and the southern USA; it is unknown whether this species is present in between these areas. Taxon classification Animalia Diptera Bombyliidae Hemipenthes sinuosa (Wiedemann, 1821) Figure 12d Material examined. REC: Apr (3 F); REE: (Feb (1 F), Apr (2 F); RLC: Jul (1 F, 1 M), Sep (1 M); RPA: Sep (2 F). Known Nearctic records. Mexico (Coahuila, Morelos); USA (Alabama, Arizona, Arkansas, Connecticut, Delaware, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maryland, Massachusetts, Minnesota, Mississippi, Missouri, Nebraska, New Jersey, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Vermont, Virginia, West Virginia, Wisconsin). Known host. Neodiprion sertifer Geoff. ( Diprionidae ). Comments. Hemipenthes sinuosa is only known from Morelos in the center of Mexico and Coahuila in the northeast, but can be found almost in all of the USA. It is clearly undersampled in Mexico. Material examined. REC: Apr (3 F); REE: (Feb (1 F), Apr (2 F); RLC: Jul (1 F, 1 M), Sep (1 M); RPA: Sep (2 F). Known Nearctic records. Mexico (Coahuila, Morelos); USA (Alabama, Arizona, Arkansas, Connecticut, Delaware, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maryland, Massachusetts, Minnesota, Mississippi, Missouri, Nebraska, New Jersey, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Vermont, Virginia, West Virginia, Wisconsin). Known host. Neodiprion sertifer Geoff. ( Diprionidae ). Comments. Hemipenthes sinuosa is only known from Morelos in the center of Mexico and Coahuila in the northeast, but can be found almost in all of the USA. It is clearly undersampled in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Lepidanthrax Osten Sacken Remarks. Forty seven of the 52 species of Lepidanthrax are from the Nearctic region. Hall (1976) published a revision of this genus including keys for species. Remarks. Forty seven of the 52 species of Lepidanthrax are from the Nearctic region. Hall (1976) published a revision of this genus including keys for species. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax arizonensis Hall, 1976 Figure 13a Material examined. EEO: Mar (1 F); Oct (2 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona). Comments. Lepidanthrax arizonensis has a restricted distribution, being present only in Arizona and Coahuila, but probably is also present in Chihuahua, Texas and New Mexico. Figure 13. Lepidanthrax . a Lepidanthrax arizonensis , female (CNIN 1352) dorsal view b Lepidanthrax disiunctus female (CNIN 334) dorsal view c Lepidanthrax hesperus , male (CNIN 1339) dorsal view d Lepidanthrax hyposcelus , male (CNIN 369) dorsal view e Lepidanthrax proboscideus , male (CNIN 357) dorsal view. All scale bars: 3 mm. Material examined. EEO: Mar (1 F); Oct (2 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona). Comments. Lepidanthrax arizonensis has a restricted distribution, being present only in Arizona and Coahuila, but probably is also present in Chihuahua, Texas and New Mexico. Figure 13. Lepidanthrax . a Lepidanthrax arizonensis , female (CNIN 1352) dorsal view b Lepidanthrax disiunctus female (CNIN 334) dorsal view c Lepidanthrax hesperus , male (CNIN 1339) dorsal view d Lepidanthrax hyposcelus , male (CNIN 369) dorsal view e Lepidanthrax proboscideus , male (CNIN 357) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax disiunctus (Wiedemann, 1830) Figure 13b Material examined. REC: Aug (2 F, 1 M). Known Nearctic records. Mexico (Coahuila, Distrito Federal, Guerrero, Veracruz); USA (Arizona). Comments. The distribution of Lepidanthrax disiunctus has its northern extreme in Arizona and its southern extreme in Oaxaca, in the southeast of Mexico. It seems this species is rarely collected, but widely distributed. Material examined. REC: Aug (2 F, 1 M). Known Nearctic records. Mexico (Coahuila, Distrito Federal, Guerrero, Veracruz); USA (Arizona). Comments. The distribution of Lepidanthrax disiunctus has its northern extreme in Arizona and its southern extreme in Oaxaca, in the southeast of Mexico. It seems this species is rarely collected, but widely distributed. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hesperus Hall, 1976 Figure 13c Material examined. EAM: Apr (2 M); CHU: Apr (2 F, 5 M); ROR: Apr (1 F, 3 M), May (1 F, 1 M); RPA: Apr (1 F, 14 M). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, New Mexico, Texas). Comments. This record is the first in northeastern Mexico. Material examined. EAM: Apr (2 M); CHU: Apr (2 F, 5 M); ROR: Apr (1 F, 3 M), May (1 F, 1 M); RPA: Apr (1 F, 14 M). Known Nearctic records. Mexico (Baja California, Coahuila, Sinaloa, Sonora); USA (Arizona, California, New Mexico, Texas). Comments. This record is the first in northeastern Mexico. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax hyposcelus Hall, 1976 Figure 13d Material examined. RLC: Sep (4 F, 15 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos, Puebla). Comments. Lepidanthrax hyposcelus is endemic to Mexico, previously only known from the southwest of the country; this record extends its distribution to the northeast of the country. Material examined. RLC: Sep (4 F, 15 M). Known Nearctic records. Mexico (Coahuila, Guerrero, Morelos, Puebla). Comments. Lepidanthrax hyposcelus is endemic to Mexico, previously only known from the southwest of the country; this record extends its distribution to the northeast of the country. Taxon classification Animalia Diptera Bombyliidae Lepidanthrax proboscideus (Loew, 1869) Figure 13e Material examined. ECA: Sep (1 F, 2 M); EEO: Apr (1 F), Aug (1 F, 1 M), Oct (4 F, 15 M); ROR: Sep (2 M); RPA: Sep (2 M), Oct (1 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila, Durango, Guerrero, Morelos, Sonora); USA (Arizona, California, Nevada, New Mexico, Utah). Comments. Lepidanthrax proboscideus , Lepidanthrax fuscipennis Hall, 1976 and Lepidanthrax disiunctus are the only species of this genus distributed in both the Nearctic and Neotropical regions. Of these Lepidanthrax proboscideus extends as far as El Salvador, the most southern distribution for a Nearctic species of this genus. This is the first record of this species in the northeast of Mexico. Material examined. ECA: Sep (1 F, 2 M); EEO: Apr (1 F), Aug (1 F, 1 M), Oct (4 F, 15 M); ROR: Sep (2 M); RPA: Sep (2 M), Oct (1 M). Known Nearctic records. Mexico (Baja California, Baja California Sur, Coahuila, Durango, Guerrero, Morelos, Sonora); USA (Arizona, California, Nevada, New Mexico, Utah). Comments. Lepidanthrax proboscideus , Lepidanthrax fuscipennis Hall, 1976 and Lepidanthrax disiunctus are the only species of this genus distributed in both the Nearctic and Neotropical regions. Of these Lepidanthrax proboscideus extends as far as El Salvador, the most southern distribution for a Nearctic species of this genus. This is the first record of this species in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Neodiplocampta Curran Remarks. Neodiplocampta is a small American genus, more diverse in the Neotropical than the Nearctic region. Hull and Martin (1974) described seven of the 16 species and published a key for all species of the genus. Remarks. Neodiplocampta is a small American genus, more diverse in the Neotropical than the Nearctic region. Hull and Martin (1974) described seven of the 16 species and published a key for all species of the genus. Taxon classification Animalia Diptera Bombyliidae Neodiplocampta ( Neodiplocampta ) miranda Hull & Martin, 1974 Figure 14 Material examined. CHU: Aug (1 F); EEO: Aug (1 F, 1 M); ROR: Jul (1 F); Oct (1 M); RPA: Aug (1 F, 2 M). Known Nearctic records. Mexico (Coahuila, Guerrero, San Luis Potosi, Sinaloa, Sonora); USA (Arizona, California, Florida, Texas). Comments. Neodiplocampta (Neodiplocampta) miranda and Neodiplocampta (Agitonia) sepia Hull, 1966 are the only two species distributed in both biogeographic regions (Nearctic and Neotropical). This species is distributed from the south of the USA to Nicaragua, but has not been collected in most Mexican states. This lack of records is possibly due its low abundance. Figure 14. Neodiplocampta (Neodiplocampta) miranda , female (CNIN 225) dorsal view. Scale bar: 3 mm. Material examined. CHU: Aug (1 F); EEO: Aug (1 F, 1 M); ROR: Jul (1 F); Oct (1 M); RPA: Aug (1 F, 2 M). Known Nearctic records. Mexico (Coahuila, Guerrero, San Luis Potosi, Sinaloa, Sonora); USA (Arizona, California, Florida, Texas). Comments. Neodiplocampta (Neodiplocampta) miranda and Neodiplocampta (Agitonia) sepia Hull, 1966 are the only two species distributed in both biogeographic regions (Nearctic and Neotropical). This species is distributed from the south of the USA to Nicaragua, but has not been collected in most Mexican states. This lack of records is possibly due its low abundance. Figure 14. Neodiplocampta (Neodiplocampta) miranda , female (CNIN 225) dorsal view. Scale bar: 3 mm. Taxon classification Animalia Diptera Bombyliidae Genus Paravilla Painter Remarks. Fifty five of the 58 species of the genus are Nearctic. All species of Paravilla collected in Coahuila were exclusively collected in the summer months from April to July. Hall (1981a) reviewed this genus and presented a key for species and description of new species. Remarks. Fifty five of the 58 species of the genus are Nearctic. All species of Paravilla collected in Coahuila were exclusively collected in the summer months from April to July. Hall (1981a) reviewed this genus and presented a key for species and description of new species. Taxon classification Animalia Diptera Bombyliidae Paravilla edititoides (Painter, 1933) Figure 15a Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Jul (2 F, 1 M); ECA: Apr (1 F, 1 M), Jul (1 M); EEO: Apr (1 F, 10 M), May (1 F, 2 M), Jul (9 M); REE: Apr (1 M); RLC: Jun (2 F, 5 M), Jul (1 F); ROR: Jul (1 F, 3 M); RPA: Oct (1 M). Known Nearctic records. Canada (Saskatchewan); Mexico (Chihuahua, Coahuila, Durango, Jalisco, México, Zacatecas); USA (Arizona, Colorado, Idaho, Kansas, Montana, Nebraska, New Mexico, Oklahoma, Utah, Texas, Wyoming). Comments. This species is very abundant and present in most of North America, from Canada as far as Jalisco in central Mexico. Figure 15. Paravilla . a Paravilla edititoides , male (CNIN 1272) dorsal view b Paravilla flavipilosa , male (CNIN 1125) dorsal view c Paravilla parvula , female (CNIN 884) dorsal view d Paravilla separata , female (CNIN 898) dorsal view. All scale bars: 3 mm. Material examined. EAM: Jun (1 M); CHU: Apr (1 F), Jul (2 F, 1 M); ECA: Apr (1 F, 1 M), Jul (1 M); EEO: Apr (1 F, 10 M), May (1 F, 2 M), Jul (9 M); REE: Apr (1 M); RLC: Jun (2 F, 5 M), Jul (1 F); ROR: Jul (1 F, 3 M); RPA: Oct (1 M). Known Nearctic records. Canada (Saskatchewan); Mexico (Chihuahua, Coahuila, Durango, Jalisco, México, Zacatecas); USA (Arizona, Colorado, Idaho, Kansas, Montana, Nebraska, New Mexico, Oklahoma, Utah, Texas, Wyoming). Comments. This species is very abundant and present in most of North America, from Canada as far as Jalisco in central Mexico. Figure 15. Paravilla . a Paravilla edititoides , male (CNIN 1272) dorsal view b Paravilla flavipilosa , male (CNIN 1125) dorsal view c Paravilla parvula , female (CNIN 884) dorsal view d Paravilla separata , female (CNIN 898) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Paravilla flavipilosa (Cole, 1923) Figure 15b Material examined. CHU: Apr (1 M); Jul (1 M); ECA: Apr (1 M); EEO: Apr (7 M), May (11 M); ROR: Apr (2 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Nuevo León); USA (Arizona, California, Colorado, Texas). Comments. Paravilla flavipilosa is abundant and restricted to the south of the USA and north of Mexico. Material examined. CHU: Apr (1 M); Jul (1 M); ECA: Apr (1 M); EEO: Apr (7 M), May (11 M); ROR: Apr (2 M); RPA: Apr (1 M). Known Nearctic records. Mexico (Baja California Sur, Coahuila, Nuevo León); USA (Arizona, California, Colorado, Texas). Comments. Paravilla flavipilosa is abundant and restricted to the south of the USA and north of Mexico. Taxon classification Animalia Diptera Bombyliidae Paravilla parvula Hall, 1981a Figure 15c Material examined. EAM: Apr (1 F); CHU: Apr (1 M); RPA: Apr (7 F, 13 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Durango, Guanajuato, Hidalgo, Jalisco, México, Michoacán, Nuevo León, San Luis Potosí, Sonora, Zacatecas), USA (Arizona, New Mexico, Texas, Utah). Comments. Paravilla parvula is relatively well collected in northern and central Mexico. Its distribution also includes the south of the USA but no farther north than Utah. Material examined. EAM: Apr (1 F); CHU: Apr (1 M); RPA: Apr (7 F, 13 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Durango, Guanajuato, Hidalgo, Jalisco, México, Michoacán, Nuevo León, San Luis Potosí, Sonora, Zacatecas), USA (Arizona, New Mexico, Texas, Utah). Comments. Paravilla parvula is relatively well collected in northern and central Mexico. Its distribution also includes the south of the USA but no farther north than Utah. Taxon classification Animalia Diptera Bombyliidae Paravilla separata (Walker, 1852) Figure 15d Material examined. CHU: Apr (1 F); EEO: Apr (3 F); REE: Apr (5 F, 3 M); RPA: Apr (1 M). Known Nearctic records. Canada (Ontario, Manitoba); Mexico (Coahuila); USA (Alabama, Florida, Georgia, Iowa, Kansas, Michigan, Minnesota, Mississippi, Nebraska, Ohio, South Dakota, Wisconsin). Comments. Paravilla separata is present mainly in the eastern half of the USA, and southeastern Canada. This record in Coahuila represents the southern extreme of the distribution of this species, and is the first in Mexico. It may also be present in Tamaulipas and Nuevo León but doubtfully in the northwest of Mexico. Material examined. CHU: Apr (1 F); EEO: Apr (3 F); REE: Apr (5 F, 3 M); RPA: Apr (1 M). Known Nearctic records. Canada (Ontario, Manitoba); Mexico (Coahuila); USA (Alabama, Florida, Georgia, Iowa, Kansas, Michigan, Minnesota, Mississippi, Nebraska, Ohio, South Dakota, Wisconsin). Comments. Paravilla separata is present mainly in the eastern half of the USA, and southeastern Canada. This record in Coahuila represents the southern extreme of the distribution of this species, and is the first in Mexico. It may also be present in Tamaulipas and Nuevo León but doubtfully in the northwest of Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Poecilanthrax Osten Sacken Remarks. Four species from this mainly Nearctic genus are recorded in Coahuila for the first time. Painter and Hall (1960) published a review of Poecilanthrax with a key and images of the species. Remarks. Four species from this mainly Nearctic genus are recorded in Coahuila for the first time. Painter and Hall (1960) published a review of Poecilanthrax with a key and images of the species. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax effrenus (Coquillett, 1887) Figure 16a Material examined. EAM: Apr (1 F, 1 M), Jun (1 F, 1 M), Sep (1 F); CHU: Jun (1 F); ROR: May (10 F, 5 M), Jul (2 F, 3 M), Aug (1 M); RPA: Jun (4 F, 1 M), Jul (6 F, 6 M), Aug (1 F, 1 M), Sep (4 F), Oct (2 F, 1 M). Known Nearctic records. Mexico (Baja California Sur, Chihuahua, Coahuila, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oklahoma, Texas). Comments. This record fills a gap in Poecilanthrax effrenus distribution between northwest and northeast populations of Mexico. This species is probably present in Baja California and Nuevo León, but has not yet been recorded. Figure 16. Poecilanthrax . a Poecilanthrax effrenus , female (CNIN 1380) dorsal view b Poecilanthrax fasciatus , male (CNIN 218) dorsal view c Poecilanthrax hyalinipennis , female (CNIN 1365) dorsal view d Poecilanthrax poecilogaster , male (CNIN 1356) dorsal view. All scale bars: 3 mm. Material examined. EAM: Apr (1 F, 1 M), Jun (1 F, 1 M), Sep (1 F); CHU: Jun (1 F); ROR: May (10 F, 5 M), Jul (2 F, 3 M), Aug (1 M); RPA: Jun (4 F, 1 M), Jul (6 F, 6 M), Aug (1 F, 1 M), Sep (4 F), Oct (2 F, 1 M). Known Nearctic records. Mexico (Baja California Sur, Chihuahua, Coahuila, Sonora, Tamaulipas); USA (Arizona, California, New Mexico, Oklahoma, Texas). Comments. This record fills a gap in Poecilanthrax effrenus distribution between northwest and northeast populations of Mexico. This species is probably present in Baja California and Nuevo León, but has not yet been recorded. Figure 16. Poecilanthrax . a Poecilanthrax effrenus , female (CNIN 1380) dorsal view b Poecilanthrax fasciatus , male (CNIN 218) dorsal view c Poecilanthrax hyalinipennis , female (CNIN 1365) dorsal view d Poecilanthrax poecilogaster , male (CNIN 1356) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax fasciatus Johnson & Johnson, 1957 Figure 16b Material examined. EAM: Sep (1 M); CHU: Oct (1 M); ROR: Oct (1 M); RPA: Oct (1 M). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Texas). Known host. Chorizagrotis auxiliaris Grote ( Noctuidae ). Comments. Poecilanthrax fasciatus is collected in Mexico for the first time, and this extends the southern limit of this species distribution. Material examined. EAM: Sep (1 M); CHU: Oct (1 M); ROR: Oct (1 M); RPA: Oct (1 M). Known Nearctic records. Mexico (Coahuila); USA (Colorado, Kansas, Texas). Known host. Chorizagrotis auxiliaris Grote ( Noctuidae ). Comments. Poecilanthrax fasciatus is collected in Mexico for the first time, and this extends the southern limit of this species distribution. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax hyalinipennis Painter & Hall, 1960 Figure 16c Material examined. EAM: Mar (3 M), Oct (1 F); CHU: Oct (1 M); ROR: Oct (4 M); RPA: Sep (1 F), Oct (2 F, 6 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Utah). Comments. This record extends the distribution of Poecilanthrax hyalinipennis into the northwest of Mexico. Considering its distribution in the USA, this species may also be present in the northeast of Mexico. Material examined. EAM: Mar (3 M), Oct (1 F); CHU: Oct (1 M); ROR: Oct (4 M); RPA: Sep (1 F), Oct (2 F, 6 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Utah). Comments. This record extends the distribution of Poecilanthrax hyalinipennis into the northwest of Mexico. Considering its distribution in the USA, this species may also be present in the northeast of Mexico. Taxon classification Animalia Diptera Bombyliidae Poecilanthrax poecilogaster (Osten Sacken, 1886b) Figure 16d Material examined. REE: Apr (2 M). Known Nearctic records. Canada (Alberta, Manitoba, Ontario, Saskatchewan); Mexico (Coahuila, Morelos, Nuevo León, Sonora); USA (Arizona, California, Colorado, Idaho, Nevada, New Mexico, Oregon, Utah). Comments. Most of the records in the USA and Mexico of this rarely collected but widespread species are from Pacific Coast states, although, there are records from Nuevo Leon and Coahuila in northeast Mexico. Material examined. REE: Apr (2 M). Known Nearctic records. Canada (Alberta, Manitoba, Ontario, Saskatchewan); Mexico (Coahuila, Morelos, Nuevo León, Sonora); USA (Arizona, California, Colorado, Idaho, Nevada, New Mexico, Oregon, Utah). Comments. Most of the records in the USA and Mexico of this rarely collected but widespread species are from Pacific Coast states, although, there are records from Nuevo Leon and Coahuila in northeast Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Rhynchanthrax Painter Remarks. Of the seven species of this exclusively Nearctic genus, six are present in Mexico, with Rhynchanthrax maria (Williston, 1901) and Rhynchanthrax nigrofimbriatus (Williston, 1901) being endemic to this country. Only Rhynchanthrax parvicornis (Loew, 1869) has not been collected in Mexico, but it is distributed across the southern USA and may also occur in the north of Mexico. Remarks. Of the seven species of this exclusively Nearctic genus, six are present in Mexico, with Rhynchanthrax maria (Williston, 1901) and Rhynchanthrax nigrofimbriatus (Williston, 1901) being endemic to this country. Only Rhynchanthrax parvicornis (Loew, 1869) has not been collected in Mexico, but it is distributed across the southern USA and may also occur in the north of Mexico. Taxon classification Animalia Diptera Bombyliidae Rhynchanthrax capreus (Coquillett, 1887) Figure 17a Material examined. CHU: Apr (1 M), Aug (13 F, 6 M), Jul (12 F, 6 M), Sep (1 F, 3 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Colorado, Nebraska, Nevada, New Mexico, Utah). Comments. This is the first record of this species in Mexico. Rhynchanthrax capreus is the only species occurring in the northwest of the USA, while the other species in the genus are present mainly in the south and east of the country. This species may also be present in the northwest of Mexico (Baja California, Sonora, Chihuahua). Figure 17. Rhynchanthrax . a Rhynchanthrax capreus , female (CNIN 940) dorsal view b Rhynachantrax texanus , male (CNIN 263) dorsal view. All scale bars: 3 mm. Material examined. CHU: Apr (1 M), Aug (13 F, 6 M), Jul (12 F, 6 M), Sep (1 F, 3 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Colorado, Nebraska, Nevada, New Mexico, Utah). Comments. This is the first record of this species in Mexico. Rhynchanthrax capreus is the only species occurring in the northwest of the USA, while the other species in the genus are present mainly in the south and east of the country. This species may also be present in the northwest of Mexico (Baja California, Sonora, Chihuahua). Figure 17. Rhynchanthrax . a Rhynchanthrax capreus , female (CNIN 940) dorsal view b Rhynachantrax texanus , male (CNIN 263) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Rhynachantrax texanus (Painter, 1933) Figure 17b Material examined. EEO: May (1 M); RLC: Jun (1 F, 11 M), Jul (3 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Kansas, New Mexico, Texas). Comments. This is the most eastern record in Mexico for this species. In the USA it is distributed in the southern-center of the country, but in Mexico it has been collected in Sonora so it probably also occurs in Arizona. Material examined. EEO: May (1 M); RLC: Jun (1 F, 11 M), Jul (3 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Kansas, New Mexico, Texas). Comments. This is the most eastern record in Mexico for this species. In the USA it is distributed in the southern-center of the country, but in Mexico it has been collected in Sonora so it probably also occurs in Arizona. Taxon classification Animalia Diptera Bombyliidae Genus Thyridanthrax Osten Sacken Remarks. Thyridanthrax has twice as many species in the Palearctic region as in the Nearctic and Neotropical regions combined. All 12 species in North America are present in the USA with five also in Mexico. These are the first records of this genus in Coahuila. The distribution of Thyridanthrax selene (Osten Sacken, 1886b) and Thyridanthrax pallidus (Coquillett, 1887) are very similar, being present in all of the southern USA and probably also in all of northern Mexico, although they have been only collected in Sonora and Coahuila to date. Both species are rare and were collected only in April. Remarks. Thyridanthrax has twice as many species in the Palearctic region as in the Nearctic and Neotropical regions combined. All 12 species in North America are present in the USA with five also in Mexico. These are the first records of this genus in Coahuila. The distribution of Thyridanthrax selene (Osten Sacken, 1886b) and Thyridanthrax pallidus (Coquillett, 1887) are very similar, being present in all of the southern USA and probably also in all of northern Mexico, although they have been only collected in Sonora and Coahuila to date. Both species are rare and were collected only in April. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax pallidus (Coquillett, 1887) Figure 18b Material examined. REE: Apr (4 F, 1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Texas, Utah). Comments. This represents the first record of this species in Mexico. Figure 18. Thyridanthrax . a Thyridanthrax selene , male (CNIN 182) dorsal view b Thyridanthrax pallidus , female (CNIN 1162) dorsal view. All scale bars: 3 mm. Material examined. REE: Apr (4 F, 1 M). Known Nearctic records. Mexico (Coahuila); USA (Arizona, California, Nevada, Texas, Utah). Comments. This represents the first record of this species in Mexico. Figure 18. Thyridanthrax . a Thyridanthrax selene , male (CNIN 182) dorsal view b Thyridanthrax pallidus , female (CNIN 1162) dorsal view. All scale bars: 3 mm. Taxon classification Animalia Diptera Bombyliidae Thyridanthrax selene (Osten Sacken, 1886b) Figure 18a Material examined. EAM: Apr (1 M); REE: Apr (2 F, 2 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Texas). Comments. This is the most eastern record in Mexico. Material examined. EAM: Apr (1 M); REE: Apr (2 F, 2 M). Known Nearctic records. Mexico (Coahuila, Sonora); USA (Arizona, California, Texas). Comments. This is the most eastern record in Mexico. Taxon classification Animalia Diptera Bombyliidae Genus Xenox Evenhuis Remarks. Of the five species that constitute this genus, four are present in Mexico. Remarks. Of the five species that constitute this genus, four are present in Mexico. Taxon classification Animalia Diptera Bombyliidae Xenox xylocopae (Marston, 1970) Figure 19 Material examined. ECA: Sep (1 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Sonora), USA (Arizona, New Mexico, Texas). Known host. Xylocopa micheneri micheneri (Hurd) ( Apidae ) as reported by Minckley (1989) . Comments. Xenox xylocopae appears to be restricted to the northeast of Mexico and south of the USA. Three of the other species also have restricted and separate distributions: Xenox delila Loew, 1869 is present in the northwest of Mexico and California; Xenox nigritus (Schaeffer, 1768) occurs from the northeast of Mexico (Veracruz and Tamaulipas without overlap with Xenox xylocopae ) to South America; and Xenox tigrinus (De Geer, 1776) is present in the eastern USA and southern Ontario. Only Xenox habrosus (Marston, 1970) has a distribution overlapping with the other four species, being present in all of Mexico and the southwest of the USA. Figure 19. Xenox xylocopae , male (CNIN 1165) dorsal view. Scale bar: 3 mm. Material examined. ECA: Sep (1 M). Known Nearctic records. Mexico (Chihuahua, Coahuila, Sonora), USA (Arizona, New Mexico, Texas). Known host. Xylocopa micheneri micheneri (Hurd) ( Apidae ) as reported by Minckley (1989) . Comments. Xenox xylocopae appears to be restricted to the northeast of Mexico and south of the USA. Three of the other species also have restricted and separate distributions: Xenox delila Loew, 1869 is present in the northwest of Mexico and California; Xenox nigritus (Schaeffer, 1768) occurs from the northeast of Mexico (Veracruz and Tamaulipas without overlap with Xenox xylocopae ) to South America; and Xenox tigrinus (De Geer, 1776) is present in the eastern USA and southern Ontario. Only Xenox habrosus (Marston, 1970) has a distribution overlapping with the other four species, being present in all of Mexico and the southwest of the USA. Figure 19. Xenox xylocopae , male (CNIN 1165) dorsal view. Scale bar: 3 mm. Discussion The data presented here increase the knowledge of Bombyliidae in Mexico but also reveals the deficiencies in sampling of the family in the country. The species list for the state increased three-fold, which demonstrates the lack of knowledge of the Bombyliidae fauna in this region. Almost all states of Mexico are in a similar situation but northern states appear to have higher diversity and should be priorities for sampling. Hull (1973) identified the northwest of Mexico as a species concentration area of Bombyliidae , but the northeast portion of the country may have the same species richness. Diversity of this family in the north of Mexico is probably much higher than recorded, as indicated by the richness in the south of USA which has similar environmental characteristics but much better sampling. Therefore northeast Mexico is possibly one of the most under sampled areas in the Nearctic region for Bombyliidae , given the great diversity of this family in the area, combined with the size of this part of the country. The study of Bombyliidae in the northern states of Mexico should be more of a priority than field work in the center or the southern states. Most of the species collected in this study have a broad distribution in the USA but Mexican records are isolated. There are probably more species yet to be recorded from Coahuila and other Mexican states, especially species present in southern border states of the USA. Some species are recorded only from Coahuila in the northeast of Mexico but are also present in the northwest of the country. More studies are required to determine if these species have a disjunct distribution or if any are represented by distinct, cryptic eastern and western species. Cuatro Ciénegas' biological and conservational importance has long been recognized for reptiles ( McCoy 1984 ), birds ( Contreras-Balderas 1984 ), plants ( Pinkava 1984 , Villarreal and Encina 2005 ), snails ( Hershler 1984 ), Crustacea ( Cole 1984 ) and particularly fishes ( Minckley 1984 ), but little is known of other groups like insects. The insects contain 53% of the described species in the planet ( Chapman 2009 ), so their distribution and diversity should be considered for conservation and natural reserve design. The diversity of insects, especially of Bombyliidae and similar arid-regions-diverse groups, increases the conservational value of Cuatro Ciénegas. Conclusions The data presented here indicates the significance of Cuatro Ciénegas for Bombyliidae diversity. Comparison with other nearby areas should be undertaken to confirm whether this area really is richer for this family. Data also reveal that true species richness of Bombyliidae is much higher than previously recorded. This could also be true for other insect groups. More funding should be destined for faunistic studies of megadiverse groups with ecological importance such as Diptera , Coleoptera , Hymenoptera and Lepidoptera . The information obtained from these studies might be used first to quantify the species richness and species exchange between areas (beta diversity) ( Whittaker 1972 ) and later to propose conservation management schemes. Supplementary Material XML Treatment for Toxophora XML Treatment for Toxophora maxima XML Treatment for Toxophora virgata XML Treatment for Bombylius XML Treatment for Bombylius ( Bombylius ) frommerorum XML Treatment for Heterostylum XML Treatment for Heterostylum croceum XML Treatment for Lordotus XML Treatment for Lordotus diplasus XML Treatment for Lordotus divisus XML Treatment for Lordotus perplexus XML Treatment for Triploechus XML Treatment for Triploechus novus XML Treatment for Ogcodocera XML Treatment for Ogcodocera analis XML Treatment for Paracosmus XML Treatment for Paracosmus ( Paracosmus ) morrisoni XML Treatment for Anthrax XML Treatment for Anthrax atriplex XML Treatment for Anthrax cybele XML Treatment for Anthrax georgicus XML Treatment for Anthrax irroratus XML Treatment for Anthrax oedipus XML Treatment for Anthrax pauper XML Treatment for Anthrax seriepunctatus XML Treatment for Dipalta XML Treatment for Dipalta serpentina XML Treatment for Hemipenthes XML Treatment for Hemipenthes jaennickeana XML Treatment for Hemipenthes lepidota XML Treatment for Hemipenthes scylla XML Treatment for Hemipenthes sinuosa XML Treatment for Lepidanthrax XML Treatment for Lepidanthrax arizonensis XML Treatment for Lepidanthrax disiunctus XML Treatment for Lepidanthrax hesperus XML Treatment for Lepidanthrax hyposcelus XML Treatment for Lepidanthrax proboscideus XML Treatment for Neodiplocampta XML Treatment for Neodiplocampta ( Neodiplocampta ) miranda XML Treatment for Paravilla XML Treatment for Paravilla edititoides XML Treatment for Paravilla flavipilosa XML Treatment for Paravilla parvula XML Treatment for Paravilla separata XML Treatment for Poecilanthrax XML Treatment for Poecilanthrax effrenus XML Treatment for Poecilanthrax fasciatus XML Treatment for Poecilanthrax hyalinipennis XML Treatment for Poecilanthrax poecilogaster XML Treatment for Rhynchanthrax XML Treatment for Rhynchanthrax capreus XML Treatment for Rhynachantrax texanus XML Treatment for Thyridanthrax XML Treatment for Thyridanthrax pallidus XML Treatment for Thyridanthrax selene XML Treatment for Xenox XML Treatment for Xenox xylocopae

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121718/

Autoimmune Disease and the Human Metagenome

The prevailing theory of autoimmune disease, that the body creates autoantibodies that attack "self," was developed during an era when culture-based methods vastly underestimated the number of microbes capable of persisting in and on Homo sapiens . Thanks to the advent of culture-independent tools, the human body is now known to harbor billions of microbes whose collective genomes work in concert with the human genome. Thus, the human genome can no longer be studied in isolation. Some of these microbes persist by slowing the activity of the vitamin D receptor nuclear receptor, affecting the expression of endogenous antimicrobials and other key components of the innate immune system. It seems that bacteria that cause autoimmune disease accumulate over a lifetime, with individuals picking up pathogens with greater ease over time, as the immune response becomes increasingly compromised. Any one autoimmune disease is likely due to many different microbes within the metagenomic microbiota. This helps explain the high levels of comorbidity observed among patients with autoimmune conditions. What are commonly believed to be autoantibodies may instead be created in response to this metagenomic microbiota when the adaptive immune system is forced to deal with disintegration of infected cells. Similarly, haplotypes associated with autoimmune conditions vary widely among individuals and populations. They are more suggestive of a regional infectious model rather than a model in which an illness is caused by inherited variation of HLA haplotypes Background In 1922, Ernst Almquist – a colleague of Louis Pasteur – commented, "Nobody can pretend to know the complete life cycle and all the varieties of even a single bacterial species. It would be an assumption to think so" (Mattman, 2000 ). While Almquist's work on idiopathic bacteria in chronic disease never received the plaudits accorded to Pasteur's work, Almquist foresaw the complexity that would later be inherent to the field of metagenomics – a field that today forces us to examine how countless microbial genomes interact with the human genome across disease states. Yet in the decades before novel genomic technology made a metagenomic understanding of disease possible, bacteria could only be cultured in vitro on a limited range of growth media. As most major diseases of the time – tuberculosis, pneumonia, leprosy, and others – were linked to the presence of a handful of acute pathogens able to grow under these constraints, a "game over" attitude toward infectious agents dominated the thinking of much of the medical community. Little consideration was given to the possible role of these pathogens in autoimmune and inflammatory disease states. Instead, for most of the twentieth century, the predominant feeling about the treatment, control, and prevention of diseases with a possible infectious etiology was optimism (Cohen, 2000 ). Between 1940 and 1960, the development and successes of antibiotics and immunizations added to this optimism and, in 1969, Surgeon General William H. Stewart told the US Congress that it was time to "close the book on infectious diseases" (Avila et al., 2008 ). With "victory" declared, increasing emphasis was directed at the "noninfectious" diseases such as cancer and heart disease. In many cases, research on infectious disease or activities on their prevention and control were de-emphasized and resources were reduced or eliminated. As recently as the 1980s, pharmaceutical companies, believing that there were already enough antibiotics, began reducing the development of new drugs or redirecting it away from antibiotics. Despite this rosy narrative, some microbiologists were never convinced that drugs like penicillin had ended the war between man and microbe. In 1932, Razumov noted a large discrepancy between the viable plate count and total direct microscopic count of bacteria taken from aquatic habitats (Razumov, 1932 ). He found higher numbers (by several orders of magnitude) by direct microscopic counting than by the plating procedure. In 1949, Winogradsky confirmed Razumov's assessment and noted that many microbes are not satisfied with laboratory cultivation conditions. He remarked that readily cultivated bacteria in natural microbial communities "draw importance to themselves, whereas the other forms, being less docile, or even resistant, escape attention" (Relman, 1998 ). In 1985, Staley and Konopka pointed to Razumov's discrepancy and called it the "Great Plate Count Anomaly" (Grice et al., 2008 ). Their review describes work in which they compared the efficacy of a fluorescent dye versus standard plating procedures in detecting bacterial species in samples of water collected from Lake Washington. They found that only approximately 0.1–1.0% of the total bacteria present in any given sample could be enumerated by the plating procedure – causing them to conclude that, unless new methods for detecting bacteria were employed, "No breakthrough in determining species diversity seems likely in the near future." Meanwhile, some microbiologists continued their best efforts to alter the pH and growth medium of their samples in an effort to look for previously undetected bacteria in chronic disease states. Over the course of a career spanning almost 50 years, Lida Mattman of Wayne State University cultured wall-less forms of bacteria from the blood samples of patients with over 20 inflammatory diagnoses including multiple sclerosis and sarcoidosis (Almenoff et al., 1996 ). She authored an entire textbook on novel approaches for in vitro cultivation of bacteria (Mattman, 2000 ). Over his 39-year career at Tulane University, Gerald Domingue published dozens of papers and book chapters devoted to the role of chronic forms of bacteria in inflammatory disease. "It is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease," he wrote. "Clearly, any patient with a history of recurrent infection and persistent disability is sending the signal that the phenomenon [infection with chronic bacteria or viruses] could be occurring. The so-called autoimmune diseases in which no organism can be identified by routine testing techniques are particularly suspect" (Domingue and Woody, 1997 ). Yet, scientists like Mattman and Domingue faced serious challenges in trying to convince the medical community that their work was valid. Other research teams using less rigorous techniques often failed to duplicate their findings. Many of their observations were dismissed on the premise that their samples could have been contaminated. However, the greatest impediment toward the acceptance of this work was a set of rules set in motion by the nineteenth-century German physician Robert Koch. These rules, known as "Koch's postulates," stipulate that in order for a microbe to be deemed a causative agent of a disease, certain criteria must be met. The same microbe must be identified in every person with a given disease; the specific microbe must be able to be grown on pure culture medium in the lab; and, when reintroduced into a healthy animal or person, must produce the disease again. While Koch's postulates may have offered certain clarity during the formative stages of the field of microbiology, the rules distracted scientists from considering the possibility that multiple species could be responsible for the onset of a single disease state. Even today, Koch's notions about disease are regularly invoked (Monaco et al., 2005 ) despite the emergence of a number of counter-examples. Neither Mycobacterium leprae , which is implicated in leprosy, nor Treponema pallidum , which causes syphilis, fulfill Koch's postulates, because these microbes cannot be grown in conventional culture media. Viruses further invalidate Koch's postulates because most require another living cell in order to replicate (Walker et al., 2006 ). In the absence of clear connections between a single microbe and a single disease, most microbiologists necessarily assumed that the body was a sterile compartment and that inflammation, which might well suggest the presence of microbes, was attributed to an idiopathic causation. Unable to grow all but a fraction of bacteria found in the human body in the confines of a Petri dish, and constrained by a lack of technology with which to detect new microbes, the theory of autoimmune disease, in which the immune system loses tolerance and generates antibodies that target self gained momentum in the 1960s. Yet over the past decade, the role of infectious agents in autoimmune disease has once again gained momentum. The 2004 International Congress on Autoimmunity in Budapest was themed "Autoimmunity and Infection" with many subsequent conferences and papers in the same vein. However, nearly all speakers discussed the role of viruses in autoimmune disease, whereas only a few contemplated bacteria. Autoimmune conditions were repeatedly attributed to easily cultured viruses such as Epstein–Barr and Herpes 6. Where bacteria were discussed, most reports centered on select pathogens such as Chlamydia pneumoniae . Yet because none of these pathogens has ever been detected in any one autoimmune disease state 100% of the time, such researchers continue to paint autoimmune diseases as a mosaic – in which the hallmarks of infection are continually present in bits and pieces but cannot be drawn into a fully cohesive picture. Yet the emerging science of metagenomics is beginning to unmask entirely new populations of microbes whose genomes allow for a means by which to bridge these gaps. The following chapter examines how this metagenomic microbiota can cause the dysfunction seen in a wide range of autoimmune conditions. Culture-Independent Methods for Identifying Microbes In 2007, a study orchestrated by NASA announced that the surfaces of the supposedly sterile "clean rooms," in which technicians assemble spacecraft, host an abundance of hardy bacteria (Moissl et al., 2007 ). Samples taken from clean rooms at the Jet Propulsion Laboratory in California, the Kennedy Space Flight Center in Florida, and the Johnson Space Center in Houston revealed the presence of almost 100 types of bacteria representing all the major bacterial phyla; 45% of the species identified were previously unknown to science. The findings came as a shock to NASA officials, who were left to wonder exactly how many unknown microbes might have been taken to the moon and Mars. These clean room bacteria had not been previously detected because they could not be characterized by standard cultivation techniques. To find them, the research team had used a genomic approach – RNA gene sequence analysis – to characterize the genetic material of the bacterial species in the rooms previously touted as sterile. Similar culture-independent tools are beginning to revolutionize our understanding of autoimmune disease by allowing for a vastly more comprehensive understanding of the microbes that persist in Homo sapiens , microbes that may cause the generation of autoantibodies. Genomic sequencing techniques, including 16S RNA sequencing, PCR and, more recently, pyrosequencing, have made it clear that only a fraction of those microbes that persist in the human body will grow on the limited medium of a Petri dish. With the advent of these technologies, the field of metagenomics was born. Rather than focusing on the study of single microbes and their genomes, metagenomics provides a means of analyzing aspects of microbial communities through their underpinning genetics. The amount of novel microbial genetic information that is generated on a daily basis by metagenomic analysis is so great that multidisciplinary approaches that integrate statistics, bioinformatics, and mathematical methods are required to assess it effectively. Today, the National Institutes of Health (NIH) estimates that a mere 10% of the cells that comprise Homo sapiens are human cells. The remaining 90% are bacterial in origin. The number of Escherichia coli in a single human is comparable to the entire human global population – approximately six billion people (Staley, 1997 ). Such knowledge has forever changed the manner in which the human organism is perceived. We may best describe the human being as a super-organism in which communities of different organisms flourish in symbiosis with the host. Yet, even with the availability of technology to explore the microbial world in depth, to date, only a fraction of the human bacterial microbiota has been genetically identified and characterized. As of late 2009, approximately 1,100 published complete bacterial genomes had been identified with 6,000 more under review (Liolios et al., 2008 ). Nevertheless, there are still huge gaps in our understanding of how the microbiota contributes to human health and disease. Viruses (the virome) and phages are also key components of the microbiota. Like bacteria, many of these microbes have yet to be fully characterized by high-throughput genome sequencing. However, molecular analysis has revealed that nearly all humans acquire multiple viruses, usually within the first years of life, viruses that generally remain with them throughout life. Polyomaviruses infect between 72 and 98% of humans, surviving in the kidney, lung, and skin (Virgin et al., 2009 ). Similarly, human herpes viruses are extremely persistent. Anelioviruses, as well as adeno-associated virus, are now recognized to infect most humans by the end of childhood. The role of these viruses is unknown, but a significant number of people who harbor them become symptomatic later in life, suggesting that they may be capable of virulence under conditions of immune dysfunction. According to Herbert Virgin of Washington University, "We carry, for good or for ill, many lifelong [viral] passengers" (Virgin et al., 2009 ). In the next 5 years, researchers associated with the NIH Human Microbiome Project (HMP) plan to use molecular genetic sequencing in an effort to catalog the bacterial component of the human microbiome. This initiative promises to increase our knowledge of bacterial diversity. The NIH has funded many more HMP projects, with the goal that the diagnosis, treatment, and prevention of many inflammatory diagnoses can be improved by examining how the microbiota differs between those people with a disease and their healthy counterparts. Thus far, targeted conditions include Crohn's disease (CD), inflammatory bowel disease, vaginosis, psoriasis, and other conditions now considered to be autoimmune. Early work has already demonstrated fundamental discrepancies in microbial composition between health and disease. Swidsinksi et al. found that patients with irritable bowel syndrome have more bacteria from diverse genera attached to their epithelial gut surfaces than do healthy controls. Some of these microbes, such as Bacteriodes , were found to penetrate the epithelial layer, at times intracellularly (Swidsinski et al., 2002 ). Enck et al. found that irritable bowel syndrome manifests with a relative decrease in populations of bifidobacteria and significant differences in a variety of other microbes, including those that cause the production of gas (Enck et al., 2009 ). Medicine has become comfortable acknowledging that bacterial populations exist in the areas of the body in contact with the external environment, such as the airways, gastrointestinal tract, mouth, skin, and vagina/penis. For example, analysis of the human oral cavity by Nasidze et al. identified 101 bacterial genera in the mouth as well as an additional 64 genera previously unknown to science (Nasidze et al., 2009 ). Yet, microbes have also been shown to persist in many other body tissues, including joints and blood vessels. Some of the same bacteria identified in the salivary microbiome, such as Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis – both of which cause tooth decay (Lamell et al., 2000 ) – have also been identified in atherosclerotic plaque (Kozarov et al., 2005 ). Bacterial DNA has been detected in the blood (Nikkari et al., 2001 ). Recently, 18 different bacterial taxa were detected in the amniotic fluid, which was previously believed to be completely sterile (DiGiulio et al., 2008 ). Analysis using 16S rRNA sequencing detected 28 distinct phylotypes on biofilm removed from prosthetic hip joints during revision arthroplasties – joints also removed from a body compartment also thought to be sterile. The prevalence of hydrothermal vent eubacteria, which were previously thought to persist only in the depths of the ocean since they were found at temperatures well above 176°F (80°C), was higher than the prevalence of Staphylococcus aureus , a common biofilm species (Fig. 12.1 ). Fig. 12.1 Bacterial species identified by 16S rRNA gene sequencing of clones from 10 prosthetic hip joints (Dempsey et al., 2007 ) It is now more prudent to assume that tissues that become inflamed in disease most probably do so because of the actions of microscopic pathogens, rather than idiopathic causation. Different microbial populations have been identified in many nongastrointestinal autoimmune conditions including sarcoidosis (el-Zaatari et al., 1996 ), ankylosing spondylitis (Liu et al., 2001 ), chronic fatigue syndrome (Lombardi et al., 2009 ), rheumatoid arthritis, multiple sclerosis, Hashimoto's thyroiditis, and others (Pordeus et al., 2008 ). These diseases share features of microbial infection including widespread inflammation and periods of relapse. Sarcoidosis and CD' are characterized by granuloma. In more than a dozen infectious diseases, granuloma is widely acknowledged to be a host-protective structure and to occur when acute inflammatory processes cannot destroy invading agents (Zumla and James, 1996 ). The Human Metagenome At only approximately 23,000 genes, the human genome is dwarfed by the thousands of genomes of the bacteria, viruses, and phages that persist in and on humans. Given the sheer number of microbial genes, it is no longer possible to study the human genome in isolation. Rather, the human genome is only one of myriad genomes that influence the Homo sapiens experience. Humans are controlled by a metagenome – a tremendous number of different genomes working in tandem. Because they are so small, thousands of microbial cells can persist inside a single infected human cell (Wirostko et al., 1989 ). The combined genetic contributions of these microbes invariably provide a vast number of gene products not encoded by our own relatively small genomes. There is considerable similarity between the functions of the bacterial organisms and the human organisms. For example, humans and E. coli metabolize glucose-6-phosphate in a similar fashion, producing almost identical metabolites (Kuroki et al., 1993 ). Thus, the transgenomic interaction between an E. coli genome and the human genome, as they exchange nutrients and toxins, increases the complexity of transcription and translation for both species. The dihydrofolate reductase (DHFR) antagonist trimethoprim is such an effective antibiotic because, like humans, bacterial species possess a folate metabolism. Bacteria in the distal intestinal tract of mice have been shown to significantly alter the composition of human blood metabolites, including amino acids, indole-3-propionic acid (IPA), and organic acids containing phenol groups, providing another example of the significant interplay between bacterial and human metabolism. A broad, drug-like phase II metabolic response of the host to metabolites generated by the gut microbiota was observed (Wikoff et al., 2009 ), suggesting that the gut microbiome has a direct impact on the host's capacity for drug metabolism. In the pre-genomic era, diseases were classified largely on the basis of symptom presentation; while in recent decades, researchers have attempted to categorize them based on common genes. Yet metagenomics dictates that we must also consider how the many microbial metabolites affect expression of these genes. Some genes and their related pathways have already been shown to influence the pathogenesis of autoimmune disease. For example, Goh et al. has shown that PTPN22 is related to rheumatoid arthritis, lupus, and diabetes mellitus (Goh et al., 2007 ). Yet expression of PTPN22 is also modified by the bacterial metagenome – it has been shown to be upregulated as part of the innate immune response to mycobacteria (Lykouras et al., 2008 ). The importance of understanding how microbes affect PTPN22 across multiple disease states has special impetus, given the increased rate of latent tuberculosis in the global population as well as studies showing high rates of infection by Mycobacterium avium among autoimmune patients (Bentley et al., 2008 ). Many of the most well-studied persistent pathogenic bacteria have evolved mechanisms to evade the immune response and survive inside macrophages and other phagocytic cells. These include Francisella tularensis (Hazlett et al., 2008 ), Mycobacterium tuberculosis (Domingue and Woody, 1997 ), Rickettsia massiliae (Monaco et al., 2005 ), Brucella spp. (Baldwin and Goenka, 2006 ), Listeria monocytogenes (Birmingham et al., 2007 ), Salmonella typhimurium (Kuijl et al., 2007 ), among others. This suggests that other disease-causing components of the microbiota may also persist in the cytoplasm of nucleated cells, where they have access to both human DNA transcription and protein translation machinery (Hall et al., 2008 ). When Shigella persists within a macrophage it modulates numerous host signaling pathways, including those that inactivate mitogen-activated protein kinases (Lutjen-Drecoll, 1992 ). Brucella spp. downregulates genes involved in cell growth and metabolism, but upregulates those associated with the inflammatory response and the complement system upon infecting a macrophage. Additionally, there appears to be an entire intra-cytoplasmic microbiota within phagocytic cells. Wirostko's team at Columbia University in the 1980s and 1990s used electron microscopy to identify entities within the cytoplasm of phagocytes from patients with juvenile rheumatoid arthritis, sarcoidosis (Wirostko et al., 1989 ), Crohn's, and other inflammatory diseases (Wirostko et al., 1987 ). The wide variety of elongated and globular formations, together with both the existence and absence of exoskeletons around the microbiota, would imply that the observed communities are metagenomic, rather than due to any one single obligate phagocytic pathogen. Microbial Complexity The HIV genome consists of a single strand of RNA comprising nine genes, from which are transcribed 19 proteins. Transcription is noncontiguous, and variations abound. For example, "Tat" is transcribed in multiple pieces that are subsequently joined. Yet 1,443 direct interactions (3,300 total interactions) have been identified between just these 19 proteins and the human metabolome (Fu et al., 2009 ). Consider that the average bacterial genome codes for hundreds or sometimes thousands of proteins. According to one recent estimate, the average human gut microbiota codes for 9 million unique genes (Yang et al., 2009 ). Factor in the proteins created by viruses and phages, and efforts to understand how these proteins affect the metabolome leave an observer with little more than stochastic noise, particularly since biological systems are replete with components showing nonlinear dynamic behavior. Subsequently, interaction between the metagenome and the human genome introduces a new level of complexity to the study of autoimmune disease – complexity that renders it nearly impossible to fully comprehend the vast number of the interactions between the human genome and those microbial genomes capable of influencing the pathogenesis of autoimmune disease. According to Bunge, the size of a gene pool for a given environmental sample can be estimated by mathematical modeling, but the size of the gene pool for a microbial biosphere, such as the human body, may be beyond any current credible model (Bunge, 2009 ). While this complexity poses a significant challenge to systems biology and to Koch's simplistic one gene–one disease model, it does not impede the emergence of a better understanding of the human super-organism and the processes that cause disease. Lifelong symbiosis between the human genome and persistent components of the metagenome has shifted the focus of microbiology away from the search for a single pathogen in a disease state. Many research teams are now striving to understand how components of the microbiota may cause disease. For example, researchers with the European MetaHIT Initiative are studying how bacteria in the gut may contribute to obesity and inflammatory bowel disease. The goal of the project is simply to examine associations between bacterial genes and human phenotypes. "We don't care if the name of the bacteria is Enterobacter or Salmonella . We want to know if there is an enzyme producing carbohydrates, an enzyme producing gas or an enzyme degrading proteins," explains Francisco Guarner of the project. Studies focused on enzymes, proteins, and carbohydrates are studies of the metabolome. Metabolomic approaches can be used to characterize entire components of the microbiome that cannot easily be seen or studied directly. Because the downstream results of gene expression manifest in the human metabolome, the metabolome can be analyzed for the presence of those unique metabolites created under the influence of the microbiota. Dumas et al. used mass spectroscopy to identify the nonhuman metabolites present in the urine of subjects living in three distinct populations – the United States, China, and Japan (Dumas et al., 2006 ). He found that subjects in each population produced very different nonhuman metabolites. Thus, genetic makeup, healthcare, nutrition, and external toxins, factors associated with the acquisition of a particular microbiota, caused the three populations to become significantly different. Moreover, when five of the Japanese subjects moved to the United States, their metabolomes changed to resemble those of the American population. This suggests that the metagenome is indeed the product of its environment, and that the composition of the microbiota is far more important than regional variations in the human genome itself. In another study, the INTERMAP epidemiological study used an 1 H NMR-based metabonomics approach to examine differences in the urine metabolite profiles for each of 4,630 participants from 17 populations in the USA, UK, Japan, and China (Stamler et al., 2003 ). Elevated blood pressure was associated with high levels of the bacterial co-metabolite formate. Interestingly, low levels of hippurate and alanine, which reflected gut microbial activities, were also found in subjects with high blood pressure (Holmes et al., 2008 ). This suggests that certain microbial metabolites may serve as useful biomarkers for a disease state. The fact that components of the microbiota are seldom found as single entities further complicates the complexity of transgenomic control in Homo sapiens . While just a few decades ago, most of the bacteria in Homo sapiens were assumed to persist on their own in a planktonic form, it is now understood that large components of the microbiota persist in communities commonly called biofilms; they are sheltered by a self-created polymeric matrix that better protects them from the immune response. Hundreds of different microbes can persist in a single biofilm community, and individual bacteria often form a niche inside the biofilm that allows them to promote their own survival and the chronic nature of the infection. For example, more virulent bacteria may protect the biofilm from outside intrusion whereas other less innocuous species inside the biofilm may focus on obtaining nutrients for the community. As the biofilm forms and then develops, the collective genetic expression of microbes in the biofilm is altered dramatically. For example, the expression of 800 genes has been shown to be altered when a single bacterial species joins a biofilm (Sauer et al., 2002 ). Biofilms are increasingly being detected in autoimmune diseases where they were not known to previously exist. For example, Wolcott recently used pyrosequencing to demonstrate that the infectious agents that drive the development of diabetic leg, foot, and pressure ulcers are almost all in a biofilm state (Dowd et al., 2008 ). Bacteria in biofilm, their planktonic counterparts, viruses, and other microbes rapidly and frequently share their DNA with other species, even distantly related species, through horizontal gene transfer. Genomic coherence is further muddled by homologous recombination. This further diversifies the variability present in the human microbiome. Horizontal gene transfer is now believed by many to occur so frequently that it has been proposed as a means by which species can acquire new genetic traits. Some argue that the number of microbes created through homologous recombination is so high that the concept of distinct bacterial species may become obsolete (Doolittle and Papke, 2006 ). Thus, the concept that a single pathogen could cause the human metabolism to fail in the myriad of ways necessary to result in an advanced, systemic autoimmune disease is increasingly recognized as an outdated nineteenth-century concept. The postulates of Koch are no longer relevant in the era of the metagenome. Brock contends in his profile of Koch that attempts to rigidly apply Koch's postulates to the diagnosis of viral diseases may have significantly impeded the early development of the field of virology (Brock, 1988 ). The same can be said for the field of bacteriology, where the postulates have long impeded researchers from considering that the genomes of many different bacteria and other pathogens interact together to cause the range of symptoms we associate with autoimmune diagnoses. Toward a More Nuanced View of the Human Microbiota In New science of metagenomics: revealing the secrets of our microbial planet , the National Research Council writes, "The billions of benign microbes that live in the human gut help us to digest food, break down toxins, and fight off disease-causing microbes" (Committee on Metagenomics and National Research Council, 2007 ). While certain components of the microbiota clearly aid humans in these and other ways, strictly classifying microbes as either commensal or pathogenic may suggest too categorical a distinction. Emerging research suggests that bacteria are no more "good" or "bad" than their human counterparts, particularly when a commensal microbe can easily acquire a plasmid or virulence factor from another microbe. According to Fredricks and Relman, "The mobile nature of virulence-associated gene islands, transported between bacteria via plasmids or phages, creates the potential for acquired virulence in previously innocuous microbes" (Fredricks and Relman, 1998 ). In September 2009, Malcolm Casadaban, an infectious disease researcher at the University of Chicago, died suddenly. An autopsy showed no obvious cause of death except Yersinia in his bloodstream. Dr. Casadaban, an associate professor at the university, was studying the bacteria to create a better vaccine for plague. Yet Casadaban was working with a strain of Yersinia that was supposed to be less virulent that those strains considered lethal. Researchers postulated that there must have been something unusual about the bacterium that caused it to be dangerous, such as a mutation. The so-called "innocuous" strain of Yersinia may have acquired a plasmid or gene that endowed it with newfound virulence. Acquired virulence via horizontal gene transfer has been studied in anthrax. Although Bacillus anthracis , which causes fatal poisoning, and B. cereus , which causes nonlethal opportunistic infections, are generally classified as separate bacterial species, Hoffmaster discovered a B. cereus mutant that also causes a deadly form of pneumonia. Analysis revealed that the B. cereus mutant ( B. cereus G9241) had acquired a plasmid with 99.6% sequence homology to pX01, B. anthracis ' most virulent, toxin-encoding plasmid. Indeed, B cereus G9241 killed mice more quickly than B. anthracis . Bacillus cereus G9241 was deemed the product of horizontal gene transfer, causing Hoffmaster to note that, depending on the extent of horizontal gene transfer, nature could produce an unlimited number of variations and combinations of any given pathogen. The distinction between commensalism and pathogenicity is further blurred by host-specific factors. For example, if a species of bacteria aids in the metabolism of carbohydrates from the human intestinal tract, the presence of the microbe in the intestines of famine victims could save lives. However, in many Western countries, where rates of obesity are rising at an alarming pace (Wang and Beydoun, 2007 ), the same microbe might contribute to excess weight gain. Returning to the gene/disease network discussed above, the ACE gene is related to myocardial infarction, renal tubular dysgenesis, Alzheimer's, the progression of SARS, diabetes mellitus, and sarcoidosis. However, Lactobacillus and Bifidobacteria , species of bacteria considered to be innocuous or "friendly," are capable of creating a number of peptides that downregulate expression of ACE (Ramchandran and Shah, 2008 ). These species of bacteria are added to many of our dairy products and are clearly present in the human body. Yet by altering the expression of ACE, these "friendly bacteria" may well affect the progression of several autoimmune and chronic inflammatory diseases, albeit in ways not yet fully understood. Pathogens Alter the Expression of Human Genes and Receptors Intracellular components create myriad metabolites that can interfere and alter the correct transcription of human proteins. Some of these metabolites can also disrupt cellular repair mechanisms, resulting in the accumulation of "junk" (e.g., proteins, enzymes, and mRNA) in the cytosol. For example, Machado et al. reported that Helicobacter pylori impairs central DNA repair mechanisms, inducing a transient mutator phenotype, rendering gastric epithelial cells vulnerable to the accumulation of genetic instability (Machado et al., 2009 ). If the accumulation of errors can exceed the capacity for cellular repair, such dysregulation not only has the potential to drive autoimmune processes, but can also result in early senescence (Muller, 2006 ), apoptosis (Knodler and Finlay, 2001 ; Yilmaz et al., 2008 ), or cancer. One of the ways in which pathogens survive is by dysregulating the activity of several of the body's key nuclear receptors. The ability of a number of pathogens to dysregulate the vitamin D receptor (VDR), a type 1 nuclear receptor, provides an excellent example of how microbes alter human gene expression so as to gain a survival advantage. Many of the nuclear receptors play a critical role in regulating immune activity and hormonal expression. The VDR expresses at least 913 genes, many connected to autoimmune conditions and cancers. The receptor also regulates expression of several families of key antimicrobial peptides (AMPs), including cathelicidin and the beta-defensins. These play a vital role in allowing the innate immune system to target intracellular pathogens. For example, vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin (Liu et al., 2007 ). The VDR also transcribes Toll-like-receptor 2 (TLR2), which recognizes bacterial polysaccharides. The TACO gene, when expressed, inhibits mycobacterial entry as well as survival. Mycobacterium tuberculosis (Mtb) downregulates the VDR, and thus expression of TACO in order to survive. Xu et al. showed that the VDR was downregulated 3.3 times in monocytic cell lines infected with Mtb (Xu et al., 2003 ). Borrelia , as assessed by BeadChip microarray, has been shown capable of downregulating VDR activity by a factor of 50-fold, with lysed Borrelia downregulating the receptor by a factor of 8 (Salazar et al., 2009 ). We have previously shown that at least one bacterial metabolite produced by gliding biofilm bacteria is also a strong VDR antagonist (Marshall, 2008 ). The HIV "tat" protein binds to the VDR in order to use this receptor to recognize its long terminal repeat (LTR) promoter region (Nevado et al., 2007 ). Thus, tat takes over the human VDR in order to transcribe HIV's own genome, so the HIV LTR can be recognized and express new HIV RNA. Tat also recruits histone acetyltransferase activity, including the CREB binding protein (CBP)/p300 complex, to acetylate the HIV LTR promoter region (Romani et al., 2009 ). Slowing the ability of the VDR to express elements of the innate immune function is such a logical survival mechanism that it is almost certain that the other less studied components of the microbiota would have also evolved ways to dysregulate the VDR, and the other nuclear receptors orchestrating the innate immune response. Eukaryotic cells respond to the presence of the microbiota by activating signaling cascades such as the NF-kappaB pathway. Induction of such pathways leads to the upregulation of gene expression mediating pro-inflammatory and anti-apoptotic effector proteins. Thus, in order for pathogens (and potentially, symbionts) to continue their life cycle, it is necessary to evade or repress these cellular responses. This is especially true because acquisition of resistance to AMPs by a sensitive microbial strain is surprisingly improbable. Furthermore, the extension of human life during the past century now offers additional opportunity for microbes to evolve their specialization in order to survive in man. Indeed, Yenamandra et al. recently showed that Epstein–Barr virus (EBV) also slows VDR activity (Yenamandra et al., 2009 ). Infection of human B cells with EBV induces metabolic activation, morphological transformation, cell proliferation, and eventual immortalization by altering the expression of a number of key nuclear receptors. The team found that the expression of 12 nuclear receptors was downregulated in the longer-lasting, younger lymphoblastoid cells. Among them were the VDR and the estrogen receptor beta (ERB), both downregulated by a factor of about 15 times (Fig. 12.2 ). Fig. 12.2 Nuclear receptors mRNA expression is downregulated upon infection of B cells with EBV (Yenamandra et al., 2009 ) EBV is found in many common chronic disease states. Indeed, EBV has been detected in a subset of patients with nearly every autoimmune diagnosis, although it has rarely been detected in 100% of patients with any given condition. In some cases, infection with the virus is described as a "precipitating factor" for autoimmune disease. That EBV downregulates VDR and ERB expression may explain this phenomenon. If a patient acquires EBV, the virus slows innate immune activity to the point where the endogenous microbiota can become dominant. This is particularly true because, in addition to reducing expression of cathelicidin and beta-defensin, VDR dysregulation opens a number of other pathways that also influence immune activity and hormonal regulation. Blockage of the VDR prevents transcription of CYP24A1, an enzyme that normally breaks down excess levels of the active vitamin D metabolite 1,25-dihydroxyvitamin-D (1,25-D). Activation of protein kinase A (PKA) by bacterial cytokines also causes increased production of the enzyme CYP27B1, resulting in increased conversion of 25-hydroxyvitamin-D (25-D) into 1,25-D. Both processes result in a rise in 1,25-D. High levels of 1,25-D in autoimmune disease have been confirmed in a clinical setting. Mawer et al. found that 1,25-D levels were particularly elevated in the synovial fluid surrounding the joints of patients with rheumatoid arthritis (Mawer et al., 1991 ). Abreu et al. found that in a cohort of 88 CD' patients, 35 patients or 40% had elevated levels of 1,25-D, which the authors defined as above 60 pg/ml (Abreu et al., 2004 ). Bell noted that patients with tuberculosis, pneumonia, AIDS, disseminated candidiasis, leprosy, rheumatoid arthritis, silicone-induced granulomas, Wegerner's granulomatosis, Hodgkin's disease, lymphoma, histocytic lymphoma, T-cell leukemia, plasma cell granuloma, leiomyoblastoma, seminoma, and subcutaneous fat necrosis all tend to manifest with higher than normal levels of 1,25-D (Bell, 1998 ). Blaney et al. found that of 100 patients with various autoimmune diagnoses, 85% had 1,25-D above the normal range (Fig. 12.3 ) (Blaney et al., 2009 ). Yoshizawa et al. reported that in VDR knockout mice, a circumstance that closely mimics extreme VDR dysregulation, 1,25-D levels increase by a factor of 10 (Yoshizawa et al., 1997 ). However, understanding 1,25-D's role in various inflammatory disease states is complicated by the fact that most researchers determining vitamin D status test subjects only for levels of the inactive vitamin D metabolite, 25-D. Fig. 12.3 25-D vs. 1,25-D in a cohort of 100 autoimmune patients (Blaney et al., 2009 ) In silico research indicates that 1,25-D has a high affinity for not just the VDR, but many of the body's other nuclear receptors (Proal et al., 2009 ). This suggests that at high concentrations it will displace their exogenous ligands. Those receptors affected by elevated 1,25-D include alpha thyroid, beta thyroid, the glucocorticoid (adrenal) receptor, and the progesterone receptor (Fig. 12.4 ). For example, 1,25-D has a very high affinity for the thyroid beta, suggesting that it can displace T3 and T4 from the binding pocket (Table 12.1 ) (Proal et al., 2009 ). Fig. 12.4 The Thyroid-alpha nuclear receptor and T3, its native ligand [PDB:2H77], with the bound conformation of 1,25-D superimposed. Since the XSCORE Kd for 1,25-D is 8.4, and for T3 is 7.2, it is apparent that 1,25-D is capable of displacing T3 from binding to key receptor residues (shown here are Arg228, Asn179, Gly290, Leu292, Leu276, Ser277, Thr275, Ala263, Leu287, Ala180, Phe218, and Arg162) (Proal et al., 2009 ) Table 12.1 Affinities of native ligands and 1,25-D for various nuclear receptors (Proal et al., 2009 ) Nuclear receptor Native ligand Native ligand (Kd) 1,25-D (Kd) Thyroid alpha T3 7.20 8.41 Thyroid beta T3 7.18 8.44 Glucocorticoid Cortisol 7.36 8.12 Androgen Testosterone 7.38 8.05 Progesterone Progesterone 7.53 8.09 If 1,25-D prevents T3 from activating thyroid beta, then genes with thyroid beta promoters will be less energetically transcribed. This would result in thyroid disease and explain why increasing levels of thyroid hormone are necessary to maintain thyroid homeostasis as chronic disease progresses. Furthermore, since the functions of type 1 nuclear receptors are largely interdependent, if transcription by thyroid beta is dysregulated, system-wide transcription is also affected. This leads to disruption of system-wide AMP production. Just as the VDR expresses cathelicidin and beta-defensin, other nuclear receptors also express AMPs. Brahmachary et al. have shown that the glucocorticoid receptor, the androgen receptor, and the VDR are, respectively, in control of 20, 17, and 16 families out of the 22 analyzed (Brahmachary et al., 2006 ). Thus, dysregulating VDR activity yields flow-on effects that potentially disable the bulk of the body's AMPs. A patient affected in this manner would become increasingly immunocompromised, allowing disease-causing components of the microbiota to proliferate with even greater ease. This supports a disease model in which key components of the microbiota responsible for autoimmune conditions gradually shut down the innate immune response over a person's lifetime as bacteria, and other pathogens, incrementally accumulate into the microbiota. CD' is already characterized by diminishing functional antimicrobial activity, particularly when it comes to expression of cathelicidin and the beta-defensins (Nuding et al., 2007 ). Eventually, genes from the accumulating microbial metagenome may instigate a clinical disease symptomology, such as one of the autoimmune diagnoses, or simply drive the inflammation associated with the aches and pains of aging. Indeed, the lifelong accumulation of an increasingly diverse microbiota directly correlates with an age-related increase in diseases and symptoms associated with inflammation. The term "inflammaging" has been coined to explain "the now widely accepted phenomenon that aging is accompanied by a low-grade chronic, systemic up-regulation of the inflammatory response, and that the underlying inflammatory changes are common to most age-associated diseases" (Giunta, 2006 ). Because 1,25-D is expressed in the human cycling endometrium and rises by 40% during early pregnancy, women may be disproportionately affected by the potential drop in AMP expression associated with VDR dysregulation (Viganò et al., 2006 ). This implies that females may more easily accumulate a more diverse microbiota than their male counterparts, which could help explain why women suffer from a higher risk of most autoimmune diagnoses. Successive Infection and Variability in Disease Onset and Presentation The makeup of a person's microbiota is unique: humans may share as little as 1% of the same species (Eckburg et al., 2005 ). Given that the human microbiome may play the principal causative role in autoimmune disease, it may not be by accident that the uniqueness with which patients' autoimmune disease symptoms develop parallels the incredible variability of the human microbiome. Traditionally, diseases have been understood to be discrete and have their own respective and distinct pathologies, hence the emphasis on diagnosis. However, if the spectrum of autoimmune disease were driven by a common factor – namely a person's microbial inhabitants – variability in disease could be explained by accounting for how the human microbiota accumulates and develops in any one person. Enck et al. recently analyzed fecal flora of stool samples from 35,292 adults whose ages ranged from 18 to 96 years of age in order to gauge the relative abundance and composition of various bacterial species over time (Enck et al., 2009 ). He found that while the number of bacteria in the fecal microbiota remained stable with age, the composition of the microbiota diversified as subjects became older, with the oldest subjects measured (over 60 years of age) representing the most profound changes. Older subjects were much more likely to have higher prevalence of microbes associated with chronic disease such as Enterococcus and E. coli . A number of microbes that slow immune activity have already been identified indicating that bacteria/viral-driven suppression of innate immune activity may occur on a much larger scale than previously imagined. Each pathogen that decreases immune activity makes it easier for the host to pick up other pathogens, which themselves may further slow immune activity, creating a snowball effect. This process is known as successive infection and offers us a framework for understanding how not only diseases of the gastrointestinal tract develop, but also any number of other autoimmune and inflammatory diseases. As human genes are upregulated or downregulated by components of the microbiota, the body shifts farther and farther away from its natural state of homeostasis. Infected cells increasingly struggle to correctly produce human metabolites in the presence of numerous proteins and enzymes being created by the pathogenic genomes. The ease with which a person acquires a pathogen from the environment, or from another person, depends largely on the state of their immune system. Those people who harbor low pathogenic loads and still have an active innate immune system could be expected to kill the acute and chronic pathogens they encounter. Conversely, those people with a compromised immune system will accumulate pathogens over time. We have previously discussed how VDR dysfunction, along with adrenal and androgen dysfunction, predispose to a weakened innate immune system, but there are many other factors in play. For example, Bukholm and team found that when the measles virus infects cell cultures, those cells are more susceptible to a secondary bacterial invasion (Bukholm et al., 1986 ). Stress has also been shown to impede immune function, by inhibiting natural killer cell activity, lymphocyte populations, lymphocyte proliferation, antibody production, and reactivation of latent viral infections (Webster Marketon and Glaser, 2008 ). Already identified consequences on health include delayed wound healing, impaired responses to vaccination, and development and progression of cancer (Boscarino, 2004 ). Depending on the variety of stressful events that occur over a lifetime, people may be more susceptible to picking up microbes at different times. The immune response could be expected to be particularly weak after traumatic events such as surgery, a car accident, or even a pregnancy (McLean et al., 2005 ). People accumulate microbiota-altering pathogens in myriad different ways, the most obvious being social contact. People in close proximity, particularly spouses and children, inevitably pick up components of each other's microbiomes (Wilhoite et al., 1993 ). Healthcare workers have higher rates of certain autoimmune and inflammatory conditions including breast cancer and malignant melanoma (Lie et al., 2007 ). Merely shaking hands causes the transfer of numerous microbes (Fierer et al., 2008 ). Genomic analysis of the bacteria on the hands of students leaving an exam room contained 332,000 genetically distinct bacteria belonging to 4,742 different species. Forty-five percent of the species detected were considered rare. This marked a 100-fold increase in the number of bacterial species detected over previous studies that had relied on purely culture-based methods to characterize the human hand microbiota. Obesity is not currently accepted as an autoimmune condition, but Christakis and Fowler recently used quantitative analysis of a densely interconnected social network to conclude that obesity is transmitted among people (Christakis and Fowler, 2007 ). A person's risk of becoming obese increases by 57% if they have a friend who becomes obese, and by 37% if their spouse becomes obese. Although, as the team concludes, people may mimic the behavior of friends or family in ways that could cause them to gain or lose weight, it is also possible that the close proximity among many of the subjects in the study would have allowed them to directly exchange microbes. Since the composition of bacteria in the gut has, in several instances, been linked to the development of obesity (Kinross et al., 2008 ) – perhaps, in some cases, obesity is literally contagious. It seems likely the same could be said for any autoimmune condition with an infectious etiology. In some cases, pathogens may be acquired in the womb, particularly if the mother already suffers from one or more autoimmune or inflammatory diagnoses. Similarly, bacterial species including Staphylococcus epidermidis , Streptococcus viridans , E. coli , Staphylococcus aureus , Streptococcus faecalis , Proteus , and others have been detected in sperm (Merino et al., 1995 ). Mycobacterium tuberculosis and influenza HSN1 have been shown to cross the placental barrier. Already implicated in implantation failure, spontaneous abortion, and preterm birth, infection with Shigella is now proposed to cause endometriosis (Kodati et al., 2008 ). DiGiulio studied ribosomal DNA (rDNA) of bacteria, fungi, and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. Fifteen percent of subjects harbored microbes that together belonged to 18 different taxa – including Sneathia sanguinegens , Leptotrichia amnionii, and an unassigned, uncultivated, and previously uncharacterized bacterium. A positive PCR was associated with histologic chorioamnionitis and funisitis. The correlation between positive PCR and preterm delivery was 100%. Pathogens can also pass from mother to child during breast-feeding. For example, Human papillomavirus type 16 (also called high-risk HPV-16), which has been linked to cervical cancer, has been detected in human breast milk collected during the early period after a woman delivers her baby (Sarkola et al., 2008 ). Pathogens can also be transmitted from person to person through bodily fluids released during coughing, sneezing, and other intimate contact and are found nearly everywhere in our environment. For example, nontuberculosis Mycobacteria and other opportunistic human pathogens are enriched to high levels in many showerhead biofilms, >100-fold above background water contents. Catheters used to treat urinary tract infections and other conditions have, in some cases, been shown to harbor copious amounts of biofilm. Early Infections Predispose a Person to Later Chronic Disease Most of the bacteria implicated in autoimmune disease are slow-growing pathogens whose effects will take decades to manifest (Davenport et al., 2009 ). In this sense, bacteria acquired earlier in life can alter the ultimate microbiota in ways that may not be recognized for decades. According to Merkler et al., "In genetically susceptible individuals, early childhood infections seem to predispose them to [such disease as] multiple sclerosis or Type 1 diabetes years or even decades before clinical onset" (Merkler et al., 2006 ). A 2006 report by the Centers for Disease Control (CDC) echoes this sentiment: "A person's age at the time of infection – from intrauterine or perinatal (the time period surrounding birth), through childhood and adolescence, to adulthood and the elder years – may further influence the risk for chronic outcome. For example, perinatal herpes virus infection dramatically increases the risk of developing adult or pediatric chronic liver disease. Recurrent infections or perhaps serial infections with certain agents might also determine a person's risk for chronic outcome" (O'Connor et al., 2006 ). Thus, while medicine generally assumes that once a person has recovered from an acute illness, they return to a state of complete health – the so-called "sterilizing immunity" – in truth, the long-term consequences of acute infection are somewhat poorly understood. Newborns who harbor certain types of bacteria in their throats, including Streptococcus pneumoniae and Haemophilus influenzae , are at increased risk for developing recurrent wheeze or asthma early in life (Bisgaard et al., 2007 ). Approximately two-thirds of patients with Guillain–Barré syndrome, a suspected autoimmune condition, have a history of an antecedent respiratory tract or gastrointestinal infection (Kuroki et al., 1993 ). Prenatal infections such as rubella, influenza, and toxoplasmosis are all associated with higher incidence of schizophrenia – with the children of those mothers exposed to influenza in the first trimester of gestation showing a seven-fold increased risk of schizophrenia (Brown, 2006 ). Reactive arthritis (Reiter's syndrome) is classically seen following infection with enteric pathogens such as Yersinia , Salmonella , Campylobacter , and Shigella (Hill Gaston and Lillicrap, 2003 ). Acute gastroenteritis, resulting from infection with the same pathogens, causes approximately 6–17% of patients to develop chronic irritable bowel syndrome. In an especially provocative experiment, a team including Doron Merkler and Nobel Laureate Rolf Zinkernagel injected cytomegalovirus (CMV) into the brains of mice that were only a few days old (Merkler et al., 2006 ). The innate immune systems of the mice were able to eliminate CMV from most of the tissues except for those of the central nervous system. As a result, the virus persisted in the brains of the mice. Later in life, when the same mice were challenged by infection with a similar virus, they developed a condition resembling a type of autoimmune disease and died. The team referred to this concept as "viral déjà vu ." Incidents of food poisoning also point to unresolved features of acute infections. Siegler et al. noted that 10% of people who suffered from E. coli food poisoning later developed a relatively infrequent life-threatening complication called hemolytic uremic syndrome (HUS) where their kidneys and other organs fail (Siegler et al., 1994 ). According to the study, 10–20 years after patients recover, between 30 and 50% of E. coli survivors will have some kidney-related problem, conditions that include high blood pressure caused by scarred kidneys, slowly failing kidneys, or even end-stage kidney failure requiring dialysis. Microbes can also be transmitted by donation of blood, bone marrow transplants, or organ donation, which, if pathogenic, can greatly disrupt the composition of the microbiota over time. The term "donor-acquired sarcoidosis" refers to the development of sarcoidosis in presumably naïve (nonsarcoidosis) transplant recipients who have received tissues or organs from donors who were not known or suspected to have active sarcoidosis (Padilla et al., 2002 ). Murphy studied over 8,500 people in the United Kingdom who underwent heart surgery between 1996 and 2003 (Murphy et al., 2007 ). Patients who had received red blood cell transfusions were about three times more likely to suffer a heart attack or stroke and were at a higher risk for infection, readmission to hospital, and death compared with heart patients who did not receive blood. The risks associated with blood transfusions were not influenced by a patient's age, hemoglobin levels, or the extent of their disability at the time of transfusion. Writing in the journal Circulation , Murphy et al. concluded: "Red blood cell transfusion appears to be harmful for almost all cardiac surgery patients and wastes a scarce commodity and other health service resources" (Murphy et al., 2007 ). Comorbidity Thus the catastrophic failure of the human metabolism we see in autoimmune disease – which at first glance appears so diverse and so different among different diagnoses – appears to be due to a single underlying mechanism: a ubiquitous microbiota, much of which has evolved to persist in the cytoplasm of nucleated cells. What differ among individuals as they gradually acquire a unique microbiota over time are the virulence, location, and combination of those pathogenic species. The high rate of comorbidity among inflammatory diagnoses (Anderson and Horvath, 2004 ) lends support for this explanation. Such comorbidity between seemingly unrelated diseases cannot be explained by laws of average – the risk of autoimmune disease is not evenly distributed. Figure 12.5 demonstrates the degree of comorbidity seen among various inflammatory diagnoses. Each "spoke" represents a study from PubMed, which has demonstrated a significant statistical relationship between patients suffering from one inflammatory disease and the next. Fig. 12.5 Comorbidities among common inflammatory diseases. Each "spoke" of this wheel represents a published study appearing in MEDLINE, which shows a significant statistical relationship between one disease and another In the case of multiple sclerosis, Barcellos et al. identified coexisting autoimmune phenotypes in patients with multiple sclerosis from families with several members with the disease and in their first-degree relatives (Barcellos et al., 2006 ). A total of 176 families (386 individuals and 1,107 first-degree relatives) were examined for a history of other autoimmune disorders. Forty-six (26%) index cases reported at least one coexisting autoimmune disorder. The most common were Hashimoto's thyroiditis (10%), psoriasis (6%), inflammatory bowel disease (3%), and rheumatoid arthritis (2%). One hundred and twelve (64%) families with a history of multiple sclerosis reported autoimmune disorders (excluding multiple sclerosis) in one or more first-degree relatives. Hashimoto's thyroiditis, psoriasis, and inflammatory bowel disease were also the most common diagnoses occurring in these family members. Such high rates of comorbidity support a model for autoimmune conditions in which no two people with the same diagnosis ever develop the exact same disease presentation; the interactions between an individual's genome and their unique metagenome are so varied that they are rarely identical. Note that Fig. 12.1 suggests that patients with autoimmune diagnoses are also much more likely to suffer from mental conditions such as depression and anxiety. Increasing clinical evidence, including that from our own study (Perez, 2008 ), confirms the involvement of microbiota in neurological disease states. This suggests that both autoimmune and neurological diagnoses, which are currently balkanized into separate medical specialties, most probably result from the same underlying dysregulation of microbial populations. Autoimmune and inflammatory conditions also suffer from specialty delineation. For example, VDR dysregulation does not just impact the autoimmune disease state. Researchers have reported epigenetic repression of VDR gene expression and activity in choriocarcinoma cell lines (Pospechova et al., 2009 ). Furthermore, the VDR expresses genes involved in both autoimmune and inflammatory processes. It transcribes insulin-like growth factor (IGFBP-3) (Wang et al., 2005 ), which influences the development of diabetes, yet also expresses metastasis suppressor protein 1 (MTSS1), which plays a vital role in repressing the cell cycle and promoting apoptosis in cancerous cells (Wang et al., 2005 ). Drawing a line between autoimmune and inflammatory disease makes these and other common mechanisms harder to recognize and study. Causation vs. Association If most autoimmune and inflammatory conditions do indeed arise from the same underlying disease process, then we must re-examine some of the cause and effect relationships postulated to exist among inflammatory conditions. For example, it is commonly believed that obesity is a causative factor in the development of diabetes (Hibbert-Jones et al., 2004 ). In fact, patients with Type 2 diabetes are so likely to become morbidly obese that the two conditions are sometimes collectively referred to as "diabesity" (Bailey, 2009 ). Obesity has been tied to microbial composition in the gut (Turnbaugh et al., 2006 ), the result of a microbial process. Roesch et al. found that the onset of Type 1 diabetes was tied to the presence of specific bacteria in the murine gut (Roesch et al., 2009 ). Additionally, at least one microbial species, Streptomyces achromogenes , secretes a substance, streptozocin, which can directly induce Type 1 diabetes (Bolzan and Bianchi, 2002 ). The diabetes disease process would also make it substantially harder for the immune system to regulate microbial gut composition. In particular, species that are extremely effective at extracting calories from food may thrive while their innocuous counterparts may find themselves out-competed. The expression of hormones that regulate appetite, such as leptin or ghrelin, could also become dysregulated by the bacterial microbiota (Fetissov et al., 2008 ). For example, H. pylori infection leads to a decrease in circulating ghrelin through a reduction in ghrelin-producing cells in the gastric mucosa (Weigt and Malfertheiner, 2009 ). In some cases, this could cause weight gain even in the absence of excess calorie consumption (English et al., 2002 ). In light of the above, obesity and diabetes might better be described as developing simultaneously. Treatments aimed at addressing those underlying factors contributing to both disease states might well prove the most effective. The same dichotomy is found in other sets of parallel conditions such as tooth decay and dementia, rheumatoid arthritis and uveitis, high cholesterol and heart disease, and others. It is far more likely that both conditions arise from a common metagenomic microbiota than that one condition is causal for the other. Microbial Interaction and Disease One of the more striking characteristics of nonobese diabetic (NOD) mice is that exposure to Mycobacteria can prevent the onset of diabetes while precipitating lupus in the same animal (Harada et al., 1990 ; Hawke et al., 2003 ). While this phenomenon is difficult to interpret by studying the murine genome alone, it may help to consider the murine metagenome. If, as in humans, the murine metagenome causes disease as it accumulates over time, then the interactions between various microbial species may be telling. Even within the context of the ultimate example of symbiotic behavior, the biofilm, bacteria have been shown to compete with one another, sometimes even "cheating" to do so (Dunny et al., 2008 ). We would not have many antibiotics if it were not for competition among bacterial species. For example, the early tetracycline antibiotics were derived from species of Streptomyces , and are toxic to a number of its competitors. With the NOD mice, introduction of a new species of bacteria into the microbiota, Mycobacteria , alters the microbiota in such a way as to wipe out, or at least diminish, the diabetes disease state. At the same time, the microbiota allows lupus to proliferate or dominate. Similar competition between microbes may also explain why lupus has been shown to inhibit the development of malaria ( Plasmodium falciparum ) (Zanini et al., 2009 ). Autism, an inflammatory condition that has been associated with several unique microbial populations (Nicolson et al., 2007 ) may have a comparable dynamic at work. In children diagnosed with autism spectrum disorder, fever associated with intercurrent bacterial or viral infections – such as upper respiratory infections – has been shown to temporarily decrease aberrant behavior such as irritability and inappropriate speech (Curran et al., 2007 ). Gastric surgery invariably alters the composition of the gastrointestinal microbiota. DePaula et al. found that after 39 diabetic type 2 patients in Brazil underwent bariatric surgery all subjects no longer required insulin therapy (DePaula et al., 2008 ). All subjects also experienced normalization of their cholesterol levels, 95.8% had their hypertension controlled, and 71% achieved targeted triglyceride levels. This correlates with data showing that the intestinal bacterial populations of normal weight individuals, morbidly obese individuals, and people who have undergone gastric bypass surgery are distinctly different. For example, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of gammaproteobacteria (Zhang et al., 2009 ). Other microbial interactions can alter the pathogenicity of one or more species involved. The pathogenic potential of Helicobacter hepaticus in a mammalian colitis model is altered by the presence of different strains of Bacteroides fragilis . When the bacterial polysaccharide PSA is expressed on the microbial cell surface of B. fragilis , it suppresses pro-inflammatory interleukin-17 production to H. hepaticus (Mazmanian et al., 2008 ). Hoffman et al. found that when the bacterial species Pseudomonas aeruginosa and S. aureus were incubated together, P. aeruginosa created a protein, HQNO, which protected S. aureus from eradication by commonly used aminoglycoside antibiotics such as tobramycin (Hoffman et al., 2006 ). Besides, in cases of P. aeruginosa and S. aureus co-infection in the presence of HQNO, small-colony variants of S. aureus are selected for, making S. aureus more difficult for the immune system to target. Although we are far from understanding the full nature of these microbial interactions, it is clear that a microbiota constantly evolves so that the symptoms of any given disease are seldom static. Familial Aggregation The common disease–common variant hypothesis suggests that chronic diseases are the product of anywhere from one to thousands of disease-causing alleles. The HapMap single nucleotide polymorphisms (SNPs) cataloging project has identified over 3.1 million SNPs, with many more expected to be found as the project continues. However, only a fraction of these SNPs confers any more than a minimal statistically increased risk for disease (Chung et al., 2010 ). For example, in cancer, for nearly all regions conclusively identified by genome-wide association studies (GWAS), the per allele effect sizes estimated are less than 1.3. While over 85 regions have been conclusively associated in over a dozen different cancers, no more than five regions have been associated with more than one distinct cancer type (Chung et al., 2010 ). According to Stephen Chanock of NIH, "Nearly every candidate SNP [associated with cancer] has failed in the long run – maybe five or six are real by rigorous standards" (personal communication). There appear to be factors at work other than just Mendelian inheritance. The increased risk of chronic disease among nonrelations in close proximity – the so-called "case clusters" – strongly implies an infectious dynamic at work. The evidence that the autoimmune disease sarcoidosis is communicable is particularly strong. A study of 215 sarcoidosis patients found that five husband-and-wife couples both had the disease – a rate 1,000 times greater than could be expected by chance (Rossman and Kreider, 2007 ). The NIH ACCESS research team also noted that the risk for sarcoidosis increased nearly five-fold in parents and siblings of people with the disease. A case-controlled study of residents of the Isle of Man found that 40% of people with sarcoidosis had been in contact with a person known to have the disease, compared with 1–2% of the control subjects (Gribbin et al., 2006 ). Another study reported three cases of sarcoidosis among 10 firefighters who apprenticed together (Kern et al., 1993 ). The literature contains many examples of unexpected familial associations among seemingly distinct disease pathologies. For example, a 2008 study of parents of children with autism found they were more likely to have been hospitalized for a mental disorder than parents of control subjects, with schizophrenia being more common among case mothers and fathers compared with respective control parents (Daniels et al., 2008 ). In the case of schizophrenia and autism, both have been associated with prenatal viral infection (Fatemi et al., 2008 ). While a fetus can acquire these and many other pathogens directly, successive infection dictates that as children age they will manifest with inflammatory symptoms that may differ from those of their parents. Major factors that would influence the development of a discrete inflammatory diagnosis include the mix of species acquired, the sequence in which the pathogens are acquired, the subsequent changes in gene expression caused by the pathogens, and the profound effect on the body's proteins, enzymes, and metabolites caused by these changes. Because the adaptive immune response in infants takes several weeks to develop, infants are particularly prone to picking up pathogens during the first weeks of life (Bisgaard et al., 2007 ). Such pathogens could be acquired from any family or friends in contact with the child, especially the grandparents, who probably harbor some of the highest pathogenic loads. Palmer et al. found that infants pick up many of the species that make up their gut flora from family members within just a few weeks of birth, suggesting that nongut bacteria may easily be acquired during this time as well (Palmer et al., 2007 ). Is Autoimmune Disease Predisposition Mendelian? Two decades ago, the attention of the research community shifted toward a new source in an attempt to explain the etiology of autoimmune disease: the human genome. Begun formally in 1990, the US Human Genome Project was a 13-year effort coordinated by the US Department of Energy and the NIH. Its primary goal was to determine the sequence of chemical base pairs that make up DNA and to identify the genes of the human genome from both a physical and a functional standpoint. A working draft of the genome was released in 2000 and a complete version in 2003, with further analyses yet to be completed and published (Collins et al., 2003 ). Meanwhile, the private company Celera Genomics conducted a parallel project (Venter et al., 2001 ). Early in the aftermath of the sequencing of the human genome, many geneticists advocated the common disease–common variant hypothesis, expressing certainty that the field would quickly determine genetic haplotypes that would correlate with and explain the bulk of chronic diseases. Dr. Francis Collins' 2001 statement was typical: "It should be possible to identify disease gene associations for many common illnesses in the next – 7 years" (Collins and McKusick, 2001 ). Researchers hoped that by dissecting the human genome, patients could be informed that they had "the gene" for breast cancer, sarcoidosis, rheumatoid arthritis, or any of the other autoimmune diagnoses. Targeted gene therapies could then be developed to effectively eradicate these conditions. It may be too early to call human genomic research an unqualified failure (Buchanan et al., 2006 ), but it is difficult to ignore a lack of utility in identification of disease. Recently, the limited progress in the genetic analysis of common diseases has begun to be acknowledged (Davey Smith et al., 2005 ; Risch, 2000 ). Certainly there have been no widely successful gene therapies to date, and genome-driven personalized medicine has yet to live up to its early promise. To identify what some researchers refer to as the "missing heritability," geneticists have proposed GWA studies with historically unprecedented sample sizes. In the past year, researchers have publicly contemplated "daunting" sample sizes exceeding 500,000 subjects in concert with studies that would be conducted over periods as long as 45 years (Burton et al., 2009 ). Ewald et al. argue that evolutionary forces that would cause a serious disease to be weeded from the population would also cause those people whose immune systems are prone to self-attack to be eliminated from the population (Cochran et al., 2000 ). An exception would occur if the disease offers a survival advantage. For example, the genetic disorder cystic fibrosis may confer resistance to tuberculosis (Poolman and Galvani, 2007 ). The Mendelian disorder sickle cell anemia is common in tropical countries because it confers resistance to malaria. With malaria, researchers can quantify the rate by generation at which the gene for sickle cell anemia is dropped from the population in the absence of an evolutionary advantage – as is the case when people migrate away from malaria-infested areas. However, no autoimmune diagnosis has been shown to confer any sort of beneficial survival trait. Under these circumstances, one would expect any faulty gene or network of genes associated with an autoimmune condition to be selected against, especially since many autoimmune conditions strike during the reproductive years. Chronic diseases have existed for thousands of years with manifestations of both arteriosclerosis (Azer, 1999 ) and cardiac disease observed in mummies of ancient Egypt (Miller et al., 2000 ). Ötzi the Neolithic Iceman who lived around 3300 BC had arthritis, allowing ample time for any alleles associated with autoimmune disease to be eliminated via natural selection (Dickson et al., 2003 ). Instead, the prevalence of autoimmune conditions seems to have remained essentially constant until quite recently. SNPs and Autoimmune Disease After noting that among his cohort of 31 patients with abdominal aortic aneurysm, SNPs in the gene BAK1 were different in aortic tissue than in blood samples from the same patients (Gottlieb et al., 2009 ) Gottlieb remarked, "Genome-wide association studies were introduced with enormous hype several years ago, and people expected tremendous breakthroughs. Unfortunately, the reality of these studies has been very disappointing, and our [own] discovery certainly could explain at least one of the reasons why." The conundrum that Gottlieb's study has exposed is that the human genome appears to vary between the tissue and plasma compartments. Medicine has always assumed that human DNA is homogeneous throughout the human body. We now need to explore the mechanisms whereby these different genetic sequences could arise through selective pressure in different tissues such as would exist if the tissue harbored a microbiota. One of the mechanisms proposed for genetic predisposition states that genetic haplotypes predispose for disease processes. Because it is a highly polymorphic genomic region, MHC has served as the preferred axis for studying susceptibility to immune diseases. Major changes have been detected within the HLA class I and class II genes related to various populations across the globe. For example, in Type 1 diabetes, the most common haplotype in the Western world is AH8.1 (HLA-A1-B8 DR3-SC01). However, this haplotype is almost nonexistent in the Indian population and has been supplanted by the variant AH8.1v, which differs from the Caucasian AH8.1 at several gene loci (Mehra et al., 2007 ). Moreover, there are additional HLA-DR3 haplotypes HLA-A26-B8-DR3, HLA-A24-B8 DR3 (AH8.3), A2-B8-DR3 (AH8.4), and A31-B8-DR3 (AH8.5) that occur largely in the Indian population alone. Similarly, the FCRL3-169T-C polymorphism, which is significantly associated with rheumatoid arthritis (RA) in East Asian populations is not associated with RA in Caucasians of European decent (Begovich et al., 2007 ). Interestingly, the frequency of the rs7528684 minor allele associated with FCRL3- varies as much within each of the two ethnic groups as it does between them. Furthermore, a recent large case-controlled study found that FCRL3-169T-C was not significantly associated with RA in Korean patients (Begovich et al., 2007 ). Thus, no diagnostic certainty can be obtained by measuring genes on the HLA axis. None of the HLA haplotypes causes disease 100% of the time and none causes any one immune disease consistently. Patterns of haplotype variation are more suggestive of a regional infectious model rather than a model in which an illness is caused by widespread inherited variation of HLA haplotypes. Potential Systematic Errors in the Interpretation of the Metagenome Primers selected for most epidemiological studies are chosen without consideration for whether they might amplify DNA from the genomes of any intracellular microbes. As artist Pablo Picasso once remarked, "Computers are useless. They can only give you answers." If a software program fails to make provision for the possibility that a metagenome might also be present, the chances of a false-positive increase significantly during the process of genomic analysis. Similarities between bacterial and human genes will likely cause the analysis software to not assemble the genomic data properly. The likelihood of error is not minuscule as there is growing evidence of molecular mimicry, homology between bacterial and human proteins. For example, significant sequence homology exists between human carbonic anhydrase II and alpha-carbonic anhydrase of H. pylori (Guarneri et al., 2005 ). Moreover, the homologous segments contain the binding motif of the HLA molecule DRB1*0405. The group A streptococcal carbohydrate antigen N -acetyl-glucosamine is able to cross react with cardiac myosin (Cunningham, 2003 ). Microbes including E. coli , H. pylori , P. aeruginosa , Cytomegalovirus, and H. influenzae share sequence homology with human pyruvate dehydrogenase complex-E2, which has been tied to the development of primary biliary cirrhosis (Bogdanos et al., 2004 ). The core oligosaccharides of low-M(r) LPSs of C. jejuni serotypes that are associated with the development of Guillain–Barré syndrome are homologous to neural gangliosides. Before we can be certain that all measured SNPs and HLA haplotypes are a product of only the human genome and not the metagenome, researchers must begin to actively choose PCR primer pairs that are unlikely to amplify microbial DNA. Primers need to be certified not only to amplify a unique sequence in the human genome, but also as not likely to amplify genes from any of the thousands of bacterial and viral genomes in the metagenomic databases. Although PCR amplification usually involves more than one stage of genomic selectivity, the increasing use of arrays of RNA probes increases the likelihood that a fragment of metagenomic RNA will unexpectedly match a probe, and increases the possibility of a false-positive being signaled for the particular SNP being sought. Antibodies in Response to Microbial DNA Autoimmune diseases are characterized largely by the presence of autoantibodies. Although autoantibodies were reported over a century ago, many scientists at the time were unwilling to accept the possibility that the immune system attacks its own cells. Ehrlich argued that autoimmunity was not possible and proposed the theory of horror autotoxicus to describe the body's innate aversion to immunological self-destruction by the production of autoantibodies. Now that humans are understood to be the product of multiple genomes, increasing evidence supports Ehrlich's view. When an innate immune system is forced to respond to a chronic microbiota, the resulting cascade of chemokines and cytokines will also stimulate an adaptive response. Antibodies are notoriously polyspecific, and the likelihood that antibodies generated to target metagenomic fragments will also target human proteins (target "self") is finite. A litany of research implies a re-evaluation of the "autoantibody." Recently researchers have shown that certain autoantibodies are created in response to several well-studied pathogens. "Lupus-specific autoantibodies" such as RO, La, or dsDNA are often generated in response to EBV (Barzilai et al., 2007 ). Similarly, anti-EBNA-1 antibodies are able to bind lupus-specific autoantigens such as Sm or Ro (Harley and James, 2006 ). Casali and Slaughter found that, in humans, EBV is a polyclonal B cell activator, and in vitro transformation with EBV results in production of rheumatoid factor (RF) (Casali et al., 1987 ; Slaughter et al., 1978 ). Possnett et al. argues that high titers of RF are not only associated with severe rheumatoid arthritis but also appear in a number of other diseases including viral, bacterial, and parasitic infections (Posnett and Edinger, 1997 ). Maturation of RF can be initiated by chronic infections (Djavad et al., 1996 ). For example, patients with subacute bacterial endocarditis, which is frequently tied to the presence of Streptococcus , also often present with high levels of RF (Russell et al., 1992 ). Williams et al. showed that once the offending infectious agent is removed with antibiotic therapy, the RF disappears (Williams and Kunkel, 1962 ). Similarly, the autoimmune disease thrombocytopenic purpura (ITP) is mediated by what are considered to be anti-platelet autoantibodies. However, Asahi et al. found that eradication of H. pylori is effective in increasing platelet count in nearly half of ITP patients infected with the bacterium (Asahi et al., 2006 ). Barzilai and team also found that Hepatitis B shares amino acid sequences with different autoantigens, further suggesting that the so-called autoantibodies may actually be created in response to pathogens (Barzilai et al., 2007 ). Autoantibodies have been detected in patients without autoimmune disease during periods of infection. Berlin et al. collected sera from 88 patients with acute infections (41 bacterial, 23 viral, 17 parasitic, and 7 rikettsial (Berlin et al., 2007 )). Elevated titers of autoantibodies including annexin-V, prothrombin, ASCA, ANA, or antiphospholipid antibodies were detected in approximately half of the subjects, with 34 individuals harboring elevated titers of at least two "autoantibodies." EBV, E. coli , Salmonella, and other pathogens discussed above are easily detected by culture-based methods that may explain why their presence has already been tied to "autoantibody" production. Yet the vast majority of the human microbiota is understudied. This means that what we now consider to be autoantibodies in many autoimmune diagnoses may also indicate the presence of pathogens, but pathogens that have yet to be fully characterized and named. Thus, in addition to looking for antibodies to well-characterized pathogens, it is also important that we look for antibodies indicating the presence of the underlying chronic microbiota, some of which we may also be mistaking for autoantibodies. Like the pathogens that may create them, many of these antibodies may not yet be detected by standard testing. If this is the case, hundreds of pathogen-induced antibodies may exist and impact the autoimmune disease state, but the possible detection and correlation of such antibodies with specific components of the microbiota remains difficult until a much larger portion of the microbiota has been characterized. Because many antibodies demonstrate a high degree of polyspecificity, it is possible that in some cases, antibodies initially directed against pathogens could also attack human tissue (Christen et al., 2010 ). According to Bozic, oxidative alterations, affecting either the hypervariable region or the receptor site of IgGs, may influence their functions (Bozic et al., 2007 ). Similarly, McIntyre reported the appearance and disappearance of antiphospholipid antibodies subsequent to oxidation reactions in human blood (McIntyre, 2004 ). Dimitrov et al. have shown that a fraction of antibodies present in all healthy individuals begin to recognize large number of self-antigens only after a transient exposure to certain protein-destabilizing conditions, including low or high pH, high salt concentration, chaotropic factors, and redox-active agents (Dimitrov et al., 2008 ). This points to at least one mechanism whereby the oxidative stress that accumulates in inflamed tissue could be at least partly responsible for the apparent polyspecificity of antibodies and autoantibodies. Molecular mimicry, in which peptides from pathogens share sequence or structural similarities with self-antigens, may also contribute to autoantibody production. Lekakh et al. found that autoantibodies with polyspecific activity in the serum of healthy donors were able to cross react with DNA and lipopolysaccharides (LPSs) of widespread species of bacteria including E. coli , P. aeruginosa , Shigella boydii , and Salmonella (Lekakh et al., 1991 ). CD' is classified as an autoimmune condition based largely on the presence of perinuclear anti-nuclear cytoplasmic antibodies (pANCAs) in patients with the disease. Yet recently two major species of proteins immunoreactive to pANCA were detected in bacteria from anaerobic libraries, implicating colonic bacteria as a possible trigger for the disease-associated immune response. We previously discussed how factors other than calorie consumption may contribute to the weight gain often associated with autoimmune or inflammatory conditions. Fetissov et al. studied healthy women for the presence of IgG or IgA autoantibodies directed against 14 key regulatory peptides and neuropeptides, including ghrelin, leptin, vasopressin, and insulin (Fetissov et al., 2008 ). They found numerous cases of sequence homology among these peptides and the protein structures of over 30 microbes including Lactobacilli , H. pylori , E. coli , Yersinia pseudotuberculosis , and Listeria monocytogenes , suggesting that the "autoantibodies" were actually the result of molecular mimicry. In the presence of certain pathogenic bacterial species, the production of IgG autoantibodies directed against ghrelin was upregulated, suggesting a complex interplay between autoantibody levels and microbial antigens. This suggested that these so-called "autoantibodies" might not only have physiologic implications in pathways that regulate hunger and satiety but also represent a key link between the gut and the brain. An increasing number of studies also show that what are currently perceived as autoantibodies can often be detected in the so-called healthy individuals years before the full presentation of an autoimmune disease state. Many researchers now espouse that early detection of these antibodies can help predict whether or not such a "healthy" person will develop an autoimmune disease. For example, in an 8-year prospective study, Swaak et al. examined the diagnostic significance of anti-double-stranded deoxyribonucleic acid (anti-dsDNA) determination in a group of 441 patients without systemic lupus erythematosus (SLE) whose sera were found to contain antibodies to dsDNA on routine screening (Swaak and Smeenk, 1985 ). Within 1 year, 69% (304) of these patients fulfilled the preliminary American Rheumatism Association (ARA) criteria for SLE. Eighty-two of the remaining 137 patients were followed up for several years. At the end of the study, 52% of these patients had also developed SLE. The team concluded that about 85% of patients without SLE with anti-dsDNA in the circulation would develop SLE within a few years. Another recent study of blood from 441 healthy Portuguese blood donors found autoantibodies for rheumatoid factor, anticyclic citrunillated peptides, anti-mitochondria, anti- Sacharomyces cerevisiae , ANA, anti-TTG, and anti-Beta2-glycoprotein (Tavares-Ratado et al., 2009 ). More than 30% of the blood contained one or more of the antibodies, 4% exhibited two antibodies, and nearly 1% had three or more antibodies present. It is clear that sub-clinical autoimmune disease is much more common than previously thought. This gradual presentation of an increasing number of the so-called "autoantibodies" in the years before a patient meets the official criteria for an autoimmune diagnosis supports the model of successive infection described earlier – pathogenic components of the microbiota gradually accumulate over the course of a lifetime until bacterial, viral, and phage load reaches a level at which a diagnosis can be made. It also supports the contention that individuals perceived as "healthy" may still harbor and accumulate pathogenic microbes that will eventually lead to an inflammatory diagnosis, or a process associated with "aging." Indeed, it is possible that any antibodies that damage "self" do so as an unintended polyspecific consequence of their activity against the metagenomic pathogens. Therapies in the Era of the Metagenome At the 2008 International Conference on Metagenomics in La Jolla, CA, James Kinross of the Imperial College of London began his speech with the following statement: "We surgeons have been operating on the gut for literally thousands of years and the microbiota has just been this extraordinary elephant in the room. We seem to have completely ignored the fact that we've co-evolved with thousands of bacteria over millions of years and that they somehow may be important to our health. As doctors, we routinely do terrible things to the microbiota and I'm sure this has implications for our health." Although most physicians are undoubtedly well intentioned, Kinross is correct in that many clinicians are generally not offered training that would keep them up to date with advances in metagenomics. The result is that many doctors still believe that nonmucosal surfaces of the body are largely sterile and that bacteria and other pathogens are not driving factors in the autoimmune processes. Instead, the standard of care for patients with autoimmune disease continues to be corticosteroids and TNF-alpha blocking medications. According to a 2008 report, TNF-alpha inhibitors accounted for 80% of RA drug sales in the United States, France, Germany, Italy, Spain, the United Kingdom, and Japan. Use of these immunosuppressants is still grounded in the theory that autoimmune disease results from an overly exuberant immune response and these drugs are administered without consideration for the presence of a metagenome. Whether helpful or harmful, there is no question that by dramatically slowing the immune response, such therapies must necessarily and profoundly affect the composition, development, and stability of the human microbiota. Despite the copious use of these immunosuppressant drugs in autoimmune conditions, they provide, at best, short-term palliation. Gottlieb et al. showed that steroid use causes relapse in sarcoidosis (Gottlieb et al., 1997 ). Additionally, there are no definitive studies showing corticosteroids improve long-term prognosis in the treatment of chronic inflammatory illness, nor is there any demonstrated reduction in mortality. Van den Bosch and Grutters write, "Remarkably, despite over 50 years of use, there is no proof of long-term (survival) benefit from corticosteroid treatment" (Grutters and van den Bosch, 2006 ). On the contrary, one of the side effects of TNF-alpha inhibitors is an increased risk of tuberculosis. Several studies have shown that TNF-alpha production is required for the proper expression of acquired specific resistance following infection with M. tuberculosis (Allie et al., 2008 ; Arend et al., 2003 ). So if we inhibit TNF-alpha expression, we would expect a long-term increase in the prevalence of not only tuberculosis, but also in any of the autoimmune or inflammatory diseases already associated with chronic forms of mycobacteria and other bacteria (Bull et al., 2003 ; Burnham et al., 1978 ). The failure of these first-line therapies to cure "autoimmunity," and the range of detrimental side effects associated with their use, suggests that slowing the immune response of patients with autoimmune disease is counterproductive, allowing microbial populations to develop unchecked. Now that autoimmune conditions are more widely understood as illnesses in which myriad pathogens may trigger or drive the disease process, efforts to target the root cause of autoimmune disease should instead be targeted toward activating the innate immune response, not suppressing it. Our own work (Perez, 2008 ) offers an example of the results of stimulating rather than suppressing the innate immune response of patients with autoimmune disease. Over the past 7 years, we have observed the effects of an experimental therapy for autoimmune disease that uses the VDR agonist olmesartan to reverse pathogen-induced VDR dysregulation. Subjects are also administered sub-inhibitory bacteriostatic antibiotics, which weaken bacterial ribosomes so that pathogens can more easily be targeted by the reactivated immune system. Nearly all of the hundreds of patients to start the therapy reported the predicted increase in specific symptoms of their autoimmune diagnosis. After months, or sometimes years, of dealing with these symptomatic flares, the very symptoms that waxed and waned in synchronism with antibiotic administration began to disappear, resulting in improvement and, in many cases, eventual resolution of the disease process. This response has been noted in the widely varying diagnoses of sarcoidosis, rheumatoid arthritis, lupus, Type 2 diabetes, uveitis, Hashimoto's thyroiditis, ankylosing spondylitis, chronic fatigue syndrome, and fibromyalgia among others. The often dramatic elevations in disease activity observed among study subjects – particularly during the early stages of therapy – cannot be attributed to side effects of the protocol medications, as individually the drugs are well known and unremarkable (Schwocho and Masonson, 2001 ). Additionally, when healthy individuals have been administered the same medications they do not suffer any similar symptoms. The most viable hypothesis for these temporary surges in disease symptoms and inflammatory markers is that treatment medications allow the immune system to mount an effective attack on an intracellular microbiota, such as the microbiota observed by Wirostko et al. It is reasonable to expect that when intraphagocytic pathogens are killed, some of the host cells will also undergo apoptosis, phagocytosis, or simply disintegration, leading to an increase in inflammation. For over 100 years, researchers have noted that the death of acute and persistent pathogens is accompanied by a surge in inflammation. They have attributed the temporary rise in inflammation to an increase in endotoxin and cytokine release upon bacterial death. Known as the Jarisch–Herxheimer reaction, or immunopathology, this phenomenon has been previously demonstrated after antibiotic administration in diseases including tuberculosis (Cheung and Chee, 2009 ), borreliosis (Vidal et al., 1998 ), tick-borne relapsing fever (Mitiku and Mengistu, 2002 ), multiple sclerosis (Kissler, 2001 ), Whipple disease (Peschard et al., 2001 ), and syphilitic alopecia (Pareek, 1977 ), among others. Zinkernagel also observed immunopathology in the mice he had infected with a persistent neuro-active virus (Zinkernagel et al., 2009 ). Similarly, immune reconstitution inflammatory syndrome (IRIS) is a condition seen in some cases of AIDS following the use of antiretroviral drugs. As the immune system begins to recover, it responds to previously acquired opportunistic infections with an overwhelming inflammatory response that, like the immunopathological reaction we observe, makes the symptoms of the infection temporarily worse (Shelburne et al., 2002 ). At this point in time, the exact species or forms of bacteria potentially killed by any one subject in our own study cohort remain unknown. As the focus of the HMP moves beyond the mucosal surfaces, and catalogs L-forms and other intracellular species within body tissues, a clearer picture of disease pathogenesis will emerge. However, as long as patients continue to report improvement and recovery, determining the exact nature of pathogens being targeted by the therapy has not been a high priority, given the limited resources currently allocated to this research team. Some subjects in the cohort have reported drops in viral titers, suggesting that once the immune system is no longer burdened by the pathogenic components of the bacterial microbiota, it may regain the ability to target chronic viruses as well. This suggests that treatments that reverse immunosuppression caused by the bacterial microbiota might also prove useful in mitigating viral virulence. Our research suggests that while some people report being "allergic" to certain bacteriostatic antibiotics, what they perceive as an "allergy" may actually be immunopathological reactions. For example, there are reports of minocycline "inducing lupus" (Geddes, 2007 ). A more logical explanation may be that certain patients harbor persistent bacterial species that predispose for sub-clinical lupus. When minocycline is administered, some of these bacteria are killed, resulting in immunopathological reactions that are mistakenly interpreted as clinical manifestation of the disease. What we have initiated needs further testing. However, the reports of profound immunopathological reactions in autoimmune subjects imply the need to re-examine whether palliative drugs actually provide long-term benefit for patients with autoimmune disease. Whether at the doctor's office or the health food store, patients with autoimmune conditions continually seek out palliative drugs or supplements that successfully reduce symptoms by lowering inflammation. Yet, if bacteria drive the pathogenesis of autoimmune inflammation, and chronic bacterial death invariably results in temporary increases in discomfort, then treatments that mitigate symptoms may well do so at the expense of proliferation in pathogenic components of the microbiota. Commonly used immunosuppressive compounds include vitamin D, which, although its immunosuppressive properties have now been identified (Arnson et al., 2007 ), is now viewed as the ultimate inexpensive wonder drug (Holick, 2008 ). Frequent use of vitamin D, as well as other substances that slow immune activity, could at least partially account for the recently increased prevalence of nearly every autoimmune disease (Luque et al., 2006 ). L-Form Bacteria: An Often Overlooked Component of the Microbiota Certain stages of the bacterial life cycle result in the loss of the cell wall. L-form bacteria are often less than 0.2 μm in diameter (Domingue and Woody, 1997 ) and are therefore difficult to view with a standard optical microscope. Not only do these L-form variants fail to succumb to antibiotics that target the bacterial cell wall, but those antibiotics also encourage the formation of L-forms. "Treatment with penicillin does not merely select for L-forms (which are penicillin-resistant) but actually induces L-form growth," states Josep Casadesus of the University of Sevilla (Casadesus, 2007 ). In fact, researchers deliberately culture classical forms of bacteria in conjunction with various beta-lactam antibiotics in order to create L-forms (Mattman, 2000 ). The ability of the L-form to flourish in the face of treatment with the beta-lactam antibiotics points to a mechanism by which acute bacterial forms can mutate into latent mutants that may cause disease at a later time. Some researchers have deemed the conversion into the L-form state to be a universal property of bacteria (Gumpert and Taubeneck, 1983 ). Joseleau-Petit et al. showed that classical forms of bacteria transform into the L-form only if they are denied the ability to form a normal cell wall (Joseleau-Petit et al., 2007 ). The beta-lactam antibiotics work toward this end by blocking the creation of penicillin-binding proteins (PBPs) – proteins responsible for forming the cross-linked chains associated with a peptidoglycan-derived cell wall. When the ability of the PBPs to create a full cell wall is blocked, the cells also become spherical and osmosensitive. Recently, Glover et al. performed the first systematic genetic evaluation of genes and pathways involved in the formation and survival of unstable L-form bacteria (Glover et al., 2009 ). Microarray analysis of L-form versus classical bacterial colonies revealed many upregulated genes of unknown function as well as multiple overexpressed stress pathways shared in common with persister cells and biofilms. Dell'Era et al. also observed cell division and changes in gene expression in stable L. monocytogenes L-forms (Dell'Era et al., 2009 ). Since the discovery of the L-forms in 1935 (Kleineberger-Nobel, 1951 ), they have been described in hundreds of publications. Yet because researchers are only just beginning to use molecular tools to study the L-form, they are still seldom factored into the mix of microbes that compose the human microbiome. However, over the years, L-forms have been implicated in dozens of diseases of unknown etiology, including RA, multiple sclerosis, sarcoidosis, glomerulonephritis, idiopathic hematuria, interstitial cystitis, rheumatic fever, and syphilis – as well as a large number of chronic and relapsing infections (Domingue and Woody, 1997 ; Mattman, 2000 ). A Research Consideration: Men Are Not Tall MiceWithout Tails The emerging role of the human microbiota implies a reconsideration of certain long-standing and frequently invoked models of disease. According to Javier Mestas of University of California, Irvine, "There has been a tendency to ignore differences and in many cases, perhaps, make the assumption that what is true in mice is necessarily true in humans. By making such assumptions we run the risk of overlooking aspects of human immunology that do not occur, or cannot be modeled, in mice" (Mestas and Hughes, 2004 ). Murine models are still used in an effort to understand most autoimmune and inflammatory conditions, despite the obvious differences between the murine and human immune systems. For example, there are major differences in the Toll-like receptors. TLR1-9 exists in both mouse and man, although TLR8 detects single-stranded RNA in man and has no known function in the mouse. TLR10 exists in humans only; it is a degenerative pseudo-gene in the mouse. TLR11, 12 and 13 in mice do not exist in man and their function is not yet well defined. Analysis of the human and murine VDR offers other examples of discord between man and mouse. Marshall's molecular dynamics emulation showed that the drug olmesartan, a putative VDR agonist, binds into a different conformation in the murine VDR to that of Homo sapiens (Marshall, 2008 ), calling into question the whole concept of drug safety testing in murine models. While the human VDR transcribes dozens of genes necessary for a robust innate immune response, including many key AMPs, the VDR does not similarly control the murine innate immune system. The murine innate immune response is dependent on a cascade of nitric oxide functions in a manner yet to be fully understood (Bogdan, 2001 ). Although mice have VDRs, the homology differs, and they express different genes than the human VDR. For example, the gene encoding the calcium-binding protein osteocalcin is "robustly" transcribed by the VDR in humans, but not in mice. Brahmachary et al. showed that the rat VDR does not express the cathelicidin AMPs, marking an important difference in the way the two species target invading pathogens (Brahmachary et al., 2006 ). Gombart et al. recently expanded on the finding by providing evidence of an evolutionarily fixed, Alu-mediated divergence in steroid hormone nuclear receptor gene regulation between humans/primates and other mammals (Gombart et al., 2009 ). This divergence, which placed the cathelicidin pathway under VDR control only in humans and closely related primates, remained under purifying selection for the past 55–60 million years, and yet even cathelicidin in primates is not identical to that in man. Eventually, the pathway evolved to become a key component of a novel innate immune response unique to human infection. Because the murine VDR does not express cathelicidin, there is less of an evolutionary incentive for components of the murine microbiota to dysregulate its expression. This suggests that the survival mechanisms employed by the human and murine microbiotas may be very different. Thus, the intermingling of murine and human biologies in the literature hinders our ability to fully understand nuclear receptor control of the AMPs and other key aspects of innate immunity. Discussion The prevailing theory of autoimmune disease, which dictates that the body creates autoantibodies that attack its own cells, was developed during an era when culture-based methods vastly underestimated the number of microbes capable of persisting in and on Homo sapiens . The advent of culture-independent tools such as 16S RNA sequencing, single cell sampling, and pyrosequencing has opened the door to an era of discovery. Rather than a sterile compartment, the human body is now known to teem with thousands of species of bacteria, viruses, and phages. In addition to persisting on the body's external surfaces, these microbes survive in the blood and in many of the tissues, which become inflamed during autoimmune disease, suggesting that what were once thought to be "autoimmune" processes may instead result from the presence of persistent microbes. Metagenomics is allowing us to study these microbes in the tissues within which they naturally persist, where they can be examined in the context of other microbes in their community. A more exact understanding of how networks of microbes can interact to cause disease has superseded Koch's postulates, which stipulate that a single microbe causes a single disease. While diseases were once categorized largely on the basis of symptom presentation, they can now be classified based on their underlying genetics. Yet the expression of key human genes is continually altered by a plethora of microbial metabolites through an almost imponderable number of interactions. These metabolites, some of which are created by bacteria considered to be "friendly" or innocuous, can directly drive the pathogenesis of autoimmune disease by altering the expression of genes such as ACE and PTN22, genes associated with diagnoses including rheumatoid arthritis, lupus, diabetes mellitus, myocardial infarction, renal tubular dysgenesis, and Alzheimer's. It is becoming apparent that autoimmune processes cannot be fully understood if the human genome is studied in isolation. An understanding of the interactions between the human genome and the metagenome calls for a more nuanced understanding of the microbiota. Classifying certain microbes as purely commensal may underrepresent the full spectrum of their actions. Indeed, harmless species of bacteria and viruses can easily acquire virulent plasmids via horizontal gene transfer or homologous recombination. The microbiota has persisted in and on the human body for millennia. It has evolved to slow the host immune response in order to ensure microbial survival. Pathogens such as M. tuberculosis , Borrelia , EBV, and HIV have evolved to dysregulate the VDR nuclear receptor, inhibiting expression of the beta-defensin and cathelicidin AMPs along with TLR2. Flow-on effects from VDR dysregulation can further alter AMP expression via (at least) the alpha-thyroid, androgen, and glucocorticoid nuclear receptors. This may result in the immunosuppression and hormonal imbalances characteristic of many autoimmune diagnoses. The bacteria that cause autoimmune disease likely accumulate over a lifetime, with individuals picking up pathogens with greater ease over time, as the immune response becomes increasingly constrained. Successive infection dictates that even people with the same autoimmune diagnosis are unlikely to present with identical clusters of symptoms and helps explain the high levels of comorbidity observed among these patients. Common autoimmune comorbidities include inflammatory conditions such as cardiovascular disease, along with mental diagnoses such as depression or anxiety, suggesting these conditions may also be driven by the microbiota. Thus, insights gained from studying microbial composition in autoimmune disease can accelerate research in other areas of medicine. Recently, several studies have shown the presence of "autoantibodies" in autism with antinuclear antibody seropositivity showing a significant positive association with disease severity, mental retardation, and electroencephalogram abnormalities. Rather than assign autism to the end of a growing list of autoimmune diagnoses, this knowledge might be better used as a basis on which to further explore the role that components of the microbiota may play in driving the pathogenesis of disease. Analyzing autoimmune disease through the lens of metagenomics calls for a re-evaluation of the autoantibody. Polyspecific autoantibodies are increasingly being associated with elements of the microbiota, making it likely that the term "autoimmune" will soon lose its diagnostic utility. When a disabled immune system is forced to respond to the presence of a chronic microbiota, the resulting cascade of cytokines and chemokines will stimulate an adaptive immune response. The adaptive immune system will then proceed to generate antibodies to fragments of DNA generated by apoptosis or phagocytosis of infected cells. This is supported by studies showing that the so-called autoantibodies such as RO, La, dsDNA, and RF can be created in response to various bacterial and viral pathogens. Autoantibodies are often observed before a patient becomes fully symptomatic with an autoimmune diagnosis, reflecting the gradual accumulation of persistent microbes. Rather than focusing on phenotypes and subsets of the metagenome, microbiome research may instead benefit from broader approaches geared toward understanding shared mechanisms of persistence. Translational medicine should aim at cutting through barriers among specialities, even between biologists and clinicians, so that more of the pieces of the emerging jigsaw of disease etiology can drop into place, and autoimmune disease patients can fully benefit from the insights gained from metagenomic science.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167786/

Non‐proteolytic functions of microbial proteases increase pathological complexity

Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non‐proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non‐proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain‐like proteases. We explore how these non‐proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non‐covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes. 1 Introduction Proteases (also known as peptidases and proteinases) are ubiquitous enzymes that cleave peptide bonds in polypeptide chains and profoundly influence protein shape, size, composition, localisation, turnover and degradation. The effects of such control include post‐cleavage gain of function, loss of function or switching between functions. Proteases are thereby responsible for a multitude of physiological processes in all organisms 1 . Classification of proteases is based on the amino acid at the catalytic site for substrate hydrolysis, which can either be serine, cysteine, threonine, aspartic or glutamic acid. For some proteases, the mode of action is mediated through coordination with a metal ion, so they are called metallopeptidases. Further classification is determined by the location of the protease cleavage site: proteases cutting at the amino terminus are called aminopeptidases, at the carboxyl terminus are called carboxypeptidases and towards the centre are called endopeptidases. Though uncommon, some proteases possess both endo‐ and exopeptidase function 2 . Microbial proteases have been implicated in prominent pathological roles and are recognised as important virulence factors. This has driven research aimed at developing inhibitors that target their catalytic sites 3 and proteomics has been used to identify promising vaccine candidates for significant human pathogens, including Streptococcus pyogenes 4 and Plasmodium falciparum 5 , and animal pathogens, such as Vibrio harveyi 6 . The virulence modulating functions of proteases are to be expected, as host immune effector and signalling molecules are often proteins or peptides. However, as proteases often function within intricate proteolytic networks, their exact targets and biological actions are often difficult to ascertain or even predict. Some proteases perform additional non‐proteolytic actions, due to either the presence of extra functional domains or alternative conformations. Proteases are macromolecules with a limited number of residues required for catalysis. The remaining structure is generally made up of non‐catalytic domains, including sorting signals that direct correct cellular localisation, autoinhibitory prodomains to prevent premature activation and ancillary domains that facilitate protein–protein interactions 7 . Alternative protease conformations may involve ligand binding, post‐translational modifications, changes in molecular environment or oligomerisation 8 . The changes to conformational structure may also be facilitated by intrinsically disordered regions, which provide structural malleability, permitting various and often opposing functions to reside within the same region 9 . Enzymes that have multiple activities, with each activity dependent on cell localisation, substrate availability or interactions with other proteins, are said to be "moonlighting". A moonlighting protein, by simplest definition, is a protein capable of performing two or more distinct biological functions within a single polypeptide chain 9 . It has been hypothesised that moonlighting functions arise from selective pressure on an advantageous novel function 10 , and may benefit an organism by increasing complexity without resorting to genome expansion 11 . To differentiate moonlighters from other multifunctional proteins, bone fide members do not encompass products of gene fusions, protein fragments, or splice variants 8 . That is, whilst a proprotein can be cleaved to produce a protease and another functional unit, a moonlighting protease can exert both functions without prior processing. Additionally, moonlighting functions are not always conserved among the same protein across different organisms 12 . Terminology used to describe the presence of more than one catalytic function in a protein molecule is not applied consistently in the literature 11 , 13 , 14 . To clarify, bifunctional enzymes have two or more catalytic sites that function independently of each other; moonlighting proteins possess catalytic activity and an additional structural or regulatory function, while promiscuous enzymes have a single catalytic domain capable of catalysing more than one chemical reaction. These enzyme classifications are not exclusive − as some moonlighting proteases also have promiscuous and/or bifunctional activities. In bacteria, the majority of moonlighting proteins identified have been cytosolic "housekeeping" proteins, including those involved in the glycolytic pathway and tricarboxylic acid cycle all of which have been reviewed extensively 15 , 16 , 17 , 18 . These intracellular proteins demonstrate additional functions when secreted or localised to the cell surface, and were originally termed anchorless or non‐classically secreted proteins as translocation occurred in the absence of any known signal peptide. The debate remains whether lysis or a novel secretion mechanism is responsible for the presence of cytosolic proteins on the cell surface 19 . Regardless, these proteins have frequently been shown to act as adhesins, invasins, and modulators of host immune responses, thereby playing important roles in colonisation, dissemination, and avoiding immune clearance 16 . Identifying which proteins have moonlighting functions has relied heavily on serendipitous empirical data 20 . Identification of additional functions of proteases and determination of their role in virulence remains a challenge. Such knowledge has implications for host–pathogen interactions and drug design strategies. Global methods for identifying moonlighting proteins include bioinformatic analysis and mass spectrometry. Bioinformatic analysis is better suited to identifying proteins that have evolved new function via the acquisition of additional domains rather than those that have acquired it via conformational changes. This is because there are currently no reliable prediction algorithms 21 , no apparent moonlighting motifs 22 and no obvious links to sequence homology between moonlighting and non‐moonlighting proteins of the same class and between species 23 . Furthermore, a moonlighting function can be introduced by as few as four amino acid substitutions 20 . Detailed genomic comparisons of high and low virulence isogenic bacteria describe the accumulation of point mutations in surface accessible proteins 16 , suggesting that minor sequence variation may have profound implications for protein function, including many moonlighting functions that are yet to be described. Mass spectrometric and proteomic analyses have been previously identified as key techniques in the search for moonlighting proteins due to their ability to identify the presence of a protein in varying cell types, locations and multiprotein complexes 24 . Bacterial moonlighting proteins with functions important in virulence would be expected to be found within microbial exoproteomes. There have been hundreds of microbial surfaceome and secretome studies published to date. Many report the presence of cytosolic proteins in extracellular fractions, providing tantalising suggestions of possible moonlighting function. Microbial proteases have established roles as virulence factors, yet the presence of traditionally cytosolic proteases within exoproteomes has generally been overlooked. Microbial proteases that have more than one function additional to proteolytic activity will form the focus of this review. Examples will be grouped into five main types of functions that the proteases have as additional to their proteolytic activity: adhesin, plasminogen‐binding, chaperone (Cpn), receptor, and non‐proteolytic catalysis. We will discuss how these additional functions may contribute to pathogenesis and point to future research directions by highlighting other potential moonlighters within microbial exoproteomes. 2 Proteases as virulence factors Although their role was once thought to be limited to degradation of unneeded proteins, proteases are now known to be mediators of many pathological processes. In these roles, their proteolytic function ranges from less specific cleavage events, such as degradation of extracellular matrix (ECM) components for nutrient acquisition 25 , to precise cleavage events. Through peptide bond cleavage many pathogens are capable of modulating host innate immune responses, including disruption of cascade systems, such as complement, coagulation and fibrinolysis, and inactivation of immunoglobulins, cytokines, chemokines and antimicrobial peptides 26 . Additionally, some proteases can affect virulence directly by acting as toxins 27 or indirectly through the processing of other virulence factors 28 . Pathogens indisputably utilise proteolysis to establish successful infections and this has driven research into developing protease inhibitors as therapeutic agents. Efficacious examples include human immunodeficiency virus aspartyl protease inhibitors, which are used to prevent the progression of acquired immunodeficiency syndrome 29 , and two serine protease inhibitors used for the treatment of chronic hepatitis C infections 30 . Like all surface accessible proteins, extracellular proteases will be under selective pressure from the host immune response and thus are likely to evolve faster that proteins strictly residing within the cytosol. The virulence attributes of proteases are not solely reliant on their enzymatic capabilities and their role in pathogenesis may be significantly underestimated. Moonlighting functions may increase pathogenic complexity and impede current drug design strategies. The literature contains many examples of microbial proteases that demonstrate additional functions independent of proteolysis, several of which have been described specifically as "moonlighting" activities (Table 1 ). Table 1 Examples of proteases with additional non‐proteolytic functions Name (Synonyms) Protease classification (Family) Other function(s) Organism Predicted localisation Found to be surface exposed Aminopeptidase S* (SgAP) Metalloaminopeptidase (M28) Hydrolyses ester bonds Streptomyces griseus 100 Unknown Unknown C5a peptidase (scpA, scpB) Serine endopeptidase (S8) Adhesin Streptococcus agalactiae 50 Cell wall Yes Cpn60.2* (chaperone 60, HSP60/5) Trypsin‐like endopeptidase Chaperone Mycobacterium leprae 90 Cytoplasmic Yes DPPIV (dipeptidyl peptidase 4, Xaa‐Pro dipeptidyl peptidase, PepX, X‐prolyl dipeptidyl aminopeptidase) Serine oligopeptidase (S9/S15) Adhesin Porphyromonas gingivalis 55 , 124 Streptococcus suis 56 Extracellular Cell wall Yes Yes Endopeptidase O (PepO, oligopeptidase 01, endopeptidase 2) Metalloendopeptidase (M13) Adhesin; accelerates plasminogen activation Streptococcus pneumonia 65 Cytoplasmic Yes FtsH* (high‐frequency lysogenisation by phage λ, hflB) Metalloendopeptidase Chaperone; AAA + ATPase Multiple organisms Outer membrane Yes Gingipain (HRgpA/RgpB/Kgp, Arg/Lys‐gingipain) Cysteine endopeptidase (C25) Adhesin; Exopeptidase Porphyromonas gingivalis 57 , 58 , 59 Outer membrane and periplasmic space Yes Glutamyl aminopeptidase* (aminopeptidase A) Metalloaminopeptidase (M42) Adhesin; Accelerates plasminogen activation Mycoplasma hyopneumoniae 66 Cytoplasmic Yes HtrA* (high‐temperature requirement A, DegP, protease Do) Serine endopeptidase (S1) ChaperoneAdhesin Multiple organisms Borrelia burgdorferi 86 Cytoplasmic/periplasmicPeriplasmic Yes Leucine aminopeptidase* (cytosol aminopeptidase, LAP3, leucyl peptidase) Metalloaminopeptidase (M17) Adhesin; Accelerates plasminogen activation; DNA‐binding proteinDNA‐binding protein M. hyopneumoniaeEscherichia coli 69 CytoplasmicCytoplasmic YesNo Lon* ATP‐ dependent serine (proteinase, endopeptidase La, LonA, Lon type1) Serine endopeptidase (S16) Chaperone; AAA + ATPase; DNA‐binding protein ssrA tagging Multiple organisms Mycoplasma spp. 74 Cytoplasmic No NS3/4A (hepacivirin, Cpro‐2) Serine endopeptidase (S29) Helicase; nucleotide‐binding protein Hepatitis C virus 125 Viral capsid, host endoplasmic reticulum Unknown Papain‐like protease (PLP) Cysteine endopeptidase (C16) DUB; deISGylating; blocks cytokine induction Coronaviridae and Arterivirdae viral families Host cytoplasm Unknown PgtE (protein E) Aspartic endopeptidase (A26) Adhesin Salmonella enterica senovar Typhimurium 42 Outer membrane Yes Prolidase (Xaa‐Pro dipeptidase, imidodipeptidase, gamma peptidase, pepQ, proline dipeptidase Metalloaminopeptidase (M24) Hydrolyses ester bonds Alteromonas haloplanktis 101 Cytoplasmic Unknown Pla* (plasminogen activator) Aspartic endopeptidase (A26) Adhesin; coagulase Yersinia pestis 8 , 42 , 44 , 47 Outer membrane Yes OmpT (protease 7, protease A) Aspartic endopeptidase (A26) Adhesin Escherichia coli 42 Outer membrane Yes SpeB (streptopain, erythrotoxin B, histase, In1β converatse, Streptococcus peptidase A) Cysteine endopeptidase (C10) Adhesin Streptococcus pyogenes 39 Extracellular Yes TAP (thioesterase I) Serine endopeptidase Thioesterase, arylesterase, esterase, lysophospholipase Escherichia coli 102 Unknown Unknown John Wiley & Sons, Ltd. This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. 2.1 Proteases with a role in adhesion Adherence to the mucosal epithelium frequently marks the first essential stage of infection and successful colonisation. This adherence is often facilitated by adhesive surface‐bound molecules called adhesins that bind to the ECM covering mucosal cells. ECM typically comprises a heterogeneous mixture of glycoproteins, such as collagen, elastin, fibronectin laminin, and glycosaminoglycans (GAGs) 31 . These molecules are targeted by microbial adhesins to aid colonisation and invasion. There is also increasing evidence suggesting that microbial binding of ECM molecules elicits more than just adhesion. For example, fibronectin not only provides a bridge between the intracellular cytoskeleton and other ECM components, but is also involved in inflammation by augmenting chemotaxis and leucocyte function, cell signalling 32 and cell invasion 33 . Fibronectin‐binding proteins are known virulence factors in important human pathogens, such as Staphylococcus aureus and group A Streptococcus 32 , and Campylobacter jejuni fibronectin‐binding proteins have been implicated in membrane ruffling stimulation and consequent host cell invasion by activating the epidermal growth factor pathway 34 . The co‐localisation of fibronectin and veterinary pathogen Mycoplasma hyopneumoniae on the ciliated lining of the porcine respiratory tract has also been demonstrated 35 . GAG binding is also associated with more than adhesion, including facilitating intracellular survival in macrophages 36 , and central nervous system entry 37 . The importance of identifying microbial adhesins is highlighted by the drive to implement anti‐adhesion methods as novel therapeutics against infections, particularly within the context of ever increasing antibiotic resistance 38 . In addition to proteins with primary adhesin functions, many examples of surface‐associated moonlighting proteins have also demonstrated adhesive interactions with ECM components. It has been proposed that this redundant adhesive activity of moonlighters is significant in host–microbe interactions 8 , 16 . The literature contains many examples of cytosolic proteins, particularly metabolic enzymes that moonlight on the cell surface, which may skew the perception of moonlighting to consideration of only multifunctional proteins found in atypical cellular compartments. Moreover, as proteases typically feature on microbial cell surfaces, those that are characteristically cytosolic may have been overlooked. Here, we examine several proteases that exhibit adhesin functions secondary to their proteolytic function. Streptococcal pyrogenic exotoxin B (SpeB), also known as streptopain, of S. pyogenes is a cysteine protease with a role as a toxin that induces apoptosis and reduces phagocytic function in human monocytes. This important virulence factor is also responsible for 'strepadhesin' activity, with capacity to bind to thyroglobulin, asialofetuin and submaxillary mucin 39 . SpeB also binds laminin, a major glycoprotein found within mammalian basement membranes (BMs). Laminin is targeted by many pathogens during the initial stages of adherence and during tissue invasion 40 . Notably, experiments using cysteine protease inhibitors and inactive forms of SpeB have demonstrated that adhesion to glycoproteins was independent of proteolytic function. These data suggested that the adhesive motif and the substrate‐binding site are distinct entities 39 . SpeB adhesive properties are essential during the initial stages of infection by group A Streptococcus ; however, after systemic infection they become problematic due to their affinity for α1 antitrypsin. SpeB binding of α1 antitrypsin mediates contact‐system mediated bacterial killing, so the protease is downregulated after dissemination into the blood stream 41 . Plasminogen activator (Pla) of Yersinia pestis is another multifunctional protease that binds ECM molecules. As its name suggests, the protease can directly cleave plasminogen at the Arg 561 ‐Val 562 bond, releasing the active serine protease plasmin. Plasmin is a proinflammatory agonist that dissolves fibrin clots, activates collagenase and gelatinase precursors, and degrades laminin 42 . Whilst laminin is not degraded by Pla, the protease binds strongly to this BM protein 43 . Thus, Pla‐mediated laminin adhesion and plasminogen activation work together to produce localised proteolysis at the mammalian BM, facilitating Y. pestis invasion. Pla also binds the GAG heparan sulphate, and collagen 44 , though the latter has been contested 45 . Pla further mediates invasiveness by utilising the lectin receptor DEC‐205 to promote macrophage uptake, thus leading to dissemination 46 . Pla also possess some weak coagulase activity but whether this function plays a role in Y. pestis pathogenesis has not been assessed 47 . The serine protease C5a peptidase of Streptococcus agalactiae (group B streptococcus) is a cell wall anchored protease with a highly specific catalytic function of degrading neutrophil chemotaxin C5a 48 . Whilst its proteolytic function has been shown to lie within the N terminus, the protease has two Arg‐Gly‐Asp motifs implicated in integrin binding and the C‐terminus possess three fibronectin type III domains 49 . Group B streptococcus C5a peptidase has a high affinity for fibronectin, an interaction shown to be biologically significant as evidenced by streptococcal strains defective in the expression of C5a peptidase having 50% less fibronectin‐binding capacity than wild type strain 50 . Furthermore, mice immunised with C5a peptidase poorly colonise the lungs 51 . Dipeptidyl peptidase IV (DPPIV, also known as cluster of differentiation 26) is a serine exopeptidase widely studied in eukaryotes largely due to its role in tumour biology 52 and in regulating blood glucose levels 53 . It is considered a moonlighting protease as it exerts numerous additional functions to proteolysis that are dependent on cell localisation. These functions include acting as an adhesin, receptor, co‐stimulatory protein and a role in apoptosis 54 . The functions of DPPIV in microbes, moonlighting or otherwise, are less well established. However, DPPIV in P. gingivalis and Streptococcus suis is known to function as an adhesin. In P. gingivalis , DPPIV binds fibronectin and this interaction is required to establish colonisation 55 . Fibronectin was not degraded by DPPIV and its binding was evident even in proteolytic inactive DPPIV mutants. Furthermore, binding fibronectin inhibited fibroblast recruitment and it was suggested that this could affect recovery from damage caused by inflammation 55 . Similarly, DPPIV in S. suis bound fibronectin and the inactivation of the DPPIV gene caused a significant decrease in the pathogens cellular adhesion ability and impaired virulence 56 . In the same study, mice that were vaccinated with purified DPPIV and challenged with a virulent strain of S. suis had a 100% survival rate after a week compared to a 100% mortality in the unvaccinated control group. The gingipains of P. gingivalis are a group of trypsin‐like cysteine proteases that contribute to periodontitis. The group consists of a lysine‐specific gingipain (Kgp) and two arginine‐specific gingipains, HRgpA and RgpB. Through their catalytic activity all three proteases are responsible for major tissue destruction and deregulation of immune cascade pathways, inducing inflammation and periodontal bone loss 57 . Incidentally, the arginine gingipains exhibit both endo‐ and exopeptidase functions 58 . HRgpA and Kgp also possess four non‐catalytic haemagglutinin/adhesin (HA) regions. These structural domains together with the proteolytic active sites work in concert to bind and degrade haemoglobin, which is fundamental for both the growth and virulence of P. gingivalis 59 . The HA domain also has a high affinity for phospholipids, fibrinogen, laminin, fibronectin and fibrin, and is thought to contribute to the greater tissue destruction capacity of HRgpA than RgpA 57 . As the HA regions enhance proteolytic activity, the gingipains differ from the other examples provided above, which appear to bind substrates that are not degraded by their protease function. 2.2 Proteases that bind plasminogen The contact activation pathway of the coagulation cascade is an important component of innate immunity. Following contact activation by antigenic stimuli, a series of events occurs that ultimately results in the deposition of a fibrin mesh that captures invading pathogens and exposes them to bactericidal peptides released from platelets and kinins 60 , 61 . Once the infection has been cleared by leukocytes, the proenzyme plasminogen is cleaved by two serine proteases, tissue plasminogen activator (tPA) and urokinase plasminogen activator, to release serine protease plasmin, which degrades the fibrin clot 62 . Despite this formidable defence system, numerous bacteria have developed strategies to avoid fibrin clot confinement and subsequent immune clearance. One common mechanism involves the expression of surface proteins that bind plasminogen. These recruit host plasminogen to the cell surface and utilise host tPA and urokinase plasminogen activator to induce localised plasmin activity, thus facilitating fibrinolysis 60 , 63 . Interestingly, some of the most extensively characterised plasminogen‐binding proteins have been cytosolic or glycolytic pathway proteins localised to the cell surface by a yet unknown secretion mechanism 63 . Several extracellular and intracellular proteases have been shown to bind plasminogen, including endopeptidase O (PepO) from Streptococcus pneumoniae , two aminopeptidases from M. hyopneumoniae and DPPIV from Pseudomonas aeruginosa 64 , 65 , 66 . PepO from S. pneumoniae 65 and glutamyl aminopeptidase 66 and leucine aminopeptidase (LAP) [67] from M. hyopneumoniae were shown to bind plasminogen in a dose‐dependent manner and that bound plasminogen was readily converted to plasmin in the presence of tPA. Notably, in the absence of tPA, these proteases did not directly cleave plasminogen to release plasmin. Adhesion was inhibited in the presence of a lysine analogue ɛ‐aminocaproic acid indicating a reliance on surface‐exposed lysine residues for plasminogen binding 68 . Using a variety of complementing methodologies, including immunofluorescence, enzymatic shaving and surface biotinylation, all three proteases were found to be surface exposed despite the absence of any transmembrane domains, secretion motifs or signal peptides. Additionally, both M. hyopneumoniae aminopeptidases bound heparin, and the LAP also bound DNA, a moonlighting function shared with LAP from E. coli in which it has been linked to site‐specific recombination and transcriptional control of the carAB operon 69 . PepO is a multifunctional adhesin by additionally binding fibronectin, and the inactivation of the pepO gene in S. pneumoniae was correlated with a significant decrease in epithelial invasiveness 65 . 2.3 Proteases with Cpn functions Molecular Cpns are proteins that assist in non‐covalent folding or unfolding of other macromolecules. They often work in conjunction with proteases to degrade malfunctioning proteins by enabling greater substrate access by the protease. In all organisms, collaborative protease activity and Cpn protein quality control is driven by ATPases associated with diverse cellular activities (AAA + ). AAA + proteins are a class of Cpn‐like ATPases involved in many vital cellular processes. Through ATP hydrolysis AAA + proteins are able to generate a mechanical force that enables the remodelling of bound substrates 70 . In bacteria, AAA + ‐mediated protein degradation is usually performed by an AAA + protein (such as ClpA or ClpX) that binds and unfolds a target substrate and then delivers it to a protease such as ClpP for proteolysis. There are currently five known AAA + ‐containing degradation pathways, comprising ClpXP, ClpAP, HsIUV, FtsH and Lon. For most systems, the AAA + domain and protease are encoded on separate polypeptides; however, both are present within a single polypeptide in FtsH and Lon 71 . Lon also possesses a non‐catalytic DNA‐binding domain 72 . As many aspects of both Lon and FtsH have been studied extensively, including the mechanics behind their functions 70 , only a summary will be provided here. Lon proteins are generally intracellular and are classified, with some contention, as belonging to the S16 family of serine proteases. Contention arises because the catalytic site is composed of a Ser–Lys dyad as opposed to the classical Ser–His–Asp triad typical of other serine proteases, and also because traditional inhibitors of serine proteases poorly impede Lon activity. Nevertheless, Lon plays multiple important roles in cells. Firstly, Lon degrades aged, misfolded or aggregated proteins 73 . In the genome‐reduced mycoplasmas, Lon mimics the function of the ClpXP proteolysis system. In most other bacteria, ClpXP is responsible for the tmRNA‐mediated aging and ribosome rescue system that appends an ssrA tag to the C‐terminus of proteins targeted for proteolysis. Having lost genes encoding ClpX and ClpP, the mycoplasmal Lon protease not only executes ssrA tagging, but does so in a more efficient manner than typically seen for ClpXP 74 . Secondly, Lon plays an important role in various regulatory circuits by processing substrates involved in cell division, capsule synthesis and DNA transcription 73 . Thirdly, Lon acts in regulating virulence factors in pathogenic bacteria. For example, in Pseudomonas aeruginosa mutational analysis has shown Lon to have roles in swimming motility, twitching motility, biofilm formation, antibiotic susceptibility, lung colonisation and quorum sensing 75 . Regulation of virulence gene expression relies on the site‐specific DNA‐binding capability of Lon 76 . Virulence genes regulated by Lon include those carried on Salmonella pathogenicity island 1 77 and the hpmBA and zapA genes of Proteus mirabilis 78 . This highlights the need to have a comprehensive understanding of the multifunctional nature of some proteases. FtsH (also known as high‐frequency lysogenisation by phage λ or hflB) is the only membrane‐bound protein classified as both an AAA + and zinc‐dependent metalloprotease 79 . Due to this localisation, identified FtsH substrates are both cytosolic and membrane bound. FtsH substrates include components involved in the transcription of the heat shock operon, protein translocation and membrane biosynthesis 80 . In Streptomyces peucetius , FtsH exerts both Cpn and protease activities upon the DrrA and DrrB proteins, which form an ABC‐type permease involved in the export of antibiotics 81 . The effects of ftsH gene knockouts appear to differ drastically between bacterial species. In E. coli, Bradyrhizobium japonicum and Helicobacter pylori ftsH mutations are lethal, yet in some bacteria they cause only mild growth impairments. However, all mutations in ftsH examined thus far have been associated with increased susceptibility to heat shock and osmotic stress 82 . Dual protease and Cpn function is not limited to members of the AAA + superfamily. High‐temperature requirement A (HtrA), also known as DegP, is a serine protease that is crucial for protein homeostasis in bacteria. Like Lon and FtsH, HtrA combines proteolytic and remodelling activities on a single polypeptide chain. The switch between the two functions is mediated by a structural loop covering the active sites and the movement of this loop appears to be temperature dependent 83 . HtrA is an important virulence factor in a number of Gram positive and Gram negative bacteria, an effect that has mainly been attributed to the protein's role in protein quality control and the heat shock response 84 . However, despite traditionally being thought to be strictly periplasmic in Gram negative bacteria, HtrA is in the growing list of proteins found to be surface exposed or secreted via an unknown pathway. On the surface, HtrA protease function has been linked to dissemination in a number of pathogens by degrading the cell junctional protein E‐cadherin 85 and in the pathogenesis of Lyme disease by binding and degrading aggrecan, a proteoglycan required for joint function 86 . One reason that molecular Cpns have been a focus of moonlighting research is that despite traditionally thought to be intracellular proteins, many are expressed extracellularly and act as cell signalling proteins or in bacterial adherence to host cells 87 . There is abundant evidence of Cpns functioning as chemoattractants and cytokine modulators 88 . In prokaryotes, only four Cpns have thus far been shown to communicate with host cells – Cpn 60 (also known as HSP60 and GroEL), Cpn10 (HSP10), DnaK (HSP70) and peptidyloprolyl isomerase 15 . Of particular interest is the Cpn60.2 mycobacterial molecular Cpn, the first identified Cpn with signalling properties 12 . Cpn60.2 moonlights as an adhesin that facilitates alveolar macrophage entry by binding the transmembrane protein CD43 found on the surface of several immune effector cells 89 . However, it has also become apparent that not all Cpn60.2 proteins possess the same moonlighting functions. A study using a murine asthma model found that out of the five Cpn60 proteins from various bacteria, only Cpn60.2 from Mycobacterium leprae had an anti‐inflammatory effect, despite 95% sequence identity with the M. tuberculosis Cnp60.2, which was also tested 12 . Inhibition of inflammation and hyper‐responsiveness induced by M. leprae Cpn60.2 may be linked to the fact that this Cpn is the only Cpn60 protein with demonstrable proteolytic activity 90 , since proteases are established modulators of innate immune responses. The proteolytic activity is catalytically related to HslV, which is the protease counterpart in the HslVU AAA + protein degradation system mentioned above. Dual functioning protease/Cpns may be more common than currently appreciated. One study illustrated that both eukaryotic and prokaryotic aspartic proteases possess structural similarities to AAA + by way of a double‐ψ barrel domain at the N‐terminus. This study also demonstrated that both pepsin and human immunodeficiency virus 1 protease exhibited inherent Cpn functions when their proteolytic action was blocked 91 . 2.4 Proteases that act as receptors Proteolytic cleavage events are well known to activate or inactivate various host receptor molecules and modulate host immune responses to microbial invasion. Several proteases are known to cleave protease‐activated receptors, Toll‐like receptors, receptors within the complement cascade and various cytokine receptors, thereby disrupting communication within the innate immune system and its signalling pathways 60 . In contrast, there are considerably fewer published examples of a protease functioning as a receptor. However, some proteases are known to function as receptors for viruses on host cells. The coronaviridae are a large family of viruses that can cause disease in both humans and other animals ranging from the common cold, the recently discovered Middle East respiratory syndrome and severe acute respiratory syndrome. Exopeptidases are the sole identified class of receptors required for the process of attachment and membrane fusion of coronaviruses within host tissues 92 . The host cell receptor for severe acute respiratory syndrome is the metallocarboxypeptidase angiotensin‐converting enzyme 2. This receptor also binds the Human (H) CoV‐NL63 virus. Fibronectin‐binding DPP4 is a receptor for Middle East respiratory syndrome 92 . Perhaps the most well‐known example of a protease viral receptor is aminopeptidase N (also known as CD13), as it is targeted by a number of human, porcine, feline and canine CoVs, as well as porcine transmissible gastroenteritis virus and feline infectious peritonitis virus 93 . It is not known whether microbial exopeptidases function as receptors for viruses. However, exopeptidase counterparts are found in the exoproteomes of a number of bacteria including S. pneumoniae, Burkholderia pseudomalleli and Moraxella catarrhalis (Table 2 ). Furthermore, synergism occurs between viral and bacterial coinfections, particularly in the respiratory tract 94 . Over the last ten years, it has been established that viral infections aid bacteria colonisation and dissemination through various means, including disrupting mucocilliary function 95 , impairing immune effector cell function 96 and providing easy access to adhesin targets and nutrients via epithelial damage 97 . As the respiratory tract is a reservoir to a number of pathobionts, one hypothesis is that these microbes secrete potential viral protease receptors or chemoattractants to enhance their own colonisation. Table 2 Examples of cytosolic/periplasmic proteases found in microbial exoproteomes Protease NCBI locus Organism Reference Additional conversed domains Aminopeptidase I BB0366 Borrelia burgdorferi 126 Aminopeptidase M XP_001682593 Leishmania major 127 Aminopeptidase N AAK99510 ZP_01769994 ADG61634 Streptococcus pneumoniae Burkholderia pseudomallei Moraxella catarrhalis 123 128 129 Aminopeptidase T NP_830199 Bacillus cereus 130 Carboxypeptidase Q18AU1_CLOD6 Clostridium difficile 131 Aminoacylase 1 C‐terminal processing peptidase BP0609 Bordetella pertussis 132 ClpB BP1198 NP_371049 Bordetella pertussis Staphylococcus aureus 132 133 AAA + ATPase ClpC AAT29160 Bacillus anthracis 134 AAA + ATPase ClpP BB2254 ZP_21019400 NP_371292 Bordetella bronchiseptica Escherichia coli Staphylococcus aureus 132 133 133 AAA + ATPase Collagenase YP_005053876 Filifactor alocis 136 Deblocking aminopeptidase NP_834277 Bacillus cereus 130 DPPIII XP_001687573 Leishmania major 127 DPPIV BP0906 AAL80949 XP_001681251 Bordetella pertussis Pyrococcus furiosus Leishmania major 132 137 127 DPPVII YP_004045754 Riemerella anatipestifer 138 Endopeptidase O AAL00295 Streptococcus pneumoniae 123 Glutamyl aminopeptidase AAL00485 Streptococcus pneumoniae 123 HtrA OE7_04918 BP2434 P0C0V0 CAB13147 CAD00819 ? BB0104 Haemophilus parasuis Bordetella pertussis Escherichia coli Bacillus subtilis Listeria monocytogenes Brucella abortus Borrelia burgdorferi 139 132 140 141 142 143 126 l ‐Aminopeptidase d ‐amidase/ d ‐esterase YP_005055390 Filifactor alocis 136 Endo‐type 6 aminohexanoate‐oligomer hydrolase Leucine aminopeptidase XP_001681531 Leishmania major 127 Lon protease YP_005054101 Filifactor alocis 136 AAA + ATPase Oligopeptidase F AAT30293/ AAT30449 Bacillus anthracis 134 O‐Sialoglycoprotein endopeptidase YP_005054583 Filifactor alocis 136 Molecular chaperone Peptidase T NP_833858 CAB15918 AAT33487 Bacillus cereus Bacillus subtilis Bacillus anthracis 130 141 134 Protease 1 PFPI_PYRFU Pyrococcus furiosus 137 Glutamine aminotransferase Protease HsIV YP_513676 Francisella tularensis 144 Pyroglutamyl peptidase YP_513002 Francisella tularensis 144 Signal peptide peptidase A YP_514244 Francisella tularensis 144 TAP P0ADA1 Escherichia coli 139 Thermostable carboxypeptidase NP_831344 CBP1_PYRFU XP_001686015 Bacillus cereus Pyrococcus furiosus Leishmania major 130 137 127 Thimet oligopeptidase XP_001684161 Leishmania major 127 Transpeptidase ZP_21022097 Escherichia coli 135 Transglycosylase Xaa‐His dipeptidase NP_372275 Staphylococcus aureus 133 Xaa‐Pro aminopeptidase CBA64059/ CBA64694 XP_003722629 Clostridium difficile Leishmania major 131 127 Creatinase Prolidase Zinc metallopeptidases BP1721 CD630_28300 XP_001685175 BB0627 Bordetella pertussis Clostridium difficile Leishmania major Borrelia burgdorferi 132 145 127 126 LysM Acetylornithine deacetylase John Wiley & Sons, Ltd. This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. 2.5 Proteases with additional catalytic functions The potential for proteases to possess additional, unexpected activities adds layers of complexity to our understanding of physiological processes. Although there are several examples of microbial proteases that catalyse reactions unrelated to proteolysis, information about their biological significance is scarce due to the intrinsic challenges faced when ascertaining specificity, redundancies and natural substrates in complex biological samples. Site‐directed mutagenesis, gene deletion studies and molecular modelling have been key in identifying unusual modes of action for proteases. For example, the papain‐like proteases (PLPs) from the viral families Coronaviridae and Arterivirdae are responsible for processing the critical replicase polyprotein into its numerous functional units 98 . Structural similarities of PLPs to deubiquitinating enzymes showed that PLPs not only function as deubiquitinating enzymes, but are also able to block induction of several endogenous proinflammatory cytokines, including IFNβ, CCL5 and CXCL10 98 . Another essential multifunctional viral protein is the hepatitis C virus NS3/4A, which possesses both chymotrypsin‐like serine protease and helicase activities in two individual domains. As this protein is vital for viral propagation, it has been the target for the small‐molecule inhibitors that have been commercially available for hepatitis C virus treatment since 2011 99 . For other multi‐catalytic proteases, biological relevance is less certain and studies often infer evolutionary and environmental advantages to an affinity for several different types of substrates. For example, there are enzymes that are able to hydrolyse different types of bonds. An aminopeptidase from the antibiotic‐producing bacterium Streptomyces griseus 100 and a prolidase from the marine bacterium Alteromonas haloplanktis 101 are capable of cleaving amide, phosphoester and phosphofluorinate bonds. This is interesting as the mechanics behind peptide and ester hydrolysis are dissimilar 100 . Another enzyme capable of mechanistically diverse activities is the thioesterase I (TAP) of E. coli , which exhibits thioesterase, arylesterase, esterase, lysophospholipase and serine protease activities 102 . However, despite an ability to interact with a great number of different substrates, the role of TAP in vivo is not yet well established. Finally, the extremophilic bacterial species Deinococcus radiodurans, Pyrococcus horikkoshii and Thermoanaerobacter tengcongensis all possess an enzyme that is both an aminoacylase (hydrolysing N‐acetylated amino acids) and a carboxypeptidase. It has been suggested that the role of these multifunctional enzymes is in enabling peptidolytic growth in these organisms 103 . 2.1 Proteases with a role in adhesion Adherence to the mucosal epithelium frequently marks the first essential stage of infection and successful colonisation. This adherence is often facilitated by adhesive surface‐bound molecules called adhesins that bind to the ECM covering mucosal cells. ECM typically comprises a heterogeneous mixture of glycoproteins, such as collagen, elastin, fibronectin laminin, and glycosaminoglycans (GAGs) 31 . These molecules are targeted by microbial adhesins to aid colonisation and invasion. There is also increasing evidence suggesting that microbial binding of ECM molecules elicits more than just adhesion. For example, fibronectin not only provides a bridge between the intracellular cytoskeleton and other ECM components, but is also involved in inflammation by augmenting chemotaxis and leucocyte function, cell signalling 32 and cell invasion 33 . Fibronectin‐binding proteins are known virulence factors in important human pathogens, such as Staphylococcus aureus and group A Streptococcus 32 , and Campylobacter jejuni fibronectin‐binding proteins have been implicated in membrane ruffling stimulation and consequent host cell invasion by activating the epidermal growth factor pathway 34 . The co‐localisation of fibronectin and veterinary pathogen Mycoplasma hyopneumoniae on the ciliated lining of the porcine respiratory tract has also been demonstrated 35 . GAG binding is also associated with more than adhesion, including facilitating intracellular survival in macrophages 36 , and central nervous system entry 37 . The importance of identifying microbial adhesins is highlighted by the drive to implement anti‐adhesion methods as novel therapeutics against infections, particularly within the context of ever increasing antibiotic resistance 38 . In addition to proteins with primary adhesin functions, many examples of surface‐associated moonlighting proteins have also demonstrated adhesive interactions with ECM components. It has been proposed that this redundant adhesive activity of moonlighters is significant in host–microbe interactions 8 , 16 . The literature contains many examples of cytosolic proteins, particularly metabolic enzymes that moonlight on the cell surface, which may skew the perception of moonlighting to consideration of only multifunctional proteins found in atypical cellular compartments. Moreover, as proteases typically feature on microbial cell surfaces, those that are characteristically cytosolic may have been overlooked. Here, we examine several proteases that exhibit adhesin functions secondary to their proteolytic function. Streptococcal pyrogenic exotoxin B (SpeB), also known as streptopain, of S. pyogenes is a cysteine protease with a role as a toxin that induces apoptosis and reduces phagocytic function in human monocytes. This important virulence factor is also responsible for 'strepadhesin' activity, with capacity to bind to thyroglobulin, asialofetuin and submaxillary mucin 39 . SpeB also binds laminin, a major glycoprotein found within mammalian basement membranes (BMs). Laminin is targeted by many pathogens during the initial stages of adherence and during tissue invasion 40 . Notably, experiments using cysteine protease inhibitors and inactive forms of SpeB have demonstrated that adhesion to glycoproteins was independent of proteolytic function. These data suggested that the adhesive motif and the substrate‐binding site are distinct entities 39 . SpeB adhesive properties are essential during the initial stages of infection by group A Streptococcus ; however, after systemic infection they become problematic due to their affinity for α1 antitrypsin. SpeB binding of α1 antitrypsin mediates contact‐system mediated bacterial killing, so the protease is downregulated after dissemination into the blood stream 41 . Plasminogen activator (Pla) of Yersinia pestis is another multifunctional protease that binds ECM molecules. As its name suggests, the protease can directly cleave plasminogen at the Arg 561 ‐Val 562 bond, releasing the active serine protease plasmin. Plasmin is a proinflammatory agonist that dissolves fibrin clots, activates collagenase and gelatinase precursors, and degrades laminin 42 . Whilst laminin is not degraded by Pla, the protease binds strongly to this BM protein 43 . Thus, Pla‐mediated laminin adhesion and plasminogen activation work together to produce localised proteolysis at the mammalian BM, facilitating Y. pestis invasion. Pla also binds the GAG heparan sulphate, and collagen 44 , though the latter has been contested 45 . Pla further mediates invasiveness by utilising the lectin receptor DEC‐205 to promote macrophage uptake, thus leading to dissemination 46 . Pla also possess some weak coagulase activity but whether this function plays a role in Y. pestis pathogenesis has not been assessed 47 . The serine protease C5a peptidase of Streptococcus agalactiae (group B streptococcus) is a cell wall anchored protease with a highly specific catalytic function of degrading neutrophil chemotaxin C5a 48 . Whilst its proteolytic function has been shown to lie within the N terminus, the protease has two Arg‐Gly‐Asp motifs implicated in integrin binding and the C‐terminus possess three fibronectin type III domains 49 . Group B streptococcus C5a peptidase has a high affinity for fibronectin, an interaction shown to be biologically significant as evidenced by streptococcal strains defective in the expression of C5a peptidase having 50% less fibronectin‐binding capacity than wild type strain 50 . Furthermore, mice immunised with C5a peptidase poorly colonise the lungs 51 . Dipeptidyl peptidase IV (DPPIV, also known as cluster of differentiation 26) is a serine exopeptidase widely studied in eukaryotes largely due to its role in tumour biology 52 and in regulating blood glucose levels 53 . It is considered a moonlighting protease as it exerts numerous additional functions to proteolysis that are dependent on cell localisation. These functions include acting as an adhesin, receptor, co‐stimulatory protein and a role in apoptosis 54 . The functions of DPPIV in microbes, moonlighting or otherwise, are less well established. However, DPPIV in P. gingivalis and Streptococcus suis is known to function as an adhesin. In P. gingivalis , DPPIV binds fibronectin and this interaction is required to establish colonisation 55 . Fibronectin was not degraded by DPPIV and its binding was evident even in proteolytic inactive DPPIV mutants. Furthermore, binding fibronectin inhibited fibroblast recruitment and it was suggested that this could affect recovery from damage caused by inflammation 55 . Similarly, DPPIV in S. suis bound fibronectin and the inactivation of the DPPIV gene caused a significant decrease in the pathogens cellular adhesion ability and impaired virulence 56 . In the same study, mice that were vaccinated with purified DPPIV and challenged with a virulent strain of S. suis had a 100% survival rate after a week compared to a 100% mortality in the unvaccinated control group. The gingipains of P. gingivalis are a group of trypsin‐like cysteine proteases that contribute to periodontitis. The group consists of a lysine‐specific gingipain (Kgp) and two arginine‐specific gingipains, HRgpA and RgpB. Through their catalytic activity all three proteases are responsible for major tissue destruction and deregulation of immune cascade pathways, inducing inflammation and periodontal bone loss 57 . Incidentally, the arginine gingipains exhibit both endo‐ and exopeptidase functions 58 . HRgpA and Kgp also possess four non‐catalytic haemagglutinin/adhesin (HA) regions. These structural domains together with the proteolytic active sites work in concert to bind and degrade haemoglobin, which is fundamental for both the growth and virulence of P. gingivalis 59 . The HA domain also has a high affinity for phospholipids, fibrinogen, laminin, fibronectin and fibrin, and is thought to contribute to the greater tissue destruction capacity of HRgpA than RgpA 57 . As the HA regions enhance proteolytic activity, the gingipains differ from the other examples provided above, which appear to bind substrates that are not degraded by their protease function. 2.2 Proteases that bind plasminogen The contact activation pathway of the coagulation cascade is an important component of innate immunity. Following contact activation by antigenic stimuli, a series of events occurs that ultimately results in the deposition of a fibrin mesh that captures invading pathogens and exposes them to bactericidal peptides released from platelets and kinins 60 , 61 . Once the infection has been cleared by leukocytes, the proenzyme plasminogen is cleaved by two serine proteases, tissue plasminogen activator (tPA) and urokinase plasminogen activator, to release serine protease plasmin, which degrades the fibrin clot 62 . Despite this formidable defence system, numerous bacteria have developed strategies to avoid fibrin clot confinement and subsequent immune clearance. One common mechanism involves the expression of surface proteins that bind plasminogen. These recruit host plasminogen to the cell surface and utilise host tPA and urokinase plasminogen activator to induce localised plasmin activity, thus facilitating fibrinolysis 60 , 63 . Interestingly, some of the most extensively characterised plasminogen‐binding proteins have been cytosolic or glycolytic pathway proteins localised to the cell surface by a yet unknown secretion mechanism 63 . Several extracellular and intracellular proteases have been shown to bind plasminogen, including endopeptidase O (PepO) from Streptococcus pneumoniae , two aminopeptidases from M. hyopneumoniae and DPPIV from Pseudomonas aeruginosa 64 , 65 , 66 . PepO from S. pneumoniae 65 and glutamyl aminopeptidase 66 and leucine aminopeptidase (LAP) [67] from M. hyopneumoniae were shown to bind plasminogen in a dose‐dependent manner and that bound plasminogen was readily converted to plasmin in the presence of tPA. Notably, in the absence of tPA, these proteases did not directly cleave plasminogen to release plasmin. Adhesion was inhibited in the presence of a lysine analogue ɛ‐aminocaproic acid indicating a reliance on surface‐exposed lysine residues for plasminogen binding 68 . Using a variety of complementing methodologies, including immunofluorescence, enzymatic shaving and surface biotinylation, all three proteases were found to be surface exposed despite the absence of any transmembrane domains, secretion motifs or signal peptides. Additionally, both M. hyopneumoniae aminopeptidases bound heparin, and the LAP also bound DNA, a moonlighting function shared with LAP from E. coli in which it has been linked to site‐specific recombination and transcriptional control of the carAB operon 69 . PepO is a multifunctional adhesin by additionally binding fibronectin, and the inactivation of the pepO gene in S. pneumoniae was correlated with a significant decrease in epithelial invasiveness 65 . 2.3 Proteases with Cpn functions Molecular Cpns are proteins that assist in non‐covalent folding or unfolding of other macromolecules. They often work in conjunction with proteases to degrade malfunctioning proteins by enabling greater substrate access by the protease. In all organisms, collaborative protease activity and Cpn protein quality control is driven by ATPases associated with diverse cellular activities (AAA + ). AAA + proteins are a class of Cpn‐like ATPases involved in many vital cellular processes. Through ATP hydrolysis AAA + proteins are able to generate a mechanical force that enables the remodelling of bound substrates 70 . In bacteria, AAA + ‐mediated protein degradation is usually performed by an AAA + protein (such as ClpA or ClpX) that binds and unfolds a target substrate and then delivers it to a protease such as ClpP for proteolysis. There are currently five known AAA + ‐containing degradation pathways, comprising ClpXP, ClpAP, HsIUV, FtsH and Lon. For most systems, the AAA + domain and protease are encoded on separate polypeptides; however, both are present within a single polypeptide in FtsH and Lon 71 . Lon also possesses a non‐catalytic DNA‐binding domain 72 . As many aspects of both Lon and FtsH have been studied extensively, including the mechanics behind their functions 70 , only a summary will be provided here. Lon proteins are generally intracellular and are classified, with some contention, as belonging to the S16 family of serine proteases. Contention arises because the catalytic site is composed of a Ser–Lys dyad as opposed to the classical Ser–His–Asp triad typical of other serine proteases, and also because traditional inhibitors of serine proteases poorly impede Lon activity. Nevertheless, Lon plays multiple important roles in cells. Firstly, Lon degrades aged, misfolded or aggregated proteins 73 . In the genome‐reduced mycoplasmas, Lon mimics the function of the ClpXP proteolysis system. In most other bacteria, ClpXP is responsible for the tmRNA‐mediated aging and ribosome rescue system that appends an ssrA tag to the C‐terminus of proteins targeted for proteolysis. Having lost genes encoding ClpX and ClpP, the mycoplasmal Lon protease not only executes ssrA tagging, but does so in a more efficient manner than typically seen for ClpXP 74 . Secondly, Lon plays an important role in various regulatory circuits by processing substrates involved in cell division, capsule synthesis and DNA transcription 73 . Thirdly, Lon acts in regulating virulence factors in pathogenic bacteria. For example, in Pseudomonas aeruginosa mutational analysis has shown Lon to have roles in swimming motility, twitching motility, biofilm formation, antibiotic susceptibility, lung colonisation and quorum sensing 75 . Regulation of virulence gene expression relies on the site‐specific DNA‐binding capability of Lon 76 . Virulence genes regulated by Lon include those carried on Salmonella pathogenicity island 1 77 and the hpmBA and zapA genes of Proteus mirabilis 78 . This highlights the need to have a comprehensive understanding of the multifunctional nature of some proteases. FtsH (also known as high‐frequency lysogenisation by phage λ or hflB) is the only membrane‐bound protein classified as both an AAA + and zinc‐dependent metalloprotease 79 . Due to this localisation, identified FtsH substrates are both cytosolic and membrane bound. FtsH substrates include components involved in the transcription of the heat shock operon, protein translocation and membrane biosynthesis 80 . In Streptomyces peucetius , FtsH exerts both Cpn and protease activities upon the DrrA and DrrB proteins, which form an ABC‐type permease involved in the export of antibiotics 81 . The effects of ftsH gene knockouts appear to differ drastically between bacterial species. In E. coli, Bradyrhizobium japonicum and Helicobacter pylori ftsH mutations are lethal, yet in some bacteria they cause only mild growth impairments. However, all mutations in ftsH examined thus far have been associated with increased susceptibility to heat shock and osmotic stress 82 . Dual protease and Cpn function is not limited to members of the AAA + superfamily. High‐temperature requirement A (HtrA), also known as DegP, is a serine protease that is crucial for protein homeostasis in bacteria. Like Lon and FtsH, HtrA combines proteolytic and remodelling activities on a single polypeptide chain. The switch between the two functions is mediated by a structural loop covering the active sites and the movement of this loop appears to be temperature dependent 83 . HtrA is an important virulence factor in a number of Gram positive and Gram negative bacteria, an effect that has mainly been attributed to the protein's role in protein quality control and the heat shock response 84 . However, despite traditionally being thought to be strictly periplasmic in Gram negative bacteria, HtrA is in the growing list of proteins found to be surface exposed or secreted via an unknown pathway. On the surface, HtrA protease function has been linked to dissemination in a number of pathogens by degrading the cell junctional protein E‐cadherin 85 and in the pathogenesis of Lyme disease by binding and degrading aggrecan, a proteoglycan required for joint function 86 . One reason that molecular Cpns have been a focus of moonlighting research is that despite traditionally thought to be intracellular proteins, many are expressed extracellularly and act as cell signalling proteins or in bacterial adherence to host cells 87 . There is abundant evidence of Cpns functioning as chemoattractants and cytokine modulators 88 . In prokaryotes, only four Cpns have thus far been shown to communicate with host cells – Cpn 60 (also known as HSP60 and GroEL), Cpn10 (HSP10), DnaK (HSP70) and peptidyloprolyl isomerase 15 . Of particular interest is the Cpn60.2 mycobacterial molecular Cpn, the first identified Cpn with signalling properties 12 . Cpn60.2 moonlights as an adhesin that facilitates alveolar macrophage entry by binding the transmembrane protein CD43 found on the surface of several immune effector cells 89 . However, it has also become apparent that not all Cpn60.2 proteins possess the same moonlighting functions. A study using a murine asthma model found that out of the five Cpn60 proteins from various bacteria, only Cpn60.2 from Mycobacterium leprae had an anti‐inflammatory effect, despite 95% sequence identity with the M. tuberculosis Cnp60.2, which was also tested 12 . Inhibition of inflammation and hyper‐responsiveness induced by M. leprae Cpn60.2 may be linked to the fact that this Cpn is the only Cpn60 protein with demonstrable proteolytic activity 90 , since proteases are established modulators of innate immune responses. The proteolytic activity is catalytically related to HslV, which is the protease counterpart in the HslVU AAA + protein degradation system mentioned above. Dual functioning protease/Cpns may be more common than currently appreciated. One study illustrated that both eukaryotic and prokaryotic aspartic proteases possess structural similarities to AAA + by way of a double‐ψ barrel domain at the N‐terminus. This study also demonstrated that both pepsin and human immunodeficiency virus 1 protease exhibited inherent Cpn functions when their proteolytic action was blocked 91 . 2.4 Proteases that act as receptors Proteolytic cleavage events are well known to activate or inactivate various host receptor molecules and modulate host immune responses to microbial invasion. Several proteases are known to cleave protease‐activated receptors, Toll‐like receptors, receptors within the complement cascade and various cytokine receptors, thereby disrupting communication within the innate immune system and its signalling pathways 60 . In contrast, there are considerably fewer published examples of a protease functioning as a receptor. However, some proteases are known to function as receptors for viruses on host cells. The coronaviridae are a large family of viruses that can cause disease in both humans and other animals ranging from the common cold, the recently discovered Middle East respiratory syndrome and severe acute respiratory syndrome. Exopeptidases are the sole identified class of receptors required for the process of attachment and membrane fusion of coronaviruses within host tissues 92 . The host cell receptor for severe acute respiratory syndrome is the metallocarboxypeptidase angiotensin‐converting enzyme 2. This receptor also binds the Human (H) CoV‐NL63 virus. Fibronectin‐binding DPP4 is a receptor for Middle East respiratory syndrome 92 . Perhaps the most well‐known example of a protease viral receptor is aminopeptidase N (also known as CD13), as it is targeted by a number of human, porcine, feline and canine CoVs, as well as porcine transmissible gastroenteritis virus and feline infectious peritonitis virus 93 . It is not known whether microbial exopeptidases function as receptors for viruses. However, exopeptidase counterparts are found in the exoproteomes of a number of bacteria including S. pneumoniae, Burkholderia pseudomalleli and Moraxella catarrhalis (Table 2 ). Furthermore, synergism occurs between viral and bacterial coinfections, particularly in the respiratory tract 94 . Over the last ten years, it has been established that viral infections aid bacteria colonisation and dissemination through various means, including disrupting mucocilliary function 95 , impairing immune effector cell function 96 and providing easy access to adhesin targets and nutrients via epithelial damage 97 . As the respiratory tract is a reservoir to a number of pathobionts, one hypothesis is that these microbes secrete potential viral protease receptors or chemoattractants to enhance their own colonisation. Table 2 Examples of cytosolic/periplasmic proteases found in microbial exoproteomes Protease NCBI locus Organism Reference Additional conversed domains Aminopeptidase I BB0366 Borrelia burgdorferi 126 Aminopeptidase M XP_001682593 Leishmania major 127 Aminopeptidase N AAK99510 ZP_01769994 ADG61634 Streptococcus pneumoniae Burkholderia pseudomallei Moraxella catarrhalis 123 128 129 Aminopeptidase T NP_830199 Bacillus cereus 130 Carboxypeptidase Q18AU1_CLOD6 Clostridium difficile 131 Aminoacylase 1 C‐terminal processing peptidase BP0609 Bordetella pertussis 132 ClpB BP1198 NP_371049 Bordetella pertussis Staphylococcus aureus 132 133 AAA + ATPase ClpC AAT29160 Bacillus anthracis 134 AAA + ATPase ClpP BB2254 ZP_21019400 NP_371292 Bordetella bronchiseptica Escherichia coli Staphylococcus aureus 132 133 133 AAA + ATPase Collagenase YP_005053876 Filifactor alocis 136 Deblocking aminopeptidase NP_834277 Bacillus cereus 130 DPPIII XP_001687573 Leishmania major 127 DPPIV BP0906 AAL80949 XP_001681251 Bordetella pertussis Pyrococcus furiosus Leishmania major 132 137 127 DPPVII YP_004045754 Riemerella anatipestifer 138 Endopeptidase O AAL00295 Streptococcus pneumoniae 123 Glutamyl aminopeptidase AAL00485 Streptococcus pneumoniae 123 HtrA OE7_04918 BP2434 P0C0V0 CAB13147 CAD00819 ? BB0104 Haemophilus parasuis Bordetella pertussis Escherichia coli Bacillus subtilis Listeria monocytogenes Brucella abortus Borrelia burgdorferi 139 132 140 141 142 143 126 l ‐Aminopeptidase d ‐amidase/ d ‐esterase YP_005055390 Filifactor alocis 136 Endo‐type 6 aminohexanoate‐oligomer hydrolase Leucine aminopeptidase XP_001681531 Leishmania major 127 Lon protease YP_005054101 Filifactor alocis 136 AAA + ATPase Oligopeptidase F AAT30293/ AAT30449 Bacillus anthracis 134 O‐Sialoglycoprotein endopeptidase YP_005054583 Filifactor alocis 136 Molecular chaperone Peptidase T NP_833858 CAB15918 AAT33487 Bacillus cereus Bacillus subtilis Bacillus anthracis 130 141 134 Protease 1 PFPI_PYRFU Pyrococcus furiosus 137 Glutamine aminotransferase Protease HsIV YP_513676 Francisella tularensis 144 Pyroglutamyl peptidase YP_513002 Francisella tularensis 144 Signal peptide peptidase A YP_514244 Francisella tularensis 144 TAP P0ADA1 Escherichia coli 139 Thermostable carboxypeptidase NP_831344 CBP1_PYRFU XP_001686015 Bacillus cereus Pyrococcus furiosus Leishmania major 130 137 127 Thimet oligopeptidase XP_001684161 Leishmania major 127 Transpeptidase ZP_21022097 Escherichia coli 135 Transglycosylase Xaa‐His dipeptidase NP_372275 Staphylococcus aureus 133 Xaa‐Pro aminopeptidase CBA64059/ CBA64694 XP_003722629 Clostridium difficile Leishmania major 131 127 Creatinase Prolidase Zinc metallopeptidases BP1721 CD630_28300 XP_001685175 BB0627 Bordetella pertussis Clostridium difficile Leishmania major Borrelia burgdorferi 132 145 127 126 LysM Acetylornithine deacetylase John Wiley & Sons, Ltd. This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. 2.5 Proteases with additional catalytic functions The potential for proteases to possess additional, unexpected activities adds layers of complexity to our understanding of physiological processes. Although there are several examples of microbial proteases that catalyse reactions unrelated to proteolysis, information about their biological significance is scarce due to the intrinsic challenges faced when ascertaining specificity, redundancies and natural substrates in complex biological samples. Site‐directed mutagenesis, gene deletion studies and molecular modelling have been key in identifying unusual modes of action for proteases. For example, the papain‐like proteases (PLPs) from the viral families Coronaviridae and Arterivirdae are responsible for processing the critical replicase polyprotein into its numerous functional units 98 . Structural similarities of PLPs to deubiquitinating enzymes showed that PLPs not only function as deubiquitinating enzymes, but are also able to block induction of several endogenous proinflammatory cytokines, including IFNβ, CCL5 and CXCL10 98 . Another essential multifunctional viral protein is the hepatitis C virus NS3/4A, which possesses both chymotrypsin‐like serine protease and helicase activities in two individual domains. As this protein is vital for viral propagation, it has been the target for the small‐molecule inhibitors that have been commercially available for hepatitis C virus treatment since 2011 99 . For other multi‐catalytic proteases, biological relevance is less certain and studies often infer evolutionary and environmental advantages to an affinity for several different types of substrates. For example, there are enzymes that are able to hydrolyse different types of bonds. An aminopeptidase from the antibiotic‐producing bacterium Streptomyces griseus 100 and a prolidase from the marine bacterium Alteromonas haloplanktis 101 are capable of cleaving amide, phosphoester and phosphofluorinate bonds. This is interesting as the mechanics behind peptide and ester hydrolysis are dissimilar 100 . Another enzyme capable of mechanistically diverse activities is the thioesterase I (TAP) of E. coli , which exhibits thioesterase, arylesterase, esterase, lysophospholipase and serine protease activities 102 . However, despite an ability to interact with a great number of different substrates, the role of TAP in vivo is not yet well established. Finally, the extremophilic bacterial species Deinococcus radiodurans, Pyrococcus horikkoshii and Thermoanaerobacter tengcongensis all possess an enzyme that is both an aminoacylase (hydrolysing N‐acetylated amino acids) and a carboxypeptidase. It has been suggested that the role of these multifunctional enzymes is in enabling peptidolytic growth in these organisms 103 . 3 Identification of moonlighting proteases Proteomic technologies have been at the forefront of efforts to characterise the identities of proteins that reside on the cell surface (surfaceome) or are secreted into the extracellular milieu (secretome) for a wide range of pathogenic Gram positive and Gram negative bacteria 18 , 104 . These hypothesis‐free methods have identified the presence of a diverse range of proteins that are localised on the cell surface or excreted into the extracellular milieu, regardless of transport mechanisms (exoproteome). The presence of proteins with well‐defined enzymatic roles in the cytosol and their detection in the exoproteome continues to challenge our understanding of the mechanisms that enable these proteins to reach these extracellular sites and the role played by cell lysis 15 , 19 . Generally, proteases need to be secreted or surface exposed to function as virulence factors. Notably, several of the known multifunctional proteases discussed in this review were initially predicted to be cytosolic proteins but were then found to be surface exposed, a location typical of the largest group of known moonlighting proteins 13 . Table 2 provides examples of typically cytosolic proteases found in recent microbial exoproteomes. One approach to identifying moonlighting proteases is to evaluate extracellular proteolytic activity in the exoproteome or via whole cell assays. Proteomic techniques including terminal amine isotopic labelling of substrates 105 have recently been developed 106 for the identification of proteolytic cleavage products and substrate, as well as proteomic identification of cleavage sites 107 , providing information about protease cleavage site specificity. Cell surface shaving with trypsin and other proteolytic enzymes and surface labelling of proteins with biotin are two powerful methodologies for identifying surface‐accessible proteins. These approaches also detect potential moonlighting proteins. Regardless of the many proteomic methodologies available for identification of surface‐exposed and secreted proteins(reviewed in 108 , 109 ), careful controls are necessary to estimate the role cell lysis has on the interpretation of these datasets. Despite extensive efforts to develop strategies to estimate cell lysis, it is difficult to determine if a protein has a bona fide role on the cell surface. Ultimately, alternative methods are required to confirm the surface location of putative moonlighting proteins identified using the high‐throughput approaches described above. The degree to which cytosolic proteins are dismissed seems to correspond to the type of analysis. For example, a surfaceome analysis implies proteins expressed at or specifically bound to the cell surface, and this results in proteins with annotated cytosolic functions being viewed as contaminants. The reporting of cytosolic proteins in surfaceome analyses varies from none in a study where 28 proteins were identified 110 , to 17% cytosolic proteins reported using a false‐positive control (50 proteins identified) 111 , to >50% cytosolic proteins reported using a combination of methodologies 112 . Experiments cataloguing the exoproteome more often acknowledge the presence of cytosolic proteins, with the caveat that cell lysis will occur in a population of cells during the analysis and this constitutes a part of the exoproteome. Furthermore, the exoproteome can be interrogated using sera from infected animals or patients to identify the immunoproteome, providing valuable biological insights. Recently, Solis et al. 113 addressed the issue of proteins that are secreted into the extracellular milieu that have the capacity to bind back to the cell surface as part of the surfaceome in S. aureus . Using cell surface shaving techniques with false‐positive controls coupled with iTRAQ labelling, a scoring approach was proposed to calculate the probability of a protein being surface‐exposed by taking into account the relative abundance of peptides identified in the shaved and control datasets. This kind of analysis is a significant move forward in the ability to identify geographical moonlighting proteins, although again there are caveats. Where proteases are present at the cell surface (whether moonlighting or not), proteolytic processing of surface proteins may occur, resulting in cleavage products, rather than intact proteins, being variably present on the cell surface or released into the extracellular milieu, and this may skew attempts at quantitative normalisation analyses. This "exoprocessing" of proteins is akin to ectodomain shedding, a common phenomenon in eukaryotes 114 , and can be seen in many pathogenic bacteria including Clostridium difficile with surface layer protein A 115 , and in M. hyopneumoniae with the P97 and P102 paralogous families of adhesins 116 , 117 , 118 , 119 , 120 , 121 , 122 . An ideal workflow for characterising suspected moonlighting proteins would include a series of orthogonal methods to interrogate the identity of proteins on the cell surface, such as trypsin shaving and surface biotinylation coupled with immunofluorescence microscopy. Functions could be inferred by characterising the immunoproteome and applying affinity chromatography using known host components (e.g. fibronectin) as bait. Functions inferred by these methods would require cloning and expression of the putative moonlighting protein followed by detailed binding studies 123 . Whilst it is not always feasible or practical to pursue these follow‐up investigations for each cytosolic protein identified in large‐scale proteomic analyses, there is good reason to focus research efforts on microbial proteases in unusual locations: proteolysis is central to a multitude of pathological processes and several microbial proteases have now been shown to have additional functions that are frequently linked to virulence. A full understanding of the pathogenic complexity of microbial proteases can contribute to combating microbial‐associated disease. The authors have declared no conflict of interest . The authors have declared no conflict of interest .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9953510/

Mint3 as a Potential Target for Cooling Down HIF-1α-Mediated Inflammation and Cancer Aggressiveness

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that plays a crucial role in cells adapting to a low-oxygen environment by facilitating a switch from oxygen-dependent ATP production to glycolysis. Mediated by membrane type-1 matrix metalloproteinase (MT1-MMP) expression, Munc-18-1 interacting protein 3 (Mint3) binds to the factor inhibiting HIF-1 (FIH-1) and inhibits its suppressive effect, leading to HIF-1α activation. Defects in Mint3 generally lead to improved acute inflammation, which is regulated by HIF-1α and subsequent glycolysis, as well as the suppression of the proliferation and metastasis of cancer cells directly through its expression in cancer cells and indirectly through its expression in macrophages or fibroblasts associated with cancer. Mint3 in inflammatory monocytes enhances the chemotaxis into metastatic sites and the production of vascular endothelial growth factors, which leads to the expression of E-selectin at the metastatic sites and the extravasation of cancer cells. Fibroblasts express L1 cell adhesion molecules in a Mint3-dependent manner and enhance integrin-mediated cancer progression. In pancreatic cancer cells, Mint3 directly promotes cancer progression. Naphthofluorescein, a Mint3 inhibitor, can disrupt the interaction between FIH-1 and Mint3 and potently suppress Mint3-mediated inflammation, cancer progression, and metastasis without causing marked adverse effects. In this review, we will introduce the potential of Mint3 as a therapeutic target for inflammatory diseases and cancers. 1. Introduction Mammalian cells generally require molecular oxygen to produce ATP as an energy source through oxidative phosphorylation (OXPHOS) in mitochondria. However, certain types of cells, such as cancer cells and macrophages, are constitutively dependent on ATP production through an alternate metabolic pathway called glycolysis, and this phenomenon is known as the Warburg effect [ 1 , 2 ]. Hypoxia-inducible factors (HIFs), especially HIF-1α, are transcription factors that have roles in immune response, angiogenesis, cell growth, metabolism, and cell death [ 3 ]. Owing to the multiple roles of HIF-1α, its activity is associated with various diseases (e.g., inflammatory and metabolic diseases and cancer proliferation and metastasis) [ 4 , 5 , 6 , 7 ]. Therefore, the proper regulation of HIF-1α is essential for maintaining healthy metabolism and homeostasis. As HIF-1α is expressed ubiquitously in various tissues, the direct inhibition of HIF-1α can cause simultaneous adverse effects. One member of the Munc-18-1 interacting protein (Mint) family, Mint3, has been identified as an indirect activator of HIF-1α. Its effect is limited because its role as an HIF-1α activator requires the expression of membrane type-1 matrix metalloproteinase (MT1-MMP), which is limited to specific cells, such as macrophages, cancer cells, and fibroblasts [ 8 , 9 , 10 , 11 ]. Herein, we summarize the key roles and influences of Mint3. 2. Overview of HIF Proteins As a key factor associated with the adaptation of cells to low-oxygen environments, HIFs are known to play crucial roles as transcription factors [ 12 , 13 ]. HIF proteins include three alpha subunits (HIF-1α, HIF-2α, and HIF-3α) and one beta subunit (HIF-1β); they are heterodimers composed of one of the three alpha subunits and HIF-1β. HIF alpha subunits are stably expressed under hypoxic conditions, whereas HIF-1β is stably expressed independent of oxygen [ 14 ]. All HIF proteins share a basic domain and a dimerization domain within the N-terminal. However, alpha subunits but not beta subunits possess an N-terminal transactivation domain (NAD) in the C-terminal, and only HIF-1α and HIF-2α have the C-terminal transactivation domain (CAD) [ 15 , 16 ]. These structural differences among the four types of HIF subunits lead to differences in protein stability and transcriptional activity regulation. The HIF subunits also show variations in their expression locus and target genes. While HIF-1α is ubiquitously expressed in human tissues and upregulates the expression of glycolysis-related genes, HIF-2α is expressed in specific tissues, such as tumor vascular cells and macrophages, and promotes erythropoietin and iron metabolism [ 17 , 18 , 19 ]. HIF-3α has an important role in adipogenesis, and its gene expression is regulated by HIF-2α activity [ 20 ]. The stability of all alpha subunits is conditionally regulated by oxygen concentration; however, HIF-1β is stably and ubiquitously expressed. HIF requires the heterodimerization of alpha and beta subunits for its activation. Therefore, alpha subunits (especially HIF-1α) are responsible for regulating HIF activity by switching between aerobic and hypoxic conditions through the upregulation of glycolysis-related genes. The protein stability and activity of HIF-1α are suppressed through two mechanisms during normoxia: (1) the proteasomal degradation of HIF-1α through NAD modification and (2) the enzymatic inhibition of HIF-1α through CAD modification. Under normal oxygen conditions, HIF-1α is hydroxylated at the proline residues P402 and P564 by proline hydroxylase domain (PHD) proteins (mainly PHD2), and these modifications are recognized by von Hippel Lindau (VHL), a component of the E3 ubiquitin ligase complex, resulting in proteasome-dependent degradation [ 21 , 22 , 23 ]. Because PHDs use oxygen for the hydroxylation of HIF-1α, the destabilization of HIF-1α by PHDs occurs only when oxygen is sufficient [ 24 ]. The transcriptional activity of HIF-1α is achieved through its stabilization during hypoxia and its interaction with the transcriptional co-activator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 protein. The factor inhibiting HIF-1 (FIH-1) protein functions as an asparaginyl hydroxylase and contains two core domains: a catalytic domain, which is homologous to the cupin protein family, and a dimerization domain in the C-terminal region [ 25 , 26 ]. Dimerized FIH-1 is involved in the hydroxylation of HIF-1α at the N803 residue within CAD and interferes with the interaction between HIF-1α and the CBP/p300 proteins [ 25 , 27 , 28 , 29 ]. Additionally, FIH-1 recruits VHL to HIF-1α by binding with its N-terminal region, which induces the destabilization of HIF-1α [ 28 ]. With these two different hydroxylase-dependent mechanisms, cells regulate the protein stability and activity of HIF-1α and adapt to a low-oxygen environment by switching the mechanism of ATP production from OXPHOS to glycolysis. 3. Mechanisms of Mint3-Mediated HIF-1 Activation 3.1. Mint3 Indirectly Upregulates HIF-1α Activity through Its Interaction with FIH-1 Mint3, also known as amyloid-beta A4 precursor protein-binding family A member 3, is a novel protein that positively regulates HIF-1α [ 30 ]. The Mint family consists of three isoforms: Mint1, Mint2, and Mint3 (also known as APBA1, APBA2, and APBA3; X11, X11-like, and X11-like2; and X11α, β, and γ, respectively). Mint1 and Mint2 bind with Munc-18-1, which is involved in neural homeostasis [ 31 ]. Mint3 shows ubiquitous expression in various tissues, with the lowest level in the testis, according to Okamoto and Sudhof [ 32 ]. Its intracellular localization is mainly at the Golgi apparatus where it interacts with furin [ 33 ]. Mint1-3 were originally identified as neural proteins that interact with the YENPTY motif within amyloid precursor proteins (APPs), which are conserved in the cytoplasmic region of APP and modulate their activities [ 32 , 34 , 35 ]. All three Mint isoforms share highly conserved structures in their C-terminal region, which comprise one phosphotyrosine-binding (PTB) domain followed by tandem PDZ domains (PDZa and PDZb). Through these two domains, Mint3 functions as an adaptor protein. With its PTB domain, Mint3 binds to proteins such as furin, N-terminal EF-hand calcium binding protein 3 (NECAB3), and Rab6 [ 32 , 33 , 36 , 37 ]. Moreover, PDZ binds with proteins such as breakpoint cluster region protein (Bcr) [ 38 ]. Amyloid precursor protein and Arf GTPase interact with both the PTB domain and the PDZb (but not PDZa) domain [ 38 , 39 ]. However, only Mint1 and Mint2 retain the N-terminal region, which contains Munc-18-1 interacting domain; because Mint-3 lacks the domain, it is unable to interact with Munc-18-1 [ 32 ] ( Figure 1 ). Additionally, a recent structural analysis of Mint3 revealed that its N-terminal region is intrinsically disordered [ 40 ]. Thus, the sequences of the N-terminal region of Mint3 differ substantially from those of the other Mint proteins, and, as a result, Mint3 has divergent binding partners at its N-terminal region. Notably, the sequences in each Mint family member are highly conserved among species [ 32 ]. Our investigation, involving a yeast two-hybrid screening with FIH-1 as a bait protein, revealed that the N-terminal region could interact with FIH-1 and that neither Mint1 nor Mint2 showed such an affinity, indicating that FIH-1 selectively binds with Mint3. Further investigation revealed that the N-terminal region of Mint3, especially amino acid residues G78–G88, and the dimerization domain of FIH-1 are required for interactions between these two proteins [ 40 ]. As a result, Mint3, especially its N-terminal region, competes with HIF-1α as an FIH-1, leading to an indirect interruption of HIF-1α hydroxylation at N803 by FIH-1. A comprehensive investigation to clarify the key mechanism of FIH-1 binding with Mint3 was performed, and the results revealed that at least two amino acid regions of the dimerized FIH-1 (78–88 and 101–110) are required [ 41 ]. In addition, the phosphorylation of the T5 and S7 amino acid residues within Mint3, mediated by the mammalian target of rapamycin complex 1 (mTORC1) but not by mTORC2, is required for HIF-1α activation [ 42 ]. These studies described the "trapping" of FIH-1 by Mint3 in the trans-Golgi network through the interaction of Mint3 with FIH-1 and furin [ 33 ] with its N-terminal and C-terminal regions, respectively, which allows HIF-1α to escape catalytic inhibition by FIH-1. 3.2. Factors That Support Mint3-Mediated HIF-1α Activation Although Mint3 is ubiquitously expressed, its effect on HIF-1α regulation is limited to specific regions where MT1-MMP is expressed simultaneously, such as fibroblasts, macrophages, and cancer cells. MT1-MMP, also known as MMP14, is a member of the MMP family. The MMP family comprises 23 members, most of which are composed of well-conserved structures. The extracellular domains include the pro-domain, catalytic (CAT) domain, which binds with zinc ions, and hemopexin (HPX) domain; in addition, a transmembrane region and an adjacent cytoplasmic region are present [ 10 ]. Among the 23 MMPs, 6 are recognized as membrane-anchored MMPs, which are further classified into two types as follows: 1) MT1-MMP (MMP14), MT2-MMP (MMP15), MT3-MMP (MMP16), and MT5 (MMP-24) are transmembrane types, which conserve the cytoplasmic region, and 2) MT4-MMP (MMP-17) and MT6-MMP (MMP-25) are glycosylphosphatidylinositol-anchored types, which lack the cytoplasmic region [ 10 , 43 ]. MT1-MMP is initially expressed in an inactive state (pro-MMP); however, it switches to the active form upon proteolytic processing in the pro-domain by furin [ 44 ]. MT1-MMP is the first transmembrane MMP type to be associated with the upregulation of invasion and the metastasis of cancer cells [ 45 ]. This is somewhat convincing because the most important substrates of MT1-MMP are the extracellular matrix (ECM) molecules, especially type-1 collagen, with MT1-MMP catalyzing various extracellular proteins, including secreted proteins (e.g., growth factors, cytokines/chemokines, and collagens) and cell surface proteins (e.g., adhesion molecules and transmembrane receptors) [ 46 , 47 ]. When MT1-MMP is expressed in macrophages or cancer cells, these cells exhibit invasive features such as the invasion of macrophages into the basement membrane and cancer metastasis [ 9 , 48 ]. Apart from the functions of MT1-MMP as a proteinase, it also plays an important role in HIF-1α activation by supporting the Mint3–FIH-1 interaction. Twenty amino acids within the cytoplasmic tail (CPT) of MT1-MMP enable FIH-1 to have direct contact with MT1-MMP, resulting in the inhibition of FIH-1′s suppression of HIF-1α activity [ 9 , 49 ]. In addition, this binding between MT1-MMP and FIH-1 is accomplished independently of the N-terminal region of MT1-MMP, which contains pro-domain, CAT, and HPX. Thus, this interaction leads to the binding of Mint3 and FIH-1, resulting in HIF-1α activation. Of note, the activation of HIF-1α mediated by the MT1-MMP–FIH-1–Mint3 axis only requires the CPT of MT1-MMP; hence, CPT that lacks affinity with FIH-1 is unable to activate HIF-1α [ 49 ]. This indicates that MT1-MMP activates HIF-1α independent of its N-terminal region. As Mint3 is mainly localized to the Golgi apparatus by binding with furin, MT1-MMP possibly catalyzes the interaction of Mint3 and FIH-1 by adhering FIH-1 to the Golgi apparatus, thus, keeping it in close contact with Mint3 instead of diffusing to the cytoplasm [ 33 , 49 ]. Overall, Mint3 requires the coordinated expression of MT1-MMP to activate HIF-1α; therefore, the critical influence of Mint3 activity is restricted to loci with sufficient levels of MT1-MMP expression, such as in macrophages, cancer cells, and fibroblasts. In addition to MT1-MMP, NECAB3 is reportedly involved in Mint3-mediated HIF-1α activation [ 37 ], indicating that other unidentified factors could affect the Mint3–FIH-1–HIF-1α axis. The cascade of HIF-1α activation through the axis of MT1-MMP, Mint3, and FIH-1 is summarized in Figure 2 . 3.1. Mint3 Indirectly Upregulates HIF-1α Activity through Its Interaction with FIH-1 Mint3, also known as amyloid-beta A4 precursor protein-binding family A member 3, is a novel protein that positively regulates HIF-1α [ 30 ]. The Mint family consists of three isoforms: Mint1, Mint2, and Mint3 (also known as APBA1, APBA2, and APBA3; X11, X11-like, and X11-like2; and X11α, β, and γ, respectively). Mint1 and Mint2 bind with Munc-18-1, which is involved in neural homeostasis [ 31 ]. Mint3 shows ubiquitous expression in various tissues, with the lowest level in the testis, according to Okamoto and Sudhof [ 32 ]. Its intracellular localization is mainly at the Golgi apparatus where it interacts with furin [ 33 ]. Mint1-3 were originally identified as neural proteins that interact with the YENPTY motif within amyloid precursor proteins (APPs), which are conserved in the cytoplasmic region of APP and modulate their activities [ 32 , 34 , 35 ]. All three Mint isoforms share highly conserved structures in their C-terminal region, which comprise one phosphotyrosine-binding (PTB) domain followed by tandem PDZ domains (PDZa and PDZb). Through these two domains, Mint3 functions as an adaptor protein. With its PTB domain, Mint3 binds to proteins such as furin, N-terminal EF-hand calcium binding protein 3 (NECAB3), and Rab6 [ 32 , 33 , 36 , 37 ]. Moreover, PDZ binds with proteins such as breakpoint cluster region protein (Bcr) [ 38 ]. Amyloid precursor protein and Arf GTPase interact with both the PTB domain and the PDZb (but not PDZa) domain [ 38 , 39 ]. However, only Mint1 and Mint2 retain the N-terminal region, which contains Munc-18-1 interacting domain; because Mint-3 lacks the domain, it is unable to interact with Munc-18-1 [ 32 ] ( Figure 1 ). Additionally, a recent structural analysis of Mint3 revealed that its N-terminal region is intrinsically disordered [ 40 ]. Thus, the sequences of the N-terminal region of Mint3 differ substantially from those of the other Mint proteins, and, as a result, Mint3 has divergent binding partners at its N-terminal region. Notably, the sequences in each Mint family member are highly conserved among species [ 32 ]. Our investigation, involving a yeast two-hybrid screening with FIH-1 as a bait protein, revealed that the N-terminal region could interact with FIH-1 and that neither Mint1 nor Mint2 showed such an affinity, indicating that FIH-1 selectively binds with Mint3. Further investigation revealed that the N-terminal region of Mint3, especially amino acid residues G78–G88, and the dimerization domain of FIH-1 are required for interactions between these two proteins [ 40 ]. As a result, Mint3, especially its N-terminal region, competes with HIF-1α as an FIH-1, leading to an indirect interruption of HIF-1α hydroxylation at N803 by FIH-1. A comprehensive investigation to clarify the key mechanism of FIH-1 binding with Mint3 was performed, and the results revealed that at least two amino acid regions of the dimerized FIH-1 (78–88 and 101–110) are required [ 41 ]. In addition, the phosphorylation of the T5 and S7 amino acid residues within Mint3, mediated by the mammalian target of rapamycin complex 1 (mTORC1) but not by mTORC2, is required for HIF-1α activation [ 42 ]. These studies described the "trapping" of FIH-1 by Mint3 in the trans-Golgi network through the interaction of Mint3 with FIH-1 and furin [ 33 ] with its N-terminal and C-terminal regions, respectively, which allows HIF-1α to escape catalytic inhibition by FIH-1. 3.2. Factors That Support Mint3-Mediated HIF-1α Activation Although Mint3 is ubiquitously expressed, its effect on HIF-1α regulation is limited to specific regions where MT1-MMP is expressed simultaneously, such as fibroblasts, macrophages, and cancer cells. MT1-MMP, also known as MMP14, is a member of the MMP family. The MMP family comprises 23 members, most of which are composed of well-conserved structures. The extracellular domains include the pro-domain, catalytic (CAT) domain, which binds with zinc ions, and hemopexin (HPX) domain; in addition, a transmembrane region and an adjacent cytoplasmic region are present [ 10 ]. Among the 23 MMPs, 6 are recognized as membrane-anchored MMPs, which are further classified into two types as follows: 1) MT1-MMP (MMP14), MT2-MMP (MMP15), MT3-MMP (MMP16), and MT5 (MMP-24) are transmembrane types, which conserve the cytoplasmic region, and 2) MT4-MMP (MMP-17) and MT6-MMP (MMP-25) are glycosylphosphatidylinositol-anchored types, which lack the cytoplasmic region [ 10 , 43 ]. MT1-MMP is initially expressed in an inactive state (pro-MMP); however, it switches to the active form upon proteolytic processing in the pro-domain by furin [ 44 ]. MT1-MMP is the first transmembrane MMP type to be associated with the upregulation of invasion and the metastasis of cancer cells [ 45 ]. This is somewhat convincing because the most important substrates of MT1-MMP are the extracellular matrix (ECM) molecules, especially type-1 collagen, with MT1-MMP catalyzing various extracellular proteins, including secreted proteins (e.g., growth factors, cytokines/chemokines, and collagens) and cell surface proteins (e.g., adhesion molecules and transmembrane receptors) [ 46 , 47 ]. When MT1-MMP is expressed in macrophages or cancer cells, these cells exhibit invasive features such as the invasion of macrophages into the basement membrane and cancer metastasis [ 9 , 48 ]. Apart from the functions of MT1-MMP as a proteinase, it also plays an important role in HIF-1α activation by supporting the Mint3–FIH-1 interaction. Twenty amino acids within the cytoplasmic tail (CPT) of MT1-MMP enable FIH-1 to have direct contact with MT1-MMP, resulting in the inhibition of FIH-1′s suppression of HIF-1α activity [ 9 , 49 ]. In addition, this binding between MT1-MMP and FIH-1 is accomplished independently of the N-terminal region of MT1-MMP, which contains pro-domain, CAT, and HPX. Thus, this interaction leads to the binding of Mint3 and FIH-1, resulting in HIF-1α activation. Of note, the activation of HIF-1α mediated by the MT1-MMP–FIH-1–Mint3 axis only requires the CPT of MT1-MMP; hence, CPT that lacks affinity with FIH-1 is unable to activate HIF-1α [ 49 ]. This indicates that MT1-MMP activates HIF-1α independent of its N-terminal region. As Mint3 is mainly localized to the Golgi apparatus by binding with furin, MT1-MMP possibly catalyzes the interaction of Mint3 and FIH-1 by adhering FIH-1 to the Golgi apparatus, thus, keeping it in close contact with Mint3 instead of diffusing to the cytoplasm [ 33 , 49 ]. Overall, Mint3 requires the coordinated expression of MT1-MMP to activate HIF-1α; therefore, the critical influence of Mint3 activity is restricted to loci with sufficient levels of MT1-MMP expression, such as in macrophages, cancer cells, and fibroblasts. In addition to MT1-MMP, NECAB3 is reportedly involved in Mint3-mediated HIF-1α activation [ 37 ], indicating that other unidentified factors could affect the Mint3–FIH-1–HIF-1α axis. The cascade of HIF-1α activation through the axis of MT1-MMP, Mint3, and FIH-1 is summarized in Figure 2 . 4. Mint3 Mediates Inflammatory Responses Macrophages are the key players in innate immunity and rely on constant HIF-1α-mediated glycolysis regardless of oxygen conditions [ 4 , 50 ], which is dependent on Mint3 activity. Upon stimulation or infection by various pathogens, macrophages are involved in producing cytokines and chemokines, growth factors, and reactive oxygen species (ROS) during inflammation to protect the host. However, the overactivation of the immune system can rather be toxic due to septic shock [ 51 ]. Stimulation by lipopolysaccharides (LPS), which are a toxic component of the outer membrane of Gram-negative bacteria, causes septic shock. This toxicity is a consequence of a cytokine storm mediated by the glycolysis-dependent secretion of cytokines associated with increased motility and invasiveness of macrophages, which involve the Mint3–FIH-1–HIF-1α axis [ 52 ]. Macrophages gain "lethal force" by Mint3 expression under LPS-driven immune reaction. In an immune response to influenza virus (IFV), Mint3 depletion efficiently improves influenza pneumonia by alleviating the production of cytokines and chemokines in macrophages (but not in dendritic cells) through two mechanisms: the upregulation of Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) α activity and the stabilization of IκB [ 53 ]. AMPK is activated through its phosphorylation on Adenosine 5'-triphosphate (ATP) starvation [ 54 ] and downregulates nuclear factor kappa B (NF-κB) activity [ 55 ]. The depletion of Mint3 downregulates glycolysis-mediated ATP production and activates AMPK and the subsequent inhibition of NF-κB. Concurrently, Mint3 deficiency leads to the glycolysis-independent stabilization of IκB, which causes the simultaneous inhibition of NF-κB. Although the precise mechanisms through which Mint3 deficiency causes IκB stabilization remain elusive, the hydroxylation of IκB by FIH-1 might regulate IκB stability [ 56 ]. However, Mint3 does not contribute to gaining immunity against IFV, as Mint3 depletion has no influence on the production of interferon (IFN)-α or anti-IFV antibodies [ 53 ]. Pyroptosis is a mechanism of programmed cell death that is associated with innate immunity and occurs when cells encounter intracellular bacterial infection. It was initially pointed out by Friedlander and was named by D'Souza to distinguish it from apoptosis [ 57 , 58 ]. Canonical pyroptosis signals involve the formation of inflammasomes, which mostly comprise Nod-like receptors (NLRs)/absent in melanoma 2 (AIM2); apoptosis-associated speck-like protein (ASC) as an adaptor protein; and the inflammatory caspase-1, whereas caspase-11/4/5 could serve as an inflammatory caspase in non-canonical pyroptosis. Inflammasomes process the pyroptosis-executing protein gasdermin D (GSDMD) and interleukin (IL)-1β/IL-18. Listeria monocytogenes (LM) is a common foodborne pathogen that causes zoonotic diseases in Western countries by infecting various types of cells. Upon innate infection by LM, the host operates a protective mechanism by clearing out the bacteria through the production of ROS and nitric oxide (NO) induced by the activation of pyroptosis [ 59 , 60 , 61 , 62 ]. However, deficient Mint3 protects mice against LM-derived lethality without affecting NO production and rather decreasing ROS production. As ROS inactivates caspase-1 by oxidization, the suppression of ROS production by Mint3 depletion stabilizes caspase-1 and activates pyroptosis [ 63 ]. Additionally, the formation of inflammasomes that comprise AIM2, NLRP3, and ASC are inhibited by glycolysis, Mint3 depletion activates pyroptosis by diminishing glycolysis and promoting the caspase-1-dependent processing of GSDMD and IL-18/IL-1β. Therefore, Mint3 depletion protects the host of LM by two mechanisms: (1) stabilization of caspase-1 by suppressing ROS production and (2) promoting the formation of inflammasomes by diminishing glycolysis. Although the mechanisms of the inhibition of ROS production in Mint3-depleted macrophages have not been elucidated, it could be mediated by the inhibition of HIF-1α and NF-κB, which plays a role in producing pro-oxidant genes, including Nicotinamideadenine-dinucleotide phosphate (NADPH) oxidase ( NOX2 ) [ 53 , 64 , 65 ]. Inconsistent with these potential effects of Mint3 on the immune system, a previous report from the group of Huai [ 66 ] showed that Mint3 potentiates an antiviral response through the expression of IFN-β upon the induction of immune signals after viral infection or LPS stimulation through Toll-like receptors (TLRs) and retinoic acid-inducible gene-I–like receptors (RLRs) [ 67 , 68 , 69 , 70 ]. In this case, Mint3, with its PDZ domains within the C-terminal regions, serves as an adaptor protein for tumor necrosis factor receptor-associated factor 3 and enhances K63-linked polyubiquitination, which activates downstream TLR- or RLR-mediated signaling [ 71 ]. This indicates that Mint3 plays an important role in maintaining the basic functions of macrophages; however, once macrophages are stimulated by various pathogens, such as LPS, viruses, and bacteria, Mint3 generally acts as an enhancer of acute inflammation. Moreover, in the case of LM infection, Mint3 attenuates the death of "sick" macrophages, implying that the environment surrounding the macrophages remains infected. When the macrophages are kept "healthy", Mint3 acts as a "housekeeper" as it is involved in ATP production by activating glycolysis; however, Mint3 can be toxic for the organisms once the immune signals are induced by the pathogens. 5. Role of Mint3 in Cancer Progression The effects of Mint3 on the innate immune system may be applicable in cancers because Mint3 has the potential to increase cancer malignancy and metastatic features through its expression in inflammatory monocytes (IMs), which are defined with the following markers: Gr-1/Ly6C + , CD11b/CD115 + [ 72 ]. In addition, a relationship between Mint3 activity in cancer cells and cancer-associated fibroblasts (CAFs) has been previously reported [ 11 , 73 , 74 ]. Because Mint3 has key roles in enhancing cancer progression and metastasis by upregulating HIF-1α, which is also related to the tumor microenvironments, it could be an attractive target for cancer treatment, as tumor-microenvironments-related factors are gaining more attraction, which is described as one of the important hallmarks of cancers [ 75 ]. In this section, we summarize the influence of Mint3 on cancer progression through its expression in Ims, cancer cells, and CAFs. 5.1. Impact of Mint3 Activity in Cancer Cells Solid tumors in human bodies are often exposed to low-oxygen environments due to the disadvantage in physical distance from the blood vessels and obtaining sufficient oxygen. In order to adapt to such circumstances, cancer cells tend to retain high HIF-1α activities and gain malignancies. Although the depletion of Mint3 in the host of xenograft models has no effects on tumor progression, its depletion in cancer cells results in HIF-1α suppression, which is associated with downregulated glycolysis, angiogenesis, and anti-proliferative effects. These Mint3-related features are commonly seen in the xenografts of various types of cancer cells (e.g., breast cancer, MDA-MB-231; fibrosarcoma, HT-1080; epidermoid carcinoma, A431; non-small cell lung cancer, A549; and urothelial carcinoma, RT-112) [ 11 , 73 , 74 , 76 ]. However, in pancreatic cancer cells, the depletion of Mint3 induces tissue-specific anti-proliferative effects in vitro and in vivo. This was mediated by the inhibition of Skp2 transcription, which promoted G1/S transition in cell cycles and suppressed the induction of metastasis and stemness [ 73 ]. In addition, the Mint3-dependent expression of Skp2 also involves the Mint3–FIH-1–HIF1α axis and is dependent on HIF-1α activity. Unlike in hypoxic conditions, pancreatic cancer cells show high expression and activity of HIF-1α-dependent Skp2 in normoxic conditions. Consistent with the known features of Skp2 and HIF-1α as metastasis enhancers in various types of cancers [ 77 , 78 , 79 , 80 ], Mint3 promotes epithelial–mesenchymal transition in a HIF-1α- and Skp2-dependent manner with an enhancing effect on the expression of the Slug protein. A recent Mint3-related study on urothelial carcinoma also indicated that the depletion of Mint3 downregulates the invasion, migration, and proliferation of cancer cells, accompanied by factors associated with suppressed HIF-1α activity, such as transcription of HIF-1α-targeted genes and glycolysis [ 74 ]. 5.2. Metastatic Ability of Cancer Cells Achieved through Mint3 Expression in IM Using the PyMT breast cancer mouse model, which develops palpable breast cancers that metastasize to the lung, Qian et al. reported that IMs, which are characterized by Gr-1/Ly6C + , CD11b/CD115 + markers, play a key role in the lung metastasis of breast cancer in a C–C motif chemokine ligand 2 (CCL2)-dependent manner [ 81 ]. Other studies using xenograft mouse models of various types of cancers have shown that Mint3 plays an important role in this mechanism of metastasis [ 72 , 82 ]. Cancer cells and the surrounding stromal cells are the sources of CCL2, and they increase the number of IMs in the peripheral blood. Both individual depletion of Mint3 or HIF-1α in IMs leads to the decreased production of vascular endothelial growth factor (VEGF) to comparable levels. These effects of Mint3 or HIF-1α depletion on VEGF expression are consistent with the fact that VEGF is one of the target genes of HIF-1α [ 72 , 83 ]. Upon Mint3-dependent VEGF secretion by IMs, E-selectin expression in endothelial cells and vascular permeabilization of cancer cells are elevated [ 72 ]. Conclusively, IMs are recruited to the lung metastatic site in a HIF-1α- or glycolysis-dependent manner, which is driven by Mint3, to release vascular endothelial growth factor (VEGF), and this secretion of VEGF induces the expression of E-selectin in the epithelial cells in the lung, which enables the extravasation of cancer cells [ 72 ]. Only macrophages/monocytes work as metastatic inducers, and T cells or B cells do not possess similar functions. Notably, IMs secrete VEGF in accordance with the existence of cancer cells; however, its mechanism remains elusive. 5.3. Supportive Effect of Mint3 Expression in CAFs on Cancer Progression CAFs are one of the factors that have a crucial role in promoting cancer malignancy through ECM remodeling, immune crosstalk, and metabolic effects [ 84 ]. These features of CAFs are achieved by functioning as a source of various secretory proteins, such as growth factors, cytokines, and exosomes, whereas the adherent molecules in CAFs could also increase tumor malignancy in a Mint3-dependent mechanism. Mint3 is also involved in the proliferation of cancer cells by regulating the expression of L1 cell adhesion molecule (L1CAM) in fibroblasts. L1CAM is a cell adhesion molecule that acts as a binding partner of heterodimeric integrins, such as α5β1, αvβ3, αIIbβ3, and αvβ5 [ 85 , 86 ], and triggers downstream signaling pathways, such as the MAPK and PI3K/Akt pathways [ 85 , 87 ]. Fibroblasts express L1CAM in a Mint3-dependent manner, which enables CAFs to promote direct contact with cancer cells expressing integrin α5β1. Therefore, Mint3 also plays a role in enhancing the proliferation of cancer cells through CAFs [ 11 ]. Because CAF-mediated ERK phosphorylation specifically occurs in the peripheral regions of breast cancers, which are surrounded by stroma, L1CAM is possibly involved in the local progression of breast cancers. The expression of Mint3 in CAFs is slightly upregulated by cytokines such as tumor necrosis factor-α and IL-1β. Indeed, CAFs express higher levels of MT1-MMP than normal fibroblasts do, which is also reflected in the activity level of Mint3 in fibroblasts. Further investigation is required to determine whether the activity level of Mint3 has an unlimited correlation with the expression level of MT1-MMP. 5.1. Impact of Mint3 Activity in Cancer Cells Solid tumors in human bodies are often exposed to low-oxygen environments due to the disadvantage in physical distance from the blood vessels and obtaining sufficient oxygen. In order to adapt to such circumstances, cancer cells tend to retain high HIF-1α activities and gain malignancies. Although the depletion of Mint3 in the host of xenograft models has no effects on tumor progression, its depletion in cancer cells results in HIF-1α suppression, which is associated with downregulated glycolysis, angiogenesis, and anti-proliferative effects. These Mint3-related features are commonly seen in the xenografts of various types of cancer cells (e.g., breast cancer, MDA-MB-231; fibrosarcoma, HT-1080; epidermoid carcinoma, A431; non-small cell lung cancer, A549; and urothelial carcinoma, RT-112) [ 11 , 73 , 74 , 76 ]. However, in pancreatic cancer cells, the depletion of Mint3 induces tissue-specific anti-proliferative effects in vitro and in vivo. This was mediated by the inhibition of Skp2 transcription, which promoted G1/S transition in cell cycles and suppressed the induction of metastasis and stemness [ 73 ]. In addition, the Mint3-dependent expression of Skp2 also involves the Mint3–FIH-1–HIF1α axis and is dependent on HIF-1α activity. Unlike in hypoxic conditions, pancreatic cancer cells show high expression and activity of HIF-1α-dependent Skp2 in normoxic conditions. Consistent with the known features of Skp2 and HIF-1α as metastasis enhancers in various types of cancers [ 77 , 78 , 79 , 80 ], Mint3 promotes epithelial–mesenchymal transition in a HIF-1α- and Skp2-dependent manner with an enhancing effect on the expression of the Slug protein. A recent Mint3-related study on urothelial carcinoma also indicated that the depletion of Mint3 downregulates the invasion, migration, and proliferation of cancer cells, accompanied by factors associated with suppressed HIF-1α activity, such as transcription of HIF-1α-targeted genes and glycolysis [ 74 ]. 5.2. Metastatic Ability of Cancer Cells Achieved through Mint3 Expression in IM Using the PyMT breast cancer mouse model, which develops palpable breast cancers that metastasize to the lung, Qian et al. reported that IMs, which are characterized by Gr-1/Ly6C + , CD11b/CD115 + markers, play a key role in the lung metastasis of breast cancer in a C–C motif chemokine ligand 2 (CCL2)-dependent manner [ 81 ]. Other studies using xenograft mouse models of various types of cancers have shown that Mint3 plays an important role in this mechanism of metastasis [ 72 , 82 ]. Cancer cells and the surrounding stromal cells are the sources of CCL2, and they increase the number of IMs in the peripheral blood. Both individual depletion of Mint3 or HIF-1α in IMs leads to the decreased production of vascular endothelial growth factor (VEGF) to comparable levels. These effects of Mint3 or HIF-1α depletion on VEGF expression are consistent with the fact that VEGF is one of the target genes of HIF-1α [ 72 , 83 ]. Upon Mint3-dependent VEGF secretion by IMs, E-selectin expression in endothelial cells and vascular permeabilization of cancer cells are elevated [ 72 ]. Conclusively, IMs are recruited to the lung metastatic site in a HIF-1α- or glycolysis-dependent manner, which is driven by Mint3, to release vascular endothelial growth factor (VEGF), and this secretion of VEGF induces the expression of E-selectin in the epithelial cells in the lung, which enables the extravasation of cancer cells [ 72 ]. Only macrophages/monocytes work as metastatic inducers, and T cells or B cells do not possess similar functions. Notably, IMs secrete VEGF in accordance with the existence of cancer cells; however, its mechanism remains elusive. 5.3. Supportive Effect of Mint3 Expression in CAFs on Cancer Progression CAFs are one of the factors that have a crucial role in promoting cancer malignancy through ECM remodeling, immune crosstalk, and metabolic effects [ 84 ]. These features of CAFs are achieved by functioning as a source of various secretory proteins, such as growth factors, cytokines, and exosomes, whereas the adherent molecules in CAFs could also increase tumor malignancy in a Mint3-dependent mechanism. Mint3 is also involved in the proliferation of cancer cells by regulating the expression of L1 cell adhesion molecule (L1CAM) in fibroblasts. L1CAM is a cell adhesion molecule that acts as a binding partner of heterodimeric integrins, such as α5β1, αvβ3, αIIbβ3, and αvβ5 [ 85 , 86 ], and triggers downstream signaling pathways, such as the MAPK and PI3K/Akt pathways [ 85 , 87 ]. Fibroblasts express L1CAM in a Mint3-dependent manner, which enables CAFs to promote direct contact with cancer cells expressing integrin α5β1. Therefore, Mint3 also plays a role in enhancing the proliferation of cancer cells through CAFs [ 11 ]. Because CAF-mediated ERK phosphorylation specifically occurs in the peripheral regions of breast cancers, which are surrounded by stroma, L1CAM is possibly involved in the local progression of breast cancers. The expression of Mint3 in CAFs is slightly upregulated by cytokines such as tumor necrosis factor-α and IL-1β. Indeed, CAFs express higher levels of MT1-MMP than normal fibroblasts do, which is also reflected in the activity level of Mint3 in fibroblasts. Further investigation is required to determine whether the activity level of Mint3 has an unlimited correlation with the expression level of MT1-MMP. 6. Therapeutic Efficacy of Targeting Mint3-Related Environments In some cases, the activity of HIF-1α is toxic in terms of its ability to promote inflammatory diseases or cancer malignancies. Thus, HIF-1α has long been regarded as a beneficial therapeutic target. Many efforts have been made to develop HIF-1α inhibitors, but none have been successful, although some clinical trials are still underway. This is probably due to the ubiquitous expression of HIF-1α, which influences various factors. The first HIF-2α inhibitor, belzutifan (Welireg), was clinically approved for patients with VHL diseases and some types of cancers in 2021. Naphthofluorescein (Naph) was first identified as a potent Mint3 inhibitor. It effectively disrupts the protein interaction between Mint3 and FIH-1, leading to the suppression of HIF-1α activity with reduced expression of HIF-1α-targeting genes, glycolysis, and ATP production [ 76 ]. The inhibitory effect of Naph on Mint3 in inflammatory diseases effectively diminishes cytokine synthesis; upon LPS stimulation, this is well reflected in the increased survival of mice with LPS-induced inflammatory diseases. Moreover, Naph retains its inhibitory potential against chemotaxis of IMs toward CCL2–expressing cancer cells and the surrounding stromal cells, thereby reducing the expression of E-selectin in metastatic lung cancers without marked adverse effects. Notably, the pharmacological effect of Naph on metastasis is not affected in Mint3-deficient mice, which implies that Naph can suppress metastasis specifically by inhibiting Mint3 [ 76 ]. Conclusively, the pharmacological inhibition of Mint3 by Naph is effective against both immune-signal-related diseases and cancer progression with minimal adverse effects. The depletion of Mint3 potentiates the efficacy of a combined therapy of gemcitabine and paclitaxel, which is currently a standard regimen for pancreatic cancers. A combined therapy of gemcitabine and Naph reportedly has a synergistic effect against the progression of urothelial carcinoma cells in vivo. These preclinical outcomes indicate that Mint3 targeting could be a therapeutic intervention for pancreatic and urothelial cancers [ 73 , 74 ]. However, we cannot ignore the fact that Naph has low solubility in water and can also bind to furin, which could potentially result in unexpected outcomes [ 88 ]. 7. Conclusions Mint3 inhibition effectively disrupts HIF-1α-mediated inflammations or cancer progressions. Although Huai et al. reported that Mint3 acts as a protective factor against inflammatory stimuli by enhancing IFN-β production associated with the positive feedback loop of Mint3 expression, Mint3 generally mediates biologically lethal effects by promoting acute inflammatory responses ( Figure 3 ). Mint3 can promote the proliferation and metastasis of cancer cells and complicate cancer treatment by employing macrophages as invasion enhancers and CAFs as cell growth drivers [ 11 , 72 ]. In some cases, Mint3 directly promotes the proliferation and metastasis of cancer cells, such as pancreatic and urothelial cancer cells [ 73 , 74 ]. Therefore, Mint3 could be a potential therapeutic target for innate immune-response-related diseases and cancer. Because of the ubiquitous expression of HIF-1α and its influence on various factors related to biological maintenance, HIF-1α deficiency can cause various adverse effects. The direct targeting of HIF-1α is challenging as a result; thus, the indirect inhibition of HIF-1α by targeting Mint3 would potentially be beneficial. This is because Mint3 requires the cooperative expression of MT1-MMP, the expression of which is limited to specific areas, such as macrophages/monocytes, cancer cells, and endothelial tips, and the intact function of Mint3 is also limited to these areas. Mint3 knockout mice models of either acute inflammation upon stimuli by various pathogens or xenografts with various cancer cells showed significant improvement in the diseases. Naph, as a Mint3 inhibitor, inhibits the secretion of inflammatory cytokines and the proliferation and metastasis of tumors in vivo without causing marked adverse effects [ 74 , 76 ]. Therefore, Naph, a potential inhibitor of Mint3 as a therapeutic target, ought to be further explored.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852055/

Ionic Cross-linked Chitosan Beads for Extended Release of Ciprofloxacin: In vitro Characterization

Chitosan beads loaded with ciprofloxacin hydrochloride were fabricated by ionic cross-linking with sodium tripolyphosphate. The beads showed an excellent water retention property. The degradation of fabricated beads was influenced by the pH of test medium. High drug load was achieved within the bead with a short curing time. Drug release was high in acidic medium (pH 1.2) vis-à -vis intestinal medium (pH 7.4). Ciprofloxacin hydrochloride release increased with an increasing concentration of ciprofloxacin and decreasing proportion of chitosan. Drug release followed both first-order and Higuchi's root time kinetics showing non-Fickian release mechanism. MATERIALS AND METHODS Ciprofloxacin hydrochloride was a generous gift of M/s Dr. Reddy's Laboratories (Hyderabad, India). Chitosan (Mol. wt. 2 × 10 5 , degree of deacetylation 82-88%) was obtained from M/s Kraeber Gmbh and Co (Ellerbek, Germany). Sodium tripolyphosphate (TPP) was procured from S. D. Fine Chemicals (Mumbai, India). All other reagents were of analytical grade and used as received. Preparation of chitosan beads: Chitosan beads were prepared by the ionic cross-linking as described by Bodemier et al. 7 with minor modifications. Briefly, chitosan was dissolved in acetic acid (2% v/v) and stirred for 6-7 h on a magnetic stirrer (Remi Equipments, Mumbai). Weighed amount of ciprofloxacin HCl ( Table 1 ) was added to the polymeric solution and stirred on a magnetic stirrer for 2 h, and allowed to stand till the removal of the entrapped air bubbles. The pH of the drug-chitosan solution was adjusted to pH 4-4.5 with dilute alkali solution (0.1 M, NaOH). Chitosan solution containing drug was added dropwise using a syringe fitted with a flat-end needle (23G, 0.7 mm id) into sodium tripolyphosphate solution (1-4% w/v, pH 5, 60°). Beads were left for 20-30 min, unless otherwise specified, and after curing, were collected by filtration, washed twice with distilled water and dried at 50° for 4 h and then at room temperature (25°) for 12 h. TABLE 1 FORMULATION COMPOSITIONS, PHYSICAL AND RELEASE CHARACTERISTICS OF THE FABRICATED BEADS Batch Chitosan (% w/v) Ciprofloxacin (% w/v) TPP (% w/v) Wet bead size (mm) Dry bead size (mm) IE (%) Release exponent (n) CB1 2 1 1 2.21 ± 0.32 0.61 ± 0.05 67.30 ± 2.34 0.51 CB2 2 1 2 2.23 ± 0.19 0.60 ± 0.09 67.21 ± 1.95 0.53 CB3 2 1 3 2.22 ± 0.09 0.62 ± 0.14 69.08 ± 3.11 0.53 CB4 2 1 4 2.23 ± 0.17 0.61 ± 0.08 68.70 ± 1.61 0.58 CB5 1 1 2 2.08 ± 0.51 0.65 ± 0.15 60.53 ± 2.09 0.51 CB6 3 1 2 2.31 ± 0.24 0.70 ± 0.03 73.67 ± 2.44 0.58 CB7 2 0.5 2 2.11 ± 0.21 0.69 ± 0.11 75.80 ± 2.39 0.59 CB8 2 2 2 2.24 ± 0.28 0.68 ± 0.08 64.42 ± 3.10 0.47 IE- Incorporation efficiency, calculated from the percentage ratio of actual drug concentration in the bead to theoretical drug concentration Particle size determination: The particle size of the prepared beads in a sample was measured with an optical micrometer fitted with a calibrated eye piece. The mean of 100 beads was noted as particle size. The sizes of both wet and dried beads were measured. All readings are average of three trials ± SD. Determination of encapsulation efficiency: About 50 mg of the beads were crushed in a glass mortar and digested in 0.1 N hydrochloric acid (pH 1.2) for 24 h in a graduated flask. The solution was filtered through a G-2 filter and an aliquot was used to assay for drug content spectrophotometrically (Jasco 7800, Japan) at 276 nm against a suitable blank. The encapsulation efficiency was calculated by expressing the actual entrapment level divided by the theoretical entrapment level, as a percentage. The values are average of three trials ± SD. Surface morphology of the beads: The surface morphology images were obtained by scanning bead surface on an electron microscope (SEM, Philips XL20, Holland) under vacuum. Beads were mounted on brass stubs using silver paste and scanned under vacuum at the required magnification at room temperature. Water uptake studies: Water uptake capacity of the beads was determined in 0.1 N HCl (pH 1.2) and pH 7.4. A weighed quantity of beads (100 mg) was immersed in SGF (0.1 N HCl, pH 1.2) and SIF (pH 7.4), and at regular intervals of time, the beads were reweighed after carefully wiping off excess of liquid with a tissue paper. The water uptake was determined from the expression; (W t – W o )/W o , where, W t and W o are the weight of the beads at time't' and under dry state, respectively. In vitro degradation study: In vitro degradation of beads was investigated in pH 1.2 and 7.4. Beads (100 mg) were placed in basket of USP-XXIII dissolution apparatus containing 900 ml of respective media at 50 rpm. At regular intervals beads were removed, excess liquid was wiped off, and oven dried at 80° till constant weight. The mass loss of the beads was calculated from the dry weights measured at the beginning and predetermined time intervals. In vitro drug release studies: In vitro release of ciprofloxacin from the beads was performed in USP XXIII dissolution apparatus II with a paddle speed of 50 rpm. The dissolution medium was 900 ml of SGF without enzyme (0.1 N HCl, pH 1.2) for first 2 h and subsequently rest of the release study was performed in SIF (phosphate buffer, pH 7.4) at 37 ± 0.2°. At regular time intervals, 5 ml aliquot was withdrawn and replenished with an equal volume of fresh dissolution medium. The drug content in the aliquot was assayed spectrophotometrically at 276 nm (Jasco 7800, Japan). A study was performed concurrently with placebo beads to record for any interference by the bead components. For analyzing drug release kinetics, in vitro release data were fitted to; zero-order equation, Q t = Q o + K o t; first-order equation 13 14 Q t = Q o e −Kt ; and Higuchi's 15 square root model Q t = K H √t, where, Q t is the amount of drug released in time't', Q o is the initial amount of drug in dissolution medium, and K o , K and K H are respective release constants. The mechanism of drug release was further analysed using the Korsmeyer-Peppas power law 16 17 M t /M ∞ = Kt n , where, M t /M ∞ is the fraction of drug released in time 't', K is structural and geometric constant, and n is the release exponent. Statistical analysis: All data were analysed by Student's t-test and one-way ANOVA, wherever necessary, using Sigma Stat 2.0 (Jandel Scientific corporation, USA) to determine the statistical difference in the results. A probability value p 50% mass loss in ∼5.5 h. Conversely, the degradation was found to be negligible at pH 7.4. The faster degradation in SGF was in contrast with the observations of Durkut et al. 20 , wherein the complete degradation was achieved in 6 months. The observed faster degradation could be due to high acid solubility of ciprofloxacin, which caused pores in the matrix leading to easy ingress of SGF and subsequent degradation. The release of ciprofloxacin in two different media (pH 1.2 and 7.4) is shown in fig. 2 . The percent of drug released at pH 1.2 was higher in comparison to that at pH 7.4 exhibiting pH-sensitivity of the beads. At the end of 4 h, 98% of drug load was depleted from CB1 (TPP- 1% w/v) in SGF (0.1 N HCl, pH 1.2), in comparison to 70% in SIF (pH 7.4). The result was similar for beads prepared with higher concentration of TPP with a release of ∼70% for CB4 (TPP- 4% w/v) at pH 1.2 and ∼50% at pH 7.4. The results indicate significant ( p 0.05). The primary reason for this observation is, increasing the cross-linking density reduces swelling of the beads hindering drug release 7 . The effect of pH of TPP solution on drug release is shown in fig. 4 . Ciprofloxacin release from the beads was influenced by the pH of TPP solution. Beads prepared in original solution (pH 8.5) released ciprofloxacin faster than those prepared after adjusting the pH of TPP solution to 5.0. The pore structure of chitosan microparticle was modified by the change in the pH of TPP solution 25 . At higher pH open porous structure with low density was reported by Ko et al. 26 . The ionization of amine group on chitosan decreases with increasing pH proceeding to weaker cross-liking density. These reasons attribute to the faster release of ciprofloxacin from beads prepared in TPP solution at pH 8.5. Fig. 4 Effect of pH of coagulation fluid on drug release. Beads were fabricated with coagulation fluid (sodium tripolyphosphate solution) at pH 5 (–●–) and 8.5(–○–). The release of ciprofloxacin depends on its concentration in the bead and chitosan ( fig. 5 ). Fast and complete drug release was observed from CB5 and CB8 containing equal ratio of ciprofloxacin and chitosan (1:1 drug:chitosan) in comparison to other batches. The drug release was reduced with decrease in ciprofloxacin and increase in chitosan concentration. Beads exhibited a burst release (∼20% in 30 min). The diffusion of drug from the surface creates a pore in the matrix which causes a channelling effect. Incorporation of higher concentration of drug causes more pore formation leading to faster and higher drug release. This phenomenon was in contrast to the reported decrease in drug release with increasing concentration of sulphadiazine, a poorly soluble drug 7 . Fig. 5 Influence of ciprofloxacin and chitosan concentration on in vitro drug release profile. Drug release profile of batches CB2 (–●–), CB5 (–○–), CB6 (–▼–), CB7 (–△–) and CB8(–■–). The regression co-efficient (r 2 ) for equation Q t = Q o e −Kt was between 0.9902 and 0.9968 for batches CB1, CB2, CB3 and CB7. This was higher than for other kinetic equations indicating that drug release followed first-order kinetics. Other fabricated batches viz., CB4, CB5, CB6 and CB8, showed higher regression co-efficient (between 0.9853 and 0.9979) for Higuchi's square root equation (Q t = K H √t) indicating time dependent release mechanism. The release exponent (n) values ( Table 1 ) were in the range of 0.47-0.59 suggesting drug release by a combination of diffusion and dissolution. The observed deviation from Fickian mechanism could be possibly due to higher molecular weight 27 of ciprofloxacin (331.4) and the polymer characteristics (solubility, pKa, tortuosity) 28 . The fabricated beads showed good reproducibility. The process being simple can be used in the formulation of sustained release formulations. A desired release profile could be achieved by modifying a few process parameters, which are discussed above. Further studies are needed to evaluate the performance of these systems in in vivo and to optimize the formulation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122042/

Mutations in the COPI coatomer subunit α-COP induce release of Aβ-42 and amyloid precursor protein intracellular domain and increase tau oligomerization and release

Alzheimer's disease (AD) is the most common cause of dementia, afflicting more than 5 million Americans; it is the 6th leading cause of death in the United States. As the population ages, the number of Americans with AD expected to increase dramatically. Understanding the cellular processes that lead to AD pathology is critical to designing meaningful therapeutic interventions. AD is characterized by the accumulation of aggregated proteins that may be toxic and may represent failure of the cell to normally process these proteins. One key to understanding sporadic AD lies in the genetics of families with highly penetrant histories of the disease. Mutations in subunits of a cellular trafficking complex known as COPI were found in families with AD and no other known AD-associated mutations. The COPI complex is involved in protein processing and trafficking within the cell. Intriguingly, several recent publications have found that components of the COPI complex can affect the metabolism of pathogenic AD proteins. We report here that reducing levels of the COPI subunit α-COP alters maturation and cleavage of amyloid precursor protein (APP), resulting in decreased release of Aβ−42 and decreased accumulation of the APP intracellular C-terminal domain. We also found that depletion of α-COP reduces uptake of proteopathic Tau seeds and reduces intracellular Tau self-association. Expression of AD-associated mutations in α-COP altered APP processing, resulting in increased release of Aβ−42 and increased intracellular Tau aggregation and release of Tau oligomers. Taken together, these results show that COPI coatomer function modulates the processing of both APP and Tau and that expression of AD-associated α-COP mutant proteins confers a toxic gain of function, which results in potentially pathogenic changes in both APP and Tau.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795356/

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Graphical abstract 1 Introduction High-throughput screening (HTS) provides a platform for the identification of small molecules capable of modulating protein function and cellular phenotypes. 1 , 2 HTS of cellular pathway assays may identify a variety of small molecule compounds with different molecular targets which can be identified in subsequent mechanistic studies. Such pathway assays are particularly attractive when studying human pathogens, since they allow HTS to occur in a nonrestrictive biosafety level environment. Recently Hobson et al. described such a cell-based pathway assay that monitors receptor-mediated endocytosis of a modified, non-toxic anthrax toxin lethal factor (LF)/protective antigen (PA) complex. 3 The technology relies upon a LF-β-lactamase fusion protein (LF-β-Lac) that co-opts the native mechanism of LF internalization, 4 whereby proteolysis of PA allows heptamerization of the cleaved PA, association with LF, internalization via endocytosis, and translocation of LF to the cytoplasm triggered by endosome acidification. 5 Once internalized, β-lactamase hydrolysis of the cephalosporin-based fluorescein/coumarin fluorogenic substrate CCF2 disrupts intramolecular CCF2 FRET and changes emission from fluorescein (EM 530 nm) to coumarin (EM 460 nm) fluorescence. The number of blue (coumarin fluorescent) cells, and their proportion relative to green (fluorescein fluorescent) cells, indicates the extent of inhibition of LF-β-lac internalization. This system accurately reports on the cellular uptake processes by which cells become intoxicated by anthrax PA and LF, which together constitute anthrax lethal toxin (LT). It should be noted that the LF-β-Lac has no toxicity. Humans contract anthrax through inhalation, ingestion, or cutaneous inoculation of endospores of the Gram-positive aerobic rod Bacillus anthraci s to cause the disease entities known, respectively, as inhalation anthrax, intestinal anthrax, and cutaneous anthrax. The endospores germinate following introduction to the body to cause a productive infection with incubation times ranging from 12 h to two weeks, and mortality rates in untreated cases ranging from 20% to more than 90%. B. anthracis forms endospores under adverse conditions that are usually present in the soil where infected animals died. The bacterium is simple to culture, and sporulation can easily be induced in a controlled laboratory setting. The endospores of B. anthraci s are highly resistant to chemical insults, heat exposure, and dehydration, and remain infectious even after prolonged storage. These properties, combined with the high morbidity and mortality of anthrax, makes B. anthraci s a significant threat as a bioterrorism agent, as illustrated by the effects of the deliberate contamination of mail with endospores in the fall of 2001. As a means to identifying chemical probes of the LF internalization process the NIH Chemical Genomics Center (NCGC), a member of the Molecular Libraries Initiative of the NIH Roadmap for Medical Research 6 was engaged. The original assay design was miniaturized and screened against 70,094 compounds in 1536-well plate format at a minimum of 7 concentrations each, utilizing the quantitative high-throughput screening (qHTS) 7 approach. Here we report the identification of several novel chemotypes from this screen which block internalization of anthrax LF and protect cells from LF action. Additionally, we provide evidence that the compounds disrupt the PA–LF constructs internalization process. 2 Results and discussion 2.1 Quantitative high-throughput screen The originally reported assay, 3 diagrammed in Figure 1 , was adapted and miniaturized to a 1536-well plate format. qHTS and data analysis were performed as previously described 7 with modifications as noted in Section 4 . A total of 70,094 compounds were tested at ⩾ 7 concentrations from 2.6 nM to 40 μM, and concentration–response curves were generated and classified for all compounds. 7 From these data, 1170 compounds showing concentration-dependent inhibition of the β-lactamase catalyzed hydrolysis of CCF2 without effects on the green fluorescence channel (a proxy for cell number) and having efficacies >50% were identified and clustered for structural similarity. The dual fluorescence output design enabled the discrimination of translocation inhibitors from cytotoxic compounds, as the latter were clearly discernable as a decrease in green fluorescence without an increase in blue fluorescence. Compounds (211) representing 44 clusters of related compounds and singletons were prioritized based on compound curve classification, potency, and when applicable, structural series cluster size. Figure 1 Principle of cell surface protease activity imaging assay. (1) PA binds to the high affinity cell surface receptors TEM8 or CMG2. (2) PA is cleaved by furin or furin-like pro-protein convertases. (3) The PA fragment that remains on the cell surface heptamerizes. (4) High-affinity binding sites for LF-β-Lac are generated by heptamerization of the PA. (5) LF-β-Lac is translocated to the cytoplasm after endocytosis of the PA–LF-β-Lac complex (not shown). (6) CCF2/AM is added to cells and diffuses into the cytoplasm where it is trapped by hydrolysis of its acetoxymethyl ester groups by non-specific cytoplasmic esterases. (7) LF-β-Lac hydrolyses the cephalosporin ring of CCF2, causing a shift in fluorescence emission from 520 nm (green light) to 447 nm (blue light) after excitation of cells at 409 nm. Blue fluorescence emission by a cell demonstrates successful translocation of LF-β-Lac to the cytoplasm. Reproduced from Ref. 3 . As an initial exploration into the numerous actives, and in order to validate the screening assay as a reliable predictor of protection against anthrax intoxication, 30 compounds with high confidence qHTS concentration–response curves (Class 1.1) 7 and/or known biological activities (niclosamide, homoharringtonine, GF 109203, PD 404182, cucurbitacin I, proscillaridin A, and bisacodyl) were chosen for follow-up. Fresh powder samples of these 30 compounds were obtained and serially diluted into 24 concentrations; EC 50 values were determined using the primary screen protocol in 1536-well plate format. The compounds were then further tested in 96-well format as a comparative experiment to the qHTS confirmatory assays, with results determined both by epifluorescence microscopy and plate reader quantification. Among the most compelling confirmed actives identified were a novel chromeno[4,3- c ]pyrazol-4(1 H )-one (NCGC00084148-01) ( 1 ), the lignan diphyllin ( 2 ) and the teniacide niclosamide ( 3 ) ( Fig. 2 ). Figure 2 Chemical structure of anthrax toxin internalization inhibitors identified through the qHTS. Structures of NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ). 2.2 Protective effect on RAW264.7 macrophages from lethal toxin Compounds (30) with confirmed activity in the qHTS assay were entered into follow-up studies to determine their native LF-protective effects on macrophages and their mechanisms of action, since the primary assay may detect compounds acting via a variety of mechanisms including inhibition of β-lactamase. 3 First, selected compounds were assayed for their ability to protect RAW264.7 macrophages from the effects of anthrax lethal toxin. RAW264.7 cells were treated with varying amounts of a combination of PA and LF in the presence of increasing concentrations of compounds. Following incubation, cell viability was assessed using a colorimetric MTT reduction assay. Of the 30 compounds tested, seven offered significant protection from LF activity. Diphyllin ( 2 ) at 3 μM afforded nearly complete protection against the LF–PA-mediated cell killing, while NCGC00084148-01 (designated 148-01) ( 1 ) and niclosamide ( 3 ) provide the same protection at ∼10 μM ( Fig. 3 ). The known kinase inhibitor GF 109203 8 was also found to have protective effects on RAW264.7 cells; however, GF 109203 did not achieve the same potency or efficacy and was noted to be cytotoxic at concentrations above 10 μM. Additionally, we assayed the alkaloid and potent myelosuppressive homoharringtonine 9 (designated 1A/61951), the KDO-8-P synthase inhibitor PD 404182 10 (designated K4607) and 2-(4-methylpyrimidin-2-yl)benzo[ d ]isothiazol-3(2 H )-one (designated 889-01). None of these other leads from the primary screen were noted as having protective effects on RAW264.7 cells. Given the strong protective effect demonstrated by diphyllin ( 2 ), we ascertained the protective effects of this agent across a concentration range of LT and after a 45 min pre-incubation of LT. The results show that diphyllin ( 2 ) is effective as a protective agent up to 1000 ng/mL concentrations of LT and demonstrates protective effects even after 45 min pre-exposure to LT ( Fig. 4 ). Figure 3 Effects of inhibitors on cytotoxicity of RAW264.7 cells by anthrax lethal toxin. RAW264.7 cells that were pre-incubated with various concentrations of the inhibitors for 30 min were treated with 100 ng/ml PA and 100 ng/ml LF for 4 h. Cell viability then was measured by adding MTT. Among 30 compounds tested (only seven shown in the figure) NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203 showed clear protection to RAW264.7 cells from LT. GF109203 displayed cytotoxicity when used at high concentrations (>10 μM)]. Figure 4 Effects of diphyllin ( 2 ) on cytotoxicity of anthrax lethal toxin toward RAW264.7 cells. Viability of RAW264.7 cells after treatment with toxin either in the absence of inhibitor, with a 1 h pre-incubation of cells with diphyllin ( 2 ) (10 μM), and with diphyllin ( 2 ) added 45 min after toxin addition. 2.3 Protective effect on CHO cells from the fusion toxin FP59 Next we determined if NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3) protected cells from killing by interfering with delivery of LF proteins to their cytoplasmic targets. For this purpose, CHO cells were incubated with PA and FP59 in the presence of the putative inhibitors. FP59 is a fusion protein consisting of the PA-binding LF amino-terminal fragment and the catalytic domain of Pseudomonas exotoxin A (PE). FP59 kills cells by ADP-ribosylating the diphthamide residue on elongation factor-2 (EF-2). Diphthamide is a unique post-translationally modified histidine residue found only in EF-2, and conserved from archaebacteria to humans, serving as the target for diphtheria toxin (DT) and PE. 11 , 12 These studies showed that NCGC00084148-01 ( 1 ) diphyllin ( 2 ) and niclosamide ( 3 ) were all capable of cell protection at micromolar concentrations (data not shown) indicating a potentially shared mechanism of action with the protective effects of these agents versus LT. 2.4 Protective effect on CHO cells from diphtheria toxin (DT) Next, NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) were studied for their ability to block the actions of DT in CHO cells. This toxin utilizes a different receptor, does not require PA for endocytosis, and exits from endosomes at an earlier stage than LF. 13 Interestingly, diphyllin ( 2 ) and niclosamide ( 3 ) provided complete protection (100% cell viability) at approximately 5 μM while NCGC00084148-01 ( 1 ) and GF 109203 produced approximately 75% and 60% cell viability at a higher concentration (10 μM), respectively ( Fig. 5 ). This strongly suggests that the mechanism by which NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) block the toxic effects of LF does not involve interfering with the binding of PA and/or LF to its receptors, but rather the common process of toxin internalization, including endosome acidification and endocytosis. Figure 5 Protective effect of the compounds on CHO cells exposed to diphtheria toxin. Viability of CHO cells in the presence of diphtheria toxin (25 ng/mL) and increasing amounts of NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203. 2.5 Blocking of PA heptamer prepore-to-pore conversion That NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) protect RAW264.7 cells from the effects of PA and LF, and CHO cells from PA + FP59 and diphtheria toxin suggests that these compounds act on the process of endocytosis, or endosomal acidification just prior to pore formation and toxin translocation. It has previously been shown that pore formation is triggered by an acidification of the endosomal pH. 14 To discriminate among these possibilities, we used PA receptor-deficient CHO PR230 cells transfected with either tumor endothelial cell marker 8 (TEM8) (PR230 TEM8-T4 cells) or capillary morphogenesis gene 2 product (CMG2) (PR230 CMG2-C4 cells). 15 , 16 Both TEM8 and CMG2 are well characterized PA receptors, but they differ significantly in their pH dependence for pore formation and LF translocation. 15 The TEM8 receptor requires a relatively mild acidic environment (pH 6.0–6.5) to allow PA pore formation, while the CMG2 receptor requires a relatively low pH (5.5–6.0). Conversion of the SDS/heat-sensitive PA heptamer prepore to the SDS/heat-resistant functional LF-conductive pore requires acidic pH in endosomes. The TEM8- and CMG2-expressing cells were treated with PA and LF in the presence of NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ), GF 109203 or the known vacuolar (H+)-ATPase (V-ATPase) inhibitor bafilomycin. PA binding, proteolytic processing, PA heptamer formation, and PA-dependent LF binding to cells were assessed by SDS–PAGE followed by Western blotting using PA and LF antibodies as described previously. 5 None of the compounds blocked cellular binding, proteolytic processing, heptamerization of PA, or PA-mediated LF binding. In contrast, all four compounds disrupted heptamer prepore-to-pore conversion in CMG2-expressing cells to a degree comparable to bafilomycin, with markedly less effects on TEM8 expressing cells ( Fig. 6 ). Interestingly, diphyllin displayed a modest effect on TEM8 cells. This trend was mirrored in the viabilities of the cells in the presence of each agent across a concentration gradient ( Fig. 7 ). These results suggest that the protective effect of these agents is the result of preventing endosome acidification (most likely through the inhibition of V-ATPase action) or blocking of toxin trafficking to endosomes of increased acidity. It is possible to differentiate between these mechanisms through the use of pH sensors that allow direct quantification of endosomal acidity (these experiments were not performed). Importantly, Collier and coworkers recently demonstrated that selective mutagenesis of phenylalanine 427 of PA (a crucial residue for membrane transport of PA) produced variable effects on PA function. 17 Selected mutants (F427K, F427R, F427D, F427G) were found to mitigate the pH-induced conformational change associated with pore formation. Other mutations (F427H, F427S, F427T) did not affect prepore-to-pore conversion but inhibited translocation of the LF construct. This study clearly demonstrates that small molecule intervention of cellular entry of the PA–LF construct can occur through different mechanisms. Figure 6 Effects of the inhibitors on anthrax toxin binding, proteolytic processing, and heptamer formation on CHO cells. Anthrax toxin receptor-deficient CHO PR230 cells transfected with either TEM8 (PR230 TEM8-T4, left panel) or CMG2 (PR230 CMG2-C4, right panel) were pre-treated with 10 μM of NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203, or 0.2 μM of Bafilomycin A1 for 30 min. Then the cells were incubated with 1 μg/ml PA and 1 μg/ml LF for 1 h at 37 °C, washed to remove unbound toxin, and lysed using modified RIPA buffer. Cell lysates boiled in SDS sample buffer were separated by SDS-PAGE, followed by Western blotting using PA antiserum (no. 5308) or LF antiserum (no. 5309) for the upper and lower images, respectively. Figure 7 Effects of inhibitors on viability of PR230 TEM8-T4 and PR230 CMG2-C4 cells treated with PA and LF. Protective effects by NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203 mirror the heptamer formation results by protecting PR230 CMG2-C4 cells, but not PR230 TEM8-T4 cells, indicating a mechanism of action that blocks late-stage vacuolar release but not early stage vacuolar release of LF complex. The cells were pre-incubated with drugs for 30 min, then 100 ng/ml PA + 100 ng/ml FP59 (final concentration) were added and incubated for 3 h. After that the toxin/durg-containing media were replaced with regular growth media containing 10 mM of ammonium chloride and incubated for 48 h as described in Section 4.5 . Interestingly, it has been demonstrated that V-ATPases are important mediators of cellular entry by a number of viruses, including Ebola and the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), 18 and the ability of bafilomycin to block endosomal acidification and limit viral entry into cells is well known. 19 , 20 Recently, Diphyllin ( 2 ) was noted to be a potent inhibitor of V-ATPase activity and was further shown to affect bone resorption in osteoclast models. 21 This study is consistent with our findings and yields insight into this particular mechanism for inhibition of LF internalization. That niclosamide ( 3 ) potentially acts as a V-ATPase inhibitor is possibility relevant to recent reports suggesting niclosamide may serve as a therapy for SARS-CoV infection. 21 Niclosamide, found here to disrupt heptamer prepore-to-pore conversion in the pH sensitive CHO-CMG2 cell line, eliminated SARS-CoV viral antigen synthesis at modest concentrations (1.56 μM). 22 The mechanism of niclosamide's effect on viral replication is unresolved; though modifications of niclosamide's core 2-chloro-4-nitroaniline substructure lead to inhibitors of the SARS 3CL protease 23 (niclosamide had only weak inhibitory activity (IC 50 40 μM) 23 , 24 against this target). The possibility that niclosamide ( 3 ) does act on V-ATPase is intriguing and may yield insight into its effect on viral entry. It should be noted that niclosamide (CID: 4477) appears as an active compound numerous times in the Pubchem online database. The discovery of the novel chromeno[4,3- c ]pyrazol-4(1 H )-one (NCGC00084148-01) ( 1 ) is also of interest. It has been hypothesized that the presence of highly basic tertiary amine moieties such as those present in NCGC00084148-01 ( 1 ) may directly neutralize endosomal pH; for example, GF109203 inhibits anthrax toxin entry into cells. 25 However, in this study, we found no such correlation between highly basic compounds and activity within the primary screen, and analogues of NCGC00084148-01 ( 1 ) that retained the piperizine moiety but differed in other significant ways were inactive in our experiments, suggesting that at least under these conditions these compounds do not act via direct neutralization of endosomal pH. 2.1 Quantitative high-throughput screen The originally reported assay, 3 diagrammed in Figure 1 , was adapted and miniaturized to a 1536-well plate format. qHTS and data analysis were performed as previously described 7 with modifications as noted in Section 4 . A total of 70,094 compounds were tested at ⩾ 7 concentrations from 2.6 nM to 40 μM, and concentration–response curves were generated and classified for all compounds. 7 From these data, 1170 compounds showing concentration-dependent inhibition of the β-lactamase catalyzed hydrolysis of CCF2 without effects on the green fluorescence channel (a proxy for cell number) and having efficacies >50% were identified and clustered for structural similarity. The dual fluorescence output design enabled the discrimination of translocation inhibitors from cytotoxic compounds, as the latter were clearly discernable as a decrease in green fluorescence without an increase in blue fluorescence. Compounds (211) representing 44 clusters of related compounds and singletons were prioritized based on compound curve classification, potency, and when applicable, structural series cluster size. Figure 1 Principle of cell surface protease activity imaging assay. (1) PA binds to the high affinity cell surface receptors TEM8 or CMG2. (2) PA is cleaved by furin or furin-like pro-protein convertases. (3) The PA fragment that remains on the cell surface heptamerizes. (4) High-affinity binding sites for LF-β-Lac are generated by heptamerization of the PA. (5) LF-β-Lac is translocated to the cytoplasm after endocytosis of the PA–LF-β-Lac complex (not shown). (6) CCF2/AM is added to cells and diffuses into the cytoplasm where it is trapped by hydrolysis of its acetoxymethyl ester groups by non-specific cytoplasmic esterases. (7) LF-β-Lac hydrolyses the cephalosporin ring of CCF2, causing a shift in fluorescence emission from 520 nm (green light) to 447 nm (blue light) after excitation of cells at 409 nm. Blue fluorescence emission by a cell demonstrates successful translocation of LF-β-Lac to the cytoplasm. Reproduced from Ref. 3 . As an initial exploration into the numerous actives, and in order to validate the screening assay as a reliable predictor of protection against anthrax intoxication, 30 compounds with high confidence qHTS concentration–response curves (Class 1.1) 7 and/or known biological activities (niclosamide, homoharringtonine, GF 109203, PD 404182, cucurbitacin I, proscillaridin A, and bisacodyl) were chosen for follow-up. Fresh powder samples of these 30 compounds were obtained and serially diluted into 24 concentrations; EC 50 values were determined using the primary screen protocol in 1536-well plate format. The compounds were then further tested in 96-well format as a comparative experiment to the qHTS confirmatory assays, with results determined both by epifluorescence microscopy and plate reader quantification. Among the most compelling confirmed actives identified were a novel chromeno[4,3- c ]pyrazol-4(1 H )-one (NCGC00084148-01) ( 1 ), the lignan diphyllin ( 2 ) and the teniacide niclosamide ( 3 ) ( Fig. 2 ). Figure 2 Chemical structure of anthrax toxin internalization inhibitors identified through the qHTS. Structures of NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ). 2.2 Protective effect on RAW264.7 macrophages from lethal toxin Compounds (30) with confirmed activity in the qHTS assay were entered into follow-up studies to determine their native LF-protective effects on macrophages and their mechanisms of action, since the primary assay may detect compounds acting via a variety of mechanisms including inhibition of β-lactamase. 3 First, selected compounds were assayed for their ability to protect RAW264.7 macrophages from the effects of anthrax lethal toxin. RAW264.7 cells were treated with varying amounts of a combination of PA and LF in the presence of increasing concentrations of compounds. Following incubation, cell viability was assessed using a colorimetric MTT reduction assay. Of the 30 compounds tested, seven offered significant protection from LF activity. Diphyllin ( 2 ) at 3 μM afforded nearly complete protection against the LF–PA-mediated cell killing, while NCGC00084148-01 (designated 148-01) ( 1 ) and niclosamide ( 3 ) provide the same protection at ∼10 μM ( Fig. 3 ). The known kinase inhibitor GF 109203 8 was also found to have protective effects on RAW264.7 cells; however, GF 109203 did not achieve the same potency or efficacy and was noted to be cytotoxic at concentrations above 10 μM. Additionally, we assayed the alkaloid and potent myelosuppressive homoharringtonine 9 (designated 1A/61951), the KDO-8-P synthase inhibitor PD 404182 10 (designated K4607) and 2-(4-methylpyrimidin-2-yl)benzo[ d ]isothiazol-3(2 H )-one (designated 889-01). None of these other leads from the primary screen were noted as having protective effects on RAW264.7 cells. Given the strong protective effect demonstrated by diphyllin ( 2 ), we ascertained the protective effects of this agent across a concentration range of LT and after a 45 min pre-incubation of LT. The results show that diphyllin ( 2 ) is effective as a protective agent up to 1000 ng/mL concentrations of LT and demonstrates protective effects even after 45 min pre-exposure to LT ( Fig. 4 ). Figure 3 Effects of inhibitors on cytotoxicity of RAW264.7 cells by anthrax lethal toxin. RAW264.7 cells that were pre-incubated with various concentrations of the inhibitors for 30 min were treated with 100 ng/ml PA and 100 ng/ml LF for 4 h. Cell viability then was measured by adding MTT. Among 30 compounds tested (only seven shown in the figure) NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203 showed clear protection to RAW264.7 cells from LT. GF109203 displayed cytotoxicity when used at high concentrations (>10 μM)]. Figure 4 Effects of diphyllin ( 2 ) on cytotoxicity of anthrax lethal toxin toward RAW264.7 cells. Viability of RAW264.7 cells after treatment with toxin either in the absence of inhibitor, with a 1 h pre-incubation of cells with diphyllin ( 2 ) (10 μM), and with diphyllin ( 2 ) added 45 min after toxin addition. 2.3 Protective effect on CHO cells from the fusion toxin FP59 Next we determined if NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3) protected cells from killing by interfering with delivery of LF proteins to their cytoplasmic targets. For this purpose, CHO cells were incubated with PA and FP59 in the presence of the putative inhibitors. FP59 is a fusion protein consisting of the PA-binding LF amino-terminal fragment and the catalytic domain of Pseudomonas exotoxin A (PE). FP59 kills cells by ADP-ribosylating the diphthamide residue on elongation factor-2 (EF-2). Diphthamide is a unique post-translationally modified histidine residue found only in EF-2, and conserved from archaebacteria to humans, serving as the target for diphtheria toxin (DT) and PE. 11 , 12 These studies showed that NCGC00084148-01 ( 1 ) diphyllin ( 2 ) and niclosamide ( 3 ) were all capable of cell protection at micromolar concentrations (data not shown) indicating a potentially shared mechanism of action with the protective effects of these agents versus LT. 2.4 Protective effect on CHO cells from diphtheria toxin (DT) Next, NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) were studied for their ability to block the actions of DT in CHO cells. This toxin utilizes a different receptor, does not require PA for endocytosis, and exits from endosomes at an earlier stage than LF. 13 Interestingly, diphyllin ( 2 ) and niclosamide ( 3 ) provided complete protection (100% cell viability) at approximately 5 μM while NCGC00084148-01 ( 1 ) and GF 109203 produced approximately 75% and 60% cell viability at a higher concentration (10 μM), respectively ( Fig. 5 ). This strongly suggests that the mechanism by which NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) block the toxic effects of LF does not involve interfering with the binding of PA and/or LF to its receptors, but rather the common process of toxin internalization, including endosome acidification and endocytosis. Figure 5 Protective effect of the compounds on CHO cells exposed to diphtheria toxin. Viability of CHO cells in the presence of diphtheria toxin (25 ng/mL) and increasing amounts of NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203. 2.5 Blocking of PA heptamer prepore-to-pore conversion That NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) protect RAW264.7 cells from the effects of PA and LF, and CHO cells from PA + FP59 and diphtheria toxin suggests that these compounds act on the process of endocytosis, or endosomal acidification just prior to pore formation and toxin translocation. It has previously been shown that pore formation is triggered by an acidification of the endosomal pH. 14 To discriminate among these possibilities, we used PA receptor-deficient CHO PR230 cells transfected with either tumor endothelial cell marker 8 (TEM8) (PR230 TEM8-T4 cells) or capillary morphogenesis gene 2 product (CMG2) (PR230 CMG2-C4 cells). 15 , 16 Both TEM8 and CMG2 are well characterized PA receptors, but they differ significantly in their pH dependence for pore formation and LF translocation. 15 The TEM8 receptor requires a relatively mild acidic environment (pH 6.0–6.5) to allow PA pore formation, while the CMG2 receptor requires a relatively low pH (5.5–6.0). Conversion of the SDS/heat-sensitive PA heptamer prepore to the SDS/heat-resistant functional LF-conductive pore requires acidic pH in endosomes. The TEM8- and CMG2-expressing cells were treated with PA and LF in the presence of NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ), GF 109203 or the known vacuolar (H+)-ATPase (V-ATPase) inhibitor bafilomycin. PA binding, proteolytic processing, PA heptamer formation, and PA-dependent LF binding to cells were assessed by SDS–PAGE followed by Western blotting using PA and LF antibodies as described previously. 5 None of the compounds blocked cellular binding, proteolytic processing, heptamerization of PA, or PA-mediated LF binding. In contrast, all four compounds disrupted heptamer prepore-to-pore conversion in CMG2-expressing cells to a degree comparable to bafilomycin, with markedly less effects on TEM8 expressing cells ( Fig. 6 ). Interestingly, diphyllin displayed a modest effect on TEM8 cells. This trend was mirrored in the viabilities of the cells in the presence of each agent across a concentration gradient ( Fig. 7 ). These results suggest that the protective effect of these agents is the result of preventing endosome acidification (most likely through the inhibition of V-ATPase action) or blocking of toxin trafficking to endosomes of increased acidity. It is possible to differentiate between these mechanisms through the use of pH sensors that allow direct quantification of endosomal acidity (these experiments were not performed). Importantly, Collier and coworkers recently demonstrated that selective mutagenesis of phenylalanine 427 of PA (a crucial residue for membrane transport of PA) produced variable effects on PA function. 17 Selected mutants (F427K, F427R, F427D, F427G) were found to mitigate the pH-induced conformational change associated with pore formation. Other mutations (F427H, F427S, F427T) did not affect prepore-to-pore conversion but inhibited translocation of the LF construct. This study clearly demonstrates that small molecule intervention of cellular entry of the PA–LF construct can occur through different mechanisms. Figure 6 Effects of the inhibitors on anthrax toxin binding, proteolytic processing, and heptamer formation on CHO cells. Anthrax toxin receptor-deficient CHO PR230 cells transfected with either TEM8 (PR230 TEM8-T4, left panel) or CMG2 (PR230 CMG2-C4, right panel) were pre-treated with 10 μM of NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203, or 0.2 μM of Bafilomycin A1 for 30 min. Then the cells were incubated with 1 μg/ml PA and 1 μg/ml LF for 1 h at 37 °C, washed to remove unbound toxin, and lysed using modified RIPA buffer. Cell lysates boiled in SDS sample buffer were separated by SDS-PAGE, followed by Western blotting using PA antiserum (no. 5308) or LF antiserum (no. 5309) for the upper and lower images, respectively. Figure 7 Effects of inhibitors on viability of PR230 TEM8-T4 and PR230 CMG2-C4 cells treated with PA and LF. Protective effects by NCGC00084148-01 (listed as 148-01) ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) and GF 109203 mirror the heptamer formation results by protecting PR230 CMG2-C4 cells, but not PR230 TEM8-T4 cells, indicating a mechanism of action that blocks late-stage vacuolar release but not early stage vacuolar release of LF complex. The cells were pre-incubated with drugs for 30 min, then 100 ng/ml PA + 100 ng/ml FP59 (final concentration) were added and incubated for 3 h. After that the toxin/durg-containing media were replaced with regular growth media containing 10 mM of ammonium chloride and incubated for 48 h as described in Section 4.5 . Interestingly, it has been demonstrated that V-ATPases are important mediators of cellular entry by a number of viruses, including Ebola and the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), 18 and the ability of bafilomycin to block endosomal acidification and limit viral entry into cells is well known. 19 , 20 Recently, Diphyllin ( 2 ) was noted to be a potent inhibitor of V-ATPase activity and was further shown to affect bone resorption in osteoclast models. 21 This study is consistent with our findings and yields insight into this particular mechanism for inhibition of LF internalization. That niclosamide ( 3 ) potentially acts as a V-ATPase inhibitor is possibility relevant to recent reports suggesting niclosamide may serve as a therapy for SARS-CoV infection. 21 Niclosamide, found here to disrupt heptamer prepore-to-pore conversion in the pH sensitive CHO-CMG2 cell line, eliminated SARS-CoV viral antigen synthesis at modest concentrations (1.56 μM). 22 The mechanism of niclosamide's effect on viral replication is unresolved; though modifications of niclosamide's core 2-chloro-4-nitroaniline substructure lead to inhibitors of the SARS 3CL protease 23 (niclosamide had only weak inhibitory activity (IC 50 40 μM) 23 , 24 against this target). The possibility that niclosamide ( 3 ) does act on V-ATPase is intriguing and may yield insight into its effect on viral entry. It should be noted that niclosamide (CID: 4477) appears as an active compound numerous times in the Pubchem online database. The discovery of the novel chromeno[4,3- c ]pyrazol-4(1 H )-one (NCGC00084148-01) ( 1 ) is also of interest. It has been hypothesized that the presence of highly basic tertiary amine moieties such as those present in NCGC00084148-01 ( 1 ) may directly neutralize endosomal pH; for example, GF109203 inhibits anthrax toxin entry into cells. 25 However, in this study, we found no such correlation between highly basic compounds and activity within the primary screen, and analogues of NCGC00084148-01 ( 1 ) that retained the piperizine moiety but differed in other significant ways were inactive in our experiments, suggesting that at least under these conditions these compounds do not act via direct neutralization of endosomal pH. 3 Conclusion Anthrax remains a significant bioterrorism threat, given its ease of propagation and the physical resistance properties of its endospores. While some vaccines and treatments are available, these are frequently ineffective or associated with significant adverse effects. Thus, effective and rapidly acting treatments for anthrax disease are urgently needed. High-throughput screening of large chemical libraries can identify leads for such programs, but is costly and cumbersome to perform in restrictive biosafety level environments. Screening of pathogen pathways reconstituted in non-pathogenic cell types, coupled with rapid follow-up testing of active compounds in the pathogenic organism, offers an attractive alternative to direct pathogen screening. The assay used here takes advantage of the complex process of anthrax intoxication ( Fig. 1 ) to allow the screening of a large chemical library for compounds that affect multiple steps of the intoxication cascade. A quantitative high-throughput screen for small molecules capable of modulating the internalization of anthrax lethal factor was accomplished. Follow-up studies allowed the rapid triage of compounds that were inactive in physiological anthrax intoxication assays. These studies successfully identified compounds with activity not only in the original assay but also on LF-induced cell killing. Of the 30 compounds identified in the primary assay and tested in native LF cell killing assays, 7 (23%) protected against LF-induced macrophage killing. The clinically used teniacide niclosamide, ( 3 ) the natural product diphyllin, ( 2 ) and a novel chromeno[4,3- c ]pyrazol-4(1 H )-one (NCGC00084148-01) ( 1 ) all protected macrophages and CHO cells from cell death induced by PA combined with LF and diphtheria toxin. Since the primary assay detects compounds acting via a variety of mechanisms, a series of mechanism of action studies were undertaken on three selected active compounds, NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ). All three compounds were found to protect CHO cells from the fusion toxin FP59 and from cell killing by diphtheria toxin (which utilizes a different receptor and does not require PA for endocytosis), suggesting that the compounds interfere with toxin internalization. Blockade of PA heptamer prepore-to-pore conversion was then measured in two cell lines where endosomal pH requirements differ. All three compounds influenced heptamer prepore-to-pore conversion at the same concentrations which affect cell viability of each cell line and to a degree comparable to the known vacuolar (H+)-ATPase (V-ATPase) inhibitor bafilomycin. These results suggest a mechanism for NCGC00084148-01 ( 1 ), diphyllin ( 2 ) and niclosamide ( 3 ) that is directly linked to endosome acidification (either inhibition of V-ATPase function or blocking of toxin internalization and/or trafficking to endosomes of increased acidity). These compounds and their derivatives promise to be useful in the study of anthrax pathogenesis and the assay will be useful for identifying additional anthrax protective compounds acting via alternative mechanisms. 4 Experimental 4.1 General procedures Anthrax toxin LF-β-Lac was generated and purified as described. 3 Recombinant B. anthracis LF, PA, and FP59 were made as previously described. 11 CCF2/AM (Coumarin cephalosporin fluorescent acetoxymethyl ester) and its loading buffers were purchased from Invitrogen (Carlsbad, CA). Cervix epithelial cancer line ME-180 was from American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% FBS (Fetal Bovine Serum, Gibco-BRL, Gaithersburg, MD) and antibiotics. 4.2 Assay miniaturization and quantitative high-throughput screen Cells and reagents were prepared as described, 3 and the assay was optimized in a miniaturized 1536-well plate format. The qHTS was performed using a fully automated screening system in a single run, according to the following protocol: 5 μL of ME-180 cells (approximately 2000 cells) were dispensed into each well of the 1536 well plates and allowed to incubate for 18 h in a 37 °C/5% CO 2 environment. The screening library of 70,094 compounds was composed of compounds collected from several sources: 1279 compounds from LOPAC (Sigma-Aldrich, St. Louis, MO), 1120 compounds from Prestwick (Prestwick Chemical Inc., Illkirch, France), 280 purified natural products from TimTec (Newark, DE), three 1000-member combinatorial libraries from Pharmacopeia (Princeton, NJ), 973 compounds from Tocris (Bristol, UK), 1981 compounds from the National Cancer Institute, 718 compounds from Boston University Center for Chemical Methodology and Library Development, and the rest were from the NIH Molecular Libraries Small Molecule Repository. The library was prepared at a minimum of 7 concentrations in DMSO as described. 7 All compounds were dispensed via pintool, to a final concentration range of 2.6 nM–40 μM. A combination of engineered LF-β-lac and PA (1 μg/mL) or engineered LF-β-lac alone (as a control) was then added, and the mixture incubated for 2 h at 37 °C/5% CO 2 . The FRET β-lactamase substrate CCF2/AM was then added to a final concentration of 5 μM, and following a 4-h incubation at room temperature, plates were read in an EnVision plate reader (PerkinElmer, Boston MA), with excitation at 405 nm and emission detection at 460 nm (blue channel) and 530 nM (green channel). 4.3 Analysis of qHTS data Screening data was processed using in-house developed software. Percent activity was computed from the median values of the uninhibited, or neutral, control set and a set of control wells containing no LF/PA. For assignment of plate concentrations and sample identifiers, ActivityBase (ID Business Solutions Ltd, Guildford, UK) was used for compound and plate registrations. An in-house database was used to track sample concentrations across plates. Correction factors were generated from control assay plates containing vehicle (DMSO) only inserted uniformly throughout the screen to monitor background systematic variation in assay signal potentially resulting from issues with reagent dispensers or decrease in enzyme specific activity. Curve fitting was performed using an in-housed developed algorithm. A four parameter Hill equation was fitted to the concentration–response data by minimizing the residual error between the modeled and observed responses. Outliers were identified and masked by modeling the Hill equation and determining if the differences exceeded those expected from the noise in the assay. The curve classification used is the same as described previously, 7 where concentration–response curves are classified based on efficacy, potency, and statistical fit. Curves are classified as negative or positive depending on whether they exhibit signal decrease (apparent inhibition) or increase (apparent activation). Translocation inhibitors are noted as negative response curves in the blue emission channel, and as flat responses in the green channel. Natively fluorescent molecules were noted to stimulate both channels. A negative response in both channels typically indicates non-specific fluorophore quenching or non-specific mechanisms of inhibition, such as cellular apoptosis. The ratio of the green and blue channels normalizes for variation in cell number, is a more robust measure of compound performance, and further allows compounds to be classified as potential inhibitors of LF translocation. Once an active set of compounds was identified, hierarchical agglomerative clustering with a 0.7 Tanimoto cutoff was performed using Leadscope (Leadscope Inc., Columbus, OH) fingerprints, which are ideally suited for two-dimensional scaffold-based based clustering. For each cluster, maximal common substructures (MCS) were extracted, and a manual step of trimming the MCSs was performed to create a list of scaffolds. This clustering step typically has overlapping compounds and thus can lead to overlapping MCSs. This list of trimmed scaffolds is abridged to a canonical set. Each scaffold is then represented as a precise definition to indicate number of attachments, ring size variability, etc. All filters were then relaxed to include all negative assay data. In the initial clustering, a set of singletons was found. These compounds were reported upon separately with their individual activity profiles. SAR series and singletons were finally ranked by their activity profile, including the average potency and efficacy of the series. The primary screening results can be viewed in the PubChem database (AIDs 912 and 942, http://pubchem.ncbi.nlm.nih.gov/ ). 4.4 Fluorescent imaging assay ME-180 cells were seeded at a density of 10,000 cells per well in black walled 96-well plates. After overnight incubation, the cells were washed twice by phenol red-free DMEM and incubated with 13 nM (1 μg/ml) PA and 18 nM (1 μg/ml) LF-β-Lac for 0.5 h at 37 °C; the compounds were then added at final concentrations of 50, 10, 2 and 0.4 μM to duplicate wells for each concentration. After a1 h incubation, the cells were washed twice with DMEM and loaded with 1.5 μM CCF2/AM using the alternative substrate loading solution from Invitrogen. Cells were incubated for 4 h at room temperature in the dark. Cells were visualized and photographed using a Zeiss Axiovert 200 M inverted microscope with Zeiss Axiovision software (Carl Zeiss, Jena, Germany). For acquisition of blue fluorescence, excitation filter HQ405/20 nm bandpass, and emitter filter HQ460/40 nm bandpass were used. For green fluorescence, HQ405/20 nm bandpass, and emitter filter HQ530/30 nm bandpass were used. All filters were purchased from Chroma Technology (Rockingham, VT). To quantify the signals, plates were then read in a Wallac Victor 2 plate reader (Perkin Elmer, Wellesley, MA). The following filters were used in dual read mode: CW-lamp filter 400DF10, emission filters F460 for blue fluorescence and F535 for green fluorescence. The ratios of blue/green were calculated for each concentration, the wells with PA plus LF-β-Lac were used as positive control and the wells with LF-β-Lac only as negative control. The blue/green ratio (B/G ratio) at each concentration was normalized to the average ratio of positive controls. 4.5 Cytotoxicity assays Cytotoxicity assays were performed as previously described. 26 RAW264.7 cells (American Type Culture Collection) were grown in DMEM with 10% FBS, 2 mM Glutamax, 2 mM Hepes, and 50 μg/ml gentamycin (all from Invitrogen) at 37 °C in a 5% CO 2 atmosphere. CHO cells were grown in as previously described. 27 Compounds in DMSO (in serial dilutions) were added to cells for 30 min, followed by addition of the combinations of PA and LF or PA and FP59 or DT as indicated. For RAW264.7 macrophages, cells were incubated for 3 h at 37 °C, followed by addition of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) to a final concentration of 0.5 mg/ml. After additional 45 min incubation, all medium was removed and cells were dissolved in 50 μL of 0.5% (wt/vol) SDS/25 mM HCl, in 90% (vol/vol) isopropyl alcohol. Plates were vortexed, and the oxidized MTT was measured at A 570 by a microplate reader. For CHO cells, cells were incubated with PA and FP59 or DT for 3 h, followed by changing the compound/toxin-containing medium with regular growth medium containing 10 mM ammonium chloride thereafter to block the toxin-entry into the cytosol of the cells. 15 After incubation for 48 h, MTT cell viability measurement was performed as described above. Percent viability was calculated as a percentage of medium-treated controls. EC 50 values for protection curves were calculated by using prism 4.0 software ( graphpad , San Diego). 4.6 Analysis of PA heptamer formation This assay was performed essentially as previously described. 5 Briefly, cells were cultured in 24-well plates to confluence, washed, and incubated in serum-free DMEM with 1 μg/ml PA. The cells were washed five times to remove unbound PA. Cells were lysed in 100 μl/well of modified radioimmune precipitation lysis buffer (RIPA buffer: 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin, and pepstatin) on ice for 10 min. Equal amounts of protein from cell lysates and equal volumes of the conditioned media were separated by PAGE using 4–20% gradient Tris-glycine gels (Novex). Western blotting was performed using a polyclonal PA antibody (no. 5308) and a polyclonal LF antibody (no. 5309). 4.1 General procedures Anthrax toxin LF-β-Lac was generated and purified as described. 3 Recombinant B. anthracis LF, PA, and FP59 were made as previously described. 11 CCF2/AM (Coumarin cephalosporin fluorescent acetoxymethyl ester) and its loading buffers were purchased from Invitrogen (Carlsbad, CA). Cervix epithelial cancer line ME-180 was from American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% FBS (Fetal Bovine Serum, Gibco-BRL, Gaithersburg, MD) and antibiotics. 4.2 Assay miniaturization and quantitative high-throughput screen Cells and reagents were prepared as described, 3 and the assay was optimized in a miniaturized 1536-well plate format. The qHTS was performed using a fully automated screening system in a single run, according to the following protocol: 5 μL of ME-180 cells (approximately 2000 cells) were dispensed into each well of the 1536 well plates and allowed to incubate for 18 h in a 37 °C/5% CO 2 environment. The screening library of 70,094 compounds was composed of compounds collected from several sources: 1279 compounds from LOPAC (Sigma-Aldrich, St. Louis, MO), 1120 compounds from Prestwick (Prestwick Chemical Inc., Illkirch, France), 280 purified natural products from TimTec (Newark, DE), three 1000-member combinatorial libraries from Pharmacopeia (Princeton, NJ), 973 compounds from Tocris (Bristol, UK), 1981 compounds from the National Cancer Institute, 718 compounds from Boston University Center for Chemical Methodology and Library Development, and the rest were from the NIH Molecular Libraries Small Molecule Repository. The library was prepared at a minimum of 7 concentrations in DMSO as described. 7 All compounds were dispensed via pintool, to a final concentration range of 2.6 nM–40 μM. A combination of engineered LF-β-lac and PA (1 μg/mL) or engineered LF-β-lac alone (as a control) was then added, and the mixture incubated for 2 h at 37 °C/5% CO 2 . The FRET β-lactamase substrate CCF2/AM was then added to a final concentration of 5 μM, and following a 4-h incubation at room temperature, plates were read in an EnVision plate reader (PerkinElmer, Boston MA), with excitation at 405 nm and emission detection at 460 nm (blue channel) and 530 nM (green channel). 4.3 Analysis of qHTS data Screening data was processed using in-house developed software. Percent activity was computed from the median values of the uninhibited, or neutral, control set and a set of control wells containing no LF/PA. For assignment of plate concentrations and sample identifiers, ActivityBase (ID Business Solutions Ltd, Guildford, UK) was used for compound and plate registrations. An in-house database was used to track sample concentrations across plates. Correction factors were generated from control assay plates containing vehicle (DMSO) only inserted uniformly throughout the screen to monitor background systematic variation in assay signal potentially resulting from issues with reagent dispensers or decrease in enzyme specific activity. Curve fitting was performed using an in-housed developed algorithm. A four parameter Hill equation was fitted to the concentration–response data by minimizing the residual error between the modeled and observed responses. Outliers were identified and masked by modeling the Hill equation and determining if the differences exceeded those expected from the noise in the assay. The curve classification used is the same as described previously, 7 where concentration–response curves are classified based on efficacy, potency, and statistical fit. Curves are classified as negative or positive depending on whether they exhibit signal decrease (apparent inhibition) or increase (apparent activation). Translocation inhibitors are noted as negative response curves in the blue emission channel, and as flat responses in the green channel. Natively fluorescent molecules were noted to stimulate both channels. A negative response in both channels typically indicates non-specific fluorophore quenching or non-specific mechanisms of inhibition, such as cellular apoptosis. The ratio of the green and blue channels normalizes for variation in cell number, is a more robust measure of compound performance, and further allows compounds to be classified as potential inhibitors of LF translocation. Once an active set of compounds was identified, hierarchical agglomerative clustering with a 0.7 Tanimoto cutoff was performed using Leadscope (Leadscope Inc., Columbus, OH) fingerprints, which are ideally suited for two-dimensional scaffold-based based clustering. For each cluster, maximal common substructures (MCS) were extracted, and a manual step of trimming the MCSs was performed to create a list of scaffolds. This clustering step typically has overlapping compounds and thus can lead to overlapping MCSs. This list of trimmed scaffolds is abridged to a canonical set. Each scaffold is then represented as a precise definition to indicate number of attachments, ring size variability, etc. All filters were then relaxed to include all negative assay data. In the initial clustering, a set of singletons was found. These compounds were reported upon separately with their individual activity profiles. SAR series and singletons were finally ranked by their activity profile, including the average potency and efficacy of the series. The primary screening results can be viewed in the PubChem database (AIDs 912 and 942, http://pubchem.ncbi.nlm.nih.gov/ ). 4.4 Fluorescent imaging assay ME-180 cells were seeded at a density of 10,000 cells per well in black walled 96-well plates. After overnight incubation, the cells were washed twice by phenol red-free DMEM and incubated with 13 nM (1 μg/ml) PA and 18 nM (1 μg/ml) LF-β-Lac for 0.5 h at 37 °C; the compounds were then added at final concentrations of 50, 10, 2 and 0.4 μM to duplicate wells for each concentration. After a1 h incubation, the cells were washed twice with DMEM and loaded with 1.5 μM CCF2/AM using the alternative substrate loading solution from Invitrogen. Cells were incubated for 4 h at room temperature in the dark. Cells were visualized and photographed using a Zeiss Axiovert 200 M inverted microscope with Zeiss Axiovision software (Carl Zeiss, Jena, Germany). For acquisition of blue fluorescence, excitation filter HQ405/20 nm bandpass, and emitter filter HQ460/40 nm bandpass were used. For green fluorescence, HQ405/20 nm bandpass, and emitter filter HQ530/30 nm bandpass were used. All filters were purchased from Chroma Technology (Rockingham, VT). To quantify the signals, plates were then read in a Wallac Victor 2 plate reader (Perkin Elmer, Wellesley, MA). The following filters were used in dual read mode: CW-lamp filter 400DF10, emission filters F460 for blue fluorescence and F535 for green fluorescence. The ratios of blue/green were calculated for each concentration, the wells with PA plus LF-β-Lac were used as positive control and the wells with LF-β-Lac only as negative control. The blue/green ratio (B/G ratio) at each concentration was normalized to the average ratio of positive controls. 4.5 Cytotoxicity assays Cytotoxicity assays were performed as previously described. 26 RAW264.7 cells (American Type Culture Collection) were grown in DMEM with 10% FBS, 2 mM Glutamax, 2 mM Hepes, and 50 μg/ml gentamycin (all from Invitrogen) at 37 °C in a 5% CO 2 atmosphere. CHO cells were grown in as previously described. 27 Compounds in DMSO (in serial dilutions) were added to cells for 30 min, followed by addition of the combinations of PA and LF or PA and FP59 or DT as indicated. For RAW264.7 macrophages, cells were incubated for 3 h at 37 °C, followed by addition of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) to a final concentration of 0.5 mg/ml. After additional 45 min incubation, all medium was removed and cells were dissolved in 50 μL of 0.5% (wt/vol) SDS/25 mM HCl, in 90% (vol/vol) isopropyl alcohol. Plates were vortexed, and the oxidized MTT was measured at A 570 by a microplate reader. For CHO cells, cells were incubated with PA and FP59 or DT for 3 h, followed by changing the compound/toxin-containing medium with regular growth medium containing 10 mM ammonium chloride thereafter to block the toxin-entry into the cytosol of the cells. 15 After incubation for 48 h, MTT cell viability measurement was performed as described above. Percent viability was calculated as a percentage of medium-treated controls. EC 50 values for protection curves were calculated by using prism 4.0 software ( graphpad , San Diego). 4.6 Analysis of PA heptamer formation This assay was performed essentially as previously described. 5 Briefly, cells were cultured in 24-well plates to confluence, washed, and incubated in serum-free DMEM with 1 μg/ml PA. The cells were washed five times to remove unbound PA. Cells were lysed in 100 μl/well of modified radioimmune precipitation lysis buffer (RIPA buffer: 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin, and pepstatin) on ice for 10 min. Equal amounts of protein from cell lysates and equal volumes of the conditioned media were separated by PAGE using 4–20% gradient Tris-glycine gels (Novex). Western blotting was performed using a polyclonal PA antibody (no. 5308) and a polyclonal LF antibody (no. 5309).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383445/

Integron associated mobile genes

Lateral gene transfer (LGT) impacts on the evolution of prokaryotes in both the short and long-term. The short-term impacts of mobilized genes are a concern to humans since LGT explains the global rise of multi drug resistant pathogens seen in the past 70 years. However, LGT has been a feature of prokaryotes from the earliest days of their existence and the concept of a bifurcating tree of life is not entirely applicable to prokaryotes since most genes in extant prokaryotic genomes have probably been acquired from other lineages. Successful transfer and maintenance of a gene in a new host is understandable if it acts independently of cell networks and confers an advantage. Antibiotic resistance provides an example of this whereby a gene can be advantageous in virtually any cell across broad species backgrounds. In a longer evolutionary context however laterally transferred genes can be assimilated into even essential cell networks. How this happens is not well understood and we discuss recent work that identifies a mobile gene, unique to a cell lineage, which is detrimental to the cell when lost. We also present some additional data and believe our emerging model will be helpful in understanding how mobile genes integrate into cell networks. Lateral Gene Transfer and Bacterial Evolution Lateral gene transfer (LGT) is a phenomenon central to prokaryotic evolution and adaptation. With the increasing use and sophistication of genomics technologies and bioinformatics tools, estimates of the extent of laterally transferred genes have tended upwards. A recent study estimated that an average of 81% of the genes in any given prokaryote had been transferred at some point in their history. 1 The existence and extent of LGT has long been a vexing issue in biology. In respect of microbiology it has led to a long debate of the species concept as it applies to prokaryotes. 2 In the broader evolutionary debate LGT has also challenged aspects of neo Darwinism especially in regards to the notion of evolution by gradual change. 3 While all microbiologists accept LGT as a fact, there are still difficulties in reconciling the observation of its large contribution to genomes in the long-term and the likely fitness impact on a genome of acquiring one or more gene units of DNA in the short-term. 3 , 4 With as little as 20% of a bacterial genome being comprised of genes that respect the classical evolutionary laws of inheritance by vertical descent it follows that most laterally acquired genes must be integrated into essential interconnected metabolic or regulatory pathways (cell networks). Neo-Darwinism argues that evolution of genes integrated into cell networks occurs by mutational changes in existing genes or duplicated genes either of which randomly generates subtle fitness advantages. Over time these can lead to networks with distinct properties in different species and, indeed, facilitate the process of speciation itself. It can also lead to the generation of completely new pathways and networks. In contrast, the sudden introduction of a new gene or genes into a bacterium is an "instantaneous" change. In contrast to a subtle point mutation in an existing or duplicated gene, this is much less likely to provide an opportunity for the new gene to co evolve with, and adapt to, existing cell networks. In the absence of other changes this would mean the transferred gene would not become established in a population. A partial explanation for this dilemma may be in the finding that mobile DNA might be silenced by host defenses preventing detrimental short-term effects. 5 Putting aside the difficulties of integrating an instantaneously acquired innovation into a cellular network, it is universally understood that some types of laterally transferred genes can have enormous positive fitness impacts on a bacterial cell. An obvious example is antibiotic resistance. With the clinical introduction of antibiotics in the mid 20th century, it was not long before resistant bacteria began to emerge. The rapid emergence and spread of antibiotic resistance was one of the earliest indicators of evolution by LGT. Today, the ubiquitous presence of multi drug resistant bacteria has led the World Health Organization to recognize the resulting decline in antibiotic efficacy as one of the great health challenges of the 21st century. Most identified antibiotic resistance genes are part of the mobile genome and the selection for antibiotic resistance has facilitated the assembly and concentration of the genes conferring resistance into a plethora of mobilizing elements. 6 This enormous resistance gene Diaspora can be attributed to strong artificial selection by human activities and is very recent. The presence of these laterally transferred genes in diverse bacteria is relatively easy to understand. First, selection for resistance is very strong, at least in some environments. Thus, in many contexts, the fitness of cells lacking resistance genes is essentially zero. Also, resistant bacteria are predicted to persist in the environment even if all uses of antibiotics ceased. 7 The reasons for persistence in the absence of selection are not all entirely clear although one explanation for the reduced fitness cost is that most acquired resistance genes or gene pathways act autonomously and outside cell metabolic networks. This in itself helps to account for the rapid dissemination of genes through diverse pathogens since a resistance gene will work in any cellular context. Thus, the same gene can be found to be mediating resistance in many disparate pathogens. Integration of Acquired Genes into Cell Networks Prior to the genomic era, the bulk of our understanding of LGT was drawn from highly mobile genes and the elements that mobilized them. LGT was then considered a marginal process which did not concern most genes, including those used to reconstruct a phylogenetic Universal Tree of Life. The compatibility of our understanding of prokaryotic evolution to neo-Darwinian concepts was not questioned, as LGT was only viewed to affect a minority of specialized genes. The microbial genomics era, which began in 1995, with every passing year and additional sequenced genome, progressively led to the incontrovertible conclusion that LGT has been a dominant force in microbial evolution. 8 Thus, rather than just adding on genes in response to strong selection that act independent of the cell network, laterally transferred genes were key integrated components in the cell. Several recent studies have shown that phylogenies of gene families that are involved in cell networks do not conform to a simple bifurcating tree type model as would be expected for vertical inheritance. 1 , 9 , 10 The likely extent of LGT for such networked genes is still unclear. Pal et al. 9 have argued that LGT has played an important role in building up complex networks via involvement in pathways that allow bacteria to niche adapt to specialized environment types. Thus, an influx of genes at the periphery is most likely to assist bacteria in adapting to new environments and not by optimization in fixed environments. This model is attractive in that it is consistent with the diverse ecological niches that prokaryotes have come to inhabit. By analogy with the complexity hypothesis first advanced by Jain et al., 11 this model predicts that metabolic genes rather than core informational genes are more likely to show evidence of LGT. There is clear evidence that this is true in a relative sense, however it is also the case that some informational genes also display evidence of frequent LGT. Even if mostly added at the periphery, it is clear that newly acquired genes need to be able to "communicate" with existing cell networks to some extent even if only, for example, to channel partially processed substrates into central metabolic networks. How this is achieved in the first instance will impact on fitness. It has thus been argued that the evolution of new integrated pathways requires the evolution or acquisition of regulatory proteins as well as enzymatic ones. 10 Understanding how networks are impacted by LGT is crucial to fully integrating this phenomenon in evolutionary theory. It also has relevance to more applied branches of science, as cellular networks are critical in the evolution of pathogenicity 12 and impact on the evolution of pathogenic bacteria. 13 Thus, although the genomics era has told us a lot about the extent of LGT and where it has occurred, as pointed out by Davids and Zhang, plausible mechanisms as to the events that lead to integration of acquired genes are lacking. 14 The Integron/Gene Cassette System For several years our laboratories have been investigating the biology of the integron/gene cassette system. 15 This system is an important component of the mobile genome in Gram-negative bacteria. It was first characterized in the context of its role in spreading antibiotic resistance genes in human pathogens. 16 In that regard, it is an exemplar of the power of the adaptive potential of mobile DNA since, although it was selected for and spread as a result of the heavy use of antibiotics by humans, the integron is an ancient structure that has been a feature of many bacterial genomes for a long period of evolutionary time. 17 The defining feature of all integrons is their ability to capture genes when the latter are part of mobilizable elements known as gene cassettes. 18 What makes integrons arguably unique in the plethora of mobile elements is that they appear to be highly adapted as a tool kit for natural experimental evolution. Their key feature is their ability to insert any gene cassette at a defined integron associated recombination site by site-specific recombination. The advantage of this process is that it allows insertion of a DNA sequence at a defined location in the genome which otherwise does not disrupt any other gene in the cell by insertional inactivation. It also allows the immediate expression of the newly acquired gene, as a promoter is located next to the insertion site. Unlike other site specific recombination systems however, insertion does not involve a single discrete DNA sequence such as a lysogenic phage but rather, a diversity of sequences most of which include defined genes, that to date has no definable upper limit in terms of numbers. 19 , 20 Thus mobilized genes acquired by LGT can be inserted and expressed in a way that does not otherwise impact on cellular gene content. Other evidence that this system is designed to facilitate adaptive innovation is the fact that the site-specific recombination reaction is genetically regulated such that the SOS response leads to an increase in mobile cassette rearrangement frequencies. 21 Therefore, this provides a mechanism for generating diversity at times when cells have to rapidly adjust to new or changing environments. Apart from the large number of mobile genes known to exist, another defining feature of the cassette metagenome is the extraordinary amount of novelty it contains. 22 This novelty extends to the point that most genes found within the cassette metagenome either possess no identified homologs or are homologous to genes encoding proteins that are identified only as "conserved hypotheticals" in databases. One of the great challenges in this area of research is in understanding what these mobile genes do, especially given the fact that they, collectively, comprise a resource that must be many orders of magnitude larger than any single bacterial genome. In our view, however, there is no question that the vast majority of cassette-encoded proteins are adaptive. For example, structural biology approaches (by obtaining high resolution crystal structures) have revealed functions for many such proteins. Thus, we have found via these approaches that some cassette proteins include putative house cleaning functions 23 and ligand binding domains commonly associated with two component transcriptional regulators 24 both of which are likely to impact on cell networks. This latter example is particularly interesting as it implies that the modular rearrangement of protein domains via cassette shuffling may be a precursor for the evolution of multi domain proteins. One could envisage a scenario whereby two adjacent cassettes providing complementary functions may become fused by loss of the cassette recombination site creating a new multi domain protein. Cassette fusion has been previously observed. 25 In any event, the notion that mobile cassettes may encode transcription regulators as well as enzymatic proteins is consistent with this genetic element constituting an adaptive toolbox. 10 Apart from potentially evolving new proteins, cassette uptake in natural environments 26 and shuffling may be a process for operon creation by bringing together functionally distinct proteins that can cooperate to form a co-regulated biochemical pathway. 27 Lineage-Specific Integration of a Recently Acquired Gene into an Essential Cell Network Other approaches besides structural biology have been used to understand what role cassette-encoded proteins play in the cell. Our major model for this is the Vibrio rotiferanus strain DAT722, the genome of which we have recently sequenced. 28 From an integron perspective, this species is typical of the vibrios in that it has large cassette arrays 29 —composed of 116 cassettes in the case of DAT722—with most of the associated proteins having no identifiable function. This bacterium is also both nonpathogenic to humans and amenable to genetic manipulation. This makes it and its close relatives useful models for testing of specific hypotheses. In a recent study we examined the impact of cassette array deletions on the metabolic capacity of the DAT722 cells. 30 This was initially done by examining the ability of the mutants to grow on a variety of carbon sources in a Biolog screening assay. Our intent was to try and identify mutants with a varied capacity to metabolize specific substrates. Surprisingly, we found that some mutants concomitantly had a greatly reduced viability in minimal media in the presence of a number of different carbon substrates including glucose. In contrast, mutant growth was normal in rich media. Furthermore, these mutants exhibited a hypermutative phenotype (Labbate M., unpublished) indicating the deletion had resulted in the loss of a significant gene which made the bacterium maladapted to its environment. This change of phenotype was ascribed to one specific protein, encoded within cassette 11 (the 11th cassette in the 116 cassette array). The cassette 11 protein was demonstrated to have a role in porin regulation and the altered growth profiles were a consequence of changes in porins. The reduction in fitness on deletion of cassette 11 is substantial to the point of making the cell nearly non viable in certain carbon-containing minimal growth media. To our knowledge this is the first experimental data that demonstrates the integration of an apparently unique mobile gene into an important cell network. One particularly interesting aspect to this is the fact that the negative impact on growth is specific for a media that closely resembles the environment in which free living vibrios are most commonly found—namely estuarine water. Results To test whether cassette 11 protein can impact on the fitness of other Vibrio strains, we introduced the cassette 11 gene containing recombinant vector pMAQ1082 30 into a V. cholerae strain designated S25 to determine whether fitness was affected. pMAQ1082 comprises the cloning vector pJAK16 into which the cassette 11 gene has been cloned under the control of an IPTG inducible promoter. S25 is an environmental nonO1/nonO139 V. cholerae strain isolated from a Sydney, Australia, estuarine environment. It has a large integron array but does not possess cassette 11 or a close homolog based on PCR using primers targeting this cassette. S25 with and without cassette 11 had identical growth characteristics ( Fig. 1 ) in complete media a result identical to that for DAT722 with and without this cassette. 30 In contrast to DAT722 however, which had greatly altered growth rates in most minimal media, including 2M + glucose, when isogenic strains with and without cassette 11 were compared, 30 the growth of S25 in the same media was unaffected by the presence or absence of this cassette and was identical to the growth seen in complete media ( Fig. 1 ). The simplest interpretation of this is that the cassette 11 protein does not interact with any S25 cell networks in stark contrast to its influence on networks in DAT722. While more data are needed, we speculate that integration of some mobile genes into cell networks may be analogous to the evolution of duplicated genes in eukaryotes. In eukaryotes duplication is most commonly via the generation of identical copies of an existing gene. In contrast, the introduction of cassette 11 into the ancestor of DAT722 initially may have had no impact on the cell as this progenitor possessed a non identical gene that nonetheless encoded a protein with a related function. As this strain evolved however incremental changes led to cassette 11 protein replacing this pre existing protein in terms of its central network role. V. cholerae S25 (pMAQ1082) may be a useful experimental evolution model for exploring this hypothesis. Figure 1. Impact of cassette 11 protein in V. cholerae S25. Growth curves of V. cholerae S25 (squares), V. cholerae S25/pJAK16 (circles) and V. cholerae S25/pMAQ1082 (triangles) induced with 0.1 mM IPTG in LB20 (A) and 2M + glucose (B). What is the cassette 11 encoded protein? At this time we do not have a definitive answer although its main target is most likely DNA and consequently it may play a role in regulation of DNA supercoiling. This potential link is inferred by the presence of two distinct domains in the protein. One of these is a C-terminal zinc finger domain commonly associated with prokaryotic DNA topoisomerase I proteins and in these proteins catalyzes the relaxation of supercoiled DNA. The second is a recently identified nuclease related NERD domain 31 inferred to have a role in DNA processing. We have found this domain is present in proteins found in diverse bacteria ( Fig. 2 ), a distribution suggestive of spread by LGT as is the case for the cassette 11 protein family overall. Interestingly this bioinformatic analysis reveals examples of this NERD domain being encoded by genes in highly mobilized elements (gene cassettes and transposons) as well as being fixed in cell lines for (presumably) longer periods of time as a result of the gene being located on a chromosome. Obtaining a crystal structure and identifying a precise biochemical function of the cassette 11 protein would be of great interest. An implied role in DNA processing is tantalizing as this type of information processing function is one of the least likely candidates for successful LGT according to the complexity hypothesis or the network evolution model advanced by Pal. 9 Understanding its precise role is likely to shed light on the forces that allow and provide for rapid integration of LGT derived genes into cell networks. It could also bring insights into speciation processes. If a gene can be integrated into a cell line specific network such that its loss is nearly fatal, this integration event may represent the first step in a process that represents sympatric speciation. Figure 2. Phylogeny of NERD proteins similar to cassette 11 protein from V. rotiferianus DAT722. Each taxon name is followed by the accession number of the protein it contains, as well as the genetic element the protein is associated with. The phylogeny has been reconstructed by maximum likelihood using RAxML. Confidence values over 80% are displayed on nodes of interest, representing the proportion of bootstrap pseudo-replicates supporting topology. Proteins containing a C4-zinc finger domain are in bold. An asterisk indicates marine/aquatic bacteria. The line marks the clade containing cassette 11 protein. Conclusions In summary, core prokaryotic genes defined by little identifiable evidence of LGT over long evolutionary periods constitute only a small minority of the genes in extant genomes. The majority of genes have at some point been acquired by LGT, including those that are now fixed on chromosomes and integrated into the cell networks that support prokaryotic life. It is these genes that have allowed niche specialization and adaptation of bacteria to novel environments. A complete understanding of the process of LGT requires an understanding of how new genes integrate into cell networks. This Systems Biology understanding will require hypothesis driven experimental approaches as well as those involving genomics and bioinformatics.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703951/

RosettaAntibody: antibody variable region homology modeling server

The RosettaAntibody server ( http://antibody.graylab.jhu.edu ) predicts the structure of an antibody variable region given the amino-acid sequences of the respective light and heavy chains. In an initial stage, the server identifies and displays the most sequence homologous template structures for the light and heavy framework regions and each of the complementarity determining region (CDR) loops. Subsequently, the most homologous templates are assembled into a side-chain optimized crude model, and the server returns a picture and coordinate file. For users requesting a high-resolution model, the server executes the full RosettaAntibody protocol which additionally models the hyper-variable CDR H3 loop. The high-resolution protocol also relieves steric clashes by optimizing the CDR backbone torsion angles and by simultaneously perturbing the relative orientation of the light and heavy chains. RosettaAntibody generates 2000 independent structures, and the server returns pictures, coordinate files, and detailed scoring information for the 10 top-scoring models. The 10 models enable users to use rational judgment in choosing the best model or to use the set as an ensemble for further studies such as docking. The high-resolution models generated by RosettaAntibody have been used for the successful prediction of antibody–antigen complex structures. INTRODUCTION Therapeutic monoclonal antibodies are a genre of biopharmaceuticals which has benefitted healthcare in various fields from oncology to immune and inflammatory disorders. Development of successful novel therapeutic antibodies requires understanding of drug and disease mechanisms and the ability to stabilize, affinity mature, and humanize antibodies. Antibody structures can help overcome these challenges by providing atomic level insights into structure–function relationships and the antibody–antigen interaction [e.g. see refs. ( 1–4 )]. However, experimental techniques for obtaining antibody structures, like X-ray crystallography and nuclear magnetic resonance, are laborious, time consuming and costly. Computational antibody structure prediction provides a fast and inexpensive route to obtain structures, including those which are not obtainable otherwise. Two antibody variable region (F V ) modeling servers are available on the Internet: the Web Antibody Modeling (WAM) ( 5 ) and Prediction of Immunoglobulin Structure (PIGS) ( 6 ) servers. WAM can require several days to output one antibody model in response to a submitted query sequence. No information on templates used for modeling the antibody is provided. Furthermore, antibody structures predicted with WAM have internal clashes and their inaccuracies can confound computational docking ( 2 , 7 ). The PIGS server returns an antibody model in about a minute and displays the antibody crystal structures that it selects as templates. The PIGS models are generated by grafting complementarity determining region (CDR) loops onto selected framework templates, even for the hyper-variable and non-canonical CDR H3 loop. Accurate CDR H3 predictions would only be expected when a similar CDR H3 loop is present in the database, which is unlikely for novel antibody sequences. The existing servers do not provide high-resolution refinement of antibody structures and do not consider thermodynamics during modeling. RosettaAntibody ( 7 ) is a homology modeling program within the Rosetta suite ( 8 ) for predicting high-resolution antibody F V structures. The prediction includes modeling CDR H3 loop conformations, and it uses a simple free energy function to relieve steric clashes by simultaneously optimizing the CDR loop backbone dihedral angles, the relative orientation of the light ( V L ) and heavy ( V H ) chains, and the side chain conformations. A crude model where all the CDRs are grafted from template structures can be provided in a few minutes, and high-resolution models can be generated by the full RosettaAntibody protocol running on a cluster of computers in about a day (sometimes users may need to wait for other jobs in the queue). The 10 top-scoring RosettaAntibody models can be used in docking techniques such as EnsembleDock ( 9 ) that can select binding competent conformers during docking. A few limitations of RosettaAntibody have been that (i) execution of multiple scripts for the identification of templates can be complex, (ii) finding the template structures that have been used in modeling can be challenging given the large number of intermediate files that are generated (iii) the Rosetta command-line interface can be difficult to use and (iv) it requires significant computational time to generate all-atom models, requiring a cluster of computers. To overcome these limitations and to make the high-resolution modeling available to a broader community, we have developed the RosettaAntibody server ( http://antibody.graylab.jhu.edu ), where the interface is simple and modest computing resources are provided. PROCESSING METHOD RosettaAntibody predicts the structure of the F V region in two stages. The first stage identifies the CDR loops and the framework regions in the input sequences, chooses the most sequence homologous templates for each respective segment, grafts the template CDRs onto template frameworks, and finally optimizes the side chains of all the residues in the assembled model. The crude model generated in the first stage is used as an input to the second stage. The second stage of RosettaAntibody is a multi-start, multi-scale Monte Carlo-plus-minimization algorithm that generates two thousand candidate structures. The second stage of the algorithm is split into a low-resolution and a high-resolution phase. The low-resolution phase represents side chains as single pseudo-atoms ( 10 ) and generates candidate CDR H3 loop conformations via fragment assembly and cyclic coordinate descent ( 11 ) in a Monte Carlo loop. Scoring in the low-resolution phase favors nonlocal properties of native protein structures such as hydrophobic burial, compactness, pairing of β-strands and closure of the chain gap during loop building ( 12 ). The high-resolution phase iteratively performs the following: (i) optimizes side chains via rotamer packing and continuous minimization ( 13 ), (ii) perturbs CDR backbone torsion angles and the relative orientation of the light and heavy chains, and (iii) uses gradient-based minimization over the CDR torsion angles and the light chain-heavy chain displacement. The high-resolution energy function includes van der Waals energy, orientation-dependent hydrogen bonding ( 14 ), implicit Gaussian solvation ( 15 ), side-chain rotamer propensities ( 16 ), and a low-weighted distance-dependent dielectric electrostatic energy ( 17 ). Complete methodological details are provided in ref. ( 7 ). INPUTS AND OUTPUTS Input Amino-acid sequences of the light and heavy chains of the F V region are submitted to the server by either pasting the sequences in the appropriate field or by uploading them as two separate FASTA formatted files. Since the RosettaAntibody server models only the F V region of the antibody, F C and leader sequences should be truncated from the input sequence prior to submission. To uniquely identify each job, the user must specify the name of the antibody and can optionally specify the user's name and an email address for notification when the modeling task has finished. Output Figure 1 shows a representative output page from the RosettaAntibody server. The top of the page summarizes the details of the respective job, e.g. the name of the antibody, the name of the user (if provided), and the date and time of submission, execution and completion. A chart shows the boundaries of the CDR loops and the framework regions. Next, a table displays the most sequence-homologous template [identified by BLAST ( 18 )] for each antibody segment (V H and V L frameworks, CDRs L1, L2, L3, H1, H2 and H3). In a series of selectable panes, the table also displays the top seven templates for each antibody segment. A picture of the crude F V model, formed by joining the top templates, is shown next on the output page with a link for downloading a file containing the coordinates and various energies. Residue numbering of all models generated by RosettaAntibody follows Chothia's antibody numbering scheme ( 19 ). Figure 1. Sample results page provided by the server for monoclonal antibody 14B7 ( 49 ). Inset shows the overlay of the 10 top-scoring RosettaAntibody models which appears further down the page (antibody framework regions, gray; light chain CDRs, green; CDRs H1 and H2, blue; CDR H3, red). Web page images are generated with MolScript ( 50 ). If requested, the next section of the page will show the 10 top-scoring high-resolution structures in rank order by energy. Each model output file includes the scoring data of individual energy terms (van der Waals, solvation, hydrogen bonding energies, etc.) for the whole F V model as well as residue-by-residue breakdowns. Finally, the web page shows a picture of a superposition of the 10 top-scoring structures from the perspective of an antigen, demonstrating the differences in the different models (figure inset). Any difficulties in processing the sequence would be shown in a 'warnings' section. Warnings sometimes arise to indicate poor matching of template structures or broken predicted CDR H3 loop conformations, resulting in a lower confidence model. Additional explanations of the warnings are provided in the website documentation. The documentation page also explains all output in detail, including a description of the scoring terms found in the coordinate files. Input Amino-acid sequences of the light and heavy chains of the F V region are submitted to the server by either pasting the sequences in the appropriate field or by uploading them as two separate FASTA formatted files. Since the RosettaAntibody server models only the F V region of the antibody, F C and leader sequences should be truncated from the input sequence prior to submission. To uniquely identify each job, the user must specify the name of the antibody and can optionally specify the user's name and an email address for notification when the modeling task has finished. Output Figure 1 shows a representative output page from the RosettaAntibody server. The top of the page summarizes the details of the respective job, e.g. the name of the antibody, the name of the user (if provided), and the date and time of submission, execution and completion. A chart shows the boundaries of the CDR loops and the framework regions. Next, a table displays the most sequence-homologous template [identified by BLAST ( 18 )] for each antibody segment (V H and V L frameworks, CDRs L1, L2, L3, H1, H2 and H3). In a series of selectable panes, the table also displays the top seven templates for each antibody segment. A picture of the crude F V model, formed by joining the top templates, is shown next on the output page with a link for downloading a file containing the coordinates and various energies. Residue numbering of all models generated by RosettaAntibody follows Chothia's antibody numbering scheme ( 19 ). Figure 1. Sample results page provided by the server for monoclonal antibody 14B7 ( 49 ). Inset shows the overlay of the 10 top-scoring RosettaAntibody models which appears further down the page (antibody framework regions, gray; light chain CDRs, green; CDRs H1 and H2, blue; CDR H3, red). Web page images are generated with MolScript ( 50 ). If requested, the next section of the page will show the 10 top-scoring high-resolution structures in rank order by energy. Each model output file includes the scoring data of individual energy terms (van der Waals, solvation, hydrogen bonding energies, etc.) for the whole F V model as well as residue-by-residue breakdowns. Finally, the web page shows a picture of a superposition of the 10 top-scoring structures from the perspective of an antigen, demonstrating the differences in the different models (figure inset). Any difficulties in processing the sequence would be shown in a 'warnings' section. Warnings sometimes arise to indicate poor matching of template structures or broken predicted CDR H3 loop conformations, resulting in a lower confidence model. Additional explanations of the warnings are provided in the website documentation. The documentation page also explains all output in detail, including a description of the scoring terms found in the coordinate files. SYSTEM ARCHITECTURE The RosettaAntibody server has a front-end web process which interfaces with the computation daemon and engine. The front-end, implemented in Python using Django ( http://www.djangoproject.com ), provides results upon request for users and enters modeling tasks into a MySQL database once an input file is submitted. A back-end daemon pulls tasks from the queue in the MySQL database, translates the modeling task into Perl wrapper scripts that detect the different segments of the antibody variable region, runs BLAST to detect templates, specifies a Rosetta++ command-line, and finally submits a job to a Condor ( http://www.cs.wisc.edu/condor ) queue. Condor runs the job on our 188-processor Linux cluster at Johns Hopkins University, as time is available. The back-end daemon periodically detects the status of the job to report, and eventually enters the complete set of results into the MySQL database. SERVER PERFORMANCE Since the RosettaAntibody web server opened in December of 2007, over 100 individuals have used the web server for more than 400 modeling jobs. Jobs typically require about 1700 processor-hours, and results are typically complete within a day of submission, although the time will vary with the current cluster load and server queue. The website is free and open to all users with no login requirement. Validation of the RosettaAntibody server algorithm In a large scale test of RosettaAntibody, the program was used to recover the native crystal structures of 54 antibodies ( 7 ). To simulate blind prediction, when database information was used in modeling, only nonrelated (less than 90% sequence identity) antibody structures in the Protein Data Bank ( 20 ) were used. For the best ranked model of each target, the median root mean square deviation (rmsd) of the antigen binding pocket comprising of all the CDR residues was 1.5 à , and 80% of the targets had an rmsd lower than 2.0 à . The loop modeling capabilities of RosettaAntibody were tested by ab initio modeling of the CDR H3 loop. The CDR H3 loop is composed of residues 95–102 of the heavy chain [Chothia numbering ( 19 )]. The median backbone heavy atom global rmsd of the CDR H3 loop prediction for the best ranked model was 1.6, 1.9, 2.4, 3.1 and 6.0 à , respectively, for very short (4–6 residues), short (7–9 residues), medium (10–11 residues), long (12–14 residues) and very long (17–22 residues) loops. Finally, a practical measure of the accuracy of the antibody structures is their utility for docking to antigens. While the inclusion of the RosettaAntibody refinement steps had a small effect on homology modeling rmsds (other than CDR H3), refinement was critical for achieving docking accuracy ( 7 ). When the set of 10 top-scoring RosettaAntibody F V homology models was used in local ensemble docking to antigen, a moderate-to-high accuracy docking prediction [rated by Critical Assessment of PRediction of Interactions criteria ( 21 )] was achieved in 7 of 15 targets ( 7 ). In a comparison of WAM and RosettaAntibody ( 7 ), for some antibodies, the CDR H3 predicted by WAM was closer to the native structure than that of the top-scoring model produced by RosettaAntibody. However, there was typically a more accurate structure among the 10 top-scoring RosettaAntibody models. Furthermore, antibody–antigen docking simulations starting with RosettaAntibody F V models consistently resulted in more accurate docking predictions than those obtained by starting with WAM generated models or unrefined RosettaAntibody models ( 7 ). Potential uses of the RosettaAntibody server Antibody structures can be used to guide rational efforts to enhance stability ( 22 , 23 ) or to humanize sequences to minimize immunological response ( 24 , 25 ). Antibody structures can also be used for docking to their antigens, either for epitope mapping ( 26 ) or for high-resolution refinement ( 27 ). For example, we docked models of monoclonal antibody 14B7 to the anthrax toxin protective antigen ( 2 ). The models helped us form hypotheses about the mechanism of affinity maturation of several variants of 14B7. Several other instances of docking antibody homology models are present in the literature ( 28–30 ). Docking calculations can be done on several publicly available servers ( 31–38 ) including the RosettaDock Server (local docking only for high-resolution refinement, http://rosettadock.graylab.jhu.edu ) ( 39 ). Docking of homology models is necessarily less accurate than docking of crystal structures. Experimental information can be used to mitigate errors. For example, we used computational mutagenesis and hotspot analysis to evaluate models of epidermal growth factor receptor binding to mAb 806 ( 1 ). In recognition of the errors present in homology models, RosettaAntibody provides 10 alternate low-energy structures. There are several new docking methods which can use multiple input structures for one of the docking partners ( 40–43 ). Our EnsembleDock program ( 9 ) can improve low-energy docking solutions, and sometimes the low-energy docking solution is formed by the component homology structure that is closest to the crystal structure. Validation of the RosettaAntibody server algorithm In a large scale test of RosettaAntibody, the program was used to recover the native crystal structures of 54 antibodies ( 7 ). To simulate blind prediction, when database information was used in modeling, only nonrelated (less than 90% sequence identity) antibody structures in the Protein Data Bank ( 20 ) were used. For the best ranked model of each target, the median root mean square deviation (rmsd) of the antigen binding pocket comprising of all the CDR residues was 1.5 à , and 80% of the targets had an rmsd lower than 2.0 à . The loop modeling capabilities of RosettaAntibody were tested by ab initio modeling of the CDR H3 loop. The CDR H3 loop is composed of residues 95–102 of the heavy chain [Chothia numbering ( 19 )]. The median backbone heavy atom global rmsd of the CDR H3 loop prediction for the best ranked model was 1.6, 1.9, 2.4, 3.1 and 6.0 à , respectively, for very short (4–6 residues), short (7–9 residues), medium (10–11 residues), long (12–14 residues) and very long (17–22 residues) loops. Finally, a practical measure of the accuracy of the antibody structures is their utility for docking to antigens. While the inclusion of the RosettaAntibody refinement steps had a small effect on homology modeling rmsds (other than CDR H3), refinement was critical for achieving docking accuracy ( 7 ). When the set of 10 top-scoring RosettaAntibody F V homology models was used in local ensemble docking to antigen, a moderate-to-high accuracy docking prediction [rated by Critical Assessment of PRediction of Interactions criteria ( 21 )] was achieved in 7 of 15 targets ( 7 ). In a comparison of WAM and RosettaAntibody ( 7 ), for some antibodies, the CDR H3 predicted by WAM was closer to the native structure than that of the top-scoring model produced by RosettaAntibody. However, there was typically a more accurate structure among the 10 top-scoring RosettaAntibody models. Furthermore, antibody–antigen docking simulations starting with RosettaAntibody F V models consistently resulted in more accurate docking predictions than those obtained by starting with WAM generated models or unrefined RosettaAntibody models ( 7 ). Potential uses of the RosettaAntibody server Antibody structures can be used to guide rational efforts to enhance stability ( 22 , 23 ) or to humanize sequences to minimize immunological response ( 24 , 25 ). Antibody structures can also be used for docking to their antigens, either for epitope mapping ( 26 ) or for high-resolution refinement ( 27 ). For example, we docked models of monoclonal antibody 14B7 to the anthrax toxin protective antigen ( 2 ). The models helped us form hypotheses about the mechanism of affinity maturation of several variants of 14B7. Several other instances of docking antibody homology models are present in the literature ( 28–30 ). Docking calculations can be done on several publicly available servers ( 31–38 ) including the RosettaDock Server (local docking only for high-resolution refinement, http://rosettadock.graylab.jhu.edu ) ( 39 ). Docking of homology models is necessarily less accurate than docking of crystal structures. Experimental information can be used to mitigate errors. For example, we used computational mutagenesis and hotspot analysis to evaluate models of epidermal growth factor receptor binding to mAb 806 ( 1 ). In recognition of the errors present in homology models, RosettaAntibody provides 10 alternate low-energy structures. There are several new docking methods which can use multiple input structures for one of the docking partners ( 40–43 ). Our EnsembleDock program ( 9 ) can improve low-energy docking solutions, and sometimes the low-energy docking solution is formed by the component homology structure that is closest to the crystal structure. FUTURE DIRECTIONS Accurate loop modeling remains one of the central challenges of antibody modeling. Thus, future improvements might be made as better and more efficient loop modeling algorithms [e.g. kinematic loop closure ( 44 ) or hierarchical local optimization ( 45 , 46 )] become available. Predictions might also be improved by inclusion of NMR constraints to bias simulations [e.g. ( 47 )]. Some researchers are pursuing therapeutics based 'heavy chain only' (VHH) antibodies discovered in the blood of camelids ( 48 ). VHHs are also easy to clone and express, and their structure might be amendable to prediction, although tests are required to assess the use of standard antibody database. Finally, we are currently developing flexible backbone antibody docking techniques which exploit the same antibody structural modeling tools as the server. These induced-fit antibody docking techniques may additionally help overcome homology modeling errors as predicted structures are used for high-resolution applications. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING National Institutes of Health ( R01-GM073151 , R01-GM078221 ) and UCB S.A. Funding for open access charge: National Institutes of Health Grant Number R01-GM078221 . Conflict of interest statement . None declared.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909601/

Toward high-resolution homology modeling of antibody F v regions and application to antibody-antigen docking

High-resolution homology models are useful in structure-based protein engineering applications, especially when a crystallographic structure is unavailable. Here, we report the development and implementation of RosettaAntibody, a protocol for homology modeling of antibody variable regions. The protocol combines comparative modeling of canonical complementarity determining region (CDR) loop conformations and de novo loop modeling of CDR H3 conformation with simultaneous optimization of V L -V H rigid-body orientation and CDR backbone and side-chain conformations. The protocol was tested on a benchmark of 54 antibody crystal structures. The median root-mean-square-deviation (rmsd) of the antigen binding pocket comprised of all the CDR residues was 1.5 à with 80% of the targets having an rmsd lower than 2.0 à . The median backbone heavy atom global rmsd of the CDR H3 loop prediction was 1.6 à , 1.9 à , 2.4 à , 3.1 à and 6.0 à for very short (4–6 residues), short (7–9), medium (10–11), long (12–14) and very long (17–22) loops respectively. When the set of ten top-scoring antibody homology models are used in local ensemble docking to antigen, a moderate to high accuracy docking prediction was achieved in seven of fifteen targets. This success in computational docking with high-resolution homology models is encouraging, but challenges still remain in modeling antibody structures for sequences with long H3 loops. This first large-scale antibody-antigen docking study using homology models reveals the level of "functional accuracy" of these structural models towards protein engineering applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152438/

The Role of Hospitals in Disaster

INTRODUCTION When the first rain began to fall in Houston, Texas, in June 2001, did hospital staff know they would soon be providing care for hundreds of patients without electrical power or running water in flooded hospital buildings? On April 19, 1995, did the emergency department staff arriving for the day shift at 13 Oklahoma City hospitals know that a former soldier was driving a rented van filled with 4000 pounds of ammonium nitrate toward the Murrah Federal Building and that they would soon be faced with 324 bombing victims? In 1984, did restaurant patrons in Wasco County, Ore., have any idea, as they selected food from salad bars, that they would soon be evaluated in hospitals for profuse, watery diarrhea from intentional food contamination by a religious cult? In March 2003, did the 11 Toronto healthcare workers who were caring for patients with respiratory symptoms know they would soon become infected with severe acute respiratory syndrome (SARS)? We can be quite certain that none of them knew. The capricious nature of disaster implies victims and responders are generally caught unaware. But we do know some things. We know there will be hurricanes, typhoons, tornadoes, earthquakes, mudslides, fires, and blizzards this year. We know people will pick up firearms, make bombs, and inflict pain and suffering on others. We know there will be casualties from train accidents, cars crumpled in chain reactions, building collapses, and explosions. We know infectious diseases will do what they do best: spread, sicken, and kill. We know terrorists have not given up their violent assaults. We know there will be mental health symptoms in accident survivors and the caregivers who respond to their needs. It is the hospital , at the heart of the health system, that will receive the injured, infected, bleeding, broken, and terrified from these events. We know the victims will seek life-saving care, comfort, and relief at hospitals, but many U.S. hospitals continue to prepare for disaster as though it will not happen to them. Hospital Capacity in the United States There are more than 5700 hospitals in the United States that form a diverse patchwork of healthcare services. U.S. hospitals vary greatly by geographic location (urban, suburban, and rural); financial and management structure (for profit, not-for-profit, private, public); type of care (general medical services or specialty care, such as psychiatric or pediatric); and government affiliation (Department of Defense, Veterans Health Administration, or Public Health Service). Any of these hospitals may be called on to respond to the next disaster or may be the victim of a disaster. Many experts believe that these hospitals are not adequately prepared to respond effectively ( Table 5-1 ). TABLE 5-1 SNAPSHOT OF 2005 U.S. HOSPITAL CAPACITY * Total U.S. Hospitals 5764 U.S. community hospitals 4845 U.S. federal government hospitals 239 Institutional hospitals (e.g., prisons, colleges) 23 Nonfederal psychiatric hospitals 477 Nonfederal long-term–care hospitals 180 Total staffed U.S. hospital beds 965,256 Staffed community hospital beds 813,307 * Source: American Hospital Association. Hospital statistics, 2005 edition. Available at: www.hospitalconnect.com/aha/resource_center/fastfacts/fast_facts_us_hospi-tals.html . Hospital Capacity in the United States There are more than 5700 hospitals in the United States that form a diverse patchwork of healthcare services. U.S. hospitals vary greatly by geographic location (urban, suburban, and rural); financial and management structure (for profit, not-for-profit, private, public); type of care (general medical services or specialty care, such as psychiatric or pediatric); and government affiliation (Department of Defense, Veterans Health Administration, or Public Health Service). Any of these hospitals may be called on to respond to the next disaster or may be the victim of a disaster. Many experts believe that these hospitals are not adequately prepared to respond effectively ( Table 5-1 ). TABLE 5-1 SNAPSHOT OF 2005 U.S. HOSPITAL CAPACITY * Total U.S. Hospitals 5764 U.S. community hospitals 4845 U.S. federal government hospitals 239 Institutional hospitals (e.g., prisons, colleges) 23 Nonfederal psychiatric hospitals 477 Nonfederal long-term–care hospitals 180 Total staffed U.S. hospital beds 965,256 Staffed community hospital beds 813,307 * Source: American Hospital Association. Hospital statistics, 2005 edition. Available at: www.hospitalconnect.com/aha/resource_center/fastfacts/fast_facts_us_hospi-tals.html . HISTORICAL PERSPECTIVE The Role of the Hospital in Society The hospital was of little significance in American healthcare before the Civil War. Only 178 hospitals existed in 1873 when the first survey was conducted—a time when no proper gentleman or lady would venture into a hospital by choice. 1 The murky medical practices of the 1800s offered little that couldn't be found in homes, and physicians had little in their armamentarium to change the course of disease and injury. However, discovery and scientific advance changed that. Effective anesthesia, surgical antisepsis, antibiotics, the x-ray, and other advances turned the hospital into a place of comfort, hope, and healing. The 20th-century hospital became a sophisticated financial institution, the core of medical education, and the site of dazzling technological display. Medical advances offered aid not only to the chronically ill but offered hope to those who suffered acute trauma or medical or psychiatric emergency. The Effect of Disaster on Hospitals Past events illuminate the variety and complexity of demands placed on a hospital in a disaster: • Hurricane Katrina . The flooding in the wake of Hurricane Katrina in 2005 left hospitals in greater New Orleans, Louisiana, and Mississippi in crisis. Patients and staff were trapped in facilities without essential services, resulting in the largest mass hospital evacuation in U.S. history. • Floods caused by tropical storm Allison in Houston, Texas . In June 2001, 3 feet of rain from tropical storm Allison fell in the Houston area, causing the flooding and complete disruption of services at the University of Texas Health Science Center and its clinical affiliate hospitals. One of the hospitals, Memorial Hermann Hospital, experienced failure of every hospital system. The main and emergency power generators and communications system failed and personnel could not come, go, or be reached. The water supply failed, and the sewer system stopped functioning. The vertical evacuation of 570 patients was conducted, and the hospital was closed for 38 days. The storm flooded an area not considered to be at risk. 2 • The Northridge, Calif., earthquake . An earthquake struck Northridge on Jan. 17, 1994, damaging a number of area hospitals. Six hospitals evacuated patients immediately: four of these evacuated all inpatients, and two evacuated some. Five of the facilities evacuated the most severely ill first, but the sixth, fearing imminent structural collapse, evacuated the healthiest patients first to permit the evacuation of the largest number of patients in the shortest period. That facility moved 334 patients from buildings to open areas in two hours. All hospitals used improvised transport devices, including backboards, blankets, and mattresses. 3 • The 2001 World Trade Center bombing . Bellevue Hospital, a level-1 trauma center in New York City, is approximately three miles from the World Trade Center. Ninety patients presented within the first five hours after the incident; a total of 194 were triaged and treated within the first 24 hours. Despite best efforts, the hospital lost track of patients, ran out of supplies, and struggled with coordination of physicians to ensure rest and safety. 4 New York University Downtown Hospital, a few blocks from the disaster, received 350 patients within the first two hours of the World Trade Center attacks. Many patients arrived on foot. 5 St. Vincent's Catholic Medical Center executed its disaster plan within minutes of the bombing, as they had done during the 1993 World Trade Center bombing. On Sept. 11, 2001, St. Vincent's Hospital treated nearly 800 victims. Because St. Vincent's shared water lines with the World Trade Center and telecommunications lines were routed through the World Trade Center, function of these systems was affected. Crisis counseling, pastoral care, and mental health services were immediately made available for victims, families, rescue workers, and staff. 6 • The Oklahoma City Bombing . After the explosion of a bomb at the Murrah Federal Building, 168 were killed and 700 were injured—388 with acute injuries. The first patients arrived in emergency departments within 15 minutes, and the hospitals within 1.5 miles of the blast site received the greatest number of victims. 7 • The Rhode Island nightclub fire . On Feb. 20, 2003, fire erupted in a crowded nightclub in West Warwick, R.I. Almost 100 were killed immediately, and nearly 200 were injured. Kent County Hospital, a 350-bed community hospital 2 miles from the site of the fire, received 40 victims within an hour; 25 were rapidly intubated. The hospital ran out of critical supplies and ventilators and struggled with supporting family members, poor communications from the scene, and pain management. 8 Ultimately, 16 hospitals in Rhode Island and Massachusetts received 196 burn victims; this included the Shriners Hospital for Children, which received and treated 16 adult victims. 9 The potential impact of disaster is staggering. The release of 40 tons of methyl isocyanate from the Union Carbide factory in Bhopal, India, in December 1984 exposed more than 500,000 to the deadly gas and killed about 6000 in the first week after the release. In September 1987, workers scavenging a dismantled cancer clinic in Goiania, Brazil, took home a source containing cesium-137. They sold it to a junkman who showed the glowing item to friends and neighbors. Once radiation exposure victims presented to hospitals, and the release became well known, hospitals were overwhelmed. Although 250 were actually exposed and 28 showed signs of radiation sickness, 112,800 people were evaluated. When the Aum Shinri Kyo cult placed sarin on five trains in the Tokyo subway system on March 20, 1995, 4000 people made their own way to hospitals, 641 were transported by authorities, and 245 hospital staff and rescue personnel were contaminated due to poor or nonexistent decontamination procedures. 10 Evolving Perspective on the Health System Role in Disasters On Sept. 11, 2001, when U.S. hospitals and healthcare professionals were confronted with the worst attack on American soil, and again during the anthrax attacks along the Eastern Seaboard, individuals and organizations responded heroically. A powerful change in thinking, also called a paradigm shift, occurred after the terror attacks: the health system came to be viewed as a foundation of national security. Another perspective has changed as well. In the event of a disaster, Emergency Medical Services (EMS), police, and fire have long been recognized as first responders. However, just recently, hospitals also have been designated as first responders—and first receivers. The value of the hospital in national security has been increased, and hospitals are recognized as safe havens in communities, the public expects hospitals to be prepared to care for their needs, and the hospital is now recognized as a first responder in emergencies. However, hospitals remain significantly underprepared to respond as effectively as the public expects. Most importantly, preparedness is at direct odds with productivity. Daily operating requirements stretch most hospitals' resources. Allocating funds to improve emergency response capabilities that may never be used could be viewed as foolhardy. Community integration is now seen as necessary, but hospitals (other than those in networks or that are government facilities) have had few reasons to build healthy relationships with other hospitals in their communities. To meet the needs of communities in a disaster, business competitors must work as partners. Public Expectations Hospitals play a vital role in the health, social structure, and economic life of a community. Patients expect hospitals, and health system workers, to be available to provide care for them in all circumstances. A level of preparedness that was viewed as adequate in the past is no longer seen as acceptable. To be more highly prepared and to be able to respond effectively, hospitals must make substantial investment in equipment, training, facilities improvements, and supplies. 11 Hospitals depend on public trust; poor performance during a disaster could be financially crippling to a facility. Rubin 12 writes that hospitals are expected to handle whatever they receive and do it right the first time. The Role of the Hospital in Society The hospital was of little significance in American healthcare before the Civil War. Only 178 hospitals existed in 1873 when the first survey was conducted—a time when no proper gentleman or lady would venture into a hospital by choice. 1 The murky medical practices of the 1800s offered little that couldn't be found in homes, and physicians had little in their armamentarium to change the course of disease and injury. However, discovery and scientific advance changed that. Effective anesthesia, surgical antisepsis, antibiotics, the x-ray, and other advances turned the hospital into a place of comfort, hope, and healing. The 20th-century hospital became a sophisticated financial institution, the core of medical education, and the site of dazzling technological display. Medical advances offered aid not only to the chronically ill but offered hope to those who suffered acute trauma or medical or psychiatric emergency. The Effect of Disaster on Hospitals Past events illuminate the variety and complexity of demands placed on a hospital in a disaster: • Hurricane Katrina . The flooding in the wake of Hurricane Katrina in 2005 left hospitals in greater New Orleans, Louisiana, and Mississippi in crisis. Patients and staff were trapped in facilities without essential services, resulting in the largest mass hospital evacuation in U.S. history. • Floods caused by tropical storm Allison in Houston, Texas . In June 2001, 3 feet of rain from tropical storm Allison fell in the Houston area, causing the flooding and complete disruption of services at the University of Texas Health Science Center and its clinical affiliate hospitals. One of the hospitals, Memorial Hermann Hospital, experienced failure of every hospital system. The main and emergency power generators and communications system failed and personnel could not come, go, or be reached. The water supply failed, and the sewer system stopped functioning. The vertical evacuation of 570 patients was conducted, and the hospital was closed for 38 days. The storm flooded an area not considered to be at risk. 2 • The Northridge, Calif., earthquake . An earthquake struck Northridge on Jan. 17, 1994, damaging a number of area hospitals. Six hospitals evacuated patients immediately: four of these evacuated all inpatients, and two evacuated some. Five of the facilities evacuated the most severely ill first, but the sixth, fearing imminent structural collapse, evacuated the healthiest patients first to permit the evacuation of the largest number of patients in the shortest period. That facility moved 334 patients from buildings to open areas in two hours. All hospitals used improvised transport devices, including backboards, blankets, and mattresses. 3 • The 2001 World Trade Center bombing . Bellevue Hospital, a level-1 trauma center in New York City, is approximately three miles from the World Trade Center. Ninety patients presented within the first five hours after the incident; a total of 194 were triaged and treated within the first 24 hours. Despite best efforts, the hospital lost track of patients, ran out of supplies, and struggled with coordination of physicians to ensure rest and safety. 4 New York University Downtown Hospital, a few blocks from the disaster, received 350 patients within the first two hours of the World Trade Center attacks. Many patients arrived on foot. 5 St. Vincent's Catholic Medical Center executed its disaster plan within minutes of the bombing, as they had done during the 1993 World Trade Center bombing. On Sept. 11, 2001, St. Vincent's Hospital treated nearly 800 victims. Because St. Vincent's shared water lines with the World Trade Center and telecommunications lines were routed through the World Trade Center, function of these systems was affected. Crisis counseling, pastoral care, and mental health services were immediately made available for victims, families, rescue workers, and staff. 6 • The Oklahoma City Bombing . After the explosion of a bomb at the Murrah Federal Building, 168 were killed and 700 were injured—388 with acute injuries. The first patients arrived in emergency departments within 15 minutes, and the hospitals within 1.5 miles of the blast site received the greatest number of victims. 7 • The Rhode Island nightclub fire . On Feb. 20, 2003, fire erupted in a crowded nightclub in West Warwick, R.I. Almost 100 were killed immediately, and nearly 200 were injured. Kent County Hospital, a 350-bed community hospital 2 miles from the site of the fire, received 40 victims within an hour; 25 were rapidly intubated. The hospital ran out of critical supplies and ventilators and struggled with supporting family members, poor communications from the scene, and pain management. 8 Ultimately, 16 hospitals in Rhode Island and Massachusetts received 196 burn victims; this included the Shriners Hospital for Children, which received and treated 16 adult victims. 9 The potential impact of disaster is staggering. The release of 40 tons of methyl isocyanate from the Union Carbide factory in Bhopal, India, in December 1984 exposed more than 500,000 to the deadly gas and killed about 6000 in the first week after the release. In September 1987, workers scavenging a dismantled cancer clinic in Goiania, Brazil, took home a source containing cesium-137. They sold it to a junkman who showed the glowing item to friends and neighbors. Once radiation exposure victims presented to hospitals, and the release became well known, hospitals were overwhelmed. Although 250 were actually exposed and 28 showed signs of radiation sickness, 112,800 people were evaluated. When the Aum Shinri Kyo cult placed sarin on five trains in the Tokyo subway system on March 20, 1995, 4000 people made their own way to hospitals, 641 were transported by authorities, and 245 hospital staff and rescue personnel were contaminated due to poor or nonexistent decontamination procedures. 10 Evolving Perspective on the Health System Role in Disasters On Sept. 11, 2001, when U.S. hospitals and healthcare professionals were confronted with the worst attack on American soil, and again during the anthrax attacks along the Eastern Seaboard, individuals and organizations responded heroically. A powerful change in thinking, also called a paradigm shift, occurred after the terror attacks: the health system came to be viewed as a foundation of national security. Another perspective has changed as well. In the event of a disaster, Emergency Medical Services (EMS), police, and fire have long been recognized as first responders. However, just recently, hospitals also have been designated as first responders—and first receivers. The value of the hospital in national security has been increased, and hospitals are recognized as safe havens in communities, the public expects hospitals to be prepared to care for their needs, and the hospital is now recognized as a first responder in emergencies. However, hospitals remain significantly underprepared to respond as effectively as the public expects. Most importantly, preparedness is at direct odds with productivity. Daily operating requirements stretch most hospitals' resources. Allocating funds to improve emergency response capabilities that may never be used could be viewed as foolhardy. Community integration is now seen as necessary, but hospitals (other than those in networks or that are government facilities) have had few reasons to build healthy relationships with other hospitals in their communities. To meet the needs of communities in a disaster, business competitors must work as partners. Public Expectations Hospitals play a vital role in the health, social structure, and economic life of a community. Patients expect hospitals, and health system workers, to be available to provide care for them in all circumstances. A level of preparedness that was viewed as adequate in the past is no longer seen as acceptable. To be more highly prepared and to be able to respond effectively, hospitals must make substantial investment in equipment, training, facilities improvements, and supplies. 11 Hospitals depend on public trust; poor performance during a disaster could be financially crippling to a facility. Rubin 12 writes that hospitals are expected to handle whatever they receive and do it right the first time. CURRENT PRACTICE Sources of Hospital Vulnerability Hospitals are vulnerable to the stresses of disaster responses due to a number of inherent characteristics: • Complexity of services: Hospitals are facilities that provide healthcare but must also function as laundromats, hotels, office buildings, laboratories, restaurants, and warehouses. • Dependence on lifelines: Hospitals are completely dependent on basic public services: water, sewer, power, medical gases, communications, fuel, and waste collection. • Hazardous materials: The hospital environment contains toxic agents and poisonous liquids and gases. • Dangerous objects: Heavy medical equipment, storage shelves, and supplies can fall or shift during an event such as an earthquake. 13 Forces Influencing Preparedness Multiple forces have placed hospitals in a precarious preparedness posture. The capacity of the health system has been scaled down to a bare minimum to cut operating costs. Emergency departments are crowded with the uninsured and the underinsured who have no other access to care. The nursing workforce has withered, and physicians have left practice due to uncontrolled liability insurance costs. Surge Capacity Many hospitals determine their surge capacity by the number of patients they could comfortably care for using standard spaces, quality care standards, and additional teams of personnel to help. In reality, a disaster is not going to comply with the limits of hospital capacity. If 300 bombing victims arrive at a 50-bed community hospital, spaces will need to be converted and used that planners may have never imagined, such as chapels, hallways, and offices. Nurses accustomed to a certain nurse-to-patient ratio may find the ratio in a disaster much higher and have to adapt practice accordingly. Surge capacity must not be viewed only as the number of beds or spaces that can be allotted to care for patients, but it must include all supporting hospital services that are involved in patient care. Critical Elements in Hospital Preparedness If hospital services fail during a disaster, the hospital fails the population depending on it. The population includes not just the victims of the disaster, but the others presenting for needed care—women preparing to give birth, patients with chronic disease exacerbation, and children with lacerations that need sutures. A vital hospital emergency management program acts as an insurance policy that increases the chances of continued operations under difficult circumstances. An effective hospital emergency management program guides the development and execution of activities that mitigate, prepare for, respond to, and recover from incidents that disrupt the normal provision of care. 14 The program should include the following components: • Emergency manager: The emergency manager is the primary point of leadership in the development, improvement, exercise, and execution of the hospital's emergency management plan. • Emergency management plan: The plan identifies the hospital's response to internal and external emergencies. Deliberate (advance) planning permits the development of strategies while the organization is not under pressure to react. • Executive leadership: Hospital executive leadership charts the course for an organization. A hospital that lacks executive leadership committed to emergency preparedness will be significantly hampered in its efforts. • Strategic planning: The hospital's strategic plan is the blueprint that guides all efforts to achieve its mission. It is critical that emergency management and preparedness efforts are woven into strategic planning. • Emergency management committee: Extremely broad membership is desired to ensure all hospital operations that will be stressed in a disaster are integrated and well prepared. • Hazard vulnerability analysis (HVA): The HVA is a tool used to assess the risks in a specific environment. The emergency management plan can be tailored to address the hazards most likely to affect hospital operations. • Vulnerability analysis: Every aspect of hospital operations that will be depended on in a disaster should be assessed to determine whether there are weaknesses present that fail when stressed. Hospitals in the U.S. Navy Medical Department and a number of civilian hospitals in New York have had their level of preparedness assessed using the Hospital Emergency Analysis Tool (HEAT). The HEAT examines more than 230 factors that contribute to effective emergency preparedness and response. After the systematic analysis by a team of experts, the hospital receives an after-action report that documents strengths and weaknesses and permits the development of a strategic plan to improve preparedness. 15 • Staff training, exercise, and continuous improvement: The Joint Commission on Accreditation of Healthcare Organizations requires hospital staff members involved in the execution of the emergency management plan to receive orientation and education relative to their role in an emergency. Exercise of the emergency plan is also required. Lessons learned should be integrated into plans to continuously revise them. Hospital Preparedness Philosophy A commitment to the following philosophies will enhance hospital emergency preparedness: • Imagine the unimaginable: When flood waters rise in a community, when a tornado touches down and demolishes an elementary school, when a disgruntled hospital employee opens fire with an automatic weapon in the emergency department, when a passing train derails and spills toxic chemicals, or when a wildfire closes in, it is too late to update an old plan, train staff to respond effectively, check phone numbers, and stock disaster supplies. Disaster complacency—believing a problem won't happen to you or your hospital—is a significant threat to effective planning and response. • Protect the staff: Only a true obsession with self-protection will ensure that staff members are not injured or become ill during disaster response. Adequate stockpiles of gloves, masks, and other equipment must be available, along with training and leadership commitment to self-protection policies. • Build in redundancy: Expect the primary plan to fail and build in alternatives to every emergency measure. • Rely on standard procedures whenever possible: People perform best in unusual situations when they perform activities that closely mirror what they do under normal conditions. • Maintain records: Patient care records are critical to obtaining reimbursement for disaster care provided. • Plan to degrade services: Normal levels of services cannot be maintained during disaster response. Identify services, such as elective surgery, that can be temporarily curtailed or minimized so that personnel and resources can be reassigned. The Federal Role in Hospital Emergency Preparedness and Response The federal government has implemented programs to augment local and state capabilities when they are overwhelmed. The National Disaster Medical System The United States has a well-established emergency medical safety net: the National Disaster Medical System (NDMS). The NDMS has two primary capabilities designed to enhance disaster medical response. The first is specialized disaster response teams who augment the medical emergency response at the site of disaster. The second NDMS capability is a plan to share the inpatient bed capacity of the civilian and federal health systems in the event either system is overwhelmed with patients requiring inpatient care. NDMS federal coordinating centers (FCCs) play a regional role in maintaining a supply of NDMS hospital members and providing training and exercises. When the NDMS is activated, FCCs coordinate patient reception and distribution of patients being evacuated. Hospitals enter into a voluntary agreement to participate in the NDMS. They must be accredited and generally have more than 100 beds. The agreement commits a hospital to provide a certain number of acute care beds to NDMS patients; however, it is recognized that hospitals may or may not be able to provide the agreed-upon number of beds. Hospitals that receive NDMS patients are reimbursed for care by the federal government. 16 The Strategic National Stockpile The Strategic National Stockpile (SNS) was established in 1999 as the National Pharmaceutical Stockpile. It is now managed by the U.S. Department of Homeland Security and serves as a national repository of antibiotics, chemical antidotes, antitoxins, intravenous therapy, airway management equipment, and medical/surgical items. The stockpile is designed to supplement local agencies that are overwhelmed by a health emergency. The Noble Training Center The Noble Training Center in Anniston, Ala., (on the site of the former Fort McClellan army base) is the only hospital facility in the United States that trains healthcare professionals in disaster preparedness and response. The Department of Homeland Security operates the Noble Training Center, which offers a variety of training programs, including one for hospital leadership. More information is available online at: http://training.fema.gov/emiweb/ntc/ . Even though the federal government has many emergency response assets that can help in the response to an emergency, experience has shown that hospitals must be prepared to be self-sufficient for 24 to 72 hours after an event. 14 Critical Elements in Hospital Disaster Preparedness A comprehensive hospital emergency management program must address a number of critical elements to adequately protect patients and staff and permit the facility to continue to operate. These are discussed in the following. Incident Command Just as one team leader is necessary for a controlled response to a cardiac arrest, an organized approach is essential to a successful hospital-wide emergency response. The Hospital Emergency Incident Command System (HEICS) is designed to provide that coordination. Developed and tested in Orange County, Calif., in 1992, it provides structure to response. HEICS uses: • A reproducible, predictable chain of command • A flexible organizational design that can be scaled to the scope of the problem • Checklists for each position to simplify response and carefully define each task • A common language that permits communication with outside agencies Emergency Operations Center (EOC) The EOC will serve as the command post for operations during an emergency response. It should be fully operational and integrated into local and county emergency operations ( Box 5-1 ). BOX 5-1 RECOMMENDED EQUIPMENT AND SUPPLIES FOR A HOSPITAL EOC Location • Secure interior space; windows not desirable • Alternative, equipped space in distant part of complex or building in the event the primary EOC is damaged or disabled Equipment • Incident command gear to identify EOC staff (vests, ball caps) • Computers with Internet access • Dedicated telephone lines • Communications-on-wheels (COWS) • Two-way communications (400 MHz, 800 MHz) • Fax • Television with cable access • Refrigerator • Radio • Bull horns • Barrier tape • Flashlights and batteries • Back-up power generator • Chalk board, dry-erase board, or other means of communicating to EOC team member Information • Hospital emergency management plan • Reference materials (emergency response, clinical references, hazardous materials) • Emergency reference contact numbers/e-mail addresses/fax numbers for local emergency response agencies (police, fire, emergency medical services, office of emergency management, department of health) • Emergency contact information for national resources, including Centers for Disease Control and Prevention (CDC), Radiation Emergency Assistance Center/Training Site (REAC/TS), Agency for Toxic Substances and Disease Registry (ATSDR), Environmental Protection Agency (EPA), and National Response Center • Staff notification information (phone contact numbers) • Memoranda of understanding/agreement (MOU/MOA) with agencies and vendors • Local and regional maps that include utility stations, hyperbaric capability, emergency medical services, police departments, fire stations, burn units, and other critical infrastructure Exercises, Drills, and Training Hospital disaster drills have often been treated as annoyances and are planned in ways to render them futile. Exercises are generally announced (unlike actual events), planned during regular business hours, and rarely include all hospital operations that will be affected by an actual event. Hospitals are encouraged to drill individual units—frequently and during nights and weekends—and then build up to full, functional exercises involving management of moulaged "casualties." Community participation is critical to identify elements that work or that need fine-tuning. Only through exercise will the plan be adequately stressed so that failure points are identified. Essential Services and Facilities Engineering The facility's structural integrity and essential services are an often overlooked part of preparedness. In 2003 a major power blackout in the northeastern United States and Canada demonstrated the impact of the loss electrical services. It is recommended that every hospital: • Possess emergency power generating capacity for 3 to 4 days' duration • Perform annual load testing on the generator(s) • Maintain the water supply and an alternative water supply in secure areas in sufficient quantity to support all services (sanitation, hygiene, laundry) for 3 to 4 days • Maintain medical gases in a secure location and have a 3- to 4-day supply for the hospital • Configure the heating-ventilation-air conditioning (HVAC) system so that it can be shut down and, ideally, so that specific zones can be manipulated to control airflow in the building in case of contamination • Maintain a fuel source for full-load demand for 3 to 4 days' duration • Develop a plan for the management and disposal of increased volumes of contaminated waste Physical Security Maintaining the physical security of the structure is important on a daily basis but becomes more of a challenge during a disaster. To ensure that the environment remains safe, egress must be controlled. Additional elements of the physical security plan should include the following: • A security force with full-time security responsibilities; the force should have undergone criminal background checks and professional law enforcement training. • All entrances and exits should be controlled, monitored, and capable of being locked. • The hospital should be able to perform perimeter security protection ("lockdown") within minutes of notification. • Hospital staff should be trained and drilled on the performance of lockdown. • Hospital leadership should know what triggers the execution of a lockdown procedure. • A plan should exist for supplementing security staff in a disaster. Situation Report (Rapid Needs Assessment) It is critical that a hospital be able to rapidly assess the impact of a disaster on its operations and communicate the status to leadership in a situation report (often referred to as a "sitrep"), or a rapid needs assessment (RAN). The assessment should, at a minimum, include the following: • The extent and magnitude of the disaster and the scope and nature of casualties • The status of operations and any disrupted critical services • The impact of disruptions on operations and the ability to sustain operations 17 Staff Notification Hospital staff must be able to receive timely and accurate notifications in a disaster, including when and where to report and for how long and other essential information. Contact information for all staff members must be continuously updated and tested. Additionally, the facility must be able to receive warnings and notifications from external agencies and be able to send warnings. Triage System Triage is performed daily in emergency departments, where the most critical are treated first. But during a disaster, triage procedures must adapt to become like what is used on the battlefield, where the greatest good is offered to the greatest number. Multiple disaster triage systems exist, including START (simple triage and rapid treatment), ID-ME (immediate, delayed, minimal, expectant), and MASS (move, assess, sort, and send). It is important that a hospital use a system that is consistent with what is being used by services delivering patients to the facility. Whatever system is selected, there must be predisaster training and exercises. Alternative Triage Area When casualties present to an emergency department in numbers that overwhelm the facility, an alternative area must be available to manage overflow. The alternative triage area should be lit so that it can be used at night, weatherproofed, and temperature-controlled. Risk Communications and Media Management Plan A plan for working with the media will be needed. It is not recommended that media personnel be permitted access to a hospital during a disaster, but rather be provided regular, factual updates on activities and the status of the facility at a predetermined meeting place. Risk communications involve using credible experts to deliver carefully worded messages to communicate most effectively in a high-stress, low-trust environment, such as a disaster. Preparing hospital leaders in risk communications principles will ensure that they are able to communicate effectively to the public via the media. Disaster Mental Health Services There is conflicting evidence about the value of certain types of mental health services in the wake of disaster, but it is clear that every disaster creates emotional trauma victims. Primary victims are those who have been directly affected by the disaster. Secondary victims are rescue workers in whom symptoms develop, and tertiary victims are relatives, friends, and others who have been affected. The critical incident disrupts a victim's sense of control as daily life is abruptly changed. 18 Hospitals must plan for providing mental health services to disaster victims but must also consider the needs—acute and long-term—of the hospital staff who attempt to respond to an overwhelming event. It is recommended that hospitals have trained crisis intervention teams that are well integrated into the emergency management plan. Evidence and Crime Scene Management In the event of an intentional act that results in mass casualties, not only must a hospital care for the victims, but it has a critical role in bringing perpetrators to justice. Hospital staff members require training in proper management of potential evidence—in both collection and preservation. Evidence collection containers, including 50-gallon drums for patient decontamination run-off, should be available as well as bags to preserve other types of evidence. Law enforcement agencies and forensic departments can provide training and guidance. Staff members should be familiar with and follow procedures for maintaining chain of custody for evidence that is collected during patient care activities. Food Services A disaster will place significant demands on the food service system of a hospital. The adequacy of food supplies for patients and staff should be evaluated. Because a hospital may need to be self-sufficient for several days in a disaster, a 3- to 4-day supply of food products is advisable. Food service personnel should be included in disaster exercises. Role of Volunteers Volunteers may or may not be of assistance, depending on their relationship with the hospital and their background. A volunteer pool that consists of individuals who serve regularly at the facility, are familiar with standard procedures, and participate in exercises can add valuable manpower to a disaster response effort. On the other hand, disasters will draw volunteers who wish to assist, a phenomenon known as "convergent volunteerism," in which unexpected and uninvited healthcare workers arrive and wish to render assistance at a large-scale incident. 19 These "freelancers" may cause problems or may even be impostors. Disaster Supplies Despite "just-in-time" supply schedules and empty warehouses, hospitals should maintain dedicated disaster supplies and arrangements for rapid resupply in the event of a disaster. Disaster response will rapidly deplete critical supplies—administrative as well as clinical. Conducting realistic exercises will help with the determination of the adequacy of stock and can be done without opening actual supplies so they can be restocked. Disaster supplies can be rotated into the daily-use stream to ensure stock does not expire. Sources of Hospital Vulnerability Hospitals are vulnerable to the stresses of disaster responses due to a number of inherent characteristics: • Complexity of services: Hospitals are facilities that provide healthcare but must also function as laundromats, hotels, office buildings, laboratories, restaurants, and warehouses. • Dependence on lifelines: Hospitals are completely dependent on basic public services: water, sewer, power, medical gases, communications, fuel, and waste collection. • Hazardous materials: The hospital environment contains toxic agents and poisonous liquids and gases. • Dangerous objects: Heavy medical equipment, storage shelves, and supplies can fall or shift during an event such as an earthquake. 13 Forces Influencing Preparedness Multiple forces have placed hospitals in a precarious preparedness posture. The capacity of the health system has been scaled down to a bare minimum to cut operating costs. Emergency departments are crowded with the uninsured and the underinsured who have no other access to care. The nursing workforce has withered, and physicians have left practice due to uncontrolled liability insurance costs. Surge Capacity Many hospitals determine their surge capacity by the number of patients they could comfortably care for using standard spaces, quality care standards, and additional teams of personnel to help. In reality, a disaster is not going to comply with the limits of hospital capacity. If 300 bombing victims arrive at a 50-bed community hospital, spaces will need to be converted and used that planners may have never imagined, such as chapels, hallways, and offices. Nurses accustomed to a certain nurse-to-patient ratio may find the ratio in a disaster much higher and have to adapt practice accordingly. Surge capacity must not be viewed only as the number of beds or spaces that can be allotted to care for patients, but it must include all supporting hospital services that are involved in patient care. Critical Elements in Hospital Preparedness If hospital services fail during a disaster, the hospital fails the population depending on it. The population includes not just the victims of the disaster, but the others presenting for needed care—women preparing to give birth, patients with chronic disease exacerbation, and children with lacerations that need sutures. A vital hospital emergency management program acts as an insurance policy that increases the chances of continued operations under difficult circumstances. An effective hospital emergency management program guides the development and execution of activities that mitigate, prepare for, respond to, and recover from incidents that disrupt the normal provision of care. 14 The program should include the following components: • Emergency manager: The emergency manager is the primary point of leadership in the development, improvement, exercise, and execution of the hospital's emergency management plan. • Emergency management plan: The plan identifies the hospital's response to internal and external emergencies. Deliberate (advance) planning permits the development of strategies while the organization is not under pressure to react. • Executive leadership: Hospital executive leadership charts the course for an organization. A hospital that lacks executive leadership committed to emergency preparedness will be significantly hampered in its efforts. • Strategic planning: The hospital's strategic plan is the blueprint that guides all efforts to achieve its mission. It is critical that emergency management and preparedness efforts are woven into strategic planning. • Emergency management committee: Extremely broad membership is desired to ensure all hospital operations that will be stressed in a disaster are integrated and well prepared. • Hazard vulnerability analysis (HVA): The HVA is a tool used to assess the risks in a specific environment. The emergency management plan can be tailored to address the hazards most likely to affect hospital operations. • Vulnerability analysis: Every aspect of hospital operations that will be depended on in a disaster should be assessed to determine whether there are weaknesses present that fail when stressed. Hospitals in the U.S. Navy Medical Department and a number of civilian hospitals in New York have had their level of preparedness assessed using the Hospital Emergency Analysis Tool (HEAT). The HEAT examines more than 230 factors that contribute to effective emergency preparedness and response. After the systematic analysis by a team of experts, the hospital receives an after-action report that documents strengths and weaknesses and permits the development of a strategic plan to improve preparedness. 15 • Staff training, exercise, and continuous improvement: The Joint Commission on Accreditation of Healthcare Organizations requires hospital staff members involved in the execution of the emergency management plan to receive orientation and education relative to their role in an emergency. Exercise of the emergency plan is also required. Lessons learned should be integrated into plans to continuously revise them. Hospital Preparedness Philosophy A commitment to the following philosophies will enhance hospital emergency preparedness: • Imagine the unimaginable: When flood waters rise in a community, when a tornado touches down and demolishes an elementary school, when a disgruntled hospital employee opens fire with an automatic weapon in the emergency department, when a passing train derails and spills toxic chemicals, or when a wildfire closes in, it is too late to update an old plan, train staff to respond effectively, check phone numbers, and stock disaster supplies. Disaster complacency—believing a problem won't happen to you or your hospital—is a significant threat to effective planning and response. • Protect the staff: Only a true obsession with self-protection will ensure that staff members are not injured or become ill during disaster response. Adequate stockpiles of gloves, masks, and other equipment must be available, along with training and leadership commitment to self-protection policies. • Build in redundancy: Expect the primary plan to fail and build in alternatives to every emergency measure. • Rely on standard procedures whenever possible: People perform best in unusual situations when they perform activities that closely mirror what they do under normal conditions. • Maintain records: Patient care records are critical to obtaining reimbursement for disaster care provided. • Plan to degrade services: Normal levels of services cannot be maintained during disaster response. Identify services, such as elective surgery, that can be temporarily curtailed or minimized so that personnel and resources can be reassigned. The Federal Role in Hospital Emergency Preparedness and Response The federal government has implemented programs to augment local and state capabilities when they are overwhelmed. The National Disaster Medical System The United States has a well-established emergency medical safety net: the National Disaster Medical System (NDMS). The NDMS has two primary capabilities designed to enhance disaster medical response. The first is specialized disaster response teams who augment the medical emergency response at the site of disaster. The second NDMS capability is a plan to share the inpatient bed capacity of the civilian and federal health systems in the event either system is overwhelmed with patients requiring inpatient care. NDMS federal coordinating centers (FCCs) play a regional role in maintaining a supply of NDMS hospital members and providing training and exercises. When the NDMS is activated, FCCs coordinate patient reception and distribution of patients being evacuated. Hospitals enter into a voluntary agreement to participate in the NDMS. They must be accredited and generally have more than 100 beds. The agreement commits a hospital to provide a certain number of acute care beds to NDMS patients; however, it is recognized that hospitals may or may not be able to provide the agreed-upon number of beds. Hospitals that receive NDMS patients are reimbursed for care by the federal government. 16 The Strategic National Stockpile The Strategic National Stockpile (SNS) was established in 1999 as the National Pharmaceutical Stockpile. It is now managed by the U.S. Department of Homeland Security and serves as a national repository of antibiotics, chemical antidotes, antitoxins, intravenous therapy, airway management equipment, and medical/surgical items. The stockpile is designed to supplement local agencies that are overwhelmed by a health emergency. The Noble Training Center The Noble Training Center in Anniston, Ala., (on the site of the former Fort McClellan army base) is the only hospital facility in the United States that trains healthcare professionals in disaster preparedness and response. The Department of Homeland Security operates the Noble Training Center, which offers a variety of training programs, including one for hospital leadership. More information is available online at: http://training.fema.gov/emiweb/ntc/ . Even though the federal government has many emergency response assets that can help in the response to an emergency, experience has shown that hospitals must be prepared to be self-sufficient for 24 to 72 hours after an event. 14 The National Disaster Medical System The United States has a well-established emergency medical safety net: the National Disaster Medical System (NDMS). The NDMS has two primary capabilities designed to enhance disaster medical response. The first is specialized disaster response teams who augment the medical emergency response at the site of disaster. The second NDMS capability is a plan to share the inpatient bed capacity of the civilian and federal health systems in the event either system is overwhelmed with patients requiring inpatient care. NDMS federal coordinating centers (FCCs) play a regional role in maintaining a supply of NDMS hospital members and providing training and exercises. When the NDMS is activated, FCCs coordinate patient reception and distribution of patients being evacuated. Hospitals enter into a voluntary agreement to participate in the NDMS. They must be accredited and generally have more than 100 beds. The agreement commits a hospital to provide a certain number of acute care beds to NDMS patients; however, it is recognized that hospitals may or may not be able to provide the agreed-upon number of beds. Hospitals that receive NDMS patients are reimbursed for care by the federal government. 16 The Strategic National Stockpile The Strategic National Stockpile (SNS) was established in 1999 as the National Pharmaceutical Stockpile. It is now managed by the U.S. Department of Homeland Security and serves as a national repository of antibiotics, chemical antidotes, antitoxins, intravenous therapy, airway management equipment, and medical/surgical items. The stockpile is designed to supplement local agencies that are overwhelmed by a health emergency. The Noble Training Center The Noble Training Center in Anniston, Ala., (on the site of the former Fort McClellan army base) is the only hospital facility in the United States that trains healthcare professionals in disaster preparedness and response. The Department of Homeland Security operates the Noble Training Center, which offers a variety of training programs, including one for hospital leadership. More information is available online at: http://training.fema.gov/emiweb/ntc/ . Even though the federal government has many emergency response assets that can help in the response to an emergency, experience has shown that hospitals must be prepared to be self-sufficient for 24 to 72 hours after an event. 14 Critical Elements in Hospital Disaster Preparedness A comprehensive hospital emergency management program must address a number of critical elements to adequately protect patients and staff and permit the facility to continue to operate. These are discussed in the following. Incident Command Just as one team leader is necessary for a controlled response to a cardiac arrest, an organized approach is essential to a successful hospital-wide emergency response. The Hospital Emergency Incident Command System (HEICS) is designed to provide that coordination. Developed and tested in Orange County, Calif., in 1992, it provides structure to response. HEICS uses: • A reproducible, predictable chain of command • A flexible organizational design that can be scaled to the scope of the problem • Checklists for each position to simplify response and carefully define each task • A common language that permits communication with outside agencies Emergency Operations Center (EOC) The EOC will serve as the command post for operations during an emergency response. It should be fully operational and integrated into local and county emergency operations ( Box 5-1 ). BOX 5-1 RECOMMENDED EQUIPMENT AND SUPPLIES FOR A HOSPITAL EOC Location • Secure interior space; windows not desirable • Alternative, equipped space in distant part of complex or building in the event the primary EOC is damaged or disabled Equipment • Incident command gear to identify EOC staff (vests, ball caps) • Computers with Internet access • Dedicated telephone lines • Communications-on-wheels (COWS) • Two-way communications (400 MHz, 800 MHz) • Fax • Television with cable access • Refrigerator • Radio • Bull horns • Barrier tape • Flashlights and batteries • Back-up power generator • Chalk board, dry-erase board, or other means of communicating to EOC team member Information • Hospital emergency management plan • Reference materials (emergency response, clinical references, hazardous materials) • Emergency reference contact numbers/e-mail addresses/fax numbers for local emergency response agencies (police, fire, emergency medical services, office of emergency management, department of health) • Emergency contact information for national resources, including Centers for Disease Control and Prevention (CDC), Radiation Emergency Assistance Center/Training Site (REAC/TS), Agency for Toxic Substances and Disease Registry (ATSDR), Environmental Protection Agency (EPA), and National Response Center • Staff notification information (phone contact numbers) • Memoranda of understanding/agreement (MOU/MOA) with agencies and vendors • Local and regional maps that include utility stations, hyperbaric capability, emergency medical services, police departments, fire stations, burn units, and other critical infrastructure Exercises, Drills, and Training Hospital disaster drills have often been treated as annoyances and are planned in ways to render them futile. Exercises are generally announced (unlike actual events), planned during regular business hours, and rarely include all hospital operations that will be affected by an actual event. Hospitals are encouraged to drill individual units—frequently and during nights and weekends—and then build up to full, functional exercises involving management of moulaged "casualties." Community participation is critical to identify elements that work or that need fine-tuning. Only through exercise will the plan be adequately stressed so that failure points are identified. Essential Services and Facilities Engineering The facility's structural integrity and essential services are an often overlooked part of preparedness. In 2003 a major power blackout in the northeastern United States and Canada demonstrated the impact of the loss electrical services. It is recommended that every hospital: • Possess emergency power generating capacity for 3 to 4 days' duration • Perform annual load testing on the generator(s) • Maintain the water supply and an alternative water supply in secure areas in sufficient quantity to support all services (sanitation, hygiene, laundry) for 3 to 4 days • Maintain medical gases in a secure location and have a 3- to 4-day supply for the hospital • Configure the heating-ventilation-air conditioning (HVAC) system so that it can be shut down and, ideally, so that specific zones can be manipulated to control airflow in the building in case of contamination • Maintain a fuel source for full-load demand for 3 to 4 days' duration • Develop a plan for the management and disposal of increased volumes of contaminated waste Physical Security Maintaining the physical security of the structure is important on a daily basis but becomes more of a challenge during a disaster. To ensure that the environment remains safe, egress must be controlled. Additional elements of the physical security plan should include the following: • A security force with full-time security responsibilities; the force should have undergone criminal background checks and professional law enforcement training. • All entrances and exits should be controlled, monitored, and capable of being locked. • The hospital should be able to perform perimeter security protection ("lockdown") within minutes of notification. • Hospital staff should be trained and drilled on the performance of lockdown. • Hospital leadership should know what triggers the execution of a lockdown procedure. • A plan should exist for supplementing security staff in a disaster. Situation Report (Rapid Needs Assessment) It is critical that a hospital be able to rapidly assess the impact of a disaster on its operations and communicate the status to leadership in a situation report (often referred to as a "sitrep"), or a rapid needs assessment (RAN). The assessment should, at a minimum, include the following: • The extent and magnitude of the disaster and the scope and nature of casualties • The status of operations and any disrupted critical services • The impact of disruptions on operations and the ability to sustain operations 17 Staff Notification Hospital staff must be able to receive timely and accurate notifications in a disaster, including when and where to report and for how long and other essential information. Contact information for all staff members must be continuously updated and tested. Additionally, the facility must be able to receive warnings and notifications from external agencies and be able to send warnings. Triage System Triage is performed daily in emergency departments, where the most critical are treated first. But during a disaster, triage procedures must adapt to become like what is used on the battlefield, where the greatest good is offered to the greatest number. Multiple disaster triage systems exist, including START (simple triage and rapid treatment), ID-ME (immediate, delayed, minimal, expectant), and MASS (move, assess, sort, and send). It is important that a hospital use a system that is consistent with what is being used by services delivering patients to the facility. Whatever system is selected, there must be predisaster training and exercises. Alternative Triage Area When casualties present to an emergency department in numbers that overwhelm the facility, an alternative area must be available to manage overflow. The alternative triage area should be lit so that it can be used at night, weatherproofed, and temperature-controlled. Risk Communications and Media Management Plan A plan for working with the media will be needed. It is not recommended that media personnel be permitted access to a hospital during a disaster, but rather be provided regular, factual updates on activities and the status of the facility at a predetermined meeting place. Risk communications involve using credible experts to deliver carefully worded messages to communicate most effectively in a high-stress, low-trust environment, such as a disaster. Preparing hospital leaders in risk communications principles will ensure that they are able to communicate effectively to the public via the media. Disaster Mental Health Services There is conflicting evidence about the value of certain types of mental health services in the wake of disaster, but it is clear that every disaster creates emotional trauma victims. Primary victims are those who have been directly affected by the disaster. Secondary victims are rescue workers in whom symptoms develop, and tertiary victims are relatives, friends, and others who have been affected. The critical incident disrupts a victim's sense of control as daily life is abruptly changed. 18 Hospitals must plan for providing mental health services to disaster victims but must also consider the needs—acute and long-term—of the hospital staff who attempt to respond to an overwhelming event. It is recommended that hospitals have trained crisis intervention teams that are well integrated into the emergency management plan. Evidence and Crime Scene Management In the event of an intentional act that results in mass casualties, not only must a hospital care for the victims, but it has a critical role in bringing perpetrators to justice. Hospital staff members require training in proper management of potential evidence—in both collection and preservation. Evidence collection containers, including 50-gallon drums for patient decontamination run-off, should be available as well as bags to preserve other types of evidence. Law enforcement agencies and forensic departments can provide training and guidance. Staff members should be familiar with and follow procedures for maintaining chain of custody for evidence that is collected during patient care activities. Food Services A disaster will place significant demands on the food service system of a hospital. The adequacy of food supplies for patients and staff should be evaluated. Because a hospital may need to be self-sufficient for several days in a disaster, a 3- to 4-day supply of food products is advisable. Food service personnel should be included in disaster exercises. Role of Volunteers Volunteers may or may not be of assistance, depending on their relationship with the hospital and their background. A volunteer pool that consists of individuals who serve regularly at the facility, are familiar with standard procedures, and participate in exercises can add valuable manpower to a disaster response effort. On the other hand, disasters will draw volunteers who wish to assist, a phenomenon known as "convergent volunteerism," in which unexpected and uninvited healthcare workers arrive and wish to render assistance at a large-scale incident. 19 These "freelancers" may cause problems or may even be impostors. Disaster Supplies Despite "just-in-time" supply schedules and empty warehouses, hospitals should maintain dedicated disaster supplies and arrangements for rapid resupply in the event of a disaster. Disaster response will rapidly deplete critical supplies—administrative as well as clinical. Conducting realistic exercises will help with the determination of the adequacy of stock and can be done without opening actual supplies so they can be restocked. Disaster supplies can be rotated into the daily-use stream to ensure stock does not expire. Incident Command Just as one team leader is necessary for a controlled response to a cardiac arrest, an organized approach is essential to a successful hospital-wide emergency response. The Hospital Emergency Incident Command System (HEICS) is designed to provide that coordination. Developed and tested in Orange County, Calif., in 1992, it provides structure to response. HEICS uses: • A reproducible, predictable chain of command • A flexible organizational design that can be scaled to the scope of the problem • Checklists for each position to simplify response and carefully define each task • A common language that permits communication with outside agencies Emergency Operations Center (EOC) The EOC will serve as the command post for operations during an emergency response. It should be fully operational and integrated into local and county emergency operations ( Box 5-1 ). BOX 5-1 RECOMMENDED EQUIPMENT AND SUPPLIES FOR A HOSPITAL EOC Location • Secure interior space; windows not desirable • Alternative, equipped space in distant part of complex or building in the event the primary EOC is damaged or disabled Equipment • Incident command gear to identify EOC staff (vests, ball caps) • Computers with Internet access • Dedicated telephone lines • Communications-on-wheels (COWS) • Two-way communications (400 MHz, 800 MHz) • Fax • Television with cable access • Refrigerator • Radio • Bull horns • Barrier tape • Flashlights and batteries • Back-up power generator • Chalk board, dry-erase board, or other means of communicating to EOC team member Information • Hospital emergency management plan • Reference materials (emergency response, clinical references, hazardous materials) • Emergency reference contact numbers/e-mail addresses/fax numbers for local emergency response agencies (police, fire, emergency medical services, office of emergency management, department of health) • Emergency contact information for national resources, including Centers for Disease Control and Prevention (CDC), Radiation Emergency Assistance Center/Training Site (REAC/TS), Agency for Toxic Substances and Disease Registry (ATSDR), Environmental Protection Agency (EPA), and National Response Center • Staff notification information (phone contact numbers) • Memoranda of understanding/agreement (MOU/MOA) with agencies and vendors • Local and regional maps that include utility stations, hyperbaric capability, emergency medical services, police departments, fire stations, burn units, and other critical infrastructure Location • Secure interior space; windows not desirable • Alternative, equipped space in distant part of complex or building in the event the primary EOC is damaged or disabled Equipment • Incident command gear to identify EOC staff (vests, ball caps) • Computers with Internet access • Dedicated telephone lines • Communications-on-wheels (COWS) • Two-way communications (400 MHz, 800 MHz) • Fax • Television with cable access • Refrigerator • Radio • Bull horns • Barrier tape • Flashlights and batteries • Back-up power generator • Chalk board, dry-erase board, or other means of communicating to EOC team member Information • Hospital emergency management plan • Reference materials (emergency response, clinical references, hazardous materials) • Emergency reference contact numbers/e-mail addresses/fax numbers for local emergency response agencies (police, fire, emergency medical services, office of emergency management, department of health) • Emergency contact information for national resources, including Centers for Disease Control and Prevention (CDC), Radiation Emergency Assistance Center/Training Site (REAC/TS), Agency for Toxic Substances and Disease Registry (ATSDR), Environmental Protection Agency (EPA), and National Response Center • Staff notification information (phone contact numbers) • Memoranda of understanding/agreement (MOU/MOA) with agencies and vendors • Local and regional maps that include utility stations, hyperbaric capability, emergency medical services, police departments, fire stations, burn units, and other critical infrastructure Exercises, Drills, and Training Hospital disaster drills have often been treated as annoyances and are planned in ways to render them futile. Exercises are generally announced (unlike actual events), planned during regular business hours, and rarely include all hospital operations that will be affected by an actual event. Hospitals are encouraged to drill individual units—frequently and during nights and weekends—and then build up to full, functional exercises involving management of moulaged "casualties." Community participation is critical to identify elements that work or that need fine-tuning. Only through exercise will the plan be adequately stressed so that failure points are identified. Essential Services and Facilities Engineering The facility's structural integrity and essential services are an often overlooked part of preparedness. In 2003 a major power blackout in the northeastern United States and Canada demonstrated the impact of the loss electrical services. It is recommended that every hospital: • Possess emergency power generating capacity for 3 to 4 days' duration • Perform annual load testing on the generator(s) • Maintain the water supply and an alternative water supply in secure areas in sufficient quantity to support all services (sanitation, hygiene, laundry) for 3 to 4 days • Maintain medical gases in a secure location and have a 3- to 4-day supply for the hospital • Configure the heating-ventilation-air conditioning (HVAC) system so that it can be shut down and, ideally, so that specific zones can be manipulated to control airflow in the building in case of contamination • Maintain a fuel source for full-load demand for 3 to 4 days' duration • Develop a plan for the management and disposal of increased volumes of contaminated waste Physical Security Maintaining the physical security of the structure is important on a daily basis but becomes more of a challenge during a disaster. To ensure that the environment remains safe, egress must be controlled. Additional elements of the physical security plan should include the following: • A security force with full-time security responsibilities; the force should have undergone criminal background checks and professional law enforcement training. • All entrances and exits should be controlled, monitored, and capable of being locked. • The hospital should be able to perform perimeter security protection ("lockdown") within minutes of notification. • Hospital staff should be trained and drilled on the performance of lockdown. • Hospital leadership should know what triggers the execution of a lockdown procedure. • A plan should exist for supplementing security staff in a disaster. Situation Report (Rapid Needs Assessment) It is critical that a hospital be able to rapidly assess the impact of a disaster on its operations and communicate the status to leadership in a situation report (often referred to as a "sitrep"), or a rapid needs assessment (RAN). The assessment should, at a minimum, include the following: • The extent and magnitude of the disaster and the scope and nature of casualties • The status of operations and any disrupted critical services • The impact of disruptions on operations and the ability to sustain operations 17 Staff Notification Hospital staff must be able to receive timely and accurate notifications in a disaster, including when and where to report and for how long and other essential information. Contact information for all staff members must be continuously updated and tested. Additionally, the facility must be able to receive warnings and notifications from external agencies and be able to send warnings. Triage System Triage is performed daily in emergency departments, where the most critical are treated first. But during a disaster, triage procedures must adapt to become like what is used on the battlefield, where the greatest good is offered to the greatest number. Multiple disaster triage systems exist, including START (simple triage and rapid treatment), ID-ME (immediate, delayed, minimal, expectant), and MASS (move, assess, sort, and send). It is important that a hospital use a system that is consistent with what is being used by services delivering patients to the facility. Whatever system is selected, there must be predisaster training and exercises. Alternative Triage Area When casualties present to an emergency department in numbers that overwhelm the facility, an alternative area must be available to manage overflow. The alternative triage area should be lit so that it can be used at night, weatherproofed, and temperature-controlled. Risk Communications and Media Management Plan A plan for working with the media will be needed. It is not recommended that media personnel be permitted access to a hospital during a disaster, but rather be provided regular, factual updates on activities and the status of the facility at a predetermined meeting place. Risk communications involve using credible experts to deliver carefully worded messages to communicate most effectively in a high-stress, low-trust environment, such as a disaster. Preparing hospital leaders in risk communications principles will ensure that they are able to communicate effectively to the public via the media. Disaster Mental Health Services There is conflicting evidence about the value of certain types of mental health services in the wake of disaster, but it is clear that every disaster creates emotional trauma victims. Primary victims are those who have been directly affected by the disaster. Secondary victims are rescue workers in whom symptoms develop, and tertiary victims are relatives, friends, and others who have been affected. The critical incident disrupts a victim's sense of control as daily life is abruptly changed. 18 Hospitals must plan for providing mental health services to disaster victims but must also consider the needs—acute and long-term—of the hospital staff who attempt to respond to an overwhelming event. It is recommended that hospitals have trained crisis intervention teams that are well integrated into the emergency management plan. Evidence and Crime Scene Management In the event of an intentional act that results in mass casualties, not only must a hospital care for the victims, but it has a critical role in bringing perpetrators to justice. Hospital staff members require training in proper management of potential evidence—in both collection and preservation. Evidence collection containers, including 50-gallon drums for patient decontamination run-off, should be available as well as bags to preserve other types of evidence. Law enforcement agencies and forensic departments can provide training and guidance. Staff members should be familiar with and follow procedures for maintaining chain of custody for evidence that is collected during patient care activities. Food Services A disaster will place significant demands on the food service system of a hospital. The adequacy of food supplies for patients and staff should be evaluated. Because a hospital may need to be self-sufficient for several days in a disaster, a 3- to 4-day supply of food products is advisable. Food service personnel should be included in disaster exercises. Role of Volunteers Volunteers may or may not be of assistance, depending on their relationship with the hospital and their background. A volunteer pool that consists of individuals who serve regularly at the facility, are familiar with standard procedures, and participate in exercises can add valuable manpower to a disaster response effort. On the other hand, disasters will draw volunteers who wish to assist, a phenomenon known as "convergent volunteerism," in which unexpected and uninvited healthcare workers arrive and wish to render assistance at a large-scale incident. 19 These "freelancers" may cause problems or may even be impostors. Disaster Supplies Despite "just-in-time" supply schedules and empty warehouses, hospitals should maintain dedicated disaster supplies and arrangements for rapid resupply in the event of a disaster. Disaster response will rapidly deplete critical supplies—administrative as well as clinical. Conducting realistic exercises will help with the determination of the adequacy of stock and can be done without opening actual supplies so they can be restocked. Disaster supplies can be rotated into the daily-use stream to ensure stock does not expire. PITFALLS Experience with disasters has demonstrated a number of predictable pitfalls that occur in hospital disaster response. Distribution of Casualties Because immediate on-scene control of a disaster is chaotic and communication is often problematic, patients will present to the closest hospital available. This often leaves other nearby facilities with capacity and personnel that go unused. Personal Protective Equipment Hospital personnel must be experts in protecting themselves, or they will become part of the problem and further stress the facility. Some controversy exists over the level of protection needed in certain environments, but it is clear that masks (N95) and gloves (latex or nonlatex) will prevent transmission of biological agents. Communications Communications failure has often been identified as a predictable failure in disaster response. Hospitals need to examine both internal communications systems (with staff and patients) and with external agencies. Multiple layers of redundancy are essential to deal with expected failures and include the use of 800-mHz radios, dedicated trunk lines in the emergency operations center, two-way communications for hospital units and essential personnel, communications-on-wheels (COWS), and access to amateur radio (Ham) operators. The last resort is using runners who carry messages. Emergency Patient Decontamination Hospitals must be able to identify and decontaminate patients who have been exposed to radiation or a compound that poses a threat to the patient's health and the safety of the facility. If the hospital depends on an external agency or has decontamination equipment that requires time to set up, an immediate alternative must be in place, such as a hose and hose bib outside of the emergency department. Consideration should be given to patient privacy, managing patient valuables and clothes, and handling weapons brought into the hospital. A trained, exercised, and well-equipped team will be the foundation of successful efforts. Child Care Hospitals will benefit from having a plan to care for children and other dependents of staff. In a disaster, staff will be called on to work extended hours, and usual family care arrangements may be unavailable. Patient Admission, Identification, and Tracking The creation of emergency patient admission packs that are maintained with disaster equipment will facilitate the admission of a large number of patients. If an automated patient tracking system is used, a back-up manual system should be available. All systems should be able to manage unidentified (John and Jane Doe) patients. Mass Fatalities Many hospitals have wholly inadequate or nonexistent plans to manage mass fatalities. Morgue space is generally limited in most facilities, so additional surge capacity must be identified in advance. Arrangements for refrigerated storage trucks, refrigerator space, and other alternatives, including ice rinks, should be addressed with socially sensitive plans. Complex cultural and religious issues may come into play in the event that there are contaminated remains and should be examined in advance. Disaster Pharmaceuticals Emergency drugs must be available at the point of care. Often they are secured in pharmacy departments or warehouses, resulting in precious minutes of life-saving time being lost as personnel try to locate and obtain critical medications. In addition to drugs needed to respond quickly to nerve agents and other emergency situations, stockpiles of antibiotics should be maintained to provide prophylaxis to patients and staff. Distribution of Casualties Because immediate on-scene control of a disaster is chaotic and communication is often problematic, patients will present to the closest hospital available. This often leaves other nearby facilities with capacity and personnel that go unused. Personal Protective Equipment Hospital personnel must be experts in protecting themselves, or they will become part of the problem and further stress the facility. Some controversy exists over the level of protection needed in certain environments, but it is clear that masks (N95) and gloves (latex or nonlatex) will prevent transmission of biological agents. Communications Communications failure has often been identified as a predictable failure in disaster response. Hospitals need to examine both internal communications systems (with staff and patients) and with external agencies. Multiple layers of redundancy are essential to deal with expected failures and include the use of 800-mHz radios, dedicated trunk lines in the emergency operations center, two-way communications for hospital units and essential personnel, communications-on-wheels (COWS), and access to amateur radio (Ham) operators. The last resort is using runners who carry messages. Emergency Patient Decontamination Hospitals must be able to identify and decontaminate patients who have been exposed to radiation or a compound that poses a threat to the patient's health and the safety of the facility. If the hospital depends on an external agency or has decontamination equipment that requires time to set up, an immediate alternative must be in place, such as a hose and hose bib outside of the emergency department. Consideration should be given to patient privacy, managing patient valuables and clothes, and handling weapons brought into the hospital. A trained, exercised, and well-equipped team will be the foundation of successful efforts. Child Care Hospitals will benefit from having a plan to care for children and other dependents of staff. In a disaster, staff will be called on to work extended hours, and usual family care arrangements may be unavailable. Patient Admission, Identification, and Tracking The creation of emergency patient admission packs that are maintained with disaster equipment will facilitate the admission of a large number of patients. If an automated patient tracking system is used, a back-up manual system should be available. All systems should be able to manage unidentified (John and Jane Doe) patients. Mass Fatalities Many hospitals have wholly inadequate or nonexistent plans to manage mass fatalities. Morgue space is generally limited in most facilities, so additional surge capacity must be identified in advance. Arrangements for refrigerated storage trucks, refrigerator space, and other alternatives, including ice rinks, should be addressed with socially sensitive plans. Complex cultural and religious issues may come into play in the event that there are contaminated remains and should be examined in advance. Disaster Pharmaceuticals Emergency drugs must be available at the point of care. Often they are secured in pharmacy departments or warehouses, resulting in precious minutes of life-saving time being lost as personnel try to locate and obtain critical medications. In addition to drugs needed to respond quickly to nerve agents and other emergency situations, stockpiles of antibiotics should be maintained to provide prophylaxis to patients and staff. CONCLUSION In a disaster, patients converge on the place they know they can obtain care—the hospital—and they arrive using any means possible. Furthermore, with the victims of disaster, come their families, loved ones, and the media—all who have very important needs that must be addressed. Hospitals can no longer approach disaster planning with a minimalist attitude that relies heavily on luck and belief that it will be someplace else that gets hit by the disaster. The hospital that received the most patients from the Rhode Island nightclub fire got lucky—the victims began arriving during a change of shift so there were two shifts of nursing staff available. However, the hospital also attributes its effective response to having drilled critical departments and procedures. Emergency planning is the backbone of preparedness, but events will occur in each disaster that demand creative responses under pressure. This ability to respond flexibly is known as planned innovation. Good plans will use general "all hazards" templates for disaster management but will permit independent initiative and a tailored response to a specific situation. 20 The U.S. health system appears to be emerging from the dark ages of emergency planning. A minimalist attitude of preparedness was acceptable in the past despite the regular occurrence of natural disasters. The threat of terrorism and the resulting health system impact have stimulated investment in research, a resurgence of disaster training in nursing and medical schools, and visionary projects such as ER One. ER One is a national prototype for a next-generation emergency department. Located in Washington, D.C., it is developing new approaches to the medical consequences of terrorist attacks, natural disasters, and emerging illnesses. More information is available online at: http://er1.org . The next phase of hospital emergency management will be a renaissance if creative planning prevails over naysayers, if resources are applied to priority preparedness activities, and if healthcare leaders are committed to ensuring that all who depend on hospitals will receive the care they need in a disaster ( Box 5-2 ). BOX 5-2 HOSPITAL PREPAREDNESS AND RESPONSE RESOURCES • Auf der Heide E. Principles of hospital disaster planning. In: Hogan DE, Burstein JL, eds. Disaster Medicine. Philadelphia: Lippincott Williams and Wilkins; 2002:57-89. • National Advisory Committee on Children and Terrorism. Recommendations to the Secretary, U.S. Department of Health and Human Services. (Includes recommendations on prehospital and hospital care.) Available at: http://www.bt.cdc.gov/children/word/working/Recommend.doc . • Preparing for the Psychological Consequences of Terrorism—A Public Health Strategy. This 2003 publication of the National Academies of Science includes an examination of current infrastructure and response strategies. Available at: http://search.nap.edu/books/0309089530/html/ . • Joint Commission on Accreditation of Healthcare Organizations. Guide to Emergency Management Planning in Health Care. Oakbrook Terrace, IL: Joint Commission Resources; 2002. • Rocky Mountain Regional Care Model for Bioterrorist Events. Available at: www.ahrq.gov/research/altsites.htm . • The Hospital Emergency Incident Command System. Available at: http://www.heics.com/ . • The American Academy of Experts in Traumatic Stress. Available at: http://www.aaets.org/ . • The International Critical Incident Stress Foundation, Inc. Available at: http://www.icisf.org/ .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796095/

Nonylphenol Induces Apoptosis through ROS/JNK Signaling in a Spermatogonia Cell Line

Nonylphenol (NP) is an endocrine-disruptor chemical that negatively affects reproductive health. Testes exposure to NP results in testicular structure disruption and a reduction in testicular size and testosterone levels. However, the effects of NP on spermatogonia in testes have not been fully elucidated. In this study, the molecular mechanisms of NP in GC-1 spermatogonia (spg) cells were investigated. We found that cell viability significantly decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to NP. Furthermore, the expression levels of the pro-apoptotic proteins increased, whereas anti-apoptosis markers decreased in NP-exposed GC-1 spg cells. We also found that NP increased reactive oxygen species (ROS) generation, suggesting that ROS-induced activation of the MAPK signaling pathway is the molecular mechanism of NP-induced apoptosis in GC-1 spg cells. Thus, NP could induce c-Jun phosphorylation; dose-dependent expression of JNK, MKK4, p53, and p38; and the subsequent inhibition of ERK1/2 and MEK1/2 phosphorylation. The genes involved in apoptosis and JNK signaling were also upregulated in GC-1 spg cells treated with NP compared to those in the controls. Our findings suggest that NP induces apoptosis through ROS/JNK signaling in GC-1 spg cells. 1. Introduction Nonylphenol (NP) is a xenobiotic used in chemical manufacturing that can accumulate in the environment. Its accumulation in water, soil, and air allows NP to enter the food chain [ 1 ]. NP is produced from the environmental degradation of nonylphenol ethoxylates, which are toxic nonylphenol metabolites [ 1 , 2 ]. In addition, NP has been detected in human biological fluids, including plasma, breast milk, and urine [ 3 , 4 , 5 ]. NP is a well-known endocrine disruptor chemical (EDC) that causes hormone imbalance; upon accumulation in the body, it can result in reproductive and developmental disorders in humans [ 6 , 7 ]. The toxic effects of NP on the reproductive systems and gonads of mammals, fish, and reptiles have been studied [ 8 , 9 ]. NP can induce oxidative stress in the epididymal sperm of rats, reduce testis size and testosterone levels, and disrupt testicular structure and spermatogenesis [ 9 , 10 , 11 ]. In Sertoli cell cultures, NP treatment induced apoptosis by inhibiting the Ca 2+ pump in the endoplasmic reticulum and increased oxidative stress [ 12 ]. NP also promotes apoptosis and autophagy in rat ovarian granulosa cells (GCs) [ 13 ]. Spermatogonia (spg) are undifferentiated male germ cells that undergo spermatogenesis to produce sperm in the testes. There is a growing concern that exposure to EDCs such as NP and bisphenol A (BPA) can affect fertility and sexual maturation. Indeed, damage by EDCs exposure at the early stage of germ-cell development, such as in the case of spermatogonia, can cause male infertility [ 14 , 15 ]. Although we previously reported that NP exposure in neonatal testes resulted in complete germ cell depletion and spermatogenic failure [ 16 ], the study did not address the specific mechanism of germ cell death. Therefore, the molecular mechanism of NP toxicity on germ cell survival and development is important for understanding the causes of male infertility and to develop novel treatment strategies. In addition, in vitro tests for assessing EDC toxicity, including that of NP, can be used to replace experiments that require animal euthanization. Furthermore, it is difficult to study the cellular and molecular mechanism of EDCs in specific cells in vivo because testes development and function is a complex process. Reactive oxygen species (ROS) are essential for the regulation of physiological activity including cell proliferation, differentiation, migration, cell cycle progression, and cell death [ 17 ]. Several environmental endocrine-disruptors can increase oxidative stress and ROS production within cells and tissues, which can eventually lead to the activation of cell-death processes such as apoptosis [ 18 , 19 ]. Diverse signaling pathways are activated by ROS, including the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/Akt, nuclear factor (erythroid-derived 2)-like 2/Kelch like-ECH-associated protein 1, nuclear factor-κB, and the tumor suppressor p53 pathways [ 20 , 21 ]. However, the signaling pathways initiated by ROS can differ depending on the stimulus and cell type. Several studies have reported that EDCs induce ROS-mediated apoptosis. For example, BPA is a widely used industrial compound that can trigger apoptosis via ROS signaling in human neuroblastoma cells [ 22 ], mouse testes [ 23 ], and rat brains [ 24 ]. Octylphenol, an alkylphenol, is another EDC that is similar to NP and can also induce apoptosis through ROS generation in mouse testicular cells, such as Leydig and Sertoli cells [ 25 , 26 ]. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin is reported to induce apoptosis through oxidative damage and mitochondrial dysfunction in human trophoblast-like JAR cells and mouse Sertoli cells [ 27 , 28 ]. Previous studies have shown that apoptotic cell death can occur in reproductive tissues and cells exposed to NP. For example, in Sertoli cells derived from 18–20-day-old male rats, NP treatment induced apoptosis via the ROS- mediated AMPK/Akt-mTOR and JNK pathways [ 29 ]. NP also induced apoptotic cell death in mouse TM4 Sertoli cells through the transient activation of ERK signaling [ 30 ]. In rat ovarian GCs, NP induced oxidative stress and apoptosis via the Akt/AMPK/mTOR pathway [ 13 ]. Additionally, an in vivo study showed that BPA and NP induced rat germ cell apoptosis mediated by the activation of p38 MAPK and ADAM17 [ 31 ]. Although these and other studies have reported the toxic effects of NP in reproductive organs [ 8 , 16 , 30 ], the molecular mechanisms of NP toxicity in GC-1 spg cells have not been characterized in detail. Therefore, we investigated the molecular mechanisms underlying NP-mediated toxicity in GC-1 spg cells. 2. Results 2.1. NP Reduces Cell Viability in the GC-1 spg Cell Line A previous study reported that NP induced germ cell apoptosis in organ-cultured neonatal testes [ 16 ]. Based on these previous results, we evaluated the effects of NP in vitro using the GC-1 spg cell line, which was derived from 10-day-old mouse testes. First, to determine the cytotoxic effect of NP on the proliferation of GC-1 spg cells, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability, which significantly decreased when the cells were exposed to 1–10 μM NP dissolved in 0.1% dimethyl sulfoxide (DMSO) for 24 h compared to the control group treated with 0.1% DMSO alone ( Figure 1 A). Based on this, the MTT results showed that the LC 50 was 5.7 μM NP, and concentrations of 1–10 μM NP were used for further experimentation ( Figure 1 A). 2.2. NP Induces Apoptotic Cell Death in GC-1 spg Cells We examined apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to evaluate the cell death mechanism in NP-treated GC-1 spg cells. As shown in Figure 1 B, TUNEL-positive cells increased in GC-1 spg cells treated with NP compared to the control. Moreover, the percentage of TUNEL-positive cells in NP-treated samples increased in a dose-dependent manner ( Figure 1 B,C). To determine the percentage of early and late apoptotic cells in NP-treated cultures, conventional flow cytometry was conducted with Annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling. Early apoptotic cells were visualized with Annexin V-FITC+/PI- staining patterns, whereas late apoptotic cells exhibited an Annexin V-FITC+/PI+ staining pattern. Our results revealed that both early and late apoptotic cells distinctly increased after NP treatment when compared with untreated control cell cultures, with approximately 20% of the cells observed to be apoptotic after treatment with 10 μM NP. The rate of apoptosis increased in an NP dose-dependent manner ( Figure 1 D,E), further suggesting that NP reduced cell viability through apoptotic mechanisms. 2.3. NP Induces the Expression of Pro-Apoptotic Proteins in GC-1 spg Cells Next, we wanted to understand the mechanism of NP-induced apoptosis in GC-1 spg cells. The protein levels of key intrinsic and extrinsic apoptotic pathways such as BAX, BID, cleaved caspase-3, cleaved caspase-8, caspase-9, cleaved PARP, and BCL2 were normalized to β-actin protein levels to quantify the changes observed between GC-1 spg cells treated with 1–10 μM NP and control conditions ( Figure 2 A,B). We found that the levels of BAX, BID, cleaved caspase-3, cleaved caspase-8, caspase-9, and cleaved PARP were upregulated by NP treatment compared to that of the control. In contrast, the expression of BCL2 was downregulated in a dose-dependent manner in NP-treated cell cultures. Stress-induced apoptosis can induce cytochrome c release from the mitochondria as well as result in caspase activation [ 31 ]. Therefore, we also examined whether NP could induce the release of cytochrome c in GC-1 spg cells. The cellular localization and protein expression of cytochrome c in GC-1 spg cells were examined using confocal immunofluorescence microscopy and Western blotting, respectively. The results showed strong cytochrome c immunofluorescence in GC-1 spg cells treated with 10 μM NP and a diffuse localization pattern in cells treated with 5–10 μM NP when compared with the untreated control. Indeed, cytochrome c was redistributed the region surrounding the LaminA/C + nucleus envelop in NP-treated cells ( Figure 3 A,B). 2.4. NP Induces ROS Production and Increases Mitochondrial Oxidative Stress in GC-1 spg Cells Based on our results, we next tested for intracellular ROS generation in GC-1 spg cells after NP treatment. The results showed that 10 μM NP triggered ROS production in GC- 1 spg cells, which was indicated by an elevated CellROX ® green signal observed in NP-treated cells compared to that of the untreated control samples ( Figure 4 A). Additionally, mitochondrial ROS production was measured in NP-treated cells that also indicated increased mitochondrial oxidative stress via an enhanced red fluorescence signal in treated cells compared with the control ( Figure 4 A). ROS production was also measured using flow cytometry and CellROX ® labeling in GC-1 spg cells after NP treatment, which showed that 1–10 μM NP in the culture medium significantly increased ROS production in a dose-dependent manner ( Figure 4 B,C). 2.5. NP Induces Cell Death through ROS-Mediated JNK-Dependent Apoptosis The MAPK-mediated pathway plays an important role in apoptosis, and it has been reported that ROS are potent inducers of the MAPK pathway through JNK activation [ 20 , 32 , 33 ]. Our results showed that NP-treated GC-1 spg cells presented apoptosis-mediated accumulation of cellular ROS ( Figure 4 ). Thus, we examined whether MAPK activation was involved in NP-induced GC-1 spg cell apoptosis. Treatment of GC-1 spg cells with NP resulted in the inhibition of ERK1/2 and MEK1/2 phosphorylation, but significantly increased the phosphorylation of c-Jun, JNK, MKK4, p53, and p38 expression in a dose-dependent manner compared to that observed in the untreated samples ( Figure 5 A,B). In addition, the gene expression levels of Mapk8, Mapk9, Map2k4, Map3k2, Tnfrsf10b, Tnfrsf1a, and Tnfrsf1b , which are related to apoptosis and JNK signaling, significantly increased in GC-1 spg cells treated with 10 μM NP compared with those in the control samples ( Figure 5 C). 2.1. NP Reduces Cell Viability in the GC-1 spg Cell Line A previous study reported that NP induced germ cell apoptosis in organ-cultured neonatal testes [ 16 ]. Based on these previous results, we evaluated the effects of NP in vitro using the GC-1 spg cell line, which was derived from 10-day-old mouse testes. First, to determine the cytotoxic effect of NP on the proliferation of GC-1 spg cells, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability, which significantly decreased when the cells were exposed to 1–10 μM NP dissolved in 0.1% dimethyl sulfoxide (DMSO) for 24 h compared to the control group treated with 0.1% DMSO alone ( Figure 1 A). Based on this, the MTT results showed that the LC 50 was 5.7 μM NP, and concentrations of 1–10 μM NP were used for further experimentation ( Figure 1 A). 2.2. NP Induces Apoptotic Cell Death in GC-1 spg Cells We examined apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to evaluate the cell death mechanism in NP-treated GC-1 spg cells. As shown in Figure 1 B, TUNEL-positive cells increased in GC-1 spg cells treated with NP compared to the control. Moreover, the percentage of TUNEL-positive cells in NP-treated samples increased in a dose-dependent manner ( Figure 1 B,C). To determine the percentage of early and late apoptotic cells in NP-treated cultures, conventional flow cytometry was conducted with Annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling. Early apoptotic cells were visualized with Annexin V-FITC+/PI- staining patterns, whereas late apoptotic cells exhibited an Annexin V-FITC+/PI+ staining pattern. Our results revealed that both early and late apoptotic cells distinctly increased after NP treatment when compared with untreated control cell cultures, with approximately 20% of the cells observed to be apoptotic after treatment with 10 μM NP. The rate of apoptosis increased in an NP dose-dependent manner ( Figure 1 D,E), further suggesting that NP reduced cell viability through apoptotic mechanisms. 2.3. NP Induces the Expression of Pro-Apoptotic Proteins in GC-1 spg Cells Next, we wanted to understand the mechanism of NP-induced apoptosis in GC-1 spg cells. The protein levels of key intrinsic and extrinsic apoptotic pathways such as BAX, BID, cleaved caspase-3, cleaved caspase-8, caspase-9, cleaved PARP, and BCL2 were normalized to β-actin protein levels to quantify the changes observed between GC-1 spg cells treated with 1–10 μM NP and control conditions ( Figure 2 A,B). We found that the levels of BAX, BID, cleaved caspase-3, cleaved caspase-8, caspase-9, and cleaved PARP were upregulated by NP treatment compared to that of the control. In contrast, the expression of BCL2 was downregulated in a dose-dependent manner in NP-treated cell cultures. Stress-induced apoptosis can induce cytochrome c release from the mitochondria as well as result in caspase activation [ 31 ]. Therefore, we also examined whether NP could induce the release of cytochrome c in GC-1 spg cells. The cellular localization and protein expression of cytochrome c in GC-1 spg cells were examined using confocal immunofluorescence microscopy and Western blotting, respectively. The results showed strong cytochrome c immunofluorescence in GC-1 spg cells treated with 10 μM NP and a diffuse localization pattern in cells treated with 5–10 μM NP when compared with the untreated control. Indeed, cytochrome c was redistributed the region surrounding the LaminA/C + nucleus envelop in NP-treated cells ( Figure 3 A,B). 2.4. NP Induces ROS Production and Increases Mitochondrial Oxidative Stress in GC-1 spg Cells Based on our results, we next tested for intracellular ROS generation in GC-1 spg cells after NP treatment. The results showed that 10 μM NP triggered ROS production in GC- 1 spg cells, which was indicated by an elevated CellROX ® green signal observed in NP-treated cells compared to that of the untreated control samples ( Figure 4 A). Additionally, mitochondrial ROS production was measured in NP-treated cells that also indicated increased mitochondrial oxidative stress via an enhanced red fluorescence signal in treated cells compared with the control ( Figure 4 A). ROS production was also measured using flow cytometry and CellROX ® labeling in GC-1 spg cells after NP treatment, which showed that 1–10 μM NP in the culture medium significantly increased ROS production in a dose-dependent manner ( Figure 4 B,C). 2.5. NP Induces Cell Death through ROS-Mediated JNK-Dependent Apoptosis The MAPK-mediated pathway plays an important role in apoptosis, and it has been reported that ROS are potent inducers of the MAPK pathway through JNK activation [ 20 , 32 , 33 ]. Our results showed that NP-treated GC-1 spg cells presented apoptosis-mediated accumulation of cellular ROS ( Figure 4 ). Thus, we examined whether MAPK activation was involved in NP-induced GC-1 spg cell apoptosis. Treatment of GC-1 spg cells with NP resulted in the inhibition of ERK1/2 and MEK1/2 phosphorylation, but significantly increased the phosphorylation of c-Jun, JNK, MKK4, p53, and p38 expression in a dose-dependent manner compared to that observed in the untreated samples ( Figure 5 A,B). In addition, the gene expression levels of Mapk8, Mapk9, Map2k4, Map3k2, Tnfrsf10b, Tnfrsf1a, and Tnfrsf1b , which are related to apoptosis and JNK signaling, significantly increased in GC-1 spg cells treated with 10 μM NP compared with those in the control samples ( Figure 5 C). 3. Discussion EDCs in the environment negatively affect vertebrate reproductive systems, including the development of reproductive organs, by interfering with hormonal systems [ 34 , 35 ]. Previous studies have demonstrated that NP exposure can induce apoptosis in various cell types, such as human epithelial intestinal cells [ 36 ], embryonic stem cells [ 37 ], thymocytes [ 38 ], and neurons [ 39 ]. Although NP induces apoptosis in cells and has been detected in the human body, its detailed mechanism of toxicity in the reproductive system has not been fully investigated. In our previous study, we showed that NP can lead to germ cell depletion in pubertal testes but not Sertoli or Leydig cells [ 16 ]. Additionally, other studies reported that NP treatment can cause abnormal reproductive function in post-pubertal female rats, disrupt gonad development in both male and female rats [ 40 ], and decrease sperm counts, testes weight, and germ cell numbers [ 40 , 41 ]. Therefore, we dissected the molecular mechanisms of NP-induced apoptosis using the GC-1 spg cell line, which was derived from prepubertal testes. The present study showed that NP induced ROS-mediated apoptosis via the JNK signaling pathway in GC-1 spg cells. Our results showed that GC-1 spg cell viability was reduced by approximately 40–50% after 10 μM NP treatment, and apoptosis rates increased by 20% with the same treatment compared with control conditions. There are major signaling pathways that can lead to cell death including apoptosis, autophagy, and necrosis. Duan et al. reported that NP induced autophagy, apoptosis, and necrosis in Sertoli cells [ 29 ], whereas our study focused on the ROS-mediated apoptotic pathway in GC-1 spg cells. It is possible that NP treatment induced GC-1 spg cell death through a mechanism other than apoptosis. Indeed, Sertoli cells that were exposed to NP showed programmed cell death via a ROS-mediated AMPK/Akt-mTOR and the JNK pathway. In our study, JNK also induced apoptosis in GC-1 spg cells treated with NP, consistent with another report that demonstrated that the JNK pathway is activated by increased ROS and induces autophagy in Sertoli cells [ 29 ]. Excess oxidative stress and ROS cause damage to DNA, proteins, lipids, and membranes, which can all lead to apoptotic cell death [ 19 ]. NP is toxic to both humans and animals [ 40 , 42 ]; the direct exposure to NP induces oxidative stress and apoptosis, previously reported in rat testicular Sertoli cells [ 8 ], ovarian GCs [ 13 ], and mouse oocytes [ 43 ] that are similar to those presented in this study. In addition to the reproductive system, NP increases ROS levels and induces oxidative damage in the liver, pancreas, and kidneys of rats [ 44 , 45 , 46 ]. GC-1 spg cells are type B spg cells that are considered to be a type of testicular germ cell, although they are not spermatogonial stem cells (SSCs). SSCs that are type A single (As) spg are located on the basement membrane of the seminiferous tubules. These cells can self-renew or produce the type A paired spg that undergo active mitosis, which further divide to produce type B spg cells that can then form primary spermatocytes that undergo meiosis [ 47 ]. Our previous study reported that NP downregulated the transcript levels of both undifferentiated and differentiated germ cell markers in neonatal mouse testes [ 16 ]. Therefore, we examined the effects of NP on apoptosis in type B spg cells, which is not a type of stem cell, in this study. Lei et al. reported that the cytotoxic effect of NP on SSCs occurs through the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. Indeed, the expression levels of SSC stemness maintenance markers, Nanog, Oct4, and Sox2, and the differentiation markers Nanos3, Stra8, Scp3, GFRα1, CD90, VASA, Nanos2, KIT, and PLZF, significantly decreased in SSCs treated with 10–30 μM NP compared with those in the controls. Moreover, the expression of Bad, cytochrome-c, and pro-Caspase 9 increased in NP-treated SSCs [ 48 ], which is consistent with our results. Therefore, although ROS are essential for the regulation of the normal physiological functions of the cell, such as proliferation and differentiation, and for maintaining normal immune responses [ 17 , 20 ], increased ROS levels can also negatively impact cell survival and development. ROS-induced apoptosis has been reported in various cell systems. During apoptosis, there are two major programs that are downstream of the death signal: the caspase pathway and mitochondria and ER dysfunction, which are known as the extrinsic and intrinsic pathways, respectively [ 49 ]. In our study, apoptosis was induced by NP exposure in GC-1 spg cells that were involved in mitochondrial ROS-mediated death signaling. In particular, the expression levels of pro-apoptotic BCL-2 family proteins, such as BAX and BID, were increased by NP treatment and promoted caspase activation. The permeabilization of the mitochondrial outer membrane by Bax/Bak during apoptosis is essential for the intrinsic pathway during apoptotic cell death because when the mitochondrial membrane permeability increases, the release of cytochrome c into the cytosol activates caspase and leads to the apoptotic phenotype [ 50 , 51 , 52 ]. These reported apoptotic processes were similar to those observed in our study. Additionally, Liu et al. reported NP toxicity in mouse Sertoli TM4 cells, which showed that cell viability decreased due to induced apoptosis and was accompanied by altered Bcl-2 family mRNA expression, as well as the activation of caspases-3, the release of Ca 2+ , and increased ROS generation. NP also increased the activation of the MAPK pathway and inhibited the Akt pathway in Sertoli TM4 cells [ 53 ]. JNK proteins belong to the MAP-kinase superfamily and were initially studied as UV-responsive protein kinases involved in the transactivation of c-Jun by phosphorylating its N-terminal Ser-63 and Ser-73 residues [ 54 , 55 ]. A number of studies have been conducted on the mechanism of ROS-meditated JNK activation and signaling in the control of diverse cellular functions, including cell differentiation, proliferation, and apoptosis [ 19 ]. JNK signaling is involved in both extrinsic pathways that initiate death receptors (TNF-α, TRAIL known as TNFSF10, and FAS-L) and intrinsic apoptotic pathways that are initiated by mitochondrial events [ 56 ]. Similar to the results presented in these previous reports, we found that the expression levels of Tnfrsf10b , Tnfrsf1a , and Tnfrsf1b were significantly increased and that mitochondrial dysfunction increased in GC-1 spg cells treated with 10 μM NP. In other words, both extrinsic and intrinsic apoptosis pathways are associated with JNK activation in response to NP exposure. In addition, apoptosis signaling through the JNK pathway indicates that these kinases function in a cell condition- and cell-type-specific manner to link these signals at different transmission points via both transcriptional and post-transcriptional mechanisms, which eventually converge on caspase activation [ 57 ]. MAPK signaling cascades include MAPK or ERK, MAPK kinase (MKK or MEK), and MAPK kinase kinase (MAPKKK or MEKK). MAPKKK/MEKK phosphorylates and activates its downstream protein kinase, MAPKK/MEK, which in turn activates MAPK [ 58 ]. There are multiple upstream components of the c-Jun N-terminal kinase (JNK/ MAPK) cascade, such as MAP3K1 (MEKK1), MAP3K2 (MEKK2), MAP2K7 (MKK7), and MAP2K4 (MKK4). In addition, the activation of JNK can lead to a multitude of downstream changes in phosphorylation, especially the JNK-potentiated transcriptional activity of c-Jun through the phosphorylation of serines 63 and 73, which modulate apoptosis [ 57 , 59 ]. Consistent with these previous studies, we also observed increased JNK, c-Jun, and MKK4 phosphorylation and Mapk8 ( JNK1 ), Mapk9 ( JNK2 ), Map2k4 ( MKK4 ), Map3k2 ( MEKK2 ), and Jun mRNA expression in NP-treated GC-1 spg cells. It has been reported that MAPK8, MAPK9, and p38 (MAPK14) are substrates for MAP2K4; however, ERK1/2 is not phosphorylated by MAP2K4 even though ERK1/2 is a MAPK family protein with typical cascade signaling characteristics [ 60 ]. Phosphorylated p44 MAPK and p42 MAPK, which are known as ERK1 and ERK2, translocate into the nucleus from the cytoplasm to regulate the transcription factors and gene expression that promote cell growth, differentiation, or mitosis in cells. Although activation of ERK/MEK is generally associated with cell proliferation, some studies have shown that ERK mediates cell death. However, the molecular mechanisms of ERK-mediated cell death are still not fully understood [ 61 ]. Our results suggested that the suppression of MEK/ERK activation by NP probably led to the inhibition of cell growth and was linked to cell death. Moreover, NP stimulated p53 and p38 phosphorylation through the JNK pathway in GC-1 spg cells. There is evidence that JNK mediates p53 activation; studies have reported that JNK can phosphorylate p53 [ 59 , 62 ]. JNK activity requires MEKK, which phosphorylates MKK4/7 and JNK on residues 183 and 185; the activated JNK phosphorylates its substrates c-Jun, ELK1, and p53 [ 59 , 63 , 64 ]. Our results revealed increased JNK pathway activation and phosphorylated p53 and p38 in NP-treated GC-1 spg cells, suggesting a correlation between JNK and p38. The p38 MAPK is a member of the MAPK subfamily that is responsive to stress stimuli, including ultraviolet irradiation, heat shock, and osmotic shock; however, it is also involved in cell apoptosis, autophagy, and cell differentiation [ 65 , 66 ]. The phosphorylation and activation of p38 can not only mediate cell apoptosis [ 67 , 68 ] but also exert protective effects on cells [ 32 , 69 ]. Both JNK and p38 MAPK cascades mediate apoptotic processes, and several Bcl-2 family proteins are under the control of JNK and/or p38 MAPK cascades at transcriptional and/or post-transcriptional levels [ 70 , 71 ]. In conclusion, although the concentration of NP is higher than physiological NP levels observed in humans [ 72 ], NP exposure resulted in decreased GC-1 spg cell viability; increased the expression of apoptosis-related proteins, BAX, BID, cleaved caspase-3 and caspase 8, caspase 9, and cleaved PARP; and decreased Bcl-2 expression. NP treatment also led to the accumulation of ROS and increased oxidative stress and apoptosis mediated by abnormal mitochondrial membrane potential in GC-1 spg cells. In addition, NP activated the MAPK family-associated proteins, JNK, c-Jun, p38, and MKK4 and also suppressed ERK1/2 and MEK1/2 activity. The results from our study suggested that these molecular mechanisms are involved in the toxic effect of NP on spermatogonia cells in vitro. 4. Material and Methods 4.1. Cell Culture and Treatments The mouse GC-1 spg (spermatogonia) cell line was purchased from the Korean Cell Line Bank (KCLB 21715, Seoul, South Korea) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C. NP (Sigma-Aldrich St. Louis, MO, USA) was dissolved in DMSO to prepare a 1 M stock solution, which was diluted to the desired concentration using the cell culture medium prior to cell culture. 4.2. Cell Viability Determination Using MTT Assay Cell viability was determined with an MTT assay using the EZ-Cytox Viability assay kit (Daeil Lab Services Co, Seoul, Korea, #EZ1000) and following the manufacturer's instructions to measure the half-maximal inhibitory concentration (IC 50 ) of NP in GC-1 spg cells. GC-1 spg cells were seeded in 96-well plates at a density of 5 × 10 3 /well in culture medium and incubated for 24 h at 37 °C. The culture medium was replaced with fresh medium containing different concentrations of NP (0.01–10 μM) and cultured for an additional 24 h. The assay reagent was added, and cultures were incubated for 60 min before the plates were read at 490 nm using a Sunrise TM spectrophotometer (TECAN, Männedorf, Switzerland). 4.3. Apoptosis Assay TUNEL assay was used to quantify DNA and chromatin morphogenic features. For TUNEL staining, an in situ TMR red cell death detection kit (Roche, Germany) was used following the manufacturer's guidelines. Cells were cultured on glass slides for 24 h and then exposed to 0, 1, 5, or 10 µM NP for 24 h. The cells grown on coverslips were washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich) and fixed with 4% paraformaldehyde in PBS for 15 min at 24 °C. Following the washes, the cells were incubated in a permeabilization solution (0.1% Triton X-100 in PBS) for 2 min on ice. The samples were incubated in 50 μL TUNEL reaction mixture (Roche, Mannheim, Germany) for 60 min at 37 °C in a humidified chamber in the dark. Samples were incubated with or without 1 µg/mL 6-diamidino-2-phenylindole (DAPI) in PBS for 5 min; coverslips were applied using the mounting solution (Dako, Carpinteria, CA, USA; S3025) and imaged using fluorescence microscopy (Nikon, Tokyo, Japan). 4.4. Flow Cytometry Annexin V-FITC staining assay was conducted using the dead cell apoptosis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), which uses Annexin V-FITC and PI, to measure apoptosis in GC-1 spg cells after NP treatment. The cells were seeded in six-well plates and treated with NP for 24 h. The cells were then collected after trypsinization and washed with PBS before being stained with Annexin V-FITC and PI for 15 min at 24 °C in the dark. Annexin V-positive cells were detected using flow cytometry (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA). ROS generation was determined using the CellROX Green flow cytometry assay kit (Thermo Fisher Scientific, San Jose, CA). Samples were stained according to the manufacturer's instructions, and the fluorescence intensity of CellROX Green was measured with a flow cytometer (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA). 4.5. Immunofluorescence Staining ROS generation was assessed in GC-1 spg cells treated with 10 µM NP using CellROX Oxidative stress reagents. GC-1 spg cells were seeded onto 12-mm glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 1 × 10 5 cells/coverslip and allowed to attach for 1 d prior to treatment with 10 µM NP for 24 h. The cells were then used for immunofluorescence staining with CellROX ® green (C10444, Life Technologies, Carlsbad, CA, USA) and MitoTracker ® red CMXRos (M7512, Life Technologies, Carlsbad, CA, USA). Twenty-four hours after NP treatment, CellROX green and DAPI were added to the cells and incubated for 30 min at 37 °C. The medium was then removed and the cells were washed three times with PBS. Cells were fixed in 4% formaldehyde for 15 min. For mitochondria staining, samples were incubated for 30 min with 100 nM MitoTracker ® red CMXRos at 37 °C and then fixed. All samples were mounted in mounting medium (Sigma-Aldrich), and images were obtained using confocal microscopy (Carl Zeiss, Oberkochen, Germany; LSM 800). 4.6. Isolation of RNA and Quantitative Real-Time PCR Analysis Total RNA was extracted from GC-1 spg cells using a RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase treatment (Qiagen). Complementary DNA (cDNA) was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) with an oligo(dT)30 primer following the manufacturer's instructions. Template cDNA was mixed with iQ SYBR Green Supermix (170–8880; Bio-Rad Laboratories), and 1 pM of each primer was used for qPCR. Primers were designed using Primer3 ( http://frodo.wi.mit.edu ). The cycle threshold values were normalized against GAPDH gene expression. Denaturation and polymerase activation steps were performed at 94 °C for 1 min followed by 40 cycles at 94 °C for 10 s, 57 °C for 10 s, and 72 °C for 20 s. The primers used to detect mouse transcripts are listed in Table 1 . 4.7. Western Blotting Western blotting was conducted as described previously [ 32 ]. GC-1 spg cell lysates were prepared using RIPA lysis buffer (Thermo Scientific™, Rockford, IL, USA) and a protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). Protein samples were separated in 4–12% gradient SDS-PAGE gels, and proteins were transferred to PVDF membranes using a transfer blotting system (Bio-Rad, Hercules, USA). Membranes were incubated in blocking buffer solution (TBS with 0.1% tween-20 (TBST) + 1% bovine serum albumin) for 1 h and then incubated with primary antibodies overnight at 4 °C. Membranes were washed with TBST for 30 min and then incubated for 1 h with secondary antibodies in TBST +1% BSA. All antibodies used in this study are listed in Table 2 . Blots were visualized using Pierce ECL solution (Thermo Fisher Scientific, Rockford, IL, USA) and X-ray film. ImageJ software was used to quantify protein expression. 4.8. Statistical Analysis All results are expressed as mean ± standard error values from at least three independent experiments and were evaluated using one-way analysis of variance. All statistical analyses were conducted using the SPSS statistical package, version 15.0 for Windows (IBM Corp, Somers, NY, USA). Values of * p < 0.05 and ** p < 0.01 were considered statistically significant. 4.1. Cell Culture and Treatments The mouse GC-1 spg (spermatogonia) cell line was purchased from the Korean Cell Line Bank (KCLB 21715, Seoul, South Korea) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C. NP (Sigma-Aldrich St. Louis, MO, USA) was dissolved in DMSO to prepare a 1 M stock solution, which was diluted to the desired concentration using the cell culture medium prior to cell culture. 4.2. Cell Viability Determination Using MTT Assay Cell viability was determined with an MTT assay using the EZ-Cytox Viability assay kit (Daeil Lab Services Co, Seoul, Korea, #EZ1000) and following the manufacturer's instructions to measure the half-maximal inhibitory concentration (IC 50 ) of NP in GC-1 spg cells. GC-1 spg cells were seeded in 96-well plates at a density of 5 × 10 3 /well in culture medium and incubated for 24 h at 37 °C. The culture medium was replaced with fresh medium containing different concentrations of NP (0.01–10 μM) and cultured for an additional 24 h. The assay reagent was added, and cultures were incubated for 60 min before the plates were read at 490 nm using a Sunrise TM spectrophotometer (TECAN, Männedorf, Switzerland). 4.3. Apoptosis Assay TUNEL assay was used to quantify DNA and chromatin morphogenic features. For TUNEL staining, an in situ TMR red cell death detection kit (Roche, Germany) was used following the manufacturer's guidelines. Cells were cultured on glass slides for 24 h and then exposed to 0, 1, 5, or 10 µM NP for 24 h. The cells grown on coverslips were washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich) and fixed with 4% paraformaldehyde in PBS for 15 min at 24 °C. Following the washes, the cells were incubated in a permeabilization solution (0.1% Triton X-100 in PBS) for 2 min on ice. The samples were incubated in 50 μL TUNEL reaction mixture (Roche, Mannheim, Germany) for 60 min at 37 °C in a humidified chamber in the dark. Samples were incubated with or without 1 µg/mL 6-diamidino-2-phenylindole (DAPI) in PBS for 5 min; coverslips were applied using the mounting solution (Dako, Carpinteria, CA, USA; S3025) and imaged using fluorescence microscopy (Nikon, Tokyo, Japan). 4.4. Flow Cytometry Annexin V-FITC staining assay was conducted using the dead cell apoptosis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), which uses Annexin V-FITC and PI, to measure apoptosis in GC-1 spg cells after NP treatment. The cells were seeded in six-well plates and treated with NP for 24 h. The cells were then collected after trypsinization and washed with PBS before being stained with Annexin V-FITC and PI for 15 min at 24 °C in the dark. Annexin V-positive cells were detected using flow cytometry (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA). ROS generation was determined using the CellROX Green flow cytometry assay kit (Thermo Fisher Scientific, San Jose, CA). Samples were stained according to the manufacturer's instructions, and the fluorescence intensity of CellROX Green was measured with a flow cytometer (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA). 4.5. Immunofluorescence Staining ROS generation was assessed in GC-1 spg cells treated with 10 µM NP using CellROX Oxidative stress reagents. GC-1 spg cells were seeded onto 12-mm glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 1 × 10 5 cells/coverslip and allowed to attach for 1 d prior to treatment with 10 µM NP for 24 h. The cells were then used for immunofluorescence staining with CellROX ® green (C10444, Life Technologies, Carlsbad, CA, USA) and MitoTracker ® red CMXRos (M7512, Life Technologies, Carlsbad, CA, USA). Twenty-four hours after NP treatment, CellROX green and DAPI were added to the cells and incubated for 30 min at 37 °C. The medium was then removed and the cells were washed three times with PBS. Cells were fixed in 4% formaldehyde for 15 min. For mitochondria staining, samples were incubated for 30 min with 100 nM MitoTracker ® red CMXRos at 37 °C and then fixed. All samples were mounted in mounting medium (Sigma-Aldrich), and images were obtained using confocal microscopy (Carl Zeiss, Oberkochen, Germany; LSM 800). 4.6. Isolation of RNA and Quantitative Real-Time PCR Analysis Total RNA was extracted from GC-1 spg cells using a RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase treatment (Qiagen). Complementary DNA (cDNA) was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) with an oligo(dT)30 primer following the manufacturer's instructions. Template cDNA was mixed with iQ SYBR Green Supermix (170–8880; Bio-Rad Laboratories), and 1 pM of each primer was used for qPCR. Primers were designed using Primer3 ( http://frodo.wi.mit.edu ). The cycle threshold values were normalized against GAPDH gene expression. Denaturation and polymerase activation steps were performed at 94 °C for 1 min followed by 40 cycles at 94 °C for 10 s, 57 °C for 10 s, and 72 °C for 20 s. The primers used to detect mouse transcripts are listed in Table 1 . 4.7. Western Blotting Western blotting was conducted as described previously [ 32 ]. GC-1 spg cell lysates were prepared using RIPA lysis buffer (Thermo Scientific™, Rockford, IL, USA) and a protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). Protein samples were separated in 4–12% gradient SDS-PAGE gels, and proteins were transferred to PVDF membranes using a transfer blotting system (Bio-Rad, Hercules, USA). Membranes were incubated in blocking buffer solution (TBS with 0.1% tween-20 (TBST) + 1% bovine serum albumin) for 1 h and then incubated with primary antibodies overnight at 4 °C. Membranes were washed with TBST for 30 min and then incubated for 1 h with secondary antibodies in TBST +1% BSA. All antibodies used in this study are listed in Table 2 . Blots were visualized using Pierce ECL solution (Thermo Fisher Scientific, Rockford, IL, USA) and X-ray film. ImageJ software was used to quantify protein expression. 4.8. Statistical Analysis All results are expressed as mean ± standard error values from at least three independent experiments and were evaluated using one-way analysis of variance. All statistical analyses were conducted using the SPSS statistical package, version 15.0 for Windows (IBM Corp, Somers, NY, USA). Values of * p < 0.05 and ** p < 0.01 were considered statistically significant.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC201196/

Species-Specific Peptide Ligands for the Detection of Bacillus anthracis Spores

Currently available detectors for spores of Bacillus anthracis , the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis . These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209696/

The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group ( sensu lato )

Bacillus toyonensis strain BCT-7112 T (NCIMB 14858 T ) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol ( catQ ) and tetracycline ( tetM ) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis . The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group ( sensu lato ). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group ( sensu lato ). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries. Introduction The Bacillus cereus group ( sensu lato ) forms an independent branch within the Bacillus genus and currently comprises eight closely related and validated species: B . cereus ( sensu stricto ), B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus , and B. toyonensis (Skerman et al., 1980 ; Lechner et al., 1998 ; Guinebretière et al., 2013 ; Jiménez et al., 2013 ). Three additional new species, B. gaemokensis, B. manliponensis , and B. bingmayongensis , have been recently reported to belong to B. cereus group, but their names are not currently validly published (Jung et al., 2010 , 2011 ; Liu et al., 2014 , 2015 ). Until relatively recently, understanding the identification and phylogenetic relationships within the B. cereus group ( sensu lato ) has proved challenging because: (a) traditionally, taxonomic distinctions among this group have relied heavily on a small number of phenotypic traits that include, in some cases, genes encoded on plasmids (e.g., anthrax toxin, insecticidal toxins, immune-evading capsules) and morphological features ( B. mycoides ; Rasko et al., 2004 ); (b) 16S rRNA gene sequence data is not sufficient to differentiate bacteria within the group due to its high degree of conservation (Bavykin et al., 2004 ). In a taxonomic study to determine whether B. toyonensis strain BCT-7112 T , previously known as B. cereus var. toyoi , strain BCT-7112, was a new species within the B. cereus group, a polyphasic approach was performed in which both phenotypic and genotypic traits were analyzed (Jiménez et al., 2013 ). Taken together, the results indicated that strain BCT-7112 T did indeed represent a new species for which the name B. toyonensis sp. nov. was proposed and approved, with BCT-7112 T (=CECT 876 T ; = NCIMB 14858 T ) as the type strain (Oren and Garrity, 2014 ). The characteristics that distinguished strain BCT-7112 T from other B. cereus species were also shared by 10 other strains (Jiménez et al., 2013 ). In a more recent taxonomic study, the affiliations of strains in the B. cereus group were established using a Genome BLAST Distance Phylogeny (GBDP) approach (Liu et al., 2015 ). This separated the 224 analyzed strains into 30 clusters; eleven known species-level clusters and 19 potentially novel species. In this analysis the 11 strains previously identified as belonging to the B. toyonensis taxonomic unit (Jiménez et al., 2013 ) formed a distinct cluster (BCG09) but was expanded to nineteen strains by the inclusion of eight additional strains. In the same study, an identical cluster was identified using a rapid typing method based on the pycA gene and, in separate studies, by multi-locus sequence analysis (MLSA) and whole-genome single nucleotide polymorphism (SNP)-based phylogeny (Böhm et al., 2015 ). B. toyonensis BCT-7112 T has been widely used for more than 30 years as the active ingredient of Toyocerin®, an additive used in animal nutrition and its non-toxigenic nature has been shown in various studies (Williams et al., 2009 ; Trapecar et al., 2011 ; Blanch et al., 2014 ). However, it is resistant to chloramphenicol and tetracycline, a trait that is generally considered inadvisable for introduction into the food chain if associated with mobile genetic elements (MGEs). We therefore set out to identify the genes responsible for these resistance phenotypes and to establish whether they were intrinsic or recent acquisitions associated with horizontal gene transfer. Unexpectedly, we not only observed homologous genes in closely related strains, but in virtually all members of the B. cereus group ( sensu lato ), irrespective of their reported susceptibility to these antibiotics. We discuss our findings in the context of regulations governing the animal feed and related industries. Methods Strains and culture conditions The strains and plasmids used in this study, together with their sources, are shown in Table 1 . When required, the following antibiotic concentrations were used: for E. coli , 100 μg/ml ampicillin and 12.5 μg/ml chloramphenicol; for Bacillus strains, 1 μg/ml erythromycin, 100 μg/ml spectinomycin, and for chloramphenicol and tetracycline concentrations ranged from 1 to 32 μg/ml, as indicated in the text. Table 1 Strains and plasmids used in this study . Strains Trait or relevant genotype * Source/Reference * Bacillus cereus ATCC 14579 T ATCC Bacillus thuringiensis ATCC 10792 ATCC Bacillus toyonensis BCT-7112 T B. toyonensis type strain, Tc R , Cm R Rubinum S.A, Spain BCT-7112Δtet BCT-7112 T with the tetM gene replaced with a spectinomycin resistance gene, Cm R , Sp R , Tc S This study BCT-7112Δcat BCT-7112 T with the tetM gene replaced with a spectinomycin resistance gene, Tc R , Sp R , Cm S This study Rock1-3 B. toyonensis strain isolated from soil in Rockville, Maryland. Previously B. cereus Rock1-3 US Naval Medical Research Center Zwick et al., 2012 Rock3-28 B. toyonensis strain isolated from soil in Rockville, Maryland. Previously B. cereus Rock3-28 US Naval Medical Research Center; Zwick et al., 2012 BCT-7112(pIGR1) BCT-7112 T with pIGR, Tc R , Cm R , Em R This study BCT-7112Δtet(pIGR1) BCT-7112Δtet with pIGR, Cm R , Sp R , Em R , Tc S This study BCT-7112Δcat(pIGR1) BCT-7112Δcat with pIGR, Tc R , Sp R , Em R , Cm S This study Rock1-3(pIGR) Rock1-3 with pIGR, Em R This study Bacillus subtilis 168 trpC2 Institute Pasteur, Paris; Anagnostopoulos and Spizizen, 1961 168tetM7112 168 with tetM from BCT-7112 T , Tc R This study 168tetM1-3 168 with tetM from Rock1-3, Tc R This study 168tetM3-28 168 with tetM from Rock3-28, Tc R This study 168catQ7112 168 with catQ from BCT-7112 T , Cm R This study 168catQ1-3 168 with catQ from Rock1-3, Cm R This study 168catQ3-28 168 with catQ from Rock3-28, Cm R This study Escherichia coli DH5α huA2 lac(del)U169 phoA glnV44 Φ80′ lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR Thermo Fisher Scientific GM48 dam − , dcm − Stratagene Plasmids pUTE583 Cm R in E. coli ; Em R and Ts in B. toyonensis Theresa M. Koehler, The University of Texas: Chen et al., 2004 pUTEΔtetM pUTE583 with tetM flanking regions and Sp R cassette This study pUTEΔcatQ pUTE583 with catQ flanking regions and Sp R cassette This study pING1 pUTE583 with the gerIC–nucB intergenic region of plasmid pBCT77 This study pDR111 B. subtilis integration/expression vector, Ap R ( E. coli ), Sp R ( B. subtilis ), amyE Δ, lacI , P hyper-spank R. Daniel, Newcastle University pDRtetM7112 pDR111 with tetM from BCT-7112 T , Ap R , Sp R , Tc R This study pDRtetM1-3 pDR111 with tetM from Rock1-3, Ap R , Sp R , Tc R This study pDRtetM3-28 pDR111 with tetM from Rock3-28, Ap R , Sp R , Tc R This study pDRcatQ7112 pDR111 with catQ from BCT-7112 T , Ap R , Sp R , Tc R This study pDRcatQ1-3 pDR111 with catQ from Rock1-3, Ap R , Sp R , Tc R This study pDRcatQ3-28 pDR111 with catQ from Rock3-28, Ap R , Sp R , Tc R This study * ATCC, American Type Culture Collection; Ap R , ampicillin resistance; Cm R , chloramphenicol resistance; Em R , erythromycin resistance; Tc R , tetracycline resistance; Sp R , spectinomycin resistance . Minimum inhibitory concentration (MIC) experiments were carried out in accordance with the European Food Safety Authority's (EFSA) "Guidance on the assessment of bacterial antimicrobial susceptibility" (EFSA-FEEDAP, 2012 ) using the microdilution method. Solutions of the required antibiotics (0, 1, 2, 4, 8, 16, and 32 μg/ml) were made up in Brain Heart Infusion broth and 200 μl added to wells in a 96-well microtitre plate (MTP). The wells were inoculated with 20 μl of the test cultures, prepared from fresh colonies grown on Brain Heart Infusion agar. The MTP was incubated in a FLUOstar Optima plate reader for 18 h at 37°C with vigorous shaking, monitoring Optical Density (OD) at 600 nm. The experiments were carried out a minimum of three times and the representative data show on a linear rather than logarithmic graph to make it easier to compare growth profiles. The MIC was determined as the lowest concentration of the antibiotic for which no growth was detected. Construction of tetM and catQ knockout strains In order to identify the genes responsible for tetracycline and chloramphenicol resistance in the genome of B. toyonensis BCT-7112 T , a gene replacement strategy was employed. This involved the replacement of the target gene with a spectinomycin gene by a reciprocal recombination event. Briefly, two DNA knockout cassettes of ~3 kb were synthesized (DNA 2.0, Newark, California) and cloned into the vector pUTE583 (Koehler et al., 1994 ) to generate pUTEΔtetM and pUTEΔcatQ. Each synthesized fragment consisted of three components, a ~1000-bp region upstream of the target gene, a 947-bp spectinomycin resistance fragment and a ~1000-bp regions downstream of the target cassette. In the case of the tetM knockout cassette, a 1102-bp upstream fragment of the B. toyonensis BCT-7112 T chromosome (nucleotides 305983–307085) was fused to the 947-bp spectinomycin resistance fragment, which in turn was fused to the 907-bp downstream fragment of the B. toyonensis BCT-7112 T chromosome (nucleotides 309075–309983; Figure 1A ). The catQ knockout cassette was similarly constructed with a 1069-bp upstream fragment (nucleotides 4932601–4933670) and 1219 downstream fragment (nucleotides 4933976–4935195) of the B. toyonensis BCT-7112 T chromosome, separated by the 947-bp spectinomycin resistance fragment (Figure 1B ). Figure 1 Regions of the B. toyonensis BCT-7112 T genome containing (A) tetracycline (Btoyo_0322, tetM ) and (B) chloramphenicol (Btoyo_4985, catQ ) resistance genes. The lower images show the locations (red arrows) of the upstream and downstream fragments and the spectinomycin resistance ( spc R ) gene that were used to replace the Btoyo_0322 or Btoyo_4985 genes by reciprocal recombination events, generating the tetM or catQ knockout strains BCT-7112Δtet and BCT-7112Δcat. The green arrows show the approximate locations of the primers used to sequence over the knockout regions. The bifunctional vector, pUTE853, is able to replicate in E. coli and members of the B. cereus group, but is lost from the latter at 37°C in the absence of antibiotic selection. The knockout plasmids, pUTEΔtetM and pUTEΔcatQ, were first passaged through a dam- minus E. coli strain (GM48) and then electro-transformed into B. toyonensis BCT-7112 T , selecting for erythromycin resistance at 30°C (Koehler et al., 1994 ). The transformants were grown in the presence of erythromycin for 2 days, diluting 1:500 at the start and end of the day with fresh medium, and then shifted to 37°C for 2 days in the presence of spectinomycin, again diluting 1:500 at the start and end of the day with fresh medium. Samples were plated onto BHI agar containing spectinomycin, incubated overnight at 37°C, and individual colonies checked for erythromycin sensitivity and spectinomycin resistance, confirming the loss of the plasmid and integration of the knockout cassette into the chromosome. In each case the target antibiotic resistance phenotype was lost, tetracycline resistance in the case of pUTEΔtetM and chloramphenicol resistance in the case of pUTEΔcatQ, generating B. toyonensis knockout strains BCT-7112Δtet and BCT-7112Δcat, respectively. The fidelity of the integration event was confirmed by sequencing over the region using primers within the flanking fragments and spectinomycin fragment (Figure 1 ). Construction of strains with additional copies of the gerIC–nucB intergenic region Previous unpublished studies indicated an involvement of the gerIC–nucB intergenic region of plasmid pBCT77 as contributing to, or enhancing, the observed tetracycline and chloramphenicol resistances in B. toyonensis BCT-7112 T . The 2003-bp gerIC–nucB intergenic region extends from nucleotide 75752–76974 on the pBCT77 genome (NC_022782). In the first place we used primers 1pBCT77Fwd (5′-GCCCCTTTAAACTTCATTATGC-3′) and 4pBCT77Rev (5′-CCCTTACCTTCAAGTATTCC-3′) to confirm that pBCT77 was still present in both knockout strains. The intergenic DNA was then synthesized and cloned into the Hin dIII and Xba I sites of pUTE853 (DNA 2.0, Newark, California). The resulting vector, pIGR1, was transformed into E. coli strain DH5α and its structure confirmed by restriction analysis and PCR. Following passage through the E. coli dam- minus strain GM48, pIGR1 was electro-transformed into B. toyonensis strains BCT-7112 T , BCT-7112Δtet, BCT-7112Δcat, and Rock1-3, selecting for erythromycin resistant transformants at 37°C. Erythromycin selection was maintained in all pre-cultures, but not during MIC experiments. Construction B. subtilis strains with tetM and catQ genes from BCT-7112 T , Rock1-3, and Rock3-28 For expression in B. subtilis , the tetM , and catQ genes from BCT-7112 T , Rock1-3, and Rock3-28 were first cloned into the bifunctional expression/integration vector pDR111 (Britton et al., 2002 ). This vector has a replication origin for E. coli but not B. subtilis , and therefore can only persist in B. subtilis by integration into the amyE locus via front-end and back-end fragments of this gene. The intervening sequences include a spectinomycin resistance genes and a copy of lacI encoding the Lac repressor. Finally, downstream of a strong P hyper-spank promoter is a multiple cloning site. The respective tetM and catQ genes were PCR amplified from the B. toyonensis strains with Sal I and Sph I sites using the following primers: (i) for the tetM genes of strains BCT-7112 T and Rock1-3 strains, tetMFwd1 (5′-GGCCGGGTCGACTGAGCATGAACTTGTCAAACTACCC-3′) and tetMRev1 (5′- GGCCGGGCATGCGTAATAGAAACACTTAAAGAAGTTGTTAGG-3′) and strain Rock3-28, tetMFwd2 (5′- GGCCGGGTCGACTGAGCATGAACTTGTCAAATTACCC-3′) and tetMRev2 (5′- GGCCGG-GCATGC-GCGCCCTCTAGTAAGGAAAAGTATC-3′); (ii) for the catQ genes of strains BCT-7112 T , Rock1-3, and Rock3-28, catQFwd1 (5′- GGCCGGGTCGACTATTAATAGTATGTGGATGGATTGC-3′) and catQRev1 (5′- GGCCGGGCATGCCAATTTTATATAATTAAAAATTTGATATTACTTAAAGCC-3′). Following ligation and transformation initially into E. coli strain DH5α, the structures of the recombinant plasmids (pDRtetM7112, pDRtetM1-3, pDRtetM3-28, pDRcatQ7112, pDRcatQ1-3, and pDRcatQ3-28, see Table 1 ) were confirmed by digestion and then transformed into B. subtilis 168 by natural transformation (Anagnostopoulos and Spizizen, 1961 ). The resulting strains were: for the tetM constructs, B. subtilis strains 168tetM7112, 168tetM1-3, and 168tetM3-28; and for the catQ constructs, B. subtilis strains 168catQ7112, 168catQ1-3, and 168catQ3-28 (Table 1 ). The growth parameters of B. subtilis with integrated copies of the tetM and catQ genes from B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 were determined in the presence of various concentrations of tetracycline and chloramphenicol. Antibiotic solutions (0, 4, 8, 16, and 32 μg/ml) were prepared in Brain Heart Infusion broth, prewarmed to 37°C and distributed to in 800 μl amounts into a 48-well FlowerPlate® (m2pLabs, Baesweiler, Germany). The individual wells were inoculated to an OD 600 of 0.05 with the required cultures (168tetM7112, 168tetM1-3, 168tetM3-28, 168catQ7112, 168catQ1-3, and 168catQ3-28), previously grown overnight in Brain Heart Infusion broth without antibiotics. The experiment was duplicated with one half of the cultures containing IPTG (1 mM) to induced expression from the upstream P hyper-spanc promoter and the other half relying on their cognate upstream regulatory regions. The cultures were incubated in a BioLector® micro bioreactor (m2pLabs) for 20 h at 37°C, with a gain of 25 and shaking at 1500 rpm. The BioLector microreactor determines the optical density of the culture by measuring the scattered light signal using an excitation light beam with a wavelength of 620 nm. The data is shown on a linear rather than logarithmic graph to make it easier to compare growth profiles. Visualization of cells by light microscopy Overnight cultures in Müller Hinton Broth (MHB) were used to inoculate fresh broth to an OD 600 of 0.05. The cells were grown at 37°C with shaking until the OD 600 reached 0.2 when chloramphenicol (2 or 32 μg/ml) was added. Samples were taken every 60 min for visualization by phase contrast microscopy. Relative quantification of tetM and catQ genes expression The expression of the tetM and catQ genes was determined by a combination of Northern Blotting and quantitative (q) PCR, the former used to establish the transcript sizes the latter for quantitation. For Northern blotting the Total RNA purification Kit (Norgen Biotek Corp) was used for the extraction of total RNA. The RNA integrity was determined by ensuring the A 260/280 ratio was between 1.8 and 2.0 and by electrophoresis on a denaturing 1.2% agarose-formaldehyde gel. Northern Blot analysis was performed as described by Homuth et al. ( 1997 ). Digoxigenin (DIG) labeled catQ - and tetM -specific RNA probes were synthesized by in vitro transcription using T7 RNA polymerase and a DIG RNA Labelling Kit (Roche Diagnostics) from specific PCR products as templates. Primer sequences used to amplify the templates for the RNA probes were as follows: catQ-FWD: 5′-GTTGATATTACGATGATGCTAGAGAAAATAC-3′, catQ-REV: 5′-CTAATACGACTCACTATAGGGAGACTTTCGGGAAGAGACCATGAAC-3′; tetM-FWD: 5′-ATGAAACGAATGTGATTAAAGAAATTGGC-3′, tetM-REV: 5′-CTAATACGACTCACTATAGGGAGAGGTGCTACTCGTTCCATACAATC-3′. The hybridization signal was developed using a digoxigenin-specific antibody conjugated to alkaline phosphatase (Roche Diagnostics) and CDP-Star (Thermo Fisher Scientific) as a chemiluminescence substrate. The images were captured on an ImageQuant LAS 4000 (GE Healthcare Life Sciences). In the case of qPCR, the unlabeled PCR primers and TaqMan® MGB probes (5′-FAM™ dye labeled) were designed using the Assays-by-Design™ service (Applied Biosystems). The primers and probes used were as follows: for the tetM gene, TETP_Fwd (5′-CGGAAATGCAGTTCAAGCTTCTAC-3′), TETP_Rev (5′-AATTTTAATATCACTCAGTCCCCACACTT-3′), and TETP_probe (5′-CTAGCGGAGAATTTT-3); for the catQ gene, CAT._Fwd (5′-CTAATGTTCATGGTCTCTTCCCGAAA-3′), CAT._Rev 5′-CTAGTCCAAGGTATCCCAGAAATCG-3′), and CAT_probe (5′-TTGGCGGAATATTTTC-3′); for the gyrB gene, GYRB_Fwd (5′-CAGCAATCGTGTCAATTAAACATCCAA-3′), GYRB_Rev (5′-CCGTAATCGTTCTCGCTTCACTATT-3′), and GYRB_probe (5′-ACGAAGACGAAACTTG-3′). The RNAprotect Bacteria Reagent (QIAGEN) was used to stabilize RNA by preventing the degradation of RNA transcripts and the induction of genes. The bacterial strains were cultured in Luria Bertani (LB) medium at 37°C. Depending on the strain and species, the incubation time required to reach exponential phase was 2–3 h before harvesting the cells. RNA extraction was performed using the RNeasy Protect Bacteria MiniKit (QIAGEN), according to the manufacturer's instructions. The expression of the housekeeping gene gyrB was used as baseline control for relative quantification. The quality and concentration of RNA was measured using a Nanodrop 1000 Spectrophotometer, as recommended in the high capacity cDNA reverse transcription kit (Applied Biosystems), and adjusted at 50 ng/μl for relative quantification and PCR normalization. The quantitative PCR was performed using the qPCR MasterMix No ROX (Eurogenetec) and the Custom TaqMan Gene Expression Assays system (Applied Biosystems). The calculation of the relative expression ratios of the target genes was based on the mathematical model of Pfaffl ( 2001 ). Bioinformatical analyses BLASTp was used to identify homologs of the B. toyonensis 7112 T CatQ and TetM proteins among sequences in the NCBI genome database (Altschul et al., 1990 ). The data was downloaded in CVS format and uploaded into Excel (Tables S1 , S2 ). The data shows the Reference Sequence IDs, individual Sequence IDs, the description of the protein in the annotation file, the bacterial source, alignment score, E -value and % identity. The BLASTp data was also used to construct Neighbor Joining trees of homologs of the B. toyonensis BCT-7112 T CatQ and TetM proteins in the NCBI genome database. The algorithm used (Saitou and Nei, 1987 ) produces an un-rooted tree. The maximum allowed fraction of mismatched bases between any pair of sequences was 0.85. The evolutionary distance between two sequences was modeled as the expected fraction of amino acid substitutions per site, based on the fraction of mismatched amino acids in the aligned region (Grishin, 1995 ). Strains and culture conditions The strains and plasmids used in this study, together with their sources, are shown in Table 1 . When required, the following antibiotic concentrations were used: for E. coli , 100 μg/ml ampicillin and 12.5 μg/ml chloramphenicol; for Bacillus strains, 1 μg/ml erythromycin, 100 μg/ml spectinomycin, and for chloramphenicol and tetracycline concentrations ranged from 1 to 32 μg/ml, as indicated in the text. Table 1 Strains and plasmids used in this study . Strains Trait or relevant genotype * Source/Reference * Bacillus cereus ATCC 14579 T ATCC Bacillus thuringiensis ATCC 10792 ATCC Bacillus toyonensis BCT-7112 T B. toyonensis type strain, Tc R , Cm R Rubinum S.A, Spain BCT-7112Δtet BCT-7112 T with the tetM gene replaced with a spectinomycin resistance gene, Cm R , Sp R , Tc S This study BCT-7112Δcat BCT-7112 T with the tetM gene replaced with a spectinomycin resistance gene, Tc R , Sp R , Cm S This study Rock1-3 B. toyonensis strain isolated from soil in Rockville, Maryland. Previously B. cereus Rock1-3 US Naval Medical Research Center Zwick et al., 2012 Rock3-28 B. toyonensis strain isolated from soil in Rockville, Maryland. Previously B. cereus Rock3-28 US Naval Medical Research Center; Zwick et al., 2012 BCT-7112(pIGR1) BCT-7112 T with pIGR, Tc R , Cm R , Em R This study BCT-7112Δtet(pIGR1) BCT-7112Δtet with pIGR, Cm R , Sp R , Em R , Tc S This study BCT-7112Δcat(pIGR1) BCT-7112Δcat with pIGR, Tc R , Sp R , Em R , Cm S This study Rock1-3(pIGR) Rock1-3 with pIGR, Em R This study Bacillus subtilis 168 trpC2 Institute Pasteur, Paris; Anagnostopoulos and Spizizen, 1961 168tetM7112 168 with tetM from BCT-7112 T , Tc R This study 168tetM1-3 168 with tetM from Rock1-3, Tc R This study 168tetM3-28 168 with tetM from Rock3-28, Tc R This study 168catQ7112 168 with catQ from BCT-7112 T , Cm R This study 168catQ1-3 168 with catQ from Rock1-3, Cm R This study 168catQ3-28 168 with catQ from Rock3-28, Cm R This study Escherichia coli DH5α huA2 lac(del)U169 phoA glnV44 Φ80′ lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR Thermo Fisher Scientific GM48 dam − , dcm − Stratagene Plasmids pUTE583 Cm R in E. coli ; Em R and Ts in B. toyonensis Theresa M. Koehler, The University of Texas: Chen et al., 2004 pUTEΔtetM pUTE583 with tetM flanking regions and Sp R cassette This study pUTEΔcatQ pUTE583 with catQ flanking regions and Sp R cassette This study pING1 pUTE583 with the gerIC–nucB intergenic region of plasmid pBCT77 This study pDR111 B. subtilis integration/expression vector, Ap R ( E. coli ), Sp R ( B. subtilis ), amyE Δ, lacI , P hyper-spank R. Daniel, Newcastle University pDRtetM7112 pDR111 with tetM from BCT-7112 T , Ap R , Sp R , Tc R This study pDRtetM1-3 pDR111 with tetM from Rock1-3, Ap R , Sp R , Tc R This study pDRtetM3-28 pDR111 with tetM from Rock3-28, Ap R , Sp R , Tc R This study pDRcatQ7112 pDR111 with catQ from BCT-7112 T , Ap R , Sp R , Tc R This study pDRcatQ1-3 pDR111 with catQ from Rock1-3, Ap R , Sp R , Tc R This study pDRcatQ3-28 pDR111 with catQ from Rock3-28, Ap R , Sp R , Tc R This study * ATCC, American Type Culture Collection; Ap R , ampicillin resistance; Cm R , chloramphenicol resistance; Em R , erythromycin resistance; Tc R , tetracycline resistance; Sp R , spectinomycin resistance . Minimum inhibitory concentration (MIC) experiments were carried out in accordance with the European Food Safety Authority's (EFSA) "Guidance on the assessment of bacterial antimicrobial susceptibility" (EFSA-FEEDAP, 2012 ) using the microdilution method. Solutions of the required antibiotics (0, 1, 2, 4, 8, 16, and 32 μg/ml) were made up in Brain Heart Infusion broth and 200 μl added to wells in a 96-well microtitre plate (MTP). The wells were inoculated with 20 μl of the test cultures, prepared from fresh colonies grown on Brain Heart Infusion agar. The MTP was incubated in a FLUOstar Optima plate reader for 18 h at 37°C with vigorous shaking, monitoring Optical Density (OD) at 600 nm. The experiments were carried out a minimum of three times and the representative data show on a linear rather than logarithmic graph to make it easier to compare growth profiles. The MIC was determined as the lowest concentration of the antibiotic for which no growth was detected. Construction of tetM and catQ knockout strains In order to identify the genes responsible for tetracycline and chloramphenicol resistance in the genome of B. toyonensis BCT-7112 T , a gene replacement strategy was employed. This involved the replacement of the target gene with a spectinomycin gene by a reciprocal recombination event. Briefly, two DNA knockout cassettes of ~3 kb were synthesized (DNA 2.0, Newark, California) and cloned into the vector pUTE583 (Koehler et al., 1994 ) to generate pUTEΔtetM and pUTEΔcatQ. Each synthesized fragment consisted of three components, a ~1000-bp region upstream of the target gene, a 947-bp spectinomycin resistance fragment and a ~1000-bp regions downstream of the target cassette. In the case of the tetM knockout cassette, a 1102-bp upstream fragment of the B. toyonensis BCT-7112 T chromosome (nucleotides 305983–307085) was fused to the 947-bp spectinomycin resistance fragment, which in turn was fused to the 907-bp downstream fragment of the B. toyonensis BCT-7112 T chromosome (nucleotides 309075–309983; Figure 1A ). The catQ knockout cassette was similarly constructed with a 1069-bp upstream fragment (nucleotides 4932601–4933670) and 1219 downstream fragment (nucleotides 4933976–4935195) of the B. toyonensis BCT-7112 T chromosome, separated by the 947-bp spectinomycin resistance fragment (Figure 1B ). Figure 1 Regions of the B. toyonensis BCT-7112 T genome containing (A) tetracycline (Btoyo_0322, tetM ) and (B) chloramphenicol (Btoyo_4985, catQ ) resistance genes. The lower images show the locations (red arrows) of the upstream and downstream fragments and the spectinomycin resistance ( spc R ) gene that were used to replace the Btoyo_0322 or Btoyo_4985 genes by reciprocal recombination events, generating the tetM or catQ knockout strains BCT-7112Δtet and BCT-7112Δcat. The green arrows show the approximate locations of the primers used to sequence over the knockout regions. The bifunctional vector, pUTE853, is able to replicate in E. coli and members of the B. cereus group, but is lost from the latter at 37°C in the absence of antibiotic selection. The knockout plasmids, pUTEΔtetM and pUTEΔcatQ, were first passaged through a dam- minus E. coli strain (GM48) and then electro-transformed into B. toyonensis BCT-7112 T , selecting for erythromycin resistance at 30°C (Koehler et al., 1994 ). The transformants were grown in the presence of erythromycin for 2 days, diluting 1:500 at the start and end of the day with fresh medium, and then shifted to 37°C for 2 days in the presence of spectinomycin, again diluting 1:500 at the start and end of the day with fresh medium. Samples were plated onto BHI agar containing spectinomycin, incubated overnight at 37°C, and individual colonies checked for erythromycin sensitivity and spectinomycin resistance, confirming the loss of the plasmid and integration of the knockout cassette into the chromosome. In each case the target antibiotic resistance phenotype was lost, tetracycline resistance in the case of pUTEΔtetM and chloramphenicol resistance in the case of pUTEΔcatQ, generating B. toyonensis knockout strains BCT-7112Δtet and BCT-7112Δcat, respectively. The fidelity of the integration event was confirmed by sequencing over the region using primers within the flanking fragments and spectinomycin fragment (Figure 1 ). Construction of strains with additional copies of the gerIC–nucB intergenic region Previous unpublished studies indicated an involvement of the gerIC–nucB intergenic region of plasmid pBCT77 as contributing to, or enhancing, the observed tetracycline and chloramphenicol resistances in B. toyonensis BCT-7112 T . The 2003-bp gerIC–nucB intergenic region extends from nucleotide 75752–76974 on the pBCT77 genome (NC_022782). In the first place we used primers 1pBCT77Fwd (5′-GCCCCTTTAAACTTCATTATGC-3′) and 4pBCT77Rev (5′-CCCTTACCTTCAAGTATTCC-3′) to confirm that pBCT77 was still present in both knockout strains. The intergenic DNA was then synthesized and cloned into the Hin dIII and Xba I sites of pUTE853 (DNA 2.0, Newark, California). The resulting vector, pIGR1, was transformed into E. coli strain DH5α and its structure confirmed by restriction analysis and PCR. Following passage through the E. coli dam- minus strain GM48, pIGR1 was electro-transformed into B. toyonensis strains BCT-7112 T , BCT-7112Δtet, BCT-7112Δcat, and Rock1-3, selecting for erythromycin resistant transformants at 37°C. Erythromycin selection was maintained in all pre-cultures, but not during MIC experiments. Construction B. subtilis strains with tetM and catQ genes from BCT-7112 T , Rock1-3, and Rock3-28 For expression in B. subtilis , the tetM , and catQ genes from BCT-7112 T , Rock1-3, and Rock3-28 were first cloned into the bifunctional expression/integration vector pDR111 (Britton et al., 2002 ). This vector has a replication origin for E. coli but not B. subtilis , and therefore can only persist in B. subtilis by integration into the amyE locus via front-end and back-end fragments of this gene. The intervening sequences include a spectinomycin resistance genes and a copy of lacI encoding the Lac repressor. Finally, downstream of a strong P hyper-spank promoter is a multiple cloning site. The respective tetM and catQ genes were PCR amplified from the B. toyonensis strains with Sal I and Sph I sites using the following primers: (i) for the tetM genes of strains BCT-7112 T and Rock1-3 strains, tetMFwd1 (5′-GGCCGGGTCGACTGAGCATGAACTTGTCAAACTACCC-3′) and tetMRev1 (5′- GGCCGGGCATGCGTAATAGAAACACTTAAAGAAGTTGTTAGG-3′) and strain Rock3-28, tetMFwd2 (5′- GGCCGGGTCGACTGAGCATGAACTTGTCAAATTACCC-3′) and tetMRev2 (5′- GGCCGG-GCATGC-GCGCCCTCTAGTAAGGAAAAGTATC-3′); (ii) for the catQ genes of strains BCT-7112 T , Rock1-3, and Rock3-28, catQFwd1 (5′- GGCCGGGTCGACTATTAATAGTATGTGGATGGATTGC-3′) and catQRev1 (5′- GGCCGGGCATGCCAATTTTATATAATTAAAAATTTGATATTACTTAAAGCC-3′). Following ligation and transformation initially into E. coli strain DH5α, the structures of the recombinant plasmids (pDRtetM7112, pDRtetM1-3, pDRtetM3-28, pDRcatQ7112, pDRcatQ1-3, and pDRcatQ3-28, see Table 1 ) were confirmed by digestion and then transformed into B. subtilis 168 by natural transformation (Anagnostopoulos and Spizizen, 1961 ). The resulting strains were: for the tetM constructs, B. subtilis strains 168tetM7112, 168tetM1-3, and 168tetM3-28; and for the catQ constructs, B. subtilis strains 168catQ7112, 168catQ1-3, and 168catQ3-28 (Table 1 ). The growth parameters of B. subtilis with integrated copies of the tetM and catQ genes from B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 were determined in the presence of various concentrations of tetracycline and chloramphenicol. Antibiotic solutions (0, 4, 8, 16, and 32 μg/ml) were prepared in Brain Heart Infusion broth, prewarmed to 37°C and distributed to in 800 μl amounts into a 48-well FlowerPlate® (m2pLabs, Baesweiler, Germany). The individual wells were inoculated to an OD 600 of 0.05 with the required cultures (168tetM7112, 168tetM1-3, 168tetM3-28, 168catQ7112, 168catQ1-3, and 168catQ3-28), previously grown overnight in Brain Heart Infusion broth without antibiotics. The experiment was duplicated with one half of the cultures containing IPTG (1 mM) to induced expression from the upstream P hyper-spanc promoter and the other half relying on their cognate upstream regulatory regions. The cultures were incubated in a BioLector® micro bioreactor (m2pLabs) for 20 h at 37°C, with a gain of 25 and shaking at 1500 rpm. The BioLector microreactor determines the optical density of the culture by measuring the scattered light signal using an excitation light beam with a wavelength of 620 nm. The data is shown on a linear rather than logarithmic graph to make it easier to compare growth profiles. Visualization of cells by light microscopy Overnight cultures in Müller Hinton Broth (MHB) were used to inoculate fresh broth to an OD 600 of 0.05. The cells were grown at 37°C with shaking until the OD 600 reached 0.2 when chloramphenicol (2 or 32 μg/ml) was added. Samples were taken every 60 min for visualization by phase contrast microscopy. Relative quantification of tetM and catQ genes expression The expression of the tetM and catQ genes was determined by a combination of Northern Blotting and quantitative (q) PCR, the former used to establish the transcript sizes the latter for quantitation. For Northern blotting the Total RNA purification Kit (Norgen Biotek Corp) was used for the extraction of total RNA. The RNA integrity was determined by ensuring the A 260/280 ratio was between 1.8 and 2.0 and by electrophoresis on a denaturing 1.2% agarose-formaldehyde gel. Northern Blot analysis was performed as described by Homuth et al. ( 1997 ). Digoxigenin (DIG) labeled catQ - and tetM -specific RNA probes were synthesized by in vitro transcription using T7 RNA polymerase and a DIG RNA Labelling Kit (Roche Diagnostics) from specific PCR products as templates. Primer sequences used to amplify the templates for the RNA probes were as follows: catQ-FWD: 5′-GTTGATATTACGATGATGCTAGAGAAAATAC-3′, catQ-REV: 5′-CTAATACGACTCACTATAGGGAGACTTTCGGGAAGAGACCATGAAC-3′; tetM-FWD: 5′-ATGAAACGAATGTGATTAAAGAAATTGGC-3′, tetM-REV: 5′-CTAATACGACTCACTATAGGGAGAGGTGCTACTCGTTCCATACAATC-3′. The hybridization signal was developed using a digoxigenin-specific antibody conjugated to alkaline phosphatase (Roche Diagnostics) and CDP-Star (Thermo Fisher Scientific) as a chemiluminescence substrate. The images were captured on an ImageQuant LAS 4000 (GE Healthcare Life Sciences). In the case of qPCR, the unlabeled PCR primers and TaqMan® MGB probes (5′-FAM™ dye labeled) were designed using the Assays-by-Design™ service (Applied Biosystems). The primers and probes used were as follows: for the tetM gene, TETP_Fwd (5′-CGGAAATGCAGTTCAAGCTTCTAC-3′), TETP_Rev (5′-AATTTTAATATCACTCAGTCCCCACACTT-3′), and TETP_probe (5′-CTAGCGGAGAATTTT-3); for the catQ gene, CAT._Fwd (5′-CTAATGTTCATGGTCTCTTCCCGAAA-3′), CAT._Rev 5′-CTAGTCCAAGGTATCCCAGAAATCG-3′), and CAT_probe (5′-TTGGCGGAATATTTTC-3′); for the gyrB gene, GYRB_Fwd (5′-CAGCAATCGTGTCAATTAAACATCCAA-3′), GYRB_Rev (5′-CCGTAATCGTTCTCGCTTCACTATT-3′), and GYRB_probe (5′-ACGAAGACGAAACTTG-3′). The RNAprotect Bacteria Reagent (QIAGEN) was used to stabilize RNA by preventing the degradation of RNA transcripts and the induction of genes. The bacterial strains were cultured in Luria Bertani (LB) medium at 37°C. Depending on the strain and species, the incubation time required to reach exponential phase was 2–3 h before harvesting the cells. RNA extraction was performed using the RNeasy Protect Bacteria MiniKit (QIAGEN), according to the manufacturer's instructions. The expression of the housekeeping gene gyrB was used as baseline control for relative quantification. The quality and concentration of RNA was measured using a Nanodrop 1000 Spectrophotometer, as recommended in the high capacity cDNA reverse transcription kit (Applied Biosystems), and adjusted at 50 ng/μl for relative quantification and PCR normalization. The quantitative PCR was performed using the qPCR MasterMix No ROX (Eurogenetec) and the Custom TaqMan Gene Expression Assays system (Applied Biosystems). The calculation of the relative expression ratios of the target genes was based on the mathematical model of Pfaffl ( 2001 ). Bioinformatical analyses BLASTp was used to identify homologs of the B. toyonensis 7112 T CatQ and TetM proteins among sequences in the NCBI genome database (Altschul et al., 1990 ). The data was downloaded in CVS format and uploaded into Excel (Tables S1 , S2 ). The data shows the Reference Sequence IDs, individual Sequence IDs, the description of the protein in the annotation file, the bacterial source, alignment score, E -value and % identity. The BLASTp data was also used to construct Neighbor Joining trees of homologs of the B. toyonensis BCT-7112 T CatQ and TetM proteins in the NCBI genome database. The algorithm used (Saitou and Nei, 1987 ) produces an un-rooted tree. The maximum allowed fraction of mismatched bases between any pair of sequences was 0.85. The evolutionary distance between two sequences was modeled as the expected fraction of amino acid substitutions per site, based on the fraction of mismatched amino acids in the aligned region (Grishin, 1995 ). Results Characterization of resistance phenotypes and the identification of resistance genes Minimum inhibitory concentrations (MICs) of B. toyonensis BCT-7112 T for tetracycline and chloramphenicol were, at 16 and 32 μg/ml respectively, higher than the resistance break points (≤8 μg/ml) identified by the European Committee on Antimicrobial Susceptibility Testing (Kahlmeter et al., 2006 ). Consequently B. toyonensis BCT-7112 T is designated as being resistant to both of these antibiotics. Analysis of the annotated B. toyonensis BCT-7112 T genome sequence (NCBI; CP006863), and plasmids pBCT77 (CP006864) and pBCT8 (CP006865), revealed the presence of genes encoding putative tetracycline and chloramphenicol resistance proteins. The most common tetracycline resistance mechanisms are efflux pumps and ribosome-protection proteins (Wilson, 2014 ). The B. toyonensis BCT-7112 T genome encodes two putative tetracycline resistance proteins, one an efflux protein and the other a putative ribosome-protection protein. Btoyo_4389 (nucleotides 4295949–4297223) is annotated as encoding a tetracycline resistance protein of the Major Facilitator Superfamily (MFS, cd06174). The MFS is a large and diverse group of secondary transporters that includes uniporters, symporters, and antiporters. MFS proteins facilitate transport across the cytoplasmic membrane of various substrates, including ions, sugar phosphates, antibiotics, amino acids, and peptides. MFS transporters are common among soil-living organisms and, without specific experimental evidence, it can be difficult to identity their substrates. BLAST analysis revealed that homologs of Btoyo_4389 are found extensively among members of the B. cereus group but are labeled, in decreasing order of frequency, "MFS transporter," "tetracycline resistance protein," major facilitator protein," and "putative multidrug resistance protein." It is not clear what, if any, evidence there is for annotating this protein specifically as a "tetracycline resistance protein" rather than as a "MFS transporter," illustrating a limitation of automated annotation programs. Btoyo_0322 (nucleotides 307023–308966) is a putative ribosome protection protein with a conserved TetM-like family domain (cd04168). TetM proteins exhibit ~45% similarity with elongation factor G (EF-G) proteins. They have conserved nucleotide-binding motifs and are members of the translation factor superfamily of GTPases that bring about the release of tetracycline from the ribosome in a GTP-dependent manner (Dönhöfer et al., 2012 ). Several genes are annotated as encoding a TetR-like protein. TetR proteins are a class of transcriptional regulators with helix-turn-helix motifs, and are so annotated because the first representative of this class was responsible for the regulation of a MFS transporter mediating tetracycline resistance on transposon Tn 10 (Beck et al., 1982 ). The association with tetracycline is therefore historic. Analysis of the B. toyonensis BCT-7112 T genome reveals the presence of two genes encoding putative chloramphenicol acetyltransferases (Cat) that inactivate chloramphenicol by acetylation. Btoyo_3723 (nucleotides 3655712–3656095), at 127 residues in length, is significantly shorter than other chloramphenicol acetyltransferases (~220 residues) and is likely to have been truncated during evolution. Although, the active site is present, it is unlikely that this gene encodes an active enzyme. In contrast, Btoyo_4985 (nucleotides 4933576–4934223) encodes a full-length CatQ-like chloramphenicol acetyltransferase. A previous unpublished transposon mutagenesis study (EFSA-FEEDAP, 2014 ; M Kato, Science Tanaka, Japan, personal communication) identified a region on pBCT77, between nucB (Btoyo_5072 [nucleotides 74482–74916]) and gerIC (Btoyo_4992 [nucleotides 250–858]) that had a minor influence on reducing the MICs of tetracycline and chloramphenicol in B. toyonensis BCT-7112 T . Two of the three genes in this region encode proteins of unknown function (Btoyo_5073 [nucleotides 74939–75190]; Btoyo_5074 [nucleotides 75202–75432]), while the third gene (Btoyo_5075 [nucleotides 75457–76029]) encodes a phage-type site-specific recombinase. There were no genes encoding known antibiotic resistance genes in this region, elsewhere on this pBCT77 or on the smaller plasmid, pBCT8. Identification of the genes encoding tetracycline and chloramphenicol resistances Btoyo_0322 and Btoyo_4389 were tentatively identified as encoding tetracycline resistance proteins, the former a TetM-like protein, the later a MFS-like transporter. Because of limitations in the identification of MFS substrates and the higher likelihood that Btoyo_4389 was incorrectly annotated, we focused on Btoyo_0322. The tetM gene encoded by Btoyo_0322 was replaced with a spectinomycin resistance gene (Section Construction of tetM and catQ Knockout Strains) and the resulting knockout strain (BCT-7112Δtet) was spectinomycin and chloramphenicol resistant, but erythromycin and tetracycline susceptible. The authenticity of the integration event was confirmed by sequencing across the entire region (see Figure 1A ). The data confirmed that Btoyo_0322 was excised from the chromosome and that this gene was solely responsible for the observed tetracycline resistance phenotype of B. toyonensis strain BCT-7112 T . A similar approach was used to identify the gene responsible for chloramphenicol resistance. Of the two putative chloramphenicol resistance genes identified in the genome of BCT-7112 T , Btoyo_3723, and Btoyo_4985, the later was chosen as the former appeared to have been truncated. The gene replacement strategy described in Section Construction of tetM and catQ Knockout Strains was employed, involving the replacement of Btoyo_4985 with a spectinomycin resistance gene. The resulting knockout strain (BCT-7112Δcat), was spectinomycin and tetracycline resistant, but erythromycin and chloramphenicol susceptible. The authenticity of the integration event was again confirmed by sequencing across the entire region (see Figure 1B ). The data confirmed that Btoyo_4985 was solely responsible for the observed chloramphenicol resistance phenotype of B. toyonensis strain BCT-7112 T . Following their identification, the %GC contents of Btoyo_0332 and Btoyo_4985 were determined to be 37.3 and 29.8%, respectively, while the genome average of B. toyonensis BCT-7112 T was 35.6%. Using a 1000-bp window and a standard deviation cut-off value of 2.5, neither the %GC of these coding sequences, nor a 3500-bp regions either side, were found to be significantly different from the running average, indicating that Btoyo_0332 and Btoyo_4985 are not recent acquisitions. The presence of homologs of tetM (Btoyo_322) and catQ (Btoyo_4985) genes in other member of the B. cereus sensu lato group In light of the recent studies revising the taxonomic status of the B. cereus group ( sensu lato ) (Jiménez et al., 2013 ; Böhm et al., 2015 ; Liu et al., 2015 ), we used BLASTp (Altschul et al., 1990 ) to search for homologs of the BCT-7112 T tetM and catQ genes in the other 18 strains within the B. toyonensis taxonomic unit. The data (Table 2 ) clearly show that homologs of TetM are present in all strains of B. toyonensis while homologs of CatQ are present in all but one strain. The failure to detect CatQ in B. toyonensis VD115 is most likely to be due to the absence of the relevant region in the partial genome sequence, which is 3884 k in length compare with 4940 k for B. toyonensis BCT-7112 T . Table 2 The percentage identity and similarity of proteins encoded by homologs of the tetM and catQ genes of B. toyonensis BCT-7112 T among other members of the B. toyonensis taxonomic unit . Strain GenBank Accession N o % Identity/Similarity to B. toyonensis BCT-7112 TetM % Identity/Similarity to B. toyonensis BCT-7112 CatQ B. toyonensis BCT-7112 T CP006863.1 100/100 100/100 B. toyonensis VD115 * JH792165.1 97/98 TS *** B. toyonensis MC28 * CP003687.1 94/97 100/100 B. toyonensis Rock3-28 * CM000730.1 93/96 100/100 B. toyonensis VD131 ** KB976660.1 93/96 100/100 B. toyonensis HuB5-5 ** JH792120.1 100/100 100/100 B. toyonensis VD148 * JH792156.1 90/95 100/100 B. toyonensis BAG2O-2 ** AHCX01000000.1 93/96 100/100 B. toyonensis Rock1-3 * CM000728.1 .1 100/100 100/100 B. toyonensis BAG1O-2 * AHCO00000000.1 100/100 100/100 B. toyonensis HuB4-10 ** AHEE00000000.1 100/100 100/100 B. toyonensis BAG5O-1 * AHDI00000000.1 99/99 100/100 B. toyonensis HuA2-3 ** AHDW00000000.1 93/97 100/100 B. toyonensis BAG6O-1 * JH804627.1 99/100 100/100 B. toyonensis BAG4X2-1 * JH804617.1 100/100 99/99 B. toyonensis VD214 ** AHFN00000000.1 100/100 99/99 B. toyonensis HuB2-9 ** AHED00000000.1 100/100 99/99 B. toyonensis Rock3-29 * CM000731.1 100/100 99/99 B. toyonensis Rock4-18 ** CM000735.1 .1 93/96 99/99 * Clustered to the B. toyonensis group by Jiménez et al. ( 2013 ); ** Clustered to the B. toyonensis group by Liu et al. ( 2015 ); *** TS, truncated sequence detected . In some cases the NCBI Reference for master record for a whole genome shotgun sequence is given . In the case of TetM, just under half of the B. toyonensis strains encode proteins with identical sequences (Table 2 ). The most variable TetM protein is encoded by B. toyonensis VD148, which shows 90% identity and 95% similarity with that of strain BCT-7112 T . Twelve of the B. toyonensis strains encode CatQ proteins that are identical to that of strain BCT-7112 T , while the remaining strains exhibit CatQ proteins with 99% identity. Moreover, the tetM and catQ genes are located in the same genomic neighborhoods in all of the B. toyonensis strains. Taken together, the data indicate that the tetM and catQ genes are ancient genomic components of the B. toyonensis taxonomic unit rather than having been acquired recently by horizontal gene transfer. The NCBI genomes database contains whole genomes, chromosomes, scaffolds and contiguous fragments (contigs) of B. anthracis, B. bombysepticus, B. cereus sensu stricto, B. mycoides, B. thuringiensis, B. toyonensis, B. weihenstephanensis , and B. wiedmannii (Table 3 ). In order to extend the homology study to other members of the B. cereus sensu lacto , BLASTp was used to search for homologs of the B. toyonensis BCT-7112 T TetM and CatQ proteins with a similarity score of 85% or greater (Table 4 ). All of the strains identified as having homologs of these TetM and CatQ proteins were members of the B. cereus group (sensu lato): no strains outside this group appeared in the analysis. Homologs of both the TetM and CatQ were found in all eight members of the B. cereus group shown in Table 3 . Genes encoding these proteins were absent from B. cytotoxicus and B. pseudomycoides , although this could be due either to the small number of DNA sequences available for these species or to changes in their taxonomic status. A list of sequences analyzed for homology with the TetM and CatQ proteins is given in Tables S1 , S2 , respectively. Table 3 Breakdown of the genomic sequences analyzed to identify homologs of the B. toyonensis BCT-7112 T TetM and CatQ proteins . Taxonomic group/ID Complete Chromosome Scaffold Contig Total B. anthracis /1392 44 10 41 42 137 B. bombysepticus /1330043 1 0 0 0 1 B. cereus (sensu stricto) /1396 32 29 154 51 266 B. mycoides /1405 2 3 3 5 13 B. thuringiensis /1428 37 16 14 12 79 B. toyonensis /155322 1 0 0 0 1 B. weihenstephanensis /86662 2 0 0 4 6 B. wiedmannii /1890302 0 0 0 11 11 Table 4 The distribution of proteins encoded by homologs (>85% similarity) of the tetM and catQ genes of B. toyonensis BCT-7112 T among other members of the B. cereus sensu lato group . Gene B. thuringiensis B. cereus (sensu stricto) B. anthracis B. mycoides B. weihen-stephanensis B. toyonensis B. wiedmannii B. bombysepticus Total CatQ 66 161 111 7 6 18 11 1 381 TetM 77 135 112 4 10 19 11 1 369 A total of 369 homologs of TetM were identified in the NBCI database and widely distributed among species of the B. cereus group, broadly reflecting the frequency of complete genomes and chromosomes in the database (Table 3 ). In the case of CatQ, 381 homologs were found, and with a distribution that was similar to that of TetM. The relationships between these TetM and CatQ sequences, in the form of Neighbor Joining trees, are shown in Figures S1 , S2 . In both cases the trees exclusively contain homologous proteins from other members of the B. cereus group ( sensu lato ), confirming the ancient origin of these proteins in this taxonomic clade. Tetracycline and chloramphenicol resistance profiles of the strains within the B. toyonensis taxonomic unit B. toyonensis BCT-7112 T is phenotypically resistant to tetracycline and chloramphenicol, and this study identified the genes responsible, namely tetM (Btoyo_0322) and catQ (Btoyo_4985). However, eight other B. toyonensis strains are phenotypically susceptible to tetracycline and chloramphenicol, despite encoding TetM and CatQ proteins that are homologous to those strain BCT-7112 T (EFSA-FEEDAP, 2014 ; L. Mocé, Eurofins Biolab, personal communication). We therefore addressed the question of why other members of the B. toyonensis taxonomic unit that encode either identical (strain Rock1-3) or similar (strain Rock3-28) resistance determinants do not exhibit the same resistance phenotypes. Both strains are susceptible to tetracycline and chloramphenicol (MIC 1 μg/ml) and have similar growth kinetics and therefore only the growth profiles of strain Rock1-3 are shown in Figure S3 , in comparison with the profiles of strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat. When B. toyonensis BCT-7112 T was grown in the presence of various concentrations of tetracycline (0, 1, 2, 4, 16, and 32 μg/ml) the MIC was 16 μg/ml (Figure S3A ). When the isogenic strain (BCT-7112Δtet) in which the tetM (Btoyo_0322) gene had been replaced with a spectinomycin resistance gene was tested, the MIC was 1 μg/ml (Figure S3B ). In contrast, when the isogenic strain (BCT-7112Δcat) in which the catQ gene was replaced with a spectinomycin resistance gene, the MIC for tetracycline remained at 16 μg/ml (Figure S3C ). In the case of the Rock1-3 strain, there was some initial growth at 1 μg/ml (Figure S3D ), but the cells eventually died and the biomass concentration declined, indicating it had an MIC for tetracycline of 1 μg/ml. Similar growth experiments were performed to determine the MICs for chloramphenicol of B. toyonensis strains BCT-7112 T , BCT-7112Δtet, BCT-7112Δcat, and Rock1-3 (Figures S3E–H ). These confirmed that catQ was responsible for the chloramphenicol resistance phenotype and that the Rock1-3 strain, with an MIC of 1 μg/ml, was susceptible to this antibiotic. Previous studies indicated an involvement of the gerIC–nucB intergenic region of plasmid pBCT77 in contributing to, or enhancing, the observed resistance of B. toyonensis BCT-7112 T to tetracycline and chloramphenicol (EFSA-FEEDAP, 2014 ; Matsumoto, Asahi Vet Japan, personal communication). We therefore confirmed the presence of the gerIC–nucB intergenic region in B. toyonensis strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat and also generated versions of these strains with additional copies of this region on plasmid pIGR19 (Section Construction of Strains with Additional Copies of the gerIC–nucB Intergenic Region). In addition, a strain of B. toyonensis Rock1-3 was generated with additional copies of the intergenic region on pIGR1. The resulting strains were grown in the presence of various concentrations of tetracycline (Figures S4A–D ) and chloramphenicol (Figures S4E–G ) to determine the influence of the intergenic region on growth and resistance. The data (Figure S4 ) indicated that the presence of additional copies of the gerIC – nucB locus do not significantly influence the growth profiles of B. toyonensis strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat on either of the antibiotics. In the case of strain Rock1-3, which encodes TetM and CatQ proteins that are identical to those in strain BCT-7112 T , but which is susceptible to tetracycline and chloramphenicol, the presence of the gerIC–nucB intergenic region had no impact it's sensitivity to these antibiotics. Taken together, these data rule out any impact of the gerIC–nucB intergenic region of pBCT77 as a factor in the resistances of B. toyonensis BCT-7112 T to these antibiotics. Why are strains encoding TetM and CatQ susceptible to tetracycline and chloramphenicol? B. toyonensis strains Rock1-3 and Rock3-28 encode identical or almost identical TetM and CatQ proteins to those encoded by strain BCT-7112 T (Table 2 ), but both are susceptible to tetracycline and chloramphenicol (MICs 1 μg/ml). We observed that strains Rock1-3 and Rock3-28 tended to aggregate at or close to their MIC and therefore monitored their morphologies while growing in LB medium in the presence of 2 μg/ml of the antibiotics, comparing them with the morphologies of strain BCT-7112 T growing in the presence of the antibiotics with either 2 and 32 μg/ml. Samples were taken every 60 min for microscopy and a representative set of data for growth in the presence of chloramphenicol is shown in Figure 2 . During the first 60 min all of the cells appeared normal. However, from ~120 min the morphologies of the Rock1-3 and Rock3-28 strains exhibited distorted cell morphologies compared with BCT-7112 T (Figure 2 ). The observed aggregation of the Rock1-3 and Rock3-28 strains is therefore likely due to this disturbed morphology that ultimately results in cell death. Figure 2 Influence of chloramphenicol on cell morphology . B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 were grown in LB medium at concentrations of chloramphenicol just above their MICs. Samples were taken every 60 min and viewed by phase-contrast microscopy. In the case of the Rock1-3 and Rock3-28, twisted cells can be seen in the samples taken at 180 min and later (white arrows). We next addressed the question of whether the tetM and catQ genes in strains Rock1-3 and Rock3-28 were expressed. To do this we carried out both Northern blot and qPCR analyses. Northern blotting analysis was carried out on B. toyonensis strains BCT-7112 T , BCT-7112Δcat, BCT-7112Δtet, Rock1-3, and Rock3-28. Cultures were grown in LB to mid-exponential phase and total RNA extracted and electrophoresed on two identical denaturing (1% formalin) agarose gels; to increase sensitivity, ~5 times more RNA was added to the tracks of strains Rock1-3 and Rock3-28 compared with that of strain BCT-7112 T . Following, hybridization with a DIG-labeled catQ probe, monocistronic catQ -specific transcripts (~0.8-kb) were detected in strains BCT-7112 T and BCT-7112Δtet, but not the other strains (Figure 3A ). In the case of the tetM- specific probe, strong monocistronic tetM -specific transcripts (~2.0-kb) were detected in strains BCT-7112 T and BCT-7112Δcat, and weak transcripts in strains Rock1-3 and Rock3-28 (Figure 3B ). As expected a catQ -specific transcript was absent from strain BCT-7112Δcat and a tetM -specific transcript from strain BCT-7112Δtet. Figure 3 Northern blot analysis for the detection of catQ and tetM transcripts in strains of B. toyonensis . (A) catQ- specific probe; (B) tetM -specific probe. The tracks contained total RNA from the following strains: 1. BCT-7112 T ; 2. BCT-7112Δcat; 2. BCT-7112Δtet; 4. Rock1–3; 5. Rock3–28. Approximately 5 times more RNA was added to the tracks containing Rock1-3 and Rock3-28 to improve sensitivity. The two prominent bands present in all the tracks are the 30S rRNA (upper) and 16S rRNA (lower). The Northern blot expression data were confirmed by qPCR using gyrB as the baseline control. B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 were included as both encode homologs of the TetM, while B. thuringiensis ATCC 10792 only encodes a copy of CatQ. The qPCR data (Table 5 ) show expression of tetM in BCT-7112 T and BCT-7112Δcat, but considerably lower or no expression in Rock1-3, BCT-7112Δtet, ATCC 14579, and ATCC 10792. In the case of the catQ qRNA data, expression was detected in BCT-7112 T , BCT-7112Δtet, but again, little or no expression of this gene in BCT-7112Δcat, Rock1-3, ATCC 14579, and ATCC 10792 (Table 5 ). Table 5 Quantitative PCR analysis of tetM and catQ transcripts in B. toyonensis strains BCT-7112 T , BCT-7112Δcat, BCT-7112Δtet, and Rock1-3, B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 . Transcript Strain R = 2 Δct tetM B. toyonensis BCT-7112 T 9.80 B. toyonensis BCT-7112Δcat 5.91 B. toyonensis BCT-7112Δtet 0.0 B. toyonensis Rock1-3 0.18 B. cereus ATCC 14579 0.59 B. thuringiensis ATCC 10792 0.0 catQ B. toyonensis BCT-7112 T 233.95 B. toyonensis BCT-7112Δcat 0.13 B. toyonensis BCT-7112Δtet 1706.9 B. toyonensis Rock1-3 3.56 B. cereus ATCC 14579 0.71 B. thuringiensis ATCC 10792 0.01 Since B. toyonensis strains Rock1-3 and Rock3-28 each contained homologs of the BCT-7112 T TetM and CatQ proteins with a high level of identity (Table 2 ), we carried out experiments to determine if their cognate genes were expressed in the related bacterium B. subtilis strain 168. The strategy involved cloning their coding sequences, together with upstream regulatory sequences, into the B. subtilis integration/expression vector pDR111. The genes and upstream sequences from B. toyonensis BCT-7112 T were also cloned as a control. In each case the tetM and catQ sequences were cloned downstream of the IPTG inducible P hyper-spank promoter in pDR111 and the resulting expression cassette was integrated at the non-essential amyE locus of B. subtilis . The resulting constructs allow for the expression of the cognate genes from their native promoters or, following IPTG induction, from the P hyper-spank promoter. The growth profiles of the B. subtilis strains grown in LB medium with various concentrations of tetracycline or chloramphenicol (0, 4, 8, 16, and 32 μg/ml) and with or without IPTG are shown in Figure 4 . Figure 4 Growth of B. subtilis strain 168 (A,H) , with tetM (A–G) or catQ (H–N) genes from BCT-7112 T (B,C,I,J) , Rock1-3 (D,E,K,L) and Rock3-28 (F,G,M,N) on various concentrations of tetracycline (A–G) or chloramphenicol (H–N) with (C,E,G,J,L,N) or without (A,B,D,F,H,I,K,M) the addition of IPTG (1 mM). The cultures were grown in a BioLector microreactor that determines optical density by measuring the scattered light signal (see Section Visualization of Cells by Light Microscopy). The data is shown on a linear rather than logarithmic graph to make it easier to compare growth profiles. The antibiotic concentrations used were (μg/ml): 0, red; 4, yellow; 8, gray; 16, orange and 32, light blue). B. subtilis is unable to grow in the presence of 4 μg/ml of tetracycline (Figure 4A ). Cloning the tetM gene from B. toyonensis BCT-7112 T into B. subtilis 168 allows this strain to grow in the presence of 32 μg/ml, albeit with an extended lag phase (Figure 4B ). The addition of IPTG to the culture medium did not have a significant influence of growth up to 32 μg/ml tetracycline, indicating that the upstream regulatory region of tetM was fully functional in this bacterium (Figure 4B ). The growth kinetics for the B. subtilis strains encoding the tetM genes from strains Rock1-3 (Figures 4C,D ) and Rock3-28 (Figures 4F,G ) were similar and are discussed together. The data show that, in the absence of IPTG, the B. subtilis strains encoding the tetM genes from these strains grew in the presence of 32 μg/ml tetracycline. However, the growth kinetics show that the lag phase increased in length with increasing concentrations of tetracycline, to the extent that growth at 32 μg/ml tetracycline is only observed after approximately 18 h. When IPTG is added, the cultures grew without a lag phase up to 8 μg/ml tetracycline and with considerably shorter lag phases at 16 and 32 μg/ml tetracycline. Taken together, the data confirm that the tetM genes in B. toyonensis strains Rock1-3 and Rock3-28 encode functional proteins, but that expression from their native promoters is weak. Similar results were observed when B. subtilis encodes the catQ genes from B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 following growth on chloramphenicol (0, 4, 8, 16, and 32 μg/ml). While B. subtilis alone is unable to grow in the presence of 4 μg/ml of chloramphenicol (Figure 4H ), the inclusion of the catQ gene from strain BCT-7112 T , together with its native promoter, facilitated growth up to 16 μg/ml (Figure 4I ). However, with increasing concentrations of chloramphenicol, there was a longer lag before the culture started to grow, indicating that the native catQ promoter was relatively efficient in B. subtilis . This was confirmed when IPTG was added to the culture; the lag phases were considerably reduced and the culture grew at 32 μg/ml (Figure 4J ). The growth kinetics for the B. subtilis strains encoding the catQ genes from B. toyonensis strains Rock1-3 (Figures 4K,L ) and Rock3-28 (Figures 4M,N ) were similar with their native promoters facilitating growth at concentrations up to 8 μg/ml, albeit with lag phases different length (Figures 4K,M ). When IPTG was included in the culture medium, the cells were able to grow in the presence of 32 μg/ml, but with longer lag phases at the higher antibiotic concentrations (Figures 4L,N ). Taken together, the data in Figure 4 clearly show that the tetM and catQ genes of B. toyonensis strains Rock1-3 and Rock3-28 encode function proteins, capable of conferring resistance to their cognate antibiotics. The data also suggests an explanation for why B. toyonensis strain BCT-7112 T exhibits resistance to these antibiotics, while strains Rock1-3 and Rock3-28 do not (Figure S3 ), namely that their upstream promoters are relatively inefficient. Alignment of the nucleotides upstream of tetM and catQ coding sequences indicates a high degree of identity between the regulatory regions of these genes from strains BCT-7112 T , Rock1-3, and Rock3-28 (Figure 5 ). Figure 5 also identifies consensus sequences for putative Sigma A (vegetative) promoters and single nucleotide polymorphisms (SNPs) with respect to BCT-7112 T . In the case of tetM , identical SNPs occur within the putative −35 sequences and within the sequences between the −35 and −10 sequences in Rock1-3 and Rock3-28, with the latter having three additional SNPs in this region (Figure 5A ). In the region upstream of catQ , three putative Sigma A promoters were identified, the most peripheral of which contains the only SNP observed in this region (Figure 5B ). Figure 5 Nucleotide sequences upstream of the (A) tetM and (B) catQ genes of B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28. Putative Sigma A (vegetative) promoter consensus sequences are shown highlighted (−35/−10) and the ribosome binding sites (RBS) and start codons are in bold. Single nucleotide polymorphisms (SNPs) in the Rock1-3 and Rock3-28 strains are also highlighted. Characterization of resistance phenotypes and the identification of resistance genes Minimum inhibitory concentrations (MICs) of B. toyonensis BCT-7112 T for tetracycline and chloramphenicol were, at 16 and 32 μg/ml respectively, higher than the resistance break points (≤8 μg/ml) identified by the European Committee on Antimicrobial Susceptibility Testing (Kahlmeter et al., 2006 ). Consequently B. toyonensis BCT-7112 T is designated as being resistant to both of these antibiotics. Analysis of the annotated B. toyonensis BCT-7112 T genome sequence (NCBI; CP006863), and plasmids pBCT77 (CP006864) and pBCT8 (CP006865), revealed the presence of genes encoding putative tetracycline and chloramphenicol resistance proteins. The most common tetracycline resistance mechanisms are efflux pumps and ribosome-protection proteins (Wilson, 2014 ). The B. toyonensis BCT-7112 T genome encodes two putative tetracycline resistance proteins, one an efflux protein and the other a putative ribosome-protection protein. Btoyo_4389 (nucleotides 4295949–4297223) is annotated as encoding a tetracycline resistance protein of the Major Facilitator Superfamily (MFS, cd06174). The MFS is a large and diverse group of secondary transporters that includes uniporters, symporters, and antiporters. MFS proteins facilitate transport across the cytoplasmic membrane of various substrates, including ions, sugar phosphates, antibiotics, amino acids, and peptides. MFS transporters are common among soil-living organisms and, without specific experimental evidence, it can be difficult to identity their substrates. BLAST analysis revealed that homologs of Btoyo_4389 are found extensively among members of the B. cereus group but are labeled, in decreasing order of frequency, "MFS transporter," "tetracycline resistance protein," major facilitator protein," and "putative multidrug resistance protein." It is not clear what, if any, evidence there is for annotating this protein specifically as a "tetracycline resistance protein" rather than as a "MFS transporter," illustrating a limitation of automated annotation programs. Btoyo_0322 (nucleotides 307023–308966) is a putative ribosome protection protein with a conserved TetM-like family domain (cd04168). TetM proteins exhibit ~45% similarity with elongation factor G (EF-G) proteins. They have conserved nucleotide-binding motifs and are members of the translation factor superfamily of GTPases that bring about the release of tetracycline from the ribosome in a GTP-dependent manner (Dönhöfer et al., 2012 ). Several genes are annotated as encoding a TetR-like protein. TetR proteins are a class of transcriptional regulators with helix-turn-helix motifs, and are so annotated because the first representative of this class was responsible for the regulation of a MFS transporter mediating tetracycline resistance on transposon Tn 10 (Beck et al., 1982 ). The association with tetracycline is therefore historic. Analysis of the B. toyonensis BCT-7112 T genome reveals the presence of two genes encoding putative chloramphenicol acetyltransferases (Cat) that inactivate chloramphenicol by acetylation. Btoyo_3723 (nucleotides 3655712–3656095), at 127 residues in length, is significantly shorter than other chloramphenicol acetyltransferases (~220 residues) and is likely to have been truncated during evolution. Although, the active site is present, it is unlikely that this gene encodes an active enzyme. In contrast, Btoyo_4985 (nucleotides 4933576–4934223) encodes a full-length CatQ-like chloramphenicol acetyltransferase. A previous unpublished transposon mutagenesis study (EFSA-FEEDAP, 2014 ; M Kato, Science Tanaka, Japan, personal communication) identified a region on pBCT77, between nucB (Btoyo_5072 [nucleotides 74482–74916]) and gerIC (Btoyo_4992 [nucleotides 250–858]) that had a minor influence on reducing the MICs of tetracycline and chloramphenicol in B. toyonensis BCT-7112 T . Two of the three genes in this region encode proteins of unknown function (Btoyo_5073 [nucleotides 74939–75190]; Btoyo_5074 [nucleotides 75202–75432]), while the third gene (Btoyo_5075 [nucleotides 75457–76029]) encodes a phage-type site-specific recombinase. There were no genes encoding known antibiotic resistance genes in this region, elsewhere on this pBCT77 or on the smaller plasmid, pBCT8. Identification of the genes encoding tetracycline and chloramphenicol resistances Btoyo_0322 and Btoyo_4389 were tentatively identified as encoding tetracycline resistance proteins, the former a TetM-like protein, the later a MFS-like transporter. Because of limitations in the identification of MFS substrates and the higher likelihood that Btoyo_4389 was incorrectly annotated, we focused on Btoyo_0322. The tetM gene encoded by Btoyo_0322 was replaced with a spectinomycin resistance gene (Section Construction of tetM and catQ Knockout Strains) and the resulting knockout strain (BCT-7112Δtet) was spectinomycin and chloramphenicol resistant, but erythromycin and tetracycline susceptible. The authenticity of the integration event was confirmed by sequencing across the entire region (see Figure 1A ). The data confirmed that Btoyo_0322 was excised from the chromosome and that this gene was solely responsible for the observed tetracycline resistance phenotype of B. toyonensis strain BCT-7112 T . A similar approach was used to identify the gene responsible for chloramphenicol resistance. Of the two putative chloramphenicol resistance genes identified in the genome of BCT-7112 T , Btoyo_3723, and Btoyo_4985, the later was chosen as the former appeared to have been truncated. The gene replacement strategy described in Section Construction of tetM and catQ Knockout Strains was employed, involving the replacement of Btoyo_4985 with a spectinomycin resistance gene. The resulting knockout strain (BCT-7112Δcat), was spectinomycin and tetracycline resistant, but erythromycin and chloramphenicol susceptible. The authenticity of the integration event was again confirmed by sequencing across the entire region (see Figure 1B ). The data confirmed that Btoyo_4985 was solely responsible for the observed chloramphenicol resistance phenotype of B. toyonensis strain BCT-7112 T . Following their identification, the %GC contents of Btoyo_0332 and Btoyo_4985 were determined to be 37.3 and 29.8%, respectively, while the genome average of B. toyonensis BCT-7112 T was 35.6%. Using a 1000-bp window and a standard deviation cut-off value of 2.5, neither the %GC of these coding sequences, nor a 3500-bp regions either side, were found to be significantly different from the running average, indicating that Btoyo_0332 and Btoyo_4985 are not recent acquisitions. The presence of homologs of tetM (Btoyo_322) and catQ (Btoyo_4985) genes in other member of the B. cereus sensu lato group In light of the recent studies revising the taxonomic status of the B. cereus group ( sensu lato ) (Jiménez et al., 2013 ; Böhm et al., 2015 ; Liu et al., 2015 ), we used BLASTp (Altschul et al., 1990 ) to search for homologs of the BCT-7112 T tetM and catQ genes in the other 18 strains within the B. toyonensis taxonomic unit. The data (Table 2 ) clearly show that homologs of TetM are present in all strains of B. toyonensis while homologs of CatQ are present in all but one strain. The failure to detect CatQ in B. toyonensis VD115 is most likely to be due to the absence of the relevant region in the partial genome sequence, which is 3884 k in length compare with 4940 k for B. toyonensis BCT-7112 T . Table 2 The percentage identity and similarity of proteins encoded by homologs of the tetM and catQ genes of B. toyonensis BCT-7112 T among other members of the B. toyonensis taxonomic unit . Strain GenBank Accession N o % Identity/Similarity to B. toyonensis BCT-7112 TetM % Identity/Similarity to B. toyonensis BCT-7112 CatQ B. toyonensis BCT-7112 T CP006863.1 100/100 100/100 B. toyonensis VD115 * JH792165.1 97/98 TS *** B. toyonensis MC28 * CP003687.1 94/97 100/100 B. toyonensis Rock3-28 * CM000730.1 93/96 100/100 B. toyonensis VD131 ** KB976660.1 93/96 100/100 B. toyonensis HuB5-5 ** JH792120.1 100/100 100/100 B. toyonensis VD148 * JH792156.1 90/95 100/100 B. toyonensis BAG2O-2 ** AHCX01000000.1 93/96 100/100 B. toyonensis Rock1-3 * CM000728.1 .1 100/100 100/100 B. toyonensis BAG1O-2 * AHCO00000000.1 100/100 100/100 B. toyonensis HuB4-10 ** AHEE00000000.1 100/100 100/100 B. toyonensis BAG5O-1 * AHDI00000000.1 99/99 100/100 B. toyonensis HuA2-3 ** AHDW00000000.1 93/97 100/100 B. toyonensis BAG6O-1 * JH804627.1 99/100 100/100 B. toyonensis BAG4X2-1 * JH804617.1 100/100 99/99 B. toyonensis VD214 ** AHFN00000000.1 100/100 99/99 B. toyonensis HuB2-9 ** AHED00000000.1 100/100 99/99 B. toyonensis Rock3-29 * CM000731.1 100/100 99/99 B. toyonensis Rock4-18 ** CM000735.1 .1 93/96 99/99 * Clustered to the B. toyonensis group by Jiménez et al. ( 2013 ); ** Clustered to the B. toyonensis group by Liu et al. ( 2015 ); *** TS, truncated sequence detected . In some cases the NCBI Reference for master record for a whole genome shotgun sequence is given . In the case of TetM, just under half of the B. toyonensis strains encode proteins with identical sequences (Table 2 ). The most variable TetM protein is encoded by B. toyonensis VD148, which shows 90% identity and 95% similarity with that of strain BCT-7112 T . Twelve of the B. toyonensis strains encode CatQ proteins that are identical to that of strain BCT-7112 T , while the remaining strains exhibit CatQ proteins with 99% identity. Moreover, the tetM and catQ genes are located in the same genomic neighborhoods in all of the B. toyonensis strains. Taken together, the data indicate that the tetM and catQ genes are ancient genomic components of the B. toyonensis taxonomic unit rather than having been acquired recently by horizontal gene transfer. The NCBI genomes database contains whole genomes, chromosomes, scaffolds and contiguous fragments (contigs) of B. anthracis, B. bombysepticus, B. cereus sensu stricto, B. mycoides, B. thuringiensis, B. toyonensis, B. weihenstephanensis , and B. wiedmannii (Table 3 ). In order to extend the homology study to other members of the B. cereus sensu lacto , BLASTp was used to search for homologs of the B. toyonensis BCT-7112 T TetM and CatQ proteins with a similarity score of 85% or greater (Table 4 ). All of the strains identified as having homologs of these TetM and CatQ proteins were members of the B. cereus group (sensu lato): no strains outside this group appeared in the analysis. Homologs of both the TetM and CatQ were found in all eight members of the B. cereus group shown in Table 3 . Genes encoding these proteins were absent from B. cytotoxicus and B. pseudomycoides , although this could be due either to the small number of DNA sequences available for these species or to changes in their taxonomic status. A list of sequences analyzed for homology with the TetM and CatQ proteins is given in Tables S1 , S2 , respectively. Table 3 Breakdown of the genomic sequences analyzed to identify homologs of the B. toyonensis BCT-7112 T TetM and CatQ proteins . Taxonomic group/ID Complete Chromosome Scaffold Contig Total B. anthracis /1392 44 10 41 42 137 B. bombysepticus /1330043 1 0 0 0 1 B. cereus (sensu stricto) /1396 32 29 154 51 266 B. mycoides /1405 2 3 3 5 13 B. thuringiensis /1428 37 16 14 12 79 B. toyonensis /155322 1 0 0 0 1 B. weihenstephanensis /86662 2 0 0 4 6 B. wiedmannii /1890302 0 0 0 11 11 Table 4 The distribution of proteins encoded by homologs (>85% similarity) of the tetM and catQ genes of B. toyonensis BCT-7112 T among other members of the B. cereus sensu lato group . Gene B. thuringiensis B. cereus (sensu stricto) B. anthracis B. mycoides B. weihen-stephanensis B. toyonensis B. wiedmannii B. bombysepticus Total CatQ 66 161 111 7 6 18 11 1 381 TetM 77 135 112 4 10 19 11 1 369 A total of 369 homologs of TetM were identified in the NBCI database and widely distributed among species of the B. cereus group, broadly reflecting the frequency of complete genomes and chromosomes in the database (Table 3 ). In the case of CatQ, 381 homologs were found, and with a distribution that was similar to that of TetM. The relationships between these TetM and CatQ sequences, in the form of Neighbor Joining trees, are shown in Figures S1 , S2 . In both cases the trees exclusively contain homologous proteins from other members of the B. cereus group ( sensu lato ), confirming the ancient origin of these proteins in this taxonomic clade. Tetracycline and chloramphenicol resistance profiles of the strains within the B. toyonensis taxonomic unit B. toyonensis BCT-7112 T is phenotypically resistant to tetracycline and chloramphenicol, and this study identified the genes responsible, namely tetM (Btoyo_0322) and catQ (Btoyo_4985). However, eight other B. toyonensis strains are phenotypically susceptible to tetracycline and chloramphenicol, despite encoding TetM and CatQ proteins that are homologous to those strain BCT-7112 T (EFSA-FEEDAP, 2014 ; L. Mocé, Eurofins Biolab, personal communication). We therefore addressed the question of why other members of the B. toyonensis taxonomic unit that encode either identical (strain Rock1-3) or similar (strain Rock3-28) resistance determinants do not exhibit the same resistance phenotypes. Both strains are susceptible to tetracycline and chloramphenicol (MIC 1 μg/ml) and have similar growth kinetics and therefore only the growth profiles of strain Rock1-3 are shown in Figure S3 , in comparison with the profiles of strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat. When B. toyonensis BCT-7112 T was grown in the presence of various concentrations of tetracycline (0, 1, 2, 4, 16, and 32 μg/ml) the MIC was 16 μg/ml (Figure S3A ). When the isogenic strain (BCT-7112Δtet) in which the tetM (Btoyo_0322) gene had been replaced with a spectinomycin resistance gene was tested, the MIC was 1 μg/ml (Figure S3B ). In contrast, when the isogenic strain (BCT-7112Δcat) in which the catQ gene was replaced with a spectinomycin resistance gene, the MIC for tetracycline remained at 16 μg/ml (Figure S3C ). In the case of the Rock1-3 strain, there was some initial growth at 1 μg/ml (Figure S3D ), but the cells eventually died and the biomass concentration declined, indicating it had an MIC for tetracycline of 1 μg/ml. Similar growth experiments were performed to determine the MICs for chloramphenicol of B. toyonensis strains BCT-7112 T , BCT-7112Δtet, BCT-7112Δcat, and Rock1-3 (Figures S3E–H ). These confirmed that catQ was responsible for the chloramphenicol resistance phenotype and that the Rock1-3 strain, with an MIC of 1 μg/ml, was susceptible to this antibiotic. Previous studies indicated an involvement of the gerIC–nucB intergenic region of plasmid pBCT77 in contributing to, or enhancing, the observed resistance of B. toyonensis BCT-7112 T to tetracycline and chloramphenicol (EFSA-FEEDAP, 2014 ; Matsumoto, Asahi Vet Japan, personal communication). We therefore confirmed the presence of the gerIC–nucB intergenic region in B. toyonensis strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat and also generated versions of these strains with additional copies of this region on plasmid pIGR19 (Section Construction of Strains with Additional Copies of the gerIC–nucB Intergenic Region). In addition, a strain of B. toyonensis Rock1-3 was generated with additional copies of the intergenic region on pIGR1. The resulting strains were grown in the presence of various concentrations of tetracycline (Figures S4A–D ) and chloramphenicol (Figures S4E–G ) to determine the influence of the intergenic region on growth and resistance. The data (Figure S4 ) indicated that the presence of additional copies of the gerIC – nucB locus do not significantly influence the growth profiles of B. toyonensis strains BCT-7112 T , BCT-7112Δtet, and BCT-7112Δcat on either of the antibiotics. In the case of strain Rock1-3, which encodes TetM and CatQ proteins that are identical to those in strain BCT-7112 T , but which is susceptible to tetracycline and chloramphenicol, the presence of the gerIC–nucB intergenic region had no impact it's sensitivity to these antibiotics. Taken together, these data rule out any impact of the gerIC–nucB intergenic region of pBCT77 as a factor in the resistances of B. toyonensis BCT-7112 T to these antibiotics. Why are strains encoding TetM and CatQ susceptible to tetracycline and chloramphenicol? B. toyonensis strains Rock1-3 and Rock3-28 encode identical or almost identical TetM and CatQ proteins to those encoded by strain BCT-7112 T (Table 2 ), but both are susceptible to tetracycline and chloramphenicol (MICs 1 μg/ml). We observed that strains Rock1-3 and Rock3-28 tended to aggregate at or close to their MIC and therefore monitored their morphologies while growing in LB medium in the presence of 2 μg/ml of the antibiotics, comparing them with the morphologies of strain BCT-7112 T growing in the presence of the antibiotics with either 2 and 32 μg/ml. Samples were taken every 60 min for microscopy and a representative set of data for growth in the presence of chloramphenicol is shown in Figure 2 . During the first 60 min all of the cells appeared normal. However, from ~120 min the morphologies of the Rock1-3 and Rock3-28 strains exhibited distorted cell morphologies compared with BCT-7112 T (Figure 2 ). The observed aggregation of the Rock1-3 and Rock3-28 strains is therefore likely due to this disturbed morphology that ultimately results in cell death. Figure 2 Influence of chloramphenicol on cell morphology . B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 were grown in LB medium at concentrations of chloramphenicol just above their MICs. Samples were taken every 60 min and viewed by phase-contrast microscopy. In the case of the Rock1-3 and Rock3-28, twisted cells can be seen in the samples taken at 180 min and later (white arrows). We next addressed the question of whether the tetM and catQ genes in strains Rock1-3 and Rock3-28 were expressed. To do this we carried out both Northern blot and qPCR analyses. Northern blotting analysis was carried out on B. toyonensis strains BCT-7112 T , BCT-7112Δcat, BCT-7112Δtet, Rock1-3, and Rock3-28. Cultures were grown in LB to mid-exponential phase and total RNA extracted and electrophoresed on two identical denaturing (1% formalin) agarose gels; to increase sensitivity, ~5 times more RNA was added to the tracks of strains Rock1-3 and Rock3-28 compared with that of strain BCT-7112 T . Following, hybridization with a DIG-labeled catQ probe, monocistronic catQ -specific transcripts (~0.8-kb) were detected in strains BCT-7112 T and BCT-7112Δtet, but not the other strains (Figure 3A ). In the case of the tetM- specific probe, strong monocistronic tetM -specific transcripts (~2.0-kb) were detected in strains BCT-7112 T and BCT-7112Δcat, and weak transcripts in strains Rock1-3 and Rock3-28 (Figure 3B ). As expected a catQ -specific transcript was absent from strain BCT-7112Δcat and a tetM -specific transcript from strain BCT-7112Δtet. Figure 3 Northern blot analysis for the detection of catQ and tetM transcripts in strains of B. toyonensis . (A) catQ- specific probe; (B) tetM -specific probe. The tracks contained total RNA from the following strains: 1. BCT-7112 T ; 2. BCT-7112Δcat; 2. BCT-7112Δtet; 4. Rock1–3; 5. Rock3–28. Approximately 5 times more RNA was added to the tracks containing Rock1-3 and Rock3-28 to improve sensitivity. The two prominent bands present in all the tracks are the 30S rRNA (upper) and 16S rRNA (lower). The Northern blot expression data were confirmed by qPCR using gyrB as the baseline control. B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 were included as both encode homologs of the TetM, while B. thuringiensis ATCC 10792 only encodes a copy of CatQ. The qPCR data (Table 5 ) show expression of tetM in BCT-7112 T and BCT-7112Δcat, but considerably lower or no expression in Rock1-3, BCT-7112Δtet, ATCC 14579, and ATCC 10792. In the case of the catQ qRNA data, expression was detected in BCT-7112 T , BCT-7112Δtet, but again, little or no expression of this gene in BCT-7112Δcat, Rock1-3, ATCC 14579, and ATCC 10792 (Table 5 ). Table 5 Quantitative PCR analysis of tetM and catQ transcripts in B. toyonensis strains BCT-7112 T , BCT-7112Δcat, BCT-7112Δtet, and Rock1-3, B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 . Transcript Strain R = 2 Δct tetM B. toyonensis BCT-7112 T 9.80 B. toyonensis BCT-7112Δcat 5.91 B. toyonensis BCT-7112Δtet 0.0 B. toyonensis Rock1-3 0.18 B. cereus ATCC 14579 0.59 B. thuringiensis ATCC 10792 0.0 catQ B. toyonensis BCT-7112 T 233.95 B. toyonensis BCT-7112Δcat 0.13 B. toyonensis BCT-7112Δtet 1706.9 B. toyonensis Rock1-3 3.56 B. cereus ATCC 14579 0.71 B. thuringiensis ATCC 10792 0.01 Since B. toyonensis strains Rock1-3 and Rock3-28 each contained homologs of the BCT-7112 T TetM and CatQ proteins with a high level of identity (Table 2 ), we carried out experiments to determine if their cognate genes were expressed in the related bacterium B. subtilis strain 168. The strategy involved cloning their coding sequences, together with upstream regulatory sequences, into the B. subtilis integration/expression vector pDR111. The genes and upstream sequences from B. toyonensis BCT-7112 T were also cloned as a control. In each case the tetM and catQ sequences were cloned downstream of the IPTG inducible P hyper-spank promoter in pDR111 and the resulting expression cassette was integrated at the non-essential amyE locus of B. subtilis . The resulting constructs allow for the expression of the cognate genes from their native promoters or, following IPTG induction, from the P hyper-spank promoter. The growth profiles of the B. subtilis strains grown in LB medium with various concentrations of tetracycline or chloramphenicol (0, 4, 8, 16, and 32 μg/ml) and with or without IPTG are shown in Figure 4 . Figure 4 Growth of B. subtilis strain 168 (A,H) , with tetM (A–G) or catQ (H–N) genes from BCT-7112 T (B,C,I,J) , Rock1-3 (D,E,K,L) and Rock3-28 (F,G,M,N) on various concentrations of tetracycline (A–G) or chloramphenicol (H–N) with (C,E,G,J,L,N) or without (A,B,D,F,H,I,K,M) the addition of IPTG (1 mM). The cultures were grown in a BioLector microreactor that determines optical density by measuring the scattered light signal (see Section Visualization of Cells by Light Microscopy). The data is shown on a linear rather than logarithmic graph to make it easier to compare growth profiles. The antibiotic concentrations used were (μg/ml): 0, red; 4, yellow; 8, gray; 16, orange and 32, light blue). B. subtilis is unable to grow in the presence of 4 μg/ml of tetracycline (Figure 4A ). Cloning the tetM gene from B. toyonensis BCT-7112 T into B. subtilis 168 allows this strain to grow in the presence of 32 μg/ml, albeit with an extended lag phase (Figure 4B ). The addition of IPTG to the culture medium did not have a significant influence of growth up to 32 μg/ml tetracycline, indicating that the upstream regulatory region of tetM was fully functional in this bacterium (Figure 4B ). The growth kinetics for the B. subtilis strains encoding the tetM genes from strains Rock1-3 (Figures 4C,D ) and Rock3-28 (Figures 4F,G ) were similar and are discussed together. The data show that, in the absence of IPTG, the B. subtilis strains encoding the tetM genes from these strains grew in the presence of 32 μg/ml tetracycline. However, the growth kinetics show that the lag phase increased in length with increasing concentrations of tetracycline, to the extent that growth at 32 μg/ml tetracycline is only observed after approximately 18 h. When IPTG is added, the cultures grew without a lag phase up to 8 μg/ml tetracycline and with considerably shorter lag phases at 16 and 32 μg/ml tetracycline. Taken together, the data confirm that the tetM genes in B. toyonensis strains Rock1-3 and Rock3-28 encode functional proteins, but that expression from their native promoters is weak. Similar results were observed when B. subtilis encodes the catQ genes from B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28 following growth on chloramphenicol (0, 4, 8, 16, and 32 μg/ml). While B. subtilis alone is unable to grow in the presence of 4 μg/ml of chloramphenicol (Figure 4H ), the inclusion of the catQ gene from strain BCT-7112 T , together with its native promoter, facilitated growth up to 16 μg/ml (Figure 4I ). However, with increasing concentrations of chloramphenicol, there was a longer lag before the culture started to grow, indicating that the native catQ promoter was relatively efficient in B. subtilis . This was confirmed when IPTG was added to the culture; the lag phases were considerably reduced and the culture grew at 32 μg/ml (Figure 4J ). The growth kinetics for the B. subtilis strains encoding the catQ genes from B. toyonensis strains Rock1-3 (Figures 4K,L ) and Rock3-28 (Figures 4M,N ) were similar with their native promoters facilitating growth at concentrations up to 8 μg/ml, albeit with lag phases different length (Figures 4K,M ). When IPTG was included in the culture medium, the cells were able to grow in the presence of 32 μg/ml, but with longer lag phases at the higher antibiotic concentrations (Figures 4L,N ). Taken together, the data in Figure 4 clearly show that the tetM and catQ genes of B. toyonensis strains Rock1-3 and Rock3-28 encode function proteins, capable of conferring resistance to their cognate antibiotics. The data also suggests an explanation for why B. toyonensis strain BCT-7112 T exhibits resistance to these antibiotics, while strains Rock1-3 and Rock3-28 do not (Figure S3 ), namely that their upstream promoters are relatively inefficient. Alignment of the nucleotides upstream of tetM and catQ coding sequences indicates a high degree of identity between the regulatory regions of these genes from strains BCT-7112 T , Rock1-3, and Rock3-28 (Figure 5 ). Figure 5 also identifies consensus sequences for putative Sigma A (vegetative) promoters and single nucleotide polymorphisms (SNPs) with respect to BCT-7112 T . In the case of tetM , identical SNPs occur within the putative −35 sequences and within the sequences between the −35 and −10 sequences in Rock1-3 and Rock3-28, with the latter having three additional SNPs in this region (Figure 5A ). In the region upstream of catQ , three putative Sigma A promoters were identified, the most peripheral of which contains the only SNP observed in this region (Figure 5B ). Figure 5 Nucleotide sequences upstream of the (A) tetM and (B) catQ genes of B. toyonensis strains BCT-7112 T , Rock1-3, and Rock3-28. Putative Sigma A (vegetative) promoter consensus sequences are shown highlighted (−35/−10) and the ribosome binding sites (RBS) and start codons are in bold. Single nucleotide polymorphisms (SNPs) in the Rock1-3 and Rock3-28 strains are also highlighted. Discussion B. toyonensis BCT-7112 T is resistant to tetracycline and chloramphenicol and the annotated genome and plasmid sequences were used to identify the putative genes involved. Reciprocal recombination was then used to replace the target resistance genes with a spectinomycin resistance gene and this unambiguously identified Btoyo_0332 ( tetM ) and Btoyo_4985 ( catQ ) as being the genes responsible for the resistance phenotypes in B. toyonensis BCT-7112 T . We addressed the question of whether the genes responsible for tetracycline and chloramphenicol resistances in BCT-7112 T were intrinsic or acquire in three ways. Firstly we established that these genes were not associated with known MGEs (e.g., transposon, transposase, insertion sequence, conjugation etc.). Secondly, we analyzed their %GC content in relation to the genome as a whole and showed that they were not significantly different. Thirdly, we showed that homologous genes were not only present in closely related species, but were also present in virtually all representative of the B. cereus group sensu lato . The data (Tables 2 , 4 ) clearly show that the tetM and catQ genes are widely distributed among members of the B. cereus group sensu lato , are located within the same genomic neighborhoods and are therefore unambiguously of ancient origin within this group, rather than having been acquired as the result of more recent horizontal gene transfer. This conclusion is supported by animal feeding experiments in which qPCR was used to quantify tetracycline and chloramphenicol resistance genes in intestinal samples from piglets and cattle (Casanovas-Massana et al., 2014 ). The conclusion from these studies is that the presence of B. toyonensis does not contribute significantly to the antibiotic resistance load already present in the intestinal tract of these animals. While B. toyonensis BCT-7112 T is resistant to tetracycline and chloramphenicol, closely related strains (e.g., Rock1-3 and Rock3-28) with identical or virtually identical genes and upstream regulatory regions are susceptible to these antibiotics. To address this apparent contradiction, we sought answers to two specific questions: are the cognate resistance genes in Rock1-3 and Rock3-28 functional and, if so, why do they not elaborate the expected resistance phenotype? Using Rock1-3 and Rock3-28 as representative strains, we integrated their tetM and catQ genes, together with their upstream regulatory sequences, into the amyE locus of B. subtilis . The data (Figure 4 ) show firstly that the products of these genes are functionally active since induction of an upstream P hyper-spank promoter conferred the cognate resistance on the B. subtilis host. More significantly, when using their native regulatory sequences, these genes were able to confer resistance to B. subtilis at levels above its MIC (~1 μg/ml), albeit with varying degrees of efficiency and extended lag phases. This suggests that the native promoters of these genes are relatively weak, and this was confirmed by analysis of their transcript levels (Figure 3 and Table 5 ). Analysis of cells taken from the non-induced cultures of B. toyonensis Rock1-3 growing on tetracycline (16 μg/ml, Figure 4D ) and chloramphenicol (8 μg/ml, Figure 4K ) revealed that the majority of the cells had become resistant to the cognate antibiotic, presumably by the selection of cells within the population with spontaneous mutations in either the native upstream regulatory sequences or elements of the P hyper-spank promoter system. It is likely that the extended lag phases in these cultures reflect the time taken for such mutants to accumulate in the population. The final question we addressed is why the B. cereus sensu lato group encodes genes that are so poorly expressed that they do not register a phenotype in the standard protocols used for regulatory purposes to identify MIC values. Although, we cannot provide a definitive answer this question, their maintenance over such a long evolutionary time-scale suggests that they must have a specific role or selective advantage in their natural environment, the soil. One possibility is that they are transcribed from non-vegetative Sigma factor promoters induced, for example, in response to specific stresses, quorum sensing pheromones or differentiation processes, and that are not encountered under the MIC test conditions. Another is that their expression has been down regulated over evolutionary time by single base-pair mutations that, as a bet-hedging strategy (Ferenci and Maharjan, 2015 ), are able to revert at a high frequency and subsequently selected under appropriate conditions. This might account for the resistance phenotypes of B. toyonensis BCT-7112 T . The results represent a potential issue for regulatory authorities who tend to rely on phenotypic data for assessing the implications of introducing antibiotic resistance organisms into the food chain. For example, in their conclusions on the antibiotic susceptibility of B. toyonensis NCIMB 14858 T (i.e., BCT-7112T) the EFSA FEEDAP Panel stated the following: " Although, there is some evidence of the presence of the catQ and tet(M) genes in eight closely related strains, none of these demonstrated the resistant phenotype. Consequently, resistance to chloramphenicol and tetracycline cannot be considered intrinsic to the newly defined species " (EFSA-FEEDAP, 2014 ). This statement raises the important issue of exactly what evidence regulatory authorities should consider before concluding whether or not a resistance factor (genotype or phenotype) is intrinsic. The traditional reliance on phenotype is clearly insufficient in the post-genomic era since the intrinsic nature of an observed phenotype can only be defined at the level of its gene. This is particularly the case with resistance determinants since the same phenotype can be encoded by entirely different mechanisms (Blair et al., 2015 ). Moreover, detecting homologous genes in both closely related and distantly related organisms, particularly if located at similar genomic neighborhoods, strengthens the case for recognizing that the gene, rather than it observable phenotype, is intrinsic. The question remains as to whether or not strains that are intrinsically resistant to antibiotic(s) should be used in any process associated with the human food chain? It is likely that antibiotics (also referred to as secondary metabolites) have been produced for over 500 million years, dating back to the Cambrian period (Baltz, 2008 ; Cox and Wright, 2013 ). Their production relates to evolutionary processes taking place millions of years before the use of antimicrobial chemotherapy, when the antibiotics encountered by bacteria were produced by competitor organisms in the same environment (Perry and Wright, 2013 ). In some cases, the evolution of impermeability barriers such as the outer membrane, conferred resistance (e.g., erythromycin resistance in Escherichia coli ), in others existing genes were duplicated and modified (e.g., the elongation factor TU in the case if TetM) or were acquired from the producers themselves (e.g., MFS efflux pumps). It would be unrealistic to exclude the use of all such strains. A realistic approach requires a detailed analysis that: (i) identifies the gene(s) responsible for the resistance phenotype, (ii), confirms that they are not associated with known MGEs (e.g., plasmids, prophages, transposons, insertion sequence, resistance cassettes, integrons etc.), and (iii) irrespective of phenotype, that homologous genes are present in related strains/species and at similar genomic locations. In conclusion our data clearly show that the tetM and catQ genes of B. toyonensis BCT-7112 T are intrinsic not only to this species, but also to the B . cereus group ( sensu lato ). They are confirmed to be of ancient origin, since genes subject to horizontal gene transfer would be associated with MGEs, have a significantly different %GC and located at a variety of genomic neighborhoods. The proven intrinsic nature and non-transferability of these antibiotic resistance genes in B. toyonensis BCT-7112 T counts in favor of recommending the safe use of this strain as an additive in animal nutrition. These studies also show that intrinsic resistance should be defined at the genomic rather than the phenotypic level and this should be taken into account whenever a scientific assessment of the nature and genetic basis of a bacterial antibiotic resistance is performed. Author contributions All authors were involved in the design of the studies and in the interpretation of the data. HG and SP carried out the majority of the experimental work at Newcastle University, under the supervision of CH. FN and EM carried out the qPCR experiments at the Hospital de la Santa Creu i Sant Pau. In addition to their involvement in the design of the studies, GJ and AB provided background information, strains and a critical input into the preparation of the manuscript. Conflict of interest statement The research was funded by Rubinum S.A. that has a commercial interest in B. toyonensis strain BCT-7112 T . However, the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AJ declared a shared affiliation, though no other collaboration, with one of the authors AB to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review. Conflict of interest statement The research was funded by Rubinum S.A. that has a commercial interest in B. toyonensis strain BCT-7112 T . However, the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AJ declared a shared affiliation, though no other collaboration, with one of the authors AB to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review. Supplementary material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.02122/full#supplementary-material Table S1 BLASTp was used to identify homologs of the B. toyonensis 7112 T TetM protein against sequences in the NCBI genome database (Altschul et al., 1990 ) . The data was downloaded in CVS format and uploaded into Excel. The data shows the Reference Sequence IDs, individual Sequence IDs, the description of the protein in the annotation file, the bacterial source, alignment score, E- value and % identity. Click here for additional data file. Table S2 BLASTp was used to identify homologs of the B. toyonensis 7112 T CatQ protein against sequences in the NCBI genome database (Altschul et al., 1990 ) . The data was downloaded in CVS format and uploaded into Excel. The data shows the Reference Sequence IDs, individual Sequence IDs, the description of the protein in the annotation file, the bacterial source, alignment score, E -value and % identity. Click here for additional data file. Figure S1 Neighbor Joining tree of homologs of the B. toyonensis BCT-7112 T TetM tetracycline resistance protein (Btoyo_0322) identified in the BLASTp analysis . The algorithm used produces an un-rooted tree (Saitou and Nei, 1987 ). The maximum allowed fraction of mismatched bases between any pair of sequences was 0.85. The evolutionary distance between two sequences was modeled as the expected fraction of amino acid substitutions per site, based on the fraction of mismatched amino acids in the aligned region (Grishin, 1995 ). Click here for additional data file. Figure S2 Neighbor Joining tree of the homologs of the B. toyonensis BCT-7112 T CatQ chloramphenicol resistance protein (Btoyo_4985) identified in the BLASTp analysis . The algorithm used produces an un-rooted tree (Saitou and Nei, 1987 ). The maximum allowed fraction of mismatched bases between any pair of sequences was 0.85. The evolutionary distance between two sequences was modeled as the expected fraction of amino acid substitutions per site, based on the fraction of mismatched amino acids in the aligned region (Grishin, 1995 ). Click here for additional data file. Figure S3 Minimum inhibitory concentrations (MICs) of strains of B. toyonensis on tetracycline (A–D) and chloramphenicol (E–H). (A/E) , BCT-7112 T ; (B/F) , BCT-7112Δtet; (C/G) , BCT-7112Δcat; (D/H) Rock1-3. The antibiotic concentrations used were (μg/ml): 0, red; 1, green; 2, dark blue; 4, yellow; 8, gray; 16, orange and 32, light blue. The MIC values are shown for each set of growth curves. The experiments were carried out a minimum of three times and representative data show on a linear rather than logarithmic graph to make it easier to compare growth profiles. Click here for additional data file. Figure S4 The impact of the gerIC–nucB intergenic region of plasmid pBCT77 on the tetracycline (A–D) and chloramphenicol (E–H) resistance profiles of the following strains: (A/E) , BCT-7112(pIGR1); (B/F) , BCT-7112Δtet(pIGR1); (C/G) , BCT-7112Δcat(pIGR1); (D/H) , Rock1-3(pIGR1). The antibiotic concentrations used were (μg/ml): 0, red; 1, green; 2, dark blue; 4, yellow; 8, gray; 16, orange and 32, light blue. The MIC values are shown for each set of growth curves. The experiments were carried out a minimum of three times and representative data show on a linear rather than logarithmic graph to make it easier to compare growth profiles. Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538665/

Wolves contribute to disease control in a multi-host system

We combine model results with field data for a system of wolves ( Canis lupus ) that prey on wild boar ( Sus scrofa ), a wildlife reservoir of tuberculosis, to examine how predation may contribute to disease control in multi-host systems. Results show that predation can lead to a marked reduction in the prevalence of infection without leading to a reduction in host population density since mortality due to predation can be compensated by a reduction in disease induced mortality. A key finding therefore is that a population that harbours a virulent infection can be regulated at a similar density by disease at high prevalence or by predation at low prevalence. Predators may therefore provide a key ecosystem service which should be recognised when considering human-carnivore conflicts and the conservation and re-establishment of carnivore populations. Introduction Infectious agents that can be transmitted to more than one host species form the majority of pathogens that infect wildlife, domestic and human systems 1 . Wildlife species play a key role in maintaining reservoirs of infection 2 and therefore disease management requires strategies to reduce transmission of pathogens from wildlife reservoirs to target hosts 1 . It has been shown that predation may contribute to disease control in multi-host systems leading to reduced spillover to livestock and human populations 3 , 4 . Therefore predators can provide a key ecosystem service that is often underappreciated by society 5 , 6 . Mathematical models have played a key role in uncovering the potential of predators to control zoonotic disease where theory has shown that predators may act to alter the epidemiological dynamics to decrease infected and increase susceptible host density and thereby reduce prevalence 4 , 7 , 8 . Furthermore, selective predation on infected individuals can reduce the force of infection and in extreme scenarios prevent pathogen establishment 9 , 10 . However, model analysis has also outlined scenarios in which predation may lead to an increase in disease prevalence – notably when the disease induces a long-lasting immune response 11 . This highlights the importance of understanding the case-specific infection dynamics of pathogens in reservoir populations that are subject to predation. Empirical evidence to underpin the theory on the interplay between predation and host infection is however limited. Hudson et al . 12 suggested that macroparasite incidence in grouse ( Lagopus lagopus scotica ) populations decreased when predator levels increased and Levi et al . 13 showed that increases in the incidence of Lyme disease correlated with a decline in small mammal predators. More recently, observational and experimental studies have indicated that parasites can increase host susceptibility to predation 8 , 14 (see 15 for a recent review). Therefore combining theory and empirical data at the system specific level has the potential to further clarify the role of predation in the control of infectious disease reservoirs in wildlife 6 . We investigate this by combining model results with field data for the case study system of wolves ( Canis lupus ) that prey on wild boar ( Sus scrofa ), a reservoir of tuberculosis, in Asturias in northern Spain. Animal tuberculosis (TB), caused by infection with Mycobacterium bovis and closely related members of the M . tuberculosis complex (MTC), is a widespread multi-host infection with a profile of moderately increasing prevalence among cattle herds in infected regions of western Europe (from 1.05% in 2010 to 1.49% herd prevalence in 2015 16 ). TB causes severe economic losses to the livestock industry due to movement restrictions and compulsory test and slaughter schemes 17 , 18 . TB also causes host mortality 19 and creates conservation concerns due to potential spillover to endangered species (e.g. to Iberian lynx) 20 , 21 . The role of wildlife reservoirs in maintaining TB is now well recognised with reservoir species including cervids in North America, European badgers ( Meles meles ) in the British Isles, brushtail possums ( Trichosurus vulpecula ) in New Zealand and buffalo ( Syncerus caffer ) in South Africa, among others 18 , 22 . In Europe, and in particular on the Iberian Peninsula, infection is maintained in a complex network of domestic and wild hosts, including abundant wild ungulates such as the Eurasian wild boar which acts as the primary reservoir of infection 18 , 23 , 24 . In multi-host settings, TB control at the wildlife-livestock interface often targets aspects such as direct and indirect contacts between host species 25 – 27 and TB control in reservoir hosts 28 . It has been shown that culling of wild boar can reduce TB prevalence in wild boar and sympatric host species 29 , 30 . However, the role of ecosystem functioning in regulating infection transmission has not been assessed in detail. The wolf is the most widely distributed top predator of the northern hemisphere 31 , 32 where wild boar and deer are its main prey 33 , 34 and wolf presence has been linked with lower ungulate prey densities 5 . It has also been found that when wolf populations decrease, wild boar populations tend to increase 35 , 36 (but see 37 ). Mathematical modelling studies have suggested that wolves may contribute to disease control in their prey in the case of Chronic Wasting Disease in North American deer ( Odocoileus sp ) 10 . Moreover, empirical evidence suggested that anthrax infection in bison ( Bison bison ) might increase wolf predation risk 38 . It has also been suggested that pathogens targeting the lung may predispose ungulate prey to wolf predation 39 , 40 . Hence, maintaining viable wolf populations might contribute to disease control in wildlife and thereby reduce transmission from wildlife reservoirs. Asturias, in north-western Spain, is an area with an established wolf population that occupies two-thirds of the region 41 . TB is present in Asturias although the current overall prevalence in wild boar (2–13%) and the level of generalised cases (17% from tests on 6 infected individuals) are lower than in TB-endemic regions of southern Spain where TB prevalence can be >50% (with 80% prevalence reported in some regions 42 ) and where a greater proportion (58%) of infected individuals are generalised 43 . Here we distinguish between individuals that are infected with TB (but not infectious) and those with generalised infection that can infect other individuals through direct contact and that shed infectious particles. Generalised individuals also suffer disease-induced mortality and their poor health increases their vulnerability to predation. Asturias is also a cattle-breeding region, with 360,735 heads in 16,312 herds in 2014 and TB is one of the main concerns of cattle farmers 44 , 45 . However, the potential role of wolf predation as a natural regulator of disease in wild ungulates is not widely recognised by farmers 46 . Asturias can therefore be used as a case study region in which to test the impact of wolf predation on TB prevalence in a wildlife reservoir species (wild boar) and on TB control in the target species (cattle). In this study we combine field observations from Asturias with mathematical modelling to test the hypothesis that TB prevalence is reduced in the presence of wolves compared to when wolves are absent. Moreover, the model findings allow us to explore the long-term impact of predation on TB control, to explain how compensatory population growth may result from a reduction in disease-induced mortality due to predation and as a consequence explain how information on prevalence and population density is necessary to assess the risk of spillover from wildlife reservoirs. The results provide important insights into the role predators can play in disease control and therefore inform on the debate related to human-carnivore conflicts and the conservation and re-establishment of carnivore populations 5 , 6 , 47 , 48 . Results Wolf population The annual number of reported wolf attacks on livestock increased from 1481 in 2000 to 3024 in 2014 (100% increase; Supplementary Information Fig. S1 ). Reports of wolf predation on livestock were unrelated to livestock numbers. Instead they correlated positively to the number of wolf packs and to wolves culled during the previous season 49 . Therefore we extrapolate wolf numbers from these wolf attack data using linear regression to ascertain the linear growth rate of the wolf population over this period (Fig. S1 ). Using the data on wolf numbers for 2003–2004 as 252 50 , we estimate the number of wolves in 2000 as 196 growing linearly to 392 in 2014. Wild boar population Areas with and without wolves had similar wild boar harvest rates in year 2000 (0.52 and 0.40 wild boar/km 2 , respectively). By 2014 harvest rates had increased to 0.85 in areas with wolves but had a greater increase to 1.32 in areas without wolves. Between 2008 and 2014, the wild boar hunting harvest grew steadily in areas without wolves but remained stable in areas with wolves. Specific hunting effort data is not available for the areas with and without wolves, however there is no known difference in the type of hunter typical to these areas as in Asturias as a whole, hunting is non-commercial and traditional among rural inhabitants 51 . The use of hunting statistics as a proxy for wild boar abundance trends is well-established in ungulates 51 – 54 . By this method we assume that wild boar density was 50% higher in areas without wolves than areas with wolves (Fig. 1 ). Therefore in the areas with wolves we estimate wild boar density as 1.65/km 2 in 2000 rising to 2.55/km 2 in 2014. In the areas without wolves we estimate wild boar density as 1.2/km 2 in 2000 rising to 3.6/km 2 in 2014. Figure 1 The mean annual wild boar hunting harvest/km 2 in Asturias, for the period 2000–2014. The dotted line represents areas where wolves are absent and the solid line those where wolves are present. Bars represent 95% confidence intervals. TB prevalence A total of 1051 wild boar sera were tested for antibodies against MTC, yielding a mean seroprevalence of 5.42% (95% CI 4.21–6.98) for the whole study period. The reduction in seroprevalence between periods was significant in sites with wolves (the southern more mountainous regions) where prevalence declined by 77% from 16.67% ± 7.47% in 2000–2007 to 3.87% ± 1.76% in 2008–2014 (Fisher's p < 0.0001). In sites without wolves prevalence was initially lower and no significant change in prevalence was recorded: 6.89% ± 10% in 2000–2007 and 3.08% ± 3.5% in 2008–2014 (Fig. S2 ). The mean annual cattle herd TB prevalence from 2005 to 2007 was 0.19%. Herd prevalence grew slightly in 2008–2014, reaching a mean of 0.22%. In areas with wolves, cattle TB herd prevalence remained almost stable during the study period (0.22% in 2005–2007; 0.19% in 2008–2014; with Yates Chi 2 = 0.45, 1 d.f., p = 0.5 indicating no significant difference between the prevalence levels). By contrast, in areas without wolves herd prevalence increased by 56% in the same period: 0.16% in 2005–2007; 0.25% in 2008–2014; with Yates Chi 2 = 7.18, 1 d.f., p = 0.0074 indicating that the difference between prevalence levels is significant (Fig. S2 ). The model comparison to data for regions with wolves The model results for the wild boar population density, TB prevalence and the percentage change in the level of pathogen in the environment in response to a linear increase in wolf density are shown in Fig. 2 for the period 2000–2014. As wolf density increases there is a decrease in TB prevalence from 17% in 2000 to 3.8% in 2014. This highlights that predation by wolves could be a key factor in reducing TB prevalence in wild boar. The level of generalised infection remains relatively constant at 29% of the total infected population throughout the study period. The reduction in prevalence leads to a more than 50% reduction in the level of pathogen in the environment by 2014. Model results indicate that wild boar density increased from 1.65/km 2 to 2.55/km 2 between 2000 and 2014 with the density starting to saturate from 2008. This is in close agreement with the observed data. Figure 2 Model results for the sub-region of Asturias inhabited by wolves. ( a ) Wolf numbers rise from 196 (2000) to 392 (2014) and then remain constant until 2050. ( b ) Wolf numbers rise from 196 (2000) to 392 (2014) and then decrease at the rate at which they increased until they die out. Initial conditions set wild boar and wolf densities to their 2000 values taken from the field data, and the initial prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) in 2000 is 17% (of which 29% are generalised). ( A ) changes in wild boar population density - total population (blue); total susceptible (green); total infected and generalised (black); infected (magenta); generalised (red); wolves (grey). ( B ) changes in total prevalence (black), infected prevalence (magenta) generalised prevalence (red). ( C ) % change in the density of environmental pathogen. For parameters see Supplementary Information. A key finding is that although wolf numbers increase, which will increase overall predation, there is also an increase in wild boar density. This increase in wild boar density can be attributed to an assumption that wild boar were below their carrying capacity in 2000 and so positive growth would be expected, but also because predation decreases TB prevalence and therefore decreases the population level mortality due to TB. Hence, the increased mortality due to predation is compensated by a reduced TB induced mortality. An implication of the approximately two-fold increase in wild boar population density and four-fold decrease in prevalence is that the level of pathogen in the environment decreases by more than 50% over the 14-year period. This is significant since a reduction in the free-living particles reduces the risk of infection in other animals, in particular livestock, which share the same environment as the wild boar. The pronounced reduction in TB prevalence (from 17% in 2000 to 3.8% in 2014) assumes selective predation by wolves on wild boar piglets and generalised individuals. In comparison (Supplementary Information and Fig. S3 ), if wolves prey indiscriminately on all wild boar classes the prevalence reduction is 17% to 8.3% but the wild boar density only grows to 2.10/km 2 in 2007 before declining to 1.93/km 2 in 2014. If wolves prey on piglets only prevalence shows a reduction from 17 to 9.5% over the 2000 to 2014 period (Fig. S4 ). The model results therefore suggest that predation on generalised individuals is key to the significant reduction of prevalence since the removal of generalised individuals reduces infection from both direct contact and environmental contamination. The impact of wolves on TB prevalence in the long-term We examine the long-term impact of predation by wolves on TB prevalence in wild boar for different trends of wolf density (Fig. 2 ). In Fig. 2a we assume wolf numbers remain constant after 2014 (reflecting that wild boar are a key component of wolf diet). There is a small increase in wild boar density due to reduced disease-induced mortality as a consequence of the further reduction in TB prevalence, but in general, predation by wolves is sufficient to stabilise wild boar numbers. TB prevalence and the level of environmental pathogen decrease to low levels. This emphasises how predation can control virulent infection in a prey species and also reduce the risk of infection to other host species. In Fig. 2b we assume wolf density will decrease and reach zero in 2042. This represents a scenario where wolves are intentionally removed. Here, as wolf numbers initially decrease there is a rise in wild boar density with TB prevalence in wild boar remaining low. However, as wolf numbers decrease further TB prevalence increases leading to a downturn in wild boar density in response to increased disease induced mortality. It is notable that the final stable wild boar numbers in the absence of wolves (Fig. 2b ) are similar to the level in the presence of wolves (Fig. 2a ). However, a key difference is that TB prevalence is low (0.1%) in the presence of wolves and high (26%) in their absence. This has significant consequences for potential environmental transmission of MTC from wild boar to other species. The underlying mechanism responsible for this difference is that wild boar density is largely regulated by the disease in the absence of wolves whereas it is regulated by predation in their presence. This is a key insight from the mathematical model. It highlights how restrictions to predator growth may have only minor impacts on prey density but a major detrimental impact on the prevalence of infection in prey species. Model comparison to data in areas of Asturias without wolves The results for the model that reflect the region of Asturias in which wolves are absent are shown in Fig. 3 . Here, there is a rapid increase in wild boar density, with close to a 3-fold increase in density between 2000 and 2014 (which reflects the increase in density observed in the field data). TB prevalence initially remains constant (at around 3%) but from 2007 onwards shows an increasing trend reaching a prevalence of 7.8% by 2014. This relatively low increase in prevalence coupled with a large increase in population density leads to a large increase (over 500%) in the level of environmental pathogen and therefore a potentially increased risk of infection spillover to co-habiting domestic and wild animals. Figure 3 Model results for the sub-region of Asturias not inhabited by wolves. Initial conditions set wild boar and wolf densities to their 2000 values, and the initial prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) in 2000 is 3% (of which 30% are generalised). ( a ) Changes in wild boar population density - total population (blue); total susceptible (green); total infected and generalised (black); infected (magenta); generalised (red); wolves (grey). ( b ) Changes in total prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) (black); infected prevalence ( I / N ) (magenta); generalised prevalence ( G / N ) (red). ( c ) % change in the density of environmental pathogen. For parameters see Supplementary Information. The potential impact of predation in regions of high TB prevalence in wild boar To represent areas of high TB prevalence we modify the baseline parameters for Asturias to reflect increased prevalence and generalised infection. In such regions wild boar density is typically high (due to management and artificial feeding) even though environmental conditions are harsh and in particular severely diminished water availability necessitates the sharing of water holes and leads to overall poor body condition 55 . This increases the level of environmental transmission and leads to a more rapid transition from the infected to the generalised class for piglets and yearlings 28 (see also Supplementary Information). We assume here that wild boar live at an endemic density 8/km 2 , and adjust K and q to reflect this (see Supplementary Information). Other parameters remain as in the set-up in Asturias and in particular note that to maintain the comparison with Asturias we do not change the background culling rate. In the absence of wolves the model results indicate a prevalence of 57% of which around 54% are individuals with generalised infection (this is in good agreement with Muñoz-Mendoza et al . 43 ). In Fig. S5 we introduce wolves at a constant density of 0.08/km 2 which represents an initial wolf to wild boar ratio of 1:100. Initial predation by wolves reduces wild boar density, but primarily affects infected and generalised individuals. This causes a reduction in TB prevalence and therefore reduced population level disease-induced mortality. This drives an increase in susceptible individuals and an increase in wild boar population density which promotes a resurgence in disease prevalence. Infection and population recovery oscillates until after 50 years the population has increased wild boar numbers (10.1/km 2 ), reduced TB prevalence to 26.5% and reduced levels of environmental pathogen by 54%. Figure 4 shows the impact of wolf density on the steady state level of wild boar density, disease prevalence and environmental contamination. In the absence of wolves the model results indicate a prevalence of 57% of which around 54% are individuals with generalised infection (this is in good agreement with 43 for wild boar TB prevalence in Mediterranean Spain). As wolf numbers increase the level of disease prevalence and risk of environmental contamination decrease. However, the density of wild boar increases as wolf density (and predation) increases. This increase in wild boar density is a direct result of the decrease in TB prevalence as the mortality from predation is lower than disease induced mortality due to TB that was experienced in the absence of wolves. There is a threshold in wolf density that leads to disease eradication and for wolf densities above this threshold there is a decrease in wild boar density (since mortality from predation is no longer compensated following eradication of the disease). Figure 4 Model results for areas with high TB prevalence showing the long-term outcome after different constant densities of wolves are introduced to a wild boar population with density steady at 8/km 2 and disease prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) of 57%. ( a ) Changes in wild boar population density - total population (blue); total susceptible (green); infected (magenta); and generalised (red). ( b ) Changes in total prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) (black); infected prevalence ( I / N ) (magenta) generalised prevalence ( G / N ) (red). ( c ) % change in the density of environmental pathogen. For parameters see Supplementary Information. Wolf population The annual number of reported wolf attacks on livestock increased from 1481 in 2000 to 3024 in 2014 (100% increase; Supplementary Information Fig. S1 ). Reports of wolf predation on livestock were unrelated to livestock numbers. Instead they correlated positively to the number of wolf packs and to wolves culled during the previous season 49 . Therefore we extrapolate wolf numbers from these wolf attack data using linear regression to ascertain the linear growth rate of the wolf population over this period (Fig. S1 ). Using the data on wolf numbers for 2003–2004 as 252 50 , we estimate the number of wolves in 2000 as 196 growing linearly to 392 in 2014. Wild boar population Areas with and without wolves had similar wild boar harvest rates in year 2000 (0.52 and 0.40 wild boar/km 2 , respectively). By 2014 harvest rates had increased to 0.85 in areas with wolves but had a greater increase to 1.32 in areas without wolves. Between 2008 and 2014, the wild boar hunting harvest grew steadily in areas without wolves but remained stable in areas with wolves. Specific hunting effort data is not available for the areas with and without wolves, however there is no known difference in the type of hunter typical to these areas as in Asturias as a whole, hunting is non-commercial and traditional among rural inhabitants 51 . The use of hunting statistics as a proxy for wild boar abundance trends is well-established in ungulates 51 – 54 . By this method we assume that wild boar density was 50% higher in areas without wolves than areas with wolves (Fig. 1 ). Therefore in the areas with wolves we estimate wild boar density as 1.65/km 2 in 2000 rising to 2.55/km 2 in 2014. In the areas without wolves we estimate wild boar density as 1.2/km 2 in 2000 rising to 3.6/km 2 in 2014. Figure 1 The mean annual wild boar hunting harvest/km 2 in Asturias, for the period 2000–2014. The dotted line represents areas where wolves are absent and the solid line those where wolves are present. Bars represent 95% confidence intervals. TB prevalence A total of 1051 wild boar sera were tested for antibodies against MTC, yielding a mean seroprevalence of 5.42% (95% CI 4.21–6.98) for the whole study period. The reduction in seroprevalence between periods was significant in sites with wolves (the southern more mountainous regions) where prevalence declined by 77% from 16.67% ± 7.47% in 2000–2007 to 3.87% ± 1.76% in 2008–2014 (Fisher's p < 0.0001). In sites without wolves prevalence was initially lower and no significant change in prevalence was recorded: 6.89% ± 10% in 2000–2007 and 3.08% ± 3.5% in 2008–2014 (Fig. S2 ). The mean annual cattle herd TB prevalence from 2005 to 2007 was 0.19%. Herd prevalence grew slightly in 2008–2014, reaching a mean of 0.22%. In areas with wolves, cattle TB herd prevalence remained almost stable during the study period (0.22% in 2005–2007; 0.19% in 2008–2014; with Yates Chi 2 = 0.45, 1 d.f., p = 0.5 indicating no significant difference between the prevalence levels). By contrast, in areas without wolves herd prevalence increased by 56% in the same period: 0.16% in 2005–2007; 0.25% in 2008–2014; with Yates Chi 2 = 7.18, 1 d.f., p = 0.0074 indicating that the difference between prevalence levels is significant (Fig. S2 ). The model comparison to data for regions with wolves The model results for the wild boar population density, TB prevalence and the percentage change in the level of pathogen in the environment in response to a linear increase in wolf density are shown in Fig. 2 for the period 2000–2014. As wolf density increases there is a decrease in TB prevalence from 17% in 2000 to 3.8% in 2014. This highlights that predation by wolves could be a key factor in reducing TB prevalence in wild boar. The level of generalised infection remains relatively constant at 29% of the total infected population throughout the study period. The reduction in prevalence leads to a more than 50% reduction in the level of pathogen in the environment by 2014. Model results indicate that wild boar density increased from 1.65/km 2 to 2.55/km 2 between 2000 and 2014 with the density starting to saturate from 2008. This is in close agreement with the observed data. Figure 2 Model results for the sub-region of Asturias inhabited by wolves. ( a ) Wolf numbers rise from 196 (2000) to 392 (2014) and then remain constant until 2050. ( b ) Wolf numbers rise from 196 (2000) to 392 (2014) and then decrease at the rate at which they increased until they die out. Initial conditions set wild boar and wolf densities to their 2000 values taken from the field data, and the initial prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) in 2000 is 17% (of which 29% are generalised). ( A ) changes in wild boar population density - total population (blue); total susceptible (green); total infected and generalised (black); infected (magenta); generalised (red); wolves (grey). ( B ) changes in total prevalence (black), infected prevalence (magenta) generalised prevalence (red). ( C ) % change in the density of environmental pathogen. For parameters see Supplementary Information. A key finding is that although wolf numbers increase, which will increase overall predation, there is also an increase in wild boar density. This increase in wild boar density can be attributed to an assumption that wild boar were below their carrying capacity in 2000 and so positive growth would be expected, but also because predation decreases TB prevalence and therefore decreases the population level mortality due to TB. Hence, the increased mortality due to predation is compensated by a reduced TB induced mortality. An implication of the approximately two-fold increase in wild boar population density and four-fold decrease in prevalence is that the level of pathogen in the environment decreases by more than 50% over the 14-year period. This is significant since a reduction in the free-living particles reduces the risk of infection in other animals, in particular livestock, which share the same environment as the wild boar. The pronounced reduction in TB prevalence (from 17% in 2000 to 3.8% in 2014) assumes selective predation by wolves on wild boar piglets and generalised individuals. In comparison (Supplementary Information and Fig. S3 ), if wolves prey indiscriminately on all wild boar classes the prevalence reduction is 17% to 8.3% but the wild boar density only grows to 2.10/km 2 in 2007 before declining to 1.93/km 2 in 2014. If wolves prey on piglets only prevalence shows a reduction from 17 to 9.5% over the 2000 to 2014 period (Fig. S4 ). The model results therefore suggest that predation on generalised individuals is key to the significant reduction of prevalence since the removal of generalised individuals reduces infection from both direct contact and environmental contamination. The impact of wolves on TB prevalence in the long-term We examine the long-term impact of predation by wolves on TB prevalence in wild boar for different trends of wolf density (Fig. 2 ). In Fig. 2a we assume wolf numbers remain constant after 2014 (reflecting that wild boar are a key component of wolf diet). There is a small increase in wild boar density due to reduced disease-induced mortality as a consequence of the further reduction in TB prevalence, but in general, predation by wolves is sufficient to stabilise wild boar numbers. TB prevalence and the level of environmental pathogen decrease to low levels. This emphasises how predation can control virulent infection in a prey species and also reduce the risk of infection to other host species. In Fig. 2b we assume wolf density will decrease and reach zero in 2042. This represents a scenario where wolves are intentionally removed. Here, as wolf numbers initially decrease there is a rise in wild boar density with TB prevalence in wild boar remaining low. However, as wolf numbers decrease further TB prevalence increases leading to a downturn in wild boar density in response to increased disease induced mortality. It is notable that the final stable wild boar numbers in the absence of wolves (Fig. 2b ) are similar to the level in the presence of wolves (Fig. 2a ). However, a key difference is that TB prevalence is low (0.1%) in the presence of wolves and high (26%) in their absence. This has significant consequences for potential environmental transmission of MTC from wild boar to other species. The underlying mechanism responsible for this difference is that wild boar density is largely regulated by the disease in the absence of wolves whereas it is regulated by predation in their presence. This is a key insight from the mathematical model. It highlights how restrictions to predator growth may have only minor impacts on prey density but a major detrimental impact on the prevalence of infection in prey species. Model comparison to data in areas of Asturias without wolves The results for the model that reflect the region of Asturias in which wolves are absent are shown in Fig. 3 . Here, there is a rapid increase in wild boar density, with close to a 3-fold increase in density between 2000 and 2014 (which reflects the increase in density observed in the field data). TB prevalence initially remains constant (at around 3%) but from 2007 onwards shows an increasing trend reaching a prevalence of 7.8% by 2014. This relatively low increase in prevalence coupled with a large increase in population density leads to a large increase (over 500%) in the level of environmental pathogen and therefore a potentially increased risk of infection spillover to co-habiting domestic and wild animals. Figure 3 Model results for the sub-region of Asturias not inhabited by wolves. Initial conditions set wild boar and wolf densities to their 2000 values, and the initial prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) in 2000 is 3% (of which 30% are generalised). ( a ) Changes in wild boar population density - total population (blue); total susceptible (green); total infected and generalised (black); infected (magenta); generalised (red); wolves (grey). ( b ) Changes in total prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) (black); infected prevalence ( I / N ) (magenta); generalised prevalence ( G / N ) (red). ( c ) % change in the density of environmental pathogen. For parameters see Supplementary Information. The potential impact of predation in regions of high TB prevalence in wild boar To represent areas of high TB prevalence we modify the baseline parameters for Asturias to reflect increased prevalence and generalised infection. In such regions wild boar density is typically high (due to management and artificial feeding) even though environmental conditions are harsh and in particular severely diminished water availability necessitates the sharing of water holes and leads to overall poor body condition 55 . This increases the level of environmental transmission and leads to a more rapid transition from the infected to the generalised class for piglets and yearlings 28 (see also Supplementary Information). We assume here that wild boar live at an endemic density 8/km 2 , and adjust K and q to reflect this (see Supplementary Information). Other parameters remain as in the set-up in Asturias and in particular note that to maintain the comparison with Asturias we do not change the background culling rate. In the absence of wolves the model results indicate a prevalence of 57% of which around 54% are individuals with generalised infection (this is in good agreement with Muñoz-Mendoza et al . 43 ). In Fig. S5 we introduce wolves at a constant density of 0.08/km 2 which represents an initial wolf to wild boar ratio of 1:100. Initial predation by wolves reduces wild boar density, but primarily affects infected and generalised individuals. This causes a reduction in TB prevalence and therefore reduced population level disease-induced mortality. This drives an increase in susceptible individuals and an increase in wild boar population density which promotes a resurgence in disease prevalence. Infection and population recovery oscillates until after 50 years the population has increased wild boar numbers (10.1/km 2 ), reduced TB prevalence to 26.5% and reduced levels of environmental pathogen by 54%. Figure 4 shows the impact of wolf density on the steady state level of wild boar density, disease prevalence and environmental contamination. In the absence of wolves the model results indicate a prevalence of 57% of which around 54% are individuals with generalised infection (this is in good agreement with 43 for wild boar TB prevalence in Mediterranean Spain). As wolf numbers increase the level of disease prevalence and risk of environmental contamination decrease. However, the density of wild boar increases as wolf density (and predation) increases. This increase in wild boar density is a direct result of the decrease in TB prevalence as the mortality from predation is lower than disease induced mortality due to TB that was experienced in the absence of wolves. There is a threshold in wolf density that leads to disease eradication and for wolf densities above this threshold there is a decrease in wild boar density (since mortality from predation is no longer compensated following eradication of the disease). Figure 4 Model results for areas with high TB prevalence showing the long-term outcome after different constant densities of wolves are introduced to a wild boar population with density steady at 8/km 2 and disease prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) of 57%. ( a ) Changes in wild boar population density - total population (blue); total susceptible (green); infected (magenta); and generalised (red). ( b ) Changes in total prevalence ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(I+G)$$\end{document} ( I + G ) / N ) (black); infected prevalence ( I / N ) (magenta) generalised prevalence ( G / N ) (red). ( c ) % change in the density of environmental pathogen. For parameters see Supplementary Information. Discussion In this study we combine field data and theory for a case study system to confirm the hypothesis that the presence of wolves can lead to a reduction in TB prevalence compared to when they are absent. Our findings indicate that wolf predation may contribute to TB control in wild boar, reducing TB prevalence and the release of MTC into the environment. These factors are likely to contribute to reduced levels of indirect transmission from the wild boar infection reservoir to other hosts. The results have wide-ranging implications that highlight how predation can play a key role in the control of infectious disease in multi-host systems. It has been postulated that MTC transmission between wild and domestic hosts is mostly indirect, mediated by contaminated vegetation, water, mud, feed or other substrates 18 , 55 . Wild boar are the primary reservoir host for MTC in Spain with infection to other host species likely to be through indirect transmission in regions where multiple hosts overlap 56 . Wild boar are relatively long-lived 57 and older age classes can mount a formidable defence against predation. Therefore wolves are likely to select generalised (severely infected) individuals (which are the class responsible for shedding pathogen to the environment 55 ) or piglets (which is an age group more likely to suffer generalised infection 58 ). Such selective predation has been suggested as a key mechanism which can decrease infection prevalence in prey 3 and was shown to lead to reduced prevalence of prion disease in cervids without a dramatic decrease in their density 10 . Our field observations and model study show that there is a reduction in wild boar disease prevalence without a consequent reduction in wild boar density in regions where wolves might selectively target piglets and generalised wild boar. Our results indicate that the decrease in prevalence would be less pronounced if predation targeted all classes indiscriminately or if it targeted only piglets. Therefore, our results support previous findings 3 , 10 that suggest the ability of predators to preferentially select the most infected prey may be key to their role in disease control. Moreover, our findings suggest that wolves could play a key role in TB control in wildlife reservoirs in Spain. In Asturias, the annual cost of compensation paid to farmers due to wolf attacks on their livestock (€1,016,860) is a quarter of the annual expenses of the cattle TB eradication scheme (€4,163,348; Regional Government 2014). The ecosystem service provided by predators in terms of disease control should form part of the debate when discussing the impact of predators since here wolves may be allies of farmers, rather than enemies. In the absence of wolves (Fig. 3 ), wild boar numbers increase significantly. Model results indicate that there is a lag between the increase in wild boar growth and the increase in TB prevalence since the increase in infected individuals has a similar increasing trend to that of the overall population. This could explain the observation that TB prevalence in wild boar in the absence of wolves has remained relatively fixed. Note, however, that while TB prevalence in wild boar has remained constant the model predicts that the density of generalised wild boar and the presence of MTC in the environment increases throughout the study period. It is notable that the empirical findings for areas of Asturias in which wolves are absent show that there is a near five-fold increase in TB detected in cattle between 2000–2014. Our model provides an explanation for how a small percentage increase in prevalence coupled with a large increase in population density in a reservoir population may lead to a large increase in environmental contamination. This could explain the observed increase in cattle TB in these regions. The model system was adapted to examine the potential impact of predation on disease control beyond the Asturias case study system (Fig. 4 ). In areas with high TB prevalence such as central and southern Spain, the observed prevalence of TB is 50% and an increased proportion of those infected exhibit generalised infection (58%). Since predators may select the most severely infected individuals there is the potential for predation to have a greater impact on disease control in such settings. More specifically, as there is a higher prevalence of generalised individuals, there will proportionately be more predation on these super-shedders and therefore the potential to have an exaggerated effect on removing the wild boar that are responsible for shedding the pathogen in the environment, thus having greater potential to reduce spillover to other wild and domestic hosts. In this scenario our model results show that predation by wolves does lead to an exaggerated reduction in disease prevalence while leading to an increase in overall population density and reduction in the level of environmental pathogen. This increase in wild boar density is a direct result of the decrease in TB prevalence as the mortality from predation is lower than disease induced mortality due to TB that was experienced in the absence of wolves. This emphasises the generality of our findings and further highlights the potential role of predators in disease control. Previous theoretical studies that have shown that, in disease regulated populations, predation can reduce the force of infection and thereby decrease the density of infected hosts, increase the density of susceptible hosts and lead to an increase in overall population density 3 , 10 . Our model study shows that increased mortality from predation is approximately balanced by a reduction in disease-induced mortality. A key result is therefore that the prey population can be regulated by the disease, with consequent high prevalence in the prey species or at a similar density by a predator but with low disease prevalence. This finding highlights how restrictions to predator growth may have only minor impacts on prey density but a major detrimental impact on the prevalence of infection in prey species. The mechanism that underlies the compensatory balance between predation and disease induced mortality has recently been explained in systems subject to culling/harvesting 59 . Tanner et al . 59 show that in systems that lack long-lived immunity to infection, population reductions from harvesting are compensated due to a population level release from disease-induced mortality. The compensatory effect increases as disease virulence increases and occurs for systems with density-dependent, frequency-dependent and environmental (free-living) modes of transmission. They explain how harvesting in systems that harbour virulent parasites can lower disease prevalence without significantly reducing, or indeed can increase population density. Our findings show how mortality from predation is compensated by a release from disease-induced mortality that can reduce TB prevalence and the potential spillover of infection to sympatric hosts 29 , 30 . Tanner et al . 59 also show that in systems in which individuals develop long-lasting immunity following infection harvesting leads to a significant reduction in population density and an increase in infected prevalence and agree with theory that examines the impact of predation in systems with long-lasting immunity 11 . This highlights the necessity to understand the system specific host infection dynamics that are subject to predation or harvesting 59 – 61 . Our results agree with earlier findings that the removal of a predator from a system that is regulated by both predator-prey interactions and virulent infection may increase disease prevalence and suppress prey abundance 3 , 12 , 13 . Our model results suggest that in the initial years of wolf removal wild boar density can increase and disease prevalence stays low. This may indicate that predator removal can be beneficial, however this is only a transitory state. When the wolf reaches sufficiently low numbers the disease is able to re-infect the increased abundance of susceptibles so that over time the population becomes regulated by disease rather than predation. This is accompanied by an increase in environmental contamination and risk of spillover to other wild and domestic hosts. This further highlights the complexity and potential negative consequences of predator removal and the need to consider disease status in predator management programmes. Our modelling results show good agreement with the field data for our case study system. We expect our general findings relating to reduced prevalence of TB and compensatory growth of wild boar in the face of predation to be robust to changes in our model assumptions. The key requirement is that TB is virulent and individuals do not recover to develop long-lasting immunity 59 . There are however specific aspects where the model and field study disagree. The model differs from field data in that it predicts a prevalence of generalised individuals of 25–30% whereas existing data for Asturias suggests 16.7% 43 . However, this lower prevalence was derived from a small data set (1 out of 6 being reported as generalised) and recent results from Asturias (personal communication, unpublished findings by the Asturias Government) would now indicate a higher prevalence of generalised in closer agreement with model findings. Also, in areas with wolves the empirical results indicated that cattle TB stayed constant rather than declining. The model results indicated that there would be an increase in wild boar density, a reduction in TB prevalence in wild boar and a reduction in generalised infected wild boar and MTC in the environment, therefore reducing the risk of transmission of MTC to livestock. This can be explained by: firstly, the wildlife reservoir in the Atlantic regions of Spain is composed of two main hosts, wild boar and badger 43 , and wolves are not likely to significantly interfere with badger population dynamics; secondly, the wildlife reservoir contributes to MTC maintenance, but is not the only driver. In Spain, the relative contribution of wildlife to cattle TB breakdowns varies between regions depending on the epidemiological circumstances 62 , 63 . Cattle movements, for instance, are likely to contribute to TB maintenance 64 . Our study has highlighted the potential of predation by wolves to reduce TB prevalence in wild boar and thereby reduce the risk of transmission from a key wildlife reservoir of infection. The model framework developed in this study was tailored to the wild boar TB wolf system but the underlying processes that represent the population and epidemiological dynamics are general and therefore we expect the results to apply more broadly. In particular, when predation can regulate a prey species that was previously regulated by virulent pathogens it is likely that infection levels will be reduced. Of course, care must be taken when considering the impact of generalist predators on disease control as they may also prey on alternative species that do not harbour virulent pathogens and therefore where mortality due to predation will not be compensated. Nevertheless, the potential of predators to control infection should be recognised more widely and be contrasted with the detrimental impact of predatory losses to domestic species. The beneficial role of predators should be given more prominence particularly given the need to manage conservation conflicts associated with predator re-establishment 65 . Methods Ethics statement All animal sampling took place post-mortem. The wildlife samples were obtained from hunter-harvested individuals that were shot in the legal hunting seasons and independently and prior to our research. According to EU and National legislation (2010/63/UE Directive and Spanish Royal Decree 53/2013) and to the University of Castilla-La Mancha guidelines, no permission or consent is required to conduct the research reported herein. Study area and target species Asturias, a province of 10,604 km 2 , is located in northwestern Spain (Fig. 5 ). Wolf population data were obtained from the Asturias Government. Wolf presence is established in two-thirds of Asturias. In the remaining third, containing the majority of coastal regions and the urban and industrial corridors in the centre-north-east of the region (Fig. 5 66 , 67 ), wolves are absent or only sporadically recorded. The annual wolf census uses simulated howling and fixed observation points to map wolf packs (methodology detailed in the Asturias Government report 50 ) and allows for an estimate of wolf population size 50 . We combine the estimate of wolf abundance for 2003–2004 50 with data on wolf attack rate on livestock to give a profile of wolf abundance from 2000 to 2014. The regional government also records the number of wild boar harvested on hunting sites annually 51 . Hunting is predominantly non-commercial and traditional among rural inhabitants, taking place in 17 game reserves and 60 municipal hunting estates covering 91% of the province 51 . After standardisation by hunting effort, hunting bag statistics can be used as reliable indices of wild boar relative abundance 68 . We use data describing the temporal variation in the number of wild boar annually hunted (Fig. 1 ) and in particular generate estimates of wild boar population abundance in 2000–01 and 2013–14. Figure 5 Wolf ( Canis lupus ) distribution maps where the distribution in the Iberian Peninsula is shown in light grey, and municipalities in Asturias, northern Spain, are expanded to show where wolves are present (dark grey) or absent (white). TB prevalence We used serum antibodies against the MTC as an indicator of TB prevalence in wild boar. Serum samples were tested by means of an indirect ELISA using bovine-purified protein derivative (bPPD) following the protocol previously described in Boadella et al . 69 . Sample results were expressed as an ELISA percentage (E%) that was calculated using the formula [Sample E% = (sample OD/2 × mean negative control OD) × 100]. Serum samples with E% values greater than 100 were considered positive. Wild boar TB prevalence was available at the municipality level from 2000 to 2014. All cattle herds are tested annually for TB by individual skin testing. This testing is performed and recorded by the Asturias Government. Individual and herd-level data on cattle TB was available from 2005 to 2014, at the municipality scale. Asturias: estimating wolf population We derive an estimate of wolf population abundance for the period 2000–2014 as follows. We use data on wolf population abundance for the period 2003–2004 50 to obtain an estimate of 252 wolves in Asturias in 2004. We fit a least squares regression on wolf attack rate data for the period 2000–2014 (Fig. S1 ) to give a rate of increase of wolf abundance. Combining this rate of increase with our wolf abundance estimate for 2004 we estimate that wolf numbers increase linearly from 196 in 2000 to 392 in 2014. Given that both wolf density and wild boar densities are rising and not saturating, and that although wild boar are a preferred prey target for wolves they do not make up the whole wolf diet 33 , we assume that the wolf attack rate on wild boar has not reached saturation levels and therefore model wolf attack rates on wild boar linearly with wolf density. Mathematical modelling We develop a mathematical model that represents the interaction between wild boar, MTC infection and predation. In the model we set disease transmission rates and the wild boar intraspecific competition parameter so the model matches observations for the prevalence of infection and for wild boar density in 2000 and 2014 for the regions of Asturias with wolves. The model findings are extended to consider the areas of Asturias in which wolves are absent, to assess the role of future wolf density in TB control and the potential impact of wolf predation on TB in regions where TB is endemic and prevalence is high. We separate the population density of wild boar into different age classes to capture distinct disease and reproductive characteristics for piglets (aged 0–1 year) P , yearlings (aged 1–2 years) Y , and adults (aged 2 years+) A . Further, the age-classes are split into susceptible, infected and generalised classes (subscripts S , I , G , respectively) to reflect the disease status of the population. Generalised individuals can also release free-living pathogen with density F into the environment. The three different age-classes are required as each class has distinct properties in terms of their demographic and infection dynamics and in the impact of predation. This model framework has been used successfully to understand the impact of vaccination and culling on TB prevalence in the wild boar TB system 28 , 59 . The model also includes predation by wolves, W , and we examine scenarios where wolf density increases from 2000–2014 according to observations and then remains constant or decreases (to represent persecution) thereafter. The population dynamics of the wild boar, TB and wolf system are represented by the following set of non-linear differential equations (which is an extension of classical disease modelling frameworks 70 , 71 ) and of a previous model of wild boar and TB interactions 28 , 59 . 1a \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{S}}{{ m{d}}t}={b}_{A}(Y+A)\,(1-qN)-m{P}_{S}-{d}_{P}{P}_{S}-{eta }_{DP}{P}_{S} rac{G}{N}-\omega {eta }_{FP}{P}_{S}F-{a}_{P}{P}_{S}W$$\end{document} d P S d t = b A ( Y + A ) ( 1 − q N ) − m P S − d P P S − β D P P S G N − ω β F P P S F − a P P S W 1b \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{I}}{{ m{d}}t}={eta }_{DP}{P}_{S} rac{G}{N}+\omega {eta }_{FP}{P}_{S}F-m{P}_{I}-{d}_{P}{P}_{I}-{ arepsilon }_{P}{P}_{I}-{a}_{P}{P}_{I}W$$\end{document} d P I d t = β D P P S G N + ω β F P P S F − m P I − d P P I − ε P P I − a P P I W 1c \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{G}}{{ m{d}}t}={ arepsilon }_{P}{P}_{I}-m{P}_{G}-lpha {P}_{G}-{d}_{P}{P}_{G}-{a}_{PG}{P}_{G}W$$\end{document} d P G d t = ε P P I − m P G − α P G − d P P G − a P G P G W 1d \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{S}}{{ m{d}}t}=m{P}_{S}-m{Y}_{S}-{d}_{Y}{Y}_{S}-{eta }_{DY}{Y}_{S} rac{G}{N}-\omega {eta }_{FY}{Y}_{S}F-c{Y}_{S}-{a}_{YA}{Y}_{S}W$$\end{document} d Y S d t = m P S − m Y S − d Y Y S − β D Y Y S G N − ω β F Y Y S F − c Y S − a Y A Y S W 1e \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{I}}{{ m{d}}t}={eta }_{DY}{Y}_{S} rac{G}{N}+\omega {eta }_{FY}{Y}_{S}F+m{P}_{I}-m{Y}_{I}-{d}_{Y}{Y}_{I}-{ arepsilon }_{Y}{Y}_{I}-c{Y}_{I}-{a}_{YA}{Y}_{I}W$$\end{document} d Y I d t = β D Y Y S G N + ω β F Y Y S F + m P I − m Y I − d Y Y I − ε Y Y I − c Y I − a Y A Y I W 1f \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{G}}{{ m{d}}t}={ arepsilon }_{Y}{Y}_{I}+m{P}_{G}-m{Y}_{G}-lpha {Y}_{G}-{d}_{Y}{Y}_{G}-c{Y}_{G}-{a}_{G}{Y}_{G}W$$\end{document} d Y G d t = ε Y Y I + m P G − m Y G − α Y G − d Y Y G − c Y G − a G Y G W 1g \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{S}}{{ m{d}}t}=m{Y}_{S}-{d}_{A}{A}_{S}-{eta }_{DA}{A}_{S} rac{G}{N}-\omega {eta }_{FA}{A}_{S}F-c{A}_{S}-{a}_{YA}{A}_{S}W$$\end{document} d A S d t = m Y S − d A A S − β D A A S G N − ω β F A A S F − c A S − a Y A A S W 1h \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{I}}{{ m{d}}t}={eta }_{DA}{A}_{S} rac{G}{N}+\omega {eta }_{FA}{A}_{S}F+m{Y}_{I}-{d}_{A}{A}_{I}-{ arepsilon }_{A}{A}_{I}-c{A}_{I}-{a}_{YA}{A}_{I}W$$\end{document} d A I d t = β D A A S G N + ω β F A A S F + m Y I − d A A I − ε A A I − c A I − a Y A A I W 1i \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{G}}{{ m{d}}t}={ arepsilon }_{A}{A}_{I}+m{Y}_{G}-lpha {A}_{G}-{d}_{A}{A}_{G}-c{A}_{G}-{a}_{G}{A}_{G}W$$\end{document} d A G d t = ε A A I + m Y G − α A G − d A A G − c A G − a G A G W 1j \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$W=W(t)$$\end{document} W = W ( t ) Here, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$N=P+Y+A$$\end{document} N = P + Y + A represents the total wild boar population where \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$P={P}_{S}+{P}_{I}+{P}_{G}$$\end{document} P = P S + P I + P G , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$Y={Y}_{S}+{Y}_{I}+{Y}_{G}$$\end{document} Y = Y S + Y I + Y G , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$A={A}_{S}+{A}_{I}+{A}_{G}$$\end{document} A = A S + A I + A G and G is the total number of generalised, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$G={P}_{G}+{Y}_{G}+{A}_{G}$$\end{document} G = P G + Y G + A G . Susceptible and infected yearlings and adults give birth to susceptible piglets at rates b Y and b A respectively. Generalised yearlings and adults give birth to piglets at rate b G . Here we assume that \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${b}_{A}={b}_{Y}={b}_{G}$$\end{document} b A = b Y = b G . The total population is regulated through a crowding parameter, q , that acts on the birth rate. Maturity from piglets to yearlings and yearlings to adults occurs at rate m and piglets, yearlings and adults may die of natural causes at rates d P , d Y , d A respectively. Here we assume d P = d Y = d A . This set-up for the demographic dynamics has previously been used to assess wild boar TB interactions 28 , 59 . We assume infection can occur through direct frequency-dependent interactions (since wild boar tend to congregate in social groups) between susceptible and generalised individuals with transmission coefficients β DP , β DY and β DA or through environmental contact with free-living MTC, with transmission coefficients β FP , β FY and β FA for the different age classes respectively. Piglets and yearlings are more likely to become infected through both direct and environmental infection than adults 58 , and we assume that they are three times more susceptible to infection than adults to ensure that the model presents sufficient levels of infection in yearlings as compared to adults. In this way we have set the model so that the yearling class is the same as the piglet class in terms of disease characteristics, but the yearling class is the same as the adult class in terms of reproductive processes. Infected individuals are not infectious but can progress to the generalised (infectious) class at rates \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}$$\end{document} ε P , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{Y}$$\end{document} ε Y and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{A}$$\end{document} ε A . In Asturias where resources are not limited we assume \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}={ arepsilon }_{Y}={ arepsilon }_{A}$$\end{document} ε P = ε Y = ε A . Later we consider regions where resources (particularly water) are scarce and overall health is impaired (similar to conditions in central and southern Spain). Co-infection can lead to a greater risk of becoming a super-shedder 72 and we assume that piglets and yearlings progress from the infected to the generalised class at three times the rate of adults ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}={ arepsilon }_{Y}=3{ arepsilon }_{A}$$\end{document} ε P = ε Y = 3 ε A ). We assume that free-living MTC is shed from generalised wild boar at rate λ and decays at rate μ . The level of environmental transmission is scaled through the parameter \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega $$\end{document} ω which increases when environmental conditions become more severe to reflect, for example, aggregation at limited water holes ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega =0.1$$\end{document} ω = 0.1 in Asturias and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega =1$$\end{document} ω = 1 in resource limited regions). We assume that wild boar suffer mortality, in addition to natural death, from three causes: individuals in the generalised class suffer an additional disease induced mortality at rate α ; all adult and yearling classes are culled due to hunting at constant rate c ; and wolves successfully prey on susceptible and infected piglets at rate a P , generalised piglets at rate a PG , generalised yearlings and adults at rate a G and susceptible and infected yearlings and adults at rate a YA . Our baseline assumption is that \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${a}_{YA}=0$$\end{document} a Y A = 0 and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${a}_{P}={a}_{PG}={a}_{G}$$\end{document} a P = a P G = a G implying that wolves prey on piglets and generalised individuals only (although we do consider alternative predation assumptions). Further parameter description and the parameter values used in this study are shown in Supplementary Information. Ethics statement All animal sampling took place post-mortem. The wildlife samples were obtained from hunter-harvested individuals that were shot in the legal hunting seasons and independently and prior to our research. According to EU and National legislation (2010/63/UE Directive and Spanish Royal Decree 53/2013) and to the University of Castilla-La Mancha guidelines, no permission or consent is required to conduct the research reported herein. Study area and target species Asturias, a province of 10,604 km 2 , is located in northwestern Spain (Fig. 5 ). Wolf population data were obtained from the Asturias Government. Wolf presence is established in two-thirds of Asturias. In the remaining third, containing the majority of coastal regions and the urban and industrial corridors in the centre-north-east of the region (Fig. 5 66 , 67 ), wolves are absent or only sporadically recorded. The annual wolf census uses simulated howling and fixed observation points to map wolf packs (methodology detailed in the Asturias Government report 50 ) and allows for an estimate of wolf population size 50 . We combine the estimate of wolf abundance for 2003–2004 50 with data on wolf attack rate on livestock to give a profile of wolf abundance from 2000 to 2014. The regional government also records the number of wild boar harvested on hunting sites annually 51 . Hunting is predominantly non-commercial and traditional among rural inhabitants, taking place in 17 game reserves and 60 municipal hunting estates covering 91% of the province 51 . After standardisation by hunting effort, hunting bag statistics can be used as reliable indices of wild boar relative abundance 68 . We use data describing the temporal variation in the number of wild boar annually hunted (Fig. 1 ) and in particular generate estimates of wild boar population abundance in 2000–01 and 2013–14. Figure 5 Wolf ( Canis lupus ) distribution maps where the distribution in the Iberian Peninsula is shown in light grey, and municipalities in Asturias, northern Spain, are expanded to show where wolves are present (dark grey) or absent (white). TB prevalence We used serum antibodies against the MTC as an indicator of TB prevalence in wild boar. Serum samples were tested by means of an indirect ELISA using bovine-purified protein derivative (bPPD) following the protocol previously described in Boadella et al . 69 . Sample results were expressed as an ELISA percentage (E%) that was calculated using the formula [Sample E% = (sample OD/2 × mean negative control OD) × 100]. Serum samples with E% values greater than 100 were considered positive. Wild boar TB prevalence was available at the municipality level from 2000 to 2014. All cattle herds are tested annually for TB by individual skin testing. This testing is performed and recorded by the Asturias Government. Individual and herd-level data on cattle TB was available from 2005 to 2014, at the municipality scale. Asturias: estimating wolf population We derive an estimate of wolf population abundance for the period 2000–2014 as follows. We use data on wolf population abundance for the period 2003–2004 50 to obtain an estimate of 252 wolves in Asturias in 2004. We fit a least squares regression on wolf attack rate data for the period 2000–2014 (Fig. S1 ) to give a rate of increase of wolf abundance. Combining this rate of increase with our wolf abundance estimate for 2004 we estimate that wolf numbers increase linearly from 196 in 2000 to 392 in 2014. Given that both wolf density and wild boar densities are rising and not saturating, and that although wild boar are a preferred prey target for wolves they do not make up the whole wolf diet 33 , we assume that the wolf attack rate on wild boar has not reached saturation levels and therefore model wolf attack rates on wild boar linearly with wolf density. Mathematical modelling We develop a mathematical model that represents the interaction between wild boar, MTC infection and predation. In the model we set disease transmission rates and the wild boar intraspecific competition parameter so the model matches observations for the prevalence of infection and for wild boar density in 2000 and 2014 for the regions of Asturias with wolves. The model findings are extended to consider the areas of Asturias in which wolves are absent, to assess the role of future wolf density in TB control and the potential impact of wolf predation on TB in regions where TB is endemic and prevalence is high. We separate the population density of wild boar into different age classes to capture distinct disease and reproductive characteristics for piglets (aged 0–1 year) P , yearlings (aged 1–2 years) Y , and adults (aged 2 years+) A . Further, the age-classes are split into susceptible, infected and generalised classes (subscripts S , I , G , respectively) to reflect the disease status of the population. Generalised individuals can also release free-living pathogen with density F into the environment. The three different age-classes are required as each class has distinct properties in terms of their demographic and infection dynamics and in the impact of predation. This model framework has been used successfully to understand the impact of vaccination and culling on TB prevalence in the wild boar TB system 28 , 59 . The model also includes predation by wolves, W , and we examine scenarios where wolf density increases from 2000–2014 according to observations and then remains constant or decreases (to represent persecution) thereafter. The population dynamics of the wild boar, TB and wolf system are represented by the following set of non-linear differential equations (which is an extension of classical disease modelling frameworks 70 , 71 ) and of a previous model of wild boar and TB interactions 28 , 59 . 1a \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{S}}{{ m{d}}t}={b}_{A}(Y+A)\,(1-qN)-m{P}_{S}-{d}_{P}{P}_{S}-{eta }_{DP}{P}_{S} rac{G}{N}-\omega {eta }_{FP}{P}_{S}F-{a}_{P}{P}_{S}W$$\end{document} d P S d t = b A ( Y + A ) ( 1 − q N ) − m P S − d P P S − β D P P S G N − ω β F P P S F − a P P S W 1b \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{I}}{{ m{d}}t}={eta }_{DP}{P}_{S} rac{G}{N}+\omega {eta }_{FP}{P}_{S}F-m{P}_{I}-{d}_{P}{P}_{I}-{ arepsilon }_{P}{P}_{I}-{a}_{P}{P}_{I}W$$\end{document} d P I d t = β D P P S G N + ω β F P P S F − m P I − d P P I − ε P P I − a P P I W 1c \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{P}_{G}}{{ m{d}}t}={ arepsilon }_{P}{P}_{I}-m{P}_{G}-lpha {P}_{G}-{d}_{P}{P}_{G}-{a}_{PG}{P}_{G}W$$\end{document} d P G d t = ε P P I − m P G − α P G − d P P G − a P G P G W 1d \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{S}}{{ m{d}}t}=m{P}_{S}-m{Y}_{S}-{d}_{Y}{Y}_{S}-{eta }_{DY}{Y}_{S} rac{G}{N}-\omega {eta }_{FY}{Y}_{S}F-c{Y}_{S}-{a}_{YA}{Y}_{S}W$$\end{document} d Y S d t = m P S − m Y S − d Y Y S − β D Y Y S G N − ω β F Y Y S F − c Y S − a Y A Y S W 1e \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{I}}{{ m{d}}t}={eta }_{DY}{Y}_{S} rac{G}{N}+\omega {eta }_{FY}{Y}_{S}F+m{P}_{I}-m{Y}_{I}-{d}_{Y}{Y}_{I}-{ arepsilon }_{Y}{Y}_{I}-c{Y}_{I}-{a}_{YA}{Y}_{I}W$$\end{document} d Y I d t = β D Y Y S G N + ω β F Y Y S F + m P I − m Y I − d Y Y I − ε Y Y I − c Y I − a Y A Y I W 1f \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{Y}_{G}}{{ m{d}}t}={ arepsilon }_{Y}{Y}_{I}+m{P}_{G}-m{Y}_{G}-lpha {Y}_{G}-{d}_{Y}{Y}_{G}-c{Y}_{G}-{a}_{G}{Y}_{G}W$$\end{document} d Y G d t = ε Y Y I + m P G − m Y G − α Y G − d Y Y G − c Y G − a G Y G W 1g \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{S}}{{ m{d}}t}=m{Y}_{S}-{d}_{A}{A}_{S}-{eta }_{DA}{A}_{S} rac{G}{N}-\omega {eta }_{FA}{A}_{S}F-c{A}_{S}-{a}_{YA}{A}_{S}W$$\end{document} d A S d t = m Y S − d A A S − β D A A S G N − ω β F A A S F − c A S − a Y A A S W 1h \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{I}}{{ m{d}}t}={eta }_{DA}{A}_{S} rac{G}{N}+\omega {eta }_{FA}{A}_{S}F+m{Y}_{I}-{d}_{A}{A}_{I}-{ arepsilon }_{A}{A}_{I}-c{A}_{I}-{a}_{YA}{A}_{I}W$$\end{document} d A I d t = β D A A S G N + ω β F A A S F + m Y I − d A A I − ε A A I − c A I − a Y A A I W 1i \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ rac{{ m{d}}{A}_{G}}{{ m{d}}t}={ arepsilon }_{A}{A}_{I}+m{Y}_{G}-lpha {A}_{G}-{d}_{A}{A}_{G}-c{A}_{G}-{a}_{G}{A}_{G}W$$\end{document} d A G d t = ε A A I + m Y G − α A G − d A A G − c A G − a G A G W 1j \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$W=W(t)$$\end{document} W = W ( t ) Here, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$N=P+Y+A$$\end{document} N = P + Y + A represents the total wild boar population where \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$P={P}_{S}+{P}_{I}+{P}_{G}$$\end{document} P = P S + P I + P G , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$Y={Y}_{S}+{Y}_{I}+{Y}_{G}$$\end{document} Y = Y S + Y I + Y G , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$A={A}_{S}+{A}_{I}+{A}_{G}$$\end{document} A = A S + A I + A G and G is the total number of generalised, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$G={P}_{G}+{Y}_{G}+{A}_{G}$$\end{document} G = P G + Y G + A G . Susceptible and infected yearlings and adults give birth to susceptible piglets at rates b Y and b A respectively. Generalised yearlings and adults give birth to piglets at rate b G . Here we assume that \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${b}_{A}={b}_{Y}={b}_{G}$$\end{document} b A = b Y = b G . The total population is regulated through a crowding parameter, q , that acts on the birth rate. Maturity from piglets to yearlings and yearlings to adults occurs at rate m and piglets, yearlings and adults may die of natural causes at rates d P , d Y , d A respectively. Here we assume d P = d Y = d A . This set-up for the demographic dynamics has previously been used to assess wild boar TB interactions 28 , 59 . We assume infection can occur through direct frequency-dependent interactions (since wild boar tend to congregate in social groups) between susceptible and generalised individuals with transmission coefficients β DP , β DY and β DA or through environmental contact with free-living MTC, with transmission coefficients β FP , β FY and β FA for the different age classes respectively. Piglets and yearlings are more likely to become infected through both direct and environmental infection than adults 58 , and we assume that they are three times more susceptible to infection than adults to ensure that the model presents sufficient levels of infection in yearlings as compared to adults. In this way we have set the model so that the yearling class is the same as the piglet class in terms of disease characteristics, but the yearling class is the same as the adult class in terms of reproductive processes. Infected individuals are not infectious but can progress to the generalised (infectious) class at rates \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}$$\end{document} ε P , \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{Y}$$\end{document} ε Y and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{A}$$\end{document} ε A . In Asturias where resources are not limited we assume \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}={ arepsilon }_{Y}={ arepsilon }_{A}$$\end{document} ε P = ε Y = ε A . Later we consider regions where resources (particularly water) are scarce and overall health is impaired (similar to conditions in central and southern Spain). Co-infection can lead to a greater risk of becoming a super-shedder 72 and we assume that piglets and yearlings progress from the infected to the generalised class at three times the rate of adults ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ arepsilon }_{P}={ arepsilon }_{Y}=3{ arepsilon }_{A}$$\end{document} ε P = ε Y = 3 ε A ). We assume that free-living MTC is shed from generalised wild boar at rate λ and decays at rate μ . The level of environmental transmission is scaled through the parameter \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega $$\end{document} ω which increases when environmental conditions become more severe to reflect, for example, aggregation at limited water holes ( \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega =0.1$$\end{document} ω = 0.1 in Asturias and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\omega =1$$\end{document} ω = 1 in resource limited regions). We assume that wild boar suffer mortality, in addition to natural death, from three causes: individuals in the generalised class suffer an additional disease induced mortality at rate α ; all adult and yearling classes are culled due to hunting at constant rate c ; and wolves successfully prey on susceptible and infected piglets at rate a P , generalised piglets at rate a PG , generalised yearlings and adults at rate a G and susceptible and infected yearlings and adults at rate a YA . Our baseline assumption is that \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${a}_{YA}=0$$\end{document} a Y A = 0 and \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${a}_{P}={a}_{PG}={a}_{G}$$\end{document} a P = a P G = a G implying that wolves prey on piglets and generalised individuals only (although we do consider alternative predation assumptions). Further parameter description and the parameter values used in this study are shown in Supplementary Information. Supplementary information Supplementary Information Supplementary Information Supplementary information Supplementary information accompanies this paper at 10.1038/s41598-019-44148-9.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841920/

Speciation Analysis of Cr(VI) and Cr(III) in Water with Surface-Enhanced Raman Spectroscopy

Identifying and quantifying chromium in water are important for the protection of precious water resources from chromium pollution. Standard methods however are unable to easily distinguish toxic hexavalent chromium, Cr(VI), from innocuous trivalent chromium, Cr(III), are time-consuming, or require large sample quantity. We show in this report that Cr(VI) and Cr(III) in water can be differentiated based on their distinct spectral features of surface-enhanced Raman scattering (SERS). Their SERS signals exhibit different pH dependences: the SERS features of Cr(VI) and Cr(III) are most prominent at pH values of 10 and 5.5, respectively. The obtained limit of detection of Cr(VI) in water is below 0.1 mg/L. Both concentration curves of their SERS signals show Langmuir sorption isotherm behavior. A procedure was developed to quantify Cr(VI) concentration based on the direct retrieval or addition method with an error of 10%. Finally, the SERS detection of Cr(VI) is shown to be insensitive to co-present Cr(III). The developed SERS procedure offers potential to monitor toxic chromium in fields. Introduction Chromium (Cr) has many oxidation states—the common ones are +3, Cr(III), and + 6, Cr(VI)—and is widely used in industry, particularly chrome plating. 1 The popular plating method is hexavalent chromium plating, while the trivalent chromium plating is less common because of its uncontrollable process and poorer outcomes. Chromate however can enter cells and cause mutagenic damage. 2 − 4 The International Agency of Research Cancer (IARC) has classified Cr(VI) compounds under Group 1 since 1990. 5 In contrast, Cr(III) was considered an essential nutrient 6 and no toxicity has been observed. 7 Because of the difficulty in differentiating chromium species with current analytical methods, a guideline value of total chromium issued by WHO is 0.05 mg/L, 5 whose health risk is however questionable. Many countries adopt higher regulated concentrations. As an example, the United States has a national primary drinking water regulation of the maximum concentration level for total chromium of 0.1 mg/L. Consequently, differentiating Cr(VI) from Cr(III) species is crucial in the protection of water resources from chromium pollution. In water, Cr(VI) is present as oxyanions: both Cr 2 O 7 2– and HCrO 4 – dominate at pH values between 0 and ∼6, while CrO 4 2– emerges at a pH value of ∼4.5, reaches its maximum at a pH value of ≥8, and remains so at higher pH values. 1 , 8 , 9 On the other hand, at least five species of Cr(III) exist in water depending on pH values: Cr 3+ , CrOH 2+ , Cr(OH) 2 + , Cr(OH) 3 0 , and Cr(OH) 4 – . 1 , 9 , 10 The knowledge above provides the fundamental understanding of equilibrated chromium species in aqueous solution. There are now several well-developed analytical methods used in environmental laboratories as standards for determining chromium concentration. They can be separated into two types. The first type is elemental analysis methods that characterize chromium according to its characteristic atomic propensities: (1) core-level absorption and emission energy—atomic adsorption spectrometry (AAS) (APHA Method 3111A, 1992) 11 with a method detection limit (MDL) of 0.02 μg/L and inductively coupled plasma-atomic emission spectrometry (USEPA 200.7, Rev. 5.0, 2007) 12 with an MDL of 4 μg/L— and (2) mass—inductively coupled plasma-mass spectrometry (ICP-MS) (USEPA Method 200.8, Rev. 5.5, 1999) 13 with an MDL of 0.08 μg/L. Only the total chromium content in aqueous samples is determined by these methods. The second type is spectrophotometric methods that can determine Cr(VI) concentration. In the standard method (APHA 3500-Cr-D-1992), 14 the concentration of Cr(VI) is determined by UV–vis absorption of a colored complex compound of Cr(VI) with 1,5-diphenyl-carbazide. The MDL of this method is from 0.1 mg/L. The disadvantages of the second-type methods are two-fold: (1) the use of additional reagents in sample preparation could alter the chromium species composition; (2) interference from sample matrices might affect the quantitative accuracy and MDL. In addition, modern hyphenated techniques, such as high-performance liquid chromatograph in combination with ICP-MS or liquid chromatography with AAS detection, can avoid those interferences and separate chromium types in the sample. However, they require expensive equipment, controlled laboratory conditions, complicated sample preparation procedures, and long analytical time that inhibit their wide applications. Most importantly, these methods cannot be used in fields to provide in-time information to quickly identify the contaminated water resource. Accordingly, there is an urgent need for a fast screening method to discriminate different chromium species—particularly, Cr(III) and Cr(VI)—in fields. Infrared absorption spectroscopy and Raman spectroscopy are two powerful tools to identify molecular species according to their characteristic vibrational spectra. The weak absorption of the vibrational modes of the analyte is often overwhelmed by a huge absorption background from water, while Raman spectroscopy, providing background-free detection, suffers from its weak signal. Thanks to the discovery of surface-enhanced Raman scattering (SERS), 15 , 16 Raman scattering probability can be enhanced by several orders of magnitude by enhancing the local field of nanostructures that interacts with analytes in proximity. 17 It has emerged as a new paradigmatic approach to assay molecules of extremely low concentration. 18 Despite its application to numerous chemical compounds, only few studies were devoted to the detection of chromium in aqueous solutions. Two types of SERS enhancers were used in these previous studies. The first type is metal colloids 19 − 23 that suffer from time-variant sensitivity. The second type is metal nanostructures on substrates 24 − 26 that often manifest poor reproducibility. Particularly, Mosier-Boss and Lieberman demonstrated the ppb sensitivity of chromate and well-behaved Frumkin isotherm with use of cation-coated roughened metal surfaces. 24 , 25 Recently, Ji et al. 27 and Bu et al. 28 separately developed functionalized nanoparticles to capture Cr(VI) species in water. Only few groups devoted research efforts to the SERS detection of Cr(III) but not quantitative analysis. For examples, the SERS spectra of Cr(III) were obtained on silver SERS-activated plates 29 and from silver-coated corrosive chromium without offering the concentration curve of the SERS signal Cr(III). 30 , 31 Ye et al. obtained the curve based on the dependence of the Au nanoparticle aggregation on the Cr(III) concentration. 32 In spite of these previous SERS studies of Cr(VI) and Cr(III), their application to identifying and quantifying Cr(VI) and Cr(III) species in actual water samples is still lacking, owing to two major hurdles: (1) poor reproducibility associated with metal colloids and roughened metal substrates and (2) perplexed influences from coated SERS enhancers. Finally, given the facts that the species of Cr(III) and Cr(VI) present in water depend on pH values, 1 , 8 − 10 there is no systematic study to show how the pH values of the chromium samples affect the SERS spectra and how the acquired relationship can be used to develop protocols for analytical applications. Two possible reasons of lack of such studies are as follows: (1) the aggregation of metal colloids could be highly sensitive to the pH value of the samples; (2) the adhesion of the functionalized linkers and their analyte-capturing capability greatly depend on the pH condition. In this work, two approaches were adopted to overcome the two hurdles encountered in the previous SERS studies of chromium, respectively: (1) well-controlled Ag nanoparticle array to enable highly repeatable, uniform SERS enhancement and (2) direct SERS detection of Cr species in water. Instead of metal colloidal nanoparticles, we have used an SERS substrate based on Ag nanoparticle array imbedded within nanochannels in anodic aluminum oxide (dubbed AgNP/AAO) developed by our group. 33 Hot spots thus created at the gap between adjacent Ag nanoparticles by plasmonic coupling firmly enhance the local optical field and amplify Raman radiation. The far-field 34 and near-field 35 optical propensities of such hot spots investigated experimentally agree with high-precision electrodynamic simulation. 36 We have applied this substrate successfully to clinical microbiology 37 and food safety 38 for their respective semiquantitative analysis. Second, the direct SERS detection of Cr species in water has two advantages. The first one is that the species is identified with characteristic SERS spectral signature, rather than relying on capturing agents. The second one is stable detection enabled by adsorption–desorption equilibrium of analytes occurring at the SERS substrate. Since the SERS signal reflects the analytes adhered on the surface of the SERS enhancer, a sorption isotherm behavior is expected to occur between the Cr species and the SERS substrate. In fact, several groups studied the sorption isotherm of species on different SERS enhancers. For example, Zhang et al. 39 showed the adsorption isotherm of an anthrax biomarker on a silver film over nanosphere substrates. Altun et al. 40 characterized the sorption isotherms of SERS signals of organic species on the Ag/HfO 2 /CNT SERS substrate. Note that the sorption isotherms obtained with conventional methods (such as volumetric analysis, gravimetric analysis, voltammetry, and calorimetry) are different from those obtained with SERS because the SERS signal is attributed to only the analytes adsorbed on the surface of the metal nanoparticles on the SERS substrate that may contain other nonmetal surfaces. In this work, a systematic study was performed to reveal how the SERS spectra of Cr(III) and Cr(VI) in water depend on pH values under such equilibrium, yielding their characteristic spectral signatures for identification and the respective concentration curves of their SERS signals for quantitative analysis. In the end, an SERS-based procedure was developed to quantify Cr(VI) in water even in the presence of Cr(III). Materials and Methods Chemicals and Sample Preparation Chromium (Cr 3+ of 1000 μg/mL in 2% HNO 3 , High-Purity Standard) was used for preparing Cr(III) samples, while potassium chromate powder (K 2 CrO 4 , 99.0%, Alfa Aesar) was used for Cr(VI) sample preparation. The pH values of alkaline Cr samples were adjusted with sodium hydroxide (Shimakyu's Pure Chemicals), while those of acid samples were adjusted with nitric acid (HNO 3 , 65% solution in water, ACROS Organic). All solutions were prepared with deionized water. To search the optimal pH values for the SERS detection of Cr species, Cr(VI) and Cr(III) aqueous solutions with respective concentrations of 10 –5 and 10 –4 M of a series of pH values were prepared. The pH value was measured with a pH meter (Seven Excellence, Mettler Toledo) with an InLab Routine Pro combined electrode and KCl solution (3.0 M) as an internal electrolyte. The electrode was calibrated with buffer solutions. The pH values were cross-checked using pH test strips (pH Fix, Macherey-Nagel). Owing to the possible dissolution of CO 2 in air into the sample solutions, the prepared samples were kept in sealed vases with minimal air containment. The pH values of the sample solutions were monitored before and after the SERS measurement. The concentrations of the prepared samples were verified if the absorption was linearly proportional to the concentration (Beer–Lambert law). 41 , 42 For the experiments of mixed Cr(VI) and Cr(III), the final pH values of the mixtures were verified to be around 10. SERS Substrate The SERS-active substrates used in the experiments were made of arrays of Ag nanoparticles imbedded in the nanochannels of anodic aluminum oxide—named as AgNP/AAO. Its fabrication was reported previously 33 and is briefed here. An aluminum-coated glass slide prepared by sputtering was anodized and then chemically etched to create two-dimensional hexagonally packed arrays of nanochannels. Silver electrochemical plating produced Ag nanoparticles in the nanochannels, yielding two-dimensional Ag-nanosphere array with an average sphere diameter of 50 nm and a mean gap of 7 nm. Individual SERS-active substrates were then cleaned by rinsing with deionized water and then vacuum-sealed in plastic bags. Each substrate was freshly used in the SERS measurement. A significant advantage of the SERS substrates is its large active area of 2.5 cm × 5 cm with a uniform enhancement factor. SERS Measurement The Raman measurements were performed with a commercial Raman microscope system. A HeNe laser served as the excitation source. The laser beam was focused using a 10× micro-objective lens onto the sample; the scattered radiation was collected backward through the same lens, dispersed using an 80-cm spectrograph, and recorded using a liquid-nitrogen cooled charge-coupled device. The spectral resolution and error were calibrated to be 7 and 0.1 cm –1 , respectively. In addition to the use of highly uniform SERS substrates, four special measures were taken to enhance the repeatability of SERS measurements. First, all the SERS measurements were performed with a designated aluminum trough where the substrate with the sample droplets was positioned. A groove at the inner bottom of the trough was filled with 0.5 mL of water. The flat crest of the trough was covered with a low-fluorescence glass plate such that the interior humidity was maintained at its saturation level. The invariance of the sample concentration was confirmed by the constant SERS signal of adenine for at least 1 h. Second, the sample droplet on the SERS substrate touched the bottom surface of the glass plate, resulting in only flat optical interfaces for the optical path between the SERS substrate and the glass plate. Third, the uniformity of the SERS substrate was checked with the SERS measurement of adenine (10 –4 M). A substrate was considered uniform as the SERS signals of four adenine droplets, located at the four corners of a 2 cm × 4 cm rectangle, varied less than 10%. The uniformity test result of an SERS substrate is shown in Figure S8 of the Supporting Information. The result shows that the standard deviation of the SERS signals of adenine (10 –4 M) at 15 different sites over the central 2 cm × 4 cm region is 15.3%, while that of the signals except the three obtained from the rightmost column is 11%. The pass rate of the substrate that has a standard deviation of less than 10% is more than 50%. The subsequent SERS measurements of the samples were performed within that rectangular region. Fourth, the sample droplets with different characteristics (concentration or pH) and three water droplets with a designated pH value were applied randomly on the substrate, further reducing the influence of the gradual variation in the enhancement factor over the selected region. Raman measurements were repeated at several randomly chosen positions within each sample droplet. Special attention was paid to check if no abnormal Raman feature appeared and the signal strength was linearly proportional to the irradiating laser power and the integration time. Spectral Analysis The inevitable SERS continuum background 43 , 44 of each measured SERS spectrum was removed by the following two steps. The first step is to subtract the measured SERS spectrum of a nearby droplet of aqueous solution with a designated pH value from that of the Cr sample with the same pH value, removing the major portion of the background. The residual background was eliminated by a de-baseline procedure (the second step): the resultant spectral data outside the frequency range of the characteristic Raman feature of interest were fit with least-order polynomial functions such that the difference between the experimental spectrum and the fitted function showed random propensity. The characteristic feature in the spectrum after the background removal was fit with multiple Gaussian peaks. The extracted area of the spectral feature was considered to reflect the amount of species of interest adsorbed on the substrate surface for the quantitative analysis. Chemicals and Sample Preparation Chromium (Cr 3+ of 1000 μg/mL in 2% HNO 3 , High-Purity Standard) was used for preparing Cr(III) samples, while potassium chromate powder (K 2 CrO 4 , 99.0%, Alfa Aesar) was used for Cr(VI) sample preparation. The pH values of alkaline Cr samples were adjusted with sodium hydroxide (Shimakyu's Pure Chemicals), while those of acid samples were adjusted with nitric acid (HNO 3 , 65% solution in water, ACROS Organic). All solutions were prepared with deionized water. To search the optimal pH values for the SERS detection of Cr species, Cr(VI) and Cr(III) aqueous solutions with respective concentrations of 10 –5 and 10 –4 M of a series of pH values were prepared. The pH value was measured with a pH meter (Seven Excellence, Mettler Toledo) with an InLab Routine Pro combined electrode and KCl solution (3.0 M) as an internal electrolyte. The electrode was calibrated with buffer solutions. The pH values were cross-checked using pH test strips (pH Fix, Macherey-Nagel). Owing to the possible dissolution of CO 2 in air into the sample solutions, the prepared samples were kept in sealed vases with minimal air containment. The pH values of the sample solutions were monitored before and after the SERS measurement. The concentrations of the prepared samples were verified if the absorption was linearly proportional to the concentration (Beer–Lambert law). 41 , 42 For the experiments of mixed Cr(VI) and Cr(III), the final pH values of the mixtures were verified to be around 10. SERS Substrate The SERS-active substrates used in the experiments were made of arrays of Ag nanoparticles imbedded in the nanochannels of anodic aluminum oxide—named as AgNP/AAO. Its fabrication was reported previously 33 and is briefed here. An aluminum-coated glass slide prepared by sputtering was anodized and then chemically etched to create two-dimensional hexagonally packed arrays of nanochannels. Silver electrochemical plating produced Ag nanoparticles in the nanochannels, yielding two-dimensional Ag-nanosphere array with an average sphere diameter of 50 nm and a mean gap of 7 nm. Individual SERS-active substrates were then cleaned by rinsing with deionized water and then vacuum-sealed in plastic bags. Each substrate was freshly used in the SERS measurement. A significant advantage of the SERS substrates is its large active area of 2.5 cm × 5 cm with a uniform enhancement factor. SERS Measurement The Raman measurements were performed with a commercial Raman microscope system. A HeNe laser served as the excitation source. The laser beam was focused using a 10× micro-objective lens onto the sample; the scattered radiation was collected backward through the same lens, dispersed using an 80-cm spectrograph, and recorded using a liquid-nitrogen cooled charge-coupled device. The spectral resolution and error were calibrated to be 7 and 0.1 cm –1 , respectively. In addition to the use of highly uniform SERS substrates, four special measures were taken to enhance the repeatability of SERS measurements. First, all the SERS measurements were performed with a designated aluminum trough where the substrate with the sample droplets was positioned. A groove at the inner bottom of the trough was filled with 0.5 mL of water. The flat crest of the trough was covered with a low-fluorescence glass plate such that the interior humidity was maintained at its saturation level. The invariance of the sample concentration was confirmed by the constant SERS signal of adenine for at least 1 h. Second, the sample droplet on the SERS substrate touched the bottom surface of the glass plate, resulting in only flat optical interfaces for the optical path between the SERS substrate and the glass plate. Third, the uniformity of the SERS substrate was checked with the SERS measurement of adenine (10 –4 M). A substrate was considered uniform as the SERS signals of four adenine droplets, located at the four corners of a 2 cm × 4 cm rectangle, varied less than 10%. The uniformity test result of an SERS substrate is shown in Figure S8 of the Supporting Information. The result shows that the standard deviation of the SERS signals of adenine (10 –4 M) at 15 different sites over the central 2 cm × 4 cm region is 15.3%, while that of the signals except the three obtained from the rightmost column is 11%. The pass rate of the substrate that has a standard deviation of less than 10% is more than 50%. The subsequent SERS measurements of the samples were performed within that rectangular region. Fourth, the sample droplets with different characteristics (concentration or pH) and three water droplets with a designated pH value were applied randomly on the substrate, further reducing the influence of the gradual variation in the enhancement factor over the selected region. Raman measurements were repeated at several randomly chosen positions within each sample droplet. Special attention was paid to check if no abnormal Raman feature appeared and the signal strength was linearly proportional to the irradiating laser power and the integration time. Spectral Analysis The inevitable SERS continuum background 43 , 44 of each measured SERS spectrum was removed by the following two steps. The first step is to subtract the measured SERS spectrum of a nearby droplet of aqueous solution with a designated pH value from that of the Cr sample with the same pH value, removing the major portion of the background. The residual background was eliminated by a de-baseline procedure (the second step): the resultant spectral data outside the frequency range of the characteristic Raman feature of interest were fit with least-order polynomial functions such that the difference between the experimental spectrum and the fitted function showed random propensity. The characteristic feature in the spectrum after the background removal was fit with multiple Gaussian peaks. The extracted area of the spectral feature was considered to reflect the amount of species of interest adsorbed on the substrate surface for the quantitative analysis. Results and Discussion The SERS measurements in this work were performed as the analyte species undertook adsorption–desorption equilibrium 45 —sorption isotherm—between the ones dissolved in water and the ones adsorbed on the surface of the SERS substrate. It is therefore crucial to understand the relationship between the equilibrated species in water and the observed SERS spectrum at a specific pH value and how this relationship is varied with the analyte concentration. In the following, the SERS spectra of Cr(VI) at different pH values are shown first. The observed spectra are interpreted with the present Cr(VI) species, resulting in an optimal pH value for the SERS speciation of Cr(VI) in water. Comparing the SERS spectrum and the normal Raman spectrum acquired under that pH condition helps identify the species responsible for the SERS spectrum and designate the spectral feature for quantitative analysis. Under such pH conditions, the extracted concentration curve of the signal area of the SERS feature is revealed. Fitting the curve with the Langmuir sorption isotherm model yields the affinity of that Cr(VI) species, providing the scientific foundation for the quantitative analysis procedure of Cr(VI) in water. The SERS analysis of Cr(III) then follows. In the end, the result of the SERS detection of Cr(VI) in the presence of Cr(III) shows the capability of this speciation protocol in identifying and quantifying Cr(VI) in water in the presence of Cr(III). pH-Dependent SERS Spectra of Cr(VI) in Water As reported previously, 1 , 8 , 9 Cr(VI) exists as Cr 2 O 7 2– and HCrO 4 – in the pH range from 0 to 6, while it appears predominantly as CrO 4 2– at pH ≥ 8. The SERS spectrum of Cr(VI) in water thus expectantly varies with the pH condition of water, as these oxyanions would confer different vibrational modes. Figure 1 a shows the typical SERS spectra of Cr(VI) in water (10 –5 M ≈ 0.5 mg/L) at pH = 3.5, 5.5, and 10 as well as the normal Raman spectrum of Cr(VI) in water (0.05 M ≈ 2.6 g/L) at pH = 10. Note that Cr(VI) in water under the three different pH conditions shows distinct SERS spectra. Moreover, the SERS spectrum at pH = 10 is almost identical to the normal Raman spectrum of Cr(VI) in water at the same pH value: one feature centered at 350 cm –1 and one compounded feature ranging from 700 to 1000 cm –1 . Since CrO 4 2– dominates at pH = 10, the agreement between the SERS spectrum and the normal spectrum indicates that CrO 4 2– species is physisorbed on the surface of the SERS substrate. The 350 cm –1 feature was attributed to symmetric and asymmetric bending modes, while the feature ranging from 700 to 1000 cm –1 was attributed to symmetric and asymmetric stretching modes. 8 The broadened features of the SERS spectrum compared with the corresponding features of the normal Raman spectrum at pH = 10 suggest that the adsorbed CrO 4 2– on the surface of the SERS substrate takes a range of different configurations. Figure 1 (a) SERS spectra of Cr(VI) in water (10 –5 M ≈ 0.5 mg/L) at pH = 3.5 (red curve), 5.5 (green), and 10 (blue curve) as well as the normal Raman spectrum (black curve) of Cr(VI) in water (0.05 M ≈ 2.6 g/L) at pH = 10; (b) normalized integrated SERS signal area from 700 to 1000 cm –1 of Cr(VI) species in water, A VI , vs pH value, where the maximal average A VI value is set to one. The line is a guide to the eye. The feature between 700 and 1000 cm –1 in the SERS spectra of Cr(VI) in water was tentatively taken as the marker of CrO 4 2– , and its signal area, A VI , was extracted with the background removal-plus-Gaussian fitting procedure described above to reflect the coverage of CrO 4 2– on the surface of the SERS substrate according to the first-layer theory of SERS. 46 Figure 1 b shows that A VI increases with the pH value as pH > 4, reaches a maximum at pH = 9.5, and decreases afterward. The increase of A VI as the pH value is increased from 4 to 8 follows approximately with the increase of CrO 4 2– in this pH range. On the other hand, although CrO 4 2– remains the single dominant species in water as pH > 8, 1 , 8 , 9 A VI decreases as pH > 9.5. The first possible reason for the disagreement is that the adsorption capability (affinity) of CrO 4 2– on the SERS substrate might be sensitive to the pH value of the sample. The second reason is that the SERS substrate could deteriorate for pH > 10. Nevertheless, the optimal pH value for the speciation analysis of Cr(VI) is between 9 and 10. Finally, in the context of the quantitative analysis, it is imperative to understand whether CrO 4 2– remains the dominant species in water for the concentration range of interest (from 10 –7 to 10 –3 M). The fractions of all the Cr(VI) species in water are governed by their equilibrium reaction equations. 47 The equations to obtain the fractions of these species ( f H 2 CrO 4 , f HCrO 4 – , f Cr 2 O 7 2– , and f CrO 4 2– ) and the calculated results are shown in Figure S1 of the Supporting Information. At pH = 10, f CrO 4 2– is dominant, while the total fraction of the other three species is less than 0.001 for C VI . Accordingly, at pH = 10, the only detectable Cr(VI) species is CrO 4 2– for the concentration range of interest, thus greatly simplifying the quantitative analysis. Quantitative Analysis of Cr(VI) in Water Before using the SERS method to quantify Cr(VI), the concentration curve of its SERS signal needs to be acquired and analyzed with a sorption isotherm model so that a quantitative analysis procedure can be developed. As described above, the SERS signal of the Cr(VI) species dissolved in water reflects its coverage on the surface of the SERS substrate, θ VI . If θ VI follows a Langmuir sorption isotherm, 48 the signal area, A VI , of the spectral marker of that species is given by 1 where A VI ∞ is A VI at infinity concentration and L VI is the affinity. Representative SERS spectra of Cr(VI) in water of different concentrations at pH = 10 are shown in Figure S2 of the Supporting Information. Figure 2 shows the concentration curve of A VI at pH = 10. Note that the curve fits eq 1 very well and the extracted L VI is 1.47 ± 0.04 L/mg. A similar concentration curve was obtained for the signal area of the 350 cm –1 feature, and the extracted affinity is 1.45 ± 0.12 L/mg. The good correspondence between the two extracted affinities confirms that the two spectral features stem from the same species CrO 4 2– and the SERS substrate is uniform. Since A VI between 700 and 1000 cm –1 is larger than that centered at 350 cm –1 , the high-frequency A VI is used as the marker to identify Cr(VI) in water and its concentration curve, Figure 2 , serves as the basis in the quantitative analysis of Cr(VI) species. Figure 2 Normalized integrated SERS signal area from 700 to 1000 cm –1 of Cr(VI) species in water, A VI , as a function of Cr(VI) concentration, C VI . The solid line is the fit to eq 1 . A VI ∞ is the signal area with the infinite C VI . The acquired concentration curve of A VI confers two messages. First, since A VI is not proportional to C VI , as shown in Figure 2 , traditional methods are not applicable 49 for the quantitative analysis of Cr(VI) in water based on its detected SERS signal. Second, the effective concentration range of SERS detection is roughly between 0.1 and 2 mg/L. Based on these two messages, we propose a two-step quantification procedure to determine the concentration of Cr(VI): (1) assessment of SERS-effective concentration and (2) determination of the concentration. In the first step, the sample with an unknown concentration C VI * is diluted by 10 and 100 times, yielding three samples with concentrations of C VI – j * = 10 – j C VI * , j = 0, 1, and 2; the SERS measurements performed on the three samples then determine the one of the lowest concentration, C VI – J * , that is detectable; if none of them is detectable, C VI * is below the minimal limit of the SERS detection. In the second step, two quantification methods can be used to determine C VI * : direct retrieval method and addition method. In the direct retrieval method, the SERS measurements of the diluted unknown sample with a concentration of C VI – J * as well as the standard Cr(VI) samples with concentrations from 0.1 to 10 mg/L are performed on the same SERS substrate to obtain their respective A VI values; the obtained A VI values of the standard samples are fit to eq 1 to extract L VI and A VI ∞ ; the A VI value of the diluted unknown sample, A VI – J , is then used to determine C VI – J * and thus C VI * . In the addition method, the SERS measurements of the diluted unknown sample with a concentration of C VI – J * mixed with the standard samples with concentrations from 0.1 to 10 mg/L are performed on the same SERS substrate; the extracted A VI values are fit to a modified Langmuir model 2 to obtain C VI – J * , where C VI is the concentration of one of the standard samples. The flow chart of this procedure is illustrated in Figure S5 of the Supporting Information. The procedure was tested on two samples with their respective concentrations of 0.52 and 10.4 mg/L. The direct retrieval method was applied to the first case to obtain a concentration of 0.586 ± 0.078 mg/L, while the addition method was applied to the second case to obtain a concentration of 12.8 ± 2.3 mg/L. The measured concentrations for the both cases are within the error bars of the measurements. The data of these two tests are shown in Figure S6 of the Supporting Information. The results demonstrate that the SERS-based procedure obtained reasonably accurate concentrations of the unknown samples. As a final note, such accurate quantification results are made possible by our stable and uniform SERS substrate (without colloidal aggregation) and the SERS detection of the CrO 4 2– species directly adsorbed on the substrate (without any linker coating). SERS Propensities of Cr(III) in Water Previous studies 1 , 9 , 10 showed that there are at least seven possible Cr(III) species existing in water—Cr 3+ , CrOH 2+ , Cr(OH) 2 + , Cr(OH) 3 0 , Cr(OH) 4 – , Cr 2 (OH) 2 4+ , and Cr 3 (OH) 4 5+ — and their fractions also depend on the pH condition. More than two species are present simultaneously in water, except that Cr 3+ and Cr(OH) 3 0 dominate at pH < 3.6 and pH = 9.5, respectively. Figure 3 shows three representative SERS spectra of Cr(III) in water (10 –4 M = 5.2 mg/L) at pH = 3.8, 5.5, and 6.8. At pH = 3.8 and 6.8, there is barely any spectral feature, while at pH = 5.5, a single feature appears at around 600 cm –1 . Since CrOH 2+ and Cr(OH) 2 + predominate at pH ∼5, this 600 cm –1 feature would be attributed to the two species though the normal Raman feature was not observed in this study. Finally, at pH = 6.8∼10, there is also no spectral feature observable probably because the precipitated Cr(OH) 3 s has weak affinity on the SERS substrate. Nevertheless, it seems that the 600 cm –1 feature can serve as the marker of Cr(III) species. The main Cr(III) species present in water are governed by seven equilibrium reaction equations with their respective reaction constants. 50 The equations to obtain the fractions of these species ( f Cr 3+ , f CrOH 2+ , f Cr(OH) 2 + , f Cr(OH) 3 0 , f Cr(OH) 4 – , f Cr 2 (OH) 2 4+ , and f Cr 3 (OH) 4 5+ ) at pH = 5.5 and 10 and the results are shown in Figure S3 of the Supporting Information. Since there is no observable feature in the SERS spectrum of Cr(III) at pH > 6.8, the dominant species Cr(OH) 3 0 ( f Cr(OH) 3 0 > 0.95) do not contribute to the SERS signal, suggesting that its affinity to the SERS substrate is very small probably owing to its charge neutrality. On the other hand, at pH = 5.5, f CrOH 2+ and f Cr(OH) 2 + are higher than 0.1 and particularly f CrOH 2+ > 0.8. The larger positive charge of CrOH 2+ than that of Cr(OH) 2 + suggests that its affinity to the SERS substrate is higher, and therefore, the single spectral feature at 600 cm –1 could be attributed to the Cr–OH stretch. 51 More evidence is needed to support this assignment. The spectral feature centered at 600 cm –1 was taken as the marker of CrOH 2+ , and its signal area, A III , was extracted. Figure 3 b shows A III as the pH value is varied from 3.8 to 6.8. Note that A III increases with the pH value, reaches a maximum at pH = 6, and decreases afterward. In comparison, the calculated fraction of CrOH 2+ increases from 0.1 at pH = 2.7 to the maximum of 0.89 at pH = 4.8 and decreases to 0.1 at pH = 6.6. Therefore, the pH dependence of A III agrees approximately with that of f CrOH 2+ . The possible reasons for the discrepancy are two-fold. First, the affinity of CrOH 2+ on the surface of the SERS substrate could be sensitive to the pH condition, similar to the case of CrO 4 2– described above. Second, CrOH 2+ might not be the only species adsorbed on the SERS substrate to confer the 600 cm –1 feature. Nevertheless, according to Figure 3 b, the optimal pH value to perform speciation analysis of Cr(III) based on SERS is 5.5–6. Similar to Cr(VI) in water, the signal area of the 600 cm –1 feature, A III , would follow a Langmuir sorption isotherm model: 3 where A III ∞ is A III at infinity concentration, C III is the concentration of Cr(III), and L III is the affinity. Representative SERS spectra of Cr(III) in water of different concentrations at pH = 5.5 are shown in Figure S4 of the Supporting Information. The experimentally obtained A III as a function of C III is shown in Figure 4 . Note that the experimental data fit eq 3 well and the extracted L III is 0.14 ± 0.04 L/mg. Presumably, the same quantitative analysis procedure for Cr(III) can be developed based on the results of Figure 3 b and Figure 4 . Since Cr(III) compounds are benign to water and soil in the environment and are not toxic, the need to detect Cr(III) on site is not pressing and the procedure is not deliberated here. Figure 3 (a) SERS spectra of Cr(III) in water (10 –4 M = 5.2 mg/L) at pH = 3.8 (red curve), 5.5 (green curve), and 6.8 (blue curve); (b) normalized integrated SERS signal area at 600 cm –1 of Cr(III) species in water, A III , vs pH value where the maximal average A III value is set to one. The line is a guide to the eye. Figure 4 Normalized integrated SERS signal area at 600 cm –1 of Cr(III) species in water, A III , as a function of Cr(III) concentration, C III . The solid line is the fit to eq 3 . A III ∞ is the signal area with the infinite C III . Quantifying Cr(VI) in the Presence of Cr(III) in Water The two-step procedure to quantify the amount of Cr(VI) species is developed based on the dependence of the SERS signal of Cr(VI) in water on its concentration and pH value. However, will the identification and the quantification of Cr(VI) species in water be affected by the presence of Cr(III) species? Finding the appropriate procedure to perform such an analysis is crucial to the use of SERS in analyzing Cr(VI) species in water. The respective dependences of the SERS signals of Cr(VI) and Cr(III) species on the pH value in water, Figure 1 b and Figure 3 b, indicate that the best pH condition to identify and quantify Cr(VI) in water is at pH ∼10, where the SERS signal of Cr(VI) is most pronounced, while that of Cr(III) is diminished. This is so because the anion CrO 4 2– appears predominantly at pH > 8, while neutral Cr(OH) 3 0 prevails at pH > 7. The SERS measurements of two Cr(VI) concentrations, 0.4 and 4 mg/L, mixed with Cr(III) of a series of concentrations were performed to examine the possible interference by the co-present Cr(III). Figure 5 shows A VI /⟨ A VI ⟩ 0 mixed with different Cr(III) concentrations, where ⟨ A VI ⟩ 0 is the average value of A VI without the mixed Cr(III). Three representative SERS spectra of Cr(VI) at 4 mg/L mixed with Cr(III) at 10 mg/L (red line), 20 mg/L (green line), and 40 mg/L (blue line) at pH = 10 are shown in Figure S7 of the Supporting information. Note that the variations of these two cases are within 20%, indicating that the present Cr(III) species virtually do not influence the SERS detection of Cr(VI) species in water if the pH value is set at 10. The slight decay in A VI /⟨ A VI ⟩ 0 with the Cr(III) concentration could be caused by the small amount of precipitated Cr(OH) 3 s remaining after centrifugation that partially covers the SERS substrate. Additional filtering would effectively remove it. Figure 5 Normalized integrated SERS signal areas from 700 to 1000 cm –1 of Cr(VI) species in water, A VI /⟨ A VI ⟩ 0 , with concentrations of 0.4 mg/L (gray columns) and 4 mg/L (black columns) mixed with Cr(III) species of five different concentrations: C III = 2.6, 10.4, 20, 30, and 40 mg/L. ⟨ A VI ⟩ 0 is the average value of A VI without mixed Cr(III). pH-Dependent SERS Spectra of Cr(VI) in Water As reported previously, 1 , 8 , 9 Cr(VI) exists as Cr 2 O 7 2– and HCrO 4 – in the pH range from 0 to 6, while it appears predominantly as CrO 4 2– at pH ≥ 8. The SERS spectrum of Cr(VI) in water thus expectantly varies with the pH condition of water, as these oxyanions would confer different vibrational modes. Figure 1 a shows the typical SERS spectra of Cr(VI) in water (10 –5 M ≈ 0.5 mg/L) at pH = 3.5, 5.5, and 10 as well as the normal Raman spectrum of Cr(VI) in water (0.05 M ≈ 2.6 g/L) at pH = 10. Note that Cr(VI) in water under the three different pH conditions shows distinct SERS spectra. Moreover, the SERS spectrum at pH = 10 is almost identical to the normal Raman spectrum of Cr(VI) in water at the same pH value: one feature centered at 350 cm –1 and one compounded feature ranging from 700 to 1000 cm –1 . Since CrO 4 2– dominates at pH = 10, the agreement between the SERS spectrum and the normal spectrum indicates that CrO 4 2– species is physisorbed on the surface of the SERS substrate. The 350 cm –1 feature was attributed to symmetric and asymmetric bending modes, while the feature ranging from 700 to 1000 cm –1 was attributed to symmetric and asymmetric stretching modes. 8 The broadened features of the SERS spectrum compared with the corresponding features of the normal Raman spectrum at pH = 10 suggest that the adsorbed CrO 4 2– on the surface of the SERS substrate takes a range of different configurations. Figure 1 (a) SERS spectra of Cr(VI) in water (10 –5 M ≈ 0.5 mg/L) at pH = 3.5 (red curve), 5.5 (green), and 10 (blue curve) as well as the normal Raman spectrum (black curve) of Cr(VI) in water (0.05 M ≈ 2.6 g/L) at pH = 10; (b) normalized integrated SERS signal area from 700 to 1000 cm –1 of Cr(VI) species in water, A VI , vs pH value, where the maximal average A VI value is set to one. The line is a guide to the eye. The feature between 700 and 1000 cm –1 in the SERS spectra of Cr(VI) in water was tentatively taken as the marker of CrO 4 2– , and its signal area, A VI , was extracted with the background removal-plus-Gaussian fitting procedure described above to reflect the coverage of CrO 4 2– on the surface of the SERS substrate according to the first-layer theory of SERS. 46 Figure 1 b shows that A VI increases with the pH value as pH > 4, reaches a maximum at pH = 9.5, and decreases afterward. The increase of A VI as the pH value is increased from 4 to 8 follows approximately with the increase of CrO 4 2– in this pH range. On the other hand, although CrO 4 2– remains the single dominant species in water as pH > 8, 1 , 8 , 9 A VI decreases as pH > 9.5. The first possible reason for the disagreement is that the adsorption capability (affinity) of CrO 4 2– on the SERS substrate might be sensitive to the pH value of the sample. The second reason is that the SERS substrate could deteriorate for pH > 10. Nevertheless, the optimal pH value for the speciation analysis of Cr(VI) is between 9 and 10. Finally, in the context of the quantitative analysis, it is imperative to understand whether CrO 4 2– remains the dominant species in water for the concentration range of interest (from 10 –7 to 10 –3 M). The fractions of all the Cr(VI) species in water are governed by their equilibrium reaction equations. 47 The equations to obtain the fractions of these species ( f H 2 CrO 4 , f HCrO 4 – , f Cr 2 O 7 2– , and f CrO 4 2– ) and the calculated results are shown in Figure S1 of the Supporting Information. At pH = 10, f CrO 4 2– is dominant, while the total fraction of the other three species is less than 0.001 for C VI . Accordingly, at pH = 10, the only detectable Cr(VI) species is CrO 4 2– for the concentration range of interest, thus greatly simplifying the quantitative analysis. Quantitative Analysis of Cr(VI) in Water Before using the SERS method to quantify Cr(VI), the concentration curve of its SERS signal needs to be acquired and analyzed with a sorption isotherm model so that a quantitative analysis procedure can be developed. As described above, the SERS signal of the Cr(VI) species dissolved in water reflects its coverage on the surface of the SERS substrate, θ VI . If θ VI follows a Langmuir sorption isotherm, 48 the signal area, A VI , of the spectral marker of that species is given by 1 where A VI ∞ is A VI at infinity concentration and L VI is the affinity. Representative SERS spectra of Cr(VI) in water of different concentrations at pH = 10 are shown in Figure S2 of the Supporting Information. Figure 2 shows the concentration curve of A VI at pH = 10. Note that the curve fits eq 1 very well and the extracted L VI is 1.47 ± 0.04 L/mg. A similar concentration curve was obtained for the signal area of the 350 cm –1 feature, and the extracted affinity is 1.45 ± 0.12 L/mg. The good correspondence between the two extracted affinities confirms that the two spectral features stem from the same species CrO 4 2– and the SERS substrate is uniform. Since A VI between 700 and 1000 cm –1 is larger than that centered at 350 cm –1 , the high-frequency A VI is used as the marker to identify Cr(VI) in water and its concentration curve, Figure 2 , serves as the basis in the quantitative analysis of Cr(VI) species. Figure 2 Normalized integrated SERS signal area from 700 to 1000 cm –1 of Cr(VI) species in water, A VI , as a function of Cr(VI) concentration, C VI . The solid line is the fit to eq 1 . A VI ∞ is the signal area with the infinite C VI . The acquired concentration curve of A VI confers two messages. First, since A VI is not proportional to C VI , as shown in Figure 2 , traditional methods are not applicable 49 for the quantitative analysis of Cr(VI) in water based on its detected SERS signal. Second, the effective concentration range of SERS detection is roughly between 0.1 and 2 mg/L. Based on these two messages, we propose a two-step quantification procedure to determine the concentration of Cr(VI): (1) assessment of SERS-effective concentration and (2) determination of the concentration. In the first step, the sample with an unknown concentration C VI * is diluted by 10 and 100 times, yielding three samples with concentrations of C VI – j * = 10 – j C VI * , j = 0, 1, and 2; the SERS measurements performed on the three samples then determine the one of the lowest concentration, C VI – J * , that is detectable; if none of them is detectable, C VI * is below the minimal limit of the SERS detection. In the second step, two quantification methods can be used to determine C VI * : direct retrieval method and addition method. In the direct retrieval method, the SERS measurements of the diluted unknown sample with a concentration of C VI – J * as well as the standard Cr(VI) samples with concentrations from 0.1 to 10 mg/L are performed on the same SERS substrate to obtain their respective A VI values; the obtained A VI values of the standard samples are fit to eq 1 to extract L VI and A VI ∞ ; the A VI value of the diluted unknown sample, A VI – J , is then used to determine C VI – J * and thus C VI * . In the addition method, the SERS measurements of the diluted unknown sample with a concentration of C VI – J * mixed with the standard samples with concentrations from 0.1 to 10 mg/L are performed on the same SERS substrate; the extracted A VI values are fit to a modified Langmuir model 2 to obtain C VI – J * , where C VI is the concentration of one of the standard samples. The flow chart of this procedure is illustrated in Figure S5 of the Supporting Information. The procedure was tested on two samples with their respective concentrations of 0.52 and 10.4 mg/L. The direct retrieval method was applied to the first case to obtain a concentration of 0.586 ± 0.078 mg/L, while the addition method was applied to the second case to obtain a concentration of 12.8 ± 2.3 mg/L. The measured concentrations for the both cases are within the error bars of the measurements. The data of these two tests are shown in Figure S6 of the Supporting Information. The results demonstrate that the SERS-based procedure obtained reasonably accurate concentrations of the unknown samples. As a final note, such accurate quantification results are made possible by our stable and uniform SERS substrate (without colloidal aggregation) and the SERS detection of the CrO 4 2– species directly adsorbed on the substrate (without any linker coating). SERS Propensities of Cr(III) in Water Previous studies 1 , 9 , 10 showed that there are at least seven possible Cr(III) species existing in water—Cr 3+ , CrOH 2+ , Cr(OH) 2 + , Cr(OH) 3 0 , Cr(OH) 4 – , Cr 2 (OH) 2 4+ , and Cr 3 (OH) 4 5+ — and their fractions also depend on the pH condition. More than two species are present simultaneously in water, except that Cr 3+ and Cr(OH) 3 0 dominate at pH < 3.6 and pH = 9.5, respectively. Figure 3 shows three representative SERS spectra of Cr(III) in water (10 –4 M = 5.2 mg/L) at pH = 3.8, 5.5, and 6.8. At pH = 3.8 and 6.8, there is barely any spectral feature, while at pH = 5.5, a single feature appears at around 600 cm –1 . Since CrOH 2+ and Cr(OH) 2 + predominate at pH ∼5, this 600 cm –1 feature would be attributed to the two species though the normal Raman feature was not observed in this study. Finally, at pH = 6.8∼10, there is also no spectral feature observable probably because the precipitated Cr(OH) 3 s has weak affinity on the SERS substrate. Nevertheless, it seems that the 600 cm –1 feature can serve as the marker of Cr(III) species. The main Cr(III) species present in water are governed by seven equilibrium reaction equations with their respective reaction constants. 50 The equations to obtain the fractions of these species ( f Cr 3+ , f CrOH 2+ , f Cr(OH) 2 + , f Cr(OH) 3 0 , f Cr(OH) 4 – , f Cr 2 (OH) 2 4+ , and f Cr 3 (OH) 4 5+ ) at pH = 5.5 and 10 and the results are shown in Figure S3 of the Supporting Information. Since there is no observable feature in the SERS spectrum of Cr(III) at pH > 6.8, the dominant species Cr(OH) 3 0 ( f Cr(OH) 3 0 > 0.95) do not contribute to the SERS signal, suggesting that its affinity to the SERS substrate is very small probably owing to its charge neutrality. On the other hand, at pH = 5.5, f CrOH 2+ and f Cr(OH) 2 + are higher than 0.1 and particularly f CrOH 2+ > 0.8. The larger positive charge of CrOH 2+ than that of Cr(OH) 2 + suggests that its affinity to the SERS substrate is higher, and therefore, the single spectral feature at 600 cm –1 could be attributed to the Cr–OH stretch. 51 More evidence is needed to support this assignment. The spectral feature centered at 600 cm –1 was taken as the marker of CrOH 2+ , and its signal area, A III , was extracted. Figure 3 b shows A III as the pH value is varied from 3.8 to 6.8. Note that A III increases with the pH value, reaches a maximum at pH = 6, and decreases afterward. In comparison, the calculated fraction of CrOH 2+ increases from 0.1 at pH = 2.7 to the maximum of 0.89 at pH = 4.8 and decreases to 0.1 at pH = 6.6. Therefore, the pH dependence of A III agrees approximately with that of f CrOH 2+ . The possible reasons for the discrepancy are two-fold. First, the affinity of CrOH 2+ on the surface of the SERS substrate could be sensitive to the pH condition, similar to the case of CrO 4 2– described above. Second, CrOH 2+ might not be the only species adsorbed on the SERS substrate to confer the 600 cm –1 feature. Nevertheless, according to Figure 3 b, the optimal pH value to perform speciation analysis of Cr(III) based on SERS is 5.5–6. Similar to Cr(VI) in water, the signal area of the 600 cm –1 feature, A III , would follow a Langmuir sorption isotherm model: 3 where A III ∞ is A III at infinity concentration, C III is the concentration of Cr(III), and L III is the affinity. Representative SERS spectra of Cr(III) in water of different concentrations at pH = 5.5 are shown in Figure S4 of the Supporting Information. The experimentally obtained A III as a function of C III is shown in Figure 4 . Note that the experimental data fit eq 3 well and the extracted L III is 0.14 ± 0.04 L/mg. Presumably, the same quantitative analysis procedure for Cr(III) can be developed based on the results of Figure 3 b and Figure 4 . Since Cr(III) compounds are benign to water and soil in the environment and are not toxic, the need to detect Cr(III) on site is not pressing and the procedure is not deliberated here. Figure 3 (a) SERS spectra of Cr(III) in water (10 –4 M = 5.2 mg/L) at pH = 3.8 (red curve), 5.5 (green curve), and 6.8 (blue curve); (b) normalized integrated SERS signal area at 600 cm –1 of Cr(III) species in water, A III , vs pH value where the maximal average A III value is set to one. The line is a guide to the eye. Figure 4 Normalized integrated SERS signal area at 600 cm –1 of Cr(III) species in water, A III , as a function of Cr(III) concentration, C III . The solid line is the fit to eq 3 . A III ∞ is the signal area with the infinite C III . Quantifying Cr(VI) in the Presence of Cr(III) in Water The two-step procedure to quantify the amount of Cr(VI) species is developed based on the dependence of the SERS signal of Cr(VI) in water on its concentration and pH value. However, will the identification and the quantification of Cr(VI) species in water be affected by the presence of Cr(III) species? Finding the appropriate procedure to perform such an analysis is crucial to the use of SERS in analyzing Cr(VI) species in water. The respective dependences of the SERS signals of Cr(VI) and Cr(III) species on the pH value in water, Figure 1 b and Figure 3 b, indicate that the best pH condition to identify and quantify Cr(VI) in water is at pH ∼10, where the SERS signal of Cr(VI) is most pronounced, while that of Cr(III) is diminished. This is so because the anion CrO 4 2– appears predominantly at pH > 8, while neutral Cr(OH) 3 0 prevails at pH > 7. The SERS measurements of two Cr(VI) concentrations, 0.4 and 4 mg/L, mixed with Cr(III) of a series of concentrations were performed to examine the possible interference by the co-present Cr(III). Figure 5 shows A VI /⟨ A VI ⟩ 0 mixed with different Cr(III) concentrations, where ⟨ A VI ⟩ 0 is the average value of A VI without the mixed Cr(III). Three representative SERS spectra of Cr(VI) at 4 mg/L mixed with Cr(III) at 10 mg/L (red line), 20 mg/L (green line), and 40 mg/L (blue line) at pH = 10 are shown in Figure S7 of the Supporting information. Note that the variations of these two cases are within 20%, indicating that the present Cr(III) species virtually do not influence the SERS detection of Cr(VI) species in water if the pH value is set at 10. The slight decay in A VI /⟨ A VI ⟩ 0 with the Cr(III) concentration could be caused by the small amount of precipitated Cr(OH) 3 s remaining after centrifugation that partially covers the SERS substrate. Additional filtering would effectively remove it. Figure 5 Normalized integrated SERS signal areas from 700 to 1000 cm –1 of Cr(VI) species in water, A VI /⟨ A VI ⟩ 0 , with concentrations of 0.4 mg/L (gray columns) and 4 mg/L (black columns) mixed with Cr(III) species of five different concentrations: C III = 2.6, 10.4, 20, 30, and 40 mg/L. ⟨ A VI ⟩ 0 is the average value of A VI without mixed Cr(III). Conclusions This study shows systematic speciation analysis of Cr(VI) and Cr(III) species in water. The pH dependence of the SERS spectra of Cr(VI) in water were examined. The equilibrium reactions among different possible Cr(VI) species provide the foundation to understand the SERS spectra under different pH conditions. Particularly, the SERS spectrum at pH = 10 is the most prominent and is consistent with its normal Raman spectrum, indicating that the dominant anion CrO 4 2– under this pH condition is physisorbed on the SERS substrate. The portrayed concentration curve of Cr(VI) follows a Langmuir sorption isotherm. A procedure was then developed to quantify Cr(VI) in water based on direct retrieval or addition methods. Similarly, the SERS spectra of Cr(III) in water were obtained at different pH values. In comparison, the equilibrium reactions of different Cr(III) species in water show that more than one species is present, thus complicating the assignment of the observed SERS spectra. Nevertheless, the prominent feature at 600 cm –1 exhibits a simple Langmuir sorption isotherm. Finally, the SERS experiments of mixed Cr(VI) and Cr(III) species show that the detected SERS signal of Cr(VI) at pH = 10 is virtually not affected by the co-present Cr(III) species, indicating that the identification and the quantification of Cr(VI) in water can be performed readily. The detection limit of this procedure is below 0.1 mg/L, making it suitable for most regulated concentrations of Cr(VI) among different countries. Although our preliminary study of applying this procedure to a water sample discharged from a factory gave an error of about 10% in the extracted concentration of Cr(VI), more studies are still needed in future to address the issue of various co-existing ions in actual effluent water. The most exciting prospect of our study is that, in principle, similar SERS-based analytic methods can be developed to perform quantitative speciation analysis of aqueous solutions of transition metal species with known equilibrium reactions among different oxyanions and oxycations. 52 , 53 Supplementary Material ao0c05020_si_001.pdf

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917824/

Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cations generates dehydroalanine at a single cysteine residue, which is easily identified by the odd electron loss of •SCH 2 CONH 2 . The presence of dehydroalanine in cyclotides provides a site-selective ring opening pathway that, in turn, generates linear cyclotide analogs in the gas-phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well suited for cyclotide species that are not abundant enough to characterize with traditional methods.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618377/

Improved Detection of Botulinum Neurotoxin Serotype A by Endopep-MS through Peptide Substrate Modification

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to man. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype specific antibodies and detecting the unique and serotype specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity five fold with toxin spiked into buffer solution or different biological matrices.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666034/

Inflammasome-Associated Nucleotide-Binding Domain, Leucine-Rich Repeat Proteins and Inflammatory Diseases 1

The nucleotide-binding domain, leucine-rich repeat (NLR) proteins are a recently discovered family of intracellular pathogen and danger signal sensors. NLRs have emerged as important contributors to innate immunity in animals. The physiological impact of these genes is increasingly evident, underscored by the genetic association of variant family members with an array of inflammatory diseases. The association of mutations in NLR genes with autoinflammatory diseases indicates an important function of these genes in inflammation in vivo. This review summarizes the role of the inflammasome NLR proteins in innate immunity and inflammatory diseases and explores the possible utility of some of these NLRs as pharmacological targets.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8564108/

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

Background High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway. Background High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway. Introduction A hallmark of acute lung injury (ALI) is pulmonary edema caused by increased vascular permeability during septic inflammation ( 1 , 2 ). The pulmonary microvascular endothelial cells play a key effect on maintaining the endothelial barrier integrity between the microvascular lumen and the lung interstitium. EC barrier function depends on the integrity of endothelial cell (EC), the coordinate expression and interplay of proteins in cellular junction complexes, including the F-actin cytoskeleton, adherens junction (AJ) and tight junction (TJ) ( 3 , 4 ). The barrier hyperpermeability is always related to the cytoskeleton rearrangement of EC and the disruption of AJ and TJ, resulting in EC contraction and intercellular gap formation ( 5 , 6 ). High mobility group box 1 (HMGB1), a potent proinflammatory cytokine, can disrupt intercellular junctions, increasing endothelial barrier permeability ( 7 , 8 ). Our previous investigation shows that HMGB1 is involved in the progression of ALI, which has been demonstrated to be associated with microvascular barrier dysfunction elicited by the AJ and TJ disruption ( 7 , 9 ). Recent studies have indicated that HMGB1 and the receptor for advanced glycation end products (RAGE) conduce to endothelial barrier dysfunction, and RAGE is the primary receptor mediating HMGB1-induced hyperpermeability and paracellular gap formation ( 10 – 12 ). HMGB1 also elicits activation of Rho small GTPases which play important effects on rearranging the F-actin cytoskeleton and the intercellular junctional proteins ( 13 , 14 ). Rho kinase (ROCK) is a downstream target of RhoA and exists in two similar isoforms: ROCK1 and ROCK2 ( 15 , 16 ). ROCK activated by the GTP-bound form of Rho can directly phosphorylate myosin light chain (MLC) and reduce the dephosphorylation of phosphorylated MLC (pMLC) ( 17 , 18 ). ROCK1 and ROCK2 play different functional roles in regulating cytoskeleton arrangement by phosphorylating different downstream proteins ( 16 ). The previous studies have demonstrated that ROCK1 was involved in TNFα-induced early endothelial hyperpermeability and ROCK1 induced actin cytoskeletal instability by regulating actomyosin contraction, whereas ROCK2 stabilized the actin cytoskeleton by regulating cofilin phosphorylation ( 15 , 16 ). To verify the hypothesis that ROCK-dependent cell contraction plays a key role in HMGB1-induced increases in pulmonary microvascular endothelial barrier permeability, we investigated the effects of HMGB1 on the structure and function of endothelial barrier and elucidated the roles of RAGE and ROCK in HMGB1-induced human pulmonary endothelial barrier disruption. Our findings indicated that HMGB1 induced F-actin rearrangement, AJ/TJ rupture, and then enhanced the EC barrier permeability through the RAGE/ROCK1 pathway in the early stage. Methods Reagents Human pulmonary microvascular endothelial cell (HPMEC) was obtained from Tongpai Biotechnology Co., Ltd (Shanghai, China). Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China). FPS-ZM1 (a high-affinity RAGE-specific inhibitor) was from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 (ROCK inhibitor) was from Selleck Chemicals (Houston, Texas, USA). CCK-8 kit was from Beyotime Technology (Shanghai, China). Fluorescein isothiocyanate-dextran was from Sigma-Aldrich (St. Louis, MO, USA). Phalloidin-iFluor 594 Conjugate was obtained from Absin Bioscience (Shanghai, China). ROCK1/2 siRNA (SC-29473/SC-29474), RAGE siRNA (SC-36374) and negative control siRNA were from Santa Cruz Biotechnology Co., Ltd (Shanghai, China). Lipofectamine ® Reagent was employed for siRNA transfection (Paisley, UK). ZO-1 (monoclonal rabbit anti-human, CST 13663S), VE-cadherin (monoclonal rabbit anti-human, CST 2500T), MLC (monoclonal rabbit anti-human, CST 3672), p-MLC (Ser-19, monoclonal rabbit anti-human, CST 3674T) and ROCK1/2 (monoclonal rabbit anti-human, CST 4035/9029) antibodies were obtained from Cell Signaling Technology (CST, Boston, USA). RAGE (polyclonal rabbit anti-human, Cat No. 16346-1-AP) and Tubblin (monoclonal mouse anti-human, Cat No. 66240-1-Ig) antibodies were from Proteintech (North America). RhoA antibody (monoclonal rabbit anti-human, Catalog # 17-294 Lot # DAM1764537) was from Millipore Corporation (Temecula, CA). Rho activation kit was purchased from Upstate Biotechnology (Millipore, Dundee, UK). Anti-mouse and anti-rabbit secondary antibodies conjugated to horse radish peroxidase were purchased from Proteintech (North America). Donkey anti-Rabbit IgG (H+L) Alexa Fluor 488 Highly Cross-Adsorbed Secondary Antibody was from Invitrogen (Carlsbad, USA). Regular Range Prestained Protein Marker was from Proteintech (North America). Enhanced chemiluminescence (ECL) was from Tanon Science & Technology Co. (Shanghai, China). Cell Culture and Treatments HPMECs were inoculated in Dulbecco's modified Eagle's culture medium with 10% fetal bovine serum at 37°C and in 5% CO 2 . The medium was replaced every 1-2 days. All experiments were conducted in confluent monolayers on the 3rd or 4th day after seeding (passages 5-7). After 24 hours of serum-free culture, the cells were treated with rhHMGB1. rhHMGB1 was trypsinized to abolish the micro-amounts of endotoxin ( 7 ). HPMECs were then stimulated with rhHMGB1 at 600 ng/ml for 10 min, 30 min, 1 h, 6 h and 24 h, or treated with rhHMGB1 at 100, 200 and 600 ng/ml for 24 h. In order to detect the toxicity of Y-27632 and FPS-ZM1 to ECs, HPMECs were treated with Y-27632 (5, 10, 15 uM) and FPS-ZM1 (0.01, 0.05, 0.1 uM) for 24 h respectively. Transfection of siRNA HPMECs were grown on dishes precoated with 4 g/ml fibronectin. To prepare siRNA-lipofectamine complexes, siRNAs were mixed with lipofectamine reagent diluted in OptiMEM ® medium for 5 min at room temperature. ECs at 60-80% confluence were treated for 4 h with 10 nM ROCK1/2 siRNA, 100 nM RAGE siRNA or the corresponding negative control siRNA through adding the siRNA lipofectamine complexes to the ECs in serum-free medium. The transfected cells were then incubated with normal medium at 37°C with 5% CO2 for 48 h. Cell Viability Assay Cell viability was examined by CCK-8 measurement. HPMECs were inoculated in 96-well plates. CCK-8 solution (10 μl/well) was added, followed by culture at 37°C for 4 h. The absorbance at 450 nm was detected with a microplate reader (Thermo Labsystems, IL, USA). Cell viability was calculated as a percentage of that of control group. Measurement of Endothelial Permeability HPMECs were inoculated on 0.4-um pore Transwell filters. FITC-dextran (MR 40,000; Sigma–Aldrich) was added into the upper chamber at a concentration of 1 mg/ml and equalized for 1h, then the culture medium sample was collected from the lower chamber to detect base permeability. After indicated treatment, the media were taken from the lower chamber. The FITC fluorescence intensity was detected by a fluorescence spectrometer (MV06744, MoleCular Devices, Shanghai). The excitation wavelength was 482 nm and the detection wavelength was 525 nm. Immunofluorescence For VE-cadherin and ZO-1 localization, ECs were fixed with ice-cold methanol on chamber slides. Cells were blockaded with serum and treated with primary antibodies (VE-cadherin and ZO-1, 1:200) and Alexa Fluor 488 donkey anti-rabbit antibody (1:200). For F-actin localization, ECs were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blockaded with 5% BSA, and treated with 5 mg/ml of fluorescein isothiocyanate conjugated phalloidin. Confocal laser scanning microscope was used for image acquisition (Zeiss, Germany). The fluorescence intensity of F-actin, VE-cadherin and ZO-1 was quantitatively analyzed by the Image J software (National Institutes of Health, Bethesda, MD, USA). Western Blot Protein concentration was measured by the BCA method, and then the samples were titrated to the same concentration. Protein samples (10 μl) were subjected to 10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, blockaded by 5% BSA at room temperature for 1 h, then treated with primary antibodies (4°C, overnight) followed by incubation with HRP-coupled anti-mouse/rabbit IgG antibody (1:8000 dilution, room temperature, 1 h). Bands were developed with SuperSignalWest Pico Chemiluminescent Substrate and images were captured by Tanon 5200 System (Tanon, Shanghai). Primary antibodies and their dilution ratios applied in this present study were as follows: anti-RhoA (1:2000), anti-ROCK1/2 (1:1000), anti-MLC (1:1000), anti-pMLC (Ser-19, 1:1000), anti-VE-cadherin (1:1000), anti-ZO-1 (1:1000), anti-RAGE (1:800) and anti-tubblin (1:8000). Anti-tubblin protein was determined as an endogenous control for other proteins. At least three different repeats were performed for quantification. Band intensity was normalized by its own endogenous control. Assessment of Activated RhoA RhoA activation was determined with a pull-down assay kit in line with the manufacturer's instructions. ECs were lysed with a Triton X-100 lysis buffer. EC lysates were centrifuged at 13,000g at 4°C for 3 min, and equal volumes of lysates were treated with rhotekin-Rho-binding domain-coated agarose beads at 4°C for 1 h, then the beads were washed three times. The content of GTP-RhoA (RhoA associated with the beads) and total RhoA in cell lysates were measured by immunoblot. The activity of RhoA was examined by normalizing the amount of rhotekin-Rho-binding domain-bound RhoA to the total amount of RhoA in cell lysates. Statistical Analysis Experiments were performed in a minimum of triplicate replications. All values were expressed as means ± standard deviation (SD). SPSS 17 for Windows was used for statistical analysis. The one-way ANOVA test followed by Dunnett's post-test was applied for comparisons between groups. P value < 0.05 was considered to indicate statistical significance. Reagents Human pulmonary microvascular endothelial cell (HPMEC) was obtained from Tongpai Biotechnology Co., Ltd (Shanghai, China). Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China). FPS-ZM1 (a high-affinity RAGE-specific inhibitor) was from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 (ROCK inhibitor) was from Selleck Chemicals (Houston, Texas, USA). CCK-8 kit was from Beyotime Technology (Shanghai, China). Fluorescein isothiocyanate-dextran was from Sigma-Aldrich (St. Louis, MO, USA). Phalloidin-iFluor 594 Conjugate was obtained from Absin Bioscience (Shanghai, China). ROCK1/2 siRNA (SC-29473/SC-29474), RAGE siRNA (SC-36374) and negative control siRNA were from Santa Cruz Biotechnology Co., Ltd (Shanghai, China). Lipofectamine ® Reagent was employed for siRNA transfection (Paisley, UK). ZO-1 (monoclonal rabbit anti-human, CST 13663S), VE-cadherin (monoclonal rabbit anti-human, CST 2500T), MLC (monoclonal rabbit anti-human, CST 3672), p-MLC (Ser-19, monoclonal rabbit anti-human, CST 3674T) and ROCK1/2 (monoclonal rabbit anti-human, CST 4035/9029) antibodies were obtained from Cell Signaling Technology (CST, Boston, USA). RAGE (polyclonal rabbit anti-human, Cat No. 16346-1-AP) and Tubblin (monoclonal mouse anti-human, Cat No. 66240-1-Ig) antibodies were from Proteintech (North America). RhoA antibody (monoclonal rabbit anti-human, Catalog # 17-294 Lot # DAM1764537) was from Millipore Corporation (Temecula, CA). Rho activation kit was purchased from Upstate Biotechnology (Millipore, Dundee, UK). Anti-mouse and anti-rabbit secondary antibodies conjugated to horse radish peroxidase were purchased from Proteintech (North America). Donkey anti-Rabbit IgG (H+L) Alexa Fluor 488 Highly Cross-Adsorbed Secondary Antibody was from Invitrogen (Carlsbad, USA). Regular Range Prestained Protein Marker was from Proteintech (North America). Enhanced chemiluminescence (ECL) was from Tanon Science & Technology Co. (Shanghai, China). Cell Culture and Treatments HPMECs were inoculated in Dulbecco's modified Eagle's culture medium with 10% fetal bovine serum at 37°C and in 5% CO 2 . The medium was replaced every 1-2 days. All experiments were conducted in confluent monolayers on the 3rd or 4th day after seeding (passages 5-7). After 24 hours of serum-free culture, the cells were treated with rhHMGB1. rhHMGB1 was trypsinized to abolish the micro-amounts of endotoxin ( 7 ). HPMECs were then stimulated with rhHMGB1 at 600 ng/ml for 10 min, 30 min, 1 h, 6 h and 24 h, or treated with rhHMGB1 at 100, 200 and 600 ng/ml for 24 h. In order to detect the toxicity of Y-27632 and FPS-ZM1 to ECs, HPMECs were treated with Y-27632 (5, 10, 15 uM) and FPS-ZM1 (0.01, 0.05, 0.1 uM) for 24 h respectively. Transfection of siRNA HPMECs were grown on dishes precoated with 4 g/ml fibronectin. To prepare siRNA-lipofectamine complexes, siRNAs were mixed with lipofectamine reagent diluted in OptiMEM ® medium for 5 min at room temperature. ECs at 60-80% confluence were treated for 4 h with 10 nM ROCK1/2 siRNA, 100 nM RAGE siRNA or the corresponding negative control siRNA through adding the siRNA lipofectamine complexes to the ECs in serum-free medium. The transfected cells were then incubated with normal medium at 37°C with 5% CO2 for 48 h. Cell Viability Assay Cell viability was examined by CCK-8 measurement. HPMECs were inoculated in 96-well plates. CCK-8 solution (10 μl/well) was added, followed by culture at 37°C for 4 h. The absorbance at 450 nm was detected with a microplate reader (Thermo Labsystems, IL, USA). Cell viability was calculated as a percentage of that of control group. Measurement of Endothelial Permeability HPMECs were inoculated on 0.4-um pore Transwell filters. FITC-dextran (MR 40,000; Sigma–Aldrich) was added into the upper chamber at a concentration of 1 mg/ml and equalized for 1h, then the culture medium sample was collected from the lower chamber to detect base permeability. After indicated treatment, the media were taken from the lower chamber. The FITC fluorescence intensity was detected by a fluorescence spectrometer (MV06744, MoleCular Devices, Shanghai). The excitation wavelength was 482 nm and the detection wavelength was 525 nm. Immunofluorescence For VE-cadherin and ZO-1 localization, ECs were fixed with ice-cold methanol on chamber slides. Cells were blockaded with serum and treated with primary antibodies (VE-cadherin and ZO-1, 1:200) and Alexa Fluor 488 donkey anti-rabbit antibody (1:200). For F-actin localization, ECs were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blockaded with 5% BSA, and treated with 5 mg/ml of fluorescein isothiocyanate conjugated phalloidin. Confocal laser scanning microscope was used for image acquisition (Zeiss, Germany). The fluorescence intensity of F-actin, VE-cadherin and ZO-1 was quantitatively analyzed by the Image J software (National Institutes of Health, Bethesda, MD, USA). Western Blot Protein concentration was measured by the BCA method, and then the samples were titrated to the same concentration. Protein samples (10 μl) were subjected to 10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, blockaded by 5% BSA at room temperature for 1 h, then treated with primary antibodies (4°C, overnight) followed by incubation with HRP-coupled anti-mouse/rabbit IgG antibody (1:8000 dilution, room temperature, 1 h). Bands were developed with SuperSignalWest Pico Chemiluminescent Substrate and images were captured by Tanon 5200 System (Tanon, Shanghai). Primary antibodies and their dilution ratios applied in this present study were as follows: anti-RhoA (1:2000), anti-ROCK1/2 (1:1000), anti-MLC (1:1000), anti-pMLC (Ser-19, 1:1000), anti-VE-cadherin (1:1000), anti-ZO-1 (1:1000), anti-RAGE (1:800) and anti-tubblin (1:8000). Anti-tubblin protein was determined as an endogenous control for other proteins. At least three different repeats were performed for quantification. Band intensity was normalized by its own endogenous control. Assessment of Activated RhoA RhoA activation was determined with a pull-down assay kit in line with the manufacturer's instructions. ECs were lysed with a Triton X-100 lysis buffer. EC lysates were centrifuged at 13,000g at 4°C for 3 min, and equal volumes of lysates were treated with rhotekin-Rho-binding domain-coated agarose beads at 4°C for 1 h, then the beads were washed three times. The content of GTP-RhoA (RhoA associated with the beads) and total RhoA in cell lysates were measured by immunoblot. The activity of RhoA was examined by normalizing the amount of rhotekin-Rho-binding domain-bound RhoA to the total amount of RhoA in cell lysates. Statistical Analysis Experiments were performed in a minimum of triplicate replications. All values were expressed as means ± standard deviation (SD). SPSS 17 for Windows was used for statistical analysis. The one-way ANOVA test followed by Dunnett's post-test was applied for comparisons between groups. P value < 0.05 was considered to indicate statistical significance. Results HMGB1-Mediated the Formation of Stress Filaments and Disruption of AJ/TJ Proteins The changes of cell viability in HPMECs treated with different concentrations of rhHMGB1, Y-27632 and FPS-ZM1 were examined by CCK-8 method ( Figures 1A , 2A and 4A ). As indicated in Figures 1B, C , rhHMGB1 stimulation upregulated FITC-dextran flux in a dose-dependent and time-dependent manner. rhHMGB1 at a dose of 600 ng/ml showed a significant effect on EC barrier permeability ( Figure 1B ). The barrier permeability of HPMEC was markedly increased 30 min after rhHMGB1 treatment, and progressively increased to 24 h ( Figure 1C ). Therefore, the selected concentration of rhHMGB1 was 600 ng/ml in the following experiments. ECs were also treated with Y-27632 (10 uM) and FPS-ZM1 (0.05 uM) for 1 h prior to stimulation with rhHMGB1 and for the last 4 h of the 24 h rhHMGB1 stimulation respectively. Immunofluorescence microscopy revealed that rhHMGB1 also induced progressive cytoskeletal changes in cultured HPMECs that were apparent after 30 min of rhHMGB1 treatment ( Figure 1D ). The 24 h exposure of rhHMGB1 elicited the formation of stress filaments and paracellular gaps ( Figures 1D, E ). After rhHMGB1 treatment for 60 min, membrane localization of VE-cadherin and ZO-1 was also significantly ruptured, indicating that AJ/TJ integrity was disrupted ( Figure 1E ). To investigate the molecular mechanisms for the rhHMGB1-mediated endothelial barrier disruption, we checked whether changes in endothelial cell barrier permeability were paralleled with changes in the expression levels of intercellular junction proteins. As shown in Figure 1F , western blot revealed that treatment with rhHMGB1 elicited a time-dependent decrease in the expressions of VE-cadherin and ZO-1, which were measurable at 6 h of rhHMGB1 stimulation in ECs, and a more significant decrease was measured at 24 h. Figure 1 rhHMGB1-induced endothelial barrier hyperpermeability and RhoA/ROCK1 expression in ECs. (A) Cell viability of HPMEC was evaluated by CCK-8 measurement after stimulated with different concentration of rhHMGB1 for 24 h. (B) HPMECs were stimulated with the indicated concentrations of rhHMGB1 for 24 h. (C) HPMECs were stimulated with 600 ng/ml rhHMGB1 for the indicated times. (D, E) Immunofluorescence location of F-actin, VE-cadherin and ZO-1 in HPMECs was detected after 600 ng/ml rhHMGB1 stimulation for the indicated times. The fluorescence intensity of F-actin, VE-cadherin and ZO-1 was quantitatively analyzed using the Image J software. (F) The concentration of 600 ng/ml rhHMGB1 could selectively downregulate the expression level of VE-cadherin and ZO-1 at 24 h. (G) Time course of rhHMGB1-mediated increase in RhoA activity. Western blots showed the content of GTP-bound RhoA and total RhoA in cell lysate. (H) rhHMGB1 (600 ng/ml) treatment significantly upregulated ROCK1 expression in HPMECs at 60 min. (I) Treatment with 600 ng/ml rhHMGB1 could transiently promote the expression of pMLC. Values were shown as mean ± SD of 3 independent trials. * p < 0.05 vs . control. Figure 2 Y-27632 pretreatment attenuated rhHMGB1-induced ROCK1/pMLC expression. (A) CCK-8 assay was performed with HPMECs for 24 h with different dosages of Y-27632 as indicated. (B) Effects of Y-27632 on changes in FITC-dextran flux in HPMECs. HPMECs were pretreated with Y-27632 and then treated with 600 ng/ml rhHMGB1 for 60 min. (C) Role of Y-27632 pretreatment in increased barrier permeability induced by rhHMGB1 at 24 h. (D) Effects of Y-27632 on rhHMGB1-mediated morphological change in endothelial F-actin, VE-cadherin and ZO-1. HPMECs were pretreated with Y-27632 for 1 h before rhHMGB1 (600 ng/ml) stimulation for 60 minutes to examine morphology of endothelial F-actin, VE-cadherin and ZO-1 by immunofluorescence. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Y-27632 pretreatment downregulated the ROCK1 expression induced by rhHMGB1 at 60 min. (F) Effects of Y-27632 treatment on rhHMGB1-induced changes in the protein expression levels of VE-cadherin and ZO-1 at 24 h. Y-27632 were added for the last 4 h of the 24 h rhHMGB1 treatment. (G) Pretreatment with Y-27632 attenuated rhHMGB1-induced MLC phosphorylation at 60 min. ECs were pretreated with Y-27632 for 1 h and then stimulated with rhHMGB1 (600 ng/ml) for 1 h. Values were indicated as mean ± SD of 3 separate trials. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group. Effects of ROCK1 on HMGB1-Mediated HPMEC Hyperpermeability To investigate the effects of ROCK activation played on the rhHMGB1-mediated EC barrier hyperpermeability, the ECs were pretreated with Y-27632 or transfected with ROCK1/2 siRNA before rhHMGB1 stimulation. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs after transfection with ROCK1/2 siRNA and there was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells ( Figures 3B, C ). As shown in Figures 1G, H , the time-dependent increases in RhoA activity and ROCK1 expression by rhHMGB1 treatment were measured. The activity of RhoA/ROCK1 was significantly upregulated at 30 min and 60 min of rhHMGB1 treatment, but the activity of RhoA/ROCK1 returned to baseline by 24 h. The rhHMGB1-induced EC hyperpermeability at 60min was significantly inhibited by Y-27632 pretreatment and ROCK1 knockdown ( Figures 2B , 3D ). However, transfection with ROCK2 siRNA alone did not reduce rhHMGB1-induced early permeability increases at 60 min ( Figure 3D ). In addition, transfection with ROCK1/2 siRNA had no significant inhibitory role in rhHMGB1-induced late permeability increases at 24 h ( Figure 3D ). When ECs were treated with Y-27632 for the last 4 h of the 24 h rhHMGB1 stimulation, the results indicated that suppression of ROCK had no marked inhibitory role in rhHMGB1-induced hyperpermeability at 24 h ( Figure 2C ), in accordance with the presence of stress filaments and intercellular gaps at 24h ( Figures 1D, E ). These data indicated that ROCK1 activation was necessary for rhHMGB1-mediated early increase in endothelial barrier permeability. Furthermore, the results of western blot showed that treatment with Y-27632 and ROCK1 siRNA had no significant effects on the expressions of VE-cadherin and ZO-1 in HPMECs after 24 h of rhHMGB1 stimulation ( Figures 2F , 3E ). Figure 3 rhHMGB1-mediated early barrier dysfunction is largely dependent on ROCK1 signaling. (A, B) ECs were transfected with ROCK1/2 siRNA. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs. (C) Cell viability was assessed by the CCK-8 assay after transfection with different concentration of ROCK1/2 siRNA for 24 h or transfection with 10 nM ROCK1/2 siRNA for the indicated times. There was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells. (D) Examination of FITC-dextran flux of HPMECs. ROCK1 knockdown ameliorated rhHMGB1-induced early permeability increases (at 60 min). (E) Effects of ROCK1 siRNA on the expression of VE-cadherin and ZO-1 induced by rhHMGB1 at 24 h. (F) ECs were transfected with ROCK1 siRNA and then stimulated with rhHMGB1 for 60 min. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was detected by fluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (G) ROCK1 knockdown attenuated the ROCK1 expression induced by rhHMGB1 at 60 min. (H) ROCK1 knockdown downregulated the rhHMGB1-induced pMLC expression in cells at 60 min. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group. NC, negative control. HMGB1 Induced MLC Activation in HPMECs It was demonstrated that Rho/ROCK signaling pathway played a key influence on increasing the level of MLC phosphorylation ( 19 ). Thus, pMLC is an important initial event for the increased paracellular flow of endothelial cell leakage ( 18 ). In the present study, some modest morphological changes occurred in the actin cytoskeleton after treatment with rhHMGB1 for 30 min ( Figure 1D ), which could reflect the enhanced Rho/ROCK activity and MLC phosphorylation. As shown in Figure 1I , rhHMGB1 stimulation (600 ng/ml) for 60 minutes obviously increased the expression level of pMLC, which was consistent with a time-dependent increase in RhoA activity and ROCK1 expression ( Figures 1G, H ). Results showed that rhHMGB1 induced an increase in Ser-19 phosphorylation at 30 min and 1 h and the expression level of Ser-19 phosphorylated MLC returned to baseline by 24 h, as shown on immunoblot ( Figure 1I ). The inhibition of ROCK1 expression with Y-27632 and ROCK1 siRNA could downregulate the phosphorylation of MLC after 60 min of rhHMGB1 treatment ( Figures 2G , 3H ), which was accompanied by decreases in the fluorescence intensity of F-actin at 60 min ( Figures 2D , 3F ). Pretreatment with FPS-ZM1 and RAGE siRNA could result in a marked downregulation of the activity of RhoA/ROCK1 and phosphorylation of MLC in HPMECs treated with rhHMGB1 for 60 minutes ( Figures 4C, F and 5E, F ), and then reduce the fluorescence intensity of stress fibers in the cell center at 60 min ( Figures 4D , 5D ). Figure 4 FPS-ZM1 treatment alleviated rhHMGB1-mediated RhoA/ROCK1 activation and barrier dysfunction. (A) Cell viability was determined by CCK-8 assay after treated with different dosage of FPS-ZM1 for 24 h. (B) FPS-ZM1 improved lung endothelial permeability at 60 min and 24 h after rhHMGB1 stimulation. (C) FPS-ZM1 significantly downregulated RhoA and ROCK1 expression in HPMECs at 60 min after rhHMGB1 stimulation. (D) Effects of FPS-ZM1 on rhHMGB1-mediated morphological changes in endothelial F-actin, VE-cadherin and ZO-1. ECs were treated with FPS-ZM1 for 1 h prior to stimulation with rhHMGB1 to evaluate morphology of endothelial cytoskeleton F-actin or for the last 4 h of the 24 h rhHMGB1 stimulation to assess morphology of endothelial VE-cadherin and ZO-1 by immunofluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (E) FPS-ZM1 significantly increased VE-cadherin and ZO-1 expression levels in HPMECs at 24 h after rhHMGB1 stimulation. ECs were treated with FPS-ZM1 for the last 4 h of the 24 h rhHMGB1 stimulation. (F) Effects of FPS-ZM1 on rhHMGB1-mediated pMLC expression in cells. ECs were pretreated with FPS-ZM1 for 1 h and then treated with rhHMGB1 for 60 minutes. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group. Figure 5 rhHMGB1-induced RhoA/ROCK1 pathway activation via RAGE in HPMECs. (A) ECs were transfected with RAGE siRNA. Western blots were used to determine the expression of RAGE in endothelial cells. (B) Cytotoxicity of RAGE siRNA was assessed by CCK-8 assay after transfected with different concentration of RAGE siRNA for 24 h or transfected with 100 nM RAGE siRNA for the different times. No evidence of cytotoxicity was found in RAGE siRNA transfected cells. (C) Treatment with RAGE siRNA ameliorated endothelial barrier dysfunction induced by rhHMGB1 at 60 min and 24 h. (D) ECs were transfected with RAGE siRNA and then stimulated with rhHMGB1 for 60 min and 24 h. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was determined by fluorescence microscopy. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Knockdown of RAGE by siRNA reduced the rhHMGB1-induced MLC phosphorylation at 60 min as detected by western blot. (F) Effects of inhibition of RAGE with siRNA on increased expression of RhoA and ROCK1 induced by rhHMGB1 at 60 min in HPMECs. (G) Role of RAGE siRNA in the VE-cadherin and ZO-1 protein expression levels in ECs at 24 h after rhHMGB1 treatment. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group. NC, negative control. RhoA/ROCK1 Pathway Mediates HMGB1-Induced EC Barrier Disruption in HPMECs As shown in Figures 1G, H , the time-dependent increases in RhoA activity and ROCK1 expression by rhHMGB1 treatment were measured. The results showed that the peak activity of RhoA appeared at 60 min and rhHMGB1 treatment also significantly up-regulated the expression of ROCK1 at 60min. Thus, binding of active GTP-bound RhoA caused unfolding and activation of ROCK1, which was accompanied by rhHMGB1-mediated MLC phosphorylation, stress filament formation, VE-cadherin and ZO-1 disruption, and the early increase in EC barrier permeability ( Figure 1 ). Pretreatment with Y-27632 and ROCK1 knockdown could partially inhibit rhHMGB1-mediated EC leakage and restore FITC-dextran flux of the endothelial barrier after 60 min of rhHMGB1 stimulation ( Figures 2B , 3D ). After pretreatment with Y-27632 and ROCK1 siRNA, the expression of ROCK1 protein at 60 min was significantly downregulated ( Figures 2E , 3G ), and the membrane location of VE-cadherin and ZO-1 in intercellular junctions was partially restored after 60 min of rhHMGB1 stimulation ( Figures 2D , 3F ). The 60 min exposure of rhHMGB1 increased the fluorescence intensity of F-actin in the cell center, and pretreatment with Y-27632 and ROCK1 siRNA decreased the fluorescence intensity of central stress filaments after 60 min of rhHMGB1 exposure ( Figures 2D , 3F ). These findings suggest that RhoA and ROCK1 may be implicated in rhHMGB1-mediated early increase in EC permeability and stress filament formation in HPMECs. RAGE Mediates the EC Barrier Leakage Induced by HMGB1 To investigate whether RAGE is implicated in the rhHMGB1-mediated increases in EC barrier permeability, the expression of RAGE was silenced by siRNA in HPMECs. In addition, HPMECs were also treated with the specific RAGE inhibitor FPS-ZM1 ( 20 ) for 60 min prior to stimulation with rhHMGB1 or for the last 4 h of the 24 h rhHMGB1 stimulation. As indicated in Figures 4B , 5C , FPS-ZM1 and RAGE knockdown significantly decreased endothelial permeability by comparison with the rhHMGB1 treatment group at 60 min and 24 h. Inhibition of RAGE with FPS-ZM1 and RAGE siRNA could also downregulate the activity of RhoA/ROCK1 and phosphorylation of MLC at 60 min ( Figures 4C, F and 5E, F ), and then partly prevent the rhHMGB1-mediated formation of stress fibers in the cell center ( Figures 4D , 5D ). Furthermore, inhibition of ROCK1 could also reduce the expression of pMLC ( Figures 2G , 3H ), and partly recover the membrane localization of VE-cadherin and ZO-1 after 60 min of rhHMGB1 stimulation ( Figures 2D , 3F ). Therefore, it seems likely that a signaling pathway proceeds from RAGE to RhoA/ROCK1 through the F-actin reorganization and disruption of AJ/TJ related proteins, which finally destroys the early EC barrier function in the present study. In addition, FPS-ZM1 and RAGE siRNA could upregulate the expression levels of VE-cadherin and ZO-1 at 24 h ( Figures 4E , 5G ). Similarly, inhibition of RAGE could also partially restore the cytomembrane location of VE-cadherin and ZO-1 after 24 h of rhHMGB1 stimulation ( Figures 4D , 5D ). HMGB1-Mediated the Formation of Stress Filaments and Disruption of AJ/TJ Proteins The changes of cell viability in HPMECs treated with different concentrations of rhHMGB1, Y-27632 and FPS-ZM1 were examined by CCK-8 method ( Figures 1A , 2A and 4A ). As indicated in Figures 1B, C , rhHMGB1 stimulation upregulated FITC-dextran flux in a dose-dependent and time-dependent manner. rhHMGB1 at a dose of 600 ng/ml showed a significant effect on EC barrier permeability ( Figure 1B ). The barrier permeability of HPMEC was markedly increased 30 min after rhHMGB1 treatment, and progressively increased to 24 h ( Figure 1C ). Therefore, the selected concentration of rhHMGB1 was 600 ng/ml in the following experiments. ECs were also treated with Y-27632 (10 uM) and FPS-ZM1 (0.05 uM) for 1 h prior to stimulation with rhHMGB1 and for the last 4 h of the 24 h rhHMGB1 stimulation respectively. Immunofluorescence microscopy revealed that rhHMGB1 also induced progressive cytoskeletal changes in cultured HPMECs that were apparent after 30 min of rhHMGB1 treatment ( Figure 1D ). The 24 h exposure of rhHMGB1 elicited the formation of stress filaments and paracellular gaps ( Figures 1D, E ). After rhHMGB1 treatment for 60 min, membrane localization of VE-cadherin and ZO-1 was also significantly ruptured, indicating that AJ/TJ integrity was disrupted ( Figure 1E ). To investigate the molecular mechanisms for the rhHMGB1-mediated endothelial barrier disruption, we checked whether changes in endothelial cell barrier permeability were paralleled with changes in the expression levels of intercellular junction proteins. As shown in Figure 1F , western blot revealed that treatment with rhHMGB1 elicited a time-dependent decrease in the expressions of VE-cadherin and ZO-1, which were measurable at 6 h of rhHMGB1 stimulation in ECs, and a more significant decrease was measured at 24 h. Figure 1 rhHMGB1-induced endothelial barrier hyperpermeability and RhoA/ROCK1 expression in ECs. (A) Cell viability of HPMEC was evaluated by CCK-8 measurement after stimulated with different concentration of rhHMGB1 for 24 h. (B) HPMECs were stimulated with the indicated concentrations of rhHMGB1 for 24 h. (C) HPMECs were stimulated with 600 ng/ml rhHMGB1 for the indicated times. (D, E) Immunofluorescence location of F-actin, VE-cadherin and ZO-1 in HPMECs was detected after 600 ng/ml rhHMGB1 stimulation for the indicated times. The fluorescence intensity of F-actin, VE-cadherin and ZO-1 was quantitatively analyzed using the Image J software. (F) The concentration of 600 ng/ml rhHMGB1 could selectively downregulate the expression level of VE-cadherin and ZO-1 at 24 h. (G) Time course of rhHMGB1-mediated increase in RhoA activity. Western blots showed the content of GTP-bound RhoA and total RhoA in cell lysate. (H) rhHMGB1 (600 ng/ml) treatment significantly upregulated ROCK1 expression in HPMECs at 60 min. (I) Treatment with 600 ng/ml rhHMGB1 could transiently promote the expression of pMLC. Values were shown as mean ± SD of 3 independent trials. * p < 0.05 vs . control. Figure 2 Y-27632 pretreatment attenuated rhHMGB1-induced ROCK1/pMLC expression. (A) CCK-8 assay was performed with HPMECs for 24 h with different dosages of Y-27632 as indicated. (B) Effects of Y-27632 on changes in FITC-dextran flux in HPMECs. HPMECs were pretreated with Y-27632 and then treated with 600 ng/ml rhHMGB1 for 60 min. (C) Role of Y-27632 pretreatment in increased barrier permeability induced by rhHMGB1 at 24 h. (D) Effects of Y-27632 on rhHMGB1-mediated morphological change in endothelial F-actin, VE-cadherin and ZO-1. HPMECs were pretreated with Y-27632 for 1 h before rhHMGB1 (600 ng/ml) stimulation for 60 minutes to examine morphology of endothelial F-actin, VE-cadherin and ZO-1 by immunofluorescence. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Y-27632 pretreatment downregulated the ROCK1 expression induced by rhHMGB1 at 60 min. (F) Effects of Y-27632 treatment on rhHMGB1-induced changes in the protein expression levels of VE-cadherin and ZO-1 at 24 h. Y-27632 were added for the last 4 h of the 24 h rhHMGB1 treatment. (G) Pretreatment with Y-27632 attenuated rhHMGB1-induced MLC phosphorylation at 60 min. ECs were pretreated with Y-27632 for 1 h and then stimulated with rhHMGB1 (600 ng/ml) for 1 h. Values were indicated as mean ± SD of 3 separate trials. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group. Effects of ROCK1 on HMGB1-Mediated HPMEC Hyperpermeability To investigate the effects of ROCK activation played on the rhHMGB1-mediated EC barrier hyperpermeability, the ECs were pretreated with Y-27632 or transfected with ROCK1/2 siRNA before rhHMGB1 stimulation. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs after transfection with ROCK1/2 siRNA and there was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells ( Figures 3B, C ). As shown in Figures 1G, H , the time-dependent increases in RhoA activity and ROCK1 expression by rhHMGB1 treatment were measured. The activity of RhoA/ROCK1 was significantly upregulated at 30 min and 60 min of rhHMGB1 treatment, but the activity of RhoA/ROCK1 returned to baseline by 24 h. The rhHMGB1-induced EC hyperpermeability at 60min was significantly inhibited by Y-27632 pretreatment and ROCK1 knockdown ( Figures 2B , 3D ). However, transfection with ROCK2 siRNA alone did not reduce rhHMGB1-induced early permeability increases at 60 min ( Figure 3D ). In addition, transfection with ROCK1/2 siRNA had no significant inhibitory role in rhHMGB1-induced late permeability increases at 24 h ( Figure 3D ). When ECs were treated with Y-27632 for the last 4 h of the 24 h rhHMGB1 stimulation, the results indicated that suppression of ROCK had no marked inhibitory role in rhHMGB1-induced hyperpermeability at 24 h ( Figure 2C ), in accordance with the presence of stress filaments and intercellular gaps at 24h ( Figures 1D, E ). These data indicated that ROCK1 activation was necessary for rhHMGB1-mediated early increase in endothelial barrier permeability. Furthermore, the results of western blot showed that treatment with Y-27632 and ROCK1 siRNA had no significant effects on the expressions of VE-cadherin and ZO-1 in HPMECs after 24 h of rhHMGB1 stimulation ( Figures 2F , 3E ). Figure 3 rhHMGB1-mediated early barrier dysfunction is largely dependent on ROCK1 signaling. (A, B) ECs were transfected with ROCK1/2 siRNA. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs. (C) Cell viability was assessed by the CCK-8 assay after transfection with different concentration of ROCK1/2 siRNA for 24 h or transfection with 10 nM ROCK1/2 siRNA for the indicated times. There was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells. (D) Examination of FITC-dextran flux of HPMECs. ROCK1 knockdown ameliorated rhHMGB1-induced early permeability increases (at 60 min). (E) Effects of ROCK1 siRNA on the expression of VE-cadherin and ZO-1 induced by rhHMGB1 at 24 h. (F) ECs were transfected with ROCK1 siRNA and then stimulated with rhHMGB1 for 60 min. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was detected by fluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (G) ROCK1 knockdown attenuated the ROCK1 expression induced by rhHMGB1 at 60 min. (H) ROCK1 knockdown downregulated the rhHMGB1-induced pMLC expression in cells at 60 min. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group. NC, negative control. HMGB1 Induced MLC Activation in HPMECs It was demonstrated that Rho/ROCK signaling pathway played a key influence on increasing the level of MLC phosphorylation ( 19 ). Thus, pMLC is an important initial event for the increased paracellular flow of endothelial cell leakage ( 18 ). In the present study, some modest morphological changes occurred in the actin cytoskeleton after treatment with rhHMGB1 for 30 min ( Figure 1D ), which could reflect the enhanced Rho/ROCK activity and MLC phosphorylation. As shown in Figure 1I , rhHMGB1 stimulation (600 ng/ml) for 60 minutes obviously increased the expression level of pMLC, which was consistent with a time-dependent increase in RhoA activity and ROCK1 expression ( Figures 1G, H ). Results showed that rhHMGB1 induced an increase in Ser-19 phosphorylation at 30 min and 1 h and the expression level of Ser-19 phosphorylated MLC returned to baseline by 24 h, as shown on immunoblot ( Figure 1I ). The inhibition of ROCK1 expression with Y-27632 and ROCK1 siRNA could downregulate the phosphorylation of MLC after 60 min of rhHMGB1 treatment ( Figures 2G , 3H ), which was accompanied by decreases in the fluorescence intensity of F-actin at 60 min ( Figures 2D , 3F ). Pretreatment with FPS-ZM1 and RAGE siRNA could result in a marked downregulation of the activity of RhoA/ROCK1 and phosphorylation of MLC in HPMECs treated with rhHMGB1 for 60 minutes ( Figures 4C, F and 5E, F ), and then reduce the fluorescence intensity of stress fibers in the cell center at 60 min ( Figures 4D , 5D ). Figure 4 FPS-ZM1 treatment alleviated rhHMGB1-mediated RhoA/ROCK1 activation and barrier dysfunction. (A) Cell viability was determined by CCK-8 assay after treated with different dosage of FPS-ZM1 for 24 h. (B) FPS-ZM1 improved lung endothelial permeability at 60 min and 24 h after rhHMGB1 stimulation. (C) FPS-ZM1 significantly downregulated RhoA and ROCK1 expression in HPMECs at 60 min after rhHMGB1 stimulation. (D) Effects of FPS-ZM1 on rhHMGB1-mediated morphological changes in endothelial F-actin, VE-cadherin and ZO-1. ECs were treated with FPS-ZM1 for 1 h prior to stimulation with rhHMGB1 to evaluate morphology of endothelial cytoskeleton F-actin or for the last 4 h of the 24 h rhHMGB1 stimulation to assess morphology of endothelial VE-cadherin and ZO-1 by immunofluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (E) FPS-ZM1 significantly increased VE-cadherin and ZO-1 expression levels in HPMECs at 24 h after rhHMGB1 stimulation. ECs were treated with FPS-ZM1 for the last 4 h of the 24 h rhHMGB1 stimulation. (F) Effects of FPS-ZM1 on rhHMGB1-mediated pMLC expression in cells. ECs were pretreated with FPS-ZM1 for 1 h and then treated with rhHMGB1 for 60 minutes. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group. Figure 5 rhHMGB1-induced RhoA/ROCK1 pathway activation via RAGE in HPMECs. (A) ECs were transfected with RAGE siRNA. Western blots were used to determine the expression of RAGE in endothelial cells. (B) Cytotoxicity of RAGE siRNA was assessed by CCK-8 assay after transfected with different concentration of RAGE siRNA for 24 h or transfected with 100 nM RAGE siRNA for the different times. No evidence of cytotoxicity was found in RAGE siRNA transfected cells. (C) Treatment with RAGE siRNA ameliorated endothelial barrier dysfunction induced by rhHMGB1 at 60 min and 24 h. (D) ECs were transfected with RAGE siRNA and then stimulated with rhHMGB1 for 60 min and 24 h. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was determined by fluorescence microscopy. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Knockdown of RAGE by siRNA reduced the rhHMGB1-induced MLC phosphorylation at 60 min as detected by western blot. (F) Effects of inhibition of RAGE with siRNA on increased expression of RhoA and ROCK1 induced by rhHMGB1 at 60 min in HPMECs. (G) Role of RAGE siRNA in the VE-cadherin and ZO-1 protein expression levels in ECs at 24 h after rhHMGB1 treatment. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group. NC, negative control. RhoA/ROCK1 Pathway Mediates HMGB1-Induced EC Barrier Disruption in HPMECs As shown in Figures 1G, H , the time-dependent increases in RhoA activity and ROCK1 expression by rhHMGB1 treatment were measured. The results showed that the peak activity of RhoA appeared at 60 min and rhHMGB1 treatment also significantly up-regulated the expression of ROCK1 at 60min. Thus, binding of active GTP-bound RhoA caused unfolding and activation of ROCK1, which was accompanied by rhHMGB1-mediated MLC phosphorylation, stress filament formation, VE-cadherin and ZO-1 disruption, and the early increase in EC barrier permeability ( Figure 1 ). Pretreatment with Y-27632 and ROCK1 knockdown could partially inhibit rhHMGB1-mediated EC leakage and restore FITC-dextran flux of the endothelial barrier after 60 min of rhHMGB1 stimulation ( Figures 2B , 3D ). After pretreatment with Y-27632 and ROCK1 siRNA, the expression of ROCK1 protein at 60 min was significantly downregulated ( Figures 2E , 3G ), and the membrane location of VE-cadherin and ZO-1 in intercellular junctions was partially restored after 60 min of rhHMGB1 stimulation ( Figures 2D , 3F ). The 60 min exposure of rhHMGB1 increased the fluorescence intensity of F-actin in the cell center, and pretreatment with Y-27632 and ROCK1 siRNA decreased the fluorescence intensity of central stress filaments after 60 min of rhHMGB1 exposure ( Figures 2D , 3F ). These findings suggest that RhoA and ROCK1 may be implicated in rhHMGB1-mediated early increase in EC permeability and stress filament formation in HPMECs. RAGE Mediates the EC Barrier Leakage Induced by HMGB1 To investigate whether RAGE is implicated in the rhHMGB1-mediated increases in EC barrier permeability, the expression of RAGE was silenced by siRNA in HPMECs. In addition, HPMECs were also treated with the specific RAGE inhibitor FPS-ZM1 ( 20 ) for 60 min prior to stimulation with rhHMGB1 or for the last 4 h of the 24 h rhHMGB1 stimulation. As indicated in Figures 4B , 5C , FPS-ZM1 and RAGE knockdown significantly decreased endothelial permeability by comparison with the rhHMGB1 treatment group at 60 min and 24 h. Inhibition of RAGE with FPS-ZM1 and RAGE siRNA could also downregulate the activity of RhoA/ROCK1 and phosphorylation of MLC at 60 min ( Figures 4C, F and 5E, F ), and then partly prevent the rhHMGB1-mediated formation of stress fibers in the cell center ( Figures 4D , 5D ). Furthermore, inhibition of ROCK1 could also reduce the expression of pMLC ( Figures 2G , 3H ), and partly recover the membrane localization of VE-cadherin and ZO-1 after 60 min of rhHMGB1 stimulation ( Figures 2D , 3F ). Therefore, it seems likely that a signaling pathway proceeds from RAGE to RhoA/ROCK1 through the F-actin reorganization and disruption of AJ/TJ related proteins, which finally destroys the early EC barrier function in the present study. In addition, FPS-ZM1 and RAGE siRNA could upregulate the expression levels of VE-cadherin and ZO-1 at 24 h ( Figures 4E , 5G ). Similarly, inhibition of RAGE could also partially restore the cytomembrane location of VE-cadherin and ZO-1 after 24 h of rhHMGB1 stimulation ( Figures 4D , 5D ). Discussion HMGB1 is released in late endotoxemia and it is closely associated with the severity and prognosis of sepsis ( 21 , 22 ). It is demonstrated that HMGB1 could elicit microvascular EC cytoskeletal rearrangement and barrier dysfunction ( 12 ). EC cytoskeleton, especially F-actin rearrangement, is main histological basis in enhanced EC barrier permeability, which elicits the increase in cell contractility and intercellular gap formation ( 23 , 24 ). To our best knowledge, this study demonstrates here that HMGB1 elicits progressive changes to the filamentous actin, intercellular junctions and increases HPMEC barrier permeability in a time-dependent and dose-dependent manner. Furthermore, RAGE and RhoA/ROCK1 were implicated in the HMGB1-mediated EC cytoskeletal reorganization and early endothelial barrier dysfunction. This present study showed that HMGB1 disrupted the microvascular endothelial cell barrier, which might be implicated in the pathogenesis of ALI in sepsis. Phosphorylation of MLC has been reported to involve in the regulation of EC barrier permeability after treatment with thrombin, histamine, and other inflammatory cytokines and so on ( 18 , 19 ). In the present study, the early rhHMGB1-induced MLC phosphorylation and subsequent formation of actin stress fibers indicated that changes in EC contractility was occurring. RhoA plays a key role in control of endothelial cellular actin cytoskeletal rearrangement and cell morphology. ROCK (a downstream target of RhoA) mediates stress filament formation by upregulating the levels of MLC phosphorylation ( 25 – 27 ). Then, phosphorylated MLC contributes to actomyosin interaction, causing EC contraction and an increased permeability ( 28 , 29 ). Furthermore, ROCK1 activation has been reported to influence cell-cell adhesion by modulating interactions between stress fibers and intercellular binding molecules (TJ and AJ) ( 30 ). It was demonstrated that ROCK1 activation induced by high glucose caused endothelial-to-mesenchymal transition with loss of CD31 and VE-cadherin, resulting in increased endothelial permeability ( 31 ). Previous study also shows that anthrax lethal toxin causes EC barrier dysfunction through actin filament formation and disruption of adherens junctions ( 32 ). This study indicated that the changes of VE-cadherin expression associated temporally with the formation of stress fibers and activation of ROCK1 ( 32 ). In this study, our findings showed that RhoA/ROCK1 was activated rapidly by rhHMGB1 in cultured HPMECs and rhHMGB1 stimulation induced rapid aggregation of actin stress filaments, intercellular gap formation and a significant increase in endothelial cell permeability by 60 minutes, indicating that the early cytoskeletal reorganization could affect permeability. To confirm the role of ROCK played in rhHMGB1-mediated EC barrier dysfunction, Y-27632 was added for 1 h prior to treatment with rhHMGB1 or for the last 4 h of the 24 h rhHMGB1 stimulation. Moreover, the ECs were transfected with ROCK1/2 siRNA before rhHMGB1 treatment. The results showed that Y-27632 and ROCK1 knockdown were able to significantly suppress ROCK1 activation, subsequent MLC Phosphorylation and the rhHMGB1-induced hyperpermeability at 60 min. In addition, Y-27632 and ROCK1 knockdown decreased the number of central stress filaments and partially restored cortical localization of F-actin and membrane location of VE-cadherin and ZO-1 at 60 min of rhHMGB1 stimulation in HPMECs. Increased endothelial barrier permeability is often related to lack or disruption of AJ/TJ proteins ( 7 , 33 ) and indeed VE-cadherin and ZO-1 expressions were downregulated following a long-term rhHMGB1 stimulation in this study. The results of western blot showed that inhibition of ROCK with Y-27632 had no influence on the expression of VE-cadherin and ZO-1 at 24 h of rhHMGB1 stimulation in HPMECs, suggesting that rhHMGB1 induced the disruption of EC barrier at 24 h independent of its effect on the RhoA/ROCK signaling. In addition, our previous study indicated that HMGB1 downregulated AJ/TJ components at 24 h through activation of the RAGE/p38 signaling pathway ( 7 ). Other study showed that HMGB1 elicited the activation of the RAGE/ERK1/2 pathway at 24 h, which correlated with barrier dysfunction in the human bronchial epithelial cells ( 34 ). So far, many evidences show that HMGB1 interacts with endothelial cell through RAGE. To study the role of RAGE in EC barrier dysfunction, we inhibited the RAGE activity using RAGE siRNA and its specific inhibitor FPS-ZM1 ( 20 , 35 ). Recent studies indicated that the inhibition of RAGE functions via FPS-ZM1 might be a meaningful therapeutic strategy for a variety of diseases, such as diabetes-related glomerular filtration barrier damage and irradiation-induced EC barrier disruption ( 10 , 36 ). Our study also demonstrated that the blockage of RAGE with FPS-ZM1 and RAGE siRNA attenuated the EC barrier hyperpermeability mediated by rhHMGB1 and blocked the rhHMGB1-induced RhoA/ROCK1 activation and MLC phosphorylation. Furthermore, our findings also confirmed FPS-ZM1 as a potential therapeutic drug for treating rhHMGB1-induced EC barrier dysfunction. In conclusion, our findings demonstrate that rhHMGB1 could induce the early EC barrier disruption, and the potential molecular mechanism may be that rhHMGB1 activates the RhoA/ROCK1 signaling pathway through RAGE, which mediates the phosphorylation of MLC inducing stress fiber formation at short time (up to 60 min), and HMGB1/RAGE disrupts the integrity of AJ/TJ at long term (up to 24 h) independently of RhoA/ROCK1 signaling pathway. These new findings will help to understand the signaling pathways of rhHMGB1-mediated increase in EC barrier permeability and contribute to establish potential therapeutic targets in the treatment of sepsis. Data Availability Statement The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors. Author Contributions M-JZ, H-RJ, J-WS, Z-AW, BH, C-RZ, X-HY, M-MC and X-CM participated in experimental design, research, data analysis and draft writing. M-JZ, H-RJ and Z-GL wrote and revised the manuscript. Z-GL and W-DZ contributed to the research concept, study design, data analysis and finalization. All authors contributed to the article and approved the submitted version. Funding The present study was supported by grants from the Natural Science Foundation of Liaoning Province, China (No. 2019-MS-09) and the Liaoning Xingliao Talent Plan Project (No. XLYC2005015). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Abbreviations HPMEC, human pulmonary microvascular endothelial cell; HMGB1, high mobility group box 1; ALI, acute lung injury; AJ, adherens junction; TJ, tight junction; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens 1; RAGE, receptor for advanced glycation end products; ROCK, Rho kinase; MLC, myosin light chain; pMLC, phosphorylated MLC; EC, endothelial cell.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583238/

One Health in Indigenous Communities: A Critical Review of the Evidence

Indigenous populations around the world face disproportionately high rates of disease related to the environment and animals. One Health is a concept that has been used effectively to understand and address these health risks. One Health refers to the relationships and interdependencies between animal, human, and environmental health and is an emerging research field that aligns with indigenous views of health. To understand the applicability of One Health in indigenous communities, a critical review was undertaken to investigate evidence of One Health research in indigenous communities internationally, assess the strength of evidence, and understand what gaps are present. This review included the appraisal of twenty-four studies based in five regions: Canada, Africa, Australia, South America, and Central America. The review found that there is a need for studies of high strength, with rigorous methods, local leadership, and active involvement of indigenous viewpoints, to be undertaken in indigenous communities internationally that focus on One Health. It highlights the need to further consider indigenous viewpoints in research to reduce limitations, increase effectiveness of findings, consider appropriateness of recommendations, and benefit communities. 1. Introduction The One Health concept can be traced back to the ancient Greek philosopher Hippocrates who recognized that human health depends on the environment [ 1 ]. However, it is likely that the ideologies surrounding One Health goes back further than this to indigenous societies and cultures that are tens of thousands of years old. The concept of One Health aligns with indigenous views encompassing a holistic view of health that recognizes traditional knowledge that links the health and wellbeing of animals, people, and the environment [ 2 ]. This approach is suitable in indigenous community settings as it aligns with cultural and community contexts, and indigenous ways of knowing, doing, and being. The One Health concept also considers health as more than the physical self, which is in line with the World Health Organization's definition of health as 'a state of complete physical, social and mental well-being, and not merely the absence of disease' [ 3 ] (p. 1). This holistic way of thinking continued into the nineteenth century with Rudolf Virchow stating that 'between animal and human medicine there is no dividing line—nor should there be' [ 4 ] (p. 100). In the 1980s 'One Medicine', which focused on bringing together the commonalities between animal and human medicine, was conceptualized by Calvin Schwabe [ 5 ]. In the early 2000s, following multiple zoonotic outbreaks, this concept evolved to 'One Health' which refers to a collaborative and interdisciplinary approach, at local, national, and global levels, to improve health for people, animals, and the environment [ 6 ]. There are similar concepts emerging from health sectors that have comparable aims with slightly different focuses. The 'One Welfare' concept has an emphasis on welfare; however, there is much overlap between these concepts, as One Welfare concentrates on the relationships between animal welfare, human wellbeing, and the environment [ 7 ]. The concept of 'Ecohealth' recognizes the inseparable linkages between ecosystems and the health of all species [ 8 ]. Similarly, the concept of 'Planetary Health' has also emerged and, driven through the human health sector, emphasizes human health systems and the linkages to political, economic, and social settings, and the Earth's natural environment [ 9 ]. This review focuses on One Health which can be used as an approach to designing and implementing programs, policies, and research in which multiple sectors work together to achieve better public health outcomes [ 10 ]. Its benefits include more timely and effective responses, accurate decision making, accountability to other sectors, shared responsibilities and resources, and advocacy for policies and programs [ 10 ]. One Health is supported internationally through the World Health Organization, the World Organization for Animal Health, and the Food and Agricultural Organization of the United Nations Tripartite Alliance that promotes the concept of One Health as the optimal method of preventing and controlling emerging and endemic zoonotic diseases. This interdisciplinary approach has been noted as an effective way of addressing health threats at the human–animal–environment interface and is the best way of preventing and responding to future zoonotic outbreaks and pandemics [ 11 ]. One Health can also take a transdisciplinary approach, which can be particularly relevant when working in indigenous community contexts as this requires a 'whole-of-society' approach and recognizes cultural knowledge [ 12 ]. Research has shown that animal and environmental factors contribute to the health and wellbeing outcomes of indigenous peoples, including the ongoing impact of environmental stress on the health of animals and people [ 13 , 14 ]. Many public health risks that may benefit from a One Health approach disproportionally affect low socio-economic communities, many of which have high indigenous populations. Such risks include exotic zoonotic disease threats, such as an increased risk of rabies incursion into remote Aboriginal communities in Northern Australia [ 15 ]. This also includes endemic diseases (including neglected zoonotic diseases) that are of particular concern for low socio-economic communities [ 16 ]. Whilst zoonotic diseases may have significant impacts on communities, they are also among the most under-diagnosed diseases in humans, with the full burden of disease not well understood [ 16 ]. Within Aboriginal communities in Australia, there is a particular need for high-quality, large-scale, and comparative studies of companion animals and people from the same household to further assess zoonotic disease risk and prevalence [ 17 ]. While there is limited evidence in Australia, findings and recommendations internationally indicate that a One Health approach has the potential to improve health within indigenous communities [ 18 , 19 , 20 ]. The One Health field is still emergent. Public health interventions remain predominantly designed and managed in siloed health sectors, with limited communication and collaboration [ 8 ]. To date, the animal health sector has largely supported and promoted the One Health concept, with much of the literature and programs emerging through the animal health field [ 21 ]. Animal programs that deliver health care and consider a One Health approach are becoming more common internationally, particularly in low-resource communities. However, many of these programs do not incorporate traditional knowledge and teachings, with indigenous involvement commonly ceasing at the engagement phase [ 22 ]. A transdisciplinary approach is likely to improve this. When designing and implementing these programs, it is important to consider that animals have integral roles in indigenous communities and can be of cultural significance through traditional values and beliefs. In some Australian Aboriginal communities, dogs can be seen as the spiritual protector of the family and the land, and integrated into the kinship system [ 23 , 24 ]. They are also commonly accepted as family members and provide a sense of purpose for owners, with many owners feeling strong bonds and connections with their dogs [ 25 ]. Companion animals can also be helpful for hunting, providing protection, companionship, and warmth, and can provide health benefits for their owners through psychological, social, and emotional benefits, and improving health outcomes [ 26 ]. Given the significance and meaning of animals in indigenous communities and the benefits of animal companionship, ensuring the health and welfare of animals should be seen as a priority. However, many indigenous communities face barriers in accessing ongoing and effective animal health care [ 27 ]. Barriers can include limited resources and funding, remoteness and accessibility, limited veterinary services and animal medicines, and climatic impacts which differ between countries [ 20 ]. For example, many Australian Aboriginal communities face tropical monsoon climates that restrict access because of flooding, whereas Canadian Indigenous communities can be cut off by ice for much of the year [ 20 ]. Low socio-economic position, language, literacy and numeracy barriers, and awareness of the benefits of animal health care also limit the use of animal medicines and veterinary services where these are available [ 24 ]. Studies have found that community animal health and management programs can be beneficial in these settings, with community-specific strategies that take a transdisciplinary approach the most effective [ 28 ]. However, the evidence of a One Health approach including culturally appropriate programs incorporating local beliefs regarding the roles and importance of animals within the indigenous health space is limited. This critical review aimed to understand One Health research that has been undertaken in indigenous communities worldwide, assess the strength of the evidence base, and identify what gaps exist. This review will be used to inform the development of a One Health model for use in Aboriginal communities with particular interest in improving animal health care and community health outcomes. 2. Materials and Methods A critical review of the evidence provides an opportunity to understand and assess current evidence and frameworks and consider the development of a new model [ 29 ]. 2.1. Indigenous Approach This review was undertaken by an Australian Aboriginal-led interdisciplinary team that considered indigenous research methodologies, recognizing the cultural knowledge surrounding the interconnectedness between animal, human, and environmental health. Rigney described the importance of privileging indigenous voices as a key component of indigenous research methodologies [ 30 ]. Indigenous research methodologies recognize the impact of colonization and the political and social structures which continue to impact indigenous peoples [ 31 ]. Following this methodology, this review concentrated mainly on countries that have settler-colonial societies and indigenous populations which have been influenced by colonization, racism, and social injustice [ 31 ]. It also adapted an existing framework to include a focus on indigenous viewpoints in the scoring of evidence to assess the manner of indigenous involvement and priorities in the evidence base. 2.2. Search Strategy Due to the differences in the use of terms relating to holistic health approaches globally, and the differing focuses of these approaches, multiple terms were included in the review. The definitions of the terms used were: Ecohealth: 'Ecohealth is committed to fostering the health of humans, animals, and ecosystems and to conducting research which recognizes the inextricable linkages between the health of all species and their environments' [ 9 ] (p. 3); One Health: 'One Health is the collaborative effort of multiple health science professions, together with their related disciplines, and institutions—working locally, nationally, and globally—to attain optimal health for people, domestic animals, wildlife, plants, and our environment' [ 9 ] (p. 2–3); One Welfare: 'One Welfare describes the inter-relationship between animal welfare, human wellbeing and the physical and social environment' [ 7 ] (p. 1); Planetary Health: 'The achievement of the highest attainable standard of health, well-being, and equity worldwide through judicious attention to the human systems—political, economic, and social—that shape the future of humanity and the Earth's natural systems that define the safe environmental limits within which humanity can flourish' [ 9 ] (p. 4). 2.3. Literature Sources and Search Terms A literature search was conducted using PubMed, the Web of Science, The Australian National University (ANU) library super search, and the Australian Indigenous Health Infonet search engines. The search terms were developed through an iterative process with input from all co-authors. The search terms used are presented in Table 1 . 2.4. Selection of Studies All documents selected from the search were screened by two reviewers (TR and JT), with 12 studies screened by a third reviewer (NA), from animal health, human health, and One Health backgrounds. Reviewers discussed conflicts until a consensus was reached with respect to inclusion criteria. Studies were screened by the third reviewer when there was uncertainty as to whether a study met the inclusion criteria. The studies identified through the search were assessed against these inclusion criteria: Original research; Written in English; Published between 2010 and 2020; Full text available; One Health, Ecohealth, Planetary Health, or One Welfare focus; Animal, human, and/or environmental health component (at least two of these); An indigenous group as the main human population of interest. Studies that met all the criteria were included in the review, with additional references included based on authors prior knowledge of the literature. All studies were stored in Endnote, and records of the search and inclusion assessment were kept in Excel. 2.5. Summarising and Assessing the Strength of Evidence (SOE) We summarized the current literature on One Health research in indigenous communities globally. Each study was summarized by the One Health sector focus (animal, human, and environmental health, or multiple) and key findings and recommendations. For the purpose of this review, we defined the One Health sectors as: animals referred to domestic animals and their health outcomes, human referred to the indigenous communities or peoples and their health outcomes, and environment referred to ecosystems and biodiversity factors including the physical environment, plants, wildlife, and invertebrates that live within an ecosystem. The analysis included calculating percentages to describe the characteristics of the evidence base including the One Health sector, Indigenous viewpoint, strength of evidence (SOE), and region. We assessed the SOE based on two reviewers' assessments of the studies (TR and JT). Reviewers assessed them independently and discussed conflicts until a consensus was reached. The review was undertaken using a modified version of the SOE framework developed by the Evidence-based Practice Centre, established by the US Agency for Healthcare Research and Quality (AHRQ), and based on the grading of recommendations, assessment, development, and evaluations (GRADE) approach [ 32 , 33 , 34 ]. The SOE framework involves assessing each study across five domains including study limitations, directness, consistency, precision, and reporting bias, with the studies scored as high, moderate, low or insufficient [ 33 ]. Given the focus on indigenous populations, studies were also assessed for an indigenous viewpoint [ 35 ] ( Table S1 ). Where a domain was not applicable to the study, this was noted as such and was not included in the overall score. There was no evidence found that any of the domains are more important than the other and should be weighted differently. Therefore, we considered each domain equal and allocated the same weighting. Each domain met was added and the studies were scored as: 0 domains met = insufficient SOE; 1–2 domains met = low SOE; 3–4 domains met = moderate SOE; 5–6 domains met = high SOE. 2.1. Indigenous Approach This review was undertaken by an Australian Aboriginal-led interdisciplinary team that considered indigenous research methodologies, recognizing the cultural knowledge surrounding the interconnectedness between animal, human, and environmental health. Rigney described the importance of privileging indigenous voices as a key component of indigenous research methodologies [ 30 ]. Indigenous research methodologies recognize the impact of colonization and the political and social structures which continue to impact indigenous peoples [ 31 ]. Following this methodology, this review concentrated mainly on countries that have settler-colonial societies and indigenous populations which have been influenced by colonization, racism, and social injustice [ 31 ]. It also adapted an existing framework to include a focus on indigenous viewpoints in the scoring of evidence to assess the manner of indigenous involvement and priorities in the evidence base. 2.2. Search Strategy Due to the differences in the use of terms relating to holistic health approaches globally, and the differing focuses of these approaches, multiple terms were included in the review. The definitions of the terms used were: Ecohealth: 'Ecohealth is committed to fostering the health of humans, animals, and ecosystems and to conducting research which recognizes the inextricable linkages between the health of all species and their environments' [ 9 ] (p. 3); One Health: 'One Health is the collaborative effort of multiple health science professions, together with their related disciplines, and institutions—working locally, nationally, and globally—to attain optimal health for people, domestic animals, wildlife, plants, and our environment' [ 9 ] (p. 2–3); One Welfare: 'One Welfare describes the inter-relationship between animal welfare, human wellbeing and the physical and social environment' [ 7 ] (p. 1); Planetary Health: 'The achievement of the highest attainable standard of health, well-being, and equity worldwide through judicious attention to the human systems—political, economic, and social—that shape the future of humanity and the Earth's natural systems that define the safe environmental limits within which humanity can flourish' [ 9 ] (p. 4). 2.3. Literature Sources and Search Terms A literature search was conducted using PubMed, the Web of Science, The Australian National University (ANU) library super search, and the Australian Indigenous Health Infonet search engines. The search terms were developed through an iterative process with input from all co-authors. The search terms used are presented in Table 1 . 2.4. Selection of Studies All documents selected from the search were screened by two reviewers (TR and JT), with 12 studies screened by a third reviewer (NA), from animal health, human health, and One Health backgrounds. Reviewers discussed conflicts until a consensus was reached with respect to inclusion criteria. Studies were screened by the third reviewer when there was uncertainty as to whether a study met the inclusion criteria. The studies identified through the search were assessed against these inclusion criteria: Original research; Written in English; Published between 2010 and 2020; Full text available; One Health, Ecohealth, Planetary Health, or One Welfare focus; Animal, human, and/or environmental health component (at least two of these); An indigenous group as the main human population of interest. Studies that met all the criteria were included in the review, with additional references included based on authors prior knowledge of the literature. All studies were stored in Endnote, and records of the search and inclusion assessment were kept in Excel. 2.5. Summarising and Assessing the Strength of Evidence (SOE) We summarized the current literature on One Health research in indigenous communities globally. Each study was summarized by the One Health sector focus (animal, human, and environmental health, or multiple) and key findings and recommendations. For the purpose of this review, we defined the One Health sectors as: animals referred to domestic animals and their health outcomes, human referred to the indigenous communities or peoples and their health outcomes, and environment referred to ecosystems and biodiversity factors including the physical environment, plants, wildlife, and invertebrates that live within an ecosystem. The analysis included calculating percentages to describe the characteristics of the evidence base including the One Health sector, Indigenous viewpoint, strength of evidence (SOE), and region. We assessed the SOE based on two reviewers' assessments of the studies (TR and JT). Reviewers assessed them independently and discussed conflicts until a consensus was reached. The review was undertaken using a modified version of the SOE framework developed by the Evidence-based Practice Centre, established by the US Agency for Healthcare Research and Quality (AHRQ), and based on the grading of recommendations, assessment, development, and evaluations (GRADE) approach [ 32 , 33 , 34 ]. The SOE framework involves assessing each study across five domains including study limitations, directness, consistency, precision, and reporting bias, with the studies scored as high, moderate, low or insufficient [ 33 ]. Given the focus on indigenous populations, studies were also assessed for an indigenous viewpoint [ 35 ] ( Table S1 ). Where a domain was not applicable to the study, this was noted as such and was not included in the overall score. There was no evidence found that any of the domains are more important than the other and should be weighted differently. Therefore, we considered each domain equal and allocated the same weighting. Each domain met was added and the studies were scored as: 0 domains met = insufficient SOE; 1–2 domains met = low SOE; 3–4 domains met = moderate SOE; 5–6 domains met = high SOE. 3. Results The literature search identified a total of 106 peer-reviewed studies. After screening, 24 references (22 from the search and 2 from authors prior knowledge) were included in the analysis ( Figure 1 ). Overall, 46% of studies were rated as having a moderate SOE ( n = 11), 29% were rated as low ( n = 7), and 25% were rated as high ( n = 6), with none rated as insufficient. The majority of studies incorporated all three One Health sectors (54%), followed by studies involving animals and people (33%), and studies involving people and the environment only (13%). The majority of studies had an indigenous viewpoint (19 of 24) including all studies scored as high, 91% of studies scored as moderate, and 43% of studies scored as low ( Table 2 ). Studies with a high SOE commonly had large sample sizes (for example, community level data or data from multiple communities), multiple sample types from different health sectors (for example, an animal and a human health variable), multiple time points for data collection (for example, repeated collection of data over multiple months or years), and indigenous viewpoints. The details of the evidence base and scoring can be seen in Table S2 . The main regions represented in the results were Canada ( n = 8), Australia ( n = 7), Africa ( n = 5), Central America ( n = 2), and South America ( n = 2). Studies from Canada and Australia were most commonly scored as moderate, Africa and South America equally had studies scored as low and high, and Central America had studies scored as low. The frequency of SOE ratings in each region is shown in Figure 2 . 4. Discussion This review found that the incorporation of all three One Health sectors does not indicate a high SOE. Overall, studies were most commonly rated as having a moderate SOE (46%), followed by those scored as low (29%), and high (25%), with none scored as insufficient. While all studies were interdisciplinary, the majority included in the review incorporated all three One Health sectors (54%), followed by those involving animals and people (33%), and studies involving people and the environment (13%). As the search focused on indigenous communities, studies with a focus on animals and the environment only were unlikely to be identified which may have limited studies that incorporated an environmental health component. When broken down by SOE, 71% of studies scored as low included all three One Health sectors, compared to 45% of studies scored as moderate, and 50% of studies scored as high. Undertaking studies that incorporate all three sectors can be more complex compared to those which focus on two sectors only; however, they can also be more beneficial. While the One Health concept commonly aims for interdisciplinary research, and this evidence base has demonstrated this, One Health within indigenous communities should strive for transdisciplinary research which takes a whole-of-society approach, values the inclusion of local community members, and prioritizes benefiting indigenous peoples [ 12 ]. While the concept has been supported by international health organizations, the practicalities of enacting it can be multifaceted and further consideration on how to truly implement the concept is needed [ 10 ]. The ownership and collaboration required between sectors can be a contested point, with many health sectors working within silos and limited examples of truly interdisciplinary work being operationalized and reported. Environmental health is commonly under-represented within One Health [ 57 ] and this review supported this with environmental health the most common aspect missing. This is particularly pertinent for research undertaken with indigenous communities, as the environment plays an important role in connecting animal and human health, and can hold significant cultural meaning. Many of the studies assessed the connection between animal and human health and noted the lack of an environmental health component as a limitation. However, more than half of the studies did include all three One Health sectors which may reflect the high value placed on the environment by indigenous populations involved in the research. Social science is also commonly neglected in One Health research with the behavioral aspects of health and man-made environmental changes often not incorporated [ 26 , 58 ]. Therefore, ways to collaborate with the environmental health and social science sectors should be considered in future work [ 18 , 19 ]. The majority of the evidence base, and all high SOE studies, had an indigenous viewpoint; however, this was not always easy to assess with this domain having many limitations. We assessed a study as having an indigenous viewpoint if it involved community engagement, involvement, partnerships, or if it included indigenous authors; however, the latter was only possible when the authorship included information about the diversity of the authors. Studies were scored as having no indigenous viewpoint if it was not obvious whether an indigenous viewpoint had been sought. This approach was appropriate as indigenous research methodologies suggest the incorporation of community involvement and genuine engagement when undertaking research in the indigenous health space [ 31 ]. It is particularly relevant for One Health research to recognize and incorporate cultural beliefs and values which emanate around the connection to animals and the environment [ 59 ]. Following indigenous research methodology principles, the involvement of indigenous peoples within the research, including indigenous authors, should be sought and described explicitly to privilege indigenous voices [ 30 ]. Indigenous engagement is commonly stated as an aim of One Health research within communities; however, there is a lack of cultural knowledge and teachings incorporated into studies and programs delivered within communities [ 22 ]. Approaches to extend the involvement of indigenous peoples from engagement to partnerships and leaders within One Health is required to ensure this work is undertaken appropriately and for the benefit of communities. This is in line with indigenous research methodologies which recognize the importance and value of partnerships with community organizations and leadership by indigenous peoples [ 31 ]. It is important that this involvement is genuine and built on trust to ensure research is undertaken appropriately, incorporates community priorities, and recognizes cultural values and beliefs [ 59 ]. Additionally, indigenous-led research projects are being increasingly recognized as important worldwide as this allows indigenous peoples ownership over ideas, methodologies, and associated data, and can increase the effectiveness and sustainability of initiatives [ 60 , 61 ]. The regions included in the evidence base were Canada, Australia, Africa, Central America, and South America. Canada and Australia most commonly had moderately scored studies, with Africa and South America equally having studies scored as low and high, and Central American studies scored as low. This indicates that there is a need to increase the SOE of One Health research in all regions. Some regions were not represented in the analysis which may be due to holistic health and indigenous terms used in the search not being applicable in other regions, or studies not meeting inclusion criteria, such as written in English. It could also demonstrate a lack of research within these regions, demonstrating multiple gaps in this field internationally. Strengths of this review included an interdisciplinary research team and the literature obtained from multiple sectors, taking a One Health approach to the review. This is also the first review that scores the evidence base of One Health in indigenous communities internationally. This review adopted an indigenous research methodology and modified an existing scoring framework to include an indigenously focused domain as part of the framework. This was important to the review as it focused on research related to indigenous communities around the world. There were common study limitations through the evidence base related to sampling methods as many studies had convenience, opportunistic or referral sampling, used secondary data, had small sample sizes, or only involved participants from a biased sample, such as only animal owners. This restricted the ability to score the evidence and to some extent diminished the overall strength of the evidence base. Many of these studies were small and, therefore, it may not have been feasible to adopt a robust study design; however, the data and findings from these studies are still valuable to the evidence base. While there can be complexities of undertaking research with an interdisciplinary or transdisciplinary approach, there is a need to improve the evidence base by conducting research that involves robust, community level, primary data collection. However, the assessment criteria within this study were limited to a framework based on human health research and may need further adaption to meet the complexity of the One Health field. Another limitation noted in the evidence base was language barriers between researchers and participants, particularly in communities that spoke indigenous languages as the primary language. This highlights the importance of working with local people from the community to assist with translation and mitigate this limitation. There was a small number of studies identified in the evidence base highlighting the gaps that exist in this field internationally; however, this is expected as One Health is an emerging research field particularly in relation to indigenous health. The search results were limited to studies written in English which may have introduced a geographical bias and excluded relevant studies written in another language. There was also differing usage of the holistic health and indigenous group terms between countries which may have further limited the results. The holistic health terms used are aiming to achieve similar outcomes and acknowledge the interconnection between animal, human, and environmental health; however, they all have slightly different focuses due to their emergence from different sectors [ 26 ]. There are reports of interventions within indigenous communities that aim to improve holistic health measures; however, these studies were not included in the review if they did not use one of the search terms. The assessment tool is also weighted heavily toward quantitative approaches to evidence. It is possible that it does not capture what qualitative elements are for evidence, and this could have affected the scoring of the evidence base. Many studies in the evidence base were prevalence, descriptive, or qualitative in nature, which were more subjective to assess and limited the ability to score the evidence. For example, if the study did not include an intervention, the directness of the study could not be scored, eliminating this domain and leading to a lower SOE. While this was common, no studies were excluded due to low SOE as they still contained valuable findings and recommendations. For the One Health approach to be effective in indigenous communities, increased collaboration and communication between the animal and human health sectors is needed, with mutual recognition of the importance of the environment and ecological factors [ 5 ]. This may be particularly pertinent for indigenous health research due to the connections to the environment which many indigenous cultures have. The incorporation of social science to account for behavioral and social factors impacting health would also be helpful in producing comprehensive findings and recommendations [ 58 ]. Without this, we may fail to see the full benefits of a One Health approach. There is a need for One Health studies within indigenous communities internationally that have a high SOE and robust sampling methods. This finding is in line with prior studies that found a need for high-quality, large-scale, and comparative studies of companion animals and people from the same household in Australian Aboriginal communities [ 17 ]. Following indigenous research methodologies, the involvement and leadership of indigenous peoples in research within community settings should be prioritized to ensure consideration of community priorities and strengthen the evidence base [ 60 ]. While evidence shows that a One Health approach would be suitable in indigenous communities and is supported internationally, there is a lack of One Health models in indigenous communities described in the evidence base. Following recommendations from the review, a strong community One Health model for animal health care should focus on prevention and have indigenous leadership and involvement to be successful and sustainable. A transdisciplinary approach should be adopted to allow the incorporation of cultural knowledge and fit within indigenous community contexts and views of health that recognize the importance of animals and the environment to health. Increased collaboration and integration of community members into health systems and organizations would be beneficial to allow the sharing of knowledge and responsibilities between human, animal, and environmental health sectors and take a whole-of-community approach. 5. Conclusions One Health is highly appropriate and applicable within indigenous communities as it adopts a holistic approach and is in line with indigenous cultural beliefs and views of health. However, this review found that there are limited One Health studies that have been undertaken in indigenous communities globally, with many gaps in the evidence base. There is a need for high SOE research to be undertaken in indigenous communities internationally to further understand how One Health approaches may be applied in this setting. The involvement and leadership of indigenous peoples in the research is vital to the effectiveness and sustainability of outcomes. The inclusion of environmental health needs further consideration to enhance understanding of the importance of the environment within health relationships. Increased collaboration and communication between health sectors are also needed to realize the benefits of One Health. This review recommends the development of an effective One Health model for animal health care within indigenous communities that is indigenous-led and transdisciplinary to address public health concerns and benefit communities.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9701380/

Extracellular signals regulate the biogenesis of extracellular vesicles

Extracellular vesicles (EVs) are naturally released membrane vesicles that act as carriers of proteins and RNAs for intercellular communication. With various biomolecules and specific ligands, EV has represented a novel form of information transfer, which possesses extremely outstanding efficiency and specificity compared to the classical signal transduction. In addition, EV has extended the concept of signal transduction to intercellular aspect by working as the collection of extracellular information. Therefore, the functions of EVs have been extensively characterized and EVs exhibit an exciting prospect for clinical applications. However, the biogenesis of EVs and, in particular, the regulation of this process by extracellular signals, which are essential to conduct further studies and support optimal utility, remain unclear. Here, we review the current understanding of the biogenesis of EVs, focus on the regulation of this process by extracellular signals and discuss their therapeutic value. Introduction Extracellular vesicles (EVs) are lipid bilayer membrane structures that are released by almost all types of cells during normal physiology and acquired abnormalities [ 1 , 2 ]. Based on their biogenesis and size, EVs are commonly classified into three subtypes: exosomes, microvesicles (MVs), and apoptotic bodies. Exosomes, 50–150 nm in diameter, are intraluminal vesicles (ILVs) formed by the inward budding of the endosomal membrane during the maturation of multivesicular bodies (MVBs), which are secreted after the fusion of MVBs with the plasma membrane [ 3 , 4 ]. MVs, about 100 to 1000 nm in diameter, are formed by the outward budding of the plasma membrane and are released directly into the extracellular milieu [ 5 ]. Apoptotic bodies are generally defined as 1 to 5 μm in diameter and as products of apoptotic cell disassembly [ 6 ]. As shown in Fig. 1 , all these three subtypes are composed of lipid bilayers and enriched in proteins, lipids, and nucleic acids (Fig. 1 ) [ 7 , 8 ]. Fig. 1 The biogenesis of the extracellular vesicle and its structure. a EVs can be divided into three subtypes: exosomes, microvesicles and apoptotic bodies. Exosomes are formed as ILVs in the MVBs. This process requires the involvement of ESCRT components, and it also occurs in ESCRT-independent pathways, including syntenin-, lipids- and tetraspanin-dependent mechanisms and others. After ILVs formation, MVBs are transported to the plasma membrane or the lysosome, which primarily involves some Rab proteins. Finally, MVBs fuse with the plasma membrane with the involvement of SNARE complex, and exosomes are released. Microvesicles are released directly after the outward budding from the plasma membrane, which primarily involves the ESCRT, ARRDC1, lipids, Rho proteins and Ca 2+ . Apoptotic bodies only generate from apoptotic cells and shed from the cell surface. Enveloped viruses that highjack the membranes for release can also be considered as a kind of EV. b The extracellular vesicle is made up of lipid bilayers and enriched in proteins, nucleic acids and lipids Although initially considered as a form of discarding waste of cells and "platelet dust", exosomes and MVs are now extensively characterized [ 9 – 12 ]. This is because they are central mediators of cell-to-cell communication and are involved in almost all physiological and pathological processes [ 13 ]. For instance, EVs play critical roles in inflammation, stem cell expansion, and diseases including cancers and neurodegenerative disorders [ 9 , 14 – 17 ]. In recent years, research on the applications of EVs in diagnoses and treatments of various diseases has been increasing. EVs are present in all body fluids, facilitating easy clinical sampling and have the potential for longitudinal sampling to track disease progression [ 2 ]. In addition, exosomes are being actively explored as drug delivery vehicles due to their small size, endogenous, less toxic, etc. [ 18 ]. Studies on the functions of EVs have attracted a lot of attention and been quite extensive over the past decade. However, relatively few studies have been conducted on the biogenesis of EVs, especially on how extracellular signals regulate the biogenesis of EVs. Compared to the classical signal transduction that works in diffusible mono-molecule form, EVs demonstrate remarkable efficiency and specificity through assembling their various and selective cargo, providing novel mechanisms of signal transfer. More importantly, the altered biogenesis of EVs reflect changes in the signaling or metabolic state of donor cells, making them versatile. Therefore, it is necessary to elucidate the regulatory mechanisms of EV biogenesis before EVs are put into application. Here, we review the development of EV research and current understanding of the biogenesis of EVs, emphasize the regulation of this process by extracellular signals, and discuss their potential therapeutic value. The development of extracellular vesicle research Extracellular vesicles were first isolated from platelets by high-speed centrifugation and were reported to inhibit plasma clotting in 1946 [ 19 ]. These vesicles are actually microvesicles as now defined. Thirty-seven years later, Harding et al. and Pan et al. reported that transferrin receptors associated with small vesicles are released from reticulocytes into extracellular space by two groups, respectively [ 20 , 21 ]. Unexpectedly, their research on the mechanisms of transferrin recycling in reticulocytes raised the possibility that cells might use MVB exocytosis as a general way to release vesicles [ 22 ]. Since then, many proteins, such as class II major histocompatibility complex, were demonstrated to be transported in MVB-exosome pathway and to play biological functions, and the understanding of the cell biology of exosome increased [ 22 , 23 ]. In 2007, it was firstly reported that exosomes contain both mRNA and microRNA, which can be delivered to another cell and exerted their functions [ 24 ]. This groundbreaking discovery introduced EVs into intercellular communication and opened up a new field of research with widespread interest. In recent years, the research on EVs has been gradually standardized under the guide from the International Society for Extracellular Vesicles (ISEV), including nomenclature, isolation method, characterization, and functional studying method. It is now universally acknowledged that exosomes are endosome-derived vesicles released from cells, while microvesicles are plasma membrane-derived, although specific markers of EV subtypes are not clear enough and it is hard to trace their origin in experiments [ 25 , 26 ]. The separation and concentration of EV, the key step in the experiment, was summarized in the guideline of ISEV [ 26 ]. The common methods include differential ultracentrifugation, density gradients, precipitation, filtration, size exclusion chromatography, and immunoisolation [ 26 ]. In addition, additional techniques and combinations of techniques were applied to get better performance, such as asymmetric flow field-flow fractionation, ion exchange chromatography [ 27 , 28 ]. Another important step is EV characterization, some methods are recommended to assess the quantification and purity of EVs, such as using light scattering technologies to count particle number, using electron microscopy to visualize EVs, and selecting EV protein compositions (CD63, CD9, CD81, TSG-101, ALIX, etc.) or non-protein components (glycosphingolipid, ceramide, etc.) as markers [ 26 ]. Given the fact that there is no ideal technology to distinguish EV subtype from each other, strict and clear definition of EV and comprehensive methods are needed. The biogenesis and release of extracellular vesicles The biogenesis of exosomes As shown in Fig. 1 , during the maturation of early endosomes into late endosomes or MVBs, endosomal membrane loaded with specific cargo buds inward and separate from the membrane, generating ILVs for exocytosis within the lumen of the endosomes (Fig. 1 ) [ 29 ]. In general, the mechanisms of exosomes biogenesis can be divided into the Endosomal Sorting Complex Required for Transport (ESCRT)-dependent pathway and ESCRT-independent pathways. ESCRT ESCRT consists of about thirty proteins that can be divided into four complexes, ESCRT-0 and ESCRT-I, II, III [ 30 – 34 ], and the discovery of them reveals the cellular mechanism underlying the biogenesis of exosomes [ 35 , 36 ]. The formation of ILVs begins with cargo loading on the limiting membrane of MVBs, which is mediated by a multivalent ubiquitin-binding complex ESCRT-0 composed of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adaptor molecule (STAM) [ 37 , 38 ]. ESCRT-0 contains ten ubiquitin-binding sites that facilitate capturing polyubiquitylated cargo [ 38 ]. When the ubiquitinated cargo binds to the ESCRT-0 complex, HRS-STAM complex recruits ESCRT-I complex to transmit cargoes through TSG-101 binding ubiquitin [ 38 ]. Subsequently, ESCRT-I recruits ESCRT-II through the connect of VPS28 and VPS36 subunit [ 39 ]. Finally, ESCRT-III is recruited by ESCRT-II to nucleate charged MVB protein 2-4 (CHMP2-4) polymers to completes ILVs budding and fission of this membrane [ 39 ]. In addition, the accessory protein AAA-ATPase VPS4 disassembles and recycles ESCRT-III [ 8 ]. Syntenin There is an alternative pathway playing key roles in the biogenesis of exosomes, which involves in syntenin, syndecan, and ESCRT accessory protein ALG-2 interacting protein X (ALIX) [ 40 ]. Syntenin binds to the cytoplasmic domain of syndecan and recruits ALIX, which allows inward budding into ILVs together with ESCRT-I, II, III components. This process relies on Src-mediated endocytosis of syndecan-1 and requires PLD2 and ARF6 GTPase activities [ 41 – 43 ]. Recently, it was reported that Src homology 2-containing protein tyrosine phosphatase 2 (Shp2) controls exosome biogenesis through dephosphorylation of syntenin, thereby regulating exosomes-mediated epithelial-macrophage crosstalk [ 44 ]. Lipids Exosome biogenesis also occurs in the absence of ESCRTs, and several studies have shown that MVBs still form despite maximal inhibition of ESCRT-dependent pathway [ 45 ]. Trajkovic et al. revealed the ESCRT-independent pathway for exosome biogenesis and showed that the transfer of exosome-associated domains into the lumen of the endosome depends on the sphingolipid ceramide in the mouse oligodendroglial cells [ 46 ]. Ceramide can induce small microdomains to coalesce into larger domains, thereby facilitating domain-induced budding [ 47 ]. Moreover, the tapering structure of ceramides lead to spontaneous negative curvature by creating area differences between the membrane leaflets [ 48 ]. Cholesterol, an important component of MVBs, is another lipid enriched in exosomes membranes [ 49 – 51 ]. It was demonstrated that induced cholesterol accumulation in late endosomal compartments of the oligodendroglial cells increases the secretion of exosomes in a flotillin-dependent way [ 52 ]. Tetraspanins In addition to lipids, proteins from tetraspanin family are involved in the regulation of cargo sorted for exosome secretion. This family is characterized by four transmembrane domains and has as many as thirty-three members in mammals, including CD9, CD37, CD51, CD53, CD63, CD81, CD82, etc. [ 53 – 55 ]. The first study showed the tetraspanin-dependent mechanism of cargo sorted to ILVs, focusing on CD63, which is involved in endosomal sorting in melanoma cells [ 56 ]. Subsequent studies demonstrated that CD63 was also involved in cargo targeting exosomes secreted by melanoma cells and in the biogenesis of exosomes in fibroblasts from patients with Down syndrome [ 57 , 58 ]. CD81 is also a member of the tetraspanin family and enriched in exosomes and was shown to target its ligands array into secreted exosomes in mouse lymphoblasts [ 59 ]. Expression of the CD9 and CD82 was reported to augment the exosome release of β-catenin from HEK293 cells [ 60 ]. Interestingly, tetraspanin Tspan8 is involved in exosome biogenesis in rat adenocarcinoma cells by contributing to selective recruitment of proteins and mRNA into exosomes without affecting the amount of secreted exosomes [ 61 , 62 ]. Other mechanisms involved in the exosome biogenesis There are other mechanisms involved in the biogenesis of MVBs. For example, soluble proteins isolated into ILVs rely on the chaperones HSC70 which recruits the transferrin receptor (TFR) to exosomes and bind cytosolic proteins containing a KFERQ-motif to be selectively transferred to ILVs in reticulocytes [ 13 , 63 , 64 ]. Exosomes also carry nucleic acids including DNA sequences and RNAs [ 24 , 65 – 67 ]. The Goldie group demonstrated that in small RNAs, the proportion of miRNAs in exosomes is higher than that of their donor cells [ 68 ]. Intriguingly, miRNAs are not randomly loaded into exosomes [ 69 ]. miRNA can be transferred into exosomes via the SUMOylated heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), the neural sphingomyelinase 2 (nSMase2) -dependent pathway, the 3'-end of the miRNA sequence-dependent pathway, and the miRNA induced silencing complex (miRISC) -related pathway [ 70 – 73 ]. It has also been proposed that the RNA packed into extracellular vesicles can also be facilitated by retroviral coat proteins such as Gag (and their silent copies present in animal genomes) in Drosophila . These proteins effectively target the RNA they recognize to the plasma membrane or MVB membrane, resulting in the release of virus-like particles [ 74 , 75 ]. In addition, post-translational modifications (PTMs) can direct cargo into exosomes. Except that ubiquitin domain of ubiquitinated proteins can be recognized by ESCRT protein TSG101, SUMOylation, ISGylation, phosphorylation, glycosylation, and acetylation of proteins can also lead to their sorting into exosomes [ 76 , 77 ]. For example, ISGylation of TSG101 induces its aggregation and degradation, impairing exosome secretion [ 78 ]. SUMOylation of α-synuclein, the major protein of pathological aggregates of Parkinson's Disease, can be sorted into MVB via the interaction between ESCRT and phosphoinositols [ 79 ]. In conclusion, the process of exosome biogenesis is complex and depends mainly on the cargo that affect the function of these exosomes. It is noteworthy that different mechanisms work simultaneously within a single cell, and therefore various subtypes of MVBs exist within a single cell. Precisely, during the maturation of MVBs, various mechanisms act simultaneously or sequentially to help release various exosomes [ 13 ]. The release of exosomes Mechanisms involved in MVB transport After formation of ILVs, MVBs are either transported to the plasma membrane for the subsequent release of ILVs (exosomes) into the extracellular environment or are degraded by fusing with lysosomes (Fig. 1 ). There is a balance between these two opposing events, and some studies suggested that this balance shifts towards exosome release in transformed cells and non-transformed cells [ 80 – 82 ]. The underlying mechanisms of this balance are largely unknown, but some have begun to emerge, such as ISGylation of the ESCRT-I component TSG101, which impair exosome release by promoting MVBs fusion with lysosomes in mice [ 78 ]. The mobilization of MVBs to the plasma membrane is similar to the general intracellular vesicles trafficking and involves the cytoskeleton, associated molecular motors, and the molecular switches [ 13 , 83 , 84 ]. Rab proteins, a subfamily of the Ras superfamily of small GTPase, are involved in different steps of intracellular vesicular trafficking, from the budding and scission of vesicles from their donor membranes, to transporting of vesicles along cytoskeletal components, and then to docking of vesicles to their target compartment[ 85 , 86 ]. Rabs work between an active GTP-bound state and an inactive GDP-bound state, and the switch requires guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) [ 87 ]. Specifically, Rabs binding to the GDP dissociation inhibitor (GDI) exist in cytosol in an inactive state, while prenylated Rabs are inserted into the membrane of their respective transport compartment. Then GDP is replaced by GTP by GEFs to form an active state [ 87 , 88 ]. Active Rabs can interact with various effector proteins, such as motor proteins involved in transport and tethering factors, then Rabs are inactivated by GAPs and enter to next cycle [ 86 ]. For example, Rab7 interacts with Rab-interacting lysosomal protein to recruit the dynein-dynactin motor complex to regulate the transport of late endosomes to lysosomes [ 86 ]. Savina et al. reported that Rab11 is involved in exosome release, which reduced the release of TfR and HSC70-containing exosomes in K562 cells when inhibited [ 89 ]. With further studies, several Rab proteins, including Rab27A/B, Rab7, Rab31 and Rab35, have been implicated in the regulation of exosome secretion. Rab35 was revealed to be necessary for PLP-bearing exosome release and allow docking of MVBs to the plasma membrane in oligodendroglial cells [ 90 ]. Rab7 was shown to be required for syntenin- and Alix-containing exosome secretion in MCF7 cells but not to affect exosome secretion from HeLa cells [ 40 , 91 ]. Recently, Rab7 was reported to be bound and recruited to MVBs by neddylated Coro1a, promoting lysosomal degradation of the MVBs and reducing exosome secretion consequently in HEK293, RAW264.7 and HeLa cells [ 92 ]. Two isoforms of Rab27, Rab27A, and Rab27B, play a role in the secretion of exosomes bearing CD63, CD81, and MHC class II in HeLa cells and docking to the plasma membrane [ 91 ]. However, depletion of Rab27A has no effect in decreasing secretion of exosomes in certain cell lines such as MDA-MB-231 [ 8 , 93 , 94 ]. Recent work identified that Rab31 marks and controls an ESCRT- independent exosome biogenesis pathway and prevents the fusion of MVBs with lysosomes in HeLa cells [ 95 ]. Interestingly, Rab31 functions as a driver for ILVs formation and determines the fates of endocytic membrane proteins by balancing with Rab7 [ 95 ]. Mechanisms involved in MVB fusion with plasma membrane Exosomes are released into the extracellular environment upon fusion of MVBs with the plasma membrane (Fig. 1 ). This process involves SNARE (soluble NSF-attachment protein receptor) proteins or complexes that allow fusion of the lipid bilayers of two different intracellular compartments [ 96 ]. VAMP7 (vesicle-associated membrane protein 7) on the lysosomes, syntaxin 7 on the plasma membrane, and the lysosomal regulatory synaptotagmin7 form a complex participating in the exocytosis of conventional lysosomes, which has been reported to be involved in the exosome secretion of K562 cells [ 97 , 98 ]. Another SNARE protein YKT6, involved in endoplasmic reticulum-to-Golgi complex transport, has been shown to be required for exosome release by depletion in HEK293 cells and A549 cells [ 99 , 100 ]. Moreover, syntaxin 5 and Ral 1 in C. elegans , syntaxin 1a in mammals and SNAP-23 in human mastocytes are regulators of the exosomal secretion process as well [ 81 , 101 , 102 ]. Similarly, different SNARE complexes may be applied to the fusion of a particular organelle with the plasma membrane in different cell type or the fusion of different subpopulations of MVBs in a single cell type [ 8 ]. As depicted in Fig. 1 , MVBs are transported to the plasma membrane, and this process primarily involves the Rab proteins. MVBs fusion with the plasma membrane is similar to the general fusion process of membranes, which requires the involvement of SNARE complex. Notably, the above studies suggest that the mechanisms of exosome secretion may be limited by the original subpopulations of endosomes or dependent on the cell type [ 13 , 103 ]. The biogenesis and release of microvesicles Unlike the fusion of MVBs with the plasma membrane, as illustrated in Fig. 1 , the shedding of MVs requires a fission process that occurs in the curved region of the plasma membrane (Fig. 1 ). Mechanisms similar to exosome biogenesis are involved in the generation of MVs. ESCRT factors are involved in this process requiring ESCRT-I, II, III, and accessory proteins [ 42 ]. Among them, the arrestin domain-containing protein 1 (ARRDC1) directly drives plasma membrane budding by recruiting TSG101 to the plasma membrane, while VPS4 is also involved [ 38 , 104 ]. Additionally, acid sphingomyelinase induces the production of ceramide-dependent MVs of astrocytes [ 105 ], while neutral sphingomyelinase in MVBs is required for the biogenesis of exosomes [ 46 ]. Another lipid-dependent mechanism is associated with cholesterol which is abundant in MVs, and the formation of MVs is impaired when cholesterol is pharmacologically depleted in activated neutrophils [ 106 ]. PTMs are also reported to participate in the MV biogenesis [ 77 ]. As a typical example, Y14-phophorylation of caveolin-1 leads to interaction with hnRNPA2B1, followed by induction of hnRNPA2B1 O-GlcNacylation, which triggers the binding between selected miRNAs and hnRNPA2B1, finally leading to selection of miRNAs into MVs [ 107 ]. Given the pattern and site of MVs biogenesis, cytoskeleton rearrangement and related molecules are critical to this process. Large increase in Ca 2+ levels induces the activation of calpain, a protease capable of cleaving cytoskeletal proteins, together with the alteration of flippase, floppase, and scramblase, drives membrane asymmetry cytoskeletal remodeling, leading to outward budding of microvesicles [ 108 – 111 ]. Consistently, the Rho family small G protein and Rho-associated protein kinase (ROCK) are required for MVs release [ 112 , 113 ]. Moreover, a recent study by Wang and Zhuang showed that Rho family small G protein Cdc42 is a convergent node of multiple regulatory signals that occur in MVs biogenesis and the binding of activated GTP-bound Cdc42 and its downstream effector, Ras GTPase-activating-like protein 1 (IQGAP1), is required for MVs shedding in breast cancer cells [ 113 ]. Also, the activation of RhoA by ARF6 or ARF1 leads to an actin-myosin-based contraction that is required for MVs budding through the kinases ROCK and ERK (extracellular signal regulated kinase) [ 114 , 115 ]. Diaphanous-related formin-3 (DRF3), a signaling protein binding small Rho family GTPases, has been reported to be associated with the formation of MVs when suppressed in prostate cancer cells [ 116 ]. Furthermore, the biogenesis of MVs in cancer cells is also associated with metabolic changes driven by the "Warburg effect", which is thought to be required for malignant transformation and cancer progression [ 117 ]. The biogenesis of exosomes As shown in Fig. 1 , during the maturation of early endosomes into late endosomes or MVBs, endosomal membrane loaded with specific cargo buds inward and separate from the membrane, generating ILVs for exocytosis within the lumen of the endosomes (Fig. 1 ) [ 29 ]. In general, the mechanisms of exosomes biogenesis can be divided into the Endosomal Sorting Complex Required for Transport (ESCRT)-dependent pathway and ESCRT-independent pathways. ESCRT ESCRT consists of about thirty proteins that can be divided into four complexes, ESCRT-0 and ESCRT-I, II, III [ 30 – 34 ], and the discovery of them reveals the cellular mechanism underlying the biogenesis of exosomes [ 35 , 36 ]. The formation of ILVs begins with cargo loading on the limiting membrane of MVBs, which is mediated by a multivalent ubiquitin-binding complex ESCRT-0 composed of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adaptor molecule (STAM) [ 37 , 38 ]. ESCRT-0 contains ten ubiquitin-binding sites that facilitate capturing polyubiquitylated cargo [ 38 ]. When the ubiquitinated cargo binds to the ESCRT-0 complex, HRS-STAM complex recruits ESCRT-I complex to transmit cargoes through TSG-101 binding ubiquitin [ 38 ]. Subsequently, ESCRT-I recruits ESCRT-II through the connect of VPS28 and VPS36 subunit [ 39 ]. Finally, ESCRT-III is recruited by ESCRT-II to nucleate charged MVB protein 2-4 (CHMP2-4) polymers to completes ILVs budding and fission of this membrane [ 39 ]. In addition, the accessory protein AAA-ATPase VPS4 disassembles and recycles ESCRT-III [ 8 ]. Syntenin There is an alternative pathway playing key roles in the biogenesis of exosomes, which involves in syntenin, syndecan, and ESCRT accessory protein ALG-2 interacting protein X (ALIX) [ 40 ]. Syntenin binds to the cytoplasmic domain of syndecan and recruits ALIX, which allows inward budding into ILVs together with ESCRT-I, II, III components. This process relies on Src-mediated endocytosis of syndecan-1 and requires PLD2 and ARF6 GTPase activities [ 41 – 43 ]. Recently, it was reported that Src homology 2-containing protein tyrosine phosphatase 2 (Shp2) controls exosome biogenesis through dephosphorylation of syntenin, thereby regulating exosomes-mediated epithelial-macrophage crosstalk [ 44 ]. Lipids Exosome biogenesis also occurs in the absence of ESCRTs, and several studies have shown that MVBs still form despite maximal inhibition of ESCRT-dependent pathway [ 45 ]. Trajkovic et al. revealed the ESCRT-independent pathway for exosome biogenesis and showed that the transfer of exosome-associated domains into the lumen of the endosome depends on the sphingolipid ceramide in the mouse oligodendroglial cells [ 46 ]. Ceramide can induce small microdomains to coalesce into larger domains, thereby facilitating domain-induced budding [ 47 ]. Moreover, the tapering structure of ceramides lead to spontaneous negative curvature by creating area differences between the membrane leaflets [ 48 ]. Cholesterol, an important component of MVBs, is another lipid enriched in exosomes membranes [ 49 – 51 ]. It was demonstrated that induced cholesterol accumulation in late endosomal compartments of the oligodendroglial cells increases the secretion of exosomes in a flotillin-dependent way [ 52 ]. Tetraspanins In addition to lipids, proteins from tetraspanin family are involved in the regulation of cargo sorted for exosome secretion. This family is characterized by four transmembrane domains and has as many as thirty-three members in mammals, including CD9, CD37, CD51, CD53, CD63, CD81, CD82, etc. [ 53 – 55 ]. The first study showed the tetraspanin-dependent mechanism of cargo sorted to ILVs, focusing on CD63, which is involved in endosomal sorting in melanoma cells [ 56 ]. Subsequent studies demonstrated that CD63 was also involved in cargo targeting exosomes secreted by melanoma cells and in the biogenesis of exosomes in fibroblasts from patients with Down syndrome [ 57 , 58 ]. CD81 is also a member of the tetraspanin family and enriched in exosomes and was shown to target its ligands array into secreted exosomes in mouse lymphoblasts [ 59 ]. Expression of the CD9 and CD82 was reported to augment the exosome release of β-catenin from HEK293 cells [ 60 ]. Interestingly, tetraspanin Tspan8 is involved in exosome biogenesis in rat adenocarcinoma cells by contributing to selective recruitment of proteins and mRNA into exosomes without affecting the amount of secreted exosomes [ 61 , 62 ]. Other mechanisms involved in the exosome biogenesis There are other mechanisms involved in the biogenesis of MVBs. For example, soluble proteins isolated into ILVs rely on the chaperones HSC70 which recruits the transferrin receptor (TFR) to exosomes and bind cytosolic proteins containing a KFERQ-motif to be selectively transferred to ILVs in reticulocytes [ 13 , 63 , 64 ]. Exosomes also carry nucleic acids including DNA sequences and RNAs [ 24 , 65 – 67 ]. The Goldie group demonstrated that in small RNAs, the proportion of miRNAs in exosomes is higher than that of their donor cells [ 68 ]. Intriguingly, miRNAs are not randomly loaded into exosomes [ 69 ]. miRNA can be transferred into exosomes via the SUMOylated heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), the neural sphingomyelinase 2 (nSMase2) -dependent pathway, the 3'-end of the miRNA sequence-dependent pathway, and the miRNA induced silencing complex (miRISC) -related pathway [ 70 – 73 ]. It has also been proposed that the RNA packed into extracellular vesicles can also be facilitated by retroviral coat proteins such as Gag (and their silent copies present in animal genomes) in Drosophila . These proteins effectively target the RNA they recognize to the plasma membrane or MVB membrane, resulting in the release of virus-like particles [ 74 , 75 ]. In addition, post-translational modifications (PTMs) can direct cargo into exosomes. Except that ubiquitin domain of ubiquitinated proteins can be recognized by ESCRT protein TSG101, SUMOylation, ISGylation, phosphorylation, glycosylation, and acetylation of proteins can also lead to their sorting into exosomes [ 76 , 77 ]. For example, ISGylation of TSG101 induces its aggregation and degradation, impairing exosome secretion [ 78 ]. SUMOylation of α-synuclein, the major protein of pathological aggregates of Parkinson's Disease, can be sorted into MVB via the interaction between ESCRT and phosphoinositols [ 79 ]. In conclusion, the process of exosome biogenesis is complex and depends mainly on the cargo that affect the function of these exosomes. It is noteworthy that different mechanisms work simultaneously within a single cell, and therefore various subtypes of MVBs exist within a single cell. Precisely, during the maturation of MVBs, various mechanisms act simultaneously or sequentially to help release various exosomes [ 13 ]. ESCRT ESCRT consists of about thirty proteins that can be divided into four complexes, ESCRT-0 and ESCRT-I, II, III [ 30 – 34 ], and the discovery of them reveals the cellular mechanism underlying the biogenesis of exosomes [ 35 , 36 ]. The formation of ILVs begins with cargo loading on the limiting membrane of MVBs, which is mediated by a multivalent ubiquitin-binding complex ESCRT-0 composed of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adaptor molecule (STAM) [ 37 , 38 ]. ESCRT-0 contains ten ubiquitin-binding sites that facilitate capturing polyubiquitylated cargo [ 38 ]. When the ubiquitinated cargo binds to the ESCRT-0 complex, HRS-STAM complex recruits ESCRT-I complex to transmit cargoes through TSG-101 binding ubiquitin [ 38 ]. Subsequently, ESCRT-I recruits ESCRT-II through the connect of VPS28 and VPS36 subunit [ 39 ]. Finally, ESCRT-III is recruited by ESCRT-II to nucleate charged MVB protein 2-4 (CHMP2-4) polymers to completes ILVs budding and fission of this membrane [ 39 ]. In addition, the accessory protein AAA-ATPase VPS4 disassembles and recycles ESCRT-III [ 8 ]. Syntenin There is an alternative pathway playing key roles in the biogenesis of exosomes, which involves in syntenin, syndecan, and ESCRT accessory protein ALG-2 interacting protein X (ALIX) [ 40 ]. Syntenin binds to the cytoplasmic domain of syndecan and recruits ALIX, which allows inward budding into ILVs together with ESCRT-I, II, III components. This process relies on Src-mediated endocytosis of syndecan-1 and requires PLD2 and ARF6 GTPase activities [ 41 – 43 ]. Recently, it was reported that Src homology 2-containing protein tyrosine phosphatase 2 (Shp2) controls exosome biogenesis through dephosphorylation of syntenin, thereby regulating exosomes-mediated epithelial-macrophage crosstalk [ 44 ]. Lipids Exosome biogenesis also occurs in the absence of ESCRTs, and several studies have shown that MVBs still form despite maximal inhibition of ESCRT-dependent pathway [ 45 ]. Trajkovic et al. revealed the ESCRT-independent pathway for exosome biogenesis and showed that the transfer of exosome-associated domains into the lumen of the endosome depends on the sphingolipid ceramide in the mouse oligodendroglial cells [ 46 ]. Ceramide can induce small microdomains to coalesce into larger domains, thereby facilitating domain-induced budding [ 47 ]. Moreover, the tapering structure of ceramides lead to spontaneous negative curvature by creating area differences between the membrane leaflets [ 48 ]. Cholesterol, an important component of MVBs, is another lipid enriched in exosomes membranes [ 49 – 51 ]. It was demonstrated that induced cholesterol accumulation in late endosomal compartments of the oligodendroglial cells increases the secretion of exosomes in a flotillin-dependent way [ 52 ]. Tetraspanins In addition to lipids, proteins from tetraspanin family are involved in the regulation of cargo sorted for exosome secretion. This family is characterized by four transmembrane domains and has as many as thirty-three members in mammals, including CD9, CD37, CD51, CD53, CD63, CD81, CD82, etc. [ 53 – 55 ]. The first study showed the tetraspanin-dependent mechanism of cargo sorted to ILVs, focusing on CD63, which is involved in endosomal sorting in melanoma cells [ 56 ]. Subsequent studies demonstrated that CD63 was also involved in cargo targeting exosomes secreted by melanoma cells and in the biogenesis of exosomes in fibroblasts from patients with Down syndrome [ 57 , 58 ]. CD81 is also a member of the tetraspanin family and enriched in exosomes and was shown to target its ligands array into secreted exosomes in mouse lymphoblasts [ 59 ]. Expression of the CD9 and CD82 was reported to augment the exosome release of β-catenin from HEK293 cells [ 60 ]. Interestingly, tetraspanin Tspan8 is involved in exosome biogenesis in rat adenocarcinoma cells by contributing to selective recruitment of proteins and mRNA into exosomes without affecting the amount of secreted exosomes [ 61 , 62 ]. Other mechanisms involved in the exosome biogenesis There are other mechanisms involved in the biogenesis of MVBs. For example, soluble proteins isolated into ILVs rely on the chaperones HSC70 which recruits the transferrin receptor (TFR) to exosomes and bind cytosolic proteins containing a KFERQ-motif to be selectively transferred to ILVs in reticulocytes [ 13 , 63 , 64 ]. Exosomes also carry nucleic acids including DNA sequences and RNAs [ 24 , 65 – 67 ]. The Goldie group demonstrated that in small RNAs, the proportion of miRNAs in exosomes is higher than that of their donor cells [ 68 ]. Intriguingly, miRNAs are not randomly loaded into exosomes [ 69 ]. miRNA can be transferred into exosomes via the SUMOylated heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), the neural sphingomyelinase 2 (nSMase2) -dependent pathway, the 3'-end of the miRNA sequence-dependent pathway, and the miRNA induced silencing complex (miRISC) -related pathway [ 70 – 73 ]. It has also been proposed that the RNA packed into extracellular vesicles can also be facilitated by retroviral coat proteins such as Gag (and their silent copies present in animal genomes) in Drosophila . These proteins effectively target the RNA they recognize to the plasma membrane or MVB membrane, resulting in the release of virus-like particles [ 74 , 75 ]. In addition, post-translational modifications (PTMs) can direct cargo into exosomes. Except that ubiquitin domain of ubiquitinated proteins can be recognized by ESCRT protein TSG101, SUMOylation, ISGylation, phosphorylation, glycosylation, and acetylation of proteins can also lead to their sorting into exosomes [ 76 , 77 ]. For example, ISGylation of TSG101 induces its aggregation and degradation, impairing exosome secretion [ 78 ]. SUMOylation of α-synuclein, the major protein of pathological aggregates of Parkinson's Disease, can be sorted into MVB via the interaction between ESCRT and phosphoinositols [ 79 ]. In conclusion, the process of exosome biogenesis is complex and depends mainly on the cargo that affect the function of these exosomes. It is noteworthy that different mechanisms work simultaneously within a single cell, and therefore various subtypes of MVBs exist within a single cell. Precisely, during the maturation of MVBs, various mechanisms act simultaneously or sequentially to help release various exosomes [ 13 ]. The release of exosomes Mechanisms involved in MVB transport After formation of ILVs, MVBs are either transported to the plasma membrane for the subsequent release of ILVs (exosomes) into the extracellular environment or are degraded by fusing with lysosomes (Fig. 1 ). There is a balance between these two opposing events, and some studies suggested that this balance shifts towards exosome release in transformed cells and non-transformed cells [ 80 – 82 ]. The underlying mechanisms of this balance are largely unknown, but some have begun to emerge, such as ISGylation of the ESCRT-I component TSG101, which impair exosome release by promoting MVBs fusion with lysosomes in mice [ 78 ]. The mobilization of MVBs to the plasma membrane is similar to the general intracellular vesicles trafficking and involves the cytoskeleton, associated molecular motors, and the molecular switches [ 13 , 83 , 84 ]. Rab proteins, a subfamily of the Ras superfamily of small GTPase, are involved in different steps of intracellular vesicular trafficking, from the budding and scission of vesicles from their donor membranes, to transporting of vesicles along cytoskeletal components, and then to docking of vesicles to their target compartment[ 85 , 86 ]. Rabs work between an active GTP-bound state and an inactive GDP-bound state, and the switch requires guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) [ 87 ]. Specifically, Rabs binding to the GDP dissociation inhibitor (GDI) exist in cytosol in an inactive state, while prenylated Rabs are inserted into the membrane of their respective transport compartment. Then GDP is replaced by GTP by GEFs to form an active state [ 87 , 88 ]. Active Rabs can interact with various effector proteins, such as motor proteins involved in transport and tethering factors, then Rabs are inactivated by GAPs and enter to next cycle [ 86 ]. For example, Rab7 interacts with Rab-interacting lysosomal protein to recruit the dynein-dynactin motor complex to regulate the transport of late endosomes to lysosomes [ 86 ]. Savina et al. reported that Rab11 is involved in exosome release, which reduced the release of TfR and HSC70-containing exosomes in K562 cells when inhibited [ 89 ]. With further studies, several Rab proteins, including Rab27A/B, Rab7, Rab31 and Rab35, have been implicated in the regulation of exosome secretion. Rab35 was revealed to be necessary for PLP-bearing exosome release and allow docking of MVBs to the plasma membrane in oligodendroglial cells [ 90 ]. Rab7 was shown to be required for syntenin- and Alix-containing exosome secretion in MCF7 cells but not to affect exosome secretion from HeLa cells [ 40 , 91 ]. Recently, Rab7 was reported to be bound and recruited to MVBs by neddylated Coro1a, promoting lysosomal degradation of the MVBs and reducing exosome secretion consequently in HEK293, RAW264.7 and HeLa cells [ 92 ]. Two isoforms of Rab27, Rab27A, and Rab27B, play a role in the secretion of exosomes bearing CD63, CD81, and MHC class II in HeLa cells and docking to the plasma membrane [ 91 ]. However, depletion of Rab27A has no effect in decreasing secretion of exosomes in certain cell lines such as MDA-MB-231 [ 8 , 93 , 94 ]. Recent work identified that Rab31 marks and controls an ESCRT- independent exosome biogenesis pathway and prevents the fusion of MVBs with lysosomes in HeLa cells [ 95 ]. Interestingly, Rab31 functions as a driver for ILVs formation and determines the fates of endocytic membrane proteins by balancing with Rab7 [ 95 ]. Mechanisms involved in MVB fusion with plasma membrane Exosomes are released into the extracellular environment upon fusion of MVBs with the plasma membrane (Fig. 1 ). This process involves SNARE (soluble NSF-attachment protein receptor) proteins or complexes that allow fusion of the lipid bilayers of two different intracellular compartments [ 96 ]. VAMP7 (vesicle-associated membrane protein 7) on the lysosomes, syntaxin 7 on the plasma membrane, and the lysosomal regulatory synaptotagmin7 form a complex participating in the exocytosis of conventional lysosomes, which has been reported to be involved in the exosome secretion of K562 cells [ 97 , 98 ]. Another SNARE protein YKT6, involved in endoplasmic reticulum-to-Golgi complex transport, has been shown to be required for exosome release by depletion in HEK293 cells and A549 cells [ 99 , 100 ]. Moreover, syntaxin 5 and Ral 1 in C. elegans , syntaxin 1a in mammals and SNAP-23 in human mastocytes are regulators of the exosomal secretion process as well [ 81 , 101 , 102 ]. Similarly, different SNARE complexes may be applied to the fusion of a particular organelle with the plasma membrane in different cell type or the fusion of different subpopulations of MVBs in a single cell type [ 8 ]. As depicted in Fig. 1 , MVBs are transported to the plasma membrane, and this process primarily involves the Rab proteins. MVBs fusion with the plasma membrane is similar to the general fusion process of membranes, which requires the involvement of SNARE complex. Notably, the above studies suggest that the mechanisms of exosome secretion may be limited by the original subpopulations of endosomes or dependent on the cell type [ 13 , 103 ]. Mechanisms involved in MVB transport After formation of ILVs, MVBs are either transported to the plasma membrane for the subsequent release of ILVs (exosomes) into the extracellular environment or are degraded by fusing with lysosomes (Fig. 1 ). There is a balance between these two opposing events, and some studies suggested that this balance shifts towards exosome release in transformed cells and non-transformed cells [ 80 – 82 ]. The underlying mechanisms of this balance are largely unknown, but some have begun to emerge, such as ISGylation of the ESCRT-I component TSG101, which impair exosome release by promoting MVBs fusion with lysosomes in mice [ 78 ]. The mobilization of MVBs to the plasma membrane is similar to the general intracellular vesicles trafficking and involves the cytoskeleton, associated molecular motors, and the molecular switches [ 13 , 83 , 84 ]. Rab proteins, a subfamily of the Ras superfamily of small GTPase, are involved in different steps of intracellular vesicular trafficking, from the budding and scission of vesicles from their donor membranes, to transporting of vesicles along cytoskeletal components, and then to docking of vesicles to their target compartment[ 85 , 86 ]. Rabs work between an active GTP-bound state and an inactive GDP-bound state, and the switch requires guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) [ 87 ]. Specifically, Rabs binding to the GDP dissociation inhibitor (GDI) exist in cytosol in an inactive state, while prenylated Rabs are inserted into the membrane of their respective transport compartment. Then GDP is replaced by GTP by GEFs to form an active state [ 87 , 88 ]. Active Rabs can interact with various effector proteins, such as motor proteins involved in transport and tethering factors, then Rabs are inactivated by GAPs and enter to next cycle [ 86 ]. For example, Rab7 interacts with Rab-interacting lysosomal protein to recruit the dynein-dynactin motor complex to regulate the transport of late endosomes to lysosomes [ 86 ]. Savina et al. reported that Rab11 is involved in exosome release, which reduced the release of TfR and HSC70-containing exosomes in K562 cells when inhibited [ 89 ]. With further studies, several Rab proteins, including Rab27A/B, Rab7, Rab31 and Rab35, have been implicated in the regulation of exosome secretion. Rab35 was revealed to be necessary for PLP-bearing exosome release and allow docking of MVBs to the plasma membrane in oligodendroglial cells [ 90 ]. Rab7 was shown to be required for syntenin- and Alix-containing exosome secretion in MCF7 cells but not to affect exosome secretion from HeLa cells [ 40 , 91 ]. Recently, Rab7 was reported to be bound and recruited to MVBs by neddylated Coro1a, promoting lysosomal degradation of the MVBs and reducing exosome secretion consequently in HEK293, RAW264.7 and HeLa cells [ 92 ]. Two isoforms of Rab27, Rab27A, and Rab27B, play a role in the secretion of exosomes bearing CD63, CD81, and MHC class II in HeLa cells and docking to the plasma membrane [ 91 ]. However, depletion of Rab27A has no effect in decreasing secretion of exosomes in certain cell lines such as MDA-MB-231 [ 8 , 93 , 94 ]. Recent work identified that Rab31 marks and controls an ESCRT- independent exosome biogenesis pathway and prevents the fusion of MVBs with lysosomes in HeLa cells [ 95 ]. Interestingly, Rab31 functions as a driver for ILVs formation and determines the fates of endocytic membrane proteins by balancing with Rab7 [ 95 ]. Mechanisms involved in MVB fusion with plasma membrane Exosomes are released into the extracellular environment upon fusion of MVBs with the plasma membrane (Fig. 1 ). This process involves SNARE (soluble NSF-attachment protein receptor) proteins or complexes that allow fusion of the lipid bilayers of two different intracellular compartments [ 96 ]. VAMP7 (vesicle-associated membrane protein 7) on the lysosomes, syntaxin 7 on the plasma membrane, and the lysosomal regulatory synaptotagmin7 form a complex participating in the exocytosis of conventional lysosomes, which has been reported to be involved in the exosome secretion of K562 cells [ 97 , 98 ]. Another SNARE protein YKT6, involved in endoplasmic reticulum-to-Golgi complex transport, has been shown to be required for exosome release by depletion in HEK293 cells and A549 cells [ 99 , 100 ]. Moreover, syntaxin 5 and Ral 1 in C. elegans , syntaxin 1a in mammals and SNAP-23 in human mastocytes are regulators of the exosomal secretion process as well [ 81 , 101 , 102 ]. Similarly, different SNARE complexes may be applied to the fusion of a particular organelle with the plasma membrane in different cell type or the fusion of different subpopulations of MVBs in a single cell type [ 8 ]. As depicted in Fig. 1 , MVBs are transported to the plasma membrane, and this process primarily involves the Rab proteins. MVBs fusion with the plasma membrane is similar to the general fusion process of membranes, which requires the involvement of SNARE complex. Notably, the above studies suggest that the mechanisms of exosome secretion may be limited by the original subpopulations of endosomes or dependent on the cell type [ 13 , 103 ]. The biogenesis and release of microvesicles Unlike the fusion of MVBs with the plasma membrane, as illustrated in Fig. 1 , the shedding of MVs requires a fission process that occurs in the curved region of the plasma membrane (Fig. 1 ). Mechanisms similar to exosome biogenesis are involved in the generation of MVs. ESCRT factors are involved in this process requiring ESCRT-I, II, III, and accessory proteins [ 42 ]. Among them, the arrestin domain-containing protein 1 (ARRDC1) directly drives plasma membrane budding by recruiting TSG101 to the plasma membrane, while VPS4 is also involved [ 38 , 104 ]. Additionally, acid sphingomyelinase induces the production of ceramide-dependent MVs of astrocytes [ 105 ], while neutral sphingomyelinase in MVBs is required for the biogenesis of exosomes [ 46 ]. Another lipid-dependent mechanism is associated with cholesterol which is abundant in MVs, and the formation of MVs is impaired when cholesterol is pharmacologically depleted in activated neutrophils [ 106 ]. PTMs are also reported to participate in the MV biogenesis [ 77 ]. As a typical example, Y14-phophorylation of caveolin-1 leads to interaction with hnRNPA2B1, followed by induction of hnRNPA2B1 O-GlcNacylation, which triggers the binding between selected miRNAs and hnRNPA2B1, finally leading to selection of miRNAs into MVs [ 107 ]. Given the pattern and site of MVs biogenesis, cytoskeleton rearrangement and related molecules are critical to this process. Large increase in Ca 2+ levels induces the activation of calpain, a protease capable of cleaving cytoskeletal proteins, together with the alteration of flippase, floppase, and scramblase, drives membrane asymmetry cytoskeletal remodeling, leading to outward budding of microvesicles [ 108 – 111 ]. Consistently, the Rho family small G protein and Rho-associated protein kinase (ROCK) are required for MVs release [ 112 , 113 ]. Moreover, a recent study by Wang and Zhuang showed that Rho family small G protein Cdc42 is a convergent node of multiple regulatory signals that occur in MVs biogenesis and the binding of activated GTP-bound Cdc42 and its downstream effector, Ras GTPase-activating-like protein 1 (IQGAP1), is required for MVs shedding in breast cancer cells [ 113 ]. Also, the activation of RhoA by ARF6 or ARF1 leads to an actin-myosin-based contraction that is required for MVs budding through the kinases ROCK and ERK (extracellular signal regulated kinase) [ 114 , 115 ]. Diaphanous-related formin-3 (DRF3), a signaling protein binding small Rho family GTPases, has been reported to be associated with the formation of MVs when suppressed in prostate cancer cells [ 116 ]. Furthermore, the biogenesis of MVs in cancer cells is also associated with metabolic changes driven by the "Warburg effect", which is thought to be required for malignant transformation and cancer progression [ 117 ]. Extracellular signals regulation of the biogenesis and release of extracellular vesicles In cells, signal transduction networks receive and transmit signals, including those from the extracellular environment, to regulate and coordinate core cellular functions, such as the release of EVs [ 118 ]. Although several pathways for the biogenesis and release of EVs and the key regulatory molecules involved have been described and characterized, extracellular signals and associated regulatory pathways have been rarely mentioned. Indeed, the extracellular signals, receptors, and downstream molecules are intertwined with the biogenesis and release mechanisms of EVs. G protein-coupled receptor regulates EV biogenesis and release G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signaling proteins that play a variety of physiological functions and roles in a wide range of diseases [ 119 ]. External stimuli, such as proteins, peptides, sugars, lipids, and neurotransmitters, can activate GPCRs, leading to the activation of subsequent effectors [ 120 ]. Previous studies have shown that many GPCR signaling components are involved in EV biogenesis and release from different cell types [ 121 – 125 ]. For instance, CXC chemokine was demonstrated to regulate hepatocyte exosome release via CXCR2 which modulates neutral sphingomyelinase activity and resultant production of ceramide [ 126 ]. Carbachol stimulates the muscarinic M1 receptor, thereby inducing the formation of ILVs in a DAG/PKD-dependent way in T lymphocytes [ 127 , 128 ]. Extracellular glutamate activates the metabotropic glutamate G-protein-coupled receptor mGluR3 to promote Rab27/CD63-dependent and mitochondrial DNA-containing EV release in breast cancer cells [ 129 ]. Moreover, Ca 2+ functions as both the second messenger and the key regulator of the release of EVs, mediating external signals regulation of EVs release as expected. External signals combine with GPCR and increase cytoplasmic Ca 2+ concentration by PLC/IP3 pathway, which triggers exosome release in microglia cells (Fig. 2 ) [ 130 ]. Meanwhile, there are some cases that the activation of multiple GPCRs stimulates EV release without raising intracellular calcium [ 131 ]. Another downstream of GPCR/PLC, Diacylglycerol/Protein Kinase C (DAG/PKC) pathway, was proved to be involved in MVBs fusion with the plasma membrane via phosphorylation of serine residue 110 of the SNAP23 in HeLa cells (Fig. 2 ) [ 132 ]. And beyond that, the pool of DAG in the Golgi determines the forming efficiency of post-trans-Golgi network secretory vesicle, and a negative regulator of DAG, diacylglycerol kinase alpha, was found to regulate secretion of bioactive exosomes in T lymphocytes [ 133 , 134 ]. In addition to above mechanisms, external signals also can regulate the release of microvesicles via RhoA signaling as reported (Fig. 3 ) [ 114 ]. Fig. 2 Extracellular signals regulate the biogenesis of exosomes. Extracellular signals regulate different events during the biogenesis of exosomes in different cell types. Activated GPCR regulates neutral sphingomyelinase activity and ceramide to effect exosome release, induces the formation of ILVs via DAG/PKD, triggers exosome release via PLC/IP3/Ca 2+ and participates in MVBs fusion with the plasma membrane via DAG/PKC. Activated EGFR activate ERK and HRS to promote cargo loading and exosome release. Inhibitory AKT/mTORC1 signals delivered by EGFR stimulate release from Rab11a compartments of exosomes. TNF-α triggers the release of exosomes which depends on sphingomyelinase. Cell death inducer stimulate TNFR, then TNFR promotes endosomal trafficking via RIPK3/MLKL pathway and enhances exosomes release. Wnt-mediated GSK3 inactivation regulates the expression of Rab27, and Wnt/β-catenin/TCF-4 activates the expression of Rab27B, therefore participating in the regulation of exosomes biogenesis. Hypoxia regulates exosome release through HIF-1α, Rab27A, Rab7, LAMP-1/2, neuraminidase-1 and ATM. Circadian clock and mechanical force also regulate exosome release, but specific mechanisms are unclear Fig. 3 Extracellular signals regulate the biogenesis of microvesicles. Microvesicles release directly from the plasma membrane and Rho signaling is one of the most important pathways mediating GPCRs signals regulation of microvesicle release. EGF activates EGFR to activate Cdc42, activated Cdc42 binds to IQGAP1 to block endocytosis and facilitate microvesicle release. Toxic lipid through TNFR activates DR5 proapoptotic signaling and ROCK1 to regulate plasma membrane-derived EV release. Wnt signaling regulates the expression of the Rab, Rho, ARF and Ca 2+ to affect microvesicle release. Hypoxia regulates Rab22A expression through HIF-1α to affect microvesicle release. Shear force induces microvesicle release through integrin signaling and Scr/Rac1 signaling EGFR regulates EV biogenesis and release Epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTKs), which plays a pivotal role in cell physiology [ 135 ]. Extracellular EGF stimulation causes receptor transphosphorylation and consequent activation of the intracellular signaling cascade, including Ras-ERK, PI3K-Akt, PLC-PKC, which ultimately results in the alteration of cell proliferation, survival, and differentiation [ 135 , 136 ]. Zhou et al. studied the correlation between exosome secretion and EGFR activation in renal tubular cells, suggesting a negative regulation of EGFR signaling in exosome secretion [ 137 ]. Consistently, it was reported that inhibition of EGFR signaling triggers a burst of release of exosomes containing EGFR in human epidermal cells [ 138 ]. However, Koistinen et al. found that epithelial to mesenchymal transition induced by EGF enhances the production of CD44 and release of EVs, and proposed CD44 as a potential marker of EVs in rat primary mesothelial cells [ 139 ]. Guan et al. reported that extracellular signal-regulated kinase (ERK) mediated phosphorylation of HRS selectively promotes PD-L1 loading to exosomes through ubiquitin-independent binding and increases exosome release in metastatic melanoma cell line [ 140 ]. Kim et al. demonstrated that heparin-binding EGF activates EGFR and ERK1/2 signaling to induce shedding of exosome-size EVs, and that loss of Diaphanous-related formin-3, a cytoskeletal regulator, also promotes the release of EVs in amoeboid prostate cancer cells [ 141 ]. Recently, EGF signaling facilitating MVs biogenesis was illustrated by the study reporting that Rho family small G protein Cdc42 activated by EGF binds to IQGAP1 to promote MVs shedding and maintains EGF signaling by blocking EGFR endocytosis and then helps MVs biogenesis (positive feedback) (Fig. 3 ) [ 113 ]. Additionally, Rab11/11a-positive compartments were identified as novel sites of exosome biogenesis and these latter exosomes appear to be preferentially released when nutrients stress or Akt/mTORC1 inhibition occurs (Fig. 2 ) [ 142 ]. TNFR regulates EV biogenesis and release TNF (tumor necrosis factor) and TNFR (TNF receptor) superfamilies, consisting of 19 ligands and 29 receptors respectively and are associated with many inflammatory disorders [ 143 ]. It has been reported that TNF-α modulates the protein and mRNA content of exosomes derived from the endothelial cells and the small intestinal epithelial cells, suggesting inflammatory signals might regulate the biogenesis of exosomes [ 144 , 145 ]. Meanwhile, some studies showed increase of exosome and microvesicle production in response to inflammatory stimuli [ 146 – 148 ]. For example, Sohda et al. demonstrated that TNF-α trigger the release of TNFR1 containing EVs from the human bronchial epithelial cells, which depends on acid- and neutral-sphingomyelinase [ 148 ]. It was reported that TNF influences the expression level of partial proteins in EVs and increases the rate of vesiculation in the brain endothelial cells [ 149 ]. In hepatocytes, toxic lipid mediates the release of EVs by death receptor 5 (DR5 or TNFRSF10B) proapoptotic signaling, which consists of a CHOP-DR5-caspase8-caspase3 signaling cascade activating ROCK1 (Fig. 3 ) [ 150 ]. Implicitly, targeting ROCK1 suggests that lipotoxicity-induced EVs are generated from the plasma membrane [ 150 ]. In addition, cell death inducers bind to TNFR and activate RIPK3, which was described to phosphorylate the pseudokinase mixed lineage kinase domain-like (MLKL) (Fig. 2 ) [ 151 ]. Mechanically, this phosphorylation was found to improve MLKL association with the endosomes and promote endosomal trafficking, resulting in enhanced release of EVs containing ph-MLKL in the colorectal adenocarcinoma cells [ 151 ]. It should be noted that this enhancement is not the result of necrosis. Furthermore, the release of exosomes in this pathway during necrosis, which is Rab27-independent, may prefer lysosomal exocytosis to MVB-mediated exocytosis [ 152 ]. Wnt signals regulate EV biogenesis and release The Wnt pathway plays key roles in short range cell–cell signaling within specific tissues, which is essential for both developmental and homeostatic processes, often divided into β-catenin dependent and independent signaling [ 153 – 155 ]. Interestingly, Wnts are involved in a complicated signaling network and crosstalk with the Notch, EGF, Hippo, and FGF pathways [ 154 ]. A genome-wide miRNA and CRISPR/Cas9 screen targeting Wnt signaling and trafficking-related genes identified multiple mediators of EVs secretion [ 121 ]. It was demonstrated that Wnt-mediated GSK3 inactivation downregulates Rab27 mRNA and protein regulating EVs release and that β-catenin/TCF-4 activates the expression of Rab27B, which is known to be required for the release of exosomes from colorectal cancer stem cells (Fig. 2 ) [ 121 , 156 ]. β-catenin-independent signaling and Wnt components affect cytoskeletal rearrangements, cell motility, induced invasiveness, and factors of EVs release such as Rabs, Rho-GTPases, Ca 2+ , and ARF6 [ 157 ]. Consistently, Wnt5A, a non-canonical Wnt protein, regulates Ca 2+ –dependent EVs release in an auto- or paracrine manner in malignant melanoma cells and enhances exosome release during Wnt5a/PI3K/miR-122 mediated hepatocyte differentiation [ 158 , 159 ]. Hypoxia affects EV biogenesis and release Oxygen is essential for energy metabolism, and hypoxia is considered as a hallmark of the tumor microenvironment due to uncontrolled proliferation of tumors [ 160 ]. Many studies have shown that hypoxia enhances the release of EVs from different cancer cells and alters cargo at the expression level [ 161 – 168 ]. Interestingly, hypoxia can promote human umbilical cord mesenchymal stem cells to release more EVs that exhibit enhanced anti-inflammation and anti-fibrosis potential, while hypoxia leads to larger EVs from adipose mesenchymal stem cells and smaller EVs from pancreatic tumor cells [ 169 – 171 ]. Such variety implies the existence of different regulation pathway in each event of EV biogenesis. In breast cancer cell lines, exposure to hypoxia significantly increases the number of exosomes in media, which may be mediated by hypoxia-inducible factor-1α (HIF-1α), a subunit of the major component of the hypoxia-related signaling pathway [ 162 ]. In ovarian cancer, exosome release increasing is facilitated by upregulating Rab27A, downregulating Rab7, LAMP-1/2, neuraminidase-1, and by promoting a more secretory lysosomal phenotype [ 172 ]. Further, the regulation of Rab proteins by hypoxia is mediated by STAT3, which indicating certain cytokines as signals also triggering STAT may affect the release of EVs [ 172 ]. Consistently, the secreted interleukin-25 from lung epithelial cells downregulates Rab27A/B expression in macrophages to suppress exosome release, and, of note, lipopolysaccharide can also stimulate marcophages to release exosomes [ 146 ]. For the balance between MVB biogenesis and degeneration, hypoxia activates the oxidized ataxia telangiectasia-mutated gene (ATM) in breast cancer-associated fibroblasts, which impairs lysosome functions by phosphorylating a subunit of proton pump in the lysosomes to enhance autophagy-associated exosome release [ 173 ]. Hypoxia increases expression and activation of some cell surface receptors through HIF, leading to induced endocytosis and exosome release [ 174 ]. There are some studies concerning hypoxia affecting the shedding and the content of microvesicles, but few mechanisms were proposed [ 166 , 175 ]. It was reported that hypoxia augments microvesicle shedding in human breast cancer cells by activating HIF and regulating the expression of Rab22a that localizes to budding microvesicles [ 176 ]. Other extracellular signals affect EV biogenesis and release Cells are in a complicated and constantly changing environment containing multiple factors such as pH, thermal and oxidative stress, nutrients, light, and physical stress [ 142 , 162 , 177 – 182 ]. There are some other extracellular signals affect EV biogenesis, and it is noteworthy that some unconventional forms of extracellular signals can regulate EV biogenesis [ 183 ]. Circadian clock, responsible for regulating many aspect of physiology in mammals, was found to regulate the proteome of small EVs in tendon fibroblasts, and the abundance of matrix metalloproteinase 14 in small EVs is regulated by flotillin-1 [ 184 ]. Extracellular matrix (ECM) constitutes the physical component of the extracellular milieu, and some studies demonstrated that ECM stiffness and mechanical force can regulate the EV secretion [ 185 – 188 ]. Intriguingly, Liu et al. found that the ECM mechanical force-induced exosomes have regulated miRNAs which can transmit the mechanical force to modulate cancer metastasis [ 189 ]. Similarly, shear force is an important signal regulating microvesicle release in vascular environment [ 179 ]. This kind of signal was demonstrated necessary for platelets to expose the phospholipid phosphatidylserine on the outer membrane surfaces and release microvesicles [ 180 ]. This process depends on integrin which serves as a mechanical sensor and a signal transducer, and downstream Src/Rac1 signaling pathway [ 180 ]. Ayers et al. have reviewed the effect of shear stress, as well as other stimuli within the cardiovascular system (acute cardiac stress, hypertriglyceridaemia, inflammation, etc.), on the release of microvesicles, and proposed that the degree of shear triggering the microvesicle release from different cells is not the same [ 179 ]. The regulatory mechanisms mentioned above are briefly illustrated in Fig. 3 and Fig. 4 , which demonstrate that information flows in, is transduced through a complex but precise pathway, and an appropriate response is delivered. Moreover, it is conceivable that these pathways intersect in a complex but precise network that is regulated on several different levels. Fig. 4 The diagram of EVs in clinical application. a EVs participate in various disease progression including cancer, infectious diseases and neurodegenerative disorders. For example, tumor-derived EVs can help pre-metastatic niche formation, act within tumor environment by educating different types of stroma cells and propagate tumor heterogeneity. EVs become potential novel targets for therapeutic intervention. b The low immunogenicity, efficiency and stability of EVs make them promising vehicles for drug delivery. Exogenous and endogenous loading approaches can be applied. For example, EVs can be loaded with specific cargo through direct transfection, and cells can be engineered to express the therapeutic of interest. In addition, EVs can also be modified to help deliver to the desired site of action. c EVs contribute significantly to drug resistance. Exosomes encapsulate and export drugs, horizontally transfer drug efflux pumps to recipient cells, and transfer biomolecules that promote drug inactivation. d EVs reflect heterogeneous biological changes related to diseases, supporting disease prediction, diagnosis, prognosis and surveillance with simplicity and stability. Created with BioRender.com G protein-coupled receptor regulates EV biogenesis and release G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signaling proteins that play a variety of physiological functions and roles in a wide range of diseases [ 119 ]. External stimuli, such as proteins, peptides, sugars, lipids, and neurotransmitters, can activate GPCRs, leading to the activation of subsequent effectors [ 120 ]. Previous studies have shown that many GPCR signaling components are involved in EV biogenesis and release from different cell types [ 121 – 125 ]. For instance, CXC chemokine was demonstrated to regulate hepatocyte exosome release via CXCR2 which modulates neutral sphingomyelinase activity and resultant production of ceramide [ 126 ]. Carbachol stimulates the muscarinic M1 receptor, thereby inducing the formation of ILVs in a DAG/PKD-dependent way in T lymphocytes [ 127 , 128 ]. Extracellular glutamate activates the metabotropic glutamate G-protein-coupled receptor mGluR3 to promote Rab27/CD63-dependent and mitochondrial DNA-containing EV release in breast cancer cells [ 129 ]. Moreover, Ca 2+ functions as both the second messenger and the key regulator of the release of EVs, mediating external signals regulation of EVs release as expected. External signals combine with GPCR and increase cytoplasmic Ca 2+ concentration by PLC/IP3 pathway, which triggers exosome release in microglia cells (Fig. 2 ) [ 130 ]. Meanwhile, there are some cases that the activation of multiple GPCRs stimulates EV release without raising intracellular calcium [ 131 ]. Another downstream of GPCR/PLC, Diacylglycerol/Protein Kinase C (DAG/PKC) pathway, was proved to be involved in MVBs fusion with the plasma membrane via phosphorylation of serine residue 110 of the SNAP23 in HeLa cells (Fig. 2 ) [ 132 ]. And beyond that, the pool of DAG in the Golgi determines the forming efficiency of post-trans-Golgi network secretory vesicle, and a negative regulator of DAG, diacylglycerol kinase alpha, was found to regulate secretion of bioactive exosomes in T lymphocytes [ 133 , 134 ]. In addition to above mechanisms, external signals also can regulate the release of microvesicles via RhoA signaling as reported (Fig. 3 ) [ 114 ]. Fig. 2 Extracellular signals regulate the biogenesis of exosomes. Extracellular signals regulate different events during the biogenesis of exosomes in different cell types. Activated GPCR regulates neutral sphingomyelinase activity and ceramide to effect exosome release, induces the formation of ILVs via DAG/PKD, triggers exosome release via PLC/IP3/Ca 2+ and participates in MVBs fusion with the plasma membrane via DAG/PKC. Activated EGFR activate ERK and HRS to promote cargo loading and exosome release. Inhibitory AKT/mTORC1 signals delivered by EGFR stimulate release from Rab11a compartments of exosomes. TNF-α triggers the release of exosomes which depends on sphingomyelinase. Cell death inducer stimulate TNFR, then TNFR promotes endosomal trafficking via RIPK3/MLKL pathway and enhances exosomes release. Wnt-mediated GSK3 inactivation regulates the expression of Rab27, and Wnt/β-catenin/TCF-4 activates the expression of Rab27B, therefore participating in the regulation of exosomes biogenesis. Hypoxia regulates exosome release through HIF-1α, Rab27A, Rab7, LAMP-1/2, neuraminidase-1 and ATM. Circadian clock and mechanical force also regulate exosome release, but specific mechanisms are unclear Fig. 3 Extracellular signals regulate the biogenesis of microvesicles. Microvesicles release directly from the plasma membrane and Rho signaling is one of the most important pathways mediating GPCRs signals regulation of microvesicle release. EGF activates EGFR to activate Cdc42, activated Cdc42 binds to IQGAP1 to block endocytosis and facilitate microvesicle release. Toxic lipid through TNFR activates DR5 proapoptotic signaling and ROCK1 to regulate plasma membrane-derived EV release. Wnt signaling regulates the expression of the Rab, Rho, ARF and Ca 2+ to affect microvesicle release. Hypoxia regulates Rab22A expression through HIF-1α to affect microvesicle release. Shear force induces microvesicle release through integrin signaling and Scr/Rac1 signaling EGFR regulates EV biogenesis and release Epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTKs), which plays a pivotal role in cell physiology [ 135 ]. Extracellular EGF stimulation causes receptor transphosphorylation and consequent activation of the intracellular signaling cascade, including Ras-ERK, PI3K-Akt, PLC-PKC, which ultimately results in the alteration of cell proliferation, survival, and differentiation [ 135 , 136 ]. Zhou et al. studied the correlation between exosome secretion and EGFR activation in renal tubular cells, suggesting a negative regulation of EGFR signaling in exosome secretion [ 137 ]. Consistently, it was reported that inhibition of EGFR signaling triggers a burst of release of exosomes containing EGFR in human epidermal cells [ 138 ]. However, Koistinen et al. found that epithelial to mesenchymal transition induced by EGF enhances the production of CD44 and release of EVs, and proposed CD44 as a potential marker of EVs in rat primary mesothelial cells [ 139 ]. Guan et al. reported that extracellular signal-regulated kinase (ERK) mediated phosphorylation of HRS selectively promotes PD-L1 loading to exosomes through ubiquitin-independent binding and increases exosome release in metastatic melanoma cell line [ 140 ]. Kim et al. demonstrated that heparin-binding EGF activates EGFR and ERK1/2 signaling to induce shedding of exosome-size EVs, and that loss of Diaphanous-related formin-3, a cytoskeletal regulator, also promotes the release of EVs in amoeboid prostate cancer cells [ 141 ]. Recently, EGF signaling facilitating MVs biogenesis was illustrated by the study reporting that Rho family small G protein Cdc42 activated by EGF binds to IQGAP1 to promote MVs shedding and maintains EGF signaling by blocking EGFR endocytosis and then helps MVs biogenesis (positive feedback) (Fig. 3 ) [ 113 ]. Additionally, Rab11/11a-positive compartments were identified as novel sites of exosome biogenesis and these latter exosomes appear to be preferentially released when nutrients stress or Akt/mTORC1 inhibition occurs (Fig. 2 ) [ 142 ]. TNFR regulates EV biogenesis and release TNF (tumor necrosis factor) and TNFR (TNF receptor) superfamilies, consisting of 19 ligands and 29 receptors respectively and are associated with many inflammatory disorders [ 143 ]. It has been reported that TNF-α modulates the protein and mRNA content of exosomes derived from the endothelial cells and the small intestinal epithelial cells, suggesting inflammatory signals might regulate the biogenesis of exosomes [ 144 , 145 ]. Meanwhile, some studies showed increase of exosome and microvesicle production in response to inflammatory stimuli [ 146 – 148 ]. For example, Sohda et al. demonstrated that TNF-α trigger the release of TNFR1 containing EVs from the human bronchial epithelial cells, which depends on acid- and neutral-sphingomyelinase [ 148 ]. It was reported that TNF influences the expression level of partial proteins in EVs and increases the rate of vesiculation in the brain endothelial cells [ 149 ]. In hepatocytes, toxic lipid mediates the release of EVs by death receptor 5 (DR5 or TNFRSF10B) proapoptotic signaling, which consists of a CHOP-DR5-caspase8-caspase3 signaling cascade activating ROCK1 (Fig. 3 ) [ 150 ]. Implicitly, targeting ROCK1 suggests that lipotoxicity-induced EVs are generated from the plasma membrane [ 150 ]. In addition, cell death inducers bind to TNFR and activate RIPK3, which was described to phosphorylate the pseudokinase mixed lineage kinase domain-like (MLKL) (Fig. 2 ) [ 151 ]. Mechanically, this phosphorylation was found to improve MLKL association with the endosomes and promote endosomal trafficking, resulting in enhanced release of EVs containing ph-MLKL in the colorectal adenocarcinoma cells [ 151 ]. It should be noted that this enhancement is not the result of necrosis. Furthermore, the release of exosomes in this pathway during necrosis, which is Rab27-independent, may prefer lysosomal exocytosis to MVB-mediated exocytosis [ 152 ]. Wnt signals regulate EV biogenesis and release The Wnt pathway plays key roles in short range cell–cell signaling within specific tissues, which is essential for both developmental and homeostatic processes, often divided into β-catenin dependent and independent signaling [ 153 – 155 ]. Interestingly, Wnts are involved in a complicated signaling network and crosstalk with the Notch, EGF, Hippo, and FGF pathways [ 154 ]. A genome-wide miRNA and CRISPR/Cas9 screen targeting Wnt signaling and trafficking-related genes identified multiple mediators of EVs secretion [ 121 ]. It was demonstrated that Wnt-mediated GSK3 inactivation downregulates Rab27 mRNA and protein regulating EVs release and that β-catenin/TCF-4 activates the expression of Rab27B, which is known to be required for the release of exosomes from colorectal cancer stem cells (Fig. 2 ) [ 121 , 156 ]. β-catenin-independent signaling and Wnt components affect cytoskeletal rearrangements, cell motility, induced invasiveness, and factors of EVs release such as Rabs, Rho-GTPases, Ca 2+ , and ARF6 [ 157 ]. Consistently, Wnt5A, a non-canonical Wnt protein, regulates Ca 2+ –dependent EVs release in an auto- or paracrine manner in malignant melanoma cells and enhances exosome release during Wnt5a/PI3K/miR-122 mediated hepatocyte differentiation [ 158 , 159 ]. Hypoxia affects EV biogenesis and release Oxygen is essential for energy metabolism, and hypoxia is considered as a hallmark of the tumor microenvironment due to uncontrolled proliferation of tumors [ 160 ]. Many studies have shown that hypoxia enhances the release of EVs from different cancer cells and alters cargo at the expression level [ 161 – 168 ]. Interestingly, hypoxia can promote human umbilical cord mesenchymal stem cells to release more EVs that exhibit enhanced anti-inflammation and anti-fibrosis potential, while hypoxia leads to larger EVs from adipose mesenchymal stem cells and smaller EVs from pancreatic tumor cells [ 169 – 171 ]. Such variety implies the existence of different regulation pathway in each event of EV biogenesis. In breast cancer cell lines, exposure to hypoxia significantly increases the number of exosomes in media, which may be mediated by hypoxia-inducible factor-1α (HIF-1α), a subunit of the major component of the hypoxia-related signaling pathway [ 162 ]. In ovarian cancer, exosome release increasing is facilitated by upregulating Rab27A, downregulating Rab7, LAMP-1/2, neuraminidase-1, and by promoting a more secretory lysosomal phenotype [ 172 ]. Further, the regulation of Rab proteins by hypoxia is mediated by STAT3, which indicating certain cytokines as signals also triggering STAT may affect the release of EVs [ 172 ]. Consistently, the secreted interleukin-25 from lung epithelial cells downregulates Rab27A/B expression in macrophages to suppress exosome release, and, of note, lipopolysaccharide can also stimulate marcophages to release exosomes [ 146 ]. For the balance between MVB biogenesis and degeneration, hypoxia activates the oxidized ataxia telangiectasia-mutated gene (ATM) in breast cancer-associated fibroblasts, which impairs lysosome functions by phosphorylating a subunit of proton pump in the lysosomes to enhance autophagy-associated exosome release [ 173 ]. Hypoxia increases expression and activation of some cell surface receptors through HIF, leading to induced endocytosis and exosome release [ 174 ]. There are some studies concerning hypoxia affecting the shedding and the content of microvesicles, but few mechanisms were proposed [ 166 , 175 ]. It was reported that hypoxia augments microvesicle shedding in human breast cancer cells by activating HIF and regulating the expression of Rab22a that localizes to budding microvesicles [ 176 ]. Other extracellular signals affect EV biogenesis and release Cells are in a complicated and constantly changing environment containing multiple factors such as pH, thermal and oxidative stress, nutrients, light, and physical stress [ 142 , 162 , 177 – 182 ]. There are some other extracellular signals affect EV biogenesis, and it is noteworthy that some unconventional forms of extracellular signals can regulate EV biogenesis [ 183 ]. Circadian clock, responsible for regulating many aspect of physiology in mammals, was found to regulate the proteome of small EVs in tendon fibroblasts, and the abundance of matrix metalloproteinase 14 in small EVs is regulated by flotillin-1 [ 184 ]. Extracellular matrix (ECM) constitutes the physical component of the extracellular milieu, and some studies demonstrated that ECM stiffness and mechanical force can regulate the EV secretion [ 185 – 188 ]. Intriguingly, Liu et al. found that the ECM mechanical force-induced exosomes have regulated miRNAs which can transmit the mechanical force to modulate cancer metastasis [ 189 ]. Similarly, shear force is an important signal regulating microvesicle release in vascular environment [ 179 ]. This kind of signal was demonstrated necessary for platelets to expose the phospholipid phosphatidylserine on the outer membrane surfaces and release microvesicles [ 180 ]. This process depends on integrin which serves as a mechanical sensor and a signal transducer, and downstream Src/Rac1 signaling pathway [ 180 ]. Ayers et al. have reviewed the effect of shear stress, as well as other stimuli within the cardiovascular system (acute cardiac stress, hypertriglyceridaemia, inflammation, etc.), on the release of microvesicles, and proposed that the degree of shear triggering the microvesicle release from different cells is not the same [ 179 ]. The regulatory mechanisms mentioned above are briefly illustrated in Fig. 3 and Fig. 4 , which demonstrate that information flows in, is transduced through a complex but precise pathway, and an appropriate response is delivered. Moreover, it is conceivable that these pathways intersect in a complex but precise network that is regulated on several different levels. Fig. 4 The diagram of EVs in clinical application. a EVs participate in various disease progression including cancer, infectious diseases and neurodegenerative disorders. For example, tumor-derived EVs can help pre-metastatic niche formation, act within tumor environment by educating different types of stroma cells and propagate tumor heterogeneity. EVs become potential novel targets for therapeutic intervention. b The low immunogenicity, efficiency and stability of EVs make them promising vehicles for drug delivery. Exogenous and endogenous loading approaches can be applied. For example, EVs can be loaded with specific cargo through direct transfection, and cells can be engineered to express the therapeutic of interest. In addition, EVs can also be modified to help deliver to the desired site of action. c EVs contribute significantly to drug resistance. Exosomes encapsulate and export drugs, horizontally transfer drug efflux pumps to recipient cells, and transfer biomolecules that promote drug inactivation. d EVs reflect heterogeneous biological changes related to diseases, supporting disease prediction, diagnosis, prognosis and surveillance with simplicity and stability. Created with BioRender.com Clinical relevance of the biogenesis and release of extracellular vesicles As research in the field of EVs intensifies, EVs show promise for clinical therapies. For example, EVs play important roles in disease pathogenesis, especially in cancer. In the studies described above, the aberrant environment caused by diseases provokes the abnormity in the biogenesis of EVs, such as increase of circulating metabolites, gene mutation at a high frequency and nutrient deficiency in cancer, inflammation, oxidative stress related to hypoxia, and mechanical force changes during cancer metastasis [ 113 , 129 , 142 , 148 , 173 , 189 ]. More significantly, it is implied that signals transformed from extracellular environment changes regulate EV biogenesis, and in turn, these EVs further deliver information to other unaffected cells to provoke extracellular signaling cascades, and finally aggravate diseases. Many studies have shown that EVs drive the formation of a pre-metastatic tumor niche and stimulate tumor progression via inducing proliferation, facilitating angiogenesis, and promoting immune escape (Fig. 4 ) [ 14 ]. In this context, strategies that inhibit the biogenesis and release of EVs are potent to treat EVs-driven diseases [ 14 ]. In addition, there is growing evidence that biomolecules in EVs can be used as biomarkers for early detection and monitoring of diseases involving the central nervous system, liver, kidney, lung, etc. (Fig. 4 ) [ 190 ]. Further, EVs have many characteristics, such as intrinsic stability and inherent targeting properties, that make them promising drug delivery vehicles (Fig. 4 ) [ 191 ]. However, with increasing expectations for the application of EVs, the mechanisms regulating the biogenesis and release of EVs have become an inevitable question. For example, the heterogeneity of EV content from different cells challenges the drug carrier development, though it is the foundation of its application to liquid biopsy. Why cells produce EVs and whether external perturbations modulate the extent of EV biogenesis, processing, and release of EVs are still unknown. Significantly, studies on the regulation by extracellular signals can be of value in directly improving the efficiency of cargo loading and EVs release in drug delivery, effectively inhibiting pathogenesis, restraining EVs-mediated drug resistance, comprehensively assessing cancer diagnosis, prognosis, and progression, and screening for therapeutic drug-induced cellular stress [ 14 , 191 – 194 ]. In recent years, technologies for EV application have been refining. As an example, exosomes were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, and treatment with these exosomes suppressed cancer in mouse model of pancreatic cancer [ 195 ]. For such drug delivery and cell-free cell therapy, EV production, isolation, purification and drug loading are general processes. The drug loading includes endogenous methods, which equip vesicles with drug or biomolecules in parent cells using nanoparticles or genetic engineering, and exogenous methods, which load drugs into isolated EVs through incubation, attachment, chemical modification (e.g. click chemistry) and permeabilization (sonication, electroporation and saponin treatment) [ 196 – 198 ]. For EV production, to produce sufficient numbers of EVs, introducing external stimuli to regulate EV release is more applicable than genetic methods since the natural secretion process is required in some cases [ 181 ]. For example, phototherapy-based LED light was applied to increase the dendritic cell-derived EVs that have been developed into clinical trials, and more importantly, the immunomodulation function, immunogenicity, oxidative stress levels, and biocompatibility of them are comparable to naturally released EVs [ 181 ]. A tubular perfusion bioreactor culture system with 3D-printed scaffolds was applied to enhance EV production, in which appropriate flow rate and produced shear stress leaded to more than 100-fold increase in endothelial cell EV production than 2D flask culture [ 199 ]. The addition of chemicals to preparing system to boost EV production has been reported. Using sodium iodoacetate and 2,4-dinitrophenol to inhibit glycolysis and oxidative phosphorylation can trigger a three- to twenty-four- fold increase in the secretion of EVs with similar property and function as those from untreated cells [ 200 ]. In practice, many methods have been considered to solve the problem of EV application, not only how to produce EVs on a large scale. However, manipulation of cargo abundance and signaling pathways using external signals should be cautious, and evaluation of the property and efficacy of produced EVs is essential to avoid risks. Conclusions Since EVs were first described to deliver mRNAs and microRNAs between cells in 2007, much advances and insights have been made in the biogenesis, release, and functions of EVs, as we discussed above [ 24 ]. As an intracellular-derived vesicular structure, the biogenesis process of EVs is consistent with the intercellular vesicles transport system, such as the pattern of cargo sorting, trafficking, and fusion with the target membrane have been described. However, studies on the biogenesis and release of EVs is just beginning and the specific molecule mechanisms remain unexplored. A single cell hosts different EVs subpopulations with different functions. EVs subpopulations and functions depend on their cargo, which in turn depends on the sorting machineries and signaling regulation. While this implies that distinct mechanisms may act simultaneously or sequentially on exosomes or microvesicles, the precise regulatory mechanisms need to be further explored. Specifically, the sorting process of certain cargo such as miRNAs, the correlation between cargo and machineries, the establishment of the balance between degradation and secretion of MVBs, and the crosstalk between different mechanisms are unclear. Studies reported that some components of classical signaling pathways were found to be involved in the biogenesis and release of EVs, which implies their necessity. Indeed, extracellular signals can regulate the biogenesis and release of EVs mainly by binding receptors and activating cascades. Major regulatory effectors are Rabs, Rhos (especially Cdc42), Ca 2+ and corresponding events are the transport of MVBs and cytoskeleton rearrangement, but other effectors regulated by extracellular signals remain to be revealed. Interestingly, the cellular signaling pathways comprise an intricate network where a single molecule can be activated by several upstream signals and can activate different effectors. It remains to be elucidated whether a single extracellular signal regulates multiple EVs subpopulations biogenesis and release, or whether a process is regulated by multiple signals. Therefore, other relevant signaling pathways should be taken into account in the study of this issue. It should be noted that several signaling cascades have been identified that utilize EVs for signaling transport in the tumor-stroma interaction, including potentially oncogenic signaling cascades and signaling cascades associated with tumor progression and metastasis, such as Wnt, TGF-β, PD-L1, EGFR [ 110 , 155 , 157 ]. Even mechanical force signals can be transmitted by exosomes [ 189 ]. In this case, EVs with signaling are themselves a form of extracellular signaling that allows signaling to transfer at the intercellular level. Thus, extracellular signaling likely promotes the biogenesis of EVs carrying components of signal cascade, which in turn promotes the biogenesis of EVs and play other roles in recipient cells. In conclusion, there are good reasons to expect that EVs will make great contributions to all biological events. EVs are not only responsible for mediating information transfer between different cells, but also extend the concept of signal transduction to intercellular aspect by working as the collection of information with extraordinary efficiency, specificity and stability. Despite the growth of therapeutic values of EVs, translating of EVs into clinical applications remains a challenge. Understanding of regulation of biogenesis and release by extracellular signals is necessary for supporting optimal utility and determines the potential of applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724183/

Innate sensors of influenza virus: clues to developing better intranasal vaccines

Mucosal immunity acquired by natural infection with influenza viruses at the respiratory tract is more effective and cross-protective against subsequent variant virus infection than systemic immunity induced by parenteral immunization with inactivated vaccines. To develop an effective influenza vaccine, it is beneficial to mimic the process of natural infection that bridges innate and adaptive immune systems. The innate immune system that recognizes influenza virus infection consists of several classes of pattern-recognition receptors, including the Toll-like receptors, the retinoic acid-inducible gene-I-like receptors and the NOD-like receptors. Here, we review our current understanding of the mechanism of innate recognition of influenza and how the signals emanating from the innate sensors control adaptive immunity. Further, we discuss the potential roles of these receptors in developing intranasal influenza vaccines.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108517/

Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium

Abstract Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin ( HA ) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA ‐activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS 2 in the human airway epithelium. In addition, we outline investigations of HA ‐activating proteases as potential drug targets for influenza treatment. Human influenza and bacterial superinfections Influenza (flu) is a highly contagious respiratory illness that affects each year millions of people. Of the three genera of influenza viruses (A, B and C), influenza A and influenza B viruses pose a continuous threat to public health. Currently, influenza A viruses of the subtypes H3N2 and H1N1 and influenza B viruses cocirculate with varying predominance in the human population and are responsible for seasonal epidemics that may result in 3–5 million cases of severe respiratory illness and up to 500 000 annual deaths worldwide. The natural reservoir of influenza A viruses are wild aquatic birds from where they are transmitted to a wide range of mammalian and avian hosts including humans, pigs and poultry. The emergence of a new influenza A virus for which there is little or no immunity in the human population may provoke an influenza pandemic, with the most devastating pandemic in 1918, the 'Spanish flu', claiming c . 40 million deaths worldwide. In addition, three more influenza pandemics occurred in the 20th and 21st centuries: the 'Asian flu' in 1957 (H2N2), the 'Hong‐Kong flu' in 1968 (H3N2) and, recently, the 2009 H1N1 pandemic. In contrast, influenza B viruses are predominantly found in humans and are not known to have an animal reservoir from which new virus variants can emerge. Human influenza viruses spread via aerosols or respiratory droplets due to sneezing and coughing, but may also spread by contact, and infect the respiratory epithelium of the upper or lower airways. Influenza is an acute respiratory illness with a rapid onset of symptoms such as fever, headache, generalized malaise and myalgias that may last for 7–14 days. Complications may be due to bronchitis, viral pneumonia or myocarditis. Moreover, viral‐bacterial pneumonia and secondary bacterial pneumonia due to concomitant or subsequent infection with bacteria, respectively, contribute significantly to morbidity and mortality of influenza infections. Most commonly isolated bacteria are Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus and Haemophilus influenzae . Pathological studies of autopsy specimens and analyses of medical reports revealed that the majority of the deaths of the 1918 influenza pandemic occurred concurrently with bacterial pneumonia predominantly by H. influenzae (McCullers, 2006 ; Morens et al ., 2008 ). Estimated 70% of severe and fatal cases during the 1957 and 1968 pandemics and c . 30% of influenza‐associated pneumonia during the 2009 H1N1 pandemic likely resulted from pneumonia due to co‐infections by S. aureus or pneumococcus. During seasonal influenza epidemics, estimated 44–57% of hospitalizations and 25% of influenza‐associated deaths are due to secondary bacterial pneumonia (Peltola et al ., 2005 ; McCullers, 2011 ). Influenza A virus morphology and viral life cycle Influenza A viruses belong to the family of Orthomyxoviridae and are enveloped viruses of pleomorphic morphology ranging from spherical to filamentous particles with a diameter of 80–120 nm. The genome consists of eight segments of single‐stranded, negative‐sense RNA, which are associated with the polymerase proteins PB1, PB2 and PA and multiple copies of the nucleoprotein (NP) to form the viral ribonucleoprotein complexes (vRNPs) and together encode up to 15 proteins (Jagger et al ., 2012 ; Muramoto et al ., 2013 ; Fig. 1 a). The viral envelope is made up of a lipid bilayer derived from the host cellular plasma membrane and contains the two major spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the proton channel protein M2. The inner side of the envelope is lined by the matrix protein M1. Based on antigenic criteria, influenza A viruses are classified into 17 HA (H1‐H17) and 10 NA (N1‐N10) subtypes to date (Tong et al ., 2012 ). Most subtypes circulate in wild aquatic birds and are sporadically transmitted to other species including humans. Figure 1 (a) Scheme of the influenza A virus particle. The virion contains a lipid envelope derived from the cellular plasma membrane. The two glycoproteins hemagglutinin (HA) and neuraminidase (NA) and the ion channel protein M2 are embedded in the envelope. The inner side is lined by the matrix protein M1. The genome consists of eight segments of single‐stranded, negative‐sense RNA, which are associated with the nucleoprotein (NP) and the polymerase subunits PB1, PB2 and PA to form viral ribonucleoprotein complexes ( vRNP s). The nuclear export protein (NEP) is also present in the virion, while the nonstructural proteins NS1, PB1‐F2, PB1‐N40, PA‐X, PA‐N155 and PA‐N182 are present in the infected cell. (b) Influenza virus replication and proteolytic activation by cellular proteases in human airway epithelial cells. Infection is initiated by the HA through binding to N‐acetyl neuraminic acid‐containing cell surface receptors. Upon receptor‐mediated endocytosis, HA mediates fusion of viral and endosomal membranes at low pH to release the vRNP s into the cytoplasm (uncoating). The vRNP s are imported into the nucleus, where transcription and replication of the viral genome occur. Translation of viral mRNA s is performed by the cellular machinery. HA, NA and M2 are synthesized into the endoplasmic reticulum (ER) and transported along the constitutive secretory pathway to the plasma membrane. The internal viral proteins are synthesized at free ribosomes, and protein components of the vRNP s are then imported into the nucleus, where assembly of new vRNP s occurs. Finally, vRNP s are exported from the nucleus and transported to the plasma membrane, where self‐assembly of viral proteins leads to budding of new virions. The NA cleaves N‐acetyl neuraminic acid from carbohydrate moieties, facilitating release of progeny virions. Proteolytic activation of HA can take place in different compartments and at different time points during viral replication and is indicated by scissors (open scissor: active protease; closed scissor: enzymatically inactive protease; truncated scissor: soluble protease). HA with multibasic cleavage site is cleaved by furin in the TGN . HA containing a monobasic cleavage site is cleaved by TMPRSS 2 in the TGN or by HAT on the plasma membrane: either during assembly and budding of progeny virus or during attachment and entry into the cell. Influenza virus replication is initiated by the HA through binding to N‐acetyl‐neuraminic acid‐containing cell surface receptors, resulting in receptor‐mediated endocytosis and subsequent fusion of the viral envelope with the endosomal membrane to release the vRNPs into the cytoplasm (Fig. 1 b). Membrane fusion requires proteolytic cleavage of HA and is induced by the acidic pH in the endosome. The vRNPs are imported into the nucleus where transcription and replication of the genome are catalysed by the viral RNA‐dependent RNA polymerase. Splicing of viral mRNAs and translation into proteins are performed by the cellular machineries. Influenza virus infection causes a shutoff of host protein synthesis, thereby enhancing translation of viral mRNAs. At the later stages of viral infection, newly synthesized vRNPs and viral proteins are transported to the apical membrane of polarized epithelial cells where assembly and budding of new progeny virions take place. Here, NA cleaves N‐acetyl‐neuraminic acid from carbohydrate moieties, facilitating release of progeny virus from the host cell and preventing virions to clump together. Proteolytic cleavage of HA is a prerequisite for membrane fusion The HA is the major surface glycoprotein of influenza viruses, mediates receptor binding and membrane fusion and is the main target for neutralizing antibodies. HA is a type I transmembrane protein and is integrated in the viral envelope as a homotrimer. HA is synthesized as a precursor protein, HA0 (75 kDa), that is transported along the constitutive secretory pathway to the plasma membrane (Fig. 1 b). During its transport, HA becomes N‐glycosylated, palmitoylated and cleaved into the subunits HA1 (55 kDa) and HA2 (25 kDa) (Fig. 2 a and b). Cleavage can take place either in the trans‐Golgi network (TGN) or on the cell surface as discussed below. The amino‐terminal cleavage fragment HA1 contains the receptor‐binding site and remains linked by a disulphide bond to the carboxy‐terminal fragment HA2, which is membrane‐anchored and responsible for fusion (Fig. 2 b). HA0 is cleaved at a specific arginine residue (R) to expose the fusion peptide consisting of about 14 hydrophobic amino acids at the N‐terminus of HA2. Cleavage is a prerequisite for conformational changes at low pH in the endosome that trigger membrane fusion and is essential for the infectivity and spread of influenza viruses. The cleavage site is located in a prominent loop that protrudes from the surface (Fig. 2 a). The amino acid sequence as well as the structure and susceptibility of the cleavage site are critical for cleavage of HA by different proteases (Klenk & Rott, 1988 ; Klenk & Garten, 1994 ; Steinhauer, 1999 ; Garten & Klenk, 2008 ). Figure 2 Cleavage of HA 0 into HA 1 and HA 2 at a specific cleavage site. (a) Structure of the monomeric HA 0 precursor of A/HongKong/68 containing the mutation R329Q to prevent cleavage determined by X‐ray crystallography (Chen et al ., 1998 ). The cleavage site is located in a prominent surface loop (yellow) highlighted by an arrow. (b) Schematic illustration of the HA 0 precursor and the cleaved form consisting of the disulphide‐linked subunits HA 1 and HA 2. The colours of HA 1 (blue) and HA 2 (red) are based on the structure shown in Fig. 1 a. The cleavage site is indicated by an arrow. FP : fusion peptide, TM : transmembrane domain. (c) Alignment of amino acid sequences at the HA cleavage site of different human and avian influenza viruses. The arrow indicates the cleavage site between HA 1 and HA 2. Basic amino acids crucial for cleavage by relevant proteases are highlighted in red. Cleavage of HA is a prime determinant of avian influenza virus pathogenicity Avian influenza A viruses are responsible for recurrent outbreaks in poultry farms worldwide and lead to serious economic losses in the poultry industry. Avian viruses differ in their pathogenicity and are classified as either low‐ or high‐pathogenic avian influenza viruses (LPAIV or HPAIV, respectively). LPAIV replicate primarily in the intestinal and also in the respiratory tract of birds, cause mild or asymptomatic infections and spread via the faecal‐oral route. In contrast, HPAIV cause systemic infections in poultry with mortality rates up to 100%, known as fowl plague or avian influenza. HPAIV are restricted to the subtypes H5 and H7 under natural settings, but not all H5 and H7 viruses are highly pathogenic. Already early studies of natural avian influenza virus isolates indicated that the cleavage site sequence connecting HA1 and HA2 is a key determinant for pathogenicity and organ tropism of the viruses. LPAIV possess a single arginine, rarely a single lysine, at the cleavage site, whereas the HA of HPAIV contains a multibasic motif with the consensus sequence R‐X‐R/K‐R (Vey et al ., 1992 ). Cleavage of HA0 into HA1 and HA2 is crucial for virus infectivity. LPAIV were found to require addition of exogenous trypsin to most tissue cultures for proteolytic activation and multicycle replication in vitro , whereas HPAIV were shown to be activated by endogenous proteases (Klenk & Rott, 1988 ; Klenk & Garten, 1994 ). HA with multibasic cleavage site is cleaved by ubiquitous subtilisin‐like endoproteases furin and the proprotein convertase 5/6 (PC5/6), supporting systemic infection (Stieneke‐Gröber et al ., 1992 ; Horimoto et al ., 1994 ; Feldmann et al ., 2000 ). Furin is a type I transmembrane protease that is predominantly localized in the TGN, but is also transported through the constitutive secretory pathway to the plasma membrane, where it can be shed or recycled and targeted back to the TGN (Schäfer et al ., 1995 ). Furin cleaves a large number of cellular protein precursors such as prohormones and growth factors at the C‐terminus of the consensus sequence R‐X‐R/K‐R into biologically active forms. Moreover, furin supports proteolytic activation of a large number of viral glycoproteins (e.g. measles virus, respiratory syncytial virus, yellow fever virus, HIV‐1, Ebola virus) and bacterial toxins (e.g. anthrax toxin, diphtheria toxin) at such basic motifs (Klenk & Garten, 1994 ; Thomas, 2002 ). Thus, furin plays important roles in numerous physiological as well as pathogenic processes. Cleavage of HA by furin takes place in the TGN (Fig. 1 b) and results in the release of infectious progeny virus containing cleaved HA from the cells, supporting spread of infection. In contrast, LPAIV possess a monobasic cleavage site and are proteolytically activated by trypsin in vitro (Klenk et al ., 1975 ). Relevant trypsin‐like proteases are present in a restricted number of tissues such as the respiratory or the intestinal tract, limiting spread of infection to these tissues. A protease homologous to blood clotting factor Xa was identified as HA‐activating protease in the chorioallantoic membrane of embryonated chicken eggs (Gotoh et al ., 1990 ), and a number of other trypsin‐like proteases, such as plasmin (Lazarowitz & Choppin, 1975 ) and tryptase Clara (Kido et al ., 1992 ), have been found to activate HA with a monobasic cleavage site in vitro . The identity of relevant proteases in the respiratory or intestinal tract of avian species, however, remains to be investigated. In 2006, the human proteases HAT and TMPRSS2 present in the human airway epithelium were identified as proteases that activate HA with monobasic cleavage (Böttcher et al ., 2006 ). A chicken protease homologous to human TMPRSS2 has been demonstrated to activate the HA at a monobasic cleavage site in vitro , suggesting that the chTMPRSS2 might be responsible for cleavage of HA also in avian hosts (Bertram et al ., 2012 ). HPAIV emerge from LPAIV by extension of the cleavage site loop due to insertion of mostly basic amino acids (Fig. 2 c). Interestingly, acquisition of a multibasic cleavage site that is activated by furin seems to be restricted to the subtypes H5 and H7 and has not been observed for any other subtype under natural settings (Klenk & Garten, 1994 ; Garten & Klenk, 2008 ). Analysis of the cleavage site of different HPAIV demonstrated that extension of the cleavage site can occur by different mechanisms: polymerase slippage at purine‐rich arginine or lysine codons, recombination with viral gene segments (NP or M genes) or 28S ribosomal RNA or still unknown mechanisms and has been reviewed in detail in the study by Garten & Klenk ( 2008 ). Already in early studies, it has been demonstrated that substitution of the multibasic cleavage site of a HPAIV by a monobasic motif results in a low‐pathogenic virus. However, insertion of a multibasic cleavage motif into a HA with a monobasic cleavage site does not automatically confer high pathogenicity to the virus, demonstrating that the emergence of a HPAIV is a multifactorial process (Stech et al ., 2009 ; Schrauwen et al ., 2011 ). In a recent study, Veits et al . ( 2012 ) introduced a multibasic cleavage site into the HA of several influenza A virus subtypes and generated reassortant viruses in the genetic background of a highly pathogenic H5N1 virus. Interestingly, reassortants with H2, H4, H8 and H14 with multibasic cleavage site caused lethal infections in chickens, while other subtypes did not. These observations demonstrate that a multibasic HA cleavage site can confer high pathogenicity to subtypes other than H5 and H7 in a suitable genetic background. Therefore, the restriction of natural HPAIV isolates to H5 and H7 subtypes seems to be due to their unique ability to acquire a multibasic cleavage site under natural settings; however, underlying mechanisms are not yet understood. Activation of human influenza viruses by host cell proteases TMPRSS2 and HAT As most avian and mammalian influenza viruses, human influenza viruses possess a monobasic cleavage site and require activation by trypsin‐like proteases. Seasonal and pandemic influenza viruses belong to the subtypes H1, H2 and H3 and influenza B virus, respectively, and are activated C‐terminal of the HA cleavage site motifs I‐Q‐S‐R, I‐E‐S‐R and K‐Q‐T‐R for H1, H2 and H3 subtypes, respectively (Fig. 2 c), and L‐K‐E‐R for influenza B viruses. A number of trypsin‐like proteases isolated from rat and swine lung, such as tryptase Clara, mini‐plasmin or tryptase TC30, were shown to support proteolytic activation and multicycle replication of human influenza viruses in vitro (Kido et al ., 1992 , 2007 ; Murakami et al ., 2001 ; Sato et al ., 2003 ). Because the genetic identity is unknown for these proteases, it still remains unclear whether they play a role in in vivo infection, and appropriate proteases that cleave HA in the human airway epithelium were unknown until recently. Activation of HA with monobasic cleavage site by plasmin, tryptase Clara or the factor Xa‐like protease in embryonated chicken eggs occurs outside the cell after release of newly synthesized virions or probably already during assembly and budding when HA is present on the plasma membrane. According to this, the classical dogma of activation of HA with monobasic cleavage site is HA cleavage by soluble proteases outside the cells. A study by Zhirnov et al . ( 2002 ), however, demonstrated that cleavage of HA in human airway epithelial cells occurs by cell‐associated proteases. Furthermore, activation of influenza viruses in human intestinal Caco‐2 cells was shown to take place intracellularly (Zhirnov et al ., 2003 ). The relevant proteases remained unknown in these studies, but indicated that cleavage of HA by human proteases may be performed by membrane‐bound proteases. In search for human HA‐activating proteases, a number of proteases that possess trypsin‐like activity were cloned from human primary tracheobronchial epithelial (HTBE) cells (Böttcher et al ., 2006 ). Among a couple of candidates, the two proteases HAT (human airway trypsin‐like protease; also designated as TMPRSS11D) and TMPRSS2 (transmembrane protease serine S1 member 2; also designated as epitheliasin) were demonstrated to cleave influenza virus HA with a monobasic cleavage site and support multicycle virus replication in vitro (Böttcher et al ., 2006 ). Later on, the TMPRSS2‐related protease TMPRSS4 was shown to activate HA with monobasic site in vitro , too (Chaipan et al ., 2009 ). By contrast, the trypsin‐like proteases prostasin, TMPRSS3, TMPRSS6 and hepsin were not able to activate HA upon co‐expression in mammalian cells (Bertram et al ., 2010 ; Böttcher‐Friebertshäuser et al ., 2010 ), emphasizing that only certain trypsin‐like proteases present in the airways support proteolytic activation of influenza viruses. TMPRSS2 and HAT belong to the family of type II transmembrane serine proteases (TTSPs) and contain an N‐terminal transmembrane domain, a highly variable stem region and a catalytic domain of the chymotrypsin S1 type (Fig. 3 a). The most prominent member of the TTSPs is the digestive enzyme enteropeptidase that originally was isolated as a soluble protease, which later was shown to be the catalytic domain of a membrane‐bound protease (Szabo & Bugge, 2008 ). TTSPs are synthesized as single‐chain zymogens that are activated by cleavage C‐terminal of a highly conserved arginine or lysine residue into a mature form (Fig. 3 a). The catalytic domain is predicted to remain linked to the membrane‐anchored domains by a disulphide bond or can be released as a soluble protease. Figure 3 Human proteases TMPRSS 2 and HAT cleave HA with monobasic cleavage site. (a) Schematic domain structures. HAT and TMPRSS 2 are synthesized as single‐chain zymogens that consist of an N‐terminal transmembrane domain ( TM ), a stem region containing, for example, sea urchin sperm protein enterokinase; agrin domain ( SEA ) for HAT or low‐density lipoprotein receptor class A domain ( LDLRA ); and scavenger receptor cysteine‐rich domain ( SRCR ) for TMPRSS 2; and a C‐terminal trypsin‐like serine (S1) protease domain with the catalytic triad histidine (H), aspartic acid (D) and serine (S). The zymogens undergo autocatalytic cleavage activation (indicated by arrows), and the catalytic domain remains linked to the transmembrane rest of the molecule by a disulphide bond. (b) Enzymatic activity of cell surface‐anchored or soluble HAT and TMPRSS 2 in MDCK ‐ HAT and MDCK ‐ TMPRSS 2 cells. Protease activity on the cell surface and in concentrated protease‐containing supernatants, respectively, was measured by incubation with the fluorogenic peptide substrate Boc‐Gly‐Pro‐Arg‐ AMC (Böttcher‐Friebertshäuser et al ., 2010 ). (c) Subcellular localization of HAT and TMPRSS 2. HAT and TMPRSS 2 on the cell surface (nonpermeabilized cells; left panels) of transient protease expressing MDCK cells and within the cell (permeabilized cells; right panels) were stained using protease‐specific antibodies and FITC ‐conjugated secondary antibodies. Cell nuclei were counterstained with DAPI . (d) Colocalization of TMPRSS 2 and furin. Huh‐7 cell with transient expression of TMPRSS 2 and furin were permeabilized, and protease expression was analysed by TMPRSS 2‐ and furin‐specific antibodies, respectively, and FITC ‐ or TRITC ‐conjugated secondary antibodies. Cell nuclei were counterstained using DAPI . The physiological roles of HAT and TMPRSS2 are still unknown. Interestingly, mice deficient in TMPRSS2 and HAT, respectively, have been shown to lack a discernible phenotype (Kim et al ., 2006 ; Sales et al ., 2011 ). HAT was originally isolated from patients with chronic airway diseases and among other functions has been demonstrated to cleave fibrinogen, to activate the protease‐activated receptor 2 (PAR‐2) and to increase mucus gene expression and to stimulate bronchial fibroblast proliferation in airway epithelial cells in vitro (Yoshinaga et al ., 1998 ; Chokki et al ., 2004 ; Matsushima et al ., 2006 ). HAT expression is prominent in the trachea and bronchi and was also detected in the gastrointestinal tract, the skin and the brain (Table 1 ). In the airway epithelium, HAT has been shown to be expressed in ciliated cells, but not in submucosal glands and mast cells (Takahashi et al ., 2001 ). TMPRSS2 is widely expressed in epithelial cells of the respiratory, gastrointestinal and urogenital tract with high expression levels in prostate and colon (Szabo & Bugge, 2008 ). Moreover, immunohistochemical studies revealed that TMPRSS2 is expressed in cardiac myocytes, suggesting that it might contribute to influenza‐associated myocarditis (Bertram et al ., 2012 ). TMPRSS2 is associated with prostate carcinogenesis in two different ways. The protease has been shown to be overexpressed in prostate cancer tissue. Moreover, fusion of the androgen‐regulated TMPRSS2 promoter to different ETS (E 26) transcription factor genes, resulting in overexpression of the respective transcription factors, is seen in nearly 50% of patients and has established as a prognostic marker of prostate carcinogenesis (Tomlins et al ., 2009 ). The role of TMPRSS2 in the airways remains to be investigated. Expression studies in Xenopus oocytes suggested that TMPRSS2 is involved in regulation of the airway surface liquid (ASL) volume by proteolytic cleavage of epithelial sodium channels (ENaCs) (Donaldson et al ., 2002 ). Table 1 HA‐activating host cell proteases, cleavage specificity and expression in human tissues Protease HA cleavage site sequence Expression in human tissues References TMPRSS2 R↓ Nasal epithelium, trachea, bronchi, lung (type II pneumocytes), larynx, tonsil, alveolar macrophages, myocardium, prostate , liver, kidney, small intestine, skin, testis, colon, pancreas Vaarala et al . ( 2001 ) Böttcher et al . ( 2006 ) Lucas et al . ( 2008 ) Szabo & Bugge ( 2008 ) Bertram et al . ( 2012 ) HAT R↓ Trachea , bronchi , oesophagus , tongue , nasal epithelium, larynx, epiglottis, tonsil, skin, brain Yasuoka et al . ( 1997 ) Böttcher et al . ( 2006 ) Szabo & Bugge ( 2008 ) Bertram et al . ( 2012 ) TMPRSS4 R↓ Oesophagus, lung, small intestine, stomach, colon, bladder, kidney, pancreas Szabo & Bugge ( 2008 ) Chaipan et al . ( 2009 ) Matriptase R/K‐X‐X/S‐R↓ Widespread expression in epithelial tissues: nasal epithelium, trachea, bronchi, salivary gland, oesophagus, kidney, small intestine, stomach, prostate, skin, hair follicle , monocytes, macrophages, mast cells, B cells, spinal neurons Oberst et al . ( 2003 ) Szabo & Bugge ( 2008 ) Baron et al . ( 2013 ) TMPRSS13/MSPL (splice isoforms) R/K‐K‐K‐R↓ Widespread tissue distribution: lung, brain, kidney, liver, spleen, prostate, pancreas, skin, small intestine, colon, testis, thymus, placenta, endothelial cells, T cells, monocytes Szabo & Bugge ( 2008 ) Okumura et al . ( 2010 ) Furin R‐X‐R/K‐R↓ Ubiquitous expression Stieneke‐Gröber et al . ( 1992 ) Thomas ( 2002 ) PC5 (also known as PC6) R‐X‐R/K‐R↓ Widespread tissue distribution Feldmann et al . ( 2000 ) Seidah et al . ( 2008 ) Cleavage of HA at the indicated amino acid sequence is shown by an arrow. Protease expression in different human tissues is listed; prominent expression levels are highlighted in bold. John Wiley & Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. TTSPs are predicted to be situated in the plasma membrane with an extracellular catalytic domain. In addition, soluble forms of HAT and TMPRSS2 have been described. HAT was originally purified as an active soluble protease from sputum of patients with chronic bronchitis and bronchial asthma (Yoshinaga et al ., 1998 ). TMPRSS2 was shown to be released from prostate and prostate cancer cells (Afar et al ., 2001 ). The subcellular localization and enzymatic activity of HAT and TMPRSS2 in airway epithelial cells have not been investigated in more detail so far however, have been studied in Madin‐Darby canine kidney (MDCK) cells with doxycycline‐induced expression of either protease that proved to be a suitable model system (Böttcher et al ., 2009 ). Initial studies on inhibition of influenza virus activation in MDCK‐HAT and MDCK‐TMPRSS2 cells using natural and synthetic protease inhibitors demonstrated that proteolytic activation of HA by HAT can be efficiently inhibited by exogenous protease inhibitors (Böttcher et al ., 2009 ; Böttcher‐Friebertshäuser et al ., 2010 ; Sielaff et al ., 2011 ). In contrast, inhibition of virus activation by TMPRSS2 required higher inhibitor concentrations. Interestingly, modification of a protease inhibitor by attachment of a fatty acid to improve cellular uptake allowed efficient inhibition of HA cleavage in TMPRSS2‐expressing MDCK cells. These data indicated that cleavage by TMPRSS2 takes place within the cell and inhibition requires cellular uptake of the inhibitor, whereas cleavage of HA by HAT occurs on the plasma membrane and therefore is easy accessible to exogenous inhibitors (Fig. 1 b). HAT and TMPRSS2 were shown to be expressed on the cell surface of MDCK cells both as zymogen and mature form (Böttcher‐Friebertshäuser et al ., 2010 ). Incubation of HAT‐ or TMPRSS2‐expressing cells with fluorogenic peptides to measure the protease activity on the cell surface revealed that HAT is present as an enzymatically active enzyme on the cell surface, while TMPRSS2 shows poor if any protease activity (Fig. 3 b). The reasons for the lack of TMPRSS2 activity on the cell surface is unknown but might be related to the presence of protease inhibitors, missing cofactors or lower amounts of TMPRSS2 on the cell surface compared with HAT. Immunofluorescence microscopy of transient TMPRSS2‐ or HAT‐expressing cells demonstrates that both HAT and TMPRSS2 are expressed on the cell surface; however, TMPRSS2 accumulates in the Golgi apparatus and TGN and colocalizes with furin, while HAT is expressed predominantly in the plasma membrane (Fig. 3 c and d). These data strongly suggest that TMPRSS2 cleaves HA in the TGN. Moreover, these observations demonstrate that cleavage of HA with monobasic and multibasic cleavage site can be performed in the same compartment but by different proteases (cf. Fig. 1 b). HA cleavage by HAT has been demonstrated to take place on the cell surface (Böttcher‐Friebertshäuser et al ., 2010 ). Interestingly, HAT on the one hand is capable of cleaving newly synthesized HA0, probably during assembly and budding of new virions on the plasma membrane. Thus, infectious virus containing cleaved HA is released from HAT‐expressing cells. Furthermore, HAT can cleave the HA0 of incoming virus at the stage of entry during attachment to the cell, facilitating infection of HAT‐expressing cells with virus containing noncleaved HA0 (Figs 1 and 5). Activation of HA at two different steps during virus replication seems to be redundant at the first few. But considering that different cell types in the airway epithelium express a partially different protease repertoire, activation of incoming virus by HAT may enable proteolytic activation of progeny viruses released from cells that lack expression of appropriate proteases (Fig. 5 ). This concept is supported by the observation that the WSN virus is activated upon entry into MDBK cells (Boycott et al ., 1994 ). It is still unknown whether autocatalytic activation of HAT occurs on the plasma membrane or already during its transit through the constitutive secretory pathway to the plasma membrane (Fig. 1 b). It therefore remains to be analysed whether HAT could cleave newly synthesized HA0 also within the cell in addition to HA cleavage on the plasma membrane. As mentioned above, soluble forms of HAT and TMPRSS2 have been described. Shedding of soluble HAT and TMPRSS2 into the cell supernatant has also been observed in MDCK‐HAT and MDCK‐TMPRSS2 cells (Böttcher‐Friebertshäuser et al ., 2010 ). But the soluble forms showed only marginal enzymatic activity (Fig. 3 b, right panel) and were not sufficient to support cleavage of influenza virus HA in these cells, indicating that HA cleavage in human airway epithelial cells occurs by cell‐associated and not by soluble proteases. Interestingly, enhanced shedding of TMPRSS2 and HAT from differentiated human nasal epithelial cells (NECs) and hence enhanced influenza virus replication were observed upon exposure to ozone (Kesic et al ., 2012 ). Thus, enhanced shedding of TMPRSS2 and HAT under stress conditions or in chronic airway diseases might play a role in influenza infections in the human airways. However, further studies are needed to prove this hypothesis. Recent studies demonstrated that HAT and TMPRSS2 support proteolytic activation of influenza B viruses, the SARS coronavirus and the human metapneumovirus at single arginines in vitro and therefore may play a role in activation and spread of different respiratory viruses in the human airways (Shirogane et al ., 2008 ; Matsuyama et al ., 2010 ; Shulla et al ., 2011 ; Böttcher‐Friebertshäuser et al ., 2012 ). Activation of HA at unusual cleavage site sequences Commonly, influenza virus HA is activated either at a single arginine by trypsin‐like proteases such as TMPRSS2 or HAT or at a multibasic motif by ubiquitous furin and PC5/6. However, a number of studies demonstrated that such a strict separation does not apply to all influenza isolates and that some influenza viruses can possess uncommon cleavage site motifs that may facilitate activation by other or additional proteases. One of the best studied exceptions is the influenza virus A/WSN/33 (H1N1), which is neurotropic in mice. The HA of A/WSN/33 contains the unusual HA cleavage site I‐Q‐Y‐R instead of I‐Q‐S‐R (cf. Fig. 2 c) with a tyrosine (Y) in P2 that was shown to facilitate efficient cleavage by plasmin, supporting proteolytic activation and virus spread to other tissues including the brain (Lazarowitz & Choppin, 1975 ; Goto & Kawaoka, 1998 ; Sun et al ., 2010 ). During a H5N2 avian influenza virus outbreak in Pennsylvania in 1983, loss of a carbohydrate side chain of HA1 due to a single point mutation was shown to be responsible for the appearance of high virulence in chickens. The influenza virus A/chick/Pennsylvania/1/83 (H5N2) possessed the multibasic cleavage site motif K‐K‐K‐R, but the low‐pathogenic isolates contained an oligosaccharide at Asn‐11 of HA1 that was demonstrated to interfere with cleavage of HA by steric hindrance (Kawaoka et al ., 1984 ). Interestingly, upon serial passage of a nonpathogenic H5N2 isolate in chicken embryo cells, pathogenic mutants emerged that had retained the carbohydrate side chain, but instead possessed an increased number of basic amino acids at the cleavage site (Ohuchi et al ., 1989 ). Thus, masking of the cleavage site by an oligosaccharide can be overcome by extension of the cleavage site. The multibasic motif K‐K‐K‐R, however, is not cleaved by furin because of the lysine (K) in P4. In a recent study, the type II transmembrane protease TMPRSS13/MSPL that is widely expressed in several tissues (Table 1 ) has been shown to cleave the HA at such a motif and to enable systemic infection independent of furin (Okumura et al ., 2010 ). Another example for an unusual HA cleavage has recently been described for influenza A viruses of the subtype H9N2 that circulate worldwide and have become highly prevalent in poultry in many countries. H9N2 viruses are associated with repeated outbreaks of severe illness in poultry farms in the Middle East and are occasionally transmitted to pigs and humans, raising concern about their pandemic potential. In contrast to most other subtypes, H9N2 viruses vary remarkably in the amino acid sequence at the HA cleavage site. While H9N2 viruses in America, Europe and Africa contain diverse monobasic HA cleavage site motifs, many recent H9N2 isolates from Asia and the Middle East possess di‐ or tribasic HA cleavage sites of the sequence R‐S‐S‐R or R‐S‐R‐R. Importantly, the di‐ or tribasic cleavage sites of H9N2 viruses evolved by substitution and not by insertion of basic amino acids as in case of HPAIV of subtypes H5 and H7 (Fig. 2 c) and are not cleaved by ubiquitous furin or PC5/6 (Gohrbandt et al ., 2011 ; Soda et al ., 2011 ). HAT and TMPRSS2 have been demonstrated to cleave H9 with mono‐, di‐ and tribasic cleavage site motifs (Baron et al ., 2013 ). Moreover, the TTSP matriptase was shown to cleave H9 with R‐S‐S‐R and R‐S‐R‐R cleavage sites (Baron et al ., 2013 ). Matriptase is expressed in a wide range of tissues with high expression levels in the kidney (Table 1 ). Interestingly, nephrotropism has been observed in H9N2 outbreaks in chickens. According to this, H9N2 viruses with R‐S‐S‐R and R‐S‐R‐R cleavage sites were able to undergo multicycle replication in primary chicken embryo kidney (CEK) cells due to proteolytic activation of HA, while H9N2 isolates containing a single R were not. HA activation in CEK cells was inhibited by matriptase inhibitors, suggesting that matriptase may contribute to the nephrotropism of H9N2 viruses in chickens (Baron et al ., 2013 ). However, it remains to be investigated whether expression of matriptase in a wide range of tissues affects organ tropism and pathogenicity of H9N2 viruses in vivo . Bacterial proteases can cleave HA during co‐infections Bacterial co‐infections contribute significantly to morbidity and mortality of influenza infections during seasonal epidemics and pandemic outbreaks (McCullers & Rehg, 2002 ; McCullers, 2006 ; Morens et al ., 2008 ; Metersky et al ., 2012 ). Bacterial infections can occur concomitant or secondary to an influenza infection. Influenza virus has been shown to increase the susceptibility to secondary bacterial infections by multiple factors: exposure of bacterial attachment sites and receptors due to virus‐induced tissue damage and removal of sialic acids by the viral NA; decreased mucociliary clearance of bacteria; and modulation of the local immune response. The synergism between influenza virus and bacteria has been reviewed in detail (McCullers, 2006 , 2011 ). In 1987, Tashiro et al . ( 1987a , b ) demonstrated that bacterial infection may also be for the benefit of the virus by producing proteases that facilitate cleavage of HA. Some strains of Staphylococcus aureus ( S. aureus) were shown to secrete proteases that are capable of cleaving the HA of certain influenza strains in vitro (Fig. 4 a). Interestingly, activation of HA was demonstrated to be specific for both bacteria and virus strains. Thus, for example, a protease secreted by S. aureus Wood 46 was able to cleave the HA of A/swine/1976/31 (H1N1), but not HA of A/chicken/Germany/N/49 (H10N7) (Fig. 4 a). By contrast, a protease produced by S. aureus M/86/86 was not capable of activating the HA of A/swine/1976/31 (H1N1), but cleaved the HA of several other influenza A virus isolates. Remarkably, intranasal co‐infection of mice with S. aureus Wood 46 and A/swine/1976/31 (H1N1) resulted in fatal disease with extended lesions in the lungs (Fig. 4 b and c left diagram), while infection with either the virus or S. aureus Wood 46 alone did not cause significant symptoms of disease and relevant pathological alterations in the lung (Fig. 4 b). Low virus titres were found in lungs of mice infected with A/Swine/1976/31 (H1N1) alone, whereas high titres were present in lung homogenates of mice co‐infected with S. aureus Wood 46 (Fig. 4 c, left diagram). Importantly, the infectivity of virus from lung homogenates of mice infected only with virus was markedly enhanced by trypsin treatment prior to plaque titration, indicating inefficient HA cleavage in the mice. In contrast, the infectivity of progeny virus from mice co‐infected with A/swine/1976/31 (H1N1) and S. aureus Wood 46 could not be increased by treatment with trypsin, suggesting that HA was already efficiently cleaved. Thus, co‐infection with influenza virus and S. aureus strains secreting appropriate HA‐cleaving proteases may promote severe pneumonia due to enhanced activation and spread of the virus. Accordingly, co‐infection of mice with influenza viruses and S. aureus strains producing proteases that failed to cleave the HA of the virus in vitro did not lead to significant symptoms of disease in the animals (Fig. 4 a and c). Low virus titres were present in lungs of mice co‐infected with virus and bacteria or infected with virus alone, and infectivity of the progeny virus was increased by trypsin treatment. In agreement with the studies by Tashiro et al ., a protease secreted by Aerococcus viridans was demonstrated to cleave the HA of several influenza A virus isolates, and co‐infection of mice resulted in severe pneumonia with a fatal outcome (Scheiblauer et al ., 1992 ). However, the HA‐activating proteases from S. aureus and A. viridans have not been identified yet, and mechanisms underlying strain‐specific differences in HA activation remain unknown. In addition, HA‐activating proteases from H. influenzae or S. pneumoniae strains have not been described so far. Attempts to isolate HA‐activating proteases from bacteria present in the respiratory tract of pigs or in the lower digestive tract of waterfowl failed (Callan et al ., 1997 ; King et al ., 2009 ). Figure 4 Activation of influenza viruses by proteases from bacteria. (a) Cleavage of HA of A/swine/1976/31 (H1N1) by proteases from S. aureus . Metabolically S 35 ‐labelled A/swine/1976/31 (H1N1) containing noncleaved HA 0 was treated with the indicated proteases or remained untreated (w/o protease). Proteins were separated by SDS ‐ PAGE and visualized by autoradiography (Tashiro et al ., 1987a ). (b) Pathological alterations in the lungs of mice. Mice were infected with either S. aureus Wood 46 or influenza virus A/swine/1976/31 (H1N1) or co‐infected with both pathogens. Noninfected mice were used as a control. Lungs were taken at 5 days p.i. (Tashiro et al ., 1987a ). Mice indicate survival or fatal infection of the animals. (c) Virus titres in mice lungs. Virus titres in lung homogenates of mice infected with certain influenza viruses and S. aureus strains are shown as plaque‐forming units (p.f.u.) per lung. Lung homogenates were either treated with trypsin or remained untreated before plaque titration. Infections with virus alone without (▵) and with (▲) trypsin treatment; co‐infections without (○) and with (●) trypsin treatment (Tashiro et al ., 1987a , 1987b , 1987c ). Mice indicate survival and fatal infection, respectively. Interestingly, indirect mechanisms of influenza virus activation upon co‐infection with bacteria have been proposed to contribute to secondary bacterial pneumonia. Scheiblauer et al . ( 1992 ) observed that Pseudomonas aeruginosa , a frequent complication in patients with cystic fibrosis, secretes a protease that is not capable of cleaving HA, but simultaneous administration of the protease and influenza virus to mice resulted in severe infections with high virus titres in the lungs and a mortality rate of 50%. Analysis of bronchoalveolar lavage (BAL) fluid of the mice revealed an increased trypsin‐like protease activity compared with BAL of mice infected in the absence of P. aeruginosa protease. The increase in protease activity was assumed to be a result of inflammatory‐induced release of trypsin‐like (host) proteases, but remains to be analysed in more detail. Moreover, generation of plasmin from its inactive zymogen plasminogen by plasminogen activators from bacteria such as staphylokinase and streptokinase, from S. aureus and various streptococci, respectively, has been suggested to facilitate enhanced HA activation in co‐infections with influenza virus (Scheiblauer et al ., 1992 ; Tse et al ., 2013 ). Infection of mice with influenza strains that can be activated by plasmin and simultaneously treatment with plasminogen and either staphylokinase or streptokinase developed severe pneumonia with high virus titres in the lung (Scheiblauer et al ., 1992 ). The contribution of certain bacteria to proteolytic activation of influenza viruses in the human airways and the development of pneumonia still remain to be demonstrated. However, bacterial proteases might provide novel drug targets for the treatment of viral‐bacterial co‐infections, and further effort in their identification and characterization might support the development of appropriate inhibitors. Outlook: HA‐activating proteases provide potential drug targets Currently available measures for the control of influenza in humans are vaccination and antiviral medications, which target the viral NA or the M2 protein. But treatment options become more limited due to the development of drug‐resistant viruses for both M2 and NA inhibitors. Furthermore, the production of influenza vaccines against a newly emerged virus so far requires 4–6 months, highlighting the urgent need for novel drug targets. HA‐activating host cell proteases provide potential drug targets due to their crucial role for virus infectivity. Early studies using the broad‐range serine protease inhibitor aprotinin from bovine lungs demonstrated that influenza virus replication in embryonated chicken eggs and mice can be markedly suppressed by inhibiting HA cleavage (Zhirnov et al ., 1982 , 2011 ). Aprotinin aerosol treatment was shown to protect mice from an otherwise lethal influenza infection and significantly reduced virus titres and lesions in the lung. Moreover, in a clinical trial, inhalations of aerosolized aprotinin in patients with seasonal influenza and parainfluenza markedly reduced the duration of symptoms without causing side effects (Zhirnov et al ., 2011 ). Inhibition of host factors such as HA‐activating proteases is a novel approach for influenza treatment, and besides a lack of knowledge about relevant proteases until recently, concerns about possible side effects or toxicity have limited the development of drugs that target them. The identification of HAT and TMPRSS2 as HA‐activating proteases from human airways, however, provided potential drug targets, and recent progress in the development of protease inhibitors is considerable. Interestingly, mice deficient in TMPRSS2 and HAT activity, respectively, have been shown to lack a discernible phenotype, indicating functional redundancy or compensation of physiological functions by other host proteases (Kim et al ., 2006 ; Sales et al ., 2011 ). Knockdown of TMPRSS2 expression using an antisense peptide‐conjugated morpholino oligomer (PPMO) strongly suppressed influenza virus replication in human airway epithelial cells without affecting cell viability (Böttcher‐Friebertshäuser et al ., 2011 ). Substrate analogue peptide mimetic inhibitors of HAT containing a 4‐amidinobenzylamide moiety as the P1 residue have been demonstrated to efficiently suppress influenza virus replication in HAT‐expressing cells (Böttcher et al ., 2009 ; Böttcher‐Friebertshäuser et al ., 2010 ; Sielaff et al ., 2011 ). Moreover, a peptide mimetic inhibitor of TMPRSS2 was shown to drastically reduce virus titres and to delay influenza virus propagation by 24–48 h in airway epithelial cells in vitro (Böttcher‐Friebertshäuser et al ., 2012 ). Remarkably, the combination of the protease inhibitor and the current NA inhibitor oseltamivir carboxylate was highly synergistic and efficiently blocked influenza virus propagation in human airway epithelial cells. The further development of abovementioned protease inhibitors may lead to novel drugs that should be considered for influenza treatment – as monotherapy or in combination with NA inhibitors. It should be mentioned that specific inhibitors of furin have recently been developed and were demonstrated to efficiently suppress virus replication of HPAIV of subtype H7 in cell culture. Inhibition of HA cleavage therefore provides a promising concept for treatment of HPAIV infections in humans, most likely in combination with NA inhibitors, for example oseltamivir (Garten et al ., 1989 ; Becker et al ., 2010 ; Y. Lu, W. Garten, T. Steinmetzer, in preparation). The therapy of viral‐bacterial pneumonia and secondary bacterial pneumonia following influenza has been considered by both antimicrobial drugs and antivirals such as NA inhibitors and has been reviewed in detail (McCullers, 2011 ; Metersky et al ., 2012 ). Co‐infection studies in animal models suggest that the best option to prevent secondary bacterial pneumonia is the prevention of influenza infection by influenza vaccination (Huber et al ., 2010 ; McCullers, 2011 ). Moreover, vaccination against both influenza virus and superinfecting bacteria is considered reasonable, but is limited by the availability of vaccines against certain superinfecting bacteria or different strains. Interestingly, Tashiro et al . ( 1987c ) demonstrated that HA‐activating proteases secreted from different S. aureus strains are inhibited by the natural broad‐range protease inhibitor leupeptin. Treatment of mice co‐infected with A/swine/1979/31 (H1N1) and S. aureus Wood 46 with leupeptin led to reduced virus titres in the lungs and survival of the animals, whereas nontreated mice developed fatal pneumonia (cf. Fig. 4 ). In summary, HA‐activating proteases have been shown to represent potential drug targets for influenza treatment. The combination of appropriate protease inhibitors with current antivirals and/or antimicrobial drugs provides a novel and promising approach that should be considered for the treatment of both influenza and viral‐bacterial pneumonia. Conclusion Proteolytic activation of influenza virus HA is essential for virus infectivity and spread. The identification of relevant proteases in the human airways has demonstrated that HA cleavage occurs by membrane‐bound proteases and can take place in different cellular compartments: either in the TGN or on the cell surface. In addition, HA activation can occur at different time points during the viral life cycle: during transport of HA to the plasma membrane, during budding and release of progeny virus or at a very late time point upon attachment and entry into a new cell. Remarkably, bacteria such as S. aureus and S. pneumoniae have been demonstrated to secrete proteases that cleave HA directly or to produce proteins that augment and activate relevant host cell proteases and thereby may facilitate HA activation upon co‐infection. Thus, influenza viruses can be activated by different proteases and mechanisms in the human airway epithelium (Fig. 5 ). At the same time, cleavage of HA is a potent drug target for the treatment of influenza infections due to its crucial role for virus infectivity. Further identification and characterization of relevant proteases provide the basis for the development of specific protease inhibitors as novel influenza drugs. Figure 5 Activation of influenza viruses by host cellular and bacterial proteases in the human airway epithelium. Ciliated and nonciliated epithelial cells are shown. HA ‐activating proteases are shown by scissors: HAT (blue), TMPRSS 2 (red); continuous arrows indicate infection of cells, and dashed arrows, release of virus progeny. (a) Infection of HAT ‐ and/or TMPRSS 2‐expressing cells results in release of infectious progeny virus containing cleaved HA (green). (b) Infection of cell without expression of relevant HA ‐activating proteases results in progeny virus containing noncleaved HA (black). (c) Virus containing noncleaved HA is not able to infect cells expressing only TMPRSS 2 or cells without expression of any relevant protease. (d) HAT ‐expressing cells support proteolytic activation of HA of incoming virus on the cell surface, facilitating infection of the cell and release of infectious progeny virus. (e) In co‐infections of influenza virus and certain bacteria, soluble bacterial proteases (purple) may support proteolytic activation and spread of the virus. (f) Bacterial proteases or proteins may activate or augment cellular proteases (orange), which in turn cleave HA . Conflict of interest The authors declare that they have no conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728851/

Management of children after renal transplantation: highlights for general pediatricians

The number of children undergoing successful renal transplantations has been increasing steadily and as a result; general pediatricians are now more likely to encounter children with a kidney allograft in their practice. Although the medical care immediately after transplantation is mostly provided by transplant teams, more and more outpatient care will eventually be performed at the patient's local community. Medical care from general pediatricians is particularly important, especially for children who are residing far from transplant centers. As these children require prolong immunosuppressive therapies and are susceptible to various specific clinical problems, it is imperative for their primary care providers and pediatricians to be knowledgeable about their specific needs and be competent in providing care. This article highlights the roles and common practice related issues that pertain to general pediatricians in the care of pediatric renal allograft recipients.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9298270/

Precision‐cut lung slices: A powerful ex vivo model to investigate respiratory infectious diseases

Abstract Respiratory infections are a leading cause of mortality worldwide. Most of the research on the underlying disease mechanisms is based on cell culture, organoid, or surrogate animal models. Although these provide important insights, they have limitations. Cell culture models fail to recapitulate cellular interactions in the lung and animal models often do not permit high‐throughput analysis of drugs or pathogen isolates; hence, there is a need for improved, scalable models. Precision‐cut lung slices (PCLS), small, uniform tissue slices generated from animal or human lungs are increasingly recognized and employed as an ex vivo organotypic model. PCLS retain remarkable cellular complexity and the architecture of the lung, providing a platform to investigate respiratory pathogens in a near‐native environment. Here, we review the generation and features of PCLS, their use to investigate the pathogenesis of viral and bacterial pathogens, and highlight their potential to advance respiratory infection research in the future. 1 INTRODUCTION Lower respiratory tract infections such as pneumonia are among the 10 most common causes of death worldwide and are a significant burden for healthcare systems (Gibson et al., 2013 ; World Health Organization, 2020 ). The swift onset of the SARS‐CoV‐2 pandemic further highlights that new respiratory pathogens can emerge rapidly. Hence, there is an urgent need to accelerate fundamental research into respiratory infection biology and to develop technologies that allow for its translation into optimized diagnosis and treatment strategies. To date, fundamental respiratory research has mostly relied on cell culture and surrogate animal models to investigate the mechanisms underlying host‐pathogen interactions and different pathologies. Two‐dimensional (2D) cell culture has been the most employed model as it is cheap and easy‐to‐implement, but it has limitations. It predominantly relies on immortalized cell lines that often display significantly altered metabolism and gene expression profiles compared to primary cells (Sanderson, 2011 ). The use of primary cells can circumvent some of these drawbacks, as these retain more morphological and physiological characteristics of the source tissue. However, primary cells can only be retrieved in low numbers, expanded for a finite time, display limited differentiation capacity and, as the other 2D models, still lack the cellular heterogeneity, intercellular interactions, and complex structural organization of tissues. More complex three‐dimensional (3D) cell culture models address some of these limitations. For example, primary airway epithelial cells cultured at air liquid interface (ALI) develop into a differentiated, polarized airway‐like epithelium (Cao et al., 2020 ; Zscheppang et al., 2018 ). Stem/progenitor cells embedded in a matrix can self‐organize into complex lung tissue‐like organoids (Hiemstra et al., 2019 ; Nikolić & Rawlins, 2017 ). These models recapitulate many aspects of the airway epithelium, as the heterogeneous epithelial cell composition including mucus‐producing goblet and beating ciliated cells, and some degree of 3D organization. Other cells, for example macrophages, can be added to these 3D models, allowing the investigation of cellular crosstalk and immune mechanisms (Hiemstra et al., 2019 ). Integration with microfluidic "organ‐on‐a‐chip" systems, which continuously supply nutrients and remove waste, further enhances their potential to mimic in vivo conditions (Gkatzis et al., 2018 ; Hiemstra et al., 2019 ). However, current models are still unable to recapitulate the true cellular richness and spatial complexity of the lung. Surrogate animal models allow experimentation under physiological conditions in a living organism and provide invaluable knowledge about the mechanisms underlying respiratory infectious diseases. Mice are the most frequently used model, due to the large range of genetically modified strains and tools that exist to study them. Guinea pigs reflect human airway pathology better than mice (Ressmeyer et al., 2006 ) and were the first model used to study tuberculosis and diphtheria. Although there are less tools available to study these, guinea pigs remain important surrogate models for Legionella pneumophila, Brucella spp., and several viruses (Padilla‐Carlin et al., 2008 ). Pigs have been increasingly employed since porcine airway anatomy, physiology and more than 80% of immune parameters closely resemble that of humans (Meurens et al., 2012 ). In contrast, mice only share 10% of immune parameters. Still, despite best efforts, significant genetic, anatomical, and physiological differences often hinder the direct translation of animal research to humans (Bonniaud et al., 2018 ; Williams & Roman, 2016 ). To bridge this gap, ex vivo human lung tissue explants (HLTEs), which are usually obtained from resected tissue samples from patients undergoing surgery for example, for lung cancer, have been used for modeling infection with L. pneumophila (Jäger et al., 2014 ), Chlamydophila pneumoniae (Rupp et al., 2004 ), Streptococcus pneumoniae (Xu et al., 2008 ), and Haemophilus influenzae (Drömann et al., 2010 ). As usually only small areas of tissue are resected at one time, the number of HLTEs generated is small, limiting the number of experimental conditions that can be tested. Moreover, while tumor tissue itself is avoided, HLTEs are typically derived from a diseased lung environment. Low availability of human tissue, the high cost of infrastructure and maintenance of laboratory animals, and substantial efforts made in recent years to implement the 3Rs principles, Refinement, Reduction, and Replacement for animal experimentation (European Parliament and Council directive 2010/63/EU, 2010 ; Hubrecht & Carter, 2019 ) led to the development and increased use of precision‐cut tissue slices (PCTS) as a 3D organotypic ex vivo tissue model. PCTS are clearly defined tissue sections of uniform thickness generated from a single piece of tissue. They can be generated in large numbers from diverse organs including the lung (precision‐cut lung slices—PCLS), from different animals and also from live or cadaveric human tissue and organs donated for research. This enables testing of a wide range of variables in replicate, reduces the amount of source tissue or animals needed and is therefore also significantly more cost‐effective when compared to in vivo work. Here, we will describe the generation and characteristics of PCLS, review their use for studying human respiratory pathogens so far and discuss their potential for advancing respiratory infectious disease research and antimicrobial drug discovery. 2 GENERATION OF PCLS Tissue slices have been used in research since the 1920's. However, it was the development of precise vibrating microtomes that enabled PCTS in 1980, increasing the homogeneity and reproducibility of the generated slices (Krumdieck et al., 1980 ). It took seven more years until the introduction of an agar infusion method provided enough structural support to the soft and fragile honeycomb architecture of lung tissue, making it amenable for slicing (Placke & Fisher, 1987 ). This method was further developed and first applied to human lung tissue in 1994 (Fisher et al., 1994 ). Various adaptations have been made since then, and detailed step‐by‐step protocols for generating PCLS from human and mouse tissue are published (Gerckens et al., 2019 ; Wu et al., 2019 ). Central to all protocols is that the freshly retrieved whole lungs or individual lobes are infused with warm low‐melting agarose dissolved in buffer or tissue culture medium, at a concentration ranging from 0.5% to 3% (Figure 1 ). The agarose can be administered through the trachea, for example for small animal lungs, or through smaller airways of dissected lobes. It is delivered at a constant slow rate and the total volume adjusted to the natural tissue volume to prevent tissue damage. Once the tissue is inflated, rapid solidification of the agarose is achieved by immersing the tissue in cold buffer, followed by incubation at 4℃. Upon solidification, the tissue is sliced using a vibrating microtome, yielding PCLS of uniform thickness between 150 and 500 µm. Thinner slices are typically obtained from smaller animals and thicker slices from larger animals and human tissue (Alsafadi et al., 2020 ). Variations of this procedure have been recently reviewed (Alsafadi et al., 2020 ). The PCLS are kept immersed in cell culture medium in well plates for downstream applications. Serum is either absent or added in very low quantities in order to prevent cell growth and changes to the native cellular diversity (Sanderson, 2011 ). The number of slices which can be generated is limited by the size of the organ, but a small human lung lobe can yield hundreds of PCLS (hPCLS). Uniform punches fitting 96 well plate wells can be generated from PCLS using biopsy punchers, facilitating high‐throughput applications. Reports on PCLS viability vary as this is likely affected by diverse factors (e.g., method for animal sacrifice, time, and conditions of conservation from tissue retrieval to slicing) (Sanderson, 2011 ) but hPCLS have been reported to remain viable for at least 15 days (Neuhaus et al., 2017 ). FIGURE 1 Generation of precision‐cut lung slices (PCLS) for infection studies. Scheme describing the key steps for generating PCLS from human and smaller animals lung tissue 3 PCLS: A NEAR NATIVE LUNG ENVIRONMENT TO STUDY AIRWAY BIOLOGY The process of PCLS generation preserves most features of the native lung tissue, that is, its architecture and mechanical properties (Hiorns et al., 2016 ; Pybus et al., 2021 ) as well as a complex cellular composition, including resident lung cells, for example, fibroblasts, ATI and ATII epithelial cells, and immune cells, for example, macrophages, monocytes, and NK and T cells (Stegmayr et al., 2021 ). Neutrophils have been detected in murine PCLS (mPCLS) (Akram et al., 2019 ; Molina‐Torres et al., 2020 ) but considering that they are short‐lived ex vivo and that the PCLS are disconnected from blood and lymphatic circulation, their number is highly dependent on the way PCLS are generated and for how long slices are kept in culture. The absence of immune cell recruitment can be exploited to define the response of resident cells to infection and treatment. PCLS are ideally suited for high‐resolution live imaging, and ultrastructural changes, cell activity and migration, progression of infection, and drug treatments can be followed in real time using fluorescent markers and probes for different cell types, cell signaling or other processes, and/or fluorescently labeled pathogens (Figure 2 ). In parallel, cytokines, metabolites, or other molecules can be sampled from the PCLS culture medium. Like any other tissue, PCLS are also suitable for classical endpoint analysis by histology, immunohistochemistry, and immunofluorescence or transmission electron microscopy (Ebsen et al., 2002 ), metabolomics (Khan et al., 2020 ; Yilmaz et al., 2018 ), proteomics (Khan et al., 2020 ), and RNA isolation for transcriptome profiling (Niehof et al., 2017 ; Stegmayr et al., 2021 ). FIGURE 2 Comparison between human (a) and mouse (b) precision‐cut lung slices (PCLS) structure (Blue: DAPI staining of nuclei). (c) Section of an hPCLS infected with Legionella pneumophila (Red: expressing mScarlet‐I) 24 hr post infection (White: CellMask staining of cytoplasmic membranes). * mark alveolar spaces in (a) and (c). (a, c) were acquired by FV and (b) was acquired by FV and John Stegmayr Molecular mechanisms in PCLS can be probed with small molecule inhibitors, with the advantage that the amount of drug needed for well plate experiments is significantly reduced compared to in vivo studies, decreasing cost and facilitating assessment of drugs with limited availability. Moreover, genetic manipulation of PCLS by siRNA‐mediated protein knockdown has been achieved (Ruigrok et al., 2017 , 2018 ). Notably, PCLS can also be obtained from healthy and diseased tissue from both human donors or animal models, as demonstrated for idiopathic pulmonary fibrosis (Alsafadi et al., 2017 ), chronic obstructive pulmonary disease (COPD), and asthma (Mertens et al., 2017 ), enabling the assessment of the interplay between these conditions and infections. Overall, these characteristics show that PCLS combine advantages of both in vitro and in vivo models and constitute an excellent ex vivo system to study lung biology, disease, and treatments. PCLS have therefore been widely adopted for pre‐clinical drug discovery and toxicological studies (Bäckström et al., 2016 ; Fisher et al., 1994 ; Hess et al., 2016 ; Lauenstein et al., 2014 ; Maihöfer et al., 2017 ; Neuhaus et al., 2017 , 2018 ; Switalla, et al., 2010a ) including the evaluation of immune modulators (Henjakovic et al., 2008 ; Leus et al., 2016 ; Switalla, et al., 2010b ; Temann et al., 2017 ) and the induction of antigen‐specific T cell responses by new vaccine formulations (Neuhaus et al., 2013 ). Additionally, PCLS were also used to characterize tissue tropism and functional properties of gene therapy vectors and oncolytic viruses (Rosales Gerpe et al., 2018 ). 4 PCLS AS MODEL FOR HUMAN RESPIRATORY INFECTIOUS DISEASES The first report of the use of PCLS to study the molecular mechanisms of infection focused on two respiratory pathogens, the Gram‐negative bacterium C. pneumoniae and paramyxovirus Respiratory Syncytial Virus (RSV) (Ebsen et al., 2002 ). C. pneumoniae is associated with wide range of diseases, for example, pulmonary emphysema, and RSV is the most common cause of viral infections in the lower respiratory tract of infants and children. Immunofluorescence and transmission electron microscopy revealed that both pathogens established infections and replicated in the mPCLS, evidenced by the characteristic chlamydial inclusions with spherical bodies of C. pneumoniae and RSV‐containing vacuoles in infected cells. Since this pioneering study, PCLS from different species have been used to investigate a wide range of human viral and bacterial pathogens. Here, we review these studies with emphasis on the different applications and readouts. 4.1 Viral pathogenesis in PCLS 4.1.1 Paramyxoviruses Paramyxoviruses are RNA viruses transmitted via inhalation of airborne droplets and able to cause diverse diseases in humans and animals. Apart from RSV, prominent members of this group are measles virus, the causative agent of measles, and metapneumovirus, an important cause of respiratory disease in particular in children (Park & Tishkowski, 2021 ; Troy & Bosco, 2016 ). PCLS from dogs, cynomolgus macaques, ferrets, and cotton rats, were assessed for their ability to support infection and discern differences in cell tropism and replication kinetics between the three human paramyxoviruses and canine distemper virus, the cause of distemper in carnivores (de Vries et al., 2018 ; Nguyen et al., 2013 ). Measles virus replicated optimally in macaque slices, metapneumovirus and RSV in cotton rat slices, and the canine distemper virus in slices from dogs and ferrets. This work not only established effective PCLS models for these viruses but, as PCLS were infected with recombinant virus expressing fluorescent proteins, also highlighted the potential of PCLS for real‐time monitoring of infection. 4.1.2 Enteroviruses Rhinoviruses (RVs) are the cause of the common cold, the most frequent human viral infection, and severe exacerbations of asthma and COPD (Troy & Bosco, 2016 ). Quantification of the RV load by quantitative PCR in hPCLS from asthmatic and healthy donors showed no differences between the groups at infection peak. However, RV triggered higher expression of cytokines, for example, IL25, TSLP , and IL13 , in the tissue from asthmatics, indicating that RV‐induced exacerbations may be linked to an altered immune response (Kennedy et al., 2018 ). As hPCLS retain ciliary beating and are responsive to treatments with contractile stimuli such as the drug carbachol, the effect of RV infection on airway constriction was also analyzed, showing an enhanced contraction in infected asthmatic donor tissue (Parikh et al., 2020 ). Increased carbachol‐induced airway narrowing was also observed in mPCLS infected with rhinovirus C15 (RV‐C15). Both studies support a model in which altered immune responses in asthmatic airways following RV infection drive airway hyperresponsiveness. PCLS derived from a mouse model of allergic asthma showed a compromised immune response to RV, that is, increased release of IL‐4, IL‐6, and IL‐10, which could be partially inhibited by the antiviral drug Rupintrivir (Danov et al., 2019 ). Bronchobini, a homeopathic multicomponent drug preparation marketed for inflammatory respiratory diseases, primed the antiviral host response in mPCLS suppressing excessive RV‐induced inflammation (Reamon‐Buettner et al., 2019 ). These studies illustrate the value of PCLS to study the effect of RV infection on airway immunology and contractility, and to discover new antiviral treatments. 4.1.3 Adenoviruses Adenoviruses cause diverse pathologies. Type 7 Adenovirus (Ad7) is associated with severe lower tract infection and pneumonia in both healthy and immunocompromised individuals, especially among children or adults living in crowded conditions (Clementi et al., 2021 ). Infection leads to infiltration of neutrophils in the lower respiratory tract and alveoli, followed by monocytes and later lymphocytes. Bovine and human PCLS support Ad7 replication and viral protein was detected in alveolar epithelial cells by immunohistochemistry (Booth et al., 2004 ). As in 2D models, PCLS infection triggered production and release of interleukin‐8 (IL‐8), the main chemoattractant for neutrophils, via the RAS/RAF‐I/MEKI/ERK pathway. A subsequent study in hPCLS, revealed that IL‐8 is mainly produced by type I alveolar epithelial cells, whereas IP‐10, a chemokine attracting monocytes and lymphocytes, is produced by epithelial cells and macrophages and seems to require communication of both cell types (Wu et al., 2010 ). These studies evidence the potential of PCLS to dissect cell tropism of pathogens and intercellular communication in the host response. 4.1.4 Influenza viruses Influenza A virus is a major cause of global morbidity and mortality. Annual flu epidemics claim tens of thousands of lives and intermittent pandemics have an even higher death toll (Troy & Bosco, 2016 ; World Health Organization (WHO), 2018 ). Rapid evolution of the virus creates the need for effective model systems for the characterization of new isolates and the development of treatments. Liu et al. optimized mPCLS for quantifying the replication of human influenza A strains PR8 (H1N1) and HUBEI (H3N2) by viral titer measurement and a fluorometric neuraminidase (NA) activity assay (Liu et al., 2015 ). Moreover, mPCLS and bronchoalveolar lavage fluid (BALF) from infected mice displayed a similar increase of cytokines and chemokines, such as IP‐10, RANTES and MIP‐3α, and a similar response to a number of antiviral and anti‐inflammatory agents (ribavirin, oseltamivir, germacrone, U0126, EGCG, 15d‐PGJ2, and SB203580). Overall, this validated mPCLS as a predictive, time and cost‐economic model for studying pathogenesis, and treatment of human influenza A virus. The mPCLS model also enables the assessment of complex interactions of infection, drug treatments, and environmental factors, such as smoking, a main risk factor for COPD. Influenza A virus is a known driver of exacerbations of COPD and asthma. PCLS from smoke‐exposed mice revealed that smoke had no effect on clearance of H1N1 virus, but was associated with higher expression of inflammatory chemokines such as MCP‐1/‐3, KC, MIP‐2, and GCP‐2 (Bauer et al., 2010 ). The same model showed that smoke alone and in combination with Influenza A virus infection (Mem71, H3N1) impaired β‐adrenoceptor sensitivity and responsiveness of small airways to the β2‐adrenoceptor agonist bronchodilator salbutamol (SALB) (Donovan et al., 2016 ). PCLS from marmosets and rhesus and cynomolgus macaques were also validated as Influenza A (Hamburg/04/2009 (H1N1) or A/PR/8/34 (H1N1)) infection models and confirmed that the virus exploits in all models the activity of the protease TMPRSS2, which cleaves and activates the viral hemagglutinin (HA) promoting replication and spread (Zmora et al., 2017 ). While not always reproducing all human disease features, animal PCLS also offer an effective system to assess the virulence of different isolates and their potential for interspecies transmission. The most severe Influenza A pandemics are often driven by virus subtypes, which recently breached the species boundary from animals to humans. A good correlation of the virulence of swine influenza viruses in vivo and replicative capacity and ciliostatic effect in porcine PCLS (pPCLS) was observed, showing that PCLS models deliver meaningful results for the comparison of the pathogenicity of different isolates (Meng et al., 2013 ). In pPCLS, swine influenza A virus (H3N2) replicated better than two avian influenza viruses (H9N2 and H7N7), but H9N2 replicated robustly in lung epithelial cells, indicating potential for interspecies transmission (Punyadarsaniya et al., 2011 ). Adaptive evolution experiments in pPCLS showed that after three passages avian influenza virus H9N2 evolved its HA protein to bind α2,6‐linked sialic acids, typical surface receptors for swine and human viruses, in addition to the α2,3‐linked sialic acids commonly used by avian subtypes (Yang et al., 2017 ). These mutants displayed enhanced replication in mice but not in pPCLS, suggesting that other adaptive mutations might still be required for virulence in pigs. These studies demonstrate the usefulness of PCLS to model zoonotic infections and assess the risk for emergence of new human pathogens. 4.1.5 Coronaviruses The SARS‐CoV‐2 pandemic generated the urgent need for relevant preclinical drug discovery platforms. hPCLS were employed for the characterization of the antiviral effect of Camostat mesylate, an inhibitor of TMPRSS2 and related proteases, which are required for proteolytic activation of the viral S protein and cell invasion (Hoffmann et al., 2021 ), and in a drug repurposing screen for inhibitors of SARS‐CoV‐2 replication (Zimniak et al., 2021 ). SARS‐CoV‐2 replicated in hPCLS as measured by quantitative RT‐PCR and viral titer. Camostat mesylate and Fluoxetine, a selective serotonin reuptake inhibitor used for treatment of depression, reduced viral burden. Notably, Lopinavir, which repressed viral replication in in vitro 2D infection models, failed to reduce the viral titers in hPCLS. This shows the value of this more complex, ex vivo tissue‐based infection model for the effective discovery of antimicrobials and for studying the pathogenesis of SARS‐CoV‐2 in the future. 4.2 Bacterial pathogenesis in PCLS Since the first ever use of PCLS as a model for respiratory infection with C. pneumoniae (Ebsen et al., 2002 ), an increasing number of studies have exploited PCLS to investigate how bacterial pathogens cause disease in humans. Additional studies on zoonotic pathogens, for example, Bordetella bronchiseptica , that focus on aspects of pathogenesis in the natural animal hosts have been discussed elsewhere (Vötsch et al., 2021 ). 4.2.1 Staphylococcus aureus Staphylococcus aureus is a multifaceted Gram‐positive pathogen of high concern due to increasing multidrug resistance. Methicillin‐resistant S. aureus (MRSA) and Methicillin‐sensitive strains (MSSA) cause community‐pneumonia in healthy individuals, but MSSA are associated with a lower mortality rate. As murine macrophages were found to be growth permissive, it was speculated that macrophages modulate pathogenesis in humans. However, both S. aureus types survive, but do not replicate in isolated human alveolar macrophages, and in hPCLS the bacteria are mostly found in S. aureus ‐containing phagosomes in epithelial cells and interstitial regions (Brann et al., 2019 ). Only limited numbers are detected in alveolar macrophages. Thus, human alveolar macrophages are most likely not a key permissive replicative niche, pointing to other cell types as drivers of S. aureus lung infection. This study highlights the potential of PCLS to investigate cell tropism and key cellular interactions of bacterial pathogens. 4.2.2 Coxiella burnetii Coxiella burnetii is an obligate intracellular pathogen that causes Q‐fever in humans, which can progress from flu‐like symptoms into a life‐threatening endocarditis (Dragan & Voth, 2020 ). In cell culture models, the bacteria replicate inside acidic parasitophorous vacuoles (PVs) in human alveolar macrophages and also non‐phagocytic cells. hPCLS infection established that while individual bacteria could be detected in different cell types, replication occurred in PVs in alveolar macrophages (Dragan et al., 2019 ; Graham et al., 2016 ). Replication in PCLS depended on the Dot/Icm type 4 secretion system, which is essential to deliver effector proteins for manipulation of host processes and biogenesis of the PV. No substantial structural damage to the PCLS tissue was observed even after 72 hr of infection. Infection induced the production and caspase‐1‐dependent release of mature IL‐1β from PCLS. Notably, these experiments were performed with the attenuated, laboratory strain C. burnetii NMII (RSA439, avirulent clone 4) (Millar et al., 2017 ), which induces a markedly different inflammatory response than wild type bacteria. Nevertheless, this study highlighted how hPCLS represent a promising model to investigate the C. burnetii pathogenesis in a physiological context. 4.2.3 Bacillus anthracis Bacillus anthracis is a spore‐forming Gram‐positive pathogen. Pulmonary infection with B. anthracis spores causes inhalation anthrax, the most severe and deadly form of the disease. Early innate immune responses to B. anthracis spores were investigated in hPCLS, showing Mitogen‐Activated Protein Kinase (MAPK)‐driven proinflammatory cytokine and chemokine production by macrophages and lung epithelia, including IL‐8, MCP‐1 and MIP‐1α/β, potent chemoattractants for neutrophils, and monocytes (Chakrabarty et al., 2007 ). This was the first evidence of an active involvement of the lung epithelium in the innate immune response to B. anthracis spores and contrasts with the lack of cytokine production observed in a complex 3D cell model of primary small airway epithelial cells cultured in collagen matrices in presence of peripheral blood monocytes (Radyuk et al., 2003 ). While hPCLS likely represent a more accurate lung model and thus show more realistic early innate immune responses, this awaits validation by autopsy data. 4.2.4 Mycobacteria Mycobacterial infections are a global health problem (Gopalaswamy et al., 2020 ). While Mycobacterium tuberculosis (MTb) remains the leading cause of death by a single infectious agent, several species of non‐tuberculous mycobacteria (NTM) are emerging as important cause of opportunistic infections. Treatment requires multidrug therapy over several months and is associated with high failure rates. This is due to increasing multi‐drug resistance of MTb and high intrinsic antibiotic resistance of NTMs. Moreover, phenotypic heterogeneity of the bacteria in the host, for example, formation of nonreplicating persisters resistant to antibiotics, promotes infection relapses. As in vitro drug screening assays do not reflect these conditions well, PCLS have been evaluated as models for anti‐mycobacterial drug discovery. Mycobacterium abscessus (MAb) is a fast growing NTM and imminent multidrug‐resistant health threat for patients with respiratory conditions such as COPD and cystic fibrosis (CF) (Molina‐Torres et al., 2020 ). Upon infection of mPCLS, progressive tissue damage occurred, for example, alveolar edema, vascular congestion, and extravasion of lymphocytes and erythrocytes in the septa and alveolar spaces with rupture and thickening of alveolar septa by 48 hr post infection, concomitantly with infiltration of histiocytes, aggregates of foamy macrophages, and fragmentation of polymorphonuclear cells (Molina‐Torres et al., 2020 ). MAb were found in macrophages and type I and II pneumocytes. Proof‐of principle antibiotic treatment assays with imipenem and tigecycline demonstrated that tigecycline lead to a significant reduction of bacterial burden in a dose‐ and time‐dependent manner while imipenem induced only a moderate reduction. MTb is a slowly growing bacterium causing mostly chronic disease. hPCLS were employed to study infection and the immune response within the first 24 hr of MTb infection in comparison with Mycobacterium bovis BCG (Carranza‐Rosales et al., 2017 ). Bacteria were found associated with alveolar septa, alveolar light spaces, near type II pneumocytes, and resided in macrophages. Immune cells were recruited to sites where bacteria accumulated, showing that PCLS retain some of the cellular immune responses of the native tissue. No significant change of bacterial load was observed over time and infection triggered the production of TNF‐α, mirroring findings in other in vitro and in vivo studies. Overall, these two studies show that PCLS can be a useful tool for fundamental mycobacterial research and drug discovery. 4.2.5 Pseudomonas aeruginosa Pseudomonas aeruginosa is a versatile opportunistic pathogen causing acute and chronic respiratory infections, particularly in immunocompromised, COPD, and CF patients. The bacteria use different virulence factors including a flagellum and type 2 and type 3 secretion systems to secrete diverse toxins and enzymes (Jurado‐Martín et al., 2021 ). However, how both these virulence factors and pathogen‐associated molecular patterns such as LPS, drive the immune response in the human lung is not fully understood. Using mPCLS exposed to different live and heat‐killed clinical isolates, Kolbe et al. showed that infection with live but not dead bacteria results in substantial transcriptional reprogramming involving increased cytokine expression (Kolbe et al., 2020 ). Comparison of bacterial mutants in combination with pharmacological inhibition of phagocytosis, showed that this response to viable bacteria required their internalization and depended on the detection of flagellin and the T3SS. Moreover, employing PCLS from knock‐out (KO) mice, the redundant involvement of the receptors MARCO and CD200R1 in bacterial uptake was also demonstrated. This illustrates that PCLS enable parallel assessment of multiple bacterial strains in the near‐native lung tissue from the same animal. Additionally, in combination with inhibitors and KO animals, PCLS provide further insight in the molecular mechanisms of host‐pathogen interactions. 4.2.6 Yersinia pestis Yersinia pestis is the causative agent of plague. Inhalation of Y. pestis leads to pneumonic plague, which can be fatal within days if untreated. Virulence factors, for example, a type 3 secretion system that injects effectors for host manipulation and the protease Plasminogen activator (Pla), are essential for Y. pestis infection and replication, as well as for dissemination and disease progression in cellular and animal models (Demeure et al., 2019 ). The exact function of Pla however remained an enigma. An hPCLS model of pneumonic plague combined with the fluorescence‐based TEM1‐β‐lactamase effector translocation assay revealed that Pla promotes optimal translocation of effectors into phagocytes, especially alveolar macrophages (Banerjee et al., 2019 ). In contrast, Pla was dispensable for type 3 secretion into THP‐1 cells, an immortalized cell line considered as macrophage surrogate model. Moreover, in the PCLS infection model, Pla plays a vital role for the inhibition of the expression of the proinflammatory cytokines IL‐6, IL‐8, and TNFα during the first hours of infection. During this phase, it is not possible to measure cytokine levels in mouse lungs as these are below detection level. This reinforces the notion that the functions of virulence factors might only become apparent under physiological infection conditions and that PCLS are a unique system to investigate these functions and early events of infection, which are usually poorly accessible. 4.1 Viral pathogenesis in PCLS 4.1.1 Paramyxoviruses Paramyxoviruses are RNA viruses transmitted via inhalation of airborne droplets and able to cause diverse diseases in humans and animals. Apart from RSV, prominent members of this group are measles virus, the causative agent of measles, and metapneumovirus, an important cause of respiratory disease in particular in children (Park & Tishkowski, 2021 ; Troy & Bosco, 2016 ). PCLS from dogs, cynomolgus macaques, ferrets, and cotton rats, were assessed for their ability to support infection and discern differences in cell tropism and replication kinetics between the three human paramyxoviruses and canine distemper virus, the cause of distemper in carnivores (de Vries et al., 2018 ; Nguyen et al., 2013 ). Measles virus replicated optimally in macaque slices, metapneumovirus and RSV in cotton rat slices, and the canine distemper virus in slices from dogs and ferrets. This work not only established effective PCLS models for these viruses but, as PCLS were infected with recombinant virus expressing fluorescent proteins, also highlighted the potential of PCLS for real‐time monitoring of infection. 4.1.2 Enteroviruses Rhinoviruses (RVs) are the cause of the common cold, the most frequent human viral infection, and severe exacerbations of asthma and COPD (Troy & Bosco, 2016 ). Quantification of the RV load by quantitative PCR in hPCLS from asthmatic and healthy donors showed no differences between the groups at infection peak. However, RV triggered higher expression of cytokines, for example, IL25, TSLP , and IL13 , in the tissue from asthmatics, indicating that RV‐induced exacerbations may be linked to an altered immune response (Kennedy et al., 2018 ). As hPCLS retain ciliary beating and are responsive to treatments with contractile stimuli such as the drug carbachol, the effect of RV infection on airway constriction was also analyzed, showing an enhanced contraction in infected asthmatic donor tissue (Parikh et al., 2020 ). Increased carbachol‐induced airway narrowing was also observed in mPCLS infected with rhinovirus C15 (RV‐C15). Both studies support a model in which altered immune responses in asthmatic airways following RV infection drive airway hyperresponsiveness. PCLS derived from a mouse model of allergic asthma showed a compromised immune response to RV, that is, increased release of IL‐4, IL‐6, and IL‐10, which could be partially inhibited by the antiviral drug Rupintrivir (Danov et al., 2019 ). Bronchobini, a homeopathic multicomponent drug preparation marketed for inflammatory respiratory diseases, primed the antiviral host response in mPCLS suppressing excessive RV‐induced inflammation (Reamon‐Buettner et al., 2019 ). These studies illustrate the value of PCLS to study the effect of RV infection on airway immunology and contractility, and to discover new antiviral treatments. 4.1.3 Adenoviruses Adenoviruses cause diverse pathologies. Type 7 Adenovirus (Ad7) is associated with severe lower tract infection and pneumonia in both healthy and immunocompromised individuals, especially among children or adults living in crowded conditions (Clementi et al., 2021 ). Infection leads to infiltration of neutrophils in the lower respiratory tract and alveoli, followed by monocytes and later lymphocytes. Bovine and human PCLS support Ad7 replication and viral protein was detected in alveolar epithelial cells by immunohistochemistry (Booth et al., 2004 ). As in 2D models, PCLS infection triggered production and release of interleukin‐8 (IL‐8), the main chemoattractant for neutrophils, via the RAS/RAF‐I/MEKI/ERK pathway. A subsequent study in hPCLS, revealed that IL‐8 is mainly produced by type I alveolar epithelial cells, whereas IP‐10, a chemokine attracting monocytes and lymphocytes, is produced by epithelial cells and macrophages and seems to require communication of both cell types (Wu et al., 2010 ). These studies evidence the potential of PCLS to dissect cell tropism of pathogens and intercellular communication in the host response. 4.1.4 Influenza viruses Influenza A virus is a major cause of global morbidity and mortality. Annual flu epidemics claim tens of thousands of lives and intermittent pandemics have an even higher death toll (Troy & Bosco, 2016 ; World Health Organization (WHO), 2018 ). Rapid evolution of the virus creates the need for effective model systems for the characterization of new isolates and the development of treatments. Liu et al. optimized mPCLS for quantifying the replication of human influenza A strains PR8 (H1N1) and HUBEI (H3N2) by viral titer measurement and a fluorometric neuraminidase (NA) activity assay (Liu et al., 2015 ). Moreover, mPCLS and bronchoalveolar lavage fluid (BALF) from infected mice displayed a similar increase of cytokines and chemokines, such as IP‐10, RANTES and MIP‐3α, and a similar response to a number of antiviral and anti‐inflammatory agents (ribavirin, oseltamivir, germacrone, U0126, EGCG, 15d‐PGJ2, and SB203580). Overall, this validated mPCLS as a predictive, time and cost‐economic model for studying pathogenesis, and treatment of human influenza A virus. The mPCLS model also enables the assessment of complex interactions of infection, drug treatments, and environmental factors, such as smoking, a main risk factor for COPD. Influenza A virus is a known driver of exacerbations of COPD and asthma. PCLS from smoke‐exposed mice revealed that smoke had no effect on clearance of H1N1 virus, but was associated with higher expression of inflammatory chemokines such as MCP‐1/‐3, KC, MIP‐2, and GCP‐2 (Bauer et al., 2010 ). The same model showed that smoke alone and in combination with Influenza A virus infection (Mem71, H3N1) impaired β‐adrenoceptor sensitivity and responsiveness of small airways to the β2‐adrenoceptor agonist bronchodilator salbutamol (SALB) (Donovan et al., 2016 ). PCLS from marmosets and rhesus and cynomolgus macaques were also validated as Influenza A (Hamburg/04/2009 (H1N1) or A/PR/8/34 (H1N1)) infection models and confirmed that the virus exploits in all models the activity of the protease TMPRSS2, which cleaves and activates the viral hemagglutinin (HA) promoting replication and spread (Zmora et al., 2017 ). While not always reproducing all human disease features, animal PCLS also offer an effective system to assess the virulence of different isolates and their potential for interspecies transmission. The most severe Influenza A pandemics are often driven by virus subtypes, which recently breached the species boundary from animals to humans. A good correlation of the virulence of swine influenza viruses in vivo and replicative capacity and ciliostatic effect in porcine PCLS (pPCLS) was observed, showing that PCLS models deliver meaningful results for the comparison of the pathogenicity of different isolates (Meng et al., 2013 ). In pPCLS, swine influenza A virus (H3N2) replicated better than two avian influenza viruses (H9N2 and H7N7), but H9N2 replicated robustly in lung epithelial cells, indicating potential for interspecies transmission (Punyadarsaniya et al., 2011 ). Adaptive evolution experiments in pPCLS showed that after three passages avian influenza virus H9N2 evolved its HA protein to bind α2,6‐linked sialic acids, typical surface receptors for swine and human viruses, in addition to the α2,3‐linked sialic acids commonly used by avian subtypes (Yang et al., 2017 ). These mutants displayed enhanced replication in mice but not in pPCLS, suggesting that other adaptive mutations might still be required for virulence in pigs. These studies demonstrate the usefulness of PCLS to model zoonotic infections and assess the risk for emergence of new human pathogens. 4.1.5 Coronaviruses The SARS‐CoV‐2 pandemic generated the urgent need for relevant preclinical drug discovery platforms. hPCLS were employed for the characterization of the antiviral effect of Camostat mesylate, an inhibitor of TMPRSS2 and related proteases, which are required for proteolytic activation of the viral S protein and cell invasion (Hoffmann et al., 2021 ), and in a drug repurposing screen for inhibitors of SARS‐CoV‐2 replication (Zimniak et al., 2021 ). SARS‐CoV‐2 replicated in hPCLS as measured by quantitative RT‐PCR and viral titer. Camostat mesylate and Fluoxetine, a selective serotonin reuptake inhibitor used for treatment of depression, reduced viral burden. Notably, Lopinavir, which repressed viral replication in in vitro 2D infection models, failed to reduce the viral titers in hPCLS. This shows the value of this more complex, ex vivo tissue‐based infection model for the effective discovery of antimicrobials and for studying the pathogenesis of SARS‐CoV‐2 in the future. 4.1.1 Paramyxoviruses Paramyxoviruses are RNA viruses transmitted via inhalation of airborne droplets and able to cause diverse diseases in humans and animals. Apart from RSV, prominent members of this group are measles virus, the causative agent of measles, and metapneumovirus, an important cause of respiratory disease in particular in children (Park & Tishkowski, 2021 ; Troy & Bosco, 2016 ). PCLS from dogs, cynomolgus macaques, ferrets, and cotton rats, were assessed for their ability to support infection and discern differences in cell tropism and replication kinetics between the three human paramyxoviruses and canine distemper virus, the cause of distemper in carnivores (de Vries et al., 2018 ; Nguyen et al., 2013 ). Measles virus replicated optimally in macaque slices, metapneumovirus and RSV in cotton rat slices, and the canine distemper virus in slices from dogs and ferrets. This work not only established effective PCLS models for these viruses but, as PCLS were infected with recombinant virus expressing fluorescent proteins, also highlighted the potential of PCLS for real‐time monitoring of infection. 4.1.2 Enteroviruses Rhinoviruses (RVs) are the cause of the common cold, the most frequent human viral infection, and severe exacerbations of asthma and COPD (Troy & Bosco, 2016 ). Quantification of the RV load by quantitative PCR in hPCLS from asthmatic and healthy donors showed no differences between the groups at infection peak. However, RV triggered higher expression of cytokines, for example, IL25, TSLP , and IL13 , in the tissue from asthmatics, indicating that RV‐induced exacerbations may be linked to an altered immune response (Kennedy et al., 2018 ). As hPCLS retain ciliary beating and are responsive to treatments with contractile stimuli such as the drug carbachol, the effect of RV infection on airway constriction was also analyzed, showing an enhanced contraction in infected asthmatic donor tissue (Parikh et al., 2020 ). Increased carbachol‐induced airway narrowing was also observed in mPCLS infected with rhinovirus C15 (RV‐C15). Both studies support a model in which altered immune responses in asthmatic airways following RV infection drive airway hyperresponsiveness. PCLS derived from a mouse model of allergic asthma showed a compromised immune response to RV, that is, increased release of IL‐4, IL‐6, and IL‐10, which could be partially inhibited by the antiviral drug Rupintrivir (Danov et al., 2019 ). Bronchobini, a homeopathic multicomponent drug preparation marketed for inflammatory respiratory diseases, primed the antiviral host response in mPCLS suppressing excessive RV‐induced inflammation (Reamon‐Buettner et al., 2019 ). These studies illustrate the value of PCLS to study the effect of RV infection on airway immunology and contractility, and to discover new antiviral treatments. 4.1.3 Adenoviruses Adenoviruses cause diverse pathologies. Type 7 Adenovirus (Ad7) is associated with severe lower tract infection and pneumonia in both healthy and immunocompromised individuals, especially among children or adults living in crowded conditions (Clementi et al., 2021 ). Infection leads to infiltration of neutrophils in the lower respiratory tract and alveoli, followed by monocytes and later lymphocytes. Bovine and human PCLS support Ad7 replication and viral protein was detected in alveolar epithelial cells by immunohistochemistry (Booth et al., 2004 ). As in 2D models, PCLS infection triggered production and release of interleukin‐8 (IL‐8), the main chemoattractant for neutrophils, via the RAS/RAF‐I/MEKI/ERK pathway. A subsequent study in hPCLS, revealed that IL‐8 is mainly produced by type I alveolar epithelial cells, whereas IP‐10, a chemokine attracting monocytes and lymphocytes, is produced by epithelial cells and macrophages and seems to require communication of both cell types (Wu et al., 2010 ). These studies evidence the potential of PCLS to dissect cell tropism of pathogens and intercellular communication in the host response. 4.1.4 Influenza viruses Influenza A virus is a major cause of global morbidity and mortality. Annual flu epidemics claim tens of thousands of lives and intermittent pandemics have an even higher death toll (Troy & Bosco, 2016 ; World Health Organization (WHO), 2018 ). Rapid evolution of the virus creates the need for effective model systems for the characterization of new isolates and the development of treatments. Liu et al. optimized mPCLS for quantifying the replication of human influenza A strains PR8 (H1N1) and HUBEI (H3N2) by viral titer measurement and a fluorometric neuraminidase (NA) activity assay (Liu et al., 2015 ). Moreover, mPCLS and bronchoalveolar lavage fluid (BALF) from infected mice displayed a similar increase of cytokines and chemokines, such as IP‐10, RANTES and MIP‐3α, and a similar response to a number of antiviral and anti‐inflammatory agents (ribavirin, oseltamivir, germacrone, U0126, EGCG, 15d‐PGJ2, and SB203580). Overall, this validated mPCLS as a predictive, time and cost‐economic model for studying pathogenesis, and treatment of human influenza A virus. The mPCLS model also enables the assessment of complex interactions of infection, drug treatments, and environmental factors, such as smoking, a main risk factor for COPD. Influenza A virus is a known driver of exacerbations of COPD and asthma. PCLS from smoke‐exposed mice revealed that smoke had no effect on clearance of H1N1 virus, but was associated with higher expression of inflammatory chemokines such as MCP‐1/‐3, KC, MIP‐2, and GCP‐2 (Bauer et al., 2010 ). The same model showed that smoke alone and in combination with Influenza A virus infection (Mem71, H3N1) impaired β‐adrenoceptor sensitivity and responsiveness of small airways to the β2‐adrenoceptor agonist bronchodilator salbutamol (SALB) (Donovan et al., 2016 ). PCLS from marmosets and rhesus and cynomolgus macaques were also validated as Influenza A (Hamburg/04/2009 (H1N1) or A/PR/8/34 (H1N1)) infection models and confirmed that the virus exploits in all models the activity of the protease TMPRSS2, which cleaves and activates the viral hemagglutinin (HA) promoting replication and spread (Zmora et al., 2017 ). While not always reproducing all human disease features, animal PCLS also offer an effective system to assess the virulence of different isolates and their potential for interspecies transmission. The most severe Influenza A pandemics are often driven by virus subtypes, which recently breached the species boundary from animals to humans. A good correlation of the virulence of swine influenza viruses in vivo and replicative capacity and ciliostatic effect in porcine PCLS (pPCLS) was observed, showing that PCLS models deliver meaningful results for the comparison of the pathogenicity of different isolates (Meng et al., 2013 ). In pPCLS, swine influenza A virus (H3N2) replicated better than two avian influenza viruses (H9N2 and H7N7), but H9N2 replicated robustly in lung epithelial cells, indicating potential for interspecies transmission (Punyadarsaniya et al., 2011 ). Adaptive evolution experiments in pPCLS showed that after three passages avian influenza virus H9N2 evolved its HA protein to bind α2,6‐linked sialic acids, typical surface receptors for swine and human viruses, in addition to the α2,3‐linked sialic acids commonly used by avian subtypes (Yang et al., 2017 ). These mutants displayed enhanced replication in mice but not in pPCLS, suggesting that other adaptive mutations might still be required for virulence in pigs. These studies demonstrate the usefulness of PCLS to model zoonotic infections and assess the risk for emergence of new human pathogens. 4.1.5 Coronaviruses The SARS‐CoV‐2 pandemic generated the urgent need for relevant preclinical drug discovery platforms. hPCLS were employed for the characterization of the antiviral effect of Camostat mesylate, an inhibitor of TMPRSS2 and related proteases, which are required for proteolytic activation of the viral S protein and cell invasion (Hoffmann et al., 2021 ), and in a drug repurposing screen for inhibitors of SARS‐CoV‐2 replication (Zimniak et al., 2021 ). SARS‐CoV‐2 replicated in hPCLS as measured by quantitative RT‐PCR and viral titer. Camostat mesylate and Fluoxetine, a selective serotonin reuptake inhibitor used for treatment of depression, reduced viral burden. Notably, Lopinavir, which repressed viral replication in in vitro 2D infection models, failed to reduce the viral titers in hPCLS. This shows the value of this more complex, ex vivo tissue‐based infection model for the effective discovery of antimicrobials and for studying the pathogenesis of SARS‐CoV‐2 in the future. 4.2 Bacterial pathogenesis in PCLS Since the first ever use of PCLS as a model for respiratory infection with C. pneumoniae (Ebsen et al., 2002 ), an increasing number of studies have exploited PCLS to investigate how bacterial pathogens cause disease in humans. Additional studies on zoonotic pathogens, for example, Bordetella bronchiseptica , that focus on aspects of pathogenesis in the natural animal hosts have been discussed elsewhere (Vötsch et al., 2021 ). 4.2.1 Staphylococcus aureus Staphylococcus aureus is a multifaceted Gram‐positive pathogen of high concern due to increasing multidrug resistance. Methicillin‐resistant S. aureus (MRSA) and Methicillin‐sensitive strains (MSSA) cause community‐pneumonia in healthy individuals, but MSSA are associated with a lower mortality rate. As murine macrophages were found to be growth permissive, it was speculated that macrophages modulate pathogenesis in humans. However, both S. aureus types survive, but do not replicate in isolated human alveolar macrophages, and in hPCLS the bacteria are mostly found in S. aureus ‐containing phagosomes in epithelial cells and interstitial regions (Brann et al., 2019 ). Only limited numbers are detected in alveolar macrophages. Thus, human alveolar macrophages are most likely not a key permissive replicative niche, pointing to other cell types as drivers of S. aureus lung infection. This study highlights the potential of PCLS to investigate cell tropism and key cellular interactions of bacterial pathogens. 4.2.2 Coxiella burnetii Coxiella burnetii is an obligate intracellular pathogen that causes Q‐fever in humans, which can progress from flu‐like symptoms into a life‐threatening endocarditis (Dragan & Voth, 2020 ). In cell culture models, the bacteria replicate inside acidic parasitophorous vacuoles (PVs) in human alveolar macrophages and also non‐phagocytic cells. hPCLS infection established that while individual bacteria could be detected in different cell types, replication occurred in PVs in alveolar macrophages (Dragan et al., 2019 ; Graham et al., 2016 ). Replication in PCLS depended on the Dot/Icm type 4 secretion system, which is essential to deliver effector proteins for manipulation of host processes and biogenesis of the PV. No substantial structural damage to the PCLS tissue was observed even after 72 hr of infection. Infection induced the production and caspase‐1‐dependent release of mature IL‐1β from PCLS. Notably, these experiments were performed with the attenuated, laboratory strain C. burnetii NMII (RSA439, avirulent clone 4) (Millar et al., 2017 ), which induces a markedly different inflammatory response than wild type bacteria. Nevertheless, this study highlighted how hPCLS represent a promising model to investigate the C. burnetii pathogenesis in a physiological context. 4.2.3 Bacillus anthracis Bacillus anthracis is a spore‐forming Gram‐positive pathogen. Pulmonary infection with B. anthracis spores causes inhalation anthrax, the most severe and deadly form of the disease. Early innate immune responses to B. anthracis spores were investigated in hPCLS, showing Mitogen‐Activated Protein Kinase (MAPK)‐driven proinflammatory cytokine and chemokine production by macrophages and lung epithelia, including IL‐8, MCP‐1 and MIP‐1α/β, potent chemoattractants for neutrophils, and monocytes (Chakrabarty et al., 2007 ). This was the first evidence of an active involvement of the lung epithelium in the innate immune response to B. anthracis spores and contrasts with the lack of cytokine production observed in a complex 3D cell model of primary small airway epithelial cells cultured in collagen matrices in presence of peripheral blood monocytes (Radyuk et al., 2003 ). While hPCLS likely represent a more accurate lung model and thus show more realistic early innate immune responses, this awaits validation by autopsy data. 4.2.4 Mycobacteria Mycobacterial infections are a global health problem (Gopalaswamy et al., 2020 ). While Mycobacterium tuberculosis (MTb) remains the leading cause of death by a single infectious agent, several species of non‐tuberculous mycobacteria (NTM) are emerging as important cause of opportunistic infections. Treatment requires multidrug therapy over several months and is associated with high failure rates. This is due to increasing multi‐drug resistance of MTb and high intrinsic antibiotic resistance of NTMs. Moreover, phenotypic heterogeneity of the bacteria in the host, for example, formation of nonreplicating persisters resistant to antibiotics, promotes infection relapses. As in vitro drug screening assays do not reflect these conditions well, PCLS have been evaluated as models for anti‐mycobacterial drug discovery. Mycobacterium abscessus (MAb) is a fast growing NTM and imminent multidrug‐resistant health threat for patients with respiratory conditions such as COPD and cystic fibrosis (CF) (Molina‐Torres et al., 2020 ). Upon infection of mPCLS, progressive tissue damage occurred, for example, alveolar edema, vascular congestion, and extravasion of lymphocytes and erythrocytes in the septa and alveolar spaces with rupture and thickening of alveolar septa by 48 hr post infection, concomitantly with infiltration of histiocytes, aggregates of foamy macrophages, and fragmentation of polymorphonuclear cells (Molina‐Torres et al., 2020 ). MAb were found in macrophages and type I and II pneumocytes. Proof‐of principle antibiotic treatment assays with imipenem and tigecycline demonstrated that tigecycline lead to a significant reduction of bacterial burden in a dose‐ and time‐dependent manner while imipenem induced only a moderate reduction. MTb is a slowly growing bacterium causing mostly chronic disease. hPCLS were employed to study infection and the immune response within the first 24 hr of MTb infection in comparison with Mycobacterium bovis BCG (Carranza‐Rosales et al., 2017 ). Bacteria were found associated with alveolar septa, alveolar light spaces, near type II pneumocytes, and resided in macrophages. Immune cells were recruited to sites where bacteria accumulated, showing that PCLS retain some of the cellular immune responses of the native tissue. No significant change of bacterial load was observed over time and infection triggered the production of TNF‐α, mirroring findings in other in vitro and in vivo studies. Overall, these two studies show that PCLS can be a useful tool for fundamental mycobacterial research and drug discovery. 4.2.5 Pseudomonas aeruginosa Pseudomonas aeruginosa is a versatile opportunistic pathogen causing acute and chronic respiratory infections, particularly in immunocompromised, COPD, and CF patients. The bacteria use different virulence factors including a flagellum and type 2 and type 3 secretion systems to secrete diverse toxins and enzymes (Jurado‐Martín et al., 2021 ). However, how both these virulence factors and pathogen‐associated molecular patterns such as LPS, drive the immune response in the human lung is not fully understood. Using mPCLS exposed to different live and heat‐killed clinical isolates, Kolbe et al. showed that infection with live but not dead bacteria results in substantial transcriptional reprogramming involving increased cytokine expression (Kolbe et al., 2020 ). Comparison of bacterial mutants in combination with pharmacological inhibition of phagocytosis, showed that this response to viable bacteria required their internalization and depended on the detection of flagellin and the T3SS. Moreover, employing PCLS from knock‐out (KO) mice, the redundant involvement of the receptors MARCO and CD200R1 in bacterial uptake was also demonstrated. This illustrates that PCLS enable parallel assessment of multiple bacterial strains in the near‐native lung tissue from the same animal. Additionally, in combination with inhibitors and KO animals, PCLS provide further insight in the molecular mechanisms of host‐pathogen interactions. 4.2.6 Yersinia pestis Yersinia pestis is the causative agent of plague. Inhalation of Y. pestis leads to pneumonic plague, which can be fatal within days if untreated. Virulence factors, for example, a type 3 secretion system that injects effectors for host manipulation and the protease Plasminogen activator (Pla), are essential for Y. pestis infection and replication, as well as for dissemination and disease progression in cellular and animal models (Demeure et al., 2019 ). The exact function of Pla however remained an enigma. An hPCLS model of pneumonic plague combined with the fluorescence‐based TEM1‐β‐lactamase effector translocation assay revealed that Pla promotes optimal translocation of effectors into phagocytes, especially alveolar macrophages (Banerjee et al., 2019 ). In contrast, Pla was dispensable for type 3 secretion into THP‐1 cells, an immortalized cell line considered as macrophage surrogate model. Moreover, in the PCLS infection model, Pla plays a vital role for the inhibition of the expression of the proinflammatory cytokines IL‐6, IL‐8, and TNFα during the first hours of infection. During this phase, it is not possible to measure cytokine levels in mouse lungs as these are below detection level. This reinforces the notion that the functions of virulence factors might only become apparent under physiological infection conditions and that PCLS are a unique system to investigate these functions and early events of infection, which are usually poorly accessible. 4.2.1 Staphylococcus aureus Staphylococcus aureus is a multifaceted Gram‐positive pathogen of high concern due to increasing multidrug resistance. Methicillin‐resistant S. aureus (MRSA) and Methicillin‐sensitive strains (MSSA) cause community‐pneumonia in healthy individuals, but MSSA are associated with a lower mortality rate. As murine macrophages were found to be growth permissive, it was speculated that macrophages modulate pathogenesis in humans. However, both S. aureus types survive, but do not replicate in isolated human alveolar macrophages, and in hPCLS the bacteria are mostly found in S. aureus ‐containing phagosomes in epithelial cells and interstitial regions (Brann et al., 2019 ). Only limited numbers are detected in alveolar macrophages. Thus, human alveolar macrophages are most likely not a key permissive replicative niche, pointing to other cell types as drivers of S. aureus lung infection. This study highlights the potential of PCLS to investigate cell tropism and key cellular interactions of bacterial pathogens. 4.2.2 Coxiella burnetii Coxiella burnetii is an obligate intracellular pathogen that causes Q‐fever in humans, which can progress from flu‐like symptoms into a life‐threatening endocarditis (Dragan & Voth, 2020 ). In cell culture models, the bacteria replicate inside acidic parasitophorous vacuoles (PVs) in human alveolar macrophages and also non‐phagocytic cells. hPCLS infection established that while individual bacteria could be detected in different cell types, replication occurred in PVs in alveolar macrophages (Dragan et al., 2019 ; Graham et al., 2016 ). Replication in PCLS depended on the Dot/Icm type 4 secretion system, which is essential to deliver effector proteins for manipulation of host processes and biogenesis of the PV. No substantial structural damage to the PCLS tissue was observed even after 72 hr of infection. Infection induced the production and caspase‐1‐dependent release of mature IL‐1β from PCLS. Notably, these experiments were performed with the attenuated, laboratory strain C. burnetii NMII (RSA439, avirulent clone 4) (Millar et al., 2017 ), which induces a markedly different inflammatory response than wild type bacteria. Nevertheless, this study highlighted how hPCLS represent a promising model to investigate the C. burnetii pathogenesis in a physiological context. 4.2.3 Bacillus anthracis Bacillus anthracis is a spore‐forming Gram‐positive pathogen. Pulmonary infection with B. anthracis spores causes inhalation anthrax, the most severe and deadly form of the disease. Early innate immune responses to B. anthracis spores were investigated in hPCLS, showing Mitogen‐Activated Protein Kinase (MAPK)‐driven proinflammatory cytokine and chemokine production by macrophages and lung epithelia, including IL‐8, MCP‐1 and MIP‐1α/β, potent chemoattractants for neutrophils, and monocytes (Chakrabarty et al., 2007 ). This was the first evidence of an active involvement of the lung epithelium in the innate immune response to B. anthracis spores and contrasts with the lack of cytokine production observed in a complex 3D cell model of primary small airway epithelial cells cultured in collagen matrices in presence of peripheral blood monocytes (Radyuk et al., 2003 ). While hPCLS likely represent a more accurate lung model and thus show more realistic early innate immune responses, this awaits validation by autopsy data. 4.2.4 Mycobacteria Mycobacterial infections are a global health problem (Gopalaswamy et al., 2020 ). While Mycobacterium tuberculosis (MTb) remains the leading cause of death by a single infectious agent, several species of non‐tuberculous mycobacteria (NTM) are emerging as important cause of opportunistic infections. Treatment requires multidrug therapy over several months and is associated with high failure rates. This is due to increasing multi‐drug resistance of MTb and high intrinsic antibiotic resistance of NTMs. Moreover, phenotypic heterogeneity of the bacteria in the host, for example, formation of nonreplicating persisters resistant to antibiotics, promotes infection relapses. As in vitro drug screening assays do not reflect these conditions well, PCLS have been evaluated as models for anti‐mycobacterial drug discovery. Mycobacterium abscessus (MAb) is a fast growing NTM and imminent multidrug‐resistant health threat for patients with respiratory conditions such as COPD and cystic fibrosis (CF) (Molina‐Torres et al., 2020 ). Upon infection of mPCLS, progressive tissue damage occurred, for example, alveolar edema, vascular congestion, and extravasion of lymphocytes and erythrocytes in the septa and alveolar spaces with rupture and thickening of alveolar septa by 48 hr post infection, concomitantly with infiltration of histiocytes, aggregates of foamy macrophages, and fragmentation of polymorphonuclear cells (Molina‐Torres et al., 2020 ). MAb were found in macrophages and type I and II pneumocytes. Proof‐of principle antibiotic treatment assays with imipenem and tigecycline demonstrated that tigecycline lead to a significant reduction of bacterial burden in a dose‐ and time‐dependent manner while imipenem induced only a moderate reduction. MTb is a slowly growing bacterium causing mostly chronic disease. hPCLS were employed to study infection and the immune response within the first 24 hr of MTb infection in comparison with Mycobacterium bovis BCG (Carranza‐Rosales et al., 2017 ). Bacteria were found associated with alveolar septa, alveolar light spaces, near type II pneumocytes, and resided in macrophages. Immune cells were recruited to sites where bacteria accumulated, showing that PCLS retain some of the cellular immune responses of the native tissue. No significant change of bacterial load was observed over time and infection triggered the production of TNF‐α, mirroring findings in other in vitro and in vivo studies. Overall, these two studies show that PCLS can be a useful tool for fundamental mycobacterial research and drug discovery. 4.2.5 Pseudomonas aeruginosa Pseudomonas aeruginosa is a versatile opportunistic pathogen causing acute and chronic respiratory infections, particularly in immunocompromised, COPD, and CF patients. The bacteria use different virulence factors including a flagellum and type 2 and type 3 secretion systems to secrete diverse toxins and enzymes (Jurado‐Martín et al., 2021 ). However, how both these virulence factors and pathogen‐associated molecular patterns such as LPS, drive the immune response in the human lung is not fully understood. Using mPCLS exposed to different live and heat‐killed clinical isolates, Kolbe et al. showed that infection with live but not dead bacteria results in substantial transcriptional reprogramming involving increased cytokine expression (Kolbe et al., 2020 ). Comparison of bacterial mutants in combination with pharmacological inhibition of phagocytosis, showed that this response to viable bacteria required their internalization and depended on the detection of flagellin and the T3SS. Moreover, employing PCLS from knock‐out (KO) mice, the redundant involvement of the receptors MARCO and CD200R1 in bacterial uptake was also demonstrated. This illustrates that PCLS enable parallel assessment of multiple bacterial strains in the near‐native lung tissue from the same animal. Additionally, in combination with inhibitors and KO animals, PCLS provide further insight in the molecular mechanisms of host‐pathogen interactions. 4.2.6 Yersinia pestis Yersinia pestis is the causative agent of plague. Inhalation of Y. pestis leads to pneumonic plague, which can be fatal within days if untreated. Virulence factors, for example, a type 3 secretion system that injects effectors for host manipulation and the protease Plasminogen activator (Pla), are essential for Y. pestis infection and replication, as well as for dissemination and disease progression in cellular and animal models (Demeure et al., 2019 ). The exact function of Pla however remained an enigma. An hPCLS model of pneumonic plague combined with the fluorescence‐based TEM1‐β‐lactamase effector translocation assay revealed that Pla promotes optimal translocation of effectors into phagocytes, especially alveolar macrophages (Banerjee et al., 2019 ). In contrast, Pla was dispensable for type 3 secretion into THP‐1 cells, an immortalized cell line considered as macrophage surrogate model. Moreover, in the PCLS infection model, Pla plays a vital role for the inhibition of the expression of the proinflammatory cytokines IL‐6, IL‐8, and TNFα during the first hours of infection. During this phase, it is not possible to measure cytokine levels in mouse lungs as these are below detection level. This reinforces the notion that the functions of virulence factors might only become apparent under physiological infection conditions and that PCLS are a unique system to investigate these functions and early events of infection, which are usually poorly accessible. 5 CONCLUSION & OUTLOOK Bridging the gap between in vitro and in vivo models, PCLS are a reliable and powerful model to investigate respiratory infectious diseases. hPCLS in particular deliver highly relevant data reflecting human disease, eliminating the often speculative extrapolation of findings from surrogate models to humans. As many human pathogens are often unable to infect and replicate in certain animal models, hPCLS can also provide an important platform for modeling viral and bacterial co‐infections, abrogating the host‐specificity issues that hinder such studies. Some constraints remain, for example, limited genetic tractability, lack of cell infiltration, and of adaptive immunity, as well as the short life span of the slices which hinders their use for studying chronic infections, but these limitations are being actively tackled, for example, by embedding PCLS in hydrogels that expand their life span (Bailey et al., 2020 ). Given the exceptional accessibility of PCLS for live imaging and the option to compare many pathogen strains or treatments, PCLS promise to become an invaluable tool for dissecting the molecular mechanisms of host‐pathogen interactions within the physiological context of lung tissue and cellular microbiology research in the future. CONFLICT OF INTEREST FV and GNS declare that they have no conflicts of interest with the contents of this article. COK has received consultancy fees from Insmed for the treatment of pulmonary non tuberculous mycobacterial infection. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this literature review as no new data were generated. Micrographs in Figure 2 are merely included for illustrative purposes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109055/

Pathophysiology of anthrax

Infection by Bacillus anthracis in animals and humans results from accidental or intentional exposure, by oral, cutaneous or pulmonary routes, to spores, which are normally present in the soil. Treatment includes administration of antibiotics, vaccination or treatment with antibody to the toxin. A better understanding of the molecular basis of the processes involved in the pathogenesis of anthrax namely, spore germination in macrophages and biological effects of the secreted toxins on heart and blood vessels will lead to improved management of infected animals and patients. Controlling germination will be feasible by inhibiting macrophage paralysis and cell death. On the other hand, the control of terminal hypotension might be achieved by inhibition of cardiomyocyte mitogen-activated protein kinase and stimulation of vessel cAMP.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9794480/

FDG–PET findings associated with various medical procedures and treatments

[ 18 F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) is a well-established modality with high sensitivity for the diagnosis and staging of oncologic patients. FDG is taken up by the glucose transporter of the cell membrane and becomes trapped within the cell. In addition to malignant neoplasms, active inflammatory lesions and some kinds of benign tumors also accumulate FDG. Moreover, the degree of uptake into normal organs and tissues depends on various physiological conditions, which is affected by various medical procedures, treatments, and drugs. To avoid misleading interpretations, it is important to recognize possible situations of unexpected abnormal accumulation that mimic tumor lesions. In this review, we present various FDG findings associated with surgical or medical procedures and treatments. Some findings reflect the expected physiological reaction to treatment, and some show inflammation due to prior procedures. Occasionally, FDG–PET visualizes other disorders that are unrelated to the malignancy, which may be associated with the adverse effects of certain drugs that the patient is taking. Careful review of medical records and detailed interviews of patients are thus necessary. Introduction Fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) or PET/computed tomography (PET/CT) is a clinically accepted modality with high sensitivity for detecting malignant neoplastic lesions. However, increased glucose metabolism with FDG accumulation is not only specific for neoplastic lesions but is also observed in many non-tumorous sites. Such sites include pathologically benign lesions, especially inflammatory lesions, and normal organs, because most cells require glucose for energy supply. Various medical procedures and prior treatments cause unexpectedly increased accumulation that is not associated with neoplasms. These FDG findings may reflect specific drug effects that patient and clinician can expect. FDG–PET also sometimes presents unexpected reactive findings that can be associated with previous procedures. Certain adverse effects and drug-associated diseases can be visualized on FDG–PET. In this review article, these iatrogenic FDG findings were addressed using relevant medical procedures or treatments. Control of blood glucose FDG, as well as glucose, is taken up by cells from circulation through cell membrane glucose transporters (GLUT) and is phosphorylated into FDG-6-phosphatate by hexokinase, the first enzyme in the glycolysis pathway. FDG-6-P is trapped within cells, because it is a poor substitute for glucose-phosphate isomerase, an enzyme involved in the second step of glycolysis. Considering glucose metabolism, muscles play a unique role in glucose storage, in the form of glycogen, and supply glucose for metabolic demands. GLUT-4, the main GLUT in the myocytes of the skeletal muscle and myocardium, usually stays within myocytes and is transferred onto the cell membrane when glucose transport is needed. Insulin Insulin induces GLUT-4 transfer onto the cell membrane of myocytes to import glucose from the blood, after which glucose is stored in the myocytes as glycogen, which decreases excessive blood glucose levels. Therefore, more than 4 h of fasting and careful scheduling of insulin use before FDG administration are required [ 1 ]. In contrast, intrinsic insulin secretion and extrinsic high insulin levels induce strong FDG uptake in myocytes, which results in images that are unsuitable for evaluating tumor distribution with diffusely increased muscular uptake [ 2 ] (Fig. 1 ). Fig. 1 Diffuse increased accumulation in the muscles of a patient who had consumed a meal 2 h before FDG injection. An insulin injection before FDG administration causes a similar image Increased intestinal uptake caused by metformin Gontier et al. first found that increased intestinal FDG uptake is commonly observed in patients with type 2 diabetes mellitus who are using metformin [ 3 ] (Fig. 2 ). Increased intestinal FDG uptake can obscure intestinal lesions, and significant findings may be missed. Therefore, the effect of metformin discontinuation on intestinal FDG uptake has been studied [ 4 , 5 ]. These studies showed that FDG uptake in both small and large intestines decreased a few days after metformin withdrawal. Recently, Morita et al. used PET/MRI, which enables simultaneous acquisition of metabolic information with PET and morphological information with MRI. They clarified that FDG levels in the lumen of the intestinal tract is significantly greater in patients treated with metformin than in those not treated with the drug [ 6 , 7 ]. However, the amount of FDG in the intestinal wall did not significantly differ between the two groups. Their discovery using PET/MRI has provided new insights into the mechanism of action of this drug. Fig. 2 Increased bowel FDG distribution in a patient using metformin Insulin Insulin induces GLUT-4 transfer onto the cell membrane of myocytes to import glucose from the blood, after which glucose is stored in the myocytes as glycogen, which decreases excessive blood glucose levels. Therefore, more than 4 h of fasting and careful scheduling of insulin use before FDG administration are required [ 1 ]. In contrast, intrinsic insulin secretion and extrinsic high insulin levels induce strong FDG uptake in myocytes, which results in images that are unsuitable for evaluating tumor distribution with diffusely increased muscular uptake [ 2 ] (Fig. 1 ). Fig. 1 Diffuse increased accumulation in the muscles of a patient who had consumed a meal 2 h before FDG injection. An insulin injection before FDG administration causes a similar image Increased intestinal uptake caused by metformin Gontier et al. first found that increased intestinal FDG uptake is commonly observed in patients with type 2 diabetes mellitus who are using metformin [ 3 ] (Fig. 2 ). Increased intestinal FDG uptake can obscure intestinal lesions, and significant findings may be missed. Therefore, the effect of metformin discontinuation on intestinal FDG uptake has been studied [ 4 , 5 ]. These studies showed that FDG uptake in both small and large intestines decreased a few days after metformin withdrawal. Recently, Morita et al. used PET/MRI, which enables simultaneous acquisition of metabolic information with PET and morphological information with MRI. They clarified that FDG levels in the lumen of the intestinal tract is significantly greater in patients treated with metformin than in those not treated with the drug [ 6 , 7 ]. However, the amount of FDG in the intestinal wall did not significantly differ between the two groups. Their discovery using PET/MRI has provided new insights into the mechanism of action of this drug. Fig. 2 Increased bowel FDG distribution in a patient using metformin Invasive or surgical procedures The abnormal FDG accumulation associated with inflammatory processes is often observed after invasive procedures, such as at surgical incision lines, along the drainage tube, around the stoma, or at the tracheostomy site. Although aortic graft infections are visualized as intense focal accumulation, mild-to-moderate uptake along the graft is normally seen for years, even without infection (Fig. 3 ). Pleurodesis with inflammation-inducing agents, such as OK-432 or talc, results in high FDG uptake [ 8 , 9 ] (Fig. 4 ). Nishimori et al. reported that FDG uptake in non-malignant inflammation after pleurodesis with OK-432 appeared linear, while malignant lesions showed nodular and higher accumulation than benign lesions [ 10 ]. Fig. 3 FDG accumulation along the abdominal aortic graft wall (arrow) is seen, without any particular findings indicating active inflammation, 3 years after operation Fig. 4 Intense FDG accumulation spreading inside the left pleura 1 month after pleurodesis with OK-432 after bruising the chest for pleuritis carcinomatosis. a MIP, b axial fusion image) Since clinicians usually know most of these medical histories, it is not difficult to correctly interpret FDG uptake due to iatrogenic inflammation. However, there are some situations in which they do not recognize the history, such as sclerotherapy of the hemorrhoid, silicone injection for augmentation of the breast or buttock [ 11 ], or other cosmetic facial fillers [ 12 , 13 ] performed at other institutions. Patients often think that these medical procedures are unrelated to their neoplasm and may not have provided this information. Interrogating the patient is as important as reviewing medical records to avoid misleading management due to incorrect interpretation of focal abnormal accumulation after these procedures. Repeated subcutaneous injections Repeated subcutaneous injections often cause granuloma formation or reactive induration with increased FDG uptake at the injection sites [ 14 ]. This is often observed in patients with prostate cancer or breast cancer, who have been receiving luprorelin acetate (an LH–RH analog) therapy, or in patients with diabetes receiving insulin injections. External radiotherapy Referring to the radiotherapy plan, images are important when interpreting FDG–PET results for patients who received radiotherapy. External radiotherapy affects healthy organs within the irradiated area near the tumor. The physiological function of the bone marrow, brain, or tonsils within the irradiated area is impaired, and FDG accumulation in the corresponding area is usually low compared with the surrounding healthy area. Decreased vertebral uptake is frequently observed in the thoracic vertebrae in patients with esophageal cancer, lung cancer, or other mediastinal tumors and in the lumbar vertebrae and sacrum in patients with uterine or ovarian cancer. Although interpreting the post-irradiation finding for symmetrically decreased uptake is not difficult, it is sometimes difficult to interpret asymmetrical uptake in the bones or palatine tonsils after irradiation on one side. Because physiological accumulation in the bone or tonsils usually shows a wide range, careful consideration is required to judge whether the area with higher uptake is abnormal or whether the area with lower uptake indicates impaired function related to irradiation. External radiotherapy also causes localized inflammation within the field. Radiation pneumonitis frequently occurs in cases of esophageal cancer, lung cancer, mediastinal tumor, or breast cancer and requires steroid therapy [ 15 ] (Fig. 5 ). Sharply margined increased FDG uptake is seen in the abnormal attenuation area, and the distribution is irrespective of the anatomical lobe or airway section. In the early stage, only FDG uptake was observed, without abnormalities in the CT images. The soft tissue, including the muscle or subcutaneous fat in the irradiated area, also has increased FDG accumulation to some extent. Radiation-induced hepatitis is visualized as a well-defined increased FDG accumulation corresponding to the radiation field, which is not always accompanied by abnormalities on plain CT images of PET/CT [ 16 ] [ 17 ] (Fig. 6 ). Fig. 5 Axial images of a case of radiation pneumonitis that occurred in a patient with esophageal cancer. a FDG–PET/CT before treatment, b image of the irradiation field with two radiation direction (1. 40 Gy (upper) and 2. 20 Gy (lower)), c FDG–PET/CT 11 months after radiotherapy Fig. 6 Images of a patient with testicular cancer who had undergone radiotherapy for a metastatic bone tumor at his L1 spine 2 months previously [ 17 ]. The FDG–PET/CT showed a localized hot spot in the lateral segment of the left lobe of the liver ( a ). Although the contrast-enhanced CT performed 10 days previously showed no abnormality ( b ), the MRI performed 7 days later showed a well-bordered square low signal area indicating radiation-induced hepatitis ( c ). (Partly cited from reference #17) Radiation-induced myocardial damage with focally increased FDG uptake has also been reported in patients with thoracic esophageal, lung, or breast cancer [ 18 ]. The physiological uptake in the myocardium varies depending on the patient and the duration of the fasting time before the FDG–PET examination. Jingu et al. showed that areas with high FDG uptake in the basal myocardium that corresponded to the irradiated fields indicated a damaged myocardium with low 201 TlCl and 123 I-BMIPP uptake, delayed Gd-enhancement, or hypokinesia on cine-MRI studies [ 18 ]. Chemotherapy Intensive chemotherapy causes time-dependent changes in the physiological distribution of FDG in some organs. Diffuse homogeneous hyperaccumulation in the red bone marrow is commonly observed as a normal hematopoietic response for a few months after marrow suppression due to intensive chemotherapy [ 19 , 20 ]. The thymus, which has a high metabolic activity in the first years of life, gradually decreases in size with age. In pediatric, adolescent, and young adult patients, uptake in the thymus shows serial changes in association with physiological reactions after chemotherapy. The thymus, which shrinks during or soon after chemotherapy, shows hyperplasia as an immunological rebound phenomenon, which is characterized by lymph follicles with large nuclear centers and the infiltration of plasma cells following chemotherapy-induced inhibition of lymphocyte proliferation [ 21 ]. This rebound phenomenon occurs slightly later than that in the bone marrow. Symmetrical enlargement is accompanied by intense FDG uptake, which reaches a peak 10 months after the cessation of chemotherapy [ 22 ]. A similar dynamic change is also observed in the tonsils; the accumulation is low during intensive chemotherapy and then increases after the completion of chemotherapy. The high accumulation persists for a long period, as in the thymus. High tonsillar accumulation may be associated with reactive enlargement, but it is not always accompanied by a morphological change [ 23 ]. Figure 7 illustrates the serial changes in physiological uptake in the bone marrow, thymus, and tonsils of a pediatric patient with lymphoma [ 23 ]. The areas where these serial reactions occur after intensive chemotherapy are also the sites, where lymphoma is frequently involved; therefore, the physician should take the time course of the patients' treatment protocol into consideration so as not to jump to the conclusion that the high uptake area indicates recurrent tumors. Fig. 7 Serial changes in physiological FDG uptake in a pediatric patient with lymphoma: a before treatment (arrow shows the primary spot), b 1 month after the end of chemotherapy, and c 1 year after treatment. The image b shows diffusely increased uptake in the bone marrow. One year after chemotherapy, rebound uptake is seen in the thymus and tonsils, both of which seem to be enlarged. The bone marrow uptake has returned to its initial levels Administration of G-CSF Administration of granulocyte colony-stimulating factor (G-CSF) for leukocytopenia after chemotherapy increases physiological bone marrow FDG uptake, and Hanaoka et al. recommended an interval of 10 days after G-CSF administration to minimize its influence [ 24 ]. Recently, long-acting pegylated G-CSF (pegfilgrastim) has been frequently used, which causes consistent increased marrow uptake over a period of approximately 3 weeks following administration [ 25 ] (Fig. 8 ). An interval of at least 3 weeks after pegfilgrastim administration before PET/CT is now recommended. Fig. 8 Marked increased uptake in the hematopoietic bone marrow and spleen in a patient with lymphoma, who received pegfilgrastim 7 days before the FDG–PET examination Administration of G-CSF Administration of granulocyte colony-stimulating factor (G-CSF) for leukocytopenia after chemotherapy increases physiological bone marrow FDG uptake, and Hanaoka et al. recommended an interval of 10 days after G-CSF administration to minimize its influence [ 24 ]. Recently, long-acting pegylated G-CSF (pegfilgrastim) has been frequently used, which causes consistent increased marrow uptake over a period of approximately 3 weeks following administration [ 25 ] (Fig. 8 ). An interval of at least 3 weeks after pegfilgrastim administration before PET/CT is now recommended. Fig. 8 Marked increased uptake in the hematopoietic bone marrow and spleen in a patient with lymphoma, who received pegfilgrastim 7 days before the FDG–PET examination Vaccination In these 2 years, transient FDG uptake in morphologically normal or slightly enlarged axillary, supraclavicular, and lower cervical lymph nodes after vaccination of the ipsilateral deltoid muscle have been commonly observed in the context of the COVID-19 mass vaccination [ 26 – 32 ]. It is sometimes accompanied by mild splenic enlargement with high uptake (Fig. 9 a). These findings last for 4–6 weeks or longer after the most recent shot of the vaccine, and it has been recommended to ask patients for information about the date and sites of vaccination. It has also been recommended that oncologic patients, especially those with potential axillary or lower neck tumor involvement, such as breast cancer or head and neck cancer, be advised about the timing of the imaging and the site of vaccination [ 33 ]. Fig. 9 Two patients with high uptake in the left axillary and supraclavicular lymph nodes and spleen after vaccination: a 5 days after the second COVID-19 vaccination on the left deltoid muscle, b 6 days after subcutaneous influenza vaccination on the left arm in a woman with inflammatory breast cancer of the right breast Similar systemic immune responses to vaccination observed on FDG–PET have also been reported with various kinds of vaccinations, including seasonal influenza (Fig. 9 b), pneumococcal, tetanus, diphtheria, pertussis, human papilloma virus, bacilli Calmette–Guerin (BCG), measles, smallpox, and anthrax [ 34 – 39 ]. Interestingly, regarding the third or following COVID-19 vaccination, the vaccine-associated hypermetabolic lymphadenopathy is mild and does not continue for weeks. Cohen et al. reported that uptake in the ipsilateral axillary lymph node persists only within the first about 5 days of administration and does not interfere with the interpretation of FDG–PET studies [ 40 ]. The mechanism of the immune response is considered to be related to the shortened duration of the reaction after the third vaccination. The first immune response of naïve cells is elicited by the first vaccine dose, and the amnestic response of memory B and T cells is induced by the second vaccine shot. Once memory cells have undergone clonal expansion, differentiation, and affinity maturation, the amnestic immune response requires only a short lag period after the third or later vaccination. BCG-induced granuloma BCG is a vaccine for tuberculosis and sometimes causes transient vaccine-associated lymphadenopathy after shots. In addition, granuloma formation with high FDG uptake is sometimes observed after procedures using BCG. In pediatric patients, particularly those with impaired cellular immunity, multiple granulomas are formed in many organs, including the bone, liver, muscle, skin, and lungs, which may masquerade as disseminated neoplasms. BCG is also used as an immunotherapeutic agent that encourages the immune system to attack cancer cells. It is administered directly into the bladder of patients with non-muscular invasive bladder cancer. BCG-contaminated urine induces asymptomatic local granulomas anywhere along the genitourinary tract, kidneys, prostate, scrotum, and penis, which are visualized on FDG–PET in patients who received intravesical BCG therapy [ 41 ] (Fig. 10 ). Mycotic aneurysms sometimes occur as a progression of ectopic granulomas at the aortic or arterial walls [ 42 , 43 ] (Fig. 11 ), which usually grow in a short period and often rupture. Occasionally, disseminated granulomas in the lungs, kidneys, bone marrow, liver, or skin have also been discovered in PET studies that mimic the systemic involvement of hematological disorders, such as lymphoma [ 44 ] (Fig. 11 ). Fig. 10 Local granuloma in the prostate in a patient who received intravesical BCG administration for bladder cancer Fig. 11 Ectopic granuloma at the abdominal aortic wall and disseminated granuloma in the lungs, liver, bone marrow, kidneys are visualized on the FDG–PET ( a ) [ 23 ]. The patient has a history of transurethral resection of bladder tumor and 8 times intravesical BCG injection for his bladder cancer. The axial PET/CT fusion image ( b ) shows intense accumulation on wall of the dilated left common iliac artery. Five days after the PET/CT examination, this patient developed an acute abdomen with impending rupture of this aneurysm ( c ) Oii-LPDs Patients with congenital or acquired immunodeficiency have a high incidence of lymphoproliferative diseases (LPDs). The WHO classification 2016 classifies immune-deficiency-associated LPDs into four subtypes: LPD associated with primary immunodeficiency disorders, lymphomas associated with HIV, post-transplant LPD, and other iatrogenic immunodeficiency-associated LPDs (Oii-LPDs) [ 45 ]. Oii-LPDs develop in patients treated with immunosuppressive drugs. The most common primary disease is rheumatoid arthritis, followed by dermatomyositis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Most cases involve patients using methotrexate (MTX); thus, the condition was first called MTX–LPD. However, patients treated with anti-tumor necrosis factor (anti-TNF) or other drugs have also reported, and it is now recognized that various drugs other than MTX can cause this disorder. The pathological subtype of Oii-LPDs mainly consists of reactive lymphoid hyperplasia, polymorphic LPDs comprising infiltration of plasma cells, immunoblasts, and lymphocytes, and lymphomas, such as diffuse large B-cell lymphoma, Hodgkin lymphoma, Epstein–Barr virus (EBV)-positive mucocutaneous ulcer, or hepatosplenic T-cell lymphoma [ 46 ]. Although LPDs show similar FDG-positive lesions as lymphoma (Fig. 12 ), Oii-LPDs have some peculiar features, such as a high incidence of extranodal disease [ 47 ] and spontaneous regression. Approximately 70% of Oii-LPDs show spontaneous regression after the discontinuation of immunosuppressive drugs. Approximately 33% of patients who have experienced transient regression experience later relapse or recurrence [ 47 ]. Fig. 12 Patient with multiple lymphadenopathies that were diagnosed as Oii-LPD with positive EBV. He had been treated with MTX for rheumatoid arthritis. In this patient, most of the lymphadenopathies disappeared after MTX discontinuation EBV is a common gamma herpesvirus, and most of the world's population is asymptomatically infected. Although EBV infects most healthy individuals without any pathogenicity, it primarily targets B lymphocytes and can develop lymphoid malignancies [ 48 , 49 ]. EBV positivity is a major risk factor for Oii-LPDs, but EBV-encoded RNA positivity and higher lymphocyte counts in peripheral blood are predictive factors for its regression [ 47 ]. EBVMCU EBV-positive mucocutaneous ulcer (EBVMCU) is a recently recognized B-cell LPD that is driven by latent EBV infection and causes ulcerations in the oropharynx, gastrointestinal tracts, and skin [ 50 , 51 ]. Intense FDG accumulation in the lesion mimics malignant tumors [ 52 ] (Fig. 13 ). This is considered to be a specific type of Oii-LPD that shows a relatively favorable prognosis. The lesions often disappear after the discontinuation of immunosuppressive drugs. Fig. 13 Patient with EBVMCU who had been receiving MTX for rheumatoid arthritis for 2 years. a Contrast-enhanced CT, b FDG–PET/CT fusion image EBVMCU EBV-positive mucocutaneous ulcer (EBVMCU) is a recently recognized B-cell LPD that is driven by latent EBV infection and causes ulcerations in the oropharynx, gastrointestinal tracts, and skin [ 50 , 51 ]. Intense FDG accumulation in the lesion mimics malignant tumors [ 52 ] (Fig. 13 ). This is considered to be a specific type of Oii-LPD that shows a relatively favorable prognosis. The lesions often disappear after the discontinuation of immunosuppressive drugs. Fig. 13 Patient with EBVMCU who had been receiving MTX for rheumatoid arthritis for 2 years. a Contrast-enhanced CT, b FDG–PET/CT fusion image Immunotherapy-related findings Immunotherapy has recently emerged as an important advancement in cancer treatment, in addition to surgery, radiation, chemotherapy, and molecular-targeted therapies. It is based on evidence that cancer development is enabled by the dysregulation of the immune system against neoplasms. Immunotherapy relies on the reactivation of the host immune system to recognize and kill cancer cells [ 53 ]; thus, FDG–PET often visualizes systemic immune activation after immunotherapy. FDG uptake in the bone marrow is attributed to inflammatory activity, and increased uptake in the lymphoid tissue, namely, in the multiple lymph nodes and spleen, is recognized as a marker of immunotherapy effectiveness [ 32 , 54 , 55 ] (Fig. 14 ). Fig. 14 Patient with lung cancer with lymph nodes metastases ( a ). Six months after achievement of a complete response, lymph nodes metastases developed in the right lower neck and bilateral supraclavicular regions. The second PET scan ( b ), which was performed 3 weeks after starting anti-PD-L1, visualized another lymph node in the left neck (closed arrow) and increased uptake in the enlarged spleen (open arrow). The increased splenic uptake and lymphadenopathy in the left neck that had disappeared on the third PET scan ( c ) are considered immunotherapy-related findings Immunotherapy-related adverse effects (irAEs) are new toxicity profiles that involve many organs. Table 1 summarizes the FDG–PET findings of both immune reactions and irAEs. The pattern of adverse effects differs across immune checkpoint inhibitor classes and can be driven by different immune cell activation patterns. The early detection of irAEs and rapid intervention by systemic immunosuppression are important for improving patient outcomes. These events are not always associated with clinical symptoms and may only be diagnosed using imaging modalities. FDG–PET is frequently beneficial for the early identification of these events [ 56 – 58 ]. Table 1 Immunotherapy-related FDG positive findings Disorder (possible symptoms) CT findings associated with the increased FDG uptake Reactive lymphadenopathy Enlarged lymph node Sarcoid-like reaction Symmetrical mediastinal and hilar enlarged nodes Reactive splenomegaly Splenomegaly Thyroiditis (Hyper/hypo-thyroidism) Diffuse goiter/normal size Pituitary hypophysitis (various symptoms due to hormone deficiency) Enlarged pituitary gland Colitis (diarrhea) Intestinal mural thickening in a long segment with/without surrounding fat stranding Pancreatitis Swelling with/without peripancreatic fat stranding Pneumonitis (cough, fever) Non-specific findings of pneumonitis Arthritis (arthralgia) Non-specific finding of polyarthritis Myositis (myalgia) Non-specific There have been many reports of patients who experienced apparent lesion progression but subsequent late responses to treatments, which has been termed "pseudoprogression". Pseudoprogression has been frequently reported in patients with melanoma receiving anti-CTLA-4 treatment, whereas it is rare in other tumor types and with anti-PD1/PD-L1 treatment. Pseudoprogression is also observed in patients treated with CD19 specific chimeric antigen receptor T cell (CAR-T) therapy, which is also a new promising cancer immunotherapy that is now approved for the treatment of relapsed/refractory diffuse B cell lymphoma [ 59 ]. However, immunotherapy sometimes accelerates tumor growth, which is called "hyperprogression" [ 60 ]. Clinicians should interrupt the treatment if hyperprogression is suspected. Understanding these features and careful follow-up are required to assess the responses of patients to immunotherapy. MRONJ Chronic osteomyelitis or osteonecrosis of the jaw is one of the most intractable inflammatory conditions of the maxillofacial region. The incidence of medication-related osteonecrosis of the jaw (MRONJ) has recently increased. According to the American Association of Oral and Maxillofacial Surgeons, the definition of MRONJ includes all of the following criteria: (1) current or previous treatment with antiresorptive (bisphosphonate and denosumab) or antiangiogenic agents (bevacizumab and sunitinib); (2) exposed bone or bone that can be probed through an intraoral or extraoral fistula (e) in the maxillofacial region that has persisted for more than 8 weeks; and (3) no history of radiation therapy to the jaws or obvious metastatic disease of the jaws [ 61 ]. Jawbones are anatomically prone to osteomyelitis compared to other bones, because they face the oral cavity and are easily damaged by chewing food. FDG sometimes incidentally visualizes the inflammatory condition of the MRONJ [ 62 , 63 ]. MRONJ should be included in the differential diagnosis when abnormally high FDG uptake is incidentally detected in the jaws of patients treated with the above-mentioned drugs (Fig. 15 ), even if the patients are asymptomatic in the early stages. Fig. 15 Patient with lung cancer with bone metastasis who developed medication-related osteonecrosis of the jaw due to bisphosphonate. The first PET scan ( a ) revealed metastatic lesions in the mediastinum, right adrenal gland, and a lumbar vertebra. The second PET scan detected the abnormality as an intense accumulation in the mandibular bone ( b , c ) earlier than the CT ( d ) Exercise Contracting skeletal muscles accelerates carbohydrate and fat metabolism during exercise. In muscles, where the redundant glucose is transformed into glycogen to be stored after meals, glycogen is quickly catabolized to glucose-6-phosphate for energy demand during exercise. However, glycogen storage in the muscle is insufficient for persistent exercise, and glucose uptake through GLUT-4 is activated. Exercise induces the transfer of GLUT-4 onto the cell membrane of myocytes to facilitate glucose uptake. By such a mechanism, FDG distribution in the body differs at rest and after exercise [ 64 , 65 ]. After exercise, FDG accumulates in exercised muscles. For example, FDG uptake increases in the masticator muscles after chewing gum [ 66 ] and in the laryngeal muscles after speaking too much [ 67 ]. Therefore, for the evaluation of tumor metabolism, patients are asked to refrain from strenuous exercise and keep quiet, without speaking much, before FDG–PET examination. Postoperative patients with oral cancer undergo oral rehabilitation for impaired mastication, swallowing, or speaking. Patients may be unaware that they are exercising, because only localized muscles of a limited area around the mouth and larynx are trained. Hard exercise of the reconstructed tongue causes intense FDG accumulation after rehabilitation (Fig. 16 ), which may be misdiagnosed as a local recurrence. Fig. 16 Patient with post-operative-state tongue cancer. An intense FDG accumulation seen in the center of the reconstructed tongue ( a ) without any abnormality on the contrast enhanced CT ( b ) or clinical inspection. The interview revealed that he had underwent rehabilitation for postoperatively impaired oral muscular function before the PET examination Miscellaneous drug-induced findings Activated BAT visualization due to β3 adrenergic agonist It has been considered that physiological uptake in brown adipose tissue (BAT) is typically observed in pediatric or young adult patients, especially in the cold circumstances. Recently, apparent FDG accumulation in activated BAT by β3 adrenergic agonist, which is prescribed for overactive bladder in elderly patients, has been occasionally observed, even in the summer [ 68 , 69 ]. As adrenergic β3 receptors exist on BAT as well as in the bladder detrusor muscles, β3 agonists sometimes cause intense FDG uptake in BATs. The characteristic distribution of the BATs that FDG visualizes in elderly patients activated by β3 adrenergic agonist or pheochromocytoma is seen in paravertebral or retroperitoneal spaces, while the BATs in pediatric or young adult patients are usually seen in the supraclavicular or lower neck regions. It is important to check the medication that patients are taking when BATs are visualized in unusual situations. Myocardial uptake influenced by medication other than insulin GLUT-4 translocation to the muscular cell surface is, as described in the previous section, upregulated by high insulin levels. Moreover, in the myocardium, where the energy requirements are supplied by mainly fatty acids as well as glucose, and many factors including sex, age, obesity, and diabetes affect its glucose and fatty acid metabolism and influence on the myocardial FDG uptake [ 70 ]. The thyroid hormone levothyroxine upregulates GLUT-4 in the skeletal muscle but reduces the myocardial glucose uptake, and bezafibrate, which reduces serum triglyceride levels [ 71 ], cause increased myocardial uptake [ 72 , 73 ]. Although the mechanism remains unclear, it is also reported the myocardial FDG uptake can be influenced by benzodiazepine. Drug-induced gynecomastia Gynecomastia refers to the enlargement of male glandular breast tissue as a result of a hormone imbalance between estrogen and androgen. Although men aged > 50 years often experience physiological gynecomastia due to the decrease in testosterone levels with increasing age, gynecomastia may be indicative of an underlying health condition, including chronic renal failure, liver cirrhosis, thyrotoxicosis, malabsorptive states, and some particular tumors. In addition, drug-induced gynecomastia accounts for 10–25% of all gynecomastias, including estrogen, anti-androgen, antihypertensive, antifungal, antiparasitic, opioids, antiarrhythmic, proton-pump inhibitors, H2 blockers, dopamine receptor blockades, and anti-epileptic [ 74 ]. FDG–PET/CT shows soft tissue growth in the breast with increased tracer accumulation [ 75 ]. When enlargement occurs rapidly or is seen in one breast, a detailed history should be taken to determine whether the enlargement may result from a tumorous lesion. Others FDG–PET can sometimes present various kinds of non-tumorous disorders that are non-specific but associated with some medications, of which patients may experience some involving symptoms or present abnormal laboratory findings. Increased accumulation in the organs is seen in patients with drug-induced pneumonitis, nephritis, pancreatitis, or reactive lymphadenopathy, for which the physician can usually reach the diagnosis easily from clinical data. FDG–PET may visualize some rare drug-induced findings, For example, pegfilgrastim, a long-acting pegylated G-CSF, can induce vasculitis that causes antibiotic-resistant fever and high C-reactive protein levels in patients treated with pegfilgrastim for leukocytopenia due to chemotherapy [ 76 ]. Pegfilgrastim is also a causative agent of iatrogenic carotidynia, which is characterized by radiating pain and tenderness over the unilateral common carotid bifurcation. Since perivascular carotid inflammation occurs, FDG–PET reveals vasculitis in a limited area [ 77 , 78 ]. Many kinds of drugs, including hypolipidemic agents (HMG-CoA reductase), antibiotics, major and minor tranquilizers, and anticancer agents, are known to cause drug-induced rhabdomyolysis. When FDG shows marked diffuse accumulation in the skeletal muscles, such as polymyositis [ 79 ] and the patient experiences muscle weakness and pain with increased serum creatine phosphokinase or myoglobin levels, the drugs that may cause rhabdomyolysis should be identified and stopped immediately to prevent serious renal failure. Although these disorders are not specific, a thoughtful interpretation of FDG–PET could contribute to elucidating the cause of patients' problems. Activated BAT visualization due to β3 adrenergic agonist It has been considered that physiological uptake in brown adipose tissue (BAT) is typically observed in pediatric or young adult patients, especially in the cold circumstances. Recently, apparent FDG accumulation in activated BAT by β3 adrenergic agonist, which is prescribed for overactive bladder in elderly patients, has been occasionally observed, even in the summer [ 68 , 69 ]. As adrenergic β3 receptors exist on BAT as well as in the bladder detrusor muscles, β3 agonists sometimes cause intense FDG uptake in BATs. The characteristic distribution of the BATs that FDG visualizes in elderly patients activated by β3 adrenergic agonist or pheochromocytoma is seen in paravertebral or retroperitoneal spaces, while the BATs in pediatric or young adult patients are usually seen in the supraclavicular or lower neck regions. It is important to check the medication that patients are taking when BATs are visualized in unusual situations. Myocardial uptake influenced by medication other than insulin GLUT-4 translocation to the muscular cell surface is, as described in the previous section, upregulated by high insulin levels. Moreover, in the myocardium, where the energy requirements are supplied by mainly fatty acids as well as glucose, and many factors including sex, age, obesity, and diabetes affect its glucose and fatty acid metabolism and influence on the myocardial FDG uptake [ 70 ]. The thyroid hormone levothyroxine upregulates GLUT-4 in the skeletal muscle but reduces the myocardial glucose uptake, and bezafibrate, which reduces serum triglyceride levels [ 71 ], cause increased myocardial uptake [ 72 , 73 ]. Although the mechanism remains unclear, it is also reported the myocardial FDG uptake can be influenced by benzodiazepine. Drug-induced gynecomastia Gynecomastia refers to the enlargement of male glandular breast tissue as a result of a hormone imbalance between estrogen and androgen. Although men aged > 50 years often experience physiological gynecomastia due to the decrease in testosterone levels with increasing age, gynecomastia may be indicative of an underlying health condition, including chronic renal failure, liver cirrhosis, thyrotoxicosis, malabsorptive states, and some particular tumors. In addition, drug-induced gynecomastia accounts for 10–25% of all gynecomastias, including estrogen, anti-androgen, antihypertensive, antifungal, antiparasitic, opioids, antiarrhythmic, proton-pump inhibitors, H2 blockers, dopamine receptor blockades, and anti-epileptic [ 74 ]. FDG–PET/CT shows soft tissue growth in the breast with increased tracer accumulation [ 75 ]. When enlargement occurs rapidly or is seen in one breast, a detailed history should be taken to determine whether the enlargement may result from a tumorous lesion. Others FDG–PET can sometimes present various kinds of non-tumorous disorders that are non-specific but associated with some medications, of which patients may experience some involving symptoms or present abnormal laboratory findings. Increased accumulation in the organs is seen in patients with drug-induced pneumonitis, nephritis, pancreatitis, or reactive lymphadenopathy, for which the physician can usually reach the diagnosis easily from clinical data. FDG–PET may visualize some rare drug-induced findings, For example, pegfilgrastim, a long-acting pegylated G-CSF, can induce vasculitis that causes antibiotic-resistant fever and high C-reactive protein levels in patients treated with pegfilgrastim for leukocytopenia due to chemotherapy [ 76 ]. Pegfilgrastim is also a causative agent of iatrogenic carotidynia, which is characterized by radiating pain and tenderness over the unilateral common carotid bifurcation. Since perivascular carotid inflammation occurs, FDG–PET reveals vasculitis in a limited area [ 77 , 78 ]. Many kinds of drugs, including hypolipidemic agents (HMG-CoA reductase), antibiotics, major and minor tranquilizers, and anticancer agents, are known to cause drug-induced rhabdomyolysis. When FDG shows marked diffuse accumulation in the skeletal muscles, such as polymyositis [ 79 ] and the patient experiences muscle weakness and pain with increased serum creatine phosphokinase or myoglobin levels, the drugs that may cause rhabdomyolysis should be identified and stopped immediately to prevent serious renal failure. Although these disorders are not specific, a thoughtful interpretation of FDG–PET could contribute to elucidating the cause of patients' problems. Uncommon findings associated with FDG injections Stress during FDG injection Adrenal medullary catecholamine production is considered the first response to stress and anxiety, acting within seconds [ 80 ]. Plasma epinephrine levels increase immediately before vasovagal reflex onset. Therefore, vasovagal-related stress just before FDG injection is considered one of the causes of increased uptake in the bilateral adrenal glands with normal adrenal configuration [ 81 ] (Fig. 17 ). Fig. 17 Increased FDG uptake in both adrenal glands with normal configuration in a case who developed vasovagal reflex at the FDG injection. a Fusion image, b CT Tracer extravasation Radiotracer extravasation sometimes causes one or more apparent hotspots in the axillary lymph nodes on the injected side (Fig. 18 ). Regardless of intense FDG uptake, each node seems to be normal in size and has a fatty hilum [ 82 ]. A marked tracer distribution within the subcutaneous tissue of the upper limb around the injection site indicates tracer extravasation, even if the patients or the medical staff who administered the tracer have not been aware of it. This can cause physiological hot spots on the ipsilateral axillary area through the lymphatic system. Fig. 18 Patient with retrosternal-reconstructed esophageal cancer. A hot spot at the left axillary lymph node as a result of tracer extravasation in the ipsilateral arm is visualized. a MIP image, b axial section of the fusion image. The CT shows a normal lymph node configuration ( c ) One or more transient high clot artifacts in the lung parenchyma, which are considered lung microembolisms, rarely occur when there is a tracer leak or retention in the dilated vein (Fig. 19 ). It is characterized by a relatively intense hot spot, without any abnormal morphological findings on CT. Delayed images usually show that the hotspot disappears or moves within a short time interval [ 83 ]. Fig. 19 Transient hot clot in the right lung, which was considered to be a microembolism due to tracer extravasation, without abnormality on CT in the corresponding site a MIP, b axial images of fusion image (left), PET (middle) and CT (right)). The hot clot rapidly disappeared on the delayed scan that was performed 30 min later c MIP image, d axial image) Stress during FDG injection Adrenal medullary catecholamine production is considered the first response to stress and anxiety, acting within seconds [ 80 ]. Plasma epinephrine levels increase immediately before vasovagal reflex onset. Therefore, vasovagal-related stress just before FDG injection is considered one of the causes of increased uptake in the bilateral adrenal glands with normal adrenal configuration [ 81 ] (Fig. 17 ). Fig. 17 Increased FDG uptake in both adrenal glands with normal configuration in a case who developed vasovagal reflex at the FDG injection. a Fusion image, b CT Tracer extravasation Radiotracer extravasation sometimes causes one or more apparent hotspots in the axillary lymph nodes on the injected side (Fig. 18 ). Regardless of intense FDG uptake, each node seems to be normal in size and has a fatty hilum [ 82 ]. A marked tracer distribution within the subcutaneous tissue of the upper limb around the injection site indicates tracer extravasation, even if the patients or the medical staff who administered the tracer have not been aware of it. This can cause physiological hot spots on the ipsilateral axillary area through the lymphatic system. Fig. 18 Patient with retrosternal-reconstructed esophageal cancer. A hot spot at the left axillary lymph node as a result of tracer extravasation in the ipsilateral arm is visualized. a MIP image, b axial section of the fusion image. The CT shows a normal lymph node configuration ( c ) One or more transient high clot artifacts in the lung parenchyma, which are considered lung microembolisms, rarely occur when there is a tracer leak or retention in the dilated vein (Fig. 19 ). It is characterized by a relatively intense hot spot, without any abnormal morphological findings on CT. Delayed images usually show that the hotspot disappears or moves within a short time interval [ 83 ]. Fig. 19 Transient hot clot in the right lung, which was considered to be a microembolism due to tracer extravasation, without abnormality on CT in the corresponding site a MIP, b axial images of fusion image (left), PET (middle) and CT (right)). The hot clot rapidly disappeared on the delayed scan that was performed 30 min later c MIP image, d axial image) Conclusions FDG, which reflects glucose uptake, positively depicts many inflammatory and physiological activities, as well as tumors. Therefore, when interpreting FDG–PET results, it is necessary to consider various situations and influences. Recognizing the information on prior medical procedures and the treatment that the patient has received is important. It is also meaningful to consider whether there are any adverse effects of the medicines he/she is taking for cancer or other diseases. An unexpected finding may be related to the previous treatment or medical procedure that the attending physician had not previously recognized. In diagnosis of the PET/CT images, collecting information directly from the patient, in addition to a careful check of the medical records, should be performed diligently to avoid misdiagnosis.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403231/

Redox Regulation of NLRP3 Inflammasomes: ROS as Trigger or Effector?

Abstract Significance: Inflammasomes are multiprotein complexes localized within the cytoplasm of the cell that are responsible for the maturation of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18, and the activation of a highly inflammatory form of cell death, pyroptosis. In response to infection or cellular stress, inflammasomes are assembled, activated, and involved in host defense and pathophysiology of diseases. Clarification of the molecular mechanisms leading to the activation of this intracellular inflammatory machinery may provide new insights into the concept of inflammation as the root of and route to human diseases. Recent Advances: The activation of inflammasomes, specifically the most fully characterized inflammasome—the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome, is now emerging as a critical molecular mechanism for many degenerative diseases. Several models have been developed to describe how NLRP3 inflammasomes are activated, including K + efflux, lysosome function, endoplasmic reticulum (ER) stress, intracellular calcium, ubiquitination, microRNAs, and, in particular, reactive oxygen species (ROS). Critical Issues: ROS may serve as a "kindling" or triggering factor to activate NLRP3 inflammasomes as well as "bonfire" or "effector" molecules, resulting in pathological processes. Increasing evidence seeks to understand how this spatiotemporal action of ROS occurs during NLRP3 inflammasome activation, which will be a major focus of this review. Future Directions: It is imperative to know how this dual action of ROS works during NLRP3 inflammation activation on different stimuli and what relevance such spatiotemporal redox regulation of NLRP3 inflammasomes has in cell or organ functions and possible human diseases. Antioxid. Redox Signal . 22, 1111–1129.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153755/

Education and training in microbial forensics

Advances in research and development have led to new technologies, tools and analytical capabilities bolstering the field of microbial forensics. However, a parallel commitment to education and training in this field is needed to prepare the next generation of scientists. Education and training are needed to: Improve first responder preparation and awareness, essential to public safety and preservation of the evidence integrity; Provide a larger pool of trained microbial forensics practitioners and; Educate the public and policy makers on the capabilities and limitations of microbial forensics. This chapter provides suggestions for topics, core courses, laboratories, skills, training, education programs, curricular guidelines and resources for microbial forensics that have been codified from a variety of sources including the American Society of Microbiology and American Academy of Forensic Sciences. Microbial forensic curricula and training Microbial forensics is defined as a scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime, or inadvertent release of a microorganism/toxin for attribution purposes ( Budowle et al., 2003 ). It is the same as other forensic disciplines except for its focus on a particular type of crime ( Budowle et al., 2003 , Carter et al., 2017 ). Based on past history and with current technology capabilities, the potential use of biological weapons is greater than at any other time in history. Only a few semiexpert individuals are needed with access to dual-use equipment (e.g., equipment used in the pharmaceutical or food industries) to produce bioweapons inexpensively. These bioweapons will contain signatures that might be exploited to help identify the perpetrators. One may consider attribution solely to be the "DNA fingerprinting" of a pathogenic agent, but unique genomic identification of a microorganism may not always be possible because of the clonal nature of many microorganisms and, on a case-by-case basis, lack of population and phylogenetic data. Microbial forensics employs the same general practices as other forensic disciplines. Recognizing a crime scene, preserving a crime scene, chain of custody practices, evidence collection and handling, evidence shipping, analysis of evidence, and interpretation of results are carried out in the same general manner as other forensic evidence. A common exception is that evidence will be handled as a biohazard (even more so than, for example, HIV-infected blood). It is anticipated that the majority of microbial forensic evidence will fall into a category with shared characteristics, with some data being very informative and some being less informative. An understanding of the field is essential to determining what type of evidence is collected, how it is analyzed, what the significance of a result is, and what is supportive in identifying a perpetrator and for prosecution. To support a career in microbial forensics, a university microbial forensic curriculum will necessarily cover a broad range of disciplines, which may include microbiology, chemistry, statistics, epidemiology of infectious diseases, evolution, genetics, genomics, and forensics. These courses could be taught individually or merged into a few dedicated microbial forensic courses. From a practical standpoint, many microbial forensic training programs will be based in other majors or minors in epidemiology, genetics, molecular biology, or microbiology as there are numerous graduate programs already in place for epidemiology and these other fields ( publichealthonline ). A major in epidemiology or microbiology could easily become a training platform for microbial forensics with the addition of select courses that include fundamentals in forensics. Alternatively, a forensic science program with additional training in basic sciences such as microbiology and epidemiology could serve to educate microbial forensic scientists. It will be important to emphasize integration of the material toward a specific microbial forensic profession. Concurrent enrollment in microbial forensic seminars, capstone courses, and internships will be needed to provide students the contextual importance of the basic material toward their chosen discipline that will often be taught more generically or under an unrelated discipline. Research internships and forensic seminars provide important opportunities to conduct hands-on experimentation, analyses, and data interpretation and exposure to recent advances in the field. Seminars also provide a forum for student research presentations and public speaking. External workshops, conferences, and meetings ( index, 2022 , scientific, 2022 , meetings, 2022 ) augment opportunities to present research, gain further insights, network, and to become aware of emerging advances in the field from microbial forensic experts. Students that present and publish their results contribute to moving the field forward and advancing their careers. Training in the communication of science is a pivotal aspect of preparing our future science leaders and should be an integral part of any microbial forensic education program. Curricular guidelines from the American Society of Microbiology and American Academy of Forensic Sciences Recent national reports have addressed the need for changing how science courses in higher education are taught, so that students develop a deeper understanding of critical concepts and the analytical and cognitive skills needed to address future challenges. Our competitiveness and national success depend on our students' science aptitude ( edsource ). National Science Teachers Association teachers point to lack of student motivation as a major concern in US science education ( news ). The need for a new approach to improve science and math education is compelling and well supported. In the American Academy for the Advancement of Science Project 2061, "just doing more science was not the answer" ( project 2061 ). Current methods put more value on learning correct answers versus exploration, collaboration, and inquiry. They recommend "practices where the learning of science is as much about the process as the result or outcome, and where students can ask questions and are actively engaged in the learning process, refocusing student learning from knowledge and comprehension to application and analysis." ( project 2061 ). Inquiry is "central to science learning" as students develop their understanding of science concepts combining knowledge, reasoning, and thinking while enhancing comprehension through hands-on learning. The National Research Council agrees. They state "effective instruction capitalizes on students' early interest and experiences, identifies and builds on what they know, and provides them with experiences that serve to engage them in the practices of science and sustain their interest" ( NRC, 2011 ). Because discipline-specific professional societies have national stature and are often the organizations that set guidelines or standards within a discipline, they are well-suited to play a role in promoting systemic change. In 2010, the AAAS and the NSF released the report entitled "Vision and Change in Undergraduate Biology Education: A Call to Action" ( American Association for the Advancement of Science (AAAS), 2011 ). In response to these recommendations, the ASM revised its curriculum guidelines for introductory microbiology courses to emphasize a deeper "understanding of core concepts, critical thinking, and essential laboratory skills." ( American Association for the Advancement of Science (AAAS), 2011 ). In 2012, the ASM Education Board published curriculum guidelines and recommendations for education in microbiology ( Merkel & the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ). "These Guidelines incorporate many of the recommendations made in Vision and Change. They embrace the scientific process and thinking skills put forth in Vision and Change, adding microbiology-based laboratory skills. Further to adopting the five core concepts of Vision and Change, the Guidelines added a sixth core concept, Impact of Microorganisms, specific to this field. Each of the six core concepts is exemplified by four or five microbiology-specific fundamental statements, which reflect basic concepts that are important for all microbiology students to understand in depth. These 27 fundamental statements, together with the four scientific thinking skills and seven laboratory skills, form a comprehensive framework for an undergraduate microbiology course. ( Merkel and the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ). In addition, the ASM Curriculum Guidelines were designed to focus microbiology teaching on student-centered goals and priorities and to enable educators to adopt the discipline-based approach to course design for microbiology courses." ( Horak et al., 2015 ) . The American Society for Microbiology has developed a number of educational programs and resources with student-centered learning modules that may be useful in developing forensic microbiology education programs ( Merkel, 2016 ). The resources cited above are codified in Table 32.2 . ( Merkel & the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ), 32.3 ( Merkel, 2016 ), and 32.4. ( Merkel, 2016 ) ( Table 32.3 , Table 32.4 ). Table 32.2 ASM Curriculum Guidelines: list of recommended skills, core concepts, and fundamental statements. Evolution 1. Cells, organelles (e.g., mitochondria and chloroplasts), and all major metabolic pathways evolved from early prokaryotic cells. 2. Mutations and horizontal gene transfer, with the immense variety of microenvironments, have selected for a huge diversity of microorganisms. 3. Human impact on the environment influences the evolution of microorganisms (e.g., emerging diseases and the selection of antibiotic resistance). 4. The traditional concept of species is not readily applicable to microbes due to asexual reproduction and the frequent occurrence of horizontal gene transfer. 5. The evolutionary relatedness of organisms is best reflected in phylogenetic trees. Cell structure and function 6. The structure and function of microorganisms have been revealed by the use of microscopy (including bright field, phase contrast, fluorescent, and electron). 7. Bacteria have unique cell structures that can be targets for antibiotics, immunity, and phage infection. 8. Bacteria and archaea have specialized structures (e.g., flagella, endospores, and pili) that often confer critical capabilities. 9. While microscopic eukaryotes (for example, fungi, protozoa, and algae) carry out some of the same processes as bacteria, many of the cellular properties are fundamentally different. 10. The replication cycles of viruses (lytic and lysogenic) differ among viruses and are determined by their unique structures and genomes. Metabolic pathways 11. Bacteria and aarchaea exhibit extensive, and often unique, metabolic diversity (e.g., nitrogen fixation, methane production, anoxygenic photosynthesis). 12. The interactions of microorganisms among themselves and with their environment are determined by their metabolic abilities (e.g., quorum sensing, oxygen consumption, nitrogen transformations). 13. The survival and growth of any microorganism in a given environment depends on its metabolic characteristics. 14. The growth of microorganisms can be controlled by physical, chemical, mechanical, or biological means. Information flow and genetics 15. Genetic variations can impact microbial functions (e.g., in biofilm formation, pathogenicity, and drug resistance). 16. Although the central dogma is universal in all cells, the processes of replication, transcription, and translation differ in bacteria, aarchaea, and eeukaryotes. 17. The regulation of gene expression is influenced by external and internal molecular cues and/or signals. 18. The synthesis of viral genetic material and proteins is dependent on host cells. 19. Cell genomes can be manipulated to alter cell function. Microbial systems 20. Microorganisms are ubiquitous and live in diverse and dynamic ecosystems. 21. Most bacteria in nature live in biofilm communities. 22. Microorganisms and their environment interact with and modify each other. 23. Microorganisms, cellular and viral, can interact with both human and nonhuman hosts in beneficial, neutral, or detrimental ways. Impact of microorganisms 24. Microbes are essential for life as we know it and the processes that support life (e.g., in biogeochemical cycles and plant and/or animal microbiota). 25. Microorganisms provide essential models that give us fundamental knowledge about life processes. 26. Humans utilize and harness microorganisms and their products. 27. Because the true diversity of microbial life is largely unknown, its effects and potential benefits have not been fully explored. Downloaded from Susan Merkel∗ and the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology (2012). The Development of Curricular Guidelines for Introductory Microbiology that Focus on Understanding. J Microbiol Biol Educ. 2012; 13(1): 32–38. Published online 2012 May 3 Downloaded from https://www.asm.org/getattachment/1b074b9e-8522-4d9d-bbc3-c0ca9b9abf1a/FINAL_Curriculum_Guidelines_w_title_page.pdf . Table 32.3 Examples of lower-order and higher-order learning outcomes and assessments from some ASM fundamental statements. Example core concept and fundamental statement Example lower-order learning outcome: after this unit, students should be able to …. Example higher-order learning outcome: after this unit, students should be able to …. Evolution Mutations and horizontal gene transfer, with the immense variety of microenvironments, have selected for a huge diversity of microorganisms. ... describe three mechanisms of horizontal gene transfer in bacteria. ... interpret sequence data to determine if horizontal gene transfer has occurred. Cell structure and function The structure and function of microorganisms have been revealed by the use of microscopy. ... explain how the cell structure of gram-negative and gram-positive cells leads to a given gram stain result. .. .compare and contrast the effects of doing the gram stain incorrectly on gram-negative and gram-positive bacteria. Metabolic pathways Bacteria and archaea exhibit extensive, and often unique, metabolic diversity. ... draw a diagram that shows the process of nitrogen fixation in cyanobacteria. ... design a mechanism that would allow a bacterium to protect its nitrogenase from oxygen. Information flow and genetics Genetic variations can impact microbial functions. ... identify each of the following: point mutation, genetic insertion, genetic deletion and frameshift mutation. … predict whether or not a given mutation (genotypic change) would result in a change of function (phenotypic change). Microbial systems Most bacteria in nature live in biofilm communities. ... order the stages of biofilm formation and maturation. ... develop a drug that would prevent biofilm formation. Impact of microorganisms Because the true diversity of microbial life is largely unknown, its effects and potential benefits have not been fully explored. ... measure cell density using viable cell counts and microscopy methods and explain the differences. ... propose an experiment that would allow you to prospect for antibiotics in a new environment. From: Merkel, S., August 2016. FEMS Microbiol Lett. 363(16), pii: fnw172. https://doi.org/10.1093/femsle/fnw172 . Epub July 12, 2016. American Society for Microbiology resources in support of an evidence-based approach to teaching microbiology: Downloaded from https://academic.oup.com/femsle/article/363/16/fnw172/2197755 and content available at https://www.asm.org/ASM/media/Education/FINAL-Learning-Outcomes-w-title-page.pdf . Table 32.4 List of ASM resources that support evidence-based teaching and learning. Resource Description Website ASM Curriculum Guidelines for an Undergraduate Microbiology Course Concepts and competencies for an introductory undergraduate microbiology course https://www.asm.org/index.php/guidelines/curriculum-guidelines Learning outcomes for the ASM Curriculum Guidelines Examples of lower-order and higher-order learning outcomes https://www.asm.org/index.php/guidelines/curriculum-guidelines ASM Sample Questions in Microbiology (release in 2016) Collection of peer-reviewed multiple-choice and true/false questions http://www.asmscience.org Microbiology Concept inventory and Microbiology for Health Sciences Concept inventory (release in 2017) Tested questions developed to assess how well students understand critical concepts facultyprograms.org/index.php/resources/concept-inventories Journal of Microbiology and Biology Education Open access, peer-reviewed collection of research articles and activities http://www.asmscience.org/content/journal/jmbe MicrobeLibrary Peer-reviewed visual resources and laboratory protocols https://www.microbelibrary.org ASM Faculty Programs Portal to the ASM educational resources http://www.facultyprograms.org ASMCUE Interactive 4-day conference for biology educators http://www.asmcue.org Biology scholars Program Five-month hybrid courses offering a range of training in microbiology education http://www.facultyprograms.org/index.php/biology-scholars-hybrid-courses Science Teaching fellowship Program Five-month online program to prepare doctoral-trained students for science teaching positions http://facultyprograms.org/index.php/stf-program ASM webinars Online courses on teaching and research http://www.facultyprograms.org/index . Php/webinars Guidelines for biosafety in Teaching laboratories A comprehensive guidebook of best practices for safely handling BSL-1 and BSL-2 microbes in teaching labs. https://www.asm.org/index.php/guidelines/safety-guidelines Downloaded from Merkel S., August 2016. FEMS Microbiol Lett. 363(16), pii: fnw172. https://doi.org/10.1093/femsle/fnw172 . Epub July 12, 2016. American Society for Microbiology resources in support of an evidence-based approach to teaching microbiology. https://academic.oup.com/femsle/article/363/16/fnw172/2197755 . Additional guidelines have also been published for Nursing and Allied Health ( McKay and ASM MINAH Undergraduate Curriculum Guidelines Committee, 2018 ). The Undergraduate Education Committee has developed a set of curriculum guidelines for microbiology majors in "ASM's curriculum recommendations: Microbiology Majors Program"( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). In this document, ASM provides "recommendations for conceptual knowledge, recommended core and elective courses, and laboratory skills and safety as well as issues for further action and discussion … to be used by programs in their own assessment, maintenance, and formation of strong programs in microbiology." Table 32.5 contains the recommendations for UG microbiology education and core and elective courses, and Table 32.6 lists recommendations for laboratory skills, laboratory safety guidelines from ASM have also been published and are listed for BSL1 and BSL2 levels ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). Table 32.5 Recommended core and elective courses from the ASM Curricular Guidelines. Core courses for microbiology majors • Introduction to microbiology (with lab) • Microbial physiology (with lab) • Microbial genetics (with lab) • Microbial diversity and eecology (with lab) • One advanced course that includes laboratory • Capstone course (e.g., senior seminar presentation, independent research project, internship) Elective courses for microbiology majors • Immunology • Pathogenic microbiology • Food and dairy microbiology • Environmental microbiology • Marine microbiology • Industrial and applied microbiology • Biotechnology • Bioinformatics • Virology and other acellular agents • Parasitology/protozoology • Mycology • phycology • Epidemiology • Public health • Undergraduate research and internship • Careers in microbiology a • Bioethics a Support courses for microbiology majors • General biology with lab (1 year) • Cell and molecular biology • General chemistry with lab • Organic chemistry with lab • Biochemistry (1 semester) • Math (through calculus) • Physics (1 year) • Statistics (1 semester) • Scientific writing and technical communication a Bioethics and careers in microbiology could be integrated into core course material to ensure exposure of all students to the topics. Downloaded from Merkel S., the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012. The development of curricular guidelines for introductory microbiology that focus on understanding. J. Microbiol. Biol. Educ. 13(1), 32–38. Published online May 3, 2012 Downloaded from https://www.asm.org/getattachment/1b074b9e-8522-4d9d-bbc3-c0ca9b9abf1a/FINAL_Curriculum_Guidelines_w_title_page.pdf . Also available from- https://www.asm.org/Articles/Education/ASM-Recommended-Curriculum-for-Microbiology-Majors . Table 32.6 Recommended scientific thinking and laboratory skills from ASM curriculum recommendations. Scientific thinking 1. Ability to apply the process of science a. Demonstrate an ability to formulate hypotheses and design experiments based on the scientific method. b. Analyze and interpret results from a variety of microbiological methods and apply these methods to analogous situations. 2. Ability to use quantitative reasoning a. Use mathematical reasoning and graphing skills to solve problems in microbiology. 3. Ability to communicate and collaborate with other disciplines a. Effectively communicate fundamental concepts of microbiology in written and oral format. b. Identify credible scientific sources and interpret and evaluate the information therein. 4. Ability to understand the relationship between science and society a. Identify and discuss ethical issues in microbiology. Microbiology laboratory skills 5. Properly prepare and view specimens for examination using microscopy (bright field and, if possible, phase contrast). 6. Use pure culture and selective techniques to enrich for and isolate microorganisms. 7. Use appropriate methods to identify microorganisms (media-based, molecular, and serological). 8. Estimate the number of microorganisms in a sample (using, for example, direct count, viable plate count, and spectrophotometric methods). 9. Use appropriate microbiological and molecular lab equipment and methods. 10. Practice safe microbiology, using appropriate protective and emergency procedures. 11. Document and report on experimental protocols, results, and conclusions. Downloaded from https://www.asm.org/ASM/media/Education/ASM-Curriculum-Guidelines.pdf . These recommendations provide a starting framework for developing curriculum requirements fit to the goals of programs and the target students they serve. ASM prompted further discussion in its curricular recommendations stating "A major problem noted was time and resources. The list is long and it is not possible to fit all of these courses into a 4-year program nor are all courses appropriate for all students. A critical component to this issue may be in getting the students into the major early. The Introduction to Microbiology course should be designed so that it can be taken no later than the fourth semester (end of second year) of study. This will allow for two years of advanced study of microbiology" ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). Another potential solution would be to require students to double major. In this dual BS model, students would be required to complete two BS degrees. Core science and laboratory courses would be covered in the first BS science degree (to fit the target career trajectory). The BS in Microbial Forensics specialty area would therefore have additional coursework space for upper division science and elective laboratory courses. There are a few forensic science programs that use this model such as the University of Central Oklahoma. Another issue that the ASM membership identified is the recommendation that the core courses all have labs. Laboratory courses are by their nature expensive. ASM raised the following question: "How can departments with limited resources deal with this resource problem?" One suggestion is to offer a two-semester sequence of independent lab courses that teach the skills needed to be a microbiologist rather than offer a lab with every course. This approach may reduce the current recommendation of five lab courses in the core curriculum to two semesters of independent lab courses that cover the basic skills and one advanced course with laboratory ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). The depth of the curriculum will vary depending on the level and occupation of the student. High school students may have abbreviated versions that can pique their appetites to learn more. College students will need comprehensive training to prepare them for graduate school or for entering the workforce. Legal experts will require an overview to understand the limitations of the field and how to support or refute scientific findings. Curricular guidelines from the American Society of Microbiology and American Academy of Forensic Sciences Recent national reports have addressed the need for changing how science courses in higher education are taught, so that students develop a deeper understanding of critical concepts and the analytical and cognitive skills needed to address future challenges. Our competitiveness and national success depend on our students' science aptitude ( edsource ). National Science Teachers Association teachers point to lack of student motivation as a major concern in US science education ( news ). The need for a new approach to improve science and math education is compelling and well supported. In the American Academy for the Advancement of Science Project 2061, "just doing more science was not the answer" ( project 2061 ). Current methods put more value on learning correct answers versus exploration, collaboration, and inquiry. They recommend "practices where the learning of science is as much about the process as the result or outcome, and where students can ask questions and are actively engaged in the learning process, refocusing student learning from knowledge and comprehension to application and analysis." ( project 2061 ). Inquiry is "central to science learning" as students develop their understanding of science concepts combining knowledge, reasoning, and thinking while enhancing comprehension through hands-on learning. The National Research Council agrees. They state "effective instruction capitalizes on students' early interest and experiences, identifies and builds on what they know, and provides them with experiences that serve to engage them in the practices of science and sustain their interest" ( NRC, 2011 ). Because discipline-specific professional societies have national stature and are often the organizations that set guidelines or standards within a discipline, they are well-suited to play a role in promoting systemic change. In 2010, the AAAS and the NSF released the report entitled "Vision and Change in Undergraduate Biology Education: A Call to Action" ( American Association for the Advancement of Science (AAAS), 2011 ). In response to these recommendations, the ASM revised its curriculum guidelines for introductory microbiology courses to emphasize a deeper "understanding of core concepts, critical thinking, and essential laboratory skills." ( American Association for the Advancement of Science (AAAS), 2011 ). In 2012, the ASM Education Board published curriculum guidelines and recommendations for education in microbiology ( Merkel & the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ). "These Guidelines incorporate many of the recommendations made in Vision and Change. They embrace the scientific process and thinking skills put forth in Vision and Change, adding microbiology-based laboratory skills. Further to adopting the five core concepts of Vision and Change, the Guidelines added a sixth core concept, Impact of Microorganisms, specific to this field. Each of the six core concepts is exemplified by four or five microbiology-specific fundamental statements, which reflect basic concepts that are important for all microbiology students to understand in depth. These 27 fundamental statements, together with the four scientific thinking skills and seven laboratory skills, form a comprehensive framework for an undergraduate microbiology course. ( Merkel and the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ). In addition, the ASM Curriculum Guidelines were designed to focus microbiology teaching on student-centered goals and priorities and to enable educators to adopt the discipline-based approach to course design for microbiology courses." ( Horak et al., 2015 ) . The American Society for Microbiology has developed a number of educational programs and resources with student-centered learning modules that may be useful in developing forensic microbiology education programs ( Merkel, 2016 ). The resources cited above are codified in Table 32.2 . ( Merkel & the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012 ), 32.3 ( Merkel, 2016 ), and 32.4. ( Merkel, 2016 ) ( Table 32.3 , Table 32.4 ). Table 32.2 ASM Curriculum Guidelines: list of recommended skills, core concepts, and fundamental statements. Evolution 1. Cells, organelles (e.g., mitochondria and chloroplasts), and all major metabolic pathways evolved from early prokaryotic cells. 2. Mutations and horizontal gene transfer, with the immense variety of microenvironments, have selected for a huge diversity of microorganisms. 3. Human impact on the environment influences the evolution of microorganisms (e.g., emerging diseases and the selection of antibiotic resistance). 4. The traditional concept of species is not readily applicable to microbes due to asexual reproduction and the frequent occurrence of horizontal gene transfer. 5. The evolutionary relatedness of organisms is best reflected in phylogenetic trees. Cell structure and function 6. The structure and function of microorganisms have been revealed by the use of microscopy (including bright field, phase contrast, fluorescent, and electron). 7. Bacteria have unique cell structures that can be targets for antibiotics, immunity, and phage infection. 8. Bacteria and archaea have specialized structures (e.g., flagella, endospores, and pili) that often confer critical capabilities. 9. While microscopic eukaryotes (for example, fungi, protozoa, and algae) carry out some of the same processes as bacteria, many of the cellular properties are fundamentally different. 10. The replication cycles of viruses (lytic and lysogenic) differ among viruses and are determined by their unique structures and genomes. Metabolic pathways 11. Bacteria and aarchaea exhibit extensive, and often unique, metabolic diversity (e.g., nitrogen fixation, methane production, anoxygenic photosynthesis). 12. The interactions of microorganisms among themselves and with their environment are determined by their metabolic abilities (e.g., quorum sensing, oxygen consumption, nitrogen transformations). 13. The survival and growth of any microorganism in a given environment depends on its metabolic characteristics. 14. The growth of microorganisms can be controlled by physical, chemical, mechanical, or biological means. Information flow and genetics 15. Genetic variations can impact microbial functions (e.g., in biofilm formation, pathogenicity, and drug resistance). 16. Although the central dogma is universal in all cells, the processes of replication, transcription, and translation differ in bacteria, aarchaea, and eeukaryotes. 17. The regulation of gene expression is influenced by external and internal molecular cues and/or signals. 18. The synthesis of viral genetic material and proteins is dependent on host cells. 19. Cell genomes can be manipulated to alter cell function. Microbial systems 20. Microorganisms are ubiquitous and live in diverse and dynamic ecosystems. 21. Most bacteria in nature live in biofilm communities. 22. Microorganisms and their environment interact with and modify each other. 23. Microorganisms, cellular and viral, can interact with both human and nonhuman hosts in beneficial, neutral, or detrimental ways. Impact of microorganisms 24. Microbes are essential for life as we know it and the processes that support life (e.g., in biogeochemical cycles and plant and/or animal microbiota). 25. Microorganisms provide essential models that give us fundamental knowledge about life processes. 26. Humans utilize and harness microorganisms and their products. 27. Because the true diversity of microbial life is largely unknown, its effects and potential benefits have not been fully explored. Downloaded from Susan Merkel∗ and the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology (2012). The Development of Curricular Guidelines for Introductory Microbiology that Focus on Understanding. J Microbiol Biol Educ. 2012; 13(1): 32–38. Published online 2012 May 3 Downloaded from https://www.asm.org/getattachment/1b074b9e-8522-4d9d-bbc3-c0ca9b9abf1a/FINAL_Curriculum_Guidelines_w_title_page.pdf . Table 32.3 Examples of lower-order and higher-order learning outcomes and assessments from some ASM fundamental statements. Example core concept and fundamental statement Example lower-order learning outcome: after this unit, students should be able to …. Example higher-order learning outcome: after this unit, students should be able to …. Evolution Mutations and horizontal gene transfer, with the immense variety of microenvironments, have selected for a huge diversity of microorganisms. ... describe three mechanisms of horizontal gene transfer in bacteria. ... interpret sequence data to determine if horizontal gene transfer has occurred. Cell structure and function The structure and function of microorganisms have been revealed by the use of microscopy. ... explain how the cell structure of gram-negative and gram-positive cells leads to a given gram stain result. .. .compare and contrast the effects of doing the gram stain incorrectly on gram-negative and gram-positive bacteria. Metabolic pathways Bacteria and archaea exhibit extensive, and often unique, metabolic diversity. ... draw a diagram that shows the process of nitrogen fixation in cyanobacteria. ... design a mechanism that would allow a bacterium to protect its nitrogenase from oxygen. Information flow and genetics Genetic variations can impact microbial functions. ... identify each of the following: point mutation, genetic insertion, genetic deletion and frameshift mutation. … predict whether or not a given mutation (genotypic change) would result in a change of function (phenotypic change). Microbial systems Most bacteria in nature live in biofilm communities. ... order the stages of biofilm formation and maturation. ... develop a drug that would prevent biofilm formation. Impact of microorganisms Because the true diversity of microbial life is largely unknown, its effects and potential benefits have not been fully explored. ... measure cell density using viable cell counts and microscopy methods and explain the differences. ... propose an experiment that would allow you to prospect for antibiotics in a new environment. From: Merkel, S., August 2016. FEMS Microbiol Lett. 363(16), pii: fnw172. https://doi.org/10.1093/femsle/fnw172 . Epub July 12, 2016. American Society for Microbiology resources in support of an evidence-based approach to teaching microbiology: Downloaded from https://academic.oup.com/femsle/article/363/16/fnw172/2197755 and content available at https://www.asm.org/ASM/media/Education/FINAL-Learning-Outcomes-w-title-page.pdf . Table 32.4 List of ASM resources that support evidence-based teaching and learning. Resource Description Website ASM Curriculum Guidelines for an Undergraduate Microbiology Course Concepts and competencies for an introductory undergraduate microbiology course https://www.asm.org/index.php/guidelines/curriculum-guidelines Learning outcomes for the ASM Curriculum Guidelines Examples of lower-order and higher-order learning outcomes https://www.asm.org/index.php/guidelines/curriculum-guidelines ASM Sample Questions in Microbiology (release in 2016) Collection of peer-reviewed multiple-choice and true/false questions http://www.asmscience.org Microbiology Concept inventory and Microbiology for Health Sciences Concept inventory (release in 2017) Tested questions developed to assess how well students understand critical concepts facultyprograms.org/index.php/resources/concept-inventories Journal of Microbiology and Biology Education Open access, peer-reviewed collection of research articles and activities http://www.asmscience.org/content/journal/jmbe MicrobeLibrary Peer-reviewed visual resources and laboratory protocols https://www.microbelibrary.org ASM Faculty Programs Portal to the ASM educational resources http://www.facultyprograms.org ASMCUE Interactive 4-day conference for biology educators http://www.asmcue.org Biology scholars Program Five-month hybrid courses offering a range of training in microbiology education http://www.facultyprograms.org/index.php/biology-scholars-hybrid-courses Science Teaching fellowship Program Five-month online program to prepare doctoral-trained students for science teaching positions http://facultyprograms.org/index.php/stf-program ASM webinars Online courses on teaching and research http://www.facultyprograms.org/index . Php/webinars Guidelines for biosafety in Teaching laboratories A comprehensive guidebook of best practices for safely handling BSL-1 and BSL-2 microbes in teaching labs. https://www.asm.org/index.php/guidelines/safety-guidelines Downloaded from Merkel S., August 2016. FEMS Microbiol Lett. 363(16), pii: fnw172. https://doi.org/10.1093/femsle/fnw172 . Epub July 12, 2016. American Society for Microbiology resources in support of an evidence-based approach to teaching microbiology. https://academic.oup.com/femsle/article/363/16/fnw172/2197755 . Additional guidelines have also been published for Nursing and Allied Health ( McKay and ASM MINAH Undergraduate Curriculum Guidelines Committee, 2018 ). The Undergraduate Education Committee has developed a set of curriculum guidelines for microbiology majors in "ASM's curriculum recommendations: Microbiology Majors Program"( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). In this document, ASM provides "recommendations for conceptual knowledge, recommended core and elective courses, and laboratory skills and safety as well as issues for further action and discussion … to be used by programs in their own assessment, maintenance, and formation of strong programs in microbiology." Table 32.5 contains the recommendations for UG microbiology education and core and elective courses, and Table 32.6 lists recommendations for laboratory skills, laboratory safety guidelines from ASM have also been published and are listed for BSL1 and BSL2 levels ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). Table 32.5 Recommended core and elective courses from the ASM Curricular Guidelines. Core courses for microbiology majors • Introduction to microbiology (with lab) • Microbial physiology (with lab) • Microbial genetics (with lab) • Microbial diversity and eecology (with lab) • One advanced course that includes laboratory • Capstone course (e.g., senior seminar presentation, independent research project, internship) Elective courses for microbiology majors • Immunology • Pathogenic microbiology • Food and dairy microbiology • Environmental microbiology • Marine microbiology • Industrial and applied microbiology • Biotechnology • Bioinformatics • Virology and other acellular agents • Parasitology/protozoology • Mycology • phycology • Epidemiology • Public health • Undergraduate research and internship • Careers in microbiology a • Bioethics a Support courses for microbiology majors • General biology with lab (1 year) • Cell and molecular biology • General chemistry with lab • Organic chemistry with lab • Biochemistry (1 semester) • Math (through calculus) • Physics (1 year) • Statistics (1 semester) • Scientific writing and technical communication a Bioethics and careers in microbiology could be integrated into core course material to ensure exposure of all students to the topics. Downloaded from Merkel S., the ASM Task Force on Curriculum Guidelines for Undergraduate Microbiology, 2012. The development of curricular guidelines for introductory microbiology that focus on understanding. J. Microbiol. Biol. Educ. 13(1), 32–38. Published online May 3, 2012 Downloaded from https://www.asm.org/getattachment/1b074b9e-8522-4d9d-bbc3-c0ca9b9abf1a/FINAL_Curriculum_Guidelines_w_title_page.pdf . Also available from- https://www.asm.org/Articles/Education/ASM-Recommended-Curriculum-for-Microbiology-Majors . Table 32.6 Recommended scientific thinking and laboratory skills from ASM curriculum recommendations. Scientific thinking 1. Ability to apply the process of science a. Demonstrate an ability to formulate hypotheses and design experiments based on the scientific method. b. Analyze and interpret results from a variety of microbiological methods and apply these methods to analogous situations. 2. Ability to use quantitative reasoning a. Use mathematical reasoning and graphing skills to solve problems in microbiology. 3. Ability to communicate and collaborate with other disciplines a. Effectively communicate fundamental concepts of microbiology in written and oral format. b. Identify credible scientific sources and interpret and evaluate the information therein. 4. Ability to understand the relationship between science and society a. Identify and discuss ethical issues in microbiology. Microbiology laboratory skills 5. Properly prepare and view specimens for examination using microscopy (bright field and, if possible, phase contrast). 6. Use pure culture and selective techniques to enrich for and isolate microorganisms. 7. Use appropriate methods to identify microorganisms (media-based, molecular, and serological). 8. Estimate the number of microorganisms in a sample (using, for example, direct count, viable plate count, and spectrophotometric methods). 9. Use appropriate microbiological and molecular lab equipment and methods. 10. Practice safe microbiology, using appropriate protective and emergency procedures. 11. Document and report on experimental protocols, results, and conclusions. Downloaded from https://www.asm.org/ASM/media/Education/ASM-Curriculum-Guidelines.pdf . These recommendations provide a starting framework for developing curriculum requirements fit to the goals of programs and the target students they serve. ASM prompted further discussion in its curricular recommendations stating "A major problem noted was time and resources. The list is long and it is not possible to fit all of these courses into a 4-year program nor are all courses appropriate for all students. A critical component to this issue may be in getting the students into the major early. The Introduction to Microbiology course should be designed so that it can be taken no later than the fourth semester (end of second year) of study. This will allow for two years of advanced study of microbiology" ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). Another potential solution would be to require students to double major. In this dual BS model, students would be required to complete two BS degrees. Core science and laboratory courses would be covered in the first BS science degree (to fit the target career trajectory). The BS in Microbial Forensics specialty area would therefore have additional coursework space for upper division science and elective laboratory courses. There are a few forensic science programs that use this model such as the University of Central Oklahoma. Another issue that the ASM membership identified is the recommendation that the core courses all have labs. Laboratory courses are by their nature expensive. ASM raised the following question: "How can departments with limited resources deal with this resource problem?" One suggestion is to offer a two-semester sequence of independent lab courses that teach the skills needed to be a microbiologist rather than offer a lab with every course. This approach may reduce the current recommendation of five lab courses in the core curriculum to two semesters of independent lab courses that cover the basic skills and one advanced course with laboratory ( Emmert and the ASM Task Committee on Laboratory Biosafety, 2013 ). The depth of the curriculum will vary depending on the level and occupation of the student. High school students may have abbreviated versions that can pique their appetites to learn more. College students will need comprehensive training to prepare them for graduate school or for entering the workforce. Legal experts will require an overview to understand the limitations of the field and how to support or refute scientific findings. Basic epidemiology Epidemiology is a cornerstone of public health and is critical to microbial forensics. One goal of epidemiology is to recognize infectious disease outbreaks and to attribute the outbreak to a source to prevent additional cases (see Chapter 16 for further detailed information). In many aspects, microbial forensics employs the same tools as those used in epidemiology. A training program in microbial forensics will parallel many parts of current programs in epidemiology. Models can be obtained from epidemiology curricula, and experience from natural outbreaks will help guide how microbial forensic scientists will perform investigations of biocrimes. Tracing the course of an outbreak will assist in identifying the index case, cause, and/or time of the outbreak. With many disease outbreaks, as well as cases of unusual infections (e.g., monkeypox), the recurring question will be: Is this a natural event or an intentional attack? Epidemiological factors will help distinguish between natural or intentional events and enable more effective responses in either event. A biocrime may be recognized through surveillance linking multiple unusual disease occurrences in contiguous or noncontiguous geographic areas. A microbial forensic investigation may be based on initial public health findings and then proceed further to address attribution to identify the perpetrator(s) of a biocrime or bioterrorist act. Molecular epidemiology Molecular epidemiology focuses on the contribution of potential genetic, identified at the molecular level, and environmental risk factors to the etiology, distribution, and prevention of disease within families and across populations ( Riley, 2004 , Eybpoosh et al., 2017 , Jagielski et al., 2016 ) [(3) also see the Genomics section below]. The field provides a good example where application of newer technologies may help overcome many of the same problems encountered with traditional epidemiology with respect to study design and interpretation (4). Molecular tools can be employed to characterize and potentially individualize samples and isolates to address forensically relevant questions. This subdivision of epidemiology has special importance in microbial forensics because it is desirable to determine the source of a particular microbe used in a crime. Highly discriminating assays can precisely identify strains and isolates, resulting in a more focused and effective investigation. These types of data could associate a sample with a single geographic area, even possibly a particular laboratory or flask, or with the specific conditions and nutrients used to culture the microorganism. Some of these aspects are discussed in the chapters on anthrax. Microbes and their products as biological weapons Agents that can be used in biocrimes span the microbial world of viruses, bacteria, fungi, eukaryotic parasites, and toxins. It is important to have a basic understanding of each type of microorganism to appreciate the factors that make a particular microbe a serious threat as a weapon. These factors include accessibility, stability, transmissibility, associated history with weapons programs, and the capacity to produce disease with transient or sustained consequences, including death. Different technologies are needed to culture bacteria and viruses, as they differ greatly in growth requirements. Indeed, some microbes are difficult or impossible to culture. Such information may help an investigator understand what microbes should be considered as a high threat and how they might have been used in a particular circumstance. A basic understanding of different microbial classes and their products would include human, animal, and plant pathogens ( Schutzer et al., 2005 , Schmedes et al., 2016 , Teshome, 2016 ). Host factors including immune responses It is important to understand how the host responds to microbes and the unique signatures that can be found, including those in response to exposure to a particular microbe or for timing the exposure to a pathogen [( Tomkovich and Jobin, 2016 ) and see Chapter 20]. For forensic purposes, an immediate goal is to distinguish a potential victim from a perpetrator and to distinguish between a natural or intentional event. A basic understanding of the immune system, how antibodies are generated, when different classes of antibodies appear, and what cell types and their signatures are generated may assist in criminal investigations. Processes and technology Sophisticated equipment (technology) that resides in the laboratory is only part of the process for obtaining reliable and meaningful information. The process begins with sample acquisition and proceeds with packaging, storage, and analysis and ultimately ends up with interpreting the results. All aspects are important and must be integrated effectively to have high confidence in results. Crime scenes and chain of custody After recognizing that a bioterrorist act or biocrime has occurred, defining the crime scene is the first important part of an investigation. Depending on the nature of the crime, there may be multiple crime scenes requiring different sets of skills, knowledge, equipment, and abilities (see Chapters 21 and 22Chapter 21Chapter 22 for more details). Once the crime scene(s) has (have) been identified and delimited, a plan is needed to properly collect and maintain integrity of the evidence that may be subsequently analyzed. Practices are needed to maintain the integrity and that minimize contamination of the evidence. The handling and storage of evidence is integral to minimizing degradation of the target analytes. Microbial contamination may be somewhat different from other types of contamination because the contaminating organisms can replicate thus confounding results. The nuances of a microbial forensic investigation add a layer on top of traditional crime scene investigations, particularly because of the hazardous nature of the evidence. The need for proper documentation may seem obvious but it is a very important part of maintaining the integrity of the evidence. Crime scenes are chaotic and missteps can occur. To minimize missteps in handling documentation procedures should be established so the crime scene can be reconstructed at a later date for investigators or in a court of law. It is likely that biocrimes and acts of bioterrorism will add another dimension of complexity because (i) there is less experience in crime scene collection due to (fortunately) fewer cases, (ii) addressing the safety of victims will not be trivial, (iii) investigators will be wearing cumbersome but absolutely necessary personal protective equipment (PPE), (iv) the best approaches for collection and preservation of evidence may have to be determined at the scene given the limited extant information available, and (v) the response to biocrime events by using forensic science in an attribution involves a complex interplay among science, policy, law, law enforcement, public health, medical, and media communities ( bioattribution ) and may require several different areas of expertise and authority. This aggregation of diverse disciplines and professions, "brought together to develop an understanding and action plan in response to a suspicious event" is challenging in a domestic context, and even more formidable if an event has global implications ( Bidwell et al., 2016 ). The historical knowledge of experts intimately involved in past events and their understanding of the dynamics of the interplay of science–law–bureaucracy–media are pivotal in developing effective and efficient responses for future attribution determinations as well as in the training of our students. Thus, crime scene investigation, safety, PPE, collection, chain of custody, and sharing important past casework experiences and examples of how to manage the interplay of the different disciplines and professionals should be included as essential parts of any curriculum. The first responder community needs to be aware of the safety issues and the methods of collection because they may become involved in performing evidence collection. Laboratorians must understand these processes because better decisions can be made as to what evidence is pertinent for analysis. Lawyers and judges will want to understand the basics of chain of custody to be assured that acceptable handling methods have been exercised to maintain the integrity of the evidence. Those who will have contact with the crime scene, as well as those in the laboratory who require downstream interoperability of collected evidence, will have to learn basic do's and don'ts of crime scene investigation ( United States Federal Bureau of Investigation Laboratory FBI, 1999 ) to effect a better systems-based process. Education about crime scene investigation will help ensure use of validated microbial identification practices that will collect the most pertinent evidence and will best preserve the integrity of the evidence for analysis in a forensic laboratory. Training resources, tools, and opportunities that may be useful to educators are available from other dedicated academic, government, and private industry groups in many of the subtopics listed in Table 32.1 . For example, there are research papers, newsletters, webinars, seminars, and guidelines at CDC, the Biological Security Countering Weapons of Mass Destruction (CWMD) US Homeland Security Office and disaster, mortuary operations response team units (DMORT) ( emergency, 2022 , topic, 2022 , Nolte, 2003 , Preparedness, 2022 ), which may be useful to educators, trainers, and students depending on the goals of the training and target audience. Sample collection and preservation of forensic evidence One must understand the tools available to collect the sample as well as the limitations posed with a collection process or tool (see Chapters 21 and 22Chapter 21Chapter 22 (∗new Budowle et al., 2005 , Budowle et al., 2006 )). While most approaches focus on collection tools, it is very important to consider sampling strategies to obtain the most relevant data, which involves strategic planning, logistics, and statistics. Conditions that are proper for collection and/or preservation of one microbe may be deleterious for another and, for that matter, to traditional forensic materials such as human DNA, fingerprints, and trace materials. For example, foodborne pathogens are particularly vexing; conditions that are intended to preserve the material may promote growth of natural bacteria in a food product, and this overgrowth may destroy or obscure the initial bioweapon. Tools for collection need to be validated for efficient collection and for determining that they do not react with the target of interest. Tools developed for powder collection may be inefficient or ineffective for collecting plant material. Sample collection is not trivial and requires substantial consideration. Issues related to the practices of sample collection, handling, transportation, and storage of microbes in the investigation of biocrimes and guidelines for the collection of evidence by physicians and medical personnel from potential victims of bioterrorism have been published and are valuable for education and training programs ( Schutzer et al., 2005 ). The same issues about evidence collection will need to be applied to preservation processes. It is imperative to prevent further degradation of the evidentiary target once collected. Conditions for preservation apply for packaging and shipping, for maintaining of the evidence in the laboratory, and for postanalysis storage. Extraction Extraction efficiency, particularly of interest to the scientist, pertains to obtaining the highest quality and quantity yield as possible of the target of interest. Yield is related to the quantity and purity of target and removal from the collection matrix. Targets can include cells, nucleic acids, proteins, nutrients, growth materials, and elements. Advanced microscopy Various forms of microscopy may be used to visualize the evidence. These tools may range from light microscopy to electron microscopy to atomic force microscopy and are available for characterization of a microbe. These approaches are rapid and can be used to identify candidate threats as well as to dismiss hoaxes. Proteomics Defining chemical and physical properties of a biological agent can provide information on how and when the agent was produced and can be used to determine if two microbial samples were produced by the same process. Proteomics is a comprehensive study of the protein composition of biological systems at a moment in time or at different stages of a microbe's growth. Many proteins are conserved and can be used for general identification, while other proteins may be expressed in response to environmental stimuli, growth state, or growth conditions. Protein profiling can provide information beyond genomic analysis about the conditions of the bioweapon before host exposure ( Gil and Monteoliva, 2014 ). Genomics One of the fastest growing areas with implications for microbial forensics is genomics. More rapid and in-depth sequencing of microbes is possible today than it was a decade ago; sequencing and targeted methods such as those used in the investigation of the anthrax-letter attack seem almost antiquated today. Genomic analyses will continue to be essential in identifying species, strains, isolates, and individual samples to assist in a microbial forensic investigation. ( Schmedes et al., 2016 , Budowle et al., 2017 , Karlsson et al., 2013 ). The rapid expansion of sequencing capabilities, where sequencing some microbes within a day at very deep coverage, has raised the importance of genetic identification. It will likely be a mainstay of the microbial forensic investigation of any attack with any microbe. The cost of whole-genome sequencing has decreased at least 100-fold in just a few years. This technology has now matured into one of the methods of choice to examine the genetic structure of a particular pathogen and to identify signatures of forensic relevance. ( Schmedes et al., 2016 , Budowle et al., 2017 , Karlsson et al., 2013 )] In addition, the legal profession should have a basic understanding of the capabilities and limitations of these technologies to be successful in the courtroom, just as has been necessary for human DNA forensics. Several chapters in this book expand on technology in detail. Interpretation, statistical analysis, and confidence Interpretation of results and the bases to support interpretation by scientists will be critical to the end-user stakeholders that rely on microbial forensic evidence for legal proceedings, for setting policy or responding to a threat or an attack. Interpretations could be as simple as positive or negative to very complex evaluations using limits of detection, complex algorithms for identifying. A host of answers and additional questions can arise from data interpretation. Central to interpretation is, when possible, a statistical analysis of the findings which should be performed to provide significance of the result, uncertainty, or to convey the strength of the evidential results. A variety of statistical approaches exists, and it is imperative to understand which ones apply to particular analyses and interpretations. One needs to consider that traditional statistical analyses may not apply to microbial evidence in some cases. With the advent and ease of massive parallel sequencing on various platforms and long-read sequencing with the MinIon (Oxford Nanopore) or PacBio (Menlo Park, CA), whole genomes of suspected biothreat agents can be quickly and easily obtained. However, the sequence differences may be limited to only informative single-nucleotide sequence (SNP) differences as represented by the genus Bacillus ( Derzelle et al., 2015 ). Genetically similar microbes are analyzed via comparative genomics tools where multiple SNP patterns (canonical SNPs (canSNPs)) emerge and can distinguish geographically unique strains from one another when compared with reference strains of known biothreat agents ( Dahiya, 2017 ). Many different phylogenetic tools and software are available now to perform such genetic comparisons. Working groups such as the AniBio Threat project ( anibiothreat ) in Europe developed standard protocols and expanded molecular databases to better assist in determining if an outbreak is natural or intentional ( Derzelle and Thierry, 2013 ). Both scientists and legal analysts need to understand (or at least appreciate) the results and their significance. Moreover, the degree of confidence that can be placed on a result must be understood so that the weight of a comparative analysis is not overstated. Basic statistics, probability, and population genetics are essential requirements of any curriculum involving the analysis of forensic biological evidence. Bioinformatics The term "bioinformatics" was developed as a result of the Human Genome Project. Because of the immense amount of data generated, it became necessary to apply more sophisticated computational techniques beyond what the average bench biologist had available. Bioinformatics requires a combination of data handling and analysis skills (including standard statistics) that connect routine biology with high-powered computation. As scientific investigations and data generation expand using high resolution, deep sequencing of genomes of microbes, and large-scale proteomics, computational analyses will be more critical than ever. This subject can be taught in a simplistic form for the biologist or a more complex form for the computationally inclined scientist. This interdisciplinary field has greatly impacted microbiology as well as forensics, medicine, agriculture, and other disciplines. Bioinformatic algorithms have simplified comparative genomics. Bioinformatics for medicine, microbial genome, and agriculture ( Dahiya, 2017 ) are able to determine biologically relevant patterns in complex datasets ( Damaso et al., 2018 ) as well as allow analyses of whole-genome shotgun sequencing of complex mixtures such as microbiomes ( Citation: Chen and Pachter, 2005 ). The information derived from complex data will have to be extracted using algorithms such as support vector machines ( Xu et al., 2015 , Schlecht et al., 2008 ), neural networks ( Vidaki et al., 2017 , Vidaki and Kayser, 2017 ), and other more complex algorithms yet to be developed. All scientists and individuals with interest in microbial forensic sciences will need to have a basic understanding of statistics and bioinformatic tools. Indicators of engineering With rapid developments in molecular biology to benefit humankind also comes a great potential for manipulating a microorganism for nefarious purposes. Microbes could be engineered to be more virulent, and difficult-to-obtain microbes may be synthesized de novo in a laboratory. There is a need to detect not only the microbe but to determine if it was genetically manipulated or perhaps is a novel chimera. Signatures indicative of manipulations or synthesis may be detected through sequencing and bioinformatic analyses. The skills and materials needed to manipulate a microbial genome may provide clues about the perpetrator and degree of sophistication used to develop the biothreat agent. This capability should be of interest to law enforcement and the intelligence community for supporting investigative leads. Population genetics A knowledge of population genetics is essential for understanding the rarity of a genetic (and sometimes protein) profile derived from an evidence sample. Molecular epidemiology is increasingly applying the principles of evolutionary and population genetics to pathogens. It is important to understand what constitutes a sample population as opposed to a sample collection, the mode of inheritance related to a genetic marker, what significance or weight to apply to a genetic marker, what the mutation rate of a marker is, and how to combine the weight of multiple markers. Training of the student in this discipline will require basic genetic courses and more advanced courses in phylogenetic analyses and other forms of comparison. Such educational material will be found in population genetics and systematic and evolutionary biology programs. The population genetics of pathogens and its importance for microbial forensics are covered elsewhere in this book. An example of a forensic application of population genetics is the human skin microbiome. Schmedes et al. assessed human skin microbiome populations and, using supervised learning algorithms, were able to associate specific human microbiome profiles with their hosts with a high degree of accuracy ( Schmedes et al., 2017 ). Recent comprehensive skin microbiome analyses ( Ross et al., 2018 ) suggests that the skin microbiota have undergone coevolution with their corresponding mammalian hosts providing support for future development of forensic applications using skin microbiomes ( Ross et al., 2018 ). Nonbiological tools This topic is broad and can encompass tools that characterize a microbe morphologically or chemically. Subjects will range from microscopy to basic chemistry to analytical chemistry. The Amerithrax investigation demonstrated the importance of nonbiological measurements on samples of biological agents. A variety of mass spectral, spectroscopic, and other instrumental methods were used in an attempt to answer questions related to how, when, and what materials were used to produce the spore powders. Such information can be used to compare evidence directly to a reference sample or indirectly to infer something about the processes used to culture, stabilize, and/or disseminate the biothreat agent. Bioelectronic scent detectors have also been developed using human olfactory and taste receptors that detect microbes in drinking water ( Son et al., 2015 , MankiSona et al., 2017 ), These human receptors have been shown to be capable of distinguishing one trillion different olfactory targets ( Bushdid et al., 2014 ). Dogs have also been used for the detection of scents to track human waste in microbial source tracking in storm drains ( Canine Scent Detection and Microbial Source Tracking of Human Waste Contamination in Storm Drains, 2014 ). Crime scenes and chain of custody After recognizing that a bioterrorist act or biocrime has occurred, defining the crime scene is the first important part of an investigation. Depending on the nature of the crime, there may be multiple crime scenes requiring different sets of skills, knowledge, equipment, and abilities (see Chapters 21 and 22Chapter 21Chapter 22 for more details). Once the crime scene(s) has (have) been identified and delimited, a plan is needed to properly collect and maintain integrity of the evidence that may be subsequently analyzed. Practices are needed to maintain the integrity and that minimize contamination of the evidence. The handling and storage of evidence is integral to minimizing degradation of the target analytes. Microbial contamination may be somewhat different from other types of contamination because the contaminating organisms can replicate thus confounding results. The nuances of a microbial forensic investigation add a layer on top of traditional crime scene investigations, particularly because of the hazardous nature of the evidence. The need for proper documentation may seem obvious but it is a very important part of maintaining the integrity of the evidence. Crime scenes are chaotic and missteps can occur. To minimize missteps in handling documentation procedures should be established so the crime scene can be reconstructed at a later date for investigators or in a court of law. It is likely that biocrimes and acts of bioterrorism will add another dimension of complexity because (i) there is less experience in crime scene collection due to (fortunately) fewer cases, (ii) addressing the safety of victims will not be trivial, (iii) investigators will be wearing cumbersome but absolutely necessary personal protective equipment (PPE), (iv) the best approaches for collection and preservation of evidence may have to be determined at the scene given the limited extant information available, and (v) the response to biocrime events by using forensic science in an attribution involves a complex interplay among science, policy, law, law enforcement, public health, medical, and media communities ( bioattribution ) and may require several different areas of expertise and authority. This aggregation of diverse disciplines and professions, "brought together to develop an understanding and action plan in response to a suspicious event" is challenging in a domestic context, and even more formidable if an event has global implications ( Bidwell et al., 2016 ). The historical knowledge of experts intimately involved in past events and their understanding of the dynamics of the interplay of science–law–bureaucracy–media are pivotal in developing effective and efficient responses for future attribution determinations as well as in the training of our students. Thus, crime scene investigation, safety, PPE, collection, chain of custody, and sharing important past casework experiences and examples of how to manage the interplay of the different disciplines and professionals should be included as essential parts of any curriculum. The first responder community needs to be aware of the safety issues and the methods of collection because they may become involved in performing evidence collection. Laboratorians must understand these processes because better decisions can be made as to what evidence is pertinent for analysis. Lawyers and judges will want to understand the basics of chain of custody to be assured that acceptable handling methods have been exercised to maintain the integrity of the evidence. Those who will have contact with the crime scene, as well as those in the laboratory who require downstream interoperability of collected evidence, will have to learn basic do's and don'ts of crime scene investigation ( United States Federal Bureau of Investigation Laboratory FBI, 1999 ) to effect a better systems-based process. Education about crime scene investigation will help ensure use of validated microbial identification practices that will collect the most pertinent evidence and will best preserve the integrity of the evidence for analysis in a forensic laboratory. Training resources, tools, and opportunities that may be useful to educators are available from other dedicated academic, government, and private industry groups in many of the subtopics listed in Table 32.1 . For example, there are research papers, newsletters, webinars, seminars, and guidelines at CDC, the Biological Security Countering Weapons of Mass Destruction (CWMD) US Homeland Security Office and disaster, mortuary operations response team units (DMORT) ( emergency, 2022 , topic, 2022 , Nolte, 2003 , Preparedness, 2022 ), which may be useful to educators, trainers, and students depending on the goals of the training and target audience. Sample collection and preservation of forensic evidence One must understand the tools available to collect the sample as well as the limitations posed with a collection process or tool (see Chapters 21 and 22Chapter 21Chapter 22 (∗new Budowle et al., 2005 , Budowle et al., 2006 )). While most approaches focus on collection tools, it is very important to consider sampling strategies to obtain the most relevant data, which involves strategic planning, logistics, and statistics. Conditions that are proper for collection and/or preservation of one microbe may be deleterious for another and, for that matter, to traditional forensic materials such as human DNA, fingerprints, and trace materials. For example, foodborne pathogens are particularly vexing; conditions that are intended to preserve the material may promote growth of natural bacteria in a food product, and this overgrowth may destroy or obscure the initial bioweapon. Tools for collection need to be validated for efficient collection and for determining that they do not react with the target of interest. Tools developed for powder collection may be inefficient or ineffective for collecting plant material. Sample collection is not trivial and requires substantial consideration. Issues related to the practices of sample collection, handling, transportation, and storage of microbes in the investigation of biocrimes and guidelines for the collection of evidence by physicians and medical personnel from potential victims of bioterrorism have been published and are valuable for education and training programs ( Schutzer et al., 2005 ). The same issues about evidence collection will need to be applied to preservation processes. It is imperative to prevent further degradation of the evidentiary target once collected. Conditions for preservation apply for packaging and shipping, for maintaining of the evidence in the laboratory, and for postanalysis storage. Extraction Extraction efficiency, particularly of interest to the scientist, pertains to obtaining the highest quality and quantity yield as possible of the target of interest. Yield is related to the quantity and purity of target and removal from the collection matrix. Targets can include cells, nucleic acids, proteins, nutrients, growth materials, and elements. Advanced microscopy Various forms of microscopy may be used to visualize the evidence. These tools may range from light microscopy to electron microscopy to atomic force microscopy and are available for characterization of a microbe. These approaches are rapid and can be used to identify candidate threats as well as to dismiss hoaxes. Proteomics Defining chemical and physical properties of a biological agent can provide information on how and when the agent was produced and can be used to determine if two microbial samples were produced by the same process. Proteomics is a comprehensive study of the protein composition of biological systems at a moment in time or at different stages of a microbe's growth. Many proteins are conserved and can be used for general identification, while other proteins may be expressed in response to environmental stimuli, growth state, or growth conditions. Protein profiling can provide information beyond genomic analysis about the conditions of the bioweapon before host exposure ( Gil and Monteoliva, 2014 ). Genomics One of the fastest growing areas with implications for microbial forensics is genomics. More rapid and in-depth sequencing of microbes is possible today than it was a decade ago; sequencing and targeted methods such as those used in the investigation of the anthrax-letter attack seem almost antiquated today. Genomic analyses will continue to be essential in identifying species, strains, isolates, and individual samples to assist in a microbial forensic investigation. ( Schmedes et al., 2016 , Budowle et al., 2017 , Karlsson et al., 2013 ). The rapid expansion of sequencing capabilities, where sequencing some microbes within a day at very deep coverage, has raised the importance of genetic identification. It will likely be a mainstay of the microbial forensic investigation of any attack with any microbe. The cost of whole-genome sequencing has decreased at least 100-fold in just a few years. This technology has now matured into one of the methods of choice to examine the genetic structure of a particular pathogen and to identify signatures of forensic relevance. ( Schmedes et al., 2016 , Budowle et al., 2017 , Karlsson et al., 2013 )] In addition, the legal profession should have a basic understanding of the capabilities and limitations of these technologies to be successful in the courtroom, just as has been necessary for human DNA forensics. Several chapters in this book expand on technology in detail. Interpretation, statistical analysis, and confidence Interpretation of results and the bases to support interpretation by scientists will be critical to the end-user stakeholders that rely on microbial forensic evidence for legal proceedings, for setting policy or responding to a threat or an attack. Interpretations could be as simple as positive or negative to very complex evaluations using limits of detection, complex algorithms for identifying. A host of answers and additional questions can arise from data interpretation. Central to interpretation is, when possible, a statistical analysis of the findings which should be performed to provide significance of the result, uncertainty, or to convey the strength of the evidential results. A variety of statistical approaches exists, and it is imperative to understand which ones apply to particular analyses and interpretations. One needs to consider that traditional statistical analyses may not apply to microbial evidence in some cases. With the advent and ease of massive parallel sequencing on various platforms and long-read sequencing with the MinIon (Oxford Nanopore) or PacBio (Menlo Park, CA), whole genomes of suspected biothreat agents can be quickly and easily obtained. However, the sequence differences may be limited to only informative single-nucleotide sequence (SNP) differences as represented by the genus Bacillus ( Derzelle et al., 2015 ). Genetically similar microbes are analyzed via comparative genomics tools where multiple SNP patterns (canonical SNPs (canSNPs)) emerge and can distinguish geographically unique strains from one another when compared with reference strains of known biothreat agents ( Dahiya, 2017 ). Many different phylogenetic tools and software are available now to perform such genetic comparisons. Working groups such as the AniBio Threat project ( anibiothreat ) in Europe developed standard protocols and expanded molecular databases to better assist in determining if an outbreak is natural or intentional ( Derzelle and Thierry, 2013 ). Both scientists and legal analysts need to understand (or at least appreciate) the results and their significance. Moreover, the degree of confidence that can be placed on a result must be understood so that the weight of a comparative analysis is not overstated. Basic statistics, probability, and population genetics are essential requirements of any curriculum involving the analysis of forensic biological evidence. Bioinformatics The term "bioinformatics" was developed as a result of the Human Genome Project. Because of the immense amount of data generated, it became necessary to apply more sophisticated computational techniques beyond what the average bench biologist had available. Bioinformatics requires a combination of data handling and analysis skills (including standard statistics) that connect routine biology with high-powered computation. As scientific investigations and data generation expand using high resolution, deep sequencing of genomes of microbes, and large-scale proteomics, computational analyses will be more critical than ever. This subject can be taught in a simplistic form for the biologist or a more complex form for the computationally inclined scientist. This interdisciplinary field has greatly impacted microbiology as well as forensics, medicine, agriculture, and other disciplines. Bioinformatic algorithms have simplified comparative genomics. Bioinformatics for medicine, microbial genome, and agriculture ( Dahiya, 2017 ) are able to determine biologically relevant patterns in complex datasets ( Damaso et al., 2018 ) as well as allow analyses of whole-genome shotgun sequencing of complex mixtures such as microbiomes ( Citation: Chen and Pachter, 2005 ). The information derived from complex data will have to be extracted using algorithms such as support vector machines ( Xu et al., 2015 , Schlecht et al., 2008 ), neural networks ( Vidaki et al., 2017 , Vidaki and Kayser, 2017 ), and other more complex algorithms yet to be developed. All scientists and individuals with interest in microbial forensic sciences will need to have a basic understanding of statistics and bioinformatic tools. Indicators of engineering With rapid developments in molecular biology to benefit humankind also comes a great potential for manipulating a microorganism for nefarious purposes. Microbes could be engineered to be more virulent, and difficult-to-obtain microbes may be synthesized de novo in a laboratory. There is a need to detect not only the microbe but to determine if it was genetically manipulated or perhaps is a novel chimera. Signatures indicative of manipulations or synthesis may be detected through sequencing and bioinformatic analyses. The skills and materials needed to manipulate a microbial genome may provide clues about the perpetrator and degree of sophistication used to develop the biothreat agent. This capability should be of interest to law enforcement and the intelligence community for supporting investigative leads. Population genetics A knowledge of population genetics is essential for understanding the rarity of a genetic (and sometimes protein) profile derived from an evidence sample. Molecular epidemiology is increasingly applying the principles of evolutionary and population genetics to pathogens. It is important to understand what constitutes a sample population as opposed to a sample collection, the mode of inheritance related to a genetic marker, what significance or weight to apply to a genetic marker, what the mutation rate of a marker is, and how to combine the weight of multiple markers. Training of the student in this discipline will require basic genetic courses and more advanced courses in phylogenetic analyses and other forms of comparison. Such educational material will be found in population genetics and systematic and evolutionary biology programs. The population genetics of pathogens and its importance for microbial forensics are covered elsewhere in this book. An example of a forensic application of population genetics is the human skin microbiome. Schmedes et al. assessed human skin microbiome populations and, using supervised learning algorithms, were able to associate specific human microbiome profiles with their hosts with a high degree of accuracy ( Schmedes et al., 2017 ). Recent comprehensive skin microbiome analyses ( Ross et al., 2018 ) suggests that the skin microbiota have undergone coevolution with their corresponding mammalian hosts providing support for future development of forensic applications using skin microbiomes ( Ross et al., 2018 ). Nonbiological tools This topic is broad and can encompass tools that characterize a microbe morphologically or chemically. Subjects will range from microscopy to basic chemistry to analytical chemistry. The Amerithrax investigation demonstrated the importance of nonbiological measurements on samples of biological agents. A variety of mass spectral, spectroscopic, and other instrumental methods were used in an attempt to answer questions related to how, when, and what materials were used to produce the spore powders. Such information can be used to compare evidence directly to a reference sample or indirectly to infer something about the processes used to culture, stabilize, and/or disseminate the biothreat agent. Bioelectronic scent detectors have also been developed using human olfactory and taste receptors that detect microbes in drinking water ( Son et al., 2015 , MankiSona et al., 2017 ), These human receptors have been shown to be capable of distinguishing one trillion different olfactory targets ( Bushdid et al., 2014 ). Dogs have also been used for the detection of scents to track human waste in microbial source tracking in storm drains ( Canine Scent Detection and Microbial Source Tracking of Human Waste Contamination in Storm Drains, 2014 ). Forensic science Forensic science is the application of science to answer questions of interest to a legal system as well as for military or state decisions (1,5,6). While science may not offer definitive solutions to the problems of society, it does serve a special investigative role, particularly in the criminal justice system. The areas of science that have been traditionally exploited are diverse, but typically include the major disciplines of biology, chemistry, physics, and geology. Within each discipline are many scientific subcategories that may be used in a forensic science investigation. For example, within the discipline of biology are the subdisciplines of medicine, pathology, molecular biology, immunology, odontology, serology, psychology, and entomology. The specific discipline(s) employed depends on the circumstances of the crime. Mathematics, especially statistics, is used to place weight or significance on observations or data retrieved from crime scene evidence. The ultimate question addressed by forensic science is usually "who committed the crime?" (i.e., attribution) or "who did not commit the crime?", and crime scene evidence can play a role in answering these questions. Evidence can be any material, physical or electronic, that can associate or exclude individuals, victim, and/or suspect with a crime. It typically comprises materials specific to the crime as well as control samples for background information. Types of evidence may be fingerprints, blood, semen, saliva, hair, fibers, documents, photos, computer files, videos, firearms, glass, metals, plastics, paint, powders, explosives, tool marks, and soil. Scientists and other practitioners need to be cognizant of the types of evidence, how these different forms of evidence interplay, and how they can be used to help reconstruct the crime and/or identify the perpetrator. Case histories A case history is a detailed account of a person or event. Studies of case histories are instructive because they provide analysis of information in the relevant context, including real complexities. The study of a variety of incidents can be tailored to the particular group learning about them (see Chapters 1, 3, 10, 17 and 39). The Amerithrax case is likely to be studied for years by many different groups ranging from scientists to law enforcement to lawyers. In addition to this case, many other cases are described in chapters of this book and the previous edition ( Budowle et al., 2005 ), as well as in specific publications [( Schutzer et al., 2005 ) ( Seth Carus, 1998 )]. Among these threats are foodborne illnesses from bacteria, such as Shigella and Salmonella , and toxins such as ricin. In addition, there have been events involving agriculture, including a foot-and-mouth outbreaks in the United Kingdom ( Jamal and Belsham, 2013 ) and mad cow disease in the United States ( Sigurdson et al., 2018 ). Environmental contamination is also an area of interest ( Yates et al., 2016 , Cano and Toranzos, 2018 ), such as microbial source tracking to detect water contamination by human sewage (see Chapters 6 and 7Chapter 6Chapter 7) or animal farming such as by the poultry industry in Arkansas. Perhaps the most common area where issues of natural versus intentional events arise is related to emerging infections. This question has arisen with the outbreaks of influenza H1N1, severe acute respiratory syndrome, monkeypox, and specific cases of HIV infection ( Jester et al., 2018 , Lucas and Nelson, 2015 , Morand et al., 2017 ). Legal issues Legal issues are of obvious importance to the legal community but are also important to the scientific community. There will be times when the evidence will be used in a court of law to prosecute an individual who has been arrested for a biocrime. There are standards for admissibility of scientific evidence in a legal setting. The scientist may be asked to provide expert testimony. These standards need to be known and appreciated so that admissibility of evidence can be achieved. The government will use microbial forensic scientists and other experts, their results, the scientific literature, and supporting validation studies to support its position. The defense will defend its client vigorously to attempt to achieve an acquittal. Because of the adversary system, challenges are expected to the credibility of the science and its practitioners in the United States and other English-based law countries ( Harmon et al., 2005 , Kirsch and Daubert 1995 ). Studying the science behind headlines can be a very instructive and creative way to interest students. Some controversial issues in forensic DNA, which can be used instructively, involve (1) the use of low quantities of template DNA ( Budowle et al., 2009 , Gilder et al., 2009 ), (2) population genetics issues and associated degrees of confidence ( NRC ), (3) potential contamination and background signals, (4) sufficient and appropriate validation studies, and (5) access to proprietary information or intellectual property. The standards and court proceedings, however, will vary for each country. For example, in the United States, possession of unauthorized material can be considered a crime by itself. Therefore, an understanding of the relevant laws for handling and possessing, for example, select agents is important. Operational and intelligence issues Evidence derived from a microbial forensic investigation may not necessarily end up in court. For example, such evidence can be used for intelligence purposes. Information can be gathered to determine the risk or probability of an individual, a group, or a state to use (or has used) a bioweapon in an attack. The primary goal is to intercede and thwart the attack before it can happen. Alternatively, if an attack has occurred, a head of state may require some evidence to determine whether to retaliate and to whom retaliation should be directed. Results from microbial evidence are far reaching and have consequences. Training individuals in understanding the strengths and limitations of scientific evidence is essential so that proper decisions and responses can be made. Understanding how information is gathered, analyzed, and acted upon is likely to be of interest to any level of student. National-level capabilities and resources Policy and decision makers need to learn about and support advances in microbial forensic strategies and capabilities, such as were described in the "National Science and Technology Council, National Research and Development Strategy for Microbial Forensics, Office of Science and Technology Policy (2009) ( National Science and Technology Council, 2009 ), and Science Needs for Microbial Forensics: Developing Initial International Research Priorities, 2014 ( Science Needs for Microbial Forensics, 2014 )." The following aspects should be addressed: (i) What and how should a country be prepared? (ii) What strategies make sense? (iii) Planning, implementing, and measuring effectiveness. (iv) Training and evaluation exercises. (v) Where can additional support be sought? (vi) Leveraging of epidemiological tools. Conclusion Education of the next generation of microbial forensic scientists, the continuing professional development and training of practitioners, and informing those in the legal community and policy makers are our collective responsibility and of paramount importance. The evolution of technology, analytical capabilities, and, equally as important, the need for incorporating advances into our education and training programs continue to evolve and expand rapidly. This chapter along with the educational resources cited indicates some resources that could be considered to educate and train those interested in the field of microbial forensics. Other forms of education should include didactic lectures, workshops, conferences, practical demonstrations, and discussions at specialty meetings. Those interested in the development of formal microbial forensic degree programs will find resources from academic, government, industry, and professional societies. The target audience may include laboratory directors, bench scientists, other practitioners, faculty, college students, law enforcement representatives, medical care and first responder personnel, lawyers, and judges. Those who fulfill teaching roles, whether by profession or indirectly as reporters and even entertainment writers, can become informed so that their writings are founded in facts that could serve as well to better educate stakeholders.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449641/

Stevens-Johnson Syndrome Due to Influenza Vaccination

Influenza is a common virus that affects millions of people every year. The influenza vaccine decreases morbidity and mortality associated with influenza and is generally well tolerated. Stevens-Johnson syndrome (SJS) is a rare disorder of the skin and mucous membranes. We report the second known case of SJS occurring after an influenza vaccination alone without any other associated drug exposure. This corroborates the possibility of the influenza vaccine alone causing SJS. Despite the initial adverse reaction, the patient made a full recovery. Although the disease can be associated with vaccinations, the benefits of receiving the vaccinations outweigh the potential harms. Introduction Stevens-Johnson syndrome (SJS) is a rare disorder of the skin and mucous membranes that occurs in about one to three cases every million persons each year [ 1 ]. This serious disorder is characterized by flu-like symptoms, red or irritated skin, and blisters. Although cases related to vaccinations have been reported, there has been only one case reported due to the influenza vaccine only in a 24-year-old female [ 1 ]. There has been a second case reporting SJS occurring after receiving the influenza vaccine, but this was given in combination with fluvoxacillin, so it does not corroborate the first case [ 2 ]. We report the second known case of SJS occurring after an influenza vaccination alone, which corroborates the possibility of the influenza vaccination alone causing SJS. Case presentation The patient was an 82-year-old male with a past medical history of successfully treated prostate cancer and hypertension. His medications included atorvastatin, candesartan, metoprolol, and aspirin taken with no dose changes for at least two years. He received the influenza vaccine Influvac® (Abbott), which he had done annually without any complications. The patient had no symptoms on the day of the injection. One day after the vaccination, he noted mild pruritus in the evening. A rash was noted in the chest and back, and he was started diphenhydramine 25 mg orally as well as diphenhydramine cream. Four days after the vaccination, the rash had spread to his arms and legs (Figures 1 , 2 ). Five days after the vaccination, the rash spread to the face, and he went to a walk-in clinic. Upon examination, his dose of diphenhydramine was increased to 50 mg. That afternoon, the patient's wife noticed facial swelling and brought him into the emergency room of a major city hospital. The doctor noted an inner lip ulcer and diagnosed it as mild Stevens-Johnson syndrome based on the morbilliform rash on his body that had spread to the patient's face. He was admitted to the hospital for two nights. A dermatologist verified the diagnosis of SJS and prescribed cetirizine (10mg daily for seven days). The patient had an estimated 85% body surface area affected with a SCORe of Toxic Epidermal Necrosis(SCORTEN) score of 1 [ 1 ]. Oral steroids were suggested, but the patient declined to take them. One day after discharge (eight days after the influenza vaccination), he noted the appearance of more ulcers on his bottom lip (Figure 3 ). Nine days after the influenza vaccination, he saw another dermatologist who recommended an oral mouthwash for symptomatic relief. Gradually, the skin rash improved, but the oral ulcers worsened until about nine days after vaccination and improved by 13 days after vaccination. The patient felt he was mostly recovered about a month after the vaccine. Other than the flu vaccine, patient had not taken any new medications, new supplements, or new foods prior to the development of the itching and skin rash. Figure 1 Thigh four days after influenza vaccination Multiple purpuric macules are present. There is a lack of serious skin detachment or blistering. Figure 2 Trunk four days after influenza vaccination Diffuse presentation of coalescing macules across the trunk. Lack of serious skin detachment. Figure 3 Lower lip eight days after vaccination Multiple coalescing papules present on oral mucosal membrane. Discussion Even though SJS is rare with an annual incidence of one to three reported cases per million people [ 1 ], there have been many reported cases of SJS since its discovery in 1922 [ 3 ]. Over 200 different medications have been identified as causes of SJS, most commonly sulfonamide antibiotics, aromatic anticonvulsants, beta-lactam antibiotics, nevirapine, abacavir, non-steroidal anti-inflammatory medications, allopurinol, lamotrigine, tetracyclines, quinolones, and others [ 4 ]. These drugs impact various physiological factors that can lead to the development of SJS. Cytotoxic T cells and natural killer cells have been associated with SJS due to the release of granulysin which destroys cells in the skin and mucous membrane. The cell death that occurs in these areas is generally identified as apoptosis as a result of dysregulation in specific transmembrane protein pathways. The most extensively investigate pathway is that of Fas. This pathway involves the binding of a soluble ligand that induces a caspase cascade leading to the destruction of the cell. Typically SJS occurs in a younger population and is a response to multiple drugs [ 4 ]. Mycoplasma pneumonia has been documented in a few cases [ 5 ]. SJS has also been associated with Herpes simplex virus [ 6 ]. Fleming in 2011 reported a case where a 65-year-old female with a breast abscess was given oral flucloxacillin, then on day 10 of the treatment, she was given the inactivated influenza vaccine Fluvirin® (Novartis) [ 2 ]. About 24 hours later, she developed SJS. Because of the abscess infection as well as dual exposures to the antibiotic and the vaccine, the definitive cause could not be determined. There are several main differential diagnoses for SJS. Acute generalized exanthematous pustulosis (AGEP) is characterized by pinhead-sized pustules that were not present in this case. AGEP is more commonly seen in middle-aged adults and only involves mucosal membranes in roughly 20%-25% of cases [ 7 ]. Erythema multiforme is another disease that presents similarly to SJS with skin lesions. These lesions, however, are most commonly associated with the herpes simplex virus and usually do not involve mucosal membranes [ 4 ]. In addition to this, the lesions are typically localized or raised atypical targets [ 8 ]. Toxic epidermal necrolysis (TEN) presents similarly to SJS, usually beginning with fever-like symptoms followed by cutaneous and mucous membrane lesions. However, the definition of each differs in the extent of skin detachment. SJS encompasses 30% of the skin [ 8 ]. This patient has <10% skin detachment which is consistent with SJS. SJS is very rarely associated with vaccination exposure only. A study of the Vaccine Adverse Event Reporting System noted over a nine-year period, there were 96,951 distinct adverse event reports noted of which six cases were either SJS or TEN following exposure to vaccination only [ 1 ]. Chopra reported SJS in a 19-year-old male military reservist who received immunizations to smallpox, anthrax, and tetanus then developed SJS twenty days after the vaccinations [ 9 ]. SJS was reported following two cases of measles vaccination [ 10 ]. To our knowledge, SJS has been previously reported in one case following exposure to only the inactivated seasonal influenza vaccine. Our case thus corroborates the possibility of SJS occurring secondary to the influenza vaccine only. Table 1 SJS/TEN cases reported after vaccination administration DTP - diphtheria, tetanus, whole-cell pertussis; Hib - Haemophilus influenzae type b bacterial; MMR - measles, mumps, rubella; OPV - oral polio vaccine, PAP - papanicolaou smear Sources: [ 1 , 2 , 9 , 10 ] Age (years) Gender Vaccines and medicine Onset Illnesses and drugs Severity 0.5 F Hepatitis B 1 day Positive eyelid culture for Branhamella catarrhalis, Staphylococcus aureus, Streptococcus pneumoniae. Acetaminophen was given after the onset of rash. Hospitalized .8 M Measles 1 day Steroids and antibiotic therapy were given one day and two days after vaccination, respectively. Hospitalized 1.3 M Hib, MMR 22 days History of torticollis and exposure to strep throat. Penicillin administered after the onset of rash. Multiple clinic visits 2.3 M Varicella 1 day Diphenhydramine one day after vaccination. Previously developed erythema multiforme after amoxicillin/clavulanate after trimethoprim-sulfamethoxazole. Hospitalized 2.3 M DTP, Hib, MMR, OPV 7 days Diphenhydramine and amoxicillin were given seen days after vaccination. Hospitalized 3.7 M Hepatitis B 1 day No previous medication or illness. Acetaminophen was given after onset of rash. Hospitalized 19 M Varicella, anthrax, and tetanus 20 days Allergy to nickel with no medications noted. Hospitalized 24.1 F Influenza Same day History of acne, abnormal PAP smear, and medroxyprogesterone injection for birth control. Hospitalized 65 F Influenza and flucloxacillin 1 day History of diabetes. Flucloxacillin was given 10 days prior to vaccination for breast abscess. Hospitalized Influenza is a common virus that affects millions of people every year. From 1976 to 2007, estimated flu-related deaths in the United States since 2010 have ranged from 12,000 to 56,000 [ 11 ]. The vaccine decreases morbidity and mortality associated with influenza. During the 2011-2012 flu season, influenza vaccination was associated with a 71% reduction in flu-related hospitalizations among adults of all ages and a 77% reduction among adults 50 years of age and older [ 12 ]. Since this case illustrates a very rare complication of the influenza vaccine, the benefits of the influenza vaccine clearly far outweigh its potential harm. Therefore this case should not deter people from receiving the influenza vaccination. Conclusions SJS is a rare condition typically associated with oral drugs and less commonly associated with infections. It is rarely associated with vaccinations and has been reported after the influenza vaccine alone in one case. This is the second reported case where SJS has been associated with an influenza vaccination alone which supports the possibility that it can result solely from the influenza vaccine. Although the disease can be associated with vaccinations, the benefits of receiving the vaccinations outweigh the potential harms.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893210/

A comprehensive analysis of Italian web pages mentioning squalene-based influenza vaccine adjuvants reveals a high prevalence of misinformation

ABSTRACT Squalene-based adjuvants have been included in influenza vaccines since 1997. Despite several advantages of adjuvanted seasonal and pandemic influenza vaccines, laypeople's perception of such formulations may be hesitant or even negative under certain circumstances. Moreover, in Italian, the term "squalene" has the same root as such common words as "shark" ( squalo ), "squalid" and "squalidness" that tend to have negative connotations. This study aimed to quantitatively and qualitatively analyze a representative sample of Italian web pages mentioning squalene-based adjuvants used in influenza vaccines. Every effort was made to limit the subjectivity of judgments. Eighty-four unique web pages were assessed. A high prevalence (47.6%) of pages with negative or ambiguous attitudes toward squalene-based adjuvants was established. Compared with web pages reporting balanced information on squalene-based adjuvants, those categorized as negative/ambiguous had significantly lower odds of belonging to a professional institution [adjusted odds ratio (aOR) = 0.12, p = .004], and significantly higher odds of containing pictures (aOR = 1.91, p = .034) and being more readable (aOR = 1.34, p = .006). Some differences in wording between positive/neutral and negative/ambiguous web pages were also observed. The most common scientifically unsound claims concerned safety issues and, in particular, claims linking squalene-based adjuvants to the Gulf War Syndrome and autoimmune disorders. Italian users searching the web for information on vaccine adjuvants have a high likelihood of finding unbalanced and misleading material. Information provided by institutional websites should be not only evidence-based but also carefully targeted towards laypeople. Conversely, authors writing for non-institutional websites should avoid sensationalism and provide their readers with more balanced information. Introduction Let us consider the following Italian word family: squalo, squallido and squallore . While the first word means "shark", the other two both have negative referential meaning (cf. "squalid" and "squalidness"). This word family derived from the Latin adjective squālidus , derived in turn from the verb squālēre that means "to be dirty/rough". While the English "squalid" and "squalidness" may be considered as learned terms, the Italian counterparts are rather common. The term "squalene" also comes from the same Latin root since this natural compound was first isolated from the shark liver oil. The denotative(i.e. literal) meaning of squalene may be described as follows: a natural unsaturated 30-carbon polyprenyl compound playing the key role in cholesterol biosynthesis. 1 , 2 On the other hand, because the term "squalene" is unfamiliar to most laypeople, its connotative (i.e. emotionally charged) meaning would likely be negative through associative analogies with the Italian words squalo, squallido and squallore . Nowadays, squalene has various uses. Mostly, it is widely employed by the cosmetic industry because it has properties which make skin smooth and elastic. 3 Squalene may also be considered a potential natural chemopreventive substance; indeed, in the Mediterranean diet – the advantages of which are internationally recognized – daily intake of squalene may reach up to 400 mg, mainly due to the intake of olive oil. By contrast, in other countries, such as the US, its intake is only around 30 mg. 4 Squalene and shark liver oil are also popular dietary supplements. 5 And finally, squalene-oil-in-water emulsions (e.g. MF59®, AS03®, AF03®) have widely been used as vaccine adjuvants since 1997. The major benefits of including squalene-based adjuvants in vaccine formulation are: enhanced immunogenicity; more rapid antibody responses; more persistent antibody responses; enhanced heterologous antibody responses; and the possibility of antigen dosage sparing, which is crucial during emergencies such as influenza pandemics. Seasonal influenza vaccines adjuvanted with MF59® have been widely used to immunize elderly individuals for the last 20 years. Both MF59® and AS03® were components of the 2009 H1N1 pandemic vaccines used in all age groups. 6-9 Despite the aforementioned advantages of squalene-based adjuvants, their widespread use generated various scientifically unsound claims and myths (these are described in the Methods section). In Italy, which was the first country where MF59®-adjuvanted influenza vaccine was authorized, such myths were somewhat reinforced by the etymology of the word "squalene" described earlier. A further challenge occurred in late 2014 when the Italian Medicines Agency suspended two batches of the MF59®-adjuvanted seasonal influenza vaccine as a precautionary measure following three post-vaccination deaths. Although, following an investigation, the vaccine complied with all existing standards and no causal association was found, this event gave rise to several consequences including unprecedented clamor in both traditional media and internet, "epidemic of panic", general increase in mistrust in vaccines and vaccine hesitancy, (consequent?) decrease in the influenza vaccination coverage rate, 11 , 12 as well as a "reawakening" of early myths around squalene and adjuvants. Although this false alarm evolved in a short time frame, its repercussions were long-term and persist even until today. Indeed, both traditional and new media are powerful means of amplifying inaccurate scientific information, undermining confidence in vaccines and reducing vaccination coverage. Probably the best-known example of this phenomenon is provided by the Wakefield case: the widespread media coverage of an alleged link between measles-mumps-rubella (MMR) vaccination and autism led to a dramatic drop in the uptake of this vaccine and an increase in the number of measles cases reported. 13 In the United Kingdom, it took more than ten years to restore the MMR coverage rate to the pre-Wakefield level. 14 Because the internet is one of the main sources of health-related information, 12 it is widely used by anti-vaccination activists to disseminate misleading information on immunization, 15 and the fact that anti-vaccination websites are highly prevalent on the Italian web 16 and may be highly ranked by the common search engines, 17 we hypothesized there to be a high frequency of web-pages critical to squalene and squalene-based adjuvants among Italian online resources. The major goal of the present study was to quantitatively and qualitatively analyze a representative sample of squalene-related Italian web pages. Results Of 2,550 web pages screened, 2,280 were removed as duplicates. Of 270 texts assessed, 141 met the inclusion criteria (5 and 123 web pages had no reference to influenza and squalene in influenza vaccines, respectively, while one page was not accessible). Subsequently, 2, 7, 33 and 15 web pages were excluded meeting exclusion criteria 1–4, respectively. In total, 84 web pages were included in the analysis. Forty of 84 web pages [47.6% (95% CI: 36.6–58.8%)] were categorized as negative or ambiguous (Neg/Amb), while the remaining 44 [52.4% (95% CI: 41.2–63.4%)] as positive or neutral (Pos/Neu). Table 1 reports the main characteristics of the included web pages, broken down by the general tone. The top-level domain ".it" largely prevailed among both Pos/Neu and Neg/Amb web pages; no statistically significant difference (χ 2 (3) = 1.92, p = .59) between the distributions of top-level domains among Pos/Neu and Neg/Amb web pages was established. By contrast, many more (5.8-fold difference; χ 2 (1) = 12.02, p <.001) Pos/Neu web pages belonged to the category of institutional web sources; the effect size was medium (φ = 0.38). In comparison with Pos/Neu web pages, more than double (χ 2 (1) = 12.50, p <.001) of those categorized as Neg/Amb had at least one picture with a medium effect size (φ = 0.39). Analogously, the Mann-Whitney test highlighted that the distribution in the absolute number of pictures between Pos/Neu and Neg/Amb was not identical ( p <. 001) with a medium effect size ( r = 0.38). On the basis of the main topic, pictures were categorized into 6 classes, namely images representing the vaccination process, syringes, vaccine packaging, the influenza virus, sharks/monsters, other. Table 1. Characteristics of the web pages analyzed, by general tone. Variable Level Positive/neutral ( N = 44) Negative/ambiguous ( N = 40) Top-level domain, % (95% CI) .it 72.7 (57.2–85.0) 60.0 (43.3–75.1) .com 6.8 (1.4–18.7) 10.0 (2.8–23.7) .org 11.4 (3.8–24.6) 12.5 (4.2–26.8) Other 9.1 (2.5–21.7) 17.5 (7.3–32.8) Website source, % (95% CI) Institutional 43.2 (28.3–59.0) 7.5 (1.6–20.4) Non-institutional 56.8 (41.0–71.7) 92.5 (79.6–98.4) Web pages with at least 1 picture, % (95% CI) — 34.1 (20.5–50.0) 75.0 (58.8–87.3) Number of pictures, median (range) — 0 (0–3) 1 (0–8) GulpEASE index, mean (SD) — 42.9 (3.7) 46.4 (3.6) Common words, % (SD) — 73.6 (5.3) 76.3 (4.7) The distribution of single picture categories broken down by general tone is reported in Table 2 ; Neg/Amb pages presented more frequently images of syringes, viruses and sharks/monsters. However, no statistically significant difference between Pos/Neu and Neg/Amb web pages emerged (Fisher's exact test: p = .19). Table 2. Categories of images, by general tone. Category Positive/neutral, N Negative/ambiguous, N Vaccination 7 6 Syringe 2 10 Vaccine 3 6 Virus 0 4 Sharks/monsters 0 3 Other 9 29 Only 3 web pages had video material (one video each): two videos contained interviews with anti-vaccination activists, while the third had an interview with a Hollywood star on vaccine-related topics. Due to the paucity of videos, these were not analyzed quantitatively. With regard to the readability properties, Pos/Neu web pages could be judged to be more (| t | (82) = 4.33, p <. 001) difficult to read than Neg/Amb pages with a large effect size of | d | = 0.95 (95% CI: 0.49–1.40). Indeed, all 11 web pages with a GulpEase index < 40 were Pos/Neu (Fisher's exact test: p <. 001). The frequency of commonly used words was also higher (| t | (82) = 2.45, p = .017) in the Neg/Amb category web pages with a medium effect size (| d | = 0.53). The adjusted logistic regression model showed that web pages belonging to the Neg/Amb category had significantly lower odds of having an institution as a source, and significantly higher odds of having at least one picture and being more readable, as measured by the GulpEase index ( Table 3 ). Table 3. Multivariable logistic regression to predict the general tone of web pages analyzed. Variable aOR 95% CI p Website source (institutional vs non-institutional) 0.12 0.03–0.50 .004 N of pictures (1-unit increase) 1.91 1.05–3.48 .034 GulpEASE index (1-unit increase) 1.34 1.09–1.65 .006 % common words (1-unit increase) 1.01 0.88–1.16 .89 The terms vaccin*, squalene, influenza and adjuvant* were the most common words among both Pos/Neu and Neg/Amb web pages. However, relative to the most frequent word vaccin* , Neg/Amb pages used the root squalene about twice as much as Pos/Neu pages (ratios squalene to vaccin* of 0.41 and 0.23 for Neg/Amb and Pos/Neu web pages, respectively). Other particular features of Neg/Amb web pages included (i) higher weights for the words gulf, war, syndrome, soldier, adverse, minister, government ; (ii) higher usage of registered trademarks(e.g. Novartis, Fluad® ) and (iii) equal weight for the words health and death ( Fig. 1 ). Figure 1. Tag clouds with 50 or more common words found among positive/neutral (A) and negative/ambiguous (B) web pages. The most prevalent claims reported in Neg/Amb web pages were those relating squalene or squalene-based adjuvants to the Gulf War Syndrome (52.5%) and autoimmune pathologies (47.5%). About a third (35.0%) of web pages dealt with conspiracy theories around squalene and/or squalene-based adjuvants. Other less common claims included safety concerns (27.5%), direct link to deaths (20.0%), and efficacy concerns (5.0%) ( Table 4 ). In addition, one web page claimed that squalene causes infertility, while another linked the use of squalene with autism. Table 4. Claims around squalene and/or squalene-based adjuvants found among negative/ambiguous web pages. Myth % 95% CI Squalene is a toxic/unnatural substance 22.5 10.8–38.5 Efficacy of influenza vaccines with squalene-based adjuvants is not demonstrated 5.0 0.6–16.9 Too little/no safety data on squalene-based adjuvants are available; there are more risks than benefits regarding squalene-based adjuvants 27.5 14.6–43.9 Squalene and/or squalene-based adjuvants are/may be directly linked to deaths 20.0 9.1–35.6 Squalene and/or squalene-based adjuvants (may) cause autoimmune disorders 47.5 31.5–63.9 Squalene and/or squalene-based adjuvants (may) cause the so-called Gulf War Syndrome 52.5 36.1–68.5 Conspiracy theories around squalene and/or squalene-based adjuvants 35.0 20.6–51.7 Table 5. Myths and facts about squalene and squalene-based adjuvants in influenza vaccines. Myths Facts Ref. Squalene is a toxic/unnatural substance Squalene is a fully biocompatible and biodegradable substance, being the direct predecessor of cholesterol. About 1 g of squalene is synthetized daily in the human liver and is also introduced through the normal diet. A considerable amount of squalene can be found in human sebum and it is largely used by the cosmetic industry. 54 , 55 Efficacy of influenza vaccines with squalene-based adjuvants is not demonstrated Several meta-analyses have shown that influenza vaccines containing squalene-based adjuvants (such as MF59®) are both immunogenic and efficacious in preventing influenza and its complications. 56-58 Too little/no safety data on squalene-based adjuvants are available; there are more risks than benefits regarding squalene-based adjuvants Tens of clinical and observational studies as well as systematic reviews/meta-analyses have shown an acceptable safety profile of MF59®-adjuvanted influenza vaccine. Although in comparison with non-adjuvanted vaccines, there is some increase in the frequency of solicited adverse events (particularly local reactions), most of which are mild. On the other hand, the frequency of unsolicited and serious adverse events tend to be lower among recipients of MF59®-adjuvanted vaccine than among those receiving conventional influenza vaccines. Moreover, to date, MF59®-adjuvanted vaccine is licensed in about 30 countries and more than 100 million doses were administered worldwide from 1997 to date. 56 , 59 Squalene and/or squalene-based adjuvants are/may be directly linked to deaths This myth probably comes from the so-called "Fluad case" when during the 2014–15 season 3 deaths occurred within 48 hours from vaccination with MF59®-adjuvanted influenza vaccine. As a result two batches of the vaccine were recalled as a precautionary measure. No causal link was later established. Moreover, it has been calculated that up to 20 Italian people die every day within 48 hours of vaccination by pure chance. 10 , 11 Squalene and/or squalene-based adjuvants (may) cause autoimmune disorders In a large ( N = 27,998) safety database, no significant difference in terms of adverse events of potential autoimmune origin between people immunized with MF59®-adjuvanted vaccine and those immunized with unadjuvanted vaccines has been found. 59 Squalene and/or squalene-based adjuvants (may) cause the so-called Gulf War Syndrome In 2000, American researchers reported data on a high frequency of the presence of anti-squalene antibodies among veterans with the so-called Gulf War Syndrome. The methodology of that study was criticized and, according to the US Federal Government, vaccines administered to military personnel in that period of time did not contain squalene. Anti-squalene antibodies are very frequently detectable (at low titers) among people never immunized with vaccines containing squalene. No casual association between positivity to squalene and the development of GWS has been established. The MF59®-adjuvanted vaccine does not induce anti-squalene antibodies nor increase the quantity of pre-existing anti-squalene antibodies. 31-34 , 60 , 61 Discussion The main finding of the present study is that web pages critical to squalene-based adjuvants are highly prevalent on the Italian web: a typical internet user has an equal chance of finding reliable and unreliable information on squalene-based adjuvants. As shown by Google Trends, 18 in the past 5 years web searches around squalene peaked in the period from 23 November to 6 December 2014 (results not shown) and thus correspond exactly to the time in which two batches of MF59®-adjuvanted vaccine were blocked in Italy as a precautionary measure. 11 Considering Italy's relatively large current market share of MF59®-adjuvanted vaccine, 19 the inadequate information on squalene may have contributed to the subsequently observed decrease in vaccination coverage. Proof of this may be found in the fact that, in a single year the vaccination coverage among the Italian elderly population decreased by 12.3% (influenza vaccination coverage among elderly Italians of 55.4% and 48.6% in the seasons 2013–14 and 2014–15, respectively). 20 As we noted above, such situations undoubtedly have long-term consequences: from influenza season 2014–15 to 2015–16 the vaccination coverage increased by only 1.3 percentage points 20 (somewhat like "easy to lose, hard to regain"). Undoubtedly, unreliable information obtained from the internet, which is the main source of health-related information, 12 contributed to the observed drop in vaccine uptake. Returning to the notorious Wakefield case, it has been shown that sensational news reports (both traditional and online) linking the MMR vaccine to autism, which often recounted the emotional and dramatic stories of parents, aroused greater interest than those which simply provided factual information from scientific studies. 21 More recently, a situation somewhat similar to the Italian "Fluad case" arose in China 22 ; after some infant deaths had allegedly been linked to vaccination against hepatitis B, the vaccine in question was recalled by the authorities. Although no causal association was ever established, many parents refused to have their children vaccinated. Again, the primary source of parental information on the issue was the internet. 22 Indeed, it was experimentally documented 23 that people exposed to negative online information on a vaccine express less intention to be immunized. We then established that the website source, level of readability, and number of images are all independent predictors of the general tone of squalene-related web pages. Non-institutional websites showed an 8.6-fold increase in belonging to the category of Neg/Amb pages. This finding was expected and it is in line with previous Italian research. In particular, all institutional websites analyzed by Poscia et al. 24 were pro-vaccination, while Tozzi et al. 25 have found that web pages from governmental agencies and universities had the highest level of credibility concerning human papillomavirus immunization. Pos/Neu web pages had on average longer words and sentences that reduced their readability. Indeed, more than a quarter of them could even be judged to be difficult to read for people with a high school diploma, while no such pages were found in the Neg/Amb category. A similar pattern has been observed by Japanese researchers: in comparison with anti-influenza immunization online messages, those pro-influenza immunization were harder to read. 26 More generally, the readability of squalene-related web pages is close to those of informed consent forms written in Italian (mean GulpEase index of 45.7). 27 Neg/Amb web pages combined different content forms more frequently. In particular, they used a relatively high number of images that could improve the intelligibility of the text since the denotative meaning of the term "squalene" is rather technical. According to a theory developed by Boles, 28 words like "squalene" have a high level of concreteness (it is a "thing") but low familiarity (extremely low frequency of word usage and exposure) and imageability (i.e. hard to visualize). Accompanying the text with pictures will undoubtedly improve imageability. In cases where the referential meaning of squalene was not given (and even if given this would not be fully comprehended by most users) or was twisted, a text containing emotionally charged images (such as needles and sharks with bloody teeth, as frequently found among Neg/Amb pages) may generate or reinforce negative connotations of squalene, adjuvants, and influenza vaccines in general among people unfamiliar to the topic. Notably, Kata 15 has reported that images of scared needles are a frequent graphical attribute used by the anti-vaccination movement on the web. From the point of view of word frequency, it emerged that Pos/Neu web pages had a relatively higher focus on vaccination itself and aspects relative to health. By contrast, Neg/Amb web pages exploited more the unfamiliar word "squalene", politically-related words (conspiracy theories), 15 names of major vaccine manufacturers and other registered trademarks (could be ascribable to the so-called "Big Pharma conspiracy theory)" 29 and accentuated fatal risks (a high usage of the word "death"). The observed differences in quantitative language led us to analyze Neg/Amb pages in a more detailed way. Despite the fact that: the etiology of Gulf War Syndrome/Illness remains largely uncertain to epidemiologists (it even has no universally adopted definition) 30 ; most people have anti-squalene antibodies 31-33 ; and no Gulf War veterans received squalene-adjuvanted vaccine, 33 , 34 the thesis that squalene is a causative agent of the syndrome and other autoimmune disorders is highly prevalent. Indeed, 60% of Neg/Amb web pages made such claims. Other safety concerns were also frequently mentioned. To analyze and discuss the observed phenomena, it is useful to look at squalene-based adjuvants as "one more unnatural vaccine ingredient" and then make some parallels with preservatives and other additives used by the food industry. Consumers are very concerned about food additives, poorly informed about preservatives, colorants, and artificial sweeteners, and have difficulties in understanding the subject of food chemicals. 35 Dickson-Spillmann et al. 36 have shown that the risk perception of food chemicals was positively correlated with preference for natural food. By analogy, anti-vaccination activists are frequent supporters of herbalism, veganism, homeopathy, naturopathy and similar practices. 15 This study has both strengths and limitations. There are basically two strengths: one, the methodology of the study accurately mimics the typical online browsing and search behavior of members of the general public, and therefore, the web pages analyzed are highly representative of real life scenarios, and two, the multidimensional vision of the study outcome. In particular, throughout the Introduction to Discussion sections of this manuscript, we have highlighted issues around squalene-based adjuvants from the perspectives of linguistics, vaccinology and immunology, and public health. The main drawback is the subjective nature of our judgements. Obviously, the authors of this manuscript are experts in vaccinology; however, every effort was made to ensure that the investigators' personal attitudes towards the web pages analyzed did not bias the outcomes and conclusions of the study (e.g. by simplifying and standardizing the rules of classification, etc.). Undoubtedly, a 4-category classification of the general tone of the web page would have allowed us to provide a more detailed description of the information available. In this regard, the use of automatic analyzers of tone and emotion (enabling the variable to be expressed on a continuous or at least multi-category scale) may unveil other important associations, and thus should be considered in future research. Conclusions This study makes a comprehensive and multidiscipline analysis of the information available online to Italian internet users (about 75% of the Italian population) concerning squalene-based vaccine adjuvants. 37 Negative, unbalanced, and misleading information was very common; this is potentially dangerous from a Public Health point of view. Unproven claims that influenza vaccines enhanced with squalene-based adjuvants may cause Gulf War Syndrome, autoimmune disorders, and even death, most likely originate from mass media reports, which are later reposted and/or re-elaborated to some degree by vaccine-critical websites, whose presence is high on the Italian web. In future, the mass media must be very cautious in choosing between legitimate concerns and sensationalism, 38 in order to avoid such false alarms and the propagation of misinformation. Undoubtedly, influenza vaccination must be an informed individual choice. In the web 2.0 era this choice is highly influenced by online resources. Indeed, more than 80% of US healthcare practitioners claim to have had at least one patient who brought internet-acquired health information to a visit in order to ask the physician's opinion on the matter in question. 39 Considering the well-known phenomenon of patient-doctor information asymmetry, in cases where patients bring web-acquired material on squalene-based adjuvants, the role of physician becomes crucial in informing the patient about all benefits and risks of such vaccine formulations. Such provision of information by physicians will undoubtedly help to reassure those hesitant to be vaccinated. Governmental public health agencies and healthcare professionals need to provide reliable, easily accessible and user-friendly information on any given health technology. For instance, given the wide availability of readability formulas in common word processors, governmental public health agencies and healthcare professionals should make greater use of such tools in order to deliver material that is not only evidence-based but also well-targeted to its potential readers. Indeed, it has been demonstrated that an accurate revision of an informed consent form may on average increase the GulpEase index by 84%. 27 Analogously, considering the niche nature of the topic of squalene-based adjuvants used in influenza vaccines, balanced evidence-based texts should be accompanied by neutral/positive and self-explanatory images, animations or, better still, video testimonials with relevant stakeholders or celebrities (indeed, testimonials of anti-vaccination celebrities are rather common among websites critical of immunization). Moreover, in our previous experience with a healthcare prevention-oriented mobile application and a sister website, 40 , 41 we realized that expert-based opinion on the content, usability and other quality attributes may differ from the expectations of lay users. Active collaboration between institutions and potential users, patient organizations, etc. would therefore help to deliver consumer-focused messages. By contrast, authors contributing to non-institutional websites should communicate science in a more ethical way. Indeed, according to the Society of Professional Journalists' code of ethics, 42 a journalist should "be accurate and fair", "verify information before releasing it", "take special care not to misrepresent or oversimplify… a story", "…update and correct information", "balance the public's need for information against potential harm", "consider the long-term implications", etc. In our opinion, given that a growing body of literature suggests that the internet is a main driver of vaccination-related decision-making and the fact that we are living in the "post-truth era", 43 future studies should shift from purely observational to implementation research that is able to efficiently inform public policies. To conclude, despite thousands of deaths avoided and millions of Euro spared during the last 20 years of squalene-adjuvanted influenza vaccine use, 44 misinformation on this naturally occurring substance with an "unhappy" etymological origin makes it a victim of its name. Methods Search strategy and eligibility criteria A set of queries containing a squalene-related plus influenza- and/or vaccine-related terms was created (LA and DA); this considered various semantically close terms, grammatical numbers, spellings and suggestions made by the Google autocomplete service. In all, 85 queries were created. No explicit Boolean operators (such as "AND" or "OR") were used since most laypeople and healthcare professionals (HCPs) do not use/are unable to use these functions. 45-47 We were aware that such a search strategy would have a low specificity and be more time-consuming, with queries being very similar and therefore having a large amount of duplicate results. We, however, opted for this methodology in order to align with the web searching behavior of the typical user and thus identify as much highly ranked squalene-related web pages as possible. Google was used to locate web pages, because Google is the most widely used search engine in Italy 48 with a market share of approximately 95%. 49 For each query the first 30 search results (first 3 pages) were consulted since most users do not go beyond this point. 46 , 50 In total, 2,550 search results were screened. The search was performed over 4 consecutive days (from 9 to 12 January 2017). Once duplicates were removed, the selected web pages were screened in order to identify those potentially eligible for analysis. The following inclusion criteria were applied: (i) page availability on the search day; (ii) pages written in Italian; (iii) influenza vaccines as one of the main topics of the web page; (iv) any reference to squalene in influenza vaccines (and not in cosmetics, foods etc.). The exclusion criteria were: (i) no textual information (e.g. only pictures); (ii) small amount of text (<150 words); (iii) highly technical material (e.g. articles published in scientific journals, operating protocols, manuals) targeting HCPs and scientists; (iv) forums, chatrooms and similar. Analysis of web pages and text mining Each included web page was analyzed by two investigators (LA and DA) each working independently. All web pages included were static (i.e. content did not change) during the period of retrieval. The general tone of an article, which was the main outcome of the present study, was a priori thought to be categorized as either positive (those approving of squalene-based adjuvants), neutral (those that neither approve nor disapprove of squalene-based adjuvants), negative (those disapproving of squalene-based adjuvants), or ambiguous (those containing both approving and disapproving messages). 51 The definition of each category was discussed and comprehended by the two raters. However, in the phase of content analysis, distinguishing between the positive and neutral tones was still deemed subjective (Cohen's κ 0.60), therefore these two categories were combined for the purpose of analysis. Moreover, most web pages that could be potentially categorized as "ambiguous" reported some scientifically sound facts on squalene/squalene-based adjuvants, but at the same time raised suspicions regarding safety issues of such adjuvants (Cohen's κ 0.55). Following discussion inside the research team and after consultation with external experts, it was agreed that any uncertainty regarding squalene/squalene-based adjuvant safety issues would negatively affect laypeople's perception, and therefore, the "negative" and "ambiguous" categories should be combined. The re-categorization allowed us to have a perfect interrater agreement. A similar categorization (pro/neutral vs adverse) of websites on influenza vaccination has been previously reported. 52 The applied dichotomous rule (i.e. Pos/Neu and Neg/Amb) in the web page categorization undoubtedly allowed us to increase the classification accuracy. The top-level domain of each web page included for analysis was extracted and categorized as follows: .it, .com, .org, and "other". Furthermore, web pages were dichotomized by website source into the following categories: (1) governmental agencies/institutions/universities/hospitals/local health units/medical centers/HCP societies (henceforth referred to as "institutional source") and (2) online newspapers/news sources/informational portals/users' generated content (e.g. blogs) (henceforth referred to as "non-institutional source"). We assumed that the first category, being composed of institutions and therefore more "official", would present more reliable information on squalene/squalene-based adjuvants. Indeed, it has been shown that the quality of Italian vaccination-related websites belonging to the government or professional associations is on average higher than that of blogs and anti-vaccination movement websites. 16 To assess the readability properties of the selected squalene-related online resources, the GulpEase index 53 – specifically designed for the Italian language – was applied. The index considers the average number of characters per word, the average number of words per sentence, and is expressed as 89+[(300 × N Sentence )–(10 × N Letter )/ N Word ]; a higher score implies greater readability. It is usually considered that texts with an index <40 would be difficult to read for people with a high school-level educational background. For the automatic readability assessment, which was carried out using Microsoft Word software, each selected article was preliminarily pre-treated in order to avoid misleading results. In particular, since identification of the sentence end performed by the automatic algorithm is triggered by the presence of punctuation marks relative to the end sentence (e.g. period, exclamation mark, question mark), such punctuation marks (eventually found in the middle of sentences, numbers, abbreviations) were deleted. In the same way, phrases without the end of sentence punctuation were also removed from the score calculation. Other than text, content forms including images and video also quantified and were qualitatively described. In the qualitative part of the analysis, the lexicon of Pos/Neu and Neg/Amb web pages was compared by constructing and visually inspecting tag clouds with 50 or more common words. Subsequently, we analyzed the content of Neg/Amb web pages in a more detailed way. In the pilot study, we retrieved the first 30 web pages using the search terms "squalene" and "influenza vaccines" in order to establish main scientifically unsound information on the squalene-based adjuvants in influenza vaccines. Six common myths were identified. Such flawed statements together with evidence-based information belying these myths are reported in Table 5 . 10 , 11 , 54-61 Moreover, some conspiracy theories, which are common among anti-vaccination activists, 15 around squalene and/or squalene-based adjuvants were also present. In particular, in the pilot study we identified two such statements: (i) "politicians are personally immunized with non-adjuvanted vaccines, while laypeople are immunized with vaccines containing squalene" and (ii) "the only reason to include squalene in vaccines is for profit, by sparing the amount of antigen and selling adjuvanted vaccines for a higher price." It should be noted that we did not analyze anything other than squalene-related information (i.e. general immunization-related claims made by anti-vaccination activists were not analyzed, nor were quality aspects of the selected web pages since these topics had been already extensively studied in both Italian and international contexts). Individuals interested in the common claims made by anti-vaccination websites may wish to study the work of Kata et al. 15 The quality of Italian vaccination-related websites is assessed in work by Tafuri et al., 16 while the quality of information concerning influenza prevention on Italian and English websites is described by Maki et al. 62 Statistical analysis Approximately normally distributed variables were expressed as means with standard deviations (SDs), while ordinal as medians with ranges. Categorical variables were expressed as proportions with 95% confidence intervals (CIs). Differences in approximately normally distributed variables were evaluated by means of the t test, while those ordinal by means of the Mann-Whitney U test. Categorical variables were compared by means of χ 2 or Fisher's exact test (whichever test was most appropriate). The effect size for normally distributed data was measured by means of Cohen's d , that for the Mann-Whitney U test – as r = z /√ N , while that for 2 × 2 contingency tables – as φ = √χ 2 / N . Cohen's d was interpreted as small (0.2), medium (0.5), and large (0.8), while r and φ – as small (0.1), medium (0.3), and large (0.5). 63-65 To establish a statistical association between the main study outcome (general tone of a web page, where Neg/Amb is 1) and independent variables of interest, a multivariable logistic regression model was constructed. Variables showing an association with the general tone at α < 0.25 in the univariable analysis were included in the multivariable model. 66 All analyses were performed in the R environment. 67 Search strategy and eligibility criteria A set of queries containing a squalene-related plus influenza- and/or vaccine-related terms was created (LA and DA); this considered various semantically close terms, grammatical numbers, spellings and suggestions made by the Google autocomplete service. In all, 85 queries were created. No explicit Boolean operators (such as "AND" or "OR") were used since most laypeople and healthcare professionals (HCPs) do not use/are unable to use these functions. 45-47 We were aware that such a search strategy would have a low specificity and be more time-consuming, with queries being very similar and therefore having a large amount of duplicate results. We, however, opted for this methodology in order to align with the web searching behavior of the typical user and thus identify as much highly ranked squalene-related web pages as possible. Google was used to locate web pages, because Google is the most widely used search engine in Italy 48 with a market share of approximately 95%. 49 For each query the first 30 search results (first 3 pages) were consulted since most users do not go beyond this point. 46 , 50 In total, 2,550 search results were screened. The search was performed over 4 consecutive days (from 9 to 12 January 2017). Once duplicates were removed, the selected web pages were screened in order to identify those potentially eligible for analysis. The following inclusion criteria were applied: (i) page availability on the search day; (ii) pages written in Italian; (iii) influenza vaccines as one of the main topics of the web page; (iv) any reference to squalene in influenza vaccines (and not in cosmetics, foods etc.). The exclusion criteria were: (i) no textual information (e.g. only pictures); (ii) small amount of text (<150 words); (iii) highly technical material (e.g. articles published in scientific journals, operating protocols, manuals) targeting HCPs and scientists; (iv) forums, chatrooms and similar. Analysis of web pages and text mining Each included web page was analyzed by two investigators (LA and DA) each working independently. All web pages included were static (i.e. content did not change) during the period of retrieval. The general tone of an article, which was the main outcome of the present study, was a priori thought to be categorized as either positive (those approving of squalene-based adjuvants), neutral (those that neither approve nor disapprove of squalene-based adjuvants), negative (those disapproving of squalene-based adjuvants), or ambiguous (those containing both approving and disapproving messages). 51 The definition of each category was discussed and comprehended by the two raters. However, in the phase of content analysis, distinguishing between the positive and neutral tones was still deemed subjective (Cohen's κ 0.60), therefore these two categories were combined for the purpose of analysis. Moreover, most web pages that could be potentially categorized as "ambiguous" reported some scientifically sound facts on squalene/squalene-based adjuvants, but at the same time raised suspicions regarding safety issues of such adjuvants (Cohen's κ 0.55). Following discussion inside the research team and after consultation with external experts, it was agreed that any uncertainty regarding squalene/squalene-based adjuvant safety issues would negatively affect laypeople's perception, and therefore, the "negative" and "ambiguous" categories should be combined. The re-categorization allowed us to have a perfect interrater agreement. A similar categorization (pro/neutral vs adverse) of websites on influenza vaccination has been previously reported. 52 The applied dichotomous rule (i.e. Pos/Neu and Neg/Amb) in the web page categorization undoubtedly allowed us to increase the classification accuracy. The top-level domain of each web page included for analysis was extracted and categorized as follows: .it, .com, .org, and "other". Furthermore, web pages were dichotomized by website source into the following categories: (1) governmental agencies/institutions/universities/hospitals/local health units/medical centers/HCP societies (henceforth referred to as "institutional source") and (2) online newspapers/news sources/informational portals/users' generated content (e.g. blogs) (henceforth referred to as "non-institutional source"). We assumed that the first category, being composed of institutions and therefore more "official", would present more reliable information on squalene/squalene-based adjuvants. Indeed, it has been shown that the quality of Italian vaccination-related websites belonging to the government or professional associations is on average higher than that of blogs and anti-vaccination movement websites. 16 To assess the readability properties of the selected squalene-related online resources, the GulpEase index 53 – specifically designed for the Italian language – was applied. The index considers the average number of characters per word, the average number of words per sentence, and is expressed as 89+[(300 × N Sentence )–(10 × N Letter )/ N Word ]; a higher score implies greater readability. It is usually considered that texts with an index <40 would be difficult to read for people with a high school-level educational background. For the automatic readability assessment, which was carried out using Microsoft Word software, each selected article was preliminarily pre-treated in order to avoid misleading results. In particular, since identification of the sentence end performed by the automatic algorithm is triggered by the presence of punctuation marks relative to the end sentence (e.g. period, exclamation mark, question mark), such punctuation marks (eventually found in the middle of sentences, numbers, abbreviations) were deleted. In the same way, phrases without the end of sentence punctuation were also removed from the score calculation. Other than text, content forms including images and video also quantified and were qualitatively described. In the qualitative part of the analysis, the lexicon of Pos/Neu and Neg/Amb web pages was compared by constructing and visually inspecting tag clouds with 50 or more common words. Subsequently, we analyzed the content of Neg/Amb web pages in a more detailed way. In the pilot study, we retrieved the first 30 web pages using the search terms "squalene" and "influenza vaccines" in order to establish main scientifically unsound information on the squalene-based adjuvants in influenza vaccines. Six common myths were identified. Such flawed statements together with evidence-based information belying these myths are reported in Table 5 . 10 , 11 , 54-61 Moreover, some conspiracy theories, which are common among anti-vaccination activists, 15 around squalene and/or squalene-based adjuvants were also present. In particular, in the pilot study we identified two such statements: (i) "politicians are personally immunized with non-adjuvanted vaccines, while laypeople are immunized with vaccines containing squalene" and (ii) "the only reason to include squalene in vaccines is for profit, by sparing the amount of antigen and selling adjuvanted vaccines for a higher price." It should be noted that we did not analyze anything other than squalene-related information (i.e. general immunization-related claims made by anti-vaccination activists were not analyzed, nor were quality aspects of the selected web pages since these topics had been already extensively studied in both Italian and international contexts). Individuals interested in the common claims made by anti-vaccination websites may wish to study the work of Kata et al. 15 The quality of Italian vaccination-related websites is assessed in work by Tafuri et al., 16 while the quality of information concerning influenza prevention on Italian and English websites is described by Maki et al. 62 Statistical analysis Approximately normally distributed variables were expressed as means with standard deviations (SDs), while ordinal as medians with ranges. Categorical variables were expressed as proportions with 95% confidence intervals (CIs). Differences in approximately normally distributed variables were evaluated by means of the t test, while those ordinal by means of the Mann-Whitney U test. Categorical variables were compared by means of χ 2 or Fisher's exact test (whichever test was most appropriate). The effect size for normally distributed data was measured by means of Cohen's d , that for the Mann-Whitney U test – as r = z /√ N , while that for 2 × 2 contingency tables – as φ = √χ 2 / N . Cohen's d was interpreted as small (0.2), medium (0.5), and large (0.8), while r and φ – as small (0.1), medium (0.3), and large (0.5). 63-65 To establish a statistical association between the main study outcome (general tone of a web page, where Neg/Amb is 1) and independent variables of interest, a multivariable logistic regression model was constructed. Variables showing an association with the general tone at α < 0.25 in the univariable analysis were included in the multivariable model. 66 All analyses were performed in the R environment. 67 Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541451/

Disparate adjuvant properties among three formulations of “alum”

Aluminum adjuvants, commonly referred to as "alum," are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject® alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject® alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject® alum, Alhydrogel®, and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel® and precipitated alum was significantly greater than that induced by Imject® alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject® exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel® and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906517/

Whole-Genome Sequencing Reveals Distinct Mutational Patterns in Closely Related Laboratory and Naturally Propagated Francisella tularensis Strains

The F. tularensis type A strain FSC198 from Slovakia and a second strain FSC043, which has attenuated virulence, are both considered to be derivatives of the North American F. tularensis type A strain SCHU S4. These strains have been propagated under different conditions: the FSC198 has undergone natural propagation in the environment, while the strain FSC043 has been cultivated on artificial media in laboratories. Here, we have compared the genome sequences of FSC198, FSC043, and SCHU S4 to explore the possibility that the contrasting propagation conditions may have resulted in different mutational patterns. We found four insertion/deletion events (INDELs) in the strain FSC043, as compared to the SCHU S4, while no single nucleotide polymorphisms (SNPs) or variable number of tandem repeats (VNTRs) were identified. This result contrasts with previously reported findings for the strain FSC198, where eight SNPs and three VNTR differences, but no INDELs exist as compared to the SCHU S4 strain. The mutations detected in the laboratory and naturally propagated type A strains, respectively, demonstrate distinct patterns supporting that analysis of mutational spectra might be a useful tool to reveal differences in past growth conditions. Such information may be useful to identify leads in a microbial forensic investigation. Introduction Following the anthrax attacks of 2001, microbial forensics has emerged as a new scientific discipline dedicated to the investigation of biocrime and bioterrorism to link pathogen, crime, and perpetrator [1] . In molecular methods being developed to this end, selectively neutral genetic mutations, such as synonymous single nucleotide polymorphisms (SNPs) and extragenic tandem repeats [2] present advantages over non-neutral mutations to establish relationships between strains. Non-neutral characters are more prone to homoplasy (i.e. sharing of marker states for other reasons than ancestry) and less likely to accumulate at a constant rate, properties that may distort phylogenetic analyses. However, non-neutral mutations may also provide a different but potentially important aspect for microbial forensics. Since such mutations may reflect the selective forces experienced by a bacterium, they may also provide information on past propagation conditions. Here, we investigate this possibility by comparing mutational patterns detected in three strains designated SCHU S4 (FSC237), FSC198 (SE-219), and FSC043 of Francisella tularensis subspecies tularensis (type A1). Due to its high virulence, ease of dissemination, low infectious dose, and previous weaponisation, this pathogen has been classified by the Centers for Disease Control and Prevention among the top six 'Category A' biological threat agents [3] . Type A strains (in particular subgroup A1) demonstrate high virulence to humans [4] compared to the two other subspecies holarctica (type B) and mediasiatica, and are almost entirely restricted to North America [5] . To date, the only exception to the North American geographical confinement of F. tularensis type A is a handful of isolates recovered in Europe: in western Slovakia in 1986 [6] , and in a bordering area in Austria in 1990 (Gurycova unpublished). A recent genomic sequencing effort demonstrated that one of the recovered Slovakian strains, FSC198, is virtually identical and has been derived from SCHU S4 [7] . Data from the study also provided plausible evidence supporting that the European isolates indeed represent valid natural isolates and not events of laboratory contamination. We sequenced the strain FSC043, which is another derivative strain of the SCHU S4. In contrast to the assumed natural propagation of FSC198, the FSC043 has been cultivated repeatedly on artificial media in laboratories during which it likely lost its pathogenicity for mice [8] . Detection of different mutational patterns between these strains would therefore support the possibility to infer differences in culture conditions from mutational data. Results Correction of Francisella tularensis subsp. tularensis strain SCHU S4 genome sequence The genome sequence of F. tularensis subsp. tularensis strain SCHU S4 AJ749949.1 [9] available in GenBank [10] contained sequence errors in the form of SNPs and incorrect variable numbers of tandem repeats (VNTR), as identified recently by Chaudhuri et al. [7] . We have confirmed these errors and a corrected version of the genome sequence of SCHU S4 has been deposited in GenBank under accession number AJ749949.2. Identified mutational patterns Direct mapping of sequence reads from FSC043 on the genome sequences of FSC198 and SCHU S4 showed an average coverage of 107× separated by highly repetitive regions. The phylogenetic positions and relationships of the analyzed strains are shown in Figure 1 . Genome-wide sequence comparisons between strain FSC043 and strain SCHU S4 did not identify any SNPs between the two strains and they showed identical VNTR patterns, while three VNTRs differentiated them from FSC198. We found four insertion/deletion events differentiating FSC043 from SCHU S4 and FSC198 ( Table 1 ). Three of them (Ftind51–53) were small deletions (2, 1, and 1 bp, respectively), while the fourth was a 1,480 bp deletion and corresponded to the previously identified RD18 [11] . Ftind51 affected a putative metal ion transporter protein (FTT0615), while Ftind52 and Ftind53 were located within the duplicated Francisella Pathogenicity Island (FPI). Additional sequencing confirmed Ftind52 and Ftind53 as single mutations in both copies of the pdpC gene ( pdpC1 and pdpC2 ). The eight previously identified SNPs (S1–S8) in FSC198 [7] were all non-synonymous mutations and affected genes for UDP-N-acetylglucosamine pyrophosphorylase ( glmU ), an outer membrane protein (FTT0602), an acid phosphatase (FTT0620), a soluble pyridine nucleotide transhydrogenase ( sthA ), a lipoprotein located between lpn A and lpnB (FTT0903), a cardiolipin synthetase ( ybhO ), and a D-methionine transport protein (FTT1124). A summary of mutations in the analyzed genomes ( Table 2 ) is shown in Table 1 . The proposed strain history and an overview of the mutations are depicted in Figure 2 . 10.1371/journal.pone.0011556.g001 Figure 1 Relationships within the species F. tularensis . The evolutionary tree was inferred using the Neighbor-Joining method. Bootstrap support values (500 replicates) are shown next to branches. Scale bar indicates the number of base substitutions per site. 10.1371/journal.pone.0011556.g002 Figure 2 Overview of different paths of evolution. Strain FSC043 and strain FSC198 have been exposed to different environments since their split from the common ancestor strain SCHU S4. Strain FSC043 has experienced 'artificial' life cycles inside a laboratory while strain FSC198 has been exposed to a natural environment in Slovakia, which is reflected in their genomes by exhibiting different mutation patterns. 10.1371/journal.pone.0011556.t001 Table 1 Identified SNP, INDEL and VNTR differences between FSC198, FSC043 and SCHU S4 strains and their corresponding regions in three other genomes of F. tularensis . Region a Position b Locus b Gene b FSC198 FSC043 SCHUS4 FSC033 WY96-3418 LVS S1 390290 FTT0387 glmU T C C C C C S2 621377 FTT0602c T C C C C C S3 635510 FTT0620 A C C C C C S4 701627 FTT0684c sthA T G G G G G S5 911510 FTT0903 T C C C C C S6 1007563 FTT0997 ybhO C G G G G G S7 1008148 FTT0997 ybhO A G G G G G S8 1134416 FTT1124 metN A G G G G G Ft-M3 8266 ISFtu1 14 21 21 14 2 13 Ft-M8 308634 FTT1124 5 4 4 2 2 2 Ft-M10 1083657 ISFtu1 11 18 18 6 1 2 Ftind51 d 635249 FTT0615c DEL c Ftind52 d 1393671 FTT1354 pdpC1 DEL c Ftind53 d 1787006 FTT1709 pdpC2 DEL c RD18 928574 FTT0918, FTT0919 DEL c DEL c a Regions S1–S8, Ft-M3, Ft-M8, Ft-M10 and RD18 have been published previously [5] , [7] , [11] . b Genes and positions are given according to SCHU S4 (AJ749949.2). c DEL indicates deletion compared to other included strains. d The Ftind-numbering continues the serial presented in [39] – [41] . 10.1371/journal.pone.0011556.t002 Table 2 F. tularensis genome sequences used in this study. Species Subspecies Strain Alt. name Origin GenBank F. tularensis tularensis A1 FSC198 SE-219 Mite, Bratislava, Slovakia, 1988 AM286280.1 FSC043 Attenuated SCHU S4 phenotypic variant SCHU S4 a FSC237 SCHU phenotypic variant, 1951 AJ749949.2 FSC033 SnMF Squirrel, GA, USA, 1992 AAYE00000000.1 tularensis A2 WY96-3418 Human ulcer, WY, USA, 1996 CP000608.1 holarctica LVS ATCC 29684 Live vaccine strain, NDBR lot 11 AM233362.1 a SCHU S4 was derived from SCHU in 1951 by Henry T. Eigelsbach [42] . SCHU was originally isolated from human ulcer in Ohio 1941 by Dr. Lee Foshay [43] . Correction of Francisella tularensis subsp. tularensis strain SCHU S4 genome sequence The genome sequence of F. tularensis subsp. tularensis strain SCHU S4 AJ749949.1 [9] available in GenBank [10] contained sequence errors in the form of SNPs and incorrect variable numbers of tandem repeats (VNTR), as identified recently by Chaudhuri et al. [7] . We have confirmed these errors and a corrected version of the genome sequence of SCHU S4 has been deposited in GenBank under accession number AJ749949.2. Identified mutational patterns Direct mapping of sequence reads from FSC043 on the genome sequences of FSC198 and SCHU S4 showed an average coverage of 107× separated by highly repetitive regions. The phylogenetic positions and relationships of the analyzed strains are shown in Figure 1 . Genome-wide sequence comparisons between strain FSC043 and strain SCHU S4 did not identify any SNPs between the two strains and they showed identical VNTR patterns, while three VNTRs differentiated them from FSC198. We found four insertion/deletion events differentiating FSC043 from SCHU S4 and FSC198 ( Table 1 ). Three of them (Ftind51–53) were small deletions (2, 1, and 1 bp, respectively), while the fourth was a 1,480 bp deletion and corresponded to the previously identified RD18 [11] . Ftind51 affected a putative metal ion transporter protein (FTT0615), while Ftind52 and Ftind53 were located within the duplicated Francisella Pathogenicity Island (FPI). Additional sequencing confirmed Ftind52 and Ftind53 as single mutations in both copies of the pdpC gene ( pdpC1 and pdpC2 ). The eight previously identified SNPs (S1–S8) in FSC198 [7] were all non-synonymous mutations and affected genes for UDP-N-acetylglucosamine pyrophosphorylase ( glmU ), an outer membrane protein (FTT0602), an acid phosphatase (FTT0620), a soluble pyridine nucleotide transhydrogenase ( sthA ), a lipoprotein located between lpn A and lpnB (FTT0903), a cardiolipin synthetase ( ybhO ), and a D-methionine transport protein (FTT1124). A summary of mutations in the analyzed genomes ( Table 2 ) is shown in Table 1 . The proposed strain history and an overview of the mutations are depicted in Figure 2 . 10.1371/journal.pone.0011556.g001 Figure 1 Relationships within the species F. tularensis . The evolutionary tree was inferred using the Neighbor-Joining method. Bootstrap support values (500 replicates) are shown next to branches. Scale bar indicates the number of base substitutions per site. 10.1371/journal.pone.0011556.g002 Figure 2 Overview of different paths of evolution. Strain FSC043 and strain FSC198 have been exposed to different environments since their split from the common ancestor strain SCHU S4. Strain FSC043 has experienced 'artificial' life cycles inside a laboratory while strain FSC198 has been exposed to a natural environment in Slovakia, which is reflected in their genomes by exhibiting different mutation patterns. 10.1371/journal.pone.0011556.t001 Table 1 Identified SNP, INDEL and VNTR differences between FSC198, FSC043 and SCHU S4 strains and their corresponding regions in three other genomes of F. tularensis . Region a Position b Locus b Gene b FSC198 FSC043 SCHUS4 FSC033 WY96-3418 LVS S1 390290 FTT0387 glmU T C C C C C S2 621377 FTT0602c T C C C C C S3 635510 FTT0620 A C C C C C S4 701627 FTT0684c sthA T G G G G G S5 911510 FTT0903 T C C C C C S6 1007563 FTT0997 ybhO C G G G G G S7 1008148 FTT0997 ybhO A G G G G G S8 1134416 FTT1124 metN A G G G G G Ft-M3 8266 ISFtu1 14 21 21 14 2 13 Ft-M8 308634 FTT1124 5 4 4 2 2 2 Ft-M10 1083657 ISFtu1 11 18 18 6 1 2 Ftind51 d 635249 FTT0615c DEL c Ftind52 d 1393671 FTT1354 pdpC1 DEL c Ftind53 d 1787006 FTT1709 pdpC2 DEL c RD18 928574 FTT0918, FTT0919 DEL c DEL c a Regions S1–S8, Ft-M3, Ft-M8, Ft-M10 and RD18 have been published previously [5] , [7] , [11] . b Genes and positions are given according to SCHU S4 (AJ749949.2). c DEL indicates deletion compared to other included strains. d The Ftind-numbering continues the serial presented in [39] – [41] . 10.1371/journal.pone.0011556.t002 Table 2 F. tularensis genome sequences used in this study. Species Subspecies Strain Alt. name Origin GenBank F. tularensis tularensis A1 FSC198 SE-219 Mite, Bratislava, Slovakia, 1988 AM286280.1 FSC043 Attenuated SCHU S4 phenotypic variant SCHU S4 a FSC237 SCHU phenotypic variant, 1951 AJ749949.2 FSC033 SnMF Squirrel, GA, USA, 1992 AAYE00000000.1 tularensis A2 WY96-3418 Human ulcer, WY, USA, 1996 CP000608.1 holarctica LVS ATCC 29684 Live vaccine strain, NDBR lot 11 AM233362.1 a SCHU S4 was derived from SCHU in 1951 by Henry T. Eigelsbach [42] . SCHU was originally isolated from human ulcer in Ohio 1941 by Dr. Lee Foshay [43] . Discussion In this study, we found that different propagation conditions for the two F. tularensis strains FSC198 and FSC043 were supported by genomic data. While propagation in natural conditions has been assumed for the strain FSC198, the strain FSC043 has been extensively passaged in vitro in laboratories. Our results confirm previous findings that FSC198 differs from SCHU S4 at three VNTR loci and by eight intragenic and non-synonymous SNP mutations. In contrast, FSC043 was identical with SCHU S4 at all 25 VNTR loci, and no SNP mutations were found. Instead, all mutations in FSC043 were found to be intragenic deletion events; three micro deletions and the previously identified large deletion RD18 [11] . All four mutations found in strain FSC043 have caused disruption of gene functions: all of the disrupted genes in the strain FSC043 have been linked to virulence or have orthologs in F. novicida that have been linked to virulence. One of the two genes (FTT0918) which span the large deletion region RD18, is involved in iron uptake [12] and has been shown to be essential for virulence in the parental SCHU S4 strain [13] as well as in the attenuation of the Live Vaccine Strain [14] . Similar repeat-mediated deletions as in the RD18 locus (and another locus denoted RD19) have been identified, and seem to be characteristic for several laboratory propagated F. tularensis strains [11] . In agreement, it has frequently been observed that the genomes of laboratory strains eventually become degraded after passage on artificial media [15] . The Ftind52 and Ftind53 mutations represent identical deletion events but in different copies of the pdpC gene of the duplicated Francisella Pathogenicity Island, a locus important for F. tularensis virulence [16] . While these mutations could have occurred independently, it is likely that the mutation at one pdpC locus could have been transferred to the other pdpC locus by gene conversion (nonreciprocal recombination). High sequence homogeneity of insertion sequence elements within F. tularensis strains but divergence between subspecies suggests a strong effect of this process in F. tularensis . The pdpC gene is required for infection of F. tularensis in mammalian cells [17] but not for F. novicida infection of mosquito cells [18] . The mutation Ftind51 affects a putative metal ion transporter protein. A transposon mutant of the corresponding gene in F. novicida was negatively selected in a mouse model of infection [19] . The Ftind51 mutation may therefore also be linked to virulence in the strain FSC043. In the strain FSC198, three( sthA , ybhO and metA ) of the seven genes affected by mutation have been linked to virulence [20] – [22] . Since approximately 30% of the core genome (1162 genes) in F. tularensis [23] , have been experimentally identified as virulence genes to date [19] – [22] , [24] – [27] , it is possible that the seemingly preferential disruption of virulence genes in the FSC043 and the mutation of virulence genes in the FSC198 may be due to chance. Although certain mutations (e.g. rearrangements, large tandem repeat-polymorphisms) are not reliably detected by the sequencing methodology used, it is not unlikely that the few deletions detected completely may explain the attenuation of virulence in the strain FSC043. This hypothesis, however, needs further examination by specific phenotypic characterization of the strains studied and by experimental gene deletion and/or complementation [28] . Two evolutionary scenarios may have resulted in the gene disruptions detected in the strain FSC043. The disrupted genes may represent neutral events (i.e. genetic drift), caused by genetic bottlenecks that reduced the impact of selection, or because the mutated genes became superfluous when the bacterium was cultured on artificial media. It is also possible that the disruptions have been beneficial and therefore become positively selected. In a recent study of experimental populations of Escherichia coli [29] , where the impact of genetic drift was reduced by the use of large inoculates, the results indicated positive selection as the predominant cause of the fixation of mutations. Since the strain FSC043 is likely to have experienced reoccurring and severe genetic bottlenecks by the transfer of single colonies between agar plates, however, the fixation of the disruptive mutations could also be due to genetic drift in this strain. Regardless of whether mutations in the FSC043 are neutral (fixed by genetic drift) or non-neutral (fixed by positive selection), it is interesting that the frequency of fixed disruptive mutations (INDELs) occurred at a frequency that greatly exceeded all other mutations (SNPs, VNTRs) in the strain FSC043. This pattern contrasts sharply to that for the naturally propageted strain FSC198, where all mutations were non-synomous SNPs and VNTRs reflecting the adaptation to its propagation environment. Thus, our data agree with previous indications that the strain FSC198 has been propagated in a natural environment subsequent to its divergence from the progenitor strain SCHU S4 [7] . We find also that these results support the potential utility of analysis of mutational patterns to infer past propagation conditions. The generality and validity of these findings will require further confirmation, but may provide a new type of evidence in microbial forensics. Materials and Methods Strains The F. tularensis subsp. tularensis strain FSC043 was obtained from the Francisella Strain Collection (FSC) at the Swedish Defense Research Agency, Umeå. FSC043 was deposited to FSC by the Rocky Mountain Laboratories, Hamilton, MT, US, in 1992. The strain FSC043 represents a standard laboratory strain and it has as such been cultured extensively over the past six decades. It is uncertain precisely when the attenuating genetic mutations occurred. An overview of strains and genome sequences used is presented in Table 2 . Major relationships within the species Francisella are depicted in Figure 1 . Genome sequencing of FSC043 FSC043 was re-cultured, suspended in phosphate buffered saline and heat-killed. DNA was prepared by a chaotropic salt method [30] . Sequencing was performed by a commercial service provider (Geneservices, Cambridge, UK) using an Illumina GAII instrument with 36 bp single-end reads. Images acquired from the Illumina sequencer were processed through the Illumina pipeline to obtain sequence and quality scores for each base. Sequence reads have been deposited in the NCBI Short Read Archive [31] as SRA009329.1. Genome comparisons Genome assembly was performed by two alternative, complementary approaches. The first method was based on alignment and assembly using reference genomes from two strains within the subspecies tularensis type A1, FSC198 [7] and SCHU S4 [9] . Firstly, sequences from FSC043 were compared against the reference genomes using VAAL [32] . Additional analysis was performed in MOSAIK [33] allowing for non-unique hits in assembly, followed by identification of SNPs and small INDELs using Gigabayes [33] . Allowing non-unique mapping of reads allows identification of potential mutations within duplicated regions. Results from both VAAL and MOSAIK were inspected and confirmed in Consed [34] . Secondly, de novo assembly of short reads was performed using Velvet [35] using settings producing the highest N50 value. Constructed contigs were mapped to the same reference genomes using Exonorate [36] and nucmer in the package MUMmer [37] . Identified mutations among the three analyzed type A1 strains were further compared to the type A2 strain WY96-3418 [38] and the type B strain LVS. Sequence differences around VNTRs and RD18 in Francisella genomes ( Table 1 ) were analyzed in silico using previously published primers [5] . To confirm VNTRs in FSC043, MLVA was performed using a CEQ 8800 instrument (Beckman Coulters, Fullerton, CA), as previously described [5] . Verification of mutations within duplicated region The F. tularensis subsp. tularensis strains SCHU S4 and FSC043 were grown on modified GC-agar base at 5% [ ], suspended in water and used as PCR (Polymerase Chain Reaction) templates together with Expand Long Range polymerase (Roche Applied Science, Mannheim, Germany). Firstly, the regions FTT1353 to FTT1360 and FTT1709 to FTT1715 of the FPI were amplified using the internal FPI primer pairs FPI-1 and FPI-2, ( Table 3 ), in order to differentiate the two copies of the FPI. Each region comprised approximately 17 kbp. The resulting PCR products were purified from agarose using the High Pure PCR Purification Kit (Roche Applied Science, Mannheim, Germany) according to manufacturer's protocol. A second PCR was performed on each of the two purified PCR products, where the 5.5 kb regions surrounding pdpC1 and pdpC2 , respectively, was PCR amplified as eight sequential fragments to facilitate sequencing using primer pairs pdpC-1 to pdpC-8 ( Table 3 ). The average overlap between fragments was 118 nucleotides. Each fragment was cloned into the pCR4-TOPO TA cloning vector (Invitrogen AB, Stockholm, Sweden) and plasmids corresponding to four different clones from each of the eight combinations were purified using the QIAPrep Spin Miniprep Kit (Qiagen, Hilden, Germany) and all 32 clones were sequenced (Eurofins MWG operon, Ebersberg, Germany). A one base pair deletion was observed in both copies of pdpC in FSC043. To verify this, the region was amplified in both SCHU S4 and FSC043 using primer pair pdpC-9 (this does not allow a separation of the two FPI copies), and subsequent sequencing of the 691 bp PCR product was performed, confirming the mutation. No other differences were observed among the 5.5 kb sequenced region. 10.1371/journal.pone.0011556.t003 Table 3 Primers used to amplify regions surrounding pdpC1 and pdpC2 to confirm the Ftind52 and Ftind53 mutations. Region name Forward primer (5′ 3′) Reverse primer (5′ 3′) FPI-1 CCAGAATGACCCCGTAGAAA CTGCCTCAAAAAGCTCACCT FPI-2 CCAGAATGACCCCGTAGAAA TGCTGTAGCTCATGGTGAGG pdpC-1 TACTAAAGCACTGCACAAATTCAC GCATCGCTATTTTTGAGGGA pdpC-2 GCATGTATGAGGAGATTAAGAGC TGTCTTACGACTAGCGCGTCTA pdpC-3 CGAGGGGTTACTTGAAAATCT GAAGCCAGGAAGATAGCATACT pdpC-4 TCCTGGCTTCTTGAGCTCTGTAA TGATCGACACTATGTGCCATG pdpC-5 ACTCGGGATGGCAACTACAA TCTGAATTAGGTGTTGCGAAACT pdpC-6 GATTTCAACTTATACTCACCAGA TCTATTACTGGTTTTGAGGCTC pdpC-7 TGTGGATTAGTAGCGAAATTGTA GTTCGAATGTACTAGCTATTATTGT pdpC-8 GCCTTTCACACTAAGCTTATAAC TGATAGCATTGAATTTGATTTCC pdpC-9 TCCCTCAAAAATAGCGATGC TGTCTTACGACTAGCGCGTCTA Strains The F. tularensis subsp. tularensis strain FSC043 was obtained from the Francisella Strain Collection (FSC) at the Swedish Defense Research Agency, Umeå. FSC043 was deposited to FSC by the Rocky Mountain Laboratories, Hamilton, MT, US, in 1992. The strain FSC043 represents a standard laboratory strain and it has as such been cultured extensively over the past six decades. It is uncertain precisely when the attenuating genetic mutations occurred. An overview of strains and genome sequences used is presented in Table 2 . Major relationships within the species Francisella are depicted in Figure 1 . Genome sequencing of FSC043 FSC043 was re-cultured, suspended in phosphate buffered saline and heat-killed. DNA was prepared by a chaotropic salt method [30] . Sequencing was performed by a commercial service provider (Geneservices, Cambridge, UK) using an Illumina GAII instrument with 36 bp single-end reads. Images acquired from the Illumina sequencer were processed through the Illumina pipeline to obtain sequence and quality scores for each base. Sequence reads have been deposited in the NCBI Short Read Archive [31] as SRA009329.1. Genome comparisons Genome assembly was performed by two alternative, complementary approaches. The first method was based on alignment and assembly using reference genomes from two strains within the subspecies tularensis type A1, FSC198 [7] and SCHU S4 [9] . Firstly, sequences from FSC043 were compared against the reference genomes using VAAL [32] . Additional analysis was performed in MOSAIK [33] allowing for non-unique hits in assembly, followed by identification of SNPs and small INDELs using Gigabayes [33] . Allowing non-unique mapping of reads allows identification of potential mutations within duplicated regions. Results from both VAAL and MOSAIK were inspected and confirmed in Consed [34] . Secondly, de novo assembly of short reads was performed using Velvet [35] using settings producing the highest N50 value. Constructed contigs were mapped to the same reference genomes using Exonorate [36] and nucmer in the package MUMmer [37] . Identified mutations among the three analyzed type A1 strains were further compared to the type A2 strain WY96-3418 [38] and the type B strain LVS. Sequence differences around VNTRs and RD18 in Francisella genomes ( Table 1 ) were analyzed in silico using previously published primers [5] . To confirm VNTRs in FSC043, MLVA was performed using a CEQ 8800 instrument (Beckman Coulters, Fullerton, CA), as previously described [5] . Verification of mutations within duplicated region The F. tularensis subsp. tularensis strains SCHU S4 and FSC043 were grown on modified GC-agar base at 5% [ ], suspended in water and used as PCR (Polymerase Chain Reaction) templates together with Expand Long Range polymerase (Roche Applied Science, Mannheim, Germany). Firstly, the regions FTT1353 to FTT1360 and FTT1709 to FTT1715 of the FPI were amplified using the internal FPI primer pairs FPI-1 and FPI-2, ( Table 3 ), in order to differentiate the two copies of the FPI. Each region comprised approximately 17 kbp. The resulting PCR products were purified from agarose using the High Pure PCR Purification Kit (Roche Applied Science, Mannheim, Germany) according to manufacturer's protocol. A second PCR was performed on each of the two purified PCR products, where the 5.5 kb regions surrounding pdpC1 and pdpC2 , respectively, was PCR amplified as eight sequential fragments to facilitate sequencing using primer pairs pdpC-1 to pdpC-8 ( Table 3 ). The average overlap between fragments was 118 nucleotides. Each fragment was cloned into the pCR4-TOPO TA cloning vector (Invitrogen AB, Stockholm, Sweden) and plasmids corresponding to four different clones from each of the eight combinations were purified using the QIAPrep Spin Miniprep Kit (Qiagen, Hilden, Germany) and all 32 clones were sequenced (Eurofins MWG operon, Ebersberg, Germany). A one base pair deletion was observed in both copies of pdpC in FSC043. To verify this, the region was amplified in both SCHU S4 and FSC043 using primer pair pdpC-9 (this does not allow a separation of the two FPI copies), and subsequent sequencing of the 691 bp PCR product was performed, confirming the mutation. No other differences were observed among the 5.5 kb sequenced region. 10.1371/journal.pone.0011556.t003 Table 3 Primers used to amplify regions surrounding pdpC1 and pdpC2 to confirm the Ftind52 and Ftind53 mutations. Region name Forward primer (5′ 3′) Reverse primer (5′ 3′) FPI-1 CCAGAATGACCCCGTAGAAA CTGCCTCAAAAAGCTCACCT FPI-2 CCAGAATGACCCCGTAGAAA TGCTGTAGCTCATGGTGAGG pdpC-1 TACTAAAGCACTGCACAAATTCAC GCATCGCTATTTTTGAGGGA pdpC-2 GCATGTATGAGGAGATTAAGAGC TGTCTTACGACTAGCGCGTCTA pdpC-3 CGAGGGGTTACTTGAAAATCT GAAGCCAGGAAGATAGCATACT pdpC-4 TCCTGGCTTCTTGAGCTCTGTAA TGATCGACACTATGTGCCATG pdpC-5 ACTCGGGATGGCAACTACAA TCTGAATTAGGTGTTGCGAAACT pdpC-6 GATTTCAACTTATACTCACCAGA TCTATTACTGGTTTTGAGGCTC pdpC-7 TGTGGATTAGTAGCGAAATTGTA GTTCGAATGTACTAGCTATTATTGT pdpC-8 GCCTTTCACACTAAGCTTATAAC TGATAGCATTGAATTTGATTTCC pdpC-9 TCCCTCAAAAATAGCGATGC TGTCTTACGACTAGCGCGTCTA

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298149/

Large Crystal Toxin Formation in Chromosomally Engineered Bacillus thuringiensis subsp. aizawai Due to σ E Accumulation

Seven distinct Bacillus thuringiensis subsp. aizawai integrants were constructed that carried the chitinase ( chiBlA ) gene from B. licheniformis under the control of the cry11Aa promoter and terminator with and without p19 and p20 genes. The toxicity of B. thuringiensis subsp. aizawai integrants against second-instar Spodoptera litura larvae was increased 1.8- to 4.6-fold compared to that of the wild-type strain (BTA1). Surprisingly, the enhanced toxicity in some strains of B. thuringiensis subsp. aizawai integrants ( Bta P19CS, Bta P19CSter, and Bta CAT) correlated with an increase in toxin formation. To investigate the role of these genes in toxin production, the expression profiles of the toxin genes, cry1Aa and chiBlA , as well as their transcriptional regulators ( sigK and sigE ), were analyzed by quantitative real-time RT-PCR (qPCR) from BTA1, Bta P19CS, and Bta CAT. Expression levels of cry1Aa in these two integrants increased about 2- to 3-fold compared to those of BTA1. The expression of the transcription factor sigK also was prolonged in the integrants compared to that of the wild type; however, sigE expression was unchanged. Western blot analysis of σ E and σ K showed the prolonged accumulation of σ E in the integrants compared to that of BTA1, resulting in the increased synthesis of pro-σ K up to T 17 after the onset of sporulation in both Bta P19CS and Bta CAT compared to that of T 13 in BTA1. The results from qPCR indicate clearly that the cry1Aa promoter activity was influenced most strongly by σ E , whereas cry11Aa depended mostly on σ K . These results on large-crystal toxin formation with enhanced toxicity should provide useful information for the generation of strains with improved insecticidal activity.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722865/

A machine learning toolkit for genetic engineering attribution to facilitate biosecurity

The promise of biotechnology is tempered by its potential for accidental or deliberate misuse. Reliably identifying telltale signatures characteristic to different genetic designers, termed 'genetic engineering attribution', would deter misuse, yet is still considered unsolved. Here, we show that recurrent neural networks trained on DNA motifs and basic phenotype data can reach 70% attribution accuracy in distinguishing between over 1,300 labs. To make these models usable in practice, we introduce a framework for weighing predictions against other investigative evidence using calibration, and bring our model to within 1.6% of perfect calibration. Additionally, we demonstrate that simple models can accurately predict both the nation-state-of-origin and ancestor labs, forming the foundation of an integrated attribution toolkit which should promote responsible innovation and international security alike. Introduction After a nearly decade-long, $100 million dollar investigation into the 2001 Amerithrax anthrax attacks 1 , the National Academy of Sciences reported that "it is not possible to reach a definitive conclusion about the origins…based solely on the available scientific evidence" 2 . In the aftermath of Amerithrax, the scientific community mobilized to develop genetic 3 and phenotypic 4 methods for forensic attribution of biological attacks. However, these efforts were severely constrained by the limits of biotechnology; satisfactory sequencing of the anthrax agent's genome would have cost $500,000 dollars at the time 5 . Today, exponential improvement in tools for biotechnology have made this and other life-saving tasks increasingly inexpensive and accessible, yet these advancements have not been matched with corresponding improvements in tools to support responsible innovation in genetic engineering. In particular, attribution is still considered technically challenging and unsolved 6 – 9 . Just as programmers leave clues in code, biologists "programming" an engineered organism differ on unique design decisions (e.g., promoter choice), style (e.g., preferred codon optimization), intent (e.g., functional gene selection), and tools (e.g., cloning method). These design elements, together with evolutionary markers, form designer "signatures". Advances in high-throughput, spatial, and distributed sequencing 10 – 12 , and omic-scale phenotyping 13 make these signatures easier to collect but require complex data analysis. Recent work suggests that deep learning, a flexible paradigm for statistical models built from differentiable primitives, can facilitate complex biological data analysis 14 – 19 . We propose deploying these methods to develop a toolkit of machine-learning algorithms which can infer the attributes of genetically engineered organisms—like lab-of-origin—to support biotechnology stakeholders and enable ongoing efforts to scale and automate biosecurity 20 . A prior attempt to use deep learning for genetic engineering attribution provided evidence that it might be possible but was  accuracy, blue indicating confidence  accuracy, blue indicating confidence 1%) and was not missing, the "other" column was set to 1. A small number of plasmids (117) had sequences but no metadata. While far from perfect, our choice to use the default category of "other" to avoid introducing noisy information should prevent spurious features from being introduced by including phenotypic metadata. Given the performance boost achieved by including even this very minimal phenotype information, we are enthusiastic about future efforts to collate more standardized, expressive and descriptive phenotype information. Inferring plasmid lineage networks Many plasmids in the Addgene database reference other plasmids used in their construction. Within the metadata of each plasmid, we searched for references to other sequences in the Addgene repository, either by name or by their unique Addgene identifier. Plasmid names were unique except for 1519 plasmids that had names associated with more than one Addgene ID (331 of these also had duplicate sequences). However, none of these plasmids with duplicate names were referenced by name by some other plasmid. We considered a plasmid reference in one of the following metadata fields to be a valid reference: A portion of this plasmid was derived from a plasmid made by, Vector backbone, Backbone manufacturer , and Modifications to backbone . Self-references were not counted, and in the rare case where two plasmids referred to each other, the descendent/ancestor relationship was picked at random. We were interested in discovering networks of plasmids with shared ancestors—collectively we may call this subset of plasmids a lineage network. The problem of assigning plasmids to their associated network reduces to the problem of finding a node's connected component from an adjacency list of a directed, potentially cyclic, graph. The algorithm proceeded iteratively: in each round we picked some unvisited node. We then performed breadth-first search (plasmids were allowed to have multiple ancestors) and assigned all nodes visited in that round to a lineage network. In the case where a visited node pointed to a node that was already a member of some network, the two networks were merged. Keeping track of nodes visited in each round prevented the formation of cycles. We verified this result by reversing our adjacency list and running the same algorithm, equivalent to traversing by descendants instead of ancestors. Train-test-validation split To rigorously evaluate the performance of a predictive algorithm, strong boundaries between the datasets used for training and evaluation are needed to prevent overfitting. We follow best practices by pre-splitting the lab-of-origin Addgene data into an 80% training set, ~10% validation set for model selection, and ~10% test set held out for final model evaluation. In our vocabulary, the training set can be used for any optimization, including fitting an arbitrarily complex model. The validation set may only be used to measure the performance of an already trained model, e.g., to select architecture or hyperparameters; no direct training. Finally, the test set may only be used after the analysis is completed, the architecture and hyperparameters are finalized, as a measure of generalization performance. We addressed two additional considerations with the split: A large number of labs only deposited one or a few sequences. This is insufficient data to either train a model to predict that lab class or reasonably measure generalization error. Like many biological sequence datasets, the Addgene data are not independent and identically distributed because many plasmids are derived from others in the dataset, potentially creating biased accuracy measures due to overperformance on related plasmids used for both training and evaluation. To handle the first, we choose to pool plasmids with fewer than ten examples into an auxiliary category called "Unknown Engineered", and additionally stratify the split to ensure that every lab has at least three plasmids in the test set. For the second, we inferred lineage networks (see above). We stratified the split such that multi-lab networks were not split into multiple sets. In other words, each lineage was assigned either to the training, validation, or test set as a group, not divided between them. We used the GroupShuffleSplit function in python with sklearn ( http://scikit-learn.org/stable/ ) to randomly split given these constraints. The final split had 63,017 training points, 7466 validation points, and 11,351 test points. The larger test set is a direct result of enforcing 1/3 of rare lab's plasmids are split there for generalization measurement. We note that, because model selection is occurring on the validation set which has no representation of a number of rare labs, there is a built-in distributional shift that makes generalizing to our test set particularly challenging. However, we believe that this is appropriate to the problem setting—attribution algorithms should be penalized if they cannot detect rare labs, because in a deployment scenario the responsible lab may be unexpected. A visualization of this phenomena, and the lab distribution after splitting can be found in Supplementary Fig. 4 . We confirmed that this cleaning and splitting procedure did not dramatically change the difficulty of the task from prior work by reproducing the model architecture, hyperparameters, and training procedure of a model with known performance on a published dataset (see Baselines in "Methods") 21 . Byte-pair encoding The sequences from the training set were formatted as a newline-separated file for Byte Pair Encoding inference. The inference was performed in python on Amazon Web Services (AWS) with the sentencepiece package ( https://github.com/google/sentencepiece ) using both the BPE and Unigram 51 algorithms, with vocabulary sizes in [100, 1000, 5000, 10000], no start or end token, and full representation of all characters. The resulting model and vocabulary files were saved for model training, which used sentencepiece to tokenize batches of sequence on the fly during training. For the Unigram model, which is probabilistic, we sampled from the top ten most likely sequence configurations. For the visualization and interpretation of the 1000-token BPE vocabulary ultimately selected by our search algorithm, we took the vocabulary produced by sentencepiece, which has a list of tokens in order of merging (which is based on their frequency), and plotted this ordering vs. the length of the detected motif. We selected three example points visually for length at a given ranking. These sequence motifs were interrogated with BLAST 24 with the NCBI web tool, and additionally with BLAST tool on the iGEM Registry of Standard Biological Parts ( http://parts.igem.org/sequencing/index.cgi ). For each motif, a collection of results were compared with their plasmid maps to place the motif sequences within a plasmid component. We found, as numbered in the figure, motif 1 repeated twice in the SV40 promoter, motif 2 repeated twice in the CMV promoter, and motif 3 occurring slightly downstream of the pMB1 origin of replication. Training deep recurrent neural networks We consider a family of models based on the LSTM recurrent neural network and a DNA motif-base encoding. We formulated an architecture and hyperparameter search space based on prior experience with these models. In particular, we searched over categorical options of learning rate, batch size, bidirectionality, LSTM hidden size, LSTM number of layers, number of fully connected layers, extent of dropout, class of activations, maximum length of the input sequence and word embedding dimension. We further searched over parameters of the motif-based encoding, including whether it was Unigram or BPE based and the vocabulary size. Configurations from this categorical search space were sampled and evaluated by the Asynchronous Hyperband 52 algorithm, which evaluates a population of configurations in tandem and halts poor performing models periodically. Thus, computational resources are more efficiently allocated to the better performing models at each step in training. Our LSTM architecture was optimized with Adam using categorical cross-entropy loss in PyTorch ( https://pytorch.org/ ) and hyperparameter tuning with Asynchronous Hyperband used ray tune ( https://ray.readthedocs.io/en/latest/tune.html ). In early exploratory experiments, we found that including metadata in the initial training process caused a rapid increase but quick plateau of the Hyperband population. We noticed that these models usually had small LSTM components, suggesting that they were ignoring sequence information. This led us to hypothesize that adding metadata early in training led to an attractive local minimum for the tuning process which neglected sequence, exploiting the fact that Hyperband penalizes slow-improving algorithms. We, therefore, adopted a progressive training policy as follows. First, 250 configurations of the search space described above were evaluated with ray tune (Supplementary Figs. 2 and 3 ) over the course of ~1 week on an AWS p2.8xlarge machine with K80 GPUs. The best-performing model was selected for a stable and steadily decreasing loss curve (Supplementary Fig. 3 ) after 300 Hyperband steps, each of 300 weight updates (~90,000 updates total). This model configuration was saved and trained from scratch to ~200,000 weight updates, selected based on an early stopping heuristic on the validation loss. Next, this model was truncated to the pre-logit layer, and metadata was concatenated with the output of this sequence-only model (Supplementary Fig. 1 ), followed by one hidden layer and the logit layer. This was further trained, but with the LSTM sequence model frozen until validation loss plateaued. Finally, the full model was jointly trained until validation loss plateaued. The effect of this approach was to prevent the model converging on a metadata-focused local optimum without overfitting on the training set (which was facilitated by training only components of the model at a time). After fully training and finalizing results using our original random split (see above), three additional random splits were performed and training was repeated as before using different random seeds but the same hyperparameters as were found in the first hyperband search. We found that even without hyperparameter tuning on each newly split dataset, the results for the full and sequence-only deteRNNt models were consistent with our earlier results (Supplementary Fig. 5 ). Calibration analysis We followed the methodology of Guo et al. 32 . Prediction on the test set was binned into 15 bins, B m . The difference between the confidence of a model and the accuracy of the resulting model's predictions, termed calibration can be measured by two metrics, Expected Calibration Error (ECE) measuring the average difference between prediction confidence and ground-truth accuracy, and Maximum Calibration Error (MCE) measuring the maximum thereof. They are defined below ( n is the number of samples). 1 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${\mathrm{ECE}} = \mathop {\sum}\limits_{m = 1}^M { rac{{\left| {B_m} ight|}}{n}\left| {{\mathrm{acc}}\left( {B_m} ight) - {\mathrm{conf}}\left( {B_m} ight)} ight|},$$\end{document} ECE = ∑ m = 1 M B m n acc B m − conf B m , 2 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${\mathrm{MCE}} = \mathop {{\max }}\limits_{m \in \{ 1, \ldots ,M\} } \left| {{\mathrm{acc}}\left( {B_m} ight) - {\mathrm{conf}}\left( {B_m} ight)} ight|.$$\end{document} MCE = max m ∈ { 1 , … , M } acc B m − conf B m . Temperature scaling Temperature scaling adjusts the logits (pre-softmax output) of a multiclass classifier of c classes by dividing them by a single scalar value called the temperature. For a categorical prediction q for a single example, logits z \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\in {\Bbb R}^{oldsymbol{c}}$$\end{document} ∈ R c predicted by the network, scalar temperature T , and the softmax function σ SM we have a the temperature-scaled prediction: 3 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\hat q = \mathop {{\max }}\limits_k \sigma _{SM}\left( { rac{{{oldsymbol{z}}_i}}{T}} ight)^{(k)}.$$\end{document} q ^ = max k σ S M z i T ( k ) . The temperature is learned on the validation set after the model is fully trained. We used PyTorch and gradient descent with Adam optimizer to fit the temperature value. Subsequently, all of the logits predicted for the test set by the original model were divided by the temperature and softmaxed to get confidences as shown in Fig. 2 (right). Because the maximum of the softmaxed vector is mathematically equivalent to the maximum of a softmax on scaled logits, we concluded that the slight difference in the accuracy of the calibrated model was due to floating-point errors. Random Forest models We used scikit-learn package ( https://scikit-learn.org/stable/index.html ) implementation of Random Forest Regressor. Unless otherwise specified, we used 1000 estimators with 0.5 as the maximum proportion of features to look at while searching for the best split and with class weights inversely proportional to class frequency. For Random Forest analysis, we represented the sequences as frequencies of 1, 2, 3, and 4-grams. We constructed the n -gram vocabulary using the training set, and then only used the frequencies of n -grams included in the vocabulary to construct features ( n -gram frequencies) for the validation and test sets. Where specified, we concatenated one-hot-encoded metadata (phenotypic information) to these n -gram frequencies. We transformed the n -grams features using TF-IDF weighting 53 before using them for the Random Forest models. Nation-of-origin data Addgene has lab country information for many depositing labs ( https://www.addgene.org/browse/pi/ ). For those missing, publication links were followed to affiliation addresses, and the country of the lab was manually read off the address and cross-checked with a web search. When country information was missing from a lab, if there was conflicting information, and for very rare countries, these classes were dropped and all the corresponding plasmids for that lab were dropped from all three training split sets. No reshuffling of the train, validation and test data occurred. Lineage network analysis Lab, country, and ancestry-descendent linkage counts were obtained from the training set and plotted as described above, rank-ordering where specified. Networks were analyzed for size and graph diameter with NetworkX. Lab lineages were obtained from plasmid lineage data by considering the presence of a link from plasmid X from lab A to plasmid Y from Lab B to be a directed edge from Lab A to B. Parallel edges were not allowed and weight was not considered. Self-edges were disregarded. The country lineage network was constructed from the lab lineage network, by considering a connection between lab X in country A and lab Y in country B to be a directed edge between country A and B. This time, weights were given as the number of lab-to-lab connections. Parallel edges were not allowed, but self-edges were. For simplicity, the arrows of the directed graph were not shown in the NetworkX visualization. A version of Google PageRank 54 was computed on the directed, weighted graph with NetworkX. Ancestor lab prediction Due to the earlier biased train-validation-test split which deliberately segregated lineage networks into one of the three sets to minimize ancestry relationships that could lead to overfitting, we reconsidered the dataset for ancestor lab prediction. By definition, an ancestor plasmid and all its descendants will always be in the same set. So, if the ancestor is in the validation set, none of its descendants is available for training. Therefore, we first parsed the most recent ancestor for each plasmid from the lineage data and assigned each plasmid that ancestor's lab. We then randomly 80-10-10 re-split the data. We recognize that there is some potential for meta-overfitting by performing this reshuffle, even though ancestor lab prediction is a unique task from any of the others done so far. However, as this analysis was using a simple model intending to show tractability rather than peak performance, we decided to enable the right train-test-split was worth the chance. Interpreting the deteRNNt model To visualize the hidden states of the model, we first performed inference of the deteRNNt model on the validation set and extracted the activations of the last hidden layer (just prior to the layer which outputs logits). These hidden states were 1000-dimensional. To visualize them, we projected them into two and three dimensions using tSNE 37 in scikit-learn with default hyperparameters. We colored each point by P(True Lab) as assigned by the model for that example. Note that throughout this section when we refer to model probabilities, we mean the probabilities given by the model after temperature scaling calibration has been applied. The three-dimensional interactive plot was made using plotly and labeled with each plasmid's true lab-of-origin. For the analyses of sequence motifs (Fig. 5b–e ), we use the deteRNNt sequence-only model as phenotype information was not available for all plasmids. For the scanning-N ablation analysis (Fig. 5b, c ), we made all possible sequences with a window of 10 Ns inserted and performed standard deteRNNt inference to predict logits. We then generated per-position predicted logits by selecting all the sequences which include a given position as mutated to N , and averaging their logits together. We then apply softmax over each of these position logits to generate per-position predicted probabilities, which are indexed by the relevant labs to visualize. For the scanning subsequence analysis (Fig. 5d, e ), we made all possible subsequences of given length K and predicted logits for each, as above. Note that these are subsequences in isolation, as if they were sequences from a new plasmid, rather than padded with Ns or similar. For each position in the full sequence, we selected all the subsequences which include that position and averaged together their predicted logits, softmaxing to visualize probabilities as above. We custom-designed a gene-drive vector using the Akbari germline-Cas9 plasmid AAEL010097-Cas9 (Addgene # 100707 ) as a baseline. We modified it by removing the eGFP sequence attached to Cas9 and replacing it with the dsRed1 sequence within the same plasmid (but removing the Opie2 promoter in the process). We then identified two Cas9 guide RNAs against the Aedes aegypti AeAct-4 gene 55 (Genbank Accession Number: AY531223 ), designed to remove most of the coding region, that are predicted to have high activity using CHOPCHOP ( https://chopchop.cbu.uib.no ) 56 . These guides were placed downstream of the Akbari identified AeU6a and AeU6c promoters(Addgene # 117221 and # 117223 , respectively) 57 . We also included somArchon-GFP from pAAV-Syn-SomArchon (Addgene # 126941 , deposited by Edward Boyden's group) as a non-Akbari derived sequence. The Cas9-dsRed1_guide cassette_somArchon-GFP payload was flanked by 500 bp homology arms (upstream of 5′ guide and downstream of 3′ guide). To analyze the results of our ablation and subsequence analyses, we indexed out the positions in the sequence with the most extreme changes in predicted probability and manually examined these regions in Benchling. We performed automated annotation, used BLAST 58 , and searched various repositories for the highest-ranked fragments in order to identify restriction sites, primers sites and other features. Baselines The comparison with BLAST was performed using the blastn command line tool from NCBI 58 . At a high level, we can consider the BLAST baseline to be a nearest-neighbor algorithm, where the blast e -value is used to define neighbors in the training set. For each of the lab-of-origin and nation-of-origin prediction tasks, a fasta file of plasmid sequences from the training set was formatted as a BLAST database. Then, each test set was blasted against this database with an e -value threshold of 10. The resulting training set hits were sorted by e-value, from lowest to highest, and used to look up the training set labels for each sequence. For top 1 accuracy, the lowest e -value sequence class was compared to the true class. For top 10 accuracy, an example was marked correct if one of the labels of the lowest 10 e -value hits, after dropping duplicate hits to the same lab, corresponded to the correct test-set label. This dropping of duplicates ensured that the BLAST baseline was permitted up to 10 unique lab "guesses", which is necessary because occasionally the top-k ranked sequence results all have the same lab label. To perform the nation-of-origin and U.S. vs. foreign comparison, the same blast results were filtered to drop the U.S. or binarized so the U.S. was a positive class, respectively. The comparison with the Convolutional Neural Network (CNN) method copied the architecture and hyperparameters reported in Nielsen & Voigt (2018) 21 . The model was implemented in PyTorch. We trained for 100 epochs, as reported in previous work 21 , on an Nvidia K80 GPU using the Amazon Web Services cloud p2.xlarge instance. Training converged on a validation score of 57.1% and appeared stable (Supplementary Fig. 6 ). After training for this duration, we saved the model and evaluated performance on the held-out validation and test sets. Test-set performance was 50.2%, which was very near the previously reported accuracy of 48% 21 , leading us to conclude that this model's performance was reproducible and robust to increases in both the number of labs and number of plasmids in our dataset compared to Nielsen & Voigt (2018) (827 vs. 1314 labs, 36,764 vs. 63,017 plasmids) 21 . In other words, this replication of the architecture, hyperparameters and training procedure of previous work with everything held constant to the best of our knowledge except the dataset, suggests that the effect of having more examples (typically associated with an easier task) and more labs to distinguish between (typically associated with a more difficult task) approximately cancel out, or perhaps net to a very weak (2%) difference in the difficulty of our dataset. In lab-of-origin, U.S. vs. foreign, nation-of-origin, and ancestor lab prediction, we show comparisons with a baseline based on guessing the most abundant class, or classes (in the case of top 10 accuracy) from the training set. We also show the frequency of success based on uniformly guessing between the available labels (1/number of categories). Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Graphing and visualizations All graphs and plots were created in python using a combination of the seaborn ( https://seaborn.pydata.org/ ), matplotlib ( https://matplotlib.org/3.1.0/index.html ), plotly (for geographic visualization and interactive 3d hidden state visualization: https://plot.ly/ ) and networkx ( https://networkx.github.io/ ). The graphics in Fig. 1a were created in part with BioRender.com. Processing the Addgene dataset The dataset of deposited plasmid sequences and phenotype information was used with permission from Addgene, Inc. Scientists using Addgene may upload full or partial sequences along with metadata such as growth temperature, antibiotic resistance, vector backbone, vector manufacturer, host organism, and more. For quality control, Addgene sequences portions of deposited plasmids, and in some cases sequences entire constructs. As such, plasmid entries featured sequences categorized as addgene-full (22,937), depositor-full (32,669), addgene-partial (56,978), depositor-partial (25,654). When more than one category was listed, we prioritized plasmids in the order listed above. When there was more than one entry for a category, the longest sequence was chosen. If more than one partial sequence was present, we concatenated them into a single sequence. The final numbers by sequence category were addgene-full (22937), depositor-full (28465), addgene-partial (27185), and depositor-partial (3247). Plasmids were dropped if they did not have any registered sequences. Any Us were changed to Ts and letters other than A, T, G, C, or N were changed to Ns. The resulting dataset contained 81834 plasmids, from 3751 labs. The raw dataset contains as many as 18 metadata fields from Addgene. In the final dataset, we kept only host organism species, growth temperature, bacterial resistance, selectable markers, growth strain, and copy number. These fields were selected because they are phenotypic characteristics we expect to be easy to measure in the scenario considered here, where a sample of the organism is available for sequencing and wet-lab experimentation. For more information on what these phenotyping assays might look like, see Supplementary Table 1 . Metadata fields on Addgene are not standardized and have many irregularities as a result. To deal with the lack of standardization, our high-level approach was to default to conservatism in assigning a given plasmid some phenotype label. We used an "other" category for each field to avoid noisy or infrequent labels, in particular, we assigned any label that made up 1%) and was not missing, the "other" column was set to 1. A small number of plasmids (117) had sequences but no metadata. While far from perfect, our choice to use the default category of "other" to avoid introducing noisy information should prevent spurious features from being introduced by including phenotypic metadata. Given the performance boost achieved by including even this very minimal phenotype information, we are enthusiastic about future efforts to collate more standardized, expressive and descriptive phenotype information. Inferring plasmid lineage networks Many plasmids in the Addgene database reference other plasmids used in their construction. Within the metadata of each plasmid, we searched for references to other sequences in the Addgene repository, either by name or by their unique Addgene identifier. Plasmid names were unique except for 1519 plasmids that had names associated with more than one Addgene ID (331 of these also had duplicate sequences). However, none of these plasmids with duplicate names were referenced by name by some other plasmid. We considered a plasmid reference in one of the following metadata fields to be a valid reference: A portion of this plasmid was derived from a plasmid made by, Vector backbone, Backbone manufacturer , and Modifications to backbone . Self-references were not counted, and in the rare case where two plasmids referred to each other, the descendent/ancestor relationship was picked at random. We were interested in discovering networks of plasmids with shared ancestors—collectively we may call this subset of plasmids a lineage network. The problem of assigning plasmids to their associated network reduces to the problem of finding a node's connected component from an adjacency list of a directed, potentially cyclic, graph. The algorithm proceeded iteratively: in each round we picked some unvisited node. We then performed breadth-first search (plasmids were allowed to have multiple ancestors) and assigned all nodes visited in that round to a lineage network. In the case where a visited node pointed to a node that was already a member of some network, the two networks were merged. Keeping track of nodes visited in each round prevented the formation of cycles. We verified this result by reversing our adjacency list and running the same algorithm, equivalent to traversing by descendants instead of ancestors. Train-test-validation split To rigorously evaluate the performance of a predictive algorithm, strong boundaries between the datasets used for training and evaluation are needed to prevent overfitting. We follow best practices by pre-splitting the lab-of-origin Addgene data into an 80% training set, ~10% validation set for model selection, and ~10% test set held out for final model evaluation. In our vocabulary, the training set can be used for any optimization, including fitting an arbitrarily complex model. The validation set may only be used to measure the performance of an already trained model, e.g., to select architecture or hyperparameters; no direct training. Finally, the test set may only be used after the analysis is completed, the architecture and hyperparameters are finalized, as a measure of generalization performance. We addressed two additional considerations with the split: A large number of labs only deposited one or a few sequences. This is insufficient data to either train a model to predict that lab class or reasonably measure generalization error. Like many biological sequence datasets, the Addgene data are not independent and identically distributed because many plasmids are derived from others in the dataset, potentially creating biased accuracy measures due to overperformance on related plasmids used for both training and evaluation. To handle the first, we choose to pool plasmids with fewer than ten examples into an auxiliary category called "Unknown Engineered", and additionally stratify the split to ensure that every lab has at least three plasmids in the test set. For the second, we inferred lineage networks (see above). We stratified the split such that multi-lab networks were not split into multiple sets. In other words, each lineage was assigned either to the training, validation, or test set as a group, not divided between them. We used the GroupShuffleSplit function in python with sklearn ( http://scikit-learn.org/stable/ ) to randomly split given these constraints. The final split had 63,017 training points, 7466 validation points, and 11,351 test points. The larger test set is a direct result of enforcing 1/3 of rare lab's plasmids are split there for generalization measurement. We note that, because model selection is occurring on the validation set which has no representation of a number of rare labs, there is a built-in distributional shift that makes generalizing to our test set particularly challenging. However, we believe that this is appropriate to the problem setting—attribution algorithms should be penalized if they cannot detect rare labs, because in a deployment scenario the responsible lab may be unexpected. A visualization of this phenomena, and the lab distribution after splitting can be found in Supplementary Fig. 4 . We confirmed that this cleaning and splitting procedure did not dramatically change the difficulty of the task from prior work by reproducing the model architecture, hyperparameters, and training procedure of a model with known performance on a published dataset (see Baselines in "Methods") 21 . Byte-pair encoding The sequences from the training set were formatted as a newline-separated file for Byte Pair Encoding inference. The inference was performed in python on Amazon Web Services (AWS) with the sentencepiece package ( https://github.com/google/sentencepiece ) using both the BPE and Unigram 51 algorithms, with vocabulary sizes in [100, 1000, 5000, 10000], no start or end token, and full representation of all characters. The resulting model and vocabulary files were saved for model training, which used sentencepiece to tokenize batches of sequence on the fly during training. For the Unigram model, which is probabilistic, we sampled from the top ten most likely sequence configurations. For the visualization and interpretation of the 1000-token BPE vocabulary ultimately selected by our search algorithm, we took the vocabulary produced by sentencepiece, which has a list of tokens in order of merging (which is based on their frequency), and plotted this ordering vs. the length of the detected motif. We selected three example points visually for length at a given ranking. These sequence motifs were interrogated with BLAST 24 with the NCBI web tool, and additionally with BLAST tool on the iGEM Registry of Standard Biological Parts ( http://parts.igem.org/sequencing/index.cgi ). For each motif, a collection of results were compared with their plasmid maps to place the motif sequences within a plasmid component. We found, as numbered in the figure, motif 1 repeated twice in the SV40 promoter, motif 2 repeated twice in the CMV promoter, and motif 3 occurring slightly downstream of the pMB1 origin of replication. Training deep recurrent neural networks We consider a family of models based on the LSTM recurrent neural network and a DNA motif-base encoding. We formulated an architecture and hyperparameter search space based on prior experience with these models. In particular, we searched over categorical options of learning rate, batch size, bidirectionality, LSTM hidden size, LSTM number of layers, number of fully connected layers, extent of dropout, class of activations, maximum length of the input sequence and word embedding dimension. We further searched over parameters of the motif-based encoding, including whether it was Unigram or BPE based and the vocabulary size. Configurations from this categorical search space were sampled and evaluated by the Asynchronous Hyperband 52 algorithm, which evaluates a population of configurations in tandem and halts poor performing models periodically. Thus, computational resources are more efficiently allocated to the better performing models at each step in training. Our LSTM architecture was optimized with Adam using categorical cross-entropy loss in PyTorch ( https://pytorch.org/ ) and hyperparameter tuning with Asynchronous Hyperband used ray tune ( https://ray.readthedocs.io/en/latest/tune.html ). In early exploratory experiments, we found that including metadata in the initial training process caused a rapid increase but quick plateau of the Hyperband population. We noticed that these models usually had small LSTM components, suggesting that they were ignoring sequence information. This led us to hypothesize that adding metadata early in training led to an attractive local minimum for the tuning process which neglected sequence, exploiting the fact that Hyperband penalizes slow-improving algorithms. We, therefore, adopted a progressive training policy as follows. First, 250 configurations of the search space described above were evaluated with ray tune (Supplementary Figs. 2 and 3 ) over the course of ~1 week on an AWS p2.8xlarge machine with K80 GPUs. The best-performing model was selected for a stable and steadily decreasing loss curve (Supplementary Fig. 3 ) after 300 Hyperband steps, each of 300 weight updates (~90,000 updates total). This model configuration was saved and trained from scratch to ~200,000 weight updates, selected based on an early stopping heuristic on the validation loss. Next, this model was truncated to the pre-logit layer, and metadata was concatenated with the output of this sequence-only model (Supplementary Fig. 1 ), followed by one hidden layer and the logit layer. This was further trained, but with the LSTM sequence model frozen until validation loss plateaued. Finally, the full model was jointly trained until validation loss plateaued. The effect of this approach was to prevent the model converging on a metadata-focused local optimum without overfitting on the training set (which was facilitated by training only components of the model at a time). After fully training and finalizing results using our original random split (see above), three additional random splits were performed and training was repeated as before using different random seeds but the same hyperparameters as were found in the first hyperband search. We found that even without hyperparameter tuning on each newly split dataset, the results for the full and sequence-only deteRNNt models were consistent with our earlier results (Supplementary Fig. 5 ). Calibration analysis We followed the methodology of Guo et al. 32 . Prediction on the test set was binned into 15 bins, B m . The difference between the confidence of a model and the accuracy of the resulting model's predictions, termed calibration can be measured by two metrics, Expected Calibration Error (ECE) measuring the average difference between prediction confidence and ground-truth accuracy, and Maximum Calibration Error (MCE) measuring the maximum thereof. They are defined below ( n is the number of samples). 1 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${\mathrm{ECE}} = \mathop {\sum}\limits_{m = 1}^M { rac{{\left| {B_m} ight|}}{n}\left| {{\mathrm{acc}}\left( {B_m} ight) - {\mathrm{conf}}\left( {B_m} ight)} ight|},$$\end{document} ECE = ∑ m = 1 M B m n acc B m − conf B m , 2 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${\mathrm{MCE}} = \mathop {{\max }}\limits_{m \in \{ 1, \ldots ,M\} } \left| {{\mathrm{acc}}\left( {B_m} ight) - {\mathrm{conf}}\left( {B_m} ight)} ight|.$$\end{document} MCE = max m ∈ { 1 , … , M } acc B m − conf B m . Temperature scaling Temperature scaling adjusts the logits (pre-softmax output) of a multiclass classifier of c classes by dividing them by a single scalar value called the temperature. For a categorical prediction q for a single example, logits z \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\in {\Bbb R}^{oldsymbol{c}}$$\end{document} ∈ R c predicted by the network, scalar temperature T , and the softmax function σ SM we have a the temperature-scaled prediction: 3 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\hat q = \mathop {{\max }}\limits_k \sigma _{SM}\left( { rac{{{oldsymbol{z}}_i}}{T}} ight)^{(k)}.$$\end{document} q ^ = max k σ S M z i T ( k ) . The temperature is learned on the validation set after the model is fully trained. We used PyTorch and gradient descent with Adam optimizer to fit the temperature value. Subsequently, all of the logits predicted for the test set by the original model were divided by the temperature and softmaxed to get confidences as shown in Fig. 2 (right). Because the maximum of the softmaxed vector is mathematically equivalent to the maximum of a softmax on scaled logits, we concluded that the slight difference in the accuracy of the calibrated model was due to floating-point errors. Random Forest models We used scikit-learn package ( https://scikit-learn.org/stable/index.html ) implementation of Random Forest Regressor. Unless otherwise specified, we used 1000 estimators with 0.5 as the maximum proportion of features to look at while searching for the best split and with class weights inversely proportional to class frequency. For Random Forest analysis, we represented the sequences as frequencies of 1, 2, 3, and 4-grams. We constructed the n -gram vocabulary using the training set, and then only used the frequencies of n -grams included in the vocabulary to construct features ( n -gram frequencies) for the validation and test sets. Where specified, we concatenated one-hot-encoded metadata (phenotypic information) to these n -gram frequencies. We transformed the n -grams features using TF-IDF weighting 53 before using them for the Random Forest models. Nation-of-origin data Addgene has lab country information for many depositing labs ( https://www.addgene.org/browse/pi/ ). For those missing, publication links were followed to affiliation addresses, and the country of the lab was manually read off the address and cross-checked with a web search. When country information was missing from a lab, if there was conflicting information, and for very rare countries, these classes were dropped and all the corresponding plasmids for that lab were dropped from all three training split sets. No reshuffling of the train, validation and test data occurred. Lineage network analysis Lab, country, and ancestry-descendent linkage counts were obtained from the training set and plotted as described above, rank-ordering where specified. Networks were analyzed for size and graph diameter with NetworkX. Lab lineages were obtained from plasmid lineage data by considering the presence of a link from plasmid X from lab A to plasmid Y from Lab B to be a directed edge from Lab A to B. Parallel edges were not allowed and weight was not considered. Self-edges were disregarded. The country lineage network was constructed from the lab lineage network, by considering a connection between lab X in country A and lab Y in country B to be a directed edge between country A and B. This time, weights were given as the number of lab-to-lab connections. Parallel edges were not allowed, but self-edges were. For simplicity, the arrows of the directed graph were not shown in the NetworkX visualization. A version of Google PageRank 54 was computed on the directed, weighted graph with NetworkX. Ancestor lab prediction Due to the earlier biased train-validation-test split which deliberately segregated lineage networks into one of the three sets to minimize ancestry relationships that could lead to overfitting, we reconsidered the dataset for ancestor lab prediction. By definition, an ancestor plasmid and all its descendants will always be in the same set. So, if the ancestor is in the validation set, none of its descendants is available for training. Therefore, we first parsed the most recent ancestor for each plasmid from the lineage data and assigned each plasmid that ancestor's lab. We then randomly 80-10-10 re-split the data. We recognize that there is some potential for meta-overfitting by performing this reshuffle, even though ancestor lab prediction is a unique task from any of the others done so far. However, as this analysis was using a simple model intending to show tractability rather than peak performance, we decided to enable the right train-test-split was worth the chance. Interpreting the deteRNNt model To visualize the hidden states of the model, we first performed inference of the deteRNNt model on the validation set and extracted the activations of the last hidden layer (just prior to the layer which outputs logits). These hidden states were 1000-dimensional. To visualize them, we projected them into two and three dimensions using tSNE 37 in scikit-learn with default hyperparameters. We colored each point by P(True Lab) as assigned by the model for that example. Note that throughout this section when we refer to model probabilities, we mean the probabilities given by the model after temperature scaling calibration has been applied. The three-dimensional interactive plot was made using plotly and labeled with each plasmid's true lab-of-origin. For the analyses of sequence motifs (Fig. 5b–e ), we use the deteRNNt sequence-only model as phenotype information was not available for all plasmids. For the scanning-N ablation analysis (Fig. 5b, c ), we made all possible sequences with a window of 10 Ns inserted and performed standard deteRNNt inference to predict logits. We then generated per-position predicted logits by selecting all the sequences which include a given position as mutated to N , and averaging their logits together. We then apply softmax over each of these position logits to generate per-position predicted probabilities, which are indexed by the relevant labs to visualize. For the scanning subsequence analysis (Fig. 5d, e ), we made all possible subsequences of given length K and predicted logits for each, as above. Note that these are subsequences in isolation, as if they were sequences from a new plasmid, rather than padded with Ns or similar. For each position in the full sequence, we selected all the subsequences which include that position and averaged together their predicted logits, softmaxing to visualize probabilities as above. We custom-designed a gene-drive vector using the Akbari germline-Cas9 plasmid AAEL010097-Cas9 (Addgene # 100707 ) as a baseline. We modified it by removing the eGFP sequence attached to Cas9 and replacing it with the dsRed1 sequence within the same plasmid (but removing the Opie2 promoter in the process). We then identified two Cas9 guide RNAs against the Aedes aegypti AeAct-4 gene 55 (Genbank Accession Number: AY531223 ), designed to remove most of the coding region, that are predicted to have high activity using CHOPCHOP ( https://chopchop.cbu.uib.no ) 56 . These guides were placed downstream of the Akbari identified AeU6a and AeU6c promoters(Addgene # 117221 and # 117223 , respectively) 57 . We also included somArchon-GFP from pAAV-Syn-SomArchon (Addgene # 126941 , deposited by Edward Boyden's group) as a non-Akbari derived sequence. The Cas9-dsRed1_guide cassette_somArchon-GFP payload was flanked by 500 bp homology arms (upstream of 5′ guide and downstream of 3′ guide). To analyze the results of our ablation and subsequence analyses, we indexed out the positions in the sequence with the most extreme changes in predicted probability and manually examined these regions in Benchling. We performed automated annotation, used BLAST 58 , and searched various repositories for the highest-ranked fragments in order to identify restriction sites, primers sites and other features. Baselines The comparison with BLAST was performed using the blastn command line tool from NCBI 58 . At a high level, we can consider the BLAST baseline to be a nearest-neighbor algorithm, where the blast e -value is used to define neighbors in the training set. For each of the lab-of-origin and nation-of-origin prediction tasks, a fasta file of plasmid sequences from the training set was formatted as a BLAST database. Then, each test set was blasted against this database with an e -value threshold of 10. The resulting training set hits were sorted by e-value, from lowest to highest, and used to look up the training set labels for each sequence. For top 1 accuracy, the lowest e -value sequence class was compared to the true class. For top 10 accuracy, an example was marked correct if one of the labels of the lowest 10 e -value hits, after dropping duplicate hits to the same lab, corresponded to the correct test-set label. This dropping of duplicates ensured that the BLAST baseline was permitted up to 10 unique lab "guesses", which is necessary because occasionally the top-k ranked sequence results all have the same lab label. To perform the nation-of-origin and U.S. vs. foreign comparison, the same blast results were filtered to drop the U.S. or binarized so the U.S. was a positive class, respectively. The comparison with the Convolutional Neural Network (CNN) method copied the architecture and hyperparameters reported in Nielsen & Voigt (2018) 21 . The model was implemented in PyTorch. We trained for 100 epochs, as reported in previous work 21 , on an Nvidia K80 GPU using the Amazon Web Services cloud p2.xlarge instance. Training converged on a validation score of 57.1% and appeared stable (Supplementary Fig. 6 ). After training for this duration, we saved the model and evaluated performance on the held-out validation and test sets. Test-set performance was 50.2%, which was very near the previously reported accuracy of 48% 21 , leading us to conclude that this model's performance was reproducible and robust to increases in both the number of labs and number of plasmids in our dataset compared to Nielsen & Voigt (2018) (827 vs. 1314 labs, 36,764 vs. 63,017 plasmids) 21 . In other words, this replication of the architecture, hyperparameters and training procedure of previous work with everything held constant to the best of our knowledge except the dataset, suggests that the effect of having more examples (typically associated with an easier task) and more labs to distinguish between (typically associated with a more difficult task) approximately cancel out, or perhaps net to a very weak (2%) difference in the difficulty of our dataset. In lab-of-origin, U.S. vs. foreign, nation-of-origin, and ancestor lab prediction, we show comparisons with a baseline based on guessing the most abundant class, or classes (in the case of top 10 accuracy) from the training set. We also show the frequency of success based on uniformly guessing between the available labels (1/number of categories). Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Supplementary information Supplementary Information Reporting Summary Supplementary Information Reporting Summary Supplementary information Supplementary information is available for this paper at 10.1038/s41467-020-19612-0.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645522/

Efficient Polymer Solar Cells with Open-Circuit Voltage of 1.01 V and Power Conversion Efficiency of 8.09%

A series of polymer solar cells (PSCs) were prepared with different solvent additive 1-chloronaphthalene (CN) doping volume ratio to adjust the phase separation of active layers. The optimized PSCs exhibit a power conversion efficiency (PCE) of 8.09%, along with an open-circuit voltage of 1.01 V, a short circuit current density of 13.64 mA cm –2 , and a fill factor of 58.70%. All the key photovoltaic parameters of PSCs can be simultaneously increased by incorporating 1.0 vol % CN in blend solutions due to the optimized phase separation of active layers assisted by the volatilization of CN. Over 24% PCE improvement can be obtained by incorporating 1.0 vol % CN, indicating that the dynamic process of film forming should play the vital role in determining the performance of PSCs. 1 Introduction Polymer solar cells (PSCs) have achieved rapid development during the past decade, especially for nonfullerene materials as electron acceptors. 1 − 6 Many efforts have been made to improve the performance of PSCs from material synthesis and device engineering sides. Tandem or ternary strategy has been commonly investigated to improve the performance of PSCs by enhancing photon harvesting with two subactive layers or one active layer containing three different band gap materials. 7 In fact, ternary PSCs have been demonstrated as an efficient and potential strategy due to its simple device fabrication process. 8 − 11 Nonfullerene materials have attracted more attention due to their tunable band gap, energy levels, and high absorption coefficients, leading to the enhanced open-circuit voltage ( V OC ) and short circuit current density ( J SC ). 12 − 14 The dynamic process of film formation also plays the vital role in determining phase separation of active layers, which will greatly affect the exciton dissociation and charge transport efficiency. Solvent additives with different boiling point and selective dissolution of donor or acceptor have been used to adjust the phase separation for improving the performance of PSCs, such as 1,8-diiodooctane, 1-chloronaphthalene (CN), 1,8-octanedithiol, diphenylether, poly(dimethylsiloxane), etc. 15 − 20 Different solubilities of donor or acceptor in solvent additive are used to optimize the phase separation in the active layers, especially in the rapid thin-film formation progress during spin-coating. Thermal annealing and solvent vapor treatment were also commonly used to optimize phase separation of the active layers. Recently, upside-down thermal annealing and upside-down solvent vapor treatment were successfully developed to finely adjust the phase separation for improving the fill factor (FF) of PSCs. 21 The photovoltaic parameters of PSCs can be simultaneously improved by selecting appropriate materials and employing active layer treatments. Here, the recent progress in PSCs with V OC s close to or more than 1.0 V are summarized in Table 1 . Most PSCs exhibit relatively low power conversion efficiencies (PCEs) and FFs if the V OC s are close to or more than 1.0 V, which may be due to insufficient exciton dissociation of the relatively low offsets between the lowest unoccupied molecular orbitals levels of donor and acceptor. Meanwhile, there is plenty of room for FF improvement of PSCs with relatively high V OC . In this work, polymer material PBDB-T as the donor and nonfullerene material O-IDTBR as the acceptor were selected to prepare PSCs with 1-chloronaphthalene (CN) as solvent additive. The optimized PSCs exhibit relatively high V OC of 1.01 V and PCE of 8.09% for the active layer with 1.0 vol % CN, which are acceptable among the previous values. Compared with the PSCs without CN solvent additive, three photovoltaic parameters of PSCs can be simultaneously improved by incorporating 1.0 vol % CN, resulting from the optimized phase separation of the active layers. Figure 1 exhibits the chemical structures of the used materials, the device structure schematic diagram, and the energy levels of the used materials. Figure 1 (a) Chemical structures of the used materials and schematic diagram of the device structure. (b) The energy levels of the used materials. Table 1 Key Photovoltaic Parameters of PSCs with V OC s Close to or More than 1.0 V binary blend J SC (mA cm –2 ) V OC (V) FF (%) PCE (%) refs DR3TBDTT:O-IDTBR 11.06 1.15 50 6.36 ( 22 ) DR3:O-IDTBR 11.3 1.12 50 6.1 ( 23 ) PvBDTTAZ:O-IDTBR 16.26 1.08 63.6 11.2 ( 24 ) BDT3TR:O-IDTBR 12.31 1.08 56 7.09 ( 25 ) PBDT-DFQX1:O-IDTBR 14 1.07 57.88 8.67 ( 26 ) PBDB-T:ITCC 15.9 1.01 71 11.4 ( 27 ) PTB7-Th:O-IDTBR 15.7 1.01 64.6 9.83 ( 28 ) PBDB-T:O-IDTBR 13.64 1.01 58.7 8.09 this work PBDB-T:IFTN 7.3 1.01 42.1 3.03 ( 29 ) PBDB-T:IF-TN 6.25 1.01 41.5 2.57 ( 29 ) PBDB-T:IT-OM-3 16.38 0.98 70 10.8 ( 30 ) PfBTAZS:O-IDTBR 16.7 0.98 63 10.3 ( 31 ) PBDB-T:DICTiF 11.2 0.98 65 7.11 ( 32 ) PBDB-T:IDT-TN 11.09 0.98 41.3 4.49 ( 29 ) PBDB-T:ITC6-IC 16.41 0.97 73 11.61 ( 33 ) PBDB-T:IT-M 16.82 0.97 72 11.5 ( 34 ) PBDB-T:IT-DM 16.48 0.97 70.6 11.29 ( 35 ) PBDB-T:IT-DM 16.47 0.97 70.26 11.25 ( 36 ) PBDB-T:NTIC-Ome 13.52 0.97 66 8.47 ( 37 ) PBDB-T:IDT-TN 13.34 0.97 45.64 5.89 ( 29 ) PBDB-T:DTNIC8 12.92 0.96 72.84 9.03 ( 38 ) PBDB-T:CDTCN 11.26 0.96 57.58 6.23 ( 39 ) PBDB-T:SFTTIC 9.28 0.96 63.8 5.66 ( 40 ) PBDB-T:IT-M 17.34 0.95 73.2 12.1 ( 41 ) PBDB-T:DF-PCIC 13.38 0.95 63 8.1 ( 42 ) 2 Results and Discussion The absorption spectra of neat PBDB-T and O-IDTBR films were recorded, and the normalized absorption spectra are shown in Figure 2 a. The absorption peaks of neat PBDB-T and O-IDTBR film are about 625 and 695 nm, respectively, with a large spectral overlapping from 600 to 650 nm. The absorption spectra of blend films with different CN doping volume ratio were measured and are shown in Figure 2 b. The absorption spectra of blend films exhibit a marked absorption peak at about 630 nm, resulting from the large absorption spectral overlapping of the neat films. The effect of CN doping volume ratio on the absorption intensity of blend films can be mostly neglected, the slightly decreased absorption intensity may be due to the low concentration of mixed solutions with more CN. To investigate the effect of CN doping volume ratio on the performance of PSCs, the current density versus voltage ( J – V ) curves were measured under AM 1.5G illumination with light intensity of 100 mW cm –2 , as shown in Figure 3 a. Figure 2 (a) Normalized absorption spectra of neat PBDB-T and O-IDTBR films. (b) Absorption spectra of blend films with different CN doping volume ratio. Figure 3 (a) J – V curves of PSCs with different CN doping volume ratio in blend solutions. (b) External quantum efficiency (EQE) spectra of the corresponding PSCs. Markedly different photovoltaic parameters can be clearly observed from the J – V curves of the corresponding PSCs for the active layers with different CN doping volume ratios, although the absorption spectra of all blend films almost overlapped. The PCE of PSCs without CN is 6.48%, with a relatively low J SC of 12.95 mA cm –2 , V OC of 0.99 V, and FF of 50.35%. For the active layers with different CN doping volume ratios, the FFs and J SC s of the PSCs can be markedly improved and then slightly decreased along with the increase of the CN doping volume ratio. Meanwhile, the V OC s of the PSCs can be kept almost constant, with a rather small fluctuation of less than 0.03 V. The PCE of the optimized PSCs reaches 8.09% for the active layer with 1.0 vol % CN, along with a J SC of 13.64 mA cm –2 , a V OC of 1.01 V, and a FF of 58.70%. The marked increase in FF exhibits that the phase-separation degree can be well optimized by incorporating appropriate CN in the blend solutions. The series resistance ( R S ) and shunt resistance ( R SH ) can be calculated according to the J – V curves of PSCs without or with different CN doping volume ratios. The optimized PSCs with 1.0 vol % CN exhibits a minimal R S of 12 Ω cm 2 and a maximal R SH of 647 Ω cm 2 , which can well support the relatively high FF. The key parameters of PSCs with different CN doping volume ratios are summarized in Table 2 . The external quantum efficiency (EQE) spectra of all the PSCs were recorded and are shown in Figure 2 b. The EQE values of the PSCs with 1.0 vol % CN are slightly larger than those of other PSCs, which may result from the optimized exciton dissociation, charge transport, and collection dependence on phase separation. Table 2 Key Parameters of PSCs with Different CN Doping Volume Ratios a PCE (%) CN (vol %) J SC (mA cm –2 ) V OC (V) FF (%) best avg. R S (Ω cm 2 ) R SH (Ω cm 2 ) without 12.95 0.99 50.35 6.48 6.40 19 508 0.5 13.30 1.01 56.75 7.62 7.60 15 592 1.0 13.64 1.01 58.70 8.09 8.06 12 647 1.5 13.17 1.01 58.07 7.73 7.66 14 585 2.0 12.77 1.02 57.30 7.46 7.63 15 542 a Avg. PCE values were calculated according to 20 cells prepared from different batches. To further investigate the effect of CN doping volume ratio on the performance of PSCs, the J – V curves of the PSCs were measured in dark and under one standard simulation solar light illumination. The photocurrent density ( J PH ) can be calculated according to the equation: J PH = J L – J D , where J L and J D are the current densities under 100 mW cm –2 light illumination and in dark conditions. 43 − 45 The J PH versus effective voltage ( V eff = V 0 – V a ) curves of the PSCs without or with 1.0 vol % CN are exhibited in Figure 4 a, where V 0 is the voltage for J PH = 0 and V a is the applied bias. It is apparent that J PH of the PSCs with 1.0 vol % CN rapidly reaches the saturated states compared with the PSCs without CN under a relatively low bias, indicating the more efficient charge transport in the active layer with 1.0 vol % CN. According to the J PH versus V eff curves, the exciton dissociation and the charge collection efficiency could be evaluated by the ratios of J PH to saturation photocurrent density ( J SAT ) at short circuit or maximal power output conditions, respectively. The detailed values of the PSCs without and with 1.0 vol % CN are summarized in Table 3 . The exciton dissociation and charge collection efficiency can be simultaneously improved by adding 1.0 vol % CN, resulting from the optimized phase separation. To further investigate the positive effect of 1.0 vol % CN on the performance of PSCs, the J – V curves of PSCs without and with 1.0 vol % CN were measured under different light intensities ( P light ) from 1 to 100 mW cm –2 . According to the J – V curves, the relationship between J SC and P light are presented in Figure 4 b, which can be evaluated by the power-law formula of J SC ∝ P light α . 46 , 47 The bimolecular recombination in active layers can be negligible if the fitted parameter α is close to 1. The fitted parameter α is about 0.853 or 0.866 for the PSCs without or with 1.0 vol % CN, which are slightly far away from 1. The bimolecular recombination in both kinds of active layers cannot be neglected, resulting in the relatively low FF of PSCs. The FF of 58.70% for PSCs with 1.0 vol % CN is slightly larger than that of 50.35% for PSCs without CN, which can be well explained according to the values of the fitted parameter α. Figure 4 (a) J PH – V eff curves of PSCs processed without or with 1 vol % CN. (b) J SC dependence on light intensity of the corresponding PSCs. Table 3 J PH , J SAT , G max , and J PH / J SAT Values of the PSCs Processed without or with 1 vol % CN CN (vol %) J PH a (mA cm –2 ) J PH b (mA cm –2 ) J SAT (mA cm –2 ) J PH / J SAT a (%) J PH / J SAT b (%) without 13.17 9.72 14.91 88.3 65.2 1.0 13.40 10.99 14.51 92.3 75.8 a Short-circuit condition. b Maximal power output condition. The hole-only and electron-only devices were fabricated to investigate the effect of CN doping volume ratio on the charge mobility in the active layers with the configuration of indium tin oxide (ITO)/poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS)/active layers/MoO 3 /Ag and ITO/ZnO/active layers/2,9-bis(3-(dimethylamino)propyl)anthrax[2,1,9- def :6,5,10- d ′ e ′ f ′]diisoquinoline-1,3,8,10(2 H ,9 H ) tetraone (PDIN)/Al, respectively. The ln( Jd 3 / V 2 )–( V / d ) 0.5 curves of hole-only and electron-only devices are shown in Figure 5 a,b. The electron mobility (μ e ) and hole mobility (μ h ) can be calculated according to the ln( Jd 3 / V 2 )–( V / d ) 0.5 curves, as listed in Table 4 . It is apparent that the hole and electron mobilities in the active layers can be increased and decreased along with the increase in CN doping volume ratio. For the active layers with 1.0 vol % CN, μ e and μ h reach 3.93 × 10 –4 and 7.10 × 10 –4 cm 2 V –1 s –1 , respectively. Meanwhile, the hole and electron transport in the active layers with 1.0 vol % CN become more balanced, which is beneficial to improve the charge collection in the corresponding PSCs. The ratio of μ h to μ e reaches 1.81 for the PSCs with 1.0 vol % CN, leading to the relatively large FF of PSCs. As we known, both photogenerated hole and electron can be efficiently collected by individual electrode, leading to the performance improvement of PSCs. Figure 5 Plotted ln( Jd 3 / V 2 ) versus ( V / d ) 0.5 curves of hole-only (a) and electron-only (b) devices processed with different CN doping volume ratios. Table 4 μ h , μ e , and μ h /μ e in Blend Films with Different CN Doping Volume Ratios CN (vol %) μ h (cm 2 V –1 s –1 ) μ e (cm 2 V –1 s –1 ) μ h /μ e without 4.26 × 10 –4 1.42 × 10 –4 3.00 0.5 5.10 × 10 –4 2.29 × 10 –4 2.23 1.0 7.10 × 10 –4 3.93 × 10 –4 1.81 1.5 6.05 × 10 –4 3.19 × 10 –4 1.90 2.0 5.31 × 10 –4 2.64 × 10 –4 2.01 To further investigate the effect of CN doping volume ratios on the phase separation of the blend films, the transmission electron microscopy (TEM) images of the blend films were recorded and are shown in Figure 6 . It is known that the bright and dark zones represent the donor or the acceptor domain due to their different electron densities, respectively. 48 , 49 It is apparent that the phase-separation degree of the blend films can be markedly adjusted by incorporating different volume ratios of CN. For the blend films without CN, no distinctive characteristics can be observed from the TEM image due to the homogeneous distribution. The homogeneous distribution of PBDB-T and O-IDTBR should be beneficial to exciton dissociation due to the sufficient interface and be detrimental to charge transport in the blend films. Most of the photogenerated charge may be easily recombined in the active layers, leading to the relatively low FF. As seen from the TEM images of the blend films with different CN doping volume ratios, the relatively large bright and dark zones indicate the more efficient charge transport channels due to the large donor and acceptor domain sizes. The large domain size should be beneficial for charge transport and detrimental to exciton dissociation in the active layers. Therefore, an appropriate phase separation is very necessary to form bicontinuous interpenetrating network for efficient exciton dissociation and charge transport in the active layers. The optimized phase separation degree may be obtained for the blend films with 1.0 vol % CN, well in accordance with the relatively high FF of the corresponding PSCs. Figure 6 TEM images of the blend films processed with different CN doping volume ratios. 3 Conclusions A series of PSCs with PBDB-T as donor and O-IDTBR as acceptor were prepared with different CN doping volume ratios to adjust the phase separation degree of the active layers. The optimized PSCs exhibit a PCE of 8.09%, with a J SC of 13.64 mA cm –2 , a V OC of 1.01 V, and a FF of 58.70% for the active layers with 1.0 vol % CN. Compared with the PSCs without CN, all of the photovoltaic parameters can be simultaneously improved by incorporating appropriate CN to optimize the phase separation. More than 24% PCE improvement can be obtained by incorporating 1.0 vol % CN into the blend solutions, indicating that the dynamic process of thin film formation should play a vital role in determining the performance of PSCs. 4 Experimental Section The indium tin oxide (ITO) coated glass substrates (15 Ω sq –1 ) were continuously cleaned by sonication in detergent, deionized water, and ethanol. The cleaned ITO substrates were dried by high-purity nitrogen and treated by oxygen plasma for a minute to further improve their working function and clearance. Afterward, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS, purchased from H.C. Starck co. Ltd.), as a hole transport layer, was spin-coated on the ITO substrates at 5000 rpm for 40 s and dried at 150 °C for 10 min in atmospheric air. Then, ITO substrates coated with PEDOT:PSS films were transferred into a high-purity nitrogen-filled glovebox to fabricate active layers. The used materials, PBDB-T and O-IDTBR, were purchased from Organtecsolar Materials Inc. and Derthon Optoelectronic Materials Science Technology Co LTD, respectively. The mixed PBDB-T/O-IDTBR powder with a weight ratio of 1:1 was dissolved in chlorobenzene to prepare 20 mg/mL blend solutions and different 1-chloronaphthalene (CN) volumes (0.5, 1.0, 1.5, and 2.0 vol %) were doped into the blend solutions. The solutions were spin-coated onto the PEDOT:PSS/ITO substrates at 2300 rpm for 40 s to prepare the active layers. And then, the prepared active layers were annealed at 80° for 10 min in a high-purity nitrogen-filled glovebox. The small molecule 2,9-bis(3-(dimethylamino)propyl)anthrax[2,1,9- def :6,5,10- d ′ e ′ f ′]diisoquinoline-1,3,8,10(2 H ,9 H ) tetraone (PDIN) was dissolved in methanol with the addition of 0.28 vol % acetic acid to prepare a 2 mg/mL solution. Then, the prepared PDIN solution was spin-coated onto the active layers at 3000 rpm for 40 s to prepare the cathode interfacial layer. Finally, 100 nm aluminum (Al) was deposited with a mask by thermal evaporation under vacuum of 4 × 10 –5 Pa. The active area is approximately 3.8 mm 2 , which is defined by the overlapping area of ITO anode and Al cathode. A series of PSCs were fabricated with the configuration ITO/PEDOT:PSS/active layer/PDIN/Al, with the only difference of the CN doping volume ratios in the blend solutions. The detailed characteristics of the PSCs and the corresponding blend films are the same with that reported in our previous works. 10 , 11

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185793/

Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology

Antibody preparations have a long history of providing protection from infectious diseases. Although antibodies remain the only natural host-derived defense mechanism capable of completely preventing infection, as products, they compete against inexpensive therapeutics such as antibiotics, small molecule inhibitors and active vaccines. The continued discovery in the monoclonal antibody (mAb) field of leads with broadened cross neutralization of viruses and demonstrable synergy of antibody with antibiotics for bacterial diseases, clearly show that innovation remains. The commercial success of mAbs in chronic disease has not been paralleled in infectious diseases for several reasons. Infectious disease immunotherapeutics are limited in scope as endemic diseases necessitate active vaccine development. Also, the complexity of these small markets draws the interest of niche companies rather than big pharmaceutical corporations. Lastly, the cost of goods for mAb therapeutics is inherently high for infectious agents due to the need for antibody cocktails, which better mimic polyclonal immunoglobulin preparations and prevent antigenic escape. In cases where vaccine or convalescent populations are available, current polyclonal hyperimmune immunoglobulin preparations (pIgG), with modern and highly efficient purification technology and standardized assays for potency, can make economic sense. Recent innovations to broaden the potency of mAb therapies, while reducing cost of production, are discussed herein. On the basis of centuries of effective use of Ab treatments, and with growing immunocompromised populations, the question is not whether antibodies have a bright future for infectious agents, but rather what formats are cost effective and generate safe and efficacious treatments to satisfy regulatory approval. Anti-infective antibodies: new opportunities for a proven technology In the late 1890s Behring and Kitasato developed the first widely available and effective antimicrobial treatment by showing that transfer of immune sera could provide passive immunity to diphtheria [1] . In the next 50 years, serum transfer was used as a successful treatment of many infections including, pneumococcal pneumonia, meningococcal meningitis, and streptococcal infection [2] . However, due to both safety concerns and the discovery of antibiotics, serum therapy was largely abandoned by the late 1940s with the exception of a limited number of toxin- and viral-mediated diseases, which continued to rely on serum due to a lack of alternative options. The mainstream human immunoglobulin preparations used today are not recognizable as the old serum therapies. These products are highly purified, treated and filtered to ensure viral inactivation and removal, and use highly reproducible release assays to avoid the historical problems associated with polyclonal antibody (Ab) such as impurities, resident viruses, and lot variation. Two troubling developments in infectious diseases have led to a necessary resurgence in the development of antibody-based therapeutics. First, the rampant emergence of multi-drug resistant forms of new and old pathogens; second, the recent explosion of the world's immunocompromised population has provided an unprotected population from which combinations of complex infections are emerging. New adjunctive antibody therapeutics to every major disease of infection type may be the best strategy in both treating and preventing the new wave of drug resistant infectious diseases. Pathogenesis is linked to treatability The pathogenesis of infectious organisms can be very complex. The co-evolution of host and pathogen over evolutionary time has resulted in many types of inflammatory response. A fine balance exists between protective versus over zealous host responses, either through deregulation or through subversion by the pathogen. Although Ab therapies are either in development or in use to many viral, bacterial, fungal, and prion-mediated infections ( Table 1 , Table 2 , Table 3 ) these pathogens exhibit huge differences in pathology and virulence. In general, highly virulent and acute infections are more likely to require the immediate protection provided by antibody treatments. Moreover, in outbreak situations, the early implementation of public health measures may help to limit spread when no vaccine is available, as was shown with SARS 3 , 4 . During stretches of a dramatically increased risk of exposure, immunotherapy may provide a means of protection preferable over conventional vaccines [3] . The half-life of passive antibody therapeutics obviates the use of these types of products except when needed. Table 1 Passive Ab therapies to bacterial agents/toxins Target Name/format Details Refs Pseudomonas aeruginosa KB001; Pegylated Fab' against PCR-V made from humaneering murine mAb 166 C-Kalobios/Sanofi Pasteur protects against strains with PCR-V 92 , 93 Vibrio cholera Murine mAb 72.1 LPS specific R – Protects against Ogawa and Inaba strains in mouse infection model [94] Yersinia pestis Human Fab mAbs m252 (F1 specific) and m253 and m254 (V-specific) expressed as hu-IgG1 R – Protect mice from death in a bubonic plague model [95] Escherichia coli (EHEC) Shigatoxin Anti-Stx1 IgY; chicken egg yolk immunoglobulin (IgY) production R – Blocks the binding of Stx1 to the Hela cells and protect BALB/c mice from toxin challenges [96] Ricinus communis Ricin – Plant Toxin Murine mAb RAC18 and polyclonal murine antibody to A chain R – Inhibits cell death in vitro and protects mice from inhalational ricin exposure [97] Bacillus anthracis Purified human Polyclonal IgG (AIG) from high titer vaccinated donors (multiple epitopes/isotypes) M – Cangene Corp; protective in several animal models [98] Bacillus anthracis – PA toxin ABthrax (raxibacumab) Human IgG1/λ mAb to PA toxin; Anthim; Humanized deimmunized IgG1/k mAb to PA toxin; Valortim (MDX-1303) Human IgG1/k M – Human Genome Sciences; protective in several animal models C – Elusys; protective in rabbit inhalational spore model M – Pharmathene; protective in rabbit inhalational spore model 99 , 100 , 101 , 102 , 103 Chlamydia trachomatis Murine MP-33b (IgG3) and MP-A5d (IgA) mAbs against the major outer membrane protein delivered by hybridoma backpack R – Reduced bacterial pathology and shedding in ascending chlamydial infection in a murine genital tract model [104] S. aureus (MRSA) + E. coli , other gram negatives Human mAb F598 binds to poly-N-acetyl glucosamine (PNAG) C – Alopexx/Sanofi-Aventis; target conserved epitope and protects mice from lethal infection 105 , 56 Clostridium botulinum Neurotoxin Despeciated blended equine hyperimmune F(ab′)2 – Heptavalent M – Protects in vivo against death by all 7 serotypes of neurotoxin 98 , 106 Clostridium botulinum Neurotoxin Human scFv mAbs expressed as hu-IgG1 R/C – Individual mAbs protect mice in vivo from one serotype of neurotoxin 107 , 108 Clostridium difficile Toxins A and B Human mAbs CDA-1 (to C terminus of TcdA) and CDB-1 (to C-terminus of TcdB) C – Medarex/Merck Reduced recurrence in humans infection in phase 2 trial [5] Clostridium difficile Toxins A and B Humanized mAbs mPA-39 (to toxin A) mPA-50 (to toxin A) and mPA-41 (to N-terminus of toxin B) C – Progenics; mAb mixtures protect hamsters with higher efficacy than Merck mAbs [109] M – marketed; R – research; C – commercial, under development (excludes those which have been dropped or suspended due to clinical failure). Table 2 Passive Ab therapy to viral agents Target Format Details Refs Respiratory syncytial virus (RSV) Respigam; Human polyclonal immune globulin to RSV M – Medimmune, Astra Zeneca; prophylactic treatment of infants; 110 , 111 Varicella Zoster Virus (chicken pox) VariZIG Human polyclonal immune globulin with titer to Varicella zoster M – Cangene Corp; purified human polyclonal antibody 98 , 112 Vaccinia VIG; Human polyclonal immune globulin to vaccinia virus from immunized people M – VIGIV Cangene and VIGIV Dynport; purified human polyclonal antibody from donors with high titer; for treatment of smallpox vaccine side effects [113] Hepatitis B virus (HBV) HepaGamB; Human polyclonal immune globulin to HBV M – Cangene Cop.; used to prevent re-infection with hepatitis B disease in HBV Ag positive liver transplant patients. [98] Hepatitis C virus (HCV) Civacir; Human polyclonal immune globulin to HCV C – Nabi; prevent re-infection with hepatitis C disease in HCV Ag positive liver transplant patients. [114] Hepatitis C mAbs to Ara C epitope on Hepatitis C E2 protein R – Scripps; used Fab display library made from chronically infected donor and showed broad protection in a novel chimeric liver mouse model [84] Respiratory syncytial virus (RSV) Synagis; a humanized IgG1/k mAb derived from murine mAb 1129 M – Medimmune Astra Zeneca; NSO cell line expressed mAb for prophylactic treatment of infants 22 , 115 Dengue virus (DENV) Murine mAbs DENV1-E105, and DENV1-E106 to the E protein C – Macrogenics licensed several of the mAbs; mAbs exhibited therapeutic activity even four days after infection with heterologous virus 116 , 117 HIV Hu mAbs IgGb12, 2F5, 4E10, 2G12 R – provides protection against mucosal SHIV challenge in macaques even at low titers 118 , 119 , 120 Ebola Hu mAb KZ52, murine mAbs 13F6 13C6 6D8, Can9G1 a R – Isolated from survivors and provide protection in rodents 121 , 122 Nipah/Hendrah Human mAb m102.4 optimised from naïve library Binds to the surface glycoprotein and cross protects both Nipah and related hendrah virus [123] Influenza A infection Several new Cross reactive mAbs C – Kirin – M2e C – Cytos M2e R-C Harvard, Crossreactive mAbs to HA 124 , 125 , 63 General Infection Normal Human polyclonal immune globulin (IVIG) M – Talecris, Bayer, CSL, Baxter others; used to reduce/prevent infection in post exposure prophylaxis of some viral disease and for individuals with immunodeficiency 126 , 127 , 128 , 129 M – marketed; R – research; C – commercial, under development. a JDB manuscript in preparation. Table 3 Passive Ab therapy to fungi and yeast Target Format Details Refs Paracoccidioides brasiliensis Murine mAb E3 against the gp43 protein R – mAbs show protection in vivo via reduction in fungal burden and less inflammation in murine models; gp43 is a protective antigen [130] Cryptococcus neoformans Murine mAb 2G8 anti β-glucan R – MAb caused a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain [60] Cryptococcus neoformans Murine 3E5 MAb anti-capsular ps R – IgG1, IgG2a, and IgG2b switch variants prolong the survival of lethally infected mice, whereas the IgG3 MAb does not; all protect in c' deficient mice. [131] Asperigillius fumigatus Murine MAb A9 anti-cell wall surface of hyphae R – murine model of invasive aspergillosis [132] Candida spp. Efungumab (Mycograb) is a hu-mAb against HSP 90 C – Novartis; prevents a conformational change needed for fungal viability; shows efficacy in humans and lowered mortality; abandoned in 2010 [133] Pneumocystis carnii Murine anti-kex1 mAb 4F11 and its F(ab′)2 derivative R – reduced infectious burden in an intranasal immunoprophylaxis model [134] M – marketed; R – research; C – commercial, under development. Not all infections are suitable for antibody therapies. The development of a therapeutic can cost hundreds of millions of dollars and take years to find its way through the various stages of research and development, clinical development, and the government regulatory approval processes. At the end of this process the manufacturing costs factor into the cost per dose of the final product ( Table 4 ). Cheaper to manufacture antibiotics have an advantage in the post development and FDA approval processes. Until antibody products produced via novel recombinant expression systems gain regulatory approval, monoclonal antibody (mAb) manufacturing remains limited cell culture. Currently every FDA approved mAb has been manufactured using classical mammalian cell culture expression systems. Table 4 Therapeutics antibodies, antibiotics and small molecules Criteria Poly Ig mAbs RNAi (antivirals) Antibiotics (bacterial treatments Ease of Delivery High (IV, IM, SC) High (IV, IM, SC) ? Low–High (Oral, IV) Low–High (Oral, IV) Regulatory Approval Cost High High High High Manufacturing Cost Medium High ? Low Cost per dose Medium (100s) High (1000s) ? Low (1s) Damage to Microbiome None None ? Yes Specific Activity Yes Yes ? No Toxicity Low Low ? Low QA effort required High to prevent lot variation Low ? Low Source Humans, animals Tissue culture ? Bacterial Fermentation History of regulatory approval Yes Yes No Yes To be considered viable for the development of an antibody therapy, an infectious disease pathogen needs very specific attributes. The antibody must show efficacy in vivo ; such as help clear the infection, reduce the infectious burden, reduce the time to heal, or prevent infection/intoxication from occurring. However, before addressing the biology of the antibody, numerous issues around the target market drive commercial interests. The continued development of antibody therapeutics, due to their inherent narrow specificity and high cost of production, is largely dependent on competing antimicrobial therapies and vaccines. For example, despite significant investment in the development of protective mAbs against Clostridium difficile toxins [5] , of which several are in clinical trials, a novel toxoid vaccine from Sanofi Pasteur may convert the relatively large and growing market for C. difficile treatment to niche market for breakthrough infections, unimmunized, or the immunocompromised. By contrast, the advent of a C. difficile specific antibiotic such as fidaxomicin (under development by Optimer Pharmaceuticals), which purportedly leaves the normal gut flora intact while clearing C. difficile , would probably leave the anti-toxin therapeutic market intact as the immunotherapeutic could still be administered as an adjunctive therapy to specifically remove the effects of the toxin. A summary of the characteristics that determine the pros and cons of the various classes of competing antimicrobial drugs is shown in Table 4 . One target, numerous biological effects Monoclonal antibody is a unique class of drugs in that numerous biological effects, depending upon the isotype, can be induced through binding to a single microbial epitope ( Fig. 1 ). Many good reviews exist which discuss contemporary mAb development [6] and more specifically for infectious agents [7] . Many of these effects are dependent on other components of the immune system; including antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), opsonization, and immunomodulation. Virus neutralization can be achieved by interfering with one of the various methods used by viruses to enter host cells. In the case of human immunodeficiency virus (HIV), Abs can prevent viral attachment to the host cell by binding to either the envelope protein of the virus, or a host receptor such as CD4 or CCR5 8 , 3 . Antibodies may also neutralize the influenza A virus by preventing the low pH induced conformational change required in hemagglutinin [9] or by preventing the release of progeny virions from infected cells [10] . Interestingly, antibodies can also be directly antimicrobial, as has been described in Candida albicans [11] , and Cryptococcus neoformans [12] . In the latter, IgG1 and IgM mAbs induce fungal gene expression and metabolic changes in an animal model of cryptococcus [12] . Figure 1 Antibody effector mechanisms used to combat microorganisms mediated by the various isotypes of human immunoglobulin. Anti-infective antibodies: monoclonal or polyclonal? Immunoglobulin from high titer plasma of immune individuals (poly Ig) and bulk intravenous immune globulin (IVIG) remain predominant therapeutics used today in passive antibody therapy for treating infectious diseases. Unlike the modest antigenic differences between normal and tumor cells which warrant a monospecific approach to treating cancer, infectious agents are generally foreign to the immune system allowing the less specific and more readily available polyclonal approach to become the norm in treating infectious disease. IVIG treatment of disease has been reviewed recently and is recommended in the treatment of Kawasaki's disease, cytomegalovirus (CMV)-induced pneumonitis, neonatal sepsis, rotaviral enterocolitis, and staphylococcal toxic shock syndrome 13 , 14 . As a high titer alternative, poly Ig is used clinically in the prevention of infections caused by CMV, hepatitis A and B viruses (HBV), rabies, varicella, and measles virus while anti-tetanus Ig is used to treat tetanus [14] ; rabies hepatitis B and tetanus donors are usually vaccinated, and CMV, measles, varicella are typically screened for high titer. In this regard, stimulation of human donors with vaccines has been used to develop effective polyclonal anthrax immune globulin (AIG) for passive therapy [15] . Poly Ig is also used to prevent mother-to-child transmission of HBV and varicella-zoster virus [16] . Conversely, only a single mAb to RSV is currently licensed for the prophylactic treatment of an infectious disease [17] . Both polyclonal and monoclonal IgG preparations have been successfully used for infectious disease prevention with the majority of experience being with purified polyclonal IgG. Advances in mAb technology necessitate a movement towards the more defined but expensive to manufacture mAb-based therapy. The ability to reproduce mAb therapeutics in scalable quantities to meet demand represents a significant difference from the harvesting processes for poly Ig preparations. Furthermore, although human IgG preparations used today have the highest safety record of all biologics and a clear pathway with the FDA, there are still some lingering fears regarding the chance of human poly Ig to transmit an infection, even if exceedingly low [18] . For example, UK citizens are still not allowed to donate their blood in the USA or the UK as a fall out of the prion incidents in the early 1990s, despite the fact that resins and complexing agents which remove prions, are well entrenched in poly IgG purification protocols from the plasma supply [19] . Although those concerns could be reduced through the use of mAbs produced via tissue culture or microbial expression systems, the cost per dose of mAbs is significantly higher and may not warrant development for many niche market products ( Table 4 ). Indeed, it is not always obvious what models are relevant in qualifying a treatment or which antigens are going to be protective, so in many cases poly Ig proceeds as a cheaper alternative to mAbs. In such cases, where a vaccine is not at least through phase 1 and available, harvesting poly Ig from endemic regions may alleviate concerns of the low titer of some IVIG reagents. For instance IVIG with a titer to West Nile virus is collected from selected Israeli donors to treat infection [13] . Furthermore, mAbs typically are limited in both specificity and functionality, are fragile macromolecules which are expensive to produce, and can take considerably longer to isolate and optimize than poly Ig or IVIG. The current status of research aimed at expanding the limits of mAbs in terms of specificity and functionality, in particular to deal with infectious agents, is discussed below. One isotype, one function Inherent in the use of a single designed antibody to a protective target with a chosen isotype is that the effector functions mediated by the single isotype must alone combat the infection. A polyclonal IgG preparation is relatively resistant to antigenic variation through targeting a range of epitopes. It also has widened biological effector functions as it consists of multiple classes of antibody (which comprises typically IgG1 > IgG2 > IgG3 > IgG4). The ability to induce ADCC or CDC is dependent on both the isotype and glycosylation status of the antibody [20] , thus the variability inherent in polyclonal preparations should in many cases provide added benefit and a protective advantage. Each isotype has characteristics that make it more or less desirable during an immune response [21] and many new recombinant Fc scaffolds have been designed in recent years [22] . Among IgG isotypes, IgG1 has long been the isotype of choice for mAb cancer therapy due to its long half life, partiality for protein antigens, ability to activate complement, and affinity for Fc receptors (FcγI, II, and IIIa/b) [21] . Synagis ® , a mAb for the prevention of severe RSV in infants, remains the sole mAb for infectious agents marketed for use in humans and is an IgG1/κ, and no other mAb isotype has been approved for infectious diseases to date [2] . However, although IgG1 is effective at inducing ADCC by natural killer (NK) cells, it is a considerably less potent activator of polymorphonuclear cells (PMN) 23 , 24 . Furthermore, the activation of inflammatory pathways by IgG1 can be detrimental depending on the immune status of the individual [25] . The IgG2 class has relatively inert effector functions; however, it is generated in response to stimulation by inherently inflammatory bacterial cell wall polysaccharides making it important in protection against bacterial infection [26] . Moreover, two IgG2 formatted mAbs have been approved for use in human chronic diseases and at least eight more are currently in clinical development [21] owing to the longest half-life, fewest allotypes, and inability to activate complement. The IgG3 class has similar biological functions as IgG1 but has received little use to date as a recombinant mAb due to a considerably shorter half-life and extensive polymorphism [27] . The IgG4 class is unique in its inability to cross-link antigens in vitro , and is generated in response to persistent antigen stimulation, making it important for chronic viral and bacterial infections [28] . At least two IgG4 class mAbs are on the market with at least another nine in different phases of research and development for chronic disease indications [21] . In natural infections, the IgM class antibodies are generally produced the earliest in response to T-dependent antigens, and can persist in response to T-cell-independent antigens, such as lipopolysaccharides (LPS). The pentameric form of IgM makes it highly avid, although atypical to commercially purify, and the most potent inducer of CDC [29] . A human IgM mAb from Kenta Biotech, KBPA101, has been under development for some time and targets the surface O-polysaccharide LPS of Pseudomonas aeruginosa [30] . Provided expression of the native pentamer can be achieved at commercial scale, as has been described recently in human PER.C6 ® cells [31] , polymeric IgM may prove to be the isotype of choice for the treatment of many bacteria, particularly for local infections at mucosal surfaces. Human IgA1 has unique O-linked glycoforms but the relatively short half-life of both IgA1 and IgA2 may also be best suited to be used locally. Indeed, intranasal administration of an IgA mAb pre- or post-infection protected mice from a sublethal challenge of H5N1 influenza virus [32] . Even IgD binds respiratory Gram-negative bacteria Moraxella catarrhalis and Haemophilus influenza , mediates internalization by mast cells and basophiles, and triggers innate antimicrobial responses [33] . Clearly the choice of mAb isotype is a crucial decision that will ultimately determine its function in vivo . Other factors such as dose, route of administration, half-life, and site of infection also require attention. The choice of isotype, however, is often not crystal clear as illustrated by the recent observation that despite the potent CDC and ADCC inducing ability of IgG1 in C. neoformans mouse models, only human IgG2 and IgG4 were able to protect mice from infection when antibodies carrying the same variable region but different constant regions were passively administered [34] . Also, passively administered serum IgG has shown protection in pig-tailed macaques from rotavirus infection in a mucosal gut model [35] . These findings point out the still empirical nature of designing therapeutic antibody to treat infectious diseases. The inherent flexibility of recombinant mAb approaches permits the modification of immunoglobulin to fit specific needs. To address the problem posed by the use of a single isotype, it is possible to engineer the Fc region to increase its biological function. Targeted amino acid substitutions increase the affinity of the Fc region for FcγRIIIa and increase ADCC by natural killer cells 2 fold [36] and the glycosylation patterns of the Fc region can be further altered to increase ADCC [37] . A different approach is to engineer a bispecific mAb where one half of the Fab binds to the microbe, and the other recognizes a host receptor. Examples include mAbs specific for either P. aeruginosa [38] , or bacteriophage [39] that also bind a host complement receptor through the other Fab domain. Other groups have taken lessons learned from clinical trials involving immunotoxin based therapies in oncology, and applied them successfully to infectious disease. MAbs conjugated to radioisotopes have been used to treat experimental infections by C. neoformans [40] , Histoplasma capsulatum [41] , Streptococcus pneumoniae [42] , and HIV [43] . Monoclonal specificity: pro or con? The exquisite specificity of mAbs may be both its biggest advantage, and biggest disadvantage. The high specificity of mAb therapy results in little cross reactivity with both host cells and normal flora, which is important considering the link between chronic diseases and certain types of cancer with the use of broad spectrum antibiotics ( Table 4 ) 2 , 44 , 45 . Furthermore, mAb therapy is unlikely to select for drug resistance in extraneous microbes due to the lack of intra-microbe cross reactivity [2] . Unlike poly IgG, which carries many irrelevant specificities, mAbs can be developed which target specific microbial epitopes important in pathogenesis, thereby theoretically increasing the potency of the specific biological activity per dose, in particular if the target epitope is highly protective. Polyclonals also can have isoagglutinins (anti A and anti B) which can be undesirable in some cases. Nevertheless, the high specificity also implies that as a narrow-spectrum antimicrobial drug, a quick and accurate diagnosis is required before therapy. The resulting small market for anti-infective mAbs may limit the economical feasibility of a mAb based approach in certain instances [14] and can be overshadowed by the breadth of reactivity of poly IgG which comes from targeting multiple epitopes with multiple IgG isotypes. Many cutting edge strategies are being developed to address the narrow specificity of mAb therapy, such as broadening the utility and pathogen range and are summarized in Fig. 2 . Typically the selection of Ab to protective antigens is the key criterion in the development of an effective passive immunization strategy. Highly conserved antigens are the most valuable targets for passive immunotherapeutics as they reduce both the number of mAbs needed to make an effective cocktail and the chance of escape from antibody. These can generally be placed into several key themes. Figure 2 Exploiting recombinant technology to address monospecificity issues surrounding mAb therapy for the treatment/prevention of infectious diseases. The creation of antibody cocktails to target groups of infectious agents Certain types of infection consistently involve a predictable cadre of infectious agents. These may be associated with a particular injury (e.g. post burn infections), environmental exposure (e.g. contaminated food) or intentional biothreat release (e.g. hemorrhagic viruses). Chemically programmed mAbs, in which the unlimited chemical diversity of synthetic molecules is coupled with the effector functions of antibodies provides a means in which a single mAb may can have specificity for multiple epitopes 47 , 48 . In this fashion, effector functions can be brought in by pre-immunized circulating antibody to "tags" on the small molecule adapters. These small molecule [48] , or single chain variable fragment (scFv) [49] adapters can be expressed cheaply in Escherichia coli and are significantly less expensive to manufacture than full-length mAbs. Targeting virulence factor associated antigens/epitopes Current trends for bacterial immunotherapeutics target virulence associated factors and are promising candidates for several reasons: First, they prevent direct damage to the host caused by large bacterial exotoxins and LPS through cytolysis or inflammation; second, virulence factors lend resistance to harsh immunological conditions as seen with capsules, spores, or antigenic variation; Third, virulence factors allow adhesion and or entry into host cell environments leading to evasion and chronic infection. Targeting a single virulence factor can be very effective where disease symptoms and pathogenesis are interrupted. For example, in gastro-intestinal tract infections with C. difficile , two large cytotoxins, TcdA and Tcd B cause the associated disease and diarrhea. Recently, Merck published clinical data showing that a cocktail of two mAbs (one to TcdA another to TcdB) at 10 mg/kg can greatly reduce reoccurrence rates compared to controls, probably due to reduced damage to the intestinal lining [5] . The concept of targeting virulence factors is confounded in some cases where a bacterium expresses a plethora of virulence factors which collectively contribute to disease. For example, Staphylococcus aureas produces a broad range of virulence factors and toxins, which may have temporal roles in specific types of infection. Other approaches target antigenically variable capsules [50] clumping factor (ClfA) 51 , 52 , 53 , alpha hemolysin and finally Panton-Valentine leukocidin (PVL) [54] . In these cases, removal of one virulence factor seems to be compensated by another. When this occurs, targeting surface structures or housekeeping proteins such as lsaA [55] , as discussed below, may be more appropriate. Protection mediated by mAbs to indirect virulence factors A related strategy is to target bacterial proteins that have an indirect role in pathogenesis; hence are subject to less selective pressure for antigenic variation. Broadly protective antibodies are not common treatments for many bacteria, including one of the most disconcerting modern infections, methicillin-resistant S. aureus (MRSA). Recently Dr. Gerald Pier and Alopexx developed a human mAb against the carbohydrate backbone of poly N-acetylglucosamine (PNAG) in S. aureus [56] . The mAb was effective initially in models of MRSA by inducing opsonization and CDC. These mAbs are a promising immunotherapy for both prophylaxis and adjunctive treatment of MRSA. In a related study, mAbs targeting the housekeeping protein lsaA, a suggested lytic transglycosylase in S. aureus provided protection in a mouse model by promoting opsonization and inducing highly reactive oxygen species [55] . MAbs against quorum sensing auto-inducers protect mice from lethal challenge from S. aureus , albeit using a clinically irrelevant dosage [57] , and mAbs specific for type III secretion system components from gram-negative bacteria Yersinia species [58] , P. aeruginosa [59] , and Aeromonas salmonicida [46] have all shown protection in animal models as well. MAbs have also been used against multi-drug efflux pumps of C. difficile , and Enterococcus species as part of a combination therapy with small molecule pump inhibitors [46] . Fungal infections may be a more promising area for mAb therapy due to the presence of conserved epitopes between species. Therapy with an IgG2 mAb specific for laminarin (beta-glucan) was effective at inducing fungal phagocytosis by monocytes and preventing infection from C. neoformans , C. albicans , and Aspergillis spp. 60 , 61 . Antibody to conserved viral targets and host receptors The incredible variability of viruses poses a significant challenge to mAb therapy. One way this can be countered is by using recombinant technology to derive an antibody specific for an epitope conserved among different viral strains [62] . Using phage display, Sui et al. selected hemagglutinin-5 specific mAbs capable of neutralizing both H5N1, and H1N1 [63] . A similar approach has been used to select cross protective mAbs against SARS coronavirus 64 , 65 and Dengue virus type 1 [66] . Although potentially advantageous under outbreak conditions in which diagnoses are delayed, treatment of viruses capable of great antigenic variation using a single mAb is not ideal for the reasons discussed below. As an alternative to targeting the viral proteome, several mAbs have been developed that target host proteins that double as virus receptors and co-receptors. These mAbs inhibit entry of a diverse range of virus subtypes including New World arenaviruses through anti-transferrin receptor 1 mAbs [67] , HCV through anti-claudin-1 mAbs [68] , and several R tropic viruses through anti-CCR5 mAbs [3] . While showing potential, mAbs against host proteins are a long way from clinical use, largely due to complications inherent in blocking host receptors. For example, blocking the CCR5 receptor may impair the host's ability fight off other viral infections, such as West Nile virus, as CCR5 is involved in lymphocyte traffic to the brain 3 , 69 . Lastly, one particularly promising area of research is the use of an anti-phospholipid mAb capable of identifying virally infected cells and inducing ADCC [70] . The mAb designated Bavituximab (Peregrine Pharmaceuticals) functions by recognizing phosphatidylserine, which is normally inaccessible to serum antibodies. Upon virus-induced cell activation, phosphatidylserine is exposed on the cell surface due to a loss in lipid symmetry, allowing the mAb to bind 70 , 46 . This mAb has prevented CMV and Pichinde arenavirus infection in mice, and clinical trials are currently underway for the treatment of HCV and HIV. Design of mAb cocktails to reduce antigenic escape The potent neutralizing ability of mAbs can inadvertently result in increased antigenic escape in experimental infection with highly variant RNA viruses. Indeed, treatment with a single mAb has culminated in escape variants in the treatment of HIV, hepatitis C virus (HCV), and influenza A 71 , 72 , 73 , 74 . To counter this, scientists have devised mixtures of two or more mAbs specific for distinct protective epitopes that can greatly prevent the rise of escape mutants in models of infection [74] . Indeed, development of such cocktails is being explored for H5N1 influenza A [75] , SARS coronavirus [76] , lymphocytic choriomeningitis virus [77] , HCV [78] , HBV [79] , and rabies virus 80 , 81 , 82 . These so called "cocktails" of mAbs address two major problems posed by targeting a single epitope. First, by targeting at minimum two neutralizing epitopes, escape mutants generated from each mAb are cleared by the complementing antibody [75] . Second, by increasing the antibody density (valency) on the viral surface, which may be the most important factor in virus neutralization [83] , the neutralizing ability of the antibody is amplified considerably [3] . A promising recent development concerns antibody to the hepatitis C virus (HCV), the leading cause of liver disease leading to transplantation in the U.S.A. Antigenic variation of HCV remains a serious challenge in antibody therapy and active vaccine design. Poly Ig for HCV is one means by which this might be dealt with, as antibody would be prepared from immunizations with distinct HCV virus vaccines. To date there has been no active vaccine available for HCV so this has not been a possible route for a therapeutic. Law et al. instead identified human mAbs that neutralize genetically diverse HCV isolates using phage display [84] . The mAbs were isolated using recombinant HCV E2A glycoprotein from a Fab phage library developed from the bone marrow RNA of a donor chronically infected with HCV. These mAbs were elegantly shown to bind discontinuous epitopes that cluster in three distinct antigenic regions in the E2A protein; one of which (antigenic region 3) appears to be highly conserved among HCV genotypes. Furthermore, the mAbs show protection against heterologous HCV quasispecies challenge in a human liver–chimeric mouse model. This represents a major advancement as the lack of a suitable model has for years prevented advancement on this field. The results provide strong evidence that broadly neutralizing antibodies to HCV can protect against heterologous HCV virus infection and furthermore, suggest that a prophylactic vaccine against HCV may be achievable and may avoid any potential antibody enhancement of infectivity effects which may be due to irrelevant antibody in a poly IgG preparation. Advent of an approved phase 1 vaccine would lead rapidly to potential donor stimulation program and the development of a human polyclonal therapeutic for testing. Immunotherapy is also one of the more promising approaches for the treatment of prion diseases. White et al. were the first to show that mAbs could both inhibit prion replication and delay disease development [85] . More recently, a camelid antibody with specificity for both PrP C and PrP Sc demonstrated the ability to cross the blood brain barrier, reduce prion replication in vivo , and eliminate prion replication in N2a neuroblastoma cells in vitro [86] . Forthcoming developments It is becoming evident that treatment of an infectious disease with multiple mAbs is most probably required to provide optimal protection. While mAb cocktails are being developed to address this problem, another approach may be to couple antibody therapy with existing antimicrobials. Protocols for clinical development of new antibody therapies for infectious diseases will require clinicians to include the current standard of care which in many cases are antibiotics or antivirals. Antibody acts in synergy with antibiotics and with antivirals to provide increased protection against infection ( Table 5 ). Indeed, naturally resistant bacteria can be rendered susceptible to antibiotics by mAb therapy, and antivirals can increase the efficacy of anti-viral mAbs by inhibiting viral replication, thereby decreasing the likelihood of escape mutants [14] . Table 5 Antibody – drug synergy in the treatment of infections Infectious agents Disease Treatment Synergy Refs Pseudomonas aeruginosa Human – Colonized Cystic fibrosis patients (chronic pulmonary infection) Conventional antibiotic treatment plus Ps-ivIG a and conventional IVIG Yes – transient but improved clinical scores [135] Mixed infection Human – trauma infection Penicillin and IVIG versus albumin in controls Yes – reduced septic complications and improved serum bactericidal activity [136] Staphylococcus aureus (MRSA) and E. coli and Proteus spp. Rat – experimental models for both gram positive and gram negative organisms Mezlocillin and IVIG for gram negatives E coli and Proteus spp.; Cephalothin or Cephamandole with IVIG for S. aureus Yes – IVIG together rendered Cephalothin as effective as oxacillin in beta-lactamase postive S. aureus and made cefamandole immediately bactericidal [137] P. aeruginosa ; Klebsiella pneumonia , Seratia . marcescens , Proteus mirabilis Experimental – murine burn model Piperacillin plus human anti-LPS IgG and conventional IVIG Yes – however strain specific ( P. aeruginosa ; not K. pneumonia , S. marcescens , P. mirabilis ) [138] Escherichia coli Proteus spp. Experimental – rat granuloma pouch model Mezlocillin plus human IgG and conventional IVIG Yes – for betalactamase + E. coli and Proteus spp. no protection by individual treatments [139] Pseudomonas aeruginosa Experimental – murine thigh infection Ceftazidime plus murine mAb Ld3-2F2 to LPS. Yes – significant reduction in bacteria recovered [140] Pseudomonas aeruginosa (antibiotic resistant) Experimental – murine burn model Ceftazidime plus murine mAb Ld3-2F2 to LPS. Yes – compared to no survival with individual treatments [141] Klebsiella pneumonia Experimental – murine burn model Ceftazidime (suboptimal dose) plus locally (sc) delivered human IgG Yes –reduction in burn bacterial burden compared to either monotherapies [142] Bacillus anthracis Experimental – inhalational anthrax Ciprofloxacin plus Human mAb AVP-21D9 to PA toxin b plus Yes – 100% protection in mice, guinea pigs; mAb protected rabbits without antibiotics [143] Clostridium difficile Human – gastrointestinal infection Metronidazole/vancomycin plus two human mAbs CDA-1 and CDB-1 to toxins A and B respectively Yes – reduced recurrence rates significantly [5] Staphylococcus aureus (MRSA) Experimental – clearance of bacteremia in rabbits Vancomycin and human SA-IVIG c Yes – accelerated clearance [53] Human immunodeficiency virus (HIV-1) Human – treatment/control of infection Anti-CCR5 antibody plus small molecule ccr5 inhibitors Yes – blocking host receptor [144] Hepatitis B virus (HBV) Human – prevents post liver transplant recurrence HBIG (hyperimmune immunoglobulin) plus lamivudine (Nabi) Yes – combination with nucleoside analogue allows reduced dose of HBIG [145] a Ps-ivIG is a purified human Ig preparation from pooled plasma with elevated titer to Pseudomonas aeruginosa (pH 4.25, from Cutter Biological). b PA toxin – anthrax protective antigen. c SA-IVIG – is a purified human Ig preparation from plasma donors with elevated titers of anti-ClfA antibody selected from the general donor population (Massachusetts Public Health Biological Laboratories). Niche markets prevail in antibody therapy for infectious diseases as disease pathogens with large potential markets are generally approached from the vaccine or antibiotic standpoint. The development of immunotherapies for the prevention of severe RSV infections in young children exemplifies the complexity of developing treatments for infectious diseases which tend to have very specific target populations. Originally a poly Ig product Respigam ® , was developed by MedImmune and approved for prophylactic use in infants. While efficacious, a humanized IgG1 mAb product called Synagis ® (palivizumab) was designed to replace Respigam ® . Synagis ® remains the only mAb approved for infectious disease in humans [17] , the cost effectiveness relative to Respigam ® is still under analysis [87] ; however, it is a more defined product and can be produced in tissue culture infinitely. Despite focused efforts by MedImmune/AstraZeneca to gain regulatory approval for a third generation fully human mAb treatment, Numax (motavizumab) a higher affinity fully human replacement for Synagis ® , has long been underway. However, AstraZeneca recently announced it was discontinuing the development of Numax, despite the $445 million dollar investment, due to unforeseen complications that arose during the clinical testing and regulatory approval process. Thus, niche markets require a very different approach to biotechnology and the investment of time, expenses, and research opportunity costs. Few niche market companies in infectious disease biotechnology could endure this risk and many might have instead simply continued to manufacture more doses of the earlier generation Respigam ® or Synagis ® , rather than boldly attempt to bring forward a new product with seemingly incremental improvement. In summary, recent developments in broadening both the utility and pathogen range of mAb therapeutics, multi-drug resistance, and the expected future efficiencies in mAb production methods 88 , 89 , 90 , 91 , combined with rational use of proven polyclonal Ig methods, enable us to predict that Ab therapy for infectious pathogens will continue to be a leading treatment for emerging, difficult to contain infectious diseases.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021483/

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro . Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol. Introduction The pro-inflammatory cytokine IL-1β is thought to play a major role in host protection against Mycobacterium tuberculosis (Mtb) infection ( 1 – 8 ). Thus, mice genetically deficient in IL-1β, IL-1α, or IL-1 receptor signaling are extremely susceptible to Mtb infection ( 1 , 2 , 4 , 7 ) while in vitro stimulation of Mtb-infected macrophages with IL-1β results in reduced mycobacterial burdens ( 7 ). Several explanations for the anti-mycobacterial activity of IL-1β have been proposed. For example, IL-1β has been shown to facilitate phagolysosomal fusion, which enhances the mycobactericidal activity of host macrophages ( 9 ). In addition, IL-1β through its induction of prostaglandin E2 could influence the cell death modality of Mtb-infected macrophages favoring an apoptotic over necrotic fate thus containing bacterial spread ( 7 , 10 – 13 ). This could occur in part thru the generation of cyclooxygenase-2, which plays critical role in plasma membrane repair ( 7 , 10 ). While there is strong evidence demonstrating a beneficial role of IL-1β in Mtb infection in vitro and in vivo ( 2 , 4 , 7 , 9 ), excessive production of this cytokine has been associated with more severe tuberculosis (TB) disease and increased lung damage ( 14 – 16 ). Thus, understanding the nuances of IL-1β function in TB is important in the design of therapeutic interventions that act by altering the levels and activity of this major pro-inflammatory cytokine. In vitro , Mtb infection of macrophages has been shown to induce inflammatory responses via activation of the NLRP3-inflammasome, leading to secretion of mature IL-1β ( 5 , 17 ). Additional studies have demonstrated that Mtb can elicit NLRP3-inflammasome through antigens encoded by the mycobacterial genome known as region of difference 1 (RD-1) ( 5 , 17 , 18 ). Among the Mtb proteins included in RD-1 encoded group, the early-secreted antigen (ESAT-6) has been identified as the major mycobacterial product responsible for IL-1β induction and does so through an NLRP3-dependent pathway ( 5 ). ESAT-6 is secreted from Mtb through a specialized protein secretion system of the bacterium called ESX-1 ( 19 – 21 ) that has been proposed to mediate pore formation in the phagosomal membranes thus facillitating mycobacterial escape from that organelle ( 22 , 23 ). Several stimuli elicit NLRP3 activation leading to the recruitment of the adaptor protein ASC (apoptosis-associated speck-like protein containing a carboxy-terminal CARD), which is required for caspase-1 recruitment and activation, culminating in the NLRP3-inflammasome complex formation ( 5 , 24 ). Once assembled, the NLRP3-inflammasome complex enables the maturation of pro-IL1β and release of its mature form in a caspase-1-dependent manner. However, the exact mechanism of NLRP3-inflammasome activation by mycobacteria is poorly understood. Several studies have described the involvement of lysosomal cathepsins in NLRP3-dependent IL-1β production ( 25 – 28 ). Cathepsins are a class of proteolytic enzymes which display diversity in terms of their structural and/or functional features. The lysosomal cathepsin family can be divided in three subsets, the aspartic cathepsins (D and E), serine cathepsins (A and G), and cysteine cathepsins (B, C, F, H, K, L, O, S, V, X, and W). Cathepsin B (CTSB) expression is ubiquitous, and usually expressed in higher levels than other cathepsins in some tissues ( 29 , 30 ). Of note, CTSB can be distinguished from cathepsin family members on the basis of its activity as either an endo- or exopeptidases at different pH ranges ( 30 ). Inside cells, CTSB regulates several functions, including cytokine exocytosis, protein cleavage inside the lysosome, and the induction of cell death ( 5 , 17 , 31 – 37 ). Interestingly, CTSB activation either inside the lysosome or in the cytosol has been proposed to promote NLRP3-inflammasome activation and IL-1β production in experimental settings ( 38 – 40 ). Furthermore, CTSB has been described in some settings to directly cleave caspase-1 and caspase-11 which could further promote IL-1β maturation ( 30 – 33 , 41 ). Nonetheless, in many experimental models it has been difficult to demonstrate a direct role for CTSB cleavage in IL-1β maturation ( 30 , 36 , 42 ) suggesting that its role may be upstream of inflammasome activation. Since ESAT-6 has been associated with both membrane pore formation and IL-1β production by Mtb, we asked in the present study whether the ESAT-6 induced release of lysosomal CTSB might link these two phenomena. Using macrophages infected in vitro with Mtb, we demonstrate that when secreted into the cytosol by an ESAT-6-dependent mechanism, lysosomal cathepsin activity inhibitable by CA074-Me is crucial for NLRP3-inflammasome activation and consequently, for mature IL-1β generation. Previous studies have shown that IL-1β control of Mtb infection is inflammasome independent. Thus, the cathepsin-dependent pathway for IL-1β generation we describe could serve as a potential target for reducing Mtb induced inflammation without compromising host resistance to the pathogen. Results Mtb Infection Triggers Increased CTSB Levels and Activity We first investigated whether Mtb infection increases the expression or activity of CTSB in macrophages. To do so, we quantified levels of the protease in lung tissues in two different animal models of pulmonary TB (mice and rabbits), in plasma from patients with active TB as well as in Mtb-infected bone marrow-derived macrophages (BMDMs). CTSB enzyme activity was found increased by two-fold in the lungs from mice infected with Mtb on day 30 post-infection (Figure 1 A) and was further elevated after 6 months of infection (Figure 1 B). Furthermore, in situ analysis of CTSB activity was evaluated during BMDM infection and was enhanced in the presence of Mtb (Figure 1 C). In the latter assays, enzyme activity was detected in close association with the bacteria in lysosomal-like structures. These findings were supported by data showing increased CTSB gene expression in the lungs of Mtb-infected rabbits (Figure 1 D) and elevated protein levels in plasma from patients with active TB (Figure 1 E). Together these results demonstrated that Mtb increases CTSB expression in different host species and that this response is detectable both in vivo and in vitro . In the case of the in vivo findings, the observed elevation in CTSB levels could reflect increased cellular exocytosis and cell damage in addition to the induction of enzyme synthesis. Figure 1 Mycobacterium tuberculosis (Mtb) infection upregulates both cathepsin B (CTSB) expression and enzymatic activity. (A) CTSB activity and (B) expression of mature CTSB were measured in the lungs of H37Rv-infected mice after 1 month or 6 months post-infection (mean ± SEM, n = 5). (C) CTSB activity (red) in macrophages infected (multiplicity of infection: 3) with GFP-H37Rv Mtb strain (green) was analyzed by confocal microscopy (magnification: 600×). DAPI (blue) was used for nuclear staining. Representative images are shown. (D) CTSB messenger RNA expression was quantified in lung granulomatous lesions from rabbits infected with Mtb H37Rv at day 42 post-infection (mean ± SEM, n = 3–4). (E) Total CTSB levels in plasma from patients with active pulmonary TB (PTB; mean ± SEM, n = 13) and health controls (HC; mean ± SEM, n = 12). Data were analyzed using the Mann–Whitney U test (* p < 0.05; ** p < 0.01). Data are representative of at least two separate experiments performed. Mature CTSB Is Released From Lysosomes Into the Cytosol by an ESAT-6-Dependent Mechanism We next asked whether during Mtb infection of macrophages, CTSB was released from the lysosomes into the cytosol. Lysosomal destabilization was analyzed by staining BMDM with the LysoTracker dye, and fluorescence intensity of the dye measured by flow cytometry ( 43 , 44 ). As a positive control for lysosomal integrity loss, we stimulated cells with Leu-leu-OMe (a known inducer of lysosomal destabilization) ( 43 ) and then checked lysosomal leakage by using LysoTracker and loss of mitochondrial membrane potential by using Mitotracker. Interestingly, previous studies have also shown that Leu-leu-OMe-induced lysosomal leakage facilitate the release of CTSB into the cytosol ( 43 , 45 , 46 ), supporting the use of Leu-leu-OMe as a gold-standard positive control for lysosomal destabilization. As expected, Leu-leu-OMe treated cells displayed reduced fluorescent intensity for Lysotracker dye but not for Mitotracker (Figure 2 A). Macrophages infected with Mtb showed reduced staining with Lysotracker comparable to that triggered by Leu-leu-OMe (Figures 2 A,B) and was not accompanied by changes in mitochondrial permeability (Figure 2 B). Importantly, Mtb strains deficient in ESAT-6 or RD-1 triggered significant smaller reductions in Lysotracker staining than the wild-type (WT) H37Rv parental strain. Together the above findings indicated that Mtb induces lysosomal destabilization by a mechanism partially dependent on ESAT-6/RD-1. Figure 2 Mycobacterium tuberculosis (Mtb) infection induces lysosomal leakage dependent on ESAT6 and leads to release of mature cathepsin B (CTSB) and IL-1β production. Cells were stained with lysotracker and mitotracker to verify lysosomal leakage or outer mitochondrial membrane potential disturbance at 24 h p.i. with Mtb H37Rv strain (MOI of 3), respectively. (A) Cells were stimulated with Leu-o-Leu as a positive control of lysosomal leakage. (B) Bone marrow-derived macrophages (BMDMs) were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv Mtb strains at MOI of 3 as described in Section " Materials and Methods ." Mtb H37Rv induced lysosomal leakage dependent of ESAT6. (C) Cytosolic fraction from BMDMs infected with Mtb H37Rv, showing the presence of mature CTSB. (D,E) BMDMs were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv strains at MOI of 1 or MOI of 3 as described in Section " Materials and Methods ." (D) IL-1β production was measured by ELISA and (E) expression of mature IL-1β, activated caspases-1, and mature CTSB in the supernatant of cells was assessed by western blotting. Differences were analyzed using the Mann–Whitney U test (between two groups) or the Kruskal–Wallis test with Dunn's multiple comparisons ad hoc test. Significant differences were observed for the indicated experimental conditions (* p < 0.05; ** p < 0.01). Bars and line represent mean and SEM, respectively. Data are representative of three independent experiments using triplicate biological samples. We next investigated whether the lysosomal disruption induced by Mtb results in the release of CTSB into the cytosol. We adapted a previously described method using saponin to discriminate lysosomal versus cytosolic content and measured the presence of CTSB in the cytosol at different doses ( 43 ) (Figure S1 in Supplementary Material). We found that Mtb-infected BMDM displayed increased levels of mature CTSB inside the cytosol as compared to uninfected controls. These data confirmed that lysosomal leakage induced by Mtb is indeed associated with release of mature CTSB into the cytosol (Figure 2 C). Mycobacterium tuberculosis ESAT-6 is a highly immunogenic mycobacterial antigen, which has been described to induce pore formation in the phagosome membrane thus facilitating bacterial escape into the cytosol ( 23 ). Moreover, it has been reported that ESAT-6 is required for NLRP3-inflammasome activation and the subsequent cleavage of pro-IL-1β into its mature form ( 5 ). We also observed reduced levels of mature IL-1β in supernatants from BMDM cultured with ΔESAT6 H37Rv or ΔRD1 H37Rv strains compared to those infected with WT H37Rv (Figure 2 D). In addition, we measured mature CTSB, cleaved caspase-1, and mature IL-1β in supernatants from Mtb-infected macrophages by western blotting (WB). Following H37Rv infection, we found increased levels of mature CTSB, cleaved caspase-1, and mature IL-1β from infected macrophages, the latter observation confirming that the changes in IL-1β levels measured by ELISA (Figure 2 D) reflect alterations in the mature cytokine. These products were not detected in supernatants from cells infected with ΔESAT6 H37Rv or ΔRD1 H37Rv strains (Figure 2 E). Taken together, the above findings suggested that ESAT-6 facilitates the secretion of the activated CTSB into the cytosol which is associated with enhanced IL-1β maturation. Inhibition of Cathepsin Activity by CA074-Me Blocks IL-1β Production Induced by Either Mtb Infection or Cytosolic ESAT-6 To confirm the requirement of NLRP3-inflammasome complex for IL-1β production in our experimental system, we infected BMDM from NLRP3 −/− , ASC −/− , caspase-1 −/− , and WT mice with Mtb H37Rv and measured IL-1β in supernatants from macrophages 24 h later. As expected, IL-1β production induced by Mtb was completely abolished in the supernatants from macrophages deficient for NLRP3, ASC, and caspase-1 (Figure 3 A). Figure 3 Mycobacterium tuberculosis (Mtb) ESAT-6-induced IL-1β production is diminished by cathepsin B inhibition. Macrophages were infected with Mtb H37Rv or ΔESAT6 strains at MOI of 3 as described in Section " Materials and Methods ." (A,B) IL-1β production was measured in the supernatants collected from Mtb-infected deficient macrophages or following different treatments as indicated. (B) Mature IL-1β and cleaved caspase-1 were analyzed by western blotting. (C,D) Infected macrophages were stimulated with PA (20 µg/mL) or LFn-ESAT6/PA (20 µg/mL) and IL-1β production was quantified by ELISA after 24 h of infection. (E,F) Bone marrow-derived macrophages were primed with LPS (0.2 µg/mL) for 1 h and further stimulated or not with LFn-ESAT/PA (10 µg/mL each) for 3 h. Furthermore, cells were stimulated with eATP (1 mM) for 25 min and IL-1β production was measured in culture supernatants. Statistical differences observed are shown for each indicated groups (*** p < 0.001). The data represent the mean ± SEM of samples in triplicate. Data are from at least three independent experiments. We next investigated whether lysosomal cathepsin activity is required for release of mature IL-1β. Macrophages were infected with H37Rv in the presence or absence of CA074-Me, a potent inhibitor of CTSB activity and in certain conditions other cathepsins ( 36 , 43 ), and mature IL-1β as well as cleaved caspase-1 were measured in supernatants at 6 and 24 h after infection. We found that the release of both cleaved caspase-1 and mature IL-1β secretion was completely inhibited after CA074-Me treatment (Figure 3 B). Of note, CA074-Me treatment does not affect pro-IL-1β expression as well as cell death induction as previously reported (Figures S2A,C in Supplementary Material) ( 17 , 43 , 47 ). Interestingly, inhibition of the tyrosine kinase Src also blocked IL-1β and cleaved caspase-1 secretion. This effect correlated with a reduction in lysosomal leakage following Src inhibitor treatment (Figure S3 in Supplementary Material). The above results taken together demonstrated that Mtb ESAT-6 expression induces lysosomal perturbation, mature CTSB release into cytosol, and mature IL-1β secretion after Mtb infection. It has been previously established that ESAT-6 facilitates the escape of Mtb into the cytosol from phagolysosomes ( 23 ). Thus, it is challenging to determine whether CTSB release into the cytosol following lysosomal leakage occurs due to ESAT-6-induced pore formation on phagolysosomal membranes or directly through ESAT-6 action in the cytosol. To distinguish these two mechanisms, we performed experiments using a previously described system involving an anthrax toxin LFn-fusion protein to deliver recombinant ESAT-6 into the cytosol ( 48 – 51 ). In these experiments, we infected BMDM with H37Rv and ΔESAT6 H37Rv strains in the presence or not of the LFn-ESAT-6/PA complex ( 50 , 51 ) and then measured IL-1β production 24 h post-infection. Delivery of ESAT-6 into the cytosol enhanced IL-1β production by H37Rv-infected macrophages and importantly restored IL-1β production in ΔESAT6 H37Rv-infected cells (Figure 3 C). Moreover, the IL-1β response induced by ESAT-6 delivery into the cytosol could be abolished by treatment of the cells with the CA074-Me, a potent CTSB inhibitor (Figure 3 D). Although the expression of recombinant ESAT-6 (rESAT-6) in the cytosol resulted in IL-1β production, this effect was nonetheless dependent on Mtb infection (Figure 3 C). To investigate whether rESAT-6 can enhance IL-1β production induced by other known NLRP3-inflammasome stimuli in a non-infectious context, we used the well-known model of NLRP3-inflammasome activation through extracellular ATP stimulation. We first performed a dose titration of eATP concentration to determine non-saturating levels of IL-1β production without inducing cell death via P2X7 receptor (Figure 3 E) and determined the optimal concentration at 1 mM. We then primed macrophages with LPS (0.2 µg/mL) for 1 h (as first signal for pro-IL-1β expression), and then rESAT-6 was delivered into the cytosol by stimulating cells with LFn-ESAT6/PA (10 µg/mL) for an additional 3 h. Finally, macrophages were incubated with eATP (1 mM) for 25 min and IL-1β production measured by ELISA. We found that cytosolic delivery of ESAT-6 amplified IL-1β production induced by ATP in a CTSB-dependent manner (Figure 3 F). Thus, these results suggested that the presence of ESAT-6 inside the cytosol can drive NLRP3-inflammasome activation independent of Mtb infection and that CTSB activity is required for this activation. CA074-Me Blocks ASC Speck Formation in Mtb-Infected Macrophages We next addressed the mechanism by which cytosolic CTSB might promote IL-1β production. ASC speck formation is an ultrastructural hallmark of NLRP3-inflammasome activation ( 52 ). These structures are formed by the accumulation of ASC and cleaved caspase-1 bound to an NLRP3 platform, thus promoting the generation of mature IL-1β ( 52 ). We wondered whether lysosomal cathepsin activity is essential for ASC speck formation as a result of NLRP3-inflammasome assembly. To test this hypothesis, we infected macrophages with Mtb H37Rv and cells were incubated in the presence or absence of the CA074-Me cathepsin inhibitor. After 24 h of infection, speck of ASC was stained using specific antibodies and samples analyzed by fluorescence microscope. Numerous ASC specks were detected in Mtb-infected macrophages (Figures 4 A–C). However, these structures were barely detectable Mtb-infected macrophages treated with CA074-Me (Figures 4 A–C). Thus, these data indicated that the suppression of mature IL-1β production induced by lysosomal cathepsin inhibitor CA074-Me (Figures 2 D,E) is associated with defective NLRP3-inflammasome assembly and consequently ASC speck formation following Mtb infection. Figure 4 Cathepsin B (CTSB) activity mediates ASC speck formation. Macrophages were infected with Mycobacterium tuberculosis H37Rv MOI of 3 as described in Section " Materials and Methods " and cells were treated or not with the CTSB inhibitor CA074-Me as indicated. (A) ASC speck formation in infected macrophages treated or not with CA074-Me (25 µM) was analyzed by fluorescence microscopy. (B) The frequency of cells ASC speck positives (C) and number of ASC speck were quantified by using the Image J software. Differences were analyzed using the Mann–Whitney U test. Statistical differences observed are shown for each indicated groups (*** p < 0.001). Data represent mean ± SEM of samples ran in triplicate. Results are representative at least two independent experiments. Mtb Infection Triggers Increased CTSB Levels and Activity We first investigated whether Mtb infection increases the expression or activity of CTSB in macrophages. To do so, we quantified levels of the protease in lung tissues in two different animal models of pulmonary TB (mice and rabbits), in plasma from patients with active TB as well as in Mtb-infected bone marrow-derived macrophages (BMDMs). CTSB enzyme activity was found increased by two-fold in the lungs from mice infected with Mtb on day 30 post-infection (Figure 1 A) and was further elevated after 6 months of infection (Figure 1 B). Furthermore, in situ analysis of CTSB activity was evaluated during BMDM infection and was enhanced in the presence of Mtb (Figure 1 C). In the latter assays, enzyme activity was detected in close association with the bacteria in lysosomal-like structures. These findings were supported by data showing increased CTSB gene expression in the lungs of Mtb-infected rabbits (Figure 1 D) and elevated protein levels in plasma from patients with active TB (Figure 1 E). Together these results demonstrated that Mtb increases CTSB expression in different host species and that this response is detectable both in vivo and in vitro . In the case of the in vivo findings, the observed elevation in CTSB levels could reflect increased cellular exocytosis and cell damage in addition to the induction of enzyme synthesis. Figure 1 Mycobacterium tuberculosis (Mtb) infection upregulates both cathepsin B (CTSB) expression and enzymatic activity. (A) CTSB activity and (B) expression of mature CTSB were measured in the lungs of H37Rv-infected mice after 1 month or 6 months post-infection (mean ± SEM, n = 5). (C) CTSB activity (red) in macrophages infected (multiplicity of infection: 3) with GFP-H37Rv Mtb strain (green) was analyzed by confocal microscopy (magnification: 600×). DAPI (blue) was used for nuclear staining. Representative images are shown. (D) CTSB messenger RNA expression was quantified in lung granulomatous lesions from rabbits infected with Mtb H37Rv at day 42 post-infection (mean ± SEM, n = 3–4). (E) Total CTSB levels in plasma from patients with active pulmonary TB (PTB; mean ± SEM, n = 13) and health controls (HC; mean ± SEM, n = 12). Data were analyzed using the Mann–Whitney U test (* p < 0.05; ** p < 0.01). Data are representative of at least two separate experiments performed. Mature CTSB Is Released From Lysosomes Into the Cytosol by an ESAT-6-Dependent Mechanism We next asked whether during Mtb infection of macrophages, CTSB was released from the lysosomes into the cytosol. Lysosomal destabilization was analyzed by staining BMDM with the LysoTracker dye, and fluorescence intensity of the dye measured by flow cytometry ( 43 , 44 ). As a positive control for lysosomal integrity loss, we stimulated cells with Leu-leu-OMe (a known inducer of lysosomal destabilization) ( 43 ) and then checked lysosomal leakage by using LysoTracker and loss of mitochondrial membrane potential by using Mitotracker. Interestingly, previous studies have also shown that Leu-leu-OMe-induced lysosomal leakage facilitate the release of CTSB into the cytosol ( 43 , 45 , 46 ), supporting the use of Leu-leu-OMe as a gold-standard positive control for lysosomal destabilization. As expected, Leu-leu-OMe treated cells displayed reduced fluorescent intensity for Lysotracker dye but not for Mitotracker (Figure 2 A). Macrophages infected with Mtb showed reduced staining with Lysotracker comparable to that triggered by Leu-leu-OMe (Figures 2 A,B) and was not accompanied by changes in mitochondrial permeability (Figure 2 B). Importantly, Mtb strains deficient in ESAT-6 or RD-1 triggered significant smaller reductions in Lysotracker staining than the wild-type (WT) H37Rv parental strain. Together the above findings indicated that Mtb induces lysosomal destabilization by a mechanism partially dependent on ESAT-6/RD-1. Figure 2 Mycobacterium tuberculosis (Mtb) infection induces lysosomal leakage dependent on ESAT6 and leads to release of mature cathepsin B (CTSB) and IL-1β production. Cells were stained with lysotracker and mitotracker to verify lysosomal leakage or outer mitochondrial membrane potential disturbance at 24 h p.i. with Mtb H37Rv strain (MOI of 3), respectively. (A) Cells were stimulated with Leu-o-Leu as a positive control of lysosomal leakage. (B) Bone marrow-derived macrophages (BMDMs) were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv Mtb strains at MOI of 3 as described in Section " Materials and Methods ." Mtb H37Rv induced lysosomal leakage dependent of ESAT6. (C) Cytosolic fraction from BMDMs infected with Mtb H37Rv, showing the presence of mature CTSB. (D,E) BMDMs were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv strains at MOI of 1 or MOI of 3 as described in Section " Materials and Methods ." (D) IL-1β production was measured by ELISA and (E) expression of mature IL-1β, activated caspases-1, and mature CTSB in the supernatant of cells was assessed by western blotting. Differences were analyzed using the Mann–Whitney U test (between two groups) or the Kruskal–Wallis test with Dunn's multiple comparisons ad hoc test. Significant differences were observed for the indicated experimental conditions (* p < 0.05; ** p < 0.01). Bars and line represent mean and SEM, respectively. Data are representative of three independent experiments using triplicate biological samples. We next investigated whether the lysosomal disruption induced by Mtb results in the release of CTSB into the cytosol. We adapted a previously described method using saponin to discriminate lysosomal versus cytosolic content and measured the presence of CTSB in the cytosol at different doses ( 43 ) (Figure S1 in Supplementary Material). We found that Mtb-infected BMDM displayed increased levels of mature CTSB inside the cytosol as compared to uninfected controls. These data confirmed that lysosomal leakage induced by Mtb is indeed associated with release of mature CTSB into the cytosol (Figure 2 C). Mycobacterium tuberculosis ESAT-6 is a highly immunogenic mycobacterial antigen, which has been described to induce pore formation in the phagosome membrane thus facilitating bacterial escape into the cytosol ( 23 ). Moreover, it has been reported that ESAT-6 is required for NLRP3-inflammasome activation and the subsequent cleavage of pro-IL-1β into its mature form ( 5 ). We also observed reduced levels of mature IL-1β in supernatants from BMDM cultured with ΔESAT6 H37Rv or ΔRD1 H37Rv strains compared to those infected with WT H37Rv (Figure 2 D). In addition, we measured mature CTSB, cleaved caspase-1, and mature IL-1β in supernatants from Mtb-infected macrophages by western blotting (WB). Following H37Rv infection, we found increased levels of mature CTSB, cleaved caspase-1, and mature IL-1β from infected macrophages, the latter observation confirming that the changes in IL-1β levels measured by ELISA (Figure 2 D) reflect alterations in the mature cytokine. These products were not detected in supernatants from cells infected with ΔESAT6 H37Rv or ΔRD1 H37Rv strains (Figure 2 E). Taken together, the above findings suggested that ESAT-6 facilitates the secretion of the activated CTSB into the cytosol which is associated with enhanced IL-1β maturation. Inhibition of Cathepsin Activity by CA074-Me Blocks IL-1β Production Induced by Either Mtb Infection or Cytosolic ESAT-6 To confirm the requirement of NLRP3-inflammasome complex for IL-1β production in our experimental system, we infected BMDM from NLRP3 −/− , ASC −/− , caspase-1 −/− , and WT mice with Mtb H37Rv and measured IL-1β in supernatants from macrophages 24 h later. As expected, IL-1β production induced by Mtb was completely abolished in the supernatants from macrophages deficient for NLRP3, ASC, and caspase-1 (Figure 3 A). Figure 3 Mycobacterium tuberculosis (Mtb) ESAT-6-induced IL-1β production is diminished by cathepsin B inhibition. Macrophages were infected with Mtb H37Rv or ΔESAT6 strains at MOI of 3 as described in Section " Materials and Methods ." (A,B) IL-1β production was measured in the supernatants collected from Mtb-infected deficient macrophages or following different treatments as indicated. (B) Mature IL-1β and cleaved caspase-1 were analyzed by western blotting. (C,D) Infected macrophages were stimulated with PA (20 µg/mL) or LFn-ESAT6/PA (20 µg/mL) and IL-1β production was quantified by ELISA after 24 h of infection. (E,F) Bone marrow-derived macrophages were primed with LPS (0.2 µg/mL) for 1 h and further stimulated or not with LFn-ESAT/PA (10 µg/mL each) for 3 h. Furthermore, cells were stimulated with eATP (1 mM) for 25 min and IL-1β production was measured in culture supernatants. Statistical differences observed are shown for each indicated groups (*** p < 0.001). The data represent the mean ± SEM of samples in triplicate. Data are from at least three independent experiments. We next investigated whether lysosomal cathepsin activity is required for release of mature IL-1β. Macrophages were infected with H37Rv in the presence or absence of CA074-Me, a potent inhibitor of CTSB activity and in certain conditions other cathepsins ( 36 , 43 ), and mature IL-1β as well as cleaved caspase-1 were measured in supernatants at 6 and 24 h after infection. We found that the release of both cleaved caspase-1 and mature IL-1β secretion was completely inhibited after CA074-Me treatment (Figure 3 B). Of note, CA074-Me treatment does not affect pro-IL-1β expression as well as cell death induction as previously reported (Figures S2A,C in Supplementary Material) ( 17 , 43 , 47 ). Interestingly, inhibition of the tyrosine kinase Src also blocked IL-1β and cleaved caspase-1 secretion. This effect correlated with a reduction in lysosomal leakage following Src inhibitor treatment (Figure S3 in Supplementary Material). The above results taken together demonstrated that Mtb ESAT-6 expression induces lysosomal perturbation, mature CTSB release into cytosol, and mature IL-1β secretion after Mtb infection. It has been previously established that ESAT-6 facilitates the escape of Mtb into the cytosol from phagolysosomes ( 23 ). Thus, it is challenging to determine whether CTSB release into the cytosol following lysosomal leakage occurs due to ESAT-6-induced pore formation on phagolysosomal membranes or directly through ESAT-6 action in the cytosol. To distinguish these two mechanisms, we performed experiments using a previously described system involving an anthrax toxin LFn-fusion protein to deliver recombinant ESAT-6 into the cytosol ( 48 – 51 ). In these experiments, we infected BMDM with H37Rv and ΔESAT6 H37Rv strains in the presence or not of the LFn-ESAT-6/PA complex ( 50 , 51 ) and then measured IL-1β production 24 h post-infection. Delivery of ESAT-6 into the cytosol enhanced IL-1β production by H37Rv-infected macrophages and importantly restored IL-1β production in ΔESAT6 H37Rv-infected cells (Figure 3 C). Moreover, the IL-1β response induced by ESAT-6 delivery into the cytosol could be abolished by treatment of the cells with the CA074-Me, a potent CTSB inhibitor (Figure 3 D). Although the expression of recombinant ESAT-6 (rESAT-6) in the cytosol resulted in IL-1β production, this effect was nonetheless dependent on Mtb infection (Figure 3 C). To investigate whether rESAT-6 can enhance IL-1β production induced by other known NLRP3-inflammasome stimuli in a non-infectious context, we used the well-known model of NLRP3-inflammasome activation through extracellular ATP stimulation. We first performed a dose titration of eATP concentration to determine non-saturating levels of IL-1β production without inducing cell death via P2X7 receptor (Figure 3 E) and determined the optimal concentration at 1 mM. We then primed macrophages with LPS (0.2 µg/mL) for 1 h (as first signal for pro-IL-1β expression), and then rESAT-6 was delivered into the cytosol by stimulating cells with LFn-ESAT6/PA (10 µg/mL) for an additional 3 h. Finally, macrophages were incubated with eATP (1 mM) for 25 min and IL-1β production measured by ELISA. We found that cytosolic delivery of ESAT-6 amplified IL-1β production induced by ATP in a CTSB-dependent manner (Figure 3 F). Thus, these results suggested that the presence of ESAT-6 inside the cytosol can drive NLRP3-inflammasome activation independent of Mtb infection and that CTSB activity is required for this activation. CA074-Me Blocks ASC Speck Formation in Mtb-Infected Macrophages We next addressed the mechanism by which cytosolic CTSB might promote IL-1β production. ASC speck formation is an ultrastructural hallmark of NLRP3-inflammasome activation ( 52 ). These structures are formed by the accumulation of ASC and cleaved caspase-1 bound to an NLRP3 platform, thus promoting the generation of mature IL-1β ( 52 ). We wondered whether lysosomal cathepsin activity is essential for ASC speck formation as a result of NLRP3-inflammasome assembly. To test this hypothesis, we infected macrophages with Mtb H37Rv and cells were incubated in the presence or absence of the CA074-Me cathepsin inhibitor. After 24 h of infection, speck of ASC was stained using specific antibodies and samples analyzed by fluorescence microscope. Numerous ASC specks were detected in Mtb-infected macrophages (Figures 4 A–C). However, these structures were barely detectable Mtb-infected macrophages treated with CA074-Me (Figures 4 A–C). Thus, these data indicated that the suppression of mature IL-1β production induced by lysosomal cathepsin inhibitor CA074-Me (Figures 2 D,E) is associated with defective NLRP3-inflammasome assembly and consequently ASC speck formation following Mtb infection. Figure 4 Cathepsin B (CTSB) activity mediates ASC speck formation. Macrophages were infected with Mycobacterium tuberculosis H37Rv MOI of 3 as described in Section " Materials and Methods " and cells were treated or not with the CTSB inhibitor CA074-Me as indicated. (A) ASC speck formation in infected macrophages treated or not with CA074-Me (25 µM) was analyzed by fluorescence microscopy. (B) The frequency of cells ASC speck positives (C) and number of ASC speck were quantified by using the Image J software. Differences were analyzed using the Mann–Whitney U test. Statistical differences observed are shown for each indicated groups (*** p < 0.001). Data represent mean ± SEM of samples ran in triplicate. Results are representative at least two independent experiments. Discussion Studies in murine models have identified IL-1β production as a critical factor in host resistance to Mtb ( 1 , 2 , 7 , 9 ), but in certain settings the cytokine can also contribute to immunopathology ( 14 , 16 ). It is well established that Mtb triggers IL-1β production in infected macrophages as a consequence of NLRP3-inflammasome activation in vitro ( 5 , 17 , 53 , 54 ). Nevertheless, the specific mechanism by which NLRP3-inflammasome is activated during Mtb infection is poorly understood. The findings reported here reveal a previously unappreciated role of ESAT-6-dependent cathepsin activity in NLRP3-inflammasome assembly and IL-1β production in Mtb-infected macrophages. The immunodominant ESAT-6 virulence factor plays major roles in the Mtb host interaction. ESAT-6-deficient mycobacteria are unable to induce necrotic macrophage death leading to low degree of bacterial spread to the extracellular milieu ( 17 , 23 , 47 ). In addition, ESAT-6 has been shown to be required for bacterial escape from the phagosome into the cytosol which promotes more rapid induction of necrosis in vitro and in vivo ( 23 , 55 ). As would be expected from its function in necrosis, mice infected with ESAT-6-deficient Mtb display mild disease compared with WT Mtb-infected animals ( 18 , 19 , 56 ). This avirulent phenotype is associated with reduced levels of inflammation, oxidative stress, and cytokine signaling ( 12 , 50 , 51 ). ESAT-6 is also known to trigger mature IL-1β production, although the mechanism involved has been unclear. As shown in the present study, ESAT-6-dependent IL-1β production was completely abolished following treatment of macrophage cultures with a CTSB inhibitor, CA074-Me. In addition, we found that delivery of ESAT-6 into cytosol restores IL-1β production during macrophage infection with ΔESAT-6-Mtb and in uninfected macrophages enhances IL-1β production mediated by eATP via P2X7 activation. In both situations, the effects of ESAT-6 on IL-1β production were blocked by treatment with CA074-Me. Unexpectedly, genetic deficiency in CTSB or cathepsin L (CTSL) actually resulted in enhanced IL-1β (Figure S3 in Supplementary Material). Similar findings have been reported in CTSB-deficient or CTSL-deficient macrophages stimulated with uric acid or alum, and could result from the overexpression in the relevant deficient mouse strains of lysosomal enzymes with redundant function ( 30 , 57 ). The use of CA074-Me for inhibiting CTSB is appropriate and justified because of its properties of high plasma membrane permeability and quick conversion to its non-methylated form, CA074, that has been shown to interfere specifically with CTSB activity ( 58 – 64 ). Importantly, CA074-Me does not affect other enzymes such as caspase-1, known to be essential for pro-IL-1β cleavage ( 36 , 65 ). Finally, CTSB is found at relatively high levels and is widely distributed in the lysosomes of cells in various tissues ( 30 ), making it a prominent target for CA074-Me inhibition at the low drug concentration used here ( 64 ). While, we cannot at this stage formally rule out the participation of other related proteases, our data strongly support a major role for CTSB activation in Mtb induced IL-1β maturation. Our data also argue that the release of mature CTSB in the cytosol is a critical step for NLRP3-inflammasome activation by facilitating interaction between NLRP3 and ASC, culminating in the formation of ASC specks. While CTSB inhibition clearly blocked Mtb-induced inflammasome assembly, we also observed inhibition of NLRP3 and ASC interaction in Mtb-infected macrophages following treatment with Src inhibitor (Figure S2C in Supplementary Material). Indeed, Src activity has been reported to be involved in mediating the secretion of proteins such as CTSB and CTSD, and its inhibition also abolishes IL-1β production in macrophage cultures stimulated with LPS and monosodium urate crystals ( 66 ). In fact, we observed that Src inhibition prevents mature CTSB release into the cytosol in Mtb-infected macrophages (Figure S2D in Supplementary Material). Direct physical interaction between recombinant NLRP3 and CTSB proteins has been previously reported ( 27 ), supporting our hypothesis that CTSB can take part in NLRP3-inflammasome complex assembly as well as ASC speck formation. Whether the molecular mechanism by which CTSB promotes NLRP3-ASC complex formation by interfering the regulation of other proteins implied in the assembly of this complex such as NEK7/vimentin ( 67 ) remains to be determined. The generation of ASC speck has been associated with high levels of IL-1β production and promotion of inflammation ( 52 ). It is known that excessive production of IL-1β cytokines is detrimental for host, since it is associated with intense inflammation and increased immunopathology. Mishra and colleagues have shown that detrimental neutrophilic inflammation in severe forms of TB is associated with elevated IL-1β production, which can be repressed by nitric oxide ( 16 ). In addition, nitric oxide inhibits IL-1β-mediated inflammation by repressing the caspase-1-dependent cleavage of pro-IL-1β ( 14 ). More recently, inflammasome signaling has been described as process in TB-associated immune reconstitution inflammatory syndrome, highlighting the importance of the inflammasome in clinical settings related to TB disease ( 68 – 74 ). In experimental models in vivo , mice deficient in IL-1β or IL-1R are extremely susceptible to Mtb infection, whereas NLRP3, ASC, and caspase-1 deficient mice do not display this phenotype ( 4 , 53 , 54 ). Interestingly, ASC has also been implicated granuloma maintenance during chronic phase of Mtb infection ( 53 ). These findings indicate that another inflammasome-independent mechanism leading to post-translational processing of pro-IL-1β may operate in vivo . One possibility is that enzymes, such as metalloproteinases and/or cathepsins, secreted during inflammation or released by cells undergoing cytolysis/necrosis may be responsible for the cleavage of pro-IL-1β in the extracellular milieu as described in other experimental models ( 75 – 77 ). Interestingly, in an animal model of viral myocarditis mice deficient in CTSB showed improved survival, reduced inflammatory cell tissue infiltration, and reduced IL-1β production, suggesting the CTSB aggravates the disease through activating the inflammasome-dependent IL-1β production and promoting pyroptosis ( 78 ). The results presented here identify CTSB (and possibly other cathepsins inhibited by CA074-Me) as key inducer of inflammasome assembly and activation in the context of Mtb infection ( Figure 5 ). Our data also demonstrate an association of increased CTSB levels with active TB in both patients and animal models and our in vitro experiments with Mtb-infected macrophages indicate that these elevations may reflect increases in the intracellular levels of the mature form of the enzyme. Whether or not the latter finding is due to increased pro-enzyme synthesis was not addressed here although a previous study employing human monocyte-derived macrophages reported a decrease in cathepsin transcription following Mtb infection ( 79 ). At present, it is not clear whether this apparent discrepancy in the data reflects the host species or bone marrow versus monocyte origin of the macrophages employed in the present versus previous study. Indeed, pharmacological CTSB inhibition by dampening inflammasome-dependent IL-1β production may suppress inflammatory processes detrimental for disease outcome, such as excessive IL-1β production. Figure 5 Schematic model for mature IL-1β generation during Mycobacterium tuberculosis (Mtb) infection. The secretion of ESAT-6 in phagosomes by Mtb induces pore formation on phagosomal membranes, facilitating the leakage of ESAT-6 into cytosol and bacterial escape to cytosolic compartment (A) . Once in the cytosol, ESAT-6 may induce pore formation on cellular membranes which could contribute to lysosomal perturbation (LMP) (B) . Simultaneously, extracellular ATP is recognized by P2X7 receptor and amplify LMP-induced by Mtb or vice versa (C) . LMP releases lysosomal enzymes, such as cathepsin B (CTSB) (D) . Once in the cytosol, activated CTSB supports the interaction between NLRP3 and ASC (E) facilitating the NLRP3-inflammasome assembly following caspase-1 recruitment (F) . Several molecules of ASC and caspase-1 will be recruited to NLRP3-complex leading the formation of the ASC speck core (G) , which amplifies IL-1 β production (H) . The secretion of ASC specks and IL-1 β to extracellular milieu may exacerbate inflammation (I) . Materials and Methods Mice and Rabbits C57BL/6 mice were purchased from Taconic Farms (Hudson, NY, USA). NLRP3 −/− , ASC −/− , and caspase-1 −/− mice were kindly provided by Dr. Karina Bortolucci from Federal University of São Paulo, Brazil. CTSB −/− and CTSL −/− mouse-derived materials were kindly provided by Dr. John Misasi from Harvard University, USA. All experimental procedures were in accordance with an animal study proposal approved by the NIAID Animal Care and Use Committee. Mice and rabbits were infected as previously described ( 51 , 80 , 81 ). The lung samples employed were from infected animals described in previously published papers ( 51 , 80 , 81 ). At day 42 post-infection, different lung regions presenting with cavitation (cavity wall), granulomatous lesions, or normal lung tissue were dissected for RNA and protein analysis, and snap frozen in liquid nitrogen as previously described ( 51 , 80 ). Human Samples The CTSB concentration in plasma samples stored with ethyl-enediaminetetraacetic acid (EDTA) were assessed by ELISA. Samples were from 13 patients [7 males; median age, 33 years (range, 25–52 years)] with a diagnosis of active pulmonary TB confirmed by sputum culture and from 12 healthy blood donors [6 males; median age, 30 years (range, 10–58 years)] who were matched for age and sex and recruited between 2012 and 2014. RNA Preparation and RNA Deep Sequencing RNA was extracted by bead beating in Trizol and column purified (Qiagen, USA) as previously described ( 80 ). Fragmentation of the whole transcriptome RNA was performed by chemical induction following the manufacture's protocol (Applied Biosystem SOLiD Total RNA-Seq Kit; Applied Biosystems, USA). Fragmented RNA was purified and construction of the amplified whole transcriptome library performed following the manufacture's instruction. CTSB expression was verified as previously reported ( 80 ). Generation of BMDMs and In Vitro Infection Bone marrow-derived macrophages were generated from mouse femurs as previously described ( 82 ), with some modifications. Briefly, the femurs were flushed with 5 mL complete RPMI medium (Gibco, USA; 1 mM sodium pyruvate, 2 mM glutamine, 0.05% gentamicin, and 10% FCS) and cultivated with 30% L929 supernatant media to differentiate BMDM. An additional 10 mL of L929 cell-conditioned medium were added after 4 days of incubation. BMDMs were detached and seeded in 96-well plates at 10 5 cells/well, containing OptiMEM media (Invitrogen, USA) at 37°C in 5% CO 2 atmosphere. In some experiments, we treated the cells with CA074-Me (25 µM) and Src inhibitor (10 µM) for 1 h prior to bacterial infection. BMDMs were infected with H37Rv, ρESAT-6 H37Rv, and ρRD1 H37Rv strains (a gift from Dr. Volker Briken, University of Maryland, College Park, MD, USA) at MOI of 1 or 3 for 3 h, washed and then cultivated for 5 days. Bacterial uptake was evaluated by CFU counting in BMDM treated with 0.05% saponin (Sigma-Aldrich, USA) for 10 min at different time point. Cytotoxicity induced by bacteria was analyzed in cell culture was determined by lactate dehydrogenase cytotoxicity assay kit (Caymam Chemical, USA) according to the manufacture's protocol. In some experiments, a recombinant Mtb ESAT-6 fused with N-terminal fragment of lethal factor of Bacillus anthracis (LFn-ESAT6) was used to deliver ESAT-6 into the cytosol of infected and uninfected macrophages as previously described ( 50 , 51 ). Anthrax-protective Ag used in this procedure was also prepared as described previously ( 83 ). WB and Immunoprecipitation (IP) Samples for WB and IP were obtained from 1.5 × 10 6 cells by lysis using NP-40 lysis buffer [150 mM NaCl, 20 mM Tris–Hcl pH 8.0, 1% Nonidet P-40 (NP-40), 10% glycerol, 2 mM EDTA]. Proteins from macrophages supernatants were concentrated by methanol/chloroform precipitation method as described previously ( 84 ). For IP, a pre-clearing step was performed in the samples by incubation with an isotype control antibody and protein A/G for 2 h at 4°C, and then washed with lysis buffer by centrifugation at 3,000 rpm for 30 s. Anti-ASC antibody (Santa Cruz, CA, USA) was added to cell lysates (1 mg/mL) and samples were continuously mixed by rotation at 4°C for 1 h, followed by Protein A/G agarose (Santa Cruz Biotechnology, USA) addition and continued overnight 4°C incubation with rotation. Beads were centrifuged at 3,000 rpm for 30 s and washed with NP-40 buffer two times prior to elution of proteins using SDS loading buffer (10% SDS, 0.6 M DTT, 30% glycerol, 0.012% bromophenol blue, at 90°C, 5 min). Prior to electrophoresis, the samples in SDS loading buffer were boiled at 95°C for 5 min. Protein transference to the nitrocellulose membrane was performed using Trans-Blot ® Turbo™ Transfer System Bio-Rad machine according to the instructions of the manufacturer. Nitrocellulose membranes were blocked using 1% milk diluted in 0.02% PBS-Tween20. Western blots were performed using either anti-CTSB (1:250; sc-6493), anti-caspase-1 (1:250; sc-514), anti-IL-1β (1:1,000; AF-401), anti-ASC (1:250; sc-30153), anti-βactin (1:1,000; a5316), or anti-NLRP3 (1:1,000; sc-34411). Cytokine Quantification and CTSB Activity Cytokine levels in lungs and spleens homogenates were measured using commercial ELISA Kits (R&D Systems, USA) according to manufacturer's instructions. Human total CTSB was measured in the human serum using Human Total CTSB DuoSet ELISA Kit (R&D Systems, USA) according to manufacturer's protocol. CTSB activity in the lungs homogenates was measured using CTSB activity assay Fluorometric Kit (Abcam, USA) according to the specifications of the manufacturer. CTSB activity was visualized in Mtb-infected BMDM using a Magic Red CTSB assay Kit according to the manufacturer's instructions (Immunochemistry, USA). Samples were examined by confocal microscopy (Nikon, Japan) using an excitation filter of 550 nm and emission filter of 620 nm. ASC Speck Formation Inflammasome complex assembly was evaluated by detection of ASC speck formation using immunofluorescence microscopy. Cells were fixed with 4% paraformaldehyde buffer (pH 7.2–7.4) for 35 min, washed three times with 1× PBS and kept in ammonium chloride buffer (50 mM; pH 8.0) for 15 min, and then washed three times with 1× PBS. Fixed cells were permeabilized with 0.5% Triton-100X buffer (Sigma-Aldrich, USA) for 10 min. After permeabilization, cells were blocked with 8% milk and 10% BSA in 1× PBS to avoid nonspecific binding, followed by overnight incubation with specific primary antibody against murine ASC (1:500; Millipore) in 0.25% Tween-20 1× PBS. Cells were washed with 0.25% Tween-20 1× PBS to remove the primary antibody and incubated with anti-rabbit FITC (1:1,000; BD, USA) for 30 min. DAPI (1:1,000; Sigma-Aldrich, USA) was added to samples for nucleus staining. Images were acquired by using fluorescence microscope (Nikon, Japan). Statistical Analysis Statistical analyses were performed using GraphPad Prism 4 software (GraphPad, USA). Both one-way ANOVA test and Tukey post hoc test were used to assess the effects of only one parameter. Differences between groups were considered significant when p < 0.05. Mice and Rabbits C57BL/6 mice were purchased from Taconic Farms (Hudson, NY, USA). NLRP3 −/− , ASC −/− , and caspase-1 −/− mice were kindly provided by Dr. Karina Bortolucci from Federal University of São Paulo, Brazil. CTSB −/− and CTSL −/− mouse-derived materials were kindly provided by Dr. John Misasi from Harvard University, USA. All experimental procedures were in accordance with an animal study proposal approved by the NIAID Animal Care and Use Committee. Mice and rabbits were infected as previously described ( 51 , 80 , 81 ). The lung samples employed were from infected animals described in previously published papers ( 51 , 80 , 81 ). At day 42 post-infection, different lung regions presenting with cavitation (cavity wall), granulomatous lesions, or normal lung tissue were dissected for RNA and protein analysis, and snap frozen in liquid nitrogen as previously described ( 51 , 80 ). Human Samples The CTSB concentration in plasma samples stored with ethyl-enediaminetetraacetic acid (EDTA) were assessed by ELISA. Samples were from 13 patients [7 males; median age, 33 years (range, 25–52 years)] with a diagnosis of active pulmonary TB confirmed by sputum culture and from 12 healthy blood donors [6 males; median age, 30 years (range, 10–58 years)] who were matched for age and sex and recruited between 2012 and 2014. RNA Preparation and RNA Deep Sequencing RNA was extracted by bead beating in Trizol and column purified (Qiagen, USA) as previously described ( 80 ). Fragmentation of the whole transcriptome RNA was performed by chemical induction following the manufacture's protocol (Applied Biosystem SOLiD Total RNA-Seq Kit; Applied Biosystems, USA). Fragmented RNA was purified and construction of the amplified whole transcriptome library performed following the manufacture's instruction. CTSB expression was verified as previously reported ( 80 ). Generation of BMDMs and In Vitro Infection Bone marrow-derived macrophages were generated from mouse femurs as previously described ( 82 ), with some modifications. Briefly, the femurs were flushed with 5 mL complete RPMI medium (Gibco, USA; 1 mM sodium pyruvate, 2 mM glutamine, 0.05% gentamicin, and 10% FCS) and cultivated with 30% L929 supernatant media to differentiate BMDM. An additional 10 mL of L929 cell-conditioned medium were added after 4 days of incubation. BMDMs were detached and seeded in 96-well plates at 10 5 cells/well, containing OptiMEM media (Invitrogen, USA) at 37°C in 5% CO 2 atmosphere. In some experiments, we treated the cells with CA074-Me (25 µM) and Src inhibitor (10 µM) for 1 h prior to bacterial infection. BMDMs were infected with H37Rv, ρESAT-6 H37Rv, and ρRD1 H37Rv strains (a gift from Dr. Volker Briken, University of Maryland, College Park, MD, USA) at MOI of 1 or 3 for 3 h, washed and then cultivated for 5 days. Bacterial uptake was evaluated by CFU counting in BMDM treated with 0.05% saponin (Sigma-Aldrich, USA) for 10 min at different time point. Cytotoxicity induced by bacteria was analyzed in cell culture was determined by lactate dehydrogenase cytotoxicity assay kit (Caymam Chemical, USA) according to the manufacture's protocol. In some experiments, a recombinant Mtb ESAT-6 fused with N-terminal fragment of lethal factor of Bacillus anthracis (LFn-ESAT6) was used to deliver ESAT-6 into the cytosol of infected and uninfected macrophages as previously described ( 50 , 51 ). Anthrax-protective Ag used in this procedure was also prepared as described previously ( 83 ). WB and Immunoprecipitation (IP) Samples for WB and IP were obtained from 1.5 × 10 6 cells by lysis using NP-40 lysis buffer [150 mM NaCl, 20 mM Tris–Hcl pH 8.0, 1% Nonidet P-40 (NP-40), 10% glycerol, 2 mM EDTA]. Proteins from macrophages supernatants were concentrated by methanol/chloroform precipitation method as described previously ( 84 ). For IP, a pre-clearing step was performed in the samples by incubation with an isotype control antibody and protein A/G for 2 h at 4°C, and then washed with lysis buffer by centrifugation at 3,000 rpm for 30 s. Anti-ASC antibody (Santa Cruz, CA, USA) was added to cell lysates (1 mg/mL) and samples were continuously mixed by rotation at 4°C for 1 h, followed by Protein A/G agarose (Santa Cruz Biotechnology, USA) addition and continued overnight 4°C incubation with rotation. Beads were centrifuged at 3,000 rpm for 30 s and washed with NP-40 buffer two times prior to elution of proteins using SDS loading buffer (10% SDS, 0.6 M DTT, 30% glycerol, 0.012% bromophenol blue, at 90°C, 5 min). Prior to electrophoresis, the samples in SDS loading buffer were boiled at 95°C for 5 min. Protein transference to the nitrocellulose membrane was performed using Trans-Blot ® Turbo™ Transfer System Bio-Rad machine according to the instructions of the manufacturer. Nitrocellulose membranes were blocked using 1% milk diluted in 0.02% PBS-Tween20. Western blots were performed using either anti-CTSB (1:250; sc-6493), anti-caspase-1 (1:250; sc-514), anti-IL-1β (1:1,000; AF-401), anti-ASC (1:250; sc-30153), anti-βactin (1:1,000; a5316), or anti-NLRP3 (1:1,000; sc-34411). Cytokine Quantification and CTSB Activity Cytokine levels in lungs and spleens homogenates were measured using commercial ELISA Kits (R&D Systems, USA) according to manufacturer's instructions. Human total CTSB was measured in the human serum using Human Total CTSB DuoSet ELISA Kit (R&D Systems, USA) according to manufacturer's protocol. CTSB activity in the lungs homogenates was measured using CTSB activity assay Fluorometric Kit (Abcam, USA) according to the specifications of the manufacturer. CTSB activity was visualized in Mtb-infected BMDM using a Magic Red CTSB assay Kit according to the manufacturer's instructions (Immunochemistry, USA). Samples were examined by confocal microscopy (Nikon, Japan) using an excitation filter of 550 nm and emission filter of 620 nm. ASC Speck Formation Inflammasome complex assembly was evaluated by detection of ASC speck formation using immunofluorescence microscopy. Cells were fixed with 4% paraformaldehyde buffer (pH 7.2–7.4) for 35 min, washed three times with 1× PBS and kept in ammonium chloride buffer (50 mM; pH 8.0) for 15 min, and then washed three times with 1× PBS. Fixed cells were permeabilized with 0.5% Triton-100X buffer (Sigma-Aldrich, USA) for 10 min. After permeabilization, cells were blocked with 8% milk and 10% BSA in 1× PBS to avoid nonspecific binding, followed by overnight incubation with specific primary antibody against murine ASC (1:500; Millipore) in 0.25% Tween-20 1× PBS. Cells were washed with 0.25% Tween-20 1× PBS to remove the primary antibody and incubated with anti-rabbit FITC (1:1,000; BD, USA) for 30 min. DAPI (1:1,000; Sigma-Aldrich, USA) was added to samples for nucleus staining. Images were acquired by using fluorescence microscope (Nikon, Japan). Statistical Analysis Statistical analyses were performed using GraphPad Prism 4 software (GraphPad, USA). Both one-way ANOVA test and Tukey post hoc test were used to assess the effects of only one parameter. Differences between groups were considered significant when p < 0.05. Ethics Statement The clinical protocol was approved by the institutional review board from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD; protocol NCT01611402). Written informed consent was obtained from all study participants. The protocols, procedures, and animal care for the murine and rabbit were approved, respectively, by the Institutional Animal Care and Use Committee from the National Institutes of Health (NIH), Bethesda, MD, USA (protocol number LPD99E) and Johns Hopkins University, Baltimore, MD, USA (protocol number RB11M466). All the experiments were carried out in accordance with the recommendation of the Guide for the Care and Use of Laboratory Animals of the National Research Council of the National Academies of the United States of America (8th Edition) as mandated by the U.S. Public Health Service Policy. All animal experiments were performed in animal BSL-3 facilities maintained by the Intramural Research Program of National Institute of Allergy and Infectious Diseases (NIAID) and Johns Hopkins University. Author Contributions EA, NR, MM, NM, KM-B, RP, SL, AK, and BA performed experiments. EA and BA designed experiments. MM, KM-B, WB, MD-L, AS, and BA provided materials and infrastructural support. EA, AS, and BA wrote the manuscript. Disclaimer The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary Material The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01427/full#supplementary-material . Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044706/

NIH Disease Funding Levels and Burden of Disease

Background An analysis of NIH funding in 1996 found that the strongest predictor of funding, disability-adjusted life-years (DALYs), explained only 39% of the variance in funding. In 1998, Congress requested that the Institute of Medicine (IOM) evaluate priority-setting criteria for NIH funding; the IOM recommended greater consideration of disease burden. We examined whether the association between current burden and funding has changed since that time. Methods We analyzed public data on 2006 NIH funding for 29 common conditions. Measures of US disease burden in 2004 were obtained from the World Health Organization's Global Burden of Disease study and national databases. We assessed the relationship between disease burden and NIH funding dollars in univariate and multivariable log-linear models that evaluated all measures of disease burden. Sensitivity analyses examined associations with future US burden, current and future measures of world disease burden, and a newly standardized NIH accounting method. Results In univariate and multivariable analyses, disease-specific NIH funding levels increased with burden of disease measured in DALYs (p = 0.001), which accounted for 33% of funding level variation. No other factor predicted funding in multivariable models. Conditions receiving the most funding greater than expected based on disease burden were AIDS ($2474 M), diabetes mellitus ($390 M), and perinatal conditions ($297 M). Depression ($719 M), injuries ($691 M), and chronic obstructive pulmonary disease ($613 M) were the most underfunded. Results were similar using estimates of future US burden, current and future world disease burden, and alternate NIH accounting methods. Conclusions Current levels of NIH disease-specific research funding correlate modestly with US disease burden, and correlation has not improved in the last decade. Background An analysis of NIH funding in 1996 found that the strongest predictor of funding, disability-adjusted life-years (DALYs), explained only 39% of the variance in funding. In 1998, Congress requested that the Institute of Medicine (IOM) evaluate priority-setting criteria for NIH funding; the IOM recommended greater consideration of disease burden. We examined whether the association between current burden and funding has changed since that time. Methods We analyzed public data on 2006 NIH funding for 29 common conditions. Measures of US disease burden in 2004 were obtained from the World Health Organization's Global Burden of Disease study and national databases. We assessed the relationship between disease burden and NIH funding dollars in univariate and multivariable log-linear models that evaluated all measures of disease burden. Sensitivity analyses examined associations with future US burden, current and future measures of world disease burden, and a newly standardized NIH accounting method. Results In univariate and multivariable analyses, disease-specific NIH funding levels increased with burden of disease measured in DALYs (p = 0.001), which accounted for 33% of funding level variation. No other factor predicted funding in multivariable models. Conditions receiving the most funding greater than expected based on disease burden were AIDS ($2474 M), diabetes mellitus ($390 M), and perinatal conditions ($297 M). Depression ($719 M), injuries ($691 M), and chronic obstructive pulmonary disease ($613 M) were the most underfunded. Results were similar using estimates of future US burden, current and future world disease burden, and alternate NIH accounting methods. Conclusions Current levels of NIH disease-specific research funding correlate modestly with US disease burden, and correlation has not improved in the last decade. Introduction The National Institutes of Health (NIH) is the largest public funder of biomedical research worldwide [1] , [2] , with a budget that has grown from $11.9 billion in 1996 to $28.5 billion in 2006 [3] . In the mid-1990s, Congress and the public raised concerns that disease-specific funding allocations by the NIH failed to adequately reflect burden of disease and incorporate public input [4] . In response, Congress requested that the Institute of Medicine (IOM) assess the NIH funding apportionment processes. In its 1998 report, Scientific Opportunities and Public Needs: Improving Priority Setting and Public Input at the National Institutes of Health , the IOM recommended improved tracking of disease-specific funding and development of a new priority-setting process [4] . A landmark study comparing disease burden to NIH funding levels was published in 1999 [5] . For 29 common conditions, the study examined a variety of measures of societal burden, recognizing that none by itself completely captures relative impacts of diseases. Disease incidence and prevalence were unrelated to funding, while mortality and years of life lost (YLLs) weakly correlated with funding. Disability-adjusted life years (DALYs)—a measure that estimates the equivalent number of healthy years lost due to disability or early death [6] , [7] —were more strongly predictive. Using DALYs as the best single predictor, only 39% of the variance in NIH funding could be explained. The prior analysis was limited to evaluation of univariate predictors, and did not attempt to evaluate whether funding aligned with other measures of disease burden. The NIH Reform Act of 2006 re-emphasized the NIH's role in identifying research to meet public health challenges, and mandated submission of a biennial report to Congress regarding disease-specific funding amounts [8] . There has been no recent comprehensive study of US disease burden and NIH funding, and an analysis of only one of its institutes has been performed [9] . To determine whether the NIH has developed processes that better align funding with burden, we assessed the correlation between NIH funding and burden of disease, and compared results with those reported 10 years ago [5] . We also considered other potential predictors of funding and assessed the association of NIH funding with estimates of future and global disease burden. Methods In a cross-sectional study, we compared measures of US and world disease burden and sociopolitical factors from 2004 to NIH funding levels in 2006. The study design was modeled on methods previously established [5] , which used 1994 burden data and 1996 NIH funding to reflect an expected lag in availability of data on disease burden. Each disease was defined using pre-specified sets of International Classification of Diseases, 9 th revision, Clinical Modification (ICD-9) codes, which were applied to public sources of information on disease burden [10] . Data Sources Amounts of NIH funding for disease categories were obtained directly from the NIH for the year 2006 ( Table 1 ). These estimates were annually consolidated from figures supplied by individual NIH Institutes and Centers ( http://www.nih.gov/news/fundingresearchareas.htm ) [11] . In 2006 and prior years, NIH Institutes and Centers categorized spending in a variety of manners to satisfy diverse reporting requirements, and calculated condition-specific total funds in a non-mutually exclusive manner. Thus, funding for a particular trial or biomarker could have been attributed to multiple conditions. 10.1371/journal.pone.0016837.t001 Table 1 NIH Research Funds and Measures of Disease Burden for 29 Conditions. Condition or Disease NIH Research Funds Measure of Disease Burden in North America * thousands (rank) millions of dollars (% of total) Incidence Prevalence Mortality Years of Life Lost Disability-Adjusted Life-Years AIDS 2902 (24.3) 142 (19) 1275 (15) 14 (16) 279 (13) 583 (14) Diabetes mellitus 1038 (8.7) 1200 (9) 21663 (2) 84 (7) 563 (7) 1473 (6) Perinatal conditions 789 (6.6) 45 (21) 3516 (7) 17 (14) 578 (6) 793 (10) Breast cancer 718 (6.0) 222 (13) 1875 (11) 53 (10) 488 (8) 684 (12) Dementia 643 (5.4) 714 (10) 3108 (8) 132 (5) 306 (11) 1359 (8) Alcohol abuse 511 (4.3) 2641 (6) 9553 (4) 8 (17) 121 (16) 1837 (4) Dental and oral disorders 413 (3.5) 109774 (1) 41152 (1) 0 2 (27) 267 (18) Cirrhosis 408 (3.4) 43 (22) 303 (21) 30 (12) 360 (10) 455 (16) Ischemic heart disease 398 (3.3) 1336 (7) 2347 (10) 531 (1) 2695 (2) 3048 (3) Schizophrenia 364 (3.1) 42 (23) 1561 (13) 1 (24) 5 (25) 522 (15) Injuries 355 (3.0) 3747 (5) 241 (23) 182 (2) 3448 (1) 4484 (2) Pneumonia 351 (3.0) 4178 (4) 75 (27) 68 (9) 294 (12) 315 (17) Prostate cancer 348 (2.9) 149 (18) 1032 (16) 38 (11) 152 (14) 253 (20) Stroke 342 (2.9) 373 (12) 2733 (9) 176 (4) 791 (4) 1336 (9) Depression 335 (2.8) 16417 (3) 8207 (5) 1 (24) 3 (26) 4564 (1) Asthma 283 (2.4) 1278 (8) 19100 (3) 4 (20) 53 (19) 755 (11) Colorectal cancer 269 (2.3) 150 (17) 713 (18) 70 (8) 466 (9) 609 (13) Lung cancer 266 (2.2) 196 (15) 706 (20) 181 (3) 1331 (3) 1384 (7) Sexually transmitted diseases 264 (2.2) Not available Not available 0 2 (27) 65 (26) Parkinson's disease 208 (1.7) 90 (20) 1025 (17) 21 (13) 66 (18) 263 (19) Tuberculosis 150 (1.3) 11 (27) 11 (28) 1 (24) 7 (24) 10 (29) Multiple sclerosis 110 (0.9) 9 (28) 176 (25) 4 (20) 45 (20) 118 (24) Epilepsy 103 (0.9) 207 (14) 1677 (12) 2 (23) 31 (22) 160 (22) Ovarian cancer 102 (0.9) 24 (26) 143 (26) 17 (14) 140 (15) 161 (21) Cervical cancer 97 (0.8) 29 (24) 225 (24) 7 (18) 86 (17) 125 (23) Chronic obstructive pulmonary disorder 67 (0.6) 429 (11) 6923 (6) 132 (5) 644 (5) 1647 (5) Uterine cancer 28 (0.2) 25 (25) 301 (22) 7 (18) 45 (20) 84 (25) Peptic ulcer disease 17 (0.1) 192 (16) 712 (19) 4 (20) 24 (23) 40 (27) Otitis media 17 (0.1) 17679 (2) 1360 (14) 0 1 (29) 35 (28) Funding rates by disease were obtained from the NIH for 2006. Estimates of incidence, prevalence, mortality, disability-adjusted life-years lost, and years-of-life-lost are total annual counts, in 1000s, for North America, obtained from the 2004 update of the World Health Organization's Global Burden of Disease project. Data denoting disease burden were collected from multiple sources ( Table 1 ). First, world and North American disease-specific data were obtained from the Global Burden of Disease (GBD) Project the World Health Organization (WHO) [12] , [13] , [14] . We used GBD's North American data for US estimates. The GBD systematically collects timely disease-specific epidemiologic information and models missing data to estimate a variety of measures of burden [15] , [16] . We used 2004 GBD estimates because they were likely to be the most recent publicly available data at the time decisions were made for the 2006 funding cycle. No other centralized, systematic source of broad national disease-specific burden estimates was identified. We evaluated five disease burden categories from the GBD: incidence, prevalence, mortality, YLLs, and DALYs [14] . YLLs for each disease were calculated by totaling the differences between life expectancy and age at death. DALYs were estimated based on YLL and on standardized weighting schemes for disability applied to those surviving with disease [6] , [7] . GBD estimates for the US were based on analyses of comprehensive death certificate data, national incidence estimates, and a systematic review of published epidemiological studies. To evaluate other estimates of disease burden, we used 2004 US inpatient and outpatient healthcare databases categorized by diagnosis, each of which includes large samples with weights to generate nationally representative estimates (Supporting Table S1 ). The number of hospital discharges, total length of stay, and mean hospital charges for disease-specific principal diagnoses were derived from the National Inpatient Sample (NIS) [17] . The number of visits to emergency departments and outpatient hospital clinics were derived from the National Hospital Ambulatory Care Survey (NHAMCS) and the National Ambulatory Care Survey (NAMCS) [18] . In both NIS and NHAMCS, community hospitals were defined as non-federal, short-stay hospitals [19] , [20] . Public interest could also influence funding levels through lobbying efforts, additional funding support from private foundations, or by directly stimulating the interest of investigators, and this could influence funding levels. To begin to assess the influence of public interest, we determined the number of disease-specific newspaper articles published in the top 10 US newspapers with highest distribution, as well as broadcast television news reports from national networks, using disease-specific keyword searches of LexisNexis News [21] and Vanderbilt Television News Archive [22] . To estimate the influence of specific interest group advocacy, the total US disease-specific charity revenue was similarly calculated for public charities receiving more than $500,000 in public support [23] . Scientific productivity in a given area could stimulate further interest from researchers and the NIH, and could result in targeted funding. As surrogate measures of scientific productivity, we tallied the number of disease-specific patents submitted [24] and articles published and listed in PubMed [25] in the 10 medical journals with the highest impact factor scores using key word searches [26] . Analysis The relationship between 2006 NIH funding levels and 2004 US disease burden metrics was designated the a priori primary analysis. All predictor and outcome variables were log-transformed to reduce positive skew. Univariate linear regression was first performed to replicate the prior study design [5] . This approach was preferred to correlation to allow for adjustment and to acknowledge that funding level was the dependent variable to be predicted. Expected funding levels in 2006 were calculated with correction for the log-transformation by applying measures of disease burden to fitted models predicting funding [27] , [28] . To account for inflation, the actual and expected funding levels for 1996 were inflated to 2006 dollars [29] . Standard, forward stepwise multivariable linear regression analysis included all variables of disease burden, including those with p<0.05 in the final model. We did not evaluate interactions. F-statistics were used to estimate differences in explanatory power (significant if p<0.05). A separate forward stepwise multivariable model was constrained so that diseases with no burden would receive zero funding, mimicking a theoretical funding process that determines allocations proportional to the disease burden. For this model, we defined the dependent variable as the ratio of dollars to DALYs for each of the included conditions. Separate multivariable models included measures of public interest and scientific productivity. In additional sensitivity analyses, we evaluated the association of funding levels with worldwide disease burden in 2004, and with projections for 2015 and 2030 for both the US and worldwide [30] . A final analysis evaluated whether a new NIH accounting method for disease-specific funding introduced in 2007 produced different results. Explanatory power of the models was estimated with adjusted R 2 values; an adjusted R 2 value was also determined for the analysis of 1996 funding levels using data derived from this prior publication. The Stata statistical package (Version 10, College Station, Texas) was used for all analyses. Data Sources Amounts of NIH funding for disease categories were obtained directly from the NIH for the year 2006 ( Table 1 ). These estimates were annually consolidated from figures supplied by individual NIH Institutes and Centers ( http://www.nih.gov/news/fundingresearchareas.htm ) [11] . In 2006 and prior years, NIH Institutes and Centers categorized spending in a variety of manners to satisfy diverse reporting requirements, and calculated condition-specific total funds in a non-mutually exclusive manner. Thus, funding for a particular trial or biomarker could have been attributed to multiple conditions. 10.1371/journal.pone.0016837.t001 Table 1 NIH Research Funds and Measures of Disease Burden for 29 Conditions. Condition or Disease NIH Research Funds Measure of Disease Burden in North America * thousands (rank) millions of dollars (% of total) Incidence Prevalence Mortality Years of Life Lost Disability-Adjusted Life-Years AIDS 2902 (24.3) 142 (19) 1275 (15) 14 (16) 279 (13) 583 (14) Diabetes mellitus 1038 (8.7) 1200 (9) 21663 (2) 84 (7) 563 (7) 1473 (6) Perinatal conditions 789 (6.6) 45 (21) 3516 (7) 17 (14) 578 (6) 793 (10) Breast cancer 718 (6.0) 222 (13) 1875 (11) 53 (10) 488 (8) 684 (12) Dementia 643 (5.4) 714 (10) 3108 (8) 132 (5) 306 (11) 1359 (8) Alcohol abuse 511 (4.3) 2641 (6) 9553 (4) 8 (17) 121 (16) 1837 (4) Dental and oral disorders 413 (3.5) 109774 (1) 41152 (1) 0 2 (27) 267 (18) Cirrhosis 408 (3.4) 43 (22) 303 (21) 30 (12) 360 (10) 455 (16) Ischemic heart disease 398 (3.3) 1336 (7) 2347 (10) 531 (1) 2695 (2) 3048 (3) Schizophrenia 364 (3.1) 42 (23) 1561 (13) 1 (24) 5 (25) 522 (15) Injuries 355 (3.0) 3747 (5) 241 (23) 182 (2) 3448 (1) 4484 (2) Pneumonia 351 (3.0) 4178 (4) 75 (27) 68 (9) 294 (12) 315 (17) Prostate cancer 348 (2.9) 149 (18) 1032 (16) 38 (11) 152 (14) 253 (20) Stroke 342 (2.9) 373 (12) 2733 (9) 176 (4) 791 (4) 1336 (9) Depression 335 (2.8) 16417 (3) 8207 (5) 1 (24) 3 (26) 4564 (1) Asthma 283 (2.4) 1278 (8) 19100 (3) 4 (20) 53 (19) 755 (11) Colorectal cancer 269 (2.3) 150 (17) 713 (18) 70 (8) 466 (9) 609 (13) Lung cancer 266 (2.2) 196 (15) 706 (20) 181 (3) 1331 (3) 1384 (7) Sexually transmitted diseases 264 (2.2) Not available Not available 0 2 (27) 65 (26) Parkinson's disease 208 (1.7) 90 (20) 1025 (17) 21 (13) 66 (18) 263 (19) Tuberculosis 150 (1.3) 11 (27) 11 (28) 1 (24) 7 (24) 10 (29) Multiple sclerosis 110 (0.9) 9 (28) 176 (25) 4 (20) 45 (20) 118 (24) Epilepsy 103 (0.9) 207 (14) 1677 (12) 2 (23) 31 (22) 160 (22) Ovarian cancer 102 (0.9) 24 (26) 143 (26) 17 (14) 140 (15) 161 (21) Cervical cancer 97 (0.8) 29 (24) 225 (24) 7 (18) 86 (17) 125 (23) Chronic obstructive pulmonary disorder 67 (0.6) 429 (11) 6923 (6) 132 (5) 644 (5) 1647 (5) Uterine cancer 28 (0.2) 25 (25) 301 (22) 7 (18) 45 (20) 84 (25) Peptic ulcer disease 17 (0.1) 192 (16) 712 (19) 4 (20) 24 (23) 40 (27) Otitis media 17 (0.1) 17679 (2) 1360 (14) 0 1 (29) 35 (28) Funding rates by disease were obtained from the NIH for 2006. Estimates of incidence, prevalence, mortality, disability-adjusted life-years lost, and years-of-life-lost are total annual counts, in 1000s, for North America, obtained from the 2004 update of the World Health Organization's Global Burden of Disease project. Data denoting disease burden were collected from multiple sources ( Table 1 ). First, world and North American disease-specific data were obtained from the Global Burden of Disease (GBD) Project the World Health Organization (WHO) [12] , [13] , [14] . We used GBD's North American data for US estimates. The GBD systematically collects timely disease-specific epidemiologic information and models missing data to estimate a variety of measures of burden [15] , [16] . We used 2004 GBD estimates because they were likely to be the most recent publicly available data at the time decisions were made for the 2006 funding cycle. No other centralized, systematic source of broad national disease-specific burden estimates was identified. We evaluated five disease burden categories from the GBD: incidence, prevalence, mortality, YLLs, and DALYs [14] . YLLs for each disease were calculated by totaling the differences between life expectancy and age at death. DALYs were estimated based on YLL and on standardized weighting schemes for disability applied to those surviving with disease [6] , [7] . GBD estimates for the US were based on analyses of comprehensive death certificate data, national incidence estimates, and a systematic review of published epidemiological studies. To evaluate other estimates of disease burden, we used 2004 US inpatient and outpatient healthcare databases categorized by diagnosis, each of which includes large samples with weights to generate nationally representative estimates (Supporting Table S1 ). The number of hospital discharges, total length of stay, and mean hospital charges for disease-specific principal diagnoses were derived from the National Inpatient Sample (NIS) [17] . The number of visits to emergency departments and outpatient hospital clinics were derived from the National Hospital Ambulatory Care Survey (NHAMCS) and the National Ambulatory Care Survey (NAMCS) [18] . In both NIS and NHAMCS, community hospitals were defined as non-federal, short-stay hospitals [19] , [20] . Public interest could also influence funding levels through lobbying efforts, additional funding support from private foundations, or by directly stimulating the interest of investigators, and this could influence funding levels. To begin to assess the influence of public interest, we determined the number of disease-specific newspaper articles published in the top 10 US newspapers with highest distribution, as well as broadcast television news reports from national networks, using disease-specific keyword searches of LexisNexis News [21] and Vanderbilt Television News Archive [22] . To estimate the influence of specific interest group advocacy, the total US disease-specific charity revenue was similarly calculated for public charities receiving more than $500,000 in public support [23] . Scientific productivity in a given area could stimulate further interest from researchers and the NIH, and could result in targeted funding. As surrogate measures of scientific productivity, we tallied the number of disease-specific patents submitted [24] and articles published and listed in PubMed [25] in the 10 medical journals with the highest impact factor scores using key word searches [26] . Analysis The relationship between 2006 NIH funding levels and 2004 US disease burden metrics was designated the a priori primary analysis. All predictor and outcome variables were log-transformed to reduce positive skew. Univariate linear regression was first performed to replicate the prior study design [5] . This approach was preferred to correlation to allow for adjustment and to acknowledge that funding level was the dependent variable to be predicted. Expected funding levels in 2006 were calculated with correction for the log-transformation by applying measures of disease burden to fitted models predicting funding [27] , [28] . To account for inflation, the actual and expected funding levels for 1996 were inflated to 2006 dollars [29] . Standard, forward stepwise multivariable linear regression analysis included all variables of disease burden, including those with p<0.05 in the final model. We did not evaluate interactions. F-statistics were used to estimate differences in explanatory power (significant if p<0.05). A separate forward stepwise multivariable model was constrained so that diseases with no burden would receive zero funding, mimicking a theoretical funding process that determines allocations proportional to the disease burden. For this model, we defined the dependent variable as the ratio of dollars to DALYs for each of the included conditions. Separate multivariable models included measures of public interest and scientific productivity. In additional sensitivity analyses, we evaluated the association of funding levels with worldwide disease burden in 2004, and with projections for 2015 and 2030 for both the US and worldwide [30] . A final analysis evaluated whether a new NIH accounting method for disease-specific funding introduced in 2007 produced different results. Explanatory power of the models was estimated with adjusted R 2 values; an adjusted R 2 value was also determined for the analysis of 1996 funding levels using data derived from this prior publication. The Stata statistical package (Version 10, College Station, Texas) was used for all analyses. Results In 2006, the total NIH budget was $28.5 billion, with $11.9 billion devoted to the 29 included conditions. Disease funding ranged from $17 million (M) for peptic ulcer disease and otitis media, to $2902 M for AIDS, with a median of $335 M (±standard deviation $537 M; Table 1 ). Other metrics from the GBD ( Table 1 ); US inpatient, emergency room, and outpatient (Supporting Table S1 ); and public interest and scientific opportunity (Supporting Table S2 ) varied by disease. In the univariate analysis, NIH funding was most strongly associated with burden of disease measured in DALYs (p = 0.001; Table 2 ). YLLs (p = 0.03), inpatient hospital discharges (p = 0.05), and total hospital days (p = 0.02) were also associated with funding levels. 10.1371/journal.pone.0016837.t002 Table 2 Univariate Predictors of NIH Disease-Specific Funding in Fiscal Year 2006. Predictor Predicted change in funding associated with a 2-fold increase in the predictor * Relative Increase 95% CI P -Value Adjusted R-squared Value Disease Burden Measure Incidence 1.05 (0.91–1.20) 0.51 −0.02 Prevalence 1.15 (0.97–1.36) 0.10 0.06 Mortality 1.12 (0.98–1.27) 0.08 0.08 Disability-adjusted life-years 1.37 (1.16–1.63) 0.001 0.33 Years-of-life-lost 1.15 (1.01–1.31) 0.03 0.12 Number of hospital discharges 1.21 (1.00–1.46) 0.05 0.11 Total hospital days 1.24 (1.03–1.48) 0.02 0.15 Average hospital charges 1.49 (0.76–2.93) 0.23 0.02 Outpatient physician visits 1.11 (0.90–1.37) 0.30 0.004 Outpatient hospital and emergency room visits 1.10 (0.92–1.31) 0.27 0.01 Public Interest Measure Newspaper articles 1.13 (1.02–1.25) 0.02 0.14 Television news broadcasts 1.19 (1.04–1.36) 0.02 0.17 Charity revenue 1.07 (1.02–1.11) 0.004 0.24 *Predictors and outcome are log-transformed to reduce positive skew. In standard multivariable analysis, DALYs was the only significant predictor of NIH funding level retained in the final model, so the analysis became univariate. In 2006, the degree of correlation between NIH funding and disease burden as measured by DALYs alone was less than in 1996: Only 33% of NIH funding variance was explained in 2006 compared to 39% in 1996. Differences between actual and expected funding based on burden of disease in DALYs were estimated for 2006 and compared to 1996 funding levels ( Table 3 ; Figure 1 ). Depression received the least funding compared to expected, and AIDS the most, consistent with findings from 1996. Relative to expected funding, AIDS, diabetes, and perinatal conditions were the three diseases with the largest amounts of funding, while depression, injuries, and COPD received the least funding ( Table 3 ). The largest positive 10-year gains in actual NIH funding relative to expected were AIDS (+$809 M), perinatal conditions (+$420 M), and diabetes (+$193 M); by contrast, injuries (−$578 M), depression (−$541 M), chronic obstructive pulmonary disease (−$512 M), and ischemic heart disease (−$459 M) decreased most sharply ( Figure 1 ). 10.1371/journal.pone.0016837.g001 Figure 1 Ten-year Comparison of Differences Between Actual and Expected Disease-Specific NIH Funding Relative to US Burden of Disease in DALYs. A comparison of differences between actual and expected funding values as predicted by DALYs burden alone in 1996 ( light blue ) and 2006 ( navy ). Negative values reflect actual funding dollars less than expected and positive values represent actual funding dollars more than expected. 10.1371/journal.pone.0016837.t003 Table 3 Ranked Differences between Expected and Actual NIH Funding According to Year of Funding and United States Disease Burden Measure(s) Used. Millions of Dollars (ascending rank * ) 1996 2006 Univariate †Standard Univariate ‡ Standard Multivariable with Public Interest Variables § Constrained Multivariable ¶ Condition or Disease Depression −178 (1) −719 (1) −689 (2) −951 (2) Perinatal Conditions −124 (2) 297 (27) 114 (23) −194 (11) Stroke −121 (3) −278 (6) −288 (5) −170 (13) Injuries −113 (4) −691 (2) −123 (12) −721 (3) Chronic obstructive pulmonary disorder −101(5) −613 (3) −357(3) −554(5) Pneumonia −52(6) 27 (21) 154 (27) 270 (27) Peptic ulcer disease −46(7) −105 (14) −75 (14) −18 (19) Lung cancer −46(8) −364 (5) −328 (4) −1358 (1) Schizophrenia −37(9) −44 (18) 129 (24) 94 (24) Ischemic heart disease −30(10) −490 (4) −731 (1) −721 (4) Uterine cancer −29(11) −146 (10) −94 (13) −111 (15) Asthma −25(12) −198 (8) −235 (6) 40 (21) Otitis media −19(13) −97 (15) −70 (15) −27 (18) Colorectal cancer −16(14) −168 (9) 21 (19) −519 (7) Ovarian cancer −15(15) −135 (11) −196 (9) −337 (9) Epilepsy −10(16) −133 (12) −226 (7) −101 (16) Parkinson's disease 23(17) −90 (17) −183 (10) −363 (8) Cervical cancer 28(18) −113 (13) −44 (17) −223 (10) Prostate cancer 32 (19) 56 (22) −3 (18) 68 (22) Tuberculosis 44 (20) 89 (23) 96 (22) 97 (25) Multiple sclerosis 52 (21) −95 (16) −222 (8) −96 (17) Sexually transmitted disease 58 (22) 110 (24) 153 (26) 198 (26) Alcohol abuse 61 (23) −202 (7) 152 (25) −152 (14) Cirrhosis 67 (24) 25 (20) −55 (16) −178 (12) Dental and oral disorders 130 (25) 113 (25) 74 (20) 90 (23) Dementia 183 (26) 18 (19) −159 (11) −524 (6) Diabetes mellitus 197(27) 390 (28) 160 (28) 382 (28) Breast cancer 346 (28) 258 (26) 92 (21) 39 (20) AIDS 1664 (29) 2474 (29) 2306 (29) 1835 (29) *Ascending rank, from most underfunded disease condition (indicated by negative numbers) to most overfunded (positive numbers) as predicted by each model. †Univariate linear regression of the association between 2004 disease-specific Disability-Adjusted Life-Years (DALYs) and the outcome NIH dollars. Differences between expected and actual funding levels in 1996 are adjusted for inflation to 2006 dollar equivalents, but are otherwise unchanged from those reported by Gross et al . ‡ Standard univariate linear regression of the outcome NIH dollars as predicted by disease-specific DALYs. A stepwise forward multivariable model retained only DALYs as a predictor. § Standard multivariable linear regression of the outcome NIH dollars as predicted by disease-specific DALYs and charity revenue. ¶ The constrained model is the multivariable linear regression model where the predicted NIH dollars are obligated to be proportional to disease-specific DALYs after adjustment for total number of hospital discharges and average hospital charges. In standard multivariable regression models including measures of public interest and scientific productivity, the total charity revenue for a given disease in 2006 (p = 0.04) was also predictive of funding in addition to DALYs (p = 0.006). A model including both variables explained 41% of the variation in NIH funding levels. In multivariable models constrained to require that diseases resulting in no burden of illness receive no NIH funding (equivalent to requiring an intercept of zero-zero in the regression line, expressed in the dashed line of Figure 2 ), expected funding amounts were generally similar to those found with the unconstrained multivariable model ( Table 3 ; Figure 3 ). 10.1371/journal.pone.0016837.g002 Figure 2 NIH Funding in 2006 and US Disease Burden in DALYs in 2004 for 29 Common Medical Conditions. The solid line represents the results of a traditional multivariable analysis, showing the relationship between US disease-specific DALYs burden and actual 2006 NIH funding dollars. The dashed line projects NIH funding levels in a similar multivariable model that requires that a disease with no burden receives no funding (constrained model). Though the models produce similar results, several diseases that would be considered overfunded in one model are considered underfunded in the other. For example, cervical cancer appears to be overfunded relative to the dashed line, while it is underfunded relative to the solid one. 10.1371/journal.pone.0016837.g003 Figure 3 Differences Between Actual and Expected Disease-Specific Funding in 2006. Determinations of actual funding relative to expected funding were generally similar among separate analytic models predicting funding levels from disease burden measures. Univariate results are based on DALYs alone ( navy ), the only variable retained in a stepwise forward multivariable model. A traditional multivariable model including public interest variables ( grey-blue ) retained only DALYs and total charity revenue in the model. A constrained multivariable ( light blue ) model required an intercept of zero-zero to impose a requirement that conditions with no burden received no funding and retained DALYs, total number of US hospital discharges, and mean charge per hospitalization in 2004. To determine if NIH funding might better correlate with world or future disease burden, we performed sensitivity analyses with global measures and future projections (2015 and 2030), all derived from the GBD project (Supporting Table S3 ). When restricted to global measures, mortality (p = 0.05) and DALYs (p = 0.001) were predictive of funding in univariate analyses (adjusted R 2 values 0.11 and 0.30), but only DALYs were retained in all the multivariable models of both global measures and future predictions. Correlation of funding with disease burden was not improved when data utilizing new NIH accounting methods was used (adjusted R 2 = 0.27) compared to prior methods (adjusted R 2 = 0.34) applied to 2007 data, the first year for which the new methods were available. Discussion In the 10 years since an initial assessment of the correlation of NIH funding with disease burden [5] and an IOM report recommending new NIH funding priority-setting criteria [4] , NIH funding is no better aligned with US disease burden. Furthermore, diseases that were previously funded more than expected–such as AIDS, breast cancer, and diabetes–continue to receive funding greater than predicted by burden of disease, while most conditions that were previously underfunded remain underfunded. Adding measures of disease burden to the model and constraining it to assure that diseases with no burden would receive no funding minimally affected the overall relationship between burden and funding. Neither global nor future disease burden were more closely related to NIH funding, and newly implemented NIH disease-specific accounting practices did not improve the correlation. Although the IOM report on NIH funding recommended ongoing assessment of alignment of NIH funding with disease burden, it also recognized other important criteria for setting funding priorities [4] . These criteria included research quality, scientific innovation and opportunity, portfolio diversification, and infrastructure building. Other experts have proposed a similar framework to guide funding decisions [31] . Additional factors to consider may include transmissibility or population risk, collateral benefits to disease control, and public interest. Given these other potential contributors to decision-making about disease funding, perfect alignment with DALYs or any other measure of disease burden would not be expected. It is unclear why particular conditions remain under- or overfunded relative to disease burden. The difficulty of attributing basic science research—a large portion of the NIH portfolio—to individual diseases complicates implementation of any disease-based allocation process [32] ; however, some discrepancies are particularly dramatic. Spending for AIDS research, the disease with greatest funding compared to expected, may be justified due to the potential threat associated with its spread, to past successes in treating and even eliminating other infectious diseases, and to a greater burden in lower income countries. However, AIDS funding remained greater than predicted even when worldwide and projected burden were considered, and strong political influences may be important in maintaining high levels of funding in the US. Also, congressionally-mandated research support for rare illnesses may explain greater funding for some diseases with little burden [33] , [34] , [35] . Conditions typically associated with substance use or mental health diagnoses tended to be underfunded (e.g., lung cancer, chronic obstructive pulmonary disease, alcoholism, and depression). Charity revenue, used as a proxy for disease-specific interest-group advocacy, was associated with funding levels and may have contributed through lobbying efforts or by providing collateral support for research and training to encourage NIH submissions in specific disease areas. The availability of proven cost-effective interventions (e.g., tobacco-related prevention strategies) [36] may also influence funding since development of new interventions may be unnecessary when effective strategies have already been identified [37] . Over the 10-year interval, funding for several conditions notably increased compared to expected. For example, the National Institute of Allergy and Infectious Diseases initiated a concerted response to bioterrorism following the terrorist attacks on Sept. 11, 2001, anthrax incidents, and severe acute respiratory syndrome (SARS) outbreak in 2002, and this may have augmented support for pneumonia-related research [38] . Conversely, the relative funding increase for perinatal conditions appears primarily due to a 55% reduction in associated DALYs over the last decade since funding increases over the same period paralleled overall growth in the NIH budget [39] . Finally, an increase in relative funding for diabetes research may have been precipitated by Congressional actions in 1997 requiring development of a comprehensive diabetes research plan and allocating $150 million to a new funding program for Type I Diabetes Research [40] . Lack of improvement in alignment between funding and disease burden may not indicate neglect of the 1998 IOM recommendations by the NIH; there are several other possible explanations. First, basic science research has consistently accounted for 55% of NIH spending and it is difficult to credit specific disease for much of this research, contributing uncertainty to the analysis and reducing correlations between funding levels and disease burden. Second, NIH funds committed at the time of the 1998 IOM publication could not be redistributed until their associated projects were completed, sometimes five or more years later; as a result, reallocations would be delayed for several years. Third, the distribution of funding among NIH Institutes is determined by Congress and incorporates input from NIH itself, scientists, health care providers, and special interest groups. Thus, fiscal and political constraints likely additionally tempered the NIH's ability to implement the IOM recommendations. Finally, without regard to overall disease funding alignment, substantial financing ($1.8 billion, 6.3% 2006 NIH budget) was invested in the creation of three new centers (National Institution for Biomedical Imaging and Bioengineering, Center for Complementary and Alternative Medicine, and National Center for Minority Health and Health Disparities) [41] , establishment of the cross-cutting Roadmap Initiatives [42] , and expanded emphasis on career training awards [43] . The NIH has recently taken steps to integrate more effectively the IOM priority-setting criteria. In 2007, the Division of Program Coordination, Planning, and Strategic Initiatives (DPCPSI) was established for the purposes of identifying scientific opportunities, public health challenges, and scientific knowledge gaps, and to improve portfolio analysis and priority-setting [44] . In 2009, a more consistent and transparent system for the tracking of disease-based funding was launched. Also, as part of the NIH's $10.4 billion allocation in the American Recovery & Reinvestment Act, $400 million will be dedicated to comparative effectiveness research that specifically evaluates the effects of clinical management on comprehensive public health outcomes such as mortality, morbidity, and quality of life [45] . Our study has several limitations. First, the accounting of disease funding in 2006 by NIH is not standardized nor is the reliability known [32] . However, no alternative source of information is publicly available and historical and new accounting methods yielded similarly poor correlations with burden. Second, we did not evaluate other sources of federal, nonprofit, and industry funding. Conditions well-funded by organizations other than the NIH may justify a corresponding decrease in NIH funding [46] . However, other sources of federal funding–The Centers for Disease Control and Prevention, Agency for Healthcare Research and Quality, and the Food and Drug Administration–distributed only 3.3% of all Department of Health and Human Services dollars dedicated to life sciences research in 2006. Similarly, the total life science research dollars spent by the Department of Defense and Department of Veterans Affairs was equivalent to less than 7% of the total amount disbursed by the NIH [47] , [48] . Thus, the total dollars spent on complementary projects by other federal institutions does not fully explain the poor correlation between disease burden and NIH funding. Although biomedical and pharmaceutical industry research funding in the United States was 1.6 times the amount allocated by the NIH in 2006, fully 49% was dedicated to supporting clinical trials less likely to have a widespread public health benefit [49] . Unfortunately, funding by disease for these sectors is not available, except for limited therapeutic areas [50] , and no such figures exist for private foundations. Third, global estimates of disease burden from the WHO GBD are uncertain due to incompleteness and bias, particularly in low-income countries [51] . However, these estimates are the best available and are particularly accurate for the US. Fourth, to permit comparisons to the prior study and to make the study feasible, many diseases and conditions funded by the NIH were omitted from our analysis. Still, an assessment of the responsive of NIH to prior recommendation was central to our study and power would not be expected to dramatically impact our findings. Fifth, the prior analysis utilized estimates for world market economies [5] , while we used North American numbers. The data we used better approximates US burdens, strengthening our conclusions, but could affect the assessment of interval change. Since we found a poorer correlation with funding than previous estimates, this did not impact our conclusions. Finally, none of the measures of disease burden individually or collectively fully captures the health and economic cost of these conditions. A better metric might capture the true societal cost of disease through a comprehensive assessment of total healthcare costs and a valuation of both deaths and DALYs [52] . Overall, funding levels today are less well aligned with DALYs compared to 10 years ago, suggesting that the IOM's 1998 priority-setting recommendations have not been implemented effectively. Accounting for other measures of disease burden did not substantially improve alignment. As a recipient of substantial governmental support, clear articulation of the rationale for NIH spending may be expected by Congress and the public, and a lack of clear alignment with measures of public burden could encourage special interests to further erode the scientific independence of NIH or to raise questions about its management. The use of more consistent accounting methods for disease funding, more comprehensive measures of burden and future risk that include impact on health and expenditures, and the timely dissemination of benchmarks on the alignment of disease burden to funding could help to make NIH funding priorities more rationale and transparent. Supporting Information Table S1 Additional Measures of Disease Burden for 29 Conditions. (DOC) Click here for additional data file. Table S2 Public Interest and Other Measures for 29 Conditions. (DOC) Click here for additional data file. Table S3 World and Future Disability-Adjusted Life-Years as Predictors of NIH Disease-Specific Funding in Fiscal Year 2006. (DOC) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122909/

Surveillance and Control of Communicable Disease in Conflicts and Disasters

Objectives To describe the principles of health surveillance in conflict and disaster situations To assist in organizing a health surveillance system in conflict and disaster situations To describe the principles of control of communicable diseases in conflict and disaster situations To assist in organizing a response to outbreaks and epidemics To introduce the challenges associated with health surveillance and communicable diseases in conflict and disaster situations A - Introduction There are five fundamental principles for the control of communicable disease in emergencies: Rapid assessment – identify and quantify the main disease threats to the population and determine the population's health status Prevention – provision of basic health care, shelter, food, water, and sanitation Surveillance – monitor disease trends and detect outbreaks Outbreak control – control outbreaks of disease. Involves proper preparedness and rapid response (confirmation, investigation, implementation of controls) Disease management – prompt diagnosis and effective treatment Rapid assessment has been dealt with elsewhere in this book as have the prevention aspects of disease control (adequate shelter, clean water, sanitation, and food, together with basic individual health care). This chapter therefore covers surveillance, outbreak/epidemic control, and public health aspects of disease management. The topics are dealt with in general terms. More details can be found in references. Disasters and Disease Disasters, particularly conflicts, by damaging or destroying the infrastructures of societies (health, sanitation, food supply) and by causing displacement of populations, generally lead to increased rates of disease. Outbreaks and epidemics are not inevitable in these situations and are relatively rare after rapid-onset natural disasters, but there is a severe increase in the risk of epidemics during and after complex emergencies involving conflict, large-scale population displacement with many persons in camps and food shortages. In most wars more people die from illness than from trauma. Preventing such problems, or at least limiting their effects, falls on those responsible for the health care of the population affected by the emergency. They must be able to assess the health status of the population affected and identify the main health priorities monitor the development and determine the severity of any health emergency that develops (including monitoring the incidence of and case fatality rates from diseases, receiving early warning of epidemics and monitoring responses) plan and set up programs identify and take action to prevent or control outbreaks and epidemics monitor the progress of health interventions and their impact and modify them if required ensure the provision of appropriate aid (and prevent inappropriate aid) provide information for relevant agencies (e.g., national Ministry of Health (MOH), UN, NGOs, donors) for use in planning, funding applications, etc. At first sight, undertaking public health activities in emergencies, especially in conflicts, may seem to be difficult or impossible. The destructive nature of warfare may prevent or inhibit the provision of adequate food and shelter, of clean water and sanitation and vaccination programs. Despite the difficulties that warfare imposes, it is generally possible to undertake at least limited public health programs, including disease surveillance and control activities. In other types of disaster public health activities may be expected to be less affected by the security situation than in a war (although aid workers may be at risk if populations are severely deprived of resources such as food, shelter, water, or cash), and with limited access and damage to communication systems and other parts of the infrastructure assessment, surveillance and control activities can be severely restricted. For example, following the Pakistan earthquake late in 2005 access was severely restricted for some time and the urgent need to treat the injured and provide food and shelter meant that the limited transport available was heavily committed. Disasters and Disease Disasters, particularly conflicts, by damaging or destroying the infrastructures of societies (health, sanitation, food supply) and by causing displacement of populations, generally lead to increased rates of disease. Outbreaks and epidemics are not inevitable in these situations and are relatively rare after rapid-onset natural disasters, but there is a severe increase in the risk of epidemics during and after complex emergencies involving conflict, large-scale population displacement with many persons in camps and food shortages. In most wars more people die from illness than from trauma. Preventing such problems, or at least limiting their effects, falls on those responsible for the health care of the population affected by the emergency. They must be able to assess the health status of the population affected and identify the main health priorities monitor the development and determine the severity of any health emergency that develops (including monitoring the incidence of and case fatality rates from diseases, receiving early warning of epidemics and monitoring responses) plan and set up programs identify and take action to prevent or control outbreaks and epidemics monitor the progress of health interventions and their impact and modify them if required ensure the provision of appropriate aid (and prevent inappropriate aid) provide information for relevant agencies (e.g., national Ministry of Health (MOH), UN, NGOs, donors) for use in planning, funding applications, etc. At first sight, undertaking public health activities in emergencies, especially in conflicts, may seem to be difficult or impossible. The destructive nature of warfare may prevent or inhibit the provision of adequate food and shelter, of clean water and sanitation and vaccination programs. Despite the difficulties that warfare imposes, it is generally possible to undertake at least limited public health programs, including disease surveillance and control activities. In other types of disaster public health activities may be expected to be less affected by the security situation than in a war (although aid workers may be at risk if populations are severely deprived of resources such as food, shelter, water, or cash), and with limited access and damage to communication systems and other parts of the infrastructure assessment, surveillance and control activities can be severely restricted. For example, following the Pakistan earthquake late in 2005 access was severely restricted for some time and the urgent need to treat the injured and provide food and shelter meant that the limited transport available was heavily committed. B - Health Surveillance Features The surveillance and control of communicable disease require data which can be collected in one of three ways: Surveillance systems – covering all or at least a significant proportion of the population Surveys – in which data are collected from a small sample of the affected population considered to be representative of the whole Outbreak investigations – in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Although the latter two can provide valuable information for disease control and form part of the surveillance process, proper control of disease requires regular monitoring of the overall disease situation, which in turn requires the establishment of a properly designed health surveillance system. It is important therefore that responsibility for surveillance activities is defined at the beginning of planning for an aid mission. Generally speaking, a team will be required, including a team leader (often an aid agency health coordinator), who should ideally have surveillance experience, clinical workers, a water and sanitation specialist, and representatives of the local health services and communities. The team may also need clerical, logistic, information technology and communications specialists. The World Health Organization defines health surveillance as "the ongoing systematic collection, analysis and interpretation of data in order to plan, implement and evaluate public health interventions." Data for surveillance must be accurate, timely, relevant, representative, and easily analyzed, and the results must be disseminated in a timely manner to all who need to receive them. In addition the data collected, the methods used for collection and the output must be acceptable to those surveyed (health-care professionals and the population). In emergencies the time that can be given to surveillance by medical personnel is likely to be limited and surveillance activities will be far from the minds of most of those involved. Therefore the methods used need to be rapid, practical, and consistent, and while the greatest possible accuracy must be achieved, "the best must not be the enemy of the good." It is necessary to strike a balance between collecting large amounts of information ("what we would like to know") and collecting too little which can lead to an ineffective response. Those responsible for establishing surveillance programs must therefore try to determine what is really needed ("what we need to know"). It is better to err on the side of too much than of too little. Ideally any existing surveillance system should be used. There is no point in establishing a system if one already exists, unless the existing one is inadequate or inappropriate or has broken down irretrievably. Surveillance systems for use in conflict and disaster situations should therefore adhere as far as possible to the criteria given in Table 13.1 . Table 13.1. Criteria for surveillance systems 1 As simple and flexible as possible 2 Appropriate in terms of the information required 3 Capable of providing such information in a timely manner 4 Appropriate in terms of the resources available 5 Sustainable in the long term within local resources 6 Based on standardized sampling methods 7 Based on agreed case definitions 8 Capable of providing regular information from defined sites 9 Capable of covering the whole affected area 10 Compatible with existing systems 11 Use existing systems as far as possible 12 Use existing records as far as possible 13 Involve collaboration between agencies so as to avoid duplication 14 Involve collaboration with local services so as to avoid duplication 15 Acceptable to those surveyed Notes on these criteria: As simple and flexible as possible Complexity and inflexibility are incompatible with surveillance systems generally and particularly when operating in emergencies where collection of data may be difficult and where situations can change very fast. Appropriate in terms of the information required Defining what you "need to know" will allow you to set up the appropriate data collection methods (questionnaires, sites, etc.) and to design the system so that it can obtain and handle the information required. Capable of providing such information in a timely manner Information that is accurate but out of date is useless for immediate disease control purposes and of little value for forward planning. Communications therefore form an integral part of any surveillance system. Appropriate in terms of the resources available Do not try to overreach when setting up a system. For example, expatriate staff may best be used to recruit local staff for the system and in supervisory activities rather than in collecting data. Sustainable in the long term within local resources This criterion is certainly a goal to aim for as sustainability must be the target for all aid work. However, there may be situations where an emergency system is needed rapidly and where it cannot readily be integrated into existing systems or be developed as a new long-term system. Based on standardized sampling methods The sampling system must use the same data collection methods throughout if data are to be comparable. Ideally this should be methods that are internationally agreed and approved. Agreement should be sought for the methods from the other agencies on the ground to ensure consistency. Based on agreed case definitions Without case definitions that are agreed by all parties the likelihood of success of a surveillance system is very low. This is especially so when laboratory support is minimal or absent since clinical case definitions have to be drawn very tightly if different diseases are not to be confused. Capable of providing regular information from defined sites Routine surveillance requires more than material from ad hoc sources. Sites such as medical centers (in towns, villages, or refugee camps), hospitals, and/or public health units should be recruited. Capable of covering the whole affected area The more comprehensive the coverage of the system, the more likely is it that the data will be accurate and complete and that problems will not be missed. Such coverage can be problematic. The coverage of the different systems that can be used is discussed below. Compatible with existing systems The data collected and the methods used should ideally fit in with systems that are operating or have previously operated in the area. Use existing systems as far as possible Following from Criterion 10, if systems are already in existence or in abeyance but revivable then this should be done so as to ensure compliance by local health-care services and continuity of data collection and analysis. Use existing records as far as possible Existing records are of considerable value for predictive purposes. Knowledge of past problems makes it possible to anticipate future trends and problems and allows for early planning decisions. Involve collaboration between agencies so as to avoid duplication If several health agencies are operating it is essential to ensure collaboration among them in surveillance activities to avoid confusion and duplication of effort. Involve collaboration with local services so as to avoid duplication As above, early involvement of local health and surveillance services will reduce workloads and avoid duplication of effort. Acceptable to those surveyed If those from whom the data are collected, those who are collecting the data, and those who will receive the results are unhappy with the system, the system is unlikely to operate effectively. These criteria can be used to evaluate a plan for a surveillance system and also, with some additions, to evaluate an existing system. However, failure to fulfil all these criteria need not rule out a system. In many emergencies it can be difficult to meet such a wide range of "best case" criteria, and the question that must be asked is whether the proposed system is capable of fulfilling its purpose – can it provide sufficiently accurate essential information to those who need it when they need it ? The emphasis of an emergency surveillance program may need to be altered as the situation changes especially if a particular item emerges as being of key importance. Those running the surveillance program should use the data gathered and a continuous assessment of the general running of the system, to alter the program as required (preferably after consultation with relevant stakeholders). Designing Health Surveillance Systems When designing health surveillance systems, it is essential to do the following: Define the population under surveillance Determine what type of system can be established Set surveillance priorities Identify sources of data Set up agreed case definitions Establish data-handling systems Establish a protocol for evaluating the surveillance system as a whole Each of these is examined in more detail. Population Under Surveillance The population under surveillance may be relatively small and well defined (such as the population of a refugee camp) or a much less defined group such as mobile groups of refugees or IDPs or the population of a village, town, or region, the size of whose population may be unknown or may be fluctuating because of a disaster. Establishment of denominators may therefore be difficult. Even refugees or IDP camps may present a challenge as, while the size of the population may appear to be (or actually be) stable, its makeup may vary over time because of movements in and out. If the age or sex makeup of the camp alters, the pattern of disease may also alter. Demography: Numbers vs. Rates Both the number of cases detected and the rate of factors such as morbidity or mortality per unit of population are important values needed to inform emergency programs. Those responsible for all aspects of health care need to know what numbers of cases are involved so as to ensure adequate provision of services (amounts of medicines, numbers of hospital beds, etc.). However, simple numbers are of little value in assessing trends and patterns since increases or decreases in numbers of cases (or numbers of deaths) may reflect changes in population size (resulting, for example, from population displacement) rather than a trend due to (for example) a particular disease. In addition, several rates (such as the crude mortality rate) are key indicators in defining health emergencies (see below). Knowing the demography of the affected population is therefore important and all agencies working in an emergency should agree on and use the same population figures. The essential demographic data needed include the following: Total population size Population structure Overall sex ratio and the sex ratio in defined age groups Population under 5 years old, with age breakdown (0–4 years) – this group has special needs and is usually a key factor in planning the emergency response Age pyramid Ethnic composition and place of origin Number of vulnerable persons (e.g., pregnant and lactating women, members of female-headed households, unaccompanied children, destitute elderly, disabled and wounded persons) Average family/household size In situations where populations are displaced and extensive population movements may be occurring, it is also necessary to know the following: The number of arrivals and departures per week The predicted number of future arrivals at the sampling sites At the outset it is therefore important to establish methods to obtain demographic data. Often the best that can be managed initially is a rough estimate, but this can usually be refined later. It is helpful to use several methods and cross-check the figures to obtain the best estimate. Surrogates of the whole population (such as those attending a clinic) may be the best that can be achieved early on. The ease with which such data can be obtained usually depends on the size and scale of the population under consideration. The demography of a well-run refugee camp is quite easy to obtain but that of a larger area may be much more difficult. A lack of knowledge of the size of a displaced group can be confounded by a lack of knowledge of the size of the resident population. In many countries with poor infrastructures, accurate census data are not available. In some instances tax records may be helpful if these can be obtained. It should be noted that demographic data, especially if they involve refugees and IDPs, can be politically sensitive and interested parties may place undue weight on any figures that are given. Types of Surveillance System Comprehensive Systems Ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. A surveillance system in a refugee or IDP camp is effectively a miniature comprehensive system as it is possible to cover the whole population. Sentinel Surveillance Systems There are situations where comprehensive surveillance is not possible and these often arise in disasters. Damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. As far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. Sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. The calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. Both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. If there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. Setting Surveillance Priorities Surveillance must provide information on key health indicators, which should include the following: Morbidity Mortality Nutritional status Immunization status Vital needs Health sector activities, including local health services Activities in related sectors The selection of information sought in these categories must be done carefully. It is neither possible nor desirable to monitor everything, especially in the early stages of a disaster response. At that stage (the acute phase) the priority of surveillance is the detection of factors that can have the greatest and most rapid effect on the population. In terms of communicable disease this means diseases that affect large numbers of people and have epidemic potential. In most instances this also means diseases for which effective rapid control measures exist. While gathering data on other large-scale disease problems should not be excluded, the main surveillance and control efforts should be aimed where they can do the most immediate good. In the very early stages, only clinical information may be available since laboratory diagnostic services will probably be damaged or simply unavailable. However, this need not be a problem if the medical response is also geared to a syndromic approach. As the situation stabilizes, laboratory support becomes available, and longer term control measures can be supported, the surveillance can become more refined and additional diseases (for example, those which can cause severe morbidity and mortality in the longer term – such as tuberculosis, HIV or AIDS, and STDs) can be added to the list. Morbidity The main morbidity figures that are routinely sought are as follows: Incidence – the number of new cases of a particular disease reported over a defined period Attack rate (used in outbreaks – usually expressed as percentage) (also called incidence proportion or cumulative incidence ) – number of new cases within a specified time period/size of the population initially at risk (×100). (e.g., if 30 per 1,000 persons develop a condition over 2 weeks, the AR/IP/CI is 30/1,000 [3.0%]) Incidence rate – number of new cases per unit of person-time at risk. In the above example, the IR is 15/1,000 person-weeks. (This statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) Prevalence – the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (NB: Prevalence " rate " is the number of cases of a disease at a particular time/population at risk) There are a number of ways of estimating morbidity. Health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. Other ways of gathering morbidity data include the following: Surveys – in which data are collected from a small sample of the emergency-affected population deemed to be representative of the whole (or from a particular group for a specific purpose) Outbreak investigations – which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Mortality As with disease, changes in numbers of deaths may reflect changes in population size. Determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. Often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. All deaths occurring in the community must therefore be recorded. The following indicators can provide the essential information to define the health situation in a population: Crude mortality rate (CMR) is the most important indicator as it indicates the severity of the problem, and changes in CMR show how a medical emergency is developing. CMR is usually expressed as number of deaths per 10,000 persons per day. If the CMR rises above 1/10,000 per day (>2/10,000 per day for young children) an acute emergency is developing and the emergency phase lasts until the daily CMR falls to 1/10,000 per day or below. Age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). In children this is usually given as the number of deaths in children younger and older than 5 years/1,000 children of each age/day). NB: If population data for the under 5s are not available, an estimate of 17% of the total population may be used. Maternal mortality rate . Maternal mortality is a sensitive indicator of the effectiveness of health-care systems. A maternal death is usually defined as the death of a woman while pregnant or within 42 days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. The 42-day cut-off is recommended by WHO but some authorities use a time of up to a year. Maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × 1,000 (or ×100,000) Cause-specific death rates (case fatality rates – usually given as a percentage) . Proportion of cases of a specified condition which are fatal within a specified time. Case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × 100 Nutritional Status The following indicators must be measured: Prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) Prevalence of severe acute malnutrition in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) Prevalence of micronutrient deficiencies Estimate number of children needing to be cared for in selective feeding programs Estimate number of additional calories per day provided by selective feeding programs Immunization Immunization programs are a vital part of the public health measures undertaken following disasters. For example, measles vaccination is one of the most important health activities in such situations. The need for campaigns may be assessed on the basis of national vaccination records if they exist. In the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). The effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. In less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. Vital Needs Items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. Depending on the circumstances it may be necessary to monitor these elements in the affected population. Health Service Activities Indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. Other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. Activities in Related Sectors Activities in related sectors such as water and sanitation, shelter and security may also be included. Sources of Data The major sources of health data will be hospitals and clinics (both national and those established by aid agencies), individual medical practitioners, and other health-care workers. Specialized agencies should be able to provide data on particular needs (e.g., food, water, sanitation, and shelter). Case Definitions Case definitions are an essential part of surveillance. If the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless – changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. This is especially important when laboratory confirmation is not possible. It is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. Case definitions must be prepared for each health event or disease or syndrome. If available, the case definitions used by the host country's MOH should be used to ensure continuity of data. Several different sets of case definitions already exist, either in generalized form (for example, those produced by the Centers for Disease Control in Atlanta) or sets prepared for specific emergencies (e.g., the WHO Communicable Disease Toolkit for the Iraq Crisis in 2003). Standard case definitions may have to be adapted according to the local situation. It should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. When case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see Table 13.2 ). Table 13.2. Types of cases Type of case Criteria Suspected case Clinical signs and symptoms compatible with the disease in question but no laboratory evidence of infection (not available, negative, or pending) Probable case Clinical signs and symptoms compatible with the disease in question and also epidemiological evidence (e.g., contact with a known case) or some laboratory evidence (e.g., the results of a screening test) for the relevant disease Confirmed case Definite laboratory evidence of current or recent infection, whether or not clinical signs or symptoms are or have been present Although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. Samples can subsequently be sent to a referral laboratory for confirmation. Once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. Establish Data-Handling Systems The following issues should be considered: Methods of recording and transferring data Methods of verifying data Frequency of reporting Who will analyze the data and how often Methods for disseminating results Recording and Transferring Data Visits to surveillance sites and discussions with staff involved will help define the recording and data transmission systems required. The great advances in information technology that have been made in recent years have greatly facilitated the collection, recording, transmission, and analysis of surveillance data, but care must be taken that the systems put in place are appropriate. In areas where electricity supplies are problematical and communications poor it may be better to use a paper recording system and verbal data transmission by radio than a computerized system. Verification Data verification is essential for the credibility of a surveillance system. Those responsible for surveillance systems must ensure good adherence to case definitions if a symptom-based system is in operation and that laboratory quality control systems operate where appropriate. Regular assessments of record keeping and the accuracy of data transfer are required. Triangulation of results from several sources can sometimes help to detect anomalies. Frequency of Reporting Frequency of reporting will usually depend on the severity of the health situation. In general, daily reporting during the acute phase of an emergency will be needed, although in an acute medical emergency (such as a severe cholera outbreak) even more frequent reporting may be necessary, especially if the situation is fluctuating rapidly. The frequency may reduce to (say) weekly as the situation resolves. Data Analysis Who is to analyze the data and how it is to be analyzed must be established at the outset. In a relatively defined area such as a camp, a data analysis session may be the last of the daily activities of the person responsible for surveillance. If record keeping and analysis protocols have been carefully worked out initially this task is not necessarily a large additional burden. Surveillance systems that cover larger areas and bigger and more diffuse populations usually rely on a central data collection point where designated staff analyze the data. Use of such a system requires good data transmission systems. Output of Surveillance Systems Output is as important as input. Collecting data without dissemination of results is a sterile exercise and tends rapidly to demotivate those who are collecting the data. There are some important points to consider: The results of surveillance must be presented in a readily comprehensible form. Surveillance reports should be produced regularly and widely distributed to aid agencies, and to national and international governments and organizations. This will help those involved to understand the overall picture, rather than just that in the area where they are working, and will allow them to take informed decisions about future actions. Evaluation of Surveillance Systems Surveillance systems should be evaluated constantly to ensure that they are working properly, that the data are representative, analysis is appropriate and accurate, and that results are being disseminated to where they are needed. Features The surveillance and control of communicable disease require data which can be collected in one of three ways: Surveillance systems – covering all or at least a significant proportion of the population Surveys – in which data are collected from a small sample of the affected population considered to be representative of the whole Outbreak investigations – in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Although the latter two can provide valuable information for disease control and form part of the surveillance process, proper control of disease requires regular monitoring of the overall disease situation, which in turn requires the establishment of a properly designed health surveillance system. It is important therefore that responsibility for surveillance activities is defined at the beginning of planning for an aid mission. Generally speaking, a team will be required, including a team leader (often an aid agency health coordinator), who should ideally have surveillance experience, clinical workers, a water and sanitation specialist, and representatives of the local health services and communities. The team may also need clerical, logistic, information technology and communications specialists. The World Health Organization defines health surveillance as "the ongoing systematic collection, analysis and interpretation of data in order to plan, implement and evaluate public health interventions." Data for surveillance must be accurate, timely, relevant, representative, and easily analyzed, and the results must be disseminated in a timely manner to all who need to receive them. In addition the data collected, the methods used for collection and the output must be acceptable to those surveyed (health-care professionals and the population). In emergencies the time that can be given to surveillance by medical personnel is likely to be limited and surveillance activities will be far from the minds of most of those involved. Therefore the methods used need to be rapid, practical, and consistent, and while the greatest possible accuracy must be achieved, "the best must not be the enemy of the good." It is necessary to strike a balance between collecting large amounts of information ("what we would like to know") and collecting too little which can lead to an ineffective response. Those responsible for establishing surveillance programs must therefore try to determine what is really needed ("what we need to know"). It is better to err on the side of too much than of too little. Ideally any existing surveillance system should be used. There is no point in establishing a system if one already exists, unless the existing one is inadequate or inappropriate or has broken down irretrievably. Surveillance systems for use in conflict and disaster situations should therefore adhere as far as possible to the criteria given in Table 13.1 . Table 13.1. Criteria for surveillance systems 1 As simple and flexible as possible 2 Appropriate in terms of the information required 3 Capable of providing such information in a timely manner 4 Appropriate in terms of the resources available 5 Sustainable in the long term within local resources 6 Based on standardized sampling methods 7 Based on agreed case definitions 8 Capable of providing regular information from defined sites 9 Capable of covering the whole affected area 10 Compatible with existing systems 11 Use existing systems as far as possible 12 Use existing records as far as possible 13 Involve collaboration between agencies so as to avoid duplication 14 Involve collaboration with local services so as to avoid duplication 15 Acceptable to those surveyed Notes on these criteria: As simple and flexible as possible Complexity and inflexibility are incompatible with surveillance systems generally and particularly when operating in emergencies where collection of data may be difficult and where situations can change very fast. Appropriate in terms of the information required Defining what you "need to know" will allow you to set up the appropriate data collection methods (questionnaires, sites, etc.) and to design the system so that it can obtain and handle the information required. Capable of providing such information in a timely manner Information that is accurate but out of date is useless for immediate disease control purposes and of little value for forward planning. Communications therefore form an integral part of any surveillance system. Appropriate in terms of the resources available Do not try to overreach when setting up a system. For example, expatriate staff may best be used to recruit local staff for the system and in supervisory activities rather than in collecting data. Sustainable in the long term within local resources This criterion is certainly a goal to aim for as sustainability must be the target for all aid work. However, there may be situations where an emergency system is needed rapidly and where it cannot readily be integrated into existing systems or be developed as a new long-term system. Based on standardized sampling methods The sampling system must use the same data collection methods throughout if data are to be comparable. Ideally this should be methods that are internationally agreed and approved. Agreement should be sought for the methods from the other agencies on the ground to ensure consistency. Based on agreed case definitions Without case definitions that are agreed by all parties the likelihood of success of a surveillance system is very low. This is especially so when laboratory support is minimal or absent since clinical case definitions have to be drawn very tightly if different diseases are not to be confused. Capable of providing regular information from defined sites Routine surveillance requires more than material from ad hoc sources. Sites such as medical centers (in towns, villages, or refugee camps), hospitals, and/or public health units should be recruited. Capable of covering the whole affected area The more comprehensive the coverage of the system, the more likely is it that the data will be accurate and complete and that problems will not be missed. Such coverage can be problematic. The coverage of the different systems that can be used is discussed below. Compatible with existing systems The data collected and the methods used should ideally fit in with systems that are operating or have previously operated in the area. Use existing systems as far as possible Following from Criterion 10, if systems are already in existence or in abeyance but revivable then this should be done so as to ensure compliance by local health-care services and continuity of data collection and analysis. Use existing records as far as possible Existing records are of considerable value for predictive purposes. Knowledge of past problems makes it possible to anticipate future trends and problems and allows for early planning decisions. Involve collaboration between agencies so as to avoid duplication If several health agencies are operating it is essential to ensure collaboration among them in surveillance activities to avoid confusion and duplication of effort. Involve collaboration with local services so as to avoid duplication As above, early involvement of local health and surveillance services will reduce workloads and avoid duplication of effort. Acceptable to those surveyed If those from whom the data are collected, those who are collecting the data, and those who will receive the results are unhappy with the system, the system is unlikely to operate effectively. These criteria can be used to evaluate a plan for a surveillance system and also, with some additions, to evaluate an existing system. However, failure to fulfil all these criteria need not rule out a system. In many emergencies it can be difficult to meet such a wide range of "best case" criteria, and the question that must be asked is whether the proposed system is capable of fulfilling its purpose – can it provide sufficiently accurate essential information to those who need it when they need it ? The emphasis of an emergency surveillance program may need to be altered as the situation changes especially if a particular item emerges as being of key importance. Those running the surveillance program should use the data gathered and a continuous assessment of the general running of the system, to alter the program as required (preferably after consultation with relevant stakeholders). Designing Health Surveillance Systems When designing health surveillance systems, it is essential to do the following: Define the population under surveillance Determine what type of system can be established Set surveillance priorities Identify sources of data Set up agreed case definitions Establish data-handling systems Establish a protocol for evaluating the surveillance system as a whole Each of these is examined in more detail. Population Under Surveillance The population under surveillance may be relatively small and well defined (such as the population of a refugee camp) or a much less defined group such as mobile groups of refugees or IDPs or the population of a village, town, or region, the size of whose population may be unknown or may be fluctuating because of a disaster. Establishment of denominators may therefore be difficult. Even refugees or IDP camps may present a challenge as, while the size of the population may appear to be (or actually be) stable, its makeup may vary over time because of movements in and out. If the age or sex makeup of the camp alters, the pattern of disease may also alter. Demography: Numbers vs. Rates Both the number of cases detected and the rate of factors such as morbidity or mortality per unit of population are important values needed to inform emergency programs. Those responsible for all aspects of health care need to know what numbers of cases are involved so as to ensure adequate provision of services (amounts of medicines, numbers of hospital beds, etc.). However, simple numbers are of little value in assessing trends and patterns since increases or decreases in numbers of cases (or numbers of deaths) may reflect changes in population size (resulting, for example, from population displacement) rather than a trend due to (for example) a particular disease. In addition, several rates (such as the crude mortality rate) are key indicators in defining health emergencies (see below). Knowing the demography of the affected population is therefore important and all agencies working in an emergency should agree on and use the same population figures. The essential demographic data needed include the following: Total population size Population structure Overall sex ratio and the sex ratio in defined age groups Population under 5 years old, with age breakdown (0–4 years) – this group has special needs and is usually a key factor in planning the emergency response Age pyramid Ethnic composition and place of origin Number of vulnerable persons (e.g., pregnant and lactating women, members of female-headed households, unaccompanied children, destitute elderly, disabled and wounded persons) Average family/household size In situations where populations are displaced and extensive population movements may be occurring, it is also necessary to know the following: The number of arrivals and departures per week The predicted number of future arrivals at the sampling sites At the outset it is therefore important to establish methods to obtain demographic data. Often the best that can be managed initially is a rough estimate, but this can usually be refined later. It is helpful to use several methods and cross-check the figures to obtain the best estimate. Surrogates of the whole population (such as those attending a clinic) may be the best that can be achieved early on. The ease with which such data can be obtained usually depends on the size and scale of the population under consideration. The demography of a well-run refugee camp is quite easy to obtain but that of a larger area may be much more difficult. A lack of knowledge of the size of a displaced group can be confounded by a lack of knowledge of the size of the resident population. In many countries with poor infrastructures, accurate census data are not available. In some instances tax records may be helpful if these can be obtained. It should be noted that demographic data, especially if they involve refugees and IDPs, can be politically sensitive and interested parties may place undue weight on any figures that are given. Types of Surveillance System Comprehensive Systems Ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. A surveillance system in a refugee or IDP camp is effectively a miniature comprehensive system as it is possible to cover the whole population. Sentinel Surveillance Systems There are situations where comprehensive surveillance is not possible and these often arise in disasters. Damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. As far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. Sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. The calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. Both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. If there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. Setting Surveillance Priorities Surveillance must provide information on key health indicators, which should include the following: Morbidity Mortality Nutritional status Immunization status Vital needs Health sector activities, including local health services Activities in related sectors The selection of information sought in these categories must be done carefully. It is neither possible nor desirable to monitor everything, especially in the early stages of a disaster response. At that stage (the acute phase) the priority of surveillance is the detection of factors that can have the greatest and most rapid effect on the population. In terms of communicable disease this means diseases that affect large numbers of people and have epidemic potential. In most instances this also means diseases for which effective rapid control measures exist. While gathering data on other large-scale disease problems should not be excluded, the main surveillance and control efforts should be aimed where they can do the most immediate good. In the very early stages, only clinical information may be available since laboratory diagnostic services will probably be damaged or simply unavailable. However, this need not be a problem if the medical response is also geared to a syndromic approach. As the situation stabilizes, laboratory support becomes available, and longer term control measures can be supported, the surveillance can become more refined and additional diseases (for example, those which can cause severe morbidity and mortality in the longer term – such as tuberculosis, HIV or AIDS, and STDs) can be added to the list. Morbidity The main morbidity figures that are routinely sought are as follows: Incidence – the number of new cases of a particular disease reported over a defined period Attack rate (used in outbreaks – usually expressed as percentage) (also called incidence proportion or cumulative incidence ) – number of new cases within a specified time period/size of the population initially at risk (×100). (e.g., if 30 per 1,000 persons develop a condition over 2 weeks, the AR/IP/CI is 30/1,000 [3.0%]) Incidence rate – number of new cases per unit of person-time at risk. In the above example, the IR is 15/1,000 person-weeks. (This statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) Prevalence – the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (NB: Prevalence " rate " is the number of cases of a disease at a particular time/population at risk) There are a number of ways of estimating morbidity. Health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. Other ways of gathering morbidity data include the following: Surveys – in which data are collected from a small sample of the emergency-affected population deemed to be representative of the whole (or from a particular group for a specific purpose) Outbreak investigations – which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Mortality As with disease, changes in numbers of deaths may reflect changes in population size. Determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. Often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. All deaths occurring in the community must therefore be recorded. The following indicators can provide the essential information to define the health situation in a population: Crude mortality rate (CMR) is the most important indicator as it indicates the severity of the problem, and changes in CMR show how a medical emergency is developing. CMR is usually expressed as number of deaths per 10,000 persons per day. If the CMR rises above 1/10,000 per day (>2/10,000 per day for young children) an acute emergency is developing and the emergency phase lasts until the daily CMR falls to 1/10,000 per day or below. Age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). In children this is usually given as the number of deaths in children younger and older than 5 years/1,000 children of each age/day). NB: If population data for the under 5s are not available, an estimate of 17% of the total population may be used. Maternal mortality rate . Maternal mortality is a sensitive indicator of the effectiveness of health-care systems. A maternal death is usually defined as the death of a woman while pregnant or within 42 days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. The 42-day cut-off is recommended by WHO but some authorities use a time of up to a year. Maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × 1,000 (or ×100,000) Cause-specific death rates (case fatality rates – usually given as a percentage) . Proportion of cases of a specified condition which are fatal within a specified time. Case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × 100 Nutritional Status The following indicators must be measured: Prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) Prevalence of severe acute malnutrition in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) Prevalence of micronutrient deficiencies Estimate number of children needing to be cared for in selective feeding programs Estimate number of additional calories per day provided by selective feeding programs Immunization Immunization programs are a vital part of the public health measures undertaken following disasters. For example, measles vaccination is one of the most important health activities in such situations. The need for campaigns may be assessed on the basis of national vaccination records if they exist. In the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). The effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. In less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. Vital Needs Items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. Depending on the circumstances it may be necessary to monitor these elements in the affected population. Health Service Activities Indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. Other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. Activities in Related Sectors Activities in related sectors such as water and sanitation, shelter and security may also be included. Sources of Data The major sources of health data will be hospitals and clinics (both national and those established by aid agencies), individual medical practitioners, and other health-care workers. Specialized agencies should be able to provide data on particular needs (e.g., food, water, sanitation, and shelter). Case Definitions Case definitions are an essential part of surveillance. If the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless – changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. This is especially important when laboratory confirmation is not possible. It is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. Case definitions must be prepared for each health event or disease or syndrome. If available, the case definitions used by the host country's MOH should be used to ensure continuity of data. Several different sets of case definitions already exist, either in generalized form (for example, those produced by the Centers for Disease Control in Atlanta) or sets prepared for specific emergencies (e.g., the WHO Communicable Disease Toolkit for the Iraq Crisis in 2003). Standard case definitions may have to be adapted according to the local situation. It should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. When case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see Table 13.2 ). Table 13.2. Types of cases Type of case Criteria Suspected case Clinical signs and symptoms compatible with the disease in question but no laboratory evidence of infection (not available, negative, or pending) Probable case Clinical signs and symptoms compatible with the disease in question and also epidemiological evidence (e.g., contact with a known case) or some laboratory evidence (e.g., the results of a screening test) for the relevant disease Confirmed case Definite laboratory evidence of current or recent infection, whether or not clinical signs or symptoms are or have been present Although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. Samples can subsequently be sent to a referral laboratory for confirmation. Once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. Population Under Surveillance The population under surveillance may be relatively small and well defined (such as the population of a refugee camp) or a much less defined group such as mobile groups of refugees or IDPs or the population of a village, town, or region, the size of whose population may be unknown or may be fluctuating because of a disaster. Establishment of denominators may therefore be difficult. Even refugees or IDP camps may present a challenge as, while the size of the population may appear to be (or actually be) stable, its makeup may vary over time because of movements in and out. If the age or sex makeup of the camp alters, the pattern of disease may also alter. Demography: Numbers vs. Rates Both the number of cases detected and the rate of factors such as morbidity or mortality per unit of population are important values needed to inform emergency programs. Those responsible for all aspects of health care need to know what numbers of cases are involved so as to ensure adequate provision of services (amounts of medicines, numbers of hospital beds, etc.). However, simple numbers are of little value in assessing trends and patterns since increases or decreases in numbers of cases (or numbers of deaths) may reflect changes in population size (resulting, for example, from population displacement) rather than a trend due to (for example) a particular disease. In addition, several rates (such as the crude mortality rate) are key indicators in defining health emergencies (see below). Knowing the demography of the affected population is therefore important and all agencies working in an emergency should agree on and use the same population figures. The essential demographic data needed include the following: Total population size Population structure Overall sex ratio and the sex ratio in defined age groups Population under 5 years old, with age breakdown (0–4 years) – this group has special needs and is usually a key factor in planning the emergency response Age pyramid Ethnic composition and place of origin Number of vulnerable persons (e.g., pregnant and lactating women, members of female-headed households, unaccompanied children, destitute elderly, disabled and wounded persons) Average family/household size In situations where populations are displaced and extensive population movements may be occurring, it is also necessary to know the following: The number of arrivals and departures per week The predicted number of future arrivals at the sampling sites At the outset it is therefore important to establish methods to obtain demographic data. Often the best that can be managed initially is a rough estimate, but this can usually be refined later. It is helpful to use several methods and cross-check the figures to obtain the best estimate. Surrogates of the whole population (such as those attending a clinic) may be the best that can be achieved early on. The ease with which such data can be obtained usually depends on the size and scale of the population under consideration. The demography of a well-run refugee camp is quite easy to obtain but that of a larger area may be much more difficult. A lack of knowledge of the size of a displaced group can be confounded by a lack of knowledge of the size of the resident population. In many countries with poor infrastructures, accurate census data are not available. In some instances tax records may be helpful if these can be obtained. It should be noted that demographic data, especially if they involve refugees and IDPs, can be politically sensitive and interested parties may place undue weight on any figures that are given. Types of Surveillance System Comprehensive Systems Ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. A surveillance system in a refugee or IDP camp is effectively a miniature comprehensive system as it is possible to cover the whole population. Sentinel Surveillance Systems There are situations where comprehensive surveillance is not possible and these often arise in disasters. Damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. As far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. Sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. The calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. Both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. If there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. Comprehensive Systems Ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. A surveillance system in a refugee or IDP camp is effectively a miniature comprehensive system as it is possible to cover the whole population. Sentinel Surveillance Systems There are situations where comprehensive surveillance is not possible and these often arise in disasters. Damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. As far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. Sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. The calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. Both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. If there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. Setting Surveillance Priorities Surveillance must provide information on key health indicators, which should include the following: Morbidity Mortality Nutritional status Immunization status Vital needs Health sector activities, including local health services Activities in related sectors The selection of information sought in these categories must be done carefully. It is neither possible nor desirable to monitor everything, especially in the early stages of a disaster response. At that stage (the acute phase) the priority of surveillance is the detection of factors that can have the greatest and most rapid effect on the population. In terms of communicable disease this means diseases that affect large numbers of people and have epidemic potential. In most instances this also means diseases for which effective rapid control measures exist. While gathering data on other large-scale disease problems should not be excluded, the main surveillance and control efforts should be aimed where they can do the most immediate good. In the very early stages, only clinical information may be available since laboratory diagnostic services will probably be damaged or simply unavailable. However, this need not be a problem if the medical response is also geared to a syndromic approach. As the situation stabilizes, laboratory support becomes available, and longer term control measures can be supported, the surveillance can become more refined and additional diseases (for example, those which can cause severe morbidity and mortality in the longer term – such as tuberculosis, HIV or AIDS, and STDs) can be added to the list. Morbidity The main morbidity figures that are routinely sought are as follows: Incidence – the number of new cases of a particular disease reported over a defined period Attack rate (used in outbreaks – usually expressed as percentage) (also called incidence proportion or cumulative incidence ) – number of new cases within a specified time period/size of the population initially at risk (×100). (e.g., if 30 per 1,000 persons develop a condition over 2 weeks, the AR/IP/CI is 30/1,000 [3.0%]) Incidence rate – number of new cases per unit of person-time at risk. In the above example, the IR is 15/1,000 person-weeks. (This statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) Prevalence – the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (NB: Prevalence " rate " is the number of cases of a disease at a particular time/population at risk) There are a number of ways of estimating morbidity. Health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. Other ways of gathering morbidity data include the following: Surveys – in which data are collected from a small sample of the emergency-affected population deemed to be representative of the whole (or from a particular group for a specific purpose) Outbreak investigations – which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Mortality As with disease, changes in numbers of deaths may reflect changes in population size. Determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. Often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. All deaths occurring in the community must therefore be recorded. The following indicators can provide the essential information to define the health situation in a population: Crude mortality rate (CMR) is the most important indicator as it indicates the severity of the problem, and changes in CMR show how a medical emergency is developing. CMR is usually expressed as number of deaths per 10,000 persons per day. If the CMR rises above 1/10,000 per day (>2/10,000 per day for young children) an acute emergency is developing and the emergency phase lasts until the daily CMR falls to 1/10,000 per day or below. Age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). In children this is usually given as the number of deaths in children younger and older than 5 years/1,000 children of each age/day). NB: If population data for the under 5s are not available, an estimate of 17% of the total population may be used. Maternal mortality rate . Maternal mortality is a sensitive indicator of the effectiveness of health-care systems. A maternal death is usually defined as the death of a woman while pregnant or within 42 days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. The 42-day cut-off is recommended by WHO but some authorities use a time of up to a year. Maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × 1,000 (or ×100,000) Cause-specific death rates (case fatality rates – usually given as a percentage) . Proportion of cases of a specified condition which are fatal within a specified time. Case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × 100 Nutritional Status The following indicators must be measured: Prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) Prevalence of severe acute malnutrition in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) Prevalence of micronutrient deficiencies Estimate number of children needing to be cared for in selective feeding programs Estimate number of additional calories per day provided by selective feeding programs Immunization Immunization programs are a vital part of the public health measures undertaken following disasters. For example, measles vaccination is one of the most important health activities in such situations. The need for campaigns may be assessed on the basis of national vaccination records if they exist. In the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). The effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. In less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. Vital Needs Items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. Depending on the circumstances it may be necessary to monitor these elements in the affected population. Health Service Activities Indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. Other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. Activities in Related Sectors Activities in related sectors such as water and sanitation, shelter and security may also be included. Morbidity The main morbidity figures that are routinely sought are as follows: Incidence – the number of new cases of a particular disease reported over a defined period Attack rate (used in outbreaks – usually expressed as percentage) (also called incidence proportion or cumulative incidence ) – number of new cases within a specified time period/size of the population initially at risk (×100). (e.g., if 30 per 1,000 persons develop a condition over 2 weeks, the AR/IP/CI is 30/1,000 [3.0%]) Incidence rate – number of new cases per unit of person-time at risk. In the above example, the IR is 15/1,000 person-weeks. (This statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) Prevalence – the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (NB: Prevalence " rate " is the number of cases of a disease at a particular time/population at risk) There are a number of ways of estimating morbidity. Health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. Other ways of gathering morbidity data include the following: Surveys – in which data are collected from a small sample of the emergency-affected population deemed to be representative of the whole (or from a particular group for a specific purpose) Outbreak investigations – which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures Mortality As with disease, changes in numbers of deaths may reflect changes in population size. Determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. Often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. All deaths occurring in the community must therefore be recorded. The following indicators can provide the essential information to define the health situation in a population: Crude mortality rate (CMR) is the most important indicator as it indicates the severity of the problem, and changes in CMR show how a medical emergency is developing. CMR is usually expressed as number of deaths per 10,000 persons per day. If the CMR rises above 1/10,000 per day (>2/10,000 per day for young children) an acute emergency is developing and the emergency phase lasts until the daily CMR falls to 1/10,000 per day or below. Age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). In children this is usually given as the number of deaths in children younger and older than 5 years/1,000 children of each age/day). NB: If population data for the under 5s are not available, an estimate of 17% of the total population may be used. Maternal mortality rate . Maternal mortality is a sensitive indicator of the effectiveness of health-care systems. A maternal death is usually defined as the death of a woman while pregnant or within 42 days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. The 42-day cut-off is recommended by WHO but some authorities use a time of up to a year. Maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × 1,000 (or ×100,000) Cause-specific death rates (case fatality rates – usually given as a percentage) . Proportion of cases of a specified condition which are fatal within a specified time. Case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × 100 Nutritional Status The following indicators must be measured: Prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) Prevalence of severe acute malnutrition in children 6–59 months of age (or 60–110 cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) Prevalence of micronutrient deficiencies Estimate number of children needing to be cared for in selective feeding programs Estimate number of additional calories per day provided by selective feeding programs Immunization Immunization programs are a vital part of the public health measures undertaken following disasters. For example, measles vaccination is one of the most important health activities in such situations. The need for campaigns may be assessed on the basis of national vaccination records if they exist. In the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). The effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. In less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. Vital Needs Items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. Depending on the circumstances it may be necessary to monitor these elements in the affected population. Health Service Activities Indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. Other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. Activities in Related Sectors Activities in related sectors such as water and sanitation, shelter and security may also be included. Sources of Data The major sources of health data will be hospitals and clinics (both national and those established by aid agencies), individual medical practitioners, and other health-care workers. Specialized agencies should be able to provide data on particular needs (e.g., food, water, sanitation, and shelter). Case Definitions Case definitions are an essential part of surveillance. If the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless – changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. This is especially important when laboratory confirmation is not possible. It is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. Case definitions must be prepared for each health event or disease or syndrome. If available, the case definitions used by the host country's MOH should be used to ensure continuity of data. Several different sets of case definitions already exist, either in generalized form (for example, those produced by the Centers for Disease Control in Atlanta) or sets prepared for specific emergencies (e.g., the WHO Communicable Disease Toolkit for the Iraq Crisis in 2003). Standard case definitions may have to be adapted according to the local situation. It should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. When case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see Table 13.2 ). Table 13.2. Types of cases Type of case Criteria Suspected case Clinical signs and symptoms compatible with the disease in question but no laboratory evidence of infection (not available, negative, or pending) Probable case Clinical signs and symptoms compatible with the disease in question and also epidemiological evidence (e.g., contact with a known case) or some laboratory evidence (e.g., the results of a screening test) for the relevant disease Confirmed case Definite laboratory evidence of current or recent infection, whether or not clinical signs or symptoms are or have been present Although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. Samples can subsequently be sent to a referral laboratory for confirmation. Once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. Case Definitions Case definitions are an essential part of surveillance. If the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless – changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. This is especially important when laboratory confirmation is not possible. It is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. Case definitions must be prepared for each health event or disease or syndrome. If available, the case definitions used by the host country's MOH should be used to ensure continuity of data. Several different sets of case definitions already exist, either in generalized form (for example, those produced by the Centers for Disease Control in Atlanta) or sets prepared for specific emergencies (e.g., the WHO Communicable Disease Toolkit for the Iraq Crisis in 2003). Standard case definitions may have to be adapted according to the local situation. It should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. When case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see Table 13.2 ). Table 13.2. Types of cases Type of case Criteria Suspected case Clinical signs and symptoms compatible with the disease in question but no laboratory evidence of infection (not available, negative, or pending) Probable case Clinical signs and symptoms compatible with the disease in question and also epidemiological evidence (e.g., contact with a known case) or some laboratory evidence (e.g., the results of a screening test) for the relevant disease Confirmed case Definite laboratory evidence of current or recent infection, whether or not clinical signs or symptoms are or have been present Although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. Samples can subsequently be sent to a referral laboratory for confirmation. Once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. Establish Data-Handling Systems The following issues should be considered: Methods of recording and transferring data Methods of verifying data Frequency of reporting Who will analyze the data and how often Methods for disseminating results Recording and Transferring Data Visits to surveillance sites and discussions with staff involved will help define the recording and data transmission systems required. The great advances in information technology that have been made in recent years have greatly facilitated the collection, recording, transmission, and analysis of surveillance data, but care must be taken that the systems put in place are appropriate. In areas where electricity supplies are problematical and communications poor it may be better to use a paper recording system and verbal data transmission by radio than a computerized system. Verification Data verification is essential for the credibility of a surveillance system. Those responsible for surveillance systems must ensure good adherence to case definitions if a symptom-based system is in operation and that laboratory quality control systems operate where appropriate. Regular assessments of record keeping and the accuracy of data transfer are required. Triangulation of results from several sources can sometimes help to detect anomalies. Frequency of Reporting Frequency of reporting will usually depend on the severity of the health situation. In general, daily reporting during the acute phase of an emergency will be needed, although in an acute medical emergency (such as a severe cholera outbreak) even more frequent reporting may be necessary, especially if the situation is fluctuating rapidly. The frequency may reduce to (say) weekly as the situation resolves. Data Analysis Who is to analyze the data and how it is to be analyzed must be established at the outset. In a relatively defined area such as a camp, a data analysis session may be the last of the daily activities of the person responsible for surveillance. If record keeping and analysis protocols have been carefully worked out initially this task is not necessarily a large additional burden. Surveillance systems that cover larger areas and bigger and more diffuse populations usually rely on a central data collection point where designated staff analyze the data. Use of such a system requires good data transmission systems. Output of Surveillance Systems Output is as important as input. Collecting data without dissemination of results is a sterile exercise and tends rapidly to demotivate those who are collecting the data. There are some important points to consider: The results of surveillance must be presented in a readily comprehensible form. Surveillance reports should be produced regularly and widely distributed to aid agencies, and to national and international governments and organizations. This will help those involved to understand the overall picture, rather than just that in the area where they are working, and will allow them to take informed decisions about future actions. Recording and Transferring Data Visits to surveillance sites and discussions with staff involved will help define the recording and data transmission systems required. The great advances in information technology that have been made in recent years have greatly facilitated the collection, recording, transmission, and analysis of surveillance data, but care must be taken that the systems put in place are appropriate. In areas where electricity supplies are problematical and communications poor it may be better to use a paper recording system and verbal data transmission by radio than a computerized system. Verification Data verification is essential for the credibility of a surveillance system. Those responsible for surveillance systems must ensure good adherence to case definitions if a symptom-based system is in operation and that laboratory quality control systems operate where appropriate. Regular assessments of record keeping and the accuracy of data transfer are required. Triangulation of results from several sources can sometimes help to detect anomalies. Frequency of Reporting Frequency of reporting will usually depend on the severity of the health situation. In general, daily reporting during the acute phase of an emergency will be needed, although in an acute medical emergency (such as a severe cholera outbreak) even more frequent reporting may be necessary, especially if the situation is fluctuating rapidly. The frequency may reduce to (say) weekly as the situation resolves. Data Analysis Who is to analyze the data and how it is to be analyzed must be established at the outset. In a relatively defined area such as a camp, a data analysis session may be the last of the daily activities of the person responsible for surveillance. If record keeping and analysis protocols have been carefully worked out initially this task is not necessarily a large additional burden. Surveillance systems that cover larger areas and bigger and more diffuse populations usually rely on a central data collection point where designated staff analyze the data. Use of such a system requires good data transmission systems. Output of Surveillance Systems Output is as important as input. Collecting data without dissemination of results is a sterile exercise and tends rapidly to demotivate those who are collecting the data. There are some important points to consider: The results of surveillance must be presented in a readily comprehensible form. Surveillance reports should be produced regularly and widely distributed to aid agencies, and to national and international governments and organizations. This will help those involved to understand the overall picture, rather than just that in the area where they are working, and will allow them to take informed decisions about future actions. Evaluation of Surveillance Systems Surveillance systems should be evaluated constantly to ensure that they are working properly, that the data are representative, analysis is appropriate and accurate, and that results are being disseminated to where they are needed. C - Control of Communicable Disease Introduction The public health aspects of communicable disease control can be broadly divided into preventive activities (such as vector control and vaccination programs) and the investigation and control of outbreaks and epidemics. Experience from many emergencies and disasters has made it possible to identify a number of syndromes or diseases that are most likely to occur in such situations (Table 13.3 ). This makes it possible to plan activities and interventions on the basis of likely occurrences, even before those involved are present at the scene of the disaster, and to make initial purchases and establish stockpiles of appropriate medicines and equipment. Table 13.3. Syndromes or diseases that occur commonly in disasters Bloody diarrhea Suspected meningitis Acute watery diarrhea Acute jaundice syndrome Suspected cholera Acute hemorrhagic fever syndrome Lower respiratory tract infection Trauma/injury Measles Malnutrition Acute flaccid paralysis A few others, such as malaria and other vector-borne diseases (e.g., typhus and leish-maniasis), are also likely to occur but are region specific. TB and HIV or AIDS can also cause major problems in the longer term Prevention "Prevention is better than cure" and proper attention to preventive measures from the earliest stage of the response to the disaster will greatly reduce the risks to the health of the population from infectious disease. Provision of Appropriate Physical Conditions A key method of preventing communicable disease is the provision of shelter, adequate amounts of clean water, sufficient safe food, and proper sanitation (latrines and facilities for personal hygiene, clothes washing, and drying). Control of Disease Vectors Arthropod vectors (mosquitoes, ticks) can be controlled by appropriate spraying programs and also by habitat management (e.g., the removal of places where water can accumulate and mosquitoes breed). Provision of bed nets, particularly nets impregnated with insecticide, is effective for reducing infection with agents such as malaria and Leishmania . Control of rodents, by proper control of rubbish, by rodent proofing food stores, by attention to domestic hygiene and by use of rodenticides, will reduce the risks of transmission of rodent-borne diseases such as plague and Lassa fever. Disposal of Contaminated Materials Medical waste includes laboratory samples, needles and syringes, body tissues, and materials stained with body fluids. This requires careful handling, especially the sharps, as infectious agents such as those causing hepatitis B and C, HIV and AIDS, and viral hemorrhagic fevers can be transmitted by these materials. Used sharps should be disposed of into suitable containers (proper sharps boxes are ideal but old metal containers such as coffee or milk powder tins are adequate). Medical waste should ideally be burned in an incinerator. This should be close to the clinic or hospital but downwind of the prevailing wind. A 200-L oil drum can be used for this purpose with a metal grate half way up and a hole at the bottom to allow in air and for the removal of ash. Larger-scale and more permanent incinerators can be constructed if necessary. Burning pits can be used in emergency. If burning is not possible items should be buried at least 1.5 m deep. This is more suitable than burning for large items of human tissue such as amputated legs. Ensure there is no risk of groundwater contamination. Dealing with the Dead This is a complex process involving not just considerations of infection risk but also legal, sociocultural, and psychological factors. There are a number of specialist publications which can be of help. Health Aspects After almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. However, in sudden impact disasters (such as the Indian Ocean tsunami in 2004), the pattern and incidence of disease found in the dead will generally reflect those in the living. The situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. In fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. The diseased living are far more dangerous. The decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. Those most at risk are those handling the deceased, not the community. The most likely risks to them are as follows: Blood-borne viruses (Hepatitis B and C, HIV) Enteric pathogens (especially cholera) Respiratory pathogens (e.g. TB) Spore-forming bacteria (anthrax, tetanus) Some vector-borne diseases (plague, typhus) because the vectors may be present on the cadaver Acute hemorrhagic fevers (Ebola, Marburg, Lassa) Those handling cadavers should do the following: Take universal precautions for blood and body fluids Dispose of or disinfect used gloves Avoid contamination of personal items Wash hands after handling bodies and before eating Have hepatitis B vaccination Ensure disinfection of vehicles and equipment Mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. Cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. Alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. The dead must always be treated with dignity and respect. As far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. If the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. When an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). In general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). Bodies should be buried at least 1.5 m deep or, if more shallowly, should have earth piled at least 1 m above the ground level and 0.5 m to each side of the grave (to prevent access by scavengers and burrowing insects). Disinfectants such as chloride of lime should not be used. New burial sites should be at least 250 m from drinking water sources and at least 0.7 m above the saturated zone. Vaccination Programs Vaccination programs are an essential part of disease prevention. Information about existing vaccination programs must be obtained during the assessment process and this should include information from external assessors (e.g., WHO, UNICEF, NGOs) as to the effectiveness of the vaccination programs that have been undertaken in the past. It cannot be assumed that simply because children have received vaccines that these vaccines were effective. Vaccination Priorities in Emergencies Measles kills large number of children in developing countries and is one of the greatest causes of morbidity and mortality in children in refugee and IDP camps. Mass vaccination of children between the ages of 6 months and 15 years should be an absolute priority during the first week of activity in humanitarian situations and can be conducted with the distribution of vitamin A. A system for maintaining measles immunization must be established once the target population has been covered adequately in the initial campaign. This is necessary to ensure that children who may have been missed in the original campaign, children reaching the age of 6 months, and children first vaccinated at the age of 6–9 months who must receive a second dose at 9 months of age are all covered. Some of the children vaccinated during such a mass campaign may have been vaccinated before. This does not matter and a second dose will have no adverse effect. It is essential to ensure full coverage against measles in the population. Other EPI vaccinations for children are not generally included in the emergency phase because they can only prevent a minor proportion of the overall morbidity and mortality at that stage. However, should specific outbreaks occur then the appropriate vaccine should be considered as a control measure. Vaccination programs require the following: Appropriate types of vaccines. Appropriate amounts of these vaccines. Equipment (needles, syringes, sterilization equipment, sharps disposal). Emergency immunization kits, including cold chain equipment, are available from a number of sources, including UNICEF and some NGOs (e.g. MSF). Logistics (transport, cold chain). Staff: a vaccination team may be quite large. It must include the following personnel: A supervisor. Logistics staff. Staff to prepare and administer vaccines. Record keepers. Security staff (to maintain order and control crowds) may also be needed. The Cold Chain Maintenance of the cold chain is particularly important. This is the system of transporting and storing vaccines within a suitable temperature range from the point of manufacture to the point of administration. The effectiveness of vaccines can be reduced or lost if they are allowed to get too cold, too hot, or are exposed to direct sunlight or fluorescent light. Careful note should be taken of the conditions needed to transport different vaccines because these can vary. The essential cold chain equipment needed to transport and store vaccines within a consistent safe temperature range includes the following: Dedicated refrigerators for storing vaccines and freezers for ice packs (fridges and freezers powered by gas or kerosene are available as alternatives to electric machines, and solar-powered fridge/freezer combinations specially designed for vaccine storage are also available) A suitable thermometer and a chart for recording daily temperature readings Cold boxes for transporting and storing vaccines Ice packs to keep vaccines cool Insulating material to separate ice packs from the vaccines when in the cold boxes (e.g., bubble wrap or expended polystyrene foam) If possible, vaccines should be stored in their original packaging because removing the packaging exposes them to room temperature and light. Check the temperature to ensure the vaccines have not been exposed to temperatures outside the normal storage ranges for those vaccines (see Table 13.4 ). Table 13.4. WHO-recommended storage conditions for different vaccines Vaccine Primary Region District/health center OPV −15 to −25°C −15 to −25°C +2 to +8°C Freeze-dried vaccines (BCG, measles, MMR, MR, yellow fever, Hib freeze dried) +2 to +8°C +2 to +8°C +2 to +8°C Other vaccines (HepB, DTP-HepB, Hib liquid, DTP, DT, T T, Td +2 to +8°C +2 to +8°C +2 to +8°C Max. storage time at the different levels: primary, 6 months; region, 3 months; district, 1 month; health center, 1 month; health post, daily use – max. 1 month Diluents must never be frozen. Freeze-dried vaccines supplied packed with diluent must be stored between +2 and +8°C. Diluents supplied separately should be kept between +2 and +8°C Vaccine Storage Vaccines must be kept at the correct temperature since all are sensitive to heat and cold to some extent. All freeze-dried vaccines become much more heat-sensitive after they have been reconstituted. Vaccines sensitive to cold will lose potency if exposed to temperatures lower than optimal for their storage, particularly if they are frozen. Some vaccines (BCG, measles, MR, MMR, and rubella vaccines) are also sensitive to strong light and must always be protected against sunlight or fluorescent (neon) light. These vaccines are usually supplied in dark brown glass vials, which give them some protection against light damage, but they must still be covered and protected from strong light at all times. Only vaccine stocks that are fit for use should be kept in the vaccine cold chain. Expired or heat-damaged vials should be removed from cold storage. If unusable vaccines need to be kept for a period before disposal (e.g., until completion of accounting or auditing procedures) they should be kept outside the cold chain, separated from all usable stocks and carefully labelled to avoid mistaken use. Diluents Diluents for vaccines are less sensitive to storage temperatures than are the vaccines with which they are used (although they must be kept cool), but may be kept in the cold chain between +2 and +8°C if space permits. However, diluent vials must never be frozen (kept in a freezer or in contact with any frozen surface) as the vial may crack and become contaminated. When vaccines are reconstituted, the diluent should be at same temperature as the vaccine, so sufficient diluent for daily needs should be kept in the cold chain at the point of vaccine use (health center or vaccination post). At other levels of the cold chain (central, provincial, or district stores) it is only necessary to keep any diluent in the cold chain if it is planned to use it within the next 24 h. Freeze-dried vaccines and their diluents should always be distributed together in matching quantities. Although the diluents do not need to be kept in the cold chain (unless needed for reconstituting vaccines within the next 24 h), they must travel with the vaccine at all times, and must always be of the correct type, and from the same manufacturer as the vaccine that they are accompanying. Each vaccine requires a specific diluent, and therefore, diluents are not interchangeable (for example, diluent made for measles vaccine must not be used for reconstituting BCG, yellow fever, or any other type of vaccine). Likewise, diluent made by one manufacturer for use with a certain vaccine cannot be used for reconstituting the same type of vaccine produced by another manufacturer. Some combination vaccines comprise a freeze-dried component (such as Hib) which is designed to be reconstituted by a liquid vaccine (such as DTP or DTP-HepB liquid vaccine) instead of a normal diluent. For such combination vaccines, it is again vital that only vaccines manufactured and licensed for this purpose are combined. Note also that for combination vaccines where the diluent is itself a vaccine, all components must now be kept in the cold chain between +2 and +8°C at all times. As for all other freeze-dried vaccines, it is also essential that the "diluent" travels with the vaccine at all times. Effectiveness of Vaccination Programs The effectiveness of a vaccination program will need to be assessed. The program can be evaluated both by routinely collected data and, if necessary, by a survey of vaccination coverage. Routine data on coverage is obtained by comparing the numbers vaccinated with the estimated size of the target population (and clearly depends on accurate assessment of the latter). A coverage survey requires the use of a statistical technique called a two-stage cluster survey details of which can be found in the appropriate WHO/EPI documents. Information about the effectiveness of the campaign should be obtained from routine surveillance of communicable disease. If, for example, large number of measles cases continue to occur, or there is an outbreak, then data on coverage should be reex-amined. If this is shown to be good (over 90%) then the efficacy of the vaccine must be suspected. If the field efficacy is below the theoretical value 85% (for measles vaccine – data on efficacy of other vaccines can be obtained online) then possible causes of a breakdown in the vaccination program must be investigated (failure of the cold chain, poorly respected vaccination schedule). Methods for measuring vaccine efficacy can be found in the WHO/EPI literature. Chemoprophylaxis Mass chemoprophylaxis for bacterial infections such as cholera and meningitis is not usually recommended except on a small scale (for example, the use of Rifampicin may be considered to prevent the spread of meningococcal meningitis among immediate contacts of a case), but the difficulties of overseeing such activities and the risks of the development of antibiotic resistance outweigh any benefits that might be gained. The use of chemoprophylaxis for malaria must be undertaken with care. It may be indicated for vulnerable groups of refugees/IDPs (for example, children and pregnant women) arriving in an endemic area, particularly if they come from a nonmalarious area, but care must be taken to provide drugs to which the local strains of malaria are sensitive. The spread of resistance means that many of the standard drugs are ineffective and the replacements are both costly and may have unwanted side effects. Public Health Education Public health education and information activities play a vital role in disease prevention. Vaccination programs will not work unless there is acceptance by the public of the necessity for such programs. Individuals must be informed as to why these programs are necessary and also where and when they need to take their children for vaccination. Such activities are also essential to inform people about particular health programs (for example, feeding programs or vector control programs) and about the steps they can take to protect their health and that of their families (e.g., good hygiene). Information can be propagated in many ways: Posters Radio/TV/Film Lectures Songs/poems, etc. Leaflets Staff who are trained in this type of activity therefore play a key role in disease prevention. Heath education also requires transport and equipment (such as video or film projectors, screens, generators, blackboards, etc.). Treatment Details of the treatment of individuals for various infectious diseases and the facilities needed are covered elsewhere in this book and in many textbooks covering disasters and disease response. In terms of the population aspects of the treatment of disease, important requirements are to ensure that there are appropriate laboratories (microbiological, parasitological, hematological, biochemical) available to confirm diagnoses and monitor treatment. adequate supplies of appropriate antimicrobial agents available and the facilities to transport these, store, and distribute them under appropriate conditions (e.g., controlled temperature), together with relevant instruction for use. Laboratories and Specimen Transport The provision of laboratory facilities in emergencies is usually limited to basic tests such as those for malaria. More advanced tests, including identification of microorganisms and the determination of antimicrobial sensitivities, require more sophisticated facilities. These may be available in the affected country but are unlikely to be operating in the disaster-affected area. It is more likely that specimens will have to be transported to laboratories abroad. Collection of specimens requires appropriate equipment. This will include items such as swabs, transport media, needles, syringes, or vacum sampling systems for blood sampling, different blood collection bottles (with and without anticoagulants) and other sterile specimen tubes, and containers for faeces and urine. Transporting specimens must be done safely, and packing specimens for shipment requiring specially trained personnel. Antimicrobials Treatment of disease requires good supplies of appropriate antimicrobial agents. It is important to ensure that the agents chosen are suitable for use in the area. It is common for doctors in affected areas to ask for the latest therapeutic agents. However, these agents, although effective, are often expensive and not part of the normal treatment programs in the region. The local doctors may not therefore be familiar with the use of these agents, nor may laboratories be capable of monitoring their use. It is better to use funds, which are often limited, to supply larger amounts of older (generic) agents. One caveat is the possibility that regular use may have allowed resistance to certain agents to develop in a country. Data on this may be available from local surveillance records. Antimicrobials should always be supplied with relevant guidelines in a language that can be understood locally. If local laboratories are unable to test microbes for resistance to antimicrobials, isolates or specimens should be sent as soon as possible to appropriate reference laboratories for testing. Response to Outbreaks and Epidemics Features Outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. An epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. An outbreak is an epidemic limited to a small area (a town, village, or camp). The term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. The areas where vaccination campaigns are a priority need to be identified and campaigns started. The term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. This will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. Infections where a single case represents a potential outbreak include the following: Cholera Some viral hemorrhagic fevers (Ebola, Marburg) Yellowfever Measles Plague Typhus Infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: Shigellosis Typhoid Hepatitis A Malaria Meningococcal meningitis Human African trypanosomiasis Visceral leishmaniasis A surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. However, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. In addition to the establishment of surveillance, outbreak preparation involves the following: Preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. Ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. Stockpiling essential supplies. This includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. Identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. Identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. Ensure that vaccination supplies (needles, syringes, etc.) are adequate. Make sure the cold chain can be maintained. Identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. Confirmation of the Outbreak If the number of reported cases is rising, is this in excess of the expected number? Ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. Verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). Laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. Outbreak Control Team In the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including MOH, WHO, other UN organizations, NGOs, etc. In the case of an epidemic the MOH will probably take the lead or may ask WHO or another UN agency to do so. The team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. This may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the MOH or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. This team should meet at least once a day to review the situation and define the necessary responses. It has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. The team should also act as the point of contact for the media. A media liaison officer should be appointed and all media contact should be through this individual. This will allow team members to refer media representatives to a central point and reduce interference with their activities. It will also ensure that a consistent message based on the most complete data is given to the media. Information The appropriate national authorities should be informed of the outbreak. In addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the Revised International Health Regulations (2005) to report outbreaks of certain diseases. These include four diseases regarded as public-health emergencies of international concern: Smallpox Polio (wild-type) New strains of human influenza Severe acute respiratory syndrome (SARS) In some cases, Member States must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and West Nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, Rift Valley fever, and meningococcal disease). Investigation Once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: Produce a case definition for the outbreak. This is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. Collect and analyze descriptive data by Time, Person, and Place (time and date of onset, individual characteristics of those affected – age, sex, occupation, etc., location of cases). Plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). Determine the population that is at risk. Determine the number of cases and the size of the affected population. Calculate the attack rate. Formulate hypotheses for the pathogen about the possible source and routes of transmission. Conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). Report results and make recommendations for action. Outbreak Investigations The two main statistical tools used to investigate outbreaks are as follows: Case–control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) Cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms However the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. Control Activities Implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) Prevent infection (e.g., by vaccination programs) Prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) Treat cases Evaluation Evaluate the outbreak detection and response – were they appropriate, timely, and effective? Change/modify policies and preparedness to deal with outbreaks if required What activities are needed to prevent similar outbreaks in the future (e.g., improved vaccination programs, new water treatment facilities, public health education, etc.)? Produce and disseminate an outbreak report. The report should include details of the outbreak, including the following: Cause Duration, location, and persons involved Cumulative attack rate (number of cases/exposed population) Incidence rate Case fatality rate Vaccine efficacy (if relevant) (no. of unvaccinated ill – no. of vaccinated ill/no. of unvaccinated ill) – Proportion of vaccine-preventable cases (no. of vaccine-preventable cases/no. of cases) Recommendations Epi Info™ 6 This is an easy-to-use tool which is of great value for handling epidemiological data and for organizing study designs and results, which can be downloaded free of charge from the Internet. It is produced by the Centers for Disease Control (Atlanta) and is a series of microcomputer programs which can be used both for surveillance and for outbreak investigation and includes features used by epidemiologists in statistical programs, such as SAS or SPSS, and database programs such as dBase. Introduction The public health aspects of communicable disease control can be broadly divided into preventive activities (such as vector control and vaccination programs) and the investigation and control of outbreaks and epidemics. Experience from many emergencies and disasters has made it possible to identify a number of syndromes or diseases that are most likely to occur in such situations (Table 13.3 ). This makes it possible to plan activities and interventions on the basis of likely occurrences, even before those involved are present at the scene of the disaster, and to make initial purchases and establish stockpiles of appropriate medicines and equipment. Table 13.3. Syndromes or diseases that occur commonly in disasters Bloody diarrhea Suspected meningitis Acute watery diarrhea Acute jaundice syndrome Suspected cholera Acute hemorrhagic fever syndrome Lower respiratory tract infection Trauma/injury Measles Malnutrition Acute flaccid paralysis A few others, such as malaria and other vector-borne diseases (e.g., typhus and leish-maniasis), are also likely to occur but are region specific. TB and HIV or AIDS can also cause major problems in the longer term Prevention "Prevention is better than cure" and proper attention to preventive measures from the earliest stage of the response to the disaster will greatly reduce the risks to the health of the population from infectious disease. Provision of Appropriate Physical Conditions A key method of preventing communicable disease is the provision of shelter, adequate amounts of clean water, sufficient safe food, and proper sanitation (latrines and facilities for personal hygiene, clothes washing, and drying). Control of Disease Vectors Arthropod vectors (mosquitoes, ticks) can be controlled by appropriate spraying programs and also by habitat management (e.g., the removal of places where water can accumulate and mosquitoes breed). Provision of bed nets, particularly nets impregnated with insecticide, is effective for reducing infection with agents such as malaria and Leishmania . Control of rodents, by proper control of rubbish, by rodent proofing food stores, by attention to domestic hygiene and by use of rodenticides, will reduce the risks of transmission of rodent-borne diseases such as plague and Lassa fever. Disposal of Contaminated Materials Medical waste includes laboratory samples, needles and syringes, body tissues, and materials stained with body fluids. This requires careful handling, especially the sharps, as infectious agents such as those causing hepatitis B and C, HIV and AIDS, and viral hemorrhagic fevers can be transmitted by these materials. Used sharps should be disposed of into suitable containers (proper sharps boxes are ideal but old metal containers such as coffee or milk powder tins are adequate). Medical waste should ideally be burned in an incinerator. This should be close to the clinic or hospital but downwind of the prevailing wind. A 200-L oil drum can be used for this purpose with a metal grate half way up and a hole at the bottom to allow in air and for the removal of ash. Larger-scale and more permanent incinerators can be constructed if necessary. Burning pits can be used in emergency. If burning is not possible items should be buried at least 1.5 m deep. This is more suitable than burning for large items of human tissue such as amputated legs. Ensure there is no risk of groundwater contamination. Dealing with the Dead This is a complex process involving not just considerations of infection risk but also legal, sociocultural, and psychological factors. There are a number of specialist publications which can be of help. Health Aspects After almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. However, in sudden impact disasters (such as the Indian Ocean tsunami in 2004), the pattern and incidence of disease found in the dead will generally reflect those in the living. The situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. In fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. The diseased living are far more dangerous. The decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. Those most at risk are those handling the deceased, not the community. The most likely risks to them are as follows: Blood-borne viruses (Hepatitis B and C, HIV) Enteric pathogens (especially cholera) Respiratory pathogens (e.g. TB) Spore-forming bacteria (anthrax, tetanus) Some vector-borne diseases (plague, typhus) because the vectors may be present on the cadaver Acute hemorrhagic fevers (Ebola, Marburg, Lassa) Those handling cadavers should do the following: Take universal precautions for blood and body fluids Dispose of or disinfect used gloves Avoid contamination of personal items Wash hands after handling bodies and before eating Have hepatitis B vaccination Ensure disinfection of vehicles and equipment Mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. Cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. Alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. The dead must always be treated with dignity and respect. As far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. If the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. When an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). In general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). Bodies should be buried at least 1.5 m deep or, if more shallowly, should have earth piled at least 1 m above the ground level and 0.5 m to each side of the grave (to prevent access by scavengers and burrowing insects). Disinfectants such as chloride of lime should not be used. New burial sites should be at least 250 m from drinking water sources and at least 0.7 m above the saturated zone. Vaccination Programs Vaccination programs are an essential part of disease prevention. Information about existing vaccination programs must be obtained during the assessment process and this should include information from external assessors (e.g., WHO, UNICEF, NGOs) as to the effectiveness of the vaccination programs that have been undertaken in the past. It cannot be assumed that simply because children have received vaccines that these vaccines were effective. Vaccination Priorities in Emergencies Measles kills large number of children in developing countries and is one of the greatest causes of morbidity and mortality in children in refugee and IDP camps. Mass vaccination of children between the ages of 6 months and 15 years should be an absolute priority during the first week of activity in humanitarian situations and can be conducted with the distribution of vitamin A. A system for maintaining measles immunization must be established once the target population has been covered adequately in the initial campaign. This is necessary to ensure that children who may have been missed in the original campaign, children reaching the age of 6 months, and children first vaccinated at the age of 6–9 months who must receive a second dose at 9 months of age are all covered. Some of the children vaccinated during such a mass campaign may have been vaccinated before. This does not matter and a second dose will have no adverse effect. It is essential to ensure full coverage against measles in the population. Other EPI vaccinations for children are not generally included in the emergency phase because they can only prevent a minor proportion of the overall morbidity and mortality at that stage. However, should specific outbreaks occur then the appropriate vaccine should be considered as a control measure. Vaccination programs require the following: Appropriate types of vaccines. Appropriate amounts of these vaccines. Equipment (needles, syringes, sterilization equipment, sharps disposal). Emergency immunization kits, including cold chain equipment, are available from a number of sources, including UNICEF and some NGOs (e.g. MSF). Logistics (transport, cold chain). Staff: a vaccination team may be quite large. It must include the following personnel: A supervisor. Logistics staff. Staff to prepare and administer vaccines. Record keepers. Security staff (to maintain order and control crowds) may also be needed. The Cold Chain Maintenance of the cold chain is particularly important. This is the system of transporting and storing vaccines within a suitable temperature range from the point of manufacture to the point of administration. The effectiveness of vaccines can be reduced or lost if they are allowed to get too cold, too hot, or are exposed to direct sunlight or fluorescent light. Careful note should be taken of the conditions needed to transport different vaccines because these can vary. The essential cold chain equipment needed to transport and store vaccines within a consistent safe temperature range includes the following: Dedicated refrigerators for storing vaccines and freezers for ice packs (fridges and freezers powered by gas or kerosene are available as alternatives to electric machines, and solar-powered fridge/freezer combinations specially designed for vaccine storage are also available) A suitable thermometer and a chart for recording daily temperature readings Cold boxes for transporting and storing vaccines Ice packs to keep vaccines cool Insulating material to separate ice packs from the vaccines when in the cold boxes (e.g., bubble wrap or expended polystyrene foam) If possible, vaccines should be stored in their original packaging because removing the packaging exposes them to room temperature and light. Check the temperature to ensure the vaccines have not been exposed to temperatures outside the normal storage ranges for those vaccines (see Table 13.4 ). Table 13.4. WHO-recommended storage conditions for different vaccines Vaccine Primary Region District/health center OPV −15 to −25°C −15 to −25°C +2 to +8°C Freeze-dried vaccines (BCG, measles, MMR, MR, yellow fever, Hib freeze dried) +2 to +8°C +2 to +8°C +2 to +8°C Other vaccines (HepB, DTP-HepB, Hib liquid, DTP, DT, T T, Td +2 to +8°C +2 to +8°C +2 to +8°C Max. storage time at the different levels: primary, 6 months; region, 3 months; district, 1 month; health center, 1 month; health post, daily use – max. 1 month Diluents must never be frozen. Freeze-dried vaccines supplied packed with diluent must be stored between +2 and +8°C. Diluents supplied separately should be kept between +2 and +8°C Vaccine Storage Vaccines must be kept at the correct temperature since all are sensitive to heat and cold to some extent. All freeze-dried vaccines become much more heat-sensitive after they have been reconstituted. Vaccines sensitive to cold will lose potency if exposed to temperatures lower than optimal for their storage, particularly if they are frozen. Some vaccines (BCG, measles, MR, MMR, and rubella vaccines) are also sensitive to strong light and must always be protected against sunlight or fluorescent (neon) light. These vaccines are usually supplied in dark brown glass vials, which give them some protection against light damage, but they must still be covered and protected from strong light at all times. Only vaccine stocks that are fit for use should be kept in the vaccine cold chain. Expired or heat-damaged vials should be removed from cold storage. If unusable vaccines need to be kept for a period before disposal (e.g., until completion of accounting or auditing procedures) they should be kept outside the cold chain, separated from all usable stocks and carefully labelled to avoid mistaken use. Diluents Diluents for vaccines are less sensitive to storage temperatures than are the vaccines with which they are used (although they must be kept cool), but may be kept in the cold chain between +2 and +8°C if space permits. However, diluent vials must never be frozen (kept in a freezer or in contact with any frozen surface) as the vial may crack and become contaminated. When vaccines are reconstituted, the diluent should be at same temperature as the vaccine, so sufficient diluent for daily needs should be kept in the cold chain at the point of vaccine use (health center or vaccination post). At other levels of the cold chain (central, provincial, or district stores) it is only necessary to keep any diluent in the cold chain if it is planned to use it within the next 24 h. Freeze-dried vaccines and their diluents should always be distributed together in matching quantities. Although the diluents do not need to be kept in the cold chain (unless needed for reconstituting vaccines within the next 24 h), they must travel with the vaccine at all times, and must always be of the correct type, and from the same manufacturer as the vaccine that they are accompanying. Each vaccine requires a specific diluent, and therefore, diluents are not interchangeable (for example, diluent made for measles vaccine must not be used for reconstituting BCG, yellow fever, or any other type of vaccine). Likewise, diluent made by one manufacturer for use with a certain vaccine cannot be used for reconstituting the same type of vaccine produced by another manufacturer. Some combination vaccines comprise a freeze-dried component (such as Hib) which is designed to be reconstituted by a liquid vaccine (such as DTP or DTP-HepB liquid vaccine) instead of a normal diluent. For such combination vaccines, it is again vital that only vaccines manufactured and licensed for this purpose are combined. Note also that for combination vaccines where the diluent is itself a vaccine, all components must now be kept in the cold chain between +2 and +8°C at all times. As for all other freeze-dried vaccines, it is also essential that the "diluent" travels with the vaccine at all times. Effectiveness of Vaccination Programs The effectiveness of a vaccination program will need to be assessed. The program can be evaluated both by routinely collected data and, if necessary, by a survey of vaccination coverage. Routine data on coverage is obtained by comparing the numbers vaccinated with the estimated size of the target population (and clearly depends on accurate assessment of the latter). A coverage survey requires the use of a statistical technique called a two-stage cluster survey details of which can be found in the appropriate WHO/EPI documents. Information about the effectiveness of the campaign should be obtained from routine surveillance of communicable disease. If, for example, large number of measles cases continue to occur, or there is an outbreak, then data on coverage should be reex-amined. If this is shown to be good (over 90%) then the efficacy of the vaccine must be suspected. If the field efficacy is below the theoretical value 85% (for measles vaccine – data on efficacy of other vaccines can be obtained online) then possible causes of a breakdown in the vaccination program must be investigated (failure of the cold chain, poorly respected vaccination schedule). Methods for measuring vaccine efficacy can be found in the WHO/EPI literature. Chemoprophylaxis Mass chemoprophylaxis for bacterial infections such as cholera and meningitis is not usually recommended except on a small scale (for example, the use of Rifampicin may be considered to prevent the spread of meningococcal meningitis among immediate contacts of a case), but the difficulties of overseeing such activities and the risks of the development of antibiotic resistance outweigh any benefits that might be gained. The use of chemoprophylaxis for malaria must be undertaken with care. It may be indicated for vulnerable groups of refugees/IDPs (for example, children and pregnant women) arriving in an endemic area, particularly if they come from a nonmalarious area, but care must be taken to provide drugs to which the local strains of malaria are sensitive. The spread of resistance means that many of the standard drugs are ineffective and the replacements are both costly and may have unwanted side effects. Public Health Education Public health education and information activities play a vital role in disease prevention. Vaccination programs will not work unless there is acceptance by the public of the necessity for such programs. Individuals must be informed as to why these programs are necessary and also where and when they need to take their children for vaccination. Such activities are also essential to inform people about particular health programs (for example, feeding programs or vector control programs) and about the steps they can take to protect their health and that of their families (e.g., good hygiene). Information can be propagated in many ways: Posters Radio/TV/Film Lectures Songs/poems, etc. Leaflets Staff who are trained in this type of activity therefore play a key role in disease prevention. Heath education also requires transport and equipment (such as video or film projectors, screens, generators, blackboards, etc.). Provision of Appropriate Physical Conditions A key method of preventing communicable disease is the provision of shelter, adequate amounts of clean water, sufficient safe food, and proper sanitation (latrines and facilities for personal hygiene, clothes washing, and drying). Control of Disease Vectors Arthropod vectors (mosquitoes, ticks) can be controlled by appropriate spraying programs and also by habitat management (e.g., the removal of places where water can accumulate and mosquitoes breed). Provision of bed nets, particularly nets impregnated with insecticide, is effective for reducing infection with agents such as malaria and Leishmania . Control of rodents, by proper control of rubbish, by rodent proofing food stores, by attention to domestic hygiene and by use of rodenticides, will reduce the risks of transmission of rodent-borne diseases such as plague and Lassa fever. Disposal of Contaminated Materials Medical waste includes laboratory samples, needles and syringes, body tissues, and materials stained with body fluids. This requires careful handling, especially the sharps, as infectious agents such as those causing hepatitis B and C, HIV and AIDS, and viral hemorrhagic fevers can be transmitted by these materials. Used sharps should be disposed of into suitable containers (proper sharps boxes are ideal but old metal containers such as coffee or milk powder tins are adequate). Medical waste should ideally be burned in an incinerator. This should be close to the clinic or hospital but downwind of the prevailing wind. A 200-L oil drum can be used for this purpose with a metal grate half way up and a hole at the bottom to allow in air and for the removal of ash. Larger-scale and more permanent incinerators can be constructed if necessary. Burning pits can be used in emergency. If burning is not possible items should be buried at least 1.5 m deep. This is more suitable than burning for large items of human tissue such as amputated legs. Ensure there is no risk of groundwater contamination. Dealing with the Dead This is a complex process involving not just considerations of infection risk but also legal, sociocultural, and psychological factors. There are a number of specialist publications which can be of help. Health Aspects After almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. However, in sudden impact disasters (such as the Indian Ocean tsunami in 2004), the pattern and incidence of disease found in the dead will generally reflect those in the living. The situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. In fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. The diseased living are far more dangerous. The decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. Those most at risk are those handling the deceased, not the community. The most likely risks to them are as follows: Blood-borne viruses (Hepatitis B and C, HIV) Enteric pathogens (especially cholera) Respiratory pathogens (e.g. TB) Spore-forming bacteria (anthrax, tetanus) Some vector-borne diseases (plague, typhus) because the vectors may be present on the cadaver Acute hemorrhagic fevers (Ebola, Marburg, Lassa) Those handling cadavers should do the following: Take universal precautions for blood and body fluids Dispose of or disinfect used gloves Avoid contamination of personal items Wash hands after handling bodies and before eating Have hepatitis B vaccination Ensure disinfection of vehicles and equipment Mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. Cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. Alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. The dead must always be treated with dignity and respect. As far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. If the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. When an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). In general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). Bodies should be buried at least 1.5 m deep or, if more shallowly, should have earth piled at least 1 m above the ground level and 0.5 m to each side of the grave (to prevent access by scavengers and burrowing insects). Disinfectants such as chloride of lime should not be used. New burial sites should be at least 250 m from drinking water sources and at least 0.7 m above the saturated zone. Health Aspects After almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. However, in sudden impact disasters (such as the Indian Ocean tsunami in 2004), the pattern and incidence of disease found in the dead will generally reflect those in the living. The situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. In fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. The diseased living are far more dangerous. The decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. Those most at risk are those handling the deceased, not the community. The most likely risks to them are as follows: Blood-borne viruses (Hepatitis B and C, HIV) Enteric pathogens (especially cholera) Respiratory pathogens (e.g. TB) Spore-forming bacteria (anthrax, tetanus) Some vector-borne diseases (plague, typhus) because the vectors may be present on the cadaver Acute hemorrhagic fevers (Ebola, Marburg, Lassa) Those handling cadavers should do the following: Take universal precautions for blood and body fluids Dispose of or disinfect used gloves Avoid contamination of personal items Wash hands after handling bodies and before eating Have hepatitis B vaccination Ensure disinfection of vehicles and equipment Mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. Cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. Alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. The dead must always be treated with dignity and respect. As far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. If the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. When an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). In general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). Bodies should be buried at least 1.5 m deep or, if more shallowly, should have earth piled at least 1 m above the ground level and 0.5 m to each side of the grave (to prevent access by scavengers and burrowing insects). Disinfectants such as chloride of lime should not be used. New burial sites should be at least 250 m from drinking water sources and at least 0.7 m above the saturated zone. Vaccination Programs Vaccination programs are an essential part of disease prevention. Information about existing vaccination programs must be obtained during the assessment process and this should include information from external assessors (e.g., WHO, UNICEF, NGOs) as to the effectiveness of the vaccination programs that have been undertaken in the past. It cannot be assumed that simply because children have received vaccines that these vaccines were effective. Vaccination Priorities in Emergencies Measles kills large number of children in developing countries and is one of the greatest causes of morbidity and mortality in children in refugee and IDP camps. Mass vaccination of children between the ages of 6 months and 15 years should be an absolute priority during the first week of activity in humanitarian situations and can be conducted with the distribution of vitamin A. A system for maintaining measles immunization must be established once the target population has been covered adequately in the initial campaign. This is necessary to ensure that children who may have been missed in the original campaign, children reaching the age of 6 months, and children first vaccinated at the age of 6–9 months who must receive a second dose at 9 months of age are all covered. Some of the children vaccinated during such a mass campaign may have been vaccinated before. This does not matter and a second dose will have no adverse effect. It is essential to ensure full coverage against measles in the population. Other EPI vaccinations for children are not generally included in the emergency phase because they can only prevent a minor proportion of the overall morbidity and mortality at that stage. However, should specific outbreaks occur then the appropriate vaccine should be considered as a control measure. Vaccination programs require the following: Appropriate types of vaccines. Appropriate amounts of these vaccines. Equipment (needles, syringes, sterilization equipment, sharps disposal). Emergency immunization kits, including cold chain equipment, are available from a number of sources, including UNICEF and some NGOs (e.g. MSF). Logistics (transport, cold chain). Staff: a vaccination team may be quite large. It must include the following personnel: A supervisor. Logistics staff. Staff to prepare and administer vaccines. Record keepers. Security staff (to maintain order and control crowds) may also be needed. The Cold Chain Maintenance of the cold chain is particularly important. This is the system of transporting and storing vaccines within a suitable temperature range from the point of manufacture to the point of administration. The effectiveness of vaccines can be reduced or lost if they are allowed to get too cold, too hot, or are exposed to direct sunlight or fluorescent light. Careful note should be taken of the conditions needed to transport different vaccines because these can vary. The essential cold chain equipment needed to transport and store vaccines within a consistent safe temperature range includes the following: Dedicated refrigerators for storing vaccines and freezers for ice packs (fridges and freezers powered by gas or kerosene are available as alternatives to electric machines, and solar-powered fridge/freezer combinations specially designed for vaccine storage are also available) A suitable thermometer and a chart for recording daily temperature readings Cold boxes for transporting and storing vaccines Ice packs to keep vaccines cool Insulating material to separate ice packs from the vaccines when in the cold boxes (e.g., bubble wrap or expended polystyrene foam) If possible, vaccines should be stored in their original packaging because removing the packaging exposes them to room temperature and light. Check the temperature to ensure the vaccines have not been exposed to temperatures outside the normal storage ranges for those vaccines (see Table 13.4 ). Table 13.4. WHO-recommended storage conditions for different vaccines Vaccine Primary Region District/health center OPV −15 to −25°C −15 to −25°C +2 to +8°C Freeze-dried vaccines (BCG, measles, MMR, MR, yellow fever, Hib freeze dried) +2 to +8°C +2 to +8°C +2 to +8°C Other vaccines (HepB, DTP-HepB, Hib liquid, DTP, DT, T T, Td +2 to +8°C +2 to +8°C +2 to +8°C Max. storage time at the different levels: primary, 6 months; region, 3 months; district, 1 month; health center, 1 month; health post, daily use – max. 1 month Diluents must never be frozen. Freeze-dried vaccines supplied packed with diluent must be stored between +2 and +8°C. Diluents supplied separately should be kept between +2 and +8°C Vaccine Storage Vaccines must be kept at the correct temperature since all are sensitive to heat and cold to some extent. All freeze-dried vaccines become much more heat-sensitive after they have been reconstituted. Vaccines sensitive to cold will lose potency if exposed to temperatures lower than optimal for their storage, particularly if they are frozen. Some vaccines (BCG, measles, MR, MMR, and rubella vaccines) are also sensitive to strong light and must always be protected against sunlight or fluorescent (neon) light. These vaccines are usually supplied in dark brown glass vials, which give them some protection against light damage, but they must still be covered and protected from strong light at all times. Only vaccine stocks that are fit for use should be kept in the vaccine cold chain. Expired or heat-damaged vials should be removed from cold storage. If unusable vaccines need to be kept for a period before disposal (e.g., until completion of accounting or auditing procedures) they should be kept outside the cold chain, separated from all usable stocks and carefully labelled to avoid mistaken use. Diluents Diluents for vaccines are less sensitive to storage temperatures than are the vaccines with which they are used (although they must be kept cool), but may be kept in the cold chain between +2 and +8°C if space permits. However, diluent vials must never be frozen (kept in a freezer or in contact with any frozen surface) as the vial may crack and become contaminated. When vaccines are reconstituted, the diluent should be at same temperature as the vaccine, so sufficient diluent for daily needs should be kept in the cold chain at the point of vaccine use (health center or vaccination post). At other levels of the cold chain (central, provincial, or district stores) it is only necessary to keep any diluent in the cold chain if it is planned to use it within the next 24 h. Freeze-dried vaccines and their diluents should always be distributed together in matching quantities. Although the diluents do not need to be kept in the cold chain (unless needed for reconstituting vaccines within the next 24 h), they must travel with the vaccine at all times, and must always be of the correct type, and from the same manufacturer as the vaccine that they are accompanying. Each vaccine requires a specific diluent, and therefore, diluents are not interchangeable (for example, diluent made for measles vaccine must not be used for reconstituting BCG, yellow fever, or any other type of vaccine). Likewise, diluent made by one manufacturer for use with a certain vaccine cannot be used for reconstituting the same type of vaccine produced by another manufacturer. Some combination vaccines comprise a freeze-dried component (such as Hib) which is designed to be reconstituted by a liquid vaccine (such as DTP or DTP-HepB liquid vaccine) instead of a normal diluent. For such combination vaccines, it is again vital that only vaccines manufactured and licensed for this purpose are combined. Note also that for combination vaccines where the diluent is itself a vaccine, all components must now be kept in the cold chain between +2 and +8°C at all times. As for all other freeze-dried vaccines, it is also essential that the "diluent" travels with the vaccine at all times. Effectiveness of Vaccination Programs The effectiveness of a vaccination program will need to be assessed. The program can be evaluated both by routinely collected data and, if necessary, by a survey of vaccination coverage. Routine data on coverage is obtained by comparing the numbers vaccinated with the estimated size of the target population (and clearly depends on accurate assessment of the latter). A coverage survey requires the use of a statistical technique called a two-stage cluster survey details of which can be found in the appropriate WHO/EPI documents. Information about the effectiveness of the campaign should be obtained from routine surveillance of communicable disease. If, for example, large number of measles cases continue to occur, or there is an outbreak, then data on coverage should be reex-amined. If this is shown to be good (over 90%) then the efficacy of the vaccine must be suspected. If the field efficacy is below the theoretical value 85% (for measles vaccine – data on efficacy of other vaccines can be obtained online) then possible causes of a breakdown in the vaccination program must be investigated (failure of the cold chain, poorly respected vaccination schedule). Methods for measuring vaccine efficacy can be found in the WHO/EPI literature. Chemoprophylaxis Mass chemoprophylaxis for bacterial infections such as cholera and meningitis is not usually recommended except on a small scale (for example, the use of Rifampicin may be considered to prevent the spread of meningococcal meningitis among immediate contacts of a case), but the difficulties of overseeing such activities and the risks of the development of antibiotic resistance outweigh any benefits that might be gained. The use of chemoprophylaxis for malaria must be undertaken with care. It may be indicated for vulnerable groups of refugees/IDPs (for example, children and pregnant women) arriving in an endemic area, particularly if they come from a nonmalarious area, but care must be taken to provide drugs to which the local strains of malaria are sensitive. The spread of resistance means that many of the standard drugs are ineffective and the replacements are both costly and may have unwanted side effects. Public Health Education Public health education and information activities play a vital role in disease prevention. Vaccination programs will not work unless there is acceptance by the public of the necessity for such programs. Individuals must be informed as to why these programs are necessary and also where and when they need to take their children for vaccination. Such activities are also essential to inform people about particular health programs (for example, feeding programs or vector control programs) and about the steps they can take to protect their health and that of their families (e.g., good hygiene). Information can be propagated in many ways: Posters Radio/TV/Film Lectures Songs/poems, etc. Leaflets Staff who are trained in this type of activity therefore play a key role in disease prevention. Heath education also requires transport and equipment (such as video or film projectors, screens, generators, blackboards, etc.). Treatment Details of the treatment of individuals for various infectious diseases and the facilities needed are covered elsewhere in this book and in many textbooks covering disasters and disease response. In terms of the population aspects of the treatment of disease, important requirements are to ensure that there are appropriate laboratories (microbiological, parasitological, hematological, biochemical) available to confirm diagnoses and monitor treatment. adequate supplies of appropriate antimicrobial agents available and the facilities to transport these, store, and distribute them under appropriate conditions (e.g., controlled temperature), together with relevant instruction for use. Laboratories and Specimen Transport The provision of laboratory facilities in emergencies is usually limited to basic tests such as those for malaria. More advanced tests, including identification of microorganisms and the determination of antimicrobial sensitivities, require more sophisticated facilities. These may be available in the affected country but are unlikely to be operating in the disaster-affected area. It is more likely that specimens will have to be transported to laboratories abroad. Collection of specimens requires appropriate equipment. This will include items such as swabs, transport media, needles, syringes, or vacum sampling systems for blood sampling, different blood collection bottles (with and without anticoagulants) and other sterile specimen tubes, and containers for faeces and urine. Transporting specimens must be done safely, and packing specimens for shipment requiring specially trained personnel. Antimicrobials Treatment of disease requires good supplies of appropriate antimicrobial agents. It is important to ensure that the agents chosen are suitable for use in the area. It is common for doctors in affected areas to ask for the latest therapeutic agents. However, these agents, although effective, are often expensive and not part of the normal treatment programs in the region. The local doctors may not therefore be familiar with the use of these agents, nor may laboratories be capable of monitoring their use. It is better to use funds, which are often limited, to supply larger amounts of older (generic) agents. One caveat is the possibility that regular use may have allowed resistance to certain agents to develop in a country. Data on this may be available from local surveillance records. Antimicrobials should always be supplied with relevant guidelines in a language that can be understood locally. If local laboratories are unable to test microbes for resistance to antimicrobials, isolates or specimens should be sent as soon as possible to appropriate reference laboratories for testing. Response to Outbreaks and Epidemics Features Outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. An epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. An outbreak is an epidemic limited to a small area (a town, village, or camp). The term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. The areas where vaccination campaigns are a priority need to be identified and campaigns started. The term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. This will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. Infections where a single case represents a potential outbreak include the following: Cholera Some viral hemorrhagic fevers (Ebola, Marburg) Yellowfever Measles Plague Typhus Infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: Shigellosis Typhoid Hepatitis A Malaria Meningococcal meningitis Human African trypanosomiasis Visceral leishmaniasis A surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. However, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. In addition to the establishment of surveillance, outbreak preparation involves the following: Preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. Ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. Stockpiling essential supplies. This includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. Identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. Identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. Ensure that vaccination supplies (needles, syringes, etc.) are adequate. Make sure the cold chain can be maintained. Identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. Confirmation of the Outbreak If the number of reported cases is rising, is this in excess of the expected number? Ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. Verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). Laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. Outbreak Control Team In the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including MOH, WHO, other UN organizations, NGOs, etc. In the case of an epidemic the MOH will probably take the lead or may ask WHO or another UN agency to do so. The team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. This may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the MOH or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. This team should meet at least once a day to review the situation and define the necessary responses. It has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. The team should also act as the point of contact for the media. A media liaison officer should be appointed and all media contact should be through this individual. This will allow team members to refer media representatives to a central point and reduce interference with their activities. It will also ensure that a consistent message based on the most complete data is given to the media. Information The appropriate national authorities should be informed of the outbreak. In addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the Revised International Health Regulations (2005) to report outbreaks of certain diseases. These include four diseases regarded as public-health emergencies of international concern: Smallpox Polio (wild-type) New strains of human influenza Severe acute respiratory syndrome (SARS) In some cases, Member States must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and West Nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, Rift Valley fever, and meningococcal disease). Investigation Once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: Produce a case definition for the outbreak. This is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. Collect and analyze descriptive data by Time, Person, and Place (time and date of onset, individual characteristics of those affected – age, sex, occupation, etc., location of cases). Plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). Determine the population that is at risk. Determine the number of cases and the size of the affected population. Calculate the attack rate. Formulate hypotheses for the pathogen about the possible source and routes of transmission. Conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). Report results and make recommendations for action. Outbreak Investigations The two main statistical tools used to investigate outbreaks are as follows: Case–control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) Cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms However the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. Control Activities Implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) Prevent infection (e.g., by vaccination programs) Prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) Treat cases Evaluation Evaluate the outbreak detection and response – were they appropriate, timely, and effective? Change/modify policies and preparedness to deal with outbreaks if required What activities are needed to prevent similar outbreaks in the future (e.g., improved vaccination programs, new water treatment facilities, public health education, etc.)? Produce and disseminate an outbreak report. The report should include details of the outbreak, including the following: Cause Duration, location, and persons involved Cumulative attack rate (number of cases/exposed population) Incidence rate Case fatality rate Vaccine efficacy (if relevant) (no. of unvaccinated ill – no. of vaccinated ill/no. of unvaccinated ill) – Proportion of vaccine-preventable cases (no. of vaccine-preventable cases/no. of cases) Recommendations Laboratories and Specimen Transport The provision of laboratory facilities in emergencies is usually limited to basic tests such as those for malaria. More advanced tests, including identification of microorganisms and the determination of antimicrobial sensitivities, require more sophisticated facilities. These may be available in the affected country but are unlikely to be operating in the disaster-affected area. It is more likely that specimens will have to be transported to laboratories abroad. Collection of specimens requires appropriate equipment. This will include items such as swabs, transport media, needles, syringes, or vacum sampling systems for blood sampling, different blood collection bottles (with and without anticoagulants) and other sterile specimen tubes, and containers for faeces and urine. Transporting specimens must be done safely, and packing specimens for shipment requiring specially trained personnel. Antimicrobials Treatment of disease requires good supplies of appropriate antimicrobial agents. It is important to ensure that the agents chosen are suitable for use in the area. It is common for doctors in affected areas to ask for the latest therapeutic agents. However, these agents, although effective, are often expensive and not part of the normal treatment programs in the region. The local doctors may not therefore be familiar with the use of these agents, nor may laboratories be capable of monitoring their use. It is better to use funds, which are often limited, to supply larger amounts of older (generic) agents. One caveat is the possibility that regular use may have allowed resistance to certain agents to develop in a country. Data on this may be available from local surveillance records. Antimicrobials should always be supplied with relevant guidelines in a language that can be understood locally. If local laboratories are unable to test microbes for resistance to antimicrobials, isolates or specimens should be sent as soon as possible to appropriate reference laboratories for testing. Response to Outbreaks and Epidemics Features Outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. An epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. An outbreak is an epidemic limited to a small area (a town, village, or camp). The term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. The areas where vaccination campaigns are a priority need to be identified and campaigns started. The term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. This will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. Infections where a single case represents a potential outbreak include the following: Cholera Some viral hemorrhagic fevers (Ebola, Marburg) Yellowfever Measles Plague Typhus Infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: Shigellosis Typhoid Hepatitis A Malaria Meningococcal meningitis Human African trypanosomiasis Visceral leishmaniasis A surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. However, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. In addition to the establishment of surveillance, outbreak preparation involves the following: Preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. Ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. Stockpiling essential supplies. This includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. Identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. Identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. Ensure that vaccination supplies (needles, syringes, etc.) are adequate. Make sure the cold chain can be maintained. Identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. Confirmation of the Outbreak If the number of reported cases is rising, is this in excess of the expected number? Ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. Verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). Laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. Outbreak Control Team In the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including MOH, WHO, other UN organizations, NGOs, etc. In the case of an epidemic the MOH will probably take the lead or may ask WHO or another UN agency to do so. The team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. This may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the MOH or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. This team should meet at least once a day to review the situation and define the necessary responses. It has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. The team should also act as the point of contact for the media. A media liaison officer should be appointed and all media contact should be through this individual. This will allow team members to refer media representatives to a central point and reduce interference with their activities. It will also ensure that a consistent message based on the most complete data is given to the media. Information The appropriate national authorities should be informed of the outbreak. In addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the Revised International Health Regulations (2005) to report outbreaks of certain diseases. These include four diseases regarded as public-health emergencies of international concern: Smallpox Polio (wild-type) New strains of human influenza Severe acute respiratory syndrome (SARS) In some cases, Member States must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and West Nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, Rift Valley fever, and meningococcal disease). Investigation Once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: Produce a case definition for the outbreak. This is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. Collect and analyze descriptive data by Time, Person, and Place (time and date of onset, individual characteristics of those affected – age, sex, occupation, etc., location of cases). Plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). Determine the population that is at risk. Determine the number of cases and the size of the affected population. Calculate the attack rate. Formulate hypotheses for the pathogen about the possible source and routes of transmission. Conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). Report results and make recommendations for action. Outbreak Investigations The two main statistical tools used to investigate outbreaks are as follows: Case–control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) Cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms However the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. Control Activities Implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) Prevent infection (e.g., by vaccination programs) Prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) Treat cases Features Outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. An epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. An outbreak is an epidemic limited to a small area (a town, village, or camp). The term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. The areas where vaccination campaigns are a priority need to be identified and campaigns started. The term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. This will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. Infections where a single case represents a potential outbreak include the following: Cholera Some viral hemorrhagic fevers (Ebola, Marburg) Yellowfever Measles Plague Typhus Infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: Shigellosis Typhoid Hepatitis A Malaria Meningococcal meningitis Human African trypanosomiasis Visceral leishmaniasis A surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. However, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. In addition to the establishment of surveillance, outbreak preparation involves the following: Preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. Ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. Stockpiling essential supplies. This includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. Identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. Identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. Ensure that vaccination supplies (needles, syringes, etc.) are adequate. Make sure the cold chain can be maintained. Identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. Confirmation of the Outbreak If the number of reported cases is rising, is this in excess of the expected number? Ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. Verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). Laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. Outbreak Control Team In the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including MOH, WHO, other UN organizations, NGOs, etc. In the case of an epidemic the MOH will probably take the lead or may ask WHO or another UN agency to do so. The team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. This may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the MOH or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. This team should meet at least once a day to review the situation and define the necessary responses. It has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. The team should also act as the point of contact for the media. A media liaison officer should be appointed and all media contact should be through this individual. This will allow team members to refer media representatives to a central point and reduce interference with their activities. It will also ensure that a consistent message based on the most complete data is given to the media. Information The appropriate national authorities should be informed of the outbreak. In addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the Revised International Health Regulations (2005) to report outbreaks of certain diseases. These include four diseases regarded as public-health emergencies of international concern: Smallpox Polio (wild-type) New strains of human influenza Severe acute respiratory syndrome (SARS) In some cases, Member States must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and West Nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, Rift Valley fever, and meningococcal disease). Investigation Once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: Produce a case definition for the outbreak. This is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. Collect and analyze descriptive data by Time, Person, and Place (time and date of onset, individual characteristics of those affected – age, sex, occupation, etc., location of cases). Plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). Determine the population that is at risk. Determine the number of cases and the size of the affected population. Calculate the attack rate. Formulate hypotheses for the pathogen about the possible source and routes of transmission. Conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). Report results and make recommendations for action. Outbreak Investigations The two main statistical tools used to investigate outbreaks are as follows: Case–control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) Cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms However the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. Control Activities Implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) Prevent infection (e.g., by vaccination programs) Prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) Treat cases Evaluation Evaluate the outbreak detection and response – were they appropriate, timely, and effective? Change/modify policies and preparedness to deal with outbreaks if required What activities are needed to prevent similar outbreaks in the future (e.g., improved vaccination programs, new water treatment facilities, public health education, etc.)? Produce and disseminate an outbreak report. The report should include details of the outbreak, including the following: Cause Duration, location, and persons involved Cumulative attack rate (number of cases/exposed population) Incidence rate Case fatality rate Vaccine efficacy (if relevant) (no. of unvaccinated ill – no. of vaccinated ill/no. of unvaccinated ill) – Proportion of vaccine-preventable cases (no. of vaccine-preventable cases/no. of cases) Recommendations Epi Info™ 6 This is an easy-to-use tool which is of great value for handling epidemiological data and for organizing study designs and results, which can be downloaded free of charge from the Internet. It is produced by the Centers for Disease Control (Atlanta) and is a series of microcomputer programs which can be used both for surveillance and for outbreak investigation and includes features used by epidemiologists in statistical programs, such as SAS or SPSS, and database programs such as dBase.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413682/

Arginine-Specific Mono ADP-Ribosylation In Vitro of Antimicrobial Peptides by ADP-Ribosylating Toxins

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro . Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues. Introduction Human defensins are cationic multifunctional arginine-rich peptides (molecular masses ranging from 3.5 to 6 kDa) characterized by three intramolecular disulfide bridges that stabilize their structure [1] – [4] . Defensins display microbicidal activity against a wide spectrum of Gram-negative and Gram-positive bacteria, fungi and viruses [5] . They are also cytotoxic for epithelial cells and chemotactic for T-cells. Based on the presence of six conserved cysteine residues and sequence homology, human defensins are grouped into α- and β- defensins. The first group (α-defensins) includes human neutrophil peptides (HNP)-1 to 4, major components of the azurophilic granules of neutrophils, and two enteric human defensins, HD-5 and HD-6, isolated from the granules of Paneth cells in the small intestine, [6] . The second group (β-defensins), is mainly expressed in epithelial cells of various organs [7] – [9] . It has been shown that ADP-ribosylation of HNP-1 on arginine 14 reduces its antimicrobial and cytotoxic activities [10] . Mono ADP-ribosylation consists in the enzymatic transfer of the single ADP-ribose moiety of NAD to specific amino-acid residues of acceptor proteins coupled to the release of nicotinamide (nam) [11] . In mammals this reaction is catalyzed by a family of ADP-ribosyltransferases (ART1-5) [12] , [13] , while the best studied ADP-ribosylation reactions are those catalyzed by bacterial ADP-ribosylating toxins. The ADP-ribosylation of a large panel of host proteins catalyzed by bacterial toxins leads to the interruption of cellular metabolic and regulatory pathways causing severe diseases [14] . Vibrio cholerae toxin (CT) [15] , Escherichia coli heat labile enterotoxin (LT) [16] , Pseudomonas aeruginosa exoenzyme S (ExoS) [17] and the recently discovered NarE, a toxin-like protein from Neisseria meningitidis [18] , recognize arginine as an ADP-ribose acceptor in a similar fashion to ART1 and ART5 [13] , [19] . Arginine specificity is conferred to ARTs by the presence of the R-S-EXE triad signature in the active site [20] . Recent studies indicated that α-defensins display a novel biological function consisting in the ability to neutralize the activity of potent bacterial toxins like lethal factor, a metalloprotease produced by Bacillus anthracis [21] , and toxin B produced by Clostridium difficile [22] . Moreover it has been shown that HNP1-3 neutralize the cytotoxic effects exerted by diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), while they were inactive on CT and pertussis toxin (PT) [23] . The neutralization of toxins with selected amino-acid specificity prompted us to hypothesize that mono ADP-ribosylation of specific amino-acids may block defensin ability to inhibit the activities of toxins. Therefore, we evaluated whether HNP-1 could be recognized by arginine-specific bacterial ARTs. In the present paper we provide evidence that CT and LT ADP-ribosylated α- and β- defensins, which thus represent novel substrates for these bacterial ARTs. On the other hand, NarE and ExoS did not modify either α- or β- defensins. Interestingly, unmodified HNP-1 exerted inhibition on NarE transferase activity suggesting a regulatory role. While the ADP-ribosyltransferase activity was inhibited by HNP-1, the NAD-glycohydrolase (NADase) activity remained unaltered. Furthermore, HNP-1 strongly enhanced the auto-ADP-ribosylation of NarE, a recently discovered catalytic activity of this toxin. Overall, our data highlight the interplay between ADP-ribosylating toxins and human defensins. Results To establish whether arginine-specific bacterial ARTs can ADP-ribosylate HNP-1, we incubated HNP-1 with the catalytic A subunit of CT (CTA), LT (LTA), ExoS or NarE individually. As shown in Fig 1A , CTA and LTA catalyzed the transfer of the biotin-ADP-ribose from biotin-NAD to HNP-1 with an efficiency that was comparable to that of ART1 ( Fig. 1 B). This incorporation was strongly reduced after heat-inactivation of the toxins ( Fig. 1 A). CTA and LTA have both transferase, and NADase activity [24] , [16] . The latter produces ADP-ribose that can react non-enzymatically with lysine residues in proteins [25] . However, since the incorporation of biotin-ADP-ribose on HNP-1 was strongly reduced in the presence of 2 mM unlabelled NAD (200-fold excess) but not with 2 mM ADP-ribose, we could rule out that the reaction was non-enzymatic. The enzymatic nature of the reaction was further confirmed in the dose dependent ( Fig. 1 D) and time-course experiments ( Fig. 1 E), showing that the increase of modified peptide is dependent on the level of free substrate and by the incubation time. In this respect the purification grade of the toxins ( Fig. 1 C) is shown, to exclude the possibility of a blockage of the peptide by contaminating proteins. Under the same conditions, HNP-1 was a poor substrate for NarE ( Fig. 1 A) compared to ART1 ( Fig. 1 B). ExoS was completely inactive towards HNP-1 (data not shown), in agreement with a previous report [26] . ADP-ribosylation of antimicrobials by CT and LT is not restricted to HNP-1. Also HBD1, which contains only one arginine at position 29 and is constitutively expressed by epithelial cells in the airway [27] , was ADP-ribosylated ( Fig. 2 A). As for HNP-1, labelling did not occur in the presence of heat-inactivated toxins. The addition of an excess of unlabelled NAD to the reaction mixture decreased the incorporation of an ADP-ribose moiety on HBD1, while the incorporation of biotin-ADP-ribose on HBD1 was not reduced by the presence of 2 mM ADP-ribose. Dose-dependent reactions and time course experiments support the enzymatic nature of the modification also in the case of HBD1 ( Fig. 2 C, D). NarE and ExoS did not modify HBD1 (data not shown). In contrast with a previous report [26] , HBD1 was modified by ART1 to the same extent of HNP-1 ( Fig. 2 B). To confirm that the observed modifications corresponded to the addition of the ADP-ribose unit, the products of the reaction of CTA with HNP-1 in the presence of NAD were identified by MALDI-TOF MS. As shown in Fig. 3 , these included a peptide of 3442.12 Da, consistent with unmodified HNP-1 (theoretical mass: 3442.1 Da) and a peptide of 3983.15 Da. Although the amount of the modified peptide was low, we can conclude that the reaction is specific since we observed a mass increase consistent with mono ADP-ribosylated HNP-1 (theoretical mass: 3983.1 Da). Similar results were obtained with the LT catalyzed reaction (data not shown). To identify the preferred arginine residue of HNP-1 modified by CTA and LTA, we used two variants of HNP-1 in which a lysine replaced the arginines at positions 14 (HNP-1-R14K) or 15 (HNP-1-R15K). We found that CTA and LTA selectively ADP-ribosylated HNP-1 at R14 ( Fig. 4 ). Recent studies have shown that when HNP-1 is not recognized as a substrate, it is able to inhibit the ART activity of bacterial toxins such as ETA and DT [23] and also the eukaryotic ART5 multiple catalytic activities [26] . Therefore, since HNP-1 is only weakly modified by NarE, we investigated whether HNP-1 exerts a similar effect on NarE activities. The addition of HNP-1 to the reaction mixture seems to reduce the ADP-ribosyltransferase activity in a concentration dependent fashion ( Fig. 5 , grey bars) while the NADase activity was not greatly affected ( Fig. 5 , white bars). In contrast, HNP-1 enhanced the auto-ADP-ribosylation of NarE ( Fig. 6 upper panel), a recently discovered activity of this toxin (Picchianti et al. manuscript in preparation). 10.1371/journal.pone.0041417.g001 Figure 1 Modification of HNP-1 by selected ADP-ribosyltransferases. ( A ) HNP-1 is ADP-ribosylated by CTA and LTA but only weakly by NarE. HNP-1 (3 µg, 43.56 µM) was incubated with CTA (2.5 U), LTA (8.9 U) or NarE (2 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h (Toxin). The same reactions were performed with heat-inactivated toxins (HI-Toxin), in the presence of 2 mM NAD (Toxin + NAD), or 2 mM ADP-ribose (Toxin + ADP-ribose). The ADP-ribosylated peptides were resolved by SDS-PAGE in a 10% NuPAGE gel, using MES as running buffer and transferred to nitrocellulose. After blocking with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 h, the blot was incubated with streptavidin-HRP conjugated (1∶10000 dilution) for 1 h at RT in the same buffer. The biotin-ADP-ribose labeled bands were visualized by chemiluminescence. ( B ) ART1 ADP-ribosylated HNP-1. HNP-1 (3 µg, 43.56 µM) was incubated with ART1 (6.8 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate, pH 7.5 at 30°C for 1 h (ART1). A control reaction with heat-inactivated ART1 is also shown (HI-ART1). ( C ) SDS-PAGE analysis of the purification grade of 2 µg each of CTA, LTA and NarE. ( D ) HNP-1 is ADP-ribosylated in a dose and response dependent manner by CTA and LTA. HNP-1 at the concentration shown in the Figure was incubated with CTA (2.5 U), or LTA (8.9 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. ( E ) HNP-1 is ADP-ribosylated in time dependent fashion. HNP-1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described for the times of incubation indicated in the Figure. Molecular markers are on the left. Data shown are representative of several experiments performed in the same conditions. 10.1371/journal.pone.0041417.g002 Figure 2 Modification of β -defensin by selected ADP-ribosyltransferases. ( A ) HBD1 is ADP-ribosylated by CTA and LTA. HBD1 (3 µg, 38.18 µM)) was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h (Toxin). Reactions were also performed in the presence of 2 mM NAD (Toxin + NAD) or 2 mM ADP-ribose (Toxin + ADP-ribose). Control reactions performed with heat-inactivated CTA or LTA (HI-Toxin) or in the absence of toxins (-Toxin) are also shown. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. The membrane was treated as previously described, incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. ( B ) ADP-ribosylation of HBD1 by ART1. HBD1 (3 µg, 38.18 µM) was incubated with 6.8 U of ART1 (ART1) or heat-inactivated ART1 (HI-ART1) and 10 µM biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. ( C ) HBD1 is ADP-ribosylated in a dose response fashion. HBD1 at the concentration shown in the Figure was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. ( D ) Time dependent ADP-ribosylation of HBD1. HBD1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described. Times of incubation are indicated in the Figure. Molecular markers are on the left. Data shown are representative of two independent experiments. 10.1371/journal.pone.0041417.g003 Figure 3 MALDI-TOF mass spectra of HNP-1 reaction with CTA. Mass spectra analysis confirmed the mono-ADP-ribosylation of HNP-1 by CTA after incubation at 30°C for 1h in the presence of 2 mM NAD. Upper panels (left side: spectrum of m/z 2500 – 5300, right side: zoomed spectrum of m/z 3800 – 4300) show the mass of the control reaction, i.e. HNP-1 incubated only with NAD without toxin ( m/z 3442.12). Lower panels (left side: spectrum of m/z 2500 – 5300, right side: zoomed spectrum of m/z 3800–4300) represent the unmodified HNP-1 peptide and the product of ADP-ribosylation peptide by CTA ( m/z 3983.15). Stars (*) correspond to Sinapinic Acid adducts (+206 Da). 10.1371/journal.pone.0041417.g004 Figure 4 HNP-1 is ADP-ribosylated at R14. HNP-1 R14K and HNP-1 R15K protein variants (3 µg, 43.85 µM) were incubated with CTA (2.5 U), LTA (8.9 U) or ART1 (5.5 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. Membranes treated as previously described, were incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. Here shown in comparison with the modification of HNP-1 wild-type in the same reaction conditions. 10.1371/journal.pone.0041417.g005 Figure 5 HNP-1 inhibited the ADP-ribosyltransferase, but not the NAD-glycohydrolase activity of NarE. NarE (20 µg) was incubated with 0.1 mM NAD [ adenine -U- 14 C]NAD (0.05 µCi), in 50 mM potassium phosphate buffer, pH 7.5, 20 mM agmatine as an ADP-ribose acceptor at 30°C for 18 h in the presence of the indicated concentrations of HNP-1. The synthesized ADP-ribosylagmatine was purified through Dowex AG 1-X2 and the incorporated radioactivity was eluted in water and counted in a counter (grey bars). To monitor the NADase activity (white bars), similar reactions in the absence of agmatine and with 0.1 mM [ carbonyl - 14 C]NAD (0.05 µCi) replacing [ adenine -U- 14 C]NAD (0.05 µCi) were performed. The radioactive nam was eluted in water in Dowex AG 1-X2 and counted. Values represent the means ± S.D. of two independent assays. 10.1371/journal.pone.0041417.g006 Figure 6 HNP-1 enhanced the auto-ADP-ribosylation of NarE. Purified NarE (0.4 µg) was auto-ADP-ribosylated with 10 µM biotin-NAD in 50 mM potassium phosphate pH 7.5 at 30°C for 3 and 6 h in the presence of the indicated concentrations of HNP-1. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. To detect biotin-ADP-ribosylated NarE, the membrane was incubated with streptavidin-HRP (upper panel). For western blotting the nitrocellulose membrane was incubated with rabbit polyclonal α-NarE (1∶10000 dilution) and with α-rabbit HRP-conjugated (lower panel). Labeled bands were detected by chemioluminescence. Data shown are representative of two independent experiments. Discussion ADP-ribosylating toxins are usually secreted by bacterial pathogens in the host environment. Some of them, which possess arginine-specificity, could recognize arginine-rich peptides such as α- and β- defensins as substrates. Both α- and β- defensins are released by neutrophils and epithelial cells respectively in high amounts at inflammatory sites. In this report we present evidence that synthetic HNP-1 and HBD1 are ADP-ribosylated in vitro by CTA and LTA. In contrast they are not recognized as substrates by ExoS and only poorly by NarE, suggesting specificity for both bacterial toxins and substrates. The artificial kemptide (PKA peptide substrate), which contains a di-arginine motif, was modified by CT on the first arginine of the motif while a mammalian ART recognized the second arginine within the R-R motif [28] , [29] . In contrast, our data indicate R14 as the preferred modification site, since the HNP-1-R14K was not ADP-ribosylated by the ADP-ribosylating toxins used in this study. Our findings are in contrast with studies performed by other groups, which failed to show toxin-catalyzed incorporation of the ADP-ribose unit on defensins [23] , [26] . Others evaluated the presence of the ADP-ribosylated-HNP-1 by monitoring the absorbance of the modified peptide in reversed-phase chromatography, but not being successful in identifying it [23] , [26] . Therefore we chose a chemiluminescence assay to detect ADP-ribosylation because of the higher sensitivity, allowing the detection of small amounts of modified HNP-1. In agreement with previous findings we did not observe incorporation of ADP-ribose with PT [23] and ExoS [26] . Labelling of proteins can also result from the covalent non-catalyzed reaction of NAD [30] or free ADP-ribose with the ε-amino groups of lysines [25] . ADP-ribosylation of HNP-1, in which lysine residues are absent ( Table 1 ), was not blocked by the addition of free ADP-ribose, while a reduction of incorporation was noticed when unlabelled NAD was added to the reaction mixture. Comparable results were obtained with HBD1 that contains four lysine residues. These data, further supported by mass spectrometry analysis, strongly indicate that an enzymatic ADP-ribosylation, and not a secondary reaction with NAD or ADP-ribose, was responsible for the modification. Defensins belonging to α- and β- group contain several conserved arginines ( Table 1 ), which are recognized by CT and LT. However they are devoid of diphtamide and asparagine residues, which are the target amino-acid of DT, ETA and clostridial toxins. Furthermore, cysteines, present in α- and β- defensins and recognized by PT as ADP-ribose acceptors, are engaged in disulphide bridges. It is well known that defensins have a variety of activities, but the antimicrobial function is by far the most important. Thus ADP-ribosylation of selected arginines might well correlate with recent discoveries, which show that antibacterial activity strictly depends on cationicity [31] and that only selective arginines support this activity [32] . Interestingly, toxic activities are not decreased in the case of CT [23] , which ADP-ribosylates HNP-1 at an arginine residue. By contrast, HNP-1, which is devoid of the target amino-acids is not modified by ETA and DT, thus protecting cells from DT- or ETA-mediated cell death [23] . Besides its antimicrobial activity, several lines of evidence suggest an additional regulatory role for HNP-1 when it is not recognized as a substrate, as described for ART5 and ART1 [26] . Here we showed that HNP-1 is able to reduce NarE transferase activity. This reduction is more evident at high concentrations of HNP-1, likely to be present in the site of inflammation. Of note the NADase activity, a reaction not usually involved in the toxicity process, is not affected. On the other hand, auto-ADP-ribosylation, which could be an intramolecular mechanism regulating the two activities (Picchianti et al. manuscript in preparation) is enhanced. Although physiological substrates for CT and LT are well known and the extent of modification is limited, members of the antimicrobial peptide family may serve as novel substrates for these ADP-ribosylating toxins. At the onset of infection, bacterial pathogens have evolved different countermeasures to limit the effectiveness of antimicrobials [33] and to counteract the immune system. The modification of antimicrobial components of the innate immune system by bacterial ADP-ribosylating toxins may represent a mechanism that could facilitate bacterial colonization. In the context of the primary immune response, the ADP-ribosylation of HNP-1 may affect both the anti-microbial weapons released by neutrophils and the interplay between inflammatory cells, eventually facilitating the onset of an infectious disease. Hence, our study showing arginine-specific ADP-ribosylation of human defensins catalyzed by some bacterial toxins may be relevant in the onset of infectious diseases. 10.1371/journal.pone.0041417.t001 Table 1 Human defensins. Human defensins α DEF1_HUMAN Neutrophil defensin 1 ACYC R IPACIAGE RR YGTCIYQG R LWAFCC DEF2_HUMAN Neutrophil defensin 2 CYC R IPACIAGE RR YGTCIYQG R LWAFCC DEF3_HUMAN Neutrophil defensin 3 DCYC R IPACIAGE RR YGTCIYQG R LWAFCC DEF4_HUMAN Neutrophil defensin 4 VCSC R LVFC RR TEL R VGNCLIGGVSFTYCCT R VD DEF5_HUMAN Neutrophil defensin 5 ATCYC R TG R CAT R ESLSGVCEISG R LY R LCC R DEF6_HUMAN Neutrophil defensin 6 TCHC RR SCYSTEYSYGTCTVMGINH R FCCL β DEFB1_HUMAN β-defensin 1 DHYNCVSSGGQCLYSACPIFTKIQGTCY R GKAKCCK DEFB2_HUMAN β-defensin 2 GIGDPVTCLKSGAICHPVFCP RR YKQIGTCGLPGTKCCKKP DEFB3_HUMAN β-defensin 3 GIINTLQKYYC R V R GG R CAVLSCLPKEEQIGKCST R G R KCC RR KK DEFB4_HUMAN β-defensin 4 ELD R ICGYGTA R C R KKC R SQEY R IG R CPNTYACCL R K Materials and Methods Reagents [ adenine -U- 14 C]NAD (274 mCi/mmol) and [ carbonyl - 14 C]NAD (53 mCi/mmol) were purchased from Amersham (Glattbrugg, Switzerland); Dowex AG 1-X2, the Bradford reagent for protein quantification and the immunoblotting detection system were purchased from Bio-Rad (Hercules, CA), while standard bovine serum albumin (BSA) was obtained from Pierce (Rockford, IL). Isopropyl-1-thio-β-D-galactopyranoside was purchased from Calbiochem (Darmstadt, Germany). SimplyBlue SafeStain was ordered from Invitrogen (Carlsbad, CA), 6-biotin-17-NAD from Trevigen (Gaithersburg, MD), synthetic defensins from Bachem (Weil am Rheim, Germany) and streptavidin HRP-conjugate from SouthernBiothech (Birmingham, AL). All other reagents used in this study were from Sigma Aldrich (Saint Louis, MO). Preparation of ART1 and bacterial toxins Chinese hamster fibroblast V79 cells transfected with pTet-ON-ART1 cDNA were kindly provided by Dr. J. Mac Dermot [34] . Human ART1 was synthesized as a GPI-linked protein on the surface of V79 cells after induction for 48 h with 2 µg/ml final concentration of doxycicline. The soluble form of ART1 was collected following treatment of intact V79 cells with 1 U/ml of phosphatidylinositol-specific phospholipase C for 1 hr at 37°C [34] . NarE was produced as previously described [18] . Briefly, the NarE gene cloned in the pET21b+ expression vector with a carboxyl-terminal 6× histidine tag was used to transform E. coli BL21 (DE3) competent cells (Invitrogen). Transformed cells were grown overnight in LB broth at 37°C with gentle shaking (180 rpm) and protein synthesis occurred after addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 3 h at 25°C. Following induction, bacteria were harvested, centrifuged and lysed in B-PER (bacterial-protein extraction reagent, Pierce) in the presence of Mg ++ , DNase, lysozyme and phenyl-methyl sulfonyl fluoride (PMSF) as a protease inhibitor. After centrifugation to discard debris and membranes, the soluble fraction was loaded on a Ni-NTA (Pharmacia, Biotech, Stockholm, Sweden) affinity resin and the protein was eluted according to manufacturer's instructions. CTA was purchased from Sigma Aldrich, while LTA was cloned and purified by Drs. Paolo Ruggiero and Laura Pancotto (Novartis Vaccines & Diagnostics, Siena Italy). Since all of the above toxins need sulphydril agents like dithiothreitol (DTT) to exert full activity, we performed their activation as previously described [35] , [36] . Since DTT causes reduction of disulphide bridges causing linearization of HNP-1 and facilitating access of ADP-ribose to arginine acceptors, the activation mixture was extensively dialyzed in the same buffer lacking DTT using Microcon centrifugal filter devices (Amicon, Houston, TX). Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase (transferase domain) and its activator FAS (Factor activating exoenzyme S) were kindly provided by Dr. Joseph Barbieri (Medical College of Wisconsin, Milwaukee WI). ADP-ribosyltransferase enzymatic assay Some of the ADP-ribosylating enzymes used in this study ART1, CTA, LTA, and NarE were tested by monitoring the transfer of ADP-ribose to agmatine using a standard assay [37] . The assay was carried out in a final volume of 0.3 ml containing 50 mM potassium phosphate, pH 7.5, 20 mM agmatine and 0.1 mM [ adenine -U- 14 C]NAD (0.05 µCi). After incubation at 30°C, duplicate samples (100 µl each) were applied to 1 ml columns of Dowex AG 1-X2. [ adenine- 14 C]ADP-ribosylagmatine was eluted for radio assay with 5 ml of H 2 O and the radioactivity counted in a Packard mod counter. The ART1 fraction exerted an activity of 6.8 nmol/h (U), while the activated CTA and LTA showed values of 2.5 nmol/h (U) and 1.8 nmol/h (U) respectively. NarE 0.9 nmol/h, Uwas used shortly after purification. Specific activities of the toxins used were 2.5 nmol/mg/h for CTA, 6.25 nmol/mg/h for LTA and 0.4 nmol/mg/h for NarE. ExoS activity was monitored using an auto-ADP-ribosylation assay in the presence of FAS ligand [38] . NAD-glycohydrolase assay An analogous assay based on nam release was employed to measure the NADase activity of NarE [24] . This assay was carried out in the same conditions described above for the transferase assay with 0.1 mM [ carbonyl - 14 C]NAD (0.05 µCi) replacing [ adenine -U- 14 C]NAD and without agmatine as ADP-ribose acceptor. Detection of biotinylated ADP-ribose-HNP-1 and immunoblot analysis HNP-1 (3 µg, 43,56 µM) and HBD1 (3 µg, 38.18 µM) were ADP-ribosylated for 1 hour at 30°C in 50 mM potassium phosphate buffer (pH 7.4), with ART1, CTA, LTA, or NarE and 10 µM of biotinylated-NAD (biotin-NAD) in a final volume of 20 µl. In control experiments 2 mM NAD or 2 mM ADP-ribose were added individually to each assay mixture. In negative controls the ADP-ribosylating toxin was omitted from the reaction mixture or heat-inactivated. The ADP-ribosylated peptides were resolved by SDS-PAGE in a 10% NuPAGE gel, using MES as running buffer and transferred to nitrocellulose using a dry system apparatus (I-Blot, Invitrogen). The membrane was blocked for 1 h with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T), extensively washed and then incubated in the same buffer containing streptavidin-HRP conjugated (1∶10000 dilution) and mixed for 1 h on a nutator at room temperature (RT). After several washings with PBS-T, bound streptavidin was detected using an ECL immunoblotting detection system (Bio-Rad) according to the manufacturer's instructions. Molecular masses were estimated from the calibration standard included in each gel. Mass-Spectrometry analysis of ADP-ribosylated-HNP-1 Peptide molecular masses were determined using a MALDI-TOF/TOF mass spectrometer UltraFlex (Bruker Daltonics, Bremen, GmbH). Ions generated by laser desorption at 337 nm (N 2 laser) were recorded at an acceleration voltage of 20 kV in linear mode. In general, about 200 single spectra were accumulated to improve the signal/noise ratio and analyzed by FlexAnalysis version 2.4 (Bruker Daltonics). Briefly, 1 µl of reaction solution (20–60 pmoles) was added to 1 µl of a saturated solution of sinapinic acid (3,5-dimethoxy-4-hydroxy-trans-cinnamic acid) in 30% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA). Then 2 µl of peptide/matrix mixture was spotted on a stainless steel sample target and air-dried at room temperature. Peptide mass spectra were calibrated using external peptide calibration standard (Bruker Daltonics). Protein assay Protein content was determined by the Bradford protein assay kit (Bio-Rad) using BSA for standardization. Reagents [ adenine -U- 14 C]NAD (274 mCi/mmol) and [ carbonyl - 14 C]NAD (53 mCi/mmol) were purchased from Amersham (Glattbrugg, Switzerland); Dowex AG 1-X2, the Bradford reagent for protein quantification and the immunoblotting detection system were purchased from Bio-Rad (Hercules, CA), while standard bovine serum albumin (BSA) was obtained from Pierce (Rockford, IL). Isopropyl-1-thio-β-D-galactopyranoside was purchased from Calbiochem (Darmstadt, Germany). SimplyBlue SafeStain was ordered from Invitrogen (Carlsbad, CA), 6-biotin-17-NAD from Trevigen (Gaithersburg, MD), synthetic defensins from Bachem (Weil am Rheim, Germany) and streptavidin HRP-conjugate from SouthernBiothech (Birmingham, AL). All other reagents used in this study were from Sigma Aldrich (Saint Louis, MO). Preparation of ART1 and bacterial toxins Chinese hamster fibroblast V79 cells transfected with pTet-ON-ART1 cDNA were kindly provided by Dr. J. Mac Dermot [34] . Human ART1 was synthesized as a GPI-linked protein on the surface of V79 cells after induction for 48 h with 2 µg/ml final concentration of doxycicline. The soluble form of ART1 was collected following treatment of intact V79 cells with 1 U/ml of phosphatidylinositol-specific phospholipase C for 1 hr at 37°C [34] . NarE was produced as previously described [18] . Briefly, the NarE gene cloned in the pET21b+ expression vector with a carboxyl-terminal 6× histidine tag was used to transform E. coli BL21 (DE3) competent cells (Invitrogen). Transformed cells were grown overnight in LB broth at 37°C with gentle shaking (180 rpm) and protein synthesis occurred after addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 3 h at 25°C. Following induction, bacteria were harvested, centrifuged and lysed in B-PER (bacterial-protein extraction reagent, Pierce) in the presence of Mg ++ , DNase, lysozyme and phenyl-methyl sulfonyl fluoride (PMSF) as a protease inhibitor. After centrifugation to discard debris and membranes, the soluble fraction was loaded on a Ni-NTA (Pharmacia, Biotech, Stockholm, Sweden) affinity resin and the protein was eluted according to manufacturer's instructions. CTA was purchased from Sigma Aldrich, while LTA was cloned and purified by Drs. Paolo Ruggiero and Laura Pancotto (Novartis Vaccines & Diagnostics, Siena Italy). Since all of the above toxins need sulphydril agents like dithiothreitol (DTT) to exert full activity, we performed their activation as previously described [35] , [36] . Since DTT causes reduction of disulphide bridges causing linearization of HNP-1 and facilitating access of ADP-ribose to arginine acceptors, the activation mixture was extensively dialyzed in the same buffer lacking DTT using Microcon centrifugal filter devices (Amicon, Houston, TX). Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase (transferase domain) and its activator FAS (Factor activating exoenzyme S) were kindly provided by Dr. Joseph Barbieri (Medical College of Wisconsin, Milwaukee WI). ADP-ribosyltransferase enzymatic assay Some of the ADP-ribosylating enzymes used in this study ART1, CTA, LTA, and NarE were tested by monitoring the transfer of ADP-ribose to agmatine using a standard assay [37] . The assay was carried out in a final volume of 0.3 ml containing 50 mM potassium phosphate, pH 7.5, 20 mM agmatine and 0.1 mM [ adenine -U- 14 C]NAD (0.05 µCi). After incubation at 30°C, duplicate samples (100 µl each) were applied to 1 ml columns of Dowex AG 1-X2. [ adenine- 14 C]ADP-ribosylagmatine was eluted for radio assay with 5 ml of H 2 O and the radioactivity counted in a Packard mod counter. The ART1 fraction exerted an activity of 6.8 nmol/h (U), while the activated CTA and LTA showed values of 2.5 nmol/h (U) and 1.8 nmol/h (U) respectively. NarE 0.9 nmol/h, Uwas used shortly after purification. Specific activities of the toxins used were 2.5 nmol/mg/h for CTA, 6.25 nmol/mg/h for LTA and 0.4 nmol/mg/h for NarE. ExoS activity was monitored using an auto-ADP-ribosylation assay in the presence of FAS ligand [38] . NAD-glycohydrolase assay An analogous assay based on nam release was employed to measure the NADase activity of NarE [24] . This assay was carried out in the same conditions described above for the transferase assay with 0.1 mM [ carbonyl - 14 C]NAD (0.05 µCi) replacing [ adenine -U- 14 C]NAD and without agmatine as ADP-ribose acceptor. Detection of biotinylated ADP-ribose-HNP-1 and immunoblot analysis HNP-1 (3 µg, 43,56 µM) and HBD1 (3 µg, 38.18 µM) were ADP-ribosylated for 1 hour at 30°C in 50 mM potassium phosphate buffer (pH 7.4), with ART1, CTA, LTA, or NarE and 10 µM of biotinylated-NAD (biotin-NAD) in a final volume of 20 µl. In control experiments 2 mM NAD or 2 mM ADP-ribose were added individually to each assay mixture. In negative controls the ADP-ribosylating toxin was omitted from the reaction mixture or heat-inactivated. The ADP-ribosylated peptides were resolved by SDS-PAGE in a 10% NuPAGE gel, using MES as running buffer and transferred to nitrocellulose using a dry system apparatus (I-Blot, Invitrogen). The membrane was blocked for 1 h with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T), extensively washed and then incubated in the same buffer containing streptavidin-HRP conjugated (1∶10000 dilution) and mixed for 1 h on a nutator at room temperature (RT). After several washings with PBS-T, bound streptavidin was detected using an ECL immunoblotting detection system (Bio-Rad) according to the manufacturer's instructions. Molecular masses were estimated from the calibration standard included in each gel. Mass-Spectrometry analysis of ADP-ribosylated-HNP-1 Peptide molecular masses were determined using a MALDI-TOF/TOF mass spectrometer UltraFlex (Bruker Daltonics, Bremen, GmbH). Ions generated by laser desorption at 337 nm (N 2 laser) were recorded at an acceleration voltage of 20 kV in linear mode. In general, about 200 single spectra were accumulated to improve the signal/noise ratio and analyzed by FlexAnalysis version 2.4 (Bruker Daltonics). Briefly, 1 µl of reaction solution (20–60 pmoles) was added to 1 µl of a saturated solution of sinapinic acid (3,5-dimethoxy-4-hydroxy-trans-cinnamic acid) in 30% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA). Then 2 µl of peptide/matrix mixture was spotted on a stainless steel sample target and air-dried at room temperature. Peptide mass spectra were calibrated using external peptide calibration standard (Bruker Daltonics). Protein assay Protein content was determined by the Bradford protein assay kit (Bio-Rad) using BSA for standardization.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4251056/

Divergence of protein-coding capacity and regulation in the Bacillus cereus sensu lato group

Background The Bacillus cereus sensu lato group contains ubiquitous facultative anaerobic soil-borne Gram-positive spore-forming bacilli. Molecular phylogeny and comparative genome sequencing have suggested that these organisms should be classified as a single species. While clonal in nature, there do not appear to be species-specific clonal lineages, excepting B. anthracis , in spite of the wide array of phenotypes displayed by these organisms. Results We compared the protein-coding content of 201 B. cereus sensu lato genomes to characterize differences and understand the consequences of these differences on biological function. From this larger group we selected a subset consisting of 25 whole genomes for deeper analysis. Cluster analysis of orthologous proteins grouped these genomes into five distinct clades. Each clade could be characterized by unique genes shared among the group, with consequences for the phenotype of each clade. Surprisingly, this population structure recapitulates our recent observations on the divergence of the generalized stress response (SigB) regulons in these organisms. Divergence of the SigB regulon among these organisms is primarily due to the placement of SigB-dependent promoters that bring genes from a common gene pool into/out of the SigB regulon. Conclusions Collectively, our observations suggest the hypothesis that the evolution of these closely related bacteria is a consequence of two distinct processes. Horizontal gene transfer, gene duplication/divergence and deletion dictate the underlying coding capacity in these genomes. Regulatory divergence overlays this protein coding reservoir and shapes the expression of both the unique and shared coding capacity of these organisms, resulting in phenotypic divergence. Data from other organisms suggests that this is likely a common pattern in prokaryotic evolution. Background The Bacillus cereus sensu lato group contains ubiquitous facultative anaerobic soil-borne Gram-positive spore-forming bacilli. Molecular phylogeny and comparative genome sequencing have suggested that these organisms should be classified as a single species. While clonal in nature, there do not appear to be species-specific clonal lineages, excepting B. anthracis , in spite of the wide array of phenotypes displayed by these organisms. Results We compared the protein-coding content of 201 B. cereus sensu lato genomes to characterize differences and understand the consequences of these differences on biological function. From this larger group we selected a subset consisting of 25 whole genomes for deeper analysis. Cluster analysis of orthologous proteins grouped these genomes into five distinct clades. Each clade could be characterized by unique genes shared among the group, with consequences for the phenotype of each clade. Surprisingly, this population structure recapitulates our recent observations on the divergence of the generalized stress response (SigB) regulons in these organisms. Divergence of the SigB regulon among these organisms is primarily due to the placement of SigB-dependent promoters that bring genes from a common gene pool into/out of the SigB regulon. Conclusions Collectively, our observations suggest the hypothesis that the evolution of these closely related bacteria is a consequence of two distinct processes. Horizontal gene transfer, gene duplication/divergence and deletion dictate the underlying coding capacity in these genomes. Regulatory divergence overlays this protein coding reservoir and shapes the expression of both the unique and shared coding capacity of these organisms, resulting in phenotypic divergence. Data from other organisms suggests that this is likely a common pattern in prokaryotic evolution. Backgound The Bacillus cereus sensu lato group contains a variety of facultative anaerobic soil-borne Gram-positive spore-forming bacilli that are ubiquitous in nature. This group consists of at least seven species ( B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus ) that new molecular phylogenies and comparative genome sequencing have suggested should be classified as a single species [ 1 ]. These various species are phylogenetically interspersed among one another in a variety of phylogenies. While clonal in nature, there do not appear to be species-specific clonal lineages in this assemblage, with the exception of the B. anthracis lineage [ 2 ]. Paradoxically, these organisms display a wide array of biological behaviors, despite their close taxonomic and phylogenetic relationship. B. anthracis is the cause of the acute and often lethal disease anthrax that can infect a wide variety of mammalian hosts, with differing virulence characteristics [ 3 ]. B. thuringiensis is a useful source of insecticidal toxins, often in the form of spore-containing preparations of crystal protein toxins ( cry toxins); however, some B. thuringiensis strains have been isolated from severe human infections [ 4 - 6 ]. B. cereus is often isolated as an opportunistic pathogen, and causes contamination problems in the dairy industry and paper mills [ 7 ]. Food poisoning isolates rarely are associated with invasive disease, and appear predominantly to cause enterointoxication without overt colonization. Other B. cereus strains are part of the normal gut microflora [ 4 ]. B. weihenstephanensis is a psychrophile, as are several B. cereus strains suggested to actually be B. weihenstephanensis [ 8 ]. Recent observations have suggested the addition of two additional species to the B. cereus sensu lato group, tentatively designated as B. gaemokensis and B. manlipoensis [ 9 ]. However, little is currently known about the genomic basis for the taxonomic affiliation of these newly described organisms. Studies seeking to understand the underlying basis of these varied phenotypes have often focused on the complement of extrachromosomal elements harbored by these organisms, with good reason [ 10 ]. The capacity to cause invasive toxigenic disease by B. anthracis is intimately rooted in the presence of the anthracis virulence plasmids pXO1 and pXO2 [ 11 ]. Similarly, the only clearly established differences between B. cereus and B. thuringiensis strains is the presence of genes coding for the cry toxins, typically plasmid-borne, although sometimes chromosomally encoded [ 6 ]. If the cry plasmids are lost, B. thuringiensis can no longer be distinguished from B. cereus [ 4 ]. The origins of virulent anthrax-like strains from multiple nonpathogenic ancestors offer an opportunity to better understand the origins of pathogenicity in this Bacillus group. It has been proposed that pathogens in this group have high virulence potential, but that their origin is limited solely by the chance horizontal gene transfer (HGT) of toxin-expressing plasmids [ 11 ]. Alternatively, it has also been proposed that multiple historical origins of pathogenic strains have occurred because of existing pre-adaptations or newly arising adaptive changes in the genomes of nonpathogenic ancestors [ 12 ]. The reconciliation of contradictory relationships exhibited by members of this phylogenetic group of species is still ongoing [ 10 ]. Some studies have clearly demonstrated that chromosomal-encoded determinants, such as plcR and enterotoxins play an important role in biological behavior [ 13 ]. Zwick et al . examined pathogenic and non-pathogenic B. cereus sensu lato draft and whole genome sequences to test whether B. anthracis biovar anthracis CI, or other similar strains were unique in terms of the gain or loss of specific genes or whether they had DNA signatures suggestive of a newly emerged pathogen. They found that little evidence for adaptive changes in the B. anthracis genome that uniquely predispose it for a virulent lifestyle [ 12 ]. In a related study, Papazisi et al . utilized comparative genome hybridization using DNA microarrays to gain insights into the unique genomic features encoded in the B. anthracis genome in comparison to other B. cereus group members. They identified genomic events associated with the emergence of B. anthracis as a distinct lineage within the B. cereus group [ 10 ]. However, despite several studies [ 14 , 15 ], the mechanisms leading to the evolution and emergence of pathogenesis in the B. cereus sensu lato group remain unclear. Our intent is to begin to deduce the phenomic differences between these organisms, beginning with genome sequence data. Although 'phenomics' would at first appear to be yet another of the '-omics' terms that has been coined in the last 10-15 years, the term 'phenome' was originally coined in 1949 [ 16 ], long before the current '-omics' revolution began. The phenome is essentially information that describes the total phenotypic potential of the organism, and is dependent on all other components of the organism, including the genome sequence, the regulatory relationships encoded in the genome, etc. For example, discussions led by A.Varki among those who had used the term up to 2003 suggested the following definition: "The body of information describing an organism's phenotypes, under the influences of genetic and environmental factors" [ 17 ]. Thus, our initial goal in this study was to begin to define the collective contribution of the chromosomally-encoded protein set of a given genome to the phenotypic variation in these organisms. Materials and methods Genomes and Annotations used in this study 201 draft and whole genome sequences of the Bacillus cereus sensu lato group were collected (Additional file 1 ). From this list, we parsed out 25 whole genome sequences and submitted them as a fasta file to the University of Maryland Institute for Genome Sciences (UMd) for re-annotation using their IGS analysis engine [ 18 ]; subsequently, these will be referred to as the UMd annotations. These re-annotations were received on January 20 and October 8, 2012 respectively (Table 1 ). Our database was frozen after October 8 so that this resource would remain stable for this analysis; whole genomes that appeared after this point were excluded for this practical reason. Table 1 Whole genomes used in this study Genome Locus Tag NCBI Accession # UMd identifier Annotation Date Sequencing group B. anthracis A0248 BAA NC_012659.1 bac1 04/2005 LANL B. cereus biovar anthracis CI BACI NC_014335.1 bac2 06/2006 GGL B. anthracis Ames BA NC_003997.3 bac3 02/1999 TIGR B. thuringiensis Al. Hakam BALH NC_008600.1 bac4 10/2002 JGI B. anthracis CDC 684 BAMEG NC_012581.1 bac5 03/2005 LANL B. anthracis Sterne BAS NC_005945.1 bac6 12/1999 JGI B. cereus AH187 BCAH187 NC_011658.1 bac7 11/2004 JCVI B. cereus AH820 BCAH820 NC_011773.1 bac8 09/2004 JCVI B. cereus B4264 BCB4264 NC_011725.1 bac9 11/2004 JCVI B. cereus ATCC 10987 BCE NC_003909.8 bac10 08/2000 TIGR B. cereus ATCC 14579 BC NC_004722.1 bac11 02/1999 INRAGM B. cereus G9842 BCG9842 NC_011772.1 bac12 09/2004 JCVI B. cereus Q1 BCQ NC_011969.1 bac13 12/2004 MGCC B. thuringiensis BMB171 BMB171 NC_014171.1 bac14 04/2006 HAU B. weihenstephanensis- KBAB4 BcerKBAB4 NC_010184.1 bac15 04/2006 HAU B. cereus 03BB102 BCA NC_012472.1 bac16 02/2005 LANL B. cereus E33L BCZK NC_006274.1 bac17 10/2000 JGI B. thuringiensis serovar- konkukian str 97-27 BT9727 NC_005957.1 bac18 11/2000 JGI B. anthracis Ames Ancestor GBAA NC_007530.2 bac19 02/1999 TIGR B. cytotoxicus NVH391-98 Bcer98 NC_009674.1 bac 20 06/2003 JGI B. anthracis H9401 BAH9401 NC_017729.1 bac 21 06/2012 KCDC B. cereus F837/76 BF83776 NC_016779.1 bac 22 06/2012 INRAGM B. cereus NC7401 BNC7401 NC_016771.1 bac 23 09/2012 KILS B. thuringiensis serovar- chinensis CT43 BCT43 NC_017208.1 bac 24 09/2012 HAU B. thuringiensis serovar-finitimus YBT-020 BYBT020 NC_017200.1 bac 25 09/2012 HAU Dates/location of original annotations. Full name for sequencing group are as follows: LANL: Los Alamos National Labs, JCVI: J. Craig Venter Institute, INRAGM: INRA Genetique Microbienne, MGCC: Microbial Genome Center of Chinese Ministry of Public Health, HAU: Huazhong Agricultural University, GGL: Goettingen Genomics Laboratory, UMd: University of Maryland Institute for Genome Sciences, KCDC: Korea Center for Disease Control, KILS: Kitasato Institute for Life Sciences Orthologous protein clustering and hierarchical genome clustering Clusters of putative orthologous proteins were generated for the 201 draft and whole genomes from NCBI. From this group of genomes, we selected a subset consisting of 25 whole genomes and generated clusters of orthologous proteins from the group of 25 original annotations available at NCBI and for the 25 UMd annotations, using CD-hit [ 19 ] with a cutoff of 85/85. In our experience, CD-hit offered comparable results to other orthology search programs such as OrthoMCL [ 20 ] and was computationally much faster. Similar to previously described methods performed by our group [ 21 ], this cutoff required an 85% sequence identity across 85% sequence length for CD-hit while all other parameters remained at the default values. Manually inspecting this output, we confirmed that a representative protein from each organism was grouped with other similar proteins to form the orthologous clusters. From the CD-hit output, two separate datasets were generated. The first one had a total of 92,806 clusters with sizes ranges from 1-201 genomes; the second dataset had a total of 21,288 clusters with cluster sizes ranging from 1-50 genes. For the 21,288 clusters, the total number of genes comprising the core genomes was calculated using all clusters ranging from 25-50 genes in size. Files were extracted and stored in a MySQL database. Next, for both sets of cd-hit outputs, orthologous clusters were parsed out for each organism and separate tables were created using Perl scripts. These tables were then imported into an excel spreadsheet and calculations were done for the presence (assigned a 1) or absence (assigned a 0) of genomes within a specific cluster using Visual Basic/macros coding. The final file was uploaded into Matlab. We then applied the 'Heatmap' and 'clustergram' scripts using Matlab [ 22 ] to render a 2-d color image of the data showing the cluster numbers on the y-axis and the organisms on the x-axis. To organize this data and identify potential relationships among the genome-specific orthologous clusters, we utilized a hierarchical clustering with Euclidean distance metric and average linkage to generate the hierarchical tree. This type of clustering enabled us to find the similarity or dissimilarity between every pair of objects in the data set, group the objects into a binary, hierarchical cluster tree, and determine where to differentiate the hierarchical tree into clusters. Clusters of Orthologous Genes (COGS) analysis We extracted all clusters from the whole genomes dataset that contained 24 and 25 members of orthologous proteins with one representative member from each organism as derived from CD-hit analysis. The 24 member clusters allowed us to include clusters with and without B. cytotoxicus as it was an outlier from our previous analysis. This yielded 2,674 protein sequences total for COGs analysis. We generated a list of proteins found in each organism per a given clade. Using these individual lists, we then proceeded to extract all the nucleotide sequences from the genes using Perl scripts to create new fasta files. We then imported these fasta sequences from NCBI and performed blastX. The blastX output files were extracted, saved, and uploaded into MEGAN v 5_1_4 [ 23 ] for COGs analysis. MEGAN was only able to successfully run ~200 genes at one time so we separated the genes into several lists and performed multiple runs to generate the COGS data. We selected the COGS analysis feature in MEGAN and then selected the file type we were using. The output generated by MEGAN showed a tree with the biological categories and our genes with respective ids located within the branches. These calculations and raw data were then extracted and saved as text files. Metabolic pathway analysis The original (NCBI) and UMd annotations for each of the 25 B. cereus sensu lato genomes were uploaded into an in-house Pathway Tools database [ 24 ]. Pathway predictions were then made based on these annotations for each genome using SRI's software algorithm. This data was downloaded, and organized into an external database for each organism. KEGG global metabolic maps [ 25 ] were constructed using the KEGG identifiers for enzymes located in the KEGG database. The KEGG database enzyme list was downloaded and saved as a csv file. Enzyme lists for each B. cereus sensu lato clades were parsed from the database into a csv file. These two lists were then compared using Visual Basic/macros coding. We then uploaded this information into the KEGG MAPPER and utilized the advance pathway mapping utility feature to search and color the individual enzymes. Global comparisons of total protein coding content and metabolic pathway predictions were compared and statistically evaluated using GraphPad Prism v.4.03 [ 26 ]. Analysis of conserved/hypothetical proteins We examined potential structure features of the conserved hypothetical and hypothetical proteins from our whole genomes dataset. Conserved/hypothetical protein sequences were extracted from a master file using a Perl script. Each ID and sequence were paired in FASTA format and saved to separate files for each of the four clades. The sequences in all four clades were then compared to the Pfam database [ 27 ], and analyzed using SignalP [ 28 ] and TmHmm [ 28 ]. Raw data from the Pfam database was evaluated based on three stringency levels [ 27 ]. The most significant hits were those with e-values of <0.001, followed by e-values between 0.1-0.001, and lastly those with e-values higher than 0.1. Pfam also provided assigned family names and clan numbers where available. All fields were kept at default values for the SignalP searches with the exception that the organism field was selected as "gram-positive bacteria". The TmHmm output format chosen was "extensive with graphics" [ 28 ]. All data generated from Pfam, SignalP, TmHmm were extracted and saved as text files. All graphical data generated from SignalP and TmHmm were also extracted and saved as individual files. For additional analysis the text files were then converted to excel spreadsheets and Visual Basic scripts were utilized to parse the raw data to create summaries of the findings for each clade. Genomes and Annotations used in this study 201 draft and whole genome sequences of the Bacillus cereus sensu lato group were collected (Additional file 1 ). From this list, we parsed out 25 whole genome sequences and submitted them as a fasta file to the University of Maryland Institute for Genome Sciences (UMd) for re-annotation using their IGS analysis engine [ 18 ]; subsequently, these will be referred to as the UMd annotations. These re-annotations were received on January 20 and October 8, 2012 respectively (Table 1 ). Our database was frozen after October 8 so that this resource would remain stable for this analysis; whole genomes that appeared after this point were excluded for this practical reason. Table 1 Whole genomes used in this study Genome Locus Tag NCBI Accession # UMd identifier Annotation Date Sequencing group B. anthracis A0248 BAA NC_012659.1 bac1 04/2005 LANL B. cereus biovar anthracis CI BACI NC_014335.1 bac2 06/2006 GGL B. anthracis Ames BA NC_003997.3 bac3 02/1999 TIGR B. thuringiensis Al. Hakam BALH NC_008600.1 bac4 10/2002 JGI B. anthracis CDC 684 BAMEG NC_012581.1 bac5 03/2005 LANL B. anthracis Sterne BAS NC_005945.1 bac6 12/1999 JGI B. cereus AH187 BCAH187 NC_011658.1 bac7 11/2004 JCVI B. cereus AH820 BCAH820 NC_011773.1 bac8 09/2004 JCVI B. cereus B4264 BCB4264 NC_011725.1 bac9 11/2004 JCVI B. cereus ATCC 10987 BCE NC_003909.8 bac10 08/2000 TIGR B. cereus ATCC 14579 BC NC_004722.1 bac11 02/1999 INRAGM B. cereus G9842 BCG9842 NC_011772.1 bac12 09/2004 JCVI B. cereus Q1 BCQ NC_011969.1 bac13 12/2004 MGCC B. thuringiensis BMB171 BMB171 NC_014171.1 bac14 04/2006 HAU B. weihenstephanensis- KBAB4 BcerKBAB4 NC_010184.1 bac15 04/2006 HAU B. cereus 03BB102 BCA NC_012472.1 bac16 02/2005 LANL B. cereus E33L BCZK NC_006274.1 bac17 10/2000 JGI B. thuringiensis serovar- konkukian str 97-27 BT9727 NC_005957.1 bac18 11/2000 JGI B. anthracis Ames Ancestor GBAA NC_007530.2 bac19 02/1999 TIGR B. cytotoxicus NVH391-98 Bcer98 NC_009674.1 bac 20 06/2003 JGI B. anthracis H9401 BAH9401 NC_017729.1 bac 21 06/2012 KCDC B. cereus F837/76 BF83776 NC_016779.1 bac 22 06/2012 INRAGM B. cereus NC7401 BNC7401 NC_016771.1 bac 23 09/2012 KILS B. thuringiensis serovar- chinensis CT43 BCT43 NC_017208.1 bac 24 09/2012 HAU B. thuringiensis serovar-finitimus YBT-020 BYBT020 NC_017200.1 bac 25 09/2012 HAU Dates/location of original annotations. Full name for sequencing group are as follows: LANL: Los Alamos National Labs, JCVI: J. Craig Venter Institute, INRAGM: INRA Genetique Microbienne, MGCC: Microbial Genome Center of Chinese Ministry of Public Health, HAU: Huazhong Agricultural University, GGL: Goettingen Genomics Laboratory, UMd: University of Maryland Institute for Genome Sciences, KCDC: Korea Center for Disease Control, KILS: Kitasato Institute for Life Sciences Orthologous protein clustering and hierarchical genome clustering Clusters of putative orthologous proteins were generated for the 201 draft and whole genomes from NCBI. From this group of genomes, we selected a subset consisting of 25 whole genomes and generated clusters of orthologous proteins from the group of 25 original annotations available at NCBI and for the 25 UMd annotations, using CD-hit [ 19 ] with a cutoff of 85/85. In our experience, CD-hit offered comparable results to other orthology search programs such as OrthoMCL [ 20 ] and was computationally much faster. Similar to previously described methods performed by our group [ 21 ], this cutoff required an 85% sequence identity across 85% sequence length for CD-hit while all other parameters remained at the default values. Manually inspecting this output, we confirmed that a representative protein from each organism was grouped with other similar proteins to form the orthologous clusters. From the CD-hit output, two separate datasets were generated. The first one had a total of 92,806 clusters with sizes ranges from 1-201 genomes; the second dataset had a total of 21,288 clusters with cluster sizes ranging from 1-50 genes. For the 21,288 clusters, the total number of genes comprising the core genomes was calculated using all clusters ranging from 25-50 genes in size. Files were extracted and stored in a MySQL database. Next, for both sets of cd-hit outputs, orthologous clusters were parsed out for each organism and separate tables were created using Perl scripts. These tables were then imported into an excel spreadsheet and calculations were done for the presence (assigned a 1) or absence (assigned a 0) of genomes within a specific cluster using Visual Basic/macros coding. The final file was uploaded into Matlab. We then applied the 'Heatmap' and 'clustergram' scripts using Matlab [ 22 ] to render a 2-d color image of the data showing the cluster numbers on the y-axis and the organisms on the x-axis. To organize this data and identify potential relationships among the genome-specific orthologous clusters, we utilized a hierarchical clustering with Euclidean distance metric and average linkage to generate the hierarchical tree. This type of clustering enabled us to find the similarity or dissimilarity between every pair of objects in the data set, group the objects into a binary, hierarchical cluster tree, and determine where to differentiate the hierarchical tree into clusters. Clusters of Orthologous Genes (COGS) analysis We extracted all clusters from the whole genomes dataset that contained 24 and 25 members of orthologous proteins with one representative member from each organism as derived from CD-hit analysis. The 24 member clusters allowed us to include clusters with and without B. cytotoxicus as it was an outlier from our previous analysis. This yielded 2,674 protein sequences total for COGs analysis. We generated a list of proteins found in each organism per a given clade. Using these individual lists, we then proceeded to extract all the nucleotide sequences from the genes using Perl scripts to create new fasta files. We then imported these fasta sequences from NCBI and performed blastX. The blastX output files were extracted, saved, and uploaded into MEGAN v 5_1_4 [ 23 ] for COGs analysis. MEGAN was only able to successfully run ~200 genes at one time so we separated the genes into several lists and performed multiple runs to generate the COGS data. We selected the COGS analysis feature in MEGAN and then selected the file type we were using. The output generated by MEGAN showed a tree with the biological categories and our genes with respective ids located within the branches. These calculations and raw data were then extracted and saved as text files. Metabolic pathway analysis The original (NCBI) and UMd annotations for each of the 25 B. cereus sensu lato genomes were uploaded into an in-house Pathway Tools database [ 24 ]. Pathway predictions were then made based on these annotations for each genome using SRI's software algorithm. This data was downloaded, and organized into an external database for each organism. KEGG global metabolic maps [ 25 ] were constructed using the KEGG identifiers for enzymes located in the KEGG database. The KEGG database enzyme list was downloaded and saved as a csv file. Enzyme lists for each B. cereus sensu lato clades were parsed from the database into a csv file. These two lists were then compared using Visual Basic/macros coding. We then uploaded this information into the KEGG MAPPER and utilized the advance pathway mapping utility feature to search and color the individual enzymes. Global comparisons of total protein coding content and metabolic pathway predictions were compared and statistically evaluated using GraphPad Prism v.4.03 [ 26 ]. Analysis of conserved/hypothetical proteins We examined potential structure features of the conserved hypothetical and hypothetical proteins from our whole genomes dataset. Conserved/hypothetical protein sequences were extracted from a master file using a Perl script. Each ID and sequence were paired in FASTA format and saved to separate files for each of the four clades. The sequences in all four clades were then compared to the Pfam database [ 27 ], and analyzed using SignalP [ 28 ] and TmHmm [ 28 ]. Raw data from the Pfam database was evaluated based on three stringency levels [ 27 ]. The most significant hits were those with e-values of <0.001, followed by e-values between 0.1-0.001, and lastly those with e-values higher than 0.1. Pfam also provided assigned family names and clan numbers where available. All fields were kept at default values for the SignalP searches with the exception that the organism field was selected as "gram-positive bacteria". The TmHmm output format chosen was "extensive with graphics" [ 28 ]. All data generated from Pfam, SignalP, TmHmm were extracted and saved as text files. All graphical data generated from SignalP and TmHmm were also extracted and saved as individual files. For additional analysis the text files were then converted to excel spreadsheets and Visual Basic scripts were utilized to parse the raw data to create summaries of the findings for each clade. Results and discussion Whole-genome comparisons of protein-coding content suggest that the B. cereus sensu lato organisms comprise four distinct clades Our first analysis employed the dataset of all available B. cereus sensu lato genomes (draft and whole genomes, Additional file 1 ). These genomes were analyzed for shared orthologous protein clusters using Cd-hit [ 18 ]. As will be discussed below, we parsed the completed genomes for a separate analysis. In each instance, this was followed by performing a Hierarchical Euclidean cluster analysis [ 22 ], expressing the results in a simple heatmap and dendrogram (Figure 1 ). This analysis clustered potential contributors to the phenomotypic differences between these organisms, by virtue of the phenomotypic effectors (proteins) that each genome harbored. Analysis of the dataset, consisting of 201 whole and draft genomes was illustrated by, a dendrogram, a tree-structured graph used in heat maps to visualize the result of the hierarchical clustering calculation (Figure 1 , Additional files 2 and 3 ). Here we show the row dendrogram which illustrates the distance or similarity between rows and which nodes each row belongs to, as a result of clustering [ 22 ]. On the x-axis, we show the relative location of 25 whole genomes within this larger group of genomes (Figure 1 , Additional file 1 ). From this analysis, patterns of the relationships between these genomes (see below) and their phenotypic potential began to emerge. However, these patterns were not as clear-cut as could be discerned from the comparisons of the completed genomes alone (see below and Figure 2 ), likely for several reasons. Since majority (almost 90%) of these genomes were draft sequences, the variable quality of these sequences and their annotation could be complicating factors. For instance, lack of a protein coding sequence in a draft genome annotation could be due to deletion of this coding sequence from the genome, lack of sampling of this region during the sequencing for the draft assembly, low sequence quality that may have excluded certain proteins from a given orthologous cluster, or differences in annotation. The annotations associated with these genomes may be somewhat uneven between sequencing groups, likely due in part to the different annotation methods and sequencing centers from which these data were obtained (e.g., see below for annotation comparisons of the completed genomes). And, for at least 10% of the genomes that we analyzed, the sequencing center provided no information describing the source or origin of these isolates (see Additional file 1 ). Associating these cryptic-source strains with a specific biological phenomotype was essentially impossible. In the face of these potential and real sources of variation, we selected a subset of these strains (all complete genome sequences) for separate analysis, and for comparison back to the total genome dataset. We also reannotated these whole genome sequences to provide a uniform annotation that eliminated much of the variability that may have affected the cluster analysis shown in Figure 1 . The analysis of the whole genomes where we looked at the presence/absence of a particular orthologous cluster was informative, even if that cluster was comprised of proteins annotated as conserved hypothetical or hypothetical proteins (Figure 2 , Additional file 4 ). In this cluster analysis, the heatmap shown is highlighting the presence of a genome within a particular orthologous protein cluster which was assigned a 1 (red in Figure 2 ), while the absence of a genome within a cluster was assigned a 0 (black in Figure 2 ). Genomes are represented by the x-axis while the clusters are represented by the y-axis (0-21,288 clusters starting from the bottom to top). Excluding B. cytotoxicus (as noted above), we found four distinct clades from this cluster analysis, designated clades A-D (the rationale for these clade designations will be presented below); this data is summarized in Table 2 . When we identified the position of these completed genomes in the clustergram shown in Figure 1 , these genomes were clustered together similarly in both clustergrams (compare Figure 1 and 2 ). This suggests that in general, the biological relationships (see next) deduced from the clustergram and heatmap shown in Figure 2 also reflects the larger dataset shown in Figure 1 . Figure 1 Orthologous protein cluster comparison of 201 whole and draft B . Cereus Sensu lato organisms highlights specific clades. Clustergram was derived using hierarchical clustering with Euclidean distance. The full list of genomes and the order for which they appear on the clustergram can be found in Additional file 1 . The relative locations and clade designations of the 25 whole genomes from Figure 2 have been labeled on the x-axis of this figure. Genome names colored in red denote Clade C, blue denote Clade D, green denote Clade B, black denote Clade A and orange denote Clade E. Figure 2 B. cereus sensu lato organisms form 5 distinct clades. Clade structure derived using hiearchical clustering with Euclidean distance. 5 clades were derived, Clades A-E. Plasmids found in each organism are denoted by the following: •-pXO1, •-pXO2, •-cry toxin, •-pBC, u-pAH, n-pBT, p-pBMB, p-pCT, n-pF837, n-BAP, u-pNC, u-pE33, u-p03BB102, n-pALH, p-pBWB, p-pG9842. Table 2 Differences in protein coding capacity mirror the stress response of the organism. Genome clusters (from Figure 2 ) Organisms Source or location of isolation SigB clade (from Reference #19) SigB regulon constituents Clade A BCB4264 Bloodstream of pneumonia patient A Core SigB regulon BMB171 Soil A regulatory proteins, BC Dairy product A cardiolipin biosynthesis, BCG9842 Stool sample from food poisoning outbreak A efflux pumps, SOS BCT43 insecticide N/A functions BcerKBAB4 Soil A Clade B BCAH187 Dairy product B Core SigB regulon only BNC7401 Food poisoning N/A BCQ Deep oil reservoir B BYBT020 insecticide N/A BCE Cheese spoilage B Clade C GBAA Bovine carcass C Core SigB regulon BA Bovine carcass C regulatory proteins, BAA Human disease C S-layer protein, BAS Vaccine strain C GalNac biosynthesis, BAH9401 Human disease C spore germination protein BAMEG CDC isolate C Clade D BACl Chimpanzee carcass D Core SigB regulon BALH Iraq bioweapons facility D other additions BCA Human blood isolate D similar to SigB Clade C BF83776 Human prostate wound isolate N/A BCAH820 Human periodontitis D BT9727 Human tissue necrosis D BCZK Zebra carcass D Comparison of our clade organisms to previously published clade structure from SigB. N/A denotes genome sequences not available at time of sig B analysis. Previously we have shown that the clades listed correspond to differences in stress responses of sigma factor B across the B. cereus sensu lato group of organisms. These differences in stress response also correlate with protein coding capacity of these organisms. Data from Figure 2 suggests that Clade A appears to be primarily comprised of organisms that were either environmental isolates or associated with food poisoning outbreaks, although in one case, a Clade A organism was isolated from a case of human septicemia. Clade B organisms appear to be derived from either soil or food poisoning episodes, and may represent a group with the least potential to cause serious mammalian disease, In general, Clades A and B appear less capable of initiating invasive infections than organisms found in Clades C and D. This impression that the four clades identified in Figure 2 correlate well with mammalian virulence potential is reinforced by a comparison with the presence of plasmid DNAs harbored in these organisms (lower half of Figure 2 ). As noted above, with the exception of the pXO1/pXO2 plasmids found in Clade C and the single B. cereus biovar anthracis Cl strain in Clade D, there is no clear pattern to the relationship between plasmid content and the mammalian virulence potential of the clades shown in Figure 2 . Clade C consisted solely of B. anthracis strains. As with many other comparative analyses that have been performed [ 12 ], these organisms appear to be comprise a clonal group easily separable from other B. cereus sensu lato organisms. While the pXO1 and pXO2 plasmids that encode these virulence factors in B. anthracis strains are clearly critical for the ability to cause septicemic disease, the observation that aggregate genomic protein coding content of Clade C organisms clusters separately from other B. cereus sensu lato organisms argues that other components of the genome of this organism also participate in the unique biology of the anthrax organism. Similarly, Clade D organisms were typically isolated from episodes of invasive disease. Indeed, one of these, B. cereus biovar anthracis Cl (isolated from a chimpanzee carcass), was shown to carry the anthrax-associated plasmids pXO1 and pXO2 [ 11 ]. This organism was part of a group of closely related isolates that caused deadly anthrax-like infections in primates in the Côte d'Ivoire in 2001-2002 and Cameroon in 2004 [ 29 ]. Other clade D organisms include a strain isolated from a case of invasive human disease, ( B. cereus 03BB102) that did not harbor the pXO1/pXO2 virulence plasmids. Clade D also includes a human tissue necrosis isolate ( B. thuringiensis konkukian 97-17) speciated with organisms that typically cause disease in insects but not in mammals [ 7 , 30 ]. Thus, Clade D has members with a propensity to cause serious and sometimes invasive mammalian disease, although these organisms may not be as virulent as the anthrax organisms in Clade C, unless they harbor the pXO1/pXO2 plasmids. This suggests that the virulence associated with Clade D organisms, including B. cereus biovar anthracis Cl, relies not only on the virulence plasmids pXO1/pXO2, but the underlying genomic coding content shared by Clade D organisms, and that this genomic content is distinct from Clade C organisms. Clade D organisms may possess unique pathogenic traits that differentiate these organisms from B. anthracis . Genome summaries and pathway predictions from the UMd and original NCBI annotations Whole genome sequences from the B. cereus sensu lato group that were used in this study are listed in Table 1 . These genome sequences have accumulated over a 10-year period, and have been provided by a number of different sequencing centers. Due both to the differences in annotation methodology and the improvements in annotation that have occurred over the last decade, we suspected that comparisons of these annotations might be somewhat uneven. Consequently, we wanted to ensure that these genomes were annotated to a single standard. We employed the UMIGS [ 18 ] automated annotation pipeline for this process. This was an arbitrary selection, and we did not compare the annotation outputs that could have been obtained from several publicly-available annotation pipelines [ 31 - 33 ]. Our purpose was not to compete different annotation pipelines against one another for evaluation, but merely to ensure that these annotations met a common standard prior to subsequent analyses. Indeed, reannotation substantially changed the interpretation of these genome sequences. In general, re-annotation suggested that the protein coding content of these genomes was even more closely related than has appeared in recent analyses [ 10 , 12 ]. [We will exclude B. cytotoxicus from the remainder of this discussion, as this organism has a substantially smaller genome than the remainder of these organisms [ 34 ], and skews the interpretation of these comparisons.] The average number of protein coding sequences predicted from these genomes increased from 5268 to 5430, and the range of predicted protein coding sequences in these genomes increased from 4737-5602 (original annotations) to 5340-5613 (UMd annotations; Figure 3a ); these were statistically significant increases. The predicted aggregate metabolism derived from these genome sequence annotations also was much more similar after reannotation (Figure 3b ); the average number of recognizable metabolic pathways extracted from these genomes increased from 231 to 247, and the range of predicted metabolic pathways went from 194-264 to 239-265 (original vs. UMd annotations, respectively). Notably, these changes increased the estimated cluster of core proteins encoded by all 25 organisms from 1483 to 1544, an increase of 4% (Additional file 5 ). Figure 3A and B There was an increase in core proteins after reannotation. Figure 3A, The average number of protein coding sequences predicted from these genomes increased significantly with reannotation and these were statistically significant increases, p = 0.0008. Figure 3B, The predicted aggregate metabolism derived from these genome sequence annotations also was much more similar after reannotation and this was also significant p = 0.0011. There are a variety of reasons for these differences. The original annotations of these genomes accumulated over a period of ten years (Table 1 ), offered from nine different sequencing groups. There are currently no set guidelines or standards for genome annotation, and as a result genome annotations vary from one source to another [ 35 ]. Despite the number of annotations available in the public domain, a surprisingly large number of genes are still not annotated [ 36 ]. Missed genes have a particularly strong impact on the delineation of the core genome for a large and diverse group of organisms, and on identification of strain- or species-specific genes. The percentage of missed genes in a genome has been estimated at 5-10%, which is consistent with the collective results of this reannotation [ 37 ]. Core genome COGS analysis, basal metabolism, and conserved hypothetical proteins amongst the clades Results from MEGAN analysis of the core genome which comprised of the 24 and 25 membered clusters, showed there were a total of 850 genes associated with the COGs category metabolism, which was comprised of these subset of functions for the given number of genes: Energy production and conversion-137, Carbohydrate transport and metabolism-100, Amino acid transport and metabolism- 206, Nucleotide transport and metabolism-69, Coenzyme transport and metabolism-101, Lipid transport and metabolism-60, Inorganic ion transport and metabolism-139, Secondary metabolites biosynthesis, transport and catabolism-38; 461 genes were associated with information, storage and processing, which included the following subset of functions: Translation, ribosomal structure and biogenesis-140, Transcription-188, Replication, recombination and repair-132, and chromatin structure and dynamics-1; 332 genes were associated with cellular processes and signaling, which included the following subset of functions: Cell cycle control, cell division, and chromosome partitioning-25, Signal transduction mechanisms-84, Cell wall/membrane/envelope biogenesis-79, Intracellular trafficking, secretion, and vesicular transport-29, Posttranslational modification, protein turnover, and chaperones-60, Defense mechanisms-49, and cell motility-6; 344 genes were unassigned due to lack of experimental information about them, and 705 genes were assigned as poorly characterized since there was limited information available for them in order to be placed confidently into a functional category (Additional file 6 Figure 4 ). Since there were a large number of genes in the metabolism-related subset of functional groups, we next examined the basal metabolism in these organisms. We found that the differences in basal metabolism were slight, with only one unique enzyme, tagatose 6-phosphate kinase, found in clade A and no other unique intermediary metabolic enzymes found in clades B, C and D. However, it is unclear how this enzyme may contribute to clade A-specific phenotypes, since little work has been reported in this group of organisms. We also encountered some heterogeneity within each clade: enzymes unique to single organisms within each clade though not present in all organisms of the given clade (Additional file 7 ). While there is little difference between these clades in terms of intermediary metabolism, we speculate that the regulatory control of these proteins is likely different in different organisms, and could contribute to phenotypic differences between these clades, even though coding content is highly similar. Figure 4 The largest groups of genes were those with metabolism-related functions . Subsets of clusters of orthologous genes (COGs) categories from core genes common to clades A-D. The subsets of COGs categories are shown here as functional groups as denoted by MEGAN. Genes not found by the COGS analysis tool were placed in non-characterized/function and unknown category. We also parsed the dataset for conserved/hypothetical proteins shared by all members of a given clade while not being found in any other clade organism. With the exception of Clade C, the contribution of clade-specific conserved hypotheticals to genomic coding capacity was minimal (Additional file 8 ). The majority of Clade C-specific conserved hypothetical proteins appear to be contained in 4 bacteriophage lysogens that had earlier been shown to be unique to B. anthracis [ 38 , 39 ]. Five of the clade C proteins were predicted by SignalP to have a signal peptide and could therefore potentially be cell wall or secreted proteins that may be important for anthrax pathogenesis. Consequently, these conserved/hypothetical proteins may contain additional information about some of the unique biology that contributes specifically to clade C organisms. However, currently these proteins have yet to be studied and characterized therefore additional experiments which are beyond the scope of this current study would be necessary to establish a precise function for these proteins (Additional file 5 ). Comparison of whole genome protein coding content to B. cereus sensu lato sigB regulons Strikingly, the genomic relationships between these organisms, shown in Figure 2 , were identical to those that we had previously identified, based on the predicted structure of the generalized stress response regulon controlled by SigB [ 21 ]. Those observations are summarized in Table 2 for comparison. Organisms in the present Clade C appear to encode a generalized stress response, controlled by SigB, that has diverged from other members of this group to include additional functions that we hypothesized are uniquely involved in invasive disease; Clade D organisms have a SigB regulon of similar structure. By contrast, organisms in Clades A and B of the present study, many of which were associated with food poisoning outbreaks, carry a SigB regulon that either harbors only the core SigB regulon genes found in all B. cereus sensu lato organisms (Clade B), or have added to this core SigB regulon additional genes (Clade A) that may act enhance the stress response of these organisms during the response to deleterious environmental conditions (e.g., cold temperatures, UV light) that may be found in food processing facilities. Importantly, differences between the SigB regulon structure in these four clades are primarily a consequence of whether components of the core genome are/are not driven by a SigB promoter [ 21 ], rather than differences in gene content between these clades; the majority of genes contained in the SigB regulons of these organisms are included in the core genomes of these organisms, and their differences lie in whether their transcription is controlled by SigB. That we arrived at the same genomic relationships between these organisms using two entirely different approaches suggests an underlying organization in this population that has not been previously noted. The four clades that we identified in Figure 2 appear to be a result of at least two processes acting in parallel: 1) divergence in genomic coding capacity (protein coding genes), and 2) divergence in the generalized stress response regulon. As noted above, these processes appear to be independent of the extrachromosomal DNA content of these organisms (see Figure 2 ). This suggests the more general hypothesis that divergence among the members of the B. cereus sensu lato group relies on a pattern of regulatory divergence overlaying the protein coding divergence seen in Figure 2 . Obviously, further work is necessary to test this hypothesis. In particular, does additional regulatory divergence in these organisms follow this same pattern, or is this restricted to the divergence of the SigB-controlled generalized stress response? Our recent informatics analysis (Scott EJ and Dyer DW, manuscript in preparation) suggests that the SigM ECF sigma factor regulons in these organisms appear to diverge in the same manner as we have observed here; the SigM regulons can be sorted into four clusters that also correspond to those described here. Thus, our finding that coupling protein coding divergence and regulatory divergence may be a more general phenomenon that contributes to the phenomotype of any given strain. Whole-genome comparisons of protein-coding content suggest that the B. cereus sensu lato organisms comprise four distinct clades Our first analysis employed the dataset of all available B. cereus sensu lato genomes (draft and whole genomes, Additional file 1 ). These genomes were analyzed for shared orthologous protein clusters using Cd-hit [ 18 ]. As will be discussed below, we parsed the completed genomes for a separate analysis. In each instance, this was followed by performing a Hierarchical Euclidean cluster analysis [ 22 ], expressing the results in a simple heatmap and dendrogram (Figure 1 ). This analysis clustered potential contributors to the phenomotypic differences between these organisms, by virtue of the phenomotypic effectors (proteins) that each genome harbored. Analysis of the dataset, consisting of 201 whole and draft genomes was illustrated by, a dendrogram, a tree-structured graph used in heat maps to visualize the result of the hierarchical clustering calculation (Figure 1 , Additional files 2 and 3 ). Here we show the row dendrogram which illustrates the distance or similarity between rows and which nodes each row belongs to, as a result of clustering [ 22 ]. On the x-axis, we show the relative location of 25 whole genomes within this larger group of genomes (Figure 1 , Additional file 1 ). From this analysis, patterns of the relationships between these genomes (see below) and their phenotypic potential began to emerge. However, these patterns were not as clear-cut as could be discerned from the comparisons of the completed genomes alone (see below and Figure 2 ), likely for several reasons. Since majority (almost 90%) of these genomes were draft sequences, the variable quality of these sequences and their annotation could be complicating factors. For instance, lack of a protein coding sequence in a draft genome annotation could be due to deletion of this coding sequence from the genome, lack of sampling of this region during the sequencing for the draft assembly, low sequence quality that may have excluded certain proteins from a given orthologous cluster, or differences in annotation. The annotations associated with these genomes may be somewhat uneven between sequencing groups, likely due in part to the different annotation methods and sequencing centers from which these data were obtained (e.g., see below for annotation comparisons of the completed genomes). And, for at least 10% of the genomes that we analyzed, the sequencing center provided no information describing the source or origin of these isolates (see Additional file 1 ). Associating these cryptic-source strains with a specific biological phenomotype was essentially impossible. In the face of these potential and real sources of variation, we selected a subset of these strains (all complete genome sequences) for separate analysis, and for comparison back to the total genome dataset. We also reannotated these whole genome sequences to provide a uniform annotation that eliminated much of the variability that may have affected the cluster analysis shown in Figure 1 . The analysis of the whole genomes where we looked at the presence/absence of a particular orthologous cluster was informative, even if that cluster was comprised of proteins annotated as conserved hypothetical or hypothetical proteins (Figure 2 , Additional file 4 ). In this cluster analysis, the heatmap shown is highlighting the presence of a genome within a particular orthologous protein cluster which was assigned a 1 (red in Figure 2 ), while the absence of a genome within a cluster was assigned a 0 (black in Figure 2 ). Genomes are represented by the x-axis while the clusters are represented by the y-axis (0-21,288 clusters starting from the bottom to top). Excluding B. cytotoxicus (as noted above), we found four distinct clades from this cluster analysis, designated clades A-D (the rationale for these clade designations will be presented below); this data is summarized in Table 2 . When we identified the position of these completed genomes in the clustergram shown in Figure 1 , these genomes were clustered together similarly in both clustergrams (compare Figure 1 and 2 ). This suggests that in general, the biological relationships (see next) deduced from the clustergram and heatmap shown in Figure 2 also reflects the larger dataset shown in Figure 1 . Figure 1 Orthologous protein cluster comparison of 201 whole and draft B . Cereus Sensu lato organisms highlights specific clades. Clustergram was derived using hierarchical clustering with Euclidean distance. The full list of genomes and the order for which they appear on the clustergram can be found in Additional file 1 . The relative locations and clade designations of the 25 whole genomes from Figure 2 have been labeled on the x-axis of this figure. Genome names colored in red denote Clade C, blue denote Clade D, green denote Clade B, black denote Clade A and orange denote Clade E. Figure 2 B. cereus sensu lato organisms form 5 distinct clades. Clade structure derived using hiearchical clustering with Euclidean distance. 5 clades were derived, Clades A-E. Plasmids found in each organism are denoted by the following: •-pXO1, •-pXO2, •-cry toxin, •-pBC, u-pAH, n-pBT, p-pBMB, p-pCT, n-pF837, n-BAP, u-pNC, u-pE33, u-p03BB102, n-pALH, p-pBWB, p-pG9842. Table 2 Differences in protein coding capacity mirror the stress response of the organism. Genome clusters (from Figure 2 ) Organisms Source or location of isolation SigB clade (from Reference #19) SigB regulon constituents Clade A BCB4264 Bloodstream of pneumonia patient A Core SigB regulon BMB171 Soil A regulatory proteins, BC Dairy product A cardiolipin biosynthesis, BCG9842 Stool sample from food poisoning outbreak A efflux pumps, SOS BCT43 insecticide N/A functions BcerKBAB4 Soil A Clade B BCAH187 Dairy product B Core SigB regulon only BNC7401 Food poisoning N/A BCQ Deep oil reservoir B BYBT020 insecticide N/A BCE Cheese spoilage B Clade C GBAA Bovine carcass C Core SigB regulon BA Bovine carcass C regulatory proteins, BAA Human disease C S-layer protein, BAS Vaccine strain C GalNac biosynthesis, BAH9401 Human disease C spore germination protein BAMEG CDC isolate C Clade D BACl Chimpanzee carcass D Core SigB regulon BALH Iraq bioweapons facility D other additions BCA Human blood isolate D similar to SigB Clade C BF83776 Human prostate wound isolate N/A BCAH820 Human periodontitis D BT9727 Human tissue necrosis D BCZK Zebra carcass D Comparison of our clade organisms to previously published clade structure from SigB. N/A denotes genome sequences not available at time of sig B analysis. Previously we have shown that the clades listed correspond to differences in stress responses of sigma factor B across the B. cereus sensu lato group of organisms. These differences in stress response also correlate with protein coding capacity of these organisms. Data from Figure 2 suggests that Clade A appears to be primarily comprised of organisms that were either environmental isolates or associated with food poisoning outbreaks, although in one case, a Clade A organism was isolated from a case of human septicemia. Clade B organisms appear to be derived from either soil or food poisoning episodes, and may represent a group with the least potential to cause serious mammalian disease, In general, Clades A and B appear less capable of initiating invasive infections than organisms found in Clades C and D. This impression that the four clades identified in Figure 2 correlate well with mammalian virulence potential is reinforced by a comparison with the presence of plasmid DNAs harbored in these organisms (lower half of Figure 2 ). As noted above, with the exception of the pXO1/pXO2 plasmids found in Clade C and the single B. cereus biovar anthracis Cl strain in Clade D, there is no clear pattern to the relationship between plasmid content and the mammalian virulence potential of the clades shown in Figure 2 . Clade C consisted solely of B. anthracis strains. As with many other comparative analyses that have been performed [ 12 ], these organisms appear to be comprise a clonal group easily separable from other B. cereus sensu lato organisms. While the pXO1 and pXO2 plasmids that encode these virulence factors in B. anthracis strains are clearly critical for the ability to cause septicemic disease, the observation that aggregate genomic protein coding content of Clade C organisms clusters separately from other B. cereus sensu lato organisms argues that other components of the genome of this organism also participate in the unique biology of the anthrax organism. Similarly, Clade D organisms were typically isolated from episodes of invasive disease. Indeed, one of these, B. cereus biovar anthracis Cl (isolated from a chimpanzee carcass), was shown to carry the anthrax-associated plasmids pXO1 and pXO2 [ 11 ]. This organism was part of a group of closely related isolates that caused deadly anthrax-like infections in primates in the Côte d'Ivoire in 2001-2002 and Cameroon in 2004 [ 29 ]. Other clade D organisms include a strain isolated from a case of invasive human disease, ( B. cereus 03BB102) that did not harbor the pXO1/pXO2 virulence plasmids. Clade D also includes a human tissue necrosis isolate ( B. thuringiensis konkukian 97-17) speciated with organisms that typically cause disease in insects but not in mammals [ 7 , 30 ]. Thus, Clade D has members with a propensity to cause serious and sometimes invasive mammalian disease, although these organisms may not be as virulent as the anthrax organisms in Clade C, unless they harbor the pXO1/pXO2 plasmids. This suggests that the virulence associated with Clade D organisms, including B. cereus biovar anthracis Cl, relies not only on the virulence plasmids pXO1/pXO2, but the underlying genomic coding content shared by Clade D organisms, and that this genomic content is distinct from Clade C organisms. Clade D organisms may possess unique pathogenic traits that differentiate these organisms from B. anthracis . Genome summaries and pathway predictions from the UMd and original NCBI annotations Whole genome sequences from the B. cereus sensu lato group that were used in this study are listed in Table 1 . These genome sequences have accumulated over a 10-year period, and have been provided by a number of different sequencing centers. Due both to the differences in annotation methodology and the improvements in annotation that have occurred over the last decade, we suspected that comparisons of these annotations might be somewhat uneven. Consequently, we wanted to ensure that these genomes were annotated to a single standard. We employed the UMIGS [ 18 ] automated annotation pipeline for this process. This was an arbitrary selection, and we did not compare the annotation outputs that could have been obtained from several publicly-available annotation pipelines [ 31 - 33 ]. Our purpose was not to compete different annotation pipelines against one another for evaluation, but merely to ensure that these annotations met a common standard prior to subsequent analyses. Indeed, reannotation substantially changed the interpretation of these genome sequences. In general, re-annotation suggested that the protein coding content of these genomes was even more closely related than has appeared in recent analyses [ 10 , 12 ]. [We will exclude B. cytotoxicus from the remainder of this discussion, as this organism has a substantially smaller genome than the remainder of these organisms [ 34 ], and skews the interpretation of these comparisons.] The average number of protein coding sequences predicted from these genomes increased from 5268 to 5430, and the range of predicted protein coding sequences in these genomes increased from 4737-5602 (original annotations) to 5340-5613 (UMd annotations; Figure 3a ); these were statistically significant increases. The predicted aggregate metabolism derived from these genome sequence annotations also was much more similar after reannotation (Figure 3b ); the average number of recognizable metabolic pathways extracted from these genomes increased from 231 to 247, and the range of predicted metabolic pathways went from 194-264 to 239-265 (original vs. UMd annotations, respectively). Notably, these changes increased the estimated cluster of core proteins encoded by all 25 organisms from 1483 to 1544, an increase of 4% (Additional file 5 ). Figure 3A and B There was an increase in core proteins after reannotation. Figure 3A, The average number of protein coding sequences predicted from these genomes increased significantly with reannotation and these were statistically significant increases, p = 0.0008. Figure 3B, The predicted aggregate metabolism derived from these genome sequence annotations also was much more similar after reannotation and this was also significant p = 0.0011. There are a variety of reasons for these differences. The original annotations of these genomes accumulated over a period of ten years (Table 1 ), offered from nine different sequencing groups. There are currently no set guidelines or standards for genome annotation, and as a result genome annotations vary from one source to another [ 35 ]. Despite the number of annotations available in the public domain, a surprisingly large number of genes are still not annotated [ 36 ]. Missed genes have a particularly strong impact on the delineation of the core genome for a large and diverse group of organisms, and on identification of strain- or species-specific genes. The percentage of missed genes in a genome has been estimated at 5-10%, which is consistent with the collective results of this reannotation [ 37 ]. Core genome COGS analysis, basal metabolism, and conserved hypothetical proteins amongst the clades Results from MEGAN analysis of the core genome which comprised of the 24 and 25 membered clusters, showed there were a total of 850 genes associated with the COGs category metabolism, which was comprised of these subset of functions for the given number of genes: Energy production and conversion-137, Carbohydrate transport and metabolism-100, Amino acid transport and metabolism- 206, Nucleotide transport and metabolism-69, Coenzyme transport and metabolism-101, Lipid transport and metabolism-60, Inorganic ion transport and metabolism-139, Secondary metabolites biosynthesis, transport and catabolism-38; 461 genes were associated with information, storage and processing, which included the following subset of functions: Translation, ribosomal structure and biogenesis-140, Transcription-188, Replication, recombination and repair-132, and chromatin structure and dynamics-1; 332 genes were associated with cellular processes and signaling, which included the following subset of functions: Cell cycle control, cell division, and chromosome partitioning-25, Signal transduction mechanisms-84, Cell wall/membrane/envelope biogenesis-79, Intracellular trafficking, secretion, and vesicular transport-29, Posttranslational modification, protein turnover, and chaperones-60, Defense mechanisms-49, and cell motility-6; 344 genes were unassigned due to lack of experimental information about them, and 705 genes were assigned as poorly characterized since there was limited information available for them in order to be placed confidently into a functional category (Additional file 6 Figure 4 ). Since there were a large number of genes in the metabolism-related subset of functional groups, we next examined the basal metabolism in these organisms. We found that the differences in basal metabolism were slight, with only one unique enzyme, tagatose 6-phosphate kinase, found in clade A and no other unique intermediary metabolic enzymes found in clades B, C and D. However, it is unclear how this enzyme may contribute to clade A-specific phenotypes, since little work has been reported in this group of organisms. We also encountered some heterogeneity within each clade: enzymes unique to single organisms within each clade though not present in all organisms of the given clade (Additional file 7 ). While there is little difference between these clades in terms of intermediary metabolism, we speculate that the regulatory control of these proteins is likely different in different organisms, and could contribute to phenotypic differences between these clades, even though coding content is highly similar. Figure 4 The largest groups of genes were those with metabolism-related functions . Subsets of clusters of orthologous genes (COGs) categories from core genes common to clades A-D. The subsets of COGs categories are shown here as functional groups as denoted by MEGAN. Genes not found by the COGS analysis tool were placed in non-characterized/function and unknown category. We also parsed the dataset for conserved/hypothetical proteins shared by all members of a given clade while not being found in any other clade organism. With the exception of Clade C, the contribution of clade-specific conserved hypotheticals to genomic coding capacity was minimal (Additional file 8 ). The majority of Clade C-specific conserved hypothetical proteins appear to be contained in 4 bacteriophage lysogens that had earlier been shown to be unique to B. anthracis [ 38 , 39 ]. Five of the clade C proteins were predicted by SignalP to have a signal peptide and could therefore potentially be cell wall or secreted proteins that may be important for anthrax pathogenesis. Consequently, these conserved/hypothetical proteins may contain additional information about some of the unique biology that contributes specifically to clade C organisms. However, currently these proteins have yet to be studied and characterized therefore additional experiments which are beyond the scope of this current study would be necessary to establish a precise function for these proteins (Additional file 5 ). Comparison of whole genome protein coding content to B. cereus sensu lato sigB regulons Strikingly, the genomic relationships between these organisms, shown in Figure 2 , were identical to those that we had previously identified, based on the predicted structure of the generalized stress response regulon controlled by SigB [ 21 ]. Those observations are summarized in Table 2 for comparison. Organisms in the present Clade C appear to encode a generalized stress response, controlled by SigB, that has diverged from other members of this group to include additional functions that we hypothesized are uniquely involved in invasive disease; Clade D organisms have a SigB regulon of similar structure. By contrast, organisms in Clades A and B of the present study, many of which were associated with food poisoning outbreaks, carry a SigB regulon that either harbors only the core SigB regulon genes found in all B. cereus sensu lato organisms (Clade B), or have added to this core SigB regulon additional genes (Clade A) that may act enhance the stress response of these organisms during the response to deleterious environmental conditions (e.g., cold temperatures, UV light) that may be found in food processing facilities. Importantly, differences between the SigB regulon structure in these four clades are primarily a consequence of whether components of the core genome are/are not driven by a SigB promoter [ 21 ], rather than differences in gene content between these clades; the majority of genes contained in the SigB regulons of these organisms are included in the core genomes of these organisms, and their differences lie in whether their transcription is controlled by SigB. That we arrived at the same genomic relationships between these organisms using two entirely different approaches suggests an underlying organization in this population that has not been previously noted. The four clades that we identified in Figure 2 appear to be a result of at least two processes acting in parallel: 1) divergence in genomic coding capacity (protein coding genes), and 2) divergence in the generalized stress response regulon. As noted above, these processes appear to be independent of the extrachromosomal DNA content of these organisms (see Figure 2 ). This suggests the more general hypothesis that divergence among the members of the B. cereus sensu lato group relies on a pattern of regulatory divergence overlaying the protein coding divergence seen in Figure 2 . Obviously, further work is necessary to test this hypothesis. In particular, does additional regulatory divergence in these organisms follow this same pattern, or is this restricted to the divergence of the SigB-controlled generalized stress response? Our recent informatics analysis (Scott EJ and Dyer DW, manuscript in preparation) suggests that the SigM ECF sigma factor regulons in these organisms appear to diverge in the same manner as we have observed here; the SigM regulons can be sorted into four clusters that also correspond to those described here. Thus, our finding that coupling protein coding divergence and regulatory divergence may be a more general phenomenon that contributes to the phenomotype of any given strain. Conclusion Thus, the biological divergence in the four clades of B. cereus sensu lato organisms shown in Figure 2 seems to be a consequence of three evolutionary strategies that contribute in different ways. HGT of extrachromosomal elements (e.g., pXO1 and pXO2 in anthrax-like disease, the cry toxin plasmids in B. thuringiensis strains) is obviously important, but the combined forces of HGT and gene duplication/divergence acting at the genomic level appear to have promoted the divergence of these B. cereus sensu lato organisms into four separable clades that are not solely defined by plasmid inheritance, as illustrated in Figure 2 . Previous studies have reported similar clade structures to ours [ 10 , 12 , 21 ]. However, the main differences between our study and others is that we focused solely on genomic content in order to understand the phenomotypic differences between Bacillus anthracis strains and other strains from the Bacillus cereus sensu lato group. We also utilized all chromosomal proteins (both core and unique proteins) for our study. Comparison of the draft genome clusters to the clade structure from the whole genomes heatmap, suggested similar relationships. Future studies will include strategies to determine how much of an influence the variation in annotation may have on the output of the data; perhaps this allow us select for those genomes with less noise, to enhance the comparisons of draft and whole genome datasets. Lastly, divergence of the SigB generalized stress response regulons in these organisms (summarized in Table 2 ) mirrors the population structure arising from bulk protein coding sequence comparisons. This suggests the hypothesis that patterns of regulatory divergence between these four clades are superimposed over the minimal differential genomic coding capacity found in these genomes. This level of divergence could fine-tune the protein expression patterns of the clade-specific gene sets, to increase fitness in specific environments. Further work to examine the divergence of transcriptional regulatory networks in these organisms is necessary to test this hypothesis. Availability of supporting data "The data sets supporting the results of this article are included within the article and its additional files" List of abbreviations used COGs- Clusters of Orthologous Genes, KEGGS- Kyoto Encyclopedia of Genes and Genomes, SRI- Stanford Research Institute, UMd- University of Maryland Competing interests The authors declare that they have no competing interests. Authors' contributions IT performed whole genome comparative analysis including pathway, metabolism, and COGS analysis. DW co-wrote this manuscript and advised on experiments to include in the paper. JW performed analysis on the conserved/hypothetical proteins and co-wrote that section of the paper. Supplementary Material Additional File 1 Table S1.xls; This file contains a list of all 201 draft and whole genomes used in the study and source/location of isolation information where available. Genomes with no information available to determine source or location of isolation are denoted with "N/A". In this table, the genome names are written in the order for which they appear in Figure 1 . Click here for file Additional File 2 Table S2 .xls; This file contains sheets A-D which show the Cd-hit clusters #0-35,000 from the Clusters of Orthologous Proteins for 201 draft and whole genomes. Click here for file Additional File 3 Table S3 .xls: This file contains a sheet showing Cd-hit clusters #35001-92805 from the Clusters of Orthologous Proteins for 201 draft and whole genomes. Click here for file Additional File 4 Table S4 .xls; This file contains Cd-hit output data from 25 whole genomes. This file contains a list of all clusters of orthologous proteins generated during our analysis. Click here for file Additional File 5 Table S5 .xls; This table shows number of pathways, total gene counts, average gene length and percent coding. Click here for file Additional File 6 Table S6 .xls; COGS categories and subset of COGS categories. Number of genes found for subset of COGs categories. Click here for file Additional File 7 Table S7 .xls; This table consists of parts A-D with data available in 4 spreadsheets. Each spreadsheet contains subsets of data from the KEGGs Enzyme analysis files. Also within this table are all enzymes from the KEGGs database as compared to our clade organisms. Click here for file Additional File 8 Table S8 .xls; Analysis of conserved/hypothetical proteins. List of conserved/hypothetical proteins genome id # with known hits in pfam, signal p, and TmHmm databases. Click here for file Acknowledgements The authors wish to acknowledge Mr. Edgar Scott for sharing his work on the sigB and sigM stress response in Bacillus cereus sensu lato group. These studies and publication were supported by Grant # P20RR016478 from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health (NIH). This article has been published as part of BMC Bioinformatics Volume 15 Supplement 11, 2014: Proceedings of the 11th Annual MCBIOS Conference. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/15/S11 .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9649426/

Risk Factors for Death or Meningitis in Adults Hospitalized for Cutaneous Anthrax, 1950–2018: A Systematic Review

Abstract Background Cutaneous anthrax accounts for approximately 95% of anthrax cases worldwide. About 24% of untreated patients die, and many cases are complicated by meningitis. Here, we explore clinical features of cutaneous disease associated with poor outcomes. Methods A systematic review identified 303 full-text articles published from 1950 through 2018 that met predefined inclusion criteria. Cases were abstracted, and descriptive analyses and univariate logistic regression were conducted to identify prognostic indicators for cutaneous anthrax. Results Of 182 included patients, 47 (25.8%) died. Previously reported independent predictors for death or meningitis that we confirmed included fever or chills; nausea or vomiting; headache; severe headache; nonheadache, nonmeningeal signs; leukocytosis; and bacteremia. Newly identified predictors included anxiety, abdominal pain, diastolic hypotension, skin trauma, thoracic edema, malignant pustule edema, lymphadenopathy, and evidence of coagulopathy (all with P < .05). Conclusions We identified patient presentations not previously associated with poor outcomes. Background Cutaneous anthrax accounts for approximately 95% of anthrax cases worldwide. About 24% of untreated patients die, and many cases are complicated by meningitis. Here, we explore clinical features of cutaneous disease associated with poor outcomes. Methods A systematic review identified 303 full-text articles published from 1950 through 2018 that met predefined inclusion criteria. Cases were abstracted, and descriptive analyses and univariate logistic regression were conducted to identify prognostic indicators for cutaneous anthrax. Results Of 182 included patients, 47 (25.8%) died. Previously reported independent predictors for death or meningitis that we confirmed included fever or chills; nausea or vomiting; headache; severe headache; nonheadache, nonmeningeal signs; leukocytosis; and bacteremia. Newly identified predictors included anxiety, abdominal pain, diastolic hypotension, skin trauma, thoracic edema, malignant pustule edema, lymphadenopathy, and evidence of coagulopathy (all with P < .05). Conclusions We identified patient presentations not previously associated with poor outcomes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9686932/

An Overview of the Potentialities of Antimicrobial Peptides Derived from Natural Sources

Antimicrobial peptides (AMPs) are constituents of the innate immune system in every kind of living organism. They can act by disrupting the microbial membrane or without affecting membrane stability. Interest in these small peptides stems from the fear of antibiotics and the emergence of microorganisms resistant to antibiotics. Through membrane or metabolic disruption, they defend an organism against invading bacteria, viruses, protozoa, and fungi. High efficacy and specificity, low drug interaction and toxicity, thermostability, solubility in water, and biological diversity suggest their applications in food, medicine, agriculture, animal husbandry, and aquaculture. Nanocarriers can be used to protect, deliver, and improve their bioavailability effectiveness. High cost of production could limit their use. This review summarizes the natural sources, structures, modes of action, and applications of microbial peptides in the food and pharmaceutical industries. Any restrictions on AMPs' large-scale production are also taken into consideration. 1. Introduction Antimicrobial peptides (AMPs) are the oldest known innate immune defense molecules. They are abundant in plants, arthropods, microorganisms, and animals [ 1 ]. Eukaryotes and prokaryotes synthesize AMPs in ribosomes, fungi, and bacteria, turning them into cytosol [ 2 ]. AMPs can have broad-spectrum or specific activity against pathogenic bacteria (both Gram-positive and Gram-negative), viruses, fungi, and other parasites [ 3 ]. AMPs differ in length and composition of amino acids [ 4 ]. Defensins, puroindolines, snakins, cyclotides, glycine-rich proteins, hevein, α-hairpin, knottin, and lipid transfer proteins are some natural classes of AMPs [ 5 ]. Their activity is bound by helical structure, charge, hydrophobicity, and amphipathicity [ 4 ]. The food industry employs AMPs as biopreservants and in food packaging (alone or with other antimicrobials and essential oils) to improve product shelf-life [ 6 ]. Antimicrobial peptides are considered potential drugs for treating infections caused by microorganisms that are untreatable with antibiotics on the market today [ 7 , 8 ]. They can reduce the development of antimicrobial resistance, affecting multiple low-affinity targets [ 9 ]. Some AMPs are subjected to peptide engineering and mutagenesis to make compounds with improved bioactivity and reduced cytotoxicity [ 10 , 11 ]. This review offers an overview of structures, sources, modes of action, and applications of AMPs in the food and pharmaceutical fields. 2. Antimicrobial Peptides' Natural Source Antimicrobial peptides are made by lower and higher organisms responding to pathogenic challenges [ 12 ]. AMPs kill the invading pathogens and modulate the innate immune response. They are commonly classified according to their sources, amino-acid-rich species, structural characteristics, and activities [ 13 ]. In multicellular organisms and humans, they are localized into specific sites commonly exposed to microbes (i.e., mucosa epithelia and skin) [ 13 ] ( Figure 1 ). 2.1. Viral AMPs Some phage proteins, including lysins, depolymerases, virion-associated peptidoglycan hydrolases (VAPGHs), and holins, show antibacterial activity [ 14 ]. They are defined as "enzybiotics" to indicate their use as antibacterial materials as alternatives to standard antibiotics [ 15 ]. The two types of phage AMPs are known as phage-encoded lytic factors and phage-tail complexes [ 16 ]. Phage lysines (size range from 25 to 40 kDa) are peptidoglycan-hydrolyzing enzymes [ 17 ], which can hydrolyze the microbial cell wall, permitting bacteriophage progeny release [ 16 ]. Lysins have rapid bactericidal activity (against Gram-positive and Gram-negative bacteria) and other desirable characteristics, such as synergy with cell-wall-reducing antibiotics, anti-biofilm action, heat stability up to ~50 °C, and the possibility of lyophilization [ 18 , 19 , 20 ]. Peptidoglycan hydrolases (VAPGHs), encoded mainly by double-stranded DNA phages, have high thermal stability. They infect Gram-positive and Gram-negative bacteria. VAPGHs have a C -terminal cell-wall-binding domain, which can link them to receptors on the bacterial cell surface. They inject genetic materials into bacterial cells after partially and locally damaging bacterial cell wall peptidoglycans [ 21 ]. They can be classified into three categories: glycosidases that cut glycosidic bonds in the peptidoglycan chain, amidases that cut amide bonds (between N-acetylmuramic acid lactyl and stem peptide l-alanines), and endopeptidases that cleave peptide bonds within either the stem peptide or cross-link [ 22 ]. Phage-tail-like AMPs are high-molecular-weight cylindrical peptides with a structure like a phage tail [ 23 , 24 ]. They can be classified into two classes: R-type (related to Myoviridae phage tails) and F-type (related to Siphoviridae phage tails) [ 23 ]. R-type phage-tail-like bacteriocins are nonflexible and have tubes surrounded by contractile sheaths [ 25 ]. They initially make a channel in the cell membrane and successively drive their internal core into the cell. This process determines rapid cell death by decoupling cellular ion gradients [ 23 ], interfering with oxygen uptake, and affecting macromolecule synthesis [ 26 ]. F-type phage-tail-like bacteriocins are flexible and noncontractile [ 25 ]. They act similarly to R-type bacteriocins [ 27 ]. 2.2. Bacterial AMPs 2.2.1. AMPs Made by Gram-Positive Bacteria Gram-positive bacteria can produce AMPs in ribosomes (ribosomal AMPs) or enzymatically (non-ribosomal AMPs) [ 28 , 29 ] ( Figure 2 ). Twenty-sixty amino acids (hydrophobic and cationic) can make up ribosomally synthesized bacterial AMPs (bacteriocins) [ 30 ]. Bacteriocins can be classified into bacteriocins produced by Gram-positive and Gram-negative bacteria [ 31 ]. 2.2.2. AMPs Made by Gram-Positive Bacteria Gram-positive organisms make bacteriocins that can be grouped into lantibiotics (class I), non-lantibiotics (class II), large-sized bacteriocins (class III), and uniquely structured bacteriocins (class IV) [ 32 ] ( Figure 2 ). Lantibiotics are active against primarily Gram-positive bacteria [ 32 ]. They are small peptides (30 kDa), heat-labile peptides [ 32 ] ( Figure 2 ). Class IV AMPs containing lipids or carbohydrates are susceptible to lipolytic and glycolytic enzymes [ 40 ] ( Figure 2 ). Non-ribosomally synthesized AMPs are made from peptide synthetases produced by Gram-positive and Gram-negative bacteria [ 9 ]. Bacteriocins can decrease food spoilage [ 31 ]. 2.2.3. AMPs Made by Gram-Negative Bacteria Gram-negative organisms make bacteriocins that can be grouped into microcins, colicins, colicin-like bacteriocins, and phage-tail-like bacteriocins [ 41 ] ( Figure 3 ). Microcins are made by Enterobacteriaceae . Microcins interact with some cellular targets. They can format pores that determine membrane disruption [ 23 ] or decrease the functionality of enzymes (the ATP synthase complex, DNA gyrase, RNA polymerase, and aspartyl-t RNA synthetase) [ 32 ]. They are grouped into two subclasses: subclass I (molecular weight lower than 5 kDa) and subclass II (molecular weight ranging from 5 to 10 kDa) [ 42 , 43 ] ( Figure 3 ). Colicins (MW > 10 kDa) are made mainly by Enterobacteriaceae (mainly E. coli ) [ 44 ]. They can form pores in the cell wall or degrade bacteria nucleic acid structures (RNAses, DNAses, or t RNAses) [ 32 ]. Colicins can be grouped into four subclasses: colicins forming channels in the cytoplasmic membrane, colicins degrading DNA, colicins targeting rRNA or tRNA, and colicins inhibiting murein and lipopolysaccharide biosynthesis [ 45 , 46 ] ( Figure 3 ). Colicin-like bacteriocins are made by the Klebsiella genus (klebicins) and P. aeruginosa (S-type pyocins) [ 46 ]. They are similar in size, structure, and function to colicins. Phage-tail-like bacteriocins have structures similar to phage tails. They are cylindrical peptides with high molecular weights [ 23 ]. They are grouped into the R-type and F-type subclasses [ 23 ] ( Figure 3 ). R-type phage-tail-like bacteriocins bind to cell surface receptors, force the internal core into the microbial cell envelope, and determine rapid cell death [ 23 ]. They also affect macromolecule synthesis and oxygen uptake [ 9 ]. F-type phage-tail-like bacteriocins have a mechanism of action similar to R-type, but do not have contractile movement [ 27 ]. 2.3. Fungal AMPs Fungi produce peptaibols and fungal defensins [ 47 , 48 ] ( Figure 4 ). The term "peptaibol" is linked to structural characteristics. It is a combination of the words "peptide," " α -aminoisobutyrate," and "amino alcohol" [ 49 ]. Peptaibols are mainly made by Trichoderma fungi [ 50 ]. They are short peptides (containing 5–21 amino acids) with a high proportion of non-proteinogenic amino acids (i.e., α-aminoisobutyric acid), acylated N -terminal residue, and amino alcohol (i.e., leucenol or phenylalaninol) linked to the C -terminal [ 51 ]. Their three-dimensional structures consist of α -helix and β -bend patterns [ 52 ]. They are classified based on sequence length as "long" (18−20 residues), "short" (11−16 amino acids) ( Figure 4 ), and founded on modification types on the terminal groups, "lipo" peptaibols (i.e., N -terminal acylated by decanoic) [ 53 ]. Different mechanisms have been proposed to describe their action. Concerning large peptaibols, it was hypothesized that their helical structures oligomerize and can form ion channels in the membrane. Instead, short peptaibols can form a pore via helical bundles (within the bilayer or by a barrel-stave mechanism) and interact with diverse molecular targets [ 9 ]. Peptaibols' modes of action that do not involve interaction with the bacterial membrane include the inhibition of cell wall synthesis, DNA, protein synthesis, and that of relevant enzymes [ 10 ]. Eukaryotes and bacteria can produce defensins. Defensins are a class of cysteine-rich AMPs with short, cationic disulfide bridges [ 54 ]. They can be grouped into two superfamilies (cis and trans) ( Figure 4 ). Fungi can produce cis -defensins with α-helical (cysteine-stabilized) or β-sheet folds. Defensins can disrupt the microbial cytoplasmic membrane, bind the bacterial precursor lipid II of the cell wall, or prevent cell wall biosynthesis [ 55 ]. 2.4. Plant AMPs Plant AMPs are the first line of defense against infections produced by pathogenic microorganisms. They can have diverse structures and action mechanisms. Their classification is based on their tridimensional structures and amino acid sequence similarity, including thionins, hevein-like peptides, defensins, knottins, stable-like peptides, snakins, lipid transfer proteins, and cyclotides [ 56 ] ( Figure 5 ). 2.4.1. Thionins Thionins are classified into five types indicated by Roman numerals, have sizes ranging from 45 to 48, and are found in monocots and dicots. They include two distinct superfamilies: α/β-thionins and γ-thionins [ 57 ]. α/β thionins have similar structures (homologous amino acid sequences) [ 58 ] and are rich in arginine, cysteine, and lysine. γ-thionins are similar to defensins, so some authors classify them in this group [ 59 ]. Thionins have a broad spectrum of activities. They act against Gram-positive and Gram-negative bacteria, yeast, fungi, insect larvae, and nematodes [ 60 , 61 , 62 ] and present cytotoxic effects against mammal cells in vitro [ 63 ]. 2.4.2. Hevein-like peptides Hevein-like peptides can contain 29–45 amino acids with glycine (6), cysteine (8–10), and aromatic residues. They have a chitin-binding domain responsible for their antifungal activity [ 64 ] and 3–5 disulfide bonds that stabilize the antiparallel β-sheets and short α-helix [ 65 ]. The factors that favor chitin-binding are the three aromatic amino acids that give stability to the hydrophobic C-H group, the π electron system that determines van der Waals forces, and the hydrogen bonds between serine and N-acetylglucosamine [ 64 ]. Hevein-like peptides damage the fungal cell wall by interacting with hydrophobic residues and chitin present in the fungal cell [ 5 ]. They can constrain some enzymes' activities by linking them with disulfide bonds [ 66 ]. 2.4.3. Defensins Defensins can comprise 45–54 amino acids and four disulfide bridges. They have an antiparallel β sheet, are enclosed by an α-helix, and are limited by intramolecular disulfide bonds [ 67 ] called cysteine-stabilized αβ (CSαβ) motifs [ 68 ]. Defensins are resistant to proteolysis and are stable to variations in temperature and pH. They prevent microbial growth, trypsin, and α-amylase activities, decrease abiotic stress, and change the redox state of ascorbic acid [ 56 ]. 2.4.4. Knottins Knottins, also called "cysteine-knot peptides", are formed by 39 amino acids (of which six are cysteine residues), have three disulfide bonds (cysteine-knot motifs), and can be found in two conformations (cyclic and linear) [ 5 , 69 ]. They have high thermal stability and resistance to proteolytic action and can inhibit α-amylase, trypsin, carboxypeptidase, and cysteine protease [ 70 , 71 ]. They differ from protease inhibitors and defensins regarding cysteine space [ 5 ]. They are amphipathic peptides whose cationic portions can bind cell membranes, acid-sensing channels, and K + and Na + channels in membranes. Once they enter a cell, they attack intracellular targets (i.e., carboxypeptidases) and promote resistance [ 61 ]. Unfortunately, knottins are highly cytotoxic to human cells since their contact with membranes is not selective. 2.4.5. Stable-like Peptides Stable-like peptides are a class of small peptides that form a helix-loop-helix structure with a typical Cys motif of XnC1X3C2XnC3X3C4Xn (-X is an amino acid residue different from cysteine). Although their amino acid sequence is highly variable, the three-dimensional structure of stable-like peptides is conserved. They can have antifungal, antibacterial, ribosome-inactivating, and trypsin inhibiting activities [ 72 ]. Their bacteriostatic effect is due to binding with DNA, which decreases RNA and protein synthesis [ 73 ]. Their activity relates to the loop region that connects the two α-helices [ 74 ]. 2.4.6. Snakins Snakins are generally small (~7 kDa), cysteine-rich, and positively charged proteins with antimicrobial, antinematode, and antifungal properties [ 75 ]. The mechanism of action is not precise. More than one hypothesis has been developed to explain it. Some authors believe they can promote immune responses by destabilizing the site of action through interaction with the negatively charged component [ 76 , 77 ]. Other authors hypothesized that they can act on phytohormone biosynthesis and transduction processes [ 78 ]. 2.4.7. Lipid Transfer Proteins Lipid transfer proteins (LTPs) are small, cysteine-rich proteins (containing 100 aa) having 4 to 5 helices in their structure that are stabilized by hydrogen bonds. They can transfer lipids (i.e., fatty acids, phospholipids, acyl CoA fatty acids, and sterols) between membranes. In this way, they form pores and determine cell death. They can be classified into two subfamilies, LTP1 (relative molecular weight of 9  kDa) and LTP2 (relative molecular weight of 7 kDa), or into five types (LTP1, LTP2, LTPc, LTPd, and LTPg) based on the position of the conserved intron, the space between the cysteine residues, and the identity of the amino acid sequence [ 69 ]. 2.4.8. Cyclotides Cyclotides are macrocyclic with cyclic cystine knot (CCK) structural motif peptides [ 79 ]. Disulfide bridges stabilize the head-to-tail cyclo. They can be classified into two subfamilies: Möbius and bracelets [ 80 ]. Their action depends on the cystine knot structural motif that promotes hydrophobic residue surface contact, some of which form a hydrophobic patch [ 81 ]. Cyclotides can act against bacteria, helminths, insects, and mollusks and have ecbolic anti-HIV and anticancer properties [ 81 ]. 2.5. Animal AMPs Vertebrate defensins are synthesized as "prepropeptides" and classified into α, β, and θ defensins [ 82 ]. They have short polypeptide sequences (18–45 amino acids), cationic net charges (+1 to +11), and three intramolecular disulfide bonds. In human α-defensins, the characteristic connections of disulfide bridges are Cys 1 –Cys 6 , Cys 2 –Cys 4 and Cys 3 –Cys 5 [ 83 ]. They are synthesized by promyelocytes and intestinal Paneth cells [ 84 ]. β-defensins differ from α-defensins in disulfide bond distributions and cysteine residues. The disulfide bridges in human β-defensins are Cys 1 –Cys 5 , Cys 2 –Cys 4 and Cys 3 –Cys 6 [ 83 ]. θ-defensins are cyclic octadecapeptides not expressed in humans and are active against B. anthrax , S. aureus , and C. albicans [ 85 , 86 , 87 ]. They contain a macrocyclic backbone and are structurally dissimilar to α- and β-defensins [ 88 ]. Invertebrates synthesize AMPs as components of humoral defense [ 89 ]. They are cationic peptides that can contain six or eight cysteine residues and show a cysteine-stabilized α/β motif [ 90 ]. The defensins produced by insects, arthropods, and mollusks contain six cysteines. Eight cysteines form defensins made by mollusks and nematodes [ 91 ]. Invertebrate defensins are phylogenetically and structurally associated with vertebrate β-defensins. They have a hydrophobic domain ( N -terminal) that can act against Gram-positive bacteria and a cationic domain ( C -terminal containing six cysteines) that can act against Gram-negative bacteria [ 92 ]. Crustins (cationic cysteine-rich peptides that form a tightly packed structure) are found in crustaceans [ 93 ]. They have an N -terminal multidomain (rich in glycine, cysteine, and proline) and a C -terminal (with four C -terminal disulfide bridges) ( Table 1 ) [ 94 ]. In fish, reptiles, amphibians, birds, and mammalians, AMPs ( size range of 15–200 residues) play an essential role in the immediate response to microorganisms [ 9 ]. Fish produce β-defensins, cathelicidins, hepicidins, histone-derived peptides, and piscidins [ 95 ] ( Table 1 ). Fish defensins are β-defensin-like proteins containing six cysteine motifs [ 96 ]. Cathelicidins are cationic proteins activated by elastase and other proteases discovered in the secretory granules of immune cells [ 97 ]. They act against Gram-positive and Gram-negative bacteria, parasites, fungi, and enveloped viruses [ 98 , 99 , 100 , 101 , 102 ]. Cathelicidins can bind and disrupt negatively charged membranes, alter RNA and DNA synthesis, damage the functions of enzymes and chaperones, and promote protein degradation [ 103 ]. Fish hepcidins are cysteine-rich peptides similar to human hepcidin with a hairpin structure linked via four disulfide bonds. They are iron-regulating antimicrobial hormones [ 104 , 105 ]. Hepcidins are grouped into HAMP1 and HAMP2 [ 95 ]. They act against bacteria (Gram-positive and Gram-negative) and fish pathogens and induce the internalization and degradation of ferroportin [ 106 ]. Piscidins are linear amphipathic AMPs. They have histidine residue and an α-helix that can interact with lipid bilayers [ 107 ]. They are classified into piscidins 1–7 based on their biological activity, amino acid sequence, and length [ 107 ]. Reptiles and avians produce cathelicidins and defensins (α-, β-, and θ-defensins) [ 108 ]. Cathelicidins are small-sized proteins made by macrophages and neutrophils [ 109 ]. Amphibians can produce magainin and cancrin (GSAQPYKQLHKVVNWDPYG) [ 13 ]. Mammalians make cathelicidin, defensin, platelet antimicrobial protein, dermcidin, and hepcidin AMPs [ 110 ]. Mammalian cathelicidins are cationic peptides with an amphipathic structure that assume α-helical, elongated conformations or β-hairpin forms [ 9 ]. 2.1. Viral AMPs Some phage proteins, including lysins, depolymerases, virion-associated peptidoglycan hydrolases (VAPGHs), and holins, show antibacterial activity [ 14 ]. They are defined as "enzybiotics" to indicate their use as antibacterial materials as alternatives to standard antibiotics [ 15 ]. The two types of phage AMPs are known as phage-encoded lytic factors and phage-tail complexes [ 16 ]. Phage lysines (size range from 25 to 40 kDa) are peptidoglycan-hydrolyzing enzymes [ 17 ], which can hydrolyze the microbial cell wall, permitting bacteriophage progeny release [ 16 ]. Lysins have rapid bactericidal activity (against Gram-positive and Gram-negative bacteria) and other desirable characteristics, such as synergy with cell-wall-reducing antibiotics, anti-biofilm action, heat stability up to ~50 °C, and the possibility of lyophilization [ 18 , 19 , 20 ]. Peptidoglycan hydrolases (VAPGHs), encoded mainly by double-stranded DNA phages, have high thermal stability. They infect Gram-positive and Gram-negative bacteria. VAPGHs have a C -terminal cell-wall-binding domain, which can link them to receptors on the bacterial cell surface. They inject genetic materials into bacterial cells after partially and locally damaging bacterial cell wall peptidoglycans [ 21 ]. They can be classified into three categories: glycosidases that cut glycosidic bonds in the peptidoglycan chain, amidases that cut amide bonds (between N-acetylmuramic acid lactyl and stem peptide l-alanines), and endopeptidases that cleave peptide bonds within either the stem peptide or cross-link [ 22 ]. Phage-tail-like AMPs are high-molecular-weight cylindrical peptides with a structure like a phage tail [ 23 , 24 ]. They can be classified into two classes: R-type (related to Myoviridae phage tails) and F-type (related to Siphoviridae phage tails) [ 23 ]. R-type phage-tail-like bacteriocins are nonflexible and have tubes surrounded by contractile sheaths [ 25 ]. They initially make a channel in the cell membrane and successively drive their internal core into the cell. This process determines rapid cell death by decoupling cellular ion gradients [ 23 ], interfering with oxygen uptake, and affecting macromolecule synthesis [ 26 ]. F-type phage-tail-like bacteriocins are flexible and noncontractile [ 25 ]. They act similarly to R-type bacteriocins [ 27 ]. 2.2. Bacterial AMPs 2.2.1. AMPs Made by Gram-Positive Bacteria Gram-positive bacteria can produce AMPs in ribosomes (ribosomal AMPs) or enzymatically (non-ribosomal AMPs) [ 28 , 29 ] ( Figure 2 ). Twenty-sixty amino acids (hydrophobic and cationic) can make up ribosomally synthesized bacterial AMPs (bacteriocins) [ 30 ]. Bacteriocins can be classified into bacteriocins produced by Gram-positive and Gram-negative bacteria [ 31 ]. 2.2.2. AMPs Made by Gram-Positive Bacteria Gram-positive organisms make bacteriocins that can be grouped into lantibiotics (class I), non-lantibiotics (class II), large-sized bacteriocins (class III), and uniquely structured bacteriocins (class IV) [ 32 ] ( Figure 2 ). Lantibiotics are active against primarily Gram-positive bacteria [ 32 ]. They are small peptides (30 kDa), heat-labile peptides [ 32 ] ( Figure 2 ). Class IV AMPs containing lipids or carbohydrates are susceptible to lipolytic and glycolytic enzymes [ 40 ] ( Figure 2 ). Non-ribosomally synthesized AMPs are made from peptide synthetases produced by Gram-positive and Gram-negative bacteria [ 9 ]. Bacteriocins can decrease food spoilage [ 31 ]. 2.2.3. AMPs Made by Gram-Negative Bacteria Gram-negative organisms make bacteriocins that can be grouped into microcins, colicins, colicin-like bacteriocins, and phage-tail-like bacteriocins [ 41 ] ( Figure 3 ). Microcins are made by Enterobacteriaceae . Microcins interact with some cellular targets. They can format pores that determine membrane disruption [ 23 ] or decrease the functionality of enzymes (the ATP synthase complex, DNA gyrase, RNA polymerase, and aspartyl-t RNA synthetase) [ 32 ]. They are grouped into two subclasses: subclass I (molecular weight lower than 5 kDa) and subclass II (molecular weight ranging from 5 to 10 kDa) [ 42 , 43 ] ( Figure 3 ). Colicins (MW > 10 kDa) are made mainly by Enterobacteriaceae (mainly E. coli ) [ 44 ]. They can form pores in the cell wall or degrade bacteria nucleic acid structures (RNAses, DNAses, or t RNAses) [ 32 ]. Colicins can be grouped into four subclasses: colicins forming channels in the cytoplasmic membrane, colicins degrading DNA, colicins targeting rRNA or tRNA, and colicins inhibiting murein and lipopolysaccharide biosynthesis [ 45 , 46 ] ( Figure 3 ). Colicin-like bacteriocins are made by the Klebsiella genus (klebicins) and P. aeruginosa (S-type pyocins) [ 46 ]. They are similar in size, structure, and function to colicins. Phage-tail-like bacteriocins have structures similar to phage tails. They are cylindrical peptides with high molecular weights [ 23 ]. They are grouped into the R-type and F-type subclasses [ 23 ] ( Figure 3 ). R-type phage-tail-like bacteriocins bind to cell surface receptors, force the internal core into the microbial cell envelope, and determine rapid cell death [ 23 ]. They also affect macromolecule synthesis and oxygen uptake [ 9 ]. F-type phage-tail-like bacteriocins have a mechanism of action similar to R-type, but do not have contractile movement [ 27 ]. 2.2.1. AMPs Made by Gram-Positive Bacteria Gram-positive bacteria can produce AMPs in ribosomes (ribosomal AMPs) or enzymatically (non-ribosomal AMPs) [ 28 , 29 ] ( Figure 2 ). Twenty-sixty amino acids (hydrophobic and cationic) can make up ribosomally synthesized bacterial AMPs (bacteriocins) [ 30 ]. Bacteriocins can be classified into bacteriocins produced by Gram-positive and Gram-negative bacteria [ 31 ]. 2.2.2. AMPs Made by Gram-Positive Bacteria Gram-positive organisms make bacteriocins that can be grouped into lantibiotics (class I), non-lantibiotics (class II), large-sized bacteriocins (class III), and uniquely structured bacteriocins (class IV) [ 32 ] ( Figure 2 ). Lantibiotics are active against primarily Gram-positive bacteria [ 32 ]. They are small peptides (30 kDa), heat-labile peptides [ 32 ] ( Figure 2 ). Class IV AMPs containing lipids or carbohydrates are susceptible to lipolytic and glycolytic enzymes [ 40 ] ( Figure 2 ). Non-ribosomally synthesized AMPs are made from peptide synthetases produced by Gram-positive and Gram-negative bacteria [ 9 ]. Bacteriocins can decrease food spoilage [ 31 ]. 2.2.3. AMPs Made by Gram-Negative Bacteria Gram-negative organisms make bacteriocins that can be grouped into microcins, colicins, colicin-like bacteriocins, and phage-tail-like bacteriocins [ 41 ] ( Figure 3 ). Microcins are made by Enterobacteriaceae . Microcins interact with some cellular targets. They can format pores that determine membrane disruption [ 23 ] or decrease the functionality of enzymes (the ATP synthase complex, DNA gyrase, RNA polymerase, and aspartyl-t RNA synthetase) [ 32 ]. They are grouped into two subclasses: subclass I (molecular weight lower than 5 kDa) and subclass II (molecular weight ranging from 5 to 10 kDa) [ 42 , 43 ] ( Figure 3 ). Colicins (MW > 10 kDa) are made mainly by Enterobacteriaceae (mainly E. coli ) [ 44 ]. They can form pores in the cell wall or degrade bacteria nucleic acid structures (RNAses, DNAses, or t RNAses) [ 32 ]. Colicins can be grouped into four subclasses: colicins forming channels in the cytoplasmic membrane, colicins degrading DNA, colicins targeting rRNA or tRNA, and colicins inhibiting murein and lipopolysaccharide biosynthesis [ 45 , 46 ] ( Figure 3 ). Colicin-like bacteriocins are made by the Klebsiella genus (klebicins) and P. aeruginosa (S-type pyocins) [ 46 ]. They are similar in size, structure, and function to colicins. Phage-tail-like bacteriocins have structures similar to phage tails. They are cylindrical peptides with high molecular weights [ 23 ]. They are grouped into the R-type and F-type subclasses [ 23 ] ( Figure 3 ). R-type phage-tail-like bacteriocins bind to cell surface receptors, force the internal core into the microbial cell envelope, and determine rapid cell death [ 23 ]. They also affect macromolecule synthesis and oxygen uptake [ 9 ]. F-type phage-tail-like bacteriocins have a mechanism of action similar to R-type, but do not have contractile movement [ 27 ]. 2.3. Fungal AMPs Fungi produce peptaibols and fungal defensins [ 47 , 48 ] ( Figure 4 ). The term "peptaibol" is linked to structural characteristics. It is a combination of the words "peptide," " α -aminoisobutyrate," and "amino alcohol" [ 49 ]. Peptaibols are mainly made by Trichoderma fungi [ 50 ]. They are short peptides (containing 5–21 amino acids) with a high proportion of non-proteinogenic amino acids (i.e., α-aminoisobutyric acid), acylated N -terminal residue, and amino alcohol (i.e., leucenol or phenylalaninol) linked to the C -terminal [ 51 ]. Their three-dimensional structures consist of α -helix and β -bend patterns [ 52 ]. They are classified based on sequence length as "long" (18−20 residues), "short" (11−16 amino acids) ( Figure 4 ), and founded on modification types on the terminal groups, "lipo" peptaibols (i.e., N -terminal acylated by decanoic) [ 53 ]. Different mechanisms have been proposed to describe their action. Concerning large peptaibols, it was hypothesized that their helical structures oligomerize and can form ion channels in the membrane. Instead, short peptaibols can form a pore via helical bundles (within the bilayer or by a barrel-stave mechanism) and interact with diverse molecular targets [ 9 ]. Peptaibols' modes of action that do not involve interaction with the bacterial membrane include the inhibition of cell wall synthesis, DNA, protein synthesis, and that of relevant enzymes [ 10 ]. Eukaryotes and bacteria can produce defensins. Defensins are a class of cysteine-rich AMPs with short, cationic disulfide bridges [ 54 ]. They can be grouped into two superfamilies (cis and trans) ( Figure 4 ). Fungi can produce cis -defensins with α-helical (cysteine-stabilized) or β-sheet folds. Defensins can disrupt the microbial cytoplasmic membrane, bind the bacterial precursor lipid II of the cell wall, or prevent cell wall biosynthesis [ 55 ]. 2.4. Plant AMPs Plant AMPs are the first line of defense against infections produced by pathogenic microorganisms. They can have diverse structures and action mechanisms. Their classification is based on their tridimensional structures and amino acid sequence similarity, including thionins, hevein-like peptides, defensins, knottins, stable-like peptides, snakins, lipid transfer proteins, and cyclotides [ 56 ] ( Figure 5 ). 2.4.1. Thionins Thionins are classified into five types indicated by Roman numerals, have sizes ranging from 45 to 48, and are found in monocots and dicots. They include two distinct superfamilies: α/β-thionins and γ-thionins [ 57 ]. α/β thionins have similar structures (homologous amino acid sequences) [ 58 ] and are rich in arginine, cysteine, and lysine. γ-thionins are similar to defensins, so some authors classify them in this group [ 59 ]. Thionins have a broad spectrum of activities. They act against Gram-positive and Gram-negative bacteria, yeast, fungi, insect larvae, and nematodes [ 60 , 61 , 62 ] and present cytotoxic effects against mammal cells in vitro [ 63 ]. 2.4.2. Hevein-like peptides Hevein-like peptides can contain 29–45 amino acids with glycine (6), cysteine (8–10), and aromatic residues. They have a chitin-binding domain responsible for their antifungal activity [ 64 ] and 3–5 disulfide bonds that stabilize the antiparallel β-sheets and short α-helix [ 65 ]. The factors that favor chitin-binding are the three aromatic amino acids that give stability to the hydrophobic C-H group, the π electron system that determines van der Waals forces, and the hydrogen bonds between serine and N-acetylglucosamine [ 64 ]. Hevein-like peptides damage the fungal cell wall by interacting with hydrophobic residues and chitin present in the fungal cell [ 5 ]. They can constrain some enzymes' activities by linking them with disulfide bonds [ 66 ]. 2.4.3. Defensins Defensins can comprise 45–54 amino acids and four disulfide bridges. They have an antiparallel β sheet, are enclosed by an α-helix, and are limited by intramolecular disulfide bonds [ 67 ] called cysteine-stabilized αβ (CSαβ) motifs [ 68 ]. Defensins are resistant to proteolysis and are stable to variations in temperature and pH. They prevent microbial growth, trypsin, and α-amylase activities, decrease abiotic stress, and change the redox state of ascorbic acid [ 56 ]. 2.4.4. Knottins Knottins, also called "cysteine-knot peptides", are formed by 39 amino acids (of which six are cysteine residues), have three disulfide bonds (cysteine-knot motifs), and can be found in two conformations (cyclic and linear) [ 5 , 69 ]. They have high thermal stability and resistance to proteolytic action and can inhibit α-amylase, trypsin, carboxypeptidase, and cysteine protease [ 70 , 71 ]. They differ from protease inhibitors and defensins regarding cysteine space [ 5 ]. They are amphipathic peptides whose cationic portions can bind cell membranes, acid-sensing channels, and K + and Na + channels in membranes. Once they enter a cell, they attack intracellular targets (i.e., carboxypeptidases) and promote resistance [ 61 ]. Unfortunately, knottins are highly cytotoxic to human cells since their contact with membranes is not selective. 2.4.5. Stable-like Peptides Stable-like peptides are a class of small peptides that form a helix-loop-helix structure with a typical Cys motif of XnC1X3C2XnC3X3C4Xn (-X is an amino acid residue different from cysteine). Although their amino acid sequence is highly variable, the three-dimensional structure of stable-like peptides is conserved. They can have antifungal, antibacterial, ribosome-inactivating, and trypsin inhibiting activities [ 72 ]. Their bacteriostatic effect is due to binding with DNA, which decreases RNA and protein synthesis [ 73 ]. Their activity relates to the loop region that connects the two α-helices [ 74 ]. 2.4.6. Snakins Snakins are generally small (~7 kDa), cysteine-rich, and positively charged proteins with antimicrobial, antinematode, and antifungal properties [ 75 ]. The mechanism of action is not precise. More than one hypothesis has been developed to explain it. Some authors believe they can promote immune responses by destabilizing the site of action through interaction with the negatively charged component [ 76 , 77 ]. Other authors hypothesized that they can act on phytohormone biosynthesis and transduction processes [ 78 ]. 2.4.7. Lipid Transfer Proteins Lipid transfer proteins (LTPs) are small, cysteine-rich proteins (containing 100 aa) having 4 to 5 helices in their structure that are stabilized by hydrogen bonds. They can transfer lipids (i.e., fatty acids, phospholipids, acyl CoA fatty acids, and sterols) between membranes. In this way, they form pores and determine cell death. They can be classified into two subfamilies, LTP1 (relative molecular weight of 9  kDa) and LTP2 (relative molecular weight of 7 kDa), or into five types (LTP1, LTP2, LTPc, LTPd, and LTPg) based on the position of the conserved intron, the space between the cysteine residues, and the identity of the amino acid sequence [ 69 ]. 2.4.8. Cyclotides Cyclotides are macrocyclic with cyclic cystine knot (CCK) structural motif peptides [ 79 ]. Disulfide bridges stabilize the head-to-tail cyclo. They can be classified into two subfamilies: Möbius and bracelets [ 80 ]. Their action depends on the cystine knot structural motif that promotes hydrophobic residue surface contact, some of which form a hydrophobic patch [ 81 ]. Cyclotides can act against bacteria, helminths, insects, and mollusks and have ecbolic anti-HIV and anticancer properties [ 81 ]. 2.4.1. Thionins Thionins are classified into five types indicated by Roman numerals, have sizes ranging from 45 to 48, and are found in monocots and dicots. They include two distinct superfamilies: α/β-thionins and γ-thionins [ 57 ]. α/β thionins have similar structures (homologous amino acid sequences) [ 58 ] and are rich in arginine, cysteine, and lysine. γ-thionins are similar to defensins, so some authors classify them in this group [ 59 ]. Thionins have a broad spectrum of activities. They act against Gram-positive and Gram-negative bacteria, yeast, fungi, insect larvae, and nematodes [ 60 , 61 , 62 ] and present cytotoxic effects against mammal cells in vitro [ 63 ]. 2.4.2. Hevein-like peptides Hevein-like peptides can contain 29–45 amino acids with glycine (6), cysteine (8–10), and aromatic residues. They have a chitin-binding domain responsible for their antifungal activity [ 64 ] and 3–5 disulfide bonds that stabilize the antiparallel β-sheets and short α-helix [ 65 ]. The factors that favor chitin-binding are the three aromatic amino acids that give stability to the hydrophobic C-H group, the π electron system that determines van der Waals forces, and the hydrogen bonds between serine and N-acetylglucosamine [ 64 ]. Hevein-like peptides damage the fungal cell wall by interacting with hydrophobic residues and chitin present in the fungal cell [ 5 ]. They can constrain some enzymes' activities by linking them with disulfide bonds [ 66 ]. 2.4.3. Defensins Defensins can comprise 45–54 amino acids and four disulfide bridges. They have an antiparallel β sheet, are enclosed by an α-helix, and are limited by intramolecular disulfide bonds [ 67 ] called cysteine-stabilized αβ (CSαβ) motifs [ 68 ]. Defensins are resistant to proteolysis and are stable to variations in temperature and pH. They prevent microbial growth, trypsin, and α-amylase activities, decrease abiotic stress, and change the redox state of ascorbic acid [ 56 ]. 2.4.4. Knottins Knottins, also called "cysteine-knot peptides", are formed by 39 amino acids (of which six are cysteine residues), have three disulfide bonds (cysteine-knot motifs), and can be found in two conformations (cyclic and linear) [ 5 , 69 ]. They have high thermal stability and resistance to proteolytic action and can inhibit α-amylase, trypsin, carboxypeptidase, and cysteine protease [ 70 , 71 ]. They differ from protease inhibitors and defensins regarding cysteine space [ 5 ]. They are amphipathic peptides whose cationic portions can bind cell membranes, acid-sensing channels, and K + and Na + channels in membranes. Once they enter a cell, they attack intracellular targets (i.e., carboxypeptidases) and promote resistance [ 61 ]. Unfortunately, knottins are highly cytotoxic to human cells since their contact with membranes is not selective. 2.4.5. Stable-like Peptides Stable-like peptides are a class of small peptides that form a helix-loop-helix structure with a typical Cys motif of XnC1X3C2XnC3X3C4Xn (-X is an amino acid residue different from cysteine). Although their amino acid sequence is highly variable, the three-dimensional structure of stable-like peptides is conserved. They can have antifungal, antibacterial, ribosome-inactivating, and trypsin inhibiting activities [ 72 ]. Their bacteriostatic effect is due to binding with DNA, which decreases RNA and protein synthesis [ 73 ]. Their activity relates to the loop region that connects the two α-helices [ 74 ]. 2.4.6. Snakins Snakins are generally small (~7 kDa), cysteine-rich, and positively charged proteins with antimicrobial, antinematode, and antifungal properties [ 75 ]. The mechanism of action is not precise. More than one hypothesis has been developed to explain it. Some authors believe they can promote immune responses by destabilizing the site of action through interaction with the negatively charged component [ 76 , 77 ]. Other authors hypothesized that they can act on phytohormone biosynthesis and transduction processes [ 78 ]. 2.4.7. Lipid Transfer Proteins Lipid transfer proteins (LTPs) are small, cysteine-rich proteins (containing 100 aa) having 4 to 5 helices in their structure that are stabilized by hydrogen bonds. They can transfer lipids (i.e., fatty acids, phospholipids, acyl CoA fatty acids, and sterols) between membranes. In this way, they form pores and determine cell death. They can be classified into two subfamilies, LTP1 (relative molecular weight of 9  kDa) and LTP2 (relative molecular weight of 7 kDa), or into five types (LTP1, LTP2, LTPc, LTPd, and LTPg) based on the position of the conserved intron, the space between the cysteine residues, and the identity of the amino acid sequence [ 69 ]. 2.4.8. Cyclotides Cyclotides are macrocyclic with cyclic cystine knot (CCK) structural motif peptides [ 79 ]. Disulfide bridges stabilize the head-to-tail cyclo. They can be classified into two subfamilies: Möbius and bracelets [ 80 ]. Their action depends on the cystine knot structural motif that promotes hydrophobic residue surface contact, some of which form a hydrophobic patch [ 81 ]. Cyclotides can act against bacteria, helminths, insects, and mollusks and have ecbolic anti-HIV and anticancer properties [ 81 ]. 2.5. Animal AMPs Vertebrate defensins are synthesized as "prepropeptides" and classified into α, β, and θ defensins [ 82 ]. They have short polypeptide sequences (18–45 amino acids), cationic net charges (+1 to +11), and three intramolecular disulfide bonds. In human α-defensins, the characteristic connections of disulfide bridges are Cys 1 –Cys 6 , Cys 2 –Cys 4 and Cys 3 –Cys 5 [ 83 ]. They are synthesized by promyelocytes and intestinal Paneth cells [ 84 ]. β-defensins differ from α-defensins in disulfide bond distributions and cysteine residues. The disulfide bridges in human β-defensins are Cys 1 –Cys 5 , Cys 2 –Cys 4 and Cys 3 –Cys 6 [ 83 ]. θ-defensins are cyclic octadecapeptides not expressed in humans and are active against B. anthrax , S. aureus , and C. albicans [ 85 , 86 , 87 ]. They contain a macrocyclic backbone and are structurally dissimilar to α- and β-defensins [ 88 ]. Invertebrates synthesize AMPs as components of humoral defense [ 89 ]. They are cationic peptides that can contain six or eight cysteine residues and show a cysteine-stabilized α/β motif [ 90 ]. The defensins produced by insects, arthropods, and mollusks contain six cysteines. Eight cysteines form defensins made by mollusks and nematodes [ 91 ]. Invertebrate defensins are phylogenetically and structurally associated with vertebrate β-defensins. They have a hydrophobic domain ( N -terminal) that can act against Gram-positive bacteria and a cationic domain ( C -terminal containing six cysteines) that can act against Gram-negative bacteria [ 92 ]. Crustins (cationic cysteine-rich peptides that form a tightly packed structure) are found in crustaceans [ 93 ]. They have an N -terminal multidomain (rich in glycine, cysteine, and proline) and a C -terminal (with four C -terminal disulfide bridges) ( Table 1 ) [ 94 ]. In fish, reptiles, amphibians, birds, and mammalians, AMPs ( size range of 15–200 residues) play an essential role in the immediate response to microorganisms [ 9 ]. Fish produce β-defensins, cathelicidins, hepicidins, histone-derived peptides, and piscidins [ 95 ] ( Table 1 ). Fish defensins are β-defensin-like proteins containing six cysteine motifs [ 96 ]. Cathelicidins are cationic proteins activated by elastase and other proteases discovered in the secretory granules of immune cells [ 97 ]. They act against Gram-positive and Gram-negative bacteria, parasites, fungi, and enveloped viruses [ 98 , 99 , 100 , 101 , 102 ]. Cathelicidins can bind and disrupt negatively charged membranes, alter RNA and DNA synthesis, damage the functions of enzymes and chaperones, and promote protein degradation [ 103 ]. Fish hepcidins are cysteine-rich peptides similar to human hepcidin with a hairpin structure linked via four disulfide bonds. They are iron-regulating antimicrobial hormones [ 104 , 105 ]. Hepcidins are grouped into HAMP1 and HAMP2 [ 95 ]. They act against bacteria (Gram-positive and Gram-negative) and fish pathogens and induce the internalization and degradation of ferroportin [ 106 ]. Piscidins are linear amphipathic AMPs. They have histidine residue and an α-helix that can interact with lipid bilayers [ 107 ]. They are classified into piscidins 1–7 based on their biological activity, amino acid sequence, and length [ 107 ]. Reptiles and avians produce cathelicidins and defensins (α-, β-, and θ-defensins) [ 108 ]. Cathelicidins are small-sized proteins made by macrophages and neutrophils [ 109 ]. Amphibians can produce magainin and cancrin (GSAQPYKQLHKVVNWDPYG) [ 13 ]. Mammalians make cathelicidin, defensin, platelet antimicrobial protein, dermcidin, and hepcidin AMPs [ 110 ]. Mammalian cathelicidins are cationic peptides with an amphipathic structure that assume α-helical, elongated conformations or β-hairpin forms [ 9 ]. 3. Antimicrobial Peptide Structures and Activities Most AMPs are made up of from 5 to 100 amino acids and have a positive net charge (generally lysine, arginine, and histidine amino acids; +2 to +11), with about 50% hydrophobic residues (generally aliphatic and aromatic amino acids) placed in variable sequence lengths [ 111 , 112 ]. AMPs can adapt to various structural changes when contacting microbe membranes [ 113 ]. Their amino acid compositions determine their charges, hydrophobic, and amphiphilic properties [ 114 ]. The number and quality of amino acids determine an AMP's pharmacological applications. Generally, shorter AMPs are more antibacterial than long-chain linear peptides, which exhibit more hemolytic and cytotoxic activity [ 115 ]. Peptides with extremely short lengths have reduced antimicrobial potency since they have difficulty forming the amphipathic secondary structures responsible for the membrane-disruption capacity [ 116 ]. Generally, small amino acids, such as glycine, increase an AMP's activity [ 117 ]. Glycine-rich peptides have high selectivity and antimicrobial ability (especially against Gram-negative bacteria) [ 118 ], as well as and antimycotic and anticancer activities [ 119 , 120 ]. Glycine-rich AMPs with net charges ranging from −1 to −2 that require cations as cofactors (i.e., Zn 2+ ) have biocidal activity obtained by improving the eukaryotic innate immune response [ 121 ]. Proline-rich peptides can enter through membrane protein channels in the bacterial cytosol and modulate the immune system via angiogenesis or cytokine activity [ 122 , 123 ]. Cysteine-rich peptides can form pores in membranes [ 124 ]. The Cys residues can improve the AMP antimicrobial activity by stabilizing sheet or β-hairpin structures [ 125 ]. Aromatic-amino-acid-rich peptides cross the microbial membrane and disrupt it [ 126 ]. Trypsin-rich peptides stabilize the AMP tertiary structure since trypsin–trypsin interactions give a cross-strand contact [ 112 ]. Phenylalanine-rich peptides are highly hydrophobic molecules with intense antimicrobial activity against bacteria (Gram-positive and Gram-negative) and yeast [ 127 ]. They do not exhibit hemolytic activity [ 128 ]. Some lipopeptides (i.e., daptomycin, polymyxins B and E) and glycopeptides (i.e., teicoplanin, vancomycin, dalbavancin, telavancin, and oritavancin) are currently used for clinical purposes [ 129 ]. AMP secondary structures can be α-helices, β-sheets, non- α- or β- structures, or mixed structures [ 130 ]. Usually, amino acids with high helical propensity (i.e., alanine, arginine, leucine, lysine, etc.) synthesize novel antimicrobial peptides since α-helical structures promote interaction with membranes and determine membrane lysis [ 131 , 132 , 133 ]. Other characteristics that affect AMP activity are hydrophobicity and amphipathicity. AMPs with low hydrophobicity have antimicrobial activities since the self-association of peptides stops peptide passage through the cell wall [ 134 ]. Amphipathic AMPs have bactericidal and cytotoxic activities linked to their aptitude to form an α-helix [ 135 ]. They can interact with intracellular targets, damaging the membrane structure or making transient pores [ 134 ]. AMPs with high hydrophobicity have antimicrobial and hemolytic activities [ 136 ]. The high hydrophobicity of the α-helix improves the antimicrobial activity since the self-association of peptides stops peptide passage across the microbial cell wall [ 137 ] and enhances hemolytic activity, inducing peptides to penetrate deeper into the hydrophobic core of red blood cells [ 138 ]. In addition, amphipathic characteristics affect AMP activities. Imperfect amphiphilic peptides have more significant antimicrobial activity than perfect ones [ 139 ]. 4. Antimicrobial Peptide Action Mostly AMPs have a short half-life. They can act by disrupting the microbial membrane or without affecting membrane stability [ 9 ]. 4.1. AMPs with Action on Cell Membranes AMPs can make electrostatic interactions between their positive charges and the microbial cell surface's negative ones, as well as hydrophobic relations between their amphipathic domain and the microbic membrane phospholipids [ 140 ]. The physical–chemical interactions and the interfacial properties determine the destabilization and permeabilization of the microbial membrane [ 8 , 141 ]. Both vertebrates and invertebrates produce AMPs (active in vitro at micromolar levels), which can affect the cell membrane by manipulating its components [ 142 ]. Gram-positive bacteria have a dense peptidoglycan layer, while Gram-negative ones have a fine peptidoglycan layer and an extra outer membrane [ 143 ]. Teichoic acid and lipopolysaccharides provide electronegative charges on the bacterial surface. On the contrary, mammalian cell membranes do not have a net charge since the outer leaflet is formed by zwitterionic phospholipids (i.e., phosphatidylcholine, phosphatydylethanolamine, and sphingomyelin) [ 144 ] and the phospholipid bilayer is stabilized by cholesterol [ 145 ]. Thus, positively charged AMPs are significantly attracted by the negative charge (i.e., phospholipids, cardiolipin, phosphatidylglycerol, and phosphatidylserine) on bacterial membranes; instead, only weak hydrophobic interactions between AMPS and mammalian cell membranes can occur. Therefore, AMPs give selective antimicrobial effects without harming normal cells since the eukaryotic cell membranes have uncharged neutral residues (generally phospholipids, cholesterol, and sphingomyelins), which cannot interact with AMPs. Highly cationic and anionic peptides have no antimicrobial activity [ 146 , 147 ]. Pore formation can be achieved by barrel-stave, toroidal pore [ 148 ], and carpet-like [ 149 ] mechanisms, the clustering of anionic lipids [ 150 ], aggregated channels [ 151 ], or more than one mechanism [ 9 ]. The barrel-stave model hypothesizes that AMPs place themself alongside a membrane and penetrate the lipid bilayer. The pore external face is made by aligning the hydrophobic region of AMPs with the lipid bilayer's central lipid region. Instead, the pore interior is made by the peptide hydrophilic contribution (by a peptide–peptide interaction) [ 152 ]. Barrel-like pores can determine cytoplasmic outflow, membrane collapse, and cell death [ 153 ] ( Figure 6 ). The toroidal pore model hypothesizes that AMPs vertically cross a lipid membrane without peptide–peptide interactions in the lipid membrane [ 152 ]. The pores are transient and less stable than barrel-stave formations [ 154 ] ( Figure 6 ). The carpet or detergent-like model assumes that AMPs are adsorbed parallel to the lipid membrane until wholly covered (like a carpet), inducing membrane disruption. In this process, no peptides across the membrane, peptide–peptide interactions, or peptide structures are made [ 155 ] ( Figure 6 ). The AMP hydrophobic regions interact with the cell membrane, and the hydrophilicity ends with an aqueous solution [ 156 ]. Anionic lipid-clustering activity is obtained by forming phase-boundary defects between lipid domains due to interaction between cationic AMPs and anionic-charged lipids [ 150 ]. 4.2. AMPs with No Action on Cell Membranes Some AMPs can kill bacteria interfering with DNA (replication, transcription, and translation), cell division, and the blocking of protein biosynthesis and folding [ 113 , 157 , 158 ]. They can also interfere with the immune system, activating white blood cells, improving angiogenesis, blocking reactive oxygen and nitrogen species [ 159 ], suppressing toll-like receptors, reducing anti-endotoxin activity, interfering with cytokine-mediated production of cytokines [ 160 ], and influencing T- and B-cell activities [ 161 ]. Moreover, AMPs can bind cell membrane receptors (alternate ligand model) or affect receptor activation (membrane disruption model) by altering a receptor's site or releasing a membrane-bound factor (transactivation model) that binds the receptor. Finally, AMPs can interfere with lipopolysaccharides, preventing inflammation [ 162 ]. 4.1. AMPs with Action on Cell Membranes AMPs can make electrostatic interactions between their positive charges and the microbial cell surface's negative ones, as well as hydrophobic relations between their amphipathic domain and the microbic membrane phospholipids [ 140 ]. The physical–chemical interactions and the interfacial properties determine the destabilization and permeabilization of the microbial membrane [ 8 , 141 ]. Both vertebrates and invertebrates produce AMPs (active in vitro at micromolar levels), which can affect the cell membrane by manipulating its components [ 142 ]. Gram-positive bacteria have a dense peptidoglycan layer, while Gram-negative ones have a fine peptidoglycan layer and an extra outer membrane [ 143 ]. Teichoic acid and lipopolysaccharides provide electronegative charges on the bacterial surface. On the contrary, mammalian cell membranes do not have a net charge since the outer leaflet is formed by zwitterionic phospholipids (i.e., phosphatidylcholine, phosphatydylethanolamine, and sphingomyelin) [ 144 ] and the phospholipid bilayer is stabilized by cholesterol [ 145 ]. Thus, positively charged AMPs are significantly attracted by the negative charge (i.e., phospholipids, cardiolipin, phosphatidylglycerol, and phosphatidylserine) on bacterial membranes; instead, only weak hydrophobic interactions between AMPS and mammalian cell membranes can occur. Therefore, AMPs give selective antimicrobial effects without harming normal cells since the eukaryotic cell membranes have uncharged neutral residues (generally phospholipids, cholesterol, and sphingomyelins), which cannot interact with AMPs. Highly cationic and anionic peptides have no antimicrobial activity [ 146 , 147 ]. Pore formation can be achieved by barrel-stave, toroidal pore [ 148 ], and carpet-like [ 149 ] mechanisms, the clustering of anionic lipids [ 150 ], aggregated channels [ 151 ], or more than one mechanism [ 9 ]. The barrel-stave model hypothesizes that AMPs place themself alongside a membrane and penetrate the lipid bilayer. The pore external face is made by aligning the hydrophobic region of AMPs with the lipid bilayer's central lipid region. Instead, the pore interior is made by the peptide hydrophilic contribution (by a peptide–peptide interaction) [ 152 ]. Barrel-like pores can determine cytoplasmic outflow, membrane collapse, and cell death [ 153 ] ( Figure 6 ). The toroidal pore model hypothesizes that AMPs vertically cross a lipid membrane without peptide–peptide interactions in the lipid membrane [ 152 ]. The pores are transient and less stable than barrel-stave formations [ 154 ] ( Figure 6 ). The carpet or detergent-like model assumes that AMPs are adsorbed parallel to the lipid membrane until wholly covered (like a carpet), inducing membrane disruption. In this process, no peptides across the membrane, peptide–peptide interactions, or peptide structures are made [ 155 ] ( Figure 6 ). The AMP hydrophobic regions interact with the cell membrane, and the hydrophilicity ends with an aqueous solution [ 156 ]. Anionic lipid-clustering activity is obtained by forming phase-boundary defects between lipid domains due to interaction between cationic AMPs and anionic-charged lipids [ 150 ]. 4.2. AMPs with No Action on Cell Membranes Some AMPs can kill bacteria interfering with DNA (replication, transcription, and translation), cell division, and the blocking of protein biosynthesis and folding [ 113 , 157 , 158 ]. They can also interfere with the immune system, activating white blood cells, improving angiogenesis, blocking reactive oxygen and nitrogen species [ 159 ], suppressing toll-like receptors, reducing anti-endotoxin activity, interfering with cytokine-mediated production of cytokines [ 160 ], and influencing T- and B-cell activities [ 161 ]. Moreover, AMPs can bind cell membrane receptors (alternate ligand model) or affect receptor activation (membrane disruption model) by altering a receptor's site or releasing a membrane-bound factor (transactivation model) that binds the receptor. Finally, AMPs can interfere with lipopolysaccharides, preventing inflammation [ 162 ]. 5. AMP Potential in the Food Field 5.1. AMPs in Food Preservation AMP application in food preservation is under review since they have a broad spectrum of activity (bacteria, fungi, and protozoa), good water solubility, and are thermostable, but the high cost of large-scale production limits their use [ 163 , 164 ]. AMP selection for food incorporation depends on their spectrum of activity and an AMP's specificity toward microorganisms in a food product. For example, fermenticins produced by Lactobacillus fermentum [ 165 ] and defensins, which act on lipid II and lipid A at the bacterial membrane, show broad spectra of activity, pH, and temperature stability [ 166 ]. Some AMPs prolong the shelf-life of food by acting as antimicrobials and inhibiting lipid oxidation, such as peptides from Cynoscion guatucupa protein hydrolysate obtained by enzymatic hydrolysis with Alcalase and Protamex [ 167 ]. AMPs, stable at diverse ranges of pH levels, temperatures, and proteases, have been studied because, in food technology, temperature variations are used to increase the preservation of food, and proteases can be added to foods to decrease a food's allergy power and alter its taste [ 168 , 169 , 170 ]. Tolerance to diverse pH conditions can be obtained by changing the sequence of AMPs. For example, adding histidine at the carboxyl terminus of a piscidin-like AMP allowed a more significant antimicrobial activity against S. aureus at pH 10.5 [ 171 ]. AMP stability can be improved by modifying an AMP's geometrical properties (i.e., the radius of gyration, lipophilicity, ovality, polar surface area, and surface area). For example, the stability of Protegrin-1 was attributed to the high number of hydrogen bonds (distances <2.5 à ) [ 172 ]. The presence of free amino, sulfur, and carbonyl functional groups affected AMP bioactivity [ 173 ]. The concurrent addition of the additives ascorbate and nitrite could increase the carbonyl compounds in proteins, altering their functionality and technological properties [ 174 ]. Similarly, sulfites used as antioxidants and antiseptics could react with the disulfide bonds of AMPs to form irreversibly bound forms of S-sulfonates [ 175 ]. Thus, AMPs can be added to low-reactive foods such as fiber-rich food (whole-grain bread, cereals, pseudocereals, legumes, nuts, fruits, and vegetables) [ 176 ] and should not be inserted into high-reactivity food, such as liquid-based food formulations [ 177 ]. Nanoparticles, nanofibers, and nanoliposomes have been examined to protect AMP antimicrobial activity [ 178 , 179 , 180 ]. For example, nisin was placed into multifunctional soy-soluble polysaccharide-based nanocarriers to enhance its stability and preserve antioxidant and antimicrobial activity [ 181 ]. In raw and pasteurized milk, nisin-loaded chitosan/alginate nanoparticles were employed to prevent the growth of S. aureus during long incubation periods [ 182 ]. The nano-encapsulation of temporin B into chitosan nanoparticles enhanced the peptide's antibacterial activity [ 183 ]. 5.2. AMPs in Food Packaging Active packaging systems have been developed to control the release of AMPs and decrease their interactions with food components. Pentocina MS1 and MS2 from Lactobacillus pentosus MS031 isolated from Chinese Sichuan paocai were added to fresh-cut fruits in cold packaging to decrease the growth of Salmonella typhi , Listeria monocytogenes , and E. coli [ 184 ]. Partially purified Gt2 peptides active against E. coli and Salmonella typhi were put into packages to preserve tomatoes [ 185 ]. The peptide MTP1 was employed in meat and dairy product packaging [ 186 ]. Nisin, which can inhibit the growth of Listeria monocytogenes, Staphylococcus aureus, Penicillium sp., and Geotrichum sp., was used to preserve mozzarella cheese. [ 187 ]. A fish protein hydrolysate was added to preserve fish flounder fillets [ 188 ]. Nisin preventing the growth of Listeria monocytogenes was employed to preserve cold-smoked salmon [ 189 ]. 5.1. AMPs in Food Preservation AMP application in food preservation is under review since they have a broad spectrum of activity (bacteria, fungi, and protozoa), good water solubility, and are thermostable, but the high cost of large-scale production limits their use [ 163 , 164 ]. AMP selection for food incorporation depends on their spectrum of activity and an AMP's specificity toward microorganisms in a food product. For example, fermenticins produced by Lactobacillus fermentum [ 165 ] and defensins, which act on lipid II and lipid A at the bacterial membrane, show broad spectra of activity, pH, and temperature stability [ 166 ]. Some AMPs prolong the shelf-life of food by acting as antimicrobials and inhibiting lipid oxidation, such as peptides from Cynoscion guatucupa protein hydrolysate obtained by enzymatic hydrolysis with Alcalase and Protamex [ 167 ]. AMPs, stable at diverse ranges of pH levels, temperatures, and proteases, have been studied because, in food technology, temperature variations are used to increase the preservation of food, and proteases can be added to foods to decrease a food's allergy power and alter its taste [ 168 , 169 , 170 ]. Tolerance to diverse pH conditions can be obtained by changing the sequence of AMPs. For example, adding histidine at the carboxyl terminus of a piscidin-like AMP allowed a more significant antimicrobial activity against S. aureus at pH 10.5 [ 171 ]. AMP stability can be improved by modifying an AMP's geometrical properties (i.e., the radius of gyration, lipophilicity, ovality, polar surface area, and surface area). For example, the stability of Protegrin-1 was attributed to the high number of hydrogen bonds (distances <2.5 à ) [ 172 ]. The presence of free amino, sulfur, and carbonyl functional groups affected AMP bioactivity [ 173 ]. The concurrent addition of the additives ascorbate and nitrite could increase the carbonyl compounds in proteins, altering their functionality and technological properties [ 174 ]. Similarly, sulfites used as antioxidants and antiseptics could react with the disulfide bonds of AMPs to form irreversibly bound forms of S-sulfonates [ 175 ]. Thus, AMPs can be added to low-reactive foods such as fiber-rich food (whole-grain bread, cereals, pseudocereals, legumes, nuts, fruits, and vegetables) [ 176 ] and should not be inserted into high-reactivity food, such as liquid-based food formulations [ 177 ]. Nanoparticles, nanofibers, and nanoliposomes have been examined to protect AMP antimicrobial activity [ 178 , 179 , 180 ]. For example, nisin was placed into multifunctional soy-soluble polysaccharide-based nanocarriers to enhance its stability and preserve antioxidant and antimicrobial activity [ 181 ]. In raw and pasteurized milk, nisin-loaded chitosan/alginate nanoparticles were employed to prevent the growth of S. aureus during long incubation periods [ 182 ]. The nano-encapsulation of temporin B into chitosan nanoparticles enhanced the peptide's antibacterial activity [ 183 ]. 5.2. AMPs in Food Packaging Active packaging systems have been developed to control the release of AMPs and decrease their interactions with food components. Pentocina MS1 and MS2 from Lactobacillus pentosus MS031 isolated from Chinese Sichuan paocai were added to fresh-cut fruits in cold packaging to decrease the growth of Salmonella typhi , Listeria monocytogenes , and E. coli [ 184 ]. Partially purified Gt2 peptides active against E. coli and Salmonella typhi were put into packages to preserve tomatoes [ 185 ]. The peptide MTP1 was employed in meat and dairy product packaging [ 186 ]. Nisin, which can inhibit the growth of Listeria monocytogenes, Staphylococcus aureus, Penicillium sp., and Geotrichum sp., was used to preserve mozzarella cheese. [ 187 ]. A fish protein hydrolysate was added to preserve fish flounder fillets [ 188 ]. Nisin preventing the growth of Listeria monocytogenes was employed to preserve cold-smoked salmon [ 189 ]. 6. AMP Potential in the Pharmaceutical Field 6.1. AMP Antioxidant Potential Some AMPs can act as free-radical scavengers, reduce lipid peroxidation, have metal ion chelation activity, and impact antioxidant enzyme activity (i.e., SOD, PPO, CAT, and GSH-Px) [ 190 ]. The presence of isoleucine, leucine, and histidine amino acids [ 191 ], as well as the number of active hydrogen sites, are essential for antioxidant activity [ 192 ]. 6.2. Antineoplastic Agent Currently, cancer is a leading cause of death worldwide. AMPs have some characteristics that make them potential drugs for cancer therapy, such as high activity, specificity and affinity, small size, slight drug–drug interaction, aptitude to cross membranes, and low toxic side effects since they do not accumulate in vital organs (i.e., the liver and kidneys) [ 193 ]. Moreover, they are easily modified and synthesized [ 194 ] and are less immunogenic than recombinant antibodies [ 195 ]. Therapeutic peptides are classified into three groups: antimicrobial or pore-forming peptides (anticancer peptides, or ACPs, naturally produced by all living creatures), cell-permeable peptides, and tumor-targeting peptides [ 195 ]. 6.3. AMP Potential against Respiratory Diseases Some natural and modified AMPs appear to have potential as drugs to cure respiratory diseases and as infection markers. Pyocins, which can inhibit the growth of P. aeruginosa , could be used to cure fibrosis patients [ 196 ]. Esc (1−21)-c, a partial D-derivative of esculentin-1 that can decrease P. aeruginosa infection and has excellent resistance to degradation due to the elastase enzyme [ 197 ], could be employed to promote bronchial epithelium repair [ 198 ]. α-and β-defensins were potential infection markers of upper respiratory tract infection [ 199 ]. 6.4. AMP Potential against Hypertension Some AMPs (SAGGYIW and APATPSFW) could inhibit angiotensin-converting I (ACE), blocking the active site via weak interactions (i.e., electrostatic interaction, hydrogen bonds, and Van Der Waals interactions) [ 200 ]. ACE is an enzyme that can convert decapeptide angiotensin I (inactive) into octapeptide angiotensin II (vasoconstrictor), which is involved in hypertension and atherosclerosis [ 201 ]. 6.5. AMP Potential against Obesity EITPEKNPQLR, CQPHPGQTC, and RKQEEDEDEEQQRE are AMPs preventing pancreatic lipase activity. Pancreatic lipase is an enzyme that can hydrolyze 50–70% of food-derived fat in human organisms. Therefore, its inhibition is helpful in obesity treatment [ 202 ]. 6.6. AMP Potential against Intestine Infection and Inflammation α-defensins and C-type lectins (AMPs) are expressed in the gastrointestinal tract to sustain intestine symbiosis and protect it from pathological bacterial translocation [ 203 ]. 6.7. AMP Potential against Viral Infections Some AMPs can act against DNA and RNA viruses [ 204 , 205 ]. They can act on the viral envelope or after adsorption on the viral surface [ 206 ]. AMP positively charged residues can interact electrostatically with negatively charged cell surface molecules, such as heparan sulfate (glycosaminoglycans) [ 207 ], prevent the spread across tight junctions of the virus from one cell to another cell (cell-to-cell spread), or prevent the formation of giant cells (syncytium) [ 13 ]. Lactoferrin (iron-binding glycoprotein) can act as an antiviral material by inhibiting the replication of a wide range of DNA and RNA viruses or preventing virus entry into a host cell through direct binding to virus particles or blocking cellular receptors [ 208 ]. Defensins (α- and β-) can act against human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), and SARS-CoV [ 209 ]. It has also been hypothesized that an infusion of defensins during Cytomegalovirus infections may be helpful in the treatment of COVID-19 in pregnant women [ 210 , 211 ]. Frog-skin-derived peptide AR-23 and some of its derivatives can act against the viral surfaces of all enveloped viruses (i.e., coronaviruses, including SARS-CoV-2; paramyxoviruses; and herpesvirus) [ 212 , 213 ]. 6.8. AMP Potential against Skin Infections AMPs can be considered as a therapeutic option since they have a broad spectrum of biological activities against microbes; remain on an application site when topically administrated; and support wound healing by controlling angiogenesis, cell migration, and cytokine release chemotaxis [ 214 ]. Human keratinocytes and the granular skin layer make and store AMPs and lipids within secretory granules (lamellar bodies) [ 215 ]. The lamellar bodies make a physical barrier in superficial layers of the epidermis that can inhibit microbial growth and water loss. RNase 5 and RNase 7 are AMPs present in healthy human skin. They are active on Gram-negative and Gram-positive bacteria [ 216 ]. Other AMPs involved in skin wellbeing are psoriasin; calprotectin (iron- and zinc-binding S100 proteins) expressed by keratinocytes; β-defensins; the cathelicidin hCAP18, which must be converted to the active form; LL-37; histone 4 (active against Gram-positive bacteria) and dermcidin (active against antibacterial and antifungal mechanisms) produced by pilosebaceous follicles and eccrine glands, respectively; and α-defensins and LL-37 formed by neutrophils and natural killer cells [ 217 ]. Bee venom peptides can be helpful as a topical agent to promote skin regeneration and acne treatment. [ 218 , 219 , 220 ]. 6.1. AMP Antioxidant Potential Some AMPs can act as free-radical scavengers, reduce lipid peroxidation, have metal ion chelation activity, and impact antioxidant enzyme activity (i.e., SOD, PPO, CAT, and GSH-Px) [ 190 ]. The presence of isoleucine, leucine, and histidine amino acids [ 191 ], as well as the number of active hydrogen sites, are essential for antioxidant activity [ 192 ]. 6.2. Antineoplastic Agent Currently, cancer is a leading cause of death worldwide. AMPs have some characteristics that make them potential drugs for cancer therapy, such as high activity, specificity and affinity, small size, slight drug–drug interaction, aptitude to cross membranes, and low toxic side effects since they do not accumulate in vital organs (i.e., the liver and kidneys) [ 193 ]. Moreover, they are easily modified and synthesized [ 194 ] and are less immunogenic than recombinant antibodies [ 195 ]. Therapeutic peptides are classified into three groups: antimicrobial or pore-forming peptides (anticancer peptides, or ACPs, naturally produced by all living creatures), cell-permeable peptides, and tumor-targeting peptides [ 195 ]. 6.3. AMP Potential against Respiratory Diseases Some natural and modified AMPs appear to have potential as drugs to cure respiratory diseases and as infection markers. Pyocins, which can inhibit the growth of P. aeruginosa , could be used to cure fibrosis patients [ 196 ]. Esc (1−21)-c, a partial D-derivative of esculentin-1 that can decrease P. aeruginosa infection and has excellent resistance to degradation due to the elastase enzyme [ 197 ], could be employed to promote bronchial epithelium repair [ 198 ]. α-and β-defensins were potential infection markers of upper respiratory tract infection [ 199 ]. 6.4. AMP Potential against Hypertension Some AMPs (SAGGYIW and APATPSFW) could inhibit angiotensin-converting I (ACE), blocking the active site via weak interactions (i.e., electrostatic interaction, hydrogen bonds, and Van Der Waals interactions) [ 200 ]. ACE is an enzyme that can convert decapeptide angiotensin I (inactive) into octapeptide angiotensin II (vasoconstrictor), which is involved in hypertension and atherosclerosis [ 201 ]. 6.5. AMP Potential against Obesity EITPEKNPQLR, CQPHPGQTC, and RKQEEDEDEEQQRE are AMPs preventing pancreatic lipase activity. Pancreatic lipase is an enzyme that can hydrolyze 50–70% of food-derived fat in human organisms. Therefore, its inhibition is helpful in obesity treatment [ 202 ]. 6.6. AMP Potential against Intestine Infection and Inflammation α-defensins and C-type lectins (AMPs) are expressed in the gastrointestinal tract to sustain intestine symbiosis and protect it from pathological bacterial translocation [ 203 ]. 6.7. AMP Potential against Viral Infections Some AMPs can act against DNA and RNA viruses [ 204 , 205 ]. They can act on the viral envelope or after adsorption on the viral surface [ 206 ]. AMP positively charged residues can interact electrostatically with negatively charged cell surface molecules, such as heparan sulfate (glycosaminoglycans) [ 207 ], prevent the spread across tight junctions of the virus from one cell to another cell (cell-to-cell spread), or prevent the formation of giant cells (syncytium) [ 13 ]. Lactoferrin (iron-binding glycoprotein) can act as an antiviral material by inhibiting the replication of a wide range of DNA and RNA viruses or preventing virus entry into a host cell through direct binding to virus particles or blocking cellular receptors [ 208 ]. Defensins (α- and β-) can act against human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), and SARS-CoV [ 209 ]. It has also been hypothesized that an infusion of defensins during Cytomegalovirus infections may be helpful in the treatment of COVID-19 in pregnant women [ 210 , 211 ]. Frog-skin-derived peptide AR-23 and some of its derivatives can act against the viral surfaces of all enveloped viruses (i.e., coronaviruses, including SARS-CoV-2; paramyxoviruses; and herpesvirus) [ 212 , 213 ]. 6.8. AMP Potential against Skin Infections AMPs can be considered as a therapeutic option since they have a broad spectrum of biological activities against microbes; remain on an application site when topically administrated; and support wound healing by controlling angiogenesis, cell migration, and cytokine release chemotaxis [ 214 ]. Human keratinocytes and the granular skin layer make and store AMPs and lipids within secretory granules (lamellar bodies) [ 215 ]. The lamellar bodies make a physical barrier in superficial layers of the epidermis that can inhibit microbial growth and water loss. RNase 5 and RNase 7 are AMPs present in healthy human skin. They are active on Gram-negative and Gram-positive bacteria [ 216 ]. Other AMPs involved in skin wellbeing are psoriasin; calprotectin (iron- and zinc-binding S100 proteins) expressed by keratinocytes; β-defensins; the cathelicidin hCAP18, which must be converted to the active form; LL-37; histone 4 (active against Gram-positive bacteria) and dermcidin (active against antibacterial and antifungal mechanisms) produced by pilosebaceous follicles and eccrine glands, respectively; and α-defensins and LL-37 formed by neutrophils and natural killer cells [ 217 ]. Bee venom peptides can be helpful as a topical agent to promote skin regeneration and acne treatment. [ 218 , 219 , 220 ]. 7. Conclusions This work summarized the current knowledge regarding antimicrobial biopeptides to highlight their potential applications in the industrial field. Researchers are examining new sources of bioactive materials to use as natural preservatives in foods and to reduce the emergence of antibiotic drug resistance. AMPs seem to have good prospects as natural preservatives incorporated in food and food packaging, as well as for antioxidant, antineoplastic, antiobesity, antihypertensive, anti-inflammatory, antiviral, and dermatological agent drugs. Nanocarriers can be used to improve their bioavailability. Nevertheless, large-scale production and high cost of production could limit their use.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379018/

Adult Non-Cystic Fibrosis Bronchiectasis Is Characterised by Airway Luminal Th17 Pathway Activation

Background Non-cystic fibrosis (CF) bronchiectasis is characterised by chronic airway infection and neutrophilic inflammation, which we hypothesised would be associated with Th17 pathway activation. Methods Th17 pathway cytokines were quantified in bronchoalveolar lavage fluid (BALF), and gene expression of IL-17A, IL-1β, IL-8 and IL-23 determined from endobronchial biopsies (EBx) in 41 stable bronchiectasis subjects and 20 healthy controls. Relationships between IL-17A levels and infection status, important clinical measures and subsequent Pseudomonas aeruginosa infection were determined. Results BALF levels of all Th17 cytokines (median (IQR) pg/mL) were significantly higher in bronchiectasis than control subjects, including IL-17A (1.73 (1.19, 3.23) vs. 0.27 (0.24, 0.35), 95% CI 1.05 to 2.21, p0.83 for all except IL-1α 0.68) with the strongest correlations to IL-23 (r 0.91, p H . influenzae >others >normal flora only). Intermittent infection was defined where the organism was not cultured in every such specimen. 'Baseline' infection status was defined as per the BLESS study[ 14 ]—subjects with P . aeruginosa cultured at either the screening or visit 1 sputum sample were classified baseline P . aeruginosa positive, similarly for H . influenzae (if no P . aeruginosa cultured) and if no pathogens cultured in either then baseline 'normal flora'. These investigations raise the possibility of a divergence of inflammatory stimulation according to specific microbiology. BALF IL-8 levels in subjects with chronic pulmonary infection by P . aeruginosa (n = 8) (63.08 (38.45, 73.4) did not differ from those with intermittent P . aeruginosa (n = 7) (46.43 (12.78, 92.22), but were significantly higher than levels in subjects without sputum P . aeruginosa (n = 26) (45.40 (3.44, 57.75), 95% CI 3.64 to 59.81, p = 0.043). IL-8 levels were significantly higher in subjects who cultured P . aeruginosa at any time during the 12 month study (n = 20)(53.82 (30.33,82.18) than those who did not (n = 21) (43.82 (3.44, 40.05), 95% CI 2.76 to 52.18, p = 0.03), however levels did not predict subsequent P . aeruginosa infection in subjects without chronic P . aeruginosa at baseline (subsequent P . aeruginosa (n = 10) 54.15 (6.84, 92.22) vs no subsequent P . aeruginosa (n = 23) 12.78 (3.44, 44.04), p = 0.21). IL-1α levels were significantly higher in subjects with H . influenzae (n = 11) (35.86 (29.2, 79.3) than subjects with P . aeruginosa (n = 12) (19.87 (15.7, 31.55), 95% CI 2.98 to 46.9, p = 0.019) at baseline and in subjects with chronic H . influenzae infection (n = 8) (31.58 (29.21, 38.03) compared with chronic P . aeruginosa (n = 9) (19.87 (16.95, 27.88), 95% CI 1.21 to 42.05, p = 0.036). BALF IL-8 levels were significantly correlated with BALF neutrophil counts (r = 0.67, p0.83 for all except IL-1α 0.68) with the strongest correlations to IL-23 (r 0.91, p H . influenzae >others >normal flora only). Intermittent infection was defined where the organism was not cultured in every such specimen. 'Baseline' infection status was defined as per the BLESS study[ 14 ]—subjects with P . aeruginosa cultured at either the screening or visit 1 sputum sample were classified baseline P . aeruginosa positive, similarly for H . influenzae (if no P . aeruginosa cultured) and if no pathogens cultured in either then baseline 'normal flora'. These investigations raise the possibility of a divergence of inflammatory stimulation according to specific microbiology. BALF IL-8 levels in subjects with chronic pulmonary infection by P . aeruginosa (n = 8) (63.08 (38.45, 73.4) did not differ from those with intermittent P . aeruginosa (n = 7) (46.43 (12.78, 92.22), but were significantly higher than levels in subjects without sputum P . aeruginosa (n = 26) (45.40 (3.44, 57.75), 95% CI 3.64 to 59.81, p = 0.043). IL-8 levels were significantly higher in subjects who cultured P . aeruginosa at any time during the 12 month study (n = 20)(53.82 (30.33,82.18) than those who did not (n = 21) (43.82 (3.44, 40.05), 95% CI 2.76 to 52.18, p = 0.03), however levels did not predict subsequent P . aeruginosa infection in subjects without chronic P . aeruginosa at baseline (subsequent P . aeruginosa (n = 10) 54.15 (6.84, 92.22) vs no subsequent P . aeruginosa (n = 23) 12.78 (3.44, 44.04), p = 0.21). IL-1α levels were significantly higher in subjects with H . influenzae (n = 11) (35.86 (29.2, 79.3) than subjects with P . aeruginosa (n = 12) (19.87 (15.7, 31.55), 95% CI 2.98 to 46.9, p = 0.019) at baseline and in subjects with chronic H . influenzae infection (n = 8) (31.58 (29.21, 38.03) compared with chronic P . aeruginosa (n = 9) (19.87 (16.95, 27.88), 95% CI 1.21 to 42.05, p = 0.036). BALF IL-8 levels were significantly correlated with BALF neutrophil counts (r = 0.67, p<0.0001), SGRQ symptoms scores (r = -0.48, p = 0.002) and LCS (r = 0.4, p = 0.01). BALF IL-1α levels were significantly correlated with SGRQ total scores (r = -0.33, p = 0.03), SGRQ symptoms (r = -0.38, p = 0.015) and LCS (r = 0.37, p = 0.017). Neither cytokine showed significant correlations with FEV 1 or exacerbation frequency. Endobronchial biopsies Gene expression of IL-17A in endobronchial biopsies did not differ between bronchiectasis and normal control subjects (median difference 0.03, 95% CI -0.66 to 0.77, p = 0.95, see Fig 1 ). However bronchiectasis subjects had significantly higher gene expression relative to the median of healthy controls for IL1β (4.12 (1.24, 8.05) vs 1 (0.13, 2.95), 95% CI 0.05 to 4.07, p = 0.04) and IL-8 (3.75 (1.64, 11.27) vs 1 (0.54, 3.89), 95% CI 0.32 to 4.87, p = 0.02, Fig 3 ). Discussion The current study confirms, for the first time, significant airway luminal activation of the Th17 pathway in established adult non-CF bronchiectasis, in particular demonstrating significant elevations of BALF IL-17A and IL-23. However, in contrast to the recent publication in children with non-CF bronchiectasis, adults with established disease did not demonstrate significantly increased expression of IL-17A in endobronchial biopsies (assessed by gene expression rather than immunohistochemistry) [ 11 ]. Additionally, in contrast to data in CF children, BALF IL-17A levels did not predict subsequent P . aeruginosa infection [ 12 ]. Furthermore, we did not observe significant relationships between IL-17A levels and concurrent airway microbiology. Rather, our results showed increased IL-8 levels in association with P . aeruginosa infection and increased IL-1α levels in those with H . influenzae infection, suggesting both greater importance of these neutrophil-related mediators than IL-17A in established, chronic airway infection, and the possibility of divergent inflammatory activation according to specific microbiology. Our data support the proposal that innate, neutrophil-rich inflammation is a more important contributor to airway mucosal inflammatory pathophysiology in established adult non-CF bronchiectasis than a specific Th17 pathway response. It is possible that our failure to demonstrate a more important role for the Th17 pathway represents relative underpowering; however, we were able to demonstrate the importance of IL-8 and IL-1 with this sample. Furthermore, these data are cross-sectional in nature and the disease and control groups were not matched, with significant differences for both age and inhaled corticosteroid use. However multivariate analyses did not identify any relationships between either of these variables and BALF IL-17A levels, with presence of bronchiectasis the only variable that predicted IL-17a levels. Additionally, we sampled a relatively large patient population for a study of this kind, and our bronchiectasis subjects, nested within our rigorously conducted, double-blind, randomised controlled BLESS study, were very well clinically characterized. Other strengths of the study include the evidence of internal validity provided by the strong relationships between all Th17 pathway cytokines. Elevated levels of sputum IL-17A have previously been described in adult CF subjects [ 16 , 17 ], although only one of those studies had a comparator control group [ 16 ]. In that study, while sputum IL-17A gene expression was readily detected, the protein itself was unable to be measured in any of the controls and 3 (of 16) CF subjects. Additionally, IL-17 levels in bronchial mucosal tissue were not evaluated, and IL-23 was unable to be quantified at protein level [ 16 ]. Difficulty in measuring IL-23 protein levels in respiratory secretions has been reported recently [ 18 , 19 ]. Whether our ability to detect these cytokines in all healthy and bronchiectasis subjects reflects differences in sample processing methods (eg our use of protease inhibition) is not clear. Nonetheless, the demonstration of significantly elevated BALF IL-23 protein levels compared to controls is, to our knowledge, the first such data in human airways disease and confirms involvement of this key Th17 cytokine in airway inflammation in adult non-CF bronchiectasis. Additionally, our finding that the cytokine most strongly correlated with IL-17 levels was IL-23 is consistent with the 'central role' that IL-23 is believed to play in the Th17 pathway and Th17 cell maturation within the lung [ 20 ]. Our findings extend those of prior studies establishing the importance of neutrophilic inflammation in adult bronchiectasis [ 5 , 6 , 21 , 22 ]. The most comprehensive BALF study in adult non-CF bronchiectasis [ 5 ] demonstrated increased levels of neutrophil elastase, myeloperoxidase, TNF-α, IL-6 and IL-8 but not IL1β compared to controls. In addition to revealing upregulation of IL-1α, IL-17A and IL-23 and confirming increased BALF levels of IL-6, IL-8 and TNF-α, our data also demonstrate highly significant increases in BALF IL-1β in contrast to that earlier study. This difference may reflect the milder disease of subjects in that study, with an average FEV 1 79% predicted and a higher proportion of subjects negative for potential pathogens (52% vs 34% in our study). The current data demonstrate highly significant increases in all Th17 pathway-associated chemokines and cytokines measured in the airways of adult bronchiectasis subjects, including pathway effectors (IL-17A, IL-6, IL-8 and TNF-α) and regulators (IL-1α, IL-1β, IL-6 and IL-23). However, while gene expression of both IL-1β and IL-8 was significantly upregulated in the bronchial mucosa of non-CF bronchiectasis subjects, IL-17A was not, possibly reflecting the scarce relative abundance of the cells of origin in the mucosa. Interestingly, in spite of demonstrating significant increases in the abundance of Th17 T cells in biopsies, Tan and colleagues did not demonstrate significant increases in BALF IL-17 in non-CF bronchiectasis compared to control children [ 11 ]. Differences in findings between the 2 studies are likely to reflect differences in clinical status (our subjects were all clinically stable, versus all paediatric bronchiectasis subjects being sampled at the time of exacerbation), age (young children vs old adults) and degree of chronicity of airway infection. In addition to production by Th17 T cells, γδ cells, innate lymphoid cells and NK cells, IL-17 can be secreted by neutrophils [ 9 , 11 , 23 ]. Luminal neutrophils might be expected to be a relatively much greater source of IL-17A in old subjects with stable but established bronchiectasis, while mucosal IL-17A appears to predominate in childhood bronchiectasis subjects with earlier infection [ 11 ]. Consistent with this, we observed substantially higher neutrophil counts in BALF from our subjects (mean 21 X 10 6 /mL) than reported in the Tan paper (from Figure 5(C), approx. 150/mL)[ 11 ]. Furthermore, BALF IL-17A levels correlated significantly with BALF neutrophils but not with gene expression of IL-17A in endobronchial biopsies. Compartmentalization of IL-17A production (luminal vs. epithelial) in these subjects with established bronchiectasis also explains the apparent discrepancy between elevated protein levels of IL-17A in BALF but not gene expression in biopsies. Prominent neutrophilia is part of the intense cellular infiltration of bronchial mucosa that has been shown in adult bronchiectasis [ 21 ], and neutrophilic inflammation is characteristic of acute Th17 pathway activation in the lungs [ 24 ]. Interestingly however, an IL-17-overexpressing murine model assessed from 5 months showed significant lung inflammation characterised by increases in lung macrophage and lymphocyte, but not neutrophil, infiltration [ 25 ]. IL-17A release in response to acute infection is predominantly from innate immune cells rather than Th17 cells [ 23 ] and neutrophils were shown to be the main cellular source of IL-17 in a murine model of inhalational anthrax [ 26 ]. Hence, we speculate that neutrophils are the primary source of airway IL-17A in the chronic airway inflammation characterizing adult non-CF bronchiectasis. Our results suggest relatively greater importance of the neutrophil-related mediators IL-8 and IL-1α, than IL-17A, in adult non-CF bronchiectasis airway mucosal pathophysiology. Both IL-8 and IL1α, but not IL-17A, correlated with cough symptoms and quality of life measures. Furthermore, while there were no significant differences in BALF IL-17A levels between subjects according to important clinical markers, IL-8 levels were significantly increased in those with the presence of any potentially pathogenic microorganism and also P . aeruginosa infection specifically. Interestingly, those with H . influenzae had levels of IL-1α that were significantly higher than those with P . aeruginosa . The demonstration in sputum of significantly higher levels of neutrophilic inflammatory mediators in subjects with P . aeruginosa infection has recently been described in a large study [ 6 ]. However, we are unaware of any data suggesting either decreased IL-1α levels in subjects with P . aeruginosa (compared to H . influenzae ) infection or the possibility of a divergence in the specific inflammatory response according to specific microbiology. This divergent inflammatory activation might explain the significantly higher rates of pulmonary exacerbation seen in subjects with P . aeruginosa than H . influenzae in the BLESS study [ 13 , 27 , 28 ]. For example, perhaps P . aeruginosa minimizes epithelial IL-1α (an intracellular cytokine mainly released following cell necrosis) activation and release in order to evade mucosal immune systems, analogous to its ability to inhibit macrophage IL-1β production via components of its Type 3 secretory system [ 29 ]. It must be recognised that some of these analyses (based upon concomitant sputum bacterial infection status) were post-hoc, performed when our initial analysis suggested the possibility of such a relationship according to BALF infection status. Further investigation of the specific inflammatory responses of bronchiectasis model systems to different microbial communities will be informative in this regard. There is significant airway luminal activation of the Th-17 pathway in established, adult non-CF bronchiectasis. However, our results suggest relatively greater importance of non-antigen-specific innate neutrophil-dominated inflammation in the airway pathophysiology of this condition. We speculate that specific activation of the Th17 pathway is less important in the longstanding, chronic infection and inflammation that characterises adult bronchiectasis once neutrophilic inflammation has become established. Supporting Information S1 Supporting Information Data Supplement. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8527739/

Different characteristics of microbial diversity and special functional microbes in rainwater and topsoil before and after 2019 new coronavirus epidemic in Inner Mongolia Grassland

Grassland ecosystems are vital terrestrial ecosystems. As areas sensitive to climate change, they are critical for assessing the effects of global climate change. In China, grasslands account for over 40% of the land area. There is currently limited information on microbial diversity evolution in different grassland areas, particularly microorganisms with ice nucleation activity (INA) and their potential resources with potential influence to regulate regional precipitation and climate. We used Illumina MiSeq to sequence the 16S rRNA V3–V4 hypervariable region and performed a simple droplet freezing experiment to determine the variation in the grassland microbial community species composition and community structure. Rainwater and topsoil samples from the Hulunbuir Grassland in Inner Mongolia collected over three years were characterized. The dominant bacterial genus in the rainwater was Massilia , and the dominant fungus was Cladosporium . Additionally, the dominant bacteria in the soil were Sphingomonas , and the dominant fungus was Gibberella . There were differences in the microbial communities before and after the coronavirus disease epidemic. Pathogenic microorganisms exhibited inconsistent responses to environmental changes. The low relative abundance of known high-INA microorganisms and the higher freezing temperature indicated that unknown high-efficiency biological ice nucleating particles may be present. We found significant differences in species diversity and richness between the rainwater and soil populations in grassland areas by analyzing the sample community structures. Our research results revealed the species composition and structure of the microbiota in grassland ecosystems in China, indicating that environmental media and human activities may affect the microbiota in the grassland area and indicating underlying microorganisms with high INA. 1 Introduction Environmental microbiota exhibit unique relationships with their environmental media and local ecological conditions. Many factors, such as moisture, temperature, and environmental pollutants, can affect the community structure ( Cao et al., 2014 ; Du et al., 2018c ; Sun et al., 2018 ; Azua-Bustos et al., 2020 ). Advances in genomics have renewed these relationships, indicating the adaptation of microbiota to the living environment ( Schulze-Makuch et al., 2018 ; Chen et al., 2021 ). Environmental microbiota play a vital role in biogeochemical cycles, such as the water, carbon, and nitrogen cycles. Soil microorganisms play a critical function as decomposers in the ecosystem, promoting the conversion of soil nutrients ( Griffiths et al., 2003 ). Therefore, changes in the microbial community affect the nutrient cycling process and function. Microorganisms can act as biological ice nuclei to facilitate water cycle processes ( Morris et al., 2008 ). Biological particles in the Earth's atmosphere are unique types of ice nucleating particles (INPs) because they can promote ice crystal formation in clouds at warm temperatures ( Huang et al., 2021 ). Biological INPs (e.g., pollen, bacteria, fungal spores, and plankton), most of which contain ice nucleation proteins, can induce freezing at warmer temperatures (mostly ≥−15 °C) ( Murray et al., 2012 ). Above this temperature, the only materials known to nucleate ice are biological. S. Zhang et al. (2020) denoted INPs with freezing temperatures of ≥−10 °C efficient INPs. Ice crystals play critical roles in modulating cloud microphysical properties and chemical compositions in the troposphere through precipitation formation processes, cloud radiative forcing, cloud electrification, etc. Thus, they indirectly influence the global hydrological cycle and climate change ( DeMott et al., 2010 ; Mulmenstadt et al., 2015 ). Microbes well-known to exhibit ice nucleating activity include four bacterial genera ( Pseudomonas , Lysinibacillus , Erwinia , and Xanthomonas ) ( Morris et al., 2004 ; Failor et al., 2017 ; Amato et al., 2015 ; Santl-Temkiv et al., 2015 ), and two fungal genera ( Fusarium and Mortierella ) ( Froehlich-Nowoisky et al., 2015 ; Froehlich-Nowoisky and Poeschl, 2013 ; Pouleur et al., 1992 ). Morris et al. (2008) analyzed the bacterial population composition in precipitation samples from different regions and explained the relationship between P. syringae and the water cycle, and then proposed a hypothetical model of the life history of P. syringae driven by the environmental water cycle process. Delort et al. (2010) reviewed the analysis results of microbes in major cloud water and rainwater samples. The concentration of total bacteria is 10 3 –10 5 cell mL −1 ; the proportion of cultivable bacteria was low, almost less than 1%. Ahern et al. (2007) showed that over 60 fluorescent Pseudomonas strains were isolated from Hebridean cloud and rain samples. In general, there are few investigations on microbial populations and community structure in atmospheric precipitation, and further research is needed. Society has been greatly affected by the coronavirus disease (COVID-19), which continues to affect the public. Bioaerosols have been reported as potential transmission routes for COVID-19 ( Zhang et al., 2021 ). Due to the movement of people between cities and countries, COVID-19 infection has spread globally. Efforts have been made at the local, regional, and national levels to reduce the movement of people and quarantine the infected people to stop the spread of COVID-19. Traffic, markets, and small industries were temporarily shut down, which caused environmental changes. Venter et al. (2020) analyzed the concentration of pollutants during the COVID-19 epidemic in 34 global regions based on satellite remote sensing and on-ground air quality monitoring data. The results demonstrated that after considering the impact of meteorological conditions, the control measures during the epidemic reduced ρ(NOx), ρ(PM 2.5 ), and ρ(O 3 ) by 60%, 31%, and 4%, respectively. Chen et al. (2020) reported that during the epidemic period in China, traffic source pollutant emissions decreased significantly, residential heating and industrial emissions remained stable or slightly decreased, and air quality in most areas improved significantly in Wuhan and throughout the country. Research on most domestic regions in China, such as the Yangtze River Delta ( L. Li et al., 2020 ; Lu et al., 2021 ) and the eastern region ( R. Zhang et al., 2020 ), illustrates that pollutant emissions were significantly reduced during the national epidemic control period. The ambient air ρ(SO 2 ), ρ(PM 2.5 ), ρ(PM 10 ), ρ(NO 2 ), and ρ(CO) also significantly decreased, while ρ(O 3 ) rose in some areas ( Lu et al., 2021 ; L. Li et al., 2020 ; Le et al., 2020 ). However, few studies have compared the microbiome before and after the COVID-19 epidemic. Considering the significant role of microorganisms in the environment, this area needs further exploration. In our previous study ( Du et al., 2017 ) on INPs in rainwater in the HunlunBuir grassland ecosystem, samples were collected from May–August for the years 2011–2013. The median freezing temperature (T 50 ) ranged from −7.7 °C to −10.3 °C. Therefore, we postulated that certain unknown efficient INPs are likely to exist in the regional precipitation. We further investigated the abundance, distribution, and sources of the efficient INPs in an additional study ( S. Zhang et al., 2020 ), wherein we focused on efficient INPs in rainwater and soil. We found that biological INPs dominated the efficient INP population. The distinct distribution of specified INPs and known ice nucleation activity (INA) genera between rainwater and soils indicated that efficient INPs in rainwater may originate from remote sources instead of local ones. This study aims to solve the following scientific questions: 1) What are the main bacteria and fungi in different environmental media in the grassland ecosystem and is there a difference? 2) Further study the scope of effective INP in the environment, determine the distribution of well-known biological INP and explore which species may be potentially undiscovered biological INP. 3) Research pathogenic microorganisms in the environment. Especially, whether the aerosol composition of environmental microorganisms be affected by human activities? To answer these questions, the droplet freezing experiment was used to determine the freezing temperature of rainwater samples, also, high-throughput genetic sequencing was used to characterize the microbiota of rainwater and soil. The results are expected to provide important references for exploring potential biological efficient ice nuclei in rainwater and their possible sources, the impact of human activities on outdoor air microorganisms, biological pollution, and biosafety related issues. 2 Materials and methods 2.1 Sampling site All samples ( Table 1 ) were collected from the Hulunbuir Grassland Ecosystem Research Station of the Chinese Academy of Agricultural Sciences at Xiertala Farm. The farm is located in the center of the Hulunbuir meadow steppe (49°19′ N, 120°03′ E, altitude: 628 m) in Inner Mongolia in the eastern part of the Eurasian steppe (total area: 88,000 km 2 ). The sampling site is a grass-covered area with an ecosystem that features chestnut soil. The region is characterized by a semi-arid climate with an annual precipitation of 400 mm. There is a large variation in precipitation, which mostly occurs from June to August. Our collection campaigns were conducted in June, July and August in summer at temperatures of approximately 20 °C during the daytime and 12 °C at nighttime. Table 1 Sampling information and geographic coordinates for rainwater and topsoil. Table 1 Sample type Sample ID Sampling time (China Standard Time UT + 8:00) and duration of the rain Rainfall intensity Location Rainwater rainwater201808 3rd Aug 2018 (2 h) Heavy rain (25–49.9 mm/24 h) 49°19′N 120°03′E rainwater201906 26th Jun 2019 (6 h) Moderate rain (10–24.9 mm/24 h) rainwater201907 15th Jul 2019 (6 h) Drizzle (<10 mm/24 h) rainwater201908 20th Aug 2019 (3 h) Heavy rain (25–49.9 mm/24 h) rainwater202007 30th Jul 2020 (3 h) Drizzle (<10 mm/24 h) rainwater202008 2nd Aug 2020 (<1 h) Heavy rain(hail) (25–49.9 mm/24 h) Topsoil soil201808 2nd Aug 2018 soil201906 25th Jun 2019 soil201907 14th Jul 2019 soil201908 19th Aug 2019 soil202007 29th Jul 2020 soil202008 1st Aug 2020 2.2 Sample collection and preparation Rainwater samples were collected in several sterilized disposable plastic bags with an open area of 1 m 2 to ensure adequate rainwater volume collection. The plastic bags were held in barrels to form cylinders. The sampling equipment was placed in an open area at a suitable height (1.5–2 m) to avoid splashing from plants and the ground. The equipment was washed once with rainwater and then used to collect the rainwater. The volume of each sample was at least 4 L. Each sample was immediately stored in a refrigerator at 4 °C prior to processing. Part of the crude rainwater (volume of 0.5 L) was filtered through a sterile polytetrafluoroethylene filter with a pore diameter of 0.22 μm (Millipore, USA). It was used for DNA extraction and microbial community composition analysis. The crude rainwater samples were then transferred to sterile plastic containers. We took 10 mL of the crude rainwater sample and the filtrate and heated them to 100 °C with a water bath for 10 min to inactivate the proteinaceous INPs by disrupting the structure of membrane-bound proteins ( Hill et al., 2016 ). Subsequently, the samples were subjected to an immersion freezing test. A thin layer of topsoil (3 cm in depth) was collected from 20 collection spots scattered in an S-shape. The soil samples were collected one day before the precipitation event. The soils were ground and passed through sieves with pore sizes of 0.150 mm and 0.045 mm for pretreatment. Then, 250 mg of ground soil was weighed for DNA extraction. All of the materials used for sampling were rinsed with ultrapure Milli–Q water and then sterilized via autoclaving to ensure that no contaminants remained. 2.3 Drop-freezing assay Immersion freezing tests were performed using a modified device based on the Vali method ( Vali, 1971 ). Immersion freezing, which is considered the predominant mode in the atmosphere, was tested to determine the ice nucleation temperature ( Hande and Hoose, 2017 ). Forty-seven 10-μL droplets were equally distributed on a sterile plate with a cooling rate of 2 °C min −1 and for five repetitions for a total of 235 droplets tested in each sample. The initial temperature was 0 °C, which then declined at a rate of 2 °C min −1 until all the droplets froze. The modified device automatically detected the frozen drop signals and processed the experimental data ( Yang and Feng, 2007 ). A study by Du et al. (2017) describes the parameters of the instrument. 2.4 Ice nucleation data analysis In this manuscript, T 10 refers to the temperature at which 10% of the droplets froze, and T 50 refers to the temperature at which 50% of the droplets froze. The cumulative INP concentration at each temperature was calculated using the following equation ( Vali, 1971 ): K T = ln N 0 − ln N T / V . where K(T) is the concentration of INPs at temperature T, N 0 is the number of droplets tested, N(T) is the number of unfrozen droplets at a given temperature T and V is the volume of the droplet. 2.5 DNA extraction and PCR amplification Microbial DNA was extracted using a PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer protocol. The tools used in the experiments were clean and sterile. The extracted DNA was quantified using a Q-bit nucleic acid protein analyzer (Thermo Fisher Scientific). The extracted DNA samples were stored at −80 °C until further analysis. The V3–V4 region of 16S rRNA was amplified using the bacterial universal PCR primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The internal transcribed spacer (ITS)1 region of the fungal rRNA gene was amplified using the primers 1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and 2043R (GCTGCGTTCTGCATCGATGC). PCR amplification was performed in a 25-μL reaction mixture containing 0.5 μL of dNTP, 1 μL of primer, 2.5 μL of PCR buffer, 0.125 units of Taq DNA polymerase, and 2 μL of DNA template. PCR was performed as follows: 94 °C for 5 min, 30 cycles of 94 °C for 5 s, 50 °C for 30 s (for bacteria) or 56 °C for 30 s (for fungi), 72 °C for 45 s, 72 °C for 5 min, and then the temperature was maintained at 4 °C ( Du et al., 2018b ; Du et al., 2018c ). The PCR products were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by the Majorbio Company (Shanghai, China). 2.6 Sequence analysis The gene sequences were processed and analyzed using the open-source software package Mothur. Sequences less than 400 bp, greater than 470 bp, containing homopolymer stretches of over 8 bp, or containing ambiguous bases were removed. PCR chimeras were filtered using the Chimera.uchime command in Mothur ( http://www.mothur.org/wiki/Chimera.uchime ). The 16S rRNA gene was processed and analyzed as described by ( Kozich et al., 2013 ). Sequences of the ITS rRNA gene were aligned using MAFFT version 7 ( https://mafft.cbrc.jp/alignment/software ). After quality control, the relative abundances were calculated using a series of processing protocols documented by ( Du et al., 2018a ). The sequences were clustered into operational taxonomic units (OTUs) by setting a distance of 0.03. The bacterial sequences were assigned to phylotypes using the Bayesian approach and the Ribosomal Database Project 16S rRNA gene training database with a confidence threshold of 70%. The method and database employed in the taxonomic classification of the fungal sequences were the K-nearest neighbor algorithm and the UNITE ITS database, respectively ( Abarenkov et al., 2010 ; Wang et al., 2007 ). Raw sequencing data were deposited in the National Center for Biotechnology Information Sequence Read Archive by accession number PRJNA749903. IBM SPSS Statistics 26 software was used for all data analyses, and drawings were created using Origin 2019b. 2.1 Sampling site All samples ( Table 1 ) were collected from the Hulunbuir Grassland Ecosystem Research Station of the Chinese Academy of Agricultural Sciences at Xiertala Farm. The farm is located in the center of the Hulunbuir meadow steppe (49°19′ N, 120°03′ E, altitude: 628 m) in Inner Mongolia in the eastern part of the Eurasian steppe (total area: 88,000 km 2 ). The sampling site is a grass-covered area with an ecosystem that features chestnut soil. The region is characterized by a semi-arid climate with an annual precipitation of 400 mm. There is a large variation in precipitation, which mostly occurs from June to August. Our collection campaigns were conducted in June, July and August in summer at temperatures of approximately 20 °C during the daytime and 12 °C at nighttime. Table 1 Sampling information and geographic coordinates for rainwater and topsoil. Table 1 Sample type Sample ID Sampling time (China Standard Time UT + 8:00) and duration of the rain Rainfall intensity Location Rainwater rainwater201808 3rd Aug 2018 (2 h) Heavy rain (25–49.9 mm/24 h) 49°19′N 120°03′E rainwater201906 26th Jun 2019 (6 h) Moderate rain (10–24.9 mm/24 h) rainwater201907 15th Jul 2019 (6 h) Drizzle (<10 mm/24 h) rainwater201908 20th Aug 2019 (3 h) Heavy rain (25–49.9 mm/24 h) rainwater202007 30th Jul 2020 (3 h) Drizzle (<10 mm/24 h) rainwater202008 2nd Aug 2020 (<1 h) Heavy rain(hail) (25–49.9 mm/24 h) Topsoil soil201808 2nd Aug 2018 soil201906 25th Jun 2019 soil201907 14th Jul 2019 soil201908 19th Aug 2019 soil202007 29th Jul 2020 soil202008 1st Aug 2020 2.2 Sample collection and preparation Rainwater samples were collected in several sterilized disposable plastic bags with an open area of 1 m 2 to ensure adequate rainwater volume collection. The plastic bags were held in barrels to form cylinders. The sampling equipment was placed in an open area at a suitable height (1.5–2 m) to avoid splashing from plants and the ground. The equipment was washed once with rainwater and then used to collect the rainwater. The volume of each sample was at least 4 L. Each sample was immediately stored in a refrigerator at 4 °C prior to processing. Part of the crude rainwater (volume of 0.5 L) was filtered through a sterile polytetrafluoroethylene filter with a pore diameter of 0.22 μm (Millipore, USA). It was used for DNA extraction and microbial community composition analysis. The crude rainwater samples were then transferred to sterile plastic containers. We took 10 mL of the crude rainwater sample and the filtrate and heated them to 100 °C with a water bath for 10 min to inactivate the proteinaceous INPs by disrupting the structure of membrane-bound proteins ( Hill et al., 2016 ). Subsequently, the samples were subjected to an immersion freezing test. A thin layer of topsoil (3 cm in depth) was collected from 20 collection spots scattered in an S-shape. The soil samples were collected one day before the precipitation event. The soils were ground and passed through sieves with pore sizes of 0.150 mm and 0.045 mm for pretreatment. Then, 250 mg of ground soil was weighed for DNA extraction. All of the materials used for sampling were rinsed with ultrapure Milli–Q water and then sterilized via autoclaving to ensure that no contaminants remained. 2.3 Drop-freezing assay Immersion freezing tests were performed using a modified device based on the Vali method ( Vali, 1971 ). Immersion freezing, which is considered the predominant mode in the atmosphere, was tested to determine the ice nucleation temperature ( Hande and Hoose, 2017 ). Forty-seven 10-μL droplets were equally distributed on a sterile plate with a cooling rate of 2 °C min −1 and for five repetitions for a total of 235 droplets tested in each sample. The initial temperature was 0 °C, which then declined at a rate of 2 °C min −1 until all the droplets froze. The modified device automatically detected the frozen drop signals and processed the experimental data ( Yang and Feng, 2007 ). A study by Du et al. (2017) describes the parameters of the instrument. 2.4 Ice nucleation data analysis In this manuscript, T 10 refers to the temperature at which 10% of the droplets froze, and T 50 refers to the temperature at which 50% of the droplets froze. The cumulative INP concentration at each temperature was calculated using the following equation ( Vali, 1971 ): K T = ln N 0 − ln N T / V . where K(T) is the concentration of INPs at temperature T, N 0 is the number of droplets tested, N(T) is the number of unfrozen droplets at a given temperature T and V is the volume of the droplet. 2.5 DNA extraction and PCR amplification Microbial DNA was extracted using a PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer protocol. The tools used in the experiments were clean and sterile. The extracted DNA was quantified using a Q-bit nucleic acid protein analyzer (Thermo Fisher Scientific). The extracted DNA samples were stored at −80 °C until further analysis. The V3–V4 region of 16S rRNA was amplified using the bacterial universal PCR primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The internal transcribed spacer (ITS)1 region of the fungal rRNA gene was amplified using the primers 1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and 2043R (GCTGCGTTCTGCATCGATGC). PCR amplification was performed in a 25-μL reaction mixture containing 0.5 μL of dNTP, 1 μL of primer, 2.5 μL of PCR buffer, 0.125 units of Taq DNA polymerase, and 2 μL of DNA template. PCR was performed as follows: 94 °C for 5 min, 30 cycles of 94 °C for 5 s, 50 °C for 30 s (for bacteria) or 56 °C for 30 s (for fungi), 72 °C for 45 s, 72 °C for 5 min, and then the temperature was maintained at 4 °C ( Du et al., 2018b ; Du et al., 2018c ). The PCR products were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by the Majorbio Company (Shanghai, China). 2.6 Sequence analysis The gene sequences were processed and analyzed using the open-source software package Mothur. Sequences less than 400 bp, greater than 470 bp, containing homopolymer stretches of over 8 bp, or containing ambiguous bases were removed. PCR chimeras were filtered using the Chimera.uchime command in Mothur ( http://www.mothur.org/wiki/Chimera.uchime ). The 16S rRNA gene was processed and analyzed as described by ( Kozich et al., 2013 ). Sequences of the ITS rRNA gene were aligned using MAFFT version 7 ( https://mafft.cbrc.jp/alignment/software ). After quality control, the relative abundances were calculated using a series of processing protocols documented by ( Du et al., 2018a ). The sequences were clustered into operational taxonomic units (OTUs) by setting a distance of 0.03. The bacterial sequences were assigned to phylotypes using the Bayesian approach and the Ribosomal Database Project 16S rRNA gene training database with a confidence threshold of 70%. The method and database employed in the taxonomic classification of the fungal sequences were the K-nearest neighbor algorithm and the UNITE ITS database, respectively ( Abarenkov et al., 2010 ; Wang et al., 2007 ). Raw sequencing data were deposited in the National Center for Biotechnology Information Sequence Read Archive by accession number PRJNA749903. IBM SPSS Statistics 26 software was used for all data analyses, and drawings were created using Origin 2019b. 3 Results and discussion 3.1 Variation in microbic community diversity in two environmental media during sampling period By sequencing the DNA of rainwater and soil samples collected during the summer from August 2018 to August 2020, approximately 515,040 sequences of the bacterial 16S rRNA gene and 840,349 sequences of the fungal ITS gene were obtained after excluding non-qualified gene sequences and chimeric sequences ( Table 2 ). Normalization was performed by randomly selecting 26,886 bacterial sequences and 44,197 fungal sequences from each sample to compare the species richness and community diversity objectively. A 97% similarity cutoff was adopted to delineate the OTUs, and 2561 and 2905 OTUs were identified for the bacteria and fungi, respectively. Table 2 Comparison of diversity estimators of the bacterial and fungal communities in rainwater and topsoil from 2018 to 2020. Table 2 Sequences a OTUs b ACE Chao 1 Shannon Coverage Bacteria rainwater201808 70,049 193 269 277 2.649 0.998 rainwater201906 35,897 161 221 216 2.666 0.998 rainwater201907 41,928 165 231 254 2.236 0.998 rainwater201908 33,632 95 131 126 2.177 0.999 rainwater202007 38,480 411 560 534 3.384 0.995 rainwater202008 40,488 829 973 972 5.279 0.994 soil201808 44,626 1436 1699 1699 5.648 0.988 soil201906 26,886 1326 1586 1592 5.381 0.988 soil201907 35,423 1448 1720 1749 5.705 0.987 soil201908 62,879 1152 1441 1427 5.085 0.988 soil202007 49,768 1490 1758 1794 5.925 0.987 soil202008 34,984 1523 1804 1840 5.879 0.987 Fungi rainwater201808 53,824 612 735 724 3.665 0.997 rainwater201906 44,197 340 403 400 1.809 0.998 rainwater201907 95,639 282 376 349 1.810 0.998 rainwater201908 52,272 679 786 780 3.520 0.997 rainwater202007 66,002 510 650 663 2.706 0.997 rainwater202008 117,957 520 725 693 3.116 0.996 soil201808 95,821 733 831 824 4.122 0.997 soil201906 50,031 1081 1248 1216 4.191 0.995 soil201907 54,749 544 704 689 2.725 0.996 soil201908 67,408 532 601 583 3.177 0.998 soil202007 73,689 687 749 756 4.190 0.998 soil202008 68,760 881 1050 1046 4.257 0.995 a Reads after quality controls and chimera removal. For each samples, 26,886 of 16S rRNA gene sequences and 44,197 of ITS gene sequences were randomly selected for calculating the species richness indexes (ACE and Chao 1) and diversity index (Shannon). b The operational taxonomic units (OTU) were defined with 3% dissimilarity. The Abundance-based Coverage Estimator and Chao1 indices in the alpha diversity index reflect the abundance of the microbial community. The higher the value, the higher the abundance. The Shannon index characterizes the diversity of microbial communities. The higher the value of the Shannon index, the greater the number of species and uniformity. The Shannon index diversity dilution curve gradually flattens, indicating that the sequence is sufficiently representative, and the diversity data obtained is credible (Figs. S1 and S2). The statistical test found that in the three years, the species richness and community diversity of bacterial communities and the community diversity of fungal communities exhibited significant differences between the rainwater and soil samples, while the species richness of the fungal communities did not exhibit such differences ( Table 3 ). Table 3 Analysis of different bacterial and fungal communities in different environmental media. Table 3 Alpha-diversity index Analysis of variance Mann-Whitney test Bacteria Chao 1 P = 0.000 ⁎ – Ace P = 0.000 ⁎ – Shannon – P = 0.006 ⁎ Fungi Chao 1 P = 0.067 – Ace P = 0.066 – Shannon P = 0.041 ⁎ – Both non-biological and biological particles can accumulate by scrubbing, including in-cloud and below-cloud scavenging, as precipitation droplets fall. Therefore, some primary biological aerosol particles (PBAPs) in the atmosphere can ultimately be deposited in rainwater through wet deposition ( Lu et al., 2016 ). Rainfall can promote the release of bioaerosol particles from the soil into the atmosphere. When raindrops hit the soil surface, soil bacteria-containing aerosols are formed. One drop of rain can release 0.01% of the soil bacteria into the air. Joung et al. (2017) comprehensively considered factors such as the aerosolization rate, average surface density of bacteria, global land area, and precipitation type. They estimated that the total number of bacteria spread in raindrops worldwide each year is (1.2–85.0) × 10 22 . The microbial community in the soil is characterized by extremely rich species diversity ( Veresoglou and Rillig, 2014 ). The soil is rich in organic matter and is a natural medium suitable for the growth and reproduction of microorganisms. Studies have demonstrated that in forest soil, the number of prokaryotic soil cells (non-nucleated organisms, including bacteria and archaea) is approximately 4 × 10 7 cells·g −1 , while in other soils (including desert and agricultural soils), the number of prokaryotic cells is approximately 2 × 10 9 cells·g −1 ( Whitman et al., 1998 ). Many soil microorganisms can be aerosolized and released into the atmosphere by the wind. Cao et al. (2014) illustrated that the relative abundance of Geodermat obscurus in winter air in Beijing is high, and the bacteria often exist in dry soil environments. Mu et al. (2020) found that leaf surface is the main local source of airborne bacteria, and the correlation between leaf surface samples and air samples is greater than the correlation between surface soil and air samples. The atmospheric environment rarely contains the nutritional conditions required for the survival of microorganisms and is often only a temporary residence for microorganisms. Therefore, the microbial content and community composition of the air largely depend on the source of the bioaerosols. This may lead to differences in the alpha diversity index of bacteria in various environmental media. Fungi can form a dormant spore structure, which can help in its survival for an extended period when the external environment is unsuitable for growth. The spore structures of the fungi can also spread in the air. Qi et al. (2020) analyzed the source distribution of airborne fungi in Xi'an from the summer of 2018 to the spring of 2019 and found that 63.5% of airborne fungi originated from unknown sources. Long-distance transportation may also be a critical source of biological aerosols. In addition, airborne microorganisms primarily originate from the surface of leaves throughout the year, and their contribution accounts for 26.8% of the total fungi. In contrast, the contribution of soil is small, accounting for only about 6% of the fungi. Fungi are typically easily dispersed, can colonize many substrata, and can tolerate diverse environmental conditions ( Santiago et al., 2018 ). Bioaerosols come from many different sources on the earth's surface ( X. Li et al., 2020 ). The complexity of the source of fungi and the small proportion of fungi from the soil may lead to no significant difference in microbial species richness indices between rainwater and soil. In the Bray–Curtis similarity matrix, if different samples in the coordinate system are closer, the community similarity is higher. The matrix results for this study are displayed in Fig. 1 . In the two different environmental media, the samples exhibit clusters, indicating that the microbial community composition in the same environmental media was similar. There are some differences between the rainwater samples from 2018 to 2019 and those from 2020. Therefore, the bacterial and fungal community composition of Hulunbuir Grassland exhibits distribution characteristics according to environmental media. Fig. 1 Principal coordinates analysis (PCoA) of bacterial (a) and fungal (b) communities of different environmental media at the genus level in Hulunbuir, Inner Mongolia. Fig. 1 In summary, during the sampling period, the bacterial community in the Hulunbuir Grassland of Inner Mongolia exhibited significant differences in species abundance, community composition, and community diversity among different environmental media. Additionally, the differences between fungal communities in different environmental media were less than those between bacterial communities. 3.2 Difference of community composition in rainwater and topsoil over three years According to taxonomic analysis, 29 phyla and 635 genera of bacteria and 14 phyla and 690 genera of fungi were identified. Of these, 416 and 576 bacterial genera and 496 and 534 fungal genera were identified in the rainwater and soil samples, respectively. The number of species in the rainwater was less than that in the soil. Based on the results displayed in Fig. S3, 34 genera of bacteria can simultaneously exist in the three-year rainwater samples, accounting for 8.17% of the total bacteria and 44 genera of fungi, accounting for 8.87% of the total fungi. This result demonstrates that few species can exist in the local atmosphere for an extended period and that the species composition of bacteria and fungi in rainwater has changed significantly, which may be because the atmospheric environment is unsuitable for the survival of microorganisms. There are 276 genera of bacteria that can persist in the soil samples for three years, accounting for 47.92% of the total bacteria, and 98 genera of fungi, accounting for 18.35% of the total fungi (Fig. S4). This result demonstrates that a large proportion of bacteria can exist in the local soil environment for a long time, and the proportion of fungi is small. There are 22 genera of bacteria that simultaneously existed in the three-year rainwater and soil samples, accounting for 3.46% of the total bacteria, and 31 genera of fungi, accounting for 4.49% of the total fungi. We define these microorganisms that can exist in different environmental media for a long time as "core species." Thus, we inferred that microorganisms in the atmosphere washed by rainwater may partly originate from the local soil. The 22 core species of bacteria accounted for 5.29% and 3.82% of the bacteria in the rainwater and soil samples, respectively. The 31 core species of fungi accounted for 6.25% and 5.81% of the fungi in the rainwater and soil samples, respectively ( Fig. 2 ). These species may originate from local sources and participate in local hydrological cycles. Among them, dominant species with a relative abundance greater than 1% were Massilia (14.11%), Sphingomonas (3.11%), Arthrobacter (2.82%), Janthinobacterium (2.69%), Pseudomonas (2.23%), Noviherbaspirillum (1.78%), and Methylobacterium (1.21%), which belong to the Proteobacteria and Actinobacteria phyla, respectively. Existing studies have shown that Proteobacteria and Actinobacteria are widely present in the soil, water, and atmosphere, and they can survive in extreme environments and endure harsh environments such as low temperatures, dryness, and strong ultraviolet rays ( Wei et al., 2020 ; Maki et al., 2015 ). Therefore, it is speculated that species with sufficient resistance can exist in various environmental media in the Hulunbuir Grassland for a long period. Fig. 2 A Venn diagram of all the 12 samples (rainwater and soils) for bacteria (a) and fungi (b) at the genus level. Fig. 2 The dominant fungal species with a relative abundance greater than 1% were Gibberella (15.49%), Cladosporium (7.51%), Fusarium (4.69%), Alternaria (4.35%), Ophiobolopsis (2.08%), Preussia (1.92%), Didymella (1.81%), Microdochium (1.63%), Knufia (1.34%), Epicoccum (1.09%), which belong to the Ascomycota phylum. Previous studies have demonstrated that Ascomycetes are the most dominant fungal phylum. We detected Fusarium in the core species, which is a known high-INA genus ( Pouleur et al., 1992 ; Frohlich-Nowoisky and Poschl, 2013 ). As illustrated in Fig. 3 , by calculating and comparing the relative abundance of the top 20 species with abundance levels in different samples, Massilia (26.83%) was identified as the genus with the highest relative abundance of bacteria in the rainwater samples for three years; however, its relative abundance in the soil was only 1.38%. Sphingomonas (5.23%) was the genus with the highest relative abundance in the soil, while its relative abundance in rainwater was only 0.99%. Compared with the rainwater samples, the relative abundance of species was more evenly distributed in the soil samples. Cladosporium (12.54%) was the most abundant genus among the rainwater samples with a three-year timespan for fungi, while in the soil, its relative abundance was only 2.47%. Gibberella (30.41%) was the genus with the highest relative abundance in the three-year soil samples; however, its relative abundance was only 0.57% in the rainwater samples. Fig. 3 Phylogenetic classification of the bacterial communities (a) at the genus level and fungal communities (b) at the genus level. Fig. 3 We found that in both the rainwater and soil samples, the dominant bacteria changed significantly over time. Plants spread spores, pollen, and debris into the atmosphere. The discharged plant particles and microorganisms in the atmosphere return to the soil through sedimentation (dry sedimentation) and/or precipitation (wet sedimentation) and form part of the global material cycle. This process includes the biological precipitation cycle, in which microorganisms related to plants and soil are transported to the height of the clouds as aerosols and trigger precipitation through INA ( Dong et al., 2019 ; Woo and Yamamoto, 2020 ). PBAPs play a vital role in atmospheric chemistry and physics. During rainfall, the entrapment of suspended particulates in the atmosphere leads to a particle removal process. This process leads rainwater to contain both ice nuclei particulate matter that freezes in high-altitude clouds and atmospheric particulate matter carried by atmospheric washing that clears under and near the clouds, regardless of whether the particle is an interstitial aerosol or not. Therefore, the biological INPs in cloud water or PBAPs in the atmosphere may eventually be deposited into the rain. As the water falls, both non-biological and biological particles may accumulate through scouring. Therefore, microorganisms in the soil and rainwater samples have a certain similarity. Precipitation is an effective way to remove airborne particles, including bacteria and fungi. The microorganisms in rainwater may originate from clouds and particles in the air. According to previous studies conducted worldwide, the total number of bacteria in rainwater is approximately 10 3 –10 5 cells mL −1 ( Herlihy et al., 1987 ; Sattler et al., 2001 ; Amato et al., 2005 ). Microorganisms can also be transported at high altitudes via clouds, which increases the complexity of the sources of microorganisms in rainwater. DasSarma et al. (2020) stated that from the top of the troposphere (approximately 10 km) to a height of 50 km is the stratosphere. In the thin and dry air of the stratosphere, the temperature can drop to −60 °F (approximately −51 °C). The stratosphere contains a group of small but tenacious microorganisms, such as Pseudomonas syringae . In addition to their potential impact on the weather system, these high-altitude residents may also spread allergens and diseases. Thus, we must investigate the transmission process of microorganisms in the atmosphere in more detail and study their survival mechanisms. In summary, from the microbial community perspective, few species in the rainwater can exist stably for a long time. In contrast, most of the species in the soil can exist in the local grassland environment for an extended period. Some core species can exist stably in both rainwater and soil for a long time. We speculate that in the grassland environment, some microorganisms, such as Massilia and Gibberella , can adapt to the local environment and participate in the hydrological cycle, which may impact the local climate. The diversity of microbial communities in different environmental media exhibited significant differences over the three years. Subsequent analysis of the sample composition demonstrated that only a small number of species can exist in different environmental media for an extended period, which explains the significant differences in the composition of microbial communities between the various environmental media. The varying abundance of the above microorganisms in different environmental media further demonstrates significant differences in the compositions of the microbial communities of different environmental media. 3.3 Difference of efficient biological INPs during the different sample years By analyzing three years of sequences, we investigated genera known to have high-efficiency INA. We detected four known genera of high-INA microorganisms, including Pseudomonas , Lysinibacillus , Mortierella and Fusarium . In contrast, Erwinia and Xanthomonas were not identified in any of the samples. These two high-INA genera were also not identified in our previous research ( S. Zhang et al., 2020 ). This result is presented in Table 4 . Lysinibacillus has rarely been detected in rainwater, and it only accounted for 0.01% of the rainwater-202008 sample. Its relative abundance in the soil sample was only 0.02%. The relative abundance of the genus Pseudomonas was 3.91%, 12.33%, and 9.13% regarding the bacterial genera in the rainwater-201906, rainwater-201908, and rainwater-202007 samples, respectively. The maximum relative abundance among the other rainwater and soil samples was only 0.62%. The abundance distribution of Mortierella in the rainwater was similar to that of Lysinibacillus , with only a 0.09% abundance in the rainwater-201907 sample and a 0.02% abundance in the rainwater-202008 sample. Mortierella accounted for 1.34%, 3.12%, and 3.00% of the fungal genera in the soil-201808, soil-202007, and soil-202008 samples, respectively. The maximum relative abundance among the other soil samples was 0.64%. By investigating the known INA fungi, we found that the genus Fusarium accounted for 9.29%, 7.18%, 17.73%, 3.87% and 10.11% of the fungal genera in the soil-201906, soil-201907, soil-201908, soil-202007, and soil-202008 samples, respectively. The maximum relative abundance among the other soil and rainwater samples was 1.80%. Overall, the abundance distribution of the above microorganisms exhibited differences between the rainwater and soil samples. Table 4 The relative abundance of certain known efficient INA bacterial and fungal genera in the rainwater and topsoil samples. Table 4 Sample ID Relative abundance Bacterial genera Fungal genera Lysinibacillus Pseudomonas Mortierella Fusarium rainwater201808 – 0.62% 0.00% 0.42% rainwater201906 – 3.91% – 0.01% rainwater201907 – 0.34% 0.09% 1.14% rainwater201908 – 12.33% – 1.42% rainwater202007 – 9.13% 0.00% 1.76% rainwater202008 0.01% 0.12% 0.02% 0.15% soil201808 0.02% 0.00% 1.34% 1.80% soil201906 0.00% 0.01% 0.64% 9.29% soil201907 0.15% 0.00% 0.06% 7.18% soil201908 0.02% 0.15% 0.14% 17.73% soil202007 0.15% 0.06% 3.12% 3.87% soil202008 0.02% 0.06% 3.00% 10.11% – The genus was not detected in this sample. Biological INPs (e.g., pollen, bacteria, fungal spores, and plankton), most of which contain ice nucleation proteins, can trigger freezing at warmer temperatures (predominately ≥−15 °C) ( Murray et al., 2012 ). Above this temperature, the only materials known to nucleate ice are biological. S. Zhang et al. (2020) denoted INPs with freezing temperatures ≥−10 °C efficient INPs. For the rainwater samples from 2018 to 2020, two samples of crude rainwater (August 2018 and June 2019) were active at approximately −3.8 °C, while the other samples induced freezing at −4.0 °C to −6.4 °C. T 50 values of crude rainwater were −9.8 °C (August 2018), −9.2 °C (June 2019), −10.4 °C (July 2019), −10.4 °C (August 2019), −6.8 °C (July 2020), and −6.6 °C (August 2020) (Table S1). All T 50 values were ≥−15 °C, and the T 10 values (−7.2 °C, −6.4 °C, −7.8 °C, −8.2 °C, −6 °C, and −6 °C, respectively) of the crude rainwater were ≥−10 °C. On average, more than 228 INP cm −3 were active at −10 °C ( Fig. 4 ). We hypothesize that these findings are due to the widespread distribution of known INA bacterial and fungal species in the rainwater ( Christner et al., 2008a ; Christner et al., 2008b ). Fig. 4 Cumulative INP spectra of ultrapure water, a Pseudomonas syringae suspension and rainwater samples collected from a single precipitation event in the Hulun Buir grassland in the summers of 2018, 2019 and 2020. The symbol * means that the sample received heat treatment. Fig. 4 Protein has been proposed as an effective biological INP, and INA can easily be reduced by heat treatment. The size of the INP directly affects the nucleation ability of ice ( Zobrist et al., 2007 ; Pummer et al., 2015 ). Filtration should eliminate INA bacteria or the INA of intact cells. The freezing temperature of heated or filtered rainwater was shown in Tables S2 and S3, respectively. For the 2020 rainwater samples, the T 50 of the filtered rainwater with particles less than 220 nm after heating were −10.2 °C and −9.8 °C (Table S4), and the freezing temperatures of the droplets were primarily between −10 °C and −6 °C. The average cumulative ice nucleus concentration at −10 °C was 71 IN/mL. These INs are not sensitive to heat and are not known efficient IN. Moreover, the heat-resistant Lysinibacillus was rarely detected in the rainwater samples, and it only accounted for 0.01% of the bacteria in the rainwater-202008 sample. Therefore, we can infer that there is probably a biological or non-biological heat-resistant INP <220 nm in the 2020 rainwater samples. This result can help us understand the possible ice nucleus formation mechanism of sub-micron or nano-scale particles, expand our understanding of high-efficiency INPs in the natural environment, and contribute to the meteorological field by proposing candidate materials for efficient IN. These results are consistent with previous research results ( Du et al., 2017 ), where rainwater samples were collected at the same site in August 2011, 2012, and 2013. Furthermore, the disparity in the concentrations of efficient INPs between the two samples may have resulted from variations in meteorological conditions and rainfall intensity. Rainfall for the rainwater-202007 sample occurred as a drizzle (<10 mm, 24 h −1 ) and lasted nearly 3 h. However, the rainfall for the rainwater-202008 sample occurred as heavy rainfall in the form of hail (25–49.9 mm, 24 h −1 ) and lasted less than 1 h. Because the relationship between INPs and the distribution, occurrence, and intensity of precipitation was not explicitly due to the lack of quantitative data ( Stopelli et al., 2016 ), more research must be conducted to reveal a correlation. The rainwater samples from 2018 to 2019 exhibited similar ice nucleus concentration ranges after different treatments. Compared with crude rainwater, heat treatment reduced the INP concentration by 66–100% at −6 °C, 24–100% at −8 °C, and 3–98% at −10 °C. The filtration treatment reduced the IN concentration by 0–100% at −6 °C, 24–93% at −8 °C, and 40–96% at −10 °C. After the filtering and heating treatments (Table S4), the initial freezing temperature remained above −10 °C, T 50 was −13.7 ± 1.7 °C, and the freezing temperatures of the droplets were primarily between −15 °C and −10 °C, which further verified the existence of heat-resistant INPs. The low relative abundance of INA genera contrasted with the INA of biological INPs in the rainwater. This result indicated that there may be unknown efficient biological INPs likely derived from other unknown INA microbes. Therefore, there are unknown biological or non-biological INPs that are heat-resistant and efficient in precipitation in this area. We found that Massilia exhibited the highest relative abundance of the bacteria genera in the three-year rainwater samples by assessing the species composition and community structure. It is also the genus with the highest relative abundance of core species existing in different environmental media for long periods. In our previous research ( S. Zhang et al., 2020 ; Du et al., 2017 ), Massilia was also the genus with the highest relative abundance among the rainwater bacteria. In another study ( Lu et al., 2016 ), Massilia was the second-most abundant genus among rainwater bacteria. Massilia is a gram-negative bacterium, which is consistent with known high-efficiency IN bacteria. The genus used to be separated in ice and water environments ( Shen et al., 2015 ; Gallego et al., 2006 ). Fig. 3 illustrates how Massilia can survive in different environmental media, that it is a core species, and that it is the genus with the highest relative abundance in the rainwater samples. Based on its abundant presence in rainwater, we suspect that Massilia may affect the formation of rainwater, and it may be a genus of bacteria with high-efficiency INA. However, we do not currently understand the corresponding mechanism, which warrants a scope for further investigation. Cladosporium and Gibberella were the top two genera in relative abundance among the core species. Through indoor simulation experiments, Iannone et al. (2011) found that Cladosporium , the most abundant fungus in the atmosphere, does not exhibit effective INA for its spores, and its freezing temperature ranges from −25 °C to −35 °C. There are few studies on the INA of Gibberella . There is currently a lack of knowledge of the INA of potential bacterial species, and our experimental conditions cannot be used for isolation and culture determination. Therefore, this may be a direction for future research. To explore the role of Pseudomonas in precipitation and topsoil over a period of three years, the 16S rRNA gene sequence of the typical Pseudomonas OTU and the verified INAs of Pseudomonas P. syringae , P. fluorescens , and P. meridian were used to construct phylogenetic trees for Pseudomonas fluorescens and Pseudomonas meridian ( Fig. 5 ). The Pseudomonas OTU with the highest sequence richness was OTU26, followed by OTU23, OTU19, and OTU892. OTU26 was similar to the Pseudomonas fluorescens strain H40 16S ribosomal RNA gene (partial sequence, EU862079.2). Phylogenetic studies based on 16S rRNA genes illustrated that OTU23, OTU675, OTU19, OTU892, OTU860, and OTU1852 appeared in new branches. These results indicated that some representative OTU sequences may not exhibit INA. However, the distribution sequence of OTU26 was much greater than that of the other representative OTUs, and it appears to have the ability to catalyze ice formation. Therefore, additional sequences related to OTU26 were involved in precipitation, and the relative abundance of OTU26 in the soil was only 0.15% at the maximum. As the relative abundance of sequences belonging to OTU26 increased, the T 50 and T 10 values also significantly increased. The increased abundance of Pseudomonas may help increase INA during precipitation. Unfortunately, due to a lack of in-situ microbial culture experiments on rainwater and soil samples and the limitations of high-throughput sequencing technologies, we were unable to identify potentially effective strains. According to the phylogenetic tree analysis, we can infer that not every strain of bacterial genera with potentially effective INA exhibit INA. Among Pseudomonas , Erwinia , and Xanthomonas , only 20 species and variants of the bacteria were INA bacteria. Comparing the relative abundance of well-known INA bacterial sequences for particulate matter in the air can help clarify the source of effective INPs in rainwater. Based on the bacterial abundance distribution in the rainwater and soil samples, the effective biological INPs are likely depleted during precipitation, including Pseudomonas , and they may be transported to the troposphere from remote areas. Fig. 5 Neighbor-joining phylogenetic tree constructed on the basis of 16S rRNA gene sequences with similarities to those of the known INA bacterial strains of genus Pseudomonas. Fig. 5 3.4 Evolution of pathogenic microorganism community composition before and after COVID-19 epidemic The principal coordinates analysis results ( Fig. 1 ) demonstrate that the microbial communities of the rainwater samples from 2018 to 2019 are clustered, indicating that the microbial community structure in these two years is similar. However, the 2020 rainwater samples are farther from those of the previous two years and cannot be clustered, indicating that there are significant differences in the composition of the 2020 sample microbial communities. Therefore, the microbial communities in the rainwater have changed. The soil samples for the three years exhibited apparent clusters, indicating that the soil microbial community composition in the area was highly stable. This also demonstrates the accuracy of our experiments and reliability of the results. Both bacteria and fungi displayed the same trends. We combined the rainwater samples from 2018 and 2019 into a "before" sample group, and the 2020 samples constituted the "after" sample group. A total of 181,506 bacterial sequences were obtained from the before samples (rainwater samples from 2018 and 2019). The most abundant bacterial genera were Massilia (39.08%), Comamonas (11.48%), Duganella (10.65%) and Janthinobacterium (7.85%). The 2020 sample group contained 78,968 bacterial sequences. The four most abundant bacterial genera were Prosthecobacter (17.47%), Pedobacter (8.78%), Pseudomonas (4.63%) and Novosphingobium (3.12%). Sphingomonas (5.23%), Arthrobacter (5.06%), Microvirga (3.26%) and Rubrobacter (2.58%) were the four most abundant genera in the soil group. A total of 245,932 and 183,959 fungal sequences were identified in the before and after sample groups, respectively. Sporobolomyces (16.14%), Bullera (16.02%), Cystofilobasidium (15.33%) and Ophiobolopsis (6.08%) were the four most abundant genera in the before sample group. The most abundant fungal in the after sample group were Cladosporium (31.11%), Alternaria (1.32%) and Schizothecium (1.17%), which differed from the before sample group. Gibberella (30.41%), Fusarium (8.79%), Alternaria (6.14%) and Preussia (3.78%) were the four most abundant genera in the soil group. The Venn diagram in Fig. S5 illustrates that the number and proportion of overlapping parts of the genera are significantly different between the sample groups. After the epidemic, the number of bacterial genera increased compared with that before, and the proportion of overlapping parts increased from 26.3% to 52.6%. This result indicates that the microbial community structure changes with the degree of human activity. Human activities may introduce many foreign microorganisms to the local environment and alter the structure of the local microbial community. For fungi, the number of genera declined, and the proportion of overlapping parts almost remained unchanged. The epidemic limited the flow of people and resulted in a cleaner air environment. Through the STAMP analysis (Welch's t -test) ( Fig. 6 ), the differences of the dominant microorganisms at phylum and family levels in the rainwater samples before and after the epidemic were shown. It was found that at phylum level for bacterial, Proteobacteria , Bacteroidota , Cyanobacteria ; and Oxalobacteraceae and Caulobacteraceae at the family level had significant differences ( P < 0.05). The fungus Basidiomycota at the phylum level and Sporidiobolaceae at the family level were significantly different ( P < 0.05). We speculate that certain microbial groups were affected by human activity. However, the relative abundance of some microorganisms did not change significantly before and after the epidemic, indicating that different microorganisms may respond differently to changes in the external environment. Fig. 6 Comparison of microbial communities before and after the COVID-19 pandemic. Welch's t- tests were performed between rainwater samples before and after the COVID-19 pandemic at phylum ((a) bacteria and (c) fungi) and family ((b) bacteria and (d) fungi) levels. Dominant phyla and families are shown. Fig. 6 We found that the rainwater microbial community in 2018 and 2019 was significantly different from that in 2020. Since December 2019, COVID-19 has spread rapidly worldwide. To control the spread of the virus, the Chinese government has implemented a nationwide blockade policy, large-scale industrial companies have suspended operations, and the national government has advised people to remain at home ( Tian et al., 2020 ; Wang et al., 2020 ). These actions have led to a sharp reduction in human activities and a subsequent reduction in the emission of major air pollutants, improving the air quality. Studies have found that during the epidemic, the primary pollutants PM 2.5 , PM 10 , SO 2 , NO X , and CO all demonstrated a significant decline during the COVID-19 epidemic ( Berman and Ebisu, 2020 ; Chauhan and Singh, 2020 ; Collivignarelli et al., 2020 ; L. Li et al., 2020 ; Wang et al., 2020 ). Humans continuously produce and release large amounts of bioaerosols. Additionally, many interior and exterior parts of the human body carry many types of bacteria ( Costello et al., 2009 ). For example, the skin and digestive pathways of the human body carry approximately (1.0–100.0) × 10 12 microorganisms ( Luckey, 1972 ). Human activities will affect the microbiota in the air, which in turn affect the local human health and ecosystem ( X. Li et al., 2020 ). We can infer that the changes in the environment and the reduction in human activities may cause shifts in the microbial community. Our sampling site was located in the Hulunbuir Grassland in Inner Mongolia, a remote area and a tourist attraction in China. Air quality during the sampling period was excellent. Rainwater fully washed the atmosphere, and we used the microorganisms in the rainwater to represent the atmosphere. At present, extensive research has focused on atmospheric environmental research under severe pollution conditions ( Cao et al., 2014 ; Li et al., 2015 ; Xu et al., 2017 ). However, although long-term exposure to these conditions poses a risk to human health, little consideration has been given to the impact of the atmospheric composition on human health under suitable air conditions (Air Quality Index <100). For example, Shi et al. (2016) has demonstrated that short-term and long-term exposure to PM 2.5 is associated with all-cause mortality (total mortality), even if the exposure level is less than 10 μg/m 3 . Zhang et al. (2019) investigated the microbial activity under non-severely polluted conditions in the suburbs of Beijing and found that the air contained the pathogenic bacterium Streptococcus , which can cause wound infections and pinkeye, with a relative abundance of 0.23%. Three pathogenic bacteria were identified in the rainwater samples in 2018, 2019, and 2020, including Rickettsia (0.005%), Acinetobacter lupus (0.015%), and pathogenic Escherichia coli (0.010%). Two pathogens were identified in the soil samples: Bacillus anthracis (0.023%) and Acinetobacter rouxii (0.002%). In 2018, 2019, and 2020, pathogenic bacteria in rainwater samples accounted for 0.019%, 0.002%, and 0.078% of all bacteria, respectively. The proportions of pathogenic bacteria in the soil samples were 0.022%, 0.024%, and 0.028%, respectively. Five pathogenic fungi were identified in rainwater samples in 2018, 2019, and 2020, including Alternaria (2.559%), Cephalosporium (0.006%), Mucor (0.008%), Botrytis (0.001%), and Trichoderma (0.002%). Six pathogenic fungi were identified in the soil samples: Alternaria (6.142%), Cephalosporium (0.003%), Mucor (0.021%), Botrytis (0.026%), and Trichoderma (0.090%) and Trichothecium (0.002%). In 2018, 2019, and 2020, pathogenic fungi in the rainwater samples accounted for 6.276%, 2.171%, and 1.329% of all fungi, respectively. The proportions of pathogenic fungi in the soil samples were 6.403%, 5.551%, and 7.327% in 2018, 2019, and 2020, respectively. In addition, other pathogens have been found in different studies. For example, ( Du et al., 2018c ) found four pathogens ( Streptococcus , Prevotella , Erysipelas , and Rickettsia ) and five pathogens ( Trichothecium , Stachybotrys , Alternaria , Trichoderma , and Arthrinium ) in the suburbs of Beijing. Cao et al. (2014) used metagenomic sequencing technology to study the composition of atmospheric microorganisms under severely polluted weather conditions in Beijing and found three human pathogens, including Streptococcus pneumoniae , Aspergillus fumigatus , and Human Adenovirus C . Hurtado et al. (2014) used culture methods to isolate Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa and Enterococcus faecalis in Mexico. Acinetobacter lwoffii , a key opportunistic pathogen, typically causes sepsis and gastroenteritis ( Ku et al., 2000 ; Regalado et al., 2009 ). Bacillus anthracis is highly pathogenic and can cause anthrax, a zoonotic acute infectious disease. People can be infected by contact with herbivores and their pollutants, and severe infections can cause anthrax meningitis and even death ( Beyer and Turnbull, 2009 ; Hicks et al., 2011 ). Rickettsia may cause epidemic typhus, spotted fever, and Brill-Zinsser disease ( Kelly et al., 2002 ; Perlman et al., 2006 ). Pathogenic Escherichia coli is a gram-negative bacterium that causes disease outbreaks by polluting drinking water, food, and recreational waters. It can cause severe diarrhea and sepsis and can be life-threatening in severe cases ( Kaper et al., 2004 ). Pathogenic fungi, such as Trichothecene , Botrytis , and Trichoderma can produce metabolites, such as trichothecenes, that can inhibit immune regulation and protein synthesis ( Sudakin, 2003 ). Alternaria can cause skin tinea, onychomycosis, jaw osteomyelitis and other diseases ( Woudenberg et al., 2015 ; Fraeyman et al., 2017 ). Certain mycotoxins produced by Alternaria alternata are critical carcinogens; the spores of this fungus are spread through air. We compared the relative abundance of pathogenic bacteria before and after the SARS-CoV-2 outbreak. The outbreak led to the COVID-19 pandemic, during which the relative abundance of pathogenic bacteria and pathogenic fungi in the soil remained stable. However, in rainwater, we found that among the detected pathogenic microorganisms, Rickettsia , Acinetobacter reuteri , pathogenic Escherichia coli , Mucor , and Botrytis spp. showed an increase in relative abundance, while the relative abundances of Alternaria , Cephalosporium , and Trichoderma exhibited a downward trend. Overall, the COVID-19 epidemic has led to a substantial reduction in human activities and pollutant discharge, improving environmental conditions. However, various pathogens may respond differently to environmental changes. The outbreak of COVID-19 reminds us that we must quickly find and establish a national environmental and biological monitoring and defense system suitable for national and regional conditions to improve biosafety. This can help us solve biological pollution in the environment and control and prevent the spread of diseases. The results of this study provide an important reference for achieving these goals and a theoretical basis for a comprehensive understanding of the potential risks of bioaerosols to human health. In addition, we must also monitor microorganisms under excellent and good weather conditions. 3.1 Variation in microbic community diversity in two environmental media during sampling period By sequencing the DNA of rainwater and soil samples collected during the summer from August 2018 to August 2020, approximately 515,040 sequences of the bacterial 16S rRNA gene and 840,349 sequences of the fungal ITS gene were obtained after excluding non-qualified gene sequences and chimeric sequences ( Table 2 ). Normalization was performed by randomly selecting 26,886 bacterial sequences and 44,197 fungal sequences from each sample to compare the species richness and community diversity objectively. A 97% similarity cutoff was adopted to delineate the OTUs, and 2561 and 2905 OTUs were identified for the bacteria and fungi, respectively. Table 2 Comparison of diversity estimators of the bacterial and fungal communities in rainwater and topsoil from 2018 to 2020. Table 2 Sequences a OTUs b ACE Chao 1 Shannon Coverage Bacteria rainwater201808 70,049 193 269 277 2.649 0.998 rainwater201906 35,897 161 221 216 2.666 0.998 rainwater201907 41,928 165 231 254 2.236 0.998 rainwater201908 33,632 95 131 126 2.177 0.999 rainwater202007 38,480 411 560 534 3.384 0.995 rainwater202008 40,488 829 973 972 5.279 0.994 soil201808 44,626 1436 1699 1699 5.648 0.988 soil201906 26,886 1326 1586 1592 5.381 0.988 soil201907 35,423 1448 1720 1749 5.705 0.987 soil201908 62,879 1152 1441 1427 5.085 0.988 soil202007 49,768 1490 1758 1794 5.925 0.987 soil202008 34,984 1523 1804 1840 5.879 0.987 Fungi rainwater201808 53,824 612 735 724 3.665 0.997 rainwater201906 44,197 340 403 400 1.809 0.998 rainwater201907 95,639 282 376 349 1.810 0.998 rainwater201908 52,272 679 786 780 3.520 0.997 rainwater202007 66,002 510 650 663 2.706 0.997 rainwater202008 117,957 520 725 693 3.116 0.996 soil201808 95,821 733 831 824 4.122 0.997 soil201906 50,031 1081 1248 1216 4.191 0.995 soil201907 54,749 544 704 689 2.725 0.996 soil201908 67,408 532 601 583 3.177 0.998 soil202007 73,689 687 749 756 4.190 0.998 soil202008 68,760 881 1050 1046 4.257 0.995 a Reads after quality controls and chimera removal. For each samples, 26,886 of 16S rRNA gene sequences and 44,197 of ITS gene sequences were randomly selected for calculating the species richness indexes (ACE and Chao 1) and diversity index (Shannon). b The operational taxonomic units (OTU) were defined with 3% dissimilarity. The Abundance-based Coverage Estimator and Chao1 indices in the alpha diversity index reflect the abundance of the microbial community. The higher the value, the higher the abundance. The Shannon index characterizes the diversity of microbial communities. The higher the value of the Shannon index, the greater the number of species and uniformity. The Shannon index diversity dilution curve gradually flattens, indicating that the sequence is sufficiently representative, and the diversity data obtained is credible (Figs. S1 and S2). The statistical test found that in the three years, the species richness and community diversity of bacterial communities and the community diversity of fungal communities exhibited significant differences between the rainwater and soil samples, while the species richness of the fungal communities did not exhibit such differences ( Table 3 ). Table 3 Analysis of different bacterial and fungal communities in different environmental media. Table 3 Alpha-diversity index Analysis of variance Mann-Whitney test Bacteria Chao 1 P = 0.000 ⁎ – Ace P = 0.000 ⁎ – Shannon – P = 0.006 ⁎ Fungi Chao 1 P = 0.067 – Ace P = 0.066 – Shannon P = 0.041 ⁎ – Both non-biological and biological particles can accumulate by scrubbing, including in-cloud and below-cloud scavenging, as precipitation droplets fall. Therefore, some primary biological aerosol particles (PBAPs) in the atmosphere can ultimately be deposited in rainwater through wet deposition ( Lu et al., 2016 ). Rainfall can promote the release of bioaerosol particles from the soil into the atmosphere. When raindrops hit the soil surface, soil bacteria-containing aerosols are formed. One drop of rain can release 0.01% of the soil bacteria into the air. Joung et al. (2017) comprehensively considered factors such as the aerosolization rate, average surface density of bacteria, global land area, and precipitation type. They estimated that the total number of bacteria spread in raindrops worldwide each year is (1.2–85.0) × 10 22 . The microbial community in the soil is characterized by extremely rich species diversity ( Veresoglou and Rillig, 2014 ). The soil is rich in organic matter and is a natural medium suitable for the growth and reproduction of microorganisms. Studies have demonstrated that in forest soil, the number of prokaryotic soil cells (non-nucleated organisms, including bacteria and archaea) is approximately 4 × 10 7 cells·g −1 , while in other soils (including desert and agricultural soils), the number of prokaryotic cells is approximately 2 × 10 9 cells·g −1 ( Whitman et al., 1998 ). Many soil microorganisms can be aerosolized and released into the atmosphere by the wind. Cao et al. (2014) illustrated that the relative abundance of Geodermat obscurus in winter air in Beijing is high, and the bacteria often exist in dry soil environments. Mu et al. (2020) found that leaf surface is the main local source of airborne bacteria, and the correlation between leaf surface samples and air samples is greater than the correlation between surface soil and air samples. The atmospheric environment rarely contains the nutritional conditions required for the survival of microorganisms and is often only a temporary residence for microorganisms. Therefore, the microbial content and community composition of the air largely depend on the source of the bioaerosols. This may lead to differences in the alpha diversity index of bacteria in various environmental media. Fungi can form a dormant spore structure, which can help in its survival for an extended period when the external environment is unsuitable for growth. The spore structures of the fungi can also spread in the air. Qi et al. (2020) analyzed the source distribution of airborne fungi in Xi'an from the summer of 2018 to the spring of 2019 and found that 63.5% of airborne fungi originated from unknown sources. Long-distance transportation may also be a critical source of biological aerosols. In addition, airborne microorganisms primarily originate from the surface of leaves throughout the year, and their contribution accounts for 26.8% of the total fungi. In contrast, the contribution of soil is small, accounting for only about 6% of the fungi. Fungi are typically easily dispersed, can colonize many substrata, and can tolerate diverse environmental conditions ( Santiago et al., 2018 ). Bioaerosols come from many different sources on the earth's surface ( X. Li et al., 2020 ). The complexity of the source of fungi and the small proportion of fungi from the soil may lead to no significant difference in microbial species richness indices between rainwater and soil. In the Bray–Curtis similarity matrix, if different samples in the coordinate system are closer, the community similarity is higher. The matrix results for this study are displayed in Fig. 1 . In the two different environmental media, the samples exhibit clusters, indicating that the microbial community composition in the same environmental media was similar. There are some differences between the rainwater samples from 2018 to 2019 and those from 2020. Therefore, the bacterial and fungal community composition of Hulunbuir Grassland exhibits distribution characteristics according to environmental media. Fig. 1 Principal coordinates analysis (PCoA) of bacterial (a) and fungal (b) communities of different environmental media at the genus level in Hulunbuir, Inner Mongolia. Fig. 1 In summary, during the sampling period, the bacterial community in the Hulunbuir Grassland of Inner Mongolia exhibited significant differences in species abundance, community composition, and community diversity among different environmental media. Additionally, the differences between fungal communities in different environmental media were less than those between bacterial communities. 3.2 Difference of community composition in rainwater and topsoil over three years According to taxonomic analysis, 29 phyla and 635 genera of bacteria and 14 phyla and 690 genera of fungi were identified. Of these, 416 and 576 bacterial genera and 496 and 534 fungal genera were identified in the rainwater and soil samples, respectively. The number of species in the rainwater was less than that in the soil. Based on the results displayed in Fig. S3, 34 genera of bacteria can simultaneously exist in the three-year rainwater samples, accounting for 8.17% of the total bacteria and 44 genera of fungi, accounting for 8.87% of the total fungi. This result demonstrates that few species can exist in the local atmosphere for an extended period and that the species composition of bacteria and fungi in rainwater has changed significantly, which may be because the atmospheric environment is unsuitable for the survival of microorganisms. There are 276 genera of bacteria that can persist in the soil samples for three years, accounting for 47.92% of the total bacteria, and 98 genera of fungi, accounting for 18.35% of the total fungi (Fig. S4). This result demonstrates that a large proportion of bacteria can exist in the local soil environment for a long time, and the proportion of fungi is small. There are 22 genera of bacteria that simultaneously existed in the three-year rainwater and soil samples, accounting for 3.46% of the total bacteria, and 31 genera of fungi, accounting for 4.49% of the total fungi. We define these microorganisms that can exist in different environmental media for a long time as "core species." Thus, we inferred that microorganisms in the atmosphere washed by rainwater may partly originate from the local soil. The 22 core species of bacteria accounted for 5.29% and 3.82% of the bacteria in the rainwater and soil samples, respectively. The 31 core species of fungi accounted for 6.25% and 5.81% of the fungi in the rainwater and soil samples, respectively ( Fig. 2 ). These species may originate from local sources and participate in local hydrological cycles. Among them, dominant species with a relative abundance greater than 1% were Massilia (14.11%), Sphingomonas (3.11%), Arthrobacter (2.82%), Janthinobacterium (2.69%), Pseudomonas (2.23%), Noviherbaspirillum (1.78%), and Methylobacterium (1.21%), which belong to the Proteobacteria and Actinobacteria phyla, respectively. Existing studies have shown that Proteobacteria and Actinobacteria are widely present in the soil, water, and atmosphere, and they can survive in extreme environments and endure harsh environments such as low temperatures, dryness, and strong ultraviolet rays ( Wei et al., 2020 ; Maki et al., 2015 ). Therefore, it is speculated that species with sufficient resistance can exist in various environmental media in the Hulunbuir Grassland for a long period. Fig. 2 A Venn diagram of all the 12 samples (rainwater and soils) for bacteria (a) and fungi (b) at the genus level. Fig. 2 The dominant fungal species with a relative abundance greater than 1% were Gibberella (15.49%), Cladosporium (7.51%), Fusarium (4.69%), Alternaria (4.35%), Ophiobolopsis (2.08%), Preussia (1.92%), Didymella (1.81%), Microdochium (1.63%), Knufia (1.34%), Epicoccum (1.09%), which belong to the Ascomycota phylum. Previous studies have demonstrated that Ascomycetes are the most dominant fungal phylum. We detected Fusarium in the core species, which is a known high-INA genus ( Pouleur et al., 1992 ; Frohlich-Nowoisky and Poschl, 2013 ). As illustrated in Fig. 3 , by calculating and comparing the relative abundance of the top 20 species with abundance levels in different samples, Massilia (26.83%) was identified as the genus with the highest relative abundance of bacteria in the rainwater samples for three years; however, its relative abundance in the soil was only 1.38%. Sphingomonas (5.23%) was the genus with the highest relative abundance in the soil, while its relative abundance in rainwater was only 0.99%. Compared with the rainwater samples, the relative abundance of species was more evenly distributed in the soil samples. Cladosporium (12.54%) was the most abundant genus among the rainwater samples with a three-year timespan for fungi, while in the soil, its relative abundance was only 2.47%. Gibberella (30.41%) was the genus with the highest relative abundance in the three-year soil samples; however, its relative abundance was only 0.57% in the rainwater samples. Fig. 3 Phylogenetic classification of the bacterial communities (a) at the genus level and fungal communities (b) at the genus level. Fig. 3 We found that in both the rainwater and soil samples, the dominant bacteria changed significantly over time. Plants spread spores, pollen, and debris into the atmosphere. The discharged plant particles and microorganisms in the atmosphere return to the soil through sedimentation (dry sedimentation) and/or precipitation (wet sedimentation) and form part of the global material cycle. This process includes the biological precipitation cycle, in which microorganisms related to plants and soil are transported to the height of the clouds as aerosols and trigger precipitation through INA ( Dong et al., 2019 ; Woo and Yamamoto, 2020 ). PBAPs play a vital role in atmospheric chemistry and physics. During rainfall, the entrapment of suspended particulates in the atmosphere leads to a particle removal process. This process leads rainwater to contain both ice nuclei particulate matter that freezes in high-altitude clouds and atmospheric particulate matter carried by atmospheric washing that clears under and near the clouds, regardless of whether the particle is an interstitial aerosol or not. Therefore, the biological INPs in cloud water or PBAPs in the atmosphere may eventually be deposited into the rain. As the water falls, both non-biological and biological particles may accumulate through scouring. Therefore, microorganisms in the soil and rainwater samples have a certain similarity. Precipitation is an effective way to remove airborne particles, including bacteria and fungi. The microorganisms in rainwater may originate from clouds and particles in the air. According to previous studies conducted worldwide, the total number of bacteria in rainwater is approximately 10 3 –10 5 cells mL −1 ( Herlihy et al., 1987 ; Sattler et al., 2001 ; Amato et al., 2005 ). Microorganisms can also be transported at high altitudes via clouds, which increases the complexity of the sources of microorganisms in rainwater. DasSarma et al. (2020) stated that from the top of the troposphere (approximately 10 km) to a height of 50 km is the stratosphere. In the thin and dry air of the stratosphere, the temperature can drop to −60 °F (approximately −51 °C). The stratosphere contains a group of small but tenacious microorganisms, such as Pseudomonas syringae . In addition to their potential impact on the weather system, these high-altitude residents may also spread allergens and diseases. Thus, we must investigate the transmission process of microorganisms in the atmosphere in more detail and study their survival mechanisms. In summary, from the microbial community perspective, few species in the rainwater can exist stably for a long time. In contrast, most of the species in the soil can exist in the local grassland environment for an extended period. Some core species can exist stably in both rainwater and soil for a long time. We speculate that in the grassland environment, some microorganisms, such as Massilia and Gibberella , can adapt to the local environment and participate in the hydrological cycle, which may impact the local climate. The diversity of microbial communities in different environmental media exhibited significant differences over the three years. Subsequent analysis of the sample composition demonstrated that only a small number of species can exist in different environmental media for an extended period, which explains the significant differences in the composition of microbial communities between the various environmental media. The varying abundance of the above microorganisms in different environmental media further demonstrates significant differences in the compositions of the microbial communities of different environmental media. 3.3 Difference of efficient biological INPs during the different sample years By analyzing three years of sequences, we investigated genera known to have high-efficiency INA. We detected four known genera of high-INA microorganisms, including Pseudomonas , Lysinibacillus , Mortierella and Fusarium . In contrast, Erwinia and Xanthomonas were not identified in any of the samples. These two high-INA genera were also not identified in our previous research ( S. Zhang et al., 2020 ). This result is presented in Table 4 . Lysinibacillus has rarely been detected in rainwater, and it only accounted for 0.01% of the rainwater-202008 sample. Its relative abundance in the soil sample was only 0.02%. The relative abundance of the genus Pseudomonas was 3.91%, 12.33%, and 9.13% regarding the bacterial genera in the rainwater-201906, rainwater-201908, and rainwater-202007 samples, respectively. The maximum relative abundance among the other rainwater and soil samples was only 0.62%. The abundance distribution of Mortierella in the rainwater was similar to that of Lysinibacillus , with only a 0.09% abundance in the rainwater-201907 sample and a 0.02% abundance in the rainwater-202008 sample. Mortierella accounted for 1.34%, 3.12%, and 3.00% of the fungal genera in the soil-201808, soil-202007, and soil-202008 samples, respectively. The maximum relative abundance among the other soil samples was 0.64%. By investigating the known INA fungi, we found that the genus Fusarium accounted for 9.29%, 7.18%, 17.73%, 3.87% and 10.11% of the fungal genera in the soil-201906, soil-201907, soil-201908, soil-202007, and soil-202008 samples, respectively. The maximum relative abundance among the other soil and rainwater samples was 1.80%. Overall, the abundance distribution of the above microorganisms exhibited differences between the rainwater and soil samples. Table 4 The relative abundance of certain known efficient INA bacterial and fungal genera in the rainwater and topsoil samples. Table 4 Sample ID Relative abundance Bacterial genera Fungal genera Lysinibacillus Pseudomonas Mortierella Fusarium rainwater201808 – 0.62% 0.00% 0.42% rainwater201906 – 3.91% – 0.01% rainwater201907 – 0.34% 0.09% 1.14% rainwater201908 – 12.33% – 1.42% rainwater202007 – 9.13% 0.00% 1.76% rainwater202008 0.01% 0.12% 0.02% 0.15% soil201808 0.02% 0.00% 1.34% 1.80% soil201906 0.00% 0.01% 0.64% 9.29% soil201907 0.15% 0.00% 0.06% 7.18% soil201908 0.02% 0.15% 0.14% 17.73% soil202007 0.15% 0.06% 3.12% 3.87% soil202008 0.02% 0.06% 3.00% 10.11% – The genus was not detected in this sample. Biological INPs (e.g., pollen, bacteria, fungal spores, and plankton), most of which contain ice nucleation proteins, can trigger freezing at warmer temperatures (predominately ≥−15 °C) ( Murray et al., 2012 ). Above this temperature, the only materials known to nucleate ice are biological. S. Zhang et al. (2020) denoted INPs with freezing temperatures ≥−10 °C efficient INPs. For the rainwater samples from 2018 to 2020, two samples of crude rainwater (August 2018 and June 2019) were active at approximately −3.8 °C, while the other samples induced freezing at −4.0 °C to −6.4 °C. T 50 values of crude rainwater were −9.8 °C (August 2018), −9.2 °C (June 2019), −10.4 °C (July 2019), −10.4 °C (August 2019), −6.8 °C (July 2020), and −6.6 °C (August 2020) (Table S1). All T 50 values were ≥−15 °C, and the T 10 values (−7.2 °C, −6.4 °C, −7.8 °C, −8.2 °C, −6 °C, and −6 °C, respectively) of the crude rainwater were ≥−10 °C. On average, more than 228 INP cm −3 were active at −10 °C ( Fig. 4 ). We hypothesize that these findings are due to the widespread distribution of known INA bacterial and fungal species in the rainwater ( Christner et al., 2008a ; Christner et al., 2008b ). Fig. 4 Cumulative INP spectra of ultrapure water, a Pseudomonas syringae suspension and rainwater samples collected from a single precipitation event in the Hulun Buir grassland in the summers of 2018, 2019 and 2020. The symbol * means that the sample received heat treatment. Fig. 4 Protein has been proposed as an effective biological INP, and INA can easily be reduced by heat treatment. The size of the INP directly affects the nucleation ability of ice ( Zobrist et al., 2007 ; Pummer et al., 2015 ). Filtration should eliminate INA bacteria or the INA of intact cells. The freezing temperature of heated or filtered rainwater was shown in Tables S2 and S3, respectively. For the 2020 rainwater samples, the T 50 of the filtered rainwater with particles less than 220 nm after heating were −10.2 °C and −9.8 °C (Table S4), and the freezing temperatures of the droplets were primarily between −10 °C and −6 °C. The average cumulative ice nucleus concentration at −10 °C was 71 IN/mL. These INs are not sensitive to heat and are not known efficient IN. Moreover, the heat-resistant Lysinibacillus was rarely detected in the rainwater samples, and it only accounted for 0.01% of the bacteria in the rainwater-202008 sample. Therefore, we can infer that there is probably a biological or non-biological heat-resistant INP <220 nm in the 2020 rainwater samples. This result can help us understand the possible ice nucleus formation mechanism of sub-micron or nano-scale particles, expand our understanding of high-efficiency INPs in the natural environment, and contribute to the meteorological field by proposing candidate materials for efficient IN. These results are consistent with previous research results ( Du et al., 2017 ), where rainwater samples were collected at the same site in August 2011, 2012, and 2013. Furthermore, the disparity in the concentrations of efficient INPs between the two samples may have resulted from variations in meteorological conditions and rainfall intensity. Rainfall for the rainwater-202007 sample occurred as a drizzle (<10 mm, 24 h −1 ) and lasted nearly 3 h. However, the rainfall for the rainwater-202008 sample occurred as heavy rainfall in the form of hail (25–49.9 mm, 24 h −1 ) and lasted less than 1 h. Because the relationship between INPs and the distribution, occurrence, and intensity of precipitation was not explicitly due to the lack of quantitative data ( Stopelli et al., 2016 ), more research must be conducted to reveal a correlation. The rainwater samples from 2018 to 2019 exhibited similar ice nucleus concentration ranges after different treatments. Compared with crude rainwater, heat treatment reduced the INP concentration by 66–100% at −6 °C, 24–100% at −8 °C, and 3–98% at −10 °C. The filtration treatment reduced the IN concentration by 0–100% at −6 °C, 24–93% at −8 °C, and 40–96% at −10 °C. After the filtering and heating treatments (Table S4), the initial freezing temperature remained above −10 °C, T 50 was −13.7 ± 1.7 °C, and the freezing temperatures of the droplets were primarily between −15 °C and −10 °C, which further verified the existence of heat-resistant INPs. The low relative abundance of INA genera contrasted with the INA of biological INPs in the rainwater. This result indicated that there may be unknown efficient biological INPs likely derived from other unknown INA microbes. Therefore, there are unknown biological or non-biological INPs that are heat-resistant and efficient in precipitation in this area. We found that Massilia exhibited the highest relative abundance of the bacteria genera in the three-year rainwater samples by assessing the species composition and community structure. It is also the genus with the highest relative abundance of core species existing in different environmental media for long periods. In our previous research ( S. Zhang et al., 2020 ; Du et al., 2017 ), Massilia was also the genus with the highest relative abundance among the rainwater bacteria. In another study ( Lu et al., 2016 ), Massilia was the second-most abundant genus among rainwater bacteria. Massilia is a gram-negative bacterium, which is consistent with known high-efficiency IN bacteria. The genus used to be separated in ice and water environments ( Shen et al., 2015 ; Gallego et al., 2006 ). Fig. 3 illustrates how Massilia can survive in different environmental media, that it is a core species, and that it is the genus with the highest relative abundance in the rainwater samples. Based on its abundant presence in rainwater, we suspect that Massilia may affect the formation of rainwater, and it may be a genus of bacteria with high-efficiency INA. However, we do not currently understand the corresponding mechanism, which warrants a scope for further investigation. Cladosporium and Gibberella were the top two genera in relative abundance among the core species. Through indoor simulation experiments, Iannone et al. (2011) found that Cladosporium , the most abundant fungus in the atmosphere, does not exhibit effective INA for its spores, and its freezing temperature ranges from −25 °C to −35 °C. There are few studies on the INA of Gibberella . There is currently a lack of knowledge of the INA of potential bacterial species, and our experimental conditions cannot be used for isolation and culture determination. Therefore, this may be a direction for future research. To explore the role of Pseudomonas in precipitation and topsoil over a period of three years, the 16S rRNA gene sequence of the typical Pseudomonas OTU and the verified INAs of Pseudomonas P. syringae , P. fluorescens , and P. meridian were used to construct phylogenetic trees for Pseudomonas fluorescens and Pseudomonas meridian ( Fig. 5 ). The Pseudomonas OTU with the highest sequence richness was OTU26, followed by OTU23, OTU19, and OTU892. OTU26 was similar to the Pseudomonas fluorescens strain H40 16S ribosomal RNA gene (partial sequence, EU862079.2). Phylogenetic studies based on 16S rRNA genes illustrated that OTU23, OTU675, OTU19, OTU892, OTU860, and OTU1852 appeared in new branches. These results indicated that some representative OTU sequences may not exhibit INA. However, the distribution sequence of OTU26 was much greater than that of the other representative OTUs, and it appears to have the ability to catalyze ice formation. Therefore, additional sequences related to OTU26 were involved in precipitation, and the relative abundance of OTU26 in the soil was only 0.15% at the maximum. As the relative abundance of sequences belonging to OTU26 increased, the T 50 and T 10 values also significantly increased. The increased abundance of Pseudomonas may help increase INA during precipitation. Unfortunately, due to a lack of in-situ microbial culture experiments on rainwater and soil samples and the limitations of high-throughput sequencing technologies, we were unable to identify potentially effective strains. According to the phylogenetic tree analysis, we can infer that not every strain of bacterial genera with potentially effective INA exhibit INA. Among Pseudomonas , Erwinia , and Xanthomonas , only 20 species and variants of the bacteria were INA bacteria. Comparing the relative abundance of well-known INA bacterial sequences for particulate matter in the air can help clarify the source of effective INPs in rainwater. Based on the bacterial abundance distribution in the rainwater and soil samples, the effective biological INPs are likely depleted during precipitation, including Pseudomonas , and they may be transported to the troposphere from remote areas. Fig. 5 Neighbor-joining phylogenetic tree constructed on the basis of 16S rRNA gene sequences with similarities to those of the known INA bacterial strains of genus Pseudomonas. Fig. 5 3.4 Evolution of pathogenic microorganism community composition before and after COVID-19 epidemic The principal coordinates analysis results ( Fig. 1 ) demonstrate that the microbial communities of the rainwater samples from 2018 to 2019 are clustered, indicating that the microbial community structure in these two years is similar. However, the 2020 rainwater samples are farther from those of the previous two years and cannot be clustered, indicating that there are significant differences in the composition of the 2020 sample microbial communities. Therefore, the microbial communities in the rainwater have changed. The soil samples for the three years exhibited apparent clusters, indicating that the soil microbial community composition in the area was highly stable. This also demonstrates the accuracy of our experiments and reliability of the results. Both bacteria and fungi displayed the same trends. We combined the rainwater samples from 2018 and 2019 into a "before" sample group, and the 2020 samples constituted the "after" sample group. A total of 181,506 bacterial sequences were obtained from the before samples (rainwater samples from 2018 and 2019). The most abundant bacterial genera were Massilia (39.08%), Comamonas (11.48%), Duganella (10.65%) and Janthinobacterium (7.85%). The 2020 sample group contained 78,968 bacterial sequences. The four most abundant bacterial genera were Prosthecobacter (17.47%), Pedobacter (8.78%), Pseudomonas (4.63%) and Novosphingobium (3.12%). Sphingomonas (5.23%), Arthrobacter (5.06%), Microvirga (3.26%) and Rubrobacter (2.58%) were the four most abundant genera in the soil group. A total of 245,932 and 183,959 fungal sequences were identified in the before and after sample groups, respectively. Sporobolomyces (16.14%), Bullera (16.02%), Cystofilobasidium (15.33%) and Ophiobolopsis (6.08%) were the four most abundant genera in the before sample group. The most abundant fungal in the after sample group were Cladosporium (31.11%), Alternaria (1.32%) and Schizothecium (1.17%), which differed from the before sample group. Gibberella (30.41%), Fusarium (8.79%), Alternaria (6.14%) and Preussia (3.78%) were the four most abundant genera in the soil group. The Venn diagram in Fig. S5 illustrates that the number and proportion of overlapping parts of the genera are significantly different between the sample groups. After the epidemic, the number of bacterial genera increased compared with that before, and the proportion of overlapping parts increased from 26.3% to 52.6%. This result indicates that the microbial community structure changes with the degree of human activity. Human activities may introduce many foreign microorganisms to the local environment and alter the structure of the local microbial community. For fungi, the number of genera declined, and the proportion of overlapping parts almost remained unchanged. The epidemic limited the flow of people and resulted in a cleaner air environment. Through the STAMP analysis (Welch's t -test) ( Fig. 6 ), the differences of the dominant microorganisms at phylum and family levels in the rainwater samples before and after the epidemic were shown. It was found that at phylum level for bacterial, Proteobacteria , Bacteroidota , Cyanobacteria ; and Oxalobacteraceae and Caulobacteraceae at the family level had significant differences ( P < 0.05). The fungus Basidiomycota at the phylum level and Sporidiobolaceae at the family level were significantly different ( P < 0.05). We speculate that certain microbial groups were affected by human activity. However, the relative abundance of some microorganisms did not change significantly before and after the epidemic, indicating that different microorganisms may respond differently to changes in the external environment. Fig. 6 Comparison of microbial communities before and after the COVID-19 pandemic. Welch's t- tests were performed between rainwater samples before and after the COVID-19 pandemic at phylum ((a) bacteria and (c) fungi) and family ((b) bacteria and (d) fungi) levels. Dominant phyla and families are shown. Fig. 6 We found that the rainwater microbial community in 2018 and 2019 was significantly different from that in 2020. Since December 2019, COVID-19 has spread rapidly worldwide. To control the spread of the virus, the Chinese government has implemented a nationwide blockade policy, large-scale industrial companies have suspended operations, and the national government has advised people to remain at home ( Tian et al., 2020 ; Wang et al., 2020 ). These actions have led to a sharp reduction in human activities and a subsequent reduction in the emission of major air pollutants, improving the air quality. Studies have found that during the epidemic, the primary pollutants PM 2.5 , PM 10 , SO 2 , NO X , and CO all demonstrated a significant decline during the COVID-19 epidemic ( Berman and Ebisu, 2020 ; Chauhan and Singh, 2020 ; Collivignarelli et al., 2020 ; L. Li et al., 2020 ; Wang et al., 2020 ). Humans continuously produce and release large amounts of bioaerosols. Additionally, many interior and exterior parts of the human body carry many types of bacteria ( Costello et al., 2009 ). For example, the skin and digestive pathways of the human body carry approximately (1.0–100.0) × 10 12 microorganisms ( Luckey, 1972 ). Human activities will affect the microbiota in the air, which in turn affect the local human health and ecosystem ( X. Li et al., 2020 ). We can infer that the changes in the environment and the reduction in human activities may cause shifts in the microbial community. Our sampling site was located in the Hulunbuir Grassland in Inner Mongolia, a remote area and a tourist attraction in China. Air quality during the sampling period was excellent. Rainwater fully washed the atmosphere, and we used the microorganisms in the rainwater to represent the atmosphere. At present, extensive research has focused on atmospheric environmental research under severe pollution conditions ( Cao et al., 2014 ; Li et al., 2015 ; Xu et al., 2017 ). However, although long-term exposure to these conditions poses a risk to human health, little consideration has been given to the impact of the atmospheric composition on human health under suitable air conditions (Air Quality Index <100). For example, Shi et al. (2016) has demonstrated that short-term and long-term exposure to PM 2.5 is associated with all-cause mortality (total mortality), even if the exposure level is less than 10 μg/m 3 . Zhang et al. (2019) investigated the microbial activity under non-severely polluted conditions in the suburbs of Beijing and found that the air contained the pathogenic bacterium Streptococcus , which can cause wound infections and pinkeye, with a relative abundance of 0.23%. Three pathogenic bacteria were identified in the rainwater samples in 2018, 2019, and 2020, including Rickettsia (0.005%), Acinetobacter lupus (0.015%), and pathogenic Escherichia coli (0.010%). Two pathogens were identified in the soil samples: Bacillus anthracis (0.023%) and Acinetobacter rouxii (0.002%). In 2018, 2019, and 2020, pathogenic bacteria in rainwater samples accounted for 0.019%, 0.002%, and 0.078% of all bacteria, respectively. The proportions of pathogenic bacteria in the soil samples were 0.022%, 0.024%, and 0.028%, respectively. Five pathogenic fungi were identified in rainwater samples in 2018, 2019, and 2020, including Alternaria (2.559%), Cephalosporium (0.006%), Mucor (0.008%), Botrytis (0.001%), and Trichoderma (0.002%). Six pathogenic fungi were identified in the soil samples: Alternaria (6.142%), Cephalosporium (0.003%), Mucor (0.021%), Botrytis (0.026%), and Trichoderma (0.090%) and Trichothecium (0.002%). In 2018, 2019, and 2020, pathogenic fungi in the rainwater samples accounted for 6.276%, 2.171%, and 1.329% of all fungi, respectively. The proportions of pathogenic fungi in the soil samples were 6.403%, 5.551%, and 7.327% in 2018, 2019, and 2020, respectively. In addition, other pathogens have been found in different studies. For example, ( Du et al., 2018c ) found four pathogens ( Streptococcus , Prevotella , Erysipelas , and Rickettsia ) and five pathogens ( Trichothecium , Stachybotrys , Alternaria , Trichoderma , and Arthrinium ) in the suburbs of Beijing. Cao et al. (2014) used metagenomic sequencing technology to study the composition of atmospheric microorganisms under severely polluted weather conditions in Beijing and found three human pathogens, including Streptococcus pneumoniae , Aspergillus fumigatus , and Human Adenovirus C . Hurtado et al. (2014) used culture methods to isolate Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa and Enterococcus faecalis in Mexico. Acinetobacter lwoffii , a key opportunistic pathogen, typically causes sepsis and gastroenteritis ( Ku et al., 2000 ; Regalado et al., 2009 ). Bacillus anthracis is highly pathogenic and can cause anthrax, a zoonotic acute infectious disease. People can be infected by contact with herbivores and their pollutants, and severe infections can cause anthrax meningitis and even death ( Beyer and Turnbull, 2009 ; Hicks et al., 2011 ). Rickettsia may cause epidemic typhus, spotted fever, and Brill-Zinsser disease ( Kelly et al., 2002 ; Perlman et al., 2006 ). Pathogenic Escherichia coli is a gram-negative bacterium that causes disease outbreaks by polluting drinking water, food, and recreational waters. It can cause severe diarrhea and sepsis and can be life-threatening in severe cases ( Kaper et al., 2004 ). Pathogenic fungi, such as Trichothecene , Botrytis , and Trichoderma can produce metabolites, such as trichothecenes, that can inhibit immune regulation and protein synthesis ( Sudakin, 2003 ). Alternaria can cause skin tinea, onychomycosis, jaw osteomyelitis and other diseases ( Woudenberg et al., 2015 ; Fraeyman et al., 2017 ). Certain mycotoxins produced by Alternaria alternata are critical carcinogens; the spores of this fungus are spread through air. We compared the relative abundance of pathogenic bacteria before and after the SARS-CoV-2 outbreak. The outbreak led to the COVID-19 pandemic, during which the relative abundance of pathogenic bacteria and pathogenic fungi in the soil remained stable. However, in rainwater, we found that among the detected pathogenic microorganisms, Rickettsia , Acinetobacter reuteri , pathogenic Escherichia coli , Mucor , and Botrytis spp. showed an increase in relative abundance, while the relative abundances of Alternaria , Cephalosporium , and Trichoderma exhibited a downward trend. Overall, the COVID-19 epidemic has led to a substantial reduction in human activities and pollutant discharge, improving environmental conditions. However, various pathogens may respond differently to environmental changes. The outbreak of COVID-19 reminds us that we must quickly find and establish a national environmental and biological monitoring and defense system suitable for national and regional conditions to improve biosafety. This can help us solve biological pollution in the environment and control and prevent the spread of diseases. The results of this study provide an important reference for achieving these goals and a theoretical basis for a comprehensive understanding of the potential risks of bioaerosols to human health. In addition, we must also monitor microorganisms under excellent and good weather conditions. 4 Conclusions In this study, we identified shared and unique bacterial taxa present in the environment from two different environmental media. Human activities may significantly affect species richness and microbial community diversity. The known high-INA genus is low in abundance, and we suspect that the high-abundance species ( Massilia and Gibberella ) present in various local environmental media may be unknown high-efficiency biological ice nuclei. However, due to the limitation of experimental conditions, the strains were not isolated and cultured, and the INA of the potential microbial genus was not measured. Further research is still required to determine whether a specific genus exhibits a valid INA to verify this postulation. The relative abundance of different pathogenic bacteria demonstrated different trends before and after the COVID-19 epidemic. Our study provides insights into the composition and structure of the environmental microbiota in grasslands, enhances our understanding of the effect of environmental media on the microbiota species diversity, and highlights the importance of biological pollution and safety. CRediT authorship contribution statement Yongtao Zhang: Writing – original draft, Formal analysis, Investigation, Validation, Data curation, Writing – review & editing. Rui Du: Conceptualization, Methodology, Writing – review & editing, Supervision, Validation, Investigation, Resources. Hanlin Chen: Investigation, Resources. Pengrui Du: Investigation, Software. Sujian Zhang: Investigation, Resources. Weishan Ren: Resources. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A Supplementary data Supplementary material Image 1

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567325/

An analysis of hospital preparedness capacity for public health emergency in four regions of China: Beijing, Shandong, Guangxi, and Hainan

Background Hospital preparedness is critical for the early detection and management of public health emergency (PHE). Understanding the current status of PHE preparedness is the first step in planning to enhance hospitals' capacities for emergency response. The objective of this study is to understand the current status of hospital PHE preparedness in China. Methods Four hundred hospitals in four city and provinces of China were surveyed using a standardized questionnaire. Data related to hospital demographic data; PHE preparation; response to PHE in community; stockpiles of drugs and materials; detection and identification of PHE; procedures for medical treatment; laboratory diagnosis and management; staff training; and risk communication were collected and analyzed. Results Valid responses were received from 318 (79.5%) of the 400 hospitals surveyed. Of the valid responses, 264 (85.2%) hospitals had emergency plans; 93.3% had command centres and personnel for PHE; 22.9% included community organisations during the training for PHE; 97.4% could transport needed medical staff to a PHE; 53.1% had evaluated stockpiles of drugs; 61.5% had evaluated their supply systems; 55.5% had developed surveillance systems; and 74.6% could monitor the abnormity(See in appendix). Physicians in 80.2% of the analyzed hospitals reported up-to-date knowledge of their institution's PHE protocol. Of the 318 respondents, 97.4% followed strict laboratory regulations, however, only about 33.5% had protocols for suspected samples. Furthermore, only 59.0% could isolate and identify salmonella and staphylococcus and less than 5% could isolate and identify human H5N1 avian flu and SARS. Staff training or drill programs were reported in 94.5% of the institutions; 50.3% periodically assessed the efficacy of staff training; 45% had experts to provide psychological counselling; 12.1% had provided training for their medical staff to assess PHE-related stress. All of the above capacities related to the demographic characteristics of hospitals and will be discussed in-depth in this paper. Conclusion Our survey suggested that, at the time of the survey, hospital preparedness for PHE in China was at an early stage of development. Comprehensive measures should be taken to enhance hospital capacity in the prevention and management of PHE. Background Hospital preparedness is critical for the early detection and management of public health emergency (PHE). Understanding the current status of PHE preparedness is the first step in planning to enhance hospitals' capacities for emergency response. The objective of this study is to understand the current status of hospital PHE preparedness in China. Methods Four hundred hospitals in four city and provinces of China were surveyed using a standardized questionnaire. Data related to hospital demographic data; PHE preparation; response to PHE in community; stockpiles of drugs and materials; detection and identification of PHE; procedures for medical treatment; laboratory diagnosis and management; staff training; and risk communication were collected and analyzed. Results Valid responses were received from 318 (79.5%) of the 400 hospitals surveyed. Of the valid responses, 264 (85.2%) hospitals had emergency plans; 93.3% had command centres and personnel for PHE; 22.9% included community organisations during the training for PHE; 97.4% could transport needed medical staff to a PHE; 53.1% had evaluated stockpiles of drugs; 61.5% had evaluated their supply systems; 55.5% had developed surveillance systems; and 74.6% could monitor the abnormity(See in appendix). Physicians in 80.2% of the analyzed hospitals reported up-to-date knowledge of their institution's PHE protocol. Of the 318 respondents, 97.4% followed strict laboratory regulations, however, only about 33.5% had protocols for suspected samples. Furthermore, only 59.0% could isolate and identify salmonella and staphylococcus and less than 5% could isolate and identify human H5N1 avian flu and SARS. Staff training or drill programs were reported in 94.5% of the institutions; 50.3% periodically assessed the efficacy of staff training; 45% had experts to provide psychological counselling; 12.1% had provided training for their medical staff to assess PHE-related stress. All of the above capacities related to the demographic characteristics of hospitals and will be discussed in-depth in this paper. Conclusion Our survey suggested that, at the time of the survey, hospital preparedness for PHE in China was at an early stage of development. Comprehensive measures should be taken to enhance hospital capacity in the prevention and management of PHE. Background Public health emergency (PHE) is an event or events that cause or may cause harm to the health of a community or nation [ 1 ]. To prevent and/or minimize the harm caused by PHE, early detection and management are necessary. As hospitals are the main location for PHE surveillance and treatment, their preparedness is critical for PHE's early detection and management [ 2 ]. Evaluating the current status of PHE preparedness within the hospital system is the first step in improving a nation's preparedness for a PHE. Yet, there is no national data on China's hospital PHE preparedness capacity aside from two studies that addressed the issues at local level [ 3 , 4 ]. To understand the current status of hospital PHE preparedness in China, a sample survey of hospitals in four representative city/provinces were conducted between November 2004 and March 2005. Methods Study design The survey used a cross-sectional study design to survey hospitals in different regions of China. Respondents were all secondary and tertiary hospitals(the detail of hospital classification see in appendix) in the city of Beijing and provinces of Shandong, Guangxi, and Hainan. The selection of hospitals in these four regions is intended to represent a variety of regional economic status. Broadly speaking, Beijing and Shandong are economically well developed, Hainan moderately developed, and Guangxi developing [ 5 ]. According to the Hospital Classification Method issued by the National Bureau of Statistics of China, the surveyed hospitals included general hospitals, hospitals of traditional Chinese medicine (TCM), hospitals of integrated traditional Chinese medicine and western medicine (TCM-WM), specialized hospitals, community health center, and medical emergency center (the definition of community health center and medical emergency center see in appendix) [ 6 ]. Four hundred secondary and tertiary hospitals were surveyed. The study was approved by the Institutional Review Board (IRB) of the School of Basic Medicine, Peking Union Medical College in Beijing, China. Survey instruments Based on a literature and government document review, a detailed methodological approach for research framework and questionnaire development was followed to inform the development of this study [ 3 ]. An indicator system framework was created and questionnaire designed based on the framework. The questionnaire consists of 17 sections and 192 items. The questionnaire and the survey protocol (including field work manual and quality control procedures) were tested by a pilot study. For the purpose of this study, we analyzed the data focused on the following nine areas of interest: (1) hospital's demographic data (including region, SARS crisis experience, teaching function, hospital type, and number of medical staff in related departments); (2) hospital PHE preparation (emergency plans, response initiating time, accessibility, and revision and implementation of emergency plan); (3) response to a community PHE (cooperation with local organizations, relationship with the community PHE network, medical treatment, and rescue work in the community); (4) stockpiles of drugs and materials (stockpiles of drugs and other resources and personal protective equipment); (5)PHE detection and identification (syndrome surveillance); (6) procedures for medical treatment (protocol for diagnosis, treatment, and transfer of PHE victims); (7) laboratory diagnosis and management (laboratory regulation and management system, sample disposal and evaluation system, collection and disposal of suspected samples, and diagnosis of pathogen/etiology); (8) staff training (organization of PHE training, current training of medical staff, curriculum development and training effectiveness assessment); and (9) risk communication (organization for communication of risk psychological counseling to victim and medical staff, and communication with public). Excluding aspect 1, items 2–9 (covering 88 survey questions) represent 8 types of PHE preparedness capacities. Each answered item was scored 1 for "yes" and 0 for "no" or "unknown". Item scores were calculated by adding together "yes" answers. Items scores were used as a proxy for measuring PHE preparedness in an institution. A total item score was measured by calculating the score across all 8 items. The higher the total item score, the better the hospital PHE preparedness capacity. Further analyses were conducted to understand the correlation between preparedness capacity and demographic information. The distribution of the related preparedness capacities across 10 categories of PHE [ 1 ] and 15 types of etiology was also assessed. Data collection procedures A computerized questionnaire stored in a CD was sent to the targeted hospitals accompanied by an official letter from each of the four city and provincial health departments stating the importance of the survey and requiring that each hospital designates a department director to be responsible for coordinating the completion of the questionnaire. Each returned questionnaire was carefully reviewed for its completeness and consistency. For those questionnaires with incomplete and/or inconsistent responses, one or two follow-up telephone calls were made to ensure completeness and consistency. The data from returned questionnaires were then transferred into a database for analysis. Data analysis A database was set up using Microsoft Excel 2003. Data was checked, cleaned, and analyzed using SPSS software version 11.5. Ninety-five percent confidence interval of means (95% CI) was used to describe PHE preparedness capacities. Categorical variables were analyzed with frequency and percentage. Comparisons of mean score of each of eight PHE preparedness capacities among different types of hospitals were performed with P < 0.05 as statistical significance using parameter test (Independent-Samples T Test (two-tailed) or One-way Analysis of Variance) and/or non-parameter test (Mann-Whitney Test or Kruskal-Wallis Test) based on data distribution characteristics and homogeneity. Study design The survey used a cross-sectional study design to survey hospitals in different regions of China. Respondents were all secondary and tertiary hospitals(the detail of hospital classification see in appendix) in the city of Beijing and provinces of Shandong, Guangxi, and Hainan. The selection of hospitals in these four regions is intended to represent a variety of regional economic status. Broadly speaking, Beijing and Shandong are economically well developed, Hainan moderately developed, and Guangxi developing [ 5 ]. According to the Hospital Classification Method issued by the National Bureau of Statistics of China, the surveyed hospitals included general hospitals, hospitals of traditional Chinese medicine (TCM), hospitals of integrated traditional Chinese medicine and western medicine (TCM-WM), specialized hospitals, community health center, and medical emergency center (the definition of community health center and medical emergency center see in appendix) [ 6 ]. Four hundred secondary and tertiary hospitals were surveyed. The study was approved by the Institutional Review Board (IRB) of the School of Basic Medicine, Peking Union Medical College in Beijing, China. Survey instruments Based on a literature and government document review, a detailed methodological approach for research framework and questionnaire development was followed to inform the development of this study [ 3 ]. An indicator system framework was created and questionnaire designed based on the framework. The questionnaire consists of 17 sections and 192 items. The questionnaire and the survey protocol (including field work manual and quality control procedures) were tested by a pilot study. For the purpose of this study, we analyzed the data focused on the following nine areas of interest: (1) hospital's demographic data (including region, SARS crisis experience, teaching function, hospital type, and number of medical staff in related departments); (2) hospital PHE preparation (emergency plans, response initiating time, accessibility, and revision and implementation of emergency plan); (3) response to a community PHE (cooperation with local organizations, relationship with the community PHE network, medical treatment, and rescue work in the community); (4) stockpiles of drugs and materials (stockpiles of drugs and other resources and personal protective equipment); (5)PHE detection and identification (syndrome surveillance); (6) procedures for medical treatment (protocol for diagnosis, treatment, and transfer of PHE victims); (7) laboratory diagnosis and management (laboratory regulation and management system, sample disposal and evaluation system, collection and disposal of suspected samples, and diagnosis of pathogen/etiology); (8) staff training (organization of PHE training, current training of medical staff, curriculum development and training effectiveness assessment); and (9) risk communication (organization for communication of risk psychological counseling to victim and medical staff, and communication with public). Excluding aspect 1, items 2–9 (covering 88 survey questions) represent 8 types of PHE preparedness capacities. Each answered item was scored 1 for "yes" and 0 for "no" or "unknown". Item scores were calculated by adding together "yes" answers. Items scores were used as a proxy for measuring PHE preparedness in an institution. A total item score was measured by calculating the score across all 8 items. The higher the total item score, the better the hospital PHE preparedness capacity. Further analyses were conducted to understand the correlation between preparedness capacity and demographic information. The distribution of the related preparedness capacities across 10 categories of PHE [ 1 ] and 15 types of etiology was also assessed. Data collection procedures A computerized questionnaire stored in a CD was sent to the targeted hospitals accompanied by an official letter from each of the four city and provincial health departments stating the importance of the survey and requiring that each hospital designates a department director to be responsible for coordinating the completion of the questionnaire. Each returned questionnaire was carefully reviewed for its completeness and consistency. For those questionnaires with incomplete and/or inconsistent responses, one or two follow-up telephone calls were made to ensure completeness and consistency. The data from returned questionnaires were then transferred into a database for analysis. Data analysis A database was set up using Microsoft Excel 2003. Data was checked, cleaned, and analyzed using SPSS software version 11.5. Ninety-five percent confidence interval of means (95% CI) was used to describe PHE preparedness capacities. Categorical variables were analyzed with frequency and percentage. Comparisons of mean score of each of eight PHE preparedness capacities among different types of hospitals were performed with P < 0.05 as statistical significance using parameter test (Independent-Samples T Test (two-tailed) or One-way Analysis of Variance) and/or non-parameter test (Mann-Whitney Test or Kruskal-Wallis Test) based on data distribution characteristics and homogeneity. Results Four hundred hospitals responded, with a response rate of 100%. However, seventy-seven questionnaires were excluded from analysis due to one of the following reasons: (1) if less than 50% of items in the questionnaire were not answered, or (2) hospital did not meet secondary and/or tertiary hospital standard according to the hospital classification system. Therefore, the valid response rate was 79.5%. Hospital demographic information Of analyzed hospitals (318), 29.9% were in Beijing, 24.5% in Shandong, 40.6% in Guangxi and 5.0% in Hainan. In terms of hospital type, 72.4% were teaching hospitals. The mean number of physicians and nurses per hospital was 174.5, and the mean number of total medical staff per hospital was 206.1. The mean number of physicians and nurses in emergency department and infectious-disease department were 24.3 and 12.0, respectively. Table 1 shows the demographic characteristics of the analyzed hospitals. Table 1 Demographic characteristics of the surveyed hospital, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Variables Tertiary grade A 1 Tertiary grade B 1 Secondary grade A 1 Secondary grade B 1 Total 4 Region 318 Beijing 34 3 53 5 95 Hainan 5 0 6 5 16 Shandong 16 10 38 14 78 Guangxi 20 8 91 10 129 Fever clinics 316 Yes, designated 50 17 119 25 211 Yes, not designated 4 1 29 3 37 No 20 3 39 6 68 SARS patients admitted 2 313 Yes 25 5 38 0 68 No 48 16 147 34 245 Teaching hospitals 315 Yes 68 18 125 17 228 No 6 3 61 17 87 Types of hospital 3 297 General hospital 41 12 117 19 189 TCM hospital 8 4 25 4 41 TCM-WM hospital 1 1 5 0 7 Specialized hospital 18 4 30 6 58 Community health center 0 0 1 0 1 Emergency center 1 0 0 0 1 1 The hospital classification system, see in the study design section. 2 SARS patient admitted means the status whether the hospital admitted SARS patients during SARS crisis in 2003. 3 Types of hospital, see in the study design section. 4 Some total number of hospitals may not be 318 due to the missing values. Hospital PHE preparation (Capacity 1) Of 318 hospitals, 264(85.2%) had an emergency plan. Among the 264 hospitals that had an emergency plan, 92.6% reported that the institution possessed a protocol to initiate the emergency plan, 75.5% had a classification system for different PHE events, 55.3% had evaluated and revised their emergency plan at least once, and 79.6% reported that their emergency plan was accessible to all medical staff. As for organizational preparation, 93.3% of hospitals with an active emergency plan had a command center and designated personnel for PHE situations. There were statistical significance among tertiary grade A hospitals (95% CI: 9.8,10.9) and secondary grade B ones(95% CI:7.0,9.3), tertiary grade B hospitals(95% CI:9.8,11.9) and secondary grade B ones. Comparison of eight aspects PHE preparedness capacities among different types of hospitals are showed in table 2 and table 3 . Table 2 Comparison of eight aspects PHE preparedness capacities (capacity 1 to 4) among different characteristics of hospitals, Beijing, Guangxi, and Shandong, Hainan, China, 2004–2005 (N = 318) Variables Number Capacity 1 95% CI Capacity 2 95% CI Capacity 3 95% CI Capacity 4 95% CI Region Beijing 95 9.6,10.6 5.9,6.9* 5.8,6.9** 5.4,6.3 Hainan 16 6.9,9.8 5.4,8.0* 4.8,7.8** 3.9,6.9 Shandong 78 9.0,10.3 7.0,7.9* 6.6,7.9** 5.2,6.3 Guangxi 129 9.2,10.2 6.7,7.4* 5.4,6.4** 5.4,6.2 Classification Tertiary grade A 75 9.8,10.9** 6.8,8.0* 6.6,7.9** 5.6,6.6* Tertiary grade B 21 9.8,11.9** 6.7,8.4* 6.3,9.0** 5.6,7.3* Secondary grade A 188 9.3,10.1** 6.5,7.1* 5.8,6.6** 5.4,6.0* Secondary grade B 34 7.0,9.3** 5.4,6.9* 3.9,6.0** 4.1,5.9* Teaching hospital Yes 228 9.6,10.3** 7.0,7.5** 6.4,7.1 5.6,6.2 No 87 8.7,9.9** 5.7,6.6** 5.0,6.1 5.1,6.0 Type General hospital 189 9.7,10.5** 7.0,7.6 6.2,7.0 5.5,6.2 TCM hospital 41 8.4,10.2** 5.5,6.9 5.1,6.8 5.3,6.7 TCM-WM hospital 7 7.7,14.5** 5.6,9.8 5.1,11.7 6.0,8.3 Specialized hospital 58 8.5,10.0** 5.8,6.8 5.2,6.8 4.7,6.0 Community health center 1 --** -- -- -- Emergency center 1 --** -- -- -- SARS patients admitted Yes 68 9.8,10.9 6.3,7.4 6.0,7.3 5.3,6.4 No 245 9.3,10.0 6.7,7.3 6.0,6.7 5.5,6.1 †*0.01 < p < 0.05, **p ≤ 0.01; 95% CI: 95% confidence interval of means. ‡ Capacity1: Hospital PHE preparation (highest score = 13); Capacity2: Response to PHE in community (highest score = 11); Capacity 3: Stockpiles of drugs and materials (highest score = 12); Capacity4: Detection and identification of PHE (highest score = 8). §The post-hoc multiple significant comparison shows that: Capacity1 (Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B; Secondary grade A vs Secondary grade B; General hospital vs Specialized hospital); Capacity2 (Beijing vs Shandong; Beijing vs Guangxi; Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B); Capacity3 (Beijing vs Shandong; Tertiary grade A vs Secondary grade A; Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade A; Tertiary grade B vs Secondary grade B; Secondary grade A vs Secondary grade B); Capacity4 (Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade B). Table 3 Comparison of eight aspects PHE preparedness capacities (capacity 5 to 8) among different characteristics of hospitals, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Variables Number Capacity 5 95% CI Capacity 6 95% CI Capacity 7 95% CI Capacity 8 95% CI Region Beijing 95 6.5,8.1** 4.0,4.9 5.7,6.6* 3.4,4.4** Hainan 16 5.4,9.9** 3.6,5.6 4.2,7.5* 2.2,5.3** Shandong 78 8.8,10.7** 4.4,5.4 5.9,7.0* 4.6,6.2** Guangxi 129 8.5,9.8** 4.0,4.6 5.1,6.0* 3.2,4.1** Classification Tertiary grade A 75 8.2,10.2* 4.6,5.7** 5.9,7.0 4.7,6.0** Tertiary grade B 21 8.6,11.4* 4.4,5.9** 4.9,7.1 4.2,6.9** Secondary grade A 188 8.0,9.2* 4.1,4.6** 5.6,6.2 3.2,4.0** Secondary grade B 34 5.5,8.3* 2.6,3.9** 4.3,6.1 2.8,4.8** Teaching hospital Yes 228 8.6,9.6* 4.4,5.0 5.8,6.4 4.0,4.8 No 87 6.8,8.5* 3.6,4.4 5.2,6.2 3.1,4.3 Type General hospital 189 8.7,9.9** 4.5,5.0 5.9,6.5 3.7,4.5 TCM hospital 41 7.3,9.6** 3.7,4.7 4.6,6.2 2.9,4.7 TCM-WM hospital 7 8.9,13.7** 1.6,7.0 3.6,8.7 1.9,8.7 Specialized hospital 58 5.5,7.7** 3.4,4.4 5.0,6.4 3.9,5.5 Community health center 1 --** -- -- -- Emergency center 1 --** -- -- -- SARS patients admitted Yes 68 7.6,9.6 4.3,5.3* 5.9,6.8 3.1,4.3 No 245 8.2,9.3 4.2,4.7* 5.6,6.2 3.9,4.7 †*0.01 < p < 0.05, **p < 0.01; 95% CI: 95% confidence interval of means. ‡ Capacity 5: Procedures for medical treatment (highest score = 15); Capacity 6: Laboratory diagnosis and management (highest score = 9); Capacity 7: Staff training (highest score = 9); Capacity 8: Risk communication (highest score = 11). § The post-hoc multiple significant comparison shows that: Capacity5 (Beijing vs Shandong; Beijing vs Guangxi; Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B; Secondary grade A vs Secondary grade B; General hospital vs Specialized hospital; TCM hospital vs Specialized hospital; TCM-WM hospital vs Specialized hospital); Capacity6 (Tertiary grade A vs Secondary grade A; Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade B); Capacity7 (Shandong vs Guangxi);Capacity8 (Beijing vs Shandong; Hainan vs Shandong; Guangxi vs Shandong; Tertiary grade A vs Secondary grade A; Secondary grade B vs Tertiary grade A; Tertiary grade B vs Secondary grade A; Tertiary grade B vs Secondary grade B). Response to PHE in community (Capacity 2) Of all analyzed respondents, 64.2% were designated as the local emergency hospital for PHE patient admissions and 53.0% of them were the designated hospitals to provide medical rescue services during a national disaster. Of all analyzed respondents, 97.4% could promptly transport needed medical staff to the PHE field, 84.5% reported that they were prepared to respond to the needs of vulnerable people (including women, children, pregnant women and the disabled) during a PHE, however, only 49.8% had evaluated their ability to increase beds and equipment for PHE. When performing a PHE preparedness drill, 22.9% of respondents reported that they would invite relevant community organizations to participate. With regard to capacity comparison, the statistics test showed: the total item score of hospitals in Beijing(95% CI:5.9,6.9) was lower than that of hospitals in Shandong (95% CI:7.0,7.9) and Guangxi(95% CI:6.7,7.4); the score of teaching hospitals(95% CI:7.0,7.5) was higher than that of non-teaching hospitals(95% CI:5.7,6.6); and the score of tertiary grade A (95% CI:6.8,8.0) and B (95% CI:6.7,8.4) hospitals was higher than that of secondary grade B ones(95% CI:5.4,6.9), respectively. Among all types of hospitals, community health center scored highest on this aspect. Stockpiles of drugs and materials (Capacity 3) Our results revealed that 53.1% of respondents had evaluated their stockpiles of drugs, and 61.5% had established a relationship with suppliers to provide emergency drug-supplies, however, only 43.2% had signed written contracts with suppliers. Of all analyzed respondents, 47.8% had drug-distribution plans, and 21.5% knew where the national or local pharmacy distribution centers were located. In regards to other medical materials, 80.1% had stockpiles of materials for responding to PHE. As for the stockpiles of drugs for infectious diseases, about 93.2%, 91.9% and 43.5% of responding hospitals had drug stockpiles for treating infectious diarrhea, influenza and botulismo toxin, respectively. When hospitals were compared on this item, statistical analysis showed that institutions in Beijing (95% CI:5.8,6.9) had a higher score than that of Shandong (95% CI:6.6,7.9). Tertiary hospitals generally had a higher score than secondary ones. PHE detection and identification (Capacity 4) Among all the respondents, 55.5% reported that they had developed syndromic surveillance systems for certain diseases and 84.4% required that physicians on duty should report any abnormity to the hospital's presidents (the definition of abnormity see in appendix). Abnormity in admission diagnosis, routine microbiological tests, emergency room patients, and death with unknown causes were systematically monitored by 74.6% of institutions and 47.4% of hospitals shared their surveillance information with the local health authority. There were statistically significant differences between tertiary grade hospitals (Grade A 95% CI: 5.6,6.6; Grade B 95% CI: 5.6,7.3) and secondary grade B hospitals (95% CI:4.1,5.9) for this capacity, with tertiary hospitals scoring higher on their ability to detect and identify a PHE. Procedures for medical treatment (Capacity 5) Physicians in 80.2% of the responding institutions reported being familiarized with the latest treatment protocol for a PHE, 92.8% could transfer PHE victims to corresponding medical agencies for appropriate treatment, and 98.0% could provide training on the protocol system. However, only 69.0% had specific procedures for patient transfer in a PHE. As for infectious disease treatment protocol, 80.1% had protocols for SARS, but only 37.3% for brucellosis. With regard to the capacity comparison between evaluated hospitals, statistical analysis revealed that hospitals in Shandong (95% CI:8.8,10.7) and Guangxi (95% CI:8.5,9.8) scored higher than those of Beijing (95% CI:6.5,8.1). Furthermore, TCM-WM hospitals (95% CI:8.9,13.7) scored higher than all other types of institutions. Tertiary grade hospitals (Grade A 95% CI:8.2,10.2; Grade B 95% CI: 8.6,11.4) and teaching hospitals (95% CI:8.6,9.6) had better score than secondary grade B (95% CI:5.5,8.3)and non-teaching institutions (95% CI:6.8,8.5), respectively. Laboratory diagnosis and management (Capacity 6) We selected 15 kinds of infectious diseases/etiologies on which to assess laboratory diagnosis capacity, medical treatment procedures, and drug stockpile for infectious disease control. Our results showed that 59.0% of responding hospitals could isolate and identify salmonella and staphylococcus, but less than 5% reported that they could isolate and identify human H5N1 avian flu and SARS. The results are listed in table 4 . As for the management of laboratory of results, 97.4% of respondents had strict laboratory operational regulations and 96.4% had personnel specially assigned to laboratory management. When faced with an emergency, 76.7% could promptly enlarge the capacity of sample disposal, while only 33.5% had protocols to collect suspected samples. Disposal and transportation of suspected samples capabilities were 33.5% and 32.6%, respectively, but once laboratories were contaminated, only 9.1% had alternatives. Statistical analysis showed that tertiary-grade A (95% CI:4.6,5.7) and B (95% CI:4.4,5.9) hospitals and hospitals with experience of SARS patients (95% CI:4.3,5.3) scored higher than those secondary-grade and without experience (95% CI:4.2,4.7), respectively. Table 4 The laboratory diagnosis capacity, medical treatment procedures and drug stockpile for 15 types of etiology in respondent hospitals, Beijing, Shandong, Guangxi, Hainan, China, 2004–2005 (N = 318) Varieties of etiology Laboratory diagnosis capacity Medical treatment procedures Drug stockpile No. "yes" % No. "yes" % No. "yes" % SARS 9 3.1 241 80.1 252 80.8 Plague 28 9.8 136 47.6 149 49.0 Cholera 145 49.3 175 60.8 194 63.6 Anthrax 49 16.9 110 38.6 140 46.4 Brucellosis 43 15.0 106 37.3 134 44.8 Meningococcal meningitis 98 33.6 203 68.4 237 77.2 Japanese encephalitis B 43 14.8 203 69.0 242 79.1 Influenza 43 14.6 234 78.8 285 91.9 Human H5N1 avian flu 6 2.1 197 67.0 182 60.1 Infectious diarrhea 77 60.4 225 77.1 288 93.2 Food poisoning of staphylococcus 172 58.7 187 64.7 259 84.6 Food poisoning of salmonella 174 59.6 184 63.7 255 83.3 Acute organophosphorus poisoning 111 37.8 226 76.6 272 88.3 Botulism toxin poisoning 24 8.3 126 44.5 131 43.5 Tetramine poisoning 18 6.3 162 56.1 162 53.6 Staff training (Capacity 7) Among all the respondents, 94.5% reported that they had a training program for the following medical staff: infection managers (56.3%); emergency department physicians and nurses (92.2%); and infectious disease ward physicians and nurses (71.8%). Staff training was supervised by a designated person in 82.3% of institutions and 65.8% had training curriculums, 66.5% of which was updated regularly. Effectiveness of PHE training was periodically assessed in 50.3% of respondents. For this capacity, statistical significance indicated that respondents in Shandong (95% CI:5.9,7.0) scored higher than participating institutions in Guangxi (95% CI:5.1,6.0). Risk communication (Capacity 8) Of all respondents, 45.0% possessed expert panels to advise on PHE psychological counseling for medical staff and PHE victims. Medical staff in 12.1% of the analyzed hospitals were trained to assess the psychological impact of PHE. If a PHE occurred, participating hospitals could: counsel victims and their family members (43.6%); offer teaching materials (51.0%); access additional psychological consultants (19.3%). With regards to communication capabilities, 39.4% of hospitals reported a mass media communication protocol, 43.9% possessed a designated spokesperson to deliver PHE information to the public, and 30.8% possessed personnel specially assigned to communicate information to the media, public, and local governments. Statistical analysis showed that hospitals in Shandong (95% CI:4.6,6.2) scored higher than those in Beijing (95% CI:3.4,4.4), Guangxi (95% CI:3.2,4.1) and Hainan (95% CI:2.2,5.3) on this capacity, with tertiary-grade hospitals (Grade A 95% CI: 4.7,6.0; Grade B 95% CI: 4.2,6.9) reporting better score than secondary ones (Grade A 95% CI: 3.2,4.0; Grade B 95% CI: 2.8,4.8). Comparisons of various PHE contents Five aspects of preparedness capacities and various PHE events were described in crosstab, as shown in table 5 . Among all the respondents, 277 hospitals (95.5%) had emergency plans for infectious epidemics, and 50 ones (20.5%) for biochemical and nuclear terrorism. Evaluation on infectious epidemic was performed by 93.7%, the percentage was relatively lower for bio-terrorism and nuclear terrorism threats (30.5%), In regards to expert consultation, 28.2% had attended local agency meetings on regulation and revision of emergency plans for infectious epidemic control and 93.3% had the available expert list for consultation on infectious epidemic, however, for terrorism, the percentage was only 23.1%. Projects for admitting and treating infectious epidemic victims were common (71%), although only 13.3% of hospitals were involved in similar plans for dealing with bio-terror and nuclear threats. Table 5 Comparisons of various PHE events and related capacities of all types of hospitals, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Varieties of PHE Emergency plans included Assessing response to PHE Attending regulation and revision of emergency plans in local agencies Having expert lists for following situation Having projects admitting and treating following victims No. "yes" % No. "yes" % No. "yes" % No. "yes" % No. "yes" % Infectious diseases incidence 277 95.5 133 93.7 81 28.2 293 93.3 210 70.7 Unidentified population diseases 167 62.8 110 79.1 54 19.0 231 75.0 141 48.8 Mass food poisoning and water pollution 200 73.5 116 82.3 57 20.1 243 79.2 173 59.5 Mass occupational poisoning 125 49.2 75 56.8 45 16.0 166 56.5 100 35.3 Outbreak of nosocomial infection 162 62.8 104 77.0 56 19.9 222 73.8 143 49.8 Mass abnormal reaction or death resulted from drugs or vaccination 93 38.0 79 59.8 41 14.5 160 54.8 91 32.4 Incident of radioactive or poisonous material contamination 103 41.4 60 45.8 32 11.4 125 43.1 73 25.9 Biochemical and nuclear terrorism 50 20.5 40 30.5 25 8.9 66 23.1 37 13.3 Grave medicine accident 203 76.0 115 83.9 58 20.4 237 77.7 158 54.7 Natural disaster 173 67.8 97 70.8 49 17.4 214 71.1 138 48.6 Hospital demographic information Of analyzed hospitals (318), 29.9% were in Beijing, 24.5% in Shandong, 40.6% in Guangxi and 5.0% in Hainan. In terms of hospital type, 72.4% were teaching hospitals. The mean number of physicians and nurses per hospital was 174.5, and the mean number of total medical staff per hospital was 206.1. The mean number of physicians and nurses in emergency department and infectious-disease department were 24.3 and 12.0, respectively. Table 1 shows the demographic characteristics of the analyzed hospitals. Table 1 Demographic characteristics of the surveyed hospital, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Variables Tertiary grade A 1 Tertiary grade B 1 Secondary grade A 1 Secondary grade B 1 Total 4 Region 318 Beijing 34 3 53 5 95 Hainan 5 0 6 5 16 Shandong 16 10 38 14 78 Guangxi 20 8 91 10 129 Fever clinics 316 Yes, designated 50 17 119 25 211 Yes, not designated 4 1 29 3 37 No 20 3 39 6 68 SARS patients admitted 2 313 Yes 25 5 38 0 68 No 48 16 147 34 245 Teaching hospitals 315 Yes 68 18 125 17 228 No 6 3 61 17 87 Types of hospital 3 297 General hospital 41 12 117 19 189 TCM hospital 8 4 25 4 41 TCM-WM hospital 1 1 5 0 7 Specialized hospital 18 4 30 6 58 Community health center 0 0 1 0 1 Emergency center 1 0 0 0 1 1 The hospital classification system, see in the study design section. 2 SARS patient admitted means the status whether the hospital admitted SARS patients during SARS crisis in 2003. 3 Types of hospital, see in the study design section. 4 Some total number of hospitals may not be 318 due to the missing values. Hospital PHE preparation (Capacity 1) Of 318 hospitals, 264(85.2%) had an emergency plan. Among the 264 hospitals that had an emergency plan, 92.6% reported that the institution possessed a protocol to initiate the emergency plan, 75.5% had a classification system for different PHE events, 55.3% had evaluated and revised their emergency plan at least once, and 79.6% reported that their emergency plan was accessible to all medical staff. As for organizational preparation, 93.3% of hospitals with an active emergency plan had a command center and designated personnel for PHE situations. There were statistical significance among tertiary grade A hospitals (95% CI: 9.8,10.9) and secondary grade B ones(95% CI:7.0,9.3), tertiary grade B hospitals(95% CI:9.8,11.9) and secondary grade B ones. Comparison of eight aspects PHE preparedness capacities among different types of hospitals are showed in table 2 and table 3 . Table 2 Comparison of eight aspects PHE preparedness capacities (capacity 1 to 4) among different characteristics of hospitals, Beijing, Guangxi, and Shandong, Hainan, China, 2004–2005 (N = 318) Variables Number Capacity 1 95% CI Capacity 2 95% CI Capacity 3 95% CI Capacity 4 95% CI Region Beijing 95 9.6,10.6 5.9,6.9* 5.8,6.9** 5.4,6.3 Hainan 16 6.9,9.8 5.4,8.0* 4.8,7.8** 3.9,6.9 Shandong 78 9.0,10.3 7.0,7.9* 6.6,7.9** 5.2,6.3 Guangxi 129 9.2,10.2 6.7,7.4* 5.4,6.4** 5.4,6.2 Classification Tertiary grade A 75 9.8,10.9** 6.8,8.0* 6.6,7.9** 5.6,6.6* Tertiary grade B 21 9.8,11.9** 6.7,8.4* 6.3,9.0** 5.6,7.3* Secondary grade A 188 9.3,10.1** 6.5,7.1* 5.8,6.6** 5.4,6.0* Secondary grade B 34 7.0,9.3** 5.4,6.9* 3.9,6.0** 4.1,5.9* Teaching hospital Yes 228 9.6,10.3** 7.0,7.5** 6.4,7.1 5.6,6.2 No 87 8.7,9.9** 5.7,6.6** 5.0,6.1 5.1,6.0 Type General hospital 189 9.7,10.5** 7.0,7.6 6.2,7.0 5.5,6.2 TCM hospital 41 8.4,10.2** 5.5,6.9 5.1,6.8 5.3,6.7 TCM-WM hospital 7 7.7,14.5** 5.6,9.8 5.1,11.7 6.0,8.3 Specialized hospital 58 8.5,10.0** 5.8,6.8 5.2,6.8 4.7,6.0 Community health center 1 --** -- -- -- Emergency center 1 --** -- -- -- SARS patients admitted Yes 68 9.8,10.9 6.3,7.4 6.0,7.3 5.3,6.4 No 245 9.3,10.0 6.7,7.3 6.0,6.7 5.5,6.1 †*0.01 < p < 0.05, **p ≤ 0.01; 95% CI: 95% confidence interval of means. ‡ Capacity1: Hospital PHE preparation (highest score = 13); Capacity2: Response to PHE in community (highest score = 11); Capacity 3: Stockpiles of drugs and materials (highest score = 12); Capacity4: Detection and identification of PHE (highest score = 8). §The post-hoc multiple significant comparison shows that: Capacity1 (Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B; Secondary grade A vs Secondary grade B; General hospital vs Specialized hospital); Capacity2 (Beijing vs Shandong; Beijing vs Guangxi; Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B); Capacity3 (Beijing vs Shandong; Tertiary grade A vs Secondary grade A; Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade A; Tertiary grade B vs Secondary grade B; Secondary grade A vs Secondary grade B); Capacity4 (Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade B). Table 3 Comparison of eight aspects PHE preparedness capacities (capacity 5 to 8) among different characteristics of hospitals, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Variables Number Capacity 5 95% CI Capacity 6 95% CI Capacity 7 95% CI Capacity 8 95% CI Region Beijing 95 6.5,8.1** 4.0,4.9 5.7,6.6* 3.4,4.4** Hainan 16 5.4,9.9** 3.6,5.6 4.2,7.5* 2.2,5.3** Shandong 78 8.8,10.7** 4.4,5.4 5.9,7.0* 4.6,6.2** Guangxi 129 8.5,9.8** 4.0,4.6 5.1,6.0* 3.2,4.1** Classification Tertiary grade A 75 8.2,10.2* 4.6,5.7** 5.9,7.0 4.7,6.0** Tertiary grade B 21 8.6,11.4* 4.4,5.9** 4.9,7.1 4.2,6.9** Secondary grade A 188 8.0,9.2* 4.1,4.6** 5.6,6.2 3.2,4.0** Secondary grade B 34 5.5,8.3* 2.6,3.9** 4.3,6.1 2.8,4.8** Teaching hospital Yes 228 8.6,9.6* 4.4,5.0 5.8,6.4 4.0,4.8 No 87 6.8,8.5* 3.6,4.4 5.2,6.2 3.1,4.3 Type General hospital 189 8.7,9.9** 4.5,5.0 5.9,6.5 3.7,4.5 TCM hospital 41 7.3,9.6** 3.7,4.7 4.6,6.2 2.9,4.7 TCM-WM hospital 7 8.9,13.7** 1.6,7.0 3.6,8.7 1.9,8.7 Specialized hospital 58 5.5,7.7** 3.4,4.4 5.0,6.4 3.9,5.5 Community health center 1 --** -- -- -- Emergency center 1 --** -- -- -- SARS patients admitted Yes 68 7.6,9.6 4.3,5.3* 5.9,6.8 3.1,4.3 No 245 8.2,9.3 4.2,4.7* 5.6,6.2 3.9,4.7 †*0.01 < p < 0.05, **p < 0.01; 95% CI: 95% confidence interval of means. ‡ Capacity 5: Procedures for medical treatment (highest score = 15); Capacity 6: Laboratory diagnosis and management (highest score = 9); Capacity 7: Staff training (highest score = 9); Capacity 8: Risk communication (highest score = 11). § The post-hoc multiple significant comparison shows that: Capacity5 (Beijing vs Shandong; Beijing vs Guangxi; Tertiary grade A vs Secondary grade B; Secondary grade B vs Tertiary grade B; Secondary grade A vs Secondary grade B; General hospital vs Specialized hospital; TCM hospital vs Specialized hospital; TCM-WM hospital vs Specialized hospital); Capacity6 (Tertiary grade A vs Secondary grade A; Tertiary grade A vs Secondary grade B; Tertiary grade B vs Secondary grade B); Capacity7 (Shandong vs Guangxi);Capacity8 (Beijing vs Shandong; Hainan vs Shandong; Guangxi vs Shandong; Tertiary grade A vs Secondary grade A; Secondary grade B vs Tertiary grade A; Tertiary grade B vs Secondary grade A; Tertiary grade B vs Secondary grade B). Response to PHE in community (Capacity 2) Of all analyzed respondents, 64.2% were designated as the local emergency hospital for PHE patient admissions and 53.0% of them were the designated hospitals to provide medical rescue services during a national disaster. Of all analyzed respondents, 97.4% could promptly transport needed medical staff to the PHE field, 84.5% reported that they were prepared to respond to the needs of vulnerable people (including women, children, pregnant women and the disabled) during a PHE, however, only 49.8% had evaluated their ability to increase beds and equipment for PHE. When performing a PHE preparedness drill, 22.9% of respondents reported that they would invite relevant community organizations to participate. With regard to capacity comparison, the statistics test showed: the total item score of hospitals in Beijing(95% CI:5.9,6.9) was lower than that of hospitals in Shandong (95% CI:7.0,7.9) and Guangxi(95% CI:6.7,7.4); the score of teaching hospitals(95% CI:7.0,7.5) was higher than that of non-teaching hospitals(95% CI:5.7,6.6); and the score of tertiary grade A (95% CI:6.8,8.0) and B (95% CI:6.7,8.4) hospitals was higher than that of secondary grade B ones(95% CI:5.4,6.9), respectively. Among all types of hospitals, community health center scored highest on this aspect. Stockpiles of drugs and materials (Capacity 3) Our results revealed that 53.1% of respondents had evaluated their stockpiles of drugs, and 61.5% had established a relationship with suppliers to provide emergency drug-supplies, however, only 43.2% had signed written contracts with suppliers. Of all analyzed respondents, 47.8% had drug-distribution plans, and 21.5% knew where the national or local pharmacy distribution centers were located. In regards to other medical materials, 80.1% had stockpiles of materials for responding to PHE. As for the stockpiles of drugs for infectious diseases, about 93.2%, 91.9% and 43.5% of responding hospitals had drug stockpiles for treating infectious diarrhea, influenza and botulismo toxin, respectively. When hospitals were compared on this item, statistical analysis showed that institutions in Beijing (95% CI:5.8,6.9) had a higher score than that of Shandong (95% CI:6.6,7.9). Tertiary hospitals generally had a higher score than secondary ones. PHE detection and identification (Capacity 4) Among all the respondents, 55.5% reported that they had developed syndromic surveillance systems for certain diseases and 84.4% required that physicians on duty should report any abnormity to the hospital's presidents (the definition of abnormity see in appendix). Abnormity in admission diagnosis, routine microbiological tests, emergency room patients, and death with unknown causes were systematically monitored by 74.6% of institutions and 47.4% of hospitals shared their surveillance information with the local health authority. There were statistically significant differences between tertiary grade hospitals (Grade A 95% CI: 5.6,6.6; Grade B 95% CI: 5.6,7.3) and secondary grade B hospitals (95% CI:4.1,5.9) for this capacity, with tertiary hospitals scoring higher on their ability to detect and identify a PHE. Procedures for medical treatment (Capacity 5) Physicians in 80.2% of the responding institutions reported being familiarized with the latest treatment protocol for a PHE, 92.8% could transfer PHE victims to corresponding medical agencies for appropriate treatment, and 98.0% could provide training on the protocol system. However, only 69.0% had specific procedures for patient transfer in a PHE. As for infectious disease treatment protocol, 80.1% had protocols for SARS, but only 37.3% for brucellosis. With regard to the capacity comparison between evaluated hospitals, statistical analysis revealed that hospitals in Shandong (95% CI:8.8,10.7) and Guangxi (95% CI:8.5,9.8) scored higher than those of Beijing (95% CI:6.5,8.1). Furthermore, TCM-WM hospitals (95% CI:8.9,13.7) scored higher than all other types of institutions. Tertiary grade hospitals (Grade A 95% CI:8.2,10.2; Grade B 95% CI: 8.6,11.4) and teaching hospitals (95% CI:8.6,9.6) had better score than secondary grade B (95% CI:5.5,8.3)and non-teaching institutions (95% CI:6.8,8.5), respectively. Laboratory diagnosis and management (Capacity 6) We selected 15 kinds of infectious diseases/etiologies on which to assess laboratory diagnosis capacity, medical treatment procedures, and drug stockpile for infectious disease control. Our results showed that 59.0% of responding hospitals could isolate and identify salmonella and staphylococcus, but less than 5% reported that they could isolate and identify human H5N1 avian flu and SARS. The results are listed in table 4 . As for the management of laboratory of results, 97.4% of respondents had strict laboratory operational regulations and 96.4% had personnel specially assigned to laboratory management. When faced with an emergency, 76.7% could promptly enlarge the capacity of sample disposal, while only 33.5% had protocols to collect suspected samples. Disposal and transportation of suspected samples capabilities were 33.5% and 32.6%, respectively, but once laboratories were contaminated, only 9.1% had alternatives. Statistical analysis showed that tertiary-grade A (95% CI:4.6,5.7) and B (95% CI:4.4,5.9) hospitals and hospitals with experience of SARS patients (95% CI:4.3,5.3) scored higher than those secondary-grade and without experience (95% CI:4.2,4.7), respectively. Table 4 The laboratory diagnosis capacity, medical treatment procedures and drug stockpile for 15 types of etiology in respondent hospitals, Beijing, Shandong, Guangxi, Hainan, China, 2004–2005 (N = 318) Varieties of etiology Laboratory diagnosis capacity Medical treatment procedures Drug stockpile No. "yes" % No. "yes" % No. "yes" % SARS 9 3.1 241 80.1 252 80.8 Plague 28 9.8 136 47.6 149 49.0 Cholera 145 49.3 175 60.8 194 63.6 Anthrax 49 16.9 110 38.6 140 46.4 Brucellosis 43 15.0 106 37.3 134 44.8 Meningococcal meningitis 98 33.6 203 68.4 237 77.2 Japanese encephalitis B 43 14.8 203 69.0 242 79.1 Influenza 43 14.6 234 78.8 285 91.9 Human H5N1 avian flu 6 2.1 197 67.0 182 60.1 Infectious diarrhea 77 60.4 225 77.1 288 93.2 Food poisoning of staphylococcus 172 58.7 187 64.7 259 84.6 Food poisoning of salmonella 174 59.6 184 63.7 255 83.3 Acute organophosphorus poisoning 111 37.8 226 76.6 272 88.3 Botulism toxin poisoning 24 8.3 126 44.5 131 43.5 Tetramine poisoning 18 6.3 162 56.1 162 53.6 Staff training (Capacity 7) Among all the respondents, 94.5% reported that they had a training program for the following medical staff: infection managers (56.3%); emergency department physicians and nurses (92.2%); and infectious disease ward physicians and nurses (71.8%). Staff training was supervised by a designated person in 82.3% of institutions and 65.8% had training curriculums, 66.5% of which was updated regularly. Effectiveness of PHE training was periodically assessed in 50.3% of respondents. For this capacity, statistical significance indicated that respondents in Shandong (95% CI:5.9,7.0) scored higher than participating institutions in Guangxi (95% CI:5.1,6.0). Risk communication (Capacity 8) Of all respondents, 45.0% possessed expert panels to advise on PHE psychological counseling for medical staff and PHE victims. Medical staff in 12.1% of the analyzed hospitals were trained to assess the psychological impact of PHE. If a PHE occurred, participating hospitals could: counsel victims and their family members (43.6%); offer teaching materials (51.0%); access additional psychological consultants (19.3%). With regards to communication capabilities, 39.4% of hospitals reported a mass media communication protocol, 43.9% possessed a designated spokesperson to deliver PHE information to the public, and 30.8% possessed personnel specially assigned to communicate information to the media, public, and local governments. Statistical analysis showed that hospitals in Shandong (95% CI:4.6,6.2) scored higher than those in Beijing (95% CI:3.4,4.4), Guangxi (95% CI:3.2,4.1) and Hainan (95% CI:2.2,5.3) on this capacity, with tertiary-grade hospitals (Grade A 95% CI: 4.7,6.0; Grade B 95% CI: 4.2,6.9) reporting better score than secondary ones (Grade A 95% CI: 3.2,4.0; Grade B 95% CI: 2.8,4.8). Comparisons of various PHE contents Five aspects of preparedness capacities and various PHE events were described in crosstab, as shown in table 5 . Among all the respondents, 277 hospitals (95.5%) had emergency plans for infectious epidemics, and 50 ones (20.5%) for biochemical and nuclear terrorism. Evaluation on infectious epidemic was performed by 93.7%, the percentage was relatively lower for bio-terrorism and nuclear terrorism threats (30.5%), In regards to expert consultation, 28.2% had attended local agency meetings on regulation and revision of emergency plans for infectious epidemic control and 93.3% had the available expert list for consultation on infectious epidemic, however, for terrorism, the percentage was only 23.1%. Projects for admitting and treating infectious epidemic victims were common (71%), although only 13.3% of hospitals were involved in similar plans for dealing with bio-terror and nuclear threats. Table 5 Comparisons of various PHE events and related capacities of all types of hospitals, Beijing, Shandong, Guangxi, and Hainan, China, 2004–2005 (N = 318) Varieties of PHE Emergency plans included Assessing response to PHE Attending regulation and revision of emergency plans in local agencies Having expert lists for following situation Having projects admitting and treating following victims No. "yes" % No. "yes" % No. "yes" % No. "yes" % No. "yes" % Infectious diseases incidence 277 95.5 133 93.7 81 28.2 293 93.3 210 70.7 Unidentified population diseases 167 62.8 110 79.1 54 19.0 231 75.0 141 48.8 Mass food poisoning and water pollution 200 73.5 116 82.3 57 20.1 243 79.2 173 59.5 Mass occupational poisoning 125 49.2 75 56.8 45 16.0 166 56.5 100 35.3 Outbreak of nosocomial infection 162 62.8 104 77.0 56 19.9 222 73.8 143 49.8 Mass abnormal reaction or death resulted from drugs or vaccination 93 38.0 79 59.8 41 14.5 160 54.8 91 32.4 Incident of radioactive or poisonous material contamination 103 41.4 60 45.8 32 11.4 125 43.1 73 25.9 Biochemical and nuclear terrorism 50 20.5 40 30.5 25 8.9 66 23.1 37 13.3 Grave medicine accident 203 76.0 115 83.9 58 20.4 237 77.7 158 54.7 Natural disaster 173 67.8 97 70.8 49 17.4 214 71.1 138 48.6 Discussion Serious PHE concerns were raised in China during the 2003 SARS crisis when it became apparent that hospitals possessed poor emergency preparedness [ 7 ]. Even the up-coming 2008 Olympics Game in Beijing and the 5.12 Earthquake Disaster in China have dramatically evoked the awareness of PHE preparedness capacity for hospital. Based on the experience of the SARS pandemic, all hospitals should possess fundamental PHE programs, including preparedness of drugs, equipment, staff, emergency education and staff training [ 3 , 8 , 9 ], coordination with relevant community bodies [ 10 ], medical treatment [ 11 ], early detection and warning [ 12 ], laboratory diagnosis [ 13 - 15 ] and psychological intervention [ 8 ]. Since the SARS crisis, the central Chinese government has become more active in the construction of public health system, especially in regards to the medical emergency response system [ 16 ]. One major effort involved a 11.4 billion RMB investment in local governments to initiate the construction of regional PHE medical treatment systems [ 17 ]. In order to offer some insight into the development of hospital PHE preparedness capacity, this study examined the current status of hospital preparedness in Beijing, Shandong, Guangxi, and Hainan. Emergency preparedness refers to the processes involved in ensuring an institution: (1) has complied with the preventive measures; (2) is in a state of readiness to contain the effects of a forecasted disastrous event in order to minimize loss of life, injury, and damage to property; (3) can provide rescue, relief, rehabilitation, and other services in the aftermath of the disaster; and (4) holds the capability and resources to continue to sustain its essential functions during a PHE [ 18 ]. An emergency preparedness systems primarily composed of emergency plans and organizational structures and lays the foundation for dealing with PHE [ 19 ]. Emergency plans establish the protocol for operation under a PHE [ 16 ]. For a hospital to mobilize all PHE resources in a short period of time, contingency plans must be issued in advance [ 9 ]. In addition, periodic review and updating of emergency plans enhance an institution's emergency response capacity [ 3 ]. Our study showed that most hospitals had emergency plans and that these plans focused on infectious diseases control with less attention to preparedness for biological, nuclear radiation and other terrorism attacks. Most of the hospitals had PHE command departments and emergency response teams, however, only 55.3% of hospitals with emergency plans reported they had evaluated and revised their PHE systems. Overall, tertiary hospitals performed better in PHE preparation than secondary hospitals. Meanwhile, no statistical significance was found between hospitals that had admitted SARS patients and those that had not, suggesting that after the SARS crisis, all hospitals raised awareness of emergency plans and implementation. No hospital or medical system can manage a public health emergency without community networks and public involvement. Therefore, hospitals need to communicate and cooperate with other local health agencies, functioning as a networked public health provider. Problems like lack of communication and coordination between hospital departments and inter-agency networks hinder the availability of resources in a community and limit timely forecasting, public communication and effective regulation of a PHE [ 10 ]. Our survey revealed that if a PHE occurred, most of hospitals reported that they could take responsibility for PHE rescue service, transport the medical staff in a timely manner, and provide priority health services to vulnerable populations. Yet, less than one third of respondents attended regulation and revision workshops for emergency plans for infectious epidemic control held by local agencies. This lack of cross-institutional interaction indicated that the ability of hospitals to coordinate with community agencies in preparation for, or in the event of a PHE was generally poor. The survey showed that among all the types of respondents community health center were best able to respond to PHE and the respondents with multiple functions performed better suggesting that communication and coordination between hospitals and community agencies should be strengthened. Characteristics of a PHE include suddenness and unpredictability [ 9 ]. For most hospitals, medicine storage may be in great demand when faced with a sudden increase in patients. Therefore, hospitals must have programs to ensure appropriate levels of emergency supplies including drugs, medical equipment, electricity, water and oxygen, disinfectant, etc. Our survey suggested that most of the hospitals could establish an emergency-drug-supply system for most of the infectious diseases we addressed in the questionnaire except anthrax, brucellosis, botulism toxin poisoning and tetramine poisoning. For most of surveyed hospitals possessed emergency resource reserves, but less than half of them had corresponding drug distribution programs. In addition, hospital capacity was affected by economic level and classification of the hospital, suggesting that the importance of local economic development strengthens hospital ability to provide PHE. Early detection and identification of a PHE are amongst the most important objectives for prompt and effective public health response to a PHE [ 12 ] as well as an essential precondition for selecting appropriate prevention and treatment measures. This study showed that most of the hospitals could regularly train medical staff on how to report and identify suspicious PHE and that the institutions possessed surveillance systems to monitor various aspects of abnormity. Approximately half of the respondents could share surveillance information with the local health authorities. There were statistically significant differences among various classification of the respondents, which demonstrated that after the SARS crisis, hospitals at all levels attached high importance to PHE monitoring and early warning system, however, the capacity was affected by the comprehensive strength of hospital. PHE happens suddenly and its incidence rate is relatively low, which leaves most medical staff inexperienced and unprepared [ 11 ]. Therefore, it is important that hospitals develop emergency plans for PHE treatment programs. In this survey, more than half of respondents showed that their physicians were aware of current PHE protocols. Most hospitals had transfer and treating procedures for infectious diseases, including SARS, influenza, and infectious diarrhea, but less held these procedures for biochemical incidents, leakage of nuclear, and terrorist attacks. Because they are easily used as biological terrorist attacks materials [ 20 ], therefore, the prevention and control of these emergencies become very important. Our statistical analyses showed that tertiary-grade, teaching and TCM-WM hospitals performed better on medical treatment procedures preparedness, which might reflect the fact that different types of hospitals have different functions and mission in the community, however, for this capacity, the statistical significance among different regions showed the important role that economic factor plays. Hospital laboratories not only have the task of clinical diagnosis, but take some responsibility in the surveillance of public health [ 13 , 14 ]. Therefore, laboratory information plays an important role in detection of the PHE [ 13 , 15 ]. Detecting PHE related pathogen/etiology can not only confirm clinical diagnosis, but also identify newly emerging infectious diseases [ 15 , 21 ]. The presence of SARS in China in 2003, and the slow response to its emergence, revealed that China's public health laboratory systems were weak [ 13 ]. This survey indicated that many of the hospitals did not report adequate laboratory diagnostic capacities. Although hospital laboratory regulations seemed relatively good, only one-third of hospital laboratories had programs for dealing with suspicious samples collecting, disposal and delivery. Only half of the surveyed hospitals could detect food-borne pathogens, including cholera vibrio, infectious diarrhea, staphylococcus and salmonella. Few hospitals had the capacity to detect the airborne pathogens, including brucella, influenza virus, anthrax bacteria, the H5N1 avian flu virus, plague bacillus, meningitis B virus, SARS virus and other pathogens. Hospitals with admitting SARS patients performed better showed the importance of the experience of SARS disposal. Prior to the 2003 SARS crisis, China had not experiences a large-scale PHE outbreak for some time. As PHE is a high-risk event with little probability [ 22 ], medical staff often possess limited awareness of appropriate response and this contributed to the under-detection of SARS nosocomial infections in 2003 [ 23 ]. When PHE occurs, hospital medical staff are usually the first responders and information providers, therefore, education and training are key measures to enhance PHE response [ 24 ]. Our survey suggested that after SARS crisis, most hospitals re-evaluated the importance of medical staff training for PHE. The majority of respondents offered training programs to their related medical staff. However, the effectiveness of these training programs needs to be periodically evaluated. PHE can cause psychological as well as physical problems for the public and medical staff attending to victims [ 9 , 25 ]. In a public health crisis or emergency, effective risk communication can help people cope, make decisions, and return their lives to normal. Crisis communication, as an important part of a PHE response [ 8 ], is key to ensuring complete, transparent and prompt information exchange, and to help hospitals make timely responses and reduce the serious consequences [ 26 ]. The results of this survey revealed that medical staff in 12.1% of the hospitals underwent training for evaluation of PHE-related stress and only one-third of respondents had specific programs and spokespersons for communicating critical messages and information to the media, public, governments and stakeholders. These results indicated that most of the surveyed hospitals do not understand the importance of psychological care in a PHE emergency, do not have the resources to deal with it, or presume that it is not their place to do so. Indeed, this capacity evaluation revealed that when a PHE occurred, most hospitals' response plans focused on physiological medical treatment, but health education, psychological counseling, and crisis communication plans were rare. However, for this capacity, the statistical significance among different regions and levels showed the important role that economic factor and comprehensive level play. Limitations The study has several limitations. First of all, the surveyed hospitals were restricted to four city and provinces, even some types of hospitals were rare (the number of the surveyed community health center and emergency center was just one, respectively), therefore, the results may not fully represent the PHE capacity of all hospitals in China. Secondly, because of self-report method there may be a respondent reporting bias. The inclusion of official documents from respective Health Bureaus, for example, may have encouraged respondents to complete survey but have also been interpreted as an official assessment of capacity leading some hospital representatives to overestimate PHE capacity. Thirdly, only quantitative data were collected to measure certain capacities of PHE preparedness. Most questions required a "yes" "no" or "unknown" answer which restricts the collated data to these three categories. Finally, this data set is not complete as some hospitals did not respond and others had to be excluded on the basis of incomplete answers or for ineligibility for hospital classification. To a certain extent, this loss of respondents caused a loss of information. Limitations The study has several limitations. First of all, the surveyed hospitals were restricted to four city and provinces, even some types of hospitals were rare (the number of the surveyed community health center and emergency center was just one, respectively), therefore, the results may not fully represent the PHE capacity of all hospitals in China. Secondly, because of self-report method there may be a respondent reporting bias. The inclusion of official documents from respective Health Bureaus, for example, may have encouraged respondents to complete survey but have also been interpreted as an official assessment of capacity leading some hospital representatives to overestimate PHE capacity. Thirdly, only quantitative data were collected to measure certain capacities of PHE preparedness. Most questions required a "yes" "no" or "unknown" answer which restricts the collated data to these three categories. Finally, this data set is not complete as some hospitals did not respond and others had to be excluded on the basis of incomplete answers or for ineligibility for hospital classification. To a certain extent, this loss of respondents caused a loss of information. Conclusion After several years of construction and development, the capability of hospitals in China to deal with PHE, in particular infectious diseases control, has improved greatly [ 3 , 4 ]. Nevertheless, this research suggests that China has more progress to make before PHE preparedness is satisfactory. To enhance hospital preparation for dealing with PHE, governments at all levels should increase investment in the construction of infrastructure to create and sustain appropriate PHE capacity. On the other hand, hospitals at all levels should enhance their management, including updating and revising of emergency plans; strengthening communication and cooperation with other local agencies; enhancing the capacity of abnormity monitoring and laboratory diagnostic capability for infectious diseases; improving the treatment program for various PHE scenarios; and strengthening psychological intervention and risk communication capabilities. Finally PHE preparedness in relation to terrorism caused by nuclear radiation and bio-chemical substance was low in this study and should be further assessed for areas of need and improvement. Appendix Abnormity: Abnormity means the rapid increase of emergency room patients with acute asthma, flu, fever of unknown causes. Hospital classification: According to "the hospital classification system" of the Ministry of Health of People's Republic of China, all hospitals in China are classified into primary, secondary, and tertiary hospitals based on their functions in providing medical care, medical education, and conducting medical research. A secondary hospital is defined as a regional hospital that provides comprehensive medical care, medical education, and medical research for the region. A tertiary hospital is defined as cross-regional, providing comprehensive and specialized medical care with a high level of medical education and research functions. Secondary and tertiary hospitals are further classified into subgroups: Grade A, Grade B, and Grade C according to their service levels, size, medical technology, medical equipment, and management and medical quality [ 27 ]. Community health center: community health center is a kind of primary health care delivery in China, most of which are transferred from secondary grade hospitals, and provide preventive, curative care, maternal and child care, rehabilitation and health education to local inhabitants by general practitioners, community nurses and public health workers. Medical emergency center: medical emergency center (First Aid Station) is a kind of emergency health care delivery in China, which provide emergency care, first aid, monitoring and treatment for all those patients with pre-hospital emergencies. Competing interests The authors declare that they have no competing interests. Authors' contributions JSH designed the study and developed the tools. HZH participated in design of the study and development of the tools, and supervised the data collection and data entry. XML performed data checkup, data analysis and drafted the manuscript. All authors participated in discussion, revision and approved of the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We appreciate all 400 hospitals who participated in this survey. Specifically we would extend our thanks to the following persons who offered great assistants in the process of data collection, data analysis, and manuscript drafting. They are Peng Lv (Beijing); Lixin Ma (Shandong); Faqing Chen (Guangxi); and Wenli Pan (Hainan).

PMC

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171460/

Steps in the selection of protective clothing materials

Publisher Summary Technological advances in the field of technical or high performance textiles have made it possible to engineer materials designed to meet specific needs. However, there is no ideal fabric that will provide protection against all hazards. Careful selection of appropriate textiles is crucial for the performance, use, care, and maintenance of protective clothing. Broadly, a clear understanding of the work environment and performance requirements, as well as knowledge of the textiles is essential for the decision making process. According to the OSHA Technical Manual, the greater the level of chemical protective clothing, the greater are the associated risks. For any given situation, equipment and clothing should be selected that provide an adequate level of protection. Overprotection as well as under-protection can be hazardous and should be avoided. In addition to protection, the selection of textiles takes into consideration factors such as the impact of protective clothing on job performance; comfort; durability; availability, and cost; use, care, and maintenance, and cultural factors. To select protective clothing materials, relevant factors have to be identified and prioritized in a multi-step process. This chapter presents a model of the decision-making process and provides some examples of the level of detail necessary in each step. It also describes an online system as a case study to illustrate how technology can be used to assist in the selection of textile materials for protective clothing. 4.1 Introduction Technological advances in the field of technical or high performance textiles have made it possible to engineer materials designed to meet specific needs. However, there is no 'ideal' fabric that will provide protection against all hazards. Careful selection of appropriate textiles is crucial for the performance, use, care, and maintenance of protective clothing. Broadly, a clear understanding of the work environment and performance requirements as well as knowledge of the textiles, is essential for the decision making process. According to the OSHA Technical Manual, 'In general, the greater the level of chemical protective clothing, the greater the associated risks. For any given situation, equipment and clothing should be selected that provide an adequate level of protection. Overprotection as well as under-protection can be hazardous and should be avoided'. 1 The above statements are also applicable to other types of protective clothing. Heat, physical and psychological stress, as well as reduced dexterity and mobility, are examples of additional hazards that may be a result of the use of protective clothing. In addition to protection, the selection of textiles takes into consideration factors such as the impact of protective clothing on job performance; comfort; durability; availability and cost; use, care, and maintenance, and cultural factors. To select protective clothing materials, relevant factors have to be identified and prioritized in a multi-step process. It is imperative that such a holistic approach be taken while selecting materials for protective clothing. Basing the decision on just the performance properties of the material may result in low user acceptance. For example, a glove that protects a surgeon may be rejected if it hinders the surgeon's ability to operate effectively. This chapter presents a model of the decision-making process and gives some examples of the level of detail necessary in each step. In addition, it describes an online system as a case study to illustrate how technology can be used to assist in the selection of textile materials for protective clothing. The scope of this chapter is limited to the selection of protective clothing materials and not the entire assembly. It is important to note that while careful selection of suitable materials is crucial for the performance of the clothing ensemble, testing of just the materials is not sufficient to determine the performance of the ensemble, especially for multi-component and fully encapsulating suits. Part II includes the performance of clothing ensembles for selected end uses. The selection of textile materials requires a step-by-step approach in which the potential hazard is clearly defined, and the material is selected in accordance with the existing standards or guidelines. If no standards or guidelines are available, relevant test methods must be identified and used. This initial screening, based on protection provided by the material against potential hazard, is used to narrow the choices. Factors other than protection are then considered to make the final selection. Figure 4.1 provides a model of the selection process discussed in this chapter. 4.1 Recommended steps in the selection of textiles for protective clothing. 4.2 Assess hazards The hazards, as well as a scenario that includes the work environment, are used to define potential risks to the individual. Hazards and scenarios to assist in the selection of materials are very well defined in some sectors, but in other sectors there may not be such well defined scenarios. In general, well defined risk scenarios and performance specifications based on the risks are available for sectors where the use of protective clothing is crucial for the safety of the individuals. For example, selection of protective clothing for firefighters and police is based upon well developed scenarios and requirements. With increased risk of terror attacks, the work scenarios for police and firefighters have changed in countries such as the United States, and the new risks impact the guidelines, standards, and selection of protective clothing materials. These new risks may also require the development of new materials that provide additional protection to such individuals. With the change in work scenario, firefighters and other professionals who are now categorized as 'first responders' need protection against other hazards. The United States Department of Homeland Security has adopted personal protective equipment (PPE) standards developed by the National Institute for Occupational Safety and Health (NIOSH) and the National Fire Protection Association (NFPA) to protect first responders against chemical, biological, radiological, nuclear, and explosive (CBRNE) threats. In case of serious new threats, decisions regarding protective clothing may have to be made in a very short time frame. For example, decisions had to be made immediately regarding selection of appropriate personal protective equipment for protection against viruses such as severe acute respiratory syndrome (SARS) and anthrax. In those two scenarios, assessing the hazards was the crucial first step in the selection of the protective clothing. 4.2.1 Type of hazard (s) The types of hazards are broadly classified as chemical, biological, physical/mechanical, radiological, and flame and thermal. This classification is used by national and international standards organizations as a framework for organizing their committees and for related publications regarding terminology, standards, and performance specifications. 2 , 3 , 4 , 5 , 6 Limited information is available regarding protective clothing against radiological hazards due to the nature of the hazard. The current approach is to incorporate detecting devices to warn individuals of the hazard and thus prevent them from being exposed. The ASTM F23 Committee on Protective Clothing has recently established the F23.70 Radiological Hazards subcommittee to address issues related to radiological hazards. Factors such as composition and physical form of the hazard are used to further categorize each type of hazard. Given below are the common sub-categories for each of the main types of hazards. Chemical hazards are categorized by the chemical composition, physical form, and toxicity. They are commonly identified by the common name, the Chemical Abstract Registry Service (CAS) number, and the chemical class to which they belong. The CAS numbers are numerical unique identifiers assigned to each substance. Chemicals from the same class, in general, have similar chemical composition. Thus, some commonly used chemicals from the various classes are used to determine the performance of materials against a broad range of chemicals. In cases such as pesticides where mixtures are used, it is recommended that the performance be measured against a representative mixture, rather than determining the performance based on a broad range of chemicals. The chemicals have different physical forms – solids, liquids, aerosols, and gases. The tests used to determine the performance are often determined by the physical form and the severity of the hazard. Additional information is included in section 4.4.1 . Biological hazards are divided by the type of microorganism and the mode of contamination. In the medical field, the primary potential risk to the individual is exposure to hazardous bacteria and viruses through contact with contaminated blood and other potentially hazardous body fluids. Surrogate microbes are commonly used to assess the penetration of extremely hazardous substances such as Hepatitis (B and C) and Human Immunodeficiency Viruses. The other risk is exposure to hazardous airborne pathogens such as influenza and the SARS viruses. Airborne pathogens may pose a risk to the general public, in addition to medical personnel. The hazard may be due to natural causes or as a result of bio-terrorism. As the use of biological agents poses a threat in wars, protective clothing materials are also used to protect military personnel. Physical hazards, categorized by the type of contact of the object with the material, can be broadly divided into cut, ballistic, puncture, and abrasion. Cut injuries occur in work environments where individuals work with sharp, high-speed objects such as chainsaws. In most cases, resistant materials are used to protect the hands and legs. Ballistic protection is required to protect individuals from bullets. Bullets may also cause blunt trauma injury by deforming the body armor. Thus, body armor designed to protect individuals is evaluated for ballistic protection as well as the ability to prevent blunt trauma injury. Puncture or stab resistance is required to protect individuals from sharp objects such as knives, as well as comparatively blunt objects commonly used by inmates in correctional facilities. In addition to the above mentioned hazards, protection against abrasion is also used as a criterion for selecting the material. Protection against multiple physical hazards is commonly required for individuals in the military, police force or correctional institutions. Body armor is commonly used to provide protection against physical hazards. Puncture resistance tests are conducted on numerous fabrics that provide protection from hazards such as radiological and biological protection, as the individuals may be exposed to other hazards, if the material is punctured by a sharp object. Flame and thermal hazards are grouped by the source of flame or heat. The most common types of hazards are exposure to open flame, radiant heat, and molten material. Individuals such as firefighters are susceptible to the potential of burn as a result of exposure to open flame and radiant heat. Individuals working in industries where they are exposed to molten metal need materials which reduce burn injuries by shedding molten metal into the floor. 4.2.2 Severity of the hazard The toxicity of the hazardous material, duration of exposure, and level of exposure are used to determine the severity of the hazard. All three factors are interrelated and should be considered as a set. For all categories, toxicity of the materials and the by-products are the primary concern. Highly toxic substances or hazardous conditions have to be handled with extreme care in all categories, regardless of the duration and level of exposure. The level of exposure and the duration of exposure are considered with reference to the toxicity. For example, exposure to even a small amount of a chemical agent for a short duration may be far more harmful than exposure to a Class IV pesticide while spraying for an entire work day. For many end uses, PPE is rated based on the severity of the hazard. For example, body armor classification developed by the National Institute of Justice (NIJ) is used for the selection of the body armor based on the potential workplace hazard. European Union certification for PPE uses Type I-VI categories that are based on very broad hazard scenarios. Although there are ratings or classifications for the various types of hazards, there is no universal system to rate the severity of all hazards. In the future, there may be a system to assess the severity and rate it regardless of the type of hazard. For example, the extremely hazardous category may be defined to include all hazard scenarios that are life threatening in very small amounts. Thus, the extremely hazardous category may include SARS and anthrax from the biological category, nerve gas from chemical, ballistic from physical, and radioactive materials from radiological hazards. This type of rating would help in the testing and selection of PPE materials. For example, when the severity of hazard is extreme, stringent rules would apply for the handling and testing of fabric, and thorough testing of the whole garment ensemble would be necessary. 4.2.1 Type of hazard (s) The types of hazards are broadly classified as chemical, biological, physical/mechanical, radiological, and flame and thermal. This classification is used by national and international standards organizations as a framework for organizing their committees and for related publications regarding terminology, standards, and performance specifications. 2 , 3 , 4 , 5 , 6 Limited information is available regarding protective clothing against radiological hazards due to the nature of the hazard. The current approach is to incorporate detecting devices to warn individuals of the hazard and thus prevent them from being exposed. The ASTM F23 Committee on Protective Clothing has recently established the F23.70 Radiological Hazards subcommittee to address issues related to radiological hazards. Factors such as composition and physical form of the hazard are used to further categorize each type of hazard. Given below are the common sub-categories for each of the main types of hazards. Chemical hazards are categorized by the chemical composition, physical form, and toxicity. They are commonly identified by the common name, the Chemical Abstract Registry Service (CAS) number, and the chemical class to which they belong. The CAS numbers are numerical unique identifiers assigned to each substance. Chemicals from the same class, in general, have similar chemical composition. Thus, some commonly used chemicals from the various classes are used to determine the performance of materials against a broad range of chemicals. In cases such as pesticides where mixtures are used, it is recommended that the performance be measured against a representative mixture, rather than determining the performance based on a broad range of chemicals. The chemicals have different physical forms – solids, liquids, aerosols, and gases. The tests used to determine the performance are often determined by the physical form and the severity of the hazard. Additional information is included in section 4.4.1 . Biological hazards are divided by the type of microorganism and the mode of contamination. In the medical field, the primary potential risk to the individual is exposure to hazardous bacteria and viruses through contact with contaminated blood and other potentially hazardous body fluids. Surrogate microbes are commonly used to assess the penetration of extremely hazardous substances such as Hepatitis (B and C) and Human Immunodeficiency Viruses. The other risk is exposure to hazardous airborne pathogens such as influenza and the SARS viruses. Airborne pathogens may pose a risk to the general public, in addition to medical personnel. The hazard may be due to natural causes or as a result of bio-terrorism. As the use of biological agents poses a threat in wars, protective clothing materials are also used to protect military personnel. Physical hazards, categorized by the type of contact of the object with the material, can be broadly divided into cut, ballistic, puncture, and abrasion. Cut injuries occur in work environments where individuals work with sharp, high-speed objects such as chainsaws. In most cases, resistant materials are used to protect the hands and legs. Ballistic protection is required to protect individuals from bullets. Bullets may also cause blunt trauma injury by deforming the body armor. Thus, body armor designed to protect individuals is evaluated for ballistic protection as well as the ability to prevent blunt trauma injury. Puncture or stab resistance is required to protect individuals from sharp objects such as knives, as well as comparatively blunt objects commonly used by inmates in correctional facilities. In addition to the above mentioned hazards, protection against abrasion is also used as a criterion for selecting the material. Protection against multiple physical hazards is commonly required for individuals in the military, police force or correctional institutions. Body armor is commonly used to provide protection against physical hazards. Puncture resistance tests are conducted on numerous fabrics that provide protection from hazards such as radiological and biological protection, as the individuals may be exposed to other hazards, if the material is punctured by a sharp object. Flame and thermal hazards are grouped by the source of flame or heat. The most common types of hazards are exposure to open flame, radiant heat, and molten material. Individuals such as firefighters are susceptible to the potential of burn as a result of exposure to open flame and radiant heat. Individuals working in industries where they are exposed to molten metal need materials which reduce burn injuries by shedding molten metal into the floor. 4.2.2 Severity of the hazard The toxicity of the hazardous material, duration of exposure, and level of exposure are used to determine the severity of the hazard. All three factors are interrelated and should be considered as a set. For all categories, toxicity of the materials and the by-products are the primary concern. Highly toxic substances or hazardous conditions have to be handled with extreme care in all categories, regardless of the duration and level of exposure. The level of exposure and the duration of exposure are considered with reference to the toxicity. For example, exposure to even a small amount of a chemical agent for a short duration may be far more harmful than exposure to a Class IV pesticide while spraying for an entire work day. For many end uses, PPE is rated based on the severity of the hazard. For example, body armor classification developed by the National Institute of Justice (NIJ) is used for the selection of the body armor based on the potential workplace hazard. European Union certification for PPE uses Type I-VI categories that are based on very broad hazard scenarios. Although there are ratings or classifications for the various types of hazards, there is no universal system to rate the severity of all hazards. In the future, there may be a system to assess the severity and rate it regardless of the type of hazard. For example, the extremely hazardous category may be defined to include all hazard scenarios that are life threatening in very small amounts. Thus, the extremely hazardous category may include SARS and anthrax from the biological category, nerve gas from chemical, ballistic from physical, and radioactive materials from radiological hazards. This type of rating would help in the testing and selection of PPE materials. For example, when the severity of hazard is extreme, stringent rules would apply for the handling and testing of fabric, and thorough testing of the whole garment ensemble would be necessary. 4.3 Identify relevant standards, specifications or guidelines Regulatory standards, performance specifications, guidelines, and test standards established by national and international standards organizations, governmental agencies, as well as associations for various professions are available to determine performance and assist in the selection of the textile materials. 2 , 3 , 4 , 5 , 6 Regulatory standards are often mandated by governmental agencies as well as other organizations that have jurisdiction for a particular group. Knowledge of the performance specifications and standards is essential. Often companies that manufacture materials and protective clothing may have to comply with more than one set of requirements, as these may vary by geographic locations. For example, due to differences in minimum specifications as well as the required test methods, garments may meet the requirements in the United States but not in the European Union or vice versa. Thus, it is not unusual to find different products being marketed by the same manufacturer in different countries. The specificity of the regulations and standards varies considerably. They range from very clearly defined selection, use, care, and maintenance criteria for personal protective equipment (PPE) to requirements in which the employer is responsible for providing 'appropriate protective clothing.' In some cases there is either no performance standard or guideline, or they are in the process of being established. Given below are examples of standards and specifications for different scenarios and the impact they have on the selection process for the particular hazard. 4.3.1 Standards with well defined tests standards and performance specifications Protection of Industrial Personnel against Flash Fire: The NFPA 2112 Standard for Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire and NFPA 2113 Standard on Selection, Care, Use, and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire are standards established by NFPA International. 7 These standards have clearly defined test methods and minimum requirements that assist in the selection of protective clothing materials. The manufacturers of the materials often provide data to guide product selection. Well defined standards, performance specifications, and guidelines that specify minimum performance requirements are very beneficial in the selection process, as they allow the individual responsible for selection to be able to compare the performance characteristics as well as other factors that play an important role in choosing the best material for the end use. 4.3.2 Standards with no tests standard and performance specification defined The PPE standard mandated by the U.S. Occupational Safety and Health Administration (OSHA) for occupational exposure to blood and other potentially infectious materials is applicable to a wide range of scenarios. According to the standard, the employer is required to provide 'appropriate' personal protective equipment (PPE) for the employees. 8 However, the test method and the minimum requirements are not specified. It is thus the responsibility of the employer to determine the hazard and select appropriate PPE to protect the employees at work. As the regulations do not provide guidelines on the types of test or material to be used, it is up to the manufacturer to determine the test method to evaluate the performance of their materials and up to the employers to select the materials suitable for their end uses. Standards with no test method and minimum requirements pose a major problem in the selection of suitable materials, often resulting in over or under protection of the individuals. 4.3.3 Standard with recommendations based on garment design and not performance Requirements for PPE for pesticide applicators in the United States are currently based on garment design and not on the performance of the garments. Test results show that the penetration of pesticides through garment materials in the same category varies considerably. Thus, recommendations such as use of long sleeve shirts and pants, commonly seen as part of the safety instruction on a pesticide label, are not sufficient. 9 Basing the recommendations on just garment design may result in over or under protection of the individuals. A task force has been formed as part of ASTM-F23 Committee for Protective Clothing to develop performance specifications for protective clothing during pesticide application. 4.3.1 Standards with well defined tests standards and performance specifications Protection of Industrial Personnel against Flash Fire: The NFPA 2112 Standard for Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire and NFPA 2113 Standard on Selection, Care, Use, and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire are standards established by NFPA International. 7 These standards have clearly defined test methods and minimum requirements that assist in the selection of protective clothing materials. The manufacturers of the materials often provide data to guide product selection. Well defined standards, performance specifications, and guidelines that specify minimum performance requirements are very beneficial in the selection process, as they allow the individual responsible for selection to be able to compare the performance characteristics as well as other factors that play an important role in choosing the best material for the end use. 4.3.2 Standards with no tests standard and performance specification defined The PPE standard mandated by the U.S. Occupational Safety and Health Administration (OSHA) for occupational exposure to blood and other potentially infectious materials is applicable to a wide range of scenarios. According to the standard, the employer is required to provide 'appropriate' personal protective equipment (PPE) for the employees. 8 However, the test method and the minimum requirements are not specified. It is thus the responsibility of the employer to determine the hazard and select appropriate PPE to protect the employees at work. As the regulations do not provide guidelines on the types of test or material to be used, it is up to the manufacturer to determine the test method to evaluate the performance of their materials and up to the employers to select the materials suitable for their end uses. Standards with no test method and minimum requirements pose a major problem in the selection of suitable materials, often resulting in over or under protection of the individuals. 4.3.3 Standard with recommendations based on garment design and not performance Requirements for PPE for pesticide applicators in the United States are currently based on garment design and not on the performance of the garments. Test results show that the penetration of pesticides through garment materials in the same category varies considerably. Thus, recommendations such as use of long sleeve shirts and pants, commonly seen as part of the safety instruction on a pesticide label, are not sufficient. 9 Basing the recommendations on just garment design may result in over or under protection of the individuals. A task force has been formed as part of ASTM-F23 Committee for Protective Clothing to develop performance specifications for protective clothing during pesticide application. 4.4 Screen materials based on protection performance The specificity of the standards, performance specifications, and guidelines determines the tests required to assess protection performance of the materials. For end uses where the hazard and minimum requirements are clearly defined, test data is usually available through fabric and garment manufacturers. Compliance of the material and garments with relevant standards is also commonly posted on the manufacturer's website. 10 , 11 , 12 , 13 In situations where regulations do not specify the test methods, or there is no performance specification, the individual responsible for selecting the protective clothing has the added responsibility of determining the scenario and test method suitable for assessing the performance of the materials. Often the appropriate test method will have to be selected. A clear understanding of the exposure scenario that includes type, level, and duration of exposure, and environmental conditions is essential. The information should be used to identify the standard that represents the scenario of the proposed end use. Often the test methods allow for testing at different levels of severity and/or duration of exposure. Defining the potential scenario is helpful in selecting the appropriate parameters for testing. In some cases, the method may have to be modified if the test parameters and procedures are not adequate to test for the potential scenarios. As there are many probable scenarios, several test standards have been developed for each of the broad hazard categories. It is not unusual to find two or more test methods developed by different groups to measure the same end use performance. The results from the test methods may be similar or may vary considerably depending on the scenario or the basis for the test method. For example, three national and/or international standards are used to measure the penetration of liquid pesticides. Comparison of the three methods showed major differences in the pesticide penetration values for the same materials. 14 This was due to the fact that the scenario or the basis of the test method ranged from simulation of accidental spill to fine spray. Given below is information regarding selection of materials based on protection performance against the broad categories of hazards. The categories and examples used are not all inclusive as there are numerous scenarios within each of the categories. The purpose of this detail is to illuminate the level of complex analysis needed. 4.4.1 Chemical protection performance The route of entry into the body determines the type of protection required. For example, face masks or respirators with different filters are used to protect individuals against exposure due to inhalation. Chemical protective clothing is used to protect against dermal exposure. Chemical protective clothing materials are broadly divided into woven; nonwoven; laminated to microporous or hydrophilic membranes; coated, bonded, or laminated with plastic films or rubber; and films, sheets, or moulded plastic or rubber. The performance of the chemical protective materials varies considerably based on the air permeability, chemical composition, and material characteristics. Penetration, permeation, and/or degradation of the materials are measured to determine the chemical resistance. In general, the physical form of the chemical that poses a potential risk and the type of material determine whether penetration, permeation, and/or degradation tests have to be conducted. Permeation is 'the process by which a chemical moves through a protective clothing material on a molecular level. Permeation involves (i) sorption of molecules of the chemical into the contacted (challenge side) surface of the material; (ii) diffusion of the sorbed molecules in the material, and (iii) desorption of the molecules from the opposite surface of the material'. 15 The permeation rate and breakthrough time are important for measuring permeation. Permeation tests are conducted to determine the performance of non-porous materials such as monolithic films against volatile and non-volatile liquids and gases. It is not used to measure the performance of woven or knitted fabrics. For such porous materials, penetration tests are used to measure performance. Penetration is defined as the 'process by which a solid, liquid, or gas moves through closures, seams, interstices, and pinholes or other imperfections on a non-molecular level, in a protective clothing material or item.' 15 Penetration through a material can be measured by several national and international standards. The selection of a suitable standard depends on the exposure scenario. For example, there are three national and international test methods to measure the penetration of pesticide through textiles. 14 Degradation is defined as 'a deleterious change in one or more properties of a material'. 15 Materials that have degraded due to chemical exposure, use or exposure to environmental conditions such as sunlight should be discarded, as these affect the fabric performance. Given below are the ASTM, ISO and EN test standards used to evaluate performance of materials against chemical hazards. In addition to the test methods stated below, national and international standards are also used to measure the performance of whole body ensemble. • ASTM F1186-03 Standard Classification System for Chemicals According to Functional Groups. • ASTM F1001-99a Standard Guide for Selection of Chemicals to Evaluate Protective Clothing Materials. • ASTM F739-99a Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Continuous Contact. • ASTM F1383-99a Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Intermittent Contact. • ASTM F1407-99a Standard Test Method for Resistance of Chemical Protective Clothing Materials to Liquid Permeation-Permeation Cup Method. • ASTM F1194-99 Standard Guide for Documenting the Results of Chemical Permeation Testing of Materials Used in Protective Clothing. • ASTM F903-03 (2004) Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Liquids. • ASTM F2053-00 Standard Guide for Documenting the Results of Airborne Particle Penetration Testing of Protective Clothing Materials. • ASTM F2130-01 Standard Test Method for Measuring Repellency, Retention, and Penetration of Liquid Pesticide Formulation through Protective Clothing Materials. • EN 943-1:2002 Protective clothing against liquid and gaseous chemicals, including liquid aerosols and solid particles – Part 1: Performance requirements. • EN 467:1995 Protective Clothing – Protection against liquid chemicals – Performance requirements for garments providing protection to parts of the body. • EN374-3:1994 Protective gloves against chemicals and micro-organisms – Part 3: Determination of resistance to permeation by chemicals. • EN ISO 6529 Protective clothing – Protection against chemicals – Determination of resistance of protective clothing materials to permeation by liquids and gases. • ISO 6530 (1990) Protective clothing – Protection against liquid chemicals – Determination of resistance of materials to penetration by liquids. • ISO 13994 (1998) Clothing for protection against liquid chemicals – Determination of the resistance of protective clothing materials to penetration by liquids under pressure. • ISO 17491(2002) Protective clothing – Protection against gaseous and liquid chemicals – Determination of resistance of protective clothing to penetration by liquids and gases. • ISO 22608 (2004) Protective clothing – Protection against liquid chemicals – Measurement of repellency, retention, and penetration of liquid pesticide formulations through protective clothing materials. Permeation data is generally available for commonly used chemical protective clothing materials. Breakthrough time, the time taken by the chemical to pass through the material, is commonly used to measure permeation. In addition, permeation rate, which is the rate at which the chemical moves through the material, is also recorded. Chemical resistance data can be obtained from published guidelines as well as from manufacturers of the chemical protective clothing. 'Guidelines for the Selection of Chemical Protective Clothing' and 'Quick Selection Guide to Chemical Protective Clothing' 15 , 16 include color-coded recommendations for sixteen commonly used barrier materials. The color codes and corresponding breakthrough times used in the guidelines are green for breakthrough detection time of >4 hours of continuous contact (a >8 is used when the breakthrough is greater than eight hours); yellow with breakthrough detection time between 1–4 hours; red with breakthrough times of less than one hour. White is used when no data is available for the material. Testing the material against the challenge liquid is recommended prior to use. Contact between the material and the chemical may result in degradation of the material or the breakthrough may be a result of the degradation of the material. It is important to note that the data reported in the guidelines is a compilation of published and unpublished permeation test data obtained from various sources. The authors of the publication state that at least 90% of the tests were conducted using ASTM F739-99, Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Continuous Contact. 15 The majority of the data for generic materials is a summary of results from different sources. According to the OSHA technical manual, 'The major limitation for these guidelines are their presentation of recommendations by generic material class. Numerous test results have shown that similar materials from different manufacturers may give widely different performance. That is to say manufacturer A's butyl rubber glove may protect against chemical X, but a butyl glove made by manufacturer B may not'. 1 A majority of manufacturers test the materials against a battery of liquid and gaseous chemicals, which represent a wide range of commonly used chemicals in different classes. Often the recommended chemicals published by national and international standards organizations are used by the manufacturers as a common group of chemicals. The recommended list of challenge chemicals as well as the chemical class that is published by ASTM International is included in Table 4.1 . 17 The performance data, based on tests conducted by independent laboratories, is publicly available through a manufacturer's website. This data is specific to the material manufactured by the company, and so may be different from that obtained from the above mentioned publications. Often information regarding compliance with minimum requirements is also provided by the manufacturers. Table 4.1 List of Recommended Gaseous and Liquid Test Chemicals (extracted with permission, from F 1001-99a Standard Guide for Selection of Chemicals to Evaluate Protective Clothing Materials, copyrig ht ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 19462.) Chemical Class Gaseous test chemicals Ammonia Strong base 1,3-Butadiene Olefin Chlorine Inorganic gas Ethyl oxide Oxygen heterocyclic gas Hydrogen chloride Acid gas Methyl chloride Chlorinated hydrocarbon Liquid test chemicals Acetone Ketone Acetonitrile Nitrile Carbon disulfide Sulfur-containing organic Dichloromethane Chlorinated hydrocarbon Diethylamine Amine Dimethylformamide Amide Ethyl acetate Ester n-Hexane Aliphatic hydrocarbon Methanol Alcohol Nitrobenzene Nitrogen-containing organic Sodium hydroxide Inorganic base Sulfuric acid Inorganic acid Tetrachloroethylene Chlorinated hydrocarbon Tetrahydrofuran Oxygen heterocyclic Toluene Aromatic hydrocarbon However, judicious use of such data is important, as there are numerous protective clothing and chemical combinations. The data from the recommended list should not be a basis for selecting material for protection against chemicals not represented on the list or against a mixture of chemicals. To the extent possible, the selection of textile materials should be based on test data for the potential challenge. Materials manufactured to provide protection against specific chemical hazards require the use of challenge liquids that represent the potential risk. For example, a pharmacist working with drugs for chemotherapy needs protective clothing and accessories that protect the individual from those drugs. A garment that provides general protection against commonly used chemicals may not be recommended for this use. Special testing would have to be conducted for the proposed end use. 4.4.2 Biological protection performance The selection of protective clothing that provides protection against biological hazards depends on the proposed end use of the clothing. Some of the common end uses are to protect the patient as well as medical professionals who are exposed to blood-borne and other pathogens. Until recently, the majority of biological hazard test methods and guidelines focused on applications in the medical field and for military personnel. With the current bio-terrorism threats, biological protection is important for 'first responders', workers with the potential of exposure to hazardous materials, and the general public. Biological hazards are commonly bacteria, viruses, or toxins that exist as very fine air or liquid-borne particles. Given below are examples of ASTM International and ISO standards that illustrate the specificity of the garments. • F1670-03 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Synthetic Blood. • F1671-03 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using Phi-X174 Bacteriophage Penetration as a Test System. • F1819-04 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Synthetic Blood Using a Mechanical Pressure Technique. • F1862-00a Standard Test Method for Resistance of Medical Face Masks to Penetration by Synthetic Blood (Horizontal Projection of Fixed Volume at a Known Velocity). • F2100-04 Standard Specification for Performance of Materials Used in Medical Face Masks. • F2101-01 Standard Test Method for Evaluating the Bacterial Filtration Efficiency (BFE) of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus. • F2299-03 Standard Test Method for Determining the Initial Efficiency of Materials Used in Medical Face Masks to Penetration by Particulates Using Latex Spheres. • F1868-02 Standard Test Method for Thermal and Evaporative Resistance of Clothing Materials Using a Sweating Hot Plate. • ISO 16603:2004 Clothing for protection against contact with blood and body fluids – Determination of the resistance of protective clothing materials to penetration by blood and body fluids – Test method using synthetic blood. • ISO 16604:2004 Clothing for protection against contact with blood and body fluids – Determination of resistance of protective clothing materials to penetration by blood-borne pathogens – Test method using Phi-X 174 bacteriophage. 4.4.3 Flame and thermal protection performance The selection of protective clothing that provides protection against heat and burning objects depends on the proposed end use of the clothing. Some of the common applications are to protect individuals from open flames (including flash fires), molten materials, and radiant heat. In general, materials that have inherent flame-resistant properties are used for flame and thermal protection. Often thermal protective apparel requires the use of multi-layered fabric construction, with each layer performing a specific function. Thus performance is measured for individual layers as well as for the whole garment assembly. Protective apparel is worn by firefighters, wildland firefighters, electrical linesmen, racing- and rally-car drivers, emergency services personnel, and many other groups. Standards and performance specifications on selection, use, care and maintenance of flame and thermal clothing, including fabric, are available through national and international standardization bodies such as NFPA International, ISO, OSHA, and ASTM, CEN/TC 162 (European Technical Committee for Protective Clothing). Due to the very specific requirements, there are many specifications for protective clothing used for protection against thermal and flame hazards. Given below are examples of ASTM, EN, and NFPA standards that illustrate the specificity of the garments. • F955-03 Standard Test Method for Evaluating Heat Transfer through Materials for Protective Clothing upon Contact with Molten Substances. • F1002-96 Standard Performance Specification for Protective Clothing for Use by Workers Exposed to Specific Molten Substances and Related Thermal Hazards. • F1060-01 Standard Test Method for Thermal Protective Performance of Materials for Protective Clothing for Hot Surface Contact. • F1358-00 Standard Test Method for Effects of Flame Impingement on Materials Used in Protective Clothing Not Designated Primarily for Flame Resistance. • F1930-00 Standard Test Method for Evaluation of Flame Resistant Clothing for Protection Against Flash Fire Simulations Using an Instrumented Manikin. • F1939-99a Standard Test Method for Radiant Protective Performance of Flame Resistant Clothing Materials. • NFPA 70E Standard for Electrical Safety Requirements for Employee Workplaces. • NFPA 2113 Selection, Care, Use and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire. • NFPA 1971, Standard on Protective Ensemble for Structural Fire Fighting, 2000 edition. • NFPA 1976, Standard on Protective Ensemble for Proximity Fire Fighting, 2000 edition. • NFPA 1977, Standard on Protective Clothing and Equipment for Wildland Fire Fighting, 1998 edition. • NFPA 1991, Standard on Vapor-Protective Ensembles for Hazardous Materials Emergencies, 2000 edition. • NFPA 1992, Standard on Liquid Splash-Protective Ensembles and Clothing for Hazardous Materials Emergencies, 2000 edition. • NFPA 1999, Standard on Protective Clothing for Emergency Medical Operations, 1997 edition. • NFPA 2113, Standard on Selection, Care, Use, and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel Against Flash Fire, 2001 edition. • EN 469: European standard for fire fighters' personal protective equipment. • EN 531: European standard for heat and flame protective clothing for industrial workers. • EN 659: European standard for fire fighters' gloves. • ISO 17492:2003 Clothing for protection against heat and flame – Determination of heat transmission on exposure to both flame and radiant heat. • ISO 2801:1998 Clothing for protection against heat and flame – General recommendations for selection, care and use of protective clothing. • ISO 6942:2002 Protective clothing – Protection against heat and fire – Method of test: Evaluation of materials and material assemblies when exposed to a source of radiant heat. • ISO 8194:1987 Radiation protection – Clothing for protection against radioactive contamination – Design, selection, testing and use. • ISO 9150:1988 Protective clothing – Determination of behaviour of materials on impact of small splashes of molten metal. • ISO 9151:1995 Protective clothing against heat and flame – Determination of heat transmission on exposure to flame. • ISO 9185:1990 Protective clothing – Assessment of resistance of materials to molten metal splash. • ISO 11612:1998 Clothing for protection against heat and flame – Test methods and performance requirements for heat-protective clothing. • ISO 11613:1999 Protective clothing for firefighters – Laboratory test methods and performance requirements. • ISO 12127:1996 Clothing for protection against heat and flame – Determination of contact heat transmission through protective clothing or constituent materials. • ISO 14460:1999 Protective clothing for automobile racing drivers – Protection against heat and flame – Performance requirements and test methods. • ISO 15025:2000 Protective clothing – Protection against heat and flame – Method of test for limited flame spread. • ISO 15383:2001 Protective gloves for firefighters – Laboratory test methods and performance requirements. • ISO 15384:2003 Protective clothing for firefighters – Laboratory test methods and performance requirements for wildland firefighting clothing. • ISO 15538:2001 Protective clothing for firefighters – Laboratory test methods and performance requirements for protective clothing with a reflective outer surface. 4.4.4 Mechanical protection performance Protective clothing designed to provide protection from mechanical hazards ranges from cut protection for chainsaw workers to body amour designed for bullet, stab, puncture, or impact protection for police and military personnel. Ballistics tests and performance standards, developed by the National Institute of Justice (NIJ), are examples of the specialized ballistic tests that are used to determine the performance of the materials (6). Numerous standards are available to measure the cut resistance of materials used to protect individuals from chainsaw cuts. Given below is a list of test standards and performance specifications developed by ASTM International for cut resistance. • F1790-04 Standard Test Method for Measuring Cut Resistance of Materials Used in Protective Clothing. • F1414-99 Standard Test Method for Measurement of Cut Resistance to Chain Saw in Lower Body (Legs) Protective Clothing. • F1458-98 Standard Test Method for Measurement of Cut Resistance to Chain Saw of Foot Protective Devices. • F1818-97(2003) Standard Specification for Foot Protection for Chain Saw Users. • F1897-98 Standard Specification for Leg Protection for Chain Saw Users. • F1342-91(1996)e2 Standard Test Method for Protective Clothing Material Resistance to Puncture. • F1414-99 Standard Test Method for Measurement of Cut Resistance to Chain Saw in Lower Body (Legs) Protective Clothing. • ISO 11393-1(1998) Protective clothing for users of hand-held chain-saws – Part 1: Test rig driven by a flywheel for testing resistance to cutting by a chain-saw. • ISO 11393-2 (1999) Protective clothing for users of hand-held chain-saws – Part 2: Test methods and performance requirements for leg protectors. • ISO 11393-5(2001) Protective clothing for users of hand-held chain-saws – Part 5: Test methods and performance requirements for protective gaiters. • ISO 13995:2000 Protective clothing - Mechanical properties – Test method for the determination of the resistance to puncture and dynamic tearing of materials. • ISO 13996:1999 Protective clothing – Mechanical properties – Determination of resistance to puncture. • ISO 13997:1999 Protective clothing – Mechanical properties – Determination of resistance to cutting by sharp objects. • ISO 13998:2003 Protective clothing – Aprons, trousers and vests protecting against cuts and stabs by hand knives. • ISO 14877:2002 Protective clothing for abrasive blasting operations using granular abrasives. • NIJ Standard-0101.03 – Ballistic Resistance of Police Body Armor. • NIJ Standard-0101.04 – Ballistic Resistance of Personal Body Armor. • NIJ Standard-0115.00 – Stab Resistance of Personal Body Armor. Knowledge of how the fabric provides the protection as well as the limitations of the material is important. Materials may have similar barrier performance; however, the manner in which the protection is provided may vary considerably. One may provide protection due to the inherent fiber properties, whereas another from a finish applied to the fabric. It would be important to obtain additional information on the durability of the finish to determine the level of protection that will be provided over the life of the garment. The durability of the finish may be a limitation for the material. The above discussion provides a sense of the level of complex analysis that must be applied for each type of hazard (chemical, biological, physical, radiological, or thermal). After such an analysis, a list is prepared of materials that meet the minimum protection requirements. This list is then used to select potential materials based on other major factors discussed in the following section. Some manufacturers provide online interactive software to assist in the selection of suitable clothing. DuPont™ SafeSPEC™ 10 is an example of an interactive tool that assists the user with hazard assessment and selection of apparel. As stated in the software, the responses are based on the information provided by the user. Thus, it is crucial for the individual entering the information to provide accurate information. With systems that are manufacturer specific, it is difficult to compare products from different manufacturers. 4.4.1 Chemical protection performance The route of entry into the body determines the type of protection required. For example, face masks or respirators with different filters are used to protect individuals against exposure due to inhalation. Chemical protective clothing is used to protect against dermal exposure. Chemical protective clothing materials are broadly divided into woven; nonwoven; laminated to microporous or hydrophilic membranes; coated, bonded, or laminated with plastic films or rubber; and films, sheets, or moulded plastic or rubber. The performance of the chemical protective materials varies considerably based on the air permeability, chemical composition, and material characteristics. Penetration, permeation, and/or degradation of the materials are measured to determine the chemical resistance. In general, the physical form of the chemical that poses a potential risk and the type of material determine whether penetration, permeation, and/or degradation tests have to be conducted. Permeation is 'the process by which a chemical moves through a protective clothing material on a molecular level. Permeation involves (i) sorption of molecules of the chemical into the contacted (challenge side) surface of the material; (ii) diffusion of the sorbed molecules in the material, and (iii) desorption of the molecules from the opposite surface of the material'. 15 The permeation rate and breakthrough time are important for measuring permeation. Permeation tests are conducted to determine the performance of non-porous materials such as monolithic films against volatile and non-volatile liquids and gases. It is not used to measure the performance of woven or knitted fabrics. For such porous materials, penetration tests are used to measure performance. Penetration is defined as the 'process by which a solid, liquid, or gas moves through closures, seams, interstices, and pinholes or other imperfections on a non-molecular level, in a protective clothing material or item.' 15 Penetration through a material can be measured by several national and international standards. The selection of a suitable standard depends on the exposure scenario. For example, there are three national and international test methods to measure the penetration of pesticide through textiles. 14 Degradation is defined as 'a deleterious change in one or more properties of a material'. 15 Materials that have degraded due to chemical exposure, use or exposure to environmental conditions such as sunlight should be discarded, as these affect the fabric performance. Given below are the ASTM, ISO and EN test standards used to evaluate performance of materials against chemical hazards. In addition to the test methods stated below, national and international standards are also used to measure the performance of whole body ensemble. • ASTM F1186-03 Standard Classification System for Chemicals According to Functional Groups. • ASTM F1001-99a Standard Guide for Selection of Chemicals to Evaluate Protective Clothing Materials. • ASTM F739-99a Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Continuous Contact. • ASTM F1383-99a Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Intermittent Contact. • ASTM F1407-99a Standard Test Method for Resistance of Chemical Protective Clothing Materials to Liquid Permeation-Permeation Cup Method. • ASTM F1194-99 Standard Guide for Documenting the Results of Chemical Permeation Testing of Materials Used in Protective Clothing. • ASTM F903-03 (2004) Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Liquids. • ASTM F2053-00 Standard Guide for Documenting the Results of Airborne Particle Penetration Testing of Protective Clothing Materials. • ASTM F2130-01 Standard Test Method for Measuring Repellency, Retention, and Penetration of Liquid Pesticide Formulation through Protective Clothing Materials. • EN 943-1:2002 Protective clothing against liquid and gaseous chemicals, including liquid aerosols and solid particles – Part 1: Performance requirements. • EN 467:1995 Protective Clothing – Protection against liquid chemicals – Performance requirements for garments providing protection to parts of the body. • EN374-3:1994 Protective gloves against chemicals and micro-organisms – Part 3: Determination of resistance to permeation by chemicals. • EN ISO 6529 Protective clothing – Protection against chemicals – Determination of resistance of protective clothing materials to permeation by liquids and gases. • ISO 6530 (1990) Protective clothing – Protection against liquid chemicals – Determination of resistance of materials to penetration by liquids. • ISO 13994 (1998) Clothing for protection against liquid chemicals – Determination of the resistance of protective clothing materials to penetration by liquids under pressure. • ISO 17491(2002) Protective clothing – Protection against gaseous and liquid chemicals – Determination of resistance of protective clothing to penetration by liquids and gases. • ISO 22608 (2004) Protective clothing – Protection against liquid chemicals – Measurement of repellency, retention, and penetration of liquid pesticide formulations through protective clothing materials. Permeation data is generally available for commonly used chemical protective clothing materials. Breakthrough time, the time taken by the chemical to pass through the material, is commonly used to measure permeation. In addition, permeation rate, which is the rate at which the chemical moves through the material, is also recorded. Chemical resistance data can be obtained from published guidelines as well as from manufacturers of the chemical protective clothing. 'Guidelines for the Selection of Chemical Protective Clothing' and 'Quick Selection Guide to Chemical Protective Clothing' 15 , 16 include color-coded recommendations for sixteen commonly used barrier materials. The color codes and corresponding breakthrough times used in the guidelines are green for breakthrough detection time of >4 hours of continuous contact (a >8 is used when the breakthrough is greater than eight hours); yellow with breakthrough detection time between 1–4 hours; red with breakthrough times of less than one hour. White is used when no data is available for the material. Testing the material against the challenge liquid is recommended prior to use. Contact between the material and the chemical may result in degradation of the material or the breakthrough may be a result of the degradation of the material. It is important to note that the data reported in the guidelines is a compilation of published and unpublished permeation test data obtained from various sources. The authors of the publication state that at least 90% of the tests were conducted using ASTM F739-99, Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Continuous Contact. 15 The majority of the data for generic materials is a summary of results from different sources. According to the OSHA technical manual, 'The major limitation for these guidelines are their presentation of recommendations by generic material class. Numerous test results have shown that similar materials from different manufacturers may give widely different performance. That is to say manufacturer A's butyl rubber glove may protect against chemical X, but a butyl glove made by manufacturer B may not'. 1 A majority of manufacturers test the materials against a battery of liquid and gaseous chemicals, which represent a wide range of commonly used chemicals in different classes. Often the recommended chemicals published by national and international standards organizations are used by the manufacturers as a common group of chemicals. The recommended list of challenge chemicals as well as the chemical class that is published by ASTM International is included in Table 4.1 . 17 The performance data, based on tests conducted by independent laboratories, is publicly available through a manufacturer's website. This data is specific to the material manufactured by the company, and so may be different from that obtained from the above mentioned publications. Often information regarding compliance with minimum requirements is also provided by the manufacturers. Table 4.1 List of Recommended Gaseous and Liquid Test Chemicals (extracted with permission, from F 1001-99a Standard Guide for Selection of Chemicals to Evaluate Protective Clothing Materials, copyrig ht ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 19462.) Chemical Class Gaseous test chemicals Ammonia Strong base 1,3-Butadiene Olefin Chlorine Inorganic gas Ethyl oxide Oxygen heterocyclic gas Hydrogen chloride Acid gas Methyl chloride Chlorinated hydrocarbon Liquid test chemicals Acetone Ketone Acetonitrile Nitrile Carbon disulfide Sulfur-containing organic Dichloromethane Chlorinated hydrocarbon Diethylamine Amine Dimethylformamide Amide Ethyl acetate Ester n-Hexane Aliphatic hydrocarbon Methanol Alcohol Nitrobenzene Nitrogen-containing organic Sodium hydroxide Inorganic base Sulfuric acid Inorganic acid Tetrachloroethylene Chlorinated hydrocarbon Tetrahydrofuran Oxygen heterocyclic Toluene Aromatic hydrocarbon However, judicious use of such data is important, as there are numerous protective clothing and chemical combinations. The data from the recommended list should not be a basis for selecting material for protection against chemicals not represented on the list or against a mixture of chemicals. To the extent possible, the selection of textile materials should be based on test data for the potential challenge. Materials manufactured to provide protection against specific chemical hazards require the use of challenge liquids that represent the potential risk. For example, a pharmacist working with drugs for chemotherapy needs protective clothing and accessories that protect the individual from those drugs. A garment that provides general protection against commonly used chemicals may not be recommended for this use. Special testing would have to be conducted for the proposed end use. 4.4.2 Biological protection performance The selection of protective clothing that provides protection against biological hazards depends on the proposed end use of the clothing. Some of the common end uses are to protect the patient as well as medical professionals who are exposed to blood-borne and other pathogens. Until recently, the majority of biological hazard test methods and guidelines focused on applications in the medical field and for military personnel. With the current bio-terrorism threats, biological protection is important for 'first responders', workers with the potential of exposure to hazardous materials, and the general public. Biological hazards are commonly bacteria, viruses, or toxins that exist as very fine air or liquid-borne particles. Given below are examples of ASTM International and ISO standards that illustrate the specificity of the garments. • F1670-03 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Synthetic Blood. • F1671-03 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using Phi-X174 Bacteriophage Penetration as a Test System. • F1819-04 Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Synthetic Blood Using a Mechanical Pressure Technique. • F1862-00a Standard Test Method for Resistance of Medical Face Masks to Penetration by Synthetic Blood (Horizontal Projection of Fixed Volume at a Known Velocity). • F2100-04 Standard Specification for Performance of Materials Used in Medical Face Masks. • F2101-01 Standard Test Method for Evaluating the Bacterial Filtration Efficiency (BFE) of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus. • F2299-03 Standard Test Method for Determining the Initial Efficiency of Materials Used in Medical Face Masks to Penetration by Particulates Using Latex Spheres. • F1868-02 Standard Test Method for Thermal and Evaporative Resistance of Clothing Materials Using a Sweating Hot Plate. • ISO 16603:2004 Clothing for protection against contact with blood and body fluids – Determination of the resistance of protective clothing materials to penetration by blood and body fluids – Test method using synthetic blood. • ISO 16604:2004 Clothing for protection against contact with blood and body fluids – Determination of resistance of protective clothing materials to penetration by blood-borne pathogens – Test method using Phi-X 174 bacteriophage. 4.4.3 Flame and thermal protection performance The selection of protective clothing that provides protection against heat and burning objects depends on the proposed end use of the clothing. Some of the common applications are to protect individuals from open flames (including flash fires), molten materials, and radiant heat. In general, materials that have inherent flame-resistant properties are used for flame and thermal protection. Often thermal protective apparel requires the use of multi-layered fabric construction, with each layer performing a specific function. Thus performance is measured for individual layers as well as for the whole garment assembly. Protective apparel is worn by firefighters, wildland firefighters, electrical linesmen, racing- and rally-car drivers, emergency services personnel, and many other groups. Standards and performance specifications on selection, use, care and maintenance of flame and thermal clothing, including fabric, are available through national and international standardization bodies such as NFPA International, ISO, OSHA, and ASTM, CEN/TC 162 (European Technical Committee for Protective Clothing). Due to the very specific requirements, there are many specifications for protective clothing used for protection against thermal and flame hazards. Given below are examples of ASTM, EN, and NFPA standards that illustrate the specificity of the garments. • F955-03 Standard Test Method for Evaluating Heat Transfer through Materials for Protective Clothing upon Contact with Molten Substances. • F1002-96 Standard Performance Specification for Protective Clothing for Use by Workers Exposed to Specific Molten Substances and Related Thermal Hazards. • F1060-01 Standard Test Method for Thermal Protective Performance of Materials for Protective Clothing for Hot Surface Contact. • F1358-00 Standard Test Method for Effects of Flame Impingement on Materials Used in Protective Clothing Not Designated Primarily for Flame Resistance. • F1930-00 Standard Test Method for Evaluation of Flame Resistant Clothing for Protection Against Flash Fire Simulations Using an Instrumented Manikin. • F1939-99a Standard Test Method for Radiant Protective Performance of Flame Resistant Clothing Materials. • NFPA 70E Standard for Electrical Safety Requirements for Employee Workplaces. • NFPA 2113 Selection, Care, Use and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel against Flash Fire. • NFPA 1971, Standard on Protective Ensemble for Structural Fire Fighting, 2000 edition. • NFPA 1976, Standard on Protective Ensemble for Proximity Fire Fighting, 2000 edition. • NFPA 1977, Standard on Protective Clothing and Equipment for Wildland Fire Fighting, 1998 edition. • NFPA 1991, Standard on Vapor-Protective Ensembles for Hazardous Materials Emergencies, 2000 edition. • NFPA 1992, Standard on Liquid Splash-Protective Ensembles and Clothing for Hazardous Materials Emergencies, 2000 edition. • NFPA 1999, Standard on Protective Clothing for Emergency Medical Operations, 1997 edition. • NFPA 2113, Standard on Selection, Care, Use, and Maintenance of Flame-Resistant Garments for Protection of Industrial Personnel Against Flash Fire, 2001 edition. • EN 469: European standard for fire fighters' personal protective equipment. • EN 531: European standard for heat and flame protective clothing for industrial workers. • EN 659: European standard for fire fighters' gloves. • ISO 17492:2003 Clothing for protection against heat and flame – Determination of heat transmission on exposure to both flame and radiant heat. • ISO 2801:1998 Clothing for protection against heat and flame – General recommendations for selection, care and use of protective clothing. • ISO 6942:2002 Protective clothing – Protection against heat and fire – Method of test: Evaluation of materials and material assemblies when exposed to a source of radiant heat. • ISO 8194:1987 Radiation protection – Clothing for protection against radioactive contamination – Design, selection, testing and use. • ISO 9150:1988 Protective clothing – Determination of behaviour of materials on impact of small splashes of molten metal. • ISO 9151:1995 Protective clothing against heat and flame – Determination of heat transmission on exposure to flame. • ISO 9185:1990 Protective clothing – Assessment of resistance of materials to molten metal splash. • ISO 11612:1998 Clothing for protection against heat and flame – Test methods and performance requirements for heat-protective clothing. • ISO 11613:1999 Protective clothing for firefighters – Laboratory test methods and performance requirements. • ISO 12127:1996 Clothing for protection against heat and flame – Determination of contact heat transmission through protective clothing or constituent materials. • ISO 14460:1999 Protective clothing for automobile racing drivers – Protection against heat and flame – Performance requirements and test methods. • ISO 15025:2000 Protective clothing – Protection against heat and flame – Method of test for limited flame spread. • ISO 15383:2001 Protective gloves for firefighters – Laboratory test methods and performance requirements. • ISO 15384:2003 Protective clothing for firefighters – Laboratory test methods and performance requirements for wildland firefighting clothing. • ISO 15538:2001 Protective clothing for firefighters – Laboratory test methods and performance requirements for protective clothing with a reflective outer surface. 4.4.4 Mechanical protection performance Protective clothing designed to provide protection from mechanical hazards ranges from cut protection for chainsaw workers to body amour designed for bullet, stab, puncture, or impact protection for police and military personnel. Ballistics tests and performance standards, developed by the National Institute of Justice (NIJ), are examples of the specialized ballistic tests that are used to determine the performance of the materials (6). Numerous standards are available to measure the cut resistance of materials used to protect individuals from chainsaw cuts. Given below is a list of test standards and performance specifications developed by ASTM International for cut resistance. • F1790-04 Standard Test Method for Measuring Cut Resistance of Materials Used in Protective Clothing. • F1414-99 Standard Test Method for Measurement of Cut Resistance to Chain Saw in Lower Body (Legs) Protective Clothing. • F1458-98 Standard Test Method for Measurement of Cut Resistance to Chain Saw of Foot Protective Devices. • F1818-97(2003) Standard Specification for Foot Protection for Chain Saw Users. • F1897-98 Standard Specification for Leg Protection for Chain Saw Users. • F1342-91(1996)e2 Standard Test Method for Protective Clothing Material Resistance to Puncture. • F1414-99 Standard Test Method for Measurement of Cut Resistance to Chain Saw in Lower Body (Legs) Protective Clothing. • ISO 11393-1(1998) Protective clothing for users of hand-held chain-saws – Part 1: Test rig driven by a flywheel for testing resistance to cutting by a chain-saw. • ISO 11393-2 (1999) Protective clothing for users of hand-held chain-saws – Part 2: Test methods and performance requirements for leg protectors. • ISO 11393-5(2001) Protective clothing for users of hand-held chain-saws – Part 5: Test methods and performance requirements for protective gaiters. • ISO 13995:2000 Protective clothing - Mechanical properties – Test method for the determination of the resistance to puncture and dynamic tearing of materials. • ISO 13996:1999 Protective clothing – Mechanical properties – Determination of resistance to puncture. • ISO 13997:1999 Protective clothing – Mechanical properties – Determination of resistance to cutting by sharp objects. • ISO 13998:2003 Protective clothing – Aprons, trousers and vests protecting against cuts and stabs by hand knives. • ISO 14877:2002 Protective clothing for abrasive blasting operations using granular abrasives. • NIJ Standard-0101.03 – Ballistic Resistance of Police Body Armor. • NIJ Standard-0101.04 – Ballistic Resistance of Personal Body Armor. • NIJ Standard-0115.00 – Stab Resistance of Personal Body Armor. Knowledge of how the fabric provides the protection as well as the limitations of the material is important. Materials may have similar barrier performance; however, the manner in which the protection is provided may vary considerably. One may provide protection due to the inherent fiber properties, whereas another from a finish applied to the fabric. It would be important to obtain additional information on the durability of the finish to determine the level of protection that will be provided over the life of the garment. The durability of the finish may be a limitation for the material. The above discussion provides a sense of the level of complex analysis that must be applied for each type of hazard (chemical, biological, physical, radiological, or thermal). After such an analysis, a list is prepared of materials that meet the minimum protection requirements. This list is then used to select potential materials based on other major factors discussed in the following section. Some manufacturers provide online interactive software to assist in the selection of suitable clothing. DuPont™ SafeSPEC™ 10 is an example of an interactive tool that assists the user with hazard assessment and selection of apparel. As stated in the software, the responses are based on the information provided by the user. Thus, it is crucial for the individual entering the information to provide accurate information. With systems that are manufacturer specific, it is difficult to compare products from different manufacturers. 4.5 Selection of materials based on other major factors For protective clothing, protection provided by the material is the primary factor in its selection. In addition, there are several other factors that have to be considered while selecting the materials. As with any other selection processes, the various factors have to be weighted in order to select the appropriate material. Impact of the use of PPE on job performance is an important factor to be considered. In addition, factors such as comfort, durability, maintenance, cost, and design considerations may apply. The major selection categories with applicable sub-categories are discussed below. 4.5.1 Job performance Problems with dexterity, added weight and bulk of the PPE, and heat stress are examples of factors that may affect job performance. The type of burden the use of material will pose is dependent on the part of the body that is covered with the textile material as well as the type of job. For example, the weight of the protective gear may be a factor for firefighters fighting wildland fires; whereas, dexterity may be crucial for protective gloves worn by surgeons and other health professionals. Dexterity is an important factor while selecting glove materials for various end uses. Test standards have been developed to measure the dexterity of gloves. F2010-00 Standard Test Method for Evaluation of Glove Effects on Wearer Hand Dexterity Using a Modified Pegboard Test is an example of a test standard used to measure dexterity of the glove. The suitability of standards to assess dexterity of the material for a specific job should be considered carefully as the type of hand movements required to do the job may not be similar to those used in the standard. In situations where the garment has to be worn on a regular basis for an extended period of time, bulk and weight often become major factors in the selection of protective clothing materials. The problem is compounded by the use of multiple layer clothing and additional equipment that has to be carried to perform the job. Protective clothing materials for military personnel and firefighters fighting wildland fires are examples of end uses where weight and bulk of protective clothing and equipment would be a factor in selection. Appropriate body armor worn by law enforcement personnel is an excellent example of an end use where bulk, weight, and flexibility of the body armor are very important to material selection. Given below is information on selection of body armor published as part of the body armor standard 18 (see also Chapter 19 on ballistic protection): Type I body armor, which was issued during the NIJ demonstration project, is the minimum level of protection that any officer should have, and is totally suitable for full-time wear. A number of departments desiring more than minimum protection wear type II-A armor, which has been found sufficiently comfortable for full-time wear where the threat warrants it, particularly for those departments that use lower velocity 357 Magnum service weapons. Type II armor, heavier and more bulky than type II-A, is worn full time by some departments, but may not be considered suitable for full-time use in hot, humid climates. Type III-A armor, which provided the highest level of protection available as soft body armor, is generally considered to be unsuitable for routine wear, however, individuals confronted with a terrorist threat may be willing to tolerate the weight and bulk of such armor while on duty. Type III and IV armor are clearly intended for use only in tactical situations when the threat warrants such protection. Job performance and comfort are closely related. Often discomfort affects the ability of the individual to perform his/her job efficiently and effectively. Comfort-related examples are included in the next section. 4.5.2 Comfort In many cases the use of PPE may negatively impact the comfort of the user. Reduced comfort results in lower user compliance and potential for heat stress injuries. Environmental or climatic conditions as well as the type and level of activity are major factors that contribute to the potential for heat stress. Thus, adequate comfort is a primary concern in hot and humid climates, as well as in environments where the individual is exposed to a heat source. According to the guidelines published by NIOSH: 19 … the frequency of accidents, in general appears to be higher in hot environments than in more moderate environmental conditions. One reason is that working in a hot environment lowers the mental alertness and physical performance of an individual. Increased body temperature and physical discomfort promote irritability, anger, and other emotional states which sometimes cause workers to overlook safety procedures or to divert attention from hazardous tasks. To the extent possible, material should be carefully selected to balance the protection and comfort properties. In end uses where suitable protective clothing materials cannot provide the required protection and comfort, solutions such as the use of cooling devices, or use of work and rest cycles reduce the risk of heat stress. For example, cooling devices are used to reduce the potential for heat stress for firefighters. They are generally designed to provide the required comfort for a limited period of time. Willingness to use protective clothing materials that provide protection but are uncomfortable depends on factors such as awareness of risk and consequences for non-compliance. An individual may be willing to bear the discomfort in order to remain protected or comply with the requirements if he/she sees the benefit in wearing the protective clothing. The measurement of comfort is very complex as many factors contribute to the comfort provided by a garment to an individual. A variety of tests are conducted to measure these properties. Material characteristics such as air permeability and moisture vapor transmission are used to quantify comfort characteristics. However, these do not take into consideration factors such as heat generated as a result of physical activity. Human subject studies that include physiological monitoring as well as self-reported questionnaires are also used to measure comfort. Factors such as perceived comfort, physical condition of the individuals, and duration of the test are some of the factors that affect the results of human subject studies. A newer method measures the micro-environment that is produced between the garment and the skin as an indicator of comfort. Thermal, sweating, and movable manikins are also being used to measure comfort. 4.5.3 Cost Cost is often an important concern for the individuals procuring the garments. It is not unusual for the procurement department, especially government departments, to purchase PPE based on the cost, using competitive tendering processes. The type of garment plays a major role in determining the cost of the garment. For some end uses, limited use or reusable PPE may be selected. In these situations the added cost of cleaning, maintaining and disposing of PPE should be considered while selecting the materials. Often the initial cost of a limited use garment may be fairly low, but the added cost of purchasing new garments and disposal of the contaminated garments might outweigh the initial cost benefit. On the other hand, the cleaning cost of reusable garments may justify the use of limited-use garments. 4.5.4 Durability Durability of the material determines the performance or the wear life of the garment. The extent to which durability is a concern is dependent on the proposed end use. The material should be able to withstand the normal wear and tear expected for the job that is being performed. Physical characteristics that are commonly measured and reported for protective clothing materials are tensile strength, tearing strength, bursting strength, abrasion resistance, puncture resistance, and cut resistance. In addition fabric weight, flexibility, flammability, and degradation due to environmental conditions may also be measured. Often the performance specifications include minimum requirements for limited use and reusable materials. For materials in which a finish is applied to provide protection, determination of the durability of the finish is very important. The protective properties and thus the wear life of these materials may be reduced. 4.5.5 Use, care, and maintenance Intended use, decontamination, maintenance, storage, and disposal factors should be considered prior to selecting a material. The impact the material will have on the use, care, and maintenance of the garment needs to be considered. The compatibility of the various materials in the manufacture of multi-component materials should also be considered. 4.5.6 Cultural factors In addition to the above factors, selection of protective clothing materials may be affected by cultural factors, especially for comparatively lower risk applications. Thus, it is difficult to develop protective clothing materials that are accepted globally. It is also important for the individual responsible for making decisions to have an understanding of the culture. Decisions made by individuals solely on performance specifications have resulted in non-compliance due to low user acceptance of the recommended PPE for cultural reasons. User acceptance of protective clothing worn by pesticide applicators is used as an example of how factors such as color and materials can be important in the selection of protective clothing. A report published on the Safe Use project in Kenya explains: 20 Persuading farmers to use protective clothing was one of the biggest challenges. They see it as uncomfortable and expensive. Locally designed and manufactured clothing is addressing these problems. More specifically, women in Africa have a cultural aversion to being seen in trousers. Kenyan women were therefore asked to design their own protective clothing and the results have been widely publicised. 4.5.1 Job performance Problems with dexterity, added weight and bulk of the PPE, and heat stress are examples of factors that may affect job performance. The type of burden the use of material will pose is dependent on the part of the body that is covered with the textile material as well as the type of job. For example, the weight of the protective gear may be a factor for firefighters fighting wildland fires; whereas, dexterity may be crucial for protective gloves worn by surgeons and other health professionals. Dexterity is an important factor while selecting glove materials for various end uses. Test standards have been developed to measure the dexterity of gloves. F2010-00 Standard Test Method for Evaluation of Glove Effects on Wearer Hand Dexterity Using a Modified Pegboard Test is an example of a test standard used to measure dexterity of the glove. The suitability of standards to assess dexterity of the material for a specific job should be considered carefully as the type of hand movements required to do the job may not be similar to those used in the standard. In situations where the garment has to be worn on a regular basis for an extended period of time, bulk and weight often become major factors in the selection of protective clothing materials. The problem is compounded by the use of multiple layer clothing and additional equipment that has to be carried to perform the job. Protective clothing materials for military personnel and firefighters fighting wildland fires are examples of end uses where weight and bulk of protective clothing and equipment would be a factor in selection. Appropriate body armor worn by law enforcement personnel is an excellent example of an end use where bulk, weight, and flexibility of the body armor are very important to material selection. Given below is information on selection of body armor published as part of the body armor standard 18 (see also Chapter 19 on ballistic protection): Type I body armor, which was issued during the NIJ demonstration project, is the minimum level of protection that any officer should have, and is totally suitable for full-time wear. A number of departments desiring more than minimum protection wear type II-A armor, which has been found sufficiently comfortable for full-time wear where the threat warrants it, particularly for those departments that use lower velocity 357 Magnum service weapons. Type II armor, heavier and more bulky than type II-A, is worn full time by some departments, but may not be considered suitable for full-time use in hot, humid climates. Type III-A armor, which provided the highest level of protection available as soft body armor, is generally considered to be unsuitable for routine wear, however, individuals confronted with a terrorist threat may be willing to tolerate the weight and bulk of such armor while on duty. Type III and IV armor are clearly intended for use only in tactical situations when the threat warrants such protection. Job performance and comfort are closely related. Often discomfort affects the ability of the individual to perform his/her job efficiently and effectively. Comfort-related examples are included in the next section. 4.5.2 Comfort In many cases the use of PPE may negatively impact the comfort of the user. Reduced comfort results in lower user compliance and potential for heat stress injuries. Environmental or climatic conditions as well as the type and level of activity are major factors that contribute to the potential for heat stress. Thus, adequate comfort is a primary concern in hot and humid climates, as well as in environments where the individual is exposed to a heat source. According to the guidelines published by NIOSH: 19 … the frequency of accidents, in general appears to be higher in hot environments than in more moderate environmental conditions. One reason is that working in a hot environment lowers the mental alertness and physical performance of an individual. Increased body temperature and physical discomfort promote irritability, anger, and other emotional states which sometimes cause workers to overlook safety procedures or to divert attention from hazardous tasks. To the extent possible, material should be carefully selected to balance the protection and comfort properties. In end uses where suitable protective clothing materials cannot provide the required protection and comfort, solutions such as the use of cooling devices, or use of work and rest cycles reduce the risk of heat stress. For example, cooling devices are used to reduce the potential for heat stress for firefighters. They are generally designed to provide the required comfort for a limited period of time. Willingness to use protective clothing materials that provide protection but are uncomfortable depends on factors such as awareness of risk and consequences for non-compliance. An individual may be willing to bear the discomfort in order to remain protected or comply with the requirements if he/she sees the benefit in wearing the protective clothing. The measurement of comfort is very complex as many factors contribute to the comfort provided by a garment to an individual. A variety of tests are conducted to measure these properties. Material characteristics such as air permeability and moisture vapor transmission are used to quantify comfort characteristics. However, these do not take into consideration factors such as heat generated as a result of physical activity. Human subject studies that include physiological monitoring as well as self-reported questionnaires are also used to measure comfort. Factors such as perceived comfort, physical condition of the individuals, and duration of the test are some of the factors that affect the results of human subject studies. A newer method measures the micro-environment that is produced between the garment and the skin as an indicator of comfort. Thermal, sweating, and movable manikins are also being used to measure comfort. 4.5.3 Cost Cost is often an important concern for the individuals procuring the garments. It is not unusual for the procurement department, especially government departments, to purchase PPE based on the cost, using competitive tendering processes. The type of garment plays a major role in determining the cost of the garment. For some end uses, limited use or reusable PPE may be selected. In these situations the added cost of cleaning, maintaining and disposing of PPE should be considered while selecting the materials. Often the initial cost of a limited use garment may be fairly low, but the added cost of purchasing new garments and disposal of the contaminated garments might outweigh the initial cost benefit. On the other hand, the cleaning cost of reusable garments may justify the use of limited-use garments. 4.5.4 Durability Durability of the material determines the performance or the wear life of the garment. The extent to which durability is a concern is dependent on the proposed end use. The material should be able to withstand the normal wear and tear expected for the job that is being performed. Physical characteristics that are commonly measured and reported for protective clothing materials are tensile strength, tearing strength, bursting strength, abrasion resistance, puncture resistance, and cut resistance. In addition fabric weight, flexibility, flammability, and degradation due to environmental conditions may also be measured. Often the performance specifications include minimum requirements for limited use and reusable materials. For materials in which a finish is applied to provide protection, determination of the durability of the finish is very important. The protective properties and thus the wear life of these materials may be reduced. 4.5.5 Use, care, and maintenance Intended use, decontamination, maintenance, storage, and disposal factors should be considered prior to selecting a material. The impact the material will have on the use, care, and maintenance of the garment needs to be considered. The compatibility of the various materials in the manufacture of multi-component materials should also be considered. 4.5.6 Cultural factors In addition to the above factors, selection of protective clothing materials may be affected by cultural factors, especially for comparatively lower risk applications. Thus, it is difficult to develop protective clothing materials that are accepted globally. It is also important for the individual responsible for making decisions to have an understanding of the culture. Decisions made by individuals solely on performance specifications have resulted in non-compliance due to low user acceptance of the recommended PPE for cultural reasons. User acceptance of protective clothing worn by pesticide applicators is used as an example of how factors such as color and materials can be important in the selection of protective clothing. A report published on the Safe Use project in Kenya explains: 20 Persuading farmers to use protective clothing was one of the biggest challenges. They see it as uncomfortable and expensive. Locally designed and manufactured clothing is addressing these problems. More specifically, women in Africa have a cultural aversion to being seen in trousers. Kenyan women were therefore asked to design their own protective clothing and the results have been widely publicised. 4.6 Future trends The current trend of standards organizations focusing on the development of performance based specifications will continue. Comprehensive specifications will include information on selection, use, care, and maintenance (SUCM). The development of these specifications will assist in the selection of appropriate protective clothing materials. The need to protect individuals from multiple hazards such as chemical, biological, and thermal will require streamlining of the selection process. The use of online systems, databases, and knowledge bases will continue to expand. In the future online systems pertaining to protective clothing will, in all probability, include global mega-knowledge bases with built-in information on compliance requirements. Selection of protective clothing materials based on performance based specifications will be an integral part of the system. In building these systems, it will be important to ensure that the system is used to assist but not replace the decision makers. The individual selecting the materials(s) should be involved with each step of the selection process. An online system entitled 'Work and Protective Clothing for Agricultural Workers' is being used as an example to illustrate the use of databases and the Internet as tools for the selection of protective clothing materials. 21 A brief description of this system is included in section 4.6.1 . 4.6.1 Sample online system The system provides a structured systematic process for data collection, analysis, and dissemination. Online data entry forms are used to enter the raw data and material information. The raw data is used to calculate the values in accordance with applicable standards. These are stored for access through easy to use dropdown menus. 4.6.2 Data entry and calculations The first step in the data entry process is the auto generation of a fabric code based on information regarding fiber content, country, and fabric construction ( Fig. 4.2 ). The next step is entry of information regarding type of garment, cost, source, etc. ( Fig. 4.3 ). Then the fabric is tested and the raw data entered by the operator using online forms. The hard copies of the raw data are retained for verification. The system uses the raw data to calculate results such as mean and standard deviation. These results are stored in a table for later use. 4.2 Screen to auto generate fabric codes (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.3 Data entry screen for fabric weight (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.3 Statistical model Data from over sixty fabrics in the database was used to develop a statistical model to predict percent pesticide penetration through the material. Data was analyzed to identify the key factors that affect pesticide penetration. Currently the scope of the statistical model is limited to estimating pesticide penetration of three formulations through woven fabrics ( Fig. 4.4 ). 4.4 Predictive model screen (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.4 Data dissemination Easy to use dropdown menus are used to disseminate textile material and penetration data sorted into various categories ( Fig. 4.5 ). Individuals can also use the system to customize their search by defining the search criteria ( Fig. 4.6 ). Once performance specifications have been established, individuals will be able to select materials that meet the performance specifications. 4.5 Dropdown menu for accessing information (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6 Screen to enter search criteria (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.5 Suggestions model for future systems The flowchart that outlines the steps involved in the selection process is given in Fig. 4.7 . The work scenario(s) and hazard(s) will be entered by the user through the online input screen. Based on the information entered, the system will list relevant standards. If relevant standards are available, the user will select the standard and proceed to the next step. If no relevant standards are available, the user will have the option to use the system to select the test methods, and/or enter data, and then move to the next step (see Fig. 4.7 ). In the next step, the user will enter the information regarding other factors such as durability, cost, etc., that are important in the decision making process. This information will be used to identify the fabrics that meet all requirements. If fabrics meet the requirements, final selection will be made. If fabrics do not meet all requirements, the requirements will have to be adjusted prior to final selection. 4.7 Recommended steps in the selection of textiles for protective clothing. 4.6.1 Sample online system The system provides a structured systematic process for data collection, analysis, and dissemination. Online data entry forms are used to enter the raw data and material information. The raw data is used to calculate the values in accordance with applicable standards. These are stored for access through easy to use dropdown menus. 4.6.2 Data entry and calculations The first step in the data entry process is the auto generation of a fabric code based on information regarding fiber content, country, and fabric construction ( Fig. 4.2 ). The next step is entry of information regarding type of garment, cost, source, etc. ( Fig. 4.3 ). Then the fabric is tested and the raw data entered by the operator using online forms. The hard copies of the raw data are retained for verification. The system uses the raw data to calculate results such as mean and standard deviation. These results are stored in a table for later use. 4.2 Screen to auto generate fabric codes (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.3 Data entry screen for fabric weight (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.3 Statistical model Data from over sixty fabrics in the database was used to develop a statistical model to predict percent pesticide penetration through the material. Data was analyzed to identify the key factors that affect pesticide penetration. Currently the scope of the statistical model is limited to estimating pesticide penetration of three formulations through woven fabrics ( Fig. 4.4 ). 4.4 Predictive model screen (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.4 Data dissemination Easy to use dropdown menus are used to disseminate textile material and penetration data sorted into various categories ( Fig. 4.5 ). Individuals can also use the system to customize their search by defining the search criteria ( Fig. 4.6 ). Once performance specifications have been established, individuals will be able to select materials that meet the performance specifications. 4.5 Dropdown menu for accessing information (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6 Screen to enter search criteria (Source: Work and Protective Clothing for Agricultural Workers, University of Maryland Eastern Shore). 4.6.5 Suggestions model for future systems The flowchart that outlines the steps involved in the selection process is given in Fig. 4.7 . The work scenario(s) and hazard(s) will be entered by the user through the online input screen. Based on the information entered, the system will list relevant standards. If relevant standards are available, the user will select the standard and proceed to the next step. If no relevant standards are available, the user will have the option to use the system to select the test methods, and/or enter data, and then move to the next step (see Fig. 4.7 ). In the next step, the user will enter the information regarding other factors such as durability, cost, etc., that are important in the decision making process. This information will be used to identify the fabrics that meet all requirements. If fabrics meet the requirements, final selection will be made. If fabrics do not meet all requirements, the requirements will have to be adjusted prior to final selection. 4.7 Recommended steps in the selection of textiles for protective clothing. 4.7 Sources of further information and advice Book of Papers, 2nd European Conference on Protective Clothing (ECPC) and NOKOBETEF 7, Challenges for Protective Clothing, Montreux, Switzerland, 21–24 May 2003. Guidance Manual for Selecting Protective Clothing for Agricultural Pesticides Operations, Risk Reduction Engineering Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, USA, 1991. Kohloff, F. H., 1999, PPE Guidelines for Melting and Pouring Operations, Modern Casting 89, (5) pp 57–59. Protective Clothing Systems and Materials, M. Raheel (ed.), Marcel Dekker 1994. STP 1237 – Performance of Protective Clothing, Fifth Volume, J. S. Johnson and S. Z. Mansdorf (eds), ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428, USA. STP 1273 – Performance of Protective Clothing, Sixth Volume, J.O. Stull and A.D. Schwope (eds), ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428, USA. STP 1386 – Performance of Protective Clothing: Issues and Priorities for the 21st Century, Seventh Volume, C. N. Nelson and N.W. Henry (eds), ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428, USA. Stull, J.O., 2004, A Suggested Approach to the Selection of Chemical and Biological Protective Clothing – Meeting Industry Needs for Protection Against a Variety of Hazards, International Journal of Occupational Safety and Ergonomics, Vol. 10, No. 3, pp. 271–290. Sun, G., H.S. Yoo, X.S. Zhang, and N. Pan, 2000, Radiant protective and transport properties of fabrics used by wildland firefighters, Textile Research Journal, Vol. 70, No. 7, pp. 567–573. Walsh, D. L., M. R. Schwerin, R. W. Kisielewski, R. M. Kotz, M. P. Chaput, G. W. Varney, and T. M, 2004, Abrasion Resistance of Medical Glove Materials, Journal of Biomedical Materials Research, Vol. 68B, No. 1, pp. 81–87. Whitford, F., Stone, J., and MacMillian, T., Chapter 11 – Personal Protective Equipment: Selection, Care, and Use, Whitford, F. (ed.), The Complete Book of Pesticide Management, John Wiley & Sons, Inc., 2001.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165481/

The Poly-γ- d -Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity ▿

The poly-γ- d -glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis , which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674197/

Anthrax Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

ABSTRACT Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP ( n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272130/

A Low Dimensional Dynamical Model of the Initial Pulmonary Innate Response to Infection

In order to gain a deeper understanding of the onset and progression of pulmonary infections we present and analyze a low dimensional, phenomenological model of infection and the innate immune response in the lungs. Because pulmonary innate immunity has features unique to itself, general mathematical models of the immune system may not be appropriate. The differential equations model that we propose is based on current knowledge of the biology of pulmonary innate immunity and accurately reproduces known features of the initial phase of the dynamics of pulmonary innate system as exhibited in recent experiments. Further, we propose to use the model as a starting point for interrogation with clinical data from a new noninvasive technique for sampling alveolar lining fluid.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496671/

Antibacterial property of Ag nanoparticle-impregnated N-doped titania films under visible light

Photocatalysts produce free radicals upon receiving light energy; thus, they possess antibacterial properties. Silver (Ag) is an antibacterial material that disrupts bacterial physiology. Our previous study reported that the high antibacterial property of silver nanoparticles on the surfaces of visible light-responsive nitrogen-doped TiO 2 photocatalysts [TiO 2 (N)] could be further enhanced by visible light illumination. However, the major limitation of this Ag-TiO 2 composite material is its durability; the antibacterial property decreased markedly after repeated use. To overcome this limitation, we developed TiO 2 (N)/Ag/TiO 2 (N) sandwich films in which the silver is embedded between two TiO 2 (N) layers. Various characteristics, including silver and nitrogen amounts, were examined in the composite materials. Various analyses, including electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and ultraviolet–visible absorption spectrum and methylene blue degradation rate analyses, were performed. The antibacterial properties of the composite materials were investigated. Here we revealed that the antibacterial durability of these thin films is substantially improved in both the dark and visible light, by which bacteria, such as Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, and Acinetobacter baumannii , could be efficiently eliminated. This study demonstrated a feasible approach to improve the visible-light responsiveness and durability of antibacterial materials that contain silver nanoparticles impregnated in TiO 2 (N) films. Results Effects of silver level on photocatalytic and antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films To regulate the amount of Ag deposition in the middle layer of sandwich films, various sputtering periods, namely 0, 15, 30, 60, and 120 s, were examined. X-ray diffraction patterns of the sandwich films revealed that the anatase phase of titania was formed after annealing ( Supplementary Fig. S1 ). Energy-dispersive X-ray spectroscopy analysis indicated that the amount of Ag deposition increased with time ( Supplementary Fig. S2 ). Furthermore, a field-emission scanning electron microscopy analysis revealed that the thickness of the sandwiched thin films was nearly 700 ± 50 nm, in which the thickness for the silver layers are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively ( Fig. 1A–J ). In addition, various amounts of Ag nanoparticles were observed on the TiO 2 (N) surfaces of some films before and after annealing ( Fig. 1K–T , white spots). An ultraviolet–visible (UV–Vis) absorption spectroscopy analysis revealed no significant difference after annealing; whereas the Ag-containing samples showed a redshift, indicating an increased visible light absorption ( Fig. 2A,B ). The visible-light-responsive photocatalytic property was determined using a methylene blue (MB) degradation analysis as described 18 . Data revealed that Ag doping did not markedly increase the visible-light-responsive photocatalysis ( Fig. 2C ). Despite this, the antibacterial property was determined. Because of the lack of the visible-light-induced photocatalytic activity, no significant difference was observed between the groups with or without visible light illumination ( Fig. 2D ). Because these thin films contained Ag, the antibacterial property was detectable even in the dark conditions. The NTA120s sample, which contained the highest Ag levels ( Supplementary Fig. S1 ) exhibited the most antibacterial activity compared with the other tested samples ( Fig. 2D ). Effect of nitrogen level on photocatalytic and antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films To increase the visible-light responsiveness, N doping is critical 6 . The nitrogen content in these films was minimal when oxygen flow was high [5 sccm (standard cubic centimeters per minute)] during film deposition. We hypothesized that this was caused by an excess supply of oxygen, rendering N unable to be efficiently integrated into the lattice structure of TiO 2 . Thus, various low-O 2 -supplying conditions, including the O 2 flow rate of 2, 3, 4, and 5 sccm, were tested ( Supplementary Table 1 ). Consistent with our hypothesis, an X-ray photoelectron spectroscopy analysis revealed that reduced O 2 supply improved N levels in composite thin films ( Fig. 3A ). However, the Ti-to-O ratio was also changed, with the N(4)TA and N(5)TA samples exhibiting a relatively 1:2 ratio, whereas the N(2)TA and N(3)TA samples exhibited a ratio of approximately 1:1 ( Fig. 3A ; the amount of oxygen supplied was indicated in brackets, e.g., N(4)TA was prepared with 4 sccm O 2 ). These results indicated that 2 and 3 sccm O 2 supply were insufficient to oxidize Ti and produce TiO 2 ; instead, TiN or TiON was formed. Consistently, X-ray photoelectron spectroscopy analysis for the 1 s atomic orbital of N indicated a significant increase in the formation of TiN ( Fig. 3B ). X-ray diffraction analysis further revealed the production of TiN in N(2)TA and N(3)TA samples before and after annealing ( Fig. 3C,D ). Because TiO 2 was not efficiently produced in the N(2)TA and N(3)TA samples, the rutile and anatase TiO 2 signals were observed only in the N(4)TA and N(5)TA thin films after annealing ( Fig. 3D ). The field-emission scanning electron microscopy analysis indicated that the N(2)TA and N(3)TA samples formed thicker films, with less surface-exposed Ag nanoparticles compared with the N(4)TA and N(5)TA samples ( Fig. 4A–E ). A UV–Vis absorption spectroscopy analysis indicated that reduced O 2 supply caused remarkable redshifts in the samples ( Fig. 5A,B ). The band gaps were calculated using the UV–Vis spectroscopy and Tauc plots 19 . However, the band gaps for the N(2)TA and N(3)TA were not derived, because the two samples produced under 2 and 3 sccm O 2 supply were insufficient to form TiO 2 ; instead, TiN or TiON was formed with high amount of nitrogen of 18.8% and 13.5% and thus, with high absorbance ( Fig. 3A,B ). These results indicated that the increased N content reduced the band gap of the thin films ( Fig. 5C , Supplementary Table S1 ). Analyses of MB and Hoechst dye degradation rates further revealed that the N(4)TA sample exhibited a superior visible-light-induced photocatalytic ability than did the other samples ( Fig. 5D ; Supplementary Fig. S3 ), suggesting that the balance of the N-doping amount and TiO 2 production was critical. In accordance with the aforementioned analyses, antibacterial experiments demonstrated that the N(4)TA sample exhibited the highest antibacterial activity among the thin film samples under visible light illumination ( Fig. 5E , N(4)TA-N500). In addition, visible light illuminating the N(4)TA sample exerted higher bactericidal activity than did the control groups with the N(4)TA sample in the dark ( Fig. 5E , N(4)TA-N500; *P < 0.05). Durability of visible-light-responsive antibacterial property The visible-light-responsive antibacterial property of these novel sandwich films after repeated usages was compared with that of our previously developed single-layer thin films containing Ag nanoparticles 6 . Although the first use of the single-layer thin films could achieve an approximately 5-log reduction in bacteria 6 , the data indicated that these thin films could not efficiently eliminate the bacteria after frequently use ( Fig. 6 , second- and third-cycle use of single-layer groups). By contrast, favorable performance was observed after these multilayer films were repeated used, which eliminated E. coli efficiently after the second and third use ( Fig. 6 , second- and third-cycle use of multilayer groups). Scanning electron microscopy was employed to investigate the E. coli cell damage caused by the sandwich films. We observed that if the bacterial cells were not treated with Ag present in the N-doped TiO 2 sandwich films with or without visible light illumination, they displayed relatively smooth surfaces ( Fig. 7A,B ). Because of the antibacterial property of Ag, bacterial cells displayed rough surfaces after being treated with a sandwich film in the dark ( Fig. 7C ). More vigorous changes, which were showed as unique cracks, such as structures on their surfaces, were observed after the bacterial cells were illuminated with visible light on a sandwich film ( Fig. 7D , arrows). Pathogen analyses To investigate the performance of TiO 2 (N)/Ag/TiO 2 (N) sandwich films in eradicating pathogenic bacteria and human pathogens, including S. pyogenes , S. aureus , and A. baumannii , the films were subjected to visible-light-induced catalysis. Among these, S. aureus and A. baumannii are pathogenic bacteria with a high antibiotic resistance rate, causing increased incidence of nosocomial infections 20 . We demonstrated that all the tested pathogens were efficiently eliminated after exposure to the sandwich films illuminated with visible light ( Fig. 8 , dark vs. light groups). The effectiveness showed a nearly 1-log reduction of the bacterial population. In addition, the TiO 2 (N)/Ag/TiO 2 (N) sandwich films appeared equally potent in eliminating the bacteria when applied to both the Gram-positive ( S. aureus and S. pyogenes ) and Gram-negative ( E. coli and A. baumannii ) bacteria ( Fig. 8 ). Effects of silver level on photocatalytic and antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films To regulate the amount of Ag deposition in the middle layer of sandwich films, various sputtering periods, namely 0, 15, 30, 60, and 120 s, were examined. X-ray diffraction patterns of the sandwich films revealed that the anatase phase of titania was formed after annealing ( Supplementary Fig. S1 ). Energy-dispersive X-ray spectroscopy analysis indicated that the amount of Ag deposition increased with time ( Supplementary Fig. S2 ). Furthermore, a field-emission scanning electron microscopy analysis revealed that the thickness of the sandwiched thin films was nearly 700 ± 50 nm, in which the thickness for the silver layers are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively ( Fig. 1A–J ). In addition, various amounts of Ag nanoparticles were observed on the TiO 2 (N) surfaces of some films before and after annealing ( Fig. 1K–T , white spots). An ultraviolet–visible (UV–Vis) absorption spectroscopy analysis revealed no significant difference after annealing; whereas the Ag-containing samples showed a redshift, indicating an increased visible light absorption ( Fig. 2A,B ). The visible-light-responsive photocatalytic property was determined using a methylene blue (MB) degradation analysis as described 18 . Data revealed that Ag doping did not markedly increase the visible-light-responsive photocatalysis ( Fig. 2C ). Despite this, the antibacterial property was determined. Because of the lack of the visible-light-induced photocatalytic activity, no significant difference was observed between the groups with or without visible light illumination ( Fig. 2D ). Because these thin films contained Ag, the antibacterial property was detectable even in the dark conditions. The NTA120s sample, which contained the highest Ag levels ( Supplementary Fig. S1 ) exhibited the most antibacterial activity compared with the other tested samples ( Fig. 2D ). Effect of nitrogen level on photocatalytic and antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films To increase the visible-light responsiveness, N doping is critical 6 . The nitrogen content in these films was minimal when oxygen flow was high [5 sccm (standard cubic centimeters per minute)] during film deposition. We hypothesized that this was caused by an excess supply of oxygen, rendering N unable to be efficiently integrated into the lattice structure of TiO 2 . Thus, various low-O 2 -supplying conditions, including the O 2 flow rate of 2, 3, 4, and 5 sccm, were tested ( Supplementary Table 1 ). Consistent with our hypothesis, an X-ray photoelectron spectroscopy analysis revealed that reduced O 2 supply improved N levels in composite thin films ( Fig. 3A ). However, the Ti-to-O ratio was also changed, with the N(4)TA and N(5)TA samples exhibiting a relatively 1:2 ratio, whereas the N(2)TA and N(3)TA samples exhibited a ratio of approximately 1:1 ( Fig. 3A ; the amount of oxygen supplied was indicated in brackets, e.g., N(4)TA was prepared with 4 sccm O 2 ). These results indicated that 2 and 3 sccm O 2 supply were insufficient to oxidize Ti and produce TiO 2 ; instead, TiN or TiON was formed. Consistently, X-ray photoelectron spectroscopy analysis for the 1 s atomic orbital of N indicated a significant increase in the formation of TiN ( Fig. 3B ). X-ray diffraction analysis further revealed the production of TiN in N(2)TA and N(3)TA samples before and after annealing ( Fig. 3C,D ). Because TiO 2 was not efficiently produced in the N(2)TA and N(3)TA samples, the rutile and anatase TiO 2 signals were observed only in the N(4)TA and N(5)TA thin films after annealing ( Fig. 3D ). The field-emission scanning electron microscopy analysis indicated that the N(2)TA and N(3)TA samples formed thicker films, with less surface-exposed Ag nanoparticles compared with the N(4)TA and N(5)TA samples ( Fig. 4A–E ). A UV–Vis absorption spectroscopy analysis indicated that reduced O 2 supply caused remarkable redshifts in the samples ( Fig. 5A,B ). The band gaps were calculated using the UV–Vis spectroscopy and Tauc plots 19 . However, the band gaps for the N(2)TA and N(3)TA were not derived, because the two samples produced under 2 and 3 sccm O 2 supply were insufficient to form TiO 2 ; instead, TiN or TiON was formed with high amount of nitrogen of 18.8% and 13.5% and thus, with high absorbance ( Fig. 3A,B ). These results indicated that the increased N content reduced the band gap of the thin films ( Fig. 5C , Supplementary Table S1 ). Analyses of MB and Hoechst dye degradation rates further revealed that the N(4)TA sample exhibited a superior visible-light-induced photocatalytic ability than did the other samples ( Fig. 5D ; Supplementary Fig. S3 ), suggesting that the balance of the N-doping amount and TiO 2 production was critical. In accordance with the aforementioned analyses, antibacterial experiments demonstrated that the N(4)TA sample exhibited the highest antibacterial activity among the thin film samples under visible light illumination ( Fig. 5E , N(4)TA-N500). In addition, visible light illuminating the N(4)TA sample exerted higher bactericidal activity than did the control groups with the N(4)TA sample in the dark ( Fig. 5E , N(4)TA-N500; *P < 0.05). Durability of visible-light-responsive antibacterial property The visible-light-responsive antibacterial property of these novel sandwich films after repeated usages was compared with that of our previously developed single-layer thin films containing Ag nanoparticles 6 . Although the first use of the single-layer thin films could achieve an approximately 5-log reduction in bacteria 6 , the data indicated that these thin films could not efficiently eliminate the bacteria after frequently use ( Fig. 6 , second- and third-cycle use of single-layer groups). By contrast, favorable performance was observed after these multilayer films were repeated used, which eliminated E. coli efficiently after the second and third use ( Fig. 6 , second- and third-cycle use of multilayer groups). Scanning electron microscopy was employed to investigate the E. coli cell damage caused by the sandwich films. We observed that if the bacterial cells were not treated with Ag present in the N-doped TiO 2 sandwich films with or without visible light illumination, they displayed relatively smooth surfaces ( Fig. 7A,B ). Because of the antibacterial property of Ag, bacterial cells displayed rough surfaces after being treated with a sandwich film in the dark ( Fig. 7C ). More vigorous changes, which were showed as unique cracks, such as structures on their surfaces, were observed after the bacterial cells were illuminated with visible light on a sandwich film ( Fig. 7D , arrows). Pathogen analyses To investigate the performance of TiO 2 (N)/Ag/TiO 2 (N) sandwich films in eradicating pathogenic bacteria and human pathogens, including S. pyogenes , S. aureus , and A. baumannii , the films were subjected to visible-light-induced catalysis. Among these, S. aureus and A. baumannii are pathogenic bacteria with a high antibiotic resistance rate, causing increased incidence of nosocomial infections 20 . We demonstrated that all the tested pathogens were efficiently eliminated after exposure to the sandwich films illuminated with visible light ( Fig. 8 , dark vs. light groups). The effectiveness showed a nearly 1-log reduction of the bacterial population. In addition, the TiO 2 (N)/Ag/TiO 2 (N) sandwich films appeared equally potent in eliminating the bacteria when applied to both the Gram-positive ( S. aureus and S. pyogenes ) and Gram-negative ( E. coli and A. baumannii ) bacteria ( Fig. 8 ). Discussion Silver has been used to prevent infections for thousands of years 21 , with Hippocrates describing the antimicrobial properties of the metal in 400 BC. Antibacterial silver products have been widely used for the handling and cleaning of burn, trauma, catheter, and dental amalgam 22 23 . Previous studies have demonstrated that both Ag + ions and Ag nanoparticles possess antibacterial properties 22 23 24 . Silver disrupts various bacterial physiologies, including disulfide bond formation, metabolism, and iron homeostasis; these changes increase production of ROS and membrane permeability and disrupt membrane respiratory electron transport chains and DNA replication components 23 25 . Thus, silver is widely used as tableware and a hygiene product. In these cases, the durability of the antibacterial property is not a problem because the entire metal product is able to release a sufficient amount of Ag ions and nanoparticles with time. However, this is not the case when only a nanoscaled layer of silver is used. Previous studies have indicated that Ag particles or their coating on TiO 2 surfaces can enhance the light-driven photoinactivation of bacteria 2 6 26 27 28 29 30 . However, the antibacterial activity of such thin films after repeated use has not been investigated thoroughly. In addition, the antibacterial property of Ag-TiO 2 composite thin films remains to be elucidated. For example, our previous study reported that TiO 2 containing nanoscale Ag wires exhibited strong visible-light enhanced antibacterial properties 6 . The durability of these thin films is always a problem; because their antibacterial properties rapidly decline after repeated use ( Fig. 6 , second- and third-cycle use of single-layer groups). Therefore, we described the influence of the various amounts of silver and nitrogen on the antibacterial property of the sandwich films. Analysis results indicated that silver depositions improved the antibacterial activity of the thin films ( Fig. 2 ). In addition, the amount of nitrogen supplied is crucial for the formation of high-performance antibacterial thin films under visible light illumination ( Fig. 5 ). Thus, we developed the TiO 2 (N)/Ag/TiO 2 (N) sandwich films, which exhibited sustainable antibacterial properties after repeated use. Nanoscaled thin films have been used to achieve a controlled release of embedded materials 31 32 33 . In the original experimental design, the silver layer was embedded in the middle of TiO 2 (N)/Ag/TiO 2 (N) sandwich films. Notably, in addition to the embedded Ag layer, we observed certain scattered Ag nanoparticles on the surface of the as-deposited sandwich films and a higher density of Ag nanoparticles on annealed films. Ag nanoparticles appeared to emerge on the film surface by atomic diffusion through the grain boundaries of the upper TiO 2 (N) layer. In addition, this could happen when silver atoms gain sufficient energy from the bombardment of atoms and ions during sputtering growth or from thermal annealing. In either case, the Ag nanoparticles are impregnated in the TiO 2 (N) films and not merely placed or attached on the film surface, which could be the main reason for the high durability of the photocatalytic TiO 2 (N)/Ag/TiO 2 (N) sandwich films. The silver-induced bactericidal effect is a complex response, involving the disruption of bacterial physiologies and abnormal elicitation of ROS in bacteria 23 25 34 . Combined treatment with exogenous ROS and silver revealed a synergistic antibacterial effect 35 . ROS production is the main antibacterial mechanism of photocatalysts 2 36 37 . Thus, it is reasonable that the silver synergizes with the photocatalytic components of the sandwich films to efficiently eradicate pathogenic bacteria under the visible light ( Fig. 8 ). However, nanoscale Ag particles and TiO 2 appear to exert a synergistic impact on the environment 38 39 . The increased production and use of silver nanoparticles in products leads to the inevitable increase in the release of these particles into the environment through the lives of these products, from the raw material stage to disposal 40 . Likewise, the increased disposal of TiO 2 nanoparticles exerts a considerable impact on the ecosystem 41 . Although the mechanism remains to be further elucidated, the coexistence of Ag and TiO 2 nanoparticles has been shown to exert increased environmental impact under sunlight 38 39 . Furthermore, exposure to Ag nanoparticles was shown to influence the immune system 42 . However, previously reported Ag–TiO 2 thin films have typically had a surface-exposed Ag layer 6 43 44 45 46 . Thus, the increase reusability of the nanoscaled Ag-TiO 2 composite material with a TiO 2 -covered layer will reduce the environmental impact of products and will benefit human health. In conclusion, we successfully demonstrated the antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films, which could be optimized through Ag and N depositions on titania substrates. The silver and TiO 2 (N) composite materials exhibited synergistic antibacterial activity under visible light illumination. These findings suggest that the concepts used in this study and multilayer TiO 2 (N)/Ag/TiO 2 (N) composite materials have potential applications in the developing alternative disinfectants. Methods Various thin films, including single layers of Ag, TiO 2 , and TiO 2 (N), and TiO 2 (N)/Ag/TiO 2 (N) sandwich layers were prepared in a reactive magnetron sputtering system As Supplementary Fig. S4 illustrates, five sputtering targets exist in the deposition system, but only two titanium (Ti) targets and one silver (Ag) target were used, and the base pressure was below 1.3 × 10 −5 Pa. To prepare pure TiO 2 or TiO 2 (N) and Ag, two Ti targets and one Ag target were used, respectively. Before sputtering, we used argon plasma to etch the surface of the substrate for 10 min to remove residual particles on the surface. The substrate holder was rotated at a speed of 5 rpm without applying substrate bias during deposition. Silicon wafer (100) and glass substrates were used. The substrate was nearly at room temperature without external heating. The target powers were set at 250 W each and 20 W in the DC mode for the two Ti targets and one Ag target, respectively. For pure TiO 2 deposition, the gas contained argon and oxygen with the fixed flow rates of Ar at 20 sccm and O 2 at 7 sccm, and the total pressure was approximately 4.7 × 10 −1 Pa (3.5 × 10 −3 Torr). For the TiO 2 (N) films, the gas contained Ar, O 2 , and N 2 at the flow rates of 20, 5–2, and 8 sccm, respectively, whereas for the Ag film, the gas used contained only Ar with a flow rate of 20 sccm without adding other gases. In addition, for forming a monolithic layer of Ag, TiO 2 , and TiO 2 (N), two series of sandwiched TiO 2 (N)Ag/TiO 2 (N) films were prepared. Furthermore, one series was prepared with various amounts of Ag in the sandwiched films, whereas the other series was prepared with a fixed amount of Ag but with various nitrogen contents in the sandwiched films. In the first series, the amount of Ag was regulated by changing the deposition time from 30 s to 60, 90, and 120 s, and the corresponding sample IDs were denoted as NTAxxxs, where xxx was the Ag deposition time. In the second series, the amount of Ag was fixed with the deposition time of 120 s, and the nitrogen contents in the TiO 2 (N) films were regulated by reducing the oxygen flow rates from 5 sccm to 4, 3, and then 2 sccm, and the corresponding sample IDs were denoted as N (x)TA, where x was the oxygen flow rate. The as-deposited films were further annealed at 500 °C for 1 h in the nitrogen atmosphere by using a conventional vacuum furnace. The deposition rate of TiO 2 (N) films varied with the oxygen flow rate and was approximately 350 nm/h with the oxygen flow rate of 4 sccm. The deposition rate of silver was approximately 7.5 nm/min. The deposition times of the typical sandwich TiO 2 (N)/Ag/TiO 2 (N) film used in this study were 1 h and 2 min, and 1 h. Thus, an average thickness of the Ag impregnated TiO 2 (N) films was 700 ± 50 nm. The thickness for the silver layer are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively. Characterization analysis The structure and crystallinity of films were analyzed using X-ray diffraction measurements recorded using the Rigaku X-ray diffractometer D/MAX-2500 V with a Cu Kα radiation (40 kV, 100 mA) source. The surface morphology and the cross-sectional view of the films were observed using the JEOL JEM-6500 F field-emission scanning electron microscopy. The UV–Vis absorption spectra of the films were recorded using a JASCO V-650 spectrophotometer ranging from 300 to 900 nm. The composition of samples was determined using an energy dispersive spectroscopy (Horiba, EMAX-ENERGY) and a K-Alpha™ X-ray photoelectron spectrometer using an AlK α X-ray radiation source to estimate elements semi-quantitatively. Antibacterial experiment Bacterial culturing and plating were performed following the previously described standard methods 5 47 48 49 . The bacterial concentration was determined using the standard plating method or from optical density readings at 595 nm (OD 595 ). For example, the conversion factor for E. coli BL 21 was calculated to be 1 × 10 9 colony-forming unit (CFU)/mL at OD 595 , and the cultures were diluted with the culture medium to 1 × 10 7 CFU/mL. The 1 × 10 6 CFU culture was allowed to drip on the sample (approximately 6.25 cm 2 ) and was then placed in a dark room or exposed under the visible light and at room temperature. The visible light source was an incandescent lamp (Classictone incandescent lamp, 60 W, Philips Taiwan; Taipei, Taiwan), and the illumination density was recorded using a light meter (model LX-102; Lutron Electronic Enterprises, Taipei, Taiwan). In the photocatalytic reaction, the illumination distance between the sample and lamp was approximately 10 cm, which was exposed for 30 min, and the light intensity on the sample surface was nearly 1.2 × 103 lux (lumen/m 2 ) (30 mW/cm 2 ). After illumination, 100 μL of the bacterial solution was recovered from the sample. Finally, the bacterial concentration was determined using standard dilution and plating methods, and the percentage of surviving bacteria was calculated 18 19 . S. pyogenes (strain M29588), pandrug-resistant A. baumannii (strain M36788), and S. aureus (strain SA02) were the clinical isolates provided by Buddhist Tzu-Chi General Hospital in Hualien, Taiwan 5 . E. coli and A. baumannii were grown and maintained in the lysogeny broth (LB) medium or LB agar (BD Diagnostics, Sparks, MD, USA) at 37 °C. By contrast, S. pyogenes and S. aureus were grown in the trypticase soy broth with yeast extract (TSBY) medium or TSBY agar (MDBio, Inc., Taipei, Taiwan) at 37 °C. When the visible light was used to elicit the photocatalysis reaction, a UV cut-off filter (400 nm; Edmund Optics, Barrington, NJ, USA) was used to prevent the illumination of small fractions with UV-range wavelength in the photocatalytic experiments. Photocatalytic properties Analysis methods were based on recently reported literatures 4 50 51 , which used the MB degradation rate to analyze the photocatalytic performance of impurity-doped TiO 2, in accordance with the generally accepted method and reported format of MB photodecomposition. Photocatalytic efficiency was evaluated by examining the decomposition of 10 ppm MB (Sigma-Aldrich, St. Louis, MO, USA). The MB concentration was determined according to the intensity of the light-absorption peak at 664 nm wavelength, as measured using a UV–Vis spectrometer. A fixed size (1 cm × 1 cm) of the sample sank in 2 mL of the MB aqueous solution. The visible light illumination was conducted using a fluorescent lamp (Philips; P-LF27W/865) with a wavelength distribution of approximately 400–750 nm and a maximum intensity at the range of 543–611 nm, producing an average power density of 4.2 mW/cm 2 at a distance of 8 cm between the visible light source and sample. Statistical analysis The means, standard deviations, and statistics of the quantifiable data were calculated using Microsoft Office Excel 2003, SigmaPlot 10, and SPSS 19. The significance of data was determined using a one-way analysis of variance followed by a post hoc Bonferroni corrected test. The probability of type I error α = 0.05 was recognized as statistically significant. Various thin films, including single layers of Ag, TiO 2 , and TiO 2 (N), and TiO 2 (N)/Ag/TiO 2 (N) sandwich layers were prepared in a reactive magnetron sputtering system As Supplementary Fig. S4 illustrates, five sputtering targets exist in the deposition system, but only two titanium (Ti) targets and one silver (Ag) target were used, and the base pressure was below 1.3 × 10 −5 Pa. To prepare pure TiO 2 or TiO 2 (N) and Ag, two Ti targets and one Ag target were used, respectively. Before sputtering, we used argon plasma to etch the surface of the substrate for 10 min to remove residual particles on the surface. The substrate holder was rotated at a speed of 5 rpm without applying substrate bias during deposition. Silicon wafer (100) and glass substrates were used. The substrate was nearly at room temperature without external heating. The target powers were set at 250 W each and 20 W in the DC mode for the two Ti targets and one Ag target, respectively. For pure TiO 2 deposition, the gas contained argon and oxygen with the fixed flow rates of Ar at 20 sccm and O 2 at 7 sccm, and the total pressure was approximately 4.7 × 10 −1 Pa (3.5 × 10 −3 Torr). For the TiO 2 (N) films, the gas contained Ar, O 2 , and N 2 at the flow rates of 20, 5–2, and 8 sccm, respectively, whereas for the Ag film, the gas used contained only Ar with a flow rate of 20 sccm without adding other gases. In addition, for forming a monolithic layer of Ag, TiO 2 , and TiO 2 (N), two series of sandwiched TiO 2 (N)Ag/TiO 2 (N) films were prepared. Furthermore, one series was prepared with various amounts of Ag in the sandwiched films, whereas the other series was prepared with a fixed amount of Ag but with various nitrogen contents in the sandwiched films. In the first series, the amount of Ag was regulated by changing the deposition time from 30 s to 60, 90, and 120 s, and the corresponding sample IDs were denoted as NTAxxxs, where xxx was the Ag deposition time. In the second series, the amount of Ag was fixed with the deposition time of 120 s, and the nitrogen contents in the TiO 2 (N) films were regulated by reducing the oxygen flow rates from 5 sccm to 4, 3, and then 2 sccm, and the corresponding sample IDs were denoted as N (x)TA, where x was the oxygen flow rate. The as-deposited films were further annealed at 500 °C for 1 h in the nitrogen atmosphere by using a conventional vacuum furnace. The deposition rate of TiO 2 (N) films varied with the oxygen flow rate and was approximately 350 nm/h with the oxygen flow rate of 4 sccm. The deposition rate of silver was approximately 7.5 nm/min. The deposition times of the typical sandwich TiO 2 (N)/Ag/TiO 2 (N) film used in this study were 1 h and 2 min, and 1 h. Thus, an average thickness of the Ag impregnated TiO 2 (N) films was 700 ± 50 nm. The thickness for the silver layer are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively. Characterization analysis The structure and crystallinity of films were analyzed using X-ray diffraction measurements recorded using the Rigaku X-ray diffractometer D/MAX-2500 V with a Cu Kα radiation (40 kV, 100 mA) source. The surface morphology and the cross-sectional view of the films were observed using the JEOL JEM-6500 F field-emission scanning electron microscopy. The UV–Vis absorption spectra of the films were recorded using a JASCO V-650 spectrophotometer ranging from 300 to 900 nm. The composition of samples was determined using an energy dispersive spectroscopy (Horiba, EMAX-ENERGY) and a K-Alpha™ X-ray photoelectron spectrometer using an AlK α X-ray radiation source to estimate elements semi-quantitatively. Antibacterial experiment Bacterial culturing and plating were performed following the previously described standard methods 5 47 48 49 . The bacterial concentration was determined using the standard plating method or from optical density readings at 595 nm (OD 595 ). For example, the conversion factor for E. coli BL 21 was calculated to be 1 × 10 9 colony-forming unit (CFU)/mL at OD 595 , and the cultures were diluted with the culture medium to 1 × 10 7 CFU/mL. The 1 × 10 6 CFU culture was allowed to drip on the sample (approximately 6.25 cm 2 ) and was then placed in a dark room or exposed under the visible light and at room temperature. The visible light source was an incandescent lamp (Classictone incandescent lamp, 60 W, Philips Taiwan; Taipei, Taiwan), and the illumination density was recorded using a light meter (model LX-102; Lutron Electronic Enterprises, Taipei, Taiwan). In the photocatalytic reaction, the illumination distance between the sample and lamp was approximately 10 cm, which was exposed for 30 min, and the light intensity on the sample surface was nearly 1.2 × 103 lux (lumen/m 2 ) (30 mW/cm 2 ). After illumination, 100 μL of the bacterial solution was recovered from the sample. Finally, the bacterial concentration was determined using standard dilution and plating methods, and the percentage of surviving bacteria was calculated 18 19 . S. pyogenes (strain M29588), pandrug-resistant A. baumannii (strain M36788), and S. aureus (strain SA02) were the clinical isolates provided by Buddhist Tzu-Chi General Hospital in Hualien, Taiwan 5 . E. coli and A. baumannii were grown and maintained in the lysogeny broth (LB) medium or LB agar (BD Diagnostics, Sparks, MD, USA) at 37 °C. By contrast, S. pyogenes and S. aureus were grown in the trypticase soy broth with yeast extract (TSBY) medium or TSBY agar (MDBio, Inc., Taipei, Taiwan) at 37 °C. When the visible light was used to elicit the photocatalysis reaction, a UV cut-off filter (400 nm; Edmund Optics, Barrington, NJ, USA) was used to prevent the illumination of small fractions with UV-range wavelength in the photocatalytic experiments. Photocatalytic properties Analysis methods were based on recently reported literatures 4 50 51 , which used the MB degradation rate to analyze the photocatalytic performance of impurity-doped TiO 2, in accordance with the generally accepted method and reported format of MB photodecomposition. Photocatalytic efficiency was evaluated by examining the decomposition of 10 ppm MB (Sigma-Aldrich, St. Louis, MO, USA). The MB concentration was determined according to the intensity of the light-absorption peak at 664 nm wavelength, as measured using a UV–Vis spectrometer. A fixed size (1 cm × 1 cm) of the sample sank in 2 mL of the MB aqueous solution. The visible light illumination was conducted using a fluorescent lamp (Philips; P-LF27W/865) with a wavelength distribution of approximately 400–750 nm and a maximum intensity at the range of 543–611 nm, producing an average power density of 4.2 mW/cm 2 at a distance of 8 cm between the visible light source and sample. Statistical analysis The means, standard deviations, and statistics of the quantifiable data were calculated using Microsoft Office Excel 2003, SigmaPlot 10, and SPSS 19. The significance of data was determined using a one-way analysis of variance followed by a post hoc Bonferroni corrected test. The probability of type I error α = 0.05 was recognized as statistically significant. Additional Information How to cite this article : Wong, M.-S. et al. Antibacterial property of Ag nanoparticle-impregnated N-doped titania films under visible light. Sci. Rep. 5 , 11978; doi: 10.1038/srep11978 (2015). Supplementary Material Supplementary Information

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394085/

Discovery of Selenocysteine as a Potential Nanomedicine Promotes Cartilage Regeneration With Enhanced Immune Response by Text Mining and Biomedical Databases

Background Unlike bone tissue, little progress has been made regarding cartilage regeneration, and many challenges remain. Furthermore, the key roles of cartilage lesion caused by traumas, focal lesion, or articular overstress remain unclear. Traumatic injuries to the meniscus as well as its degeneration are important risk factors for long-term joint dysfunction, degenerative joint lesions, and knee osteoarthritis (OA) a chronic joint disease characterized by degeneration of articular cartilage and hyperosteogeny. Nearly 50% of the individuals with meniscus injuries develop OA over time. Due to the limited inherent self-repair capacity of cartilage lesion, the Biomaterial drug-nanomedicine is considered to be a promising alternative. Therefore, it is important to elucidate the gene potential regeneration mechanisms and discover novel precise medication, which are identified through this study to investigate their function and role in pathogenesis. Methods We downloaded the mRNA microarray statistics GSE117999, involving paired cartilage lesion tissue samples from 12 OA patients and 12 patients from a control group. First, we analyzed these statistics to recognize the differentially expressed genes (DEGs). We then exposed the gene ontology (GO) annotation and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses for these DEGs. Protein-protein interaction (PPI) networks were then constructed, from which we attained eight significant genes after a functional interaction analysis. Finally, we identified a potential nanomedicine attained from this assay set, using a wide range of inhibitor information archived in the Search Tool for the Retrieval of Interacting Genes (STRING) database. Results Sixty-six DEGs were identified with our standards for meaning (adjusted P-value 0.900 (maximum confidence) were chosen to create the PPI networks: CAMP and TOLLIP , DEFA3 , HLA-DQA2 , SLC38A6 , SLC3A1 , FAM20A and ANO8 ( Figure 5 ). CAMP translates the set of an antimicrobial peptide family, characterized by chemoattractant, immune mediator induction, and immunoreaction regulation ( Snoussi et al., 2018 ; Uysal et al., 2019 ). Toll interacting protein ( TOLLIP ) encodes a ubiquitin-binding protein that regulates inflammatory signalling ( Diao et al., 2016 ; Shah et al., 2017 ). Defensin alpha 3 ( DEFA3 ) belongs to antimicrobial and cytotoxic peptides involved in host defence ( Okazaki et al., 2007 ; Froy and Sthoeger, 2009 ). Major histocompatibility complex, class II, DQ alpha 2 ( HLA-DQA2 ) are a family of the HLA class II alpha family. Many investigators suggest this involved in the release of CLIP molecule ( Rudy and Lew, 1994 ). Solute carrier family 38 member 6 ( SLC38A6 ) has possibly a relation with the glutamate-glutamine cycle regulation, responsible for preventing excitotoxicity ( Schioth et al., 2013 ; Bagchi et al., 2014 ). Solute carrier family 3 member 1 ( SLC3A1 ) translates a type II membrane glycoprotein that encodes neutral amino acids associated with cystinuria ( Ma et al., 2018 ). FAM20A golgi is associated with the secretory pathway pseudokinase ( FAM20A ), which encodes a protein that might function in haematopoiesis and is associated with amelogenesis imperfecta and gingival hyperplasia syndrome ( Beres et al., 2018 ; Koruyucu et al., 2018 ). Anoctamin 8 ( ANO8 ) is associated with a human disorder that is often overexpressed in diverse cancers ( Katoh and Katoh, 2005 ; Ousingsawat et al., 2011 ). Figure 5 PPI networks of DEGs. Sixty-six genes were clarified into the PPI network using the STRING databank. ↑ indicates up-Genes; ↓ indicates down-Genes. Drug-Gene Interactions The functional enrichment investigation was conducted to identify the last prospective HUB genes with the sifter stricture, p value 0.900 (maximum confidence) were chosen to create the PPI networks: CAMP and TOLLIP , DEFA3 , HLA-DQA2 , SLC38A6 , SLC3A1 , FAM20A and ANO8 ( Figure 5 ). CAMP translates the set of an antimicrobial peptide family, characterized by chemoattractant, immune mediator induction, and immunoreaction regulation ( Snoussi et al., 2018 ; Uysal et al., 2019 ). Toll interacting protein ( TOLLIP ) encodes a ubiquitin-binding protein that regulates inflammatory signalling ( Diao et al., 2016 ; Shah et al., 2017 ). Defensin alpha 3 ( DEFA3 ) belongs to antimicrobial and cytotoxic peptides involved in host defence ( Okazaki et al., 2007 ; Froy and Sthoeger, 2009 ). Major histocompatibility complex, class II, DQ alpha 2 ( HLA-DQA2 ) are a family of the HLA class II alpha family. Many investigators suggest this involved in the release of CLIP molecule ( Rudy and Lew, 1994 ). Solute carrier family 38 member 6 ( SLC38A6 ) has possibly a relation with the glutamate-glutamine cycle regulation, responsible for preventing excitotoxicity ( Schioth et al., 2013 ; Bagchi et al., 2014 ). Solute carrier family 3 member 1 ( SLC3A1 ) translates a type II membrane glycoprotein that encodes neutral amino acids associated with cystinuria ( Ma et al., 2018 ). FAM20A golgi is associated with the secretory pathway pseudokinase ( FAM20A ), which encodes a protein that might function in haematopoiesis and is associated with amelogenesis imperfecta and gingival hyperplasia syndrome ( Beres et al., 2018 ; Koruyucu et al., 2018 ). Anoctamin 8 ( ANO8 ) is associated with a human disorder that is often overexpressed in diverse cancers ( Katoh and Katoh, 2005 ; Ousingsawat et al., 2011 ). Figure 5 PPI networks of DEGs. Sixty-six genes were clarified into the PPI network using the STRING databank. ↑ indicates up-Genes; ↓ indicates down-Genes. Drug-Gene Interactions The functional enrichment investigation was conducted to identify the last prospective HUB genes with the sifter stricture, p value <1X10^10, as the edge in the GenCLiP 2.0 website ( Wang et al., 2019 ) ( Figure 6 ). DGIdb database was used to hunt the potential targets CAMP and its small organic compounds Selenocysteine. Selenocysteine is the main form of selenium in proteins. The study of selenocysteine biosynthesis and the mechanism of protein participation is a classic protein biochemical. This important supplement of molecular biology is also the basis for further research into the biological functions and applications of selenoproteins ( Stadtman, 1996 ). The structure of Selenocysteine is shown in Figure 7 . Figure 6 The functional enrichment analysis of eight applicant cartilage regeneration - associated genes (green shows that the gene is enhanced in the related pathway, black shows not on the contrary). Figure 7 The schematic structure of Selenocysteine. The Expression of CAMP was Inverse in OA Cartilage Tissues To examine the expression of mRNAs in the articular cartilage, qRT-PCR was performed in 3 samples. We found that the expression of CAMP was significantly upregulated in the OA group compared with the normal group ( Figure 8 ). Figure 8 The expression of CAMP transcripts differentially expressed between normal and osteoarthritis was validated by real-time PCR. The data is presented as mean with standard error of the mean. Discussion This analysis was designed to recognize prospective cartilage regeneration - associated genes, by comparing cartilage tissues in patients without osteoarthritis (arthroscopic partial meniscectomy). Thirty-five UP and 31 DOWN DEGs were recognized. We accomplished GO and KEGG comment examinations. Next, a PPI network was created, and eight significant genes were recognized. Finally, eight genes, CAMP and TOLLIP , DEFA3 , HLA-DQA2 , SLC38A6 , SLC3A1 , FAM20A , and ANO8 were identified as being significantly associated with immune response, immune mediator induction, and cell chemotaxis. The CAMP inhibitor Selenocysteine may be a nanomedicine potential candidate for cartilage regeneration. In summary, only the CAMP gene has its commercial inhibitor of the eight hub genes, there have been many studies into the role of transcription factors in regulating the promoter region of the CAMP gene ( Horibe et al., 2013 ). These studies have revealed that many biomolecules and factors can regulate the expression of the human antibacterial peptide CAMP gene, and that different signalling pathways can also affect the expression of the gene ( Hancock and Diamond, 2000 ). Due to the important role that the CAMP protein plays in infectious diseases, tumors, and other diseases, it may be a target for the diagnosis and treatment of these diseases ( Kovach et al., 2012 ). It is expected that the molecular regulatory mechanism of the CAMP gene during the occurrence of diseases, especially infectious diseases, will become a topic of significant further research ( Rosenberger et al., 2004 ). Selenocysteine is the main form of selenium in proteins. The determination of its codon UGA increases the number of amino acids that make up the biosynthetic protein, from 20 to 21 ( Mousa et al., 2017 ). It is also the only amino acid that contains a metalloid element. It is mostly located in the active center of selenoprotein or selenoenzyme (especially in antioxidant enzymes). At the same time, as an essential trace element of the human body, it has a very significant role in anti-oxidation, immune regulation, and anti-tumor roles ( Hatfield et al., 2014 ; Varlamova and Cheremushkina, 2017 ). This important supplement of molecular biology is also the basis for further research into the biological functions and applications of selenoproteins ( Stadtman, 1996 ). It is one of the hotspots of research in the fields of protein biochemistry and molecular biology ( Ren et al., 2018 ). Interestingly, Se deficiency has been proposed as an underlying contributing factor for the chronic osteochondral disease Kashin-Beck disease, an important but neglected disease in parts of China. It was previously shown that Se(IV), as well as superoxide dismutase, can prevent damage done to cultured human embryonic cartilage cells caused by various etiological environmental substrates, and increase the activity of GSHpx while decreasing the production of lipid peroxides ( Peng and Yang, 1991 ). In another report, the disease was associated with the incidence of Se deficiency in regions where the disease is prevalent ( Peng et al., 1992 ). Finally, a mixture containing glycosaminoglycans, selenium, and vitamin E was shown to be exceptionally capable of promoting osteochondral repair in a rabbit model of knee osteochondral defect after 6 weeks of treatment ( Handl et al., 2007 ). These past studies provide some evidence to support our current finding that selenocysteine may be effective drug to support cartilage regeneration. To conclude, 66 prospective candidate cartilage regeneration - associated genes, which have earlier been involved in numerous pathways related to pathogenesis. All of these DEG applicant cartilage regeneration -associated genes should be further established through biological trials. Moreover, CAMP , TOLLIP , DEFA3 , HLA-DQA2 , SLC38A6 , SLC3A1 , FAM20A , and ANO8 , as prospective markers for cartilage regeneration; they have not been linked previously, either in diagnosis or in research. Thus, the eight targets were considered to be potential therapeutic targets of cartilage regeneration. Thus, the CAMP inhibitor selenocysteine is considered to be a potential nanomedicine candidate for regenerative medicine. Data Availability Statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Author Contributions All authors contributed to the data analysis. JYe and BX completed the analysis using bioinformatics instruments. All authors contributed to the article and approved the submitted version. Funding This work was supported by the National Natural Science Foundation of China (ID. 5192010500) and the National Key R&D Program of China (No.2017YFB1303000). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020.01138/full#supplementary-material Click here for additional data file. Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705234/

Applying Science: Opportunities to Inform Disease Management Policy with Cooperative Research within a One Health Framework

The ongoing Ebola outbreak in West Africa and the current saiga antelope die off in Kazakhstan each represent very real and difficult to manage public or veterinary health crises. They also illustrate the importance of stable and funded surveillance and sound policy for intervention or disease control. While these two events highlight extreme cases of infectious disease (Ebola) or (possible) environmental exposure (saiga), diseases such as anthrax, brucellosis, tularemia, and plague are all zoonoses that pose risks and present surveillance challenges at the wildlife-livestock–human interfaces. These four diseases are also considered important actors in the threat of biological terror activities and have a long history as legacy biowarfare pathogens. This paper reviews recent studies done cooperatively between American and institutions within nations of the Former Soviet Union (FSU) focused on spatiotemporal, epidemiological, and ecological patterns of these four zoonoses. We examine recent studies and discuss the possible ways in which techniques, including ecological niche modeling, disease risk modeling, and spatiotemporal cluster analysis, can inform disease surveillance, control efforts, and impact policy. Our focus is to posit ways to apply science to disease management policy and actual management or mitigation practices. Across these examples, we illustrate the value of cooperative studies that bring together modern geospatial and epidemiological analyses to improve our understanding of the distribution of pathogens and diseases in livestock, wildlife, and humans. For example, ecological niche modeling can provide national level maps of pathogen distributions for surveillance planning, while space-time models can identify the timing and location of significant outbreak events for defining active control strategies. We advocate for the need to bring the results and the researchers from cooperative studies into the meeting rooms where policy is negotiated and use these results to inform future disease surveillance and control or eradication campaigns. Introduction The recent Ebola outbreak in West Africa ( 1 , 2 ) has been a shocking reminder of the ever present risk of rapidly spreading disease outbreaks and the reality of the difficulties involved in outbreak response ( 3 ) and surveillance. That outbreak, resulting in more than 10,000 human deaths (and still ongoing at the time of this writing), highlights the severity that re-emerging diseases can pose. The difficulties in identifying the potential zoonotic source of this infection ( 4 ) highlight the importance of understanding interactions at the human–wildlife interface ( 5 ) and of the importance of wildlife surveillance. The ongoing saiga antelope, Saiga tatarica ) die off in Kazakhstan, where more than 100,000 antelope have died in <8 weeks 1 (representing approximately 1/2 of all remaining saiga), presents another example of a severe loss of animals to unknown environmental contamination or pathogen exposure. While the ongoing Ebola outbreak and saiga die off represents the extreme of outbreak consequences (high human or wildlife mortality), several other important zoonoses have been re-emerging or maintaining with high incidence in known endemic areas. Diseases such as anthrax, brucellosis, tularemia, and plague are all zoonoses that pose risks and present surveillance challenges at the wildlife-livestock–human interfaces. These four diseases are also considered the most important pathogens for use in biological terror activities and have a long history as legacy biowarfare pathogens. Inter-specific transmission of each of these diseases demands that surveillance should include coordination between veterinary and human health personnel. This paper will review recent studies done cooperatively between American and institutions within nations of the Former Soviet Union (FSU) focused on spatiotemporal, epidemiological, and ecological patterns of these four zoonoses. These cooperative studies were all funded by the Defense Threat Reduction Agency's Cooperative Biological Engagement Program (or previous iterations of the program such as the Biological Threat Reduction Program). The goal of those efforts was to bring local and American scientists together to apply contemporary geospatial and epidemiological techniques to the issues of zoonotic disease persistence and transmission in the FSU with an emphasis on pathogens on the US Federal Select Agent Program pathogen list 2 . These projects were designed to be aimed at understanding disease spatiotemporal patterns and ecology. While the disease systems, study sites, and geospatial techniques differed, each of the studies highlighted here are related in that they applied geospatial modeling techniques to better understand disease patterns in humans, livestock, or wildlife. From these examples, we identify how study results may be use to inform national and international policy making to improve disease surveillance and inform control strategies. Broadly, these techniques rely on surveillance-derived datasets describing either case reports or serological evidence of disease presence at a spatial resolution smaller than the national level; most data were either farm or village locations mapped as GPS coordinate pairs or data aggregated to the rayon (district equivalent). Across the studies that we survey in this paper, it is noteworthy to point out a significant DTRA investment in data development for these projects in terms of data access, data compilation, and personnel time required. Such efforts are instrumental in cooperative biological engagements and are an important and often undervalued part of disease ecology studies. This paper illustrates how these techniques can inform disease surveillance, control efforts and impact policy. Our focus is to posit ways to apply science to disease management policy and actual management or mitigation practices. As part of this, we emphasize the importance of data sharing between human and veterinary health professionals and suggest actionable ways of improving data sharing. The objective of this paper were to review four diseases (anthrax, brucellosis, tularemia, and plague), that are important to cooperative biological engagement programs, in relation to published research studies and the resulting impact on policy and management decisions. Pathogens and Policies Anthrax Globally, anthrax is an important zoonosis with rapid onset and high mortality in wildlife and livestock, a cause of secondary human cases, and a security risk as a bioterror agent ( 6 ). The disease, caused by the Gram-positive, spore-forming bacterium Bacillus anthracis , can exert significant impacts on wildlife and livestock populations ( 7 , 8 ). Human cases are most commonly associated with slaughtering infected animals ( 9 ). However, anthrax ecology and transmission remain poorly understood and understudied ( 10 ). Broadly, outbreaks in enzootic regions arise in specific environmental conditions ( 11 ), such as semi-arid grasslands and steppes, and at times of seasonal transitions in climate ( 12 – 14 ). This is true for livestock and wildlife populations. Reports of wildlife cases are becoming more common in North America. Wildlife outbreaks are regular in white-tailed deer, Odocoileus virginianus , in Texas ( 10 ) and bison, Bison bison ssp., in Canada ( 15 ). Globally, livestock outbreaks have also been associated with contaminated animal feed ( 16 ). Anthrax occurs nearly worldwide with the heaviest livestock and human disease burden in resource limited countries ( 17 ). Animal cases occur across much of the globe, but human cases are most concentrated in Central Asia, Southern Africa, and the Caucasus. Over the last two decades, based on reviews from Promed Mail, Kyrgyzstan had some of the highest reported human case numbers. Additionally, Turkey ( 18 ) and Georgia ( 19 ) have both reported high numbers of human cases; Georgia has seen significant increases in human anthrax in the last few years ( 20 ). Disease control in these areas requires approaches that provide livestock control, such as vaccination campaigns and increased animal surveillance ( 21 ) and education programs targeting animal handlers and local slaughter operations. Anthrax in Azerbaijan and Georgia A recent cooperative study by Kracalik et al. ( 22 ) examined the spatial patterns of human anthrax in Azerbaijan looking at three time periods: Soviet (1984–1991), post-Soviet (1992–1995), and post-independence following livestock vaccination campaigns (1996–2010). Generally, the rate of human anthrax increased from the Soviet to the post-Soviet periods, which was most likely due to the drastic decrease in funding and changes in livestock management associated with Azerbaijani independence. From the post-Soviet through the post-independence period, there was a drastic decrease in the overall human incidence rate and a geographic shift in the concentration of reporting. These results suggest that livestock-associated human anthrax can be controlled with livestock vaccination campaigns. Furthermore, these findings indicate that surveillance cannot focus solely on areas of historic disease presence, but rather needs to be dynamic and sensitive to changes in livestock distributions and socioeconomic shifts associated with agricultural production. Like Azerbaijan, Georgia has seen an increase in human anthrax cases over the last decade, characterized by a drastic increase over the past 5 years ( 20 ). In recent years, Georgia has seen some of the highest rates of human anthrax globally. Cooperative research has shown the during the period 2000–2009, clusters of human anthrax cases in eastern and west central Georgia were associated ecological conditions that promote pathogen persistence ( 19 ). Consistent with research in Azerbaijan, these studies identified spatial heterogeneity across the Georgia landscape suggesting control efforts should be targeted to prioritize high risk areas. One major contrast between the two countries is the change in livestock vaccination policy, while Azerbaijan maintained vaccination through the last decade, Georgia ended compulsory vaccination in 2007, which lead to a drastic decrease in the number of animals vaccinated. This compulsory policy was nominally reinstated in 2012 following a historical rise in cases ( 20 ), though the total coverage of animals remains low. In both countries, anthrax surveillance remains highly anthropocentric, making it difficult to identify livestock populations at greatest risk. The use of spatial analyses in these studies provides a starting point for identifying areas where livestock surveillance should be prioritized. Another important finding of the cooperative research in Georgia is related to the human populations at risk. Classically, anthrax is considered a rural disease. However, the recent increase in Georgian anthrax has been associated with an increase in peri-urban and urban dwellers likely associated with the selling of contaminated meat ( 19 , 20 ). From a policy perspective, these results indicate a need for increased inspection and regulation of meat markets and sources of meat. Likewise, marketplaces may serve as points of outreach for public health education campaigns directed at both meat consumers and meat producers. Anthrax in Kazakhstan The Central Asian Steppe has a long history of livestock and human anthrax ( 23 ). Using data compiled from 1930 to 2006, several cooperative studies have increased our knowledge of the spatiotemporal, and ecological distribution of anthrax in Kazakhstan ( 21 , 24 – 27 ). These studies have implemented predictive modeling approaches to map the distribution of anthrax and B. anthracis in Kazakhstan. Research by Joyner et al. ( 26 ) used livestock anthrax outbreak locations reported between 1960 and 2000 to develop an ecological niche model. A subsequent study refined those predictions to identify areas of anthrax risk by using a combination of spatial analysis and generalized linear modeling ( 21 ). Both the ecological niche and risk models identify areas that can be used to define surveillance areas, with risk models best used to prioritize areas for preemptive annual livestock vaccination campaigns. From a policy perspective, passive surveillance zones require laboratory infrastructure and veterinary training to identify and test for anthrax should spring or summer time livestock die offs present. These studies illustrate specific examples of how maps of pathogens (ecological niche models) or disease risk (predicting clusters) can be used to prioritize surveillance and control. Such approaches can be introduced into other countries where anthrax surveillance and control remains a challenge. Anthrax in Ukraine Like Kazakhstan, Ukraine has a long documented history of livestock and human anthrax ( 28 ). Two cooperative studies have recently been published that illustrate the changing distribution of livestock anthrax in Ukraine. First, Bezymennyi et al. ( 28 ) examined spatial patterns of livestock anthrax (1913–2012) in pre- and post-Soviet Ukraine. Like many countries, during the last 50 years, Ukraine saw a drastic reduction in the overall reports of anthrax and a contraction of the geographic area where anthrax persists. Following the dissolution of the Soviet Union, livestock anthrax increased briefly (1992–1997). During the last 17-years, there has been a reduction in reporting and a contraction of anthrax foci on the landscape, although compulsory vaccination in Ukraine extends only to state owned livestock operations. Independent livestock owners are less likely to vaccinate, as they bear the burden of cost. A recent outbreak in eastern Ukraine illustrated the ongoing risk when an unvaccinated cow died of anthrax, and was subsequently fed to a domestic dog that also succumbed to infection ( 29 ). Furthermore, there was an attempt to sell the infected meat at a market, which could have resulted in human cases had the public health service not halted its sale ( 28 ). From a policy perspective, these studies identify the need to examine definitions of compulsory vaccination and the cost benefits of requiring private owners to bear vaccination costs. Similar to the situation in Georgia, these studies also identify the need to evaluate meat inspection processes and to meet regulatory infrastructure to better identify potential sources of contamination meat being sold to the public. An additional published collaborative study highlights the importance of including wildlife and surveillance for zoonotic diseases ( 30 ). In that study, the serological survey of wild boar collected from hunter check stations confirmed that at least some boars are exposed to B. anthracis in Ukraine. Most interesting was the fact that positive boars were identified in proximity to historical hotspots of anthrax but not directly overlapping with them. From a policy perspective, these results suggest surveillance should not be limited to livestock and should include wildlife populations. A similar strategy could be employed in Kazakhstan where there is potential overlap between saiga antelope and livestock ( 31 ) and may also be useful in the Caucasus, particularly Georgia, where livestock anthrax has been increasing. Brucellosis Brucellosis is one of the most widespread zoonotic diseases worldwide and is regarded as an emerging and re-emerging threat to public and veterinary health worldwide ( 32 ). Controlling brucellosis in humans is dependent upon limiting or reducing infection in livestock. Despite the availability of effective livestock vaccines for Brucella spp ., the disease continues to pose a global public health threat. Regions most heavily burdened by the disease include countries of the Mediterranean, Central Asia, Middle East, Latin America, Sub-Saharan African, and Balkan Peninsula ( 33 ). The causative agents of the disease are a group of pathogenic bacteria in the genus Brucella , which primarily infect animal reservoirs. Humans are often secondarily infected through the consumption of unpasteurized dairy products or coming into contact with infected animals during animal husbandry or meat processing ( 32 ). The primary agents of infection in humans are Brucella abortus (cattle), Brucella melitensis (sheep and goats), Brucella suis (swine), and Brucella canis (dogs) ( 34 ). Brucellosis in Azerbaijan The disease was first reported from Azerbaijan in 1922 and quickly spread to more than two-thirds of the rayons in <30 years ( 35 ). Recent governmental changes brought on by the collapse of the Soviet Union have likely contributed the persistence of the disease, due primarily to decreased funding for surveillance and eradication programs ( 33 ). A recent cooperative study evaluated changes in the distribution of human brucellosis over each of three 5-year intervals from 1995 to 2009 ( 36 ). That study documented rayon-level disease persistence in humans that can direct contemporary surveillance efforts. As was suggested for Georgia, these data can be used to evaluate the cost-effectiveness and human health impacts of livestock disease control programs. Brucellosis in Georgia Brucellosis is an endemic livestock disease in Georgia with a relatively high human burden ( 37 ). In a recent study, data from a contemporary livestock serological survey were used to estimate true disease prevalence in a Bayesian framework comparing each of three important areas within Georgia (Imereti – west central, Kvemo Kartli – southern, and Kakheti – eastern) ( 38 ). In total, these three regions represent approximately 45% of George's milk production and total livestock population. Results from this study of livestock match a recent study of human brucellosis rates in Georgia ( 39 ). From a policy perspective, the results of the livestock surveillance study can be used to inform brucellosis control and eradication campaigns focusing first on the areas with greatest livestock brucellosis prevalence. It is likely that control in these areas would result in immediate and measurable improvements in human brucellosis incidents. As was illustrated in the anthrax work of Kracalik et al. ( 20 , 22 ) in Azerbaijan, surveillance data from humans and animals could be analyzed in a one health framework that directly measure the impact of livestock control on human disease burden. Plague Plague is a flea borne zoonosis caused by the Gram-negative bacterium Yersinia pestis ( 40 ). Since the onset of the most recent pandemic, which started in China during the mid-nineteenth century, the geographic range of plague has greatly expanded ( 40 ). Classically, Y. pestis is maintained a sylvatic transmission cycle between partially resistant rodent hosts and adult hematophagous fleas ( 40 ) and foci can be maintained indefinitely in enzootic or maintenance cycles as long as sufficient numbers of rodent hosts and flea vectors are present. Natural plague reservoirs are active in Asia, and parts of the Russian Federation ( 41 ). Human plague cases have recently reemerged in this region ( 42 ). Plague in Azerbaijan Morris et al. ( 43 ) used historical maps of plague hosts derived annually between 1972 and 1985 to identify areas on the Azerbaijani landscape were plague carrying mammal densities were high and stable across years. The study digitized historical maps into active GIS layers and applied modern spatial analyses to evaluate areas of disease stability. The goal of the effort was to identify environmental conditions and geographic areas of historical sampling that may identify priority areas for contemporary sampling. In the years following Soviet independence, funds for plague surveillance were limited creating a gap in surveillance needing to be filled. That study identified a few key areas on the Azerbaijani landscape where plague may reemerge today. Beyond identifying those locations, Morris et al. ( 43 ) also identified which of those areas were in closest proximity or directly overlapping with increasing human populations. A similar effort field survey in neighboring Iran went a step further and use serological screening of dogs and small rodents and confirmed that historically defined foci could be active as many as two decades after the most recent zoological surveillance ( 44 ). From a policy perspective, the Morris et al. ( 43 ) all study in Azerbaijan can be used to prioritize exploratory surveillance in historically defined plague foci using serological testing or PCR-based methods. The areas identified as having increasing human population can further be used to prioritize those areas most important for surveillance. In this example, those areas with historically high rodent populations that saw little development until recently should be considered areas of highest likelihood of overlap between those contemporary rodent populations and human encroachment into those habitats. Tularemia Francisella tularensis , the causative agent of tularemia, is a zoonotic, Gram-negative bacterium that is broadly distributed across the Northern Hemisphere ( 45 ). Human exposure may occur through various pathways including arthropod bites, ingesting contaminated food products or liquids, inhaling aerosolized bacteria, or handling infected animals ( 45 ). Despite a global decline in reported human cases ( 46 ), tularemia has recently (re)emerged in several countries [summarized by Hightower et al. ( 47 )] Historically, outbreaks in the FSU were linked to small mammals and arthropods (ticks), possibly related to increases host or vector population abundance or density or water-borne outbreaks. Tularemia foci were previously described in the 1960s across a limited geography in the south of Ukraine where several arthropods and small mammals were recognized as competent vectors and hosts ( 48 ; 49 ). However, contemporary characterizations of the spatial distribution and composition of vectors and hosts remains incomplete and should remain a priority in countries with known tularemia outbreaks ( 47 ). Tularemia in Ukraine Tularemia has a long history in Ukraine. In an effort to understand the historical distribution and identify possible areas of contemporary surveillance, Hightower et al. ( 47 ) mapped the spatial patterns of historical F. tularensis isolates from the Ukrainian Central Sanitation and Epidemiological Station (CSES; now Ukrainian Center for Disease Control) and tested for space-time clusters on a database spanning more than 60 years. That study identified several historical foci that may serve as areas of persistence where disease reemergence is likely in humans. Additionally, that study defined tick vector and mammalian host species that should be priorities for sylvatic surveillance efforts. Hightower ( 50 ) use those data to construct small mammal and tick species-specific ecological niche models to estimate the potential geographic distribution of the pathogen across Ukraine. From a policy perspective, these studies provide Ukraine with specific local areas where disease surveillance should be focused. The space-time clusters defined can also serve as a baseline for comparing contemporary surveillance results. Additionally, Hightower et al. ( 47 ) identified areas where potential environmental exposure to contaminated crops may serve as an important transmission source that may not be detected from small mammal surveillance. These areas require additional infrastructure for testing such samples in the absence of human cases. A Call for One Health Strategies for Improved Disease Surveillance and Control As illustrated in the examples presented here, these zoonoses cross the human/livestock/wildlife interface. Because of this, effective surveillance and control strategies require a one health approach. These strategies should target different populations (human, livestock, wildlife) across the geography of the diseases. Central Asia and the Caucasus require improved livestock surveillance and vaccination strategies aimed at reducing the livestock burden of disease; this is true for both anthrax and brucellosis. Such strategies should have significant livestock and human health benefits. In contrast, the wildlife situation of anthrax in wild boar in Ukraine poses a different challenge. Anthrax vaccination is untenable in wildlife ( 11 , 51 ). In the absence of vaccination, rapid carcass cleanup during outbreaks is the only apparent means of reducing the size of outbreaks ( 51 ). However, it is important that burial efforts result in deep burial to reduce potential for inadvertent digging that exposes carcasses, as bone can remain infectious for long periods of time. Because of this, there is a need to better understand the timing and spatial distribution of epizootics; such information comes from increased surveillance and environmental sampling. This would allow managers to stage preemptive surveillance and control efforts. Ecological niche models (also referred to as species distribution models) can be used to broadly define the geographic range of B. anthracis, Y. pestis , and F. tularensis to better inform surveillance efforts. When coupled with spatial analyses of outbreaks, we can identify areas of high risk (where the clusters occur) and areas where passive surveillance should increase (where niche models predict in under investigated areas). Specific to anthrax, expanded surveillance and wildlife telemetry studies can assist in understanding the relationship between individual animals in a herd and their use of the landscape during anthrax risk periods ( 11 ). Such studies can shed light on the role of animal behavior in contacting the environmental reservoir for the pathogen. This could greatly improve our understanding of anthrax in Ukrainian boar populations. Much of the recent spatial modeling of anthrax has relied on mortality data to understand the disease, which likely underestimates the extent and intensity of the disease ( 52 ). The data from boars suggest that pathogen exposure occurs beyond known foci, even without reported mortality events. Coupling data from across temporal and spatial scales and across host species in a modeling framework should provide better information on the disease that can be shared with wildlife managers, regional public health officers, and policy makers. We have also illustrated that disease surveillance in these countries is anthropocentric and requires greater data sharing, cooperation, and funding to support joint human and veterinary surveillance and disease control. The work on anthrax in Azerbaijan highlights the role of livestock disease control for improving human health. Future efforts should expand this type of research to brucellosis studies across these countries. Regional approaches to zoonotic and transboundary diseases often require regional efforts in mitigation and vaccination strategies that are effective in reducing the propagation of the infectious diseases. Through the above collaborations discussed in this paper, the countries of Azerbaijan, Georgia, Kazakhstan, and Ukraine have developed a Regional Disease Surveillance Working Group (RDSWG) to foster communication and collaboration in disease surveillance of these pathogens. This is in direct response to the cooperative engagements with the countries and the results of research studies that show the importance of sharing of data and communication in reducing the impact of transboundary diseases. The examples of plague and tularemia presented here illustrate the importance of continued small mammal and associated vector surveys across these countries. Each of these diseases is maintained in small mammal populations and is likely to maintain over long periods of time. Ultimately, surveillance is time-consuming and expensive and must be balanced against risk. In the work in Azerbaijan, Morris et al. ( 43 ) illustrated the use of high resolution spatial data mapping human populations can be compared to areas of historical disease foci to focus what are realistically limited surveillance dollars to those areas of greatest likelihood of human infection. Anthrax Globally, anthrax is an important zoonosis with rapid onset and high mortality in wildlife and livestock, a cause of secondary human cases, and a security risk as a bioterror agent ( 6 ). The disease, caused by the Gram-positive, spore-forming bacterium Bacillus anthracis , can exert significant impacts on wildlife and livestock populations ( 7 , 8 ). Human cases are most commonly associated with slaughtering infected animals ( 9 ). However, anthrax ecology and transmission remain poorly understood and understudied ( 10 ). Broadly, outbreaks in enzootic regions arise in specific environmental conditions ( 11 ), such as semi-arid grasslands and steppes, and at times of seasonal transitions in climate ( 12 – 14 ). This is true for livestock and wildlife populations. Reports of wildlife cases are becoming more common in North America. Wildlife outbreaks are regular in white-tailed deer, Odocoileus virginianus , in Texas ( 10 ) and bison, Bison bison ssp., in Canada ( 15 ). Globally, livestock outbreaks have also been associated with contaminated animal feed ( 16 ). Anthrax occurs nearly worldwide with the heaviest livestock and human disease burden in resource limited countries ( 17 ). Animal cases occur across much of the globe, but human cases are most concentrated in Central Asia, Southern Africa, and the Caucasus. Over the last two decades, based on reviews from Promed Mail, Kyrgyzstan had some of the highest reported human case numbers. Additionally, Turkey ( 18 ) and Georgia ( 19 ) have both reported high numbers of human cases; Georgia has seen significant increases in human anthrax in the last few years ( 20 ). Disease control in these areas requires approaches that provide livestock control, such as vaccination campaigns and increased animal surveillance ( 21 ) and education programs targeting animal handlers and local slaughter operations. Anthrax in Azerbaijan and Georgia A recent cooperative study by Kracalik et al. ( 22 ) examined the spatial patterns of human anthrax in Azerbaijan looking at three time periods: Soviet (1984–1991), post-Soviet (1992–1995), and post-independence following livestock vaccination campaigns (1996–2010). Generally, the rate of human anthrax increased from the Soviet to the post-Soviet periods, which was most likely due to the drastic decrease in funding and changes in livestock management associated with Azerbaijani independence. From the post-Soviet through the post-independence period, there was a drastic decrease in the overall human incidence rate and a geographic shift in the concentration of reporting. These results suggest that livestock-associated human anthrax can be controlled with livestock vaccination campaigns. Furthermore, these findings indicate that surveillance cannot focus solely on areas of historic disease presence, but rather needs to be dynamic and sensitive to changes in livestock distributions and socioeconomic shifts associated with agricultural production. Like Azerbaijan, Georgia has seen an increase in human anthrax cases over the last decade, characterized by a drastic increase over the past 5 years ( 20 ). In recent years, Georgia has seen some of the highest rates of human anthrax globally. Cooperative research has shown the during the period 2000–2009, clusters of human anthrax cases in eastern and west central Georgia were associated ecological conditions that promote pathogen persistence ( 19 ). Consistent with research in Azerbaijan, these studies identified spatial heterogeneity across the Georgia landscape suggesting control efforts should be targeted to prioritize high risk areas. One major contrast between the two countries is the change in livestock vaccination policy, while Azerbaijan maintained vaccination through the last decade, Georgia ended compulsory vaccination in 2007, which lead to a drastic decrease in the number of animals vaccinated. This compulsory policy was nominally reinstated in 2012 following a historical rise in cases ( 20 ), though the total coverage of animals remains low. In both countries, anthrax surveillance remains highly anthropocentric, making it difficult to identify livestock populations at greatest risk. The use of spatial analyses in these studies provides a starting point for identifying areas where livestock surveillance should be prioritized. Another important finding of the cooperative research in Georgia is related to the human populations at risk. Classically, anthrax is considered a rural disease. However, the recent increase in Georgian anthrax has been associated with an increase in peri-urban and urban dwellers likely associated with the selling of contaminated meat ( 19 , 20 ). From a policy perspective, these results indicate a need for increased inspection and regulation of meat markets and sources of meat. Likewise, marketplaces may serve as points of outreach for public health education campaigns directed at both meat consumers and meat producers. Anthrax in Kazakhstan The Central Asian Steppe has a long history of livestock and human anthrax ( 23 ). Using data compiled from 1930 to 2006, several cooperative studies have increased our knowledge of the spatiotemporal, and ecological distribution of anthrax in Kazakhstan ( 21 , 24 – 27 ). These studies have implemented predictive modeling approaches to map the distribution of anthrax and B. anthracis in Kazakhstan. Research by Joyner et al. ( 26 ) used livestock anthrax outbreak locations reported between 1960 and 2000 to develop an ecological niche model. A subsequent study refined those predictions to identify areas of anthrax risk by using a combination of spatial analysis and generalized linear modeling ( 21 ). Both the ecological niche and risk models identify areas that can be used to define surveillance areas, with risk models best used to prioritize areas for preemptive annual livestock vaccination campaigns. From a policy perspective, passive surveillance zones require laboratory infrastructure and veterinary training to identify and test for anthrax should spring or summer time livestock die offs present. These studies illustrate specific examples of how maps of pathogens (ecological niche models) or disease risk (predicting clusters) can be used to prioritize surveillance and control. Such approaches can be introduced into other countries where anthrax surveillance and control remains a challenge. Anthrax in Ukraine Like Kazakhstan, Ukraine has a long documented history of livestock and human anthrax ( 28 ). Two cooperative studies have recently been published that illustrate the changing distribution of livestock anthrax in Ukraine. First, Bezymennyi et al. ( 28 ) examined spatial patterns of livestock anthrax (1913–2012) in pre- and post-Soviet Ukraine. Like many countries, during the last 50 years, Ukraine saw a drastic reduction in the overall reports of anthrax and a contraction of the geographic area where anthrax persists. Following the dissolution of the Soviet Union, livestock anthrax increased briefly (1992–1997). During the last 17-years, there has been a reduction in reporting and a contraction of anthrax foci on the landscape, although compulsory vaccination in Ukraine extends only to state owned livestock operations. Independent livestock owners are less likely to vaccinate, as they bear the burden of cost. A recent outbreak in eastern Ukraine illustrated the ongoing risk when an unvaccinated cow died of anthrax, and was subsequently fed to a domestic dog that also succumbed to infection ( 29 ). Furthermore, there was an attempt to sell the infected meat at a market, which could have resulted in human cases had the public health service not halted its sale ( 28 ). From a policy perspective, these studies identify the need to examine definitions of compulsory vaccination and the cost benefits of requiring private owners to bear vaccination costs. Similar to the situation in Georgia, these studies also identify the need to evaluate meat inspection processes and to meet regulatory infrastructure to better identify potential sources of contamination meat being sold to the public. An additional published collaborative study highlights the importance of including wildlife and surveillance for zoonotic diseases ( 30 ). In that study, the serological survey of wild boar collected from hunter check stations confirmed that at least some boars are exposed to B. anthracis in Ukraine. Most interesting was the fact that positive boars were identified in proximity to historical hotspots of anthrax but not directly overlapping with them. From a policy perspective, these results suggest surveillance should not be limited to livestock and should include wildlife populations. A similar strategy could be employed in Kazakhstan where there is potential overlap between saiga antelope and livestock ( 31 ) and may also be useful in the Caucasus, particularly Georgia, where livestock anthrax has been increasing. Brucellosis Brucellosis is one of the most widespread zoonotic diseases worldwide and is regarded as an emerging and re-emerging threat to public and veterinary health worldwide ( 32 ). Controlling brucellosis in humans is dependent upon limiting or reducing infection in livestock. Despite the availability of effective livestock vaccines for Brucella spp ., the disease continues to pose a global public health threat. Regions most heavily burdened by the disease include countries of the Mediterranean, Central Asia, Middle East, Latin America, Sub-Saharan African, and Balkan Peninsula ( 33 ). The causative agents of the disease are a group of pathogenic bacteria in the genus Brucella , which primarily infect animal reservoirs. Humans are often secondarily infected through the consumption of unpasteurized dairy products or coming into contact with infected animals during animal husbandry or meat processing ( 32 ). The primary agents of infection in humans are Brucella abortus (cattle), Brucella melitensis (sheep and goats), Brucella suis (swine), and Brucella canis (dogs) ( 34 ). Brucellosis in Azerbaijan The disease was first reported from Azerbaijan in 1922 and quickly spread to more than two-thirds of the rayons in <30 years ( 35 ). Recent governmental changes brought on by the collapse of the Soviet Union have likely contributed the persistence of the disease, due primarily to decreased funding for surveillance and eradication programs ( 33 ). A recent cooperative study evaluated changes in the distribution of human brucellosis over each of three 5-year intervals from 1995 to 2009 ( 36 ). That study documented rayon-level disease persistence in humans that can direct contemporary surveillance efforts. As was suggested for Georgia, these data can be used to evaluate the cost-effectiveness and human health impacts of livestock disease control programs. Brucellosis in Georgia Brucellosis is an endemic livestock disease in Georgia with a relatively high human burden ( 37 ). In a recent study, data from a contemporary livestock serological survey were used to estimate true disease prevalence in a Bayesian framework comparing each of three important areas within Georgia (Imereti – west central, Kvemo Kartli – southern, and Kakheti – eastern) ( 38 ). In total, these three regions represent approximately 45% of George's milk production and total livestock population. Results from this study of livestock match a recent study of human brucellosis rates in Georgia ( 39 ). From a policy perspective, the results of the livestock surveillance study can be used to inform brucellosis control and eradication campaigns focusing first on the areas with greatest livestock brucellosis prevalence. It is likely that control in these areas would result in immediate and measurable improvements in human brucellosis incidents. As was illustrated in the anthrax work of Kracalik et al. ( 20 , 22 ) in Azerbaijan, surveillance data from humans and animals could be analyzed in a one health framework that directly measure the impact of livestock control on human disease burden. Plague Plague is a flea borne zoonosis caused by the Gram-negative bacterium Yersinia pestis ( 40 ). Since the onset of the most recent pandemic, which started in China during the mid-nineteenth century, the geographic range of plague has greatly expanded ( 40 ). Classically, Y. pestis is maintained a sylvatic transmission cycle between partially resistant rodent hosts and adult hematophagous fleas ( 40 ) and foci can be maintained indefinitely in enzootic or maintenance cycles as long as sufficient numbers of rodent hosts and flea vectors are present. Natural plague reservoirs are active in Asia, and parts of the Russian Federation ( 41 ). Human plague cases have recently reemerged in this region ( 42 ). Plague in Azerbaijan Morris et al. ( 43 ) used historical maps of plague hosts derived annually between 1972 and 1985 to identify areas on the Azerbaijani landscape were plague carrying mammal densities were high and stable across years. The study digitized historical maps into active GIS layers and applied modern spatial analyses to evaluate areas of disease stability. The goal of the effort was to identify environmental conditions and geographic areas of historical sampling that may identify priority areas for contemporary sampling. In the years following Soviet independence, funds for plague surveillance were limited creating a gap in surveillance needing to be filled. That study identified a few key areas on the Azerbaijani landscape where plague may reemerge today. Beyond identifying those locations, Morris et al. ( 43 ) also identified which of those areas were in closest proximity or directly overlapping with increasing human populations. A similar effort field survey in neighboring Iran went a step further and use serological screening of dogs and small rodents and confirmed that historically defined foci could be active as many as two decades after the most recent zoological surveillance ( 44 ). From a policy perspective, the Morris et al. ( 43 ) all study in Azerbaijan can be used to prioritize exploratory surveillance in historically defined plague foci using serological testing or PCR-based methods. The areas identified as having increasing human population can further be used to prioritize those areas most important for surveillance. In this example, those areas with historically high rodent populations that saw little development until recently should be considered areas of highest likelihood of overlap between those contemporary rodent populations and human encroachment into those habitats. Tularemia Francisella tularensis , the causative agent of tularemia, is a zoonotic, Gram-negative bacterium that is broadly distributed across the Northern Hemisphere ( 45 ). Human exposure may occur through various pathways including arthropod bites, ingesting contaminated food products or liquids, inhaling aerosolized bacteria, or handling infected animals ( 45 ). Despite a global decline in reported human cases ( 46 ), tularemia has recently (re)emerged in several countries [summarized by Hightower et al. ( 47 )] Historically, outbreaks in the FSU were linked to small mammals and arthropods (ticks), possibly related to increases host or vector population abundance or density or water-borne outbreaks. Tularemia foci were previously described in the 1960s across a limited geography in the south of Ukraine where several arthropods and small mammals were recognized as competent vectors and hosts ( 48 ; 49 ). However, contemporary characterizations of the spatial distribution and composition of vectors and hosts remains incomplete and should remain a priority in countries with known tularemia outbreaks ( 47 ). Tularemia in Ukraine Tularemia has a long history in Ukraine. In an effort to understand the historical distribution and identify possible areas of contemporary surveillance, Hightower et al. ( 47 ) mapped the spatial patterns of historical F. tularensis isolates from the Ukrainian Central Sanitation and Epidemiological Station (CSES; now Ukrainian Center for Disease Control) and tested for space-time clusters on a database spanning more than 60 years. That study identified several historical foci that may serve as areas of persistence where disease reemergence is likely in humans. Additionally, that study defined tick vector and mammalian host species that should be priorities for sylvatic surveillance efforts. Hightower ( 50 ) use those data to construct small mammal and tick species-specific ecological niche models to estimate the potential geographic distribution of the pathogen across Ukraine. From a policy perspective, these studies provide Ukraine with specific local areas where disease surveillance should be focused. The space-time clusters defined can also serve as a baseline for comparing contemporary surveillance results. Additionally, Hightower et al. ( 47 ) identified areas where potential environmental exposure to contaminated crops may serve as an important transmission source that may not be detected from small mammal surveillance. These areas require additional infrastructure for testing such samples in the absence of human cases. A Call for One Health Strategies for Improved Disease Surveillance and Control As illustrated in the examples presented here, these zoonoses cross the human/livestock/wildlife interface. Because of this, effective surveillance and control strategies require a one health approach. These strategies should target different populations (human, livestock, wildlife) across the geography of the diseases. Central Asia and the Caucasus require improved livestock surveillance and vaccination strategies aimed at reducing the livestock burden of disease; this is true for both anthrax and brucellosis. Such strategies should have significant livestock and human health benefits. In contrast, the wildlife situation of anthrax in wild boar in Ukraine poses a different challenge. Anthrax vaccination is untenable in wildlife ( 11 , 51 ). In the absence of vaccination, rapid carcass cleanup during outbreaks is the only apparent means of reducing the size of outbreaks ( 51 ). However, it is important that burial efforts result in deep burial to reduce potential for inadvertent digging that exposes carcasses, as bone can remain infectious for long periods of time. Because of this, there is a need to better understand the timing and spatial distribution of epizootics; such information comes from increased surveillance and environmental sampling. This would allow managers to stage preemptive surveillance and control efforts. Ecological niche models (also referred to as species distribution models) can be used to broadly define the geographic range of B. anthracis, Y. pestis , and F. tularensis to better inform surveillance efforts. When coupled with spatial analyses of outbreaks, we can identify areas of high risk (where the clusters occur) and areas where passive surveillance should increase (where niche models predict in under investigated areas). Specific to anthrax, expanded surveillance and wildlife telemetry studies can assist in understanding the relationship between individual animals in a herd and their use of the landscape during anthrax risk periods ( 11 ). Such studies can shed light on the role of animal behavior in contacting the environmental reservoir for the pathogen. This could greatly improve our understanding of anthrax in Ukrainian boar populations. Much of the recent spatial modeling of anthrax has relied on mortality data to understand the disease, which likely underestimates the extent and intensity of the disease ( 52 ). The data from boars suggest that pathogen exposure occurs beyond known foci, even without reported mortality events. Coupling data from across temporal and spatial scales and across host species in a modeling framework should provide better information on the disease that can be shared with wildlife managers, regional public health officers, and policy makers. We have also illustrated that disease surveillance in these countries is anthropocentric and requires greater data sharing, cooperation, and funding to support joint human and veterinary surveillance and disease control. The work on anthrax in Azerbaijan highlights the role of livestock disease control for improving human health. Future efforts should expand this type of research to brucellosis studies across these countries. Regional approaches to zoonotic and transboundary diseases often require regional efforts in mitigation and vaccination strategies that are effective in reducing the propagation of the infectious diseases. Through the above collaborations discussed in this paper, the countries of Azerbaijan, Georgia, Kazakhstan, and Ukraine have developed a Regional Disease Surveillance Working Group (RDSWG) to foster communication and collaboration in disease surveillance of these pathogens. This is in direct response to the cooperative engagements with the countries and the results of research studies that show the importance of sharing of data and communication in reducing the impact of transboundary diseases. The examples of plague and tularemia presented here illustrate the importance of continued small mammal and associated vector surveys across these countries. Each of these diseases is maintained in small mammal populations and is likely to maintain over long periods of time. Ultimately, surveillance is time-consuming and expensive and must be balanced against risk. In the work in Azerbaijan, Morris et al. ( 43 ) illustrated the use of high resolution spatial data mapping human populations can be compared to areas of historical disease foci to focus what are realistically limited surveillance dollars to those areas of greatest likelihood of human infection. Conclusion Across these examples, we have illustrated the value of cooperative studies that bring together modern geospatial and epidemiological analyses with historical and contemporary disease surveillance to improve our understanding of the distribution of pathogens and diseases in livestock, wildlife, and humans. The results of these efforts illustrated in this paper are all available as peer-reviewed studies. We advocate for the need to bring the results and the researchers from cooperative studies into meetings where policy is negotiated to best use these results to inform future disease surveillance and control or eradication campaigns. Each of the studies highlighted here identify local spatial heterogeneity in the distributions of these diseases. Such information should be considered critical for policymakers when considering strategies for reducing eradicating these diseases. Conflict of Interest Statement Dr. Jeanne Fair is currently a program officer with the Defense Threat Reduction Agency. She was not involved with the studies reviewed in this paper, nor involved in this decisions to fund those projects. Jason K. Blackburn and Ian T. Kracalik have no conflict of interest to declare. Funding JB and IK were partially funded by the Defense Threat Reduction Agency through the Academic Engagement Program administered by Penn State University. Support for JB was also provided by the Emerging Pathogens Institute at the University of Florida.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3532170/

Modeling rapidly disseminating infectious disease during mass gatherings

We discuss models for rapidly disseminating infectious diseases during mass gatherings (MGs), using influenza as a case study. Recent innovations in modeling and forecasting influenza transmission dynamics at local, regional, and global scales have made influenza a particularly attractive model scenario for MG. We discuss the behavioral, medical, and population factors for modeling MG disease transmission, review existing model formulations, and highlight key data and modeling gaps related to modeling MG disease transmission. We argue that the proposed improvements will help integrate infectious-disease models in MG health contingency plans in the near future, echoing modeling efforts that have helped shape influenza pandemic preparedness plans in recent years. Background Mass gatherings (MGs) occur around the world on a relatively frequent basis, and include events as diverse as sport, religious, and educational activities [ 1 ]. MGs are typically defined as the influx of a large number of people at a specific location, for a specific purpose, and for a defined period of time; much of the available literature refers to gatherings exceeding 25,000 individuals [ 1 ]. Some MGs are spontaneous, whereas others will have been planned several years in advance, and include events as varied as royal weddings, the Olympic Games, or the Muslim Hajj pilgrimage [ 1 ]. A range of respiratory and waterborne diseases outbreaks have been reported at previous MGs, and are responsible for an estimated 14 out of 21 documented events, with occasional onward dissemination beyond the initial location of the MG [ 2 ]. A comprehensive review of infectious diseases at MGs highlights that influenza is the acute upper respiratory tract pathogen most commonly reported in these settings, partly because of its short incubation period and ubiquitous nature [ 2 - 4 ]. Influenza outbreaks have been reported in settings of varying scale, ranging from outbreaks in close living conditions (including troop ships [ 5 ] and airplanes [ 6 , 7 ]) to outbreaks in large public gatherings (such as the Winter Olympics in 2002 in Salt Lake City, USA[ 8 ] and the World Youth Day in July 2008 in Sydney [ 9 ]). Further, respiratory illness was the most common diagnosis made at a surveillance clinic during the 2008 Olympic and Paralympic Games held in Beijing and other cities of China [ 10 ]. More recently, increased 2009 pandemic influenza virus activity has been reported during several music festivals [ 11 ], and the pandemic transmission risk at the 2009 Hajj pilgrimage and the Asian South Games was deemed sufficiently important to prompt a strengthening disease surveillance systems and implementation of vaccination programs [ 2 , 12 ]. In addition, outbreaks of vaccine-preventable diseases can occur during MGs; for instance, measles outbreaks were reported during the 2008 European Football Championship in Austria and Switzerland, with onward transmission to France, Germany, and Spain [ 13 ]. Similarly, recurrent outbreaks of meningococcal meningitis have been well publicized during past Hajj pilgrimages, which attract millions of pilgrims, prompting mandatory vaccination of all participating visitors by the Government of Saudi Arabia. Olympic events are perhaps the largest and most anticipated of all MGs, and yet, despite their scale, experience suggests that the probability of a large-scale infectious disease event is typically low. Indeed, the proportion of healthcare visits in Sydney during the 2000 Olympics for infectious diseases was less than 1%. In the 2006 winter Olympics in Torino, Italy, surveillance for acute gastroenteritis, influenza-like illnesses, measles, and other infections found incidences similar to non-Olympics time periods [ 14 ]. Similar experiences were reported in the earlier Summer Olympics in Atlanta (1996) and Los Angeles (1984) [ 10 ]. However, epidemiological alerts can happen, as shown by an anthrax alert at the Salt Lake City airport on the first night of the 2002 Winter Olympics, which was found to be due to an environmental sample that falsely tested positive [ 15 ]. The relatively low frequency of outbreaks reported during MGs could stem from several factors. MG events are naturally ideal settings for infectious-disease transmission because of the large numbers of dense contacts. However, the probability of observing a large-scale outbreak given the introduction of a particular infectious disease introduction is relatively small, owing to the high disease-extinction rates associated with high stochasticity in heterogeneous populations typical of MGs (for example, heterogeneity in background susceptibility, infectiousness, and vaccination rates) [ 16 ]. In addition, limited or overwhelmed disease surveillance systems can complicate early outbreak detection, reporting, and control during MGs. Moreover, for infectious diseases with long incubation periods such as tuberculosis, transmission may not be noticed during the time course of an MG event [ 2 ]. Overall, although the probability of large-scale epidemics arising from MGs seems, based on previous experience, to be low, such events do have the potential to generate unprecedented rates of morbidity and mortality at local and global levels (that is, these are low probability, high-impact events, similar to the risk of emergence of a novel pandemic virus). A review of key data and methodological needs is useful to improve assessment of epidemic risk during MGs and to guide public-health interventions. In this article, we discuss modeling aspects and data requirements relating to modeling respiratory-disease outbreaks and responses during MG, with a specific focus on influenza and other respiratory diseases. The focus on respiratory diseases, particularly influenza, is based on the observations that respiratory infections are the most commonly reported diseases at MGs, and that influenza has been the subject of a rich modeling literature that can be used as a model for other pathogens. In particular, the A/H5N1 avian influenza threat and the 2009 A/H1N1 pandemic have helped improved influenza models and forecasts [ 17 - 22 ]. Because disease transmission during MG events is often tightly connected to the community at large via local and global transportation networks, we also considered onward transmission at broader spatial scales (city, region, world) and the relevant public-health control interventions. In the first section of this paper, we review key modeling concepts, and characterize the population and social network of MG participants. In the second section, we discuss how to incorporate these specificities into existing disease-transmission models. In the third section, we highlight key data gaps related to models of respiratory diseases at MG, and suggest innovative approaches to fill those gaps and better inform future models. Key disease-model concepts The risk of infectious-disease transmission during MGs is directly related to the characteristics of the participants and their environment [ 2 ]. The effects of these factors on the risk of disease transmission can be integrated in key epidemiological quantities: the basic reproduction number, R 0 , and the effective reproduction number, R [ 23 - 25 ]. R 0 measures the average number of secondary cases generated by a primary infectious individual in a completely susceptible population. A more practical quantity is the effective reproduction number, R , which quantifies the potential for infectious-disease transmission in a population that may be only partially susceptible owing to prior exposure or vaccination [ 26 ]. From a probabilistic perspective, R and R 0 denote the mean of the distribution of secondary cases for each single primary case in the population to account for individual-level variation in, for instance, infectiousness and contact rates [ 16 ]. R can be formulated as the product of three quantities: the contact rate, the conditional probability of transmission per contact, and the duration of the infectious period [ 23 , 24 ], hence we can expect higher values for R in crowded or confined conditions associated with MGs. Overall, R of less than 1 indicates that a major epidemic is likely to occur whereas R of greater than 1 indicates that transmission chains cannot be sustained. Respiratory infections cover a wide range of transmission potentials, with R being estimated at 1.2 to 1.6 for seasonal influenza [ 27 ], 1.4 to 5.2 for pandemics [ 28 - 32 ], 15 for pertussis, 17 for measles [ 23 ], and 1.2 to 1.3 for meningococcal meningitis [ 33 ]. Another key quantity for disease control is the serial interval, which measures the time interval between successive cases and sets the time scale for epidemic growth, and hence the speed with which intervention measures need to be initiated [ 34 ]. Despite the relatively low transmission potential of influenza, outbreaks are difficult to control in real time because of its short serial interval of 2 to 3 days and the fact that a substantial fraction of transmission events occur before a case becomes symptomatic [ 34 ]. Characterizing population at risk and contact networks during mass gatherings Structure of disease-relevant contact network Perhaps the most challenging aspect of modeling infectious-disease transmission in the context of MG lies in appropriately capturing the complexity of dynamic human interactions and contact networks to provide valid and reliable predictions of transmission potential and attack rates. The dynamic social contact networks during MGs will depend on a number of factors, including the type (for example, confined versus open setting), size and duration of the event, the schedule of activities, the capacity of the corresponding locations in which the activities take place (for example, Olympic stadium, aquatic center), and specific crowd behavior (for example, in the case of diseases spread by aerosols, the use and sharing of plastic blowing horns by sports fans to provide audible support for their team [ 35 ]). Most contact-network surveys have been based on cumbersome questionnaires with arbitrary physical definitions of 'social' contacts between individuals, but recent technological advances in wearable sensing devices allows unobtrusive and unsupervised quantification of contact intensity and duration. Radiofrequency identification devices were recently used to monitor in great detail the face-to-face contact patterns relevant to the spread of infectious diseases [ 36 ], particularly in primary schools [ 37 , 38 ]. Such detailed analysis of contact patterns highlighted important departures from homogeneous mixing assumptions, which should be integrated in disease models focused on outbreaks in schools or on childhood infections [ 38 - 41 ]. Another recent study analyzed real-time close contact interactions between conference participants, and found that the duration of contacts between the participants provided a good approximation to the epidemic dynamics compared with results obtained by modeling the full dynamic contact network [ 42 , 43 ]. To our knowledge, no detailed survey of contacts has been performed at MG events. A key avenue for future research would be to gain more information on contact networks in this context, perhaps by distributing sensing devices to a sample of MG participants. Although it could be challenging to enroll representative populations of MGs, recent efforts have achieved 30% participation rates for conference settings [ 42 ]. Crowd modeling is another interesting research area that offers useful tools for modeling pedestrian flow and crowd dynamics at the individual level, particularly during MGs [ 44 ]. Of note, electronic devices such as mobile phones have improved the estimation of the sizes of crowds compared with capture-recapture methods [ 45 ]. Demographic characteristics The demographic characteristics and particularly the age distribution of the MG participants could inform the parameterization of age-specific contact rates and pre-existing immunity [ 46 , 47 ], and thus the risk of severe disease outcomes [ 48 , 49 ]. For instance, school-age children tend to have high contact rates, are more susceptible to influenza infection, and have increased viral shedding relative to other age groups [ 50 ]. By contrast, populations of seniors experience relatively low influenza attack rates, but they are at higher risk of severe disease outcomes during seasonal influenza epidemics [ 51 ]. During pandemic seasons, however, senior populations may benefit from significant residual immunity to infection [ 52 - 54 ]. During the 2008 Olympic and Paralympic Games in Beijing, most (76%) of the patients at a surveillance clinic were between the ages of 16 and 54 years [ 10 ], suggesting a relatively low susceptibility of older MG populations to influenza infection and severe outcomes, relative to other age groups. Finally, rates of hand hygiene and disease reporting are likely to be reduced during MGs, but relevant data on this behavioral aspect is lacking. Susceptibility levels and vaccination status Susceptibility of the MG population to the occurrence of outbreaks is a function of the collective vaccination status, previous disease exposure history, and resulting immunity. It is important to take into account the country of origin of participants, as populations from different geographical areas are exposed to different pathogens. In addition, exposure and co-infections with multiple pathogens in populations from low-income and middle-income countries could also affect immune status to common infections, such as influenza [ 55 ]. Hence, it is important to know the expected composition of participants based on country of origin and expected immunization status in order to assess susceptibility of the total MG population, which could significantly differ from that of the local population. During the 2008 Olympic and Paralympic Games in Beijing, the foreign visitors came from 46 countries, but the great majority arrived from high-income temperate regions including the USA (24%), the Netherlands (19%), Australia (9%), and the UK (9%) [ 10 ]. In addition, only 9% of foreign visitors at a surveillance clinic reported having received the influenza vaccine in their country of origin [ 10 ]. Timing of MG in relation to travel patterns, climatic conditions, and school cycles The risk of influenza transmission at MG is connected to incoming travel patterns, local climatic conditions, and school cycles. In particular, the transmissibility of influenza has been shown to be associated with environmental conditions, as low absolute humidity has been shown to favor virus transmission and survival in the laboratory [ 56 - 58 ]. Influenza has marked winter seasonal patterns in temperate areas of the world, with viruses being reintroduced every winter and causing large and intense outbreaks, followed by fade-out periods in warmer months, during which little influenza activity is detected [ 59 ]. By contrast, in the Tropics, the seasonality of influenza is less defined, and the timing of virus activity varies between locales [ 60 , 61 ]. As an example, the influenza outbreaks identified during the World Youth Day occurred during the regular influenza season in Australia in winter 2008 [ 62 ]. Similarly, schools have been associated with increased rates of influenza transmission at the community level [ 50 ], and the celebration of MGs during school activity periods could significantly increase the risk of epidemic events. Modeling infectious-disease transmission during mass gatherings Stochastic versus deterministic models Mathematical models of infectious-disease transmission are typically expressed as deterministic dynamical systems that capture the average epidemic behavior and are often amenable to mathematical analysis [ 23 , 24 , 63 , 64 ]. By contrast, the models most appropriate for MGs should include probabilistic components to integrate stochasticity in the risk of infection, especially in the case of smaller populations, in which demographic stochasticity will affect the risk of outbreak emergence. We produced stochastic simulations of the classic SEIR (susceptible, exposed, infectious, recovered) transmission model tailored to the epidemiology of influenza [ 65 ] (Figure 1 ). It is also important to use probabilistic models to consider shorter temporal scales during MGs and compare these with community-level transmission. Moreover, stochastic models allow the estimation of the probability that introduction of initial case(s) will trigger a major epidemic, which is also significantly affected by host heterogeneity (for example, age, vaccination status) and mixing patterns (for example, confined space in airplane, large conference hall). Figure 1 Effect of demographic stochasticity on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, basic reproduction number ( R 0 ) of 1.5, and an assumption of homogenous mixing of the population, was used to generate 100 stochastic simulations in two population sizes of n = 1,000 (left panels) and 11,000 individuals (right panels). Simulations were initialized with five infectious individuals. Histograms show a higher probability of epidemic extinction in the lower population setting. Stochastic epidemic realizations are shown in light blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Mass gathering contact structure In general, infectious-disease models using explicit contact-network approaches can be classified as agent-based, individual-based, and spatially structured models, according to the level of detail used to model disease transmission. Agent-based models are very flexible, and can incorporate heterogeneities in the interactions, behaviors, and susceptibility of individuals, which make them particularly suitable for the analysis of collective dynamics of complex systems [ 66 ]. Models relying on detailed information on individual-level activities have been used to model the spread of rapidly disseminating infectious diseases, including influenza, and to help assess intervention strategies [ 19 , 20 , 67 - 73 ]. A slightly cruder approach is provided by individual-based models, which are often based on static networks of individual interactions [ 74 , 75 ]. Spatially structured epidemic models consider subsets of the population categorized by geographic location, with interactions between these subpopulations based on human mobility patterns [ 76 - 78 ] (for example, governed by gravity laws, whereby larger population centers tend to interact with higher probability) and age-specific contact rates based on contact survey data [ 46 ]. The choice of the underlying assumptions about the contact-structure profile will depend on the MG population. For instance, in the case of infectious-disease transmission in small populations within confined settings, such as disease transmission in Navy ships [ 79 ] and correctional facilities [ 64 ], assuming a well-mixed population could be reasonable. However, recent work has identified significant departures from homogenous mixing assumptions during specific MGs, including conference settings [ 42 ]. The role of the contact-network structure on transmission dynamics is illustrated in Figure 2 ; faster disease-transmission rates are seen in random-mixing structures than in small-world network structures [ 74 ]. Figure 2 Effect of contact-network structure on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model was tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed transmission probability per contact, simulated using two different contact networks: 1) a small-world contact network based on the model of Watts and Strogatz [ 74 ], with an average degree of 4 and disorder parameter (p) of 0.1 in a population of 1,000 individuals (left panels) and 2) in a random network model with an average degree of 4 (right panels) with the same population size. Histograms show the distribution of outbreak sizes for both network topologies when everything else is kept fixed. Epidemic realizations are shown in blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Airborne transmission in confined spaces Quantitative microbial risk-assessment models have been successful in modeling airborne transmission of respiratory pathogens in confined spaces, such as influenza and tuberculosis on airplanes [ 6 , 80 - 82 ], and quantifying infection risk as a function of host, pathogen, and environmental factors [ 83 - 86 ]. In particular, the Wells-Riley model [ 87 - 90 ] has been shown to be useful for well-mixed confined spaces, which can readily incorporate environmental and pathogen-specific variables such as room ventilation rates, pulmonary respiratory rates, number of infectious individuals, and infectivity, as a function of virus concentration exhaled by infectious individuals [ 91 ]. This model has been widely used in the microbial dose-response modeling literature [ 92 ], and was successful in assessing the risk of transmission in different areas of a closed-space environment and calculating the potential number of new infections generated over a specific time period. Table 1 summarizes the differences between the quantitative risk-assessment and the dynamic transmission models. Dose-response models are traditionally used to understand dose-response mechanisms and allowable microbial concentration in foods and water, but they can also be combined with other epidemiological models. A typical example is a model for point source outbreaks caused by certain exposure doses (for example, the Sverdlovsk anthrax leak), which can provide estimates for the total number of cases, the time of exposure, and the dose [ 92 , 93 ]. Another useful approach is to account for the dose-response nature of the risk of infection in traditional disease-transmission models, allowing adjustment for different transmission probabilities by route of transmission (aerosols, droplets) [ 94 ]. Table 1 Contrasting quantitative microbial risk-assessment models and infectious disease-transmission models. Modeling aspects Quantitative microbial risk-assessment model Dynamic transmission model Non-linear dynamics Usually no Usually yes Environmental sources Yes Usually no Inclusion of uncertainty/stochasticity Yes Case-by-case basis (deterministic dynamical systems, stochastic, hybrid models) Time scale Days Weeks to months Population size Thousands (music festival) to millions (Hajj pilgrimage) Hundred thousands to millions Population density High Low to high Model structure Spatial-temporal network (Summer Olympics); confined space (Army barracks) Age-structured, random-mixing populations; patch models; household-level models; large-scale individual-level models Stochastic disease extinction Yes Unlikely Endemicity No Yes Contribution of super-spreading events High Low to moderate The role of multiple initial infectious sources An important modeling consideration is the initial number of infectious individuals, and their geographical location and contact networks, particularly in the context of highly heterogeneous MG populations. Indeed, the probability of an epidemic unfolding and the rate of growth rate in the number of infections will depend upon the number of initial infectious individuals and their spatial location within the MG contact network [ 65 ]. This also has relevance to the potential deliberate release of infectious diseases during MGs. To illustrate this point, we show how outbreak size increases with the initial number of infectious individuals, using a small-world contact-network structure of 1,000 individuals (Figure 3 ). Figure 3 Effect of the initial number of infectious individuals . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed probability of transmission per contact, was simulated on small-world contact networks based on the Watts and Strogatz network model [ 74 ] with an average degree of 4 and disorder parameter (p) of 0.1 in a populations of 1,000 individuals. Initially infectious individuals were selected uniformly at random from the population. Histograms show how the distribution of outbreak sizes shifts to larger epidemics as the initial number of infectious individuals increases. Spatial scale considerations and global transmission models Disease transmission during MG events cannot be disconnected from the rest of the population, because of the tight connections to the community at large via local, regional, and global transportation networks. For instance, in the context of large MGs such as the Olympics Games or the Soccer World Cups, given the scale of the event and the many neighborhoods and sometimes cities involved, models appropriate for a large city or a network of cities may be more appropriate than models limited to a confined space. Hence, large-scale transmission models and population-wide public-health control interventions are also useful to discuss in the context of MG events. In the event of an infectious-disease outbreak during a MG, worldwide travel patterns originating from the MG population could disseminate the outbreak on a global scale within a matter of weeks [ 95 ]. For instance, following the identification of the 2009 A/H1N1 influenza pandemic in Mexico and California in late March 2009, the novel pandemic virus was detected within a few weeks in the 20 countries with highest volume of passengers arriving from Mexico [ 96 ]. Large-scale computational transmission models parameterized with high-volume air-traffic data and country-level seasonality factors are being increasingly used to assess the global transmission patterns of emerging infectious diseases and the effectiveness of control measures [ 18 , 77 , 97 , 98 ]. Such large-scale modeling efforts predicted that an early peak of pandemic influenza A/H1N1 virus activity would occur in October/November 2009 in the Northern hemisphere, several weeks before vaccination campaigns could be carried out, and that antiviral use could delay peak pandemic timing. In regards to the effects of interventions on global transmission patterns, multiple studies have concluded that substantial reductions in air travel could only provide a short delay in pandemic progression [ 99 - 102 ]. Overall, network-based approaches have become useful tools to model the transmission dynamics of infectious diseases and control interventions on city, regional, and global scales [ 19 , 20 , 67 - 69 , 74 , 103 , 104 ]. These simulation models are currently underused in the context of MGs. To our knowledge, only a single network-based modeling study has examined how outbreak size may depend on the timing of an MG event, relative to the temporal course of an influenza pandemic [ 105 ]. The MG event was modeled as a change in population mixing. Key data needs for large-scale simulation models Global transmission models rely on large amounts of data on demographics, population movements, and age-specific contact rates. High-resolution demographic and age-specific contact data has become available for a number of areas, including the USA [ 19 , 106 ], and southeast Asia [ 20 , 107 ], while age-specific contact rates have been derived from population surveys for a number of European countries [ 46 ]. However, travel patterns before and after the MG event are naturally difficult to ascertain well in advance of the event, but are essential data for anticipating the expected composition of the MG and the potential global infectious disease-transmission patterns. Analysis of international travel data from previous Summer Olympics suggests that changes in travel patterns to the host city are difficult to predict months in advance [ 12 ]. Moreover, high-resolution contact rate data and within-country connectivity data are not available for most countries. Equally important is the need for country-specific historical immunization coverage data to assess the risk of importation to and from different locations. Finally, additional information on the temperature, humidity, ventilation settings, and capacity of confined environments specific to MGs, such as indoor stadiums (Olympics) and mosques (the Hajj), would be useful to tailor environmental risk models to MGs. Public-health interventions during mass gatherings The decision-making process on the type and intensity of interventions to put in place to control a MG outbreak will depend on our ability to detect early cases, characterize the transmission potential and severity of the associated pathogen, and implement interventions rapidly [ 108 ]. Overall, the timing of start of interventions will depend on the epidemiology of the infectious disease and the availability of surveillance data that is representative of the general population. Cancellation of large public gatherings has been successfully implemented during past influenza pandemics [ 4 , 9 , 25 , 109 ]; however, cancellation of a major MG event such as the Summer Olympics as a result of perceived infectious-disease transmission risk could be counterproductive, because of potential for rapid global spread. Instead, recommendations to use face-masks and increase hygiene measures could prove to be effective mitigation strategies, together with preventive or reactive vaccination in the early stages of the outbreak, as shown in past epidemics of influenza, meningococcal meningitis, and measles [ 69 , 110 - 114 ]. This is particularly important in light of outbreaks of measles and mumps reported at past MGs [ 2 ], and the difficulty in reaching the critical vaccination coverage rate against childhood infectious diseases in many European countries [ 115 ]. The importance of putting in place targeted vaccination strategies against influenza and other respiratory pathogens prior to any MG event cannot be overemphasized. In the extreme situation in which evidence suggests the potential for increased rates of hospitalization and mortality, stringent interventions could be justifiable, including imposing movement restrictions on MG participants to avoid or slow the importation of cases [ 86 ] to high-risk countries, concurrent with a reactive vaccination strategy [ 116 ]. The composition and dynamics of MG events as large as the Olympics are complex. Recent data indicate that influxes of several million visitors are typically expected, including tens of thousands of journalists and athletes from a couple of hundred nationalities. Moreover, competitions take place in a number of open and confined settings, with capacities ranging from a few thousand to tens of thousands. The expected age distribution of participants is relatively young, with the majority being young and middle-aged adults [ 10 ], which is the age group that experienced the highest death rates during the most recent influenza pandemic [ 117 ]. Moreover, recent data indicate that only a small fraction of the participant population is expected to have received the seasonal influenza vaccine in their country of origin before the start of the competitions [ 10 , 118 ]. Public-health preparedness planning is very intense during Olympic Games. This was illustrated by the 2012 London Summer Olympics, for which the UK Health Protection Agency set up an enhanced disease-monitoring system including laboratory surveillance, clinical case reporting, and syndromic surveillance based on patient symptoms [ 119 - 121 ], and surveillance was conducted using a lower than usual detection threshold. However, to our knowledge, infectious disease-transmission models were not integrated in any of these preparedness efforts. Future directions Below we summarize the data and modeling gaps that we have identified throughout this review, which must be filled to improve infectious-disease models for MGs. Because no detailed survey of MG contact networks has been carried out, a key avenue for future research would be to gain more information on the patterns and duration of human interactions during MG events, perhaps by distributing innovative contact-sensing devices to a sample of MG participants [ 36 ]. The Olympic Games, Soccer World Cups, and annual Hajj pilgrimages offer an interesting opportunity to study large crowds in the context of infectious-disease transmission. Although it is not feasible to monitor contact patterns in the entire MG population, information from a representative sample would be useful, and participation in previous contact-sensing device studies has been high. Ideally, such studies should be combined with enhanced monitoring of disease activity, including simultaneously testing for several pathogens (for example, using multiplex PCR) and incorporating innovative approaches for disease surveillance (for example, exploiting web-based technologies, and data-gathering and dissemination methods via smart phones) [ 122 ]. Comprehensive modeling studies are lacking to assess the cost-effectiveness of intervention strategies such as movement restrictions and reactive vaccination in the context of infectious-disease transmission during MGs. This is partly because of our limited knowledge of crucial demographic and susceptibility characteristics of the population and the relevant contact structure for disease transmission. In parallel, the higher levels of computational power now available is facilitating the development of extremely detailed transmission models at multiple spatial scales ([ 18 , 19 , 22 , 107 , 123 ]. Another key data gap is the difficulty in ascertaining up-to-date air-travel patterns relating to MG events as well as country-specific repositories of demographic, contact rates, and immunization data for childhood and other infectious diseases, which would be needed for appropriate calibration of large-scale transmission models involving large numbers of international visitors. Finally, it would be useful to validate infectious-disease models for MGs against historical outbreaks that have been well documented in the literature, especially given the stochastic nature of these outbreaks in heterogeneous populations [ 2 ]. We note that the number of efforts to integrate microbial risk-assessment modeling and dynamic population-level transmission modeling approaches remains limited [ 92 - 94 ]. Hence, the integration of quantitative risk models into large-scale dynamic transmission models has the potential to improve predictive capabilities in relation to epidemic transmission patterns and prospects for outbreak control, particularly in the context of disease transmission at MGs. Such modeling approaches could follow a hierarchical structure by connecting disease-transmission processes on different spatial scales [ 86 ]. Key disease-model concepts The risk of infectious-disease transmission during MGs is directly related to the characteristics of the participants and their environment [ 2 ]. The effects of these factors on the risk of disease transmission can be integrated in key epidemiological quantities: the basic reproduction number, R 0 , and the effective reproduction number, R [ 23 - 25 ]. R 0 measures the average number of secondary cases generated by a primary infectious individual in a completely susceptible population. A more practical quantity is the effective reproduction number, R , which quantifies the potential for infectious-disease transmission in a population that may be only partially susceptible owing to prior exposure or vaccination [ 26 ]. From a probabilistic perspective, R and R 0 denote the mean of the distribution of secondary cases for each single primary case in the population to account for individual-level variation in, for instance, infectiousness and contact rates [ 16 ]. R can be formulated as the product of three quantities: the contact rate, the conditional probability of transmission per contact, and the duration of the infectious period [ 23 , 24 ], hence we can expect higher values for R in crowded or confined conditions associated with MGs. Overall, R of less than 1 indicates that a major epidemic is likely to occur whereas R of greater than 1 indicates that transmission chains cannot be sustained. Respiratory infections cover a wide range of transmission potentials, with R being estimated at 1.2 to 1.6 for seasonal influenza [ 27 ], 1.4 to 5.2 for pandemics [ 28 - 32 ], 15 for pertussis, 17 for measles [ 23 ], and 1.2 to 1.3 for meningococcal meningitis [ 33 ]. Another key quantity for disease control is the serial interval, which measures the time interval between successive cases and sets the time scale for epidemic growth, and hence the speed with which intervention measures need to be initiated [ 34 ]. Despite the relatively low transmission potential of influenza, outbreaks are difficult to control in real time because of its short serial interval of 2 to 3 days and the fact that a substantial fraction of transmission events occur before a case becomes symptomatic [ 34 ]. Characterizing population at risk and contact networks during mass gatherings Structure of disease-relevant contact network Perhaps the most challenging aspect of modeling infectious-disease transmission in the context of MG lies in appropriately capturing the complexity of dynamic human interactions and contact networks to provide valid and reliable predictions of transmission potential and attack rates. The dynamic social contact networks during MGs will depend on a number of factors, including the type (for example, confined versus open setting), size and duration of the event, the schedule of activities, the capacity of the corresponding locations in which the activities take place (for example, Olympic stadium, aquatic center), and specific crowd behavior (for example, in the case of diseases spread by aerosols, the use and sharing of plastic blowing horns by sports fans to provide audible support for their team [ 35 ]). Most contact-network surveys have been based on cumbersome questionnaires with arbitrary physical definitions of 'social' contacts between individuals, but recent technological advances in wearable sensing devices allows unobtrusive and unsupervised quantification of contact intensity and duration. Radiofrequency identification devices were recently used to monitor in great detail the face-to-face contact patterns relevant to the spread of infectious diseases [ 36 ], particularly in primary schools [ 37 , 38 ]. Such detailed analysis of contact patterns highlighted important departures from homogeneous mixing assumptions, which should be integrated in disease models focused on outbreaks in schools or on childhood infections [ 38 - 41 ]. Another recent study analyzed real-time close contact interactions between conference participants, and found that the duration of contacts between the participants provided a good approximation to the epidemic dynamics compared with results obtained by modeling the full dynamic contact network [ 42 , 43 ]. To our knowledge, no detailed survey of contacts has been performed at MG events. A key avenue for future research would be to gain more information on contact networks in this context, perhaps by distributing sensing devices to a sample of MG participants. Although it could be challenging to enroll representative populations of MGs, recent efforts have achieved 30% participation rates for conference settings [ 42 ]. Crowd modeling is another interesting research area that offers useful tools for modeling pedestrian flow and crowd dynamics at the individual level, particularly during MGs [ 44 ]. Of note, electronic devices such as mobile phones have improved the estimation of the sizes of crowds compared with capture-recapture methods [ 45 ]. Demographic characteristics The demographic characteristics and particularly the age distribution of the MG participants could inform the parameterization of age-specific contact rates and pre-existing immunity [ 46 , 47 ], and thus the risk of severe disease outcomes [ 48 , 49 ]. For instance, school-age children tend to have high contact rates, are more susceptible to influenza infection, and have increased viral shedding relative to other age groups [ 50 ]. By contrast, populations of seniors experience relatively low influenza attack rates, but they are at higher risk of severe disease outcomes during seasonal influenza epidemics [ 51 ]. During pandemic seasons, however, senior populations may benefit from significant residual immunity to infection [ 52 - 54 ]. During the 2008 Olympic and Paralympic Games in Beijing, most (76%) of the patients at a surveillance clinic were between the ages of 16 and 54 years [ 10 ], suggesting a relatively low susceptibility of older MG populations to influenza infection and severe outcomes, relative to other age groups. Finally, rates of hand hygiene and disease reporting are likely to be reduced during MGs, but relevant data on this behavioral aspect is lacking. Susceptibility levels and vaccination status Susceptibility of the MG population to the occurrence of outbreaks is a function of the collective vaccination status, previous disease exposure history, and resulting immunity. It is important to take into account the country of origin of participants, as populations from different geographical areas are exposed to different pathogens. In addition, exposure and co-infections with multiple pathogens in populations from low-income and middle-income countries could also affect immune status to common infections, such as influenza [ 55 ]. Hence, it is important to know the expected composition of participants based on country of origin and expected immunization status in order to assess susceptibility of the total MG population, which could significantly differ from that of the local population. During the 2008 Olympic and Paralympic Games in Beijing, the foreign visitors came from 46 countries, but the great majority arrived from high-income temperate regions including the USA (24%), the Netherlands (19%), Australia (9%), and the UK (9%) [ 10 ]. In addition, only 9% of foreign visitors at a surveillance clinic reported having received the influenza vaccine in their country of origin [ 10 ]. Timing of MG in relation to travel patterns, climatic conditions, and school cycles The risk of influenza transmission at MG is connected to incoming travel patterns, local climatic conditions, and school cycles. In particular, the transmissibility of influenza has been shown to be associated with environmental conditions, as low absolute humidity has been shown to favor virus transmission and survival in the laboratory [ 56 - 58 ]. Influenza has marked winter seasonal patterns in temperate areas of the world, with viruses being reintroduced every winter and causing large and intense outbreaks, followed by fade-out periods in warmer months, during which little influenza activity is detected [ 59 ]. By contrast, in the Tropics, the seasonality of influenza is less defined, and the timing of virus activity varies between locales [ 60 , 61 ]. As an example, the influenza outbreaks identified during the World Youth Day occurred during the regular influenza season in Australia in winter 2008 [ 62 ]. Similarly, schools have been associated with increased rates of influenza transmission at the community level [ 50 ], and the celebration of MGs during school activity periods could significantly increase the risk of epidemic events. Structure of disease-relevant contact network Perhaps the most challenging aspect of modeling infectious-disease transmission in the context of MG lies in appropriately capturing the complexity of dynamic human interactions and contact networks to provide valid and reliable predictions of transmission potential and attack rates. The dynamic social contact networks during MGs will depend on a number of factors, including the type (for example, confined versus open setting), size and duration of the event, the schedule of activities, the capacity of the corresponding locations in which the activities take place (for example, Olympic stadium, aquatic center), and specific crowd behavior (for example, in the case of diseases spread by aerosols, the use and sharing of plastic blowing horns by sports fans to provide audible support for their team [ 35 ]). Most contact-network surveys have been based on cumbersome questionnaires with arbitrary physical definitions of 'social' contacts between individuals, but recent technological advances in wearable sensing devices allows unobtrusive and unsupervised quantification of contact intensity and duration. Radiofrequency identification devices were recently used to monitor in great detail the face-to-face contact patterns relevant to the spread of infectious diseases [ 36 ], particularly in primary schools [ 37 , 38 ]. Such detailed analysis of contact patterns highlighted important departures from homogeneous mixing assumptions, which should be integrated in disease models focused on outbreaks in schools or on childhood infections [ 38 - 41 ]. Another recent study analyzed real-time close contact interactions between conference participants, and found that the duration of contacts between the participants provided a good approximation to the epidemic dynamics compared with results obtained by modeling the full dynamic contact network [ 42 , 43 ]. To our knowledge, no detailed survey of contacts has been performed at MG events. A key avenue for future research would be to gain more information on contact networks in this context, perhaps by distributing sensing devices to a sample of MG participants. Although it could be challenging to enroll representative populations of MGs, recent efforts have achieved 30% participation rates for conference settings [ 42 ]. Crowd modeling is another interesting research area that offers useful tools for modeling pedestrian flow and crowd dynamics at the individual level, particularly during MGs [ 44 ]. Of note, electronic devices such as mobile phones have improved the estimation of the sizes of crowds compared with capture-recapture methods [ 45 ]. Demographic characteristics The demographic characteristics and particularly the age distribution of the MG participants could inform the parameterization of age-specific contact rates and pre-existing immunity [ 46 , 47 ], and thus the risk of severe disease outcomes [ 48 , 49 ]. For instance, school-age children tend to have high contact rates, are more susceptible to influenza infection, and have increased viral shedding relative to other age groups [ 50 ]. By contrast, populations of seniors experience relatively low influenza attack rates, but they are at higher risk of severe disease outcomes during seasonal influenza epidemics [ 51 ]. During pandemic seasons, however, senior populations may benefit from significant residual immunity to infection [ 52 - 54 ]. During the 2008 Olympic and Paralympic Games in Beijing, most (76%) of the patients at a surveillance clinic were between the ages of 16 and 54 years [ 10 ], suggesting a relatively low susceptibility of older MG populations to influenza infection and severe outcomes, relative to other age groups. Finally, rates of hand hygiene and disease reporting are likely to be reduced during MGs, but relevant data on this behavioral aspect is lacking. Susceptibility levels and vaccination status Susceptibility of the MG population to the occurrence of outbreaks is a function of the collective vaccination status, previous disease exposure history, and resulting immunity. It is important to take into account the country of origin of participants, as populations from different geographical areas are exposed to different pathogens. In addition, exposure and co-infections with multiple pathogens in populations from low-income and middle-income countries could also affect immune status to common infections, such as influenza [ 55 ]. Hence, it is important to know the expected composition of participants based on country of origin and expected immunization status in order to assess susceptibility of the total MG population, which could significantly differ from that of the local population. During the 2008 Olympic and Paralympic Games in Beijing, the foreign visitors came from 46 countries, but the great majority arrived from high-income temperate regions including the USA (24%), the Netherlands (19%), Australia (9%), and the UK (9%) [ 10 ]. In addition, only 9% of foreign visitors at a surveillance clinic reported having received the influenza vaccine in their country of origin [ 10 ]. Timing of MG in relation to travel patterns, climatic conditions, and school cycles The risk of influenza transmission at MG is connected to incoming travel patterns, local climatic conditions, and school cycles. In particular, the transmissibility of influenza has been shown to be associated with environmental conditions, as low absolute humidity has been shown to favor virus transmission and survival in the laboratory [ 56 - 58 ]. Influenza has marked winter seasonal patterns in temperate areas of the world, with viruses being reintroduced every winter and causing large and intense outbreaks, followed by fade-out periods in warmer months, during which little influenza activity is detected [ 59 ]. By contrast, in the Tropics, the seasonality of influenza is less defined, and the timing of virus activity varies between locales [ 60 , 61 ]. As an example, the influenza outbreaks identified during the World Youth Day occurred during the regular influenza season in Australia in winter 2008 [ 62 ]. Similarly, schools have been associated with increased rates of influenza transmission at the community level [ 50 ], and the celebration of MGs during school activity periods could significantly increase the risk of epidemic events. Modeling infectious-disease transmission during mass gatherings Stochastic versus deterministic models Mathematical models of infectious-disease transmission are typically expressed as deterministic dynamical systems that capture the average epidemic behavior and are often amenable to mathematical analysis [ 23 , 24 , 63 , 64 ]. By contrast, the models most appropriate for MGs should include probabilistic components to integrate stochasticity in the risk of infection, especially in the case of smaller populations, in which demographic stochasticity will affect the risk of outbreak emergence. We produced stochastic simulations of the classic SEIR (susceptible, exposed, infectious, recovered) transmission model tailored to the epidemiology of influenza [ 65 ] (Figure 1 ). It is also important to use probabilistic models to consider shorter temporal scales during MGs and compare these with community-level transmission. Moreover, stochastic models allow the estimation of the probability that introduction of initial case(s) will trigger a major epidemic, which is also significantly affected by host heterogeneity (for example, age, vaccination status) and mixing patterns (for example, confined space in airplane, large conference hall). Figure 1 Effect of demographic stochasticity on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, basic reproduction number ( R 0 ) of 1.5, and an assumption of homogenous mixing of the population, was used to generate 100 stochastic simulations in two population sizes of n = 1,000 (left panels) and 11,000 individuals (right panels). Simulations were initialized with five infectious individuals. Histograms show a higher probability of epidemic extinction in the lower population setting. Stochastic epidemic realizations are shown in light blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Mass gathering contact structure In general, infectious-disease models using explicit contact-network approaches can be classified as agent-based, individual-based, and spatially structured models, according to the level of detail used to model disease transmission. Agent-based models are very flexible, and can incorporate heterogeneities in the interactions, behaviors, and susceptibility of individuals, which make them particularly suitable for the analysis of collective dynamics of complex systems [ 66 ]. Models relying on detailed information on individual-level activities have been used to model the spread of rapidly disseminating infectious diseases, including influenza, and to help assess intervention strategies [ 19 , 20 , 67 - 73 ]. A slightly cruder approach is provided by individual-based models, which are often based on static networks of individual interactions [ 74 , 75 ]. Spatially structured epidemic models consider subsets of the population categorized by geographic location, with interactions between these subpopulations based on human mobility patterns [ 76 - 78 ] (for example, governed by gravity laws, whereby larger population centers tend to interact with higher probability) and age-specific contact rates based on contact survey data [ 46 ]. The choice of the underlying assumptions about the contact-structure profile will depend on the MG population. For instance, in the case of infectious-disease transmission in small populations within confined settings, such as disease transmission in Navy ships [ 79 ] and correctional facilities [ 64 ], assuming a well-mixed population could be reasonable. However, recent work has identified significant departures from homogenous mixing assumptions during specific MGs, including conference settings [ 42 ]. The role of the contact-network structure on transmission dynamics is illustrated in Figure 2 ; faster disease-transmission rates are seen in random-mixing structures than in small-world network structures [ 74 ]. Figure 2 Effect of contact-network structure on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model was tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed transmission probability per contact, simulated using two different contact networks: 1) a small-world contact network based on the model of Watts and Strogatz [ 74 ], with an average degree of 4 and disorder parameter (p) of 0.1 in a population of 1,000 individuals (left panels) and 2) in a random network model with an average degree of 4 (right panels) with the same population size. Histograms show the distribution of outbreak sizes for both network topologies when everything else is kept fixed. Epidemic realizations are shown in blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Airborne transmission in confined spaces Quantitative microbial risk-assessment models have been successful in modeling airborne transmission of respiratory pathogens in confined spaces, such as influenza and tuberculosis on airplanes [ 6 , 80 - 82 ], and quantifying infection risk as a function of host, pathogen, and environmental factors [ 83 - 86 ]. In particular, the Wells-Riley model [ 87 - 90 ] has been shown to be useful for well-mixed confined spaces, which can readily incorporate environmental and pathogen-specific variables such as room ventilation rates, pulmonary respiratory rates, number of infectious individuals, and infectivity, as a function of virus concentration exhaled by infectious individuals [ 91 ]. This model has been widely used in the microbial dose-response modeling literature [ 92 ], and was successful in assessing the risk of transmission in different areas of a closed-space environment and calculating the potential number of new infections generated over a specific time period. Table 1 summarizes the differences between the quantitative risk-assessment and the dynamic transmission models. Dose-response models are traditionally used to understand dose-response mechanisms and allowable microbial concentration in foods and water, but they can also be combined with other epidemiological models. A typical example is a model for point source outbreaks caused by certain exposure doses (for example, the Sverdlovsk anthrax leak), which can provide estimates for the total number of cases, the time of exposure, and the dose [ 92 , 93 ]. Another useful approach is to account for the dose-response nature of the risk of infection in traditional disease-transmission models, allowing adjustment for different transmission probabilities by route of transmission (aerosols, droplets) [ 94 ]. Table 1 Contrasting quantitative microbial risk-assessment models and infectious disease-transmission models. Modeling aspects Quantitative microbial risk-assessment model Dynamic transmission model Non-linear dynamics Usually no Usually yes Environmental sources Yes Usually no Inclusion of uncertainty/stochasticity Yes Case-by-case basis (deterministic dynamical systems, stochastic, hybrid models) Time scale Days Weeks to months Population size Thousands (music festival) to millions (Hajj pilgrimage) Hundred thousands to millions Population density High Low to high Model structure Spatial-temporal network (Summer Olympics); confined space (Army barracks) Age-structured, random-mixing populations; patch models; household-level models; large-scale individual-level models Stochastic disease extinction Yes Unlikely Endemicity No Yes Contribution of super-spreading events High Low to moderate The role of multiple initial infectious sources An important modeling consideration is the initial number of infectious individuals, and their geographical location and contact networks, particularly in the context of highly heterogeneous MG populations. Indeed, the probability of an epidemic unfolding and the rate of growth rate in the number of infections will depend upon the number of initial infectious individuals and their spatial location within the MG contact network [ 65 ]. This also has relevance to the potential deliberate release of infectious diseases during MGs. To illustrate this point, we show how outbreak size increases with the initial number of infectious individuals, using a small-world contact-network structure of 1,000 individuals (Figure 3 ). Figure 3 Effect of the initial number of infectious individuals . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed probability of transmission per contact, was simulated on small-world contact networks based on the Watts and Strogatz network model [ 74 ] with an average degree of 4 and disorder parameter (p) of 0.1 in a populations of 1,000 individuals. Initially infectious individuals were selected uniformly at random from the population. Histograms show how the distribution of outbreak sizes shifts to larger epidemics as the initial number of infectious individuals increases. Spatial scale considerations and global transmission models Disease transmission during MG events cannot be disconnected from the rest of the population, because of the tight connections to the community at large via local, regional, and global transportation networks. For instance, in the context of large MGs such as the Olympics Games or the Soccer World Cups, given the scale of the event and the many neighborhoods and sometimes cities involved, models appropriate for a large city or a network of cities may be more appropriate than models limited to a confined space. Hence, large-scale transmission models and population-wide public-health control interventions are also useful to discuss in the context of MG events. In the event of an infectious-disease outbreak during a MG, worldwide travel patterns originating from the MG population could disseminate the outbreak on a global scale within a matter of weeks [ 95 ]. For instance, following the identification of the 2009 A/H1N1 influenza pandemic in Mexico and California in late March 2009, the novel pandemic virus was detected within a few weeks in the 20 countries with highest volume of passengers arriving from Mexico [ 96 ]. Large-scale computational transmission models parameterized with high-volume air-traffic data and country-level seasonality factors are being increasingly used to assess the global transmission patterns of emerging infectious diseases and the effectiveness of control measures [ 18 , 77 , 97 , 98 ]. Such large-scale modeling efforts predicted that an early peak of pandemic influenza A/H1N1 virus activity would occur in October/November 2009 in the Northern hemisphere, several weeks before vaccination campaigns could be carried out, and that antiviral use could delay peak pandemic timing. In regards to the effects of interventions on global transmission patterns, multiple studies have concluded that substantial reductions in air travel could only provide a short delay in pandemic progression [ 99 - 102 ]. Overall, network-based approaches have become useful tools to model the transmission dynamics of infectious diseases and control interventions on city, regional, and global scales [ 19 , 20 , 67 - 69 , 74 , 103 , 104 ]. These simulation models are currently underused in the context of MGs. To our knowledge, only a single network-based modeling study has examined how outbreak size may depend on the timing of an MG event, relative to the temporal course of an influenza pandemic [ 105 ]. The MG event was modeled as a change in population mixing. Stochastic versus deterministic models Mathematical models of infectious-disease transmission are typically expressed as deterministic dynamical systems that capture the average epidemic behavior and are often amenable to mathematical analysis [ 23 , 24 , 63 , 64 ]. By contrast, the models most appropriate for MGs should include probabilistic components to integrate stochasticity in the risk of infection, especially in the case of smaller populations, in which demographic stochasticity will affect the risk of outbreak emergence. We produced stochastic simulations of the classic SEIR (susceptible, exposed, infectious, recovered) transmission model tailored to the epidemiology of influenza [ 65 ] (Figure 1 ). It is also important to use probabilistic models to consider shorter temporal scales during MGs and compare these with community-level transmission. Moreover, stochastic models allow the estimation of the probability that introduction of initial case(s) will trigger a major epidemic, which is also significantly affected by host heterogeneity (for example, age, vaccination status) and mixing patterns (for example, confined space in airplane, large conference hall). Figure 1 Effect of demographic stochasticity on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, basic reproduction number ( R 0 ) of 1.5, and an assumption of homogenous mixing of the population, was used to generate 100 stochastic simulations in two population sizes of n = 1,000 (left panels) and 11,000 individuals (right panels). Simulations were initialized with five infectious individuals. Histograms show a higher probability of epidemic extinction in the lower population setting. Stochastic epidemic realizations are shown in light blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Mass gathering contact structure In general, infectious-disease models using explicit contact-network approaches can be classified as agent-based, individual-based, and spatially structured models, according to the level of detail used to model disease transmission. Agent-based models are very flexible, and can incorporate heterogeneities in the interactions, behaviors, and susceptibility of individuals, which make them particularly suitable for the analysis of collective dynamics of complex systems [ 66 ]. Models relying on detailed information on individual-level activities have been used to model the spread of rapidly disseminating infectious diseases, including influenza, and to help assess intervention strategies [ 19 , 20 , 67 - 73 ]. A slightly cruder approach is provided by individual-based models, which are often based on static networks of individual interactions [ 74 , 75 ]. Spatially structured epidemic models consider subsets of the population categorized by geographic location, with interactions between these subpopulations based on human mobility patterns [ 76 - 78 ] (for example, governed by gravity laws, whereby larger population centers tend to interact with higher probability) and age-specific contact rates based on contact survey data [ 46 ]. The choice of the underlying assumptions about the contact-structure profile will depend on the MG population. For instance, in the case of infectious-disease transmission in small populations within confined settings, such as disease transmission in Navy ships [ 79 ] and correctional facilities [ 64 ], assuming a well-mixed population could be reasonable. However, recent work has identified significant departures from homogenous mixing assumptions during specific MGs, including conference settings [ 42 ]. The role of the contact-network structure on transmission dynamics is illustrated in Figure 2 ; faster disease-transmission rates are seen in random-mixing structures than in small-world network structures [ 74 ]. Figure 2 Effect of contact-network structure on influenza epidemics . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model was tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed transmission probability per contact, simulated using two different contact networks: 1) a small-world contact network based on the model of Watts and Strogatz [ 74 ], with an average degree of 4 and disorder parameter (p) of 0.1 in a population of 1,000 individuals (left panels) and 2) in a random network model with an average degree of 4 (right panels) with the same population size. Histograms show the distribution of outbreak sizes for both network topologies when everything else is kept fixed. Epidemic realizations are shown in blue, while the red solid line curve corresponds to the average of the stochastic realizations that resulted in epidemics. Airborne transmission in confined spaces Quantitative microbial risk-assessment models have been successful in modeling airborne transmission of respiratory pathogens in confined spaces, such as influenza and tuberculosis on airplanes [ 6 , 80 - 82 ], and quantifying infection risk as a function of host, pathogen, and environmental factors [ 83 - 86 ]. In particular, the Wells-Riley model [ 87 - 90 ] has been shown to be useful for well-mixed confined spaces, which can readily incorporate environmental and pathogen-specific variables such as room ventilation rates, pulmonary respiratory rates, number of infectious individuals, and infectivity, as a function of virus concentration exhaled by infectious individuals [ 91 ]. This model has been widely used in the microbial dose-response modeling literature [ 92 ], and was successful in assessing the risk of transmission in different areas of a closed-space environment and calculating the potential number of new infections generated over a specific time period. Table 1 summarizes the differences between the quantitative risk-assessment and the dynamic transmission models. Dose-response models are traditionally used to understand dose-response mechanisms and allowable microbial concentration in foods and water, but they can also be combined with other epidemiological models. A typical example is a model for point source outbreaks caused by certain exposure doses (for example, the Sverdlovsk anthrax leak), which can provide estimates for the total number of cases, the time of exposure, and the dose [ 92 , 93 ]. Another useful approach is to account for the dose-response nature of the risk of infection in traditional disease-transmission models, allowing adjustment for different transmission probabilities by route of transmission (aerosols, droplets) [ 94 ]. Table 1 Contrasting quantitative microbial risk-assessment models and infectious disease-transmission models. Modeling aspects Quantitative microbial risk-assessment model Dynamic transmission model Non-linear dynamics Usually no Usually yes Environmental sources Yes Usually no Inclusion of uncertainty/stochasticity Yes Case-by-case basis (deterministic dynamical systems, stochastic, hybrid models) Time scale Days Weeks to months Population size Thousands (music festival) to millions (Hajj pilgrimage) Hundred thousands to millions Population density High Low to high Model structure Spatial-temporal network (Summer Olympics); confined space (Army barracks) Age-structured, random-mixing populations; patch models; household-level models; large-scale individual-level models Stochastic disease extinction Yes Unlikely Endemicity No Yes Contribution of super-spreading events High Low to moderate The role of multiple initial infectious sources An important modeling consideration is the initial number of infectious individuals, and their geographical location and contact networks, particularly in the context of highly heterogeneous MG populations. Indeed, the probability of an epidemic unfolding and the rate of growth rate in the number of infections will depend upon the number of initial infectious individuals and their spatial location within the MG contact network [ 65 ]. This also has relevance to the potential deliberate release of infectious diseases during MGs. To illustrate this point, we show how outbreak size increases with the initial number of infectious individuals, using a small-world contact-network structure of 1,000 individuals (Figure 3 ). Figure 3 Effect of the initial number of infectious individuals . The classic stochastic SEIR (susceptible, exposed, infectious, recovered) model tailored to the epidemiology of influenza, based on a latent period of 1.5 days, an infectious period of 3 days, and fixed probability of transmission per contact, was simulated on small-world contact networks based on the Watts and Strogatz network model [ 74 ] with an average degree of 4 and disorder parameter (p) of 0.1 in a populations of 1,000 individuals. Initially infectious individuals were selected uniformly at random from the population. Histograms show how the distribution of outbreak sizes shifts to larger epidemics as the initial number of infectious individuals increases. Spatial scale considerations and global transmission models Disease transmission during MG events cannot be disconnected from the rest of the population, because of the tight connections to the community at large via local, regional, and global transportation networks. For instance, in the context of large MGs such as the Olympics Games or the Soccer World Cups, given the scale of the event and the many neighborhoods and sometimes cities involved, models appropriate for a large city or a network of cities may be more appropriate than models limited to a confined space. Hence, large-scale transmission models and population-wide public-health control interventions are also useful to discuss in the context of MG events. In the event of an infectious-disease outbreak during a MG, worldwide travel patterns originating from the MG population could disseminate the outbreak on a global scale within a matter of weeks [ 95 ]. For instance, following the identification of the 2009 A/H1N1 influenza pandemic in Mexico and California in late March 2009, the novel pandemic virus was detected within a few weeks in the 20 countries with highest volume of passengers arriving from Mexico [ 96 ]. Large-scale computational transmission models parameterized with high-volume air-traffic data and country-level seasonality factors are being increasingly used to assess the global transmission patterns of emerging infectious diseases and the effectiveness of control measures [ 18 , 77 , 97 , 98 ]. Such large-scale modeling efforts predicted that an early peak of pandemic influenza A/H1N1 virus activity would occur in October/November 2009 in the Northern hemisphere, several weeks before vaccination campaigns could be carried out, and that antiviral use could delay peak pandemic timing. In regards to the effects of interventions on global transmission patterns, multiple studies have concluded that substantial reductions in air travel could only provide a short delay in pandemic progression [ 99 - 102 ]. Overall, network-based approaches have become useful tools to model the transmission dynamics of infectious diseases and control interventions on city, regional, and global scales [ 19 , 20 , 67 - 69 , 74 , 103 , 104 ]. These simulation models are currently underused in the context of MGs. To our knowledge, only a single network-based modeling study has examined how outbreak size may depend on the timing of an MG event, relative to the temporal course of an influenza pandemic [ 105 ]. The MG event was modeled as a change in population mixing. Key data needs for large-scale simulation models Global transmission models rely on large amounts of data on demographics, population movements, and age-specific contact rates. High-resolution demographic and age-specific contact data has become available for a number of areas, including the USA [ 19 , 106 ], and southeast Asia [ 20 , 107 ], while age-specific contact rates have been derived from population surveys for a number of European countries [ 46 ]. However, travel patterns before and after the MG event are naturally difficult to ascertain well in advance of the event, but are essential data for anticipating the expected composition of the MG and the potential global infectious disease-transmission patterns. Analysis of international travel data from previous Summer Olympics suggests that changes in travel patterns to the host city are difficult to predict months in advance [ 12 ]. Moreover, high-resolution contact rate data and within-country connectivity data are not available for most countries. Equally important is the need for country-specific historical immunization coverage data to assess the risk of importation to and from different locations. Finally, additional information on the temperature, humidity, ventilation settings, and capacity of confined environments specific to MGs, such as indoor stadiums (Olympics) and mosques (the Hajj), would be useful to tailor environmental risk models to MGs. Public-health interventions during mass gatherings The decision-making process on the type and intensity of interventions to put in place to control a MG outbreak will depend on our ability to detect early cases, characterize the transmission potential and severity of the associated pathogen, and implement interventions rapidly [ 108 ]. Overall, the timing of start of interventions will depend on the epidemiology of the infectious disease and the availability of surveillance data that is representative of the general population. Cancellation of large public gatherings has been successfully implemented during past influenza pandemics [ 4 , 9 , 25 , 109 ]; however, cancellation of a major MG event such as the Summer Olympics as a result of perceived infectious-disease transmission risk could be counterproductive, because of potential for rapid global spread. Instead, recommendations to use face-masks and increase hygiene measures could prove to be effective mitigation strategies, together with preventive or reactive vaccination in the early stages of the outbreak, as shown in past epidemics of influenza, meningococcal meningitis, and measles [ 69 , 110 - 114 ]. This is particularly important in light of outbreaks of measles and mumps reported at past MGs [ 2 ], and the difficulty in reaching the critical vaccination coverage rate against childhood infectious diseases in many European countries [ 115 ]. The importance of putting in place targeted vaccination strategies against influenza and other respiratory pathogens prior to any MG event cannot be overemphasized. In the extreme situation in which evidence suggests the potential for increased rates of hospitalization and mortality, stringent interventions could be justifiable, including imposing movement restrictions on MG participants to avoid or slow the importation of cases [ 86 ] to high-risk countries, concurrent with a reactive vaccination strategy [ 116 ]. The composition and dynamics of MG events as large as the Olympics are complex. Recent data indicate that influxes of several million visitors are typically expected, including tens of thousands of journalists and athletes from a couple of hundred nationalities. Moreover, competitions take place in a number of open and confined settings, with capacities ranging from a few thousand to tens of thousands. The expected age distribution of participants is relatively young, with the majority being young and middle-aged adults [ 10 ], which is the age group that experienced the highest death rates during the most recent influenza pandemic [ 117 ]. Moreover, recent data indicate that only a small fraction of the participant population is expected to have received the seasonal influenza vaccine in their country of origin before the start of the competitions [ 10 , 118 ]. Public-health preparedness planning is very intense during Olympic Games. This was illustrated by the 2012 London Summer Olympics, for which the UK Health Protection Agency set up an enhanced disease-monitoring system including laboratory surveillance, clinical case reporting, and syndromic surveillance based on patient symptoms [ 119 - 121 ], and surveillance was conducted using a lower than usual detection threshold. However, to our knowledge, infectious disease-transmission models were not integrated in any of these preparedness efforts. Future directions Below we summarize the data and modeling gaps that we have identified throughout this review, which must be filled to improve infectious-disease models for MGs. Because no detailed survey of MG contact networks has been carried out, a key avenue for future research would be to gain more information on the patterns and duration of human interactions during MG events, perhaps by distributing innovative contact-sensing devices to a sample of MG participants [ 36 ]. The Olympic Games, Soccer World Cups, and annual Hajj pilgrimages offer an interesting opportunity to study large crowds in the context of infectious-disease transmission. Although it is not feasible to monitor contact patterns in the entire MG population, information from a representative sample would be useful, and participation in previous contact-sensing device studies has been high. Ideally, such studies should be combined with enhanced monitoring of disease activity, including simultaneously testing for several pathogens (for example, using multiplex PCR) and incorporating innovative approaches for disease surveillance (for example, exploiting web-based technologies, and data-gathering and dissemination methods via smart phones) [ 122 ]. Comprehensive modeling studies are lacking to assess the cost-effectiveness of intervention strategies such as movement restrictions and reactive vaccination in the context of infectious-disease transmission during MGs. This is partly because of our limited knowledge of crucial demographic and susceptibility characteristics of the population and the relevant contact structure for disease transmission. In parallel, the higher levels of computational power now available is facilitating the development of extremely detailed transmission models at multiple spatial scales ([ 18 , 19 , 22 , 107 , 123 ]. Another key data gap is the difficulty in ascertaining up-to-date air-travel patterns relating to MG events as well as country-specific repositories of demographic, contact rates, and immunization data for childhood and other infectious diseases, which would be needed for appropriate calibration of large-scale transmission models involving large numbers of international visitors. Finally, it would be useful to validate infectious-disease models for MGs against historical outbreaks that have been well documented in the literature, especially given the stochastic nature of these outbreaks in heterogeneous populations [ 2 ]. We note that the number of efforts to integrate microbial risk-assessment modeling and dynamic population-level transmission modeling approaches remains limited [ 92 - 94 ]. Hence, the integration of quantitative risk models into large-scale dynamic transmission models has the potential to improve predictive capabilities in relation to epidemic transmission patterns and prospects for outbreak control, particularly in the context of disease transmission at MGs. Such modeling approaches could follow a hierarchical structure by connecting disease-transmission processes on different spatial scales [ 86 ]. Conclusions Comprehensive modeling studies are needed to assess the cost-effectiveness of intervention strategies against infectious disease arising during large MGs such as the Summer Olympics and the annual Hajj events. These studies will heavily rely on our ability to quantify population mixing characteristics during MG events; anticipate air-travel patterns before and after the MG event; gather country-specific repositories of demographic factors, contact rates, and immunization rates for childhood and other infectious diseases; and estimate the potential effect of collective behavioral changes during MG events. Further, development of novel mathematical and statistical approaches specific to MGs, and integration of existing approaches, would be useful to provide more appropriate models, which could be tested against historical events [ 124 ]. Finally, MG preparedness and contingency intervention plans to mitigate infectious-disease transmission could incorporate some of these modeling research, inspired by influenza modeling efforts that have helped shape pandemic preparedness plans in recent years [ 18 , 19 , 22 , 99 - 102 , 107 , 123 ]. Competing interests The authors declare that they have no competing interests. Authors' contributions All the authors contributed to the writing and editing of the manuscript. All the authors have read and approved the manuscript for publication. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1741-7015/10/159/prepub

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711331/

Assessment of Local Public Health Workers' Willingness to Respond to Pandemic Influenza through Application of the Extended Parallel Process Model

Background Local public health agencies play a central role in response to an influenza pandemic, and understanding the willingness of their employees to report to work is therefore a critically relevant concern for pandemic influenza planning efforts. Witte's Extended Parallel Process Model (EPPM) has been found useful for understanding adaptive behavior in the face of unknown risk, and thus offers a framework for examining scenario-specific willingness to respond among local public health workers. We thus aim to use the EPPM as a lens for examining the influences of perceived threat and efficacy on local public health workers' response willingness to pandemic influenza. Methodology/Principal Findings We administered an online, EPPM-based survey about attitudes/beliefs toward emergency response ( Johns Hopkins∼Public Health Infrastructure Response Survey Tool ), to local public health employees in three states between November 2006 – December 2007. A total of 1835 responses were collected for an overall response rate of 83%. With some regional variation, overall 16% of the workers in 2006-7 were not willing to "respond to a pandemic flu emergency regardless of its severity". Local health department employees with a perception of high threat and high efficacy – i.e., those fitting a 'concerned and confident' profile in the EPPM analysis – had the highest declared rates of willingness to respond to an influenza pandemic if required by their agency, which was 31.7 times higher than those fitting a 'low threat/low efficacy' EPPM profile. Conclusions/Significance In the context of pandemic influenza planning, the EPPM provides a useful framework to inform nuanced understanding of baseline levels of – and gaps in – local public health workers' response willingness. Within local health departments, 'concerned and confident' employees are most likely to be willing to respond. This finding may allow public health agencies to design, implement, and evaluate training programs focused on emergency response attitudes in health departments. Background Local public health agencies play a central role in response to an influenza pandemic, and understanding the willingness of their employees to report to work is therefore a critically relevant concern for pandemic influenza planning efforts. Witte's Extended Parallel Process Model (EPPM) has been found useful for understanding adaptive behavior in the face of unknown risk, and thus offers a framework for examining scenario-specific willingness to respond among local public health workers. We thus aim to use the EPPM as a lens for examining the influences of perceived threat and efficacy on local public health workers' response willingness to pandemic influenza. Methodology/Principal Findings We administered an online, EPPM-based survey about attitudes/beliefs toward emergency response ( Johns Hopkins∼Public Health Infrastructure Response Survey Tool ), to local public health employees in three states between November 2006 – December 2007. A total of 1835 responses were collected for an overall response rate of 83%. With some regional variation, overall 16% of the workers in 2006-7 were not willing to "respond to a pandemic flu emergency regardless of its severity". Local health department employees with a perception of high threat and high efficacy – i.e., those fitting a 'concerned and confident' profile in the EPPM analysis – had the highest declared rates of willingness to respond to an influenza pandemic if required by their agency, which was 31.7 times higher than those fitting a 'low threat/low efficacy' EPPM profile. Conclusions/Significance In the context of pandemic influenza planning, the EPPM provides a useful framework to inform nuanced understanding of baseline levels of – and gaps in – local public health workers' response willingness. Within local health departments, 'concerned and confident' employees are most likely to be willing to respond. This finding may allow public health agencies to design, implement, and evaluate training programs focused on emergency response attitudes in health departments. Introduction The anticipated worldwide morbidity, mortality, and social disruption from an influenza pandemic [1] require detailed and tested approaches to staffing and resource allocation in public health systems [2] . The willingness of health responders to report to duty during an influenza pandemic is a highly salient concern given the "inevitable"nature of this threat [3] and its associated challenges. Scant margin exists in the nation's public health system for local health department workers – the backbone of public health system readiness – to "opt out" of response duties, given limitations of health system surge capacity [4] , public health personnel shortages [5] , and continued steep learning curves associated with relatively new 24/7 response expectations for health department employees. The unwillingness of some health workers to place themselves at risk of exposure to emerging infectious diseases was observed during the 2003 SARS epidemic and the early years of the HIV/AIDS epidemic [6] . In the aftermath of the terror attacks of September 11, 2001 and the ensuing anthrax bioterrorism attacks, a growing body of research literature has examined willingness to respond to large-scale emergencies among a variety of health-related cohorts [7] – [14] . Despite the evidence for fundamental distinctions between ability and willingness to respond [7] , [13] , there remains a gap in the public health preparedness literature on training approaches that explicitly address response willingness (attitude) as a discrete outcome. Based on the principle that "all disasters begin locally", these observations underscore a fundamental need to understand root causes of local public health workers' barriers to response willingness, as a basis for identifying and addressing public health response system gaps in this domain. A variety of risk perception theories have been suggested and may help to identify barriers to health personnel adopting an emergency responder role. One prominent model conceptualizes risk perception as the sum of "hazard" and "outrage", where hazard is a product of risk magnitude and probability, and outrage is a function of other peripheral influences independent of the actual risk, such as perceived authority, trust, and situational control [15] . Among the public health workforce, recent applications of this "Risk = Hazard+Outrage" model have uncovered a variety of potential peripheral risk perception influences on health department workers' response willingness apart from the actual hazard [16] . For example, in a 2005 pilot study conducted in three local health departments in Maryland, we found that a health department employee's individual perceived level of importance in their agency's response efforts was a particularly strong peripheral influence on response willingness toward an influenza pandemic [8] . The cumulative evidence from these studies suggests that willingness to respond is multidimensional. Specifically, its dimensions appear to include: 1) perceived threat , as evidenced by findings of scenario-specific response willingness rates; and 2) perceived efficacy , as highlighted by the powerful influences of response efficacy ("My response makes a difference") and self-efficacy ("I can do what is expected of me as a responder"). Further, preparedness training for public health workers is a form of risk communication in itself, intended to build health department workers' efficacy in the face of a variety of hazards. To build a public health workforce that is not only able to respond, but also willing to do so, the above observations suggest the need for a unifying paradigm that can address both the threat and efficacy dimensions of willingness to respond. To date, the research literature on public health emergency response willingness has lacked such a paradigm. The Extended Parallel Process Model (EPPM) [ Figure 1 ] has been found to be useful for understanding adaptive behavior in the face of unknown risk [17] . First proposed by Witte in the early 1990s [18] , the EPPM represents an integration and expansion of previous psychosocial models of "fear appeal." The model focuses on messages that are received by both individuals and collectively by groups. Importantly, while the model was first developed to explain individual behavior [18] , it has since been directly applied to the analysis of collective behavior [19] . 10.1371/journal.pone.0006365.g001 Figure 1 Extended Parallel Process Model. Witte's Extended Parallel Process Model (EPPM) describes how people, when faced with a potential hazard, will sequentially appraise the threat and efficacy content of related health and safety protection messages, and will respond accordingly. The first appraisal is for threat [threat appraisal]. The threat appraisal has two components: severity and susceptibility . If, in the threat appraisal, the message recipient personally perceives the hazard to be of negligible consequence (low severity) or improbable (low susceptibility), any related message content encouraging a desired protection-oriented response or behavior will be rejected. If, however, the message passes the threat appraisal, the message recipient will next process the message's content for efficacy [efficacy appraisal]. The efficacy appraisal contains two components: self-efficacy and response efficacy . If the message recipient does not find the message's targeted behavior to be achievable (low self-efficacy) or efficacious (low response efficacy), the message recipient will engage in undesirable responses such as denial and avoidance in order to manage fear (described as "fear control" in the EPPM); this will be accompanied by message rejection. If, however, the efficacy appraisal is also passed, message acceptance will result, leading to adoption of the message's intended protective behavior change outcomes by taking desirable steps to minimize personal risk against the actual hazard (described as "danger control" in the EPPM). According to the EPPM, in order to be effective, messages must contain two parts: threat and efficacy. Message receivers sequentially perform two appraisals, first of threat and then of efficacy. The first portion of the message must convincingly transmit the existence of a threat, leading to concern on the part of the message receiver(s). The second portion of the message must convincingly transmit the existence of efficacious interventions/mitigations, especially those that are self-efficacious (i.e., able to be performed by the message receivers), leading to confidence. According to this model, the threat and efficacy components must be accepted by the message receivers to achieve the desired behavior or practice (at both individual and collective levels); this is termed "danger control" per the EPPM. If the threat portion is not accepted, the message is rejected. If the threat portion is accepted, but the efficacy portion is not, the acceptance of the threat portion triggers fear, which the message receivers attempt to manage (by rejecting the entire message); such a reaction is referred to as "fear control" per the EPPM. Through the design and administration of an EPPM-centered survey of local health department personnel in three states, we aim to examine the relative influences of perceived threat and efficacy on public health workers' response willingness to pandemic influenza. Methods Ethics Statement Research ethics approval for the Johns Hopkins∼Public Health Infrastructure Response Survey Tool (JH∼PHIRST) survey and its administration was received from the Johns Hopkins Bloomberg School of Public Health Institutional Review Board (JHSPH IRB) (exempt status # 45 CFR 46.101 (b) (2)). Per JHSPH IRB approval, written consent was not obtained, as the research presented no more than minimal risk to subjects and involved no procedures for which written consent is normally required. The JHSPH IRB-approved study materials included a written disclosure describing the study and emphasizing voluntary participation; verbal consent was not requested or required by JHSPH IRB for this approved study. Survey Instrument The JH∼PHIRST is an anonymous online survey instrument consisting of demographic and attitude/belief sections focusing on health department workers' attitudes and beliefs toward public health emergency response. The demographic information includes gender, highest education level, role in an emergency response, responsibility for a family member, and categories of age, professional classification, years in present organization, and years in profession. For each of four emergency scenarios (weather-related emergency, pandemic influenza, 'dirty bomb' radiological terrorism event, and inhalational anthrax bioterrorism event) the same 20 attitudes and beliefs were presented for level of agreement along with two open-ended questions. Responses to the attitude and belief questions were based on a 10-point Likert scale with a response of '1' indicating strong agreement with the question and a response of '10' indicating strong disagreement with the question. Respondents could also indicate "don't know". The online JH∼PHIRST instrument used in the current study evolved from an earlier, paper-based pilot survey instrument that we implemented in three Maryland local health departments in 2005 to assess the willingness of these employees to respond to an influenza pandemic [8] . Of note, while this earlier pilot version did incorporate some aspects of risk perception, it did not use the threat and efficacy measures from EPPM that have become commonplace in risk communication studies over the past decade. We incorporated the EPPM content to generate the online JH∼PHIRST instrument based on a series of relevant observations from the 2005 Maryland-based pilot study – namely, that local public health workers' willingness to respond in a pandemic flu scenario was hampered by uncertainties, fears and lack of confidence [8] that are reflected in the standard batteries of questions used by EPPM studies. The online JH∼PHIRST survey's EPPM-based threat and efficacy measures have been widely validated by numerous studies in multiple countries, cultural settings, and health contexts [20] . The "non-EPPM" constructs in the online JH∼PHIRST survey were derived from our original paper-based Maryland 2005 pilot study [8] , which itself was based on validated risk communication theory [15] , [21] in the context of an identified set of potential peripheral risk perception influences from emergency preparedness training experiences in local health departments [16] . Study participants Four clusters of local health departments from three states in the Midwestern and Eastern U.S. participated in the JH∼PHIRST survey. Each region (cluster) had access to the online version of JH∼PHIRST via the SurveyMonkey (SurveyMonkey.com, Portland OR) web site for 4 to 6 weeks, and each region's survey results were logged separately. The health departments were responsible for encouraging all of their employees to respond to this survey and individual participation was voluntary. During the survey administration, completion rates by health department were intermittently provided to pre-designated administrative points of contact at the participating agencies within the cluster to encourage agency-wide survey participation. Statistical analysis This survey scale did not include a neutral point, and the option of a "don't know" response may have carried the ambivalence stance. Suspecting that the use of the "don't know" response was not random, a sensitivity analysis was performed to determine how the "don't know" responses could best be incorporated into the analysis approach. Subsequently and prior to analysis, "don't know" responses were assigned the construct-specific (or attitude/belief-specific) median value of the Likert-scale responses. These responses were then dichotomized into categories of ≤5 ('positive response' or agreement) versus >5 ('negative response' or disagreement). Four scenario-specific categories for the EPPM were also created, based on level of perceived threat and level of perceived efficacy. These categories include: low threat and low efficacy (LT/LE), low threat and high efficacy (LT/HE), high threat and low efficacy (HT/LE), and finally high threat and high efficacy (HT/HE). Using the Likert-scale responses, the 'threat' variable was determined as the product of the participant's response to the perceived likelihood of the occurrence of the given public health threat and the perceived severity of the event constructs, while the 'efficacy' variable was calculated as the product of the participant's response to their perceived ability to perform their duty (Self Efficacy) and their perceived impact on combating the given public health threat (Response Efficacy) constructs. Low and high categories of perceived threat and efficacy were determined by the median value of each product, respectively. Pearson chi-square tests were used to compare regions on demographic characteristics and on agreement with the dichotomized questions. Multinomial logistic regression analysis was performed to evaluate relationships between the pandemic flu EPPM categories and demographic factors. In addition, logistic regression analysis was utilized to evaluate relationships between these EPPM categories and the attitude and belief responses to the 16 questions not considered in assigning the EPPM categories. In this analysis the EPPM categories were evaluated as predictors with and without adjustment for demographic characteristics. Missing responses were excluded from the analyses. All analyses were performed using STATA version 10.0 (Stata Corporation College Station, TX) and SAS version 9.1 (SAS Institute, Cary, NC). Ethics Statement Research ethics approval for the Johns Hopkins∼Public Health Infrastructure Response Survey Tool (JH∼PHIRST) survey and its administration was received from the Johns Hopkins Bloomberg School of Public Health Institutional Review Board (JHSPH IRB) (exempt status # 45 CFR 46.101 (b) (2)). Per JHSPH IRB approval, written consent was not obtained, as the research presented no more than minimal risk to subjects and involved no procedures for which written consent is normally required. The JHSPH IRB-approved study materials included a written disclosure describing the study and emphasizing voluntary participation; verbal consent was not requested or required by JHSPH IRB for this approved study. Survey Instrument The JH∼PHIRST is an anonymous online survey instrument consisting of demographic and attitude/belief sections focusing on health department workers' attitudes and beliefs toward public health emergency response. The demographic information includes gender, highest education level, role in an emergency response, responsibility for a family member, and categories of age, professional classification, years in present organization, and years in profession. For each of four emergency scenarios (weather-related emergency, pandemic influenza, 'dirty bomb' radiological terrorism event, and inhalational anthrax bioterrorism event) the same 20 attitudes and beliefs were presented for level of agreement along with two open-ended questions. Responses to the attitude and belief questions were based on a 10-point Likert scale with a response of '1' indicating strong agreement with the question and a response of '10' indicating strong disagreement with the question. Respondents could also indicate "don't know". The online JH∼PHIRST instrument used in the current study evolved from an earlier, paper-based pilot survey instrument that we implemented in three Maryland local health departments in 2005 to assess the willingness of these employees to respond to an influenza pandemic [8] . Of note, while this earlier pilot version did incorporate some aspects of risk perception, it did not use the threat and efficacy measures from EPPM that have become commonplace in risk communication studies over the past decade. We incorporated the EPPM content to generate the online JH∼PHIRST instrument based on a series of relevant observations from the 2005 Maryland-based pilot study – namely, that local public health workers' willingness to respond in a pandemic flu scenario was hampered by uncertainties, fears and lack of confidence [8] that are reflected in the standard batteries of questions used by EPPM studies. The online JH∼PHIRST survey's EPPM-based threat and efficacy measures have been widely validated by numerous studies in multiple countries, cultural settings, and health contexts [20] . The "non-EPPM" constructs in the online JH∼PHIRST survey were derived from our original paper-based Maryland 2005 pilot study [8] , which itself was based on validated risk communication theory [15] , [21] in the context of an identified set of potential peripheral risk perception influences from emergency preparedness training experiences in local health departments [16] . Study participants Four clusters of local health departments from three states in the Midwestern and Eastern U.S. participated in the JH∼PHIRST survey. Each region (cluster) had access to the online version of JH∼PHIRST via the SurveyMonkey (SurveyMonkey.com, Portland OR) web site for 4 to 6 weeks, and each region's survey results were logged separately. The health departments were responsible for encouraging all of their employees to respond to this survey and individual participation was voluntary. During the survey administration, completion rates by health department were intermittently provided to pre-designated administrative points of contact at the participating agencies within the cluster to encourage agency-wide survey participation. Statistical analysis This survey scale did not include a neutral point, and the option of a "don't know" response may have carried the ambivalence stance. Suspecting that the use of the "don't know" response was not random, a sensitivity analysis was performed to determine how the "don't know" responses could best be incorporated into the analysis approach. Subsequently and prior to analysis, "don't know" responses were assigned the construct-specific (or attitude/belief-specific) median value of the Likert-scale responses. These responses were then dichotomized into categories of ≤5 ('positive response' or agreement) versus >5 ('negative response' or disagreement). Four scenario-specific categories for the EPPM were also created, based on level of perceived threat and level of perceived efficacy. These categories include: low threat and low efficacy (LT/LE), low threat and high efficacy (LT/HE), high threat and low efficacy (HT/LE), and finally high threat and high efficacy (HT/HE). Using the Likert-scale responses, the 'threat' variable was determined as the product of the participant's response to the perceived likelihood of the occurrence of the given public health threat and the perceived severity of the event constructs, while the 'efficacy' variable was calculated as the product of the participant's response to their perceived ability to perform their duty (Self Efficacy) and their perceived impact on combating the given public health threat (Response Efficacy) constructs. Low and high categories of perceived threat and efficacy were determined by the median value of each product, respectively. Pearson chi-square tests were used to compare regions on demographic characteristics and on agreement with the dichotomized questions. Multinomial logistic regression analysis was performed to evaluate relationships between the pandemic flu EPPM categories and demographic factors. In addition, logistic regression analysis was utilized to evaluate relationships between these EPPM categories and the attitude and belief responses to the 16 questions not considered in assigning the EPPM categories. In this analysis the EPPM categories were evaluated as predictors with and without adjustment for demographic characteristics. Missing responses were excluded from the analyses. All analyses were performed using STATA version 10.0 (Stata Corporation College Station, TX) and SAS version 9.1 (SAS Institute, Cary, NC). Results Across the four health department regions, 1835 responses were collected for an overall response rate of 83%. Table 1 describes the composition of the four regions of respondents, and their catchment regional demographics based on U.S. Census data [22] . Most respondents were female (81%), over 40 years of age (72%), had at least a Bachelors' degree (73%), worked in their present organization for at least five years (64%), were in their profession for 10 or more years (58%), perceived having a role in responding to a public health emergency (84%), and had a family member dependent on them (67%). The regions were significantly different on most of these characteristics. The level of non-responses ranged from 1 to 2% across the four scenarios, considering all constructs and potential respondents for a given scenario. Similarly, the level of "don't know" responses ranged from 1.1 to 2.6%. Based on the criteria considered and the sensitivity analysis of how best to incorporate the "don't know" responses as a measure of attitudinal ambivalence in subsequent analyses, imputing these responses with the construct-specific "sample" median.was determined to be a reasonable approach. 10.1371/journal.pone.0006365.t001 Table 1 Comparison of population and respondent characteristics by survey region. Region 1 a Region 2 a Region 3 a Region 4 a p-value b Total Catchment (population-weighted) Population 1,727,938 1,133,212 1,073,513 1,055,578 NA Median Income $62,075 $44,513 $43,552 $34,842 NA % with Bachelors degree or higher 32 21 21 15 NA % minority 15 13 17 5 NA Respondent characteristics Number responding 668 354 532 281 1835 Response rate 89 88 82 67 83 % female 88 77 77 78  = 40 years ( = 5 years ( = 10 years (999.9) c If required: self-reported willingness to respond 18.14 (5.70–57.66) 2.61 (1.63–4.18) 31.7 (10.00–100.51) If asked but not required: self-reported willingness to respond 5.73 (3.25–10.12) 1.53 (1.08–2.16) 9.52 (5.52–16.44) Perceived knowledge about the public health impact 5.39 (3.16–9.22) 1.84 (1.29–2.62) 17.43 (8.80–34.52) Perceived awareness of role-specific responsibilities 3.76 (2.59–5.45) 1.41 (1.06–1.87) 7.93 (5.45–11.54) Perceived skills for role-specific responsibilities 7.25 (4.12–12.75) 1.45 (1.05–2.00) 11.29 (6.66–19.13) Perception of psychological preparedness 4.78 (2.90–7.87) 1.15 (0.84–1.57) 8.64 (5.29–14.12) Perceived ability to safely get to work 9.65 (4.85–19.21) 1.52 (1.08–2.13) 6.48 (4.11–10.21) Confidence in personal safety at work 4.89 (3.20–7.47) 1.27 (0.96–1.70) 6.19 (4.31–8.88) Perception that family is prepared to function in absence 4.19 (2.76–6.37) 1.38 (1.02–1.85) 4.16 (2.98–5.81) Perceived ability of Health Department to provide timely information 5.08 (2.82–9.16) 2.01 (1.35–2.99) 10.78 (5.75–20.20) Perceived ability to address public questions 4.74 (3.12–7.19) 1.56 (1.16–2.10) 8.74 (5.80–13.15) Perception of the importance of one's role in the agency's overall response 13.72 (6.92–27.22) 2.03 (1.47–2.82) 111.08 (27.43–449.92) Perceived need for pre-event preparation and training 4.98 (2.27–10.97) 3.71 (1.94–7.09) 20.63 (6.46–65.85) Perceived need for post-event psychological support 1.52 (1.08–2.13) 2.60 (1.81–3.74) 3.60 (2.55–5.08) Willingness to respond regardless of severity 10.87 (5.65–20.92) 1.79 (1.29–2.49) 11.22 (6.71–18.74) a The odds ratio compares this category to the Low Threat/Low Efficacy category as the Reference. b The number of participants included in the analysis for each question was approximately 1680. c All responses in the High Threat/High Efficacy category were positive. In order to provide an accurate yet reasonable representation of the relationship between this and the Low Threat/Low Efficacy category, a weighted logistic regression analysis (SAS) was performed adding 0.1 to each cell count. The odds ratio and confidence interval indicate that the odds of a positive response to the attitude/belief is exceedingly greater for the High Threat/High Efficacy group than for the Low Threat/Low Efficacy group. 10.1371/journal.pone.0006365.t004 Table 4 Associations of categories of the Extended Parallel Process Model with attitudes and beliefs regarding a pandemic influenza emergency (adjusted for demographic characteristics). Low Threat, High Efficacy High Threat, Low Efficacy High Threat, High Efficacy Attitudes and Beliefs Odds Ratio a,b (95%CI) Odds Ratio a,b (95%CI) Odds Ratio a,b (95%CI) Perceived likelihood of being asked to report to duty 20.95 (5.09–86.16) 2.79 (1.60–4.87) >999.9 (999.9) c If required: self-reported willingness to respond 16.48 (5.16–52.65) 2.39 (1.48–3.87) 41.58 (10.15–170.40) If asked but not required: self-reported willingness to respond 5.31 (2.93–9.61) 1.43 (1.00–2.04) 8.46 (4.77–15.01) Perceived knowledge about the public health impact 5.93 (3.31–10.60) 1.73 (1.19–2.52) 14.34 (7.16–28.74) Perceived awareness of role-specific responsibilities 3.61 (2.43–5.35) 1.37 (1.01–1.84) 6.55 (4.45–9.64) Perceived skills for role-specific responsibilities 7.71 (4.19–14.20) 1.35 (0.97–1.88) 10.18 (5.94–17.43) Perceived psychological preparedness 5.02 (2.96–8.52) 1.07 (0.78–1.48) 8.56 (5.12–14.30) Perceived ability to safely get to work 10.17 (4.90–21.14) 1.43 (1.01–2.03) 5.95 (3.72–9.50) Confidence in personal safety at work 4.63 (2.99–7.16) 1.25 (0.93–1.69) 6.30 (4.31–9.22) Perception that family is prepared to function in absence 4.38 (2.81–6.83) 1.45 (1.06–2.00) 4.61 (3.23–6.58) Perceived ability of Health Department to provide timely information 5.14 (2.78–9.51) 1.99 (1.32–2.98) 11.3 (5.81–21.99) Perceived ability to address public questions 5.04 (3.23–7.86) 1.52 (1.11–2.07) 8.84 (5.67–13.79) Perception of the importance of one's role in the agency's overall response 12.80 (6.40–25.60) 1.93 (1.37–2.71) 88.88 (21.86–361.40) Perceived need for pre-event preparation and training 6.80 (2.70–17.16) 3.83 (1.94–7.56) 19.27 (5.98–62.12) Perceived need for post-event psychological support 1.52 (1.06–2.17) 2.69 (1.84–3.93) 3.44 (2.39–4.96) Willingness to respond regardless of severity 10.89 (5.46–21.75) 1.74 (1.24–2.44) 11.00 (6.43–18.84) a The odd ratios compares this category to the Low Threat/Low Efficacy category as the Reference. b The number of participants included in the analysis for each question was approximately 1600. c The Odds Ratio and 95%CI could not be calculated with adjustments for the demographic characteristics because all responses were positive. This odds ratio and CI indicate that the odds of a positive response to the attitude/belief is exceedingly greater for the High Threat/High Efficacy group than for the Low Threat/Low Efficacy group. A trend previously seen in region-specific analyses (reports to the agencies) and reiterated in Tables 3 and 4 was that the EPPM efficacy dimension tended to have a larger impact on a positive response to an attitude/belief question than the EPPM threat dimension. With respect to the reference (LT/LE) category, for example, the LT/HE category for self-reported willingness to respond if required had a higher OR of a positive response than the HT/LE category. This pattern, observed in 12 of the 16 construct questions, may suggest that the efficacy dimension has a larger impact on positive responses to the attitude and belief questions than the threat dimension. Three of the 16 questions specifically addressed a respondent's willingness to respond. The OR (95%CI) of answering positively to self-reported willingness to respond if required was 31.7 (10.0, 100.51) times higher for the HT/HE group than for the LT/LE group. The comparable OR (95%CI) for self-reported willingness to respond if asked but not required was 9.52 (5.52, 16.44) and for willingness to respond regardless of severity was 11.22 (6.71, 18.74). The vast majority (94%) of the respondents believed they will be called upon to respond to duty during an influenza pandemic ( Table S1 ). However, follow up analyses indicate that health department employees who considered their individual roles to be important in the context of overall agency response efforts were, after adjustment for demographic characteristics, 8.45 (95%CI: 6.06, 11.77) times more likely to indicate they would report to duty during a pandemic even if it were severe. Ninety-one percent of the clinical staff felt their job during an influenza pandemic would be important, as compared with 85% among non-clinical staff, while only 73% of the non-clinical staff are aware of their role-specific responsibilities, as compared with 77% in the clinical staff. Discussion Willingness to respond is a critical component of effective public health system readiness and sustainability in emergencies. So far, on a national level, public health readiness and response efforts have focused nearly exclusively on enhancing ability, without specifically attending to willingness issues. Our study results suggest that this response willingness is not to be taken for granted in the public health arena. With some regional variation, overall 16% of the workers in 2006-7 were not willing to "respond to a pandemic flu emergency regardless of its severity". This number is reassuring in contrast to that reported in previous studies, where higher percentages indicated they would not be willing to respond to an influenza pandemic [8] , [10] . However, the workload in public health agencies during a pandemic will be so immense that "all hands on deck" will be required to tackle the resulting challenges, and significant changes in roles and responsibilities will be required. Reported unwillingness to respond by approximately 1 in 6 means that additional efforts are required to increase and sustain the proportion of local health department employees willing to respond. It simultaneously highlights the critical importance of understanding the reasons why some public health employees are unwilling to respond to a pandemic threat. In this study, we address some of these gaps through systematic application of a behavioral model that addresses cognitive and emotional dynamics of response willingness attitudes. As a theoretical model based on decades of prior research on fear campaigns and health risk messaging, the EPPM describes how a sequence of threat appraisals (perceived severity and susceptibility) and efficacy appraisals (perceived response efficacy and self-efficacy) may influence behavioral responses to messages with fear content. Using the EPPM, we can see how public health workers' individual degrees of perceived threat ('concern') and perceived efficacy ('confidence') influence their willingness to respond. Indeed, in line with our basic hypothesis, we have found that individuals who had a perception of high threat and high efficacy – i.e., those who fit a 'concerned and confident' profile in the EPPM analysis – had the highest declared self-reported willingness to respond (if required) rates to pandemic flu, which was 31.7 times higher than those fitting a 'low threat/low efficacy' EPPM profile. Of the two basic components of the EPPM – perceived threat and perceived efficacy – the latter proves to be the more significant component in determining willingness in this scenario. Compared to the low threat/low efficacy reference category, low threat/high efficacy increases the willingness to respond if required almost 18-fold, while high threat/low efficacy is associated with less than a 3-fold increase. These results, assessed in 2006-7 following several years of increased awareness and high profile pandemic flu preparedness efforts at federal, state and local agencies, reveal a unique opportunity to induce change. As any amount of additional assistance will make a difference in response to an influenza pandemic, the first step is to better educate public health workers as to their designated roles during this emergency scenario, and then motivate them with an understanding of why this role makes a difference. If a specific designated role cannot be predetermined, a set of potential roles have to be defined and adequately introduced to all relevant workers, up to a point at which they will feel confident in their ability to perform their duty, and perceive it as important. Our results also suggest that downplaying the threat of the scenario to 'calm' the fears of the workers is not an advisable approach. A sense of threat is an important component in the worker's motivation to prepare for the event and to respond to it. It is important to note that 24% of the respondents did not perceive their work environment as safe, and 15% of the respondents felt they could not safely arrive to work. These issues must be an important component of the workers' training, and some assurance to their personal safety can and should be provided. Certain limitations to the current study must be acknowledged. First, while we have strived to minimize social desirability bias in the construction and phrasing of the JH∼PHIRST instrument content, this survey-based study is not necessarily predictive of actual behavior during an event. Second, the findings from our study of local health department personnel may not necessarily translate to similar findings among responders in other cohorts such as hospital employees, EMS, or police; indeed, this is an area worthy of further comparative research study. Third, the imputation for "don't know" responses may result in narrower confidence intervals for the odds ratio estimates; however, our intent is to provide a relative perspective on willingness to respond and the factors that influence it for a given health department and to identify patterns of attitudes that may influence effectiveness of health departments in emergency situations. Despite these caveats, however, ascertaining local health department workers' dispositions toward fulfilling pandemic flu response expectations nonetheless has value for current local public health agency readiness and response efforts and related training needs assessments. In a pilot study of local Maryland public health personnel in 2005 [8] , we illustrated that risk perception influences peripheral to the actual event – such as perceived importance of one's role in an agency response – can markedly affect willingness-to-respond rates. The results at the time indicated that nearly half of the workers noted they are unlikely to respond to duty during a pandemic emergency. In the US, efforts put into planning, training, exercising and increasing awareness of public health roles in disaster response since the introduction of the Pandemic and All-Hazards Preparedness Act [23] , as well as the federal, state, and local resources provided in support of the same, may explain some of the improvement in the willingness to respond rates between the surveys. It should be noted, though, that significant changes in the phrasing of the questions from the pilot study do not allow direct comparison of the responses to the current larger-scale multi-state online survey. In conclusion, our findings point to the EPPM as a useful framework to inform nuanced assessments of levels of – and gaps in – willingness to respond within the local public health infrastructure. Our data indicate that 'concerned and confident' local public health employees are most likely to be willing to respond to an influenza pandemic. This finding may allow public health agencies to design, implement, and evaluate training programs focused on emergency response willingness in health departments. Supporting Information Table S1 Percent positive responses to attitude and belief questions and percent in EPPM categories by region (0.06 MB DOC) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797153/

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257868/

Radioimmunotherapy of Fungal Diseases: The Therapeutic Potential of Cytocidal Radiation Delivered by Antibody Targeting Fungal Cell Surface Antigens

Radioimmunotherapy is the targeted delivery of cytocidal radiation to cells via specific antibody. Although mature for the treatment of cancer, RIT of infectious diseases is in pre-clinical development. However, as there is an obvious and urgent need for novel approaches to treat infectious diseases, RIT can provide us with a powerful approach to combat serious diseases, including invasive fungal infections. For example, RIT has proven more effective than standard amphotericin B for the treatment of experimental cryptococcosis. This review will discuss the concepts of RIT, its applications for infectious diseases, and the strides made to date to bring RIT of infectious diseases to fruition. Finally, we will discuss the potential of PAN-FUNGAL RIT, the targeting of conserved fungal cell surface antigens by RIT, as a treatment modality for fungi prior to the formal microbiological identification of the specific pathogen. In sum, RIT provides a mechanism for the targeted killing of drug susceptible or resistant fungi irrespective of the host immune status and may dramatically reduce the length of therapy currently required for many invasive fungal diseases. Introduction Fungal diseases are increasingly common major causes of human disease, especially in immunocompromised individuals and hospitalized patients with underlying medical conditions (Patterson, 2005 ; Caston-Osorio et al., 2008 ; Richardson and Lass-Florl, 2008 ; Pfaller and Diekema, 2010 ). In fact, since 1979 there has been a >200% increase in the annual number of cases of invasive fungal infections (IFI) in the United States. IFIs rates have risen in large part due enhanced interventions in intensive care units (ICUs), increased survival rates of individuals with immunodeficiencies, and the increased administration of potent therapeutics, chemotherapeutics, and biologicals, that compromise the immune responses of our patients. This increase is best exemplified by the example of Candida spp.: Candida were once infrequent causes of invasive disease, whereas they are currently the fourth leading cause of nosocomial bloodstream infection in the United States, responsible for 8–15% of all such hospital acquired infections. However, despite the increased prevalence of many mycotic diseases, there remains an enormous gap in knowledge and our current therapeutic armamentarium all too often fails to eradicate these insidious pathogens. Although they have powerful activities, the number of available medications for mycoses is significantly less than for bacterial diseases. At present, there are three main medication categories for IFI: azoles (fluconazole, itraconazole, voriconazole, and posaconazole), polyenes (primarily formulations of amphotericin B), and echinocandins (caspofungin, micafungin, and anidulafungin). Notably, both the azoles and polyenes target cell membrane sterols, with azoles inhibiting sterol synthesis and the polyenes purportedly disrupting the membrane structure. The echinocandins inhibit cell wall production by interfering with beta-1,3-glucan synthesis. In addition to these drugs, flucytosine, an antimetabolite, is utilized primarily in combination with amphotericin B for the treatment of cryptococcosis. Notably, the echinocandins are the last new class of antifungal drug, with caspofungin gaining FDA approval in by the FDA in 2001. Unfortunately, there is no antifungal medication poised to enter clinical medicine for the foreseeable future. Hence, there is a consensus that new approaches are needed to combat IFI. Radioimmunotherapy (RIT) uses antigen–antibody interactions to deliver cytocidal amounts of ionizing radiation to specific cell targets. Currently, RIT is clinically utilized in the treatment of primary, refractory, and recurrent non-Hodgkin lymphoma using the radiolabeled mAbs Zevalin ® and Bexxar ® . It is important to note that RIT offers several significant advantages over standard antifungal therapy. Firstly, RIT delivers lethal radiation, such that it does not merely interfere with a single cellular pathway but completely destroys targeted cells. As such, RIT is less subject to drug resistance mechanisms. Moreover, RIT is cidal in immunologically compromised individuals as the nuclides are equally able to destroy cell targets in immunologically intact individuals or those with HIV or other immunodeficiencies, either primary or drug induced. RIT does not suffer the drug–drug interactions that clinically trouble clinicians caring for complex patients, such as azole or echinocandin interactions with commonly prescribed immunosuppressive drugs, like cyclosporine or tacrolimus. Finally, in contrast to weeks, months, or years required for the treatment of certain mycoses with standard antifungals, RIT may permit single dose or a limited number of doses to combat fungal diseases. What are the barriers for translating RIT into treatment approaches for infectious diseases? Cell surface antigens are well defined for diverse pathogens, including viruses, bacteria, parasites, and fungi. Moreover, monoclonal antibodies exist that target microbial cell surface antigens. Additionally, the technology for linking radionuclides to mAbs is well established, so the approaches can be readily translated from oncology into infectious diseases. Additionally, the US hospitals that are now regularly using RIT to treating cancer patients are fully equipped for initiating Infectious Diseases RIT. Included in this ability, imaging of patients receiving RIT to ascertain the targeting of radiolabeled mAbs in Infectious Diseases RIT can be readily achieved using portable imaging equipment that is standard in these hospitals. Hence, the time is "now" for developing RIT to combat IFI. RIT of Infectious Diseases Our laboratories were the first to demonstrate that microorganism-specific mAb-RIT is highly effective for the treatment of experimental fungal, bacterial, and viral infections, as well as virally induced cancers (Table 1 ). Although the initial RIT work utilized Cryptococcus neoformans for proof-of-principle studies in 2003 (Dadachova et al., 2003 ), RIT of bacterial and viral pathogens also has rapidly progressed. In 2004, we established the feasibility of RIT for invasive bacterial infection using a mouse pneumococcal disease model (Dadachova et al., 2004a ). An IgM isotype mAb to serotype 8 Streptococcus pneumoniae capsular polysaccharide was conjugated to the alpha-particle emitter Bismuth-213 ( 213 Bi) and we showed that an 80-μCi dose was sufficient to protect 60% of animals from an otherwise lethal challenge. More recently, in 2009, mAbs to the protective or lethal antigens of Bacillus anthracis labeled with either 213 Bi or the beta-particle emitter rhenium-188 ( 188 Re) were shown to prolong the survival of mice infected with B. anthracis Stern bacterial cells, but not spores (Rivera et al., 2009 ). Treatment with 213 Bi was more effective than when less powerful beta-particles were used. Table 1 List of monoclonal antibodies that have been used in experimental studies of radioimmunotherapy for infectious diseases . Antibody Isotype Antigen recognized Reference or source D11 IgM, human Serotype 8 pneumococcal polysaccharide Dadachova et al. ( 2004a ), Zhong et al. ( 1999 ) 7.5G IgG2b, mouse B. anthracis protective antigen Rivera et al. ( 2006 , 2009 ) 10F4 IgG1, mouse B. anthracis protective antigen Rivera et al. ( 2006 , 2009 ) 14FA IgG2b, mouse B. anthracis lethal antigen Rivera et al. ( 2009 ) 18B7 IgG1, mouse GXM, cryptococcal polysaccharide Dadachova et al. ( 2003 , 2004b , 2006b ), Bryan et al. ( 2009 , 2010 ) 246-D (cluster I) IgG1, human HIV gp41 Dadachova et al. ( 2006a ) C1-P5 IgG1, mouse HPV E6 Wang et al. ( 2007 ), Phaeton et al. ( 2010a , b ), Harris et al. ( 2011 ) 2G8 IgG2b, mouse Beta (1,3) glucan Torosantucci et al. ( 2009 ), Rachini et al. ( 2007 ) 4E12 IgG2a, mouse Hsp60 Guimaraes et al. ( 2009 ) 6D2 IgM, mouse Melanin Rosas et al. ( 2000 ) B11 IgM Fungal glucosyl ceramide Rhome et al. ( 2011 ) In 2006, HIV infected cells were found to be effectively targeted by RIT (Dadachova et al., 2006a ). Radiolabeling of antibody to the HIV-1 envelope glycoproteins gp120 and gp41 with 213 Bi or 188 Re selectively killed HIV infected cells in vitro and eliminated the majority of HIV infected cells injected into SCID mice. Notably, the mAb to gp41 is fully human. It is possible that, used in combination with anti-retroviral drugs, that RIT may provide a means to eradicate HIV infection (Casadevall et al., 2007 ). RIT has also proven to be highly effective in the treatment of virally induced cancers (Dadachova et al., 2007 ). Using a human papillomavirus (HPV) type 16-associated cervical cancer model in mice, RIT with a human mAb targeting the HPV16 E6 antigen significantly impeded tumor growth compared to cancers in control animals (Wang et al., 2007 ). Interestingly, RIT targeting cancer cells expressing low levels of HPV16 E6 was also successful (Phaeton et al., 2010a ). Furthermore, pre-treatment of the cervical tumors with a proteosome inhibitor, MG-132, and unlabeled antibody to E6 resulted in an increase in the levels of E6 for targeting by 188 Re-labeled mAb and enhanced the efficacy of RIT (Phaeton et al., 2010b ). These studies also suggest that RIT could potentially eradicate infected cells prior to their malignant transformation. In addition to cervical cancer, RIT proof-of-principle experiments with HPV-associated head and neck squamous cell carcinoma suggest that this modality may be effective for this difficult to combat disease (Harris et al., 2011 ). RIT of Cryptococcosis Cryptococcus neoformans is a yeast-like encapsulated fungus with a global distribution that is responsible for ∼600,000 deaths annually (Park et al., 2009 ), particularly in individuals with HIV infection. As noted above, the first RIT experiments published in 2003 utilized C. neoformans as a model organism (Dadachova et al., 2003 ). The initial studies with C. neoformans demonstrated that a mAb specific for glucuronoxylomannan (GXM), the major component of the pathogen's polysaccharide capsule, radiolabeled with either 213 Bi or 188 Re efficiently killed cryptococcal cells in vitro and significantly reduced fungal burdens in and prolonged the survival of lethally infected mice (Dadachova et al., 2003 ). Most impressively, 60% of infected mice treated with 100 μCi 213 Bi were alive at day 75 after RIT. Importantly, systemically infected mice tolerated activities of up to 150 μCi of mAb labeled with either nuclide with platelet counts normalizing by 14 days post-treatment (Dadachova et al., 2004b ). The reduction in platelets in the infected mice was similar to that seen in the setting of cancer therapy (Sharkey et al., 1997 ; Behr et al., 1999 ). Additionally, these mice had no evidence of pulmonary fibrosis 5 months after receiving RIT. The lack of severe toxicities due to radiation in the mice with cryptococcosis treated with RIT is presumably due to the high specificity of the mAb. Chemotherapy for severe cryptococcosis is based on the administration of amphotericin B. However, RIT was recently shown to be more effective than amphotericin B for the treatment of disseminated cryptococcosis (Bryan et al., 2010 ). In the murine model examined, amphotericin B was able to prolong survival, but fungal burdens were not significantly reduced. In contrast, RIT significantly reduced the fungal burdens in the tissues examined, regardless of the melanization state of the yeast cell. This is an important observation as C. neoformans can produce melanin along the yeast cell wall and this pigment can protect the fungus from injury from external radiation (Nosanchuk and Casadevall, 2006 ; Dadachova et al., 2008 ). Interestingly, ∼10 times less mAb 18B7 conjugated to 213 Bi was required for killing melanized C. neoformans yeast cells than 188 Re conjugated antibody (Bryan et al., 2010 ), demonstrating the superior penetrating power of the alpha-particle versus the beta-particle emitter. Combining RIT with amphotericin B was more effective than amphotericin B alone (Bryan et al., 2010 ). However, combination therapy was less potent than RIT alone if melanized fungal cells were used for the infectious challenge. It is not clear whether this result is due to the potential toxicities of the amphotericin or the inflammatory responses that are induced upon infection with melanized yeast cells (Mednick et al., 2005 ). Although drug resistance is a concern in the treatment of fungal infections, there is no literature on pathogen resistance to RIT. Nevertheless, experiments have been performed to assess whether resistance can develop (Bryan et al., 2009 ). C. neoformans yeast cells were harvested from infected mice that received RIT and subjected to treatment with 213 Bi or 188 Re conjugated mAbs. Notably, the RIT and radiation naive C. neoformans yeast cells were similarly radiosensitive to the radiolabeled mAbs. Additionally, no differences in survival occurred comparing mice infected with RIT C. neoformans to radiation naive yeast cells. Mechanism Although the precise mechanism of action is not known, there are major radiobiological probabilities for the effectiveness of RIT. The first and most targeted effect occurs by a "direct hit," when the radiolabeled antibody binds to a cell and the emitted particles kill the cell (Figure 1 A). However, "cross-fire" effects, killing of a cell by particles emitted from a radiolabeled antibody bound to a distant cell, can be equally efficient (Figure 1 B). Both of these mechanisms lead to cell apoptosis and cell cycle redistribution (Macklis, 2004 ). It is also highly probable, that a radiolabeled antibody could kill by both of these processes. Notably, the "cross-fire" mechanism would be expected to play a large role in combating fungal biofilm diseases or situations where some fungi are extracellular, whilst others are intracellular. Figure 1 Proposed mechanism of RIT . (A) "Direct hit," where emitted particles kills the cell bound by radiolabeled antibody. (B) "Cross-fire," where emitted particles kill cells distant from the cell bound by radiolabeled antibody. Image is epithelial tissue infected with Candida parapsilosis . Certain mechanisms of radiation effects on fungi have been studied using C. neoformans . C. neoformans yeast cells can be directly killed by gamma, beta, and alpha radiation in a dose and time dependent manner (Bryan et al., 2008 ). Notably, although membrane permeability did occur, it does not appear to be the primary cause of death. Gamma radiation induced significantly more membrane disruption than either beta or alpha radiation. All forms of radiation induced cellular apoptosis, although external gamma radiation and 188 Re-labeled antibody induced more apoptosis than 213 Bi-labeled antibody. However, 213 Bi-labeled antibody significantly reduced the metabolic activity of the yeast cells, whereas the other forms of radiation did not. Hence, the cellular effects of radiation on yeast are dependent on dose and time as well as the form of radiation administered. In a more recent study, gamma radiation, as well as UV and visible light, were shown to affect ATP levels in C. neoformans yeast cells, and this effect was more pronounced in melanized compared to non-melanized cells (Bryan et al., 2011a ). Another study assessed whether "direct hit" or "cross-fire" predominated in the killing of C. neoformans yeast cells (Dadachova et al., 2006b ). As expected, given the power of alpha radiation, 213 Bi-labeled antibody resulted in significant "direct hit" killing. However, 213 Bi-labeled antibody bound to heat-killed yeast cells also led to "cross-fire" fungicidal effects. In contrast, the killing with the lower energy beta-particles produced by the 188 Re-labeled antibody was primarily due to "cross-fire." RIT Toxicities As briefly described above, the primary toxicity identified in our RIT experiments is a modest thrombocytopenia that resolves within 14 days post therapy, which is similar to that seen in RIT in cancer patients (Macklis and Pohlman, 2006 ). Importantly, thrombocytopenia prediction models are developed for RIT and dose-fractionated RIT strategies can be implemented to minimize the nadir and duration of the thrombocytopenia (Shen et al., 2002 ). Importantly, since microbial cells in otherwise sterile sites are foreign to hosts, the microbes display antigens that are distinct from those expressed by host cells. These distinct microbial antigens are the primary targets for infectious diseases RIT. In contrast, RIT of cancers targets tumor-associated antigens that are also expressed on normal cells, which clearly increases toxicity risks. Therefore, in addition to leading to a theoretically higher therapeutic index in infectious diseases RIT relative to that for cancer, RIT of infectious diseases should have a lower toxicity risk than RIT for cancer. There is an important issue of "free antigen" as pathogens release/secrete many substances, including proteins, carbohydrates, and lipids. Hence, antibody directed to released or potentially secreted antigen has the potential to produce off-target effects. Previously, we have studied this issue in murine cryptococcosis, since C. neoformans releases large amounts of capsular polysaccharide and we were concerned that the radiolabeled antibody to the capsule would interact with non-organism associated polysaccharide. However, we determined that radiolabeled antibody had higher affinity for mice-associated polysaccharide than for its soluble form (Dadachova et al., 2003 ). Nevertheless, unlabeled antibody can be administered prior to RIT in order to potentially clear circulating antigens. Biofilms Biofilms are complex communities of pathogens that adhere to one another and are typically surrounded by a matrix of extracellular materials, particularly exopolysaccharides. Fungal biofilms can be especially tenacious and can exacerbate disease by leading to increased drug resistance or enhanced adherence to foreign bodies or tissues. RIT using mAb to C. neoformans GXM conjugated to 213 Bi can effectively damage cryptococcal biofilms (50% reduction in metabolic activity; Martinez et al., 2006 ). Notably, beta and gamma forms of radiation did not have a significant effect on the fungal biofilms. It was not determined whether RIT of biofilms synergized with antifungal drugs, as the alterations in biofilm could enhance drug penetration facilitating the activities of the drugs on fungal cells. Hence, RIT may be a means for combating fungal biofilms with or without concomitant antifungal drugs. Concept of PAN-FUNGAL RIT To dramatically drive the treatment of infectious diseases forward with RIT, we need to make a "quantum leap" by identifying antigens shared by major IFI-causing pathogens (PAN-FUNGAL antigens) in order to deliver RIT without the need for specific mycological diagnosis or fear of drug resistance. To this end, we have been exploring the possibility of targeting common cell wall associated antigens, which also happen to constitute major virulence factors for these fungi. We have begun exploring the utility of four cell surface antigens. The majority of fungal cells, yeast, and hyphal, display beta-glucans on their cell surface and Cassone and colleagues have generated a mAb to beta-glucan that provides significant protection against Candida albicans, C. neoformans , and Aspergillus fumigatus in animal models if given prior to infection (Torosantucci et al., 2005 , 2009 ; Rachini et al., 2007 ). Heat shock protein 60 (Hsp60) is a key regulator of virulence in Histoplasma capsulatum and mAbs directed to this protein are protective in murine histoplasmosis (Guimaraes et al., 2009 ). Recently, we demonstrated that mAbs to H. capsulatum Hsp60 also bound other pathogenic fungal species (Bryan et al., 2011b ), but does not react with human Hsp60 (Guimaraes et al., 2009 ). Melanin is present in the cell wall of diverse human fungal pathogens and a monoclonal to fungal melanin has been shown to bind C. neoformans, H. capsulatum, Aspergillus spp., C. albicans, Scytalidium dimidiatum, Sporothrix schenckii, Paracoccidioides brasiliensis , Coccidioides posadasii , and Blastomyces dermatitidis (reviewed in Nosanchuk and Casadevall, 2006 ; Taborda et al., 2008 ). Glucosylceramide is another common exposed cell surface antigen on fungi and a mAb to glucosylceramide has been shown to bind glucosylceramide in C. neoformans and C. albicans , but not mammalian glucosylceramide (Rhome et al., 2011 ). Selected mAbs to these four surface antigens were used for radiolabeling and tested for their capacity to kill C. neoformans and C. albicans (Bryan et al., 2011b ). 213 Bi-labeled >90% of mAbs 2G8, IgG2b to beta-galactan, and 4E12, IgG2a to Hsp60, but the labeling efficiency of mAb 6D2, IgM to melanin, was only 30%. 213 Bi did not effectively label mAb B11, IgM to glucosylceramide, but the mAb was labeled by 188 Re. Radiolabeled mAb to beta-glucan killed 100% of C. neoformans yeast cells, whereas C. albicans pseudohyphal cells were significantly more resistant as the treatment produced a 30% reduction in CFUs. Targeting Hsp60 produced > 90% reductions in CFUs of both C. neoformans and C. albicans . The poor 213 Bi radiolabeling of the mAb to melanin resulted in the delivery of three to four times less radiation than the 213 Bi-labeled mAbs to beta-glucan and Hsp60, and ∼4 μCi/5 million yeast cells was the maximal radiation delivered resulting in only 15 and 22% reduction for C. albicans and C. neoformans , respectively, but the decrease was only significant for C. neoformans . The 188 Re-labeled mAb to glucosylceramide decreased the CFU of C. neoformans by 40%. Hence, we have demonstrated that we can radiolabel diverse antibodies to distinct conserved surface antigens and deliver cytocidal radiation to important fungal pathogens by RIT. It is noteworthy that the efficacy of killing was achieved with significantly lower (∼1,000 times) concentrations than were required for biological efficacy with unlabeled antibody, such as with mAb 2G8 (Torosantucci et al., 2009 ). Moreover, the data demonstrated that the powerful alpha particles from 213 Bi efficiently killed fungal cells even when there were relatively few binding sites on their cell surface (∼5% mAb binding). Additional experimentation is required to develop more efficient labeling of IgM isotype mAbs and perhaps improve the labeling of IgG isotype mAbs. Nevertheless, the PAN-FUNGAL concept is validated by these proof-of-principle experiments and further study is clearly warranted. Discussion The drive to develop RIT of infectious diseases is fueled by the facts that (1) our current antifungal armamentarium often fails to eradicate IFI, (2) the last new category of antifungals was FDA approved in 2001, (3) no new antifungal drugs are close to clinical availability, and (4) RIT is a means to specifically target microbes in a cytocidal manner, irrespective of the host immune status, current drug regimen, or potential resistance mechanisms of the microbe. With RIT of fungi, especially with PAN-FUNGAL RIT, we can potentially reduce treatment durations, prevent the toxicities of the prolonged administration of (possibly ineffective) antifungals, minimize concerns of drug–drug interactions, permit the preservation of current immunomodulating medications (i.e., for transplant patients), and eliminate the need for a specific mycological diagnosis. Given the well developed applications of RIT in Oncology, the barriers to initiating RIT for infectious diseases, especially IFIs that continue to have unacceptably high mortality rates, are indeed surmountable. The work to date demonstrates that we have in hand a diverse set of specific mAbs and mAbs with reactivity to several leading fungal pathogens that can be harnessed for RIT. We are hopeful that the upcoming years will see a broader acceptance of this approach and a rapid growth in pre-clinical work that will allow the translation of RIT from the basic science laboratory to our patients. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9412515/

A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people's health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/μL at the flow rate of 0.4 μL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device. 1. Introduction Food security is one of the most critical issues in modern society. People eat food not just for their appetite as humans but also for their physical and mental health. Some valuable traditional Chinese medicines (TCMs) have been widely used as tonic food for a long time, especially in some East Asian countries. However, as the demand for TCMs has risen rapidly over the past decades, some substitutions derived from the skins or bones of animals have been fraudulently labeled as true TCMs. These substitutions sometimes have little pharmaceutical effect and harm people's health seriously. Because morphological characters are not reliable enough to distinguish true TCMs, it is essential to establish an effective and applicable method to identify them to protect people's health. People have the right to choose healthy food. Therefore, the identification of the food for safety purposes is very significant [ 1 ]. It is critical to establish a reliable and convenient method for food identification. The bacterial culture method is considered the gold standard for diagnosis in clinical practice for food security. It is specific and accurate. However, it is also time-consuming, complex, and without high sensitivity [ 2 ]. Some researchers have also demonstrated various spectroscopy-based and chromatographic methods for quality control [ 3 , 4 ]. However, similar chemical properties in an adulterant sometimes make it difficult to identify species, and they have not reached the stage of practical application due to sample-processing variability. An alternative method for specific analysis in species identification is the nucleic acid-based assay [ 5 ], which became popular during the last decades. Deoxyribonucleic acid (DNA)-based molecular diagnostics have been widely applied in order to authenticate species for health and religious purposes in food security. Since Saiki et al. [ 6 ] invented polymerase chain reaction (PCR) in the 1980s, PCR has become one of the most established molecular biology techniques in recent decades. Lots of researchers utilized PCR in the biotechnology field, such as in criminal forensics, genetic analysis, and medical diagnostics, due to the exponential amplification of tiny amounts of specific DNA molecules to a detectable concentration. The PCR process goes through the following stages. Firstly, double-stranded DNA (dsDNA) is separated into two single-stranded DNA (ssDNA) at high temperatures (denaturation stage at about 95 °C or 368 K). Secondly, primers bind to their complementary site of ssDNA at low temperatures (annealing stage at about 55 °C or 328 K). Thirdly, the thermostable DNA polymerase extends the primers, complementary to the DNA template, at intermediate temperatures (extension stage at about 72 °C or 345 K). Conventional PCR machines are sizable and require a long cycling time. During the last three decades, microelectromechanical systems (MEMS) fabrication processes play an essential role in reducing these inconveniences. Implementation of such miniaturized devices enables the development of fast, easy-to-use, and portable systems with a high level of automation and functional integration for applications such as point-of-care (POC) diagnostics. Moreover, the disposable elements used in the miniaturized devices prevent the samples from cross-contamination. Many researchers divided the micro-PCR (μPCR) devices into two types based on the handling of sample mixtures. One is the static chamber μPCR devices. The injected sample and the whole reaction chamber are stationary in the static chamber device and undergo external thermal variations to accomplish the PCR process. These devices are a scaled-down version of commercial thermocyclers and perform repeated heating and cooling. Any enzyme adsorbed by the walls of the reaction volume is slight. Kaprou et al. [ 7 ] designed the embedded resistive microheaters for realization in the inner Cu layer of the commercially available PCB substrate. Huang et al. [ 8 ] developed a microfluidic chamber-based PCR-array system. The system can screen multiple respiratory pathogens in an integrated manner. There is a Peltier heating device responsible for the thermal control of PCR. On the contrary, the PCR mixture flows consecutively across the distinct heated zones corresponding to the temperature steps needed for PCR in continuous-flow (CF) PCR devices. The reaction mixture moves through the individual isothermal zones by keeping the temperatures constant over time at different locations in the device. The arrangement of isothermal zones determines the residence time and the number of thermal cycles [ 9 ]. The usual method of arranging the heater location in CFPCR devices is to place some heaters side by side. Then, the sample mixture flows through the required temperature regions along a serpentine channel to accomplish the PCR process. Ragsdale et al. [ 10 ] presented a disposable polycarbonate (PC) device that included the amplification of the foot-and-mouth disease virus (FMDV)-derived cDNA. The opposite sides of the chip demonstrate the placement of two thin-film heaters. The PCR mix was pulled through the chip using a commercial syringe pump. Li et al. [ 11 ] explored a glass-polydimethylsiloxane (PDMS) bonding chip for multi-PCR of Porphyromonas gingivalis , Treponema denticola , and Tannerella forsythia . The chip system is composed of a commercial syringe pump and two positive temperature coefficient (PTC) ceramic heaters controlled by two commercial temperature controllers. Jiang et al. [ 12 ] developed a microfluidic device combining CFPCR and DNA hybridization to detect bacterial pathogens. Using a polyimide heating membrane, they built a thermal cycler of the PDMS-based device. A syringe pump connected to the inlet and outlet of the microchannel implemented the sample injection and collection, respectively. Chang and You [ 13 ] presented an irreversible bond between a PDMS microfluidic chip and a half-cured PDMS film coated on a PCB substrate. The PCB substrate with three heating electrodes supplied the specific heating zones and amplified a 120-bp DNA fragment from BEAS-2B of human bronchial epithelial cells. Pješčić et al. [ 14 ] utilized a commercial syringe pump to inject the phage DNA template (ΦX174) sample into the microfluidic channel. The authors designed the thermal controlling system comprised of a cartridge heater, a heat sink, and a PID controller to generate high- and low-temperature regions within the glass-composite device. Schaerli et al. [ 15 ] presented an SU-8 substrate device for sample droplets. A cartridge heater and a Peltier module supported the denaturation and annealing zones of the device, respectively. An 85 bp sequence of the tyrocidine synthetase 1 gene (P09095) solution was in syringes pumped using the Harvard Apparatus pump. Fukuba et al. [ 16 ] demonstrated a microfabricated device in environmental microbiology. Six indium tin oxide (ITO) heaters on a glass substrate define three isothermal zones for amplifying 580 and 1450 bp of DNA fragments. Hsieh et al. [ 17 ] presented the integrated PMMA chip combined with a microfluidic PCR and a surface plasmon resonance sensor to detect LMP1 DNA. Two aluminum blocks with electric heating films and PID controllers defined the temperature zones. Some researchers choose the other heater arrangement and distribute the heating regions circumferentially to avoid unnecessary heating during PCR. The reactant plug moves through a spiral microchannel or tube to the outlet to accomplish the PCR process. Peham et al. [ 18 ] demonstrated a cyclic microsystem for amplifying the 16S ribosomal RNA gene. The miniaturized device consists of three Peltier elements driven by commercial PID controllers and arranged to form a triangular prism. The microfluidic tubing comprises transparent polytetrafluoroethylene (PTFE) and is coiled around the triangular prism 40 times. Chung et al. [ 19 ] presented a polymer disk equipped with a spiral microchannel. Six temperature controllers maintained the temperatures of the metal plates at different constant values. The syringe pump pushed a transport liquid containing the fragment from arylamine N-acetyltransferase DNA through a PTFE capillary tube. Shu et al. [ 20 ] proposed a segmented CD-PCR on a spiral PTFE tubing microfluidic device. The device for identifying four foodborne bacterial pathogens mainly consists of three resistance cartridge heaters, a LabVIEW-based temperature control system, and a syringe pump. Kim et al. [ 21 ] developed a cylindrical device combined with a laser-induced fluorescence detection system for real-time PCR. They chose a high-purity perfluoroalkoxy alkane (PFA) capillary as the reactor. The device includes three holes for the cartridge heaters controlled by a series of commercial controllers. Hajji et al. [ 22 ] presented a microfluidic PCR platform in which sample droplets are transported in a PTFE capillary by the syringe pump and flow through two isothermal areas. Peltier modules were used for temperature control by independent proportional–integral–derivative (PID) boards. Typically, microfluidic systems transport fluids and the fluid pump operates in most applications. However, pumping devices may lead to a costly system. Researchers seek to make a simple and easy pump and integrate it into the CFPCR system. Wu et al. [ 23 ] proposed a self-activated micropump system for applications of on-chip PCRs. A single heater achieves the thermal cycle requirement by controlling the temperature gradient of the PDMS block. Nagai and Fuchiwaki [ 24 ] described a PCR system to detect a microorganism modeling anthrax and a point mutation of the FGFR3 gene. They integrated two cartridge heaters, a self-made syringe pump, a fluorescence detector, and a tablet personal computer into a suitcase. Commercial PID controllers provide a total solution for temperature control. In portable devices, it is convenient to utilize a microcontroller which is often low-powered consumption. Han et al. [ 25 ] fabricated a PDMS-based chip and indium tin oxide (ITO) coated heaters on a CFPCR platform. The heating circuit used an ATMega 128 MCU (Microchip Technology, Chandler, AZ, USA) to control the temperature. A syringe pump injects the PCR mixture into the microchannel. Kulkarni and Goel [ 26 ] developed a PDMS–glass bonding chip with a serpentine microchannel. The NodeMCU microcontroller (an open source firmware) was responsible for the temperature control of the cartridge heater. A commercial syringe pump pushed the reaction volume through the channel. Finally, the device amplified the rat GAPDH gene of the 594-bp successfully. Talebi et al. [ 27 ] presented a CF microfluidic device for PCR and fabricated the heater electrodes based on PCB technology. They used the Arduino MEGA to control the heater temperatures. The PCR mixture was pumped into the PMMA microchannel using an ordinary syringe pump. During the last few decades, microfluidic PCR chips have gained much attention due to their small thermal masses and the high heat transfer rate inside the system. A CFPCR device can regulate the temperature of the reactants by moving the reactants through different temperature regions. The merit of establishing several isothermal areas for CFPCR simplifies the fabricated cost and reduces the research and design cycle time. Some researchers have designed a five-temperature-region chip with a serpentine channel for CFPCR [ 27 , 28 ]. It does reduce the total volume of the chip device. External pumping in the microfluidics field is an essential function for sample transportation. However, a standard syringe pump is expensive and occupies the majority of the system cost. Building a user-friendly and miniaturized syringe pump for the microfluidic device is one of the cost-saving measures in the portable CFPCR device. Due to the high demand for valuable gelatinous TCMs, bovine or swine skin was often used to make fake or adulterated Colla corii asini in recent decades. This situation exposes public health to high risk and causes unfair competition in the commercial markets. Establishing a reliable and convenient technology to develop an accurate detection method and identify fake health food before being used is essential. This work describes a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic PCR chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. The use of Arduino has increased exponentially during the last two decades due to its readability and easiness [ 29 , 30 ]. The main contribution of the current work is to integrate the thermal control and liquid pumping modules into the microfluidics system and test the PCR performance of the CFPCR device by incorporating two Arduino boards. We can greatly decrease the device volume, save the system cost, and reduce the research and design cycle time. One heater supports the denaturation zone located at the center of the chip. A Peltier element cools the annealing zones set at the two sides of the chip. A DC power supported by three serial-connected 18,650 batteries or another 12 V external DC power supplies the power for performing CFPCR. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The specific amplification products in our device and the thermal cycler are clear. In the future, we can apply the portable continuous-flow device to a low-cost PCR system. In the following, we first describe our main methods regarding the design of the microfluidic chips, the fabrication of the heating blocks and thermal control modules, the assembly of the homemade syringe pump, the development of the power supply and user interface, the surface treatment of the microchannel, and the operation of the device. Then, we express the characteristic results of the homemade pump. Next, we investigate the influences of the operational parameters of the system on the temperature uniformity of the chip. Finally, we conduct some experimental work to complete the contents of this article. 2. Materials and Experimentation The portable microfluidic PCR device comprises a microfluidic chip, a set of thermal control module, a home-made syringe pump, a power supply module, and a user interface module. The DNA mixture pumped through a serpentine channel with various widths meanders inside the chip. Only one heater supports the denaturation zone located at the center of the chip. A Peltier element cools the annealing zones set at the two sides of the chip. The temperature distribution is created within the chip so that the DNA mixture can be heated and cooled during several thermal cycles to finish the PCR process. A syringe pump continuously pushes a sample mixture of a specific volume in a glass syringe at a fixed flow rate through a Teflon capillary tube connected to the inlet of the chip. A schematic diagram and photographic image of the portable CFPCR device are shown in Figure 1 . 2.1. Design of the Microfluidic Chips The CFPCR chip that holds the sample during the PCR process comprises a PDMS layer and a glass slide with thicknesses of 1.9 and 1.1 mm, respectively, as shown in Figure 2 a. The outer dimensions of the PDMS layer are 40 mm in length and 25 mm in width. Those of the glass slide are 75 mm by 25 mm, similar to the size of a microscopy slide. The 30-loop channel of 50 μm depth is 200 μm in width, except for the extension region with a maximum width of 500 μm, as shown in Figure 2 b. The pre-denaturation part is located near the inlet at the left-hand side of the chip. The pro-extension part is near the outlet at the right, shown in Figure 2 c. After completing the SU-8 patterned master, we can use it to replicate the PDMS microchannel. It can considerably reduce the fabrication cost. Due to the low cost of the fabricated chip and the prevention of the sample cross-contamination between reactions, the reactor chip is disposable after use. In our earlier work, we have expressed the comprehensive fabrication process of the PDMS-glass bonding chip [ 31 ]. The schematic diagram of the fabrication process is illustrated in Figure 3 . Initially, a silicon wafer is cleaned and dehydrated on a hotplate. An epoxy-based negative photoresist (SU-8 3035) is spin-coated on a silicon wafer. We utilize UV exposure for the production of the channel pattern. After development, the master is washed and baked to fix the photoresist. The PDMS (Sylgard 184, Dow Silicones Corp., Midland, MI, USA) mixture thoroughly mixed with the precursor and curing agent in a 10:1 weight ratio is degassed with a mechanical vacuum pump to remove air bubbles. After pouring the PDMS mixture onto the SU-8 patterned master, we use a convection oven to cure the PDMS. The replicas are peeled off carefully from the master. The single-hole puncher punches the inlet and outlet holes on the PDMS channel chip. After surface oxidation and bonding, the designed microfluidic chips are ready. The pattern defines the number of cycles performed through the chip. The arrangement of the channel layout and the flow rate of the mixture determine the reaction time for each PCR step. 2.2. Fabrication of the Heating Blocks and Thermal Control Modules Figure 4 a shows the structure of the thermal cycling apparatus consisting of a cartridge heater, a Peltier element, aluminum fins, and thermally conductive aluminum blocks. When performing PCR, the PCR chip is attached tightly to the top side of the aluminum blocks. The aluminum heating blocks and the PDMS-based chip are assembled and fixed onto a polymethyl methacrylate (PMMA) housing as shown in Figure 4 b. A machined PMMA block holds the microfluidic chip, rectangular and U-squared aluminum heating blocks. The heating blocks are isolated by a 2 mm air gap and set at different temperatures for denaturation and annealing. In our designed chip, the denaturation region heated by one heater locates at the center of the chip, and the annealing regions supported by a Peltier element lie at the two sides of the chip. The extension regions span from the annealing region to the denaturation region. A PCR thermal cycle is successful when the reagent leaves the extension region. The symmetric management creates the five reaction regions in the chip. The five-temperature-region design requires only one half-loop per PCR cycle, as shown in Figure 4 c. The fabrication of the half-size chip volume is mandatory. The thermal control modules are detachable from the channel chip and reused. The photograph of the thermal control module is shown in Figure 5 a and the functions are illustrated in Figure 5 b. The cartridge heater (3.175 mm diameter, 38 mm length, 10 V, 14 W, C1J-9412, Watlow, St. Louis, MO, USA) inserted into the central part of the rectangular block (6 mm × 6 mm × 40 mm) keeps the chip temperature at the denaturation region. The temperature distribution inside the heating block is made highly uniform by the high thermal conductivity of aluminum. The temperature sensor, DS18B20 (Dallas Semiconductor, Dallas, TX, USA), mounted onto the aluminum heating block, is utilized to supply temperature feedback under a homemade PID controller. The contacted Peltier element module and the other sensor with a similar thermal control program regulate the annealing temperature. An aluminum block (40 mm × 5 mm × 27 mm) of a U-squared shape sticks on the surface of a Peltier element (4 mm × 4 mm × 3.5 mm, TEC1-12706, Hebei IT, Shanghai, China). An aluminum fin is placed under the Peltier element to achieve the required temperature at the annealing regions. The block is in contact with the chip via some thermal conductive adhesive. The temperature difference of the block surface at three measured points is about ±1 °C. A schematic block diagram of the temperature controller is presented in Figure 5 c. The Arduino Mega 2560 (an open source firmware) is a microcontroller board based on the ATmega2560 (Microchip Technology Inc., Chandler, AZ, USA) and is utilized for controlling the temperature of the heating blocks. An NPN (negative-positive-negative) Darlington power transistor (TIP120, STMicroelectronics, Geneva, Switzerland) regulates the heater power to heat the aluminum blocks. A single intelligent temperature sensor (DS18B20, Dallas semiconductor, Dallas, TX, USA) provides the temperature value for feedback control. Then, a 16 × 2 I2C LCD, which consists of an HD44780 (Hitachi, Tokyo, Japan)-based character LCD and an I2C LCD adapter, provides the user interface. A DC power supported by three serial-connected 18650 batteries supplies the power requirement when the system is working. The power supply part uses an LM2596S (Texas Instruments, Dallas, TX, USA) DC-DC buck converter step-down module to provide up to 3 A output current. The push buttons start the command given after entering the flow rate and timer values; the start function begins running the machine and the stop function is to stop all programs. A program in the C++ programming language is compiled in an Arduino integrated development environment (IDE) for developing ATmega2560 microcontroller offerings and applications. The thermal sensors connect to Arduino Mega 2560. The microcontroller receives the temperature signals. The measured temperature compares with the setting temperature input using the buttons. The control signal determines the power inputs to the heater and the Peltier element using a proportional–integral–derivative (PID) algorithm. After the injection of the sample, the heater and the Peltier element maintain at specific temperatures. 2.3. Assembly of the Homemade Syringe Pump A syringe pump injects the inserted fluid at a constant rate for some time. It also combines mechanical and electronic components that manipulate a standard syringe. Figure 6 a demonstrates the homemade pumping unit. It is mainly composed of a stepper motor (14PM-M011-G1ST, Minebea Co., Ltd., Nagano, Japan), a lead screw (5 mm diameter, 100 mm length), a coupling (3 mm to 5 mm), two linear bearings (6 mm I.D., 12 mm O.D., 19 mm width), and two guide rods (6 mm diameter, 110 mm length). Some 5 mm thickness PMMA plates assemble the pump frame. It can substitute for the costly external precision syringe pump. In our work, a homemade syringe pump costs less than 200 USD and pumps the DNA mixture into the chip. Figure 6 b illustrates the block diagram of the syringe pump in our research. The construction is a DC voltage source for the power required by the Arduino Nano (an open source firmware) and the stepper motor, the buttons as an input interface of parameters, the Arduino Nano as a controller of all systems, ULN2003A (STMicroelectronics, Geneva, Switzerland) as the driver of the stepper motor, stepper motor as the lead screw pusher, LCD as a display for volumetric flow rate, and PMMA plates as a box from the syringe pump. Figure 6 c shows the circuit diagram of the syringe pump based on Arduino. Three serial-connected 18650 batteries supply the power requirement for the Arduino Nano and the stepper motor when the system is working. The LM2596S can take an input voltage of 12.6 V and convert it to 5 V up to 3A of continuous current. The Arduino Nano regulates the entire work process of the system by processing the flow rate entered through a push-button. The ULN2003A is the motor driver for 14PM-M011-G1ST stepper motor (MinebeaMitsumi, Inc., Nagano, Japan). The LCD shows information about the amount of flow rate. The serial peripheral interface (SPI) is a synchronous serial communication interface specification used for short-distance communication, primarily in embedded systems. In this work, we use SPI protocol for communication between Arduino Mega and Arduino Nano. The Arduino Mega acts as Master, and the Arduino Nano acts as Slave. A homemade syringe pump continuously pushes a sample mixture of a specific volume in a glass syringe at a fixed flow rate through a Teflon capillary tube connected to the inlet of the chip. The Arduino Nano controls a continually running motor which applies a continuous force upon the plunger end of the syringe. 2.4. Development of the Power Supply and User Interface A DC power of 12.6 V supported by three serial-connected 18650 batteries is shown in Figure 7 a and utilized to supply the power requirement when the system is working. The 18650 cells with a nominal voltage of 3.7 V and a maximum voltage of 4.2 ± 0.05 V are exploited and have a discharge capacity greater than 2.05 ampere-hour (Ah). There is another choice of 12 V external DC power for our device, presented in Figure 7 b. The panel DC power jack is an electrical connector to supply the external power for a period of measuring time. Figure 7 c presents the user interface on the device. We develop an interface that sets the operational parameters by pressing the buttons and displays the current status in the LCD 1602A (OpenHacks, Rosario, Santa Fe, Argentina). The steps are as follows. The user can set the denaturation temperature, annealing temperature, and the pumping rate of the sample. When the high- and low-temperature regions reach the steady state, the device does not proceed until the user presses the enter button. 2.5. Surface Treatment of the Microchannel Due to the high surface-to-volume ratio of the PDMS microchannel, a serpentine channel design increases the possibility of enzymes in PCR mixtures sticking onto the hydrophobic surface walls. Any enzyme adsorbed by the walls of the channel reduces its efficiency. Thus, the surface of the PDMS channel is coated with Tween 20 to prevent the adsorption of DNA polymerase. Immediately after the bonding process, the PDMS channel is filled with the 20% Tween 20 solution and kept still for 1 h. This treatment is static surface modification. After removing the 20% Tween 20 solution and rinsing the PDMS channel briefly with 0.25% Tween 20 solution at the flow rate of 1.5 μL/min, the injection of airflow of 3 μL/min cleans out the channel. Countless bubbles might also form in the denaturation zone and block the microchannel. The generation of air bubbles in the sample solution under high-temperature conditions is one of the crucial topics in microfluidic systems. A volume amount of 50 μL of highly viscous mineral oil with a high boiling point flows into the microchannel just before the introduction of the sample solution, which helps increase the pressure of the sample solution in the microchannel. Then, the latter sample solution follows the flowing mineral oil into the high-temperature zone without air bubbles. A volume of 10 μL of mineral oil injects into the microchannel after the sample solution to prevent the dead volume of the reaction mixture. 2.6. Operation of the Device The DNA sample preparation has been described considerably in our previous work [ 32 , 33 ]. A segment of DNA about 200 bps is amplified. The reaction mixture for CFPCR requires 25 μL of a mixed solution, which contains 5 μL of 20 ng/μL of the DNA template, 1 μL of 0.4 μM of each forward and reverse primer, 10 μL of 5 × PCR-Mastermix (5 × reaction buffer B (400 mM Tris-HCl pH 9.4~9.5 at 25 °C, 100 mM (NH 4 ) 2 SO 4 , 0.1% w / v Tween 20), 12.5 mM MgCl 2 , 1 mM of each dATP, dGTP, dCTP, and dTTP, and Taq -DNA-Polymerase) and 9 μL of nuclease-free water. Two primers (5′-TGGAGAGAAATGGGCTACA-3′ and 5′-CATGGTTTTGTGTAATATTGTGA-3′) are used as forward and reverse primers for PCR, respectively. For our device, we set the temperature zones at 369 K for denaturation and 333 K for annealing after modifying the channel surface, sealing the PDMS surface with a film (Microseal 'B' PCR Plate Sealing Film, Bio-Rad, Hercules, CA, USA) and fixing the chip by a clamp. Pre-mixed samples are transported to the microchip using a homemade syringe pump. Then, the reagent moves through sequential temperature zones corresponding to denaturation, annealing, and extension zones. PCR amplification is also performed with a conventional thermal cycler (MJ Mini™ 48-Well Personal Thermal Cycler, Bio-Rad, Hercules, CA, USA). The cycling conditions are as follows: 40 cycles of 30 s at 367 K for denaturation, 30 s at 333 K for annealing, and 30 s at 345 K for extension; 300 s for pre-denaturation and 420 s for pro-extension are added to the initial and the final steps of PCR, respectively. The PCR products are collected in a pipette tip from the channel outlet and mixed with 1× blue dye. PCR amplified fragments are analyzed after fractionation by agarose slab gel electrophoresis (Mini-Sub Cell GT System, Bio-Rad, Hercules, CA, USA). Ten μL of each sample are loaded onto 2% agarose gel (Certified Molecular Biology Agarose, Bio-Rad, Hercules, CA, USA) and electrophoresed in 10× Tris/Boric Acid/EDTA (TBE) buffer. After electrophoresis, the gel is stained with 10 mg/mL ethidium bromide solution (Bio-Rad, Hercules, CA, USA) and visualized by UV transillumination. 2.1. Design of the Microfluidic Chips The CFPCR chip that holds the sample during the PCR process comprises a PDMS layer and a glass slide with thicknesses of 1.9 and 1.1 mm, respectively, as shown in Figure 2 a. The outer dimensions of the PDMS layer are 40 mm in length and 25 mm in width. Those of the glass slide are 75 mm by 25 mm, similar to the size of a microscopy slide. The 30-loop channel of 50 μm depth is 200 μm in width, except for the extension region with a maximum width of 500 μm, as shown in Figure 2 b. The pre-denaturation part is located near the inlet at the left-hand side of the chip. The pro-extension part is near the outlet at the right, shown in Figure 2 c. After completing the SU-8 patterned master, we can use it to replicate the PDMS microchannel. It can considerably reduce the fabrication cost. Due to the low cost of the fabricated chip and the prevention of the sample cross-contamination between reactions, the reactor chip is disposable after use. In our earlier work, we have expressed the comprehensive fabrication process of the PDMS-glass bonding chip [ 31 ]. The schematic diagram of the fabrication process is illustrated in Figure 3 . Initially, a silicon wafer is cleaned and dehydrated on a hotplate. An epoxy-based negative photoresist (SU-8 3035) is spin-coated on a silicon wafer. We utilize UV exposure for the production of the channel pattern. After development, the master is washed and baked to fix the photoresist. The PDMS (Sylgard 184, Dow Silicones Corp., Midland, MI, USA) mixture thoroughly mixed with the precursor and curing agent in a 10:1 weight ratio is degassed with a mechanical vacuum pump to remove air bubbles. After pouring the PDMS mixture onto the SU-8 patterned master, we use a convection oven to cure the PDMS. The replicas are peeled off carefully from the master. The single-hole puncher punches the inlet and outlet holes on the PDMS channel chip. After surface oxidation and bonding, the designed microfluidic chips are ready. The pattern defines the number of cycles performed through the chip. The arrangement of the channel layout and the flow rate of the mixture determine the reaction time for each PCR step. 2.2. Fabrication of the Heating Blocks and Thermal Control Modules Figure 4 a shows the structure of the thermal cycling apparatus consisting of a cartridge heater, a Peltier element, aluminum fins, and thermally conductive aluminum blocks. When performing PCR, the PCR chip is attached tightly to the top side of the aluminum blocks. The aluminum heating blocks and the PDMS-based chip are assembled and fixed onto a polymethyl methacrylate (PMMA) housing as shown in Figure 4 b. A machined PMMA block holds the microfluidic chip, rectangular and U-squared aluminum heating blocks. The heating blocks are isolated by a 2 mm air gap and set at different temperatures for denaturation and annealing. In our designed chip, the denaturation region heated by one heater locates at the center of the chip, and the annealing regions supported by a Peltier element lie at the two sides of the chip. The extension regions span from the annealing region to the denaturation region. A PCR thermal cycle is successful when the reagent leaves the extension region. The symmetric management creates the five reaction regions in the chip. The five-temperature-region design requires only one half-loop per PCR cycle, as shown in Figure 4 c. The fabrication of the half-size chip volume is mandatory. The thermal control modules are detachable from the channel chip and reused. The photograph of the thermal control module is shown in Figure 5 a and the functions are illustrated in Figure 5 b. The cartridge heater (3.175 mm diameter, 38 mm length, 10 V, 14 W, C1J-9412, Watlow, St. Louis, MO, USA) inserted into the central part of the rectangular block (6 mm × 6 mm × 40 mm) keeps the chip temperature at the denaturation region. The temperature distribution inside the heating block is made highly uniform by the high thermal conductivity of aluminum. The temperature sensor, DS18B20 (Dallas Semiconductor, Dallas, TX, USA), mounted onto the aluminum heating block, is utilized to supply temperature feedback under a homemade PID controller. The contacted Peltier element module and the other sensor with a similar thermal control program regulate the annealing temperature. An aluminum block (40 mm × 5 mm × 27 mm) of a U-squared shape sticks on the surface of a Peltier element (4 mm × 4 mm × 3.5 mm, TEC1-12706, Hebei IT, Shanghai, China). An aluminum fin is placed under the Peltier element to achieve the required temperature at the annealing regions. The block is in contact with the chip via some thermal conductive adhesive. The temperature difference of the block surface at three measured points is about ±1 °C. A schematic block diagram of the temperature controller is presented in Figure 5 c. The Arduino Mega 2560 (an open source firmware) is a microcontroller board based on the ATmega2560 (Microchip Technology Inc., Chandler, AZ, USA) and is utilized for controlling the temperature of the heating blocks. An NPN (negative-positive-negative) Darlington power transistor (TIP120, STMicroelectronics, Geneva, Switzerland) regulates the heater power to heat the aluminum blocks. A single intelligent temperature sensor (DS18B20, Dallas semiconductor, Dallas, TX, USA) provides the temperature value for feedback control. Then, a 16 × 2 I2C LCD, which consists of an HD44780 (Hitachi, Tokyo, Japan)-based character LCD and an I2C LCD adapter, provides the user interface. A DC power supported by three serial-connected 18650 batteries supplies the power requirement when the system is working. The power supply part uses an LM2596S (Texas Instruments, Dallas, TX, USA) DC-DC buck converter step-down module to provide up to 3 A output current. The push buttons start the command given after entering the flow rate and timer values; the start function begins running the machine and the stop function is to stop all programs. A program in the C++ programming language is compiled in an Arduino integrated development environment (IDE) for developing ATmega2560 microcontroller offerings and applications. The thermal sensors connect to Arduino Mega 2560. The microcontroller receives the temperature signals. The measured temperature compares with the setting temperature input using the buttons. The control signal determines the power inputs to the heater and the Peltier element using a proportional–integral–derivative (PID) algorithm. After the injection of the sample, the heater and the Peltier element maintain at specific temperatures. 2.3. Assembly of the Homemade Syringe Pump A syringe pump injects the inserted fluid at a constant rate for some time. It also combines mechanical and electronic components that manipulate a standard syringe. Figure 6 a demonstrates the homemade pumping unit. It is mainly composed of a stepper motor (14PM-M011-G1ST, Minebea Co., Ltd., Nagano, Japan), a lead screw (5 mm diameter, 100 mm length), a coupling (3 mm to 5 mm), two linear bearings (6 mm I.D., 12 mm O.D., 19 mm width), and two guide rods (6 mm diameter, 110 mm length). Some 5 mm thickness PMMA plates assemble the pump frame. It can substitute for the costly external precision syringe pump. In our work, a homemade syringe pump costs less than 200 USD and pumps the DNA mixture into the chip. Figure 6 b illustrates the block diagram of the syringe pump in our research. The construction is a DC voltage source for the power required by the Arduino Nano (an open source firmware) and the stepper motor, the buttons as an input interface of parameters, the Arduino Nano as a controller of all systems, ULN2003A (STMicroelectronics, Geneva, Switzerland) as the driver of the stepper motor, stepper motor as the lead screw pusher, LCD as a display for volumetric flow rate, and PMMA plates as a box from the syringe pump. Figure 6 c shows the circuit diagram of the syringe pump based on Arduino. Three serial-connected 18650 batteries supply the power requirement for the Arduino Nano and the stepper motor when the system is working. The LM2596S can take an input voltage of 12.6 V and convert it to 5 V up to 3A of continuous current. The Arduino Nano regulates the entire work process of the system by processing the flow rate entered through a push-button. The ULN2003A is the motor driver for 14PM-M011-G1ST stepper motor (MinebeaMitsumi, Inc., Nagano, Japan). The LCD shows information about the amount of flow rate. The serial peripheral interface (SPI) is a synchronous serial communication interface specification used for short-distance communication, primarily in embedded systems. In this work, we use SPI protocol for communication between Arduino Mega and Arduino Nano. The Arduino Mega acts as Master, and the Arduino Nano acts as Slave. A homemade syringe pump continuously pushes a sample mixture of a specific volume in a glass syringe at a fixed flow rate through a Teflon capillary tube connected to the inlet of the chip. The Arduino Nano controls a continually running motor which applies a continuous force upon the plunger end of the syringe. 2.4. Development of the Power Supply and User Interface A DC power of 12.6 V supported by three serial-connected 18650 batteries is shown in Figure 7 a and utilized to supply the power requirement when the system is working. The 18650 cells with a nominal voltage of 3.7 V and a maximum voltage of 4.2 ± 0.05 V are exploited and have a discharge capacity greater than 2.05 ampere-hour (Ah). There is another choice of 12 V external DC power for our device, presented in Figure 7 b. The panel DC power jack is an electrical connector to supply the external power for a period of measuring time. Figure 7 c presents the user interface on the device. We develop an interface that sets the operational parameters by pressing the buttons and displays the current status in the LCD 1602A (OpenHacks, Rosario, Santa Fe, Argentina). The steps are as follows. The user can set the denaturation temperature, annealing temperature, and the pumping rate of the sample. When the high- and low-temperature regions reach the steady state, the device does not proceed until the user presses the enter button. 2.5. Surface Treatment of the Microchannel Due to the high surface-to-volume ratio of the PDMS microchannel, a serpentine channel design increases the possibility of enzymes in PCR mixtures sticking onto the hydrophobic surface walls. Any enzyme adsorbed by the walls of the channel reduces its efficiency. Thus, the surface of the PDMS channel is coated with Tween 20 to prevent the adsorption of DNA polymerase. Immediately after the bonding process, the PDMS channel is filled with the 20% Tween 20 solution and kept still for 1 h. This treatment is static surface modification. After removing the 20% Tween 20 solution and rinsing the PDMS channel briefly with 0.25% Tween 20 solution at the flow rate of 1.5 μL/min, the injection of airflow of 3 μL/min cleans out the channel. Countless bubbles might also form in the denaturation zone and block the microchannel. The generation of air bubbles in the sample solution under high-temperature conditions is one of the crucial topics in microfluidic systems. A volume amount of 50 μL of highly viscous mineral oil with a high boiling point flows into the microchannel just before the introduction of the sample solution, which helps increase the pressure of the sample solution in the microchannel. Then, the latter sample solution follows the flowing mineral oil into the high-temperature zone without air bubbles. A volume of 10 μL of mineral oil injects into the microchannel after the sample solution to prevent the dead volume of the reaction mixture. 2.6. Operation of the Device The DNA sample preparation has been described considerably in our previous work [ 32 , 33 ]. A segment of DNA about 200 bps is amplified. The reaction mixture for CFPCR requires 25 μL of a mixed solution, which contains 5 μL of 20 ng/μL of the DNA template, 1 μL of 0.4 μM of each forward and reverse primer, 10 μL of 5 × PCR-Mastermix (5 × reaction buffer B (400 mM Tris-HCl pH 9.4~9.5 at 25 °C, 100 mM (NH 4 ) 2 SO 4 , 0.1% w / v Tween 20), 12.5 mM MgCl 2 , 1 mM of each dATP, dGTP, dCTP, and dTTP, and Taq -DNA-Polymerase) and 9 μL of nuclease-free water. Two primers (5′-TGGAGAGAAATGGGCTACA-3′ and 5′-CATGGTTTTGTGTAATATTGTGA-3′) are used as forward and reverse primers for PCR, respectively. For our device, we set the temperature zones at 369 K for denaturation and 333 K for annealing after modifying the channel surface, sealing the PDMS surface with a film (Microseal 'B' PCR Plate Sealing Film, Bio-Rad, Hercules, CA, USA) and fixing the chip by a clamp. Pre-mixed samples are transported to the microchip using a homemade syringe pump. Then, the reagent moves through sequential temperature zones corresponding to denaturation, annealing, and extension zones. PCR amplification is also performed with a conventional thermal cycler (MJ Mini™ 48-Well Personal Thermal Cycler, Bio-Rad, Hercules, CA, USA). The cycling conditions are as follows: 40 cycles of 30 s at 367 K for denaturation, 30 s at 333 K for annealing, and 30 s at 345 K for extension; 300 s for pre-denaturation and 420 s for pro-extension are added to the initial and the final steps of PCR, respectively. The PCR products are collected in a pipette tip from the channel outlet and mixed with 1× blue dye. PCR amplified fragments are analyzed after fractionation by agarose slab gel electrophoresis (Mini-Sub Cell GT System, Bio-Rad, Hercules, CA, USA). Ten μL of each sample are loaded onto 2% agarose gel (Certified Molecular Biology Agarose, Bio-Rad, Hercules, CA, USA) and electrophoresed in 10× Tris/Boric Acid/EDTA (TBE) buffer. After electrophoresis, the gel is stained with 10 mg/mL ethidium bromide solution (Bio-Rad, Hercules, CA, USA) and visualized by UV transillumination. 3. Research Methodology We have fabricated a fully integrated device for continuous-flow PCR. The denaturation zone is in the central part of the chip. The annealing and the extension zones are on opposite sides. Five temperature regions are located within the chip width of 25 mm. First of all, we measure the flow volume of the syringe pump and express the characteristic results of the homemade pump. Then, we investigate the influences of the operational parameters of the system on the temperature uniformity of the chip. 3.1. Characteristic Results for the Motor Our syringe pump features a software interface where a user can control the device to change the flow rate of the sample mixture. The flow rates for the liquid volumes in the glass syringes (25, 50, and 100 μL, 700 series, Hamilton Co., Reno, NV, USA) are measured. Table 1 demonstrates the measured flow rate at various setting values of RPMs with three syringes. We average three experimental results to obtain the measured flow rate at one RPM value. It is convenient for users to set the required flow rate rather than the RPM value. Table 2 presents the setting values of revolution(s) per minute (RPM) at various flow rates with three syringes. The linear relationship between the setting value of RPM and flow rate is acceptable. 3.2. Temperature Measurements Temperature uniformity is one of the critical issues influencing the amplified efficiency of PCR. In the CFPCR device, the isothermal area and the mixture flow rate determine the heating time for the DNA sample. Two main approaches ensure the mixture temperature suitable for CFPCR. Firstly, an infrared (IR) camera (TAS-G100EXD, Nippon Avionics Co., Ltd., Tokyo, Japan) characterizes the spatial temperature distribution across the surface of the PDMS-based chip and evaluates the performance of the thermal modules. After reaching a steady-state temperature distribution, the IR imager captures the IR images of the chip surface with the various sample flow rates. Figure 8 demonstrates the influence of different sample flow rates on the temperature distributions of the chip surface. The results present temperature gradients between different temperature zones. The five temperature regions become clearly distinct and separate strictly from each other when the water flow rate is 1, 2, 3, 4, and 5 μL/min, respectively, as shown in Figure 8 a. Due to the symmetric arrangement of the heating blocks, the symmetry of the temperature distribution on the chip is predictable. When the liquid flows through the serpentine microchannel, a smaller Nusselt number (Nu) of less than one corresponds to increased heat conduction rather than active convection. The water flow rate of less than 5 μL/min results in a small Nu of less than one, and the symmetry of the temperature distribution is distinct. It also shows that the present thermal arrangement is effective in generating the temperature requirement of the CFPCR chip. The water flow rate is 10, 15, 20, 25, and 30 μL/min, respectively, as shown in Figure 8 b(a–e) Due to the high convective effect resulting from the large Nu, the temperature near the denaturation region is lower than the required temperature, and the temperature near the annealing region is higher than the requisite one. The distortions of the symmetry of the temperature distributions are apparent. Inappropriate temperature distribution inside the CF device for PCR may cause either diffuse smearing upon gel electrophoresis or poor DNA amplification efficiency. The temperature distributions in the CFPCR device do not fit with the requisite PCR temperatures. Secondly, the channel temperatures are measured using thermocouples inserted into the PDMS/glass bonding chip, placed in contact with the glass substrate, and connected to a data acquisition system (Model NI 9211, National Instruments, Austin, TX, USA). Some K-type thermocouples (outer diameter of 0.254 mm, K30-2-506, Watlow Electric Manufacturing Co., St. Louis, MI, USA) measure the mixture temperatures. We immerse the thermocouple in a known temperature inside a water bath to calibrate it before usage. The marked points on the PDMS substrate are the locations of the measuring points. After reaching a steady state, the LabVIEW (National Instruments, Austin, TX, USA) program records the temperatures of the thermocouples at seventeen points shown in Figure 9 a. We can obtain the surface temperatures of the chip with the sample flow rate of 2 μL/min by the infrared imager. The locations of the marked points at the annealing, extension, and nearby regions in Figure 9 b are similar to the measuring points in Figure 9 a. The measured temperatures lined at the same horizontal positions are averaged and plotted in Figure 9 c. From Figure 9 c, the inner temperatures refer to the temperatures measured by the thermocouples, and the surface temperatures are the temperatures derived from the IR imager. The setting temperatures of the cartridge heater and Peltier element at the denaturation and annealing regions are 105 °C and 60 °C, respectively. We can find the surface temperatures of 87.3 °C and 58.5 °C and the inner temperatures of 94.8 °C and 55.3 °C near the denaturation and annealing regions. We can use the results to modify the setting temperatures to obtain the proper temperatures for PCR. 3.1. Characteristic Results for the Motor Our syringe pump features a software interface where a user can control the device to change the flow rate of the sample mixture. The flow rates for the liquid volumes in the glass syringes (25, 50, and 100 μL, 700 series, Hamilton Co., Reno, NV, USA) are measured. Table 1 demonstrates the measured flow rate at various setting values of RPMs with three syringes. We average three experimental results to obtain the measured flow rate at one RPM value. It is convenient for users to set the required flow rate rather than the RPM value. Table 2 presents the setting values of revolution(s) per minute (RPM) at various flow rates with three syringes. The linear relationship between the setting value of RPM and flow rate is acceptable. 3.2. Temperature Measurements Temperature uniformity is one of the critical issues influencing the amplified efficiency of PCR. In the CFPCR device, the isothermal area and the mixture flow rate determine the heating time for the DNA sample. Two main approaches ensure the mixture temperature suitable for CFPCR. Firstly, an infrared (IR) camera (TAS-G100EXD, Nippon Avionics Co., Ltd., Tokyo, Japan) characterizes the spatial temperature distribution across the surface of the PDMS-based chip and evaluates the performance of the thermal modules. After reaching a steady-state temperature distribution, the IR imager captures the IR images of the chip surface with the various sample flow rates. Figure 8 demonstrates the influence of different sample flow rates on the temperature distributions of the chip surface. The results present temperature gradients between different temperature zones. The five temperature regions become clearly distinct and separate strictly from each other when the water flow rate is 1, 2, 3, 4, and 5 μL/min, respectively, as shown in Figure 8 a. Due to the symmetric arrangement of the heating blocks, the symmetry of the temperature distribution on the chip is predictable. When the liquid flows through the serpentine microchannel, a smaller Nusselt number (Nu) of less than one corresponds to increased heat conduction rather than active convection. The water flow rate of less than 5 μL/min results in a small Nu of less than one, and the symmetry of the temperature distribution is distinct. It also shows that the present thermal arrangement is effective in generating the temperature requirement of the CFPCR chip. The water flow rate is 10, 15, 20, 25, and 30 μL/min, respectively, as shown in Figure 8 b(a–e) Due to the high convective effect resulting from the large Nu, the temperature near the denaturation region is lower than the required temperature, and the temperature near the annealing region is higher than the requisite one. The distortions of the symmetry of the temperature distributions are apparent. Inappropriate temperature distribution inside the CF device for PCR may cause either diffuse smearing upon gel electrophoresis or poor DNA amplification efficiency. The temperature distributions in the CFPCR device do not fit with the requisite PCR temperatures. Secondly, the channel temperatures are measured using thermocouples inserted into the PDMS/glass bonding chip, placed in contact with the glass substrate, and connected to a data acquisition system (Model NI 9211, National Instruments, Austin, TX, USA). Some K-type thermocouples (outer diameter of 0.254 mm, K30-2-506, Watlow Electric Manufacturing Co., St. Louis, MI, USA) measure the mixture temperatures. We immerse the thermocouple in a known temperature inside a water bath to calibrate it before usage. The marked points on the PDMS substrate are the locations of the measuring points. After reaching a steady state, the LabVIEW (National Instruments, Austin, TX, USA) program records the temperatures of the thermocouples at seventeen points shown in Figure 9 a. We can obtain the surface temperatures of the chip with the sample flow rate of 2 μL/min by the infrared imager. The locations of the marked points at the annealing, extension, and nearby regions in Figure 9 b are similar to the measuring points in Figure 9 a. The measured temperatures lined at the same horizontal positions are averaged and plotted in Figure 9 c. From Figure 9 c, the inner temperatures refer to the temperatures measured by the thermocouples, and the surface temperatures are the temperatures derived from the IR imager. The setting temperatures of the cartridge heater and Peltier element at the denaturation and annealing regions are 105 °C and 60 °C, respectively. We can find the surface temperatures of 87.3 °C and 58.5 °C and the inner temperatures of 94.8 °C and 55.3 °C near the denaturation and annealing regions. We can use the results to modify the setting temperatures to obtain the proper temperatures for PCR. 4. Results and Discussion The present paper conducts some experimental work to complete the contents of this article. A portable CFPCR device integrated with Arduino boards for thermal controlling and sample pumping amplifies the DNA fragment of Colla corii asini successfully. 4.1. Preparation Works for CFPCR We compare the performance of PCR amplification of the microfluidic device with that of the conventional method. A moderate amount of the mineral oil flows into the microchannel before the introduction of the PCR solution. Then the latter solution follows the flowing mineral oil into the high-temperature zone without generating air bubbles. Figure 10 demonstrates the preparation work for using the portable device for CFPCR. By injecting a small amount of black ink into the bonding chip, we can make sure that the channel pattern of the fabrication microfluidic chip is acceptable for CFPCR. The bonding result of the PDMS chip and glass substrate shows excellent leakage-free performance in Figure 10 a. We insert two thermocouples onto the annealing zone (Low Temperature Zone) and denaturation zone (High Temperature Zone) of the CFPCR chip. The measured temperatures in Figure 10 b show the chip temperatures reaching the steady state within one minute and the variation of temperature at the steady state is less than ±1 °C. 4.2. DNA Sample Amplification and Gel Electrophoresis Results The following figures present the results of gel electrophoresis analysis of the DNA products. We carry out the amplification of a 200 bp DNA fragment on the portable device as well as the commercial PCR machine. The first lane (Lane Mk) indicates the DNA ladder in Figure 11 a. The negative control without a DNA template at Lane NC expresses that the amplified product is specific inside a conventional PCR machine. The positive control inside a conventional PCR machine presents at Lane M as reference. The PCR product from the CFPCR chip is at Lane T. Using the image processing software (ImageJ, Version 1.50d, National Institutes of Health, Bethesda, MD, USA) to analyze the images, we obtain the fluorescence intensities in Figure 11 b. Despite the slightly weaker PCR band intensity of Lane T than half that of Lane M, they are sufficient for evaluations and analyses in diagnoses. The results in Figure 11 demonstrate that the specific amplification products in our device and the thermal cycler are clear. The flow rate through the microchannel determines the residence time of a fluid element in a specific temperature zone. Speeding up the flow rate can reduce the total reaction time. It might, however, cause the reaction to be inefficient due to short reaction time in the working zone. PCR performed with various flow rates from 0.3 μL/min to 1 μL/min is shown in Figure 12 . The chip system amplifies the DNA product well at a flow rate of 0.3 μL/min. When the flow rate increases, the PCR amplification time is decreased. However, the amount of amplified fragment decreased with higher flow rates from 0.3 μL/min to 0.6 μL/min. This decrease in PCR efficiency may be partly due to the short residence time in the extension zone at a high flow rate, which leads to insufficient time for DNA polymerase synthesis. Consequently, PCR products decreased with the flow rate. Our CFPCR device reduces the run time from 2 h for a conventional PCR down to 69 min at the flow rate of 0.4 μL/min. The device can further reduce its run time down to less than 55 min at the flow rate of 0.5 μL/min. The CFPCR chip performs the experiments using various initial DNA concentrations for 20 ng/μL, 2.0 ng/μL, 0.2 ng/μL, and 0.02 ng/μL. Figure 13 shows the effects of various initial DNA concentrations (20 ng/μL, Lane D; 2.0 ng/μL, Lane 10 −1 ; 0.2 ng/μL, Lane 10 −2 ; 0.02 ng/μL, Lane 10 −3 ) on PCR amplification when the DNA mixture flows through the microchannel at the flow rate of 0.4 μL/min. The negative control without a DNA template at Lane NC expresses that the amplified product from the CFPCR chip is specific. It demonstrates that the amount of CFPCR products almost decreases with the initial DNA concentration from 20 ng/μL to 1000 × dilution, as shown in Figure 13 b. The initial concentration for accomplishing the PCR process is at least 20 ng/μL in the portable CFPCR device. There are some limitations in our CFPCR chip device. Firstly, the outer dimensions of the PDMS layer are 40 mm in length and 25 mm in width. It is not easy to further reduce the volume of the chip. Secondly, the cycle number of our CFPCR reactor is fixed once the design is finalized. Obeid and Christopoulos [ 34 ] provided a CFPCR chip and collected the amplification products through the output access holes after cycles 20, 25, 30, 35, and 40. Their chip has the flexibility to change the thermal cycle numbers. However, the seal of the unused output access holes is an issue during PCR. 4.1. Preparation Works for CFPCR We compare the performance of PCR amplification of the microfluidic device with that of the conventional method. A moderate amount of the mineral oil flows into the microchannel before the introduction of the PCR solution. Then the latter solution follows the flowing mineral oil into the high-temperature zone without generating air bubbles. Figure 10 demonstrates the preparation work for using the portable device for CFPCR. By injecting a small amount of black ink into the bonding chip, we can make sure that the channel pattern of the fabrication microfluidic chip is acceptable for CFPCR. The bonding result of the PDMS chip and glass substrate shows excellent leakage-free performance in Figure 10 a. We insert two thermocouples onto the annealing zone (Low Temperature Zone) and denaturation zone (High Temperature Zone) of the CFPCR chip. The measured temperatures in Figure 10 b show the chip temperatures reaching the steady state within one minute and the variation of temperature at the steady state is less than ±1 °C. 4.2. DNA Sample Amplification and Gel Electrophoresis Results The following figures present the results of gel electrophoresis analysis of the DNA products. We carry out the amplification of a 200 bp DNA fragment on the portable device as well as the commercial PCR machine. The first lane (Lane Mk) indicates the DNA ladder in Figure 11 a. The negative control without a DNA template at Lane NC expresses that the amplified product is specific inside a conventional PCR machine. The positive control inside a conventional PCR machine presents at Lane M as reference. The PCR product from the CFPCR chip is at Lane T. Using the image processing software (ImageJ, Version 1.50d, National Institutes of Health, Bethesda, MD, USA) to analyze the images, we obtain the fluorescence intensities in Figure 11 b. Despite the slightly weaker PCR band intensity of Lane T than half that of Lane M, they are sufficient for evaluations and analyses in diagnoses. The results in Figure 11 demonstrate that the specific amplification products in our device and the thermal cycler are clear. The flow rate through the microchannel determines the residence time of a fluid element in a specific temperature zone. Speeding up the flow rate can reduce the total reaction time. It might, however, cause the reaction to be inefficient due to short reaction time in the working zone. PCR performed with various flow rates from 0.3 μL/min to 1 μL/min is shown in Figure 12 . The chip system amplifies the DNA product well at a flow rate of 0.3 μL/min. When the flow rate increases, the PCR amplification time is decreased. However, the amount of amplified fragment decreased with higher flow rates from 0.3 μL/min to 0.6 μL/min. This decrease in PCR efficiency may be partly due to the short residence time in the extension zone at a high flow rate, which leads to insufficient time for DNA polymerase synthesis. Consequently, PCR products decreased with the flow rate. Our CFPCR device reduces the run time from 2 h for a conventional PCR down to 69 min at the flow rate of 0.4 μL/min. The device can further reduce its run time down to less than 55 min at the flow rate of 0.5 μL/min. The CFPCR chip performs the experiments using various initial DNA concentrations for 20 ng/μL, 2.0 ng/μL, 0.2 ng/μL, and 0.02 ng/μL. Figure 13 shows the effects of various initial DNA concentrations (20 ng/μL, Lane D; 2.0 ng/μL, Lane 10 −1 ; 0.2 ng/μL, Lane 10 −2 ; 0.02 ng/μL, Lane 10 −3 ) on PCR amplification when the DNA mixture flows through the microchannel at the flow rate of 0.4 μL/min. The negative control without a DNA template at Lane NC expresses that the amplified product from the CFPCR chip is specific. It demonstrates that the amount of CFPCR products almost decreases with the initial DNA concentration from 20 ng/μL to 1000 × dilution, as shown in Figure 13 b. The initial concentration for accomplishing the PCR process is at least 20 ng/μL in the portable CFPCR device. There are some limitations in our CFPCR chip device. Firstly, the outer dimensions of the PDMS layer are 40 mm in length and 25 mm in width. It is not easy to further reduce the volume of the chip. Secondly, the cycle number of our CFPCR reactor is fixed once the design is finalized. Obeid and Christopoulos [ 34 ] provided a CFPCR chip and collected the amplification products through the output access holes after cycles 20, 25, 30, 35, and 40. Their chip has the flexibility to change the thermal cycle numbers. However, the seal of the unused output access holes is an issue during PCR. 5. Conclusions People eat food not only for preventing hunger but also for sustaining the body's well-being. Food security, therefore, is always the first priority when going shopping. Colla corii asini is one of the most valuable tonic traditional Chinese medicines. To establish an effective and applicable method to distinguish authentic TCMs is essential. This work describes a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a PDMS/glass bonding chip with a serpentine microchannel of various widths. The cartridge heater supports the thermal energy to sustain the denaturation temperature at the central part of the chip. The Peltier element supplies the energy to withstand the annealing temperature at two sides of the chip. It does reduce the total volume of the chip device due to the symmetrical five-temperature-region pattern, and requires only one Arduino Mega board for the thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. A DC power supported by three serial-connected 18650 batteries or another 12 V external DC power supplies the power for measuring. We build a portable CFPCR chip device with a compact modular design and the device is suitable for the usage in the remote area. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then, we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified in the microchannel under the treatment of specific concentrations of Tween 20 solutions to evaluate the PCR performance. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a PCR device. The initial concentration for accomplishing the PCR process is at least 20 ng/μL at the flow rate of 0.4 μL/min in the portable CFPCR device. In the future, we can apply the portable continuous-flow device to a low-cost PCR system. Once we finalize the design, low-cost polycarbonate chips may be a good alternative for microfluidics chips. This device not only tests Colla corii asini but also can be used for other TCMs. Our future work will optimize the volume of the sample mixture based on the design of experiments. We will also be reporting in future work on an experimental investigation in order to perform a comprehensive analysis of the amplification efficiencies using more than one sample at once in the reaction chip during PCR processes. To further reduce the system cost, we will utilize one Arduino-compatible device with proper programming for controlling the temperature of the heating blocks and a continually running motor. To make the user interface friendlier, the user can set the RPM value by pushing "+" and incrementing 0.1 RPM or pushing "−" and decrementing 0.1 RPM in our future work. Furthermore, we can integrate the battery charger circuit and an ESP32 board into our device to make it more customer-oriented.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059087/

Antidotes to anthrax lethal factor intoxication. Part 2: Structural modifications leading to improved in vivo efficacy

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2 . Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model. [BMCL ABSTRACT]

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314066/

Inflammasome-Dependent Cytokines at the Crossroads of Health and Autoinflammatory Disease

As key regulators of both innate and adaptive immunity, it is unsurprising that the activity of interleukin (IL)-1 cytokine family members is tightly controlled by decoy receptors, antagonists, and a variety of other mechanisms. Additionally, inflammasome-mediated proteolytic maturation is a prominent and distinguishing feature of two important members of this cytokine family, IL-1β and IL-18, because their full-length gene products are biologically inert. Although vital in antimicrobial host defense, deregulated inflammasome signaling is linked with a growing number of autoimmune and autoinflammatory diseases. Here, we focus on introducing the diverse inflammasome types and discussing their causal roles in periodic fever syndromes. Therapies targeting IL-1 or IL-18 show great efficacy in some of these autoinflammatory diseases, although further understanding of the molecular mechanisms leading to unregulated production of these key cytokines is required to benefit more patients.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785798/

Tumor Imaging Using Radiolabeled Matrix Metalloproteinase–Activated Anthrax Proteins

Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111 In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111 In-radiolabeled PA-L1, 111 In-PA-WT K563C , or 111 In-LF E687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1–mediated delivery of 111 In-LF E687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LF E687A . Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111 In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111 In-LF E687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue. MATERIALS AND METHODS Protein Production and Synthesis of Labeled Compounds All components of B. anthracis LT were expressed and purified as previously described ( 12 ). All protein batches were analyzed by liquid chromatography mass spectrometry analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis to confirm purity. PA-WT (83 kDa) is cleavable by furin and other furinlike enzymes ( 13 ). Here, we used a PA-WT variant containing an engineered cysteine (PA-WT K563C ; 83 kDa) whenever radiolabeled PA-WT is used ( 14 ). PA-L1 has the furin cleavage site replaced by a sequence targeted by MMPs (such as MMP2, MMP9, or MMP14) (PA-L1; 83 kDa) ( 10 ), whereas in PA-U7 an uncleavable sequence was inserted (PA-U7; 83 kDa) ( 15 ). LF variants included the fusion toxin of the N-terminal translocation domain of LF (LFn, LF amino acids 1–254) and P. aeruginosa exotoxin A domain III (FP59; 53 kDa) ( 15 ), LFn modified with a cysteine residue at the C terminus (LFn; 30 kDa) ( 16 ), and full-length mutant LF E687A (90 kDa) containing a defective catalytic domain ( 17 ). Cleavage PA protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after exposure to MMP2, furin. Full experimental details are laid out in the supplemental materials (available at http://jnm.snmjournals.org ). LFn and PA-WT K563C were site-specifically conjugated to the metal ion chelator diethylenetriamine pentaacetic acid (DTPA), using the bifunctional agent maleimide-DTPA (2,2′-(1-carboxy-2-(carboxymethyl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-2,5,8,11-tetraazatridecane-5,8-diyl)diacetic acid) to allow radiolabeling with 111 In, whereas PA-L1 or LF E687A was conjugated to p -SCN-benzyl-DTPA ( S -2-(4-isothiocyanatobenzyl)-DTPA) before radiolabeling with 111 In, using previously described methods ( 18 ). For cross-validation of the radiolabeled compound using confocal fluorescence microscopy, LF E687A was also fluorescently labeled using a Cy3- N -hydroxysuccinimide ester. Full experimental details are laid out in the supplemental materials. In Vitro Analyses MCF-7, MDA-MB-231, and HT1080 cells were originally purchased from ATCC. KPC-mouse–derived B8484 cells were provided by Professor Owen Sansom at the CRUK Beatson Institute. Cells were cultured in Dulbecco modified Eagle medium, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin, in a 37°C environment containing 5% CO 2 . Cells were authenticated by the provider, and the identities of purchased cells were corroborated by STR profiling. The cumulative length of culture was less than 3 mo after retrieval from liquid nitrogen storage. Cells were tested regularly to confirm the absence of Mycoplasma contamination. Gelatin zymography analysis was used to gauge the level of MMP2 and MMP9 secretion of cells. MMP14 and CMG2 expression was corroborated by Western blot and immunofluorescence. Full experimental details are laid out in the supplemental materials. The ability of the different PA variants to permit LF intoxication of the panel of cell lines was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after exposure of cells to PA-WT, PA-WT K563C , PA-L1, or PA-U7 in combination with FP59 ( 19 ). To investigate PA activation by MMP hydrolysis, cells were exposed to the broad-spectrum MMP inhibitor, GM6001 (30 μM; Abcam), for 30 min before adding PA/LF variants. Full experimental details are laid out in the supplemental materials. Cy3-LF E687A was added to HT1080 or MCF7 cells growing on glass cover slides, in combination with PA-WT, PA-L1, or a vehicle control. After 3 h, cells were washed, fixed, and mounted with 4′,6-diamidino-2-phenylindole, and images were acquired using a confocal microscope (TCS SP8; Leica). Full experimental details are laid out in the supplemental materials. Saturation cell binding experiments were performed to determine the apparent dissociation constant (K D ) and number of binding sites for 111 In-PA-WT K563C or 111 In-PA-L1. In vitro assays were performed to evaluate the capacity of 111 In-LF E687A or 111 In-LFn to interact with PA pores and to be delivered into cells after radiolabeling. Full experimental details are laid out in the supplemental materials. In Vivo Studies: Imaging and Biodistribution All animal procedures were performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and with local ethical committee approval. Animals were housed in individually ventilated cages in sex-matched groups of up to 6 per cage in an artificial day–night cycle facility with ad libitum access to food and water. No animals were euthanized for welfare reasons. All analyses were performed masked to experimental group assignment. To measure plasma clearance, dynamic SPECT/CT imaging of tumor-naïve SCID mice was performed after intravenous administration of 111 In-LFn (3 μg, 3 MBq; 100 μL) or 111 In-LF E687E (10 μg, 10 MBq; 100 μL) through a cannula inserted in the tail vein. SPECT/CT images were acquired for up to 3 h in list mode using a VECTor 4 CT scanner (MILabs). Three mice were used per group. Additional experimental detail is provided in the supplemental materials. To determine the pharmacokinetics of PA-L1 in vivo, dynamic SPECT images were acquired in MDA-MB-231 tumor–bearing mice over 3 h after intravenous injection of 111 In-PA-L1 or 111 In-PA-WT K563C (20 μg, 10 MBq; 100 μL). Three mice were used per group. MMP-activatable, PA-L1–mediated uptake of radiolabeled LF was evaluated in a pretargeting-inspired setup. MDA-MB-231 tumor xenograft–bearing mice were administered 111 In-LF E687E (10 μg, 10 MBq; 50 μL) by intravenous injection, together with PA-L1 (20 μg; 50 μL). To determine the PA-L1 selectivity of tumor uptake, and thus of MMP-mediated uptake, groups of control animals were administered 111 In-LF E687A alone or in combination with PA-WT (20 μg; 50 μL), PA-U7 (20 μg; 50 μL), or PA-L1 and an excess of unlabeled LF E687A (1 mg; 100 μL). SPECT imaging was performed 24 h after administration of the 111 In-LF/PA combinations. Three mice were used per group. After imaging, the mice were euthanized by cervical dislocation, and selected organs, tissues, and blood were removed. The amount of 111 In was determined per gram of tissue, normalized by the administered amount of 111 In (expressed as percentage injected dose per gram [%ID/g]). Tumor tissues were stored in 30% sucrose and, after 24 h, flash-frozen with dry ice and stored at −80°C until required for further processing. Statistical Analysis All statistical and regression analyses were performed using GraphPad Prism, version 7 (GraphPad Software). Linear regression was used to test for correlations between measurements. After testing for normality using a Shapiro–Wilk test, means were compared using a t test with the Welch correction for nonequal variances to compare 2 groups. One-way ANOVA followed by Dunnett posttests were used to compare multiple groups. Two-way ANOVA was used to analyze grouped data. All results are reported as mean ± SD of at least 3 independent replicates, unless otherwise indicated. Protein Production and Synthesis of Labeled Compounds All components of B. anthracis LT were expressed and purified as previously described ( 12 ). All protein batches were analyzed by liquid chromatography mass spectrometry analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis to confirm purity. PA-WT (83 kDa) is cleavable by furin and other furinlike enzymes ( 13 ). Here, we used a PA-WT variant containing an engineered cysteine (PA-WT K563C ; 83 kDa) whenever radiolabeled PA-WT is used ( 14 ). PA-L1 has the furin cleavage site replaced by a sequence targeted by MMPs (such as MMP2, MMP9, or MMP14) (PA-L1; 83 kDa) ( 10 ), whereas in PA-U7 an uncleavable sequence was inserted (PA-U7; 83 kDa) ( 15 ). LF variants included the fusion toxin of the N-terminal translocation domain of LF (LFn, LF amino acids 1–254) and P. aeruginosa exotoxin A domain III (FP59; 53 kDa) ( 15 ), LFn modified with a cysteine residue at the C terminus (LFn; 30 kDa) ( 16 ), and full-length mutant LF E687A (90 kDa) containing a defective catalytic domain ( 17 ). Cleavage PA protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after exposure to MMP2, furin. Full experimental details are laid out in the supplemental materials (available at http://jnm.snmjournals.org ). LFn and PA-WT K563C were site-specifically conjugated to the metal ion chelator diethylenetriamine pentaacetic acid (DTPA), using the bifunctional agent maleimide-DTPA (2,2′-(1-carboxy-2-(carboxymethyl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-2,5,8,11-tetraazatridecane-5,8-diyl)diacetic acid) to allow radiolabeling with 111 In, whereas PA-L1 or LF E687A was conjugated to p -SCN-benzyl-DTPA ( S -2-(4-isothiocyanatobenzyl)-DTPA) before radiolabeling with 111 In, using previously described methods ( 18 ). For cross-validation of the radiolabeled compound using confocal fluorescence microscopy, LF E687A was also fluorescently labeled using a Cy3- N -hydroxysuccinimide ester. Full experimental details are laid out in the supplemental materials. In Vitro Analyses MCF-7, MDA-MB-231, and HT1080 cells were originally purchased from ATCC. KPC-mouse–derived B8484 cells were provided by Professor Owen Sansom at the CRUK Beatson Institute. Cells were cultured in Dulbecco modified Eagle medium, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin, in a 37°C environment containing 5% CO 2 . Cells were authenticated by the provider, and the identities of purchased cells were corroborated by STR profiling. The cumulative length of culture was less than 3 mo after retrieval from liquid nitrogen storage. Cells were tested regularly to confirm the absence of Mycoplasma contamination. Gelatin zymography analysis was used to gauge the level of MMP2 and MMP9 secretion of cells. MMP14 and CMG2 expression was corroborated by Western blot and immunofluorescence. Full experimental details are laid out in the supplemental materials. The ability of the different PA variants to permit LF intoxication of the panel of cell lines was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after exposure of cells to PA-WT, PA-WT K563C , PA-L1, or PA-U7 in combination with FP59 ( 19 ). To investigate PA activation by MMP hydrolysis, cells were exposed to the broad-spectrum MMP inhibitor, GM6001 (30 μM; Abcam), for 30 min before adding PA/LF variants. Full experimental details are laid out in the supplemental materials. Cy3-LF E687A was added to HT1080 or MCF7 cells growing on glass cover slides, in combination with PA-WT, PA-L1, or a vehicle control. After 3 h, cells were washed, fixed, and mounted with 4′,6-diamidino-2-phenylindole, and images were acquired using a confocal microscope (TCS SP8; Leica). Full experimental details are laid out in the supplemental materials. Saturation cell binding experiments were performed to determine the apparent dissociation constant (K D ) and number of binding sites for 111 In-PA-WT K563C or 111 In-PA-L1. In vitro assays were performed to evaluate the capacity of 111 In-LF E687A or 111 In-LFn to interact with PA pores and to be delivered into cells after radiolabeling. Full experimental details are laid out in the supplemental materials. In Vivo Studies: Imaging and Biodistribution All animal procedures were performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and with local ethical committee approval. Animals were housed in individually ventilated cages in sex-matched groups of up to 6 per cage in an artificial day–night cycle facility with ad libitum access to food and water. No animals were euthanized for welfare reasons. All analyses were performed masked to experimental group assignment. To measure plasma clearance, dynamic SPECT/CT imaging of tumor-naïve SCID mice was performed after intravenous administration of 111 In-LFn (3 μg, 3 MBq; 100 μL) or 111 In-LF E687E (10 μg, 10 MBq; 100 μL) through a cannula inserted in the tail vein. SPECT/CT images were acquired for up to 3 h in list mode using a VECTor 4 CT scanner (MILabs). Three mice were used per group. Additional experimental detail is provided in the supplemental materials. To determine the pharmacokinetics of PA-L1 in vivo, dynamic SPECT images were acquired in MDA-MB-231 tumor–bearing mice over 3 h after intravenous injection of 111 In-PA-L1 or 111 In-PA-WT K563C (20 μg, 10 MBq; 100 μL). Three mice were used per group. MMP-activatable, PA-L1–mediated uptake of radiolabeled LF was evaluated in a pretargeting-inspired setup. MDA-MB-231 tumor xenograft–bearing mice were administered 111 In-LF E687E (10 μg, 10 MBq; 50 μL) by intravenous injection, together with PA-L1 (20 μg; 50 μL). To determine the PA-L1 selectivity of tumor uptake, and thus of MMP-mediated uptake, groups of control animals were administered 111 In-LF E687A alone or in combination with PA-WT (20 μg; 50 μL), PA-U7 (20 μg; 50 μL), or PA-L1 and an excess of unlabeled LF E687A (1 mg; 100 μL). SPECT imaging was performed 24 h after administration of the 111 In-LF/PA combinations. Three mice were used per group. After imaging, the mice were euthanized by cervical dislocation, and selected organs, tissues, and blood were removed. The amount of 111 In was determined per gram of tissue, normalized by the administered amount of 111 In (expressed as percentage injected dose per gram [%ID/g]). Tumor tissues were stored in 30% sucrose and, after 24 h, flash-frozen with dry ice and stored at −80°C until required for further processing. Statistical Analysis All statistical and regression analyses were performed using GraphPad Prism, version 7 (GraphPad Software). Linear regression was used to test for correlations between measurements. After testing for normality using a Shapiro–Wilk test, means were compared using a t test with the Welch correction for nonequal variances to compare 2 groups. One-way ANOVA followed by Dunnett posttests were used to compare multiple groups. Two-way ANOVA was used to analyze grouped data. All results are reported as mean ± SD of at least 3 independent replicates, unless otherwise indicated. RESULTS Cleavage and Activation of PA-L1 by MMP2 and MMP14 In a cell-free setting, recombinant human MMP2 and furin-activated MMP14, but not furin alone, were able to cleave PA-L1. Furin, but not MMP2, was able to cleave PA-WT (Supplemental Fig. 1). Cleavage of PA-L1 by MMP2 and MMP14 at the intended site was further confirmed by mass spectrometry (Supplemental Fig. 2). MMP-Mediated Delivery by PA-L1 of LF Variants into Cells A panel of cancer cell lines was characterized for the expression of various proteins involved in the Bacillus anthracis MMP-activatable LT system. All cell lines, except for HT1080, expressed relatively high levels of furin ( Fig. 2A ). Both HT1080 and MDA-MB-231 presented high levels of CMG2 and MMP14 expression. B8484 was found to have less CMG2 and MMP14 expression, whereas levels in MCF7 cells were undetectable by this technique. These findings were corroborated by immunohistochemistry in MDA-MB-231 and MCF7 cells (Supplemental Fig. 3). Gelatin zymography probing for MMP2 and MMP9 activity showed high MMP2 levels in HT1080, with far lower levels in the other cell lines in the panel ( Fig. 2B ). FIGURE 2. In vitro characterization of panel of cancer cell lines for protease and anthrax toxin receptor expression. (A) Western blots show differential MMP14, CMG2, and furin expression in whole-cell lysates. (B) MMP2 activity was measured in HT1080 cell lysates, using gelatin zymography. Broad-spectrum inhibitor GM6001 inhibited MMP2 activity. (C and D) Furin-cleavable PA-WT and MMP-cleavable PA-L1 delivered cytotoxic fusion toxin FP59 to panel of cancer cells, with variable efficiency. Controls are defined as MTT signal derived from vehicle-treated cells. * P 0.05). Similar results were obtained using a labeled LFn, 111 In-LFn, or using HT1080 and B8484 cells (Supplemental Fig. 7). Saturation binding studies of 111 In-LF E687A using fixed prepores in MDA-MB-231 cells revealed a binding constant of K D = 0.9 ± 0.09 nM (Supplemental Fig. 8). The pharmacokinetic behavior of 111 In-LFn and 111 In-LF E687A was compared in vivo. Dynamic SPECT scans were performed after intravenous bolus injection of either 111 In-LFn or 111 In-LF E687A in naïve SCID mice ( Fig. 5 ). Imaging data revealed that the truncated 111 In-LFn presented a biexponential decay in blood with a rapid distribution phase resulting in a fast half-life of 1.8 min, and an elimination phase presenting a slow half-life of 6.0 min. Thirty minutes after injection, only low concentrations of radiotracer in the blood pool were observed (1.1 ± 0.1 %ID/mL). In contrast, the radiolabeled full-length protein, 111 In-LF E687A , presented a significantly slower plasma clearance ( P 0.05). Similar results were obtained using a labeled LFn, 111 In-LFn, or using HT1080 and B8484 cells (Supplemental Fig. 7). Saturation binding studies of 111 In-LF E687A using fixed prepores in MDA-MB-231 cells revealed a binding constant of K D = 0.9 ± 0.09 nM (Supplemental Fig. 8). The pharmacokinetic behavior of 111 In-LFn and 111 In-LF E687A was compared in vivo. Dynamic SPECT scans were performed after intravenous bolus injection of either 111 In-LFn or 111 In-LF E687A in naïve SCID mice ( Fig. 5 ). Imaging data revealed that the truncated 111 In-LFn presented a biexponential decay in blood with a rapid distribution phase resulting in a fast half-life of 1.8 min, and an elimination phase presenting a slow half-life of 6.0 min. Thirty minutes after injection, only low concentrations of radiotracer in the blood pool were observed (1.1 ± 0.1 %ID/mL). In contrast, the radiolabeled full-length protein, 111 In-LF E687A , presented a significantly slower plasma clearance ( P < 0.0001) than the truncated protein, maintaining a significant amount in the blood circulation 3 h after administration (35 ± 2.6 %ID/mL). In this case, a monoexponential decay described the radiotracer pharmacokinetic, with a blood half-life of 33 min. Ex vivo biodistribution studies confirmed the in vivo imaging results, revealing low blood signal, yet high kidney uptake (29 ± 1.0 %ID/g), after administration of 111 In-LFn, possibly because of glomerular filtration (Supplemental Table 1). FIGURE 5. In vivo evaluation of 111 In-LFn and 111 In-LF E687A in tumor-naïve SCID mice. (A) Blood clearance of 111 In-LFn (inset: maximum-intensity projections over time). (B) Ex vivo biodistribution showing uptake of 111 In-LFn in selected tissues and blood, 1 h after intravenous administration. (C) Representative coronal and sagittal sections of SPECT images acquired 1 h after intravenous administration of 111 In-LFn. (D) Blood clearance of 111 In-LF E687A (inset: maximum-intensity projections over time). (E) Ex vivo biodistribution showing uptake of 111 In-LF E687A in selected tissues and blood, 3 h after intravenous administration. (F) Representative coronal and sagittal sections of SPECT images acquired 3 h after intravenous administration of 111 In-LF E687A . b = bladder; h = heart; k = kidneys; l = liver. Full-sized versions of panels A and D are available in the supplemental materials. Uptake of 111 In-PA-L1 and 111 In-PA-WT K563C by Tumor Tissue In Vivo The pharmacokinetics of radiolabeled furin–cleavable PA-WT K563C and MMP-cleavable PA-L1 were also compared, with a view to optimizing the time of injection in a 111 In-LF pretargeting system. Dynamic SPECT imaging after intravenous injection of 111 In-PA-L1 or 111 In-PA-WT K563C in MDA-MB-231 tumor–bearing mice revealed a distinct pharmacokinetic profile for 111 In-PA-WT K563C compared with 111 In-PA-L1 ( Fig. 6A ). Both presented monoexponential decay, with blood half-lives of 13.5 and 40.9 min, respectively. SPECT/CT imaging and ex vivo biodistribution showed a significantly increased uptake of 111 In-PA-L1 (5.7 ± 1.7 %ID/g) in MDA-MB-231 tumor tissue, compared with 111 In-PA-WT K563C (1.7 ± 0.52 %ID/g; P < 0.05), at 3 h after administration of the labeled compound ( Figs. 6B–6E ; Supplemental Table 2). Contrastingly, tumor-to-blood ratios were higher for 111 In-PA-WT K563C than for 111 In-PA-L1 ( P < 0.05). All in vivo SPECT and ex vivo biodistribution results were confirmed by autoradiography (Supplemental Fig. 9). FIGURE 6. In vivo evaluation of 111 In-PA-L1 and 111 In-PA-WT K563C in MDA-MB-231 tumor-bearing SCID mice. (A) Blood clearance of 111 In-PA-L1 and 111 In-PA-WT K563C . (B) Ex vivo biodistribution showing uptake of 111 In-PA-L1 or 111 In-PA-WT K563C in selected tissues and blood, 3 h after intravenous administration. (C) Tumor-to-blood ratios determined from B. (D and E) Representative coronal and sagittal maximum-intensity projections of SPECT/CT images, 3 h after administration of 111 In-PA-L1 or 111 In-PA-WT K563C . * P < 0.05. **** P < 0.0001. b = bladder; h = heart; k = kidneys; l = liver; t = tumor. MMP-Mediated Delivery by PA-L1 of 111 In-LF Variants to Tumor Tissue In Vivo On the basis of its pharmacokinetic profile, 111 In-LF E687A was considered to be the more appropriate variant to image MMP-mediated PA-L1 pore formation in tumor tissue in vivo, given the extremely fast clearance of 111 In-LFn. In addition, the slow but steady increase in tumor uptake of PA-L1 over time indicated that a coinjection of PA-L1 with 111 In-LF E687A would be advantageous. The delivery of 111 In-LF E678E by PA-L1 was evaluated in mice bearing MDA-MB-231 xenografts ( Fig. 7 ). Full ex vivo biodistribution data are summarized in Supplemental Table 3. SPECT/CT images and ex vivo biodistribution analysis at 24 h after injection revealed significantly increased delivery by PA-L1 of 111 In-LF E678E to tumor tissue (5.98 ± 0.62 %ID/g), compared with control animals that were not coinjected with PA-L1 (group V, 3.30 ± 1.12 %ID/g; P = 0.0017), indicating PA-L1–specific delivery. Imaging data at 3 h after injection showed similar results (Supplemental Fig. 10). When animals were also administered noncleavable PA-U7, instead of PA-L1, tumor uptake was significantly reduced (group III, 2.74 ± 0.24; P = 0.0004), indicating MMP activity–specific targeting of PA-L1 in vivo. Additionally, when the interaction between PA-L1 pores or prepores and 111 In-LF E678E was blocked by coadministration of an excess of unlabeled LF E678E , tumor uptake was significantly reduced to nonspecific uptake levels (group II, 2.68 ± 0.28 %ID/g; P = 0.0003). 111 In-LF E678E delivery to tumor tissue was significantly lower when coadministered with furin-cleavable PA-WT (group IV, 1.05 ± 0.21 %ID/g; P < 0.0001). This finding was in line with the rapid plasma clearance, activation by proteases in plasma ( 21 ), and lower tumor uptake of PA-WT. Kidney uptake was also affected by this effect (7.02 ± 2.15 vs. 6.31 ± 0.32 %ID/g, respectively; P < 0.0001). Contrastingly, PA-WT–mediated delivery of 111 In-LF E678E resulted in significantly higher delivery to spleen (29.84 ± 16.10 vs. 126.25 ± 42.18 %ID/g, respectively; P < 0.0001), again consistent with PA-WT cleavage and activation in blood plasma. FIGURE 7. (A) Representative coronal maximum-intensity projections of SPECT images of MDA-MB-231 tumor xenograft–bearing SCID mice, 24 h after administration of 111 In-LF E687A , alone or in combination with (left to right) PA-L1, PA-L1 and excess of unlabeled LF E687A , PA-U7, PA-WT, or no PA protein. Tumors are encircled (B) Ex vivo biodistribution showing tumor uptake of 111 In-LF E687A in different experimental groups. b = bladder; h = heart; l = liver; s = spleen; t = tumor. ** P < 0.01. **** P < 0.0001. Taken together, our results indicate the selective delivery to tumor tissue of 111 In-LF E678E by PA-L1 via binding to anthrax receptors, MMP-activation of PA-L1, and LF binding to its de novo receptor site on PA pores. DISCUSSION Molecular imaging is a promising method to visualize the clinical function of MMPs in a wide variety of diseases such as cancer, atherosclerosis, stroke, arthritis, periodontal disease, multiple sclerosis, and liver fibrosis. Here, we demonstrate a novel concept, using an MMP-activated pretargeting approach, based on the B. anthracis LT system. Previous work by Liu et al. showed in detail that PA-L1, an MMP-cleavable PA variant, allows for selective delivery of LF and related fusion toxins such as FP59 to tumor tissue in vivo for therapeutic gain ( 22 ). Here, we exploited this system to develop a molecular imaging system using a radiolabeled form of LF. The PA-L1/LF system used here is distinctly different from any previously reported MMP imaging methodology. An advantage is that the system does not rely on binding to an extracellular, potentially nonpermanent epitope but rather depends on the uptake of radiolabeled 111 In-LF E687A by cells. Notably, the LT system has the distinct additional advantage that on cleavage of PA-L1 by MMPs, the PA pore is capable of delivering multiple radiolabeled copies of 111 In-LF E687A to the cytoplasm of tumor cells. Here, we have demonstrated in a panel of cancer cell lines that MMP-activated PA-L1–mediated delivery of labeled LF variants enables selective visualization and measurement of MMP activity levels in tumor tissue in vivo. We performed detailed characterization of the system and demonstrated selectivity for anthrax receptors, mainly CMG2, high affinity of radiolabeled LF for PA-L1 pores in cells, and selective, specific delivery of 111 In-LF E687A to cancer cells in vitro and in vivo. PA-L1–mediated 111 In-LF E687A delivery to cells and tumors was blockable with an excess of LF or could be reduced by replacing PA-L1 with PA-U7, PA-WT, or vehicle only, indicating the PA-L1 and thus MMP-dependent cleavage of this interaction. Some limitations of the PA-L1/ 111 In-LF E687A system to visualize MMP activity are the inherent uptake of 111 In-LF E687A in liver and spleen, as is possibly due to ER-mediated uptake. Its pharmacokinetics were also rather slow, necessitating SPECT imaging 24 h after intravenous administration. Imaging at earlier time points after injection (we evaluated a 3-h time point) did not show the necessary tumor-to-background ratios compared with the longer interval. In contrast, the smaller size of 111 In-LFn resulted in renal clearance and therefore negligible liver uptake. 111 In-LFn performed equally well in in vitro assays but, in vivo, was cleared from the blood so fast that PA-L1–mediated tumor uptake could not be observed (Supplemental Table 4). It may be possible that variants of intermediate size may be cleared more slowly yet are still renally cleared, in analogy with radiolabeled antibody fragments and derivatives, and thus will result in more optimal imaging characteristics ( 23 ). It is also notable that because of the complexity of the MMP-activated LT system, the use of a large number of control conditions was required to demonstrate the specificity of the approach. In addition, the immunogenicity of DTPA-LF E687A and DTPA-LFn was not studied in the current work. However, it is known that nonmodified LF, when injected in the bloodstream, induces formation of neutralizing antibodies, resulting in protective immunity ( 24 , 25 ). Finally, the specificity of the PA-L1 protein variant to be cleaved and therefore activated by proteases other than its intended targets (MMP2, MMP9, and MMP14) and deliver 111 In-LF E687A to tumor cells was not evaluated here. The inherent overlap in specificity of all MMP proteins toward their substrates means that no single substrate will be specific for any given individual MMP. Similar to other proteases, MMP substrate specificity is guided by positional preferences of the residues on both the N terminus and the C terminus from the scissile bond. The substrate of MMPs contain a canonical P-X-X-↓L motif, with ↓ denoting the scissile bond ( 26 ). Depending on the nature of the other amino acids, the resulting sequence can be cleaved by all MMPs or show a preference for some individual MMPs or groups. This high level of substrate cleavage redundancy explains the functional overlap observed among members of the MMP family. Additionally, our study showed the pharmacokinetics and tissue-binding properties of radiolabeled PA-WT K563C and LF. As expected, uptake of 111 In-PA-WT K563C was observed in organs where the presence of CMG2 receptors has been previously described ( 27 , 28 ). Its fast clearance from the blood pool, when compared with that of 111 In-PA-L1, indicates that this molecule may be cleaved by proteases in the blood and that part of the observed organ uptake corresponds to labeled PA fragments rather than the interaction of the intact PA-WT K563C with its receptors ( 21 ). Renal uptake of 111 In-LF E687A in animals that also were exposed to PA-WT were similar to unspecific uptake levels. This finding may indicate that the uptake of PA-WT K563C observed in the kidneys was not due to competent PA-WT K563C pore formation. Conversely, mice treated with PA-WT and 111 In-LF E687A presented higher liver, lung, and spleen uptake of 111 In, suggesting higher levels of intoxication in these organs. These results suggest that PA-WT, and PA-WT K563C , are interacting with their cellular receptors and forming competent pores after having been cleaved, resulting in delivery of 111 In-LF E687A to these tissues. Although this tool could be used to study LT intoxication, the delivery of labeled LF did not correlate with an earlier report by Liu et al. In this study, it was demonstrated in various cell-type–specific CMG2-null mice, the key tissues responsible for LT lethality are cardiomyocytes and smooth vasculature in the heart ( 29 – 31 ), indicating that targeting and toxicity of LT are not interchangeable. CONCLUSION Taken together, our results indicate that radiolabeled forms of mutated anthrax LT hold promise for noninvasive imaging of MMP activity in tumor tissue. DISCLOSURE This research was supported by CRUK through the Oxford Institute for Radiation Oncology and the CRUK Oxford Centre, the CRUK/EPSRC Imaging Centre in Oxford, and Pancreatic Cancer U.K. Mary-Ann Elvina Xavier is funded by a Science Without Borders (CNPq) grant. Julia Baguña Torres is funded through a grant from the Pancreatic Cancer Research Fund. Thomas Bugge is funded by the NIDCR, NIH Intramural Research Program, and Samantha Hopkins is funded by the NIAID, NIH Intramural Research Program. Mass spectrometry analysis was performed at the Discovery Proteomics Facility (headed by Roman Fischer), which is part of the TDI MS Laboratory led by Professor Benedikt Kessler. No other potential conflict of interest relevant to this article was reported. Supplementary Material Click here for additional data file. KEY POINTS QUESTION: Radiolabeled engineered anthrax proteins allow detection of matrix metalloproteinases (MMPs) in mouse models of cancer, using SPECT. PERTINENT FINDINGS: Engineered anthrax proteins are activated specifically by MMP activity. A radiolabeled variant of anthrax lethal toxin was selectively delivered to MMP-active xenograft tumors in mice, presenting a 20-fold target-to-background contrast. IMPLICATIONS FOR PATIENT CARE: Imaging overactive MMPs with radiolabeled engineered anthrax proteins can aid in the prognosis of cancer patients and early detection.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403022/

Anthrax Cases Associated with Animal-Hair Shaving Brushes

During the First World War, anthrax cases in the United States and England increased greatly and seemed to be associated with use of new shaving brushes. Further investigation revealed that the source material and origin of shaving brushes had changed during the war. Cheap brushes of imported horsehair were being made to look like the preferred badger-hair brushes. Unfortunately, some of these brushes were not effectively disinfected and brought with them a nasty stowaway: Bacillus anthracis . A review of outbreak summaries, surveillance data, and case reports indicated that these cases originated from the use of ineffectively disinfected animal-hair shaving brushes. This historical information is relevant to current public health practice because renewed interest in vintage and animal-hair shaving brushes has been seen in popular culture. This information should help healthcare providers and public health officials answer questions on this topic.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961213/

IFN-induced Guanylate Binding Proteins in Inflammasome Activation and Host Defense

Traditional views of the inflammasome highlight pre-existing core components being assembled under basal conditions shortly after infection or tissue damage. Recent work, however, suggests the inflammasome machinery is also subject to tunable or inducible signals that may accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these immune signals operate downstream of interferon (IFN) receptors to elicit inflammasome regulators, including a new family of IFN-induced GTPases termed guanylate binding proteins (GBPs). Here, we examine the critical roles for IFN-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity against a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential impact of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294465/

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis . We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the d ual-filter method for a pplied r ecovery of microbial particles from e nvironmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527960/

Electrostatic Ratchet in the Protective Antigen Channel Promotes Anthrax Toxin Translocation *

Background: Ratchets are key features in molecular machines that unfold and transport biopolymers. Results: An electrostatic ratchet in the anthrax toxin protein translocase was experimentally identified and modeled. Conclusion: The anthrax toxin translocase harnesses the proton motive force with an electrostatic ratchet. Significance: This report describes an electrostatic ratchet element critical to proton motive force-driven translocation. Introduction Protein translocation is a fundamental molecular process required to transport proteins across membranes and to disassemble, denature, renature, and/or degrade proteins within the cell ( 1 , 2 ). Many biological events depend upon protein translocation ( 3 ), namely microbial toxin translocation into host cells ( 1 , 2 , 4 – 9 ), toxin secretion ( 10 ), antigen presentation ( 11 ), membrane and organelle biogenesis ( 12 ), and retrograde transport of misprocessed proteins from the endoplasmic reticulum ( 11 ). The translocase machinery is generally, but not always ( 13 ), comprised of proteinaceous components. Although diverse in mechanism, translocases utilize common driving forces such as ATP hydrolysis or the proton motive force (PMF) 2 to provide the necessary energy for unfolding and translocation ( 1 ). Brownian-ratchet (BR) ( Fig. 1 A ) and power-stroke (PS) ( Fig. 1 B ) mechanisms have been invoked to describe how molecular machines convert potential energy (the PMF or ATP) into useful work, such as unfolding and translocating proteins ( 2 ). The PS mechanism is believed to do work via a direct chemomechanical coupling of the energy source, whereas the BR mechanism does work by rectifying Brownian motion. In each case, the PS and BR mechanisms function via a cyclical dissipation of the potential energy source, creating repeated nonequilibrium fluctuations in the system. The substrate polymer is then directed to move in a unidirectional manner by means of some type of rectification or ratchet mechanism. The ratchet can be thought of energetically as an asymmetrical potential energy barrier that fluctuates as the energy source dissipates, or structurally, as a loop that forcefully pushes the peptide in one direction and/or biases against retro-translocation. The molecular bases of these ratchet features are not well understood. FIGURE 1. Models of translocation. Shown are ( A ) H + -powered BR and ( B ) ATP-driven PS protein-translocation models. The translocating peptide has its residue chemistries colored: deprotonated acidic ( red ), protonated acidic ( black ), basic ( blue ), and hydrophobic ( green ). Dynamic gates and clamps that cycle in these systems are shown as steel blue . See text for details. C , assembly and translocation mechanism of anthrax lethal toxin. The components of lethal toxin, PA ( steel blue ) ( 21 , 42 ), and LF ( magenta ) ( 24 ), assemble into heterogeneous oligomeric complexes, PA 8 LF 4 ( 4 ) and PA 7 LF 3 , which are then endocytosed upon binding a receptor ( gold ). Acidification triggers the PA oligomer to form a translocase channel ( 19 , 23 , 43 ), and the ΔpH component of the PMF drives LF unfolding and translocation into the cytosol ( 6 , 8 , 9 ). D, the PA oligomer ( gray surface ) facilitates LF ( magenta ) unfolding and translocation with several known polypeptide clamps. The α clamp ( light blue surface ) ( 42 ) binds nonspecifically to peptide helices and initiates LF N ( magenta ) translocation by binding to its first helix, α1, which is just carboxyl-terminal to the modeled amino-terminal leader sequence leading into the central lumen. The φ clamp, a ring of 7 or 8 Phe-427 residues ( red sticks ) in the PA oligomer, which is depicted here in the prechannel conformation to show its approximate location, then engages the amino-terminal leader sequence again through nonspecific interactions ( 7 ). These clamps may work in concert to bind and release substrate promoting unfolding and translocation ( 2 ). Anthrax toxin ( 1 , 2 , 14 ), the tripartite virulence factor secreted by Bacillus anthracis (the etiologic agent of anthrax), is ideally suited for biophysical studies probing the molecular mechanism of PMF-driven protein translocation ( 1 , 4 – 9 , 15 – 18 ) ( Fig. 1 C ). Using electrophysiology, the electrical potential (Δψ) and chemical potential (ΔpH) compositions of the PMF can be externally controlled ( 4 – 9 , 16 , 17 ). Lethal factor (LF) and edema factor (EF) are the two different ∼90-kDa enzyme components of the toxin, which are translocated by the oligomeric channel formed by a third component, protective antigen (PA, 83 kDa). To function, PA, LF, and EF must assemble into holotoxin complexes ( Fig. 1 C ). PA is initially cleaved by a furin-type protease. The resulting 63-kDa PA subunits assemble into either heptameric (PA 7 ) ( 19 – 21 ) or octameric (PA 8 ) ( 4 , 5 , 22 , 23 ) oligomers, or prechannels. PA 7 and PA 8 can bind up to three and four EF/LF moieties, respectively ( 4 , 5 ). Crystal structures of LF ( 24 ), EF ( 25 , 26 ), PA ( 21 ), the PA 7 ( 20 ) and PA 8 ( 5 ) prechannel oligomers, and the core of a PA 8 LF 4 holotoxin complex ( 4 ) have been described. Once assembled, toxin complexes are endocytosed and trafficked to an acidic compartment in the cell, where PA converts to a cation-selective channel ( 27 ). The channel structure as resolved by electron microscopy (EM) ( 19 ) has a putative extended tubular β-barrel architecture ( 28 , 29 ), analogous to the Staphlococcus aureus α-hemolysin toxin pore ( 30 ). The narrowness of the PA channel requires that LF and EF unfold during translocation. Some destabilization of these proteins is imparted by the acidic conditions of the endosome ( 31 ). Interestingly, some unfolding occurs when LF and EF initially form a complex with the PA oligomer. In a recent crystal structure of the core of the PA 8 LF 4 holotoxin, it was determined that the first α helix and β strand of the amino-terminal PA-binding domain of LF (LF N ) are unfolded and docked into a cleft, called the α clamp ( 4 ) ( Fig. 1 D ). The α clamp is created at the interface of adjacent PA subunits, such that the deep cleft is framed by twin Ca 2+ -ion binding sites ( 4 ). The α clamp is also a highly nonspecific binding site, and can interact with diverse sequence chemistries, binding amphipathic and nonamphipathic helices with similar affinities ( 4 ). Detailed mutagenesis studies have shown that the most force-dependent step of the translocation mechanism coincides with the unfolding of the remaining structure of LF N ( 8 ). In fact, to cross the rate-limiting barrier, a significant portion of the amino-terminal β-sheet subdomain of LF is required to unfold ( 8 ). The unfolding process appears to also require another unfoldase active site, called the φ clamp ( 7 , 8 ). The φ clamp is a ring of Phe-427 residues, which also bind nonspecifically to substrates that are dense in aromatic, hydrophobic, and cationic functional groups ( 7 ) ( Fig. 1 D ). These two unique protein-denaturation sites in the PA channel (α and φ clamps) together favor the unfolding process. Although the mechanism is uncertain, these protein-denaturation sites are not thought to be traditional protein-binding sites; rather they are believed to be dynamic, coordinated, and ratchet-like, switching between high and low affinity states to promote directional motion, where binding at one clamp site can allosterically control binding at the other clamp site ( 1 ). Although translocation can be driven by either the Δψ ( 15 ) or ΔpH ( 6 ), the ΔpH is sufficient ( 9 ) and critical to the efficient translocation of the full-length enzymes, LF and EF ( 6 ). A consensus picture is emerging that the underlying mechanism of ΔpH-driven translocation involves a charge-state BR ( 6 , 8 , 9 , 16 – 18 ). Differences in the relative rates of protonation on either side of the membrane are believed to be able to bias Brownian fluctuations and impart directionality in the translocation mechanism. Brown et al. ( 9 ) have shown that acidic residues in a protein substrate are required for ΔpH-driven translocation. These residues are effectively the molecular teeth upon which an electrostatic ratchet feature within the channel acts to produce forces during translocation. An anionic charge requirement for ΔpH-dependent protein translocation may seem unusual, as the PA channel itself is strongly cation selective (or anion repulsive) ( 27 ). However, the protonation of acidic residues is likely required to make a portion of the translocating chain within the channel near neutral or slightly cationic. Doing so allows the protein to pass through the anion-rejection site of the channel by means of Brownian motion ( Fig. 1 A ). Once the protonated portion of the translocating protein reaches the higher pH of the cytosol, these sites are more frequently deprotonated, becoming electrostatically incompatible with the channel. The same electrostatic feature that repels anion flux into the channel may then also act to ratchet and exclude retrograde efflux back into the channel. This rectification/ratchet feature is a critical aspect of BR- and PS-type molecular machines, because it can bias nonequilibrium substrate fluctuations by limiting retrograde efflux. Cycles of substrate protonation, Brownian motion, and deprotonation are likely required to pull the protein across the membrane. Analogously, with ATP-dependent systems, 100s of cycles of ATP binding and hydrolysis are required to unfold and transport a substrate protein. Several critical questions remain unanswered as to how this mechanism applies to protein translocation. What substrate sequence features allow for rapid translocation? What feature in the channel rectifies or ratchets Brownian motion and nonequilibrium fluctuations? How does the proposed charge-state BR mechanism develop forces sufficient to unfold substrate proteins? To address these questions, we investigated electrostatic requirements of the substrate and channel in PMF-driven anthrax toxin translocation. Our results and modeling studies are consistent with an electrostatic ratchet translocation model. EXPERIMENTAL PROCEDURES Proteins Recombinant wild-type (WT) PA, LF N , the amino-terminal PA-binding domain of EF (EF N ), and resulting chimeras and mutants were expressed and purified as described ( 5 , 8 ). Assembly PCR was used to construct LF N /EF N chimeras ( 4 , 9 ). The amino-terminal six-histidine affinity tags (His 6 ) were removed from LF N /EF N chimeras using bovine α thrombin ( 8 ). PA 7 prechannel oligomers were assembled as described ( 5 ). For the PA mutants PA top (containing the substitutions D276S, D335S, and E343S) and PA bot (containing the substitutions E302T, H304T, E308T, and H310T), and a WT PA control, 10 μg of each PA monomer was proteolyzed by 0.4 units of furin (New England Biolabs) in 20 m m Tris-Cl, pH 9, 150 m m NaCl, and 1 m m CaCl 2 at room temperature. After 30 min, LF N was added at a 1:1 molar ratio, and following another 30-min incubation at 25 °C, Fos-choline-14 was introduced to a final concentration of 2 m m to stabilize the PA oligomers in the channel form ( 32 ). Proper PA assembly was verified by native PAGE, SDS-PAGE, and negative stain EM. Electrophysiology Planar lipid bilayers were formed by painting ( 33 ) a membrane-forming solution (3% 1,2-diphytanoyl- sn -glycerol-3-phosphocholine in n -decane) across a 100-μm aperture in a 1-ml white Delrin or polysulfone cup ( 4 , 5 , 8 ). A capacitance test confirmed the quality of the membrane. The membrane separates the cis and trans chambers, each containing 1 ml of universal bilayer buffer (100 m m KCl, 1 m m EDTA, 10 m m oxalic acid, 10 m m MES, 10 m m phosphoric acid). Ag/AgCl electrodes bathed in saturated 3 m KCl were linked to the chambers via 3 m KCl-agar salt bridges. PA currents were recorded with an Axoclamp 200B amplifier in CLAMPEX10. Translocation Assays Bilayers were bathed in symmetrical universal bilayer buffer. PA 7 prechannels were added to the cis chamber (held at 20 mV), and conductance was blocked by the addition of substrate (LF N , EF N , or chimera) to the cis side (held at 20 mV in symmetric pH 5.6 experiments). The substrate blockade was >95% of the original current. Excess substrate was perfused by a hand-cranked, push-pull perfusion system. In Δψ-driven translocation assays, substrate translocation was initiated by increasing the Δψ; Δψ ≡ ψ cis − ψ trans (ψ trans ≡ 0). Translocation activation energy (Δ G ‡ ) was computed by RT ln t ½/ c ( 8 ). The t ½ value is the time for half the substrate to translocate; c is a 1-s reference; R is the gas constant; and T is the temperature. In ΔpH-driven experiments, the cis and trans chambers were bathed in universal bilayer buffer differing only in pH (pH cis = 5.6; pH trans = 6.6), where ΔpH ≡ pH trans − pH cis . The Δψ was −1 mV during substrate blockade and perfusion. Translocation was initiated by increasing Δψ to 20 mV. Translocation records in either case were acquired across a range of Δψ values ( n = 6 to 30). Equilibrium Stability Measurements Guanidinium chloride titrations of LF N , EF N , and chimeras were carried out as described ( 1 , 2 ) in 10 m m sodium phosphate, 1 m glucose, pH 7.5, at 20 °C. The stabilizing glucose additive was used to define the native state baseline. Each titration point was monitored after reaching equilibrium by circular dichroism (CD) spectroscopy at 222 (±2) nm using a Jasco J-810 spectropolarimeter. The CD-probed curves fit to a four-state thermodynamic model ( N ↔ I ↔ J ↔ U ), where native ( N ), two intermediates ( I and J ), and an unfolded ( U ) state are populated ( 2 ). We used the thermodynamic difference between the N and I states (Δ G NI ) to assess the stability of the protein. Reversal Potential (Δψ rev ) Measurements A planar bilayer was formed with the cis chamber bathed in 5 m m potassium phosphate, 100 m m KCl, pH 6.6, and the trans chamber bathed in unbuffered saline consisting of 100 m m KCl, pH 5.8. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side. Following channel insertion, the cis chamber was perfused thoroughly with fresh 100 m m KCl, making the system symmetrical, unbuffered KCl, pH 5.8. Residual LF N was then removed by applying a strong 100 mV Δψ to translocate it through the channel; and in some cases, a 1-unit ΔpH was established to aid in channel clearance of residual LF N . Upon stabilization, a series of 50-μl aliquots of 3 m KCl were added to the cis side, and Δψ rev was recorded as the Δψ required to drop the current to zero. All given KCl ratios of the two sides of the bilayer have been corrected for activity in water ( 34 ), and following the experiment, the chambers were weighed to confirm their volume. Ensemble Channel Blocking A planar bilayer was formed with both chambers in 10 m m potassium phosphate, pH 6.6. The cis chamber alone had an additional 100 m m equivalent of KCl. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side, and the chamber was perfused following insertion. To remove remaining LF N , 10 μl of 0.4 m phosphoric acid was added to the cis chamber to lower the pH to ∼4.4, and a Δψ of 20 mV was applied. Afterward, the cis chamber was perfused with fresh pH 6.6 buffer, and the Δψ was returned to 0 mV. LF N was added to a given concentration and allowed equilibrate. The percent blockade was determined by the equilibrium drop in current following the addition of LF N . EM Preparations of PA top , PA bot , and a WT PA control were purified by anion exchange chromatography to remove residual PA monomer and excess LF N . Fos-choline-14 was only added to a concentration of 0.05 m m to avoid reaching the critical micelle concentration. All samples were diluted to an estimated final concentration of 70 n m (based on absorbance at 280 nm). Diluted complexes were incubated for 30 s on 400-mesh copper grids (Electron Microscopy Sciences) coated with continuous carbon on nitrocellulose, stained with 1% uranyl formate, and imaged with a Tecnai 12 TEM operated at 120 kV and at ×49,000 magnification. Single particles were selected automatically using boxer (EMAN). The total numbers of particles ( n ) analyzed were: PA WT ( n = 4847), PA top ( n = 4577), and PA bot ( n = 4971). Class averages were determined iteratively using 10 successive cycles of Adapt, an automated classification program (written in house) and two-dimensional multireference alignment in IMAGIC. Molecular Models EF N and LF N domains from EF (PDB 1Y0V ( 26 )) and LF (PDB 1J7N ( 24 )), respectively, were α-carbon-(C α )-aligned in CHIMERA ( 35 ). A three-dimensional model of the 14-stranded β-barrel region of the PA channel (residues 275 to 352) was made by coaxially stacking multiple copies of the heptameric β-barrel from α hemolysin (PDB 7AHL ( 30 )). Peptide bonds were formed and residues were repopulated using COOT ( 36 ). The model was aligned to the z axis in CHIMERA ( 35 ). To obtain an electrostatic energy U ( z ) as a function of the distance moved axially through the barrel z axis, we computed the sum of all pairwise electrostatic energies in a PERL script (zforce.pl, which is available on request), using a 1-unit elementary point charge, q test , moved along the center of the barrel in 0.1-à increments, U ( z ) = q test b Σ q i cos θ i / d i , where d i is the distance between the C α of the i th charged site within the channel of elementary charge, q i , and q test ; θ i is the angle between the charges and the z axis; and b is an electrostatic energy conversion constant of 1390 kJ à mol −1 . Proteins Recombinant wild-type (WT) PA, LF N , the amino-terminal PA-binding domain of EF (EF N ), and resulting chimeras and mutants were expressed and purified as described ( 5 , 8 ). Assembly PCR was used to construct LF N /EF N chimeras ( 4 , 9 ). The amino-terminal six-histidine affinity tags (His 6 ) were removed from LF N /EF N chimeras using bovine α thrombin ( 8 ). PA 7 prechannel oligomers were assembled as described ( 5 ). For the PA mutants PA top (containing the substitutions D276S, D335S, and E343S) and PA bot (containing the substitutions E302T, H304T, E308T, and H310T), and a WT PA control, 10 μg of each PA monomer was proteolyzed by 0.4 units of furin (New England Biolabs) in 20 m m Tris-Cl, pH 9, 150 m m NaCl, and 1 m m CaCl 2 at room temperature. After 30 min, LF N was added at a 1:1 molar ratio, and following another 30-min incubation at 25 °C, Fos-choline-14 was introduced to a final concentration of 2 m m to stabilize the PA oligomers in the channel form ( 32 ). Proper PA assembly was verified by native PAGE, SDS-PAGE, and negative stain EM. Electrophysiology Planar lipid bilayers were formed by painting ( 33 ) a membrane-forming solution (3% 1,2-diphytanoyl- sn -glycerol-3-phosphocholine in n -decane) across a 100-μm aperture in a 1-ml white Delrin or polysulfone cup ( 4 , 5 , 8 ). A capacitance test confirmed the quality of the membrane. The membrane separates the cis and trans chambers, each containing 1 ml of universal bilayer buffer (100 m m KCl, 1 m m EDTA, 10 m m oxalic acid, 10 m m MES, 10 m m phosphoric acid). Ag/AgCl electrodes bathed in saturated 3 m KCl were linked to the chambers via 3 m KCl-agar salt bridges. PA currents were recorded with an Axoclamp 200B amplifier in CLAMPEX10. Translocation Assays Bilayers were bathed in symmetrical universal bilayer buffer. PA 7 prechannels were added to the cis chamber (held at 20 mV), and conductance was blocked by the addition of substrate (LF N , EF N , or chimera) to the cis side (held at 20 mV in symmetric pH 5.6 experiments). The substrate blockade was >95% of the original current. Excess substrate was perfused by a hand-cranked, push-pull perfusion system. In Δψ-driven translocation assays, substrate translocation was initiated by increasing the Δψ; Δψ ≡ ψ cis − ψ trans (ψ trans ≡ 0). Translocation activation energy (Δ G ‡ ) was computed by RT ln t ½/ c ( 8 ). The t ½ value is the time for half the substrate to translocate; c is a 1-s reference; R is the gas constant; and T is the temperature. In ΔpH-driven experiments, the cis and trans chambers were bathed in universal bilayer buffer differing only in pH (pH cis = 5.6; pH trans = 6.6), where ΔpH ≡ pH trans − pH cis . The Δψ was −1 mV during substrate blockade and perfusion. Translocation was initiated by increasing Δψ to 20 mV. Translocation records in either case were acquired across a range of Δψ values ( n = 6 to 30). Equilibrium Stability Measurements Guanidinium chloride titrations of LF N , EF N , and chimeras were carried out as described ( 1 , 2 ) in 10 m m sodium phosphate, 1 m glucose, pH 7.5, at 20 °C. The stabilizing glucose additive was used to define the native state baseline. Each titration point was monitored after reaching equilibrium by circular dichroism (CD) spectroscopy at 222 (±2) nm using a Jasco J-810 spectropolarimeter. The CD-probed curves fit to a four-state thermodynamic model ( N ↔ I ↔ J ↔ U ), where native ( N ), two intermediates ( I and J ), and an unfolded ( U ) state are populated ( 2 ). We used the thermodynamic difference between the N and I states (Δ G NI ) to assess the stability of the protein. Reversal Potential (Δψ rev ) Measurements A planar bilayer was formed with the cis chamber bathed in 5 m m potassium phosphate, 100 m m KCl, pH 6.6, and the trans chamber bathed in unbuffered saline consisting of 100 m m KCl, pH 5.8. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side. Following channel insertion, the cis chamber was perfused thoroughly with fresh 100 m m KCl, making the system symmetrical, unbuffered KCl, pH 5.8. Residual LF N was then removed by applying a strong 100 mV Δψ to translocate it through the channel; and in some cases, a 1-unit ΔpH was established to aid in channel clearance of residual LF N . Upon stabilization, a series of 50-μl aliquots of 3 m KCl were added to the cis side, and Δψ rev was recorded as the Δψ required to drop the current to zero. All given KCl ratios of the two sides of the bilayer have been corrected for activity in water ( 34 ), and following the experiment, the chambers were weighed to confirm their volume. Ensemble Channel Blocking A planar bilayer was formed with both chambers in 10 m m potassium phosphate, pH 6.6. The cis chamber alone had an additional 100 m m equivalent of KCl. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side, and the chamber was perfused following insertion. To remove remaining LF N , 10 μl of 0.4 m phosphoric acid was added to the cis chamber to lower the pH to ∼4.4, and a Δψ of 20 mV was applied. Afterward, the cis chamber was perfused with fresh pH 6.6 buffer, and the Δψ was returned to 0 mV. LF N was added to a given concentration and allowed equilibrate. The percent blockade was determined by the equilibrium drop in current following the addition of LF N . EM Preparations of PA top , PA bot , and a WT PA control were purified by anion exchange chromatography to remove residual PA monomer and excess LF N . Fos-choline-14 was only added to a concentration of 0.05 m m to avoid reaching the critical micelle concentration. All samples were diluted to an estimated final concentration of 70 n m (based on absorbance at 280 nm). Diluted complexes were incubated for 30 s on 400-mesh copper grids (Electron Microscopy Sciences) coated with continuous carbon on nitrocellulose, stained with 1% uranyl formate, and imaged with a Tecnai 12 TEM operated at 120 kV and at ×49,000 magnification. Single particles were selected automatically using boxer (EMAN). The total numbers of particles ( n ) analyzed were: PA WT ( n = 4847), PA top ( n = 4577), and PA bot ( n = 4971). Class averages were determined iteratively using 10 successive cycles of Adapt, an automated classification program (written in house) and two-dimensional multireference alignment in IMAGIC. Molecular Models EF N and LF N domains from EF (PDB 1Y0V ( 26 )) and LF (PDB 1J7N ( 24 )), respectively, were α-carbon-(C α )-aligned in CHIMERA ( 35 ). A three-dimensional model of the 14-stranded β-barrel region of the PA channel (residues 275 to 352) was made by coaxially stacking multiple copies of the heptameric β-barrel from α hemolysin (PDB 7AHL ( 30 )). Peptide bonds were formed and residues were repopulated using COOT ( 36 ). The model was aligned to the z axis in CHIMERA ( 35 ). To obtain an electrostatic energy U ( z ) as a function of the distance moved axially through the barrel z axis, we computed the sum of all pairwise electrostatic energies in a PERL script (zforce.pl, which is available on request), using a 1-unit elementary point charge, q test , moved along the center of the barrel in 0.1-à increments, U ( z ) = q test b Σ q i cos θ i / d i , where d i is the distance between the C α of the i th charged site within the channel of elementary charge, q i , and q test ; θ i is the angle between the charges and the z axis; and b is an electrostatic energy conversion constant of 1390 kJ à mol −1 . Proteins Recombinant wild-type (WT) PA, LF N , the amino-terminal PA-binding domain of EF (EF N ), and resulting chimeras and mutants were expressed and purified as described ( 5 , 8 ). Assembly PCR was used to construct LF N /EF N chimeras ( 4 , 9 ). The amino-terminal six-histidine affinity tags (His 6 ) were removed from LF N /EF N chimeras using bovine α thrombin ( 8 ). PA 7 prechannel oligomers were assembled as described ( 5 ). For the PA mutants PA top (containing the substitutions D276S, D335S, and E343S) and PA bot (containing the substitutions E302T, H304T, E308T, and H310T), and a WT PA control, 10 μg of each PA monomer was proteolyzed by 0.4 units of furin (New England Biolabs) in 20 m m Tris-Cl, pH 9, 150 m m NaCl, and 1 m m CaCl 2 at room temperature. After 30 min, LF N was added at a 1:1 molar ratio, and following another 30-min incubation at 25 °C, Fos-choline-14 was introduced to a final concentration of 2 m m to stabilize the PA oligomers in the channel form ( 32 ). Proper PA assembly was verified by native PAGE, SDS-PAGE, and negative stain EM. Electrophysiology Planar lipid bilayers were formed by painting ( 33 ) a membrane-forming solution (3% 1,2-diphytanoyl- sn -glycerol-3-phosphocholine in n -decane) across a 100-μm aperture in a 1-ml white Delrin or polysulfone cup ( 4 , 5 , 8 ). A capacitance test confirmed the quality of the membrane. The membrane separates the cis and trans chambers, each containing 1 ml of universal bilayer buffer (100 m m KCl, 1 m m EDTA, 10 m m oxalic acid, 10 m m MES, 10 m m phosphoric acid). Ag/AgCl electrodes bathed in saturated 3 m KCl were linked to the chambers via 3 m KCl-agar salt bridges. PA currents were recorded with an Axoclamp 200B amplifier in CLAMPEX10. Translocation Assays Bilayers were bathed in symmetrical universal bilayer buffer. PA 7 prechannels were added to the cis chamber (held at 20 mV), and conductance was blocked by the addition of substrate (LF N , EF N , or chimera) to the cis side (held at 20 mV in symmetric pH 5.6 experiments). The substrate blockade was >95% of the original current. Excess substrate was perfused by a hand-cranked, push-pull perfusion system. In Δψ-driven translocation assays, substrate translocation was initiated by increasing the Δψ; Δψ ≡ ψ cis − ψ trans (ψ trans ≡ 0). Translocation activation energy (Δ G ‡ ) was computed by RT ln t ½/ c ( 8 ). The t ½ value is the time for half the substrate to translocate; c is a 1-s reference; R is the gas constant; and T is the temperature. In ΔpH-driven experiments, the cis and trans chambers were bathed in universal bilayer buffer differing only in pH (pH cis = 5.6; pH trans = 6.6), where ΔpH ≡ pH trans − pH cis . The Δψ was −1 mV during substrate blockade and perfusion. Translocation was initiated by increasing Δψ to 20 mV. Translocation records in either case were acquired across a range of Δψ values ( n = 6 to 30). Equilibrium Stability Measurements Guanidinium chloride titrations of LF N , EF N , and chimeras were carried out as described ( 1 , 2 ) in 10 m m sodium phosphate, 1 m glucose, pH 7.5, at 20 °C. The stabilizing glucose additive was used to define the native state baseline. Each titration point was monitored after reaching equilibrium by circular dichroism (CD) spectroscopy at 222 (±2) nm using a Jasco J-810 spectropolarimeter. The CD-probed curves fit to a four-state thermodynamic model ( N ↔ I ↔ J ↔ U ), where native ( N ), two intermediates ( I and J ), and an unfolded ( U ) state are populated ( 2 ). We used the thermodynamic difference between the N and I states (Δ G NI ) to assess the stability of the protein. Reversal Potential (Δψ rev ) Measurements A planar bilayer was formed with the cis chamber bathed in 5 m m potassium phosphate, 100 m m KCl, pH 6.6, and the trans chamber bathed in unbuffered saline consisting of 100 m m KCl, pH 5.8. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side. Following channel insertion, the cis chamber was perfused thoroughly with fresh 100 m m KCl, making the system symmetrical, unbuffered KCl, pH 5.8. Residual LF N was then removed by applying a strong 100 mV Δψ to translocate it through the channel; and in some cases, a 1-unit ΔpH was established to aid in channel clearance of residual LF N . Upon stabilization, a series of 50-μl aliquots of 3 m KCl were added to the cis side, and Δψ rev was recorded as the Δψ required to drop the current to zero. All given KCl ratios of the two sides of the bilayer have been corrected for activity in water ( 34 ), and following the experiment, the chambers were weighed to confirm their volume. Ensemble Channel Blocking A planar bilayer was formed with both chambers in 10 m m potassium phosphate, pH 6.6. The cis chamber alone had an additional 100 m m equivalent of KCl. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side, and the chamber was perfused following insertion. To remove remaining LF N , 10 μl of 0.4 m phosphoric acid was added to the cis chamber to lower the pH to ∼4.4, and a Δψ of 20 mV was applied. Afterward, the cis chamber was perfused with fresh pH 6.6 buffer, and the Δψ was returned to 0 mV. LF N was added to a given concentration and allowed equilibrate. The percent blockade was determined by the equilibrium drop in current following the addition of LF N . EM Preparations of PA top , PA bot , and a WT PA control were purified by anion exchange chromatography to remove residual PA monomer and excess LF N . Fos-choline-14 was only added to a concentration of 0.05 m m to avoid reaching the critical micelle concentration. All samples were diluted to an estimated final concentration of 70 n m (based on absorbance at 280 nm). Diluted complexes were incubated for 30 s on 400-mesh copper grids (Electron Microscopy Sciences) coated with continuous carbon on nitrocellulose, stained with 1% uranyl formate, and imaged with a Tecnai 12 TEM operated at 120 kV and at ×49,000 magnification. Single particles were selected automatically using boxer (EMAN). The total numbers of particles ( n ) analyzed were: PA WT ( n = 4847), PA top ( n = 4577), and PA bot ( n = 4971). Class averages were determined iteratively using 10 successive cycles of Adapt, an automated classification program (written in house) and two-dimensional multireference alignment in IMAGIC. Molecular Models EF N and LF N domains from EF (PDB 1Y0V ( 26 )) and LF (PDB 1J7N ( 24 )), respectively, were α-carbon-(C α )-aligned in CHIMERA ( 35 ). A three-dimensional model of the 14-stranded β-barrel region of the PA channel (residues 275 to 352) was made by coaxially stacking multiple copies of the heptameric β-barrel from α hemolysin (PDB 7AHL ( 30 )). Peptide bonds were formed and residues were repopulated using COOT ( 36 ). The model was aligned to the z axis in CHIMERA ( 35 ). To obtain an electrostatic energy U ( z ) as a function of the distance moved axially through the barrel z axis, we computed the sum of all pairwise electrostatic energies in a PERL script (zforce.pl, which is available on request), using a 1-unit elementary point charge, q test , moved along the center of the barrel in 0.1-à increments, U ( z ) = q test b Σ q i cos θ i / d i , where d i is the distance between the C α of the i th charged site within the channel of elementary charge, q i , and q test ; θ i is the angle between the charges and the z axis; and b is an electrostatic energy conversion constant of 1390 kJ à mol −1 . Proteins Recombinant wild-type (WT) PA, LF N , the amino-terminal PA-binding domain of EF (EF N ), and resulting chimeras and mutants were expressed and purified as described ( 5 , 8 ). Assembly PCR was used to construct LF N /EF N chimeras ( 4 , 9 ). The amino-terminal six-histidine affinity tags (His 6 ) were removed from LF N /EF N chimeras using bovine α thrombin ( 8 ). PA 7 prechannel oligomers were assembled as described ( 5 ). For the PA mutants PA top (containing the substitutions D276S, D335S, and E343S) and PA bot (containing the substitutions E302T, H304T, E308T, and H310T), and a WT PA control, 10 μg of each PA monomer was proteolyzed by 0.4 units of furin (New England Biolabs) in 20 m m Tris-Cl, pH 9, 150 m m NaCl, and 1 m m CaCl 2 at room temperature. After 30 min, LF N was added at a 1:1 molar ratio, and following another 30-min incubation at 25 °C, Fos-choline-14 was introduced to a final concentration of 2 m m to stabilize the PA oligomers in the channel form ( 32 ). Proper PA assembly was verified by native PAGE, SDS-PAGE, and negative stain EM. Electrophysiology Planar lipid bilayers were formed by painting ( 33 ) a membrane-forming solution (3% 1,2-diphytanoyl- sn -glycerol-3-phosphocholine in n -decane) across a 100-μm aperture in a 1-ml white Delrin or polysulfone cup ( 4 , 5 , 8 ). A capacitance test confirmed the quality of the membrane. The membrane separates the cis and trans chambers, each containing 1 ml of universal bilayer buffer (100 m m KCl, 1 m m EDTA, 10 m m oxalic acid, 10 m m MES, 10 m m phosphoric acid). Ag/AgCl electrodes bathed in saturated 3 m KCl were linked to the chambers via 3 m KCl-agar salt bridges. PA currents were recorded with an Axoclamp 200B amplifier in CLAMPEX10. Translocation Assays Bilayers were bathed in symmetrical universal bilayer buffer. PA 7 prechannels were added to the cis chamber (held at 20 mV), and conductance was blocked by the addition of substrate (LF N , EF N , or chimera) to the cis side (held at 20 mV in symmetric pH 5.6 experiments). The substrate blockade was >95% of the original current. Excess substrate was perfused by a hand-cranked, push-pull perfusion system. In Δψ-driven translocation assays, substrate translocation was initiated by increasing the Δψ; Δψ ≡ ψ cis − ψ trans (ψ trans ≡ 0). Translocation activation energy (Δ G ‡ ) was computed by RT ln t ½/ c ( 8 ). The t ½ value is the time for half the substrate to translocate; c is a 1-s reference; R is the gas constant; and T is the temperature. In ΔpH-driven experiments, the cis and trans chambers were bathed in universal bilayer buffer differing only in pH (pH cis = 5.6; pH trans = 6.6), where ΔpH ≡ pH trans − pH cis . The Δψ was −1 mV during substrate blockade and perfusion. Translocation was initiated by increasing Δψ to 20 mV. Translocation records in either case were acquired across a range of Δψ values ( n = 6 to 30). Equilibrium Stability Measurements Guanidinium chloride titrations of LF N , EF N , and chimeras were carried out as described ( 1 , 2 ) in 10 m m sodium phosphate, 1 m glucose, pH 7.5, at 20 °C. The stabilizing glucose additive was used to define the native state baseline. Each titration point was monitored after reaching equilibrium by circular dichroism (CD) spectroscopy at 222 (±2) nm using a Jasco J-810 spectropolarimeter. The CD-probed curves fit to a four-state thermodynamic model ( N ↔ I ↔ J ↔ U ), where native ( N ), two intermediates ( I and J ), and an unfolded ( U ) state are populated ( 2 ). We used the thermodynamic difference between the N and I states (Δ G NI ) to assess the stability of the protein. Reversal Potential (Δψ rev ) Measurements A planar bilayer was formed with the cis chamber bathed in 5 m m potassium phosphate, 100 m m KCl, pH 6.6, and the trans chamber bathed in unbuffered saline consisting of 100 m m KCl, pH 5.8. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side. Following channel insertion, the cis chamber was perfused thoroughly with fresh 100 m m KCl, making the system symmetrical, unbuffered KCl, pH 5.8. Residual LF N was then removed by applying a strong 100 mV Δψ to translocate it through the channel; and in some cases, a 1-unit ΔpH was established to aid in channel clearance of residual LF N . Upon stabilization, a series of 50-μl aliquots of 3 m KCl were added to the cis side, and Δψ rev was recorded as the Δψ required to drop the current to zero. All given KCl ratios of the two sides of the bilayer have been corrected for activity in water ( 34 ), and following the experiment, the chambers were weighed to confirm their volume. Ensemble Channel Blocking A planar bilayer was formed with both chambers in 10 m m potassium phosphate, pH 6.6. The cis chamber alone had an additional 100 m m equivalent of KCl. Assembled mutant and WT PA oligomer-LF N prechannel complexes were added to the cis side, and the chamber was perfused following insertion. To remove remaining LF N , 10 μl of 0.4 m phosphoric acid was added to the cis chamber to lower the pH to ∼4.4, and a Δψ of 20 mV was applied. Afterward, the cis chamber was perfused with fresh pH 6.6 buffer, and the Δψ was returned to 0 mV. LF N was added to a given concentration and allowed equilibrate. The percent blockade was determined by the equilibrium drop in current following the addition of LF N . EM Preparations of PA top , PA bot , and a WT PA control were purified by anion exchange chromatography to remove residual PA monomer and excess LF N . Fos-choline-14 was only added to a concentration of 0.05 m m to avoid reaching the critical micelle concentration. All samples were diluted to an estimated final concentration of 70 n m (based on absorbance at 280 nm). Diluted complexes were incubated for 30 s on 400-mesh copper grids (Electron Microscopy Sciences) coated with continuous carbon on nitrocellulose, stained with 1% uranyl formate, and imaged with a Tecnai 12 TEM operated at 120 kV and at ×49,000 magnification. Single particles were selected automatically using boxer (EMAN). The total numbers of particles ( n ) analyzed were: PA WT ( n = 4847), PA top ( n = 4577), and PA bot ( n = 4971). Class averages were determined iteratively using 10 successive cycles of Adapt, an automated classification program (written in house) and two-dimensional multireference alignment in IMAGIC. Molecular Models EF N and LF N domains from EF (PDB 1Y0V ( 26 )) and LF (PDB 1J7N ( 24 )), respectively, were α-carbon-(C α )-aligned in CHIMERA ( 35 ). A three-dimensional model of the 14-stranded β-barrel region of the PA channel (residues 275 to 352) was made by coaxially stacking multiple copies of the heptameric β-barrel from α hemolysin (PDB 7AHL ( 30 )). Peptide bonds were formed and residues were repopulated using COOT ( 36 ). The model was aligned to the z axis in CHIMERA ( 35 ). To obtain an electrostatic energy U ( z ) as a function of the distance moved axially through the barrel z axis, we computed the sum of all pairwise electrostatic energies in a PERL script (zforce.pl, which is available on request), using a 1-unit elementary point charge, q test , moved along the center of the barrel in 0.1-à increments, U ( z ) = q test b Σ q i cos θ i / d i , where d i is the distance between the C α of the i th charged site within the channel of elementary charge, q i , and q test ; θ i is the angle between the charges and the z axis; and b is an electrostatic energy conversion constant of 1390 kJ à mol −1 . RESULTS EF N Translocates Slower Than LF N LF N and EF N share high levels of sequence ( 37 ) and structural homology ( 24 , 26 ); however, the most divergent sequence homology occurs on the amino terminus ( Fig. 2 A ). In planar lipid bilayer electrophysiology experiments, LF N and EF N translocate through the PA channel at remarkably different rates. Although LF N translocates with a t ½ value of ∼10 s at symmetrical pH 5.6 and a Δψ of 60 mV ( 6 , 8 ), His 6 -EF N translocates with a t ½ of ∼140 s under identical conditions ( 5 ). The His 6 tag used in affinity purification tends to have modest effects on the translocation t ½ ( 9 ), and so we re-examined these translocation differences under two different driving force extremes, a pure Δψ and a strong ΔpH, using the constructs in which the His 6 tag was removed by a protease. In our electrophysiological assay ( 6 – 8 ), a planar lipid bilayer separates two aqueous chambers ( cis and trans ). We first insert PA 7 channels into the bilayer. Either WT LF N or EF N was added to the cis side of the membrane (side to which PA 7 was added). Generally, an exponential decrease in current is observed as the amino-terminal presequence of the substrate inserts into the ion-conducting PA channel ( 38 ). A brief perfusion removes excess substrate from the cis chamber, and translocation is initiated by changing the Δψ and/or ΔpH. The subsequent current increase results from substrate translocation to the trans side of the membrane, as determined by control experiments ( 6 , 15 ). Two parameters are obtained from these "single turnover" translocation records: the t ½ and the efficiency of translocation, which is equivalent to the fraction of substrate that successfully translocates. We note that there are multiple LF N or EF N bound to each PA complex so these translocation records likely represent the turnover of several substrates. Therefore, single turnover kinetics refers to a single loaded PA complex that has translocated all of its substrates. We analyzed LF N and EF N translocation under identical conditions. Under a pure Δψ driving force, EF N translocated ∼200-fold slower than LF N ( Fig. 2 B ). Likewise, under a 1-unit ΔpH, EF N translocated ∼10-fold slower than LF N ( Fig. 2 C ). Interestingly, previous studies ( 31 ) and our more recent thermodynamic analysis ( Fig. 2 D and supplemental Table S1 ) show that the equilibrium stability of EF N , Δ G NI , is ∼2.4 kcal mol −1 less stable than LF N ( 31 ). As destabilization should in the most extreme case increase the rate of translocation due to the lowered unfolding barrier ( 8 ), it is unlikely that the weakened solution thermodynamic stability of EF N explains the observed increase in the activation energy of translocation relative to LF N . FIGURE 2. LF N /EF N chimeras are sufficient to mimic LF N -like translocation kinetics. A ( left ), sequence alignment of the first 50 amino acids of LF N and EF N . Residue pairs are shaded as follows: identity ( blue ), similarity ( light blue ), and weak similarity ( gray ). LF N /EF N chimera constructs are shown below where the increasing amounts of amino-terminal sequence from LF N ( blue ) appended to the EF N carboxyl-terminal folded domain ( green ). Right , C α -backbone alignment of EF N (1Y0V, green ) and LF N (1J7N, blue ) computed in CHIMERA ( 35 ). B , representative translocation recordings of LF N ( black ), EF N ( dashed ), and LF 1–50 EF 41–254 ( red ) under a Δψ driving force (at symmetric pH 5.6, Δψ of 50 mV). C, representative translocation records of LF N ( black ), EF N ( dashed ), and LF 1–30 EF 21–254 ( red ) under a 1-unit ΔpH driving force (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Records in panels B and C are normalized to maximal expected fraction translocated. D , representative equilibrium denaturant titrations comparing LF N ( solid ) and EF N ( dashed ) in guanidinium chloride (1 m glucose, pH 7.5, 20 °C) probed by CD at 222 nm and normalized to fraction unfolded ( f U ). Inset , equilibrium stability differences (ΔΔ G NI ) are referenced to WT LF N (where ΔΔ G NI compares EF N and chimeras to LF N ). For other chimeras, see supplemental Table S1 . Error are the mean ± S.D. for n = 3. Amino-terminal Chimeras with LF N Complement Slow EF N Translocation To determine the sequence differences responsible for the relatively slow translocation of EF N , we created a series of chimera constructs ( Fig. 2 A ). In these, we used the bulk of the EF N domain and only replaced the amino-terminal peptide with the corresponding sequence from LF N , where specifically 10, 18, 22, 26, 30, 40, or 50 LF N residues replaced equivalent positions in the EF N construct. (In our scheme, LF 1- a EF b -254 , a and b inclusively delimit the last residue of LF N and starting residue of EF N , respectively.) We found that the LF 1–50 EF 41–254 and LF 1–30 EF 21–254 chimeras represented the minimal chimera constructs ( Fig. 2 , B and C ) of all tested chimeras ( supplemental Fig. S1, A and B ) to exhibit LF N -like translocation under a pure Δψ and a 1-unit ΔpH, respectively. The sequence determinants that define the relatively slow translocation kinetics of EF N are found on its amino terminus. Thus the translocation kinetic stabilization we observe with EF N relative to LF N cannot be attributed to a phenomenon that occurs in solution (in isolation), but rather this difference manifests only in the context of the unfolding machine, the PA channel ( Fig. 2 D ). We then further explored the translocation differences of these chimeras under a variety of driving force conditions. Under pure Δψ-driven translocation at symmetric pH, we found that the more LF N sequence introduced into the chimera, the faster the rate of translocation ( supplemental Fig. S1 A ). Due to the complex nature of these ensemble translocation kinetics, a rate constant for translocation, k , was estimated using the t ½ for translocation, as k ∝ 1/ t ½, and from this we compute the Δ G ‡ . Interestingly, we found the LF 1–10 EF 1–254 chimera had similar to slightly slower translocation rates than EF N across many Δψ values ( supplemental Fig. S1 C ), indicating that these additional 10 residues in LF N are not responsible for the observed differences in translocation. To effectively recapitulate the LF N Δψ-dependence curve, the LF 1–50 EF 41–254 chimera was sufficient. We then examined the set of chimeras under a 1-unit ΔpH gradient ( supplemental Fig. S1 B ). Interestingly, the LF 1–10 EF 1–254 and LF 1–18 EF 9–254 chimeras showed slower translocation than EF N ( supplemental Fig. S1 D ), indicating potentially that these sequences, which have more densely hydrophobic amino termini ( Fig. 2 A ), may impede translocation due to the formation of an unusually tight binding interaction at the φ-clamp site. We found that the LF 1–30 EF 21–254 chimera, however, was sufficient to completely restore LF N -like translocation ( Fig. 2 C and supplemental Fig. S1 D ); and in contrast to purely Δψ driving forces, the sequence determinant for this restoration was concentrated between LF N residues 20 and 30. Two Sequence Cassettes Modulate the Translocation Stability of EF N and LF N A summary of the Δψ- and ΔpH-driven translocation results ( Fig. 3 A ) identified two sequence regions of interest, or "cassettes:" (i) the 20s cassette (residues 19–30); and (ii) the 40s cassette (residues 41–50) ( Fig. 3 B ). (Note that because EF N is 10 residues shorter than LF N on the amino-terminal end, we are applying the LF N -numbering scheme to EF N .) Under symmetric pH conditions and a Δψ driving force, there is a ∼1.3 kcal mol −1 difference in Δ G ‡ between LF 1–18 EF 9–254 and LF 1–26 EF 17–254 in the 20s cassette ( Fig. 3 A ). Under a 1-unit ΔpH gradient, there is a ∼2 kcal mol −1 difference between the same chimeras ( Fig. 3 A ). Also notable is the ∼1.5 kcal mol −1 Δ G ‡ difference between the LF 1–40 EF 31–254 and LF 1–50 EF 41–254 chimeras ( Fig. 3 A ); however, this difference was only observed under a Δψ driving force. Therefore, we hypothesize that sequence divergences in the 20s and 40s cassettes are responsible for the slow translocation kinetics of EF N . FIGURE 3. Charged residues in the 20s and 40s cassettes utilize the Δψ and ΔpH driving forces to promote unfolding and translocation. A ( left ), translocation activation energy for chimeric constructs at symmetric pH 5.6 and Δψ of 50 mV. One value was estimated by extrapolation (*) based on a larger Δψ-dependent dataset ( supplemental Fig. S1 C ) and associated fit parameters ( supplemental Table S2 ). Right, translocation Δ G ‡ for LF N , EF N , and the indicated LF N /EF N chimeras under a 1-unit ΔpH (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Brackets indicate significant differences (or "steps") in Δ G ‡ due to inclusion of the intervening LF N sequence cassette ( cass .) Additional Δψ-dependent data at a 1-unit ΔpH are given in supplemental Fig. S1 D , where associated fit parameters are given in supplemental Table S3 . B ( above ), amino-terminal 20s ( green ) and 40s ( orange ) cassette peptides are highlighted and the residue sequences in LF N and EF N are shown. Below , top / outside and inside / sagittal plane vantages of a molecular model of LF N ( blue ) in complex with the PA 8 oligomer ( gray ) (PDB 3KWV ( 4 )). Mutations in these two sequence cassettes may have destabilized the chimera and altered the unfolding step of the translocation mechanism. To test this possibility, we measured Δ G NI of the base and most highly internally mutagenized chimera constructs using standard solution unfolding procedures ( 8 , 31 ). We generally found no significant differences in Δ G NI between these chimeras and EF N ( Fig. 2 D and supplemental Table S1 ). As the bulk of the folded domain is from EF N , this result was expected. The residues differing between the chimeras are contained in the amino-terminal unstructured region and first α helix and β strand, which are highly solvent accessible. Thus we ruled out protein destabilization for these chimeras, and the amino-terminal sequence divergence in EF N likely affects the mechanisms of PA channel-dependent unfolding and translocation. Charge Content of Cassettes Controls Driving Force Dependence of Translocation To identify sequence features in the two cassettes contributing to the observed translocation Δ G ‡ differences, we introduced several point mutations within the existing chimera constructs ( Fig. 4 , A and B ). These mutations were made given the variation in net charge ( z ) observed within the cassettes. Net charge was estimated by z = n basic − n acidic , where n basic and n acidic are the number of basic and acidic residues, respectively. For the 20s cassette, we found that EF N and LF N had fairly different z values of +5 and 0, respectively. Likewise, for the 40s cassette, EF N and LF N had z values of 0 and +3, respectively. Upon our examination of their translocation kinetics, we found that correlations emerged between z values within the cassettes and their translocation Δ G ‡ values ( Fig. 4 , C and D ). Thus as expected, the subtraction of positive charge in the 20s cassette and addition of positive charge in the 40s cassette tended to generally increase the rate of translocation for EF N -based chimeras. FIGURE 4. Charged cassettes are nonspecific. A , construct design for chimeras and derivative mutants in the 20s cassette (residues 19–30) are arranged from the most positive to the most negative. Net charge given to the right of each sequence is computed using the following scoring system: D , E = −1; H , K , r = +1. Residues from native LF N ( blue ) and native EF N ( black ) are shown alongside non-native mutations ( boxed ) to either LF N or EF N . Residue-numbering scheme is according to LF N ( 24 ). B, constructs altering the 40s cassette (residues 41–50). Net charge is computed as in panel A. C ( top ), Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 20s cassette (residues 19–30 inclusive). Two-barrier model fit ( Equation 1 ): Δ G ‡ ° 1 = 3.2 (±0.5), δ 1 = −0.3 (±0.2), Δ G ‡ ° 2 = 3.2 (±0.5), and δ 2 = 0.3 (±0.1) ( n = 21, p < 0.001). Bottom, Δ G ‡ versus z at a Δψ of 20 mV, 1-unit ΔpH (5.6 pH cis , 6.6 pH trans ) for the same 20s-cassette variants. Two-barrier fit parameters: Δ G ‡ ° 1 = 0.3 (±0.5), δ 1 = −0.7 (±0.4), Δ G ‡ ° 2 = 0.9 (±0.3), and δ 2 = 1 (±0.2) ( n = 21, p < 0.001). D, Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 40s-cassette region (residues 41–50 inclusive). Single-barrier model ( Equation 2 ) fit parameters: Δ G ‡ ° = 2.7 (±0.1) and δ = −0.58 (±0.07) ( n = 8, p < 0.001). Error bars are the mean ± S.D. ( n ≥ 3). We also examined the residue identity and position dependence of these effects. When we separately introduced an Asp at positions 23 and 28 of LF 1–22 EF 13–254 (LF 1–22 EF 13–254 N23D, z = +1; LF 1–22 EF 13–254 K28D, z = 0) ( Fig. 4 A ), the rate of translocation increased relative to the parent construct ( z = +2) ( Fig. 4 C ). Furthermore, both LF 1–22 EF 13–254 K25D and LF 1–22 EF 13–254 K25E ( z = 0) increased the translocation rate similarly, indicating that there is a general requirement for negative charge, but residue identity is not critical. In general when examining all the data, translocation rates were only affected by changes in z values and not by changes in the position of the charges ( Fig. 4 C ). The rate of translocation is similar for the LF 1–22 EF 13–254 K25D/T26E and LF 1–22 EF 13–254 H24D/K25N chimeras ( z = −1). Finally, the negative charge neutralizing mutation LF 1–25 EF 16–254 D25N ( z = +2) showed slowed translocation compared with its parent construct LF 1–25 EF 16–254 ( z = +1). A similar but opposite effect can be seen in the 40s cassette, where there is a general requirement for positive charges independent of the specific positions ( Fig. 4 D ). For example, LF 1–40 EF 31–254 N41E and LF 1–40 EF 31–254 T49E ( z = −1) had similarly decreased translocation rates relative to their parent chimera ( z = 0). Thus we conclude that the 20s and 40s cassettes indeed have particular anionic and cationic charge requirements, respectively, but these requirements are highly nonspecific in terms of both position and residue identity. Although most of the charge-dependent Δ G ‡ data for the 20s cassette is linear with respect to charge, the presence of outlier data at higher negative charge density led to the hypothesis that there may be two barriers in the charge-dependent transport mechanism. Increasing negative charge can lower one barrier; however, the second barrier is either charge insensitive or somewhat inversely dependent on negative charge. To allow for partial-charge character (δ) during each respective barrier crossing ( 39 ), we used the following model, where F is Faraday's constant. For the ΔpH-dependent data ( n = 21), the fit to Equation 1 was significant ( p < 0.001) ( Fig. 4 C ). The δ parameter was obtained for each barrier as δ 1 = −0.7 (±0.4) and δ 2 = 1.0 (±0.2). The corresponding activation energies, ΔG ‡ ° 1 and ΔG ‡ ° 2 , in the absence of net charge were 0.3 (±0.5) and 0.9 (±0.3), respectively. For the Δψ-dependent translocation ( n = 21), the fit was also significant ( p < 0.001) with δ 1 = −0.3 (±0.2) and δ 2 = 0.3 (±0.1) and ΔG ‡ ° 1 = 3.2 (±0.5) and ΔG ‡ ° 2 = 3.2 (±0.5) ( Fig. 4 C ). Typically, δ values are challenging to interpret: residues may be partially charged due to p K a shifts; metal ions may bind to the translocating peptide and alter net charge; and finally, only part of the charged region in the substrate may be required to cross the rate-limiting barrier. Nevertheless, the goodness of fit suggests that indeed two unique charge-dependent barriers with inverse charge requirements are present in the translocation mechanism. In the 40s cassette region ( Fig. 4 B ), LF N possesses additional positive charge comparative to EF N at positions 41, 42, and 49. We created several point mutations in the existing chimeras to determine the effects of increasing or decreasing charge of the 40s cassette and investigated the charge-based differences in this region via translocation assays ( Fig. 4 D ). Starting with a sequence similar to EF N and increasing its positive charge to that of LF N , we again observe a direct relationship between charge and the translocation Δ G ‡ , where increasing positive charge leads to faster translocation. Also the charge dependence was again nonspecific ( Fig. 4 D ) where the position and identity of the residues did not appear to matter as much as the overall z value ( Fig. 4 B ). These charge-dependent data ( n = 8) for the 40s cassette were best fit by a single-barrier model ( 39 ). The fit was significant ( p < 0.001) with a δ of −0.58 (±0.07) and ΔG ‡ ° of 2.7 (±0.1) ( Fig. 4 D ). The type of cationic-charge preference in the 40s cassette is classical in the sense that it coincides with the direction of the electric field created by the applied membrane potential ( i.e. the field is cis -positive). Electrostatic Analysis of the PA β Barrel Given the unusual preference for anionic residues in the 20s cassette when driven by a Δψ (which is exactly opposite of the result expected for a cis -positive membrane potential), we hypothesized that the local electrostatic field produced by features within the channel, E chan , may override the electrical potential applied across the membrane, E m . The overall electric field, E , is a vector, where E = E chan + E m . The force applied upon the translocating chain is related to the sign and magnitude of the charge, q , of groups in the translocating chain and E by E × q . Because the electrical field contributed by the membrane potential relates to Δψ as E m = Δψ/ d , where d is the distance over which the potential drops, we can assume that the membrane potential will contribute unproductively to a negatively charged substrate if the membrane potential is positive in polarity. Therefore, E chan likely provides an oppositely oriented electrical field component that can apply a productive force on the substrate that aligns with the productive direction of translocation. Our hypothesis is also supported by the fact that the anion-charge preference in the 20s cassette appears independent of the makeup of the driving force; both Δψ-driven and ΔpH-driven kinetics can be accelerated by including additional negative charge in the 20s cassette of EF N . To characterize the electrostatic features within the PA channel, we initially built a model of the β barrel portion of the PA channel using the coordinates of α hemolysin ( 30 ) ( Fig. 5 A ). From this β barrel model, we calculated the sum of all pairwise electrostatic potentials for a point charge translocated along the central axis of the channel ("Experimental Procedures"). Our analysis revealed two prominent and oppositely charged electrostatic features, which were juxtaposed in the β barrel. One is a strongly anion-repulsive feature (PA residue ranges 275–283 and 343–352, generally localized to the top of the β barrel), and the other is a strongly cation-repulsive feature (PA residue ranges 287–299 and 328–340, generally localized to the middle of the β barrel) ( Fig. 5 A ). The PA residues contributing to these two features were located both inside and outside of the β barrel. Based on the same analytical model, we produced two β barrel mutants, one that would disrupt the anionic feature and one that would not. PA top disrupted the upper, cis -most portion of the β barrel, targeting its negatively charged residues by substituting them with isosteric Ser residues (D276S, D335S, and E343S). We chose Ser or Thr substitutions because the inside of the channel is hydrophilic and composed mostly of Ser and Thr residues ( 31 ). PA bot disrupted the lower trans -most portion of the β barrel and channel via the similar isosteric Thr substitutions (E302T, H304T, E308T, and H310T). The modeled electrostatic effects of these two mutant PA β barrels are shown in Fig. 5 A . FIGURE 5. Charge-selectivity filter in PA β barrel is required for efficient translocation. A ( left ), molecular model of the PA channel β barrel ( gray ), where acidic ( red ) and basic ( blue ) residues are highlighted. The outside and a sagittal section of the inside of the β-barrel structure are depicted. Right, the electrostatic energy for a negative point charge moved down the central axis of the β barrel of the channel. The origin on the distance axis is at the cis -most end of the β barrel, and increasing positive values indicates productive translocation. The potential was computed as described under "Experimental Procedures." B, relative differences in ion selectivity for WT PA ( black squares ), PA top ( red triangles ), and PA bot ( blue circles ) determined by −Δψ rev versus the KCl activity ratio ( cis : trans ). The x axis is plotted as a natural log scale marked by factors of e . The ideal cation-selective Nernstian relationship ( e -fold activity ratio per 25.2 mV at 20 °C) is indicated with a solid line . Three independent measurements assessed on three different membranes were corrected for membrane and electronics offsets. C , representative protein translocation records for WT LF N under ΔpH ( left ) and Δψ ( right ) using WT PA ( black ), PA top ( red ), and PA bot ( blue ). The Δψ and ΔpH conditions are identical to those applied in Fig. 2 , B and C . Results shown are consistent with replicates obtained on at least two separate membranes. D , ensemble bilayer recordings of WT PA ( black ), PA top ( red ), and PA bot ( blue ) channel conductance block by WT LF N at 1, 5, 25, and 1200 n m were obtained at symmetrical pH 6.6 and no Δψ. Error bars are the mean ± S.D. ( n = 2). WT and PA top were tested for significance using an unpaired t test ( p < 0.0001) for all observations ( n = 16) at each set of conditions. The Ion Selectivity Filter of the PA Channel Is Critical for Δψ- and ΔpH-driven Translocation To characterize PA top and PA bot , however, we first needed to properly assemble the monomeric PA into oligomers. The multisite mutations would not assemble using the traditional ion-exchange approach ( 27 ). Hence we developed a modified assembly procedure. We nicked the PA monomers at pH 9 with furin instead of trypsin (to avoid nonspecific tryptic degradation), co-assembled the PA at pH 9 by adding LF N ( 5 , 23 ), and finally added Fos-choline-14 detergent to convert the prechannel oligomers into stable, detergent-solubilized channels ( 32 ). As a control, we also assembled WT PA by the same procedure. Native and SDS-PAGE ( supplemental Fig. S2 A ) and negative-stain EM ( supplemental Fig. S2B ) verified the proper assembly of these samples. To monitor channel formation by planar bilayer electrophysiology, we had to remove the LF N in situ by perfusing the cis chamber and translocating the residual LF N through the channels. We found that Fos-choline-14 favorably weakened the interaction of LF N with the channel, making its removal rapid and complete. In conclusion, the three preparations had reasonable insertion activities, albeit WT PA was most optimal. To determine whether these mutations change the ion selectivity of the PA channel, we first measured Δψ rev for WT PA, PA top , and PA bot . (Δψ rev is the voltage required to reduce the ionic current to zero under asymmetrical KCl gradients.) Each of these complexes was applied to planar bilayer membranes to form stable populations of channels following the removal of excess LF N by perfusion and translocation. The removal of residual LF N was judged to be complete by the stabilization of the current. Over a range of tested KCl gradients (in unbuffered saline, pH 5.8), WT PA and PA bot possessed similar Δψ rev values and, therefore, possessed similar ion selectivity ( Fig. 5 B ). However, PA top showed a reduced magnitude of Δψ rev relative to WT PA ( Fig. 5 B ). Thus PA top disrupts a portion of the ion-selectivity filter of the channel, presumably by reducing its anionic charge character ( Fig. 5 A ). PA top and PA bot were then assayed for their ability to translocate LF N under either a Δψ or a ΔpH. We found strong translocation deficiencies for PA top with either type of driving force ( Fig. 5 C ). Under a 1-unit ΔpH (pH cis 5.6 to pH trans 6.6) with Δψ of 20 mV, translocation of LF N through PA top is slowed more than 10-fold compared with WT PA, whereas PA bot is unaffected ( Fig. 5 C, left ). With a 50 mV Δψ at symmetrical pH 5.6, PA top was also less able to translocate LF N relative to WT PA ( Fig. 5 C, right ). Under these conditions, the rate and efficiency of translocation were affected. Although WT PA and PA bot are fully translocated within 2 min, PA top achieved less than 20% efficiency after 10 min. Thus PA top reveals significant translocation deficiencies under either a Δψ or ΔpH driving force. Finally, LF N was assayed for its ability to block PA top and PA bot channels. In this experiment, we added 5 n m LF N to the channels bathed in an asymmetrical KCl gradient at symmetrical pH 6.6 and a Δψ of 0 mV. Under these conditions, we found 99.0% (±0.1) of WT PA channel current was blocked ( Fig. 5 D ). For PA bot , we observed 98.0% (±0.1) conductance blockade; however, for PA top , 88% (±1) of the conductance was blocked by LF N . The binding defect observed with PA top may indicate that the charge disruption in that region affects the ability of the amino terminus of LF N to properly dock inside the pore and block conductance. In this model ( Equation 3 ), we expect two different stages of binding. In stage one, LF N binds to the top surface of the channel, forming the (PA·LF N ) complex; and in stage 2, the amino terminus docks into the channel to block conductance, forming the (PA·LF N )* complex. To test whether stage 1 or stage 2 were affected by the PA top mutation, we determined the percent blockade as a function of LF N concentration. Although the concentration of LF N should affect the equilibrium of stage 1, the equilibrium describing stage 2 is, of course, concentration-independent. To test for these two possibilities, we altered the LF N concentration. Reducing the concentration to 1 n m resulted in small changes in channel blockade (PA WT, 98.4% (±0.1); PA bot , 97.0% (±0.3); PA top , 86% (±2)). However, increasing the concentration 5-fold to 25 n m did not appreciably change the blockade (PA WT, 99.3% (±0.0); PA bot , 98.7% (±0.2); PA top , 88% (±1)), indicating that the system is at saturating levels of LF N . Indeed, even increasing the concentration to 1.2 μ m did not appreciably affect the percent block ( Fig. 5 D ). The inability of LF N to fully saturate channel conductance blockade in the PA top mutant over a 1000-fold concentration range demonstrates that channel docking (stage two) is impaired, and the PA top mutation likely disrupts a latching or ratcheting feature within the PA channel. EF N Translocates Slower Than LF N LF N and EF N share high levels of sequence ( 37 ) and structural homology ( 24 , 26 ); however, the most divergent sequence homology occurs on the amino terminus ( Fig. 2 A ). In planar lipid bilayer electrophysiology experiments, LF N and EF N translocate through the PA channel at remarkably different rates. Although LF N translocates with a t ½ value of ∼10 s at symmetrical pH 5.6 and a Δψ of 60 mV ( 6 , 8 ), His 6 -EF N translocates with a t ½ of ∼140 s under identical conditions ( 5 ). The His 6 tag used in affinity purification tends to have modest effects on the translocation t ½ ( 9 ), and so we re-examined these translocation differences under two different driving force extremes, a pure Δψ and a strong ΔpH, using the constructs in which the His 6 tag was removed by a protease. In our electrophysiological assay ( 6 – 8 ), a planar lipid bilayer separates two aqueous chambers ( cis and trans ). We first insert PA 7 channels into the bilayer. Either WT LF N or EF N was added to the cis side of the membrane (side to which PA 7 was added). Generally, an exponential decrease in current is observed as the amino-terminal presequence of the substrate inserts into the ion-conducting PA channel ( 38 ). A brief perfusion removes excess substrate from the cis chamber, and translocation is initiated by changing the Δψ and/or ΔpH. The subsequent current increase results from substrate translocation to the trans side of the membrane, as determined by control experiments ( 6 , 15 ). Two parameters are obtained from these "single turnover" translocation records: the t ½ and the efficiency of translocation, which is equivalent to the fraction of substrate that successfully translocates. We note that there are multiple LF N or EF N bound to each PA complex so these translocation records likely represent the turnover of several substrates. Therefore, single turnover kinetics refers to a single loaded PA complex that has translocated all of its substrates. We analyzed LF N and EF N translocation under identical conditions. Under a pure Δψ driving force, EF N translocated ∼200-fold slower than LF N ( Fig. 2 B ). Likewise, under a 1-unit ΔpH, EF N translocated ∼10-fold slower than LF N ( Fig. 2 C ). Interestingly, previous studies ( 31 ) and our more recent thermodynamic analysis ( Fig. 2 D and supplemental Table S1 ) show that the equilibrium stability of EF N , Δ G NI , is ∼2.4 kcal mol −1 less stable than LF N ( 31 ). As destabilization should in the most extreme case increase the rate of translocation due to the lowered unfolding barrier ( 8 ), it is unlikely that the weakened solution thermodynamic stability of EF N explains the observed increase in the activation energy of translocation relative to LF N . FIGURE 2. LF N /EF N chimeras are sufficient to mimic LF N -like translocation kinetics. A ( left ), sequence alignment of the first 50 amino acids of LF N and EF N . Residue pairs are shaded as follows: identity ( blue ), similarity ( light blue ), and weak similarity ( gray ). LF N /EF N chimera constructs are shown below where the increasing amounts of amino-terminal sequence from LF N ( blue ) appended to the EF N carboxyl-terminal folded domain ( green ). Right , C α -backbone alignment of EF N (1Y0V, green ) and LF N (1J7N, blue ) computed in CHIMERA ( 35 ). B , representative translocation recordings of LF N ( black ), EF N ( dashed ), and LF 1–50 EF 41–254 ( red ) under a Δψ driving force (at symmetric pH 5.6, Δψ of 50 mV). C, representative translocation records of LF N ( black ), EF N ( dashed ), and LF 1–30 EF 21–254 ( red ) under a 1-unit ΔpH driving force (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Records in panels B and C are normalized to maximal expected fraction translocated. D , representative equilibrium denaturant titrations comparing LF N ( solid ) and EF N ( dashed ) in guanidinium chloride (1 m glucose, pH 7.5, 20 °C) probed by CD at 222 nm and normalized to fraction unfolded ( f U ). Inset , equilibrium stability differences (ΔΔ G NI ) are referenced to WT LF N (where ΔΔ G NI compares EF N and chimeras to LF N ). For other chimeras, see supplemental Table S1 . Error are the mean ± S.D. for n = 3. Amino-terminal Chimeras with LF N Complement Slow EF N Translocation To determine the sequence differences responsible for the relatively slow translocation of EF N , we created a series of chimera constructs ( Fig. 2 A ). In these, we used the bulk of the EF N domain and only replaced the amino-terminal peptide with the corresponding sequence from LF N , where specifically 10, 18, 22, 26, 30, 40, or 50 LF N residues replaced equivalent positions in the EF N construct. (In our scheme, LF 1- a EF b -254 , a and b inclusively delimit the last residue of LF N and starting residue of EF N , respectively.) We found that the LF 1–50 EF 41–254 and LF 1–30 EF 21–254 chimeras represented the minimal chimera constructs ( Fig. 2 , B and C ) of all tested chimeras ( supplemental Fig. S1, A and B ) to exhibit LF N -like translocation under a pure Δψ and a 1-unit ΔpH, respectively. The sequence determinants that define the relatively slow translocation kinetics of EF N are found on its amino terminus. Thus the translocation kinetic stabilization we observe with EF N relative to LF N cannot be attributed to a phenomenon that occurs in solution (in isolation), but rather this difference manifests only in the context of the unfolding machine, the PA channel ( Fig. 2 D ). We then further explored the translocation differences of these chimeras under a variety of driving force conditions. Under pure Δψ-driven translocation at symmetric pH, we found that the more LF N sequence introduced into the chimera, the faster the rate of translocation ( supplemental Fig. S1 A ). Due to the complex nature of these ensemble translocation kinetics, a rate constant for translocation, k , was estimated using the t ½ for translocation, as k ∝ 1/ t ½, and from this we compute the Δ G ‡ . Interestingly, we found the LF 1–10 EF 1–254 chimera had similar to slightly slower translocation rates than EF N across many Δψ values ( supplemental Fig. S1 C ), indicating that these additional 10 residues in LF N are not responsible for the observed differences in translocation. To effectively recapitulate the LF N Δψ-dependence curve, the LF 1–50 EF 41–254 chimera was sufficient. We then examined the set of chimeras under a 1-unit ΔpH gradient ( supplemental Fig. S1 B ). Interestingly, the LF 1–10 EF 1–254 and LF 1–18 EF 9–254 chimeras showed slower translocation than EF N ( supplemental Fig. S1 D ), indicating potentially that these sequences, which have more densely hydrophobic amino termini ( Fig. 2 A ), may impede translocation due to the formation of an unusually tight binding interaction at the φ-clamp site. We found that the LF 1–30 EF 21–254 chimera, however, was sufficient to completely restore LF N -like translocation ( Fig. 2 C and supplemental Fig. S1 D ); and in contrast to purely Δψ driving forces, the sequence determinant for this restoration was concentrated between LF N residues 20 and 30. Two Sequence Cassettes Modulate the Translocation Stability of EF N and LF N A summary of the Δψ- and ΔpH-driven translocation results ( Fig. 3 A ) identified two sequence regions of interest, or "cassettes:" (i) the 20s cassette (residues 19–30); and (ii) the 40s cassette (residues 41–50) ( Fig. 3 B ). (Note that because EF N is 10 residues shorter than LF N on the amino-terminal end, we are applying the LF N -numbering scheme to EF N .) Under symmetric pH conditions and a Δψ driving force, there is a ∼1.3 kcal mol −1 difference in Δ G ‡ between LF 1–18 EF 9–254 and LF 1–26 EF 17–254 in the 20s cassette ( Fig. 3 A ). Under a 1-unit ΔpH gradient, there is a ∼2 kcal mol −1 difference between the same chimeras ( Fig. 3 A ). Also notable is the ∼1.5 kcal mol −1 Δ G ‡ difference between the LF 1–40 EF 31–254 and LF 1–50 EF 41–254 chimeras ( Fig. 3 A ); however, this difference was only observed under a Δψ driving force. Therefore, we hypothesize that sequence divergences in the 20s and 40s cassettes are responsible for the slow translocation kinetics of EF N . FIGURE 3. Charged residues in the 20s and 40s cassettes utilize the Δψ and ΔpH driving forces to promote unfolding and translocation. A ( left ), translocation activation energy for chimeric constructs at symmetric pH 5.6 and Δψ of 50 mV. One value was estimated by extrapolation (*) based on a larger Δψ-dependent dataset ( supplemental Fig. S1 C ) and associated fit parameters ( supplemental Table S2 ). Right, translocation Δ G ‡ for LF N , EF N , and the indicated LF N /EF N chimeras under a 1-unit ΔpH (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Brackets indicate significant differences (or "steps") in Δ G ‡ due to inclusion of the intervening LF N sequence cassette ( cass .) Additional Δψ-dependent data at a 1-unit ΔpH are given in supplemental Fig. S1 D , where associated fit parameters are given in supplemental Table S3 . B ( above ), amino-terminal 20s ( green ) and 40s ( orange ) cassette peptides are highlighted and the residue sequences in LF N and EF N are shown. Below , top / outside and inside / sagittal plane vantages of a molecular model of LF N ( blue ) in complex with the PA 8 oligomer ( gray ) (PDB 3KWV ( 4 )). Mutations in these two sequence cassettes may have destabilized the chimera and altered the unfolding step of the translocation mechanism. To test this possibility, we measured Δ G NI of the base and most highly internally mutagenized chimera constructs using standard solution unfolding procedures ( 8 , 31 ). We generally found no significant differences in Δ G NI between these chimeras and EF N ( Fig. 2 D and supplemental Table S1 ). As the bulk of the folded domain is from EF N , this result was expected. The residues differing between the chimeras are contained in the amino-terminal unstructured region and first α helix and β strand, which are highly solvent accessible. Thus we ruled out protein destabilization for these chimeras, and the amino-terminal sequence divergence in EF N likely affects the mechanisms of PA channel-dependent unfolding and translocation. Charge Content of Cassettes Controls Driving Force Dependence of Translocation To identify sequence features in the two cassettes contributing to the observed translocation Δ G ‡ differences, we introduced several point mutations within the existing chimera constructs ( Fig. 4 , A and B ). These mutations were made given the variation in net charge ( z ) observed within the cassettes. Net charge was estimated by z = n basic − n acidic , where n basic and n acidic are the number of basic and acidic residues, respectively. For the 20s cassette, we found that EF N and LF N had fairly different z values of +5 and 0, respectively. Likewise, for the 40s cassette, EF N and LF N had z values of 0 and +3, respectively. Upon our examination of their translocation kinetics, we found that correlations emerged between z values within the cassettes and their translocation Δ G ‡ values ( Fig. 4 , C and D ). Thus as expected, the subtraction of positive charge in the 20s cassette and addition of positive charge in the 40s cassette tended to generally increase the rate of translocation for EF N -based chimeras. FIGURE 4. Charged cassettes are nonspecific. A , construct design for chimeras and derivative mutants in the 20s cassette (residues 19–30) are arranged from the most positive to the most negative. Net charge given to the right of each sequence is computed using the following scoring system: D , E = −1; H , K , r = +1. Residues from native LF N ( blue ) and native EF N ( black ) are shown alongside non-native mutations ( boxed ) to either LF N or EF N . Residue-numbering scheme is according to LF N ( 24 ). B, constructs altering the 40s cassette (residues 41–50). Net charge is computed as in panel A. C ( top ), Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 20s cassette (residues 19–30 inclusive). Two-barrier model fit ( Equation 1 ): Δ G ‡ ° 1 = 3.2 (±0.5), δ 1 = −0.3 (±0.2), Δ G ‡ ° 2 = 3.2 (±0.5), and δ 2 = 0.3 (±0.1) ( n = 21, p < 0.001). Bottom, Δ G ‡ versus z at a Δψ of 20 mV, 1-unit ΔpH (5.6 pH cis , 6.6 pH trans ) for the same 20s-cassette variants. Two-barrier fit parameters: Δ G ‡ ° 1 = 0.3 (±0.5), δ 1 = −0.7 (±0.4), Δ G ‡ ° 2 = 0.9 (±0.3), and δ 2 = 1 (±0.2) ( n = 21, p < 0.001). D, Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 40s-cassette region (residues 41–50 inclusive). Single-barrier model ( Equation 2 ) fit parameters: Δ G ‡ ° = 2.7 (±0.1) and δ = −0.58 (±0.07) ( n = 8, p < 0.001). Error bars are the mean ± S.D. ( n ≥ 3). We also examined the residue identity and position dependence of these effects. When we separately introduced an Asp at positions 23 and 28 of LF 1–22 EF 13–254 (LF 1–22 EF 13–254 N23D, z = +1; LF 1–22 EF 13–254 K28D, z = 0) ( Fig. 4 A ), the rate of translocation increased relative to the parent construct ( z = +2) ( Fig. 4 C ). Furthermore, both LF 1–22 EF 13–254 K25D and LF 1–22 EF 13–254 K25E ( z = 0) increased the translocation rate similarly, indicating that there is a general requirement for negative charge, but residue identity is not critical. In general when examining all the data, translocation rates were only affected by changes in z values and not by changes in the position of the charges ( Fig. 4 C ). The rate of translocation is similar for the LF 1–22 EF 13–254 K25D/T26E and LF 1–22 EF 13–254 H24D/K25N chimeras ( z = −1). Finally, the negative charge neutralizing mutation LF 1–25 EF 16–254 D25N ( z = +2) showed slowed translocation compared with its parent construct LF 1–25 EF 16–254 ( z = +1). A similar but opposite effect can be seen in the 40s cassette, where there is a general requirement for positive charges independent of the specific positions ( Fig. 4 D ). For example, LF 1–40 EF 31–254 N41E and LF 1–40 EF 31–254 T49E ( z = −1) had similarly decreased translocation rates relative to their parent chimera ( z = 0). Thus we conclude that the 20s and 40s cassettes indeed have particular anionic and cationic charge requirements, respectively, but these requirements are highly nonspecific in terms of both position and residue identity. Although most of the charge-dependent Δ G ‡ data for the 20s cassette is linear with respect to charge, the presence of outlier data at higher negative charge density led to the hypothesis that there may be two barriers in the charge-dependent transport mechanism. Increasing negative charge can lower one barrier; however, the second barrier is either charge insensitive or somewhat inversely dependent on negative charge. To allow for partial-charge character (δ) during each respective barrier crossing ( 39 ), we used the following model, where F is Faraday's constant. For the ΔpH-dependent data ( n = 21), the fit to Equation 1 was significant ( p < 0.001) ( Fig. 4 C ). The δ parameter was obtained for each barrier as δ 1 = −0.7 (±0.4) and δ 2 = 1.0 (±0.2). The corresponding activation energies, ΔG ‡ ° 1 and ΔG ‡ ° 2 , in the absence of net charge were 0.3 (±0.5) and 0.9 (±0.3), respectively. For the Δψ-dependent translocation ( n = 21), the fit was also significant ( p < 0.001) with δ 1 = −0.3 (±0.2) and δ 2 = 0.3 (±0.1) and ΔG ‡ ° 1 = 3.2 (±0.5) and ΔG ‡ ° 2 = 3.2 (±0.5) ( Fig. 4 C ). Typically, δ values are challenging to interpret: residues may be partially charged due to p K a shifts; metal ions may bind to the translocating peptide and alter net charge; and finally, only part of the charged region in the substrate may be required to cross the rate-limiting barrier. Nevertheless, the goodness of fit suggests that indeed two unique charge-dependent barriers with inverse charge requirements are present in the translocation mechanism. In the 40s cassette region ( Fig. 4 B ), LF N possesses additional positive charge comparative to EF N at positions 41, 42, and 49. We created several point mutations in the existing chimeras to determine the effects of increasing or decreasing charge of the 40s cassette and investigated the charge-based differences in this region via translocation assays ( Fig. 4 D ). Starting with a sequence similar to EF N and increasing its positive charge to that of LF N , we again observe a direct relationship between charge and the translocation Δ G ‡ , where increasing positive charge leads to faster translocation. Also the charge dependence was again nonspecific ( Fig. 4 D ) where the position and identity of the residues did not appear to matter as much as the overall z value ( Fig. 4 B ). These charge-dependent data ( n = 8) for the 40s cassette were best fit by a single-barrier model ( 39 ). The fit was significant ( p < 0.001) with a δ of −0.58 (±0.07) and ΔG ‡ ° of 2.7 (±0.1) ( Fig. 4 D ). The type of cationic-charge preference in the 40s cassette is classical in the sense that it coincides with the direction of the electric field created by the applied membrane potential ( i.e. the field is cis -positive). Electrostatic Analysis of the PA β Barrel Given the unusual preference for anionic residues in the 20s cassette when driven by a Δψ (which is exactly opposite of the result expected for a cis -positive membrane potential), we hypothesized that the local electrostatic field produced by features within the channel, E chan , may override the electrical potential applied across the membrane, E m . The overall electric field, E , is a vector, where E = E chan + E m . The force applied upon the translocating chain is related to the sign and magnitude of the charge, q , of groups in the translocating chain and E by E × q . Because the electrical field contributed by the membrane potential relates to Δψ as E m = Δψ/ d , where d is the distance over which the potential drops, we can assume that the membrane potential will contribute unproductively to a negatively charged substrate if the membrane potential is positive in polarity. Therefore, E chan likely provides an oppositely oriented electrical field component that can apply a productive force on the substrate that aligns with the productive direction of translocation. Our hypothesis is also supported by the fact that the anion-charge preference in the 20s cassette appears independent of the makeup of the driving force; both Δψ-driven and ΔpH-driven kinetics can be accelerated by including additional negative charge in the 20s cassette of EF N . To characterize the electrostatic features within the PA channel, we initially built a model of the β barrel portion of the PA channel using the coordinates of α hemolysin ( 30 ) ( Fig. 5 A ). From this β barrel model, we calculated the sum of all pairwise electrostatic potentials for a point charge translocated along the central axis of the channel ("Experimental Procedures"). Our analysis revealed two prominent and oppositely charged electrostatic features, which were juxtaposed in the β barrel. One is a strongly anion-repulsive feature (PA residue ranges 275–283 and 343–352, generally localized to the top of the β barrel), and the other is a strongly cation-repulsive feature (PA residue ranges 287–299 and 328–340, generally localized to the middle of the β barrel) ( Fig. 5 A ). The PA residues contributing to these two features were located both inside and outside of the β barrel. Based on the same analytical model, we produced two β barrel mutants, one that would disrupt the anionic feature and one that would not. PA top disrupted the upper, cis -most portion of the β barrel, targeting its negatively charged residues by substituting them with isosteric Ser residues (D276S, D335S, and E343S). We chose Ser or Thr substitutions because the inside of the channel is hydrophilic and composed mostly of Ser and Thr residues ( 31 ). PA bot disrupted the lower trans -most portion of the β barrel and channel via the similar isosteric Thr substitutions (E302T, H304T, E308T, and H310T). The modeled electrostatic effects of these two mutant PA β barrels are shown in Fig. 5 A . FIGURE 5. Charge-selectivity filter in PA β barrel is required for efficient translocation. A ( left ), molecular model of the PA channel β barrel ( gray ), where acidic ( red ) and basic ( blue ) residues are highlighted. The outside and a sagittal section of the inside of the β-barrel structure are depicted. Right, the electrostatic energy for a negative point charge moved down the central axis of the β barrel of the channel. The origin on the distance axis is at the cis -most end of the β barrel, and increasing positive values indicates productive translocation. The potential was computed as described under "Experimental Procedures." B, relative differences in ion selectivity for WT PA ( black squares ), PA top ( red triangles ), and PA bot ( blue circles ) determined by −Δψ rev versus the KCl activity ratio ( cis : trans ). The x axis is plotted as a natural log scale marked by factors of e . The ideal cation-selective Nernstian relationship ( e -fold activity ratio per 25.2 mV at 20 °C) is indicated with a solid line . Three independent measurements assessed on three different membranes were corrected for membrane and electronics offsets. C , representative protein translocation records for WT LF N under ΔpH ( left ) and Δψ ( right ) using WT PA ( black ), PA top ( red ), and PA bot ( blue ). The Δψ and ΔpH conditions are identical to those applied in Fig. 2 , B and C . Results shown are consistent with replicates obtained on at least two separate membranes. D , ensemble bilayer recordings of WT PA ( black ), PA top ( red ), and PA bot ( blue ) channel conductance block by WT LF N at 1, 5, 25, and 1200 n m were obtained at symmetrical pH 6.6 and no Δψ. Error bars are the mean ± S.D. ( n = 2). WT and PA top were tested for significance using an unpaired t test ( p < 0.0001) for all observations ( n = 16) at each set of conditions. The Ion Selectivity Filter of the PA Channel Is Critical for Δψ- and ΔpH-driven Translocation To characterize PA top and PA bot , however, we first needed to properly assemble the monomeric PA into oligomers. The multisite mutations would not assemble using the traditional ion-exchange approach ( 27 ). Hence we developed a modified assembly procedure. We nicked the PA monomers at pH 9 with furin instead of trypsin (to avoid nonspecific tryptic degradation), co-assembled the PA at pH 9 by adding LF N ( 5 , 23 ), and finally added Fos-choline-14 detergent to convert the prechannel oligomers into stable, detergent-solubilized channels ( 32 ). As a control, we also assembled WT PA by the same procedure. Native and SDS-PAGE ( supplemental Fig. S2 A ) and negative-stain EM ( supplemental Fig. S2B ) verified the proper assembly of these samples. To monitor channel formation by planar bilayer electrophysiology, we had to remove the LF N in situ by perfusing the cis chamber and translocating the residual LF N through the channels. We found that Fos-choline-14 favorably weakened the interaction of LF N with the channel, making its removal rapid and complete. In conclusion, the three preparations had reasonable insertion activities, albeit WT PA was most optimal. To determine whether these mutations change the ion selectivity of the PA channel, we first measured Δψ rev for WT PA, PA top , and PA bot . (Δψ rev is the voltage required to reduce the ionic current to zero under asymmetrical KCl gradients.) Each of these complexes was applied to planar bilayer membranes to form stable populations of channels following the removal of excess LF N by perfusion and translocation. The removal of residual LF N was judged to be complete by the stabilization of the current. Over a range of tested KCl gradients (in unbuffered saline, pH 5.8), WT PA and PA bot possessed similar Δψ rev values and, therefore, possessed similar ion selectivity ( Fig. 5 B ). However, PA top showed a reduced magnitude of Δψ rev relative to WT PA ( Fig. 5 B ). Thus PA top disrupts a portion of the ion-selectivity filter of the channel, presumably by reducing its anionic charge character ( Fig. 5 A ). PA top and PA bot were then assayed for their ability to translocate LF N under either a Δψ or a ΔpH. We found strong translocation deficiencies for PA top with either type of driving force ( Fig. 5 C ). Under a 1-unit ΔpH (pH cis 5.6 to pH trans 6.6) with Δψ of 20 mV, translocation of LF N through PA top is slowed more than 10-fold compared with WT PA, whereas PA bot is unaffected ( Fig. 5 C, left ). With a 50 mV Δψ at symmetrical pH 5.6, PA top was also less able to translocate LF N relative to WT PA ( Fig. 5 C, right ). Under these conditions, the rate and efficiency of translocation were affected. Although WT PA and PA bot are fully translocated within 2 min, PA top achieved less than 20% efficiency after 10 min. Thus PA top reveals significant translocation deficiencies under either a Δψ or ΔpH driving force. Finally, LF N was assayed for its ability to block PA top and PA bot channels. In this experiment, we added 5 n m LF N to the channels bathed in an asymmetrical KCl gradient at symmetrical pH 6.6 and a Δψ of 0 mV. Under these conditions, we found 99.0% (±0.1) of WT PA channel current was blocked ( Fig. 5 D ). For PA bot , we observed 98.0% (±0.1) conductance blockade; however, for PA top , 88% (±1) of the conductance was blocked by LF N . The binding defect observed with PA top may indicate that the charge disruption in that region affects the ability of the amino terminus of LF N to properly dock inside the pore and block conductance. In this model ( Equation 3 ), we expect two different stages of binding. In stage one, LF N binds to the top surface of the channel, forming the (PA·LF N ) complex; and in stage 2, the amino terminus docks into the channel to block conductance, forming the (PA·LF N )* complex. To test whether stage 1 or stage 2 were affected by the PA top mutation, we determined the percent blockade as a function of LF N concentration. Although the concentration of LF N should affect the equilibrium of stage 1, the equilibrium describing stage 2 is, of course, concentration-independent. To test for these two possibilities, we altered the LF N concentration. Reducing the concentration to 1 n m resulted in small changes in channel blockade (PA WT, 98.4% (±0.1); PA bot , 97.0% (±0.3); PA top , 86% (±2)). However, increasing the concentration 5-fold to 25 n m did not appreciably change the blockade (PA WT, 99.3% (±0.0); PA bot , 98.7% (±0.2); PA top , 88% (±1)), indicating that the system is at saturating levels of LF N . Indeed, even increasing the concentration to 1.2 μ m did not appreciably affect the percent block ( Fig. 5 D ). The inability of LF N to fully saturate channel conductance blockade in the PA top mutant over a 1000-fold concentration range demonstrates that channel docking (stage two) is impaired, and the PA top mutation likely disrupts a latching or ratcheting feature within the PA channel. EF N Translocates Slower Than LF N LF N and EF N share high levels of sequence ( 37 ) and structural homology ( 24 , 26 ); however, the most divergent sequence homology occurs on the amino terminus ( Fig. 2 A ). In planar lipid bilayer electrophysiology experiments, LF N and EF N translocate through the PA channel at remarkably different rates. Although LF N translocates with a t ½ value of ∼10 s at symmetrical pH 5.6 and a Δψ of 60 mV ( 6 , 8 ), His 6 -EF N translocates with a t ½ of ∼140 s under identical conditions ( 5 ). The His 6 tag used in affinity purification tends to have modest effects on the translocation t ½ ( 9 ), and so we re-examined these translocation differences under two different driving force extremes, a pure Δψ and a strong ΔpH, using the constructs in which the His 6 tag was removed by a protease. In our electrophysiological assay ( 6 – 8 ), a planar lipid bilayer separates two aqueous chambers ( cis and trans ). We first insert PA 7 channels into the bilayer. Either WT LF N or EF N was added to the cis side of the membrane (side to which PA 7 was added). Generally, an exponential decrease in current is observed as the amino-terminal presequence of the substrate inserts into the ion-conducting PA channel ( 38 ). A brief perfusion removes excess substrate from the cis chamber, and translocation is initiated by changing the Δψ and/or ΔpH. The subsequent current increase results from substrate translocation to the trans side of the membrane, as determined by control experiments ( 6 , 15 ). Two parameters are obtained from these "single turnover" translocation records: the t ½ and the efficiency of translocation, which is equivalent to the fraction of substrate that successfully translocates. We note that there are multiple LF N or EF N bound to each PA complex so these translocation records likely represent the turnover of several substrates. Therefore, single turnover kinetics refers to a single loaded PA complex that has translocated all of its substrates. We analyzed LF N and EF N translocation under identical conditions. Under a pure Δψ driving force, EF N translocated ∼200-fold slower than LF N ( Fig. 2 B ). Likewise, under a 1-unit ΔpH, EF N translocated ∼10-fold slower than LF N ( Fig. 2 C ). Interestingly, previous studies ( 31 ) and our more recent thermodynamic analysis ( Fig. 2 D and supplemental Table S1 ) show that the equilibrium stability of EF N , Δ G NI , is ∼2.4 kcal mol −1 less stable than LF N ( 31 ). As destabilization should in the most extreme case increase the rate of translocation due to the lowered unfolding barrier ( 8 ), it is unlikely that the weakened solution thermodynamic stability of EF N explains the observed increase in the activation energy of translocation relative to LF N . FIGURE 2. LF N /EF N chimeras are sufficient to mimic LF N -like translocation kinetics. A ( left ), sequence alignment of the first 50 amino acids of LF N and EF N . Residue pairs are shaded as follows: identity ( blue ), similarity ( light blue ), and weak similarity ( gray ). LF N /EF N chimera constructs are shown below where the increasing amounts of amino-terminal sequence from LF N ( blue ) appended to the EF N carboxyl-terminal folded domain ( green ). Right , C α -backbone alignment of EF N (1Y0V, green ) and LF N (1J7N, blue ) computed in CHIMERA ( 35 ). B , representative translocation recordings of LF N ( black ), EF N ( dashed ), and LF 1–50 EF 41–254 ( red ) under a Δψ driving force (at symmetric pH 5.6, Δψ of 50 mV). C, representative translocation records of LF N ( black ), EF N ( dashed ), and LF 1–30 EF 21–254 ( red ) under a 1-unit ΔpH driving force (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Records in panels B and C are normalized to maximal expected fraction translocated. D , representative equilibrium denaturant titrations comparing LF N ( solid ) and EF N ( dashed ) in guanidinium chloride (1 m glucose, pH 7.5, 20 °C) probed by CD at 222 nm and normalized to fraction unfolded ( f U ). Inset , equilibrium stability differences (ΔΔ G NI ) are referenced to WT LF N (where ΔΔ G NI compares EF N and chimeras to LF N ). For other chimeras, see supplemental Table S1 . Error are the mean ± S.D. for n = 3. Amino-terminal Chimeras with LF N Complement Slow EF N Translocation To determine the sequence differences responsible for the relatively slow translocation of EF N , we created a series of chimera constructs ( Fig. 2 A ). In these, we used the bulk of the EF N domain and only replaced the amino-terminal peptide with the corresponding sequence from LF N , where specifically 10, 18, 22, 26, 30, 40, or 50 LF N residues replaced equivalent positions in the EF N construct. (In our scheme, LF 1- a EF b -254 , a and b inclusively delimit the last residue of LF N and starting residue of EF N , respectively.) We found that the LF 1–50 EF 41–254 and LF 1–30 EF 21–254 chimeras represented the minimal chimera constructs ( Fig. 2 , B and C ) of all tested chimeras ( supplemental Fig. S1, A and B ) to exhibit LF N -like translocation under a pure Δψ and a 1-unit ΔpH, respectively. The sequence determinants that define the relatively slow translocation kinetics of EF N are found on its amino terminus. Thus the translocation kinetic stabilization we observe with EF N relative to LF N cannot be attributed to a phenomenon that occurs in solution (in isolation), but rather this difference manifests only in the context of the unfolding machine, the PA channel ( Fig. 2 D ). We then further explored the translocation differences of these chimeras under a variety of driving force conditions. Under pure Δψ-driven translocation at symmetric pH, we found that the more LF N sequence introduced into the chimera, the faster the rate of translocation ( supplemental Fig. S1 A ). Due to the complex nature of these ensemble translocation kinetics, a rate constant for translocation, k , was estimated using the t ½ for translocation, as k ∝ 1/ t ½, and from this we compute the Δ G ‡ . Interestingly, we found the LF 1–10 EF 1–254 chimera had similar to slightly slower translocation rates than EF N across many Δψ values ( supplemental Fig. S1 C ), indicating that these additional 10 residues in LF N are not responsible for the observed differences in translocation. To effectively recapitulate the LF N Δψ-dependence curve, the LF 1–50 EF 41–254 chimera was sufficient. We then examined the set of chimeras under a 1-unit ΔpH gradient ( supplemental Fig. S1 B ). Interestingly, the LF 1–10 EF 1–254 and LF 1–18 EF 9–254 chimeras showed slower translocation than EF N ( supplemental Fig. S1 D ), indicating potentially that these sequences, which have more densely hydrophobic amino termini ( Fig. 2 A ), may impede translocation due to the formation of an unusually tight binding interaction at the φ-clamp site. We found that the LF 1–30 EF 21–254 chimera, however, was sufficient to completely restore LF N -like translocation ( Fig. 2 C and supplemental Fig. S1 D ); and in contrast to purely Δψ driving forces, the sequence determinant for this restoration was concentrated between LF N residues 20 and 30. Two Sequence Cassettes Modulate the Translocation Stability of EF N and LF N A summary of the Δψ- and ΔpH-driven translocation results ( Fig. 3 A ) identified two sequence regions of interest, or "cassettes:" (i) the 20s cassette (residues 19–30); and (ii) the 40s cassette (residues 41–50) ( Fig. 3 B ). (Note that because EF N is 10 residues shorter than LF N on the amino-terminal end, we are applying the LF N -numbering scheme to EF N .) Under symmetric pH conditions and a Δψ driving force, there is a ∼1.3 kcal mol −1 difference in Δ G ‡ between LF 1–18 EF 9–254 and LF 1–26 EF 17–254 in the 20s cassette ( Fig. 3 A ). Under a 1-unit ΔpH gradient, there is a ∼2 kcal mol −1 difference between the same chimeras ( Fig. 3 A ). Also notable is the ∼1.5 kcal mol −1 Δ G ‡ difference between the LF 1–40 EF 31–254 and LF 1–50 EF 41–254 chimeras ( Fig. 3 A ); however, this difference was only observed under a Δψ driving force. Therefore, we hypothesize that sequence divergences in the 20s and 40s cassettes are responsible for the slow translocation kinetics of EF N . FIGURE 3. Charged residues in the 20s and 40s cassettes utilize the Δψ and ΔpH driving forces to promote unfolding and translocation. A ( left ), translocation activation energy for chimeric constructs at symmetric pH 5.6 and Δψ of 50 mV. One value was estimated by extrapolation (*) based on a larger Δψ-dependent dataset ( supplemental Fig. S1 C ) and associated fit parameters ( supplemental Table S2 ). Right, translocation Δ G ‡ for LF N , EF N , and the indicated LF N /EF N chimeras under a 1-unit ΔpH (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Brackets indicate significant differences (or "steps") in Δ G ‡ due to inclusion of the intervening LF N sequence cassette ( cass .) Additional Δψ-dependent data at a 1-unit ΔpH are given in supplemental Fig. S1 D , where associated fit parameters are given in supplemental Table S3 . B ( above ), amino-terminal 20s ( green ) and 40s ( orange ) cassette peptides are highlighted and the residue sequences in LF N and EF N are shown. Below , top / outside and inside / sagittal plane vantages of a molecular model of LF N ( blue ) in complex with the PA 8 oligomer ( gray ) (PDB 3KWV ( 4 )). Mutations in these two sequence cassettes may have destabilized the chimera and altered the unfolding step of the translocation mechanism. To test this possibility, we measured Δ G NI of the base and most highly internally mutagenized chimera constructs using standard solution unfolding procedures ( 8 , 31 ). We generally found no significant differences in Δ G NI between these chimeras and EF N ( Fig. 2 D and supplemental Table S1 ). As the bulk of the folded domain is from EF N , this result was expected. The residues differing between the chimeras are contained in the amino-terminal unstructured region and first α helix and β strand, which are highly solvent accessible. Thus we ruled out protein destabilization for these chimeras, and the amino-terminal sequence divergence in EF N likely affects the mechanisms of PA channel-dependent unfolding and translocation. Charge Content of Cassettes Controls Driving Force Dependence of Translocation To identify sequence features in the two cassettes contributing to the observed translocation Δ G ‡ differences, we introduced several point mutations within the existing chimera constructs ( Fig. 4 , A and B ). These mutations were made given the variation in net charge ( z ) observed within the cassettes. Net charge was estimated by z = n basic − n acidic , where n basic and n acidic are the number of basic and acidic residues, respectively. For the 20s cassette, we found that EF N and LF N had fairly different z values of +5 and 0, respectively. Likewise, for the 40s cassette, EF N and LF N had z values of 0 and +3, respectively. Upon our examination of their translocation kinetics, we found that correlations emerged between z values within the cassettes and their translocation Δ G ‡ values ( Fig. 4 , C and D ). Thus as expected, the subtraction of positive charge in the 20s cassette and addition of positive charge in the 40s cassette tended to generally increase the rate of translocation for EF N -based chimeras. FIGURE 4. Charged cassettes are nonspecific. A , construct design for chimeras and derivative mutants in the 20s cassette (residues 19–30) are arranged from the most positive to the most negative. Net charge given to the right of each sequence is computed using the following scoring system: D , E = −1; H , K , r = +1. Residues from native LF N ( blue ) and native EF N ( black ) are shown alongside non-native mutations ( boxed ) to either LF N or EF N . Residue-numbering scheme is according to LF N ( 24 ). B, constructs altering the 40s cassette (residues 41–50). Net charge is computed as in panel A. C ( top ), Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 20s cassette (residues 19–30 inclusive). Two-barrier model fit ( Equation 1 ): Δ G ‡ ° 1 = 3.2 (±0.5), δ 1 = −0.3 (±0.2), Δ G ‡ ° 2 = 3.2 (±0.5), and δ 2 = 0.3 (±0.1) ( n = 21, p < 0.001). Bottom, Δ G ‡ versus z at a Δψ of 20 mV, 1-unit ΔpH (5.6 pH cis , 6.6 pH trans ) for the same 20s-cassette variants. Two-barrier fit parameters: Δ G ‡ ° 1 = 0.3 (±0.5), δ 1 = −0.7 (±0.4), Δ G ‡ ° 2 = 0.9 (±0.3), and δ 2 = 1 (±0.2) ( n = 21, p < 0.001). D, Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 40s-cassette region (residues 41–50 inclusive). Single-barrier model ( Equation 2 ) fit parameters: Δ G ‡ ° = 2.7 (±0.1) and δ = −0.58 (±0.07) ( n = 8, p < 0.001). Error bars are the mean ± S.D. ( n ≥ 3). We also examined the residue identity and position dependence of these effects. When we separately introduced an Asp at positions 23 and 28 of LF 1–22 EF 13–254 (LF 1–22 EF 13–254 N23D, z = +1; LF 1–22 EF 13–254 K28D, z = 0) ( Fig. 4 A ), the rate of translocation increased relative to the parent construct ( z = +2) ( Fig. 4 C ). Furthermore, both LF 1–22 EF 13–254 K25D and LF 1–22 EF 13–254 K25E ( z = 0) increased the translocation rate similarly, indicating that there is a general requirement for negative charge, but residue identity is not critical. In general when examining all the data, translocation rates were only affected by changes in z values and not by changes in the position of the charges ( Fig. 4 C ). The rate of translocation is similar for the LF 1–22 EF 13–254 K25D/T26E and LF 1–22 EF 13–254 H24D/K25N chimeras ( z = −1). Finally, the negative charge neutralizing mutation LF 1–25 EF 16–254 D25N ( z = +2) showed slowed translocation compared with its parent construct LF 1–25 EF 16–254 ( z = +1). A similar but opposite effect can be seen in the 40s cassette, where there is a general requirement for positive charges independent of the specific positions ( Fig. 4 D ). For example, LF 1–40 EF 31–254 N41E and LF 1–40 EF 31–254 T49E ( z = −1) had similarly decreased translocation rates relative to their parent chimera ( z = 0). Thus we conclude that the 20s and 40s cassettes indeed have particular anionic and cationic charge requirements, respectively, but these requirements are highly nonspecific in terms of both position and residue identity. Although most of the charge-dependent Δ G ‡ data for the 20s cassette is linear with respect to charge, the presence of outlier data at higher negative charge density led to the hypothesis that there may be two barriers in the charge-dependent transport mechanism. Increasing negative charge can lower one barrier; however, the second barrier is either charge insensitive or somewhat inversely dependent on negative charge. To allow for partial-charge character (δ) during each respective barrier crossing ( 39 ), we used the following model, where F is Faraday's constant. For the ΔpH-dependent data ( n = 21), the fit to Equation 1 was significant ( p < 0.001) ( Fig. 4 C ). The δ parameter was obtained for each barrier as δ 1 = −0.7 (±0.4) and δ 2 = 1.0 (±0.2). The corresponding activation energies, ΔG ‡ ° 1 and ΔG ‡ ° 2 , in the absence of net charge were 0.3 (±0.5) and 0.9 (±0.3), respectively. For the Δψ-dependent translocation ( n = 21), the fit was also significant ( p < 0.001) with δ 1 = −0.3 (±0.2) and δ 2 = 0.3 (±0.1) and ΔG ‡ ° 1 = 3.2 (±0.5) and ΔG ‡ ° 2 = 3.2 (±0.5) ( Fig. 4 C ). Typically, δ values are challenging to interpret: residues may be partially charged due to p K a shifts; metal ions may bind to the translocating peptide and alter net charge; and finally, only part of the charged region in the substrate may be required to cross the rate-limiting barrier. Nevertheless, the goodness of fit suggests that indeed two unique charge-dependent barriers with inverse charge requirements are present in the translocation mechanism. In the 40s cassette region ( Fig. 4 B ), LF N possesses additional positive charge comparative to EF N at positions 41, 42, and 49. We created several point mutations in the existing chimeras to determine the effects of increasing or decreasing charge of the 40s cassette and investigated the charge-based differences in this region via translocation assays ( Fig. 4 D ). Starting with a sequence similar to EF N and increasing its positive charge to that of LF N , we again observe a direct relationship between charge and the translocation Δ G ‡ , where increasing positive charge leads to faster translocation. Also the charge dependence was again nonspecific ( Fig. 4 D ) where the position and identity of the residues did not appear to matter as much as the overall z value ( Fig. 4 B ). These charge-dependent data ( n = 8) for the 40s cassette were best fit by a single-barrier model ( 39 ). The fit was significant ( p < 0.001) with a δ of −0.58 (±0.07) and ΔG ‡ ° of 2.7 (±0.1) ( Fig. 4 D ). The type of cationic-charge preference in the 40s cassette is classical in the sense that it coincides with the direction of the electric field created by the applied membrane potential ( i.e. the field is cis -positive). Electrostatic Analysis of the PA β Barrel Given the unusual preference for anionic residues in the 20s cassette when driven by a Δψ (which is exactly opposite of the result expected for a cis -positive membrane potential), we hypothesized that the local electrostatic field produced by features within the channel, E chan , may override the electrical potential applied across the membrane, E m . The overall electric field, E , is a vector, where E = E chan + E m . The force applied upon the translocating chain is related to the sign and magnitude of the charge, q , of groups in the translocating chain and E by E × q . Because the electrical field contributed by the membrane potential relates to Δψ as E m = Δψ/ d , where d is the distance over which the potential drops, we can assume that the membrane potential will contribute unproductively to a negatively charged substrate if the membrane potential is positive in polarity. Therefore, E chan likely provides an oppositely oriented electrical field component that can apply a productive force on the substrate that aligns with the productive direction of translocation. Our hypothesis is also supported by the fact that the anion-charge preference in the 20s cassette appears independent of the makeup of the driving force; both Δψ-driven and ΔpH-driven kinetics can be accelerated by including additional negative charge in the 20s cassette of EF N . To characterize the electrostatic features within the PA channel, we initially built a model of the β barrel portion of the PA channel using the coordinates of α hemolysin ( 30 ) ( Fig. 5 A ). From this β barrel model, we calculated the sum of all pairwise electrostatic potentials for a point charge translocated along the central axis of the channel ("Experimental Procedures"). Our analysis revealed two prominent and oppositely charged electrostatic features, which were juxtaposed in the β barrel. One is a strongly anion-repulsive feature (PA residue ranges 275–283 and 343–352, generally localized to the top of the β barrel), and the other is a strongly cation-repulsive feature (PA residue ranges 287–299 and 328–340, generally localized to the middle of the β barrel) ( Fig. 5 A ). The PA residues contributing to these two features were located both inside and outside of the β barrel. Based on the same analytical model, we produced two β barrel mutants, one that would disrupt the anionic feature and one that would not. PA top disrupted the upper, cis -most portion of the β barrel, targeting its negatively charged residues by substituting them with isosteric Ser residues (D276S, D335S, and E343S). We chose Ser or Thr substitutions because the inside of the channel is hydrophilic and composed mostly of Ser and Thr residues ( 31 ). PA bot disrupted the lower trans -most portion of the β barrel and channel via the similar isosteric Thr substitutions (E302T, H304T, E308T, and H310T). The modeled electrostatic effects of these two mutant PA β barrels are shown in Fig. 5 A . FIGURE 5. Charge-selectivity filter in PA β barrel is required for efficient translocation. A ( left ), molecular model of the PA channel β barrel ( gray ), where acidic ( red ) and basic ( blue ) residues are highlighted. The outside and a sagittal section of the inside of the β-barrel structure are depicted. Right, the electrostatic energy for a negative point charge moved down the central axis of the β barrel of the channel. The origin on the distance axis is at the cis -most end of the β barrel, and increasing positive values indicates productive translocation. The potential was computed as described under "Experimental Procedures." B, relative differences in ion selectivity for WT PA ( black squares ), PA top ( red triangles ), and PA bot ( blue circles ) determined by −Δψ rev versus the KCl activity ratio ( cis : trans ). The x axis is plotted as a natural log scale marked by factors of e . The ideal cation-selective Nernstian relationship ( e -fold activity ratio per 25.2 mV at 20 °C) is indicated with a solid line . Three independent measurements assessed on three different membranes were corrected for membrane and electronics offsets. C , representative protein translocation records for WT LF N under ΔpH ( left ) and Δψ ( right ) using WT PA ( black ), PA top ( red ), and PA bot ( blue ). The Δψ and ΔpH conditions are identical to those applied in Fig. 2 , B and C . Results shown are consistent with replicates obtained on at least two separate membranes. D , ensemble bilayer recordings of WT PA ( black ), PA top ( red ), and PA bot ( blue ) channel conductance block by WT LF N at 1, 5, 25, and 1200 n m were obtained at symmetrical pH 6.6 and no Δψ. Error bars are the mean ± S.D. ( n = 2). WT and PA top were tested for significance using an unpaired t test ( p < 0.0001) for all observations ( n = 16) at each set of conditions. The Ion Selectivity Filter of the PA Channel Is Critical for Δψ- and ΔpH-driven Translocation To characterize PA top and PA bot , however, we first needed to properly assemble the monomeric PA into oligomers. The multisite mutations would not assemble using the traditional ion-exchange approach ( 27 ). Hence we developed a modified assembly procedure. We nicked the PA monomers at pH 9 with furin instead of trypsin (to avoid nonspecific tryptic degradation), co-assembled the PA at pH 9 by adding LF N ( 5 , 23 ), and finally added Fos-choline-14 detergent to convert the prechannel oligomers into stable, detergent-solubilized channels ( 32 ). As a control, we also assembled WT PA by the same procedure. Native and SDS-PAGE ( supplemental Fig. S2 A ) and negative-stain EM ( supplemental Fig. S2B ) verified the proper assembly of these samples. To monitor channel formation by planar bilayer electrophysiology, we had to remove the LF N in situ by perfusing the cis chamber and translocating the residual LF N through the channels. We found that Fos-choline-14 favorably weakened the interaction of LF N with the channel, making its removal rapid and complete. In conclusion, the three preparations had reasonable insertion activities, albeit WT PA was most optimal. To determine whether these mutations change the ion selectivity of the PA channel, we first measured Δψ rev for WT PA, PA top , and PA bot . (Δψ rev is the voltage required to reduce the ionic current to zero under asymmetrical KCl gradients.) Each of these complexes was applied to planar bilayer membranes to form stable populations of channels following the removal of excess LF N by perfusion and translocation. The removal of residual LF N was judged to be complete by the stabilization of the current. Over a range of tested KCl gradients (in unbuffered saline, pH 5.8), WT PA and PA bot possessed similar Δψ rev values and, therefore, possessed similar ion selectivity ( Fig. 5 B ). However, PA top showed a reduced magnitude of Δψ rev relative to WT PA ( Fig. 5 B ). Thus PA top disrupts a portion of the ion-selectivity filter of the channel, presumably by reducing its anionic charge character ( Fig. 5 A ). PA top and PA bot were then assayed for their ability to translocate LF N under either a Δψ or a ΔpH. We found strong translocation deficiencies for PA top with either type of driving force ( Fig. 5 C ). Under a 1-unit ΔpH (pH cis 5.6 to pH trans 6.6) with Δψ of 20 mV, translocation of LF N through PA top is slowed more than 10-fold compared with WT PA, whereas PA bot is unaffected ( Fig. 5 C, left ). With a 50 mV Δψ at symmetrical pH 5.6, PA top was also less able to translocate LF N relative to WT PA ( Fig. 5 C, right ). Under these conditions, the rate and efficiency of translocation were affected. Although WT PA and PA bot are fully translocated within 2 min, PA top achieved less than 20% efficiency after 10 min. Thus PA top reveals significant translocation deficiencies under either a Δψ or ΔpH driving force. Finally, LF N was assayed for its ability to block PA top and PA bot channels. In this experiment, we added 5 n m LF N to the channels bathed in an asymmetrical KCl gradient at symmetrical pH 6.6 and a Δψ of 0 mV. Under these conditions, we found 99.0% (±0.1) of WT PA channel current was blocked ( Fig. 5 D ). For PA bot , we observed 98.0% (±0.1) conductance blockade; however, for PA top , 88% (±1) of the conductance was blocked by LF N . The binding defect observed with PA top may indicate that the charge disruption in that region affects the ability of the amino terminus of LF N to properly dock inside the pore and block conductance. In this model ( Equation 3 ), we expect two different stages of binding. In stage one, LF N binds to the top surface of the channel, forming the (PA·LF N ) complex; and in stage 2, the amino terminus docks into the channel to block conductance, forming the (PA·LF N )* complex. To test whether stage 1 or stage 2 were affected by the PA top mutation, we determined the percent blockade as a function of LF N concentration. Although the concentration of LF N should affect the equilibrium of stage 1, the equilibrium describing stage 2 is, of course, concentration-independent. To test for these two possibilities, we altered the LF N concentration. Reducing the concentration to 1 n m resulted in small changes in channel blockade (PA WT, 98.4% (±0.1); PA bot , 97.0% (±0.3); PA top , 86% (±2)). However, increasing the concentration 5-fold to 25 n m did not appreciably change the blockade (PA WT, 99.3% (±0.0); PA bot , 98.7% (±0.2); PA top , 88% (±1)), indicating that the system is at saturating levels of LF N . Indeed, even increasing the concentration to 1.2 μ m did not appreciably affect the percent block ( Fig. 5 D ). The inability of LF N to fully saturate channel conductance blockade in the PA top mutant over a 1000-fold concentration range demonstrates that channel docking (stage two) is impaired, and the PA top mutation likely disrupts a latching or ratcheting feature within the PA channel. EF N Translocates Slower Than LF N LF N and EF N share high levels of sequence ( 37 ) and structural homology ( 24 , 26 ); however, the most divergent sequence homology occurs on the amino terminus ( Fig. 2 A ). In planar lipid bilayer electrophysiology experiments, LF N and EF N translocate through the PA channel at remarkably different rates. Although LF N translocates with a t ½ value of ∼10 s at symmetrical pH 5.6 and a Δψ of 60 mV ( 6 , 8 ), His 6 -EF N translocates with a t ½ of ∼140 s under identical conditions ( 5 ). The His 6 tag used in affinity purification tends to have modest effects on the translocation t ½ ( 9 ), and so we re-examined these translocation differences under two different driving force extremes, a pure Δψ and a strong ΔpH, using the constructs in which the His 6 tag was removed by a protease. In our electrophysiological assay ( 6 – 8 ), a planar lipid bilayer separates two aqueous chambers ( cis and trans ). We first insert PA 7 channels into the bilayer. Either WT LF N or EF N was added to the cis side of the membrane (side to which PA 7 was added). Generally, an exponential decrease in current is observed as the amino-terminal presequence of the substrate inserts into the ion-conducting PA channel ( 38 ). A brief perfusion removes excess substrate from the cis chamber, and translocation is initiated by changing the Δψ and/or ΔpH. The subsequent current increase results from substrate translocation to the trans side of the membrane, as determined by control experiments ( 6 , 15 ). Two parameters are obtained from these "single turnover" translocation records: the t ½ and the efficiency of translocation, which is equivalent to the fraction of substrate that successfully translocates. We note that there are multiple LF N or EF N bound to each PA complex so these translocation records likely represent the turnover of several substrates. Therefore, single turnover kinetics refers to a single loaded PA complex that has translocated all of its substrates. We analyzed LF N and EF N translocation under identical conditions. Under a pure Δψ driving force, EF N translocated ∼200-fold slower than LF N ( Fig. 2 B ). Likewise, under a 1-unit ΔpH, EF N translocated ∼10-fold slower than LF N ( Fig. 2 C ). Interestingly, previous studies ( 31 ) and our more recent thermodynamic analysis ( Fig. 2 D and supplemental Table S1 ) show that the equilibrium stability of EF N , Δ G NI , is ∼2.4 kcal mol −1 less stable than LF N ( 31 ). As destabilization should in the most extreme case increase the rate of translocation due to the lowered unfolding barrier ( 8 ), it is unlikely that the weakened solution thermodynamic stability of EF N explains the observed increase in the activation energy of translocation relative to LF N . FIGURE 2. LF N /EF N chimeras are sufficient to mimic LF N -like translocation kinetics. A ( left ), sequence alignment of the first 50 amino acids of LF N and EF N . Residue pairs are shaded as follows: identity ( blue ), similarity ( light blue ), and weak similarity ( gray ). LF N /EF N chimera constructs are shown below where the increasing amounts of amino-terminal sequence from LF N ( blue ) appended to the EF N carboxyl-terminal folded domain ( green ). Right , C α -backbone alignment of EF N (1Y0V, green ) and LF N (1J7N, blue ) computed in CHIMERA ( 35 ). B , representative translocation recordings of LF N ( black ), EF N ( dashed ), and LF 1–50 EF 41–254 ( red ) under a Δψ driving force (at symmetric pH 5.6, Δψ of 50 mV). C, representative translocation records of LF N ( black ), EF N ( dashed ), and LF 1–30 EF 21–254 ( red ) under a 1-unit ΔpH driving force (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Records in panels B and C are normalized to maximal expected fraction translocated. D , representative equilibrium denaturant titrations comparing LF N ( solid ) and EF N ( dashed ) in guanidinium chloride (1 m glucose, pH 7.5, 20 °C) probed by CD at 222 nm and normalized to fraction unfolded ( f U ). Inset , equilibrium stability differences (ΔΔ G NI ) are referenced to WT LF N (where ΔΔ G NI compares EF N and chimeras to LF N ). For other chimeras, see supplemental Table S1 . Error are the mean ± S.D. for n = 3. Amino-terminal Chimeras with LF N Complement Slow EF N Translocation To determine the sequence differences responsible for the relatively slow translocation of EF N , we created a series of chimera constructs ( Fig. 2 A ). In these, we used the bulk of the EF N domain and only replaced the amino-terminal peptide with the corresponding sequence from LF N , where specifically 10, 18, 22, 26, 30, 40, or 50 LF N residues replaced equivalent positions in the EF N construct. (In our scheme, LF 1- a EF b -254 , a and b inclusively delimit the last residue of LF N and starting residue of EF N , respectively.) We found that the LF 1–50 EF 41–254 and LF 1–30 EF 21–254 chimeras represented the minimal chimera constructs ( Fig. 2 , B and C ) of all tested chimeras ( supplemental Fig. S1, A and B ) to exhibit LF N -like translocation under a pure Δψ and a 1-unit ΔpH, respectively. The sequence determinants that define the relatively slow translocation kinetics of EF N are found on its amino terminus. Thus the translocation kinetic stabilization we observe with EF N relative to LF N cannot be attributed to a phenomenon that occurs in solution (in isolation), but rather this difference manifests only in the context of the unfolding machine, the PA channel ( Fig. 2 D ). We then further explored the translocation differences of these chimeras under a variety of driving force conditions. Under pure Δψ-driven translocation at symmetric pH, we found that the more LF N sequence introduced into the chimera, the faster the rate of translocation ( supplemental Fig. S1 A ). Due to the complex nature of these ensemble translocation kinetics, a rate constant for translocation, k , was estimated using the t ½ for translocation, as k ∝ 1/ t ½, and from this we compute the Δ G ‡ . Interestingly, we found the LF 1–10 EF 1–254 chimera had similar to slightly slower translocation rates than EF N across many Δψ values ( supplemental Fig. S1 C ), indicating that these additional 10 residues in LF N are not responsible for the observed differences in translocation. To effectively recapitulate the LF N Δψ-dependence curve, the LF 1–50 EF 41–254 chimera was sufficient. We then examined the set of chimeras under a 1-unit ΔpH gradient ( supplemental Fig. S1 B ). Interestingly, the LF 1–10 EF 1–254 and LF 1–18 EF 9–254 chimeras showed slower translocation than EF N ( supplemental Fig. S1 D ), indicating potentially that these sequences, which have more densely hydrophobic amino termini ( Fig. 2 A ), may impede translocation due to the formation of an unusually tight binding interaction at the φ-clamp site. We found that the LF 1–30 EF 21–254 chimera, however, was sufficient to completely restore LF N -like translocation ( Fig. 2 C and supplemental Fig. S1 D ); and in contrast to purely Δψ driving forces, the sequence determinant for this restoration was concentrated between LF N residues 20 and 30. Two Sequence Cassettes Modulate the Translocation Stability of EF N and LF N A summary of the Δψ- and ΔpH-driven translocation results ( Fig. 3 A ) identified two sequence regions of interest, or "cassettes:" (i) the 20s cassette (residues 19–30); and (ii) the 40s cassette (residues 41–50) ( Fig. 3 B ). (Note that because EF N is 10 residues shorter than LF N on the amino-terminal end, we are applying the LF N -numbering scheme to EF N .) Under symmetric pH conditions and a Δψ driving force, there is a ∼1.3 kcal mol −1 difference in Δ G ‡ between LF 1–18 EF 9–254 and LF 1–26 EF 17–254 in the 20s cassette ( Fig. 3 A ). Under a 1-unit ΔpH gradient, there is a ∼2 kcal mol −1 difference between the same chimeras ( Fig. 3 A ). Also notable is the ∼1.5 kcal mol −1 Δ G ‡ difference between the LF 1–40 EF 31–254 and LF 1–50 EF 41–254 chimeras ( Fig. 3 A ); however, this difference was only observed under a Δψ driving force. Therefore, we hypothesize that sequence divergences in the 20s and 40s cassettes are responsible for the slow translocation kinetics of EF N . FIGURE 3. Charged residues in the 20s and 40s cassettes utilize the Δψ and ΔpH driving forces to promote unfolding and translocation. A ( left ), translocation activation energy for chimeric constructs at symmetric pH 5.6 and Δψ of 50 mV. One value was estimated by extrapolation (*) based on a larger Δψ-dependent dataset ( supplemental Fig. S1 C ) and associated fit parameters ( supplemental Table S2 ). Right, translocation Δ G ‡ for LF N , EF N , and the indicated LF N /EF N chimeras under a 1-unit ΔpH (5.6 pH cis , 6.6 pH trans , Δψ of 20 mV). Brackets indicate significant differences (or "steps") in Δ G ‡ due to inclusion of the intervening LF N sequence cassette ( cass .) Additional Δψ-dependent data at a 1-unit ΔpH are given in supplemental Fig. S1 D , where associated fit parameters are given in supplemental Table S3 . B ( above ), amino-terminal 20s ( green ) and 40s ( orange ) cassette peptides are highlighted and the residue sequences in LF N and EF N are shown. Below , top / outside and inside / sagittal plane vantages of a molecular model of LF N ( blue ) in complex with the PA 8 oligomer ( gray ) (PDB 3KWV ( 4 )). Mutations in these two sequence cassettes may have destabilized the chimera and altered the unfolding step of the translocation mechanism. To test this possibility, we measured Δ G NI of the base and most highly internally mutagenized chimera constructs using standard solution unfolding procedures ( 8 , 31 ). We generally found no significant differences in Δ G NI between these chimeras and EF N ( Fig. 2 D and supplemental Table S1 ). As the bulk of the folded domain is from EF N , this result was expected. The residues differing between the chimeras are contained in the amino-terminal unstructured region and first α helix and β strand, which are highly solvent accessible. Thus we ruled out protein destabilization for these chimeras, and the amino-terminal sequence divergence in EF N likely affects the mechanisms of PA channel-dependent unfolding and translocation. Charge Content of Cassettes Controls Driving Force Dependence of Translocation To identify sequence features in the two cassettes contributing to the observed translocation Δ G ‡ differences, we introduced several point mutations within the existing chimera constructs ( Fig. 4 , A and B ). These mutations were made given the variation in net charge ( z ) observed within the cassettes. Net charge was estimated by z = n basic − n acidic , where n basic and n acidic are the number of basic and acidic residues, respectively. For the 20s cassette, we found that EF N and LF N had fairly different z values of +5 and 0, respectively. Likewise, for the 40s cassette, EF N and LF N had z values of 0 and +3, respectively. Upon our examination of their translocation kinetics, we found that correlations emerged between z values within the cassettes and their translocation Δ G ‡ values ( Fig. 4 , C and D ). Thus as expected, the subtraction of positive charge in the 20s cassette and addition of positive charge in the 40s cassette tended to generally increase the rate of translocation for EF N -based chimeras. FIGURE 4. Charged cassettes are nonspecific. A , construct design for chimeras and derivative mutants in the 20s cassette (residues 19–30) are arranged from the most positive to the most negative. Net charge given to the right of each sequence is computed using the following scoring system: D , E = −1; H , K , r = +1. Residues from native LF N ( blue ) and native EF N ( black ) are shown alongside non-native mutations ( boxed ) to either LF N or EF N . Residue-numbering scheme is according to LF N ( 24 ). B, constructs altering the 40s cassette (residues 41–50). Net charge is computed as in panel A. C ( top ), Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 20s cassette (residues 19–30 inclusive). Two-barrier model fit ( Equation 1 ): Δ G ‡ ° 1 = 3.2 (±0.5), δ 1 = −0.3 (±0.2), Δ G ‡ ° 2 = 3.2 (±0.5), and δ 2 = 0.3 (±0.1) ( n = 21, p < 0.001). Bottom, Δ G ‡ versus z at a Δψ of 20 mV, 1-unit ΔpH (5.6 pH cis , 6.6 pH trans ) for the same 20s-cassette variants. Two-barrier fit parameters: Δ G ‡ ° 1 = 0.3 (±0.5), δ 1 = −0.7 (±0.4), Δ G ‡ ° 2 = 0.9 (±0.3), and δ 2 = 1 (±0.2) ( n = 21, p < 0.001). D, Δ G ‡ versus z at symmetric pH 5.6, Δψ of 50 mV for LF N /EF N chimeras and related mutants affecting the 40s-cassette region (residues 41–50 inclusive). Single-barrier model ( Equation 2 ) fit parameters: Δ G ‡ ° = 2.7 (±0.1) and δ = −0.58 (±0.07) ( n = 8, p < 0.001). Error bars are the mean ± S.D. ( n ≥ 3). We also examined the residue identity and position dependence of these effects. When we separately introduced an Asp at positions 23 and 28 of LF 1–22 EF 13–254 (LF 1–22 EF 13–254 N23D, z = +1; LF 1–22 EF 13–254 K28D, z = 0) ( Fig. 4 A ), the rate of translocation increased relative to the parent construct ( z = +2) ( Fig. 4 C ). Furthermore, both LF 1–22 EF 13–254 K25D and LF 1–22 EF 13–254 K25E ( z = 0) increased the translocation rate similarly, indicating that there is a general requirement for negative charge, but residue identity is not critical. In general when examining all the data, translocation rates were only affected by changes in z values and not by changes in the position of the charges ( Fig. 4 C ). The rate of translocation is similar for the LF 1–22 EF 13–254 K25D/T26E and LF 1–22 EF 13–254 H24D/K25N chimeras ( z = −1). Finally, the negative charge neutralizing mutation LF 1–25 EF 16–254 D25N ( z = +2) showed slowed translocation compared with its parent construct LF 1–25 EF 16–254 ( z = +1). A similar but opposite effect can be seen in the 40s cassette, where there is a general requirement for positive charges independent of the specific positions ( Fig. 4 D ). For example, LF 1–40 EF 31–254 N41E and LF 1–40 EF 31–254 T49E ( z = −1) had similarly decreased translocation rates relative to their parent chimera ( z = 0). Thus we conclude that the 20s and 40s cassettes indeed have particular anionic and cationic charge requirements, respectively, but these requirements are highly nonspecific in terms of both position and residue identity. Although most of the charge-dependent Δ G ‡ data for the 20s cassette is linear with respect to charge, the presence of outlier data at higher negative charge density led to the hypothesis that there may be two barriers in the charge-dependent transport mechanism. Increasing negative charge can lower one barrier; however, the second barrier is either charge insensitive or somewhat inversely dependent on negative charge. To allow for partial-charge character (δ) during each respective barrier crossing ( 39 ), we used the following model, where F is Faraday's constant. For the ΔpH-dependent data ( n = 21), the fit to Equation 1 was significant ( p < 0.001) ( Fig. 4 C ). The δ parameter was obtained for each barrier as δ 1 = −0.7 (±0.4) and δ 2 = 1.0 (±0.2). The corresponding activation energies, ΔG ‡ ° 1 and ΔG ‡ ° 2 , in the absence of net charge were 0.3 (±0.5) and 0.9 (±0.3), respectively. For the Δψ-dependent translocation ( n = 21), the fit was also significant ( p < 0.001) with δ 1 = −0.3 (±0.2) and δ 2 = 0.3 (±0.1) and ΔG ‡ ° 1 = 3.2 (±0.5) and ΔG ‡ ° 2 = 3.2 (±0.5) ( Fig. 4 C ). Typically, δ values are challenging to interpret: residues may be partially charged due to p K a shifts; metal ions may bind to the translocating peptide and alter net charge; and finally, only part of the charged region in the substrate may be required to cross the rate-limiting barrier. Nevertheless, the goodness of fit suggests that indeed two unique charge-dependent barriers with inverse charge requirements are present in the translocation mechanism. In the 40s cassette region ( Fig. 4 B ), LF N possesses additional positive charge comparative to EF N at positions 41, 42, and 49. We created several point mutations in the existing chimeras to determine the effects of increasing or decreasing charge of the 40s cassette and investigated the charge-based differences in this region via translocation assays ( Fig. 4 D ). Starting with a sequence similar to EF N and increasing its positive charge to that of LF N , we again observe a direct relationship between charge and the translocation Δ G ‡ , where increasing positive charge leads to faster translocation. Also the charge dependence was again nonspecific ( Fig. 4 D ) where the position and identity of the residues did not appear to matter as much as the overall z value ( Fig. 4 B ). These charge-dependent data ( n = 8) for the 40s cassette were best fit by a single-barrier model ( 39 ). The fit was significant ( p < 0.001) with a δ of −0.58 (±0.07) and ΔG ‡ ° of 2.7 (±0.1) ( Fig. 4 D ). The type of cationic-charge preference in the 40s cassette is classical in the sense that it coincides with the direction of the electric field created by the applied membrane potential ( i.e. the field is cis -positive). Electrostatic Analysis of the PA β Barrel Given the unusual preference for anionic residues in the 20s cassette when driven by a Δψ (which is exactly opposite of the result expected for a cis -positive membrane potential), we hypothesized that the local electrostatic field produced by features within the channel, E chan , may override the electrical potential applied across the membrane, E m . The overall electric field, E , is a vector, where E = E chan + E m . The force applied upon the translocating chain is related to the sign and magnitude of the charge, q , of groups in the translocating chain and E by E × q . Because the electrical field contributed by the membrane potential relates to Δψ as E m = Δψ/ d , where d is the distance over which the potential drops, we can assume that the membrane potential will contribute unproductively to a negatively charged substrate if the membrane potential is positive in polarity. Therefore, E chan likely provides an oppositely oriented electrical field component that can apply a productive force on the substrate that aligns with the productive direction of translocation. Our hypothesis is also supported by the fact that the anion-charge preference in the 20s cassette appears independent of the makeup of the driving force; both Δψ-driven and ΔpH-driven kinetics can be accelerated by including additional negative charge in the 20s cassette of EF N . To characterize the electrostatic features within the PA channel, we initially built a model of the β barrel portion of the PA channel using the coordinates of α hemolysin ( 30 ) ( Fig. 5 A ). From this β barrel model, we calculated the sum of all pairwise electrostatic potentials for a point charge translocated along the central axis of the channel ("Experimental Procedures"). Our analysis revealed two prominent and oppositely charged electrostatic features, which were juxtaposed in the β barrel. One is a strongly anion-repulsive feature (PA residue ranges 275–283 and 343–352, generally localized to the top of the β barrel), and the other is a strongly cation-repulsive feature (PA residue ranges 287–299 and 328–340, generally localized to the middle of the β barrel) ( Fig. 5 A ). The PA residues contributing to these two features were located both inside and outside of the β barrel. Based on the same analytical model, we produced two β barrel mutants, one that would disrupt the anionic feature and one that would not. PA top disrupted the upper, cis -most portion of the β barrel, targeting its negatively charged residues by substituting them with isosteric Ser residues (D276S, D335S, and E343S). We chose Ser or Thr substitutions because the inside of the channel is hydrophilic and composed mostly of Ser and Thr residues ( 31 ). PA bot disrupted the lower trans -most portion of the β barrel and channel via the similar isosteric Thr substitutions (E302T, H304T, E308T, and H310T). The modeled electrostatic effects of these two mutant PA β barrels are shown in Fig. 5 A . FIGURE 5. Charge-selectivity filter in PA β barrel is required for efficient translocation. A ( left ), molecular model of the PA channel β barrel ( gray ), where acidic ( red ) and basic ( blue ) residues are highlighted. The outside and a sagittal section of the inside of the β-barrel structure are depicted. Right, the electrostatic energy for a negative point charge moved down the central axis of the β barrel of the channel. The origin on the distance axis is at the cis -most end of the β barrel, and increasing positive values indicates productive translocation. The potential was computed as described under "Experimental Procedures." B, relative differences in ion selectivity for WT PA ( black squares ), PA top ( red triangles ), and PA bot ( blue circles ) determined by −Δψ rev versus the KCl activity ratio ( cis : trans ). The x axis is plotted as a natural log scale marked by factors of e . The ideal cation-selective Nernstian relationship ( e -fold activity ratio per 25.2 mV at 20 °C) is indicated with a solid line . Three independent measurements assessed on three different membranes were corrected for membrane and electronics offsets. C , representative protein translocation records for WT LF N under ΔpH ( left ) and Δψ ( right ) using WT PA ( black ), PA top ( red ), and PA bot ( blue ). The Δψ and ΔpH conditions are identical to those applied in Fig. 2 , B and C . Results shown are consistent with replicates obtained on at least two separate membranes. D , ensemble bilayer recordings of WT PA ( black ), PA top ( red ), and PA bot ( blue ) channel conductance block by WT LF N at 1, 5, 25, and 1200 n m were obtained at symmetrical pH 6.6 and no Δψ. Error bars are the mean ± S.D. ( n = 2). WT and PA top were tested for significance using an unpaired t test ( p < 0.0001) for all observations ( n = 16) at each set of conditions. The Ion Selectivity Filter of the PA Channel Is Critical for Δψ- and ΔpH-driven Translocation To characterize PA top and PA bot , however, we first needed to properly assemble the monomeric PA into oligomers. The multisite mutations would not assemble using the traditional ion-exchange approach ( 27 ). Hence we developed a modified assembly procedure. We nicked the PA monomers at pH 9 with furin instead of trypsin (to avoid nonspecific tryptic degradation), co-assembled the PA at pH 9 by adding LF N ( 5 , 23 ), and finally added Fos-choline-14 detergent to convert the prechannel oligomers into stable, detergent-solubilized channels ( 32 ). As a control, we also assembled WT PA by the same procedure. Native and SDS-PAGE ( supplemental Fig. S2 A ) and negative-stain EM ( supplemental Fig. S2B ) verified the proper assembly of these samples. To monitor channel formation by planar bilayer electrophysiology, we had to remove the LF N in situ by perfusing the cis chamber and translocating the residual LF N through the channels. We found that Fos-choline-14 favorably weakened the interaction of LF N with the channel, making its removal rapid and complete. In conclusion, the three preparations had reasonable insertion activities, albeit WT PA was most optimal. To determine whether these mutations change the ion selectivity of the PA channel, we first measured Δψ rev for WT PA, PA top , and PA bot . (Δψ rev is the voltage required to reduce the ionic current to zero under asymmetrical KCl gradients.) Each of these complexes was applied to planar bilayer membranes to form stable populations of channels following the removal of excess LF N by perfusion and translocation. The removal of residual LF N was judged to be complete by the stabilization of the current. Over a range of tested KCl gradients (in unbuffered saline, pH 5.8), WT PA and PA bot possessed similar Δψ rev values and, therefore, possessed similar ion selectivity ( Fig. 5 B ). However, PA top showed a reduced magnitude of Δψ rev relative to WT PA ( Fig. 5 B ). Thus PA top disrupts a portion of the ion-selectivity filter of the channel, presumably by reducing its anionic charge character ( Fig. 5 A ). PA top and PA bot were then assayed for their ability to translocate LF N under either a Δψ or a ΔpH. We found strong translocation deficiencies for PA top with either type of driving force ( Fig. 5 C ). Under a 1-unit ΔpH (pH cis 5.6 to pH trans 6.6) with Δψ of 20 mV, translocation of LF N through PA top is slowed more than 10-fold compared with WT PA, whereas PA bot is unaffected ( Fig. 5 C, left ). With a 50 mV Δψ at symmetrical pH 5.6, PA top was also less able to translocate LF N relative to WT PA ( Fig. 5 C, right ). Under these conditions, the rate and efficiency of translocation were affected. Although WT PA and PA bot are fully translocated within 2 min, PA top achieved less than 20% efficiency after 10 min. Thus PA top reveals significant translocation deficiencies under either a Δψ or ΔpH driving force. Finally, LF N was assayed for its ability to block PA top and PA bot channels. In this experiment, we added 5 n m LF N to the channels bathed in an asymmetrical KCl gradient at symmetrical pH 6.6 and a Δψ of 0 mV. Under these conditions, we found 99.0% (±0.1) of WT PA channel current was blocked ( Fig. 5 D ). For PA bot , we observed 98.0% (±0.1) conductance blockade; however, for PA top , 88% (±1) of the conductance was blocked by LF N . The binding defect observed with PA top may indicate that the charge disruption in that region affects the ability of the amino terminus of LF N to properly dock inside the pore and block conductance. In this model ( Equation 3 ), we expect two different stages of binding. In stage one, LF N binds to the top surface of the channel, forming the (PA·LF N ) complex; and in stage 2, the amino terminus docks into the channel to block conductance, forming the (PA·LF N )* complex. To test whether stage 1 or stage 2 were affected by the PA top mutation, we determined the percent blockade as a function of LF N concentration. Although the concentration of LF N should affect the equilibrium of stage 1, the equilibrium describing stage 2 is, of course, concentration-independent. To test for these two possibilities, we altered the LF N concentration. Reducing the concentration to 1 n m resulted in small changes in channel blockade (PA WT, 98.4% (±0.1); PA bot , 97.0% (±0.3); PA top , 86% (±2)). However, increasing the concentration 5-fold to 25 n m did not appreciably change the blockade (PA WT, 99.3% (±0.0); PA bot , 98.7% (±0.2); PA top , 88% (±1)), indicating that the system is at saturating levels of LF N . Indeed, even increasing the concentration to 1.2 μ m did not appreciably affect the percent block ( Fig. 5 D ). The inability of LF N to fully saturate channel conductance blockade in the PA top mutant over a 1000-fold concentration range demonstrates that channel docking (stage two) is impaired, and the PA top mutation likely disrupts a latching or ratcheting feature within the PA channel. DISCUSSION General Substrate Charge Requirements To address the molecular mechanism of PMF-driven translocation, we traced the source of the differences in the translocation kinetics between LF N and EF N . Previous translocation studies ( 5 , 8 ) and our more controlled re-examination here show that EF N translocates ∼200-fold slower than LF N under a Δψ alone and ∼10-fold slower than LF N under a combined Δψ and ΔpH ( Fig. 2 , B and C ). This phenomenon occurs despite the fact that LF N and EF N have ∼55% sequence similarity, adopt identical folds ( 24 , 26 ), possess similar solution stabilities ( Fig. 2 D ) ( 31 ), and bind to the same location on the PA channel ( 4 , 37 ). Interestingly, whereas LF and EF initiate translocation starting from the amino termini of their homologous LF N and EF N domains, the amino-terminal initiation sequence of these domains is the most divergent sequence in the domain. We anticipated that this region of the sequence was responsible for the differences we observed in their translocation kinetics. Swapping the 40-residue amino terminus of EF N with the homologous 50-residue amino terminus from LF N allows the chimera to translocate as rapidly as LF N . The inability of EF N to utilize the PMF as well as LF N is hence due to sequence differences in the amino-terminal presequence, and therefore, the charged presequence is critical to allowing the substrate to best capture the PMF to drive unfolding and translocation. Within the presequence, we were then able to locate two sequence cassettes, or motifs, required for efficient translocation ( Fig. 3 B ). When additional acidic residues are added within the 20s cassette of EF N , its translocation becomes more LF N -like. Previous studies by Brown et al. ( 9 ) have shown that under a ΔpH driving force, acidic residues are needed in the 20s cassette for efficient translocation, and whereas our studies here support prior observations, they also show that higher acidic residue content in the 20s cassette is favorable under a pure Δψ. Hence the acidic residue-dependent mechanism we observe is independent of the nature of the driving force. This dependence, at first glance, is most unusual because it is opposite to the effect expected for a cis -positive Δψ, and we will expand on this point in detail below. But from this unusual charge requirement, we expect that the electrostatics of the channel itself govern the overall mechanism. We also identified a 40s cassette in the presequence and found it prefers cationic residues. This preference in the 40s cassette is only observed under a pure Δψ driving force, and whereas the 40s cassette is a novel sequence feature, it was expected to exist because a productive Δψ driving force is cis -positive. Broad Sequence Specificity in Protein Translocases The broad sequence specificity we observe for these charged cassettes ( Fig. 4 , C and D ) is similar to the binding preferences of other polypeptide-clamping sites in the PA channel and in other systems ( 2 ). This observation is the case during translocation for several reasons. The sequence complexity is high, meaning the amino acid sequences, which continually pass through the channel, cover an enormous combinatorial sequence space. Also, the conformational and configuration space the translocating chain may explore during translocation is enormous. Levinthal ( 40 ) originally stated that a folding protein would be unable to sample all the possible configurations of the unfolded state in a reasonable time scale, and instead, proteins must fold via a specific pathway. The hydrophobic effect, for example, is likely a key feature that guides many folding pathways. Hence, we propose that broad sequence specificity is key for a protein translocase, because it must process unfolded protein, which may otherwise occupy too many possible states. The charged cassettes we report here again have general electrostatic requirements, but the specific details are far less critical. Other examples of these nonspecific clamping sites in the PA channel include the α clamp and the φ clamp. The φ clamp prefers hydrophobic and aromatic substrates ( 7 ), whereas the α clamp binds most optimally to α-helical structure with minimal sequence specificity ( 4 ). Each clamping site binds broadly to a different type of chemical handle in the translocating chain, where specific hydrogen bonds and salt bridges are noncritical. Polypeptide clamps are critical because forces cannot be applied to the unfolding substrate protein without a fulcrum. Also, competing diffusive and entropic forces in the system scale with the size of the unfolded state; i.e. for an n -residue long unfolded chain where each residue can sample an average of C conformations, the total number of potential configurations scales as C N . Limiting the size of the unfolded chain that is freely diffusible through nonspecific clamping allows the force-generating apparatus to focus more efficiently on producing directional motion and mechanically unfolding the substrate. The downside to nonspecifically clamping the chain becomes immediately apparent; because when interactions are too tight translocation should become impeded. However, we have proposed instead that clamping sites are dynamic, and the chain is continually bound and released during translocation, and hence such events would reduce diffusive entropic costs, improve energy transduction and force generation, and lower the overall barriers to translocation. Role of Channel Electrostatics in Translocation Previous work by Brown et al. ( 9 ) has shown that sites within the 20s cassette of LF N were optimal for the placement of acidic residues when translocation is driven by a ΔpH. The key finding in this report is that EF N chimeras also require additional acidic residue density in the 20s cassette; however, this requirement for more rapid translocation kinetics holds even under a pure Δψ driving force. The requirement is counterintuitive because the relationship expected between a purely Δψ-driven process and charge should rather be a preference for cationic residues. Because the acidic residue requirement in the 20s cassette is driving force independent, we surmised that the electric field acting on the negatively charged region is not purely derived from the Δψ (as that would create forces opposite in sign to productive translocation) but rather from charged residues residing inside the PA channel. Simplified electrostatic modeling of the PA channel β barrel reveals two strong oppositely charged electrostatic barriers/wells are present depending upon the identity of the test charge used ( Fig. 5 A ). We started with the β barrel because the structure is well supported by numerous studies ( 19 , 28 , 29 ). The electrostatic features we identified in the β barrel are produced by residues pointing into the lumen of the barrel and residues on the outside of the barrel. We mutated various residues in the β barrel in clusters to investigate their role in the translocation mechanism. The contribution of these charged residue mutations are, of course, amplified by the 7- to 8-fold nature of the oligomer. Based on our electrostatic modeling, PA bot (which removed 4 charges per monomer, 2 positive and 2 negative) will have very modest effects on the electrostatic energy landscape; however, PA top (which removed 3 negative charges) is expected to diminish the anion-repulsive barrier ( Fig. 5 A ). We hypothesized that this would shift the ion selectivity and confirmed this to be true by measuring a reduction in Δψ rev for PA top relative to WT PA and PA bot ( Fig. 5 B ). This result implies that this region is part of the ion-selectivity filter. It should also be stated that other reports have implicated the φ-clamp site as a key electrostatic filter central to ΔpH translocation, albeit it is unclear what charged residue comprises the φ-clamp filter itself ( 18 ). We report here that when the charge-selective filter is removed from the PA top mutant, both substrate docking and translocation are defective ( Fig. 5 , C and D ). The inability to properly dock LF N argues that a clamping or latching feature in the channel is disrupted in the PA top mutation, and we suspect this element in the top of the PA β barrel is a key piece of the electrostatic ratchet expected in our BR model. Model Our BR model ( Fig. 6 ) suggests that ion selectivity plays an important role in PMF-driven translocation ( 6 , 9 ). We expect that a polypeptide chain can pass through the anion-repulsive charge filter once it is partially protonated by the lower cis pH. As this chain moves through the charge filter, the chain may deprotonate in the higher trans pH and become net repulsive to the charge filter. Such changes in the protonation state may also occur in the channel itself, because the residues we have identified in the PA top mutant are also acidic, and this change would only favor the proposed model. At this stage, the filter acts like a ratchet and holds the chain in a way that limits retrotranslocation. An entropic tension develops in the leading sequence and favors further substrate unfolding of the lagging folded domain ( 9 ). In our current model, based upon the recent discovery of the helix stabilizing cleft, the α clamp, we propose that the helical structure can be stabilized inside of the channel. The transition from helix → random coil is highly favorable entropically, and thus should tend to thermodynamically drive the translocation of the chain from inside the channel to outside the chain during the deprotonation phase. Some coordination with the φ clamp site is evident in prior studies, and hence dynamics at the φ clamp site may be required for coordinated peptide movement or protonation state changes in the system ( 6 ). Brownian motion likely underlies the transitions in this system, especially when particular electrostatic barriers are lowered upon protonation/depronation cycles. Such diffusive motion is critical to driving the overall helix-to-coil transition we have proposed. This process can repeat in subsequent sequences and domains until translocation is complete. FIGURE 6. Electrostatic ratchet model. A schematic model of the PA channel ( black outline ) with the indicated α-, φ-, and charge-clamp sites ( blue moveable gates ) based on results described here and elsewhere ( 2 , 4 , 6 – 9 ). The folded substrate domains from LF are indicated as gray circles on the top surface of the channel, where its amino-terminal leader sequence is shown as a thick gray line . The α clamp may nucleate the helical structure into the channel, where the φ clamp can grip the amino-terminal leader. Protonation of the peptide on the lower pH side ( cis protonation) converts acidic, charged residues ( red squares ) to neutral ones ( black squares ), allowing for the leader to move past the charge-clamp site via Brownian motion. Deprotonation of these acidic residues on the higher pH side ( trans deprotonation) and an accompanying helix-to-coil transition in the leader are thermodynamically favorable and result in further translocation. The deprotonated sequence is, however, unable to retrotranslocate due to the charge-clamp site. Entropic tension in the upstream folded substrate maintained by the clamp sites leads to domain unfolding. Further cycles complete the translocation of the remaining domains. It is tantalizing to point out that there is also a cation-repulsive site downstream of the anion-repulsive site in the β barrel. This cation-repulsive site will be stabilizing, however, to the formation of deprotonated Glu and Asp residues, favoring their deprotonation effectively. Such an activity would reinforce our BR model. The energy landscape we have computed is consistent with the biphasic nature of the Δ G ‡ versus charge relationship observed in the 20s cassette ( Fig. 4 C ). One barrier prefers negative charge and the other prefers positive charge in the region. Based on these electrostatic features, the channel may hold amino-terminal polycationic substrates, such as His 6 tags ( 41 ), at low driving forces in a peptide-clamped or conductance-blocked stage indefinitely without actually translocating the substrate until a higher cis -positive potential is applied ( 6 , 7 , 9 , 38 ). Many phenomena involving the amino-terminal presequences of LF, EF, and other heterologous substrates likely derive their origins from their interactions with the highly charged β barrel. General Substrate Charge Requirements To address the molecular mechanism of PMF-driven translocation, we traced the source of the differences in the translocation kinetics between LF N and EF N . Previous translocation studies ( 5 , 8 ) and our more controlled re-examination here show that EF N translocates ∼200-fold slower than LF N under a Δψ alone and ∼10-fold slower than LF N under a combined Δψ and ΔpH ( Fig. 2 , B and C ). This phenomenon occurs despite the fact that LF N and EF N have ∼55% sequence similarity, adopt identical folds ( 24 , 26 ), possess similar solution stabilities ( Fig. 2 D ) ( 31 ), and bind to the same location on the PA channel ( 4 , 37 ). Interestingly, whereas LF and EF initiate translocation starting from the amino termini of their homologous LF N and EF N domains, the amino-terminal initiation sequence of these domains is the most divergent sequence in the domain. We anticipated that this region of the sequence was responsible for the differences we observed in their translocation kinetics. Swapping the 40-residue amino terminus of EF N with the homologous 50-residue amino terminus from LF N allows the chimera to translocate as rapidly as LF N . The inability of EF N to utilize the PMF as well as LF N is hence due to sequence differences in the amino-terminal presequence, and therefore, the charged presequence is critical to allowing the substrate to best capture the PMF to drive unfolding and translocation. Within the presequence, we were then able to locate two sequence cassettes, or motifs, required for efficient translocation ( Fig. 3 B ). When additional acidic residues are added within the 20s cassette of EF N , its translocation becomes more LF N -like. Previous studies by Brown et al. ( 9 ) have shown that under a ΔpH driving force, acidic residues are needed in the 20s cassette for efficient translocation, and whereas our studies here support prior observations, they also show that higher acidic residue content in the 20s cassette is favorable under a pure Δψ. Hence the acidic residue-dependent mechanism we observe is independent of the nature of the driving force. This dependence, at first glance, is most unusual because it is opposite to the effect expected for a cis -positive Δψ, and we will expand on this point in detail below. But from this unusual charge requirement, we expect that the electrostatics of the channel itself govern the overall mechanism. We also identified a 40s cassette in the presequence and found it prefers cationic residues. This preference in the 40s cassette is only observed under a pure Δψ driving force, and whereas the 40s cassette is a novel sequence feature, it was expected to exist because a productive Δψ driving force is cis -positive. Broad Sequence Specificity in Protein Translocases The broad sequence specificity we observe for these charged cassettes ( Fig. 4 , C and D ) is similar to the binding preferences of other polypeptide-clamping sites in the PA channel and in other systems ( 2 ). This observation is the case during translocation for several reasons. The sequence complexity is high, meaning the amino acid sequences, which continually pass through the channel, cover an enormous combinatorial sequence space. Also, the conformational and configuration space the translocating chain may explore during translocation is enormous. Levinthal ( 40 ) originally stated that a folding protein would be unable to sample all the possible configurations of the unfolded state in a reasonable time scale, and instead, proteins must fold via a specific pathway. The hydrophobic effect, for example, is likely a key feature that guides many folding pathways. Hence, we propose that broad sequence specificity is key for a protein translocase, because it must process unfolded protein, which may otherwise occupy too many possible states. The charged cassettes we report here again have general electrostatic requirements, but the specific details are far less critical. Other examples of these nonspecific clamping sites in the PA channel include the α clamp and the φ clamp. The φ clamp prefers hydrophobic and aromatic substrates ( 7 ), whereas the α clamp binds most optimally to α-helical structure with minimal sequence specificity ( 4 ). Each clamping site binds broadly to a different type of chemical handle in the translocating chain, where specific hydrogen bonds and salt bridges are noncritical. Polypeptide clamps are critical because forces cannot be applied to the unfolding substrate protein without a fulcrum. Also, competing diffusive and entropic forces in the system scale with the size of the unfolded state; i.e. for an n -residue long unfolded chain where each residue can sample an average of C conformations, the total number of potential configurations scales as C N . Limiting the size of the unfolded chain that is freely diffusible through nonspecific clamping allows the force-generating apparatus to focus more efficiently on producing directional motion and mechanically unfolding the substrate. The downside to nonspecifically clamping the chain becomes immediately apparent; because when interactions are too tight translocation should become impeded. However, we have proposed instead that clamping sites are dynamic, and the chain is continually bound and released during translocation, and hence such events would reduce diffusive entropic costs, improve energy transduction and force generation, and lower the overall barriers to translocation. Role of Channel Electrostatics in Translocation Previous work by Brown et al. ( 9 ) has shown that sites within the 20s cassette of LF N were optimal for the placement of acidic residues when translocation is driven by a ΔpH. The key finding in this report is that EF N chimeras also require additional acidic residue density in the 20s cassette; however, this requirement for more rapid translocation kinetics holds even under a pure Δψ driving force. The requirement is counterintuitive because the relationship expected between a purely Δψ-driven process and charge should rather be a preference for cationic residues. Because the acidic residue requirement in the 20s cassette is driving force independent, we surmised that the electric field acting on the negatively charged region is not purely derived from the Δψ (as that would create forces opposite in sign to productive translocation) but rather from charged residues residing inside the PA channel. Simplified electrostatic modeling of the PA channel β barrel reveals two strong oppositely charged electrostatic barriers/wells are present depending upon the identity of the test charge used ( Fig. 5 A ). We started with the β barrel because the structure is well supported by numerous studies ( 19 , 28 , 29 ). The electrostatic features we identified in the β barrel are produced by residues pointing into the lumen of the barrel and residues on the outside of the barrel. We mutated various residues in the β barrel in clusters to investigate their role in the translocation mechanism. The contribution of these charged residue mutations are, of course, amplified by the 7- to 8-fold nature of the oligomer. Based on our electrostatic modeling, PA bot (which removed 4 charges per monomer, 2 positive and 2 negative) will have very modest effects on the electrostatic energy landscape; however, PA top (which removed 3 negative charges) is expected to diminish the anion-repulsive barrier ( Fig. 5 A ). We hypothesized that this would shift the ion selectivity and confirmed this to be true by measuring a reduction in Δψ rev for PA top relative to WT PA and PA bot ( Fig. 5 B ). This result implies that this region is part of the ion-selectivity filter. It should also be stated that other reports have implicated the φ-clamp site as a key electrostatic filter central to ΔpH translocation, albeit it is unclear what charged residue comprises the φ-clamp filter itself ( 18 ). We report here that when the charge-selective filter is removed from the PA top mutant, both substrate docking and translocation are defective ( Fig. 5 , C and D ). The inability to properly dock LF N argues that a clamping or latching feature in the channel is disrupted in the PA top mutation, and we suspect this element in the top of the PA β barrel is a key piece of the electrostatic ratchet expected in our BR model. Model Our BR model ( Fig. 6 ) suggests that ion selectivity plays an important role in PMF-driven translocation ( 6 , 9 ). We expect that a polypeptide chain can pass through the anion-repulsive charge filter once it is partially protonated by the lower cis pH. As this chain moves through the charge filter, the chain may deprotonate in the higher trans pH and become net repulsive to the charge filter. Such changes in the protonation state may also occur in the channel itself, because the residues we have identified in the PA top mutant are also acidic, and this change would only favor the proposed model. At this stage, the filter acts like a ratchet and holds the chain in a way that limits retrotranslocation. An entropic tension develops in the leading sequence and favors further substrate unfolding of the lagging folded domain ( 9 ). In our current model, based upon the recent discovery of the helix stabilizing cleft, the α clamp, we propose that the helical structure can be stabilized inside of the channel. The transition from helix → random coil is highly favorable entropically, and thus should tend to thermodynamically drive the translocation of the chain from inside the channel to outside the chain during the deprotonation phase. Some coordination with the φ clamp site is evident in prior studies, and hence dynamics at the φ clamp site may be required for coordinated peptide movement or protonation state changes in the system ( 6 ). Brownian motion likely underlies the transitions in this system, especially when particular electrostatic barriers are lowered upon protonation/depronation cycles. Such diffusive motion is critical to driving the overall helix-to-coil transition we have proposed. This process can repeat in subsequent sequences and domains until translocation is complete. FIGURE 6. Electrostatic ratchet model. A schematic model of the PA channel ( black outline ) with the indicated α-, φ-, and charge-clamp sites ( blue moveable gates ) based on results described here and elsewhere ( 2 , 4 , 6 – 9 ). The folded substrate domains from LF are indicated as gray circles on the top surface of the channel, where its amino-terminal leader sequence is shown as a thick gray line . The α clamp may nucleate the helical structure into the channel, where the φ clamp can grip the amino-terminal leader. Protonation of the peptide on the lower pH side ( cis protonation) converts acidic, charged residues ( red squares ) to neutral ones ( black squares ), allowing for the leader to move past the charge-clamp site via Brownian motion. Deprotonation of these acidic residues on the higher pH side ( trans deprotonation) and an accompanying helix-to-coil transition in the leader are thermodynamically favorable and result in further translocation. The deprotonated sequence is, however, unable to retrotranslocate due to the charge-clamp site. Entropic tension in the upstream folded substrate maintained by the clamp sites leads to domain unfolding. Further cycles complete the translocation of the remaining domains. It is tantalizing to point out that there is also a cation-repulsive site downstream of the anion-repulsive site in the β barrel. This cation-repulsive site will be stabilizing, however, to the formation of deprotonated Glu and Asp residues, favoring their deprotonation effectively. Such an activity would reinforce our BR model. The energy landscape we have computed is consistent with the biphasic nature of the Δ G ‡ versus charge relationship observed in the 20s cassette ( Fig. 4 C ). One barrier prefers negative charge and the other prefers positive charge in the region. Based on these electrostatic features, the channel may hold amino-terminal polycationic substrates, such as His 6 tags ( 41 ), at low driving forces in a peptide-clamped or conductance-blocked stage indefinitely without actually translocating the substrate until a higher cis -positive potential is applied ( 6 , 7 , 9 , 38 ). Many phenomena involving the amino-terminal presequences of LF, EF, and other heterologous substrates likely derive their origins from their interactions with the highly charged β barrel. General Substrate Charge Requirements To address the molecular mechanism of PMF-driven translocation, we traced the source of the differences in the translocation kinetics between LF N and EF N . Previous translocation studies ( 5 , 8 ) and our more controlled re-examination here show that EF N translocates ∼200-fold slower than LF N under a Δψ alone and ∼10-fold slower than LF N under a combined Δψ and ΔpH ( Fig. 2 , B and C ). This phenomenon occurs despite the fact that LF N and EF N have ∼55% sequence similarity, adopt identical folds ( 24 , 26 ), possess similar solution stabilities ( Fig. 2 D ) ( 31 ), and bind to the same location on the PA channel ( 4 , 37 ). Interestingly, whereas LF and EF initiate translocation starting from the amino termini of their homologous LF N and EF N domains, the amino-terminal initiation sequence of these domains is the most divergent sequence in the domain. We anticipated that this region of the sequence was responsible for the differences we observed in their translocation kinetics. Swapping the 40-residue amino terminus of EF N with the homologous 50-residue amino terminus from LF N allows the chimera to translocate as rapidly as LF N . The inability of EF N to utilize the PMF as well as LF N is hence due to sequence differences in the amino-terminal presequence, and therefore, the charged presequence is critical to allowing the substrate to best capture the PMF to drive unfolding and translocation. Within the presequence, we were then able to locate two sequence cassettes, or motifs, required for efficient translocation ( Fig. 3 B ). When additional acidic residues are added within the 20s cassette of EF N , its translocation becomes more LF N -like. Previous studies by Brown et al. ( 9 ) have shown that under a ΔpH driving force, acidic residues are needed in the 20s cassette for efficient translocation, and whereas our studies here support prior observations, they also show that higher acidic residue content in the 20s cassette is favorable under a pure Δψ. Hence the acidic residue-dependent mechanism we observe is independent of the nature of the driving force. This dependence, at first glance, is most unusual because it is opposite to the effect expected for a cis -positive Δψ, and we will expand on this point in detail below. But from this unusual charge requirement, we expect that the electrostatics of the channel itself govern the overall mechanism. We also identified a 40s cassette in the presequence and found it prefers cationic residues. This preference in the 40s cassette is only observed under a pure Δψ driving force, and whereas the 40s cassette is a novel sequence feature, it was expected to exist because a productive Δψ driving force is cis -positive. Broad Sequence Specificity in Protein Translocases The broad sequence specificity we observe for these charged cassettes ( Fig. 4 , C and D ) is similar to the binding preferences of other polypeptide-clamping sites in the PA channel and in other systems ( 2 ). This observation is the case during translocation for several reasons. The sequence complexity is high, meaning the amino acid sequences, which continually pass through the channel, cover an enormous combinatorial sequence space. Also, the conformational and configuration space the translocating chain may explore during translocation is enormous. Levinthal ( 40 ) originally stated that a folding protein would be unable to sample all the possible configurations of the unfolded state in a reasonable time scale, and instead, proteins must fold via a specific pathway. The hydrophobic effect, for example, is likely a key feature that guides many folding pathways. Hence, we propose that broad sequence specificity is key for a protein translocase, because it must process unfolded protein, which may otherwise occupy too many possible states. The charged cassettes we report here again have general electrostatic requirements, but the specific details are far less critical. Other examples of these nonspecific clamping sites in the PA channel include the α clamp and the φ clamp. The φ clamp prefers hydrophobic and aromatic substrates ( 7 ), whereas the α clamp binds most optimally to α-helical structure with minimal sequence specificity ( 4 ). Each clamping site binds broadly to a different type of chemical handle in the translocating chain, where specific hydrogen bonds and salt bridges are noncritical. Polypeptide clamps are critical because forces cannot be applied to the unfolding substrate protein without a fulcrum. Also, competing diffusive and entropic forces in the system scale with the size of the unfolded state; i.e. for an n -residue long unfolded chain where each residue can sample an average of C conformations, the total number of potential configurations scales as C N . Limiting the size of the unfolded chain that is freely diffusible through nonspecific clamping allows the force-generating apparatus to focus more efficiently on producing directional motion and mechanically unfolding the substrate. The downside to nonspecifically clamping the chain becomes immediately apparent; because when interactions are too tight translocation should become impeded. However, we have proposed instead that clamping sites are dynamic, and the chain is continually bound and released during translocation, and hence such events would reduce diffusive entropic costs, improve energy transduction and force generation, and lower the overall barriers to translocation. Role of Channel Electrostatics in Translocation Previous work by Brown et al. ( 9 ) has shown that sites within the 20s cassette of LF N were optimal for the placement of acidic residues when translocation is driven by a ΔpH. The key finding in this report is that EF N chimeras also require additional acidic residue density in the 20s cassette; however, this requirement for more rapid translocation kinetics holds even under a pure Δψ driving force. The requirement is counterintuitive because the relationship expected between a purely Δψ-driven process and charge should rather be a preference for cationic residues. Because the acidic residue requirement in the 20s cassette is driving force independent, we surmised that the electric field acting on the negatively charged region is not purely derived from the Δψ (as that would create forces opposite in sign to productive translocation) but rather from charged residues residing inside the PA channel. Simplified electrostatic modeling of the PA channel β barrel reveals two strong oppositely charged electrostatic barriers/wells are present depending upon the identity of the test charge used ( Fig. 5 A ). We started with the β barrel because the structure is well supported by numerous studies ( 19 , 28 , 29 ). The electrostatic features we identified in the β barrel are produced by residues pointing into the lumen of the barrel and residues on the outside of the barrel. We mutated various residues in the β barrel in clusters to investigate their role in the translocation mechanism. The contribution of these charged residue mutations are, of course, amplified by the 7- to 8-fold nature of the oligomer. Based on our electrostatic modeling, PA bot (which removed 4 charges per monomer, 2 positive and 2 negative) will have very modest effects on the electrostatic energy landscape; however, PA top (which removed 3 negative charges) is expected to diminish the anion-repulsive barrier ( Fig. 5 A ). We hypothesized that this would shift the ion selectivity and confirmed this to be true by measuring a reduction in Δψ rev for PA top relative to WT PA and PA bot ( Fig. 5 B ). This result implies that this region is part of the ion-selectivity filter. It should also be stated that other reports have implicated the φ-clamp site as a key electrostatic filter central to ΔpH translocation, albeit it is unclear what charged residue comprises the φ-clamp filter itself ( 18 ). We report here that when the charge-selective filter is removed from the PA top mutant, both substrate docking and translocation are defective ( Fig. 5 , C and D ). The inability to properly dock LF N argues that a clamping or latching feature in the channel is disrupted in the PA top mutation, and we suspect this element in the top of the PA β barrel is a key piece of the electrostatic ratchet expected in our BR model. Model Our BR model ( Fig. 6 ) suggests that ion selectivity plays an important role in PMF-driven translocation ( 6 , 9 ). We expect that a polypeptide chain can pass through the anion-repulsive charge filter once it is partially protonated by the lower cis pH. As this chain moves through the charge filter, the chain may deprotonate in the higher trans pH and become net repulsive to the charge filter. Such changes in the protonation state may also occur in the channel itself, because the residues we have identified in the PA top mutant are also acidic, and this change would only favor the proposed model. At this stage, the filter acts like a ratchet and holds the chain in a way that limits retrotranslocation. An entropic tension develops in the leading sequence and favors further substrate unfolding of the lagging folded domain ( 9 ). In our current model, based upon the recent discovery of the helix stabilizing cleft, the α clamp, we propose that the helical structure can be stabilized inside of the channel. The transition from helix → random coil is highly favorable entropically, and thus should tend to thermodynamically drive the translocation of the chain from inside the channel to outside the chain during the deprotonation phase. Some coordination with the φ clamp site is evident in prior studies, and hence dynamics at the φ clamp site may be required for coordinated peptide movement or protonation state changes in the system ( 6 ). Brownian motion likely underlies the transitions in this system, especially when particular electrostatic barriers are lowered upon protonation/depronation cycles. Such diffusive motion is critical to driving the overall helix-to-coil transition we have proposed. This process can repeat in subsequent sequences and domains until translocation is complete. FIGURE 6. Electrostatic ratchet model. A schematic model of the PA channel ( black outline ) with the indicated α-, φ-, and charge-clamp sites ( blue moveable gates ) based on results described here and elsewhere ( 2 , 4 , 6 – 9 ). The folded substrate domains from LF are indicated as gray circles on the top surface of the channel, where its amino-terminal leader sequence is shown as a thick gray line . The α clamp may nucleate the helical structure into the channel, where the φ clamp can grip the amino-terminal leader. Protonation of the peptide on the lower pH side ( cis protonation) converts acidic, charged residues ( red squares ) to neutral ones ( black squares ), allowing for the leader to move past the charge-clamp site via Brownian motion. Deprotonation of these acidic residues on the higher pH side ( trans deprotonation) and an accompanying helix-to-coil transition in the leader are thermodynamically favorable and result in further translocation. The deprotonated sequence is, however, unable to retrotranslocate due to the charge-clamp site. Entropic tension in the upstream folded substrate maintained by the clamp sites leads to domain unfolding. Further cycles complete the translocation of the remaining domains. It is tantalizing to point out that there is also a cation-repulsive site downstream of the anion-repulsive site in the β barrel. This cation-repulsive site will be stabilizing, however, to the formation of deprotonated Glu and Asp residues, favoring their deprotonation effectively. Such an activity would reinforce our BR model. The energy landscape we have computed is consistent with the biphasic nature of the Δ G ‡ versus charge relationship observed in the 20s cassette ( Fig. 4 C ). One barrier prefers negative charge and the other prefers positive charge in the region. Based on these electrostatic features, the channel may hold amino-terminal polycationic substrates, such as His 6 tags ( 41 ), at low driving forces in a peptide-clamped or conductance-blocked stage indefinitely without actually translocating the substrate until a higher cis -positive potential is applied ( 6 , 7 , 9 , 38 ). Many phenomena involving the amino-terminal presequences of LF, EF, and other heterologous substrates likely derive their origins from their interactions with the highly charged β barrel. General Substrate Charge Requirements To address the molecular mechanism of PMF-driven translocation, we traced the source of the differences in the translocation kinetics between LF N and EF N . Previous translocation studies ( 5 , 8 ) and our more controlled re-examination here show that EF N translocates ∼200-fold slower than LF N under a Δψ alone and ∼10-fold slower than LF N under a combined Δψ and ΔpH ( Fig. 2 , B and C ). This phenomenon occurs despite the fact that LF N and EF N have ∼55% sequence similarity, adopt identical folds ( 24 , 26 ), possess similar solution stabilities ( Fig. 2 D ) ( 31 ), and bind to the same location on the PA channel ( 4 , 37 ). Interestingly, whereas LF and EF initiate translocation starting from the amino termini of their homologous LF N and EF N domains, the amino-terminal initiation sequence of these domains is the most divergent sequence in the domain. We anticipated that this region of the sequence was responsible for the differences we observed in their translocation kinetics. Swapping the 40-residue amino terminus of EF N with the homologous 50-residue amino terminus from LF N allows the chimera to translocate as rapidly as LF N . The inability of EF N to utilize the PMF as well as LF N is hence due to sequence differences in the amino-terminal presequence, and therefore, the charged presequence is critical to allowing the substrate to best capture the PMF to drive unfolding and translocation. Within the presequence, we were then able to locate two sequence cassettes, or motifs, required for efficient translocation ( Fig. 3 B ). When additional acidic residues are added within the 20s cassette of EF N , its translocation becomes more LF N -like. Previous studies by Brown et al. ( 9 ) have shown that under a ΔpH driving force, acidic residues are needed in the 20s cassette for efficient translocation, and whereas our studies here support prior observations, they also show that higher acidic residue content in the 20s cassette is favorable under a pure Δψ. Hence the acidic residue-dependent mechanism we observe is independent of the nature of the driving force. This dependence, at first glance, is most unusual because it is opposite to the effect expected for a cis -positive Δψ, and we will expand on this point in detail below. But from this unusual charge requirement, we expect that the electrostatics of the channel itself govern the overall mechanism. We also identified a 40s cassette in the presequence and found it prefers cationic residues. This preference in the 40s cassette is only observed under a pure Δψ driving force, and whereas the 40s cassette is a novel sequence feature, it was expected to exist because a productive Δψ driving force is cis -positive. Broad Sequence Specificity in Protein Translocases The broad sequence specificity we observe for these charged cassettes ( Fig. 4 , C and D ) is similar to the binding preferences of other polypeptide-clamping sites in the PA channel and in other systems ( 2 ). This observation is the case during translocation for several reasons. The sequence complexity is high, meaning the amino acid sequences, which continually pass through the channel, cover an enormous combinatorial sequence space. Also, the conformational and configuration space the translocating chain may explore during translocation is enormous. Levinthal ( 40 ) originally stated that a folding protein would be unable to sample all the possible configurations of the unfolded state in a reasonable time scale, and instead, proteins must fold via a specific pathway. The hydrophobic effect, for example, is likely a key feature that guides many folding pathways. Hence, we propose that broad sequence specificity is key for a protein translocase, because it must process unfolded protein, which may otherwise occupy too many possible states. The charged cassettes we report here again have general electrostatic requirements, but the specific details are far less critical. Other examples of these nonspecific clamping sites in the PA channel include the α clamp and the φ clamp. The φ clamp prefers hydrophobic and aromatic substrates ( 7 ), whereas the α clamp binds most optimally to α-helical structure with minimal sequence specificity ( 4 ). Each clamping site binds broadly to a different type of chemical handle in the translocating chain, where specific hydrogen bonds and salt bridges are noncritical. Polypeptide clamps are critical because forces cannot be applied to the unfolding substrate protein without a fulcrum. Also, competing diffusive and entropic forces in the system scale with the size of the unfolded state; i.e. for an n -residue long unfolded chain where each residue can sample an average of C conformations, the total number of potential configurations scales as C N . Limiting the size of the unfolded chain that is freely diffusible through nonspecific clamping allows the force-generating apparatus to focus more efficiently on producing directional motion and mechanically unfolding the substrate. The downside to nonspecifically clamping the chain becomes immediately apparent; because when interactions are too tight translocation should become impeded. However, we have proposed instead that clamping sites are dynamic, and the chain is continually bound and released during translocation, and hence such events would reduce diffusive entropic costs, improve energy transduction and force generation, and lower the overall barriers to translocation. Role of Channel Electrostatics in Translocation Previous work by Brown et al. ( 9 ) has shown that sites within the 20s cassette of LF N were optimal for the placement of acidic residues when translocation is driven by a ΔpH. The key finding in this report is that EF N chimeras also require additional acidic residue density in the 20s cassette; however, this requirement for more rapid translocation kinetics holds even under a pure Δψ driving force. The requirement is counterintuitive because the relationship expected between a purely Δψ-driven process and charge should rather be a preference for cationic residues. Because the acidic residue requirement in the 20s cassette is driving force independent, we surmised that the electric field acting on the negatively charged region is not purely derived from the Δψ (as that would create forces opposite in sign to productive translocation) but rather from charged residues residing inside the PA channel. Simplified electrostatic modeling of the PA channel β barrel reveals two strong oppositely charged electrostatic barriers/wells are present depending upon the identity of the test charge used ( Fig. 5 A ). We started with the β barrel because the structure is well supported by numerous studies ( 19 , 28 , 29 ). The electrostatic features we identified in the β barrel are produced by residues pointing into the lumen of the barrel and residues on the outside of the barrel. We mutated various residues in the β barrel in clusters to investigate their role in the translocation mechanism. The contribution of these charged residue mutations are, of course, amplified by the 7- to 8-fold nature of the oligomer. Based on our electrostatic modeling, PA bot (which removed 4 charges per monomer, 2 positive and 2 negative) will have very modest effects on the electrostatic energy landscape; however, PA top (which removed 3 negative charges) is expected to diminish the anion-repulsive barrier ( Fig. 5 A ). We hypothesized that this would shift the ion selectivity and confirmed this to be true by measuring a reduction in Δψ rev for PA top relative to WT PA and PA bot ( Fig. 5 B ). This result implies that this region is part of the ion-selectivity filter. It should also be stated that other reports have implicated the φ-clamp site as a key electrostatic filter central to ΔpH translocation, albeit it is unclear what charged residue comprises the φ-clamp filter itself ( 18 ). We report here that when the charge-selective filter is removed from the PA top mutant, both substrate docking and translocation are defective ( Fig. 5 , C and D ). The inability to properly dock LF N argues that a clamping or latching feature in the channel is disrupted in the PA top mutation, and we suspect this element in the top of the PA β barrel is a key piece of the electrostatic ratchet expected in our BR model. Model Our BR model ( Fig. 6 ) suggests that ion selectivity plays an important role in PMF-driven translocation ( 6 , 9 ). We expect that a polypeptide chain can pass through the anion-repulsive charge filter once it is partially protonated by the lower cis pH. As this chain moves through the charge filter, the chain may deprotonate in the higher trans pH and become net repulsive to the charge filter. Such changes in the protonation state may also occur in the channel itself, because the residues we have identified in the PA top mutant are also acidic, and this change would only favor the proposed model. At this stage, the filter acts like a ratchet and holds the chain in a way that limits retrotranslocation. An entropic tension develops in the leading sequence and favors further substrate unfolding of the lagging folded domain ( 9 ). In our current model, based upon the recent discovery of the helix stabilizing cleft, the α clamp, we propose that the helical structure can be stabilized inside of the channel. The transition from helix → random coil is highly favorable entropically, and thus should tend to thermodynamically drive the translocation of the chain from inside the channel to outside the chain during the deprotonation phase. Some coordination with the φ clamp site is evident in prior studies, and hence dynamics at the φ clamp site may be required for coordinated peptide movement or protonation state changes in the system ( 6 ). Brownian motion likely underlies the transitions in this system, especially when particular electrostatic barriers are lowered upon protonation/depronation cycles. Such diffusive motion is critical to driving the overall helix-to-coil transition we have proposed. This process can repeat in subsequent sequences and domains until translocation is complete. FIGURE 6. Electrostatic ratchet model. A schematic model of the PA channel ( black outline ) with the indicated α-, φ-, and charge-clamp sites ( blue moveable gates ) based on results described here and elsewhere ( 2 , 4 , 6 – 9 ). The folded substrate domains from LF are indicated as gray circles on the top surface of the channel, where its amino-terminal leader sequence is shown as a thick gray line . The α clamp may nucleate the helical structure into the channel, where the φ clamp can grip the amino-terminal leader. Protonation of the peptide on the lower pH side ( cis protonation) converts acidic, charged residues ( red squares ) to neutral ones ( black squares ), allowing for the leader to move past the charge-clamp site via Brownian motion. Deprotonation of these acidic residues on the higher pH side ( trans deprotonation) and an accompanying helix-to-coil transition in the leader are thermodynamically favorable and result in further translocation. The deprotonated sequence is, however, unable to retrotranslocate due to the charge-clamp site. Entropic tension in the upstream folded substrate maintained by the clamp sites leads to domain unfolding. Further cycles complete the translocation of the remaining domains. It is tantalizing to point out that there is also a cation-repulsive site downstream of the anion-repulsive site in the β barrel. This cation-repulsive site will be stabilizing, however, to the formation of deprotonated Glu and Asp residues, favoring their deprotonation effectively. Such an activity would reinforce our BR model. The energy landscape we have computed is consistent with the biphasic nature of the Δ G ‡ versus charge relationship observed in the 20s cassette ( Fig. 4 C ). One barrier prefers negative charge and the other prefers positive charge in the region. Based on these electrostatic features, the channel may hold amino-terminal polycationic substrates, such as His 6 tags ( 41 ), at low driving forces in a peptide-clamped or conductance-blocked stage indefinitely without actually translocating the substrate until a higher cis -positive potential is applied ( 6 , 7 , 9 , 38 ). Many phenomena involving the amino-terminal presequences of LF, EF, and other heterologous substrates likely derive their origins from their interactions with the highly charged β barrel.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248776/

Thermostable Cross-Protective Subunit Vaccine against Brucella Species

A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis ). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus , B. melitensis , and B. suis . As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10 5 CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413549/

Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system. Introduction Japanese encephalitis virus (JEV), the causal agent of Japanese encephalitis (JE), is a plus-strand RNA virus of the family Flaviviridae ( Vaughn and Hoke, 1992 ; Unni et al., 2011 ). JE is a major public health problem in Asia, causes up to 50,000 encephalitis cases and 10,000 deaths annually in humans ( Campbell et al., 2011 ; Unni et al., 2011 ; Li et al., 2014 ; Tarantola et al., 2014 ; Cappelle et al., 2016 ). With the lack of specific antiviral treatment, vaccination against JEV is crucial for prevention ( Li et al., 2014 ), and is recommended by the World Health Organization (WHO) for the at-risk populations ( WHO, 2015 ). However, the successful implementation of vaccination programs in such areas may depend largely on the cost-effectiveness and safety concerns of the vaccines, similar to the cases for a close relative of JEV, the West Nile virus ( Zohrabian et al., 2006 ; Martina et al., 2010 ; Chen, 2015 ). Currently inactivated JEV vaccines prepared from infected mouse brains (BIKEN or JEVAX) or primary hamster kidney cells and a live attenuated vaccine (SA14-14-2) have been successfully developed to control JEV infection ( Mackenzie et al., 2004 ; Ghosh and Basu, 2009 ). Nevertheless, the use of inactivated JEV vaccine does not confer sufficient long-term immunity to provide effective protection ( Mackenzie et al., 2004 ; Ghosh and Basu, 2009 ). In addition, there are also concerns of side effects ( Shlim and Solomon, 2002 ). Accordingly, WHO has designated JEV vaccines as a high-priority target for development of a new vaccine to fight against JE worldwide ( Tsai, 2000 ). The applications of plants as bioreactors to produce valuable proteins, including vaccines, have attracted considerable interests in recent years ( Takeyama et al., 2015 ). Plants can produce large volumes of products efficiently and can have significant advantages in decreasing manufacturing costs ( Thomas et al., 2011 ; Moustafa et al., 2016 ). The production of foreign proteins can be achieved through stable transformation of the nuclear or chloroplast genomes, or the transient expression mediated by Agrobacterium - or virus-based vector systems ( Lico et al., 2008 ; Chen and Lai, 2013 ). Among these commonly used approaches, virus-based transient expression vector systems are particularly promising for rapid expression of recombinant proteins at levels higher than with stable transgenic plants ( Daniell et al., 2009 ). Plant viral vector systems explore various strategies for recombinant protein expression, including gene insertion or substitution, modular or deconstructed vector design, and protein fusion (peptide display) ( Lico et al., 2008 ). The presentation of heterologous epitopes on plant virus particles is very convenient for peptide-based production of therapeutics and vaccines. The protein fusion strategy has been used extensively to display target peptides on the surface of chimeric virus particles (CVPs) to enhance immunogenicity (e.g., Gerloni et al., 2000 ; Smith et al., 2006 ; Massa et al., 2008 ; Hassani-Mehraban et al., 2015 ), and to facilitate easy antigen purification. In a previous study, we have reported the use of a bamboo mosaic virus (BaMV)-based vector as an effective epitope presentation system, and demonstrated that the foot-and-mouth disease virus (FMDV) VP1 epitopes expressed on BaMV CVPs can effectively induce humoral and cell-mediated immune responses in swine and provide full protection against FMDV challenges in that host ( Yang et al., 2007 ). This BaMV-based CVP vector system presents an alternative approach for the development of a vaccine candidate against JEV. Japanese encephalitis virus RNA contains a single open reading frame (ORF) that codes for a polyprotein which is proteolytically processed into three structural proteins designated envelope (E), membrane (M), and capsid (C) and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 ( Unni et al., 2011 ). The E protein appears to play an important role in viral attachment, membrane fusion for entry into the host cell ( Stiasny and Heinz, 2006 ), virus assembly and maturation, and most notably, inducing virus-neutralizing antibodies ( Mason et al., 1989 ; Kurane, 2002 ). The key domain of E protein, EDIII, forms a β-barrel type structure resembling the immunoglobulin constant domain and can be independently folded as an individual fragment by forming a disulfide bond (between residues 304 and 335) to maintain its conformation ( Wu K. P. et al., 2003 ). Moreover, neutralizing epitopes in the EDIII have been identified on the lateral surface ( Cecilia and Gould, 1991 ; Seif et al., 1995 ; Lin and Wu, 2003 ). Therefore, EDIII represents a potential antigen for producing vaccine candidates. The use of autonomously replicating viruses as expression vectors provides an attractive means for the transient expression of CVPs displaying JEV EDIII antigens in plants. However, the sizes of the epitope presented in our previous BaMV-based CVP vector was limited to 37 amino acids ( Yang et al., 2007 ), which is also a common barrier encountered by other CVP-based expression systems (e.g., Bendahmane et al., 1999 ; Jiang et al., 2006 ; Uhde-Holzem et al., 2010 ; Zhang et al., 2010 ). For epitopes with larger sizes, such as the EDIII epitope of JEV, or other unfavorable structural features, alternative strategies are required to improve the survival rate and the stability of the fusion proteins. In this study, we aimed to develop a BaMV-based CVP vaccine against JEV by fusing JEV EDIII to BaMV coat protein (CP) and displaying the EDIII epitopes on the surfaces of CVPs. To overcome the size-limitations of the epitope-presentation systems, we have adopted the strategy of Cruz et al. (1996) by inserting the 2A co-translational dissociation sequence from FMDV (designated 2A) to the junction of JEV EDIII and BaMV CP, providing enhanced solidity of the CVPs while retaining the presentation of EDIII epitopes on portions of virion surfaces. Detailed analysis were performed to investigate the genetic stabilities of the chimeric virus and the proportions of EDIII-2A-BaMV CP fusion proteins assembled into CVPs among those produced in plant cells. Immunization assays were also conducted to examine the effectiveness of these chimeric CVPs to stimulate the immune responses in mice. Evidence was provided to support that the BaMV-based CVP may offer an alternative vaccine candidate to elicit the generation of neutralization antibodies in mice. Materials and Methods Construction of Chimeric BaMV Infectious Clone The infectious recombinant constructs used in this study were derived from a mutant BaMV cDNA plasmid, pBS-d35CP ( Yang et al., 2007 ) ( Figure 1A ), in which the N-terminal 35 amino acids of CP have been deleted. The coding sequence of JEV (CH2195LA strain) EDIII region, from nucleotide position 874 to 1206, was amplified with primers 5′ gg actagt accatg gacaaactggccctgaaaggc 3′ and 5′ cgttccagctccagacatt gcggccgc cgtgcttcctgctttgtg 3′ (with JEV EDIII coding sequences italicized, and restriction sites for Spe I, and Not I, respectively, underlined) by PCR using plasmid pET32a/LD3 ( Wu S. C. et al., 2003 ) as the template. The PCR-amplified fragment was purified and inserted into plasmid pBS-d35CP at the Nhe I and Not I site, resulting in plasmid pBJ ( Figure 1A ). The DNA fragment coding for the FMDV 2A peptide (LLNFDLLKLAGDVESNPGP) ( Ryan et al., 1991 ) was amplified by PCR with primers 5′ g gctagcgcggccgcg c tgttgaattttga ccttcttaagcttgcggg 3′ and 5′ cct gggccc c gggtccggggttggactcgacgtct cccgcaagcttaagaagg 3′ (with FMDV 2A coding sequence italicized, restriction sites underlined for Nhe I, Not I, in conjunction, and Psp OMI, respectively, and complementary sequences in boldface). Plasmid pB2A was constructed by inserting the FMDV 2A coding sequence at the 5′-terminus of the truncated CP ORF of pBS-d35CP with proper restriction enzyme digestions ( Figure 1A ). The above-mentioned JEV EDIII coding sequence was inserted into plasmid pB2A at the Nhe I and Not I site to give plasmid pBJ2A ( Figure 1A ). The identities of all plasmids were confirmed by nucleotide sequencing. FIGURE 1 Japanese encephalitis virus (JEV) EDIII is expressed in plants infected with chimeric BaMV. (A) Schematic representation of the recombinant constructs based on BaMV genome. (B) Infectivity and symptom of various recombinant BaMV construct on Chenopodium quinoa . Leaves inoculated with H 2 O (mock) or recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A were shown. The photos were taken at 10 days post-inoculation (dpi). (C) SDS-PAGE separation and immunoblot analysis of proteins extracted from inoculated or systemically infected leaves of N. benthamiana , as indicated on top of each panel. Leaves were H 2 O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A as indicated. Total proteins extracted from inoculated leaves (accounting for 1 mg fresh weight of leaf) from each treatment were separated in a 12% SDS-PAGE (Top panel), and stained with coomassie blue (CB). The proteins were transferred to PVDF membranes and reacted with antisera against BaMV CP (anti-CP, middle panel), or JEV EDIII (anti-EDIII, bottom panel), respectively. The relative molecular weights (in kDa) are given on the left of each panel, and positions of each target proteins on the right. IB, immuno-blot. Preparation of Recombinant EDIII (rEDIII) Japanese encephalitis virus EDIII fragments were obtained from pET32a/LD3 plasmid ( Wu S. C. et al., 2003 ) by digestion with Nco I and Not I, and cloned into plasmid pET21d (Novagen) at the respective sites for over-expression in Escherichia coli . Methods used for expression and purification of rEDIII protein were as previously reported ( Seif et al., 1995 ), except that the E. coli strain BL21(DE3) (Novagen) was transformed with the rEDIII-expression plasmid and grown overnight in LB medium in the presence of ampicillin (50 μg ml -1 ). The cells were then diluted 50-fold in LB medium containing ampicillin and grown at 37°C. The rEDIII protein was further dialyzed against phosphate-buffered saline (PBS). The purified rEDIII was further subjected to raise specific antiserum in rabbits following standard procedures ( Lin and Chen, 1991 ). Protein Analysis of the Infected Plant Tissue and Stability of Chimeras during Sequential Transmission The genetic stability of BJ2A chimeric virus was tested using local-lesion host Chenopodium quinoa , while the systemic movement of the chimeric virus was tested on systemic-infection host Nicotiana benthamiana . The infectious viral cDNA clones of pBS-d35CP, pB2A, pBJ, and pBJ2A were inoculated onto N. benthamiana or C. quinoa as previously reported ( Yang et al., 2007 ). The plants were grown in a greenhouse exposed to normal daylight. After local lesions appeared on the pBJ2A-inoculated leaves of C. quinoa at 10 days post-inoculation (dpi), leaves were excised and ground in deionized H 2 O (1:10; weight:volume). The crude sap was mechanically inoculated to healthy C. quinoa . The above-mentioned procedure was repeated for nine times, and the progeny virus, BJ2A, on C. quinoa leaves was assayed each time to examine the stability of the chimeric virus during successive passages in plants. Total proteins extracted from inoculated leaves were separated by electrophoresis on a 12% polyacrylamide gel containing 1% sodium dodecyl sulfate (SDS-PAGE), and stained with coomassie blue (CB). The proteins were then transferred to PVDF membranes (Millipore) and reacted with antisera against BaMV CP ( Lin and Chen, 1991 ) or rEDIII, respectively. Detection of EDIII in Inoculated Plants by Enzyme-Linked Immunosorbent Assay (ELISA) The plant-made JEV EDIII proteins in C. quinoa leaves inoculated with pBJ2A were examined by indirect ELISA using the rabbit antiserum against rEDIII. ELISA was performed as described previously with minor modifications ( Saejung et al., 2007 ). The bound protein-antibodies were detected with biotin-conjugated goat anti- rabbit IgG using the VECTASTAIN Elite ABC kit (avidin biotinylated peroxidase; Vector Laboratories). Following color development, the absorbance at 450 nm was measured on an ELISA reader (Spectramax M2, Molecular Device, USA). Known amounts of purified JEV rEDIII protein was used to establish the standard curve for quantification. Protein extract from a healthy plant was used as a negative control. BJ2A CVP Purification Chenopodium quinoa was chosen as the host plant for the production of BJ2A CVPs to avoid the potential side effects of nicotine and other alkaloids present in N. benthamiana ( Mishra et al., 2015 ). Leaves of C. quinoa inoculated with pBJ2A were harvested at 10 dpi. The BJ2A CVPs were subsequently purified from the leaves and the yield was determined spectrophotometrically by absorbance at 280 nm as described previously ( Lin and Chen, 1991 ). Purified BJ2A CVPs were dissolved in BE buffer (50 mM Borate, pH 8.0, 1 mM EDTA), then stored at -20°C until used. Chimeric BJ2A virions were separated on a 12% SDS-PAGE. The protein bands corresponding to the EDIII-2A-CP fusion protein or cleaved CP on the gel were quantitated by using the Alpha Imager 2200 V5.04 documentation and analysis system. Immunoelectron Microscopy Methods used for the examination of chimeric BJ2A virions by immunoelectron microscopy were as previously reported ( Lin, 1984 ). Gold-labeled antibodies specific for BaMV CP ( Lin and Chen, 1991 ), JEV EDIII, and pre-immune serum were used in the respective experiments. The grids were finally negatively stained with 2% uranyl acetate and examined with transmission electron microscopy (Philips CM 100 Bio) at 80 KV. Control grids were treated with pre-immune rabbit antiserum. Mouse Immune Response Three groups of 6-week-old female BALB/c ByJ mice, six mice per group, obtained from the National Laboratory Animal Center (Taipei, Taiwan), were immunized by intraperitoneal injection. The care of the animals was provided in accordance with guidelines approved by the animal committee of the Institute of Biomedical Sciences, Academia Sinica. One group was immunized with 200 μg BJ2A CVPs. The second group was immunized with 30 μg rEDIII as the positive control. The third group was injected with saline as the negative control. All the mice were boosted with the same dose on day 12. The primary antigens were emulsified in Freund's complete adjuvant (Difco) and boosters emulsified in Freund's incomplete adjuvant (Sigma). Sera were collected on days 0 and 49. The titers and reactivity of sera were tested using indirect ELISA, indirect immunofluorescence assay and plaque reduction neutralization, described as follows. Analysis of EDIII-Specific Antibody in Mice Sera by ELISA Serum samples were collected by periorbital route and heat-inactivated at 56°C for 30 min. JEV EDIII-specific antibodies in serum samples were analyzed by indirect ELISA as described previously ( Yang et al., 2007 ), except that ELISA plates (Nunc) were coated with rEDIII (1 μg per well) as antigens, and bound antibodies were detected with biotin-conjugated goat anti-mouse IgG (H+L). Following color development, the absorbance at 450 nm was measured on an ELISA reader. For background reactions, mice pre-immune sera were used in the ELISA. Indirect Immunofluorescence Assay To analyze whether the BJ2A CVPs elicited the production of effective JEV EDIII-specific antibodies in the immunized mice, indirect immunofluorescence assay was performed as described previously ( Wu et al., 2002 ), except that BHK-21 cells were infected with JEV (RP-9 strain) and the sera obtained from the immunized mice were pooled and 100-fold diluted. Fluorescence was observed with a Leica fluorescence microscope. Cell nuclei were visualized by 4, 6-diamidino-2 -phenylindole (DAPI) staining in 0.9% sodium chloride. Pictures were taken using an inverted fluorescent microscope (Leica) by double exposure of the same fields with filters for FITC and DAPI. Neutralization Test Neutralizing antibody was assayed by plaque reduction neutralization test (PRNT) in BHK-21 cells as previously described ( Chen et al., 2005 ) with minor modifications. Briefly, serum samples were subjected to a serial twofold dilution in 5% fetal bovine serum (FBS)-PBS on ice. Then, equal volumes of infectious JEV in minimum essential medium (MEM) supplemented with FBS were mixed with the serially diluted serum sample to make a mixture containing approximately 100 pfu of virus per well. The virus-antibody complex was added to six-well plates (in triplicates) containing confluent monolayers of BHK-21 cells. The plates were incubated at 37°C for 1 hr with gentle rocking every 15 min. The wells were then overlaid with 2 ml of 1% methyl cellulose prepared in MEM, supplemented with 5% FBS and incubated at 37°C in 5% CO 2 for 4 days. Plaques were stained with naphthol blue black and counted. The neutralizing antibody titer was calculated as the reciprocal of the highest dilution resulting in a 70% reduction of plaques compared to that of a control of virus without antibody added. Construction of Chimeric BaMV Infectious Clone The infectious recombinant constructs used in this study were derived from a mutant BaMV cDNA plasmid, pBS-d35CP ( Yang et al., 2007 ) ( Figure 1A ), in which the N-terminal 35 amino acids of CP have been deleted. The coding sequence of JEV (CH2195LA strain) EDIII region, from nucleotide position 874 to 1206, was amplified with primers 5′ gg actagt accatg gacaaactggccctgaaaggc 3′ and 5′ cgttccagctccagacatt gcggccgc cgtgcttcctgctttgtg 3′ (with JEV EDIII coding sequences italicized, and restriction sites for Spe I, and Not I, respectively, underlined) by PCR using plasmid pET32a/LD3 ( Wu S. C. et al., 2003 ) as the template. The PCR-amplified fragment was purified and inserted into plasmid pBS-d35CP at the Nhe I and Not I site, resulting in plasmid pBJ ( Figure 1A ). The DNA fragment coding for the FMDV 2A peptide (LLNFDLLKLAGDVESNPGP) ( Ryan et al., 1991 ) was amplified by PCR with primers 5′ g gctagcgcggccgcg c tgttgaattttga ccttcttaagcttgcggg 3′ and 5′ cct gggccc c gggtccggggttggactcgacgtct cccgcaagcttaagaagg 3′ (with FMDV 2A coding sequence italicized, restriction sites underlined for Nhe I, Not I, in conjunction, and Psp OMI, respectively, and complementary sequences in boldface). Plasmid pB2A was constructed by inserting the FMDV 2A coding sequence at the 5′-terminus of the truncated CP ORF of pBS-d35CP with proper restriction enzyme digestions ( Figure 1A ). The above-mentioned JEV EDIII coding sequence was inserted into plasmid pB2A at the Nhe I and Not I site to give plasmid pBJ2A ( Figure 1A ). The identities of all plasmids were confirmed by nucleotide sequencing. FIGURE 1 Japanese encephalitis virus (JEV) EDIII is expressed in plants infected with chimeric BaMV. (A) Schematic representation of the recombinant constructs based on BaMV genome. (B) Infectivity and symptom of various recombinant BaMV construct on Chenopodium quinoa . Leaves inoculated with H 2 O (mock) or recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A were shown. The photos were taken at 10 days post-inoculation (dpi). (C) SDS-PAGE separation and immunoblot analysis of proteins extracted from inoculated or systemically infected leaves of N. benthamiana , as indicated on top of each panel. Leaves were H 2 O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A as indicated. Total proteins extracted from inoculated leaves (accounting for 1 mg fresh weight of leaf) from each treatment were separated in a 12% SDS-PAGE (Top panel), and stained with coomassie blue (CB). The proteins were transferred to PVDF membranes and reacted with antisera against BaMV CP (anti-CP, middle panel), or JEV EDIII (anti-EDIII, bottom panel), respectively. The relative molecular weights (in kDa) are given on the left of each panel, and positions of each target proteins on the right. IB, immuno-blot. Preparation of Recombinant EDIII (rEDIII) Japanese encephalitis virus EDIII fragments were obtained from pET32a/LD3 plasmid ( Wu S. C. et al., 2003 ) by digestion with Nco I and Not I, and cloned into plasmid pET21d (Novagen) at the respective sites for over-expression in Escherichia coli . Methods used for expression and purification of rEDIII protein were as previously reported ( Seif et al., 1995 ), except that the E. coli strain BL21(DE3) (Novagen) was transformed with the rEDIII-expression plasmid and grown overnight in LB medium in the presence of ampicillin (50 μg ml -1 ). The cells were then diluted 50-fold in LB medium containing ampicillin and grown at 37°C. The rEDIII protein was further dialyzed against phosphate-buffered saline (PBS). The purified rEDIII was further subjected to raise specific antiserum in rabbits following standard procedures ( Lin and Chen, 1991 ). Protein Analysis of the Infected Plant Tissue and Stability of Chimeras during Sequential Transmission The genetic stability of BJ2A chimeric virus was tested using local-lesion host Chenopodium quinoa , while the systemic movement of the chimeric virus was tested on systemic-infection host Nicotiana benthamiana . The infectious viral cDNA clones of pBS-d35CP, pB2A, pBJ, and pBJ2A were inoculated onto N. benthamiana or C. quinoa as previously reported ( Yang et al., 2007 ). The plants were grown in a greenhouse exposed to normal daylight. After local lesions appeared on the pBJ2A-inoculated leaves of C. quinoa at 10 days post-inoculation (dpi), leaves were excised and ground in deionized H 2 O (1:10; weight:volume). The crude sap was mechanically inoculated to healthy C. quinoa . The above-mentioned procedure was repeated for nine times, and the progeny virus, BJ2A, on C. quinoa leaves was assayed each time to examine the stability of the chimeric virus during successive passages in plants. Total proteins extracted from inoculated leaves were separated by electrophoresis on a 12% polyacrylamide gel containing 1% sodium dodecyl sulfate (SDS-PAGE), and stained with coomassie blue (CB). The proteins were then transferred to PVDF membranes (Millipore) and reacted with antisera against BaMV CP ( Lin and Chen, 1991 ) or rEDIII, respectively. Detection of EDIII in Inoculated Plants by Enzyme-Linked Immunosorbent Assay (ELISA) The plant-made JEV EDIII proteins in C. quinoa leaves inoculated with pBJ2A were examined by indirect ELISA using the rabbit antiserum against rEDIII. ELISA was performed as described previously with minor modifications ( Saejung et al., 2007 ). The bound protein-antibodies were detected with biotin-conjugated goat anti- rabbit IgG using the VECTASTAIN Elite ABC kit (avidin biotinylated peroxidase; Vector Laboratories). Following color development, the absorbance at 450 nm was measured on an ELISA reader (Spectramax M2, Molecular Device, USA). Known amounts of purified JEV rEDIII protein was used to establish the standard curve for quantification. Protein extract from a healthy plant was used as a negative control. BJ2A CVP Purification Chenopodium quinoa was chosen as the host plant for the production of BJ2A CVPs to avoid the potential side effects of nicotine and other alkaloids present in N. benthamiana ( Mishra et al., 2015 ). Leaves of C. quinoa inoculated with pBJ2A were harvested at 10 dpi. The BJ2A CVPs were subsequently purified from the leaves and the yield was determined spectrophotometrically by absorbance at 280 nm as described previously ( Lin and Chen, 1991 ). Purified BJ2A CVPs were dissolved in BE buffer (50 mM Borate, pH 8.0, 1 mM EDTA), then stored at -20°C until used. Chimeric BJ2A virions were separated on a 12% SDS-PAGE. The protein bands corresponding to the EDIII-2A-CP fusion protein or cleaved CP on the gel were quantitated by using the Alpha Imager 2200 V5.04 documentation and analysis system. Immunoelectron Microscopy Methods used for the examination of chimeric BJ2A virions by immunoelectron microscopy were as previously reported ( Lin, 1984 ). Gold-labeled antibodies specific for BaMV CP ( Lin and Chen, 1991 ), JEV EDIII, and pre-immune serum were used in the respective experiments. The grids were finally negatively stained with 2% uranyl acetate and examined with transmission electron microscopy (Philips CM 100 Bio) at 80 KV. Control grids were treated with pre-immune rabbit antiserum. Mouse Immune Response Three groups of 6-week-old female BALB/c ByJ mice, six mice per group, obtained from the National Laboratory Animal Center (Taipei, Taiwan), were immunized by intraperitoneal injection. The care of the animals was provided in accordance with guidelines approved by the animal committee of the Institute of Biomedical Sciences, Academia Sinica. One group was immunized with 200 μg BJ2A CVPs. The second group was immunized with 30 μg rEDIII as the positive control. The third group was injected with saline as the negative control. All the mice were boosted with the same dose on day 12. The primary antigens were emulsified in Freund's complete adjuvant (Difco) and boosters emulsified in Freund's incomplete adjuvant (Sigma). Sera were collected on days 0 and 49. The titers and reactivity of sera were tested using indirect ELISA, indirect immunofluorescence assay and plaque reduction neutralization, described as follows. Analysis of EDIII-Specific Antibody in Mice Sera by ELISA Serum samples were collected by periorbital route and heat-inactivated at 56°C for 30 min. JEV EDIII-specific antibodies in serum samples were analyzed by indirect ELISA as described previously ( Yang et al., 2007 ), except that ELISA plates (Nunc) were coated with rEDIII (1 μg per well) as antigens, and bound antibodies were detected with biotin-conjugated goat anti-mouse IgG (H+L). Following color development, the absorbance at 450 nm was measured on an ELISA reader. For background reactions, mice pre-immune sera were used in the ELISA. Indirect Immunofluorescence Assay To analyze whether the BJ2A CVPs elicited the production of effective JEV EDIII-specific antibodies in the immunized mice, indirect immunofluorescence assay was performed as described previously ( Wu et al., 2002 ), except that BHK-21 cells were infected with JEV (RP-9 strain) and the sera obtained from the immunized mice were pooled and 100-fold diluted. Fluorescence was observed with a Leica fluorescence microscope. Cell nuclei were visualized by 4, 6-diamidino-2 -phenylindole (DAPI) staining in 0.9% sodium chloride. Pictures were taken using an inverted fluorescent microscope (Leica) by double exposure of the same fields with filters for FITC and DAPI. Neutralization Test Neutralizing antibody was assayed by plaque reduction neutralization test (PRNT) in BHK-21 cells as previously described ( Chen et al., 2005 ) with minor modifications. Briefly, serum samples were subjected to a serial twofold dilution in 5% fetal bovine serum (FBS)-PBS on ice. Then, equal volumes of infectious JEV in minimum essential medium (MEM) supplemented with FBS were mixed with the serially diluted serum sample to make a mixture containing approximately 100 pfu of virus per well. The virus-antibody complex was added to six-well plates (in triplicates) containing confluent monolayers of BHK-21 cells. The plates were incubated at 37°C for 1 hr with gentle rocking every 15 min. The wells were then overlaid with 2 ml of 1% methyl cellulose prepared in MEM, supplemented with 5% FBS and incubated at 37°C in 5% CO 2 for 4 days. Plaques were stained with naphthol blue black and counted. The neutralizing antibody titer was calculated as the reciprocal of the highest dilution resulting in a 70% reduction of plaques compared to that of a control of virus without antibody added. Results Production of JEV EDIII Using Chimeric BaMV Vectors in Plants To achieve better yield and stability of JEV EDIII in plants, we explored two different strategies by using BaMV-based vector: (i) direct fusion of JEV EDIII to the N-terminus of truncated BaMV CP, and (ii) insertion of FMDV 2A co-translational dissociation peptide sequence in between JEV EDIII and BaMV CP. The first approach was expected to result in higher yield of the epitope, with JEV EDIII presented on every BaMV CP subunits, at the cost of losing virion stability. The second approach allowed for the production of both the JEV EDIII-2A-BaMV CP recombinant protein and the unfused BaMV CP, leading to the display of JEV EDIII on only portions of the chimeric BaMV virions, with the expected increase in stability of the CVP. Accordingly, two recombinant plasmids, pBJ and pBJ2A, were constructed based on a modified BaMV vector pBS-d35CP ( Figure 1A ). The infectivity of the recombinant viral vectors was assayed in both N. benthamiana and C. quinoa . The result revealed that infection with pBJ2A led to stronger mosaic symptoms than that with pBS-d35CP in N. benthamiana , whereas chlorotic local lesions distinct from those caused by pBS-d35CP were observed after pBJ2A inoculation in C. quinoa ( Figure 1B ). In contrast, inoculation with pBJ did not cause any visible symptom on both N. benthamiana and C. quinoa ( Figure 1B ). To determine whether fusion proteins were produced properly in plants inoculated with the chimeric viruses, total proteins from the inoculated leaves of N. benthamiana infected with distilled water (mock), pBS-d35CP, pB2A, pBJ, or pBJ2A were subjected to analyses by SDS-PAGE ( Figure 1C ) and western blotting assay using BaMV CP-specific antibodies ( Figure 1C , middle panel). As anticipated, no BaMV CP was detected in protein extract of mock-inoculated leaves ( Figure 1C , mock). The FMDV 2A-BaMV CP fusion protein and N-terminal 35-amino-acid truncated BaMV CP ( Figure 1C , middle panel, B2A) were both detected in the protein extract of pB2A-inoculated leaves, which migrated slightly faster than the chimeric CP from pBJ2A-inoculated leaves ( Figure 1C , middle panel, BJ2A). In contrast, no BaMV CP was detected in the pBJ-inoculated leaves ( Figure 1C , middle panel, BJ). To further verify that chimeric CP generated from pBJ2A-inoculated leaves harbored JEV EDIII peptide, western blotting analysis using rEDIII-specific antiserum was performed ( Figure 1C , lower panel). Two proteins of 36.8 and 14.1 kDa were detected by rEDIII-specific antiserum, corresponding to the chimeric EDIII-2A-CP fusion protein and the free JEV EDIII, respectively ( Figure 1C , lower panel, BJ2A). In contrast, no protein band was detected by the rEDIII-specific antiserum in protein extracts from leaves inoculated with pBS-d35CP, pB2A, or pBJ ( Figure 1C , lower panel). Similar results were obtained when total protein extracts from systemic leaves of the infected N. benthamiana were assayed by western blotting using either BaMV CP- or rEDIII-specific antisera, respectively ( Figure 1C , right panel). The above results suggested that the incorporation of FMDV 2A peptide did not affect the replication and systemic movement of the chimeric viruses, and indeed improved the infectivity of pBJ2A as compared to pBJ in both host plants tested. Stability of BJ2A Chimeras during Successive Passages in C. quinoa To examine the stability of the chimeric BJ2A virus during successive passages in plants, infectious recombinant pBJ2A plasmid was inoculated onto C. quinoa leaves to generate the initial inoculum, designated P0, which was then subjected to nine sequential transmissions (P1 through P9) in C. quinoa . The presence of the EDIII-2A-CP fusion protein was monitored at each transfer by western blot analysis using specific antisera. All inoculated leaves developed lesions similar in appearance and number to those observed on P0-infected plants. Results from western blot analyses using antisera specific to BaMV CP or JEV EDIII clearly identified fusion-form BJ2A CP (36.8 kDa), EDIII2A polyprotein (14.1 kDa) and free CP (22.7 kDa) in total protein extracts from all the serially inoculated C. quinoa plants ( Figure 2 ). After quantification of the proteins by using ELISA, the level of JEV EDIII expressed in the leaves of the C. quinoa plants was estimated to be 8.9 ± 4.3 μg mg -1 , corresponding to 0.89 ± 0.43% of total soluble protein (TSP). The result demonstrated that the insertion of foreign coding sequences could be stably maintained in the genome of the chimeric virus over serial passages. FIGURE 2 Analysis of the stability of the chimeric BJ2A over serial passages in C. quinoa plants by SDS-PAGE and immunoblot. Leaves were H 2 O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A, respectively. BJ2A P0 denotes the initial inoculation with the plasmid DNA as inoculum, whereas P1to P9 indicate the 1st to 9th passage using crude leaf sap from P0 as inocula, respectively. SDS-PAGE and immunoblot assays were performed as described in Figure 1C . JEV EDIII Peptides on the Outer Surfaces of CVPs Following the successful observation of the stable expression of both BJ2A fusion proteins and free CP in the inoculated plants, it is important to examine whether the CVPs could be properly assembled with the EDIII peptides presented on the outer surfaces. Results of the electron microscopy observation revealed that the BJ2A CVPs appeared typically filamentous with lengths approximately the same as those of the wild type BaMV virions (480 mm) ( Figures 3A–C ). Immunogold labeling using polyclonal antibodies against EDIII confirmed that the foreign EDIII epitopes were accessible and exposed on the surface of the CPVs ( Figure 3B ). As controls, BJ2A CVPs were labeled with gold-conjugated antiserum against BaMV CP ( Figure 3C ), but not with pre-immune serum ( Figure 3A ). The results demonstrated that the CPs and EDIII-2A-CP fusions can be properly assembled into BJ2A CVPs. FIGURE 3 Immunoelectron microscopy for the identification of JEV EDIII on the surface of BJ2A virus particles. Purified BJ2A virions were incubated with pre-immune rabbit antiserum (A) , or antisera specific for JEV EDIII (B) or BaMV CP (C) , followed by gold-labeled goat anti-rabbit IgG secondary antibody, and subjected to examination by transmission electron microscopy. Scale bars, 100 nm. Induction of Anti-JEV Antibody in Mice Immunized with Purified BJ2A CVPs To determine immunogenicity and efficacy of the CVPs with target proteins presented on the surface, immune responses in mice were assayed as describes in section "Materials and Methods." Blood samples from each group were collected from the periorbital route at days 0 and 49 after immunization. The reactivity to JEV EDIII by sera from BJ2A CVPs-immunized mice was examined by ELISA. The result showed that BJ2A-immunization elicited high levels of anti-EDIII antibodies in sera of the treated mice, similar to those observed for sera from rEDIII-immunized mice as a positive control ( Figure 4A ). The antibody reactivity was weak in the negative control group, which received a combination of saline and adjuvant throughout the experiment ( Figure 4A ). Subsequently, the reactivity of BJ2A CVP-immunized sera was tested by immunofluorescence assay in JEV infected BHK 21 cells. The results showed that sera from BJ2A CVP- or rEDIII-immunized mice recognized the JEV infected BHK 21 cells ( Figure 4B ), demonstrating their ex vivo reactivity. As a negative control, no fluorescence was detected when using sera from the group that received a combination of saline and adjuvant throughout the experiment, nor in non-infected BHK 21 cells ( Figure 4B ). FIGURE 4 Immune response of mice injected intraperitoneally with chimeric virus BJ2A as a vaccine candidate. (A) Determination of immune response by ELISA. Sera from mice immunized with saline, BJ2A, or rEDIII, were subjected to ELISA using rDEIII as the antigen. The titers of antisera were determined by blocking ELISA as described ( Yang et al., 2007 ). The columns represent the mean O.D. (at 450 nm) values obtained with sera from individual mice with standard deviations shown as error bars. (B) Analysis of effective JEV EDIII-specific antibodies by indirect immunofluorescence assay. BHK-21 cells, infected with JEV or uninfected as indicated on the top, were fixed and stained with pooled sera prepared from mice immunized with saline, BJ2A, or rEDIII and an FITC-conjugated secondary antibody, followed by examination with an inverted fluorescent microscope (Leica) (panels denoted by "FITC"). Cell nuclei were stained by DAPI (panels denoted by "DAPI"). To further demonstrate the potential of the BJ2A CVPs as a vaccine candidate, JEV-specific neutralizing antibodies were measured by PRNT, which provides a reasonable immunogenic correlation to protection ( Chen et al., 2005 ). Neutralization efficacy was determined by PRNT 70 titer (serum dilution giving a 70% plaque reduction compared with plaque formation in virus-only controls). Seroconversion was defined as a fourfold or greater increase in PRNT 70 titer ( WHO, 2005 ). Indeed, a fourfold increase of JEV-specific neutralizing antibody titers were detected in pooled sera from mice immunized with BJ2A CVPs (PRNT 70 = 1:160) than from those immunized with saline ( Table 1 ). The result suggested that the BJ2A CVPs could elicit effective immunity against JEV infections. Table 1 Plaque reduction neutralization titers of sera obtained from mice immunized with BJ2A chimeric virus particles. Immunogen Plaque neutralization titers ∗ Saline 1:40 BJ2A 1:160 rEDIII 1:320 ∗ Represented as the serum dilution yielding 70% reduction in plaque number.Pooled sera from six mice were used for the study. Sera from mice immunized with saline or rEDIII were used as negative and positive controls, respectively. Production of JEV EDIII Using Chimeric BaMV Vectors in Plants To achieve better yield and stability of JEV EDIII in plants, we explored two different strategies by using BaMV-based vector: (i) direct fusion of JEV EDIII to the N-terminus of truncated BaMV CP, and (ii) insertion of FMDV 2A co-translational dissociation peptide sequence in between JEV EDIII and BaMV CP. The first approach was expected to result in higher yield of the epitope, with JEV EDIII presented on every BaMV CP subunits, at the cost of losing virion stability. The second approach allowed for the production of both the JEV EDIII-2A-BaMV CP recombinant protein and the unfused BaMV CP, leading to the display of JEV EDIII on only portions of the chimeric BaMV virions, with the expected increase in stability of the CVP. Accordingly, two recombinant plasmids, pBJ and pBJ2A, were constructed based on a modified BaMV vector pBS-d35CP ( Figure 1A ). The infectivity of the recombinant viral vectors was assayed in both N. benthamiana and C. quinoa . The result revealed that infection with pBJ2A led to stronger mosaic symptoms than that with pBS-d35CP in N. benthamiana , whereas chlorotic local lesions distinct from those caused by pBS-d35CP were observed after pBJ2A inoculation in C. quinoa ( Figure 1B ). In contrast, inoculation with pBJ did not cause any visible symptom on both N. benthamiana and C. quinoa ( Figure 1B ). To determine whether fusion proteins were produced properly in plants inoculated with the chimeric viruses, total proteins from the inoculated leaves of N. benthamiana infected with distilled water (mock), pBS-d35CP, pB2A, pBJ, or pBJ2A were subjected to analyses by SDS-PAGE ( Figure 1C ) and western blotting assay using BaMV CP-specific antibodies ( Figure 1C , middle panel). As anticipated, no BaMV CP was detected in protein extract of mock-inoculated leaves ( Figure 1C , mock). The FMDV 2A-BaMV CP fusion protein and N-terminal 35-amino-acid truncated BaMV CP ( Figure 1C , middle panel, B2A) were both detected in the protein extract of pB2A-inoculated leaves, which migrated slightly faster than the chimeric CP from pBJ2A-inoculated leaves ( Figure 1C , middle panel, BJ2A). In contrast, no BaMV CP was detected in the pBJ-inoculated leaves ( Figure 1C , middle panel, BJ). To further verify that chimeric CP generated from pBJ2A-inoculated leaves harbored JEV EDIII peptide, western blotting analysis using rEDIII-specific antiserum was performed ( Figure 1C , lower panel). Two proteins of 36.8 and 14.1 kDa were detected by rEDIII-specific antiserum, corresponding to the chimeric EDIII-2A-CP fusion protein and the free JEV EDIII, respectively ( Figure 1C , lower panel, BJ2A). In contrast, no protein band was detected by the rEDIII-specific antiserum in protein extracts from leaves inoculated with pBS-d35CP, pB2A, or pBJ ( Figure 1C , lower panel). Similar results were obtained when total protein extracts from systemic leaves of the infected N. benthamiana were assayed by western blotting using either BaMV CP- or rEDIII-specific antisera, respectively ( Figure 1C , right panel). The above results suggested that the incorporation of FMDV 2A peptide did not affect the replication and systemic movement of the chimeric viruses, and indeed improved the infectivity of pBJ2A as compared to pBJ in both host plants tested. Stability of BJ2A Chimeras during Successive Passages in C. quinoa To examine the stability of the chimeric BJ2A virus during successive passages in plants, infectious recombinant pBJ2A plasmid was inoculated onto C. quinoa leaves to generate the initial inoculum, designated P0, which was then subjected to nine sequential transmissions (P1 through P9) in C. quinoa . The presence of the EDIII-2A-CP fusion protein was monitored at each transfer by western blot analysis using specific antisera. All inoculated leaves developed lesions similar in appearance and number to those observed on P0-infected plants. Results from western blot analyses using antisera specific to BaMV CP or JEV EDIII clearly identified fusion-form BJ2A CP (36.8 kDa), EDIII2A polyprotein (14.1 kDa) and free CP (22.7 kDa) in total protein extracts from all the serially inoculated C. quinoa plants ( Figure 2 ). After quantification of the proteins by using ELISA, the level of JEV EDIII expressed in the leaves of the C. quinoa plants was estimated to be 8.9 ± 4.3 μg mg -1 , corresponding to 0.89 ± 0.43% of total soluble protein (TSP). The result demonstrated that the insertion of foreign coding sequences could be stably maintained in the genome of the chimeric virus over serial passages. FIGURE 2 Analysis of the stability of the chimeric BJ2A over serial passages in C. quinoa plants by SDS-PAGE and immunoblot. Leaves were H 2 O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A, respectively. BJ2A P0 denotes the initial inoculation with the plasmid DNA as inoculum, whereas P1to P9 indicate the 1st to 9th passage using crude leaf sap from P0 as inocula, respectively. SDS-PAGE and immunoblot assays were performed as described in Figure 1C . JEV EDIII Peptides on the Outer Surfaces of CVPs Following the successful observation of the stable expression of both BJ2A fusion proteins and free CP in the inoculated plants, it is important to examine whether the CVPs could be properly assembled with the EDIII peptides presented on the outer surfaces. Results of the electron microscopy observation revealed that the BJ2A CVPs appeared typically filamentous with lengths approximately the same as those of the wild type BaMV virions (480 mm) ( Figures 3A–C ). Immunogold labeling using polyclonal antibodies against EDIII confirmed that the foreign EDIII epitopes were accessible and exposed on the surface of the CPVs ( Figure 3B ). As controls, BJ2A CVPs were labeled with gold-conjugated antiserum against BaMV CP ( Figure 3C ), but not with pre-immune serum ( Figure 3A ). The results demonstrated that the CPs and EDIII-2A-CP fusions can be properly assembled into BJ2A CVPs. FIGURE 3 Immunoelectron microscopy for the identification of JEV EDIII on the surface of BJ2A virus particles. Purified BJ2A virions were incubated with pre-immune rabbit antiserum (A) , or antisera specific for JEV EDIII (B) or BaMV CP (C) , followed by gold-labeled goat anti-rabbit IgG secondary antibody, and subjected to examination by transmission electron microscopy. Scale bars, 100 nm. Induction of Anti-JEV Antibody in Mice Immunized with Purified BJ2A CVPs To determine immunogenicity and efficacy of the CVPs with target proteins presented on the surface, immune responses in mice were assayed as describes in section "Materials and Methods." Blood samples from each group were collected from the periorbital route at days 0 and 49 after immunization. The reactivity to JEV EDIII by sera from BJ2A CVPs-immunized mice was examined by ELISA. The result showed that BJ2A-immunization elicited high levels of anti-EDIII antibodies in sera of the treated mice, similar to those observed for sera from rEDIII-immunized mice as a positive control ( Figure 4A ). The antibody reactivity was weak in the negative control group, which received a combination of saline and adjuvant throughout the experiment ( Figure 4A ). Subsequently, the reactivity of BJ2A CVP-immunized sera was tested by immunofluorescence assay in JEV infected BHK 21 cells. The results showed that sera from BJ2A CVP- or rEDIII-immunized mice recognized the JEV infected BHK 21 cells ( Figure 4B ), demonstrating their ex vivo reactivity. As a negative control, no fluorescence was detected when using sera from the group that received a combination of saline and adjuvant throughout the experiment, nor in non-infected BHK 21 cells ( Figure 4B ). FIGURE 4 Immune response of mice injected intraperitoneally with chimeric virus BJ2A as a vaccine candidate. (A) Determination of immune response by ELISA. Sera from mice immunized with saline, BJ2A, or rEDIII, were subjected to ELISA using rDEIII as the antigen. The titers of antisera were determined by blocking ELISA as described ( Yang et al., 2007 ). The columns represent the mean O.D. (at 450 nm) values obtained with sera from individual mice with standard deviations shown as error bars. (B) Analysis of effective JEV EDIII-specific antibodies by indirect immunofluorescence assay. BHK-21 cells, infected with JEV or uninfected as indicated on the top, were fixed and stained with pooled sera prepared from mice immunized with saline, BJ2A, or rEDIII and an FITC-conjugated secondary antibody, followed by examination with an inverted fluorescent microscope (Leica) (panels denoted by "FITC"). Cell nuclei were stained by DAPI (panels denoted by "DAPI"). To further demonstrate the potential of the BJ2A CVPs as a vaccine candidate, JEV-specific neutralizing antibodies were measured by PRNT, which provides a reasonable immunogenic correlation to protection ( Chen et al., 2005 ). Neutralization efficacy was determined by PRNT 70 titer (serum dilution giving a 70% plaque reduction compared with plaque formation in virus-only controls). Seroconversion was defined as a fourfold or greater increase in PRNT 70 titer ( WHO, 2005 ). Indeed, a fourfold increase of JEV-specific neutralizing antibody titers were detected in pooled sera from mice immunized with BJ2A CVPs (PRNT 70 = 1:160) than from those immunized with saline ( Table 1 ). The result suggested that the BJ2A CVPs could elicit effective immunity against JEV infections. Table 1 Plaque reduction neutralization titers of sera obtained from mice immunized with BJ2A chimeric virus particles. Immunogen Plaque neutralization titers ∗ Saline 1:40 BJ2A 1:160 rEDIII 1:320 ∗ Represented as the serum dilution yielding 70% reduction in plaque number.Pooled sera from six mice were used for the study. Sera from mice immunized with saline or rEDIII were used as negative and positive controls, respectively. Discussion Circumventing the Epitope Size-Limitation Problems for Virus-Based Vector Systems by the Incorporation of FMDV 2A Peptide The use of plants as safer and less expensive production systems for vaccine antigens has been actively investigated for more than 20 years ( Rybicki, 2010 ), including several plant virus-based expression systems (for excellent reviews, see Lico et al., 2008 ; Rybicki, 2014 ; Chen, 2015 ; Streatfield et al., 2015 ; Shahid and Daniell, 2016 ). The CP genes of viruses are commonly exploited for the development of various strategies, since CP genes are usually expressed with high efficiency and provide natural scaffoldings for the target proteins to be displayed on the surface of CVPs ( Lico et al., 2008 ). However, the production of antigens using plant viral vectors is hindered by several common limitations that stem from the interference of the normal biological functions of viral proteins by the fused peptides. These problems include: (1) the reliability of epitope presentation affected by the nature and sizes of foreign peptides (e.g., Bendahmane et al., 1999 ; Jiang et al., 2006 ; Uhde-Holzem et al., 2010 ; Zhang et al., 2010 ); (2) mutual restriction between encoding recombination virus RNA and the chimeric CP (e.g., Rao, 2006 ; Schneemann, 2006 ), and virus-host interactions (e.g., Porta et al., 2003 ; Ahlquist et al., 2005 ; Chen et al., 2007 ); (3) the stability of the foreign fragments over long-term successive passages (e.g., Porta and Lomonossoff, 1998 ; Porta et al., 2003 ; Lico et al., 2006 ); (4) reduced efficiency for virion assembly caused by special structural features of the chimeric CP (e.g., Canizares et al., 2005 ), and (5) the changes in virion morphology and stability due to cysteine residues in the foreign peptide (e.g., Li et al., 2007 ). Likewise, the construct pBJ, harboring direct fusion between JEV EDIII and BaMV CP, was not infectious, and the fusion protein was not detected in inoculated plants ( Figures 1C , 2 ). In this study, we presented several lines of evidence that these obstacles were circumvented by the incorporation of FMDV 2A co-translational dissociation peptide in between JEV EDIII and BaMV CP. The resulting construct, pBJ2A, was infectious, and generated chimeric virus progeny BJ2A which expressed two fusion proteins, EDIII-2A-CP and EDIII-2A, and one non-recombinant BaMV CP in ( Figures 1C , left panel, 2 ). The chimeric virus BJ2A could infect N. benthamiana systemically and produce JEV EDIII throughout whole plants ( Figure 1C , right panel). The coding sequence of the foreign peptide EDIII-2A was stably maintained in the genome of the chimeric virus BJ2A after nine serial passages in C. quinoa leaves ( Figure 2 ). The fusion protein EDIII-2A-CP and free-form BaMV CP subunits were able to assemble into filamentous BJ2A CVPs ( Figures 3A–C ). Although these JEV EDIII contain two cysteines, which potentially could cause changes in virion morphology and stability ( Li et al., 2007 ), BJ2A CVPs exhibited the same particle morphology as that of the wild type BaMV's ( Figures 3A–C ). Furthermore, JEV EDIII antibody could specifically recognize BJ2A CVPs, indicating that the JEV EDIII peptide was properly presented on the surface of BJ2A CVPs ( Figures 3B,C ). Most importantly, BJ2A CVPs elicited immuno-responses in mice to generate neutralizing antibodies against the infection of JEV ( Figure 4 and Table 1 ). Foot-and-mouth disease virus 2A peptide leads to partial dissociation of the fusion proteins with various efficiency for different fusion constructs ( Donnelly et al., 2001 ). In this study, the incorporation of FMDV 2A peptide allowed production of enough free-form BaMV CP and the EDIII-2A-Cp fusion protein for both the assembly of stable CVPs and proper display of the epitope on the surface. In contrast, the CP produced by the construct pBJ is expected to be the EDIII-CP fusion form only, which might hinder the virion assembly process and result in the loss of infectivity of pBJ (as shown in Figure 1 ). The use of FMDV 2A peptide might have facilitated the virion assembly of the chimeric viruses in planta and likely contributed to the maintenance of the foreign coding sequences over long-term successive passages ( Figure 2 ). Furthermore, the BaMV virion-based epitope-presentation system might provide an adjuvant-like function ( Gerloni et al., 2000 ; Savard et al., 2011 ) and compensated for the partial incorporation of the EDIII-2A-CP in the CVPs. The immunization assays in mice further confirmed that the EDIII peptide was presented in a biologically functional conformation on the surfaces of the CVPs, since the BJ2A CVPs elicited effective immuno-response against JEV infection in mice ( Figure 4 and Table 1 ). These results demonstrated the potential and applicability of the BaMV-based vector system in producing potent vaccine candidates in plants. Comparison with Other Plant-Based Vaccine Candidate Producing Systems against JEV or Related Viruses As mentioned above, plants have been actively explored as effective vaccine candidate-producing systems in recent years. For JEV and related flaviviruses, transgenic and transient expression approaches have been documented ( Martinez et al., 2012 ; Chen and Lai, 2013 ). JEV subunit vaccine candidate produced in transgenic rice has been reported to elicit antigen-specific neutralizing antibodies in mice previously ( Wang et al., 2009 ). However, the yields of JEV E protein expressed in the leaves of transgenic rice were relatively low, amounting to 1.1–1.9 μg mg -1 (corresponding to 0.11–0.19% of TSP) ( Wang et al., 2009 ). For transient expression approach, the EDIII of Dengue virus type 2 (D2EDIII), with only slight differences in structure from that of JEV EDIII ( Chavez et al., 2010 ), was successfully produced in N. benthamiana using tobacco mosaic virus (TMV)-based duplicated-promoter strategy. The yield of D2EDIII protein accounted for 0.28% of TSP ( Saejung et al., 2007 ). For another closely related virus, West Nile virus (WNV), Chen et al. (2011) developed a virus-like particle (VLP) vaccine by fusing the EDIII of WNV to the C-terminus of hepatitis B core antigen (HBcAg) and utilized a geminivirus-based vector to express the recombinant protein in N. benthamiana ( Chen et al., 2011 ). The assembly of the VLP and the effectiveness in inducing strong B and T-cell responses were demonstrated. The yield of the WNV EDIII-HBcAg fusion protein was estimated to be ∼0.35 μg mg -1 fresh leaf weight (FLW). By using the MagnICON vector system, the accumulation level was increased to >1 μg mg -1 FLW ( Chen, 2015 ). In contrast, the BJ2A virus expressed JEV EDIII-2A-CP fusion proteins in the leaves at levels reaching 8.9 ± 4.3 μg mg -1 FLW. Therefore, the BaMV-based vector systems have enabled rapid expression of recombinant proteins at levels comparable to or higher than those produced with previous transgenic or other transient expression approaches in plants. As for immunogenicity, the plant-made D2EDIII elicited only low level of anti-dengue virus antibodies, and no antibody induction was detected when mice were immunized without adjuvant ( Saejung et al., 2007 ), possibly due to the small size of D2EDIII fragment expressed. In comparison, the BJ2A virus displayed the peptide of interest on the surface of assembled CVPs, enhancing immunogenicity ( Table 1 ) by taking advantage of using BaMV CP as the dominant pathogen-derived antigens ( Gerloni et al., 2000 ; Massa et al., 2008 ). VLPs and CVPs have been known to induce strong protective responses in the absence of adjuvants ( Roldao et al., 2010 ). The repetitive display of the epitopes on the quasi-crystalline surface of CVPs may serve as the prime target for B-cell recognition and trigger strong B-cell responses ( Fehr et al., 1998 ). It has been shown that the immunization by using JEV EDIII can elicit the generation of neutralizing antibodies to protect against JEV infection ( Kaur et al., 2002 ). In this study, we have found that BJ2A CVPs could induce IgG-level immune responses in mice ( Figure 4A ). Moreover, the fluorescence staining results indicated that BJ2A CVPs successfully induced the anti-JEV virus antibody in mice ( Figure 4B ). As for the preparation of immunogens, the D2EDIII proteins were purified by immobilized metal ion affinity chromatography ( Saejung et al., 2007 ). In this study, the macromolecular nature of BJ2A CVPs allowed for the development of easy procedures for virion purification and the recovery of high doses of recombinant protein by simple centrifugation. Therefore, BaMV-based epitope presentation strategy provides an efficient alternative for convenient, rapid, and low-cost expression of vaccine candidates. The Advantages of BaMV-Based Epitope Presentation System Plants have been explored as bioreactors for the production of therapeutic proteins, and several plant-produced biopharmaceuticals have been through Phases II and III clinical trials in humans ( Daniell et al., 2009 ; Rybicki, 2010 , 2014 ; Thomas et al., 2011 ; Chen, 2015 ). It has also been shown that the plant-produced CVPs administered to animals intranasally, intraperitoneally or orally are able to induce strong neutralizing immune responses ( Pogue et al., 2002 ; Rybicki, 2010 ). In addition, many achievements have been made using plant virus-based vector with FMDV 2A strategy for expressing foreign proteins as vaccines (e.g., Smolenska et al., 1998 ; O'Brien et al., 2000 ; Marconi et al., 2006 ; Zelada et al., 2006 ; Uhde-Holzem et al., 2010 ). In this study, we demonstrated that the BaMV-based vector system allowed expression of longer peptide, up to 111 amino acids, on CVPs than Potato virus X -based vector did ( Marconi et al., 2006 ; Uhde-Holzem et al., 2010 ). The BaMV-based vector offered some advantages compared to other available systems. Firstly, BaMV has a narrow host range in nature, and therefore is ecologically safer for field use ( Hsu and Lin, 2004 ), minimizing the concern for environmental contaminations. Secondly, by the incorporation of FMDV 2A peptide, BaMV-based epitope-presentation vector was stable over long-term successive passages, as opposed to the previously described systems ( Porta and Lomonossoff, 1998 ; Porta et al., 2003 ; Lico et al., 2006 ). Thirdly, the plant, C. quinoa , used for the production of JEV subunit vaccine candidate is a widely cultivated crop ( Bhargava et al., 2006 ), and poses minimal safety concern in animals. Furthermore, we have resolved the atomic model of the BaMV virion structure by using cryo-electron microscopy recently ( DiMaio et al., 2015 ). This model provides the theoretical basis for the modeling of more candidate epitopes to be presented on BaMV-based vector system by using convenient in silico analyses. Circumventing the Epitope Size-Limitation Problems for Virus-Based Vector Systems by the Incorporation of FMDV 2A Peptide The use of plants as safer and less expensive production systems for vaccine antigens has been actively investigated for more than 20 years ( Rybicki, 2010 ), including several plant virus-based expression systems (for excellent reviews, see Lico et al., 2008 ; Rybicki, 2014 ; Chen, 2015 ; Streatfield et al., 2015 ; Shahid and Daniell, 2016 ). The CP genes of viruses are commonly exploited for the development of various strategies, since CP genes are usually expressed with high efficiency and provide natural scaffoldings for the target proteins to be displayed on the surface of CVPs ( Lico et al., 2008 ). However, the production of antigens using plant viral vectors is hindered by several common limitations that stem from the interference of the normal biological functions of viral proteins by the fused peptides. These problems include: (1) the reliability of epitope presentation affected by the nature and sizes of foreign peptides (e.g., Bendahmane et al., 1999 ; Jiang et al., 2006 ; Uhde-Holzem et al., 2010 ; Zhang et al., 2010 ); (2) mutual restriction between encoding recombination virus RNA and the chimeric CP (e.g., Rao, 2006 ; Schneemann, 2006 ), and virus-host interactions (e.g., Porta et al., 2003 ; Ahlquist et al., 2005 ; Chen et al., 2007 ); (3) the stability of the foreign fragments over long-term successive passages (e.g., Porta and Lomonossoff, 1998 ; Porta et al., 2003 ; Lico et al., 2006 ); (4) reduced efficiency for virion assembly caused by special structural features of the chimeric CP (e.g., Canizares et al., 2005 ), and (5) the changes in virion morphology and stability due to cysteine residues in the foreign peptide (e.g., Li et al., 2007 ). Likewise, the construct pBJ, harboring direct fusion between JEV EDIII and BaMV CP, was not infectious, and the fusion protein was not detected in inoculated plants ( Figures 1C , 2 ). In this study, we presented several lines of evidence that these obstacles were circumvented by the incorporation of FMDV 2A co-translational dissociation peptide in between JEV EDIII and BaMV CP. The resulting construct, pBJ2A, was infectious, and generated chimeric virus progeny BJ2A which expressed two fusion proteins, EDIII-2A-CP and EDIII-2A, and one non-recombinant BaMV CP in ( Figures 1C , left panel, 2 ). The chimeric virus BJ2A could infect N. benthamiana systemically and produce JEV EDIII throughout whole plants ( Figure 1C , right panel). The coding sequence of the foreign peptide EDIII-2A was stably maintained in the genome of the chimeric virus BJ2A after nine serial passages in C. quinoa leaves ( Figure 2 ). The fusion protein EDIII-2A-CP and free-form BaMV CP subunits were able to assemble into filamentous BJ2A CVPs ( Figures 3A–C ). Although these JEV EDIII contain two cysteines, which potentially could cause changes in virion morphology and stability ( Li et al., 2007 ), BJ2A CVPs exhibited the same particle morphology as that of the wild type BaMV's ( Figures 3A–C ). Furthermore, JEV EDIII antibody could specifically recognize BJ2A CVPs, indicating that the JEV EDIII peptide was properly presented on the surface of BJ2A CVPs ( Figures 3B,C ). Most importantly, BJ2A CVPs elicited immuno-responses in mice to generate neutralizing antibodies against the infection of JEV ( Figure 4 and Table 1 ). Foot-and-mouth disease virus 2A peptide leads to partial dissociation of the fusion proteins with various efficiency for different fusion constructs ( Donnelly et al., 2001 ). In this study, the incorporation of FMDV 2A peptide allowed production of enough free-form BaMV CP and the EDIII-2A-Cp fusion protein for both the assembly of stable CVPs and proper display of the epitope on the surface. In contrast, the CP produced by the construct pBJ is expected to be the EDIII-CP fusion form only, which might hinder the virion assembly process and result in the loss of infectivity of pBJ (as shown in Figure 1 ). The use of FMDV 2A peptide might have facilitated the virion assembly of the chimeric viruses in planta and likely contributed to the maintenance of the foreign coding sequences over long-term successive passages ( Figure 2 ). Furthermore, the BaMV virion-based epitope-presentation system might provide an adjuvant-like function ( Gerloni et al., 2000 ; Savard et al., 2011 ) and compensated for the partial incorporation of the EDIII-2A-CP in the CVPs. The immunization assays in mice further confirmed that the EDIII peptide was presented in a biologically functional conformation on the surfaces of the CVPs, since the BJ2A CVPs elicited effective immuno-response against JEV infection in mice ( Figure 4 and Table 1 ). These results demonstrated the potential and applicability of the BaMV-based vector system in producing potent vaccine candidates in plants. Comparison with Other Plant-Based Vaccine Candidate Producing Systems against JEV or Related Viruses As mentioned above, plants have been actively explored as effective vaccine candidate-producing systems in recent years. For JEV and related flaviviruses, transgenic and transient expression approaches have been documented ( Martinez et al., 2012 ; Chen and Lai, 2013 ). JEV subunit vaccine candidate produced in transgenic rice has been reported to elicit antigen-specific neutralizing antibodies in mice previously ( Wang et al., 2009 ). However, the yields of JEV E protein expressed in the leaves of transgenic rice were relatively low, amounting to 1.1–1.9 μg mg -1 (corresponding to 0.11–0.19% of TSP) ( Wang et al., 2009 ). For transient expression approach, the EDIII of Dengue virus type 2 (D2EDIII), with only slight differences in structure from that of JEV EDIII ( Chavez et al., 2010 ), was successfully produced in N. benthamiana using tobacco mosaic virus (TMV)-based duplicated-promoter strategy. The yield of D2EDIII protein accounted for 0.28% of TSP ( Saejung et al., 2007 ). For another closely related virus, West Nile virus (WNV), Chen et al. (2011) developed a virus-like particle (VLP) vaccine by fusing the EDIII of WNV to the C-terminus of hepatitis B core antigen (HBcAg) and utilized a geminivirus-based vector to express the recombinant protein in N. benthamiana ( Chen et al., 2011 ). The assembly of the VLP and the effectiveness in inducing strong B and T-cell responses were demonstrated. The yield of the WNV EDIII-HBcAg fusion protein was estimated to be ∼0.35 μg mg -1 fresh leaf weight (FLW). By using the MagnICON vector system, the accumulation level was increased to >1 μg mg -1 FLW ( Chen, 2015 ). In contrast, the BJ2A virus expressed JEV EDIII-2A-CP fusion proteins in the leaves at levels reaching 8.9 ± 4.3 μg mg -1 FLW. Therefore, the BaMV-based vector systems have enabled rapid expression of recombinant proteins at levels comparable to or higher than those produced with previous transgenic or other transient expression approaches in plants. As for immunogenicity, the plant-made D2EDIII elicited only low level of anti-dengue virus antibodies, and no antibody induction was detected when mice were immunized without adjuvant ( Saejung et al., 2007 ), possibly due to the small size of D2EDIII fragment expressed. In comparison, the BJ2A virus displayed the peptide of interest on the surface of assembled CVPs, enhancing immunogenicity ( Table 1 ) by taking advantage of using BaMV CP as the dominant pathogen-derived antigens ( Gerloni et al., 2000 ; Massa et al., 2008 ). VLPs and CVPs have been known to induce strong protective responses in the absence of adjuvants ( Roldao et al., 2010 ). The repetitive display of the epitopes on the quasi-crystalline surface of CVPs may serve as the prime target for B-cell recognition and trigger strong B-cell responses ( Fehr et al., 1998 ). It has been shown that the immunization by using JEV EDIII can elicit the generation of neutralizing antibodies to protect against JEV infection ( Kaur et al., 2002 ). In this study, we have found that BJ2A CVPs could induce IgG-level immune responses in mice ( Figure 4A ). Moreover, the fluorescence staining results indicated that BJ2A CVPs successfully induced the anti-JEV virus antibody in mice ( Figure 4B ). As for the preparation of immunogens, the D2EDIII proteins were purified by immobilized metal ion affinity chromatography ( Saejung et al., 2007 ). In this study, the macromolecular nature of BJ2A CVPs allowed for the development of easy procedures for virion purification and the recovery of high doses of recombinant protein by simple centrifugation. Therefore, BaMV-based epitope presentation strategy provides an efficient alternative for convenient, rapid, and low-cost expression of vaccine candidates. The Advantages of BaMV-Based Epitope Presentation System Plants have been explored as bioreactors for the production of therapeutic proteins, and several plant-produced biopharmaceuticals have been through Phases II and III clinical trials in humans ( Daniell et al., 2009 ; Rybicki, 2010 , 2014 ; Thomas et al., 2011 ; Chen, 2015 ). It has also been shown that the plant-produced CVPs administered to animals intranasally, intraperitoneally or orally are able to induce strong neutralizing immune responses ( Pogue et al., 2002 ; Rybicki, 2010 ). In addition, many achievements have been made using plant virus-based vector with FMDV 2A strategy for expressing foreign proteins as vaccines (e.g., Smolenska et al., 1998 ; O'Brien et al., 2000 ; Marconi et al., 2006 ; Zelada et al., 2006 ; Uhde-Holzem et al., 2010 ). In this study, we demonstrated that the BaMV-based vector system allowed expression of longer peptide, up to 111 amino acids, on CVPs than Potato virus X -based vector did ( Marconi et al., 2006 ; Uhde-Holzem et al., 2010 ). The BaMV-based vector offered some advantages compared to other available systems. Firstly, BaMV has a narrow host range in nature, and therefore is ecologically safer for field use ( Hsu and Lin, 2004 ), minimizing the concern for environmental contaminations. Secondly, by the incorporation of FMDV 2A peptide, BaMV-based epitope-presentation vector was stable over long-term successive passages, as opposed to the previously described systems ( Porta and Lomonossoff, 1998 ; Porta et al., 2003 ; Lico et al., 2006 ). Thirdly, the plant, C. quinoa , used for the production of JEV subunit vaccine candidate is a widely cultivated crop ( Bhargava et al., 2006 ), and poses minimal safety concern in animals. Furthermore, we have resolved the atomic model of the BaMV virion structure by using cryo-electron microscopy recently ( DiMaio et al., 2015 ). This model provides the theoretical basis for the modeling of more candidate epitopes to be presented on BaMV-based vector system by using convenient in silico analyses. Conclusion To our knowledge, this is the first report describing the production of a vaccine candidate of JEV EDIII using plant virus-based vector system. Our results also demonstrated the feasibility of using FMDV 2A peptide to circumvent some commonly encountered problems for plant virus-based epitope presentation systems. This strategy enabled the production of large quantity of both EDIII-2A-CP fusion protein and free CP in plant cells, allowing the self-assembly of stable CVPs using the two forms of CPs. As compared to the construct pBJ, which does not express non-recombinant BaMV CP, the incorporation of 2A peptide improved the infectivity of the chimeric virus BJ2A and might contribute to the enhanced stability over serial passages and the preservation of key structural features of the CVPs. The BaMV-based CVP vaccine successfully induced the generation of neutralizing antibodies against JEV infection. Together, these results demonstrated that the BaMV-based CVP system may serve as an alternative for the production of effective and useful vaccine candidates against JEV infections. Author Contributions Designed the study: T-HC, C-CH, J-TL, N-SL, Y-LLi, and Y-HH. Analyzed the data: T-HC, J-TL, Y-WH, and Y-LLe. Wrote the manuscript: T-HC, C-CH, and Y-HH. Interpreted the data, and revised the manuscript: C-CH, Y-WH, N-SL, Y-LLi, and Y-HH. All authors read, edited and approved the final manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153350/

Passive Monoclonal and Polyclonal Antibody Therapies

Passive antibody therapies have a long history of use. In the 19th century, antibodies from xenographic sources of polyclonal antibodies were used to treat infections (diphtheria). They were used often as protection from infectious agents and toxins. Complications related to their use involved development of immune complexes and severe allergic reactions. As a result, human source plasma for polyclonal antibodies became the preferential source for antibodies. They are used to treat infection, remove toxins, prevent hemolytic disease of the newborn, modify inflammatory reactions, and control autoimmune diseases. Continued improvements in processing decreased the transfusion/infusion transmission of infections. In the late 20th century (∼1986), monoclonal antibodies were developed. The first monoclonal antibodies were of xenographic source and were wrought with problems of immunogenicity. These forms of antibodies did not gain favor until chimerization took pace in the mid-1990s and in 1998 two monoclonal antibodies were approved one to treat respiratory syncytial virus and the other for breast cancers. Further development of humanized and then fully human monoclonal antibodies has led to an evolution of therapies with these agents. Monoclonal antibodies are being researched or approved to treat a multitude of diseases to include oncologic, inflammatory, autoimmune, cardiovascular, respiratory, neurologic, allergic, benign hematologic, infections, orthopedic, coagulopathy, metabolic and to decrease morbidity of disease (diminution of pain), modify disease progression, and potentially anatomic development. In this chapter, we will review the history of use of these passive antibody therapies, their mechanism of action, pharmacologic-therapeutic classification, particular medical indication, adverse reactions, and potential future use of these medications. Passive Polyclonal Antibodies Therapy Passive Polyclonal Antibody Treatment Overview Polyclonal immunoglobulins have been in use since the 19th century to protect against infectious agents, toxins, and disease conditions such as those with an autoimmune etiology. These immunoglobulin preparations are made from pools of selected human donors or animals with high titers of antibodies against viruses and toxins. These antibody treatments provide passive transfer of high titer antibodies that either reduces risk or reduces severity of infection. They are used to prevent hemolytic disease of the newborn and modify inflammatory reactions. Earlier drugs were very nonselective and patients frequently succumbed to infection due to suppression of both antibody-mediated (humoral) and cell-mediated arms of the immune system. Today, the principal approach is to alter lymphocyte function using drugs or antibodies against immune proteins. However, with the advent of human organ and tissue transplantation (e.g., kidney, heart, bone marrow, and/or peripheral blood stem cells) as treatment options, these polyclonal antibody therapies in combination with other treatment regimens are being used to lower the ability of the body's immune system to reject these transplants. However, their use is not without risk, as complications include development of immune complexes and severe allergic reactions. A summary of these polyclonal antibody therapies may be found in Table 16.2. Immunosuppressive Agents: Disease Modifying Antithymocyte globulin (rabbit)/thymoglobulin; antithymocyte globulin (equine)/Atgam Description Rabbit antithymocyte globulin (rATG) and equine antithymocyte globulin (eATG) are purified, pasteurized preparation of lymphocyte depleting polyclonal gamma immunoglobulin (IgG) raised against human thymus lymphocytes in rabbits and horses, respectively. They are used in prevention and/or treatment of renal transplant rejection worldwide. 1 , 2 , 3 , 4 , 5 , 6 , 7 History of antibody use rATG induction in combination with immunosuppressive therapy is more effective in preventing episodes of acute renal graft rejection in adult renal transplant recipients, in recurrent episodes of acute rejection, 8 , 9 and those acute rejections that are not responsive to high-dose corticosteroid therapy than other monoclonal antibody preparations. 10 , 11 rATG recipients had a lower incidence of biopsy-confirmed acute rejection episodes, 12 greater event-free survival up to 10 years posttransplantation, and greater graft survival up to 5 years posttransplantation. 13 Mechanisms of action The exact mechanism of these polyclonal antibodies has not been fully understood. 3 , 4 , 14 , 15 , 16 , 17 , 18 , 19 , 20 However, being polyclonal, they display specificity toward a wide variety of surface antigens (Ags) expressed on T and B-lymphocytes, dendritic cells, natural killer (NK) cells, and endothelial cells. However, T-cell depletion is considered to play a key role by modulating the expression of lymphocyte surface antigens involved in a wide variety of functions such as T-cell activation to endothelial adherence, activation of certain transcription factors, and interference with numerous immune cell processes, such as cytokine production, chemotaxis, endocytosis, cell stimulation, and proliferation. 14 , 15 , 16 , 17 , 18 , 19 , 20 In vitro studies indicate that binding of eATG to cells is generally nonspecific; the drug binds to visceral tissues, including thymus and testis cell membranes and nuclear and cytoplasmic components of tissues such as tonsil, kidney, and liver, 21 and is extensively bound to bone marrow cells, 22 and to other peripheral blood cells besides lymphocytes. 21 Diseases treated As mentioned earlier, both antithymocyte globulins are used for treatment and prevention of acute renal allograft rejection. 2 , 3 , 4 , 5 , 6 , 7 , 8 More rATG recipients have been reported to achieve the endpoint of successful response (return of serum creatinine levels to baseline by end of treatment or within 14 days of treatment initiation). However, among those who achieved a successful response, fewer episodes of recurrent rejection occurred with rATG within 90 days of treatment cessation. 2 eATG is also used for treating moderate-to-severe aplastic anemia in patients who are unsuitable for bone marrow transplantation. 3 , 23 , 24 Adverse effects The most common adverse effects are fever, thrombocytopenia, leukopenia, gastrointestinal disorders, and/or concurrent infection. 1 , 2 Cytomegalovirus (CMV) infection was generally higher with rATG except in high-risk patients. 1 , 25 eATG therapy may result in reactivation of or infection with CMV, herpes simplex virus, 25 or Epstein–Barr virus. 26 The incidence of malignancies is generally lower with rATG therapy. 27 This product is made of equine and human blood components, so it may carry a risk of transmitting infectious agents such as viruses, and theoretically, the Creutzfeldt–Jakob disease (CJD) agent. Update There has been recent evidence that the addition of human anti-T-lymphocyte globulin (ATLG) plus cyclosporine and methotrexate to standard graft-versus-host disease (GVHD) prophylaxis is preferred over standard GVHD prophylaxis alone because it improves the probability of survival without relapse and of chronic GVHD after myeloablative peripheral blood stem-cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor for patients with acute leukemia in remission. Additionally, this therapy provides better quality of life and shorter immunosuppressive treatment compared to standard GVHD prophylaxis without ATLG. 22 Antitoxin and Immune Globulins: Disease Modifying Tetanus immune globulin/Baytet/Hypertet Description Tetanus immune globulin (TIG) is a specific solvent-detergent-treated plasma-derived product obtained from donors immunized with tetanus toxoid. TIG contains tetanus antitoxin that provides temporary passive immunity to individuals who have low or no immunity to the toxin produced by Clostridium tetani . 28 , 29 Mechanisms of action TIG contains tetanus antitoxin antibodies, which neutralize the free form of the powerful exotoxin produced by Clostridium tetani . 28 , 30 TIG can only neutralize unbound exotoxin; it does not affect toxin already bound to nerve endings. 31 Diseases treated TIG is used to provide passive immunity to tetanus as part of a postexposure prophylaxis regimen following an injury in patients whose immunization is incomplete or uncertain or if it has been more than 10 years since last dose of tetanus toxoid. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 Adverse reaction Slight soreness at injection site, mild fever, and rarely sensitization to repeated injections of human immune globulin has been reported. 28 Antitoxin and Immune Globulins: Disease Modifying Cytomegalovirus immune Globulin/Cytogam Description Cytomegalovirus immune globulin IV (CMV-IG) is a purified immune globulin (hyperimmune globulin) that contains immunoglobulin G (IgG) derived from pooled adult human plasma selected for high titers of anti-CMV antibodies. 32 Mechanisms of action CMV-IG provides relatively high concentration of antibodies directed against CMV. It provides prophylaxis against CMV infection or disease in immunocompromised individuals. 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 Results from in vitro studies and mice indicate that anti-CMV antibodies can neutralize the pathogenic properties of CMV. 42 , 43 , 44 As CMV usually targets a population of bone marrow-derived myeloid lineage progenitor cells, antibody-neutralization of the virus alone may not be enough to prevent or make active disease less severe in already CMV-infected individuals. 42 , 44 , 45 , 46 , 47 Disease treated CMV-IG provides passive immunity to individuals who are at risk for primary CMV infection/disease, or secondary CMV disease (reactivation of CMV). 41 , 42 , 44 , 45 , 46 , 48 , 49 , 50 , 51 It is also prescribed for the prophylaxis of CMV disease associated with transplantation of kidney, lung, liver, pancreas, and heart. With the exception of CMV-seronegative recipients of kidneys from CMV-seropositive donors, CMV-IG prophylaxis should be considered in conjunction with ganciclovir. Adverse reactions Most frequent adverse reactions reported are flushing, chills, muscle cramps, back pain, fever, nausea, vomiting, arthralgia, and wheezing. 32 , 34 , 35 , 36 There is a slight risk of hemolysis, as intravenous immunoglobulin (IVIG) products can contain blood group antibodies, which may act as a hemolysin and induce in vivo coating of red blood cells with immunoglobulin, causing a positive direct antiglobulin reaction. Transfusion-related acute lung injury (noncardiogenic pulmonary edema) and thrombotic events have been reported in patients receiving IVIG preparations. 32 Similar to all other products made from human plasma, this CMV-IG also carries the possibility for transmission of blood-borne viral agents and the CJD agent. However, this IVIG is treated with a solvent detergent viral inactivation procedure to inactivate a wide spectrum of lipid-enveloped viruses, including HIV-1, HIV-2, Hepatitis B, and Hepatitis C. Antivenin [ latrodectus mactans ]/black widow spider antivenin—antivenin Micrurus fulvius /eastern and Texas coral snake antivenin—crotalidae polyvalent immune Fab/Crofab Description These antivenins are sterile, nonpyrogenic, purified, and lyophilized preparation of specific venom-neutralizing serum globulins obtained from the blood serum of healthy horses exposed to the venom of black widow spiders and eastern coral snake ( Micrurus fulvius ) venom, respectively. 52 , 53 , 54 , 55 In contrast, crofab is an antivenin made up of ovine Fab (monovalent) immunoglobulin fragments obtained from blood of healthy sheep immunized with North American Crotalinae subfamily of venomous snakes that includes rattlesnakes, copperheads, cottonmouth, or water moccasins. 56 Mechanisms of action Mode of action of these antivenins is unknown. 52 However, they probably act by neutralizing venom of black widow spiders and coral snakes. 54 Crofab is a venom-specific Fab fragment of IgG that works by binding and neutralizing venom toxins, facilitating their redistribution away from target tissues and their elimination from the body. 56 Disease treated These antivenins are indicated for patients with symptoms due to bites by black widow spider ( Latrodectus mactans ) 52 and bites of two genera of coral snakes, that is, Micrurus (including the eastern and Texas varieties) and Micruroides (the Sonoran or Arizona variety), found in southeastern Arizona and southwestern New Mexico. 52 , 57 , 58 , 59 Antivenin Micrurus fulvius (equine origin) is indicated only for treatment and management of adult and pediatric patients exposed to North American crotalid envenomation. 54 Adverse effects Immediate systemic reactions (allergic reactions or anaphylaxis) and death can occur in patients sensitive to antivenin from horse serum. 52 , 60 Most common adverse reactions to crofab are urticaria, rash, nausea, pruritus, and back pain. 61 , 62 High antibody titer influenza fresh frozen plasma Description Use of convalescent (persons who have recovered from a particular infection) donor plasma with high hemagglutination inhibition titer against certain influenza strains has been recommended as a primary therapy for severe respiratory infectious diseases including influenza, severe acute respiratory syndrome, and Middle East respiratory syndrome. 63 History of antibody use A meta-analysis of previous cohort studies during the 1918 influenza pandemic showed a case-fatality rate of 16% among subjects treated with plasma, serum, or whole blood compared to 37% among controls. Similarly, in 2009, a cohort study using convalescent plasma for the treatment of pandemic H1N1 influenza resulted in a mortality of 20% in the treatment group versus 54% in the control group. 64 Mechanisms of action Antiinfluenza convalescent plasma decreases the rate of viral shedding measured by neutralizing antibody titer and hemagglutination inhibition. 65 Both preexisting immunity (previous infections and vaccinations) as well as any immune response occurring after illness onset makes this mechanism of action more complex. Disease classifications treated Influenza, severe acute respiratory syndrome, and Middle East respiratory syndrome. 63 Adverse effects Convalescent plasma seems safe. The serious adverse events reported are related to the underlying influenza, its complications, preexisting comorbidities, and not due to the convalescent plasma usage. High antibody titer ebola fresh frozen plasma Description Antibodies to the Ebola virus (EV) in whole blood or plasma from convalescent donors may be effective in the treatment of EV infection. History of antibody use The World Health Organization (WHO) has stated that convalescent blood or plasma is an option in the treatment of Ebola. 66 In 1999, transfusion of locally collected convalescent blood helped to decrease Ebola mortality. 67 Therefore, WHO has recommended the collection of convalescent plasma to treat patients with Ebola virus infection. Mechanisms of action This fresh frozen plasma (FFP) has high titers of antibodies directed against Ebola virus. 68 Adverse effects Convalescent plasma seems safe with few adverse effects. 69 , 70 Digoxin immune Fab/DigiFab; Digibind Description Digoxin immune Fab is a sterile, purified, lyophilized monovalent preparation of bovine immunoglobulin Fab fragments that binds to digoxin. These Fab fragments are obtained from the blood of healthy sheep immunized with a digoxin derivative, digoxindicarboxymethoxylamine, a digoxin analogue that contains the functionally essential cyclopentaperhydrophenanthrene: lactone ring moiety coupled to keyhole limpet hemocyanin. The final product is prepared by taking the immunoglobulin fraction of the ovine serum, digesting it with papain, and isolating the digoxin-specific Fab fragments by affinity chromatography. 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 Mechanisms of action DigiFab or Digibind have antigen-binding fragments that bind to free digoxin molecules that results in an equilibrium shift away from binding to receptors, thereby reversing the cardiotoxic effects of the glycoside. 71 , 72 , 75 , 76 , 78 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 Subsequently, Fab-digoxin complexes are cleared by the kidney and reticuloendothelial system. Due to papain treatment, the Fab fragments lack the antigenic determinants of the Fc fragment resulting in reduced immunogenicity to patients as opposed to intact immunoglobulin products. 71 , 72 , 75 , 76 , 78 , 79 , 84 , 88 , 89 Diseases treated Digoxin immune Fab is indicated for patients with either life-threatening or potentially life-threatening digoxin toxicity or overdose. 71 , 79 , 90 , 91 , 92 , 93 , 94 , 95 Data from clinical trials have showed that both DigiFab and Digibind reduce levels of free digoxin in the serum to below the limit of assay quantitation for several hours after Fab administration. Adverse reactions Digoxin immune Fab (ovine) generally is well tolerated following intravenous (IV) administration. 71 , 72 , 73 , 76 , 78 Hypokalemia may occur, sometimes developing rapidly in patients receiving digoxin immune Fab (ovine). 71 , 72 , 79 , 96 , 97 DigiFab should not be administered to patients with a known history of hypersensitivity to papaya or papain unless the benefits outweigh the risks. Immune Globulins: Antiinfectious Hepatitis B immune globulin/HepaGam B/nabi-HB/BayHepB/HyperHEP B S/D Description Hepatitis B immune globulin (HBIG) is a specific immune globulin (hyperimmune globulin) that contains antibody to hepatitis B surface antigen (anti-HBs) prepared from plasma of healthy donors with high titer (>1:100,000) of anti-HBs antibody. It provides temporary passive immunity against hepatitis B virus (HBV). 98 , 99 , 100 , 101 , 102 , 103 , 104 HepaGam-B is a solvent/detergent-treated sterile solution of purified gamma globulin containing antibody to HBs antigen that contains high titers of anti-HBs from plasma donated by healthy screened donors. Both HBIG and HepaGam-B are manufactured by a solvent/detergent (S/D) treatment procedure that is effective in inactivating lipid-enveloped viruses such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 and type 2. However, S/D is less effective against nonlipid-enveloped viruses such as hepatitis A virus and parvovirus B-19. 100 , 101 , 104 Mechanisms of action It provides passive immunization for individuals exposed to the hepatitis B virus by binding to the surface antigen and reducing rate of hepatitis B infection. Diseases treated HBIG provides passive prophylactic immunity to HBV infection for prevention of perinatal HBV infection in neonates born to HBs antigen-positive (HBsAg-positive) mothers, 100 , 101 , 102 , 103 , 104 , 105 , 106 for postexposure prophylaxis in susceptible individuals exposed to HBV or HBsAg-positive materials (e.g., blood, plasma, serum), 100 , 101 , 102 , 103 , 104 , 107 , 108 , 109 sexual exposure to HBsAg-positive persons, for household exposure to persons with acute HBV infection, and for prevention of HBV recurrence in liver transplant recipients who are HBsAg-positive (HepaGam-B only). 104 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 HBIG is not indicated for treatment of active hepatitis B infection and is ineffective in the treatment of chronic active hepatitis B infection. 105 Adverse reactions The local adverse reactions that may occur at the site of injection after intramuscular (IM) administration are pain, tenderness, swelling, and erythema. 100 , 101 , 109 The systemic effects that may occur after IM administration are urticaria, angioedema, nausea, vomiting, myalgia, headache, flu- or cold-like symptoms, lightheadedness, and malaise have been reported. 100 , 101 , 104 Varicella zoster immune globulin/VariZIG Summary Varicella zoster immune globulin (VZIG) is a specific immune globulin (hyperimmune globulin). VZIG is prepared from plasma of donors selected for high titers of antibodies to varicella zoster virus (anti-VZV) and used to provide temporary passive immunity against VZV. 118 , 119 , 120 Mechanisms of action VZIG acts by neutralizing varicella zoster virus via high titers of IgG antibodies present in the plasma used. Diseases treated VZIG is used for postexposure prophylaxis of varicella (chickenpox) in individuals who do not have evidence of varicella immunity and are at high risk for severe varicella infection and its complications. These high risk individuals include immunocompromised patients such as neonates whose mothers have signs and symptoms of varicella around the time of delivery (i.e., 5 days before to 2 days after), premature infants born at ≥28 weeks of gestation who are exposed during the neonatal period and whose mothers do not have evidence of immunity, premature infants born at 1:100,000) of anti-HBs antibody. It provides temporary passive immunity against hepatitis B virus (HBV). 98 , 99 , 100 , 101 , 102 , 103 , 104 HepaGam-B is a solvent/detergent-treated sterile solution of purified gamma globulin containing antibody to HBs antigen that contains high titers of anti-HBs from plasma donated by healthy screened donors. Both HBIG and HepaGam-B are manufactured by a solvent/detergent (S/D) treatment procedure that is effective in inactivating lipid-enveloped viruses such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 and type 2. However, S/D is less effective against nonlipid-enveloped viruses such as hepatitis A virus and parvovirus B-19. 100 , 101 , 104 Mechanisms of action It provides passive immunization for individuals exposed to the hepatitis B virus by binding to the surface antigen and reducing rate of hepatitis B infection. Diseases treated HBIG provides passive prophylactic immunity to HBV infection for prevention of perinatal HBV infection in neonates born to HBs antigen-positive (HBsAg-positive) mothers, 100 , 101 , 102 , 103 , 104 , 105 , 106 for postexposure prophylaxis in susceptible individuals exposed to HBV or HBsAg-positive materials (e.g., blood, plasma, serum), 100 , 101 , 102 , 103 , 104 , 107 , 108 , 109 sexual exposure to HBsAg-positive persons, for household exposure to persons with acute HBV infection, and for prevention of HBV recurrence in liver transplant recipients who are HBsAg-positive (HepaGam-B only). 104 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 HBIG is not indicated for treatment of active hepatitis B infection and is ineffective in the treatment of chronic active hepatitis B infection. 105 Adverse reactions The local adverse reactions that may occur at the site of injection after intramuscular (IM) administration are pain, tenderness, swelling, and erythema. 100 , 101 , 109 The systemic effects that may occur after IM administration are urticaria, angioedema, nausea, vomiting, myalgia, headache, flu- or cold-like symptoms, lightheadedness, and malaise have been reported. 100 , 101 , 104 Varicella zoster immune globulin/VariZIG Summary Varicella zoster immune globulin (VZIG) is a specific immune globulin (hyperimmune globulin). VZIG is prepared from plasma of donors selected for high titers of antibodies to varicella zoster virus (anti-VZV) and used to provide temporary passive immunity against VZV. 118 , 119 , 120 Mechanisms of action VZIG acts by neutralizing varicella zoster virus via high titers of IgG antibodies present in the plasma used. Diseases treated VZIG is used for postexposure prophylaxis of varicella (chickenpox) in individuals who do not have evidence of varicella immunity and are at high risk for severe varicella infection and its complications. These high risk individuals include immunocompromised patients such as neonates whose mothers have signs and symptoms of varicella around the time of delivery (i.e., 5 days before to 2 days after), premature infants born at ≥28 weeks of gestation who are exposed during the neonatal period and whose mothers do not have evidence of immunity, premature infants born at 1:100,000) of anti-HBs antibody. It provides temporary passive immunity against hepatitis B virus (HBV). 98 , 99 , 100 , 101 , 102 , 103 , 104 HepaGam-B is a solvent/detergent-treated sterile solution of purified gamma globulin containing antibody to HBs antigen that contains high titers of anti-HBs from plasma donated by healthy screened donors. Both HBIG and HepaGam-B are manufactured by a solvent/detergent (S/D) treatment procedure that is effective in inactivating lipid-enveloped viruses such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 and type 2. However, S/D is less effective against nonlipid-enveloped viruses such as hepatitis A virus and parvovirus B-19. 100 , 101 , 104 Mechanisms of action It provides passive immunization for individuals exposed to the hepatitis B virus by binding to the surface antigen and reducing rate of hepatitis B infection. Diseases treated HBIG provides passive prophylactic immunity to HBV infection for prevention of perinatal HBV infection in neonates born to HBs antigen-positive (HBsAg-positive) mothers, 100 , 101 , 102 , 103 , 104 , 105 , 106 for postexposure prophylaxis in susceptible individuals exposed to HBV or HBsAg-positive materials (e.g., blood, plasma, serum), 100 , 101 , 102 , 103 , 104 , 107 , 108 , 109 sexual exposure to HBsAg-positive persons, for household exposure to persons with acute HBV infection, and for prevention of HBV recurrence in liver transplant recipients who are HBsAg-positive (HepaGam-B only). 104 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 HBIG is not indicated for treatment of active hepatitis B infection and is ineffective in the treatment of chronic active hepatitis B infection. 105 Adverse reactions The local adverse reactions that may occur at the site of injection after intramuscular (IM) administration are pain, tenderness, swelling, and erythema. 100 , 101 , 109 The systemic effects that may occur after IM administration are urticaria, angioedema, nausea, vomiting, myalgia, headache, flu- or cold-like symptoms, lightheadedness, and malaise have been reported. 100 , 101 , 104 Description Hepatitis B immune globulin (HBIG) is a specific immune globulin (hyperimmune globulin) that contains antibody to hepatitis B surface antigen (anti-HBs) prepared from plasma of healthy donors with high titer (>1:100,000) of anti-HBs antibody. It provides temporary passive immunity against hepatitis B virus (HBV). 98 , 99 , 100 , 101 , 102 , 103 , 104 HepaGam-B is a solvent/detergent-treated sterile solution of purified gamma globulin containing antibody to HBs antigen that contains high titers of anti-HBs from plasma donated by healthy screened donors. Both HBIG and HepaGam-B are manufactured by a solvent/detergent (S/D) treatment procedure that is effective in inactivating lipid-enveloped viruses such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 and type 2. However, S/D is less effective against nonlipid-enveloped viruses such as hepatitis A virus and parvovirus B-19. 100 , 101 , 104 Mechanisms of action It provides passive immunization for individuals exposed to the hepatitis B virus by binding to the surface antigen and reducing rate of hepatitis B infection. Diseases treated HBIG provides passive prophylactic immunity to HBV infection for prevention of perinatal HBV infection in neonates born to HBs antigen-positive (HBsAg-positive) mothers, 100 , 101 , 102 , 103 , 104 , 105 , 106 for postexposure prophylaxis in susceptible individuals exposed to HBV or HBsAg-positive materials (e.g., blood, plasma, serum), 100 , 101 , 102 , 103 , 104 , 107 , 108 , 109 sexual exposure to HBsAg-positive persons, for household exposure to persons with acute HBV infection, and for prevention of HBV recurrence in liver transplant recipients who are HBsAg-positive (HepaGam-B only). 104 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 HBIG is not indicated for treatment of active hepatitis B infection and is ineffective in the treatment of chronic active hepatitis B infection. 105 Adverse reactions The local adverse reactions that may occur at the site of injection after intramuscular (IM) administration are pain, tenderness, swelling, and erythema. 100 , 101 , 109 The systemic effects that may occur after IM administration are urticaria, angioedema, nausea, vomiting, myalgia, headache, flu- or cold-like symptoms, lightheadedness, and malaise have been reported. 100 , 101 , 104 Varicella zoster immune globulin/VariZIG Summary Varicella zoster immune globulin (VZIG) is a specific immune globulin (hyperimmune globulin). VZIG is prepared from plasma of donors selected for high titers of antibodies to varicella zoster virus (anti-VZV) and used to provide temporary passive immunity against VZV. 118 , 119 , 120 Mechanisms of action VZIG acts by neutralizing varicella zoster virus via high titers of IgG antibodies present in the plasma used. Diseases treated VZIG is used for postexposure prophylaxis of varicella (chickenpox) in individuals who do not have evidence of varicella immunity and are at high risk for severe varicella infection and its complications. These high risk individuals include immunocompromised patients such as neonates whose mothers have signs and symptoms of varicella around the time of delivery (i.e., 5 days before to 2 days after), premature infants born at ≥28 weeks of gestation who are exposed during the neonatal period and whose mothers do not have evidence of immunity, premature infants born at 55 years old. Up to 40% of these patients can develop relapsing disease. Brentuximab vedotin (Adcentrix) is a chimeric humanized Mab drug conjugate (Mab + linker + payload {IgG1κ +protease cleavage linker + monomethyl auristatin E [MMAE]}) with specificity to CD30 (a cell membrane protein of the tumor necrosis factor receptor superfamily member 8. MMAE is a microtubule-disrupting agent. The combination of this a Mab and drug conjugate disrupts the intracellular microtubule network causing cell cycle arrest at G2/M stage and apoptosis. This medication has a 43% PFS at 30 months. 231 Mab to look out for in the future include Camidanlumab tesirine (ADCT-301) a human Mab (IgG1κ). This Mab has specificity to CD25 (a IL-2 receptor alpha subunit) with a drug conjugate. The drug is released intracellularly and causes DNA interstrand crosslinks. This Mab is in phase I studies to be completed in 2019 for Hodgkin's and non-Hodgkin's T- and B-cell lymphomas. In addition, there are clinical phase I studies against multiple solid tumors to be completed in 2021. 232 , 233 Agents abandoned or not found to be beneficial include apolizumab, denintuzumab mafodotin (HBU-12), iratumumab (MDX060), and lucatumumab (HCD122). 212 , 234 , 235 Anaplastic large cell lymphoma Brentuximab vedotin (Adcentrix) is an FDA-approved medication for patients with refractory or relapsed anaplastic large cell lymphoma who achieved CR. This Mab had 79% OS and 57% PFS at 5 years, with median response duration not reached at time of publication. 236 Breast Cancer Atezolizumab (Tecentriq) is an FcγR binding–deficient, fully humanized Mab (IgG1κ). This Mab binds to programmed death ligand I (PD-L1) to prevent interaction with receptors PD-1 and B7.1 (a costimulatory cell-surface protein), reversing T-cell suppression. Activation of B7.1 can potentially stimulate long-term responses through development of new immunity via priming and activation of T cells in lymph nodes. A lack of FcγR binding decreases ADCC of the T cells enabling more tumor-specific T cell to remain active. This medication was approved by the FDA in 2019 to treat triple negative (estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2) unresectable or metastatic breast cancers. 237 , 238 Colorectal Cancer Bevacizumab (Avastin) is a humanized Mab (IgG1κ) with specificity to vascular endothelial growth factor-a (VEGF-A) that acts as an inhibitor of angiogenesis. It was FDA approved for treatment of colorectal cancer and has recently been approved for multiple other cancers including ovarian, fallopian cancers, renal cell carcinoma, and recurrent glioblastoma multiforme (GBM). 239 , 240 Urothelial Carcinoma Atezolizumab (Tecentriq) is FDA approved as a single agent in urothelial carcinoma and for patients with disease progression despite other chemotherapy treatment. 241 , 242 Nonsmall cell lung cancer Atezolizumab (Tecentriq) is FDA approved as a single agent for nonsmall cell lung cancer (NSCLC). Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, nonsquamous NSCLC. Nivolumab (Opdivo) is an FDA-approved human Mab (IgG4κ) immunoglobulin and blocks PD-1 preventing interaction PD-1 and its ligands PD-L1 and PD-L2. It is used to treat RCC, NSCLC, Hodgkin's lymphoma, melanoma, small cell lung cancer, colorectal cancer, and squamous cell carcinoma of the head and neck. In phase III clinical trials, nivolumab performed better than docetaxel in the treatment of NSCLC. 243 , 244 , 245 Ovarian/cervical fallopian cancer Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, ovarian, cervical, and fallopian cancers after treatment with chemotherapy regimens and surgery. 246 Merkel Cell Carcinoma Merkel cell carcinoma is a rare aggressive cutaneous malignancy caused by infection with polyoma virus and exposure to ultraviolet radiation. This cancer was classically treated with chemotherapeutic agents leading to rare durable responses. Avelumab (Bavencio) is a fully human Mab (IgG1λ) with specificity to PD-L1. This Mab was approved by the FDA for treatment of Merkel cell carcinoma in 2017. Treatment with this Mab increases response rates to about 50% and extended durable response times approximately five times. 247 , 248 This Mab is in clinical trial to treat other solid tumors including but not limited to hepatocellular, ovarian, esophagogastric, colorectal NSCLC, testicular, urothelial, and adrenocortical carcinomas. 249 Neuroblastoma Neuroblastoma is an aggressive tumor of children with a 5-year survival of about 50%. Treatment classically is high-dose intensive chemotherapy, myeloablative chemotherapy with stem cell rescue, and/or irradiation therapy. Dinutuximab (Unituxin) is a chimeric Mab (IgG1κ) with specificity to GD2 ganglioside that has mechanisms of action via CDC and ADCC. This Mab is used in patients who have had at least a partial response to classic therapy. 250 , 251 Glioblastoma Multiforme GBM is the most common malignant primary brain tumor in adults. This disease remains incurable. Bevacizumab (Avastin) is FDA approved for treatment of recurrent GBM as salvage therapy. This medication with chemotherapy increases overall survival by 4 months but as a single agent is not effective. 252 Relatlimab (BMS-986,016) is a human Mab (IgG4κ) with specificity to lymphocyte activation gene 3 (LAG3, CD223) and is in phase I clinical trials to be completed in 2020 for treatment of GBM. 253 Tanibirumab (aka Olinvacimab, TTAC-0001) is a human Mab (IgG1) with specificity to vascular endothelial growth factor receptor-2 (VEFR-2) and is in phase II studies to treat GBM to be completed in 2020. 254 , 255 , 256 Malignant Ascites Catumaxomab (Removab) is a trifunctional rat/murine hybrid antibody (IgG2a/IgG2b). Catumaxomab consists of one "half" (one heavy chain and one light chain) of an antiepithelial cell adhesion molecule (anti-EpCAM) antibody and one-half of an anti-CD3 antibody, so that each molecule of catumaxomab can bind both EpCAM and CD3. In addition, the Fc-region can bind to an Fc receptor on accessory cells such as other antibodies, which has led to calling the drug a trifunctional antibody. This antibody's mechanism of action is through ADCC. It is approved for use in Europe for malignant ascites from ovarian, gastric, colon, pancreatic, breast, and endometrial carcinoma and is a pending review for approval by the FDA. 257 , 258 , 259 , 260 Cutaneous squamous cell carcinoma Cemiplimab (Libtayo) is a human Mab (IgG4) for treatment of cutaneous squamous cell carcinoma (CSCC) that is metastatic or locally advanced and not amenable to surgery. CSCC is second only to basal cell carcinoma as the most common skin cancer. Surgical intervention is not possible in 5% of patients. This Mab offers a treatment with less morbidity than palliative radiation or surgery, and gives an ORR in 50% of these otherwise untreatable patients. There are many additional phase II studies involving this Mab to be completed from 2020 to 23. 261 , 262 Oncology Malignancies can be caused by infectious agents, toxins, or genetic mutations with changes in control of growth, proliferation, or programmed cell death. Historically these have been treated with a variety of radiation therapies to eradicate malignant cells or with chemotherapeutic agents to enhance maturity, decrease proliferation, or cause destruction of cancer cells. In some cases intense high-dose chemotherapy is used to cause cancer remission with stem cell transplants for subsequent rescue. Passive antibody therapy may replace or be additive to other pharmacology therapies and increase chances for complete remission, prolong disease-free survival, and overall survival. B-cell chronic lymphocytic leukemia Rituximab (Rituxan) is a chimeric murine/human Mab (IgG1κ) that binds to CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35 {controlling differentiation and possible calcium ion channel}). Its mechanism of action is not entirely clear and may involve CDC and ADCC. Many studies have shown this antibody to have an additive benefit to standard chemotherapy alone. This antibody has been approved by the FDA to treat chronic lymphocytic leukemia (CLL) since 1997. Nowadays, this medication is often combined with ibritumomab in treating CLL (to be discussed with non-Hodgkin's lymphoma). 202 , 203 Alemtuzumab (Campath) binds to CD52 and is a humanized rat Mab (IgG1κ) binding to receptors on both T and B cells as well as macrophages, NK cells, and neutrophils, leading to CDC and ADCC. The resultant cytopenias lead to a severe immunocompromised state. Alemtuzumab was FDA approved as a single agent in the treatment of B-cell CLL in 2001. 204 Ofatumumab (Ocrevus) is a human Mab (IgG1κ) with CDC that binds to CD20 near the cellular membrane. In phase II studies, this agent had 86% objective response rate (ORR) when used alone and with CHOP therapy had 100% ORR and 62% complete remission (CR); whereas, in phase III trials, this Mab showed ORR of 10% after rituximab relapse. This medication was approved by the FDA to treat CLL in 2009. 205 Monalizumab is a humanized Mab (IgG4κ) that binds to CD94/NKG2A (an inhibitory signal receptor transmitter) on NK cells. Monalizumab demonstrated blockade of NKG2A/HLA-E and restores the ability of NK cells to lyse B cells in vitro. In addition, this Mab was shown to be of benefit in murine models. Ongoing phase I/II studies will be completed in 2019. 206 Otlertuzumab is a humanized Mab fragment (IgG Fab') with specificity to CD37 that induces both ADCC and caspase-independent apoptosis. In a phase II study both better progression-free survival (PFS) and ORR were observed when used with bendamustine compared to bendamustine used alone. 207 Urelumab is a human Mab (IgG4κ) with specificity to CD134 (an immune checkpoint inhibitor). This antibody has completed safety phase I dosing trials. Higher doses lead to significant hepatotoxicity. Safe dosing is now established in clinical phase II studies to be completed in 2020. 208 , 209 Ulocuplumab is a human Mab with specificity to CD184 (CXCR4). In vitro studies showed apoptotic effects via production of oxygen species that was not associated with better caspase activation than AMD3100. Phase I studies were completed in 2014, no manuscripts were found for review. This medication is presently in phase II trials against acute myelocytic leukemia (AML) to be completed in 2021. 210 , 211 Other monoclonal antibodies not demonstrating benefit in clinical trials for CLL include apolizumab, dacetuzumab, and gomiliximab (aka lumiliximab) 212 , 213 , 214 , 215 Acute myelocytic leukemia AML is the leading cause of leukemic mortality in the United States (US). Over the last 10 years therapy has not changed significantly for this disease. Novel therapies have been developed in the last decade, some showing temporal success and some showing a brighter tomorrow. 216 AMG330 is a bispecific T-cell engager (BiTE) antibody with specificity for CD3 and CD33. This Mab is currently in clinical trials to be completed in 2020 for treatment of AML. A BiTE antibody stimulates ADCC (via T cells) in the presence of antigenic targets on cells of interest. In vitro studies have shown effective lysis of AML cells, while in animal studies it has demonstrated significant decrease in tumor burden. 217 IMGN632 is an anti-CD123 antibody complexed to a DNA mono-alkylating agent. In vitro studies showed it had more potency against AML cells than to normal myeloid progenitor cells. In animal models there was an excellent response rate against tumor cells. Ongoing clinical trials will be completed in 2021. 216 Talacotuzumab is a humanized monoclonal antibody (IgG1-2κ) with specificity to interleukin (IL)-3 receptor subunit-α (CD123, a growth and differentiating receptor). This antibody induces ADCC both in vitro and in animal models. Phase III clinical trials were reportedly completed in 2018; published results are forthcoming. 218 Samalizumab is a humanized Mab (IgG2/IgG4κ) with specificity to CD200 (OX-2membrane glycoprotein) is in phase II trials to be completed in 2021. 219 Ficlatuzumab is a humanized Mab (IgG1κ) in a phase I trial to treat refractory/relapsing AML to be completed in 2020. 220 Other Mab not demonstrating benefit in clinical trials or withdrawn following postmarketing for AML include gemtuzumab ozogamicin (FDA approved 2000 withdrawn 2010 secondary to venoocclusive disease) and lintuzumab ( no added benefit over standard chemotherapy). 221 , 222 , 223 B-cell chronic lymphocytic leukemia Rituximab (Rituxan) is a chimeric murine/human Mab (IgG1κ) that binds to CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35 {controlling differentiation and possible calcium ion channel}). Its mechanism of action is not entirely clear and may involve CDC and ADCC. Many studies have shown this antibody to have an additive benefit to standard chemotherapy alone. This antibody has been approved by the FDA to treat chronic lymphocytic leukemia (CLL) since 1997. Nowadays, this medication is often combined with ibritumomab in treating CLL (to be discussed with non-Hodgkin's lymphoma). 202 , 203 Alemtuzumab (Campath) binds to CD52 and is a humanized rat Mab (IgG1κ) binding to receptors on both T and B cells as well as macrophages, NK cells, and neutrophils, leading to CDC and ADCC. The resultant cytopenias lead to a severe immunocompromised state. Alemtuzumab was FDA approved as a single agent in the treatment of B-cell CLL in 2001. 204 Ofatumumab (Ocrevus) is a human Mab (IgG1κ) with CDC that binds to CD20 near the cellular membrane. In phase II studies, this agent had 86% objective response rate (ORR) when used alone and with CHOP therapy had 100% ORR and 62% complete remission (CR); whereas, in phase III trials, this Mab showed ORR of 10% after rituximab relapse. This medication was approved by the FDA to treat CLL in 2009. 205 Monalizumab is a humanized Mab (IgG4κ) that binds to CD94/NKG2A (an inhibitory signal receptor transmitter) on NK cells. Monalizumab demonstrated blockade of NKG2A/HLA-E and restores the ability of NK cells to lyse B cells in vitro. In addition, this Mab was shown to be of benefit in murine models. Ongoing phase I/II studies will be completed in 2019. 206 Otlertuzumab is a humanized Mab fragment (IgG Fab') with specificity to CD37 that induces both ADCC and caspase-independent apoptosis. In a phase II study both better progression-free survival (PFS) and ORR were observed when used with bendamustine compared to bendamustine used alone. 207 Urelumab is a human Mab (IgG4κ) with specificity to CD134 (an immune checkpoint inhibitor). This antibody has completed safety phase I dosing trials. Higher doses lead to significant hepatotoxicity. Safe dosing is now established in clinical phase II studies to be completed in 2020. 208 , 209 Ulocuplumab is a human Mab with specificity to CD184 (CXCR4). In vitro studies showed apoptotic effects via production of oxygen species that was not associated with better caspase activation than AMD3100. Phase I studies were completed in 2014, no manuscripts were found for review. This medication is presently in phase II trials against acute myelocytic leukemia (AML) to be completed in 2021. 210 , 211 Other monoclonal antibodies not demonstrating benefit in clinical trials for CLL include apolizumab, dacetuzumab, and gomiliximab (aka lumiliximab) 212 , 213 , 214 , 215 Acute myelocytic leukemia AML is the leading cause of leukemic mortality in the United States (US). Over the last 10 years therapy has not changed significantly for this disease. Novel therapies have been developed in the last decade, some showing temporal success and some showing a brighter tomorrow. 216 AMG330 is a bispecific T-cell engager (BiTE) antibody with specificity for CD3 and CD33. This Mab is currently in clinical trials to be completed in 2020 for treatment of AML. A BiTE antibody stimulates ADCC (via T cells) in the presence of antigenic targets on cells of interest. In vitro studies have shown effective lysis of AML cells, while in animal studies it has demonstrated significant decrease in tumor burden. 217 IMGN632 is an anti-CD123 antibody complexed to a DNA mono-alkylating agent. In vitro studies showed it had more potency against AML cells than to normal myeloid progenitor cells. In animal models there was an excellent response rate against tumor cells. Ongoing clinical trials will be completed in 2021. 216 Talacotuzumab is a humanized monoclonal antibody (IgG1-2κ) with specificity to interleukin (IL)-3 receptor subunit-α (CD123, a growth and differentiating receptor). This antibody induces ADCC both in vitro and in animal models. Phase III clinical trials were reportedly completed in 2018; published results are forthcoming. 218 Samalizumab is a humanized Mab (IgG2/IgG4κ) with specificity to CD200 (OX-2membrane glycoprotein) is in phase II trials to be completed in 2021. 219 Ficlatuzumab is a humanized Mab (IgG1κ) in a phase I trial to treat refractory/relapsing AML to be completed in 2020. 220 Other Mab not demonstrating benefit in clinical trials or withdrawn following postmarketing for AML include gemtuzumab ozogamicin (FDA approved 2000 withdrawn 2010 secondary to venoocclusive disease) and lintuzumab ( no added benefit over standard chemotherapy). 221 , 222 , 223 Multiple Myeloma Daratumumab (Darzalex) is a human Mab (IgG1κ) with specificity to CD38 (functions reportedly include receptor-mediated adhesion and signaling events, as well as important bifunctional ectoenzymatic activities that contribute to intracellular calcium mobilization. This Mab mechanism of action is thought to induce CDC, ADCC, antibody-dependent cellular phagocytosis, and apoptosis. This medication is used to treat refractory and recurrent multiple myeloma. 224 , 225 Silutuximab (Sylvant) is a chimeric Mab (IgG1κ) with specificity to IL-6. This medication was FDA approved in 2014 for multicentric Castleman's disease (MCD) with HIV negative and HHV-8 negative. There are ongoing studies in phase II clinical trials to be completed in 2019. 226 , 227 B-cell acute lymphoblastic leukemia (B-cell ALL) Blinatumomab (Blincyto) is a mouse double heavy-chain fragment (Murine {scFv - kappa – heavy} – {scFv - heavy – kappa}) with specificity for CD19 and CD3 known as a BiTE. This Mab's mode of action is by directing CD3 + effector memory T cells to CD19 + target cells leading to T-cell activation and B-cell apoptosis. This biologic is used to treat relapsed/refractory cell ALL. In phase III trials event-free survival almost tripled and duration of remission almost doubled. 228 , 229 , 230 Hodgkin's lymphoma Hodgkin's lymphoma is a rare malignancy affecting young adults with a peak incidence in patients >55 years old. Up to 40% of these patients can develop relapsing disease. Brentuximab vedotin (Adcentrix) is a chimeric humanized Mab drug conjugate (Mab + linker + payload {IgG1κ +protease cleavage linker + monomethyl auristatin E [MMAE]}) with specificity to CD30 (a cell membrane protein of the tumor necrosis factor receptor superfamily member 8. MMAE is a microtubule-disrupting agent. The combination of this a Mab and drug conjugate disrupts the intracellular microtubule network causing cell cycle arrest at G2/M stage and apoptosis. This medication has a 43% PFS at 30 months. 231 Mab to look out for in the future include Camidanlumab tesirine (ADCT-301) a human Mab (IgG1κ). This Mab has specificity to CD25 (a IL-2 receptor alpha subunit) with a drug conjugate. The drug is released intracellularly and causes DNA interstrand crosslinks. This Mab is in phase I studies to be completed in 2019 for Hodgkin's and non-Hodgkin's T- and B-cell lymphomas. In addition, there are clinical phase I studies against multiple solid tumors to be completed in 2021. 232 , 233 Agents abandoned or not found to be beneficial include apolizumab, denintuzumab mafodotin (HBU-12), iratumumab (MDX060), and lucatumumab (HCD122). 212 , 234 , 235 Anaplastic large cell lymphoma Brentuximab vedotin (Adcentrix) is an FDA-approved medication for patients with refractory or relapsed anaplastic large cell lymphoma who achieved CR. This Mab had 79% OS and 57% PFS at 5 years, with median response duration not reached at time of publication. 236 B-cell acute lymphoblastic leukemia (B-cell ALL) Blinatumomab (Blincyto) is a mouse double heavy-chain fragment (Murine {scFv - kappa – heavy} – {scFv - heavy – kappa}) with specificity for CD19 and CD3 known as a BiTE. This Mab's mode of action is by directing CD3 + effector memory T cells to CD19 + target cells leading to T-cell activation and B-cell apoptosis. This biologic is used to treat relapsed/refractory cell ALL. In phase III trials event-free survival almost tripled and duration of remission almost doubled. 228 , 229 , 230 Hodgkin's lymphoma Hodgkin's lymphoma is a rare malignancy affecting young adults with a peak incidence in patients >55 years old. Up to 40% of these patients can develop relapsing disease. Brentuximab vedotin (Adcentrix) is a chimeric humanized Mab drug conjugate (Mab + linker + payload {IgG1κ +protease cleavage linker + monomethyl auristatin E [MMAE]}) with specificity to CD30 (a cell membrane protein of the tumor necrosis factor receptor superfamily member 8. MMAE is a microtubule-disrupting agent. The combination of this a Mab and drug conjugate disrupts the intracellular microtubule network causing cell cycle arrest at G2/M stage and apoptosis. This medication has a 43% PFS at 30 months. 231 Mab to look out for in the future include Camidanlumab tesirine (ADCT-301) a human Mab (IgG1κ). This Mab has specificity to CD25 (a IL-2 receptor alpha subunit) with a drug conjugate. The drug is released intracellularly and causes DNA interstrand crosslinks. This Mab is in phase I studies to be completed in 2019 for Hodgkin's and non-Hodgkin's T- and B-cell lymphomas. In addition, there are clinical phase I studies against multiple solid tumors to be completed in 2021. 232 , 233 Agents abandoned or not found to be beneficial include apolizumab, denintuzumab mafodotin (HBU-12), iratumumab (MDX060), and lucatumumab (HCD122). 212 , 234 , 235 Anaplastic large cell lymphoma Brentuximab vedotin (Adcentrix) is an FDA-approved medication for patients with refractory or relapsed anaplastic large cell lymphoma who achieved CR. This Mab had 79% OS and 57% PFS at 5 years, with median response duration not reached at time of publication. 236 Breast Cancer Atezolizumab (Tecentriq) is an FcγR binding–deficient, fully humanized Mab (IgG1κ). This Mab binds to programmed death ligand I (PD-L1) to prevent interaction with receptors PD-1 and B7.1 (a costimulatory cell-surface protein), reversing T-cell suppression. Activation of B7.1 can potentially stimulate long-term responses through development of new immunity via priming and activation of T cells in lymph nodes. A lack of FcγR binding decreases ADCC of the T cells enabling more tumor-specific T cell to remain active. This medication was approved by the FDA in 2019 to treat triple negative (estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2) unresectable or metastatic breast cancers. 237 , 238 Colorectal Cancer Bevacizumab (Avastin) is a humanized Mab (IgG1κ) with specificity to vascular endothelial growth factor-a (VEGF-A) that acts as an inhibitor of angiogenesis. It was FDA approved for treatment of colorectal cancer and has recently been approved for multiple other cancers including ovarian, fallopian cancers, renal cell carcinoma, and recurrent glioblastoma multiforme (GBM). 239 , 240 Urothelial Carcinoma Atezolizumab (Tecentriq) is FDA approved as a single agent in urothelial carcinoma and for patients with disease progression despite other chemotherapy treatment. 241 , 242 Nonsmall cell lung cancer Atezolizumab (Tecentriq) is FDA approved as a single agent for nonsmall cell lung cancer (NSCLC). Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, nonsquamous NSCLC. Nivolumab (Opdivo) is an FDA-approved human Mab (IgG4κ) immunoglobulin and blocks PD-1 preventing interaction PD-1 and its ligands PD-L1 and PD-L2. It is used to treat RCC, NSCLC, Hodgkin's lymphoma, melanoma, small cell lung cancer, colorectal cancer, and squamous cell carcinoma of the head and neck. In phase III clinical trials, nivolumab performed better than docetaxel in the treatment of NSCLC. 243 , 244 , 245 Ovarian/cervical fallopian cancer Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, ovarian, cervical, and fallopian cancers after treatment with chemotherapy regimens and surgery. 246 Nonsmall cell lung cancer Atezolizumab (Tecentriq) is FDA approved as a single agent for nonsmall cell lung cancer (NSCLC). Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, nonsquamous NSCLC. Nivolumab (Opdivo) is an FDA-approved human Mab (IgG4κ) immunoglobulin and blocks PD-1 preventing interaction PD-1 and its ligands PD-L1 and PD-L2. It is used to treat RCC, NSCLC, Hodgkin's lymphoma, melanoma, small cell lung cancer, colorectal cancer, and squamous cell carcinoma of the head and neck. In phase III clinical trials, nivolumab performed better than docetaxel in the treatment of NSCLC. 243 , 244 , 245 Ovarian/cervical fallopian cancer Bevacizumab (Avastin) is FDA approved for treatment of locally advanced, recurrent or metastatic, ovarian, cervical, and fallopian cancers after treatment with chemotherapy regimens and surgery. 246 Merkel Cell Carcinoma Merkel cell carcinoma is a rare aggressive cutaneous malignancy caused by infection with polyoma virus and exposure to ultraviolet radiation. This cancer was classically treated with chemotherapeutic agents leading to rare durable responses. Avelumab (Bavencio) is a fully human Mab (IgG1λ) with specificity to PD-L1. This Mab was approved by the FDA for treatment of Merkel cell carcinoma in 2017. Treatment with this Mab increases response rates to about 50% and extended durable response times approximately five times. 247 , 248 This Mab is in clinical trial to treat other solid tumors including but not limited to hepatocellular, ovarian, esophagogastric, colorectal NSCLC, testicular, urothelial, and adrenocortical carcinomas. 249 Neuroblastoma Neuroblastoma is an aggressive tumor of children with a 5-year survival of about 50%. Treatment classically is high-dose intensive chemotherapy, myeloablative chemotherapy with stem cell rescue, and/or irradiation therapy. Dinutuximab (Unituxin) is a chimeric Mab (IgG1κ) with specificity to GD2 ganglioside that has mechanisms of action via CDC and ADCC. This Mab is used in patients who have had at least a partial response to classic therapy. 250 , 251 Glioblastoma Multiforme GBM is the most common malignant primary brain tumor in adults. This disease remains incurable. Bevacizumab (Avastin) is FDA approved for treatment of recurrent GBM as salvage therapy. This medication with chemotherapy increases overall survival by 4 months but as a single agent is not effective. 252 Relatlimab (BMS-986,016) is a human Mab (IgG4κ) with specificity to lymphocyte activation gene 3 (LAG3, CD223) and is in phase I clinical trials to be completed in 2020 for treatment of GBM. 253 Tanibirumab (aka Olinvacimab, TTAC-0001) is a human Mab (IgG1) with specificity to vascular endothelial growth factor receptor-2 (VEFR-2) and is in phase II studies to treat GBM to be completed in 2020. 254 , 255 , 256 Malignant Ascites Catumaxomab (Removab) is a trifunctional rat/murine hybrid antibody (IgG2a/IgG2b). Catumaxomab consists of one "half" (one heavy chain and one light chain) of an antiepithelial cell adhesion molecule (anti-EpCAM) antibody and one-half of an anti-CD3 antibody, so that each molecule of catumaxomab can bind both EpCAM and CD3. In addition, the Fc-region can bind to an Fc receptor on accessory cells such as other antibodies, which has led to calling the drug a trifunctional antibody. This antibody's mechanism of action is through ADCC. It is approved for use in Europe for malignant ascites from ovarian, gastric, colon, pancreatic, breast, and endometrial carcinoma and is a pending review for approval by the FDA. 257 , 258 , 259 , 260 Cutaneous squamous cell carcinoma Cemiplimab (Libtayo) is a human Mab (IgG4) for treatment of cutaneous squamous cell carcinoma (CSCC) that is metastatic or locally advanced and not amenable to surgery. CSCC is second only to basal cell carcinoma as the most common skin cancer. Surgical intervention is not possible in 5% of patients. This Mab offers a treatment with less morbidity than palliative radiation or surgery, and gives an ORR in 50% of these otherwise untreatable patients. There are many additional phase II studies involving this Mab to be completed from 2020 to 23. 261 , 262 Cutaneous squamous cell carcinoma Cemiplimab (Libtayo) is a human Mab (IgG4) for treatment of cutaneous squamous cell carcinoma (CSCC) that is metastatic or locally advanced and not amenable to surgery. CSCC is second only to basal cell carcinoma as the most common skin cancer. Surgical intervention is not possible in 5% of patients. This Mab offers a treatment with less morbidity than palliative radiation or surgery, and gives an ORR in 50% of these otherwise untreatable patients. There are many additional phase II studies involving this Mab to be completed from 2020 to 23. 261 , 262 Autoimmune/Inflammatory Diseases Inflammatory Bowel Disease Inflammatory bowel disease (IBD) pathophysiology remains unknown but may have genetic, infectious, autoimmune origins including cell-mediated immunity. These diseases may be classified as ulcerative colitis (UC), isolated to the colon, or Crohn's disease primarily found in the colon but may involve the entire gastrointestinal tract. With long-standing active disease, malignancy is much more frequent in UC than in Crohn's disease. Mild UC is treated with antiinflammatory agents such as sulfasalazine and glucocorticosteroids. For more severe disease, high-dose steroids may be used to maintain disease quiescent and low-dose steroids to keep disease in remission. Low-dose chemotherapeutic agent or immunosuppressive agent may also be added if dose of corticosteroids is too high to maintain remission. Surgery may be necessary to control disease. For Crohn's disease, medical therapy is usually less successful in managing the disease and surgery may be necessary but is not curative as in UC. For both of these disease processes, passive antibody therapy may offer not only control of disease but possible complete remission from mucosal damage. 263 , 264 Adalimumab (two formulations: Humira and Amjevita) is a recombinant human Mab (IgG1) with specificity to tumor necrosis factor alpha (TNF-α). Both forms are FDA approved to treat Crohn's disease as well as multiple types of rheumatoid arthritis. In Crohn's disease, this medication decreases signs and symptoms of disease and is able to induce clinical remissions. 265 , 266 Certolizumab (Cimzia) is a recombinant humanized m fragment with TNF-α as target. It is FDA approved for both Crohn's disease and Rheumatoid arthritis. 267 , 268 , 269 Vedolizumab (Entyvio) is a humanized Mab (IgG1κ) that has selectivity for integrin α4β7 and is FDA approved for treatment of Crohn's disease. This Mab mode of action is to selectively block trafficking of memory T cells into inflamed gut tissue by inhibiting α4β7-mucosal addressin cell adhesion molecule-1 (MAd-CAM-1) interaction with intestinal vasculature. This medication has shown a good safety profile with no cases of promyelocytic leukemia (PML), no increased risk of infections, malignancies compared with classically treated IBD, and low incidence of infusion-related reactions. This medication is also FDA approved for UC. 270 , 271 Infliximab (Remicade, Inflectra, Remsira) is a chimeric Mab (IgG1κ) with specificity to TNF-α and is FDA approved for IBD and multiple inflammatory arthritic diseases. This medication allows for steroid-free remission within months of starting therapy. 272 Natalizumab (Tysabri) is a humanized Mab (IgG2κ) with selectivity to CD62L (L selectin α4 subunit of α4β1 and α4β7 integrins of leukocytes, not neutrophils, VLA-4). This Mab is FDA approved for Crohn's disease and multiple sclerosis. This medications is effective in induction of clinical remission in moderate-to-severe Crohn's disease. This medication does have the risk of PML. 273 , 274 Other Mab being studied for Crohn's disease but not yet approved by the FDA include Ustekinumab, brazikumab, etrolizumab, risankizumab, and ontamalimab. In contrast, Mabs studied but not beneficial for Crohn's disease include andecaliximab, eldelumab, and fontolizumab. Refer to Table 16.1 . Table 16.1 Summary of Monoclonal Antibody Therapies. Generic Drug Name Brand Name Type of Antibody AHFS Classification Dosage Form(s) Target 8H9 Iodine 124 monoclonal antibody (Murine) Antineoplastic Neuroblastoma, sarcoma, metastatic brain cancers Another study Sloan Kettering using I 331 version phase I good results Intravenous B7–H3 Abagovomab Monoclonal antibody (Murine) An antiidiotypic mAb that mimics ovarian cancer CA125 protein Antineoplastic Phase II study for ovarian cancer Phase III good immune response but no increase RFS or OS no benefit Subcutaneous CA-125 Abatacept Orencia FDA 2005 EU 2010 Recombinant soluble fusion protein of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human immunoglobulin G1 (IgG1). Disease modifying Rheumatoid arthritis Juvenile and adult psoriatic arthritis (phase III) Subcutaneous or intravenous Selective costimulation modulator, inhibits T cell (T lymphocyte) activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. Abciximab c7Ec Fab ReoPro FDA 1994 EU 1995 (country-specific approval) Human-murine chimera Recombinant monoclonal IgG1 Fab Procedure modification High-risk coronary intervention Platelet aggregation inhibitor Intravenous Platelet glycoprotein IIb/IIIa receptor (CD41 7E3)/Intergrin α-IIb Abituzumab DI17E6 EMD525797 Humanized monoclonal antibody IgG2κ Antineoplastic Colorectal cancer phase I 2013, phase II 2015 primary endpoint PFS not met Sclerosing interstitial lung disease phase II terminated 2018 slow enrollment Prostate phase IIno significant increase PFS Intravenous CD51 (?integrin alpha V) Abrilumab AMG 181 Phase II study discontinued development (2016) Integrin α-4 β-7 Actoxumab Human monoclonal antibody Disease modifying Clostridium difficile Phase I and II anti-CDTB1 much better Clostridium difficile toxin A Adalimumab Humira FDA 2002 EU 2003 Amjevita FDA 2016 EU 2017 Recombinant human IgG1 monoclonal antibody Disease modifying Humira Rheumatoid arthritis; juvenile idiopathic arthritis; psoriatic arthritis; ankylosing spondylitis; Crohn's disease, plaque psoriasis Amjevita Arthritis; juvenile rheumatoid arthritis; psoriatic arthritis; rheumatoid colitis; ulcerative Crohn's disease; psoriasis; spondylitis; ankylosing Possibly hemolytic disease of newborn Injection subcutaneous TNF-α Adecatumumab MT-201 Recombinant human monoclonal antibody IgG1κ Antineoplastic Breast phase Ib+, colorectal and prostate Phase II completed Phase III soon? Intravenous EpCAM (CD326) epithelial cell adhesion molecule Aducanumab Human monoclonal antibody IgG1 Disease modifying Alzheimer's disease Phase III x 2 ongoing started 2015 Intravenous Beta-amyloid (N-terminus 3–6) soluble oligomers and insoluble fibrils Afasevikumab Human monoclonal antibody IgG1κ Disease modifying Multiple sclerosis Phase I completed Nothing in pubmed Subcutaneous IL17A and IL17F Afelimomab Murine F(ab') Antibody Fab' fragment IgG3κ Disease modifying Sepsis Phase III trial marginal benefit abandoned TNF-α Alacizumab pegol Humanized monoclonal antibody F(ab') 2 Limited information on development; Cancer VEGFR2 Alemtuzumab LDP-03 Campath-1H Lemtrada FDA 2014 EU 2013 MS Campath FDA 2001 EU 2001 CLL Humanized rat monoclonal antibody IgG1κ Antineoplastic B-Cell CLL , CTCL, T cell lymphoma Disease modifying Multiple sclerosis (phase III) Not effective for kidney transplant conditioning or rejection prevention Intravenous CD52 Alirocumab Praluent FDA 2015 EU 2015 Human monoclonal antibody IgG1 Disease modifying Decrease cholesterol Phase III Subcutaneous Proprotein convertase subtilisin kexin type 9 (PCSK9) Altumomab pentetate In 98 Hybri-ceaker Murine monoclonal antibody IgG1 Diagnostic purpose radiology colorectal cancer (diagnosis) CEA ALX-0171 Trimeric nanobody Antiinfectious RSV phase II 2020 Inhalation RSVF Amatuximab MORAb-009 Chimeric murine-human monoclonal antibody IgG1κ Antineoplastic Ovarian cancer Phase II Now research on using to treat mesotheliomas Phase I/II Pancreatic cancer Intravenous Mesothelin Prohibits binding of MSLN with antigen CA125/MUC16 AMG330 Bispecific T-cell engager (BiTE) Antineoplastic AML phase I AML 2020 Intravenous CD33 and CD3 Anatumomab mafenatox Murine monoclonal fragment Fab Antineoplastic Nonsmall cell lung carcinoma Tumor-associated glycoprotein 72 (TAG-72) Andecaliximab GS 5745 Chimeric monoclonal antibody IgG4κ Antineoplastic gastric cancer phase I, II, III ongoing or gastroesophageal junction adenocarcinoma phase III ongoing Crohn phase II no response, UC Intravenous Gelatinase B is a matrix metalloproteinase-9 (MMP-9) Anetumab ravtansine In 98 Human monoclonal antibody IgG1λ Antineoplastic ovarian phase II, lung, pancreatic phase I, breast now research on using to treat mesotheliomas Phase II Cervical cancer ?preclinical Mesothelin Prohibits binding of MSLN with antigen CA125/MUC16 Anifrolumab Human monoclonal antibody IgG1κ Disease modifying Systemic lupus erythematosus phase I and IIb 2018 Intravenous Interferon α/β receptor Anrukinzumab (=IMA-638) Humanized monoclonal antibody IgG1κ Disease modifying Asthma phase II ?results UC phase II no benefit IL-13 Apolizumab Humanized monoclonal antibody Antineoplastic non-Hodgkin's lymphoma abandoned 2009 toxic effects 2009 CLL phase I/II HLA-DRβ Arcitumomab CEA-Scan FDA 1996 EU 1996 Withdrawn EU market 2005 Murine monoclonal antibody IgG1 Fab' Diagnostic imaging Gastrointestinal cancers Colorectal cancers CEA Ascrinvacumab Human monoclonal antibody Antineoplastic mesothelioma Nothing in pub med or web search Activin receptor-like kinase 1 Aselizumab Humanized monoclonal antibody Disease modifying Severe injured patients phase II 2004, no benefit L-selectin (CD62L) Atezolizumab MPDL3280A Tecentriq FDA 2016 Fc engineered, humanized monoclonal antibody IgG1κ Antineoplastic agent, treat metastatic urothelial carcinoma , non-small cell lung cancer Phase III Bladder/urothelial cancer phase I Breast cancer phase Ib triple marker neg breast cancer Intravenous Binds to PD-L1 and blocks interactions with the PD-1 and B7.1 receptors FDA-approved atezolizumab (TECENTRIQ, Genentech, Inc.), in combination with bevacizumab, paclitaxel, and carboplatin for the first-line treatment of patients with metastatic nonsquamous, nonsmall cell lung cancer (NSq NSCLC) with no EGFR or ALK genomic tumor aberrations Atidortoxumab Human monoclonal antibody IgG1κ Limited information on use and development Negative search PubMed-internet Staph aureus alpha toxin Atinumab Human monoclonal antibody IgG4κ Disease modifying Acute spinal cord injury RTN4 Atorolimumab Developed?? Human monoclonal antibody IgG3 Disease modifying hemolytic disease of the newborn Rhesus factor Avelumab Bavencio FDA 2017 Human monoclonal antibody IgG1λ Antineoplastic Cancers, ovarian, gastric, nonsmall cell lung (NSCLC), metastatic, solid tumors phase II Studies completed metastatic Merkel cell carcinoma Intravenous PD-L1 Azintuxizumab vedotin Chimeric/humanized monoclonal antibody IgG1 Antineoplastic Nothing in PubMed CD319 BAN-2401 Humanized monoclonal antibody IgG1 Disease modifying Alzheimer A phase IIb study ongoing started 2013 Intravenous Soluble Aβ amyloid protofibrils Bapineuzumab Humanized IgG1 monoclonal antibody Disease modifying Alzheimer's disease Phase III no more studies discontinued research 2012 ARIA-E, amyloid-related imaging abnormalities–edema Intravenous Beta amyloid Fibrillary and soluble β amyloid Basiliximab Simulect FDA 1998 EU 1998 Chimeric monoclonal antibody IgG1κ Immunosuppressive agents Prophylaxis of acute rejection in allogeneic renal transplantation Intravenous CD25 (α chain of IL-2 receptor) Bavituximab Chimeric monoclonal antibody IgG1κ IgG3 (SUNRISE trial) Cancer, viral infections (Hep C) phase III NSCLC failed to improve survival Sunrise trial stopped Feb 2016, phase II/III breast cancer, phase II pancreatic cancer, phase I/II trial hepatocellular carcinoma, phase I malignant melanoma + rectal cancer good response rectal; not for prostate cancer, phase II hepatitis C not resulted? Phosphatidylserine BAY-103356 CDP 571 Humicade Senlizumab Humanized monoclonal antibody IgG4κ For research only Phase II 1995 TNF α BCD-100 Human monoclonal antibody Antineoplastic Phase II/III melanoma NCT03269565 (complete Dec 2019) Intravenous Programmed cell death-1 (PD1) Bectumomab LymphoScan Fab'-IgG2κ Antineoplastic Non-Hodgkin's lymphoma (detection) CD22 Begelomab Begedina Murine IgG2b Disease modifying GvHD phase II/III DPP4 binds CD26 on T lymphocytes Belantamab mafodotin Humanized monoclonal antibodymab Antineoplastic No studies or info on clinical trial, PubMed, FDA substance BCMA Belatacept Nulojix FDA 2011 Soluble fusion Protein consisting of the modified extracellular domain of CTLA-4 fused to Fc domain of a recombinant human monoclonal antibody IgG1 Immunosuppressive agents Prophylaxis renal transplant rejection in adults Phase III FDA approved Intravenous Selectively inhibits T-cell activation through costimulation blockade binds to both CD80 and CD86 blocking CD28 Belimumab Benlysta FDA 2011 EU 2011 LymphoStat-B Human monoclonal antibody IgG1λ Disease modifying Kidney transplant phase II Treat SLE (testing phase III for renal involvement) Phase II Rheum arthritis failure Phase II Srogren ± GVHD ongoing Intravenous Subcutaneous B-cell activating factor (BAFF), B-lymphocyte stimulator Bemarituzumab Humanized monoclonal antibody Antineoplastic FGFR2 Benralizumab Fasenra FDA 2017 EU 2017 Humanized monoclonal antibody IgG1κ Disease-Modifying Asthma phase III completed Severe asthma eosinophilic subtype Subcutaneous Interleukin-5 (IL-5α) receptor alpha subunit-directed cytolytic (CD125) Berlimatoxumab Human monoclonal antibody Staph aureus bicomponent leukocidin No studies clinical, no find creative, zero pub med Bermekimab MABp1 T2-18C3 CA-18C3 Xilonix Human monoclonal antibody IgG1κ Disease modifying psoriasis phase III x 2 2020 Ank spond II 2022 Psor arth II 2020 III 2020 Subcutaneous Intravenous IL17A Bersanlimab Human monoclonal antibody ICAM-1 Bertilimumab CAT-214 Human monoclonal antibody IgG4κ Disease modifying Severe allergic disorders phase II atopic dermititis Ongoing studies bullous pemphigoid and ulcerative colitis phase II Intravenous CCL11 (eotaxin-1) Besilesomab Scintimun EU 2010 Not FDA approved Murine monoclonal antibody IgG1κ Diagnostic use Inflammatory lesions and metastases (detection) CEA-CAM8-related antigen Bevacizumab Avastin FDA 2004 EU 2005 Humanized monoclonal antibody IgG1κ BiTE Antineoplastic agent Antiangiogenesis inhibitor Colorectal cancer 2004, NSCLC 2006, RCC 2009, GBM phase III, ovarian cancer, metastatic cervical cancer, fallopian 2014 Breast cancer (FDA removed approval for breast cancer 2010) Recurrent glioblastoma multiform Non squamous nonsmall cell lung cancer Intravenous solution or ophthalmic injection May not be so good for GBM or ovarian VEGF-A anti-angiogenesis inhibitor Bezlotoxumab Zinplava FDA 2016 EU 2017 Human monoclonal antibody IgG1 Disease modifying phase III studies done MODIFY I and II Modify III ongoing Pseudomembranous colitis Intravenous Clostridium difficile colitis anti-B toxin Biciromab FibriScint Murine monoclonal fragment Fab' IgG1κ Detect cardiovascular thromboembolism (diagnosis) Fibrin II, beta chain Bimagrumab BYM338 Human monoclonal antibody IgG1λ Disease modifying Myostatin inhibitor DM II decrease BMI phase II Sporadic inclusion body myositis phase III not meet endpoint Treat sarcopenia in older adults phase II Intravenous Activin A receptor type IIB (ACVR2B) Bimekizumab Humanized monoclonal antibody IgG1κ Disease modifying Ankylosing spondylitis (2018 II, 2022 II, +), plaque psoriasis (2021 III, 2020 III, 2019 III, +), psoriatic arthritis (2020), RA (2017 II), UC phase II Subcutaneous IL 17A and IL 17F Bivatuzumab mertansine Humanized monoclonal antibody IgG1 Antineoplastic squamous cell carcinoma, breast phase I fail x 2, head/neck or esophagus phase I fail, toxicity CD44 v6 Bleselumab Human monoclonal antibody IgG4κ Disease modifying organ transplant rejection phase II 2020 to prevent FSGS in kidney transplant patients Phase II psoriasis- medication tolerated with minimal reaction, no benefit to disease process Intravenous CD40 Blinatumomab Blincyto FDA 2014 EU 2015 Murine(scFv - kappa - heavy) - (scFv - heavy - kappa) BiTE Antineoplastic Ph chrom neg pre-B ALL (CD19+) phase II B- cell precursor acute lymphoblastic leukemia (ALL) initial or relapsed/refractory Intravenous Bispecific T-cell engager monoclonal antibody construct that directs CD-3 positive effector memory T cells to CD19-positive target cells Blontuvetmab Blontress Canine monoclonal antibody IgG2 κ/λ Veterinary treat canine B-cell lymphoma CD20 Blosozumab Humanized IgG4κ Disease modifying Osteoporosis 3 phase I and one phase 2 injection site reaction and antibodies to antibody Intravenous Subcutaneous SOST Antisclerostin Bococizumab RN316 PF-04950615 Humanized IgG2κ Disease modifying Dyslipidemia Phase III 2019 Discontinued secondary to antidrug antibodies, no primary endpoint achieved Subcutaneous intravenous Neural apoptosis-regulated proteinase 1 PCSK9 (proprotein convertase subtilisin/kexin type 9, neural apoptosis-regulated convertase 1, NARC1, NARC-1, proproteine convertase 9, PC9) Brazikumab Human monoclonal antibody IgG2λ Disease modifying Ulcerative colitis phase II 2021 Phase I/II completed Crohn. Phase III ongoing Subcutaneous IL23 Brentuximab vedotin Adcetris FDA 2013 Breakthrough therapy status by FDA 2018 Chimeric humanized monoclonal antibody IgG1κ Antineoplastic Hodgkin lymphoma Anaplastic large-cell lymphoma Intravenous CD30 (TNFRSF8) an antibody-drug conjugate (ADC) 3 parts: anti-CD30 (cAC10, a cell membrane protein of the tumor necrosis factor receptor), a microtubule disrupting agent monomethyl auristatin E (MMAE) and a protease-cleavable linker that attaches MMAE covalently to cAC10. The combination disrupts the intracellular microtubule network causing cell-cycle arrest and apoptotic cellular death Briakinumab Human monoclonal antibody Disease modifying psoriasis, Drug development stopped for psoriasis, phase IIb study in Crohn's Intravenous IL-12, IL-23 Brodalumab AMG827 Siliz FDA 2016 Human monoclonal antibody IgG2κ Disease modifying Plaque psoriasis Completed phase III Subcutaneous Receptor IL-17RA Brolucizumab RTH258 ESBA1008 FDA review 2018 Humanized single chain antibody fragment (scFv κ) Disease modifying Wet or age-related macular degeneration phase III to be completed Sept 2018, 2020 HAWK (NCT02307682) and HARRIER (NCT02434328) phase III trials good results Intravitreal https://www.novartis.com/news/media-releases/new-novartis-phase-iii-data-brolucizumab-demonstrate-reliability-12-week-treatment-interval VEGFA Brontictuzumab Humanized IgG2λ Antineoplastic Phase I Colorectal Lymphoid Adenoid cystic Solid tumors Intravenous Notch 1 Burosumab KRN23 Crysvita FDA 2018 Human monoclonal antibody IgG1κ Disease modifying X-linked hypophosphatemia Phase III completed Subcutaneous https://www.creativebiolabs.net/burosumab-overview.htm FGF 23 phosphaturic hormone fibroblast growth factor 23 Cabiralizumab Humanized monoclonal antibody IgG4κ Antineoplastic metastatic pancreatic cancer phase II 2020 Many other cancers phase I Intravenous CSF1R Camidanlumab tesirine ADCT-21 Human monoclonal antibody Antineoplastic B-cell Hodgkin's lymphoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia 2018 phase I Advanced solid tumors with literature evidence of CD25(+) treg content Head and neck Nonsmall cell lung Gastric, esophageal, Pancreas, bladder, Renal cell, melanoma, Triple-negative breast, ovarian phase I 2021 Intravenous CD25 Sinilimab Camrelizumab IBI308 China pending approval Humanized monoclonal antibody IgG4κ Antineoplastic Phase III nasopharyngeal cancer 2021 Phase III esophageal cancer 2021 Programmed cell death 1 (PDCD1) Canakinumab ACZ885 Ilaris FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Cryopyrin- associated periodic syndromes Including familial cold auto-inflammatory syndrome and Muckle –Wells syndrome ; tumor necrosis factor receptor-associated periodic syndrome (TRAPS); hyperimmunoglobulin D syndrome (HIDS)/mevalonate kinase deficiency (MKD) and familial Mediterranean fever (FMF) Systemic Juvenile idiopathic arthritis Treat Juvenile idiopathic arthritis phase III NSCLC 2025 phase III CVD rejected by FDA Behcet Subcutaneous IL-1β Cantuzumab mertansine Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase I 2007 Intravenous Mucin CanAg Cantuzumab ravtansine Humanized monoclonal antibody IgG1κ Antineoplastic Cancers MUC1 Caplacizumab-yhdp Cablivi (Nanobody program) FDA 2019 EU 2018 Humanized single variable domain antibody (bivalent nanobody) Disease modifying Inhibits interaction vWF and platelets Treat acquired TTP Phase III Hercules study completed Intravenous Subcutaneous VWF Capromab pendetide Prostascint FDA 1996 Murine monoclonal antibody Diagnostic imaging Prostatic carcinoma cells detection Intravenous Tumor surface antigen PSMA Carlumab Human monoclonal antibody IgG1κ Antineoplastic Prostate phase II no long term benefit Pulm fibrosis phase II no benefit Intravenous hMCAF/MCP-1 (human macrophage/monocyte chemotactic protein-1) Carotuximab TRC105 Chimeric monoclonal antibody IgG1κ Antineoplastic angiosarcoma Hepatocellular car phase I/II 2020 Glioblastoma multi-phase II 2014 terminated poor accrual ?results Angiosarcoma phase III 2019 TAPPAS trial Prostate ca phase II 2021 NSCLC phase I 2019 Intravenous Endoglin (CD105) Catumaxomab Removab FDA approved pend 2017) EU approved 2009 Removab: A trifunctional rat/murine hybrid antibody IgG2a/IgG2b Antineoplastic Removab Ovarian cancer phase II, malignant ascites phase II, gastric cancer phase II (ovarian, gastric, colon, pancreatic, breast, endometrial) Proxinium Head and neck cancer Intraperitoneal EpCAM, CD3 Catumaxomab consists of one "half" (one heavy chain and one light chain) of an anti-EpCAM antibody and one half of an anti-CD3 antibody, so that each molecule of catumaxomab can bind both EpCAM and CD3. In addition, the Fc-region can bind to an Fc receptor on accessory cells like other antibodies, which has led to calling the drug a trifunctional antibody. cBR96-doxorubicin immunoconjugate aka SGN-15 Humanized monoclonal antibody IgG1κ Antineoplastic Cancer Sponsorship ceased 2005 Cedelizumab CIMZIA Humanized monoclonal antibody IgG4κ Prevent organ transplant rejection CD4 Cemiplimab Libtayo FDA 2018 Human monoclonal antibody IgG4 Antineoplastic Nonsmall cell lung cancer (NSCLC) phase I 2021, phase III 2022 × 3 Oropharynx phase II 2022 Multiple myeloma phase II 2022 Ovarian ca phase II 2022 Head neck squamous cell carcinoma phase II 2020 Cutaneous squamous cell Glioblastoma multiforme phase II 2021 Lung ca phase II 2022 Cervical cancer phase III 2023 Intravenous Programmed cell death receptor PCDC1 Cergutuzumab amunaleukin Aka RO6895882, CEA-IL2v Humanized monoclonal antibody Antineoplastic phase I Dec 2018 Intravenous IL2 Certolizumab pegol CDP870 Cimzia FDA 2008 EU 2009 Recombinant, humanized antibody Fab' fragment Disease-Modifying Crohns Rheumatoid arthritis (phase IIIs completed) Psoriatic arthritis phase III Ankylosing spondylitis Subcutaneous Tumor necrosis factor α blocker Cetrelimab Relatimab Human monoclonal antibody IgG4κ Antineoplastic Nothing on PubMed or creative lab Substance is registered with FDA Programmed cell death 1 Cetuximab IMC-225 Leukeran Erbitux FDA 2004 EU 2004 Recombinant chimeric monoclonal antibody IgG1κ Antineoplastic agent Metastatic colorectal cancer and head and neck cancer NSCLC Intravenous solution EGFR Citatuzumab bogatox Humanized Fab IgG1κ Antineoplastic ovarian cancer and other solid tumors Study phase I terminated 2008 EpCAM Cixutumumab Human monoclonal antibody IgG1κ Antineoplastic Solid tumors Sarcoma phase II Esophageal cancer phase II Rhabdomyosarcoma phase II no benefit Liver cancer phase I Low antitumor effect Pancreas no benefit 2012 Intravenous IGF-1 receptor (CD221) Clazakizumab ALD−518 Humanized monoclonal antibody Disease modifying rheumatoid arthritis phase II 2015 × 3 Crohn disease phase II 2013 Highly sensitized renal transplant candidates phase II 2020 Treat post-tx rejection kidney phase II 2020 Antibody-mediated rejection phase III 2027 Subcutaneous IL6 Clenoliximab Chimeric monoclonal antibody Disease modifying Rheum Arth No study since 2003 CD4 Clivatuzumab tetraxetan (90)Y-clivatuzumab tetraxetan hPAM4-Cide Humanized monoclonal antibody IgG1κ Antineoplastic Pancreatic cancer Phase III 2017 PANCRIT-1 study. Study terminated no increase improvement of overall survival MUC1 Codrituzumab Humanized monoclonal antibody IgG1κ Antineoplastic HCC Phase Ib no response Phase II no response Glypican 3 Cofetuzumab pelidotin Humanized monoclonal antibody IgG1κ Antineoplastic Nothing on PubMed or creative lab Substance is not registered with FDA Protein tyrosine kinase 7 (PTK7) Coltuximab ravtansine SAR3419 Chimeric monoclonal antibody IgG1 conjugated to DM4 (N2′-(4-((3-carboxypropyl)dithio)-4-methyl-1-oxopentyl)-N2′-deacetylmaytansine) Antineoplastic Relapse/refractory ALL phase II 2015 low clinical response Phase II moderate response CD19 Conatumumab AMG655 Human monoclonal antibody IgG1κ Antineoplastic Phase II 2019: Advanced solid tumors Carcinoid Colorectal cancer Locally advanced Lymphoma Metastatic cancer Nonsmall cell lung cancer Sarcoma Solid tumors Colon cancer phase Ib/II no benefit Intravenous TRAIL-R2 Concizumab Humanized IgG4κ Disease modifying Hemophilia A and B phase II 2020 Subcutaneous Kunitz-type protease inhibitor 2 domain of tissue factor pathway inhibitor (TFPI) Cosfroviximab ZMapp Chimeric monoclonal antibody IgG1κ Triple monoclonal antibody cocktail Disease modifying Ebola virus Ongoing studies show benefit but not enough enrolled to power study Ebola virus glycoprotein Crenezumab RG7412 MABT5102A Humanized monoclonal antibody IgG4 Disease modifying Alzheimer's disease phase III study ongoing prodromal/mild AD 2021 Phase III 2022 Intravenous 1-40-β-amyloid Crizanlizumab SelG1 FDA review possible 2019 Humanized monoclonal antibody IgG2κ Disease modifying Sickle cell disease phase II 2022 children Phase II adults decrease pain crisis Intravenous P Selectin Crotedumab Human monoclonal antibody IgG4κ Disease modifying DM type II No results GCGR Cusatuzumab ARGX-110 Humanized monoclonal antibody IgG1 Antineoplastic Phase I completed safe Phase I/II CTCL dec 2018 Nasopharyngeal carcinoma 2018 Intravenous CD70 Dacetuzumab HU-S2C6 ASKP1240 SGN-40 Humanized monoclonal antibody IgG1 Antineoplastic Hematologic cancers Multiple myeloma phase I 2007 Large B-cell lymphoma phase II 2009 enrollment stopped no benefit CLL phase II 2006 NHL phase I Renal transplant (CIRRUS I) phase II 2022 SLE nephritis phase II 2020 Intravenous CD40 Daclizumab Zenapax Zinbryta FDA 1997 EU 1999 Zenapax withdrawn from market Apr 2009 for commercial reasons Zinbryta withdrawal 2018 secondary to risk/benefit profile Humanized monoclonal antibody IgG1κ Disease modifying Prevention of organ transplant rejections Phase IV kidney transplants, multiple sclerosis phase III 2018 pulled from market secondary inflammatory brain disorders Biogen Heart transplant phase IV 108 studies Zanapax discontinued from market by Roche (basiliximab replace) CD25 (α chain of IL-2 receptor) Dalotuzumab Humanized monoclonal antibody IgG1κ Antineoplastic Phase I multiple Phase II breast no improvement × 2 Phase III colon no improvement Ped solid phase I Intravenous IGF-1 receptor (CD221) Dapirolizumab pegol Humanized monoclonal antibody IgG1κ SLE phase II Nov 2018 Phase I safe Intravenous CD154 (CD40L) Daratumumab Darzalex FDA 2015 EU 2016 Human IgG1κ Antineoplastic agent Multiple myeloma relapse/refractory Phase III completed Intravenous solution CD38 Induces CDC, ADCC, ADCP, and apoptosis Dectrekumab QAX576 Human monoclonal antibody IgG1κ Cancers, asthma phase II, idiopathic pulmonary fibrosis, eosinophilic Esophagitis phase II some benefit but primary endpoint not achieved, Keloids, Crohn's disease phase II trials 2013 Nothing on PubMed Substance is registered with FDA, creative lab IL-13 Demcizumab Humanized monoclonal antibody IgG2κ Antineoplastic NSCLC phase II 2018 Phase I safety established with 50% tumor regression response Intravenous Delta-like ligand 4DLL4 DLL4 and Notch1, signaling stimulated by DLL4 plays a role in development of blood vessels throughout life Denintuzumab mafodotin HBU-12 SGN-CD19A Humanized monoclonal antibody IgG1κ Antibody-drug conjugate (ADC) composed of a humanized anti-CD19 monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) Antineoplastic LBCL phase II terminated study by company Acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma Phase I 2017 Phase II 2018 terminated by sponsor Intravenous CD19 Denosumab AMG162 Prolia FDA 2010 EU 2010 Xgeva FDA 2011 EU 2011 Human monoclonal antibody IgG2 Disease modifying Osteoporosis FREEDOM trial, bone metastases, etc. 186 studies Phase III completed Melanoma phase II 2022 Bone giant cell tumor phase II 2025 Subcutaneous Receptor activator of nuclear factor kappa-B ligand (RANKL) Xgeva: Prevention of skeletal-related events (SREs) in adults with bone metastases from breast and castration-resistant prostate cancer. Prolia: Osteoporosis Depatuxizumab mafodotin ABT 414 Chimeric humanized monoclonal antibody IgG1κ CONJUGATED TO AURISTATIN F Glioblastoma Phase III Nov 2019 Children phase III 2020 Intravenous EGFR Derlotuximab biotin Iodine (131 I) derlotuximab biotin Chimeric monoclonal antibody IgG1κ Immunoassays Potential for glioblastoma multiforme Histone complex Detumomab Murine monoclonal antibody IgG1 Antineoplastic B-lymphoma cell Nothing on PubMed or clinical trials Substance is not registered with FDA, is on creative lab CD3E Dezamizumab GSK-2398852 Humanized monoclonal antibody IgG1κ Disease modifying Treat amyloidosis Transthyretin cardiomyopathy amyloidosis (ATTR-CM), suspended pending data review Aug 2018 phase I x 4 Intravenous Serum amyloid P component Dinutuximab APN311 Unituxin FDA 2015 EU 2015 then withdrawn EU Chimeric monoclonal antibody IgG1κ Antineoplastic Neuroblastoma phase I 2022 SCLC phase III Nov 2019 Osteosarcoma phase II Dec 2018 Neuroblastoma phase II 2020 Intravenous GD2 ganglioside Diridavumab CR6261 Human monoclonal antibody IgG1λ Disease modifying Infectious disease/influenza A Very good response in animal study mice Phase II 2019 Intravenous Influenza A hemagglutinin Domagrozumab PF-06252616 Humanized monoclonal antibody IgG1κ Disease modifying Duchenne muscular dystrophy phase II 2018 Phase I completed GDF-8 Dorlimomab aritox F(ab') 2 Murine Nothing on PubMed or clinical trials Substance is not registered with FDA, or creative lab Dostarlimab TSR042 WBP285 FDA review pending 2019 Humanized monoclonal antibody IgG4κ Antineoplastic Solid tumor Phase I, II, III studies ongoing Ovarian CA (first study) phase III 2023 Programmed cell death protein-1 (CD279) PCDP1 Drozitumab PRO95780 rhuMAB DR5 Human monoclonal antibody IgG1λ Antineoplastic Colorectal cancer Ib 2012 Preclinical rhabdomyosarcoma 2018 Chondrosarcoma not efficacious NHL results? Intravenous Death receptor 5 (DR5) Duligotuzumab MEHD7945A Human monoclonal antibody IgG1κ Antineoplastic squamous head and neck phase II no benefit Colon ca phase II no benefit Anti-EGFR × Anti-HER3 bispecific antibody Dupilumab Dupixent FDA 2017 Human monoclonal antibody IgG4 Disease modifying asthma, atopic dermatitis Ongoing studies Subcutaneous IL4 Durvalumab Imfinzi FDA 2017 Human monoclonal antibody IgG1κ Antineoplastic agent Treat NSCLC stage III phase I, urothelial carcinoma Intravenous PD-L1 (CD274) and CD80—inhibit binding of programmed death ligand 1 to PD-1 and CD80 allowing T cell to recognize and kill tumor cells Dusigitumab MEDI 573 Human monoclonal antibody IgG2λ Antineoplastic Breast cancer phase II results? HCC phase II results? Intravenous ILGF2 Duvortuxizumab MGD011 Chimeric/humanized monoclonal antibody Antineoplastic B-cell malignancy Phase I/II Jul 2018/2020 Intravenous CD19, CD3E Ecromeximab KW2871 Chimeric monoclonal antibody IgG1κ Antineoplastic Metastatic melanoma Phase I/II clinical activity limited Intravenous GD3 ganglioside Eculizumab Soliris PNH FDA 2007 EU 2007 aHUS, and myasthenia gravis FDA 2018 EU 2018 Japan 2018 Humanized monoclonal antibody IgG1/4 Immuno-regulation Paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (HUS ) Generalized myasthenia gravis (MG) Phase II CAD Intravenous C5 Edobacomab E5 Murine monoclonal antibody No improved survival Endotoxin Edrecolomab Panorex Murine monoclonal antibody IgG2κ Antineoplastic Colorectal carcinoma phase III 2003 no improvement Intravenous Glycoprotein EpCAM/17-1A Efalizumab Raptiva FDA 2003 EU 2004 Withdrawn both markets 2009 Recombinant humanized monoclonal antibody IgG1κ Disease modifying (2003 approved) psoriasis Subcutaneous Voluntary withdrawal 2009 Human CD11a Increase risk progressive multifocal leukoencephalopathy (PML) Efungumab MYC123 Mycograb Mycograb C28Y Human scFv Antiinfectious agent Invasive Candida infection Intravenous Heat shock protein 90 (Hsp90) Eldelumab Mdx 1100 Human monoclonal antibody IgG1κ Crohn's disease phase IIa no significant response, ulcerative colitis phase IIb prim endpoint not achieved Rheum arthritis phase II Intravenous Interferon γ-induced protein CXCL 10 Elezanumab PR-1432051 ABT-555 Human monoclonal antibody IgG1λ Spinal cord injury and multiple sclerosis phase II 2021 Intravenous REPULSIVE GUIDANCE MOLECULE FAMILY MEMBER A (RGMA) Elgemtumab LJM716 Human IgG1κ Antineoplastic Breast gastric phase I Intravenous ERBB3 (HER3) Elotuzumab PDL063 Empliciti FDA 2015 EU 2016 Human IgG1κ Antineoplastic Multiple myeloma Phase III completed and ongoing Intravenous SLAMF7 Elsilimomab B-E8 Humanized monoclonal antibody IgG1 Antineoplastic multiple myeloma Not effective in mice IL-6 Emactuzumab RG7155 Humanized monoclonal antibody IgG1 ANTINEOPLASTIC Phase I 2019 solid tumors Phase II 2025 REDIRECT study ovarian, fallopian tube cancer Pancreatic phase II 2020 HUMAN MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR (CSF1R, CD115) Emapalumab NI-0501 Gamifant FDA 2018 EU pending Human monoclonal antibody IgG1λ Hemophagocytic lymphohistiocytosis Phase III 2021 Intravenous Interferon γ Emibetuzumab LA480 LY2875358 Humanized monoclonal antibody IgG4κ Bivalent antibody Antineoplastic NSCLC phase II 2020 Advanced cancer Gastric safe ?effective adenocarcinoma Gastroesophageal junction adenocarcinoma Hepatocellular cancer Renal cell carcinoma Nonsmall cell lung cancer phase II Jan 2018 Phase I safe with tumor response Intravenous Hepatocyte growth factor receptor (HHGFR) and MET signaling Emicizumab ACE910 Hemlibra FDA 2018 Humanized monoclonal antibody IgG4κ Bispecific Disease modifying Hemophilia A phase III 2020 With or without inhibitors Subcutaneous Activated F9, F10 Enapotamab vedotin Human monoclonal antibody IgG1κ Antineoplastic Nothing on PubMed or clinical trials Substance is not registered with FDA, or creative lab Human growth factor receptor AXL Enavatuzumab PDL192 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2011 No responses and liver pancreatic toxicity Intravenous TWEAK receptor Enfortumab vedotin FDA review pending 2019 Human monoclonal antibody Antineoplastic bladder cancer phase I Phase II ongoing Nectin-4 Anti-Nectin-4 Monoclonal antibody attached to a microtubule-disrupting agent, monomethyl auristatin E (MMAE) Enlimomab pegol Murine monoclonal antibodyIgG2a Disease modifying Stroke Nothing on PubMed or clinical trials Substance is not registered with FDA ICAM-1 (CD54) Enoblituzumab MGA 271 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2022 children Neuroblastoma Rhabdomyosarcoma Osteosarcoma Ewing sarcoma Wilms tumor Desmoplastic small round cell tumor Phase I melanoma, NSCLC 2018 Phase II prostate 2021 CD276 (B7–H3) Enokizumab MEDI528 Humanized monoclonal antibody IgG1κ Asthma phase II No improvement Intravenous IL9 Enoticumab REGN421 Human monoclonal antibody IgG1κ Antineoplastic Phase 1 2014 ovarian cancer + Delta-like canonical notch ligand 4 (DLL4) Ensituximab NEO-201 NPC-1C Chimeric monoclonal antibody IgG1κ Antineoplastic Phase II pancreatic and colorectal cancer 2017 Intravenous 5AC Enterecept RHU-TNFR:FC Enbril FDA 2003 1-235-Tumor necrosis factor receptor fusion protein attached to recombinant human IgG1 Fc fragment Disease modifying Antirheumatic drug Not effective for inflammatory bowel disease Subcutaneous TNFα Epitumomab cituxetan AS-1402 HuHMFG-1 Sontuzumab Humanized monoclonal antibody IgG1 Antineoplastic Breast cancer phase II 2012 no benefit Episialin MS4A1 (membrane-spanning 4-domains subfamily A member 1, CD20 (HMFG-1) Epratuzumab HLL2 AMG412 Humanized monoclonal antibody IgG1κ ADCC/CDC Antineoplastic B-ALL phase III ongoing 2018 Disease modifying SLE phase III no improvement Intravenous CD22 Eptinezumab ALD403 FDA review possible 2019 Monoclonal antibody IgG1κ Disease modifying Migraine phase III Calcitonin gene-related peptide Erenumab Aimovig FDA May 2018 Human monoclonal antibody IgG2λ Disease modifying Migraine phase III Calcitonin gene-related peptide (CGRP) Erlizumab Rhumab CD18 Humanized IgG1 F(ab') 2 fragment Antineoplastic (lab tests) Immunosuppressive drug phase I study cough up blood and phase II did not meet goals Heart attack, stroke, traumatic shock ??no successful CD18 drug to date LGL type leukemia ITGB2 (CD18) and LFA-1 block growth factor of blood vessels stop lymphocytes from moving into inflamed tissue Ertumaxomab Rexomun Rat/murine hybrid triomab, murine IgG2a HET2 target, RAT IgG2bλ CD3 target Antineoplastic Breast Gastric, esophageal Phase II studies terminated company to focus on other plans not safety concerns concentrate on catumaxomab Phase I found safe 2016 Intravenous HER2/neu, CD3 Etaracizumab or etaratuzumab MEDI-522 Abegrin Vitaxin Humanized monoclonal antibody IgG2κ Antineoplastic Melanoma phase II 2010 not beneficial, prostate cancer, ovarian cancer small and large bowel cancer phase I and II completed results unreported 2017 Intravenous Integrin αvβ3 Etigilimab Humanized monoclonal antibody IgG1κ Nothing on pubmed or clinical trials Substance is registered with FDA, or is not in creative lab TIGIT T-cell immunoreceptor with Ig and ITIM domains Etrolizumab PRO145223 RHUMAB BETA7 Humanized monoclonal antibody IgG1κ Disease modifying Inflammatory bowel disease UC phase III 2020 × 4/2023/2024/2025 Crohn phase III 2021 Subcutaneous Integrin β7 Inhibits binding of αEβ7 to E-cadherin Evinacumab REGN1500 Human monoclonal antibody IgG4κ Disease modifying Dyslipidemia Phase II 2020 Phase III 2020/2022 Angiopoietin 3 Evolocumab Repatha FDA 2015 EU 2015 Human monoclonal antibody IgG2λ Disease modifying hypercholesterolemia Completed phase III Heterozygous familial hypercholesterolemia, CVD Subcutaneous Proprotein convertase subtilisin kexin type 9 (PCSK9) Exbivirumab Humanized monoclonal antibody IgG1λ Disease modifying prevent disease Hep B Oral therapy Abstract of randomized study of 50 patients Hepatitis B surface antigen Fanolesomab RB5-IGM NeutroSpec Murine monoclonal antibody Diagnostic imaging Appendicitis (diagnosis only) CD15 Faralimomab Murine monoclonal antibody IgG1 Nothing on PubMed or clinical trials Substance is not registered with FDA, or is not in creative lab Interferon receptor Faricimab RG7716 RO6867461 Humanized monoclonal antibody IgG1mab Disease modifying angiogenesis, ocular vascular diseases STAIRWAY, BOULEVARD, RHINE, Yosemite phase II and III studies phase III 2022 for diabetes maculae edema AMD LUCERNE phase III 2022 TENAYA phase III 2022 Intravitreous ANTIVASCULAR ENDOTHELIAL GROWTH FACTOR/ANTIANGIOPOIETIN 2 BISPECIFIC ANTIBODY (VEGF-A and Ang-2) Farletuzumab MORAB-003 Humanized monoclonal antibody IgG1κ Antineoplastic Ovarian cancer phase III subgroup may benefit Intravenous Folate receptor 1 Fasinumab REGN475 SAR164877 MT5547 Human monoclonal antibody IgG4κ Disease modifying acute sciatic pain phase III Knee arthritis pain phase III 2021 Subcutaneous (auto injector) Human nerve growth factor (HNGF) FBTA05 Bi20 Lymphomun Rat IgG2b (CD3)/murine IgG2a (CD20) hybrid trifunct Antineoplastic Chronic lymphocytic leukemia trial terminated recruitment too slow Intravenous? CD20/CD3 Felvizumab Humanized monoclonal antibody IgG1κ Antiinfectious agent Respiratory syncytial virus infection Nothing on PubMed or clinical trials Substance is not registered with FDA, but is in creative lab Respiratory syncytial virus Fezakinumab Human monoclonal antibody IgG1λ Disease modifying Rheumatoid arthritis, psoriasis (not good for) Atopic dermatitis phase IIb good results Intravenous IL-22 Fibatuzumab Ifabotuzumab Humanized monoclonal antibody IgG1κ Disease modifying Myelodysplastic syndrome Research in Australia for GBM phase I Ephrin receptor A3 Ficlatuzumab SCH 900105 AV 299 Humanized monoclonal antibody IgG1κ Antineoplastic Head and neck cancer phase I 2020 Pancreatic phase I 2023 NSCLC phase I/II 2013 AML phase I 2020 Intravenous Hepatocyte growth factor (HGF) hepapoietin A Figitumumab CP751871 Human monoclonal antibody IgG2κ Ceased development in 2011 by pfizer Antineoplastic Adrenocortical carcinoma, nonsmall cell lung carcinoma etc. ?additional benefit in phase I Insulin-like growth factor receptor IGF-1 receptor (CD221) Firivumab Human monoclonal antibody IgG1κ Disease modifying Influenza A virus hemagglutinin Nothing on PubMed or clinical trials Substance is registered with FDA, and is in creative lab INFLUENZA A VIRUS HEMAGGLUTININ HA Flanvotumab IMC20D7S Human monoclonal antibody IgG1κ Antineoplastic Melanoma phase I 2012 Intravenous No published data TYRP1 (glycoprotein 75) Fletikumab Human monoclonal antibody IgG4 Disease modifying Rheumatoid arthritis phase IIa good, phase IIb no results IL 20 Flotetuzumab MGD006 S80880 Humanized di-scFv dual affinity retargeting (DART) to CD123 and CD3 Antineoplastic Hematologic malignancies (ALL, NHL) Phase II not yet recruiting 2018 Intravenous? IL 3 receptor Fontolizumab HuZAF Humanized monoclonal antibody IgG Disease modifying Treat Crohn's clinical development stopped despite some benefit phase II ustekinumab is better IFN-γ FOR46 Antibody drug conjugate Antineoplastic Phase I for multiple myeloma failed remission or relapse Phase I prostate cancer Intravenous CD46 Foralumab Human monoclonal antibody IgG1κ Disease modifying NASH phase II 2019 Oral CD3 epsilon Foravirumab Human monoclonal antibody IgG1κ Disease modifying rabies (prophylaxis) Nothing in pub med or clinical trials Rabies virus glycoprotein Fremanezumab LBR-101 RN307 Ajovy FDA 2018 Humanized monoclonal antibody IgG2κ Disease modifying migraine and cluster headache phase III 2019 Subcutaneous α-Calcitonin gene-related peptide Fresolimumab Humanized monoclonal antibody IgG4 Disease modifying Idiopathic pulmonary fibrosis (IPF), scleroderma, focal segmental glomerulosclerosis (phase 2), cancer (kidney cancer and melanoma) Need larger study for FSGS Good response scleroderma https://newdrugapprovals.org/2016/01/30/fresolimumab/ TGF β 1 Frovocimab LY3015014 Humanized monoclonal antibody IgG4κ Disease modifying hypercholesterolemia Completed phase II trials 2014 good response and safe Subcutaneous PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9 (PCSK9) Frunevetmab https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies - cite_note-WHOList 116-17 NV-02 Veterinary monoclonal antibody IgG1κ Veterinary Feline muscle nerve growth factor Fulranumab AMG403 Human monoclonal antibody IgG2κ Disease modifying Pain osteoarthritis pain phase III 2017 Subcutaneous Nerve growth factor Galcanezumab LY2951742 Emgality FDA 2018 Humanized monoclonal antibody IgG4κ Disease modifying Migraine Phase III completed Cluster HA phase III 2020 Subcutaneous Calcitonin gene-related polypeptides (CGRPs) α and β Galiximab IDEC-114 Chimeric monoclonal antibody IgG1λ ADCC/CDC Antineoplastic lymphoma phase II 2015 minimal response ORR 10.3% Intravenous CD80 Gancotamab MM-302 Human cFv Single chain fragment Antineoplastic Breast cancer phase I–III Intravenous HER2/neu Ganitumab AMG479 Human monoclonal antibody IgG1κ Antineoplastic Pancreatic phase III—no increase benefit Phase III for rhabdomyosarcoma and Ewings 2021 Not beneficial in NSCLC Intravenous IGF-1 receptor (CD221) Gantenerumab R04909832 R1450 Human monoclonal antibody IgG1κ Disease modifying Alzheimers Phase III stopped for potential futility additional studies at higher dosing (DIAN in a phase II/III trial in individuals at risk For and with early-stage autosomal-dominant AD phase III 2021 Subcutaneous Beta amyloid Gatipotuzumab PankoMab-GEX Humanized monoclonal antibody IgG1κ Antineoplastic Ovarian, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), gynecological cancers (GYN) phase I used for diag/prog now Intravenous Musculus, antimucin (MUC1) Gavilimomab ABX-CBL Murine monoclonal antibody IgM Disease modifying Graft versus host disease phase III completed 2005 less effective than antithymocyte antibody CD147 (basigin) Gedivumab RG7745 RO6876802 Human monoclonal antibody IgG1κ Disease modifying Influenza virus A No studies PubMed/clinical trials Influenza virus hemagglutinin HA Gemtuzumab ozogamicin Mylotarg AML FDA 2000 Voluntary withdrawal 2010 VOD now black box warning Returned to market with FDA approval 2017 Humanized monoclonal antibody IgG4/toxin conjugate Antineoplastic Acute myelogenous leukemia Many ongoing and completed studies Intravenous CD33 Gevokizumab XOMA 052 Humanized monoclonal antibody IgG2κ Disease modifying DM phase II (late stage) no results Other dz too 24 studies behcet uveitis failed primary end point phase III (Eyeguard _B) 2015 Subcutaneous IL-1β Gilvetmab PD1 Veterinary monoclonal antibody IgG2κ Antineoplastic No studies clinical trial, pub med CANIS FAMILIARIS PROGRAMMED CELL DEATH PROTEIN 1 (PCDC1) Gimsilumab MORAb-022 Human monoclonal antibody IgG1 Disease modifying Rheumatoid arthritis Asthma Phase I poster presentation of safety results good 2016 Intravenous HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (CSF2) Girentuximab WX-G250 CG250 Rencarex Reductane Chimeric monoclonal antibodyIgG1κ Radioactive labeled ab Antineoplastic Clear cell renal cell carcinoma for treatment and imaging Intravenous Carbonic anhydrase 9 (CA-IX) Glembatumumab vedotin CR011 Human monoclonal antibody IgG2κ Antibody drug complex Antineoplastic Melanoma phase II, breast cancer Intravenous Human glycoprotein NMB extracellular domain (GPNMB) Golimumab CNTO 148 Simponi FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis, psoriatic arthritis , juvenile rheum arth, ankylosing spondylitis many studies, UC, DM1 phase I (2020, 2021) Subcutaneous, intravenous TNF-α Gomiliximab IDEC-152 Lumiliximab ST-152 Chimeric monoclonal antibody IgG1κ ADCC/CDC Allergic asthma ? Antineoplastic CLL Phase I, phase 2/3 2014 Failed efficacy CD23 (IgE receptor) Gosuranemab BIIB092 IPN-007 FDA orphan drug status Humanized monoclonal antibody IgG4κ Progressive supranuclear palsy Phase I 2020 Alzheimer 2021 Intravenous τ protein Guselkumab CNTO 1959 Tremfya FDA ? Human monoclonal antibody IgG1λ Disease modifying Psoriasis Adenomatous polyposis Subcutaneous IL23A Hu3F8 Humanized monoclonal antibody IgG3 Antineoplastic Phase I For neuroblastoma mod tox and substantial effect on tumor Phase II ongoing Intravenous GD2 ganglioside Ianalumab Human monoclonal antibody IgG1κ Immunomodulation Autoimmune hepatitis Human cytokine receptor BAFF-R Ibalizumab Trogarzo FDA/EU approved Humanized monoclonal antibody IgG4 Disease modifying anti-HIV Phase III CD4 Ibritumomab tiuxetan IDEC-129 IDEC-IN2B8 IDEC-Y2B8 Zevalin FDA 2002 EU 2004 Murine monoclonal antibody IgG1κ YT 77 or In 98 bound Antineoplastic Follicular non-Hodgkin's lymphoma , B-cell NHL , multiple myeloma conditioning for BMT, B-cell DLCL, mantle cell Many studies CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35) Icrucumab IMC 18F1 Human monoclonal antibody IgG1κ Antineoplastic No benefit breast phase II No benefit colon phase II No benefit urothelial phase II Intravenous Vascular endothelial growth factor receptor (VEGFR-1) Idarucizumab Praxbind FDA 2015 EU 2015 Humanized monoclonal antibody Fab fragment Antidotes Drug reversal agent Reversal of anticoagulant effects of dabigatran Phase III trial RE-VERSE AD Intravenous Dabigatran etexilate Igovomab Indimacis-125 Murine F(ab') 2 Diagnostic imaging Ovarian cancer (diagnosis) Iladatuzumab vedotin RG7986 Humanized monoclonal antibody IgG1κ Antineoplastic Nothing in PubMed or clinical trials Human gene B29 protein (CD97B) Imalumab BAX69 Human monoclonal antibody IgG1κ Antineoplastic Intraperitoneal infusion, intravenous Macrophage migration inhibitory factor (MIF) Imaprelimab Humanized IgG1κ Antineoplastic Nothing in PubMed or clinical trials Melanoma cell adhesion molecule (MCAM) Imciromab pentetate Myoscint FDA approved 1996 Withdrawn from market Murine monoclonal antibody fragment Fab IgG2aκ Diagnostic Cardiac imaging Cardiac myosin Imgatuzumab RG7160 RO5083945 GA201 HUMA-B Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal 2013 Head and neck 2017 NSCLC 2017 Epidermal growth factor receptor (EGFR, HER1) IMGN632 Monoclonal antibody with antibody drug conjugate Antineoplastic AML, ALL phase I 2021 NCT03386513 Intravenous CD123 Inclacumab RG1512 RO4905417 Human monoclonal antibody IgG4κ Disease modifying Cardiovascular disease phase II Intravenous Selectin P Indatuiximab ravtansine Chimeric monoclonal antibody IgG4κ Antineoplastic Preclinical breast cancer CD138 (syndecan-1) SDC1 Indusatumab vedotin TAK-264 MLN0264 Human monoclonal antibody IgG1κ conjuagated via a mc-val-cit-PABC linker to monomethyl auristatin E {MMAE (5F9-mc-val-cit-PABC-MMAE)} Antineoplastic Gastrointestinal, pancreatic, gastroesophageal Safe phase I, phase II pancreatic min response, three studies terminated by company business Intravenous Guanylate cyclase C (GUCY2C) Inebilizumab MEDI-551 Humanized monoclonal antibody IgG2κ ADCC Antineoplastic Refractory DLBCL phase II 2016 Disease modifying systemic sclerosis, multiple sclerosis Neuromyelitis optica Intravenous CD19 Infliximab Remicade FDA 1998 EU 1999 Inflectra FDA 2016 EU 2013 Remsima EU 2013 Human-murine chimera IgG1κ Human constant, murine variable region Disease modifying Remicade Crohn's disease; ulcerative colitis; rheumatoid arthritis; ankylosing spondylitis; psoriatic arthrits; plaque psoriasis Inflectra Spondylitis; ankylosing; arthritis; rheumatoid colitis; ulcerative arthritis; psoriatic Crohn's disease; psoriasis Remsima Spondylitis; ankylosing arthritis; rheumatoid colitis; ulcerative Crohn's disease; arthritis; psoriatic psoriasis Intravenous solution TNF-α Inolimomab Murine monoclonal antibody Disease modifying GVHD phase III No better than ATG in 3 studies Abandoned not in 2017 Cochrane review CD25 (α chain of IL-2 receptor) Inotuzumab ozogamicin G544 Besponsa FDA 2017 Humanized monoclonal antibody IgG4κ ADCC/CDC Antineoplastic ALL phase II 2023 Multiple other studies Intravenous CD22 Intetumumab CNTO095 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors (prostate cancer, melanoma) Melanoma phase II possible benefit 2011 Prostate cancer no additional benefit 2013 phase II Intravenous CD51 Ipilimumab Yervoy FDA 2011 EU 2011 Human monoclonal antibody IgG1κ Antineoplastic agent Bladder carcinoma (trials ongoing) Melanoma Ipilimumab MDX010 MDX101 BMS-734016 Yervoy FDA 2011 melanoma Metastatic renal cancer/colorectal cancer 2018 Human monoclonal antibody IgG1κ Antineoplastic Melanoma (checkmate 067) Renal cell carcinoma (checkmate 214) Colorectal cancer Pancreatic? Intravenous CD152 cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and blocks interaction with its ligands CD80/CD86 Iratumumab MDX060 Human monoclonal antibody IgG1κ Antineoplastic Hodgkin's lymphoma phase II completed Clinical research discontinued 2009 Intravenous CD30 (tumor necrosis factor receptor superfamily, Member 8; TNFRSF8) aka Ki-1 Ag Isatuximab SAR650984 FDA review possible 2019 Chimeric monoclonal antibody IgG1κ Antineoplastic multiple myeloma Phase I 2019 Phase II 2022 Phase III 2025 Intravenous CD 38 Iscalimab CFZ533 Human monoclonal antibody IgG1κ Disease modifying Potential treat autoimmune disease Lupus nephritis phase II 2020 Myasthenia gravis GVHD Kidney transplant 2022 phase II Preclinicals Intravenous CD40 Istiratumab MM-005 MM-141 Human monoclonal antibody IgG1 Antineoplastic Advanced solid tumors Pancreatic cancer phase II 2018 Insulin-like growth factor I receptor/neuregulin receptor HER3 (IGF1R, CD221) Itolizumab Alzumab FDA Humanized monoclonal antibody IgG1κ Disease modifying Psoriasis GVHS phase II 2022 CD6 Ixekizumab Taltz Humanized monoclonal antibody Disease modifying Phase III radiographic axial spondyloarthritis Psoriatic arthritis Subcutaneous IL 17A Keliximab Became clenoliximab IgG4 Chimeric monoclonal antibody IgG1λ Disease modifying Chronic asthma Rheumatoid arthritis CD4 Labetuzumab hMN14 CEA-Cide Humanized monoclonal antibody IgG1 Antineoplastic Colorectal cancer Gastrointestinal phase II 2021imagin Phase II completed therapy 2003 Intravenous CEA Lacnotuzumab MCS110 Humanized monoclonal antibody IgG1κ Antineoplastic Breast, pigmented villonodular synovitis (PVNS) Squam ESOP phase II 2024 Gastric 2019 CSF1, MCSF Ladiratuzumab vedotin Humanized monoclonal antibody IgG1κ Antibody drug conjugate Antineoplastic Phase I triple-negative breast cancer 2017 ongoing study LIV-1 Lampalizumab RG7417 Humanized monoclonal fragment IgG1κ Disease modifying Geographic atrophy secondary to age-related macular degeneration phase III Jan 2018 ineffective Intravitreous Complement factor D (CFD) Lanadelumab SHP643 DX-2930 Takhzyro FDA 2018 Human monoclonal antibody IgG1κ Disease modifying Angioedema phase III 2019 Subcutaneous Kallikrein (KLKB1) Landogrozumab LY2495655 Humanized monoclonal antibody IgG4κ Disease modifying Muscle wasting disorders, i.e., after hip surgery phase II Pancreatic cancer phase II no benefit cancer, some muscle improvement but primary objective not met Intravenous Subcutaneous Human growth differentiating factor 8 (GDF-8) aka myostatin (MSTN) Laprituximab emtansine IMGN289 Chimeric monoclonal antibody No trials or PubMed or creative lab only in FDA registry ?new EGFR Larcaviximab ZMAPP Chimeric monoclonal antibody IgG1κ Disease modifying Ebola virus No studies clinical trial or PubMed Ebolavirus glycoprotein Lebrikizumab MILR1444A TNX-650 RG-3637 PRO301444 Humanized monoclonal antibody IgG4κ Disease modifying Asthma phase III Atopic dermatitis HL phase II 2007 Subcutaneous injection Interleukin-13 (IL-13) Lemalesomab Murine monoclonal antibody IgG1κ Diagnostic agent NCA-90 (granulocyte antigen) Lendalizumab Olendalizumab ALXN-1007 Humanized monoclonal antibody IgGκ Disease modifying Antiphospholipid syndrome GI GVHD Intravenous Anticomplement 5A Lenvervimab Humanized IgG1κ Disease modifying Hepatitis B No studies in clinical trials or PubMed Hepatitis B surface antigen Lenzilumab KB-003 Human monoclonal antibody Antineoplastic chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia phase I Intravenous GRANULOCYTEMACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) Lerdelimumab CAT-152 Trabio Human monoclonal antibody IgG4 Disease modifying Phase I studies ?Cancer and fibrosis Trials stopped for fibrosis after glaucoma surgery Transforming growth factor β 2 Leronlimab PRO-140 FDA review 2018 Humanized IgG4κ Disease modifying HIV phase III ongoing no results published good results phase II Subcutaneous Chemokine receptor 5 (CCR5) Lesofavumab RG70026 Human monoclonal antibody IgG1κ Disease modifying Influenza A No studies clinical trials or PubMed Hemagglutinin HA Letolizumab Humanized synthetic light chain variable region (scFv) Disease modifying inflammatory diseases No studies clinical trials or PubMed or creative labs TRAP Lexatumumab HGS1018 HGS-ETR2 Human monoclonal antibody IgG1λ Antineoplastic Breast Pancreatic Intravenous Tumor necrosis factor receptor superfamily member 10B/death receptor 5 (TRAIL-R2) Libivirumab Humanized monoclonal antibody IgG1κ Antiinfectious Prevent disease Hep B Oral therapy Abstract of randomized study of 50 patients Hepatitis B surface antigen Lifastuzumab vedotin DBNIB0600A Humanized monoclonal antibody Antineoplastic Ovarian cancer Phase 2 Intravenous Phosphate-sodium cotransporter Ligelizumab QGE031 Humanized monoclonal antibody IgG1κ Disease modifying SSevere asthma and chronic spontaneous urticaria phase II and III ongoing trial 2021 Subcutaneous Immunoglobulin E (IGHE) Lilotomab satetraxetan Betalutin Murine monoclonal antibody IgG1 Antineoplastic NHL 2020/phase II 2025 Diffuse lg B-cell lymphoma 2019 CD37 Lintuzumab SGN33 Humanized monoclonal antibody IgG1κ Antineoplastic AMLphase I/II 2022 Mult myeloma phase I 2020 Myelodysplastic phae II 2011 Intravenous CD33 Lirilumab IPH2102 Human monoclonal antibody IgG4 Antineoplastic Solid and hematological cancers No good aml, squam cell head neck no good, bladder cancer ongoing? May benefit MDS Intravenous Killer cell immunoglobulin like (KIR2D) Block the interaction between KIR2DL-1,-2,-3 inhibitory receptors and their ligands Lodelcizumab LFU720 Humanized monoclonal antibody IgG1κ Disease modifying Hypercholesterolemia Unknown studies in clinical trials and PubMed Proprotein convertase subtilisin/kexin type 9 (PCSK9) Lokivetmab Cytopoint FDA approved for dogs only Canis monoclonal antibody IgG2κ Disease modifying Veterinary Clinical signs of atopic dermatitis in dogs Canis lupus familiaris IL31 Loncastuximab tesirine ADCT-402 Chimeric monoclonal antibody IgG1κ Antineoplastic Diffuse large B-cell lymphoma phase II 2020 Intravenous CD19 Lorvotuzumab mertansine BB-10901 IMGN901 Humanized monoclonal antibody IgG1κ Antineoplastic SCLC Ovarian AML phase II Wilm, rhabdomyosarcoma, Neuroblast, MPNST, Synovial sarcoma 2018 phase II Intravenous CD56 Losatuxizumab vedotin ABBV-221 Chimeric/humanized monoclonal antibody IgG1 Antineoplastic Epidermal growth factor (EGRF, ERBB1 HER1) Lucatumumab HCD122 Discontinued development by Novartis 2013 Human monoclonal antibody IgG1κ Antineoplastic Multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma Intravenous CD40 Lulizumab pegol Humanized monoclonal antibody Disease modifying SLE Phase I safe Phase II no response Intravenous Subcutaneous CD28 Lumretuzumab RG7116 RO5479599 Humanized monoclonal antibody IgG1κ Antineoplastic Intravenous CD28; receptor for tyrosine-protein kinase(erbB-3, HER3) Lupartumab amadotin BAY-1129980 Human monoclonal antibody IgG Antineoplastic Phase I terminated Why? Intravenous GPI- anchored cell surface-associated protein C4.4A (LYPD3) Lutikizumab ABT981 Humanized monoclonal antibody Disease modifying Osteoarthritis Phase IIa no effect Subcutaneous Interleukin 1 alpha/interleukin 1 beta Mapatumumab HGS1012 Human monoclonal antibody IgG4λ Antineoplastic Hepatocellular no benefit Multiple myeloma Cervical cancer NSCLC no benefit NHL Bladder cancer may be beneficial Tumor necrosis factor receptor superfamily member 10A; cytokine receptor DR4 (death receptor 4 tumor necrosis receptor apoptosis-induced ligand (TRAIL-R1) Margetuximab MGAH22 Chimeric/Humanized monoclonal antibody IgG1κ Antineoplastic Breast cancer Gastric cancer/GEC phase Ib/II trial Intravenous erbB2/HER2 Marstacimab PF-06741086 Human monoclonal antibody IgG1λ Disease modifying Bleeding with hemophilia phase II 2020 Subcutaneous Tissue pathway factor inhibitor (TFPI) Maslimomab Murine monoclonal antibody Immunosuppressive Unknown no studies and not listed in creative lab or FDA T-cell receptor Matuzumab EMD 72000 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal, lung and stomach cancer weakly beneficial Intravenous Epidermal growth factor receptor (EGFR) Mavrilimumab CAM3001 Human monoclonal antibody IgG4λ Disease modifying rheumatoid arthritis phase IIb good Subcutaneous GMCSF receptor α-chain MEDI565 MT111 AMG211 Fab IgG1 BiTE Antineoplastic Gastrointestinal adenocarcinoma phase I 2018 Intravenous CD3 and CEA Mepolizumab SB-240563 Bosatria Nucala FDA 2015 EU 2015 Human monoclonal IgG1κ Disease modifying No benefit in eosinophilic esophagitis Beneficial allergic severe asthma Subcutaneous Interleukin-5 (IL-5) antagonist Metelimumab CAT 192 Humanized monoclonal antibody IgG4 Disease modifying Scleroderma Dropped from further development TGF β 1 Milatuzumab HLL1 IMMU-115 Humanized monoclonal antibody IgG1κ Antineoplastic Multiple myeloma Lupus Leukemia Intravenous CD74 Minretumomab MOAB CC49 Murine monoclonal antibody IgG1 Diagnostic Tumor detection/diagnostic/prognostic Failed phase I clinical trials Tumor-associated glycoprotein 72 (TAG-72) Mirikizumab LY3074828 Humanized monoclonal antibody Disease modifying Psoriasis phase III 2020 UC phase III 2023 LUCENT 1 2021 phase III LUCENT 2 2022 Intravenous IL23A Mirvetuximab soravtansine M9346A IMGN853 Chimeric monoclonal antibody IgG1 Antineoplastic Ovarian phase III 2019 Breast ca phase II 2020 Intravenous Folate receptor alpha Mitumomab BEC-2 Murine monoclonal antibody Antineoplastic SCLC phase III no benefit 2005 GD3 ganglioside Modotuximab 1024 DS Zatuximab Futuximab SYM004 Chimeric monoclonal antibody IgG1κ Antineoplastic Antineoplastic Colorectal Phase 2019 Phase III 2025 Subcutaneous EGFR extracellular domain III/HER1 Mogamulizumab AMG761 KM8761 Poteligeo FDA 2018 Humanized monoclonal antibody IgG1κ Antineoplastic Adult T-cell leukemia/lymphoma Solid tumors Many studies ongoing Intravenous CC chemokine receptor CCR4 MOR202 MOR03087 Human monoclonal antibody IgG1 Antineoplastic multiple myeloma phase I 2018 Intravenous CD38 Monalizumab NN8765 IPH2201 Humanized monoclonal antibody IgG4κ Disease modifying Rheumatoid arthritis, antineoplastic gynecologic malignancies, and other cancers phase II 2021 NSCLC phase II 2022 s/p stem cell transplant phase I 2020 CLL phase II 2019 Intravenous Killer cell lectin-like receptor subfamily C member1 (NKG2A, CD159A, CD94) that recognize nonclassical HLA (i.e., HLA-E) Morolimumab Human monoclonal antibody IgG1 ?Diagnostic No studies in pub med, creative lab or FDA substance Rhesus factor Mosunetuzumab RG7828 BTCT4465A Humanized monoclonal antibody IgG1κ bispecific Antineoplastic NHL phase II 2023 DLBCL phase II 2023 Intravenous Subcutaneous CD3E, MS4A1, CD20 Motavizumab MEDI-524 Numax FDA not approved 2010 Older drug just as effective with less side effects Humanized monoclonal antibody IgG1κ Disease modifying Respiratory syncytial virus phase III completed Safety concerns hives and allergic reactions Intramuscular Respiratory syncytial virus glycoprotein F Moxetumomab pasudotox Lumoxiti FDA 2018 Recombinant immunotoxin comprised of a variable fragment (Fv) of a Murine IgG4 anti-CD22 monoclonal antibody genetically Fused to a truncated fragment of Pseudomonas exotoxin A Antineoplastic Hairy cell leukemia Phase I ALL peds Intravenous CD22 Muromonab-CD3 Muromab Aka teplizimab/MGA031 Orthoclone OKT3 FDA 1986 EU 1986 (country specific approval) Humanized monoclonal antibody IgG2aκ Disease modifying Prevention of kidney transplant rejection Many trials GVHD, NASH and T2DM, giant cell myocarditis AbATE Intravenous Oral CD3 Nacolomab tafenatox Murine monoclonal fragment Fab Antineoplastic ?Colorectal cancer No studies in clinical trial or PubMed C242 antigen Namilumab MT203 Human monoclonal antibody IgG1κ Disease modifying Ank spond psoriasis, RA phase II Subcutaneous Colony-stimulating factor 2 (CSF2) Naptumomab estafenatox TTS-CD3 ANYARA ABR-217620 Murine monoclanl antibody fragment Fab Antineoplastic Nonsmall cell lung carcinoma, renal cell carcinoma phase III completed primary endpoint not achieved Intravenous Tumor-associated antigen 5T4 Naratuximab emtansine IMGN529 Chimeric monoclonal antibody IgG1κ Antineoplastic B-Cell lymphoma NHL Intravenous Tetraspanin-26 (CD37) Narnatumab IMC-RON-8 Ron8 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors phase I Intravenous Human cell surface receptor RON (CD 135) macrophage-stimulating 1 receptor Natalizumab Antegran Antegren Tysabri FDA 2004 EU 2006 Humanized monoclonal antibody IgG4κ Disease modifying Relapsing multiple sclerosis, Crohn's disease Intravenous L selectin (CD62L) α4-subunit of α4β1 and α4β7 integrins of leukocytes (except neutrophils) (VLA-4) Navicixizumab OMP 305B83 Humanized/chimeric monoclonal antibody IgG2κ Antineoplastic Phase I study colorectal gyn tumors Intravenous Delta-like 4 (DLL4) Vascular endothelial growth factor A (VEGF-A) Navivumab CT-P27 Human monoclonal antibody IgG1κ Disease modifying Influenza A No studies PubMed Influenza A virus hemagglutinin HA Naxitamab HU3F8 Humanized monoclonal antibody IgG3 Antineoplastic High-risk neuroblastoma and refractory osteomedullary disease study 2023 ?Intravenous c-Met Ganglioside anti-GD2 Nebacumab Humanized monoclonal antibody IgM Withdrawn for safety, Efficacy and commercial reasons Endotoxin Necitumumab IMC-11F8 Portrazza FDA 2015 EU 2016 Human monoclonal antibody IgG1κ Antineoplastic Nonsmall cell lung carcinoma Intravenous EGFR Nemolizumab CIM331 CD14152 Humanized monoclonal antibody IgG2κ Disease modifying Eczema phase I and II Subcutaneous Interleukin-31 receptor A (IL31RA) NEOD001 Birtamimab ELT1-01 HU2A4 Humanized monoclonal antibody IgG1κ Disease modifying Primary systemic amyloidosis lack clinical benefit Intravenous Amyloid A protein/amyloid light chain Nesvacumab REGN910 SAR307746 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors not as beneficial as other agents in breast cancer Disease modifying Macular degeneration Intravenous Angiopoietin 2 Netakimab Chimeric monoclonal antibody Disease modifying Psoriasis PLANETA study (Russia, future EU and China) Interleukin 17A Nimotuzumab Theracim Theraloc Humanized monoclonal antibodyIgG1κ Antineoplastic Squamous cell carcinoma, head and neck cancer, nasopharyngeal cancer, glioma Intravenous EGFR Nirsevimab MEDI8897 Human monoclonal antibody IgG1κ Disease modifying Respiratory syncytial virus phase II 2018 Intramuscular Respiratory syncytial virus fusion protein (RSVFR) Nivolumab Opdivo FDA 2015 EU 2015 Human monoclonal antibody IgG4κ immunoglobulin Antineoplastic agent Programmed death receptor-1 (PD-1) blocking antibody NSCLC, bladder cancer, renal cell cancer phase III 2021 Hodgkin lymphoma Melanoma Small cell lung cancer Squamous carcinoma head and neck Colorectal cancer GBM no added benefit 2017 Intravenous Blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2 Nofetumomab merpentan Verluma FDA 1996 No longer marketed in USA Murine monoclonal fragment IgG2bκ Fab Cancer diagnostic imaging SCLC Antitumor Membrane-spanning 4-domains, subfamily A, member 1 Obiltoxaximab ETI-204 Anthim FDA 2016 Chimeric monoclonal antibody IgG1κ Disease modifying Bacillus anthracis anthrax phase IV 2021 Intravenous Intramuscular Bacillus anthracis spores PA component of B. anthracis toxin Obinutuzumab GA101HUMAB RG7159 RO5072759 Afutuzumab Gazyvaro FDA 2013 Humanized monoclonal antibody IgG1κ Antineoplastic lymphoma phase II (MCL, DLBCL) Chronic lymphocytic leukemia Phase II 2021 Intravenous CD20 Induces B-cell apoptosis Ocaratuzumab LY2469298 AME-133V Humanized monoclonal antibody IgG1κ Antineoplastic NHL Pemphigus phase III Intravenous CD20 Ocrelizumab Ocrevus FDA 2017 Humanized monoclonal antibody IgG1κ Disease modifying Multiple sclerosis Intravenous CD20 Odulimomab Murine monoclonal antibody Disease modifying Transplant rejection Only studied in mice Lymphocyte function-associated antigen-1 (LFA-1 (CD11a)) Ofatumumab Arzerra FDA 2009 EU 2010 Human monoclonal antibody IgG1κ Complement-dependent cytotoxicity (CDC) Antineoplastic CLL Phase III 10% ORR after ritux Phase II as first line 86% ORR With CHOP 100% ORR with 62% CR Intravenous CD20 Olaratumab IMC3G3 Lartruvo FDA 2016 EU 2016 Human monoclonal antibody IgG1κ Antineoplastic Sarcoma phase II 2023 Ovarian not beneficial Intravenous Platelet derived growth factor receptor alpha (PDGF-R α) Oleclumab MEDI9447 Human monoclonal antibody IgG1λ Antineoplastic pancreatic phase II 2021 Colorectal cancer Bladder cancer phase I 2020 Breast cancer phase II 2022 NSCLC phase II 2022 Intravenous? 5′-nucleotidase CD73 Olokizumab Humanized monoclonal antibody IgG4κ Disease modifying rheumatoid arthritis Phase I 2014 phase IIb mod results IL6 Omalizumab IGE25 RG3648 Xolair Humanized monoclonal antibody IgG1κ Disease modifying allergic asthma Urticaria Subcutaneous IgE Fc region Omburtamab Murine monoclonal antibody IgG1κ Antineoplastic Neuroblastoma Phase III 2022 Intracerebroventricular treatment CD276 OMS721 Human monoclonal antibody Disease modifying Atypical hemolytic uremic syndrome phase III 2020 Lupus nephritis phase II 2018 Intravenous Mannan-binding lectin-associated serine protease-2 (MASP-2) Onartuzumab PRO143966 RO5490258 METMAB Humanized monoclonal antibody IgG Antineoplastic Intravenous Human scatter factor receptor kinase Ontuxizumab MORAB-004 Chimeric/humanized monoclonal antibody Antineoplastic No clinical response Intravenous Endosialin tumor endothelial marker-1 (TEM1) Onvatilimab Human monoclonal antibody IgG1κ Nothing in PubMed Vista (V-domain immunoglobulin suppression of T activation (VSIR) Opicinumab BIIB033 Human monoclonal antibody IgG1 Disease modifying multiple sclerosis Phase II 2020 Leucine-rich repeat and immunoglobulin domain containing neurite outgrowth inhibitor receptor interacting protein-1 (LINGO-1) LINGO-1 Oportuzumab monatox VB4-845 Vicinium Proxinium FDA 2005 EU 2005 Additional approval pending 2019 Humanized monoclonal antibody fragment scFv Antineoplastic Bladder phase III Head and neck cancer Intravescical Epithelial cell adhesion molecule (EPCAM) and tumor-associated calcium signal transducer 1 (TACSTD1) and pseudomonas exotoxin A immunotoxin fusion protein (anti-EPCAM antibody fragment-Pseudomonas exotoxin fusion protein) Oregovomab MAB-B43.13 OvaRex Murine monoclonal antibody IgG1κ Antiidiopathic antibody to ovarian antigen CA-125 Antineoplastic Ovarian cancer Not effective in achieving increase RFS or OS Ovarian phase I 2021 Phase II 2019 Subcutaneous Intravenous CA-125 Orticumab RG7418 Human monoclonal antibody fragment Fab Disease modifying Antiinflammatory Oxidized low-density lipoprotein oxLDL Otelixizumab Chimeric humanized monoclonal antibody IgG1 Disease modifying Diabetes mellitus type 1 TTEDD phase II DEFEND-1 phase III failed DEFEND-2 phase III- no real benefit Subcutaneous CD3 Otilimab MOR103 GSK3196165 Human monoclonal antibody IgG1λ Disease modifying Osteoarthritis, rheumatoid arthritis phase II 2012 Multiple sclerosis phase II 2014 Intravenous Granulocyte-macrophage colony-stimulating factor (GMCSF) Otlertuzumab TRU-016 Humanized monoclonal antibody IgG fragment Antineoplastic CLL phase I and II 2014 and 2019 Intravenous CD37 Oxelumab OX40L R4930 HUMAB OX40L Human monoclonal antibody IgG1κ Disease modifying Asthma mainly preclinical mice Many clinical studies ongoing leukemia and asthma Intravenous OX-40 (CD252) Ozanezumab GSK1223249 Humanized IgG1 Disease modifying ALS phase II 2015 ALS no good Intravenous Neurite outgrowth inhibitor (NOGO-A) Ozoralizumab ATN 103 Humanized monoclonal antibody Disease modifying Rheumatoid arthritis phase II 2012 Subcutaneous TNF-α Pagibaximab Chimeric monoclonal antibody Disease modifying Staph sepsis low birth weight infants Phase II/III studies 2010 Intravenous Lipoteichoic acid Palivizumab Synagis, Abbosynagis FDA 1998 EU 1999 Humanized monoclonal antibody IgG1κ Disease modifying RSV many phase III studies Intramuscular F protein of respiratory syncytial virus Pamrevlumab FG-3019 Human monoclonal antibody IgG1κ Disease modifying Idiopathic pulmonary fibrosis (IPF), Antineoplastic Pancreatic cancer Muscular dystrophy phase II 2021 Diabetes nephropathy Connective tissue growth factor (CTGF) Insulin-like growth factor binding protein 8 (IGFBP-8) Panitumumab ABENIX ABX-EGF https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies - cite_note-WHOList91-38 Vectibix FDA 2006 EU 2007 Human monoclonal antibody IgG2κ Antineoplastic Metastatic colorectal cancer Intravenous EGFR/erbB-1/HER1 PankoMab-GEX Gatipotuzumab Humanized monoclonal antibody IgG1κ Antineoplastic Phase IIb 2017 Phase I solid tumors 2019 Intravenous Tumor-specific glycosylation of MUC1 Panobacumab Aerumab 11 AR-101 KBPA-101 Human monoclonal antibody Antimicrobial Pseudomonas aeruginosa infection Intravenous Pseudomonas aeruginosa serotype O11 Parsatuzumab MEGF0444A RG-7414 Human monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase II 2014 no benefit Epidermal growth factor-like domain 7 (EGFL7) Pascolizumab Humanized monoclonal antibody Disease modifying Not effective trails aborted IL-4 Pasotuxizumab Chimeric/humanized monoclonal antibody fragment Antineoplastic No studies Folate hydrolase/prostate-specific membrane antigen (PSMA) Pateclizumab RG7415 PRO283698 MLTA3698A Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis Phase II not as efficacious as adalimumab but had response Subcutaneous Lymphotoxin-α Patritumab AMG888 U3-1287 Human monoclonal antibody IgG1κ Antineoplastic May not be beneficial head/neck NSCLC CT site ErbB3 (HER3) Spartalizumab PDR001 FDA review possible 2019 Humanized monoclonal antibody Antineoplastic Breast cancer phase II 2021 NSCLC 2021 Melanoma phase II 2022 Phase III 2020 Intravenous PD1, PDCD1, CD279 Pembrolizumab MK-3475 Keytruda FDA 2014 EU 2015 FDA 2018 for metastatic Merkel cell carcinoma, HCC, NSCLC Humanized monoclonal antibody IgG4κ Antineoplastic Squamous carcinoma trachea, NSCLC , urothelial (HCC phase II) Melanoma cHL, LgB cell lymph Gastric cancer Cervical cancer Hepatocellular carcinoma Intravenous Trials for multiple myeloma discontinued by FDA PD-1 Pemtumomab HMFG1 antibody labeled with 90Yttrium Theragyn Murine monoclonal antibody Antineoplastic Phase III Europe 2009/US 2013 no benefit after 3.5 years follow-up MUC1/human milk fat globule antigen 1 (HMFG1) Perakizumab Humanized monoclonal antibody IgG1κ Disease modifying psoriatic arthritis Phase I discontinued IL 17A Pertuzumab Perjeta FDA2012 EU 2013 Omnitarg Humanized monoclonal antibody IgG1 Antineoplastic agent HER2-positive metastatic breast cancer Gastric/breast cancer Phase III gastric Intravenous Extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 protein (HER2/neu) Pexelizumab Humanized scFv Disease modifying acute myocardial infarctions APEX-AMI trial negative results PRIMO-CABG I and II trials no significant benefit C5 Pidilizumab CT-011 Humanized monoclonal antibody IgG1κ Antineoplastic Mult myeloma DLBCL Pontine glioma Pancreas Melenaoma HCC Antiinfection Intravenous PD-1 Pinatuzumab vedotin Humanized monoclonal antibody ADC consisting of the microtubule-disrupting agent, monomethyl auristatin E (MMAE), conjugated to an anti-CD22 mAbvia the protease-cleavable peptide linker maleimidocaproylvaline-citrulline(vc)-p-aminobenzoyloxycarbonyl Antineoplastic B-cell NHL phase I study good response Phase II completion 2019 Intravenous CD22 Pintumomab Murine monoclonal antibody Not therapeutic Diagnostic imaging adenocarcinoma antigen Adenocarcinoma (imaging) Placulumab Human monoclonal antibody V-kappa)2 FC Disease modifying pain and inflammatory diseases Development discontinued 2012 Human TNF Plozalizumab MLN1202 HU1D9 Withdrawn by company Humanized monoclonal antibody IgG1κ Disease modifying Diabetic nephropathy and arteriovenous graft patency RA no benefit Intravenous CC chemokine receptor 2 (CCR2) Pogalizumab MOXR0916 R07021608 Vonlerolizumab Humanized monoclonal antibody IgG1κ Antineoplastic Solid tumors phase I 2019 may be safe but may not be effective No formal manuscripts yet Intravenous Tumor necrosis factor receptor superfamily member 4 (ACT35, OX40, CD134) Polatuzumab vedotin FCU2711 RO5541077-000 FDA review 2018 Humanized monoclonal antibody IgG1κ Antineoplastic NHL phase II 2019 DLBCL phase III 2023 Intravenous CD79B Ponezumab RN1219 PF-04360365 Humanized monoclonal antibody IgG2 Disease modifying Alzheimer's disease Safe but no clinical efficacy 2013 Intravenous Human beta-40-amyloid Aβ40 Porgaviximab C2G4 Chimeric monoclonal IgG1κ Antiinfectious Ebola virus disease No known ongoing studies Zaire ebolavirus glycoprotein Prasinezumab PRX002 RG7935 RO7046015 Humanized monoclonal antibody IgG1κ Disease modifying Parkinson's disease Phase II 2021 Intravenous Anti-alpha-synuclein (NACP) Prezalizumab AMG-557 MEDI5872 Humanized monoclonal antibody IgG2 Disease modifying SLE phase II 2018 Sjogren's Subcutaneous B7-related protein inducible T-cell costimulator ligand (ICOSL) Priliximab cMT 412 CEN 000029 Chimeric monoclonal antibody Disease modifying Crohn's disease, multiple sclerosis IN FDA no known studies CD4 Pritoxaximab Chimeric monoclonal antibody IgG1κ Antiinfectious E. coli shiga toxin type-1 Pritumumab Human monoclonal antibody IgG1κ Antineoplastic Brain cancer Phase II studies in Japan, could not find literature reportedly increase survivability 10 fold Vimentin Quilizumab MEMP1972A RG-7449 Anti-M1 Humanized monoclonal antibody IgG1κ Disease modifying Asthma phase II 2014 no great benefit Urticaria phase II 2014 no great benefit Allergic rhinitis Subcutaneous Intravenous M1 prime segment of membrane bound IgE (IGHE) Racotumomab Vaxira Murine monoclonal antibody IgG1κ Antineoplastic Nonsmall cell lung cancer phase III 2016 cimavax better (recombinant EGF injection) 2 more months survival over placebo Neuroblastoma phase II 2020 Intradermal Subcutaneous N-glycolylneuraminic acid gangliosides (NGNA ganglioside) Radretumab F16SIP L19SIP radiolabeled with I 331 Human monoclonal antibody Imaging study PET Antineoplastic Lymphoma brain mets 2012 phae I Stage III NSclC Fibronectin extra domain-B Rafivirumab CR57 Human monoclonal antibody IgG1λ Used in cocktail and with vaccination Antiinfectious Rabies (prophylaxis) No known studies Rabies virus glycoprotein Ralpancizumab RN317 PF-05335810 Humanized monoclonal antibody IgG2κ Disease modifying Dyslipidemia phase I 2017 PCSK9 (proprotein convertase subtilisin/kexin type 9, neural apoptosis-regulated convertase 1, NARC1, NARC-1, proprotein convertase 9, PC9) Ramucirumab LY3009806 IMC-1121B Cyramza FDA 2014 EU 2014 Human monoclonal antibody IgG1κ Antineoplastic Urothelial phase III done Adenocarcinoma stomach and GE junction phase II 2023 Colorectal cancer NSCLC HCC phase III 2017 no additional benefit Intravenous VEGFR2 Ranevetmab NV-01 Veterinary monoclonal antibody IgG1κ canine Disease modifying Osteoarthritis in dogs Nerve growth factor-β (NGF-β) Ranibizumab RBZ RG-3645 RHuFAb Lucentis FDA 2006 EU 2007 Humanized monoclonal fragment IgG1κ Fab Disease modifying Macular degeneration (wet form) post market studies phase II Intravitreal Vascular endothelial growth factor A (VEGF-A) Ravagalimab PR-1629977 ABBV-323 Humanized monoclonal antibody IgG1κ Disease modifying UC phase II 2023 Intravenous Subcutaneous CD40 Ravulizumab ALXN1210 Ultomiris FDA 2019 EU pending Humanized monoclonal antibody IgG2/IgG4κ Disease modifying Paroxysmal nocturnal hemoglobinuria (PNH) Phase III 2021 similar to eculizumab, atypical hemolytic uremic syndrome phase III 2021 Intravenous Complement C5 (C5) Raxibacumab ABthrax FDA 2012 Human monoclonal antibody IgG1λ Antiinfectious Treat inhalation anthrax Intravenous Bacillus anthracis protective antigen Refanezumab GSK249320 Humanized monoclonal antibody IgG1κ Disease modifying recovery of motor function after stroke Phase II completed 2011 no benefit Intravenous Myelin-associated glycoprotein Regavirumab MCA C23 TI-23 Human monoclonal antibody Antiinfectious Cytomegalovirus glycoprotein B ONLY STUDIES IN RATS 1994 Cytomegalovirus infection Relatlimab BMS-986016 Human monoclonal antibody IgG4κ Antineoplastic Melanoma phase II 2022 Colon cancer phase II 2022 Chordoma phase II 2020 Cannot find manuscripts but company website phase II good results Glioblastoma phase I 2020 Intravenous Lymphocyte activation gene 3 (LAG3) CD223 Remtolumab ABT-122 Human monoclonal antibody Disease modifying RA Phase II 2016 no increased benefit over adalimumab Subcutaneous Interleukin 17 alpha, TNF-α Reslizumab DCP 835 Scheme 55700 CEP-38072 Cinqair FDA 2016 EU 2016 Humanized monoclonal antibody IgG4κ Disease modifying Inflammations of the airways asthma completed and ongoing, skin and gastrointestinal tract, polyarteritis stage II 2018 Rhino sinusitis 2020 Intravenous Subcutaneous IL-5 Rilotumumab AMG-102 Human monoclonal antibody IgG2κ Antineoplastic Gastric completed phase III 2015 not effective NSCLC phase II 2014 no benefit Glioma phase II no response Intravenous Hepatocyte growth factor (HGF) Rinucumab REGN2176 Human monoclonal antibody IgG4κ Disease modifying neovascular age-related macular degeneration phase II 2014 Intravitreal Platelet-derived growth factor receptor beta Risankizumab ABBV-066 BI-655066 FDA/EU pending approval Humanized monoclonal antibody IgG1κ Disease modifying Crohn's disease phase II good, phase III ongoing psoriasis phase II response better than ustekinumab, psoriatic arthritis, and asthma Subcutaneous IL23A Rituximab GP2013 IDEC-102 RG-105 MabThera, Rituxan FDA 1997 EU 1998 Chimeric monoclonal antibody IgG1κ Antineoplastic Non-Hodgkin lymphomas, chronic lymphocytic leukemias, some autoimmune disorders, i.e., rheumatoid arthritis, >2K studies ongoing Subcutaneous CD20 Rivabazumab pegol Humanized monoclonal antibody fragment Fab' IgG1κ Antiinfectious No studies found Pseudomonas aeruginosa type III secretion system Rmab Rabishield Made in India Human monoclonal antibody Antiinfectious Postexposure prophylaxis of rabies Rabies virus G glycoprotein Robatumumab 19D12 SCH 717454 MK-7454 P04722 Human monoclonal antibody IgG1κ Antineoplastic Colorectal phase II 2009 little benefit Ewings no response 2016 Intravenous Insulin-like growth factor I (IGF-1 receptor) (CD221) Roledumab Human monoclonal antibody IgG1κ Immunomodulation Rh disease Phase III 2017 Intravenous RHD Romilkimab SAR156597 HUBTI3_2_1 Humanized chimeric monoclonal antibody IgG4 bispecific Disease modifying Systemic sclerosis phase II 2019 Pulm fibrosis phase II 2017 no benefit Subcutaneous Interleukin 13 and IL4 Romosozumab Evenity FDA pending EU pending Japan 2018 Humanized monoclonal antibody IgG2κ Disease modifying Postmenopausal osteoporosis phase III study FRAME Men phase III BRIDGE phase III Intravenous Sclerostin/scleroscin SOST Rontalizumab rhuMAb IFNalpha Humanized monoclonal antibody Disease modifying Systemic lupus erythematosus phase II 2013 end points not met Subcutaneous IFN-α Rosmantuzumab OMP-131R10 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase I 2018 Intravenous Root plate-specific spondin r-spondin-3 WNT? (wingless/integrated) Rovalpituzumab tesirine SC0002 SC16LD6.5 ABBV-181 Humanized monoclonal antibody IgG1κ Antineoplastic Small cell lung cancer phase I 2018 Phase II 2024 Intravenous Delta-like ligand-3 (DLL3) Rovelizumab Hu23F2G LeukArrest Humanized monoclonal antibody IgG1κ Disease modifying Hemorrhagic shock, MI stroke phase III goals not met 2000 CD11, CD18 Rozanolixizumab UCB7665 Chimeric/humanized monoclonal antibody IgG4κ Thrombocytopenia ITP phase II 2019 Myasthenia gravis phase II 2018 Subcutaneous Intravenous Neonatal Fc receptor (FCGRT) Ruplizumab Antova Humanized monoclonal antibody Disease modifying lupus and lupus nephritis not effective Life-threatening thromboembolism BioDrugs. 2004; 18(2):95–102. Costimulation blockade in the treatment of rheumatic diseases CD154 (CD40L) Sacituzumab govitecan IMMU-132 FDA/EU pending approval Humanized monoclonal antibody IgG1κ Antineoplastic agent Prostate cancer phase II 2021 Urothelial phase II 2020 Trip neg breast ca phase III 2020 NSCLC SCLC UC Intravenous Tumor-associated calcium signal transducer 2 (TROP-2) inhibits topoisomerase I Samalizumab ALXN6000 BAML-16-001-S1 Humanized monoclonal antibody IgG2/G4κ Antineoplastic CLL MM phase I 2010 (terminated by sponsor) AML phase II 2021 Intravenous OX-2 membrane glycoprotein (CD200) Samrotamab vedotin Chimeric/humanized monoclonal antibody IgG1κ Antineoplastic No studies found Leucine-rich repeat-containing protein 15 (LRRC15) Sarilumab REGN88 SAR153191 Kevzara FDA?EU/Japan under review approved in Canada Human monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis phase III 2015/2020/2027(preg exposure), ankylosing spondylitis Juvenile idiopathic arthritis phase II 2022 Subcutaneous IL6 Satralizumab SA237 Sapelizumab FDA review possible 2019 Humanized monoclonal antibody IgG2κ Disease modifying Neuromyelitis optica phase III 2019/2020 Subcutaneous? IL6 receptor Satumomab pendetide OncoScint CR103 FDA 1992 Murine monoclonal antibody IgGκ fragment Fab' Diagnostic imaging Detection colorectal and ovarian cancer Intravenous Tumor-associated glycoprotein (TAG-72) Secukinumab AIN457 Cosentyx FDA 2015 EU 2015 Human monoclonal antibody IgG1κ Disease modifying Uveitis, rheumatoid arthritis psoriasis phase II 2019 over 100 other studies arthritis ; psoriatic psoriasis; spondylitis; ankylosing Subcutaneous IL 17A Selicrelumab CP 870.893 RG7876 RO-7009789 Human monoclonal antibody IgG2κ Antineoplastic Solid tumors phase I 2020 Pancreatic cancer phase II 2020 Colon cancer phase II 2021 Mesothelioma phase ib 2015 Subcutaneous Intravenous Tumor necrosis factor receptor superfamily member 5 (CD40) Seribantumab MM121 SAR256212 Human monoclonal antibody IgG2λ Antineoplastic Breast phase II 2020 Ovarian phase I 2014 Intravenous Receptor tyrosine-protein kinase erbB-3 (HER3) Setoxaximab Chimeric monoclonal antibody IgG1κ Antiinfection E. coli No known studies or clinical use E. coli shiga toxin type-2 Setrusumab BPS804 MOR05813 Human monoclonal antibody IgG2 Disease modifying Osteogenesis imperfecta phase II 2020 Intravenous Sclerostin (SOST) Sevirumab MSL-109 Antiinfectious CMV retinitis early termination trial secondary to safety Phase II 2003 Cytomegalovirus infection SHP647 Ontamalimab PF-00547659 Human monoclonalantibodyIgG2κ Disease modifying Crohn's/UC phase III 2020–2025 × 7 Phase II study 2007 better response in UC than in Crohn (?more time needed to evaluate clinical significance Subcutaneous Mucosal addressin cell adhesion molecule (MADCAM) Sibrotuzumab BIBH1 F19 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase II 2003 failed Lung cancer2001 Intravenous FAP Sifalimumab MDX-1103 MEDI-545 CP145 Humanized monoclonal antibody IgG1κ Disease modifying SLE phase II 2015 dermatomyositis, polymyositis Intravenous Subcutaneous IFN-α Siltuximab CLLB8 CNTO-328 Sylvant FDA 2014 EU 2014 Chimeric monoclonal antibody IgG1κ Antineoplastic Multiple myeloma phase II 2019 DM type I phase I 2017 Schizophrenia adjunct 2020 phase II Multicentric Castleman's disease (MCD) with HIV negative and HHV-8 negative Intravenous IL-6 Simtuzumab AB0024 GS-6624 Humanized monoclonal antibody IgG4κ Disease modifying Hepatic fibrosis Phase II 2016 no benefit Pulm fibroses phase II 2017 no benefit Myelo fibr 2017 phase II Subcutaneous Intravenous Lysyl oxidase homolog 2 (LOXL2) Siplizumab MEDI-507 Humanized monoclonal antibody IgG1κ Antineoplastic CD2 T Or NK cells Sirtratumab vedotin Human monoclonal antibody Antineoplastic Nothing in PubMed or clinical trials SLITRK6 Sirukumab Human monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis Phase III done good results Subcutaneous IL-6 Sofituzumab vedotin Humanized monoclonal antibody Antineoplastic Ovarian pancreatic Phase I (2014) CA-125 Solanezumab LY2062430 Humanized monoclonal antibody IgG1 Disease modifying Alzheimer's Phase III study discontinued no effect In preclinical trial for secondary prevention 2022 Hereditary AD phase III 2021 Intravenous Beta amyloid Solitomab MT110-011 AMG110 Murine monoclonal antibody bispecific T-cell engager (BiTE) Antineoplastic Gastrointestinal, lung, and other cancers Phase I 2015 Intravenous Epithelial cell adhesion molecule (EpCAM) CD3 Sonepcizumab LT1009 iSONEP Humanized monoclonal antibody Disease modifying Choroidal and retinal neovascularization phase II 2015 not so good Antineoplastic phase II renal cancer 2017 potential Intravenous Intravitreous Sphingosine-1-phosphate (S1P) Stamulumab Humanized monoclonal antibody Disease modifying muscular dystrophy Animal studies, minimal efficacy Phase I/II studies ongoing (no improvement) Intravenous Myostatin Sulesomab IMMU-MN3 LeukoScan EU 1997 Murine monoclonal IgG1 fragment Fab′ Diagnostic Osteomyelitis (imaging) NCA-90 (granulocyte antigen) Suptavumab REGN2222 SAR438584 Human monoclonal antibody IgG1κ Antiinfectious Medically attended lower respiratory disease phase III 2017 not meet primary endpoint Another study no data yet at 30 mg/kg dose Intramuscular Resp sync virus fusion protein (RSVFR) Sutimlimab BIVV009 Chimeric/humanized monoclonal antibody IgG4κ Disease modifying cold agglutinin disease phase III 2020 Intravenous Complement C1s (C1s) Suvizumab KD-247 Humanized monoclonal antibody IgG1κ Antiinfectious HIV Phase I KD-247 2007 Intravenous Human immunodeficiency virus glycoprotein 120 third variable loop Suvratoxumab MEDI4893 Human monoclonal antibody IgG1κ Disease modifying Nosocomial pneumonia phase II 2018 Intravenous Staphylococcus aureus alpha toxin Tabalumab LY2127399 Human monoclonal lantibodyIgG4κ Antineoplastic Rheum arthr phase III 2013 no signif response SLE phase III 2015 endpoints not met Mult myelo phase I 2014 may not treat but be prognostic Subcutaneous Cytokine B-cell activating factor (BAFF) Tacatuzumab tetraxetan HAFP-31 AFP-Cide Humanized monoclonal antibody yttrium 77 Antineoplastic No studies in clinical trial or PubMed Alpha-fetoprotein Tadocizumab C4G1 YM337 Humanized monoclonal antibody fragment IgG1κ Fab' Disease modifying Percutaneous coronary intervention phase I 1999 ?not further developed Integrin αIIbβ3 Talacotuzumab CSL362 JNJ-56022473 Humanized monoclonal antibody IgG1-2κ Antineoplastic AML phase III 2018 MDS phase II 2019 SLE 2019 phase I Intravenous Interleukin 3 receptor subunit-α (IL3Rα, CD123) Talizumab C21/AL-90 TNX-901 Humanized monoclonal antibody IgG1κ Disease modifying Peanut allergy Allergic reaction Phase II 2003 good results legal issues shelved the drug Subcutaneous IgE Tamtuvetmab AT-005 Tactress Canine monoclonal antibody IgG2λ CD52 Tanezumab RN624 PF-4383119 FDA review possible 2019 Humanized monoclonal antibody IgG2 Disease modifying Pain Osteoarthritis Back pain Metastatic cancer pain Phase II ∼2008 Nerve growth factor (NGF) Tanibirumab Olinvacimab TTAC-0001 Human monoclonal antibody IgG1 Disease modifying Antineoplastic Phase I glioblastoma 2020 Breast cancer phase I 2020 AMD murine no human studies found Intravenous VEFR-2 Taplitumomab paptox Murine monoclonal antibody IgG1κ Antineoplastic No studies pub med or clinical trials CD19 Tarextumab OMP-59R5 Human monoclonal antibody IgG2 Antineoplastic Phase II trial NSCLC no benefit 2017 Pancreatic phase II 2017 Intravenous Notch2/3, Notch receptor Tavolimab MEDI0562 Chimeric/humanized monoclonal antibody IgG1κ Antineoplastic Head and neck phase I 2024 Ovarian cancer phase II 2023 Tumor necrosis factor receptor superfamily member 4 (TNFRS4) OX40L receptor (CD134) Technetium (99 mTc) acritumomab Rabbit monoclonal IgG Not for use in humans- research purpose only CEA Technicium (99 mTc) Fanolesomab NeutroSpec FDA2004 Murine monoclonal IgM radiolabeled Disease modifying osteomyelitis Sales and marketing suspended (2005) Diagnostic scans for acute appendicitis Intravenous CD15 Tefibazumab INH–H2002 Aurexis Humanized monoclonal antibody IgG1κ Antiinfectious Staphylococcus aureus infection Phase II 2006 Intravenous Clumping factor A Telimomab aritox (TAB-885) Recombinant murine monoclonal antibody Fab with ricin Antineoplastic T Cell lymphoma/leukemia No studies in pub med or clinical trials CD5 Telisotuzumab vedotin ABT-700 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2017 SCLC phase II 2022 NSCLC phase II 2021 Intravenous Hepatocyte growth factor receptor HGFR Tenatumomab Murine monoclonal antibody IgG2b Antineoplastic Phase I 2017 Phase II brain tumors 2010 Intravenous P24821, tenascin C Teneliximab Chimeric monoclonal antibody IgG1 Not in clinical trials 2009 CD40 (TNF receptor superfamily member 5) Teplizumab MGA031 PRV-031 hOKT3g1(Ala-Ala) Humanized monoclonal antibody IgG1κ Disease modifying type I DM phase II completion AbATE trial 2019 Psoriasis phase I and II completed 2010 study stopped secondary to injection reaction severe allergy Intravenous Subcutaneous CD3 Tepoditamab Human monoclonal antibody IgG1κ bispecific Antineoplastic No studies on PubMed or clinical trials c-type dendritic cell-associated lectin 2 (CLEC-2A, MCLA-117) and CD3 Teprotumumab RV001 R-1507 RO4858696 HZN-001 FDA review possible 2019 Human monoclonal antibody Disease modifying Thyroid eye disease phase II 2017 Graves phase III 2020 Intravenous Insulin-like growth factor receptor type I (IGF-1 receptor) (CD221) Tesidolumab LFG316 NOV-4 Human monoclonal antibody Phase I 2017 PNH phase II 2020 AMD phase II 2015 not beneficial Intravenous Intravitreous C5 Tetulomab tetraxetan LU-177 Betalutin Humanized monoclonal antibody Antineoplastic Animal studies 2013 CD37 Tezepelumab MEDI9929 AMG-157 Human monoclonal antibody IgG2λ Disease modifying Asthma, atopic dermatitis Phase II 2017 Subcutaneous Thymic stromal lymphopoietin (TSLP) Theralizumab TGN1412 TAB08 Humanized monoclonal antibody Antineoplastic Solid tumors phase I 2020 Disease modifying Rheum arth, SLE phase II Intravenous CD28 History of cytokine storm at higher doses 2006 Tibulizumab LY3090106 Humanized monoclonal antibody bispecific tetravalent Disease modifying Autoimmune disorder Phase I 2020 Subcutaneous Intravenous No manuscripts found specific to this antibody Human B-cell activating factor of the tumor necrosis factor family interleukin 17 (BAFF) Tigatuzumab CS-1008 TRA-8 Humanized monoclonal antibody IgG1κ Antineoplastic Colon phase II 2011no added benefit Colon phase I 2013 NSCLC phase II 2011 no benefit Pancreatic phase II 2008 benefit TN breast canc 2015 phase II no added benefit Cytokine receptor DR5 (death receptor 5) TRAIL-R2 Tildrakizumab MK-3222 SCH-900222 Ilumya Ilumetri FDA 2018 Humanized monoclonal antibody IgG1κ Immunologically mediated inflammatory disorders Mod/severe psoriasis phase III 2018-20 Subcutaneous IL23 Timigutuzumab Humanized monoclonal antibody IgG1κ Antineoplastic No studies in clinical trial or PubMed erbB2/HER2 Timolumab BTT-1023 Human monoclonal antibody Disease modifying Scler cholang phase II 2019 Intravenous AOC3 Tiragotumab MTIG-7192A RG6058 RO7092284 Human monoclonal antibody IgG1κ Antineoplastic Phase I 2020 NSCLC phase II 2021 HL phase II 2019 Intravenous Nothing published yet T-cell IG and immune-receptor tyrosine-based inhibitory motif (TIGIT) Tislelizumab China pending approval Humanized monoclonal antibody Antineoplastic NSCLC phase III 2020 Gastric phase III 2022 Esophageal cancer phase III 2021 NHL phase II 2020 Intravenous Nothing published yet 2019 PCDC1, CD279 Tisotumab vedotin Human monoclonal antibody IgG1κ Antineoplastic Ovary cancer Cervix cancer Endometrium cancer Bladder cancer Prostate cancer Esophagus cancer Lung cancer, NSCLC Squamous cell carcinoma of the head and neck Pancreatic phase II 2022/3 Intravenous Coagulation factor III Tocilizumab MRA R-1569 RG-1569 RHPM-1 RO-4877533 Atlizumab Actemra, RoActemra FDA 2010 EU 2009 Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis >100 studies Behcet syndrome Intravenous Subcutaneous IL-6 receptor Tomuzotuximabfibri Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2019 EGFR, HER1 Toralizumab E−6040 IDEC-131 Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis, lupus nephritis etc. Phase II trials failed with TE CD154 (CD40L) Tosatoxumab Human monoclonal antibody IgG1λ Antiinfectious No studies PubMed or Clin trials Staphylococcus aureus α-hemolysin Tositumomab and iodine 131 Tositumomab Bexxar FDA 2003 Murine monoclonal antibody IgG2aλ Antineoplastic Follicular lymphoma ( NHL ) >100 studies Intravenous CD20 Tovetumab MEDI-575 Human monoclonal antibody IgG2κ Antineoplastic Phase I/II 2012 Glioblastoma limited clin activity Intravenous Platelet-derived growth factor receptor α (CD140a) Tralokinumab CAT-354 Human monoclonal antibody IgG4 Disease modifying asthma phase IIb +/−, atopic dermatitis phase II 2016 Intravenous Subcutaneous IL-13 Trastuzumab 4D5v8 R-597 SYD977 Herceptin FDA 1998 EU 2000 Herceptin Hylecta FDA 2019 –trastuzumab/hyaluronidase Herzuma 2018 Humanized monoclonal antibody IgG1κ Antineoplastic Breast cancer Gastric and gastro-esophageal junction cancer HER2-positive phase III Subcutaneous Intravenous HER2/neu Trastuzumab Deruxtecan DS-8201 Hercion FDA breakthrough therapy Antibody drug conjugate humanized antibody IgG1κ with topoisomerase I inhibitor (DXd) Antineoplastic breast cancer phase I study breast, gastric, colorectal, salivary, and nonsmall cell lung cancer participated in part 2 2020 phase II DESTINY-Breast01 HER2 Trastuzumab emtansine RG-3502 PRO132365 Kadcyla FDA2013 EU 2013 Humanized monoclonal antibody IgG1κ as ADC Antineoplastic Breast cancer Intravenous HER2/neu TRBS07 Ektomab 3funct Antineoplastic Melanoma GD2 ganglioside Tribbles-related protein (TRB) family members are the mammalian orthologs of Drosophila tribbles. Tribbles was originally identified as a cell cycle regulator during Drosophila development. Tribbles genes are evolutionary conserved, and three TRB genes (TRB1, TRB2 and TRB3) have been identified in mammals. TRBs are considered pseudokinases because they lack an ATP binding site or one of the conserved catalytic motifs essential for kinase activity. Instead, TRBs play important roles in various cellular processes as scaffolds or adaptors to promote the degradation of target proteins and to regulate several key signaling pathways. Recent research has focused on the role of TRBs in tumorigenesis and neoplastic progression. In this review, we focus on the physiological roles of TRB family members in tumorigenesis through the regulation of the ubiquitin-proteasome system and discuss TRBs as biomarkers or potential therapeutic targets in cancer Tregalizumab BT-061 Humanized monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis Phase IIb no benefit Subcutaneous CD4 Tremelimumab (aka ticilimumab) ∗CP-675,206 Human monoclonal antibody IgG2 Antineoplastic agent NSCLC, small cell lung cancer, urethelial cancer phase II 2020, head and neck cancer and colon phase I 2021 Mesothelial phase IIb DETERMINE not beneficial >100 studies CTLA4 (cytotoxic T lymphocyte-associated antigen 4, CD152 Trevogrumab REGN1033 SAR391786 Human monoclonal antibody IgG4κ Disease modifying Muscle atrophy due to orthopedic disuse and sarcopenia phase II 2020 Myostatin, growth differentiation factor 8 (GDF8) TRL3d3 3D3 IgG Studies only in mice to this point Ati-G protein antibody (RSVGV) Tucotuzumab celmoleukin EMD-273066 HUKS-IL2 Humanized monoclonal antibody IgG1 Antineoplastic Ovarian phase II 2008 Lung, kidney, bladder phase I 2000 no benefit Intravenous Interleukin2 (EpCAM) Tuvirumab Humanized monoclonal antibody Antiinfectious Not effective in achieving primary efficacy as assessed by neutralization of circulating HBsAg Intravenous Hepatitis B virus surface antigen Ublituximab TG-1101 FDA review pending 2019 Chimeric monoclonal antibodyIgG1κ Antineoplastic Chronic lymphocytic leukemia, follicular cell lymphoma phase II 2020 Disease modifying Multiple sclerosis phase II 2019, phase III 2021 Awaiting result looks good prelim Intravenous CD20 MS4A1 Ulocuplumab Human monoclonal antibody IgG4 Antineoplastic CLL phase I 2014 Phase I/II Waldenstrom macroglobulinemia 2025 Phase I/II AML 2021 Intravenous CXCR4 (CD184) Urelumab BMS-663513 Human monoclonal antibody IgG4κ Antineoplastic CLL phase II 2020 Solid tumors phase II 2023 Intravenous Human receptor 4-1BB (CD137) Urtoxazumab TMA-15 Humanized monoclonal antibody IgG1κ Disease modifying EHEC animal studies Escherichia coli (EHEC) shiga toxin 2 Ustekinumab Stelara FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Crohn disease Plaque psoriasis Psoriatic arthritis Subcutaneous Intravenous p40 subunit of interleukin 12 (IL-12p40), IL-23 Utomilumab PF-05082566 Human monoclonal antibody IgG2 Antineoplastic Diffuse large B-cell lymphoma Phase I 2021 phase II 2020 Breast phase II 2025 Intravenous 4-1BB (CD137) Vadastuximab talirine H2H12EC Chimeric monoclonal antibody IgG1κ Antineoplastic Acute myeloid leukemia phase II 2017 phase III 2017 MDS phase II 2017 Intravenous CD33 Vanalimab Mitazalimab Humanized monoclonal antibody IgG1λ Antineoplastic? No studies clinical trial or PubMed Immune checkpoint receptor, tumor necrosis receptor family CD40, (TNFRSF5) Vandortuzumab vedotin Humanized monoclonal antibody Antineoplastic Prostate cancer STEAP1 Vantictumab OMP-18R5 Human monoclonal antibody IgG2mab Antineoplastic NSCLC, breast phase I 2017 Intravenous Frizzled receptor Vanucizumab RG-7221 RO5520985 Humanized monoclonal antibody IgG1κ bispecific ANG-2/VEGF-Amab Antineoplastic Phase I 2018 Intravenous Angiopoietin 2/vascular endothelial growth factor A Vapaliximab BTT-1002 HUVAP Chimeric monoclonal antibody IgG2κ No studies in PubMed or clinical trials Vascular adhesion protein AOC3 (VAP-1) Varisacumab GNR-011 R-84 Human monoclonal antibody IgG1κ No studies in PubMed or clinical trials VEGF-A Varlilumab CDX-1127 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors and hematologic malignancies Phase I 2017, phase II 2019/20 Melanoma phase II 2018/21 Intravenous CD27 Vatelizumab GBR500 SAR339658 Humanized monoclonal antibody IgG4 Disease modifying UC phase II 2016 MS phase II 2016 withdrawn lack of efficacy Α2β1 integrin I domain ITGA2 (CD49b) Vedolizumab LDP02 MLN02 Entyvio FDA 2014 EU 2014 Humanized monoclonal antibody IgG1κ Disease modifying Crohn disease Ulcerative colitis In CD resolution extraintestinal manifestations Intravenous Integrin α4β7 Selectively blocks trafficking of Memory T cells to inflamed gut tissue by inhibiting a4b7-mucosal addressin cell adhesion molecule-1 (MAd-CAM-1) interaction Veltuzumab IMMU-106 HA20 Humanized monoclonal antibody IgG1κ Antineoplastic Non-Hodgkin's lymphoma phase II 2013 ITP phase II 2016 Subcutaneous CD20 Vepalimomab Murine monoclonal antibody AOC3 (vascular adhesion protein-1) Vesencumab MNRP1685A Human monoclonal antibody IgG1mab Antineoplastic Solid malignancies Phase I 2011 proteinuria Neuropilin1 (NRP1) Visilizumab Nuvion Humanized monoclonal antibody IgG2 Disease modifying Prevent GVHD Not effective in UC CD3 Vobarilizumab Humanize monoclonal scFv Disease modifying inflammatory autoimmune diseases Nothing in PubMed IL6R Volociximab M200 Chimeric monoclonal antibody IgG4κ Antineoplastic Solid tumors NSCLC phase I/II 2010 Disease modifying phase I AMD terminated no results Integrin α5β1 Vopratelimab JTX-2011 Humanized monoclonal antibody IgG1κ Antineoplastic Solid tumors phase II 2022 Intravenous Inducible T-cell costimulator (ICOS) Vorsetuzumab mafodotin H1F6 SGN-70 Humanized monoclonal antibody Antineoplastic Phase I 2017 Intravenous CD70 Votumumab HumaSPECT Diagnostic EU 1998 Withdrawn from market 2003 Human monoclonal antibody Diagnostic Human colon cancer imaging Tumor antigen Cytokeratin tumor-associated antigen (CTAA16.88) Vunakizumab SHR-1314 Humanized monoclonal antibody IgG1 Disease modifying Psoriasis phase II 2019 Subcutaneous Nothing published Interleukin 17 alpha Xentuzumab BI-836845 Humanized monoclonal antibody Antineoplastic Breast, prostate, solid phase I 2019 Intravenous No clinical studies published Insulin-like growth factor (IGF1, IGF2) XMAB-5574 Tafasitamab MOR00208 FDA review possible 2019 Humanized monoclonal immunoglobulin fragment κ Fc Antineoplastic Diffuse large B-cell lymphoma phase II 2015/18/19/22 phase III 2022 Intravenous CD19 Zalutumumab 2F8 HUMAX-EGFR HuMax-EGFr Suspended for commercialization Human monoclonal antibody Antineoplastic Squamous cell carcinoma of the head and neck phase II 2011 phase III 2016 Intravenous EGFR Zanolimumab HuMax-CD4 (trade name) Humanized monoclonal antibody IgG1κ Antineoplastic CTCL Phase II good results Phase III suspended by company? Intravenous CD4 Zenocutuzumab Humanized monoclonal antibodyIgG1 bispecific epidermal growth factor receptors her2,her3 Antineoplastic ERBB3, HER3 Ziralimumab Human monoclonal antibody IgM Disease modifying immunosuppressive No studies clinical trials or PubMed CD147 (basigin) Zolbetuximab IMAB362 Claudiximab Chimeric monoclonal antibody IgG1κ ADCC enhance antibody Antineoplastic gastric cancer phase I, IIb Phase III 2023 Gastrointestinal adenocarcinomas and pancreatic tumor Intravenous Claudin protein (CLDN18.2) Zolimomab aritox H65-RTA ZX-CD5 Orthozyme CD 5 plus Human monoclonal antibody IgG1 Disease modifying Not effective in preventing GVHD 1994 CD5 Auristatins are water-soluble dolastatin analogs of dolastatin 10. Dolastatin 10 belongs to dolastatin family and it can powerfully bind to tubulin, thus inhibiting polymerization mediated through the binding to the vinca alkaloid-binding domain, and causes cell to accumulate in metaphase arrest. Ulcerative Colitis Mabs being studied for UC but not yet approved by the FDA include bimekizumab, etrolizumab, golimumab, mirikizumab, ravagalimab, sacituzumab govitecan, ontamalimab, and vatelizumab. Refer to Table 16.1 . Autoimmune Diseases Autoimmune diseases affect many organs and tissues including liver, gall bladder, pancreas (β islet cells in diabetes mellitus), nerve junctions (myasthenia gravis), thyroid, bone and joints, blood vessels, and multiorgan systems, systemic lupus erythematosus (SLE). Autoimmune arthritis is of multiple types including psoriatic, sclerosis, rheumatoid arthritis (RA), and SLE. Many of these diseases are mediated by antibody or cellular autoimmunity but ultimately appear to be secondary to an underlying abnormality in T-cell immune-regulatory control. These disease processes are historically controlled with antiinflammatory agents, immunosuppressive/immunomodulatory agents, or low-dose chemotherapy. Those with resultant hormone deficiencies are supplemented with hormones depleted by the disease process. It is hoped that passive antibody therapy will mitigate the sequelae of these inflammatory processes. Plaque psoriasis/psoriatic arthritis Psoriasis affects 2%–3% of the world population and is an inflammatory skin disease. Brodalumab (Siliz) is a human Mab (IgG2κ) with specificity to IL-17 receptor A (IL-17RA). It is FDA approved for treatment of plaque psoriasis, and its mechanism of action is by inhibiting IL-17A, IL-17F, IL-17C, IL-25, and IL-17A/F heterodimer cytokine-induced responses including release of proinflammatory cytokines. When compared to ustekinumab, response rates nearly doubled with brodalumab in phase II and phase III trials during induction and maintenance therapies. 275 , 276 Other therapies currently also approved or being studied for treatment of this disease include bermekimab (MABp1,T2-18C3, CA-18C3, Xilonix), bimekizumab, briakinumab, certolizumab pegol (Cimzia), etanercept (Enbrel), infliximab (Remicade, Inflectra, Remsima), itolizumab (Alzumab), adalimumab (Humira, Amjevita), ustekinumab (Stelara), secukinumab (AIN457, Cosentyx), guselkumab (Tremfya), tildrakizumab (MK-3222, SCH-900,222, Ilumya, Ilumetri), risankizumab (ABBV-066, BI-655,066), mirikizumab (LY3074828), namilumab (MT203), netakimab, and vunakizumab. Refer to Table 16.1 . Withdrawn from market or ineffective for treating psoriasis include efalizumab (Raptiva), fezakinumab, bleselumab, and teplizumab (MGA031, PRV-031, hOKT3g1(Ala-Ala)) Refer to Table 16.1 . Systemic juvenile idiopathic arthritis Abatacept (Orencia) is a recombinant soluble fusion protein of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human IgG1. Its mechanism of action is as selective costimulation modulator as it inhibits T lymphocyte activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. This medication is FDA approved for both juvenile idiopathic arthritis (JIA) and adult RA. 277 , 278 , 279 Rheumatoid Arthritis Certolizumab pegol alone or with methotrexate improves quality of life in RA and may cause disease remission and reduce joint damage. 280 Ankylosing Spondylitis Certolizumab pegolis is also approved for use with ankylosing spondylitis. Systemic Lupus Erythematosus Belimumab (Benlysta) is a human Mab (IgG1λ) that binds to B-cell activating factor and acts as a B-lymphocyte stimulator-specific inhibitor. It was approved by the FDA in 2011 for treatment of adult patients with active, autoantibody-positive SLE receiving standard therapy. This medication also decreases episodic frequency of lupus nephritis. 281 , 282 , 283 Cardiovascular Disease Despite marked improvement in survival from cardiovascular disease, this illness remains the number one cause of mortality in the US. This process causes injury to the endothelium of blood vessels of the heart secondary to toxins, accumulation of cholesterol, or chronic low-grade inflammation. Treatment has been preventive, primarily during actual injury or following injury. Therapies involve changes in behavior (diet, exercise, and cessation of tobacco use), pharmacologic to control contributing underlying illness (hypercholesterolemia, hypertension, diabetes type I and II), to diminish injury through thrombolytics, stents, vasodilators, supplemental oxygen, or to control sequelae of infarctions (cardiac dysfunction/failure). Passive antibody therapies are being tried to decrease the effects of some of the contributing factors of atherosclerotic plaque formation. Abciximab (ReoPro) is a chimeric recombinant monoclonal fragment (IgG1 Fab') with specificity to platelet glycoprotein IIb/IIIa receptor (CD41 7E3)/Intergrin α-IIb that prevents platelets from binding to fibrinogen. This Mab also prevents coagulation factor XIII from binding to platelets allowing stabilization of clots and are more easily lysed. The Fc portion of the antibody is removed to decrease thrombocytopenias. This antibody is used during high-risk coronary intervention to prevent clot formation and cardiac ischemia. 284 Alirocumab (Praluent) is a human Mab (IgG1) with specificity to proprotein convertase subtilisin/kexin type 9. This medication is used to control cholesterol levels in patients at high risk for cardiovascular events and in patients with familial hypercholesterolemia who are not controlled by other agents. 285 , 286 , 287 Evolocumab (Repatha) is a human Mab (IgG2λ) FDA approved for the treatment of hypercholesterolemia in patients with familial hypercholesterolemia or history of cardiovascular disease. This Mab has specificity to PCSK9. This medication reduced low-density lipoprotein (LDL) and cholesterol levels by 60% even after statin therapy. Hazard ratios for primary and secondary endpoints were less than one (∼0.80–0.85) with fewer cardiovascular-related death or infarction and stroke. 288 , 289 Under future watch is frovocimab (LY3015014) a humanized Mab (IgG4κ) with specificity to PCSK9 that completed phase I and II trials. There was up to 50% reduction in LDL cholesterol levels. Phase III studies have yet to be performed. 290 An additional antibody is lodelcizumab a humanized Mab (IgG1κ); however, no studies were found in clinicaltrials.gov or in Pubmed searches. Bococizumab is a humanized Mab (IgG2κ) that was in phase III trial, which was discontinued secondary to primary endpoints not being achieved. 291 Inflammatory Bowel Disease Inflammatory bowel disease (IBD) pathophysiology remains unknown but may have genetic, infectious, autoimmune origins including cell-mediated immunity. These diseases may be classified as ulcerative colitis (UC), isolated to the colon, or Crohn's disease primarily found in the colon but may involve the entire gastrointestinal tract. With long-standing active disease, malignancy is much more frequent in UC than in Crohn's disease. Mild UC is treated with antiinflammatory agents such as sulfasalazine and glucocorticosteroids. For more severe disease, high-dose steroids may be used to maintain disease quiescent and low-dose steroids to keep disease in remission. Low-dose chemotherapeutic agent or immunosuppressive agent may also be added if dose of corticosteroids is too high to maintain remission. Surgery may be necessary to control disease. For Crohn's disease, medical therapy is usually less successful in managing the disease and surgery may be necessary but is not curative as in UC. For both of these disease processes, passive antibody therapy may offer not only control of disease but possible complete remission from mucosal damage. 263 , 264 Adalimumab (two formulations: Humira and Amjevita) is a recombinant human Mab (IgG1) with specificity to tumor necrosis factor alpha (TNF-α). Both forms are FDA approved to treat Crohn's disease as well as multiple types of rheumatoid arthritis. In Crohn's disease, this medication decreases signs and symptoms of disease and is able to induce clinical remissions. 265 , 266 Certolizumab (Cimzia) is a recombinant humanized m fragment with TNF-α as target. It is FDA approved for both Crohn's disease and Rheumatoid arthritis. 267 , 268 , 269 Vedolizumab (Entyvio) is a humanized Mab (IgG1κ) that has selectivity for integrin α4β7 and is FDA approved for treatment of Crohn's disease. This Mab mode of action is to selectively block trafficking of memory T cells into inflamed gut tissue by inhibiting α4β7-mucosal addressin cell adhesion molecule-1 (MAd-CAM-1) interaction with intestinal vasculature. This medication has shown a good safety profile with no cases of promyelocytic leukemia (PML), no increased risk of infections, malignancies compared with classically treated IBD, and low incidence of infusion-related reactions. This medication is also FDA approved for UC. 270 , 271 Infliximab (Remicade, Inflectra, Remsira) is a chimeric Mab (IgG1κ) with specificity to TNF-α and is FDA approved for IBD and multiple inflammatory arthritic diseases. This medication allows for steroid-free remission within months of starting therapy. 272 Natalizumab (Tysabri) is a humanized Mab (IgG2κ) with selectivity to CD62L (L selectin α4 subunit of α4β1 and α4β7 integrins of leukocytes, not neutrophils, VLA-4). This Mab is FDA approved for Crohn's disease and multiple sclerosis. This medications is effective in induction of clinical remission in moderate-to-severe Crohn's disease. This medication does have the risk of PML. 273 , 274 Other Mab being studied for Crohn's disease but not yet approved by the FDA include Ustekinumab, brazikumab, etrolizumab, risankizumab, and ontamalimab. In contrast, Mabs studied but not beneficial for Crohn's disease include andecaliximab, eldelumab, and fontolizumab. Refer to Table 16.1 . Table 16.1 Summary of Monoclonal Antibody Therapies. Generic Drug Name Brand Name Type of Antibody AHFS Classification Dosage Form(s) Target 8H9 Iodine 124 monoclonal antibody (Murine) Antineoplastic Neuroblastoma, sarcoma, metastatic brain cancers Another study Sloan Kettering using I 331 version phase I good results Intravenous B7–H3 Abagovomab Monoclonal antibody (Murine) An antiidiotypic mAb that mimics ovarian cancer CA125 protein Antineoplastic Phase II study for ovarian cancer Phase III good immune response but no increase RFS or OS no benefit Subcutaneous CA-125 Abatacept Orencia FDA 2005 EU 2010 Recombinant soluble fusion protein of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human immunoglobulin G1 (IgG1). Disease modifying Rheumatoid arthritis Juvenile and adult psoriatic arthritis (phase III) Subcutaneous or intravenous Selective costimulation modulator, inhibits T cell (T lymphocyte) activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. Abciximab c7Ec Fab ReoPro FDA 1994 EU 1995 (country-specific approval) Human-murine chimera Recombinant monoclonal IgG1 Fab Procedure modification High-risk coronary intervention Platelet aggregation inhibitor Intravenous Platelet glycoprotein IIb/IIIa receptor (CD41 7E3)/Intergrin α-IIb Abituzumab DI17E6 EMD525797 Humanized monoclonal antibody IgG2κ Antineoplastic Colorectal cancer phase I 2013, phase II 2015 primary endpoint PFS not met Sclerosing interstitial lung disease phase II terminated 2018 slow enrollment Prostate phase IIno significant increase PFS Intravenous CD51 (?integrin alpha V) Abrilumab AMG 181 Phase II study discontinued development (2016) Integrin α-4 β-7 Actoxumab Human monoclonal antibody Disease modifying Clostridium difficile Phase I and II anti-CDTB1 much better Clostridium difficile toxin A Adalimumab Humira FDA 2002 EU 2003 Amjevita FDA 2016 EU 2017 Recombinant human IgG1 monoclonal antibody Disease modifying Humira Rheumatoid arthritis; juvenile idiopathic arthritis; psoriatic arthritis; ankylosing spondylitis; Crohn's disease, plaque psoriasis Amjevita Arthritis; juvenile rheumatoid arthritis; psoriatic arthritis; rheumatoid colitis; ulcerative Crohn's disease; psoriasis; spondylitis; ankylosing Possibly hemolytic disease of newborn Injection subcutaneous TNF-α Adecatumumab MT-201 Recombinant human monoclonal antibody IgG1κ Antineoplastic Breast phase Ib+, colorectal and prostate Phase II completed Phase III soon? Intravenous EpCAM (CD326) epithelial cell adhesion molecule Aducanumab Human monoclonal antibody IgG1 Disease modifying Alzheimer's disease Phase III x 2 ongoing started 2015 Intravenous Beta-amyloid (N-terminus 3–6) soluble oligomers and insoluble fibrils Afasevikumab Human monoclonal antibody IgG1κ Disease modifying Multiple sclerosis Phase I completed Nothing in pubmed Subcutaneous IL17A and IL17F Afelimomab Murine F(ab') Antibody Fab' fragment IgG3κ Disease modifying Sepsis Phase III trial marginal benefit abandoned TNF-α Alacizumab pegol Humanized monoclonal antibody F(ab') 2 Limited information on development; Cancer VEGFR2 Alemtuzumab LDP-03 Campath-1H Lemtrada FDA 2014 EU 2013 MS Campath FDA 2001 EU 2001 CLL Humanized rat monoclonal antibody IgG1κ Antineoplastic B-Cell CLL , CTCL, T cell lymphoma Disease modifying Multiple sclerosis (phase III) Not effective for kidney transplant conditioning or rejection prevention Intravenous CD52 Alirocumab Praluent FDA 2015 EU 2015 Human monoclonal antibody IgG1 Disease modifying Decrease cholesterol Phase III Subcutaneous Proprotein convertase subtilisin kexin type 9 (PCSK9) Altumomab pentetate In 98 Hybri-ceaker Murine monoclonal antibody IgG1 Diagnostic purpose radiology colorectal cancer (diagnosis) CEA ALX-0171 Trimeric nanobody Antiinfectious RSV phase II 2020 Inhalation RSVF Amatuximab MORAb-009 Chimeric murine-human monoclonal antibody IgG1κ Antineoplastic Ovarian cancer Phase II Now research on using to treat mesotheliomas Phase I/II Pancreatic cancer Intravenous Mesothelin Prohibits binding of MSLN with antigen CA125/MUC16 AMG330 Bispecific T-cell engager (BiTE) Antineoplastic AML phase I AML 2020 Intravenous CD33 and CD3 Anatumomab mafenatox Murine monoclonal fragment Fab Antineoplastic Nonsmall cell lung carcinoma Tumor-associated glycoprotein 72 (TAG-72) Andecaliximab GS 5745 Chimeric monoclonal antibody IgG4κ Antineoplastic gastric cancer phase I, II, III ongoing or gastroesophageal junction adenocarcinoma phase III ongoing Crohn phase II no response, UC Intravenous Gelatinase B is a matrix metalloproteinase-9 (MMP-9) Anetumab ravtansine In 98 Human monoclonal antibody IgG1λ Antineoplastic ovarian phase II, lung, pancreatic phase I, breast now research on using to treat mesotheliomas Phase II Cervical cancer ?preclinical Mesothelin Prohibits binding of MSLN with antigen CA125/MUC16 Anifrolumab Human monoclonal antibody IgG1κ Disease modifying Systemic lupus erythematosus phase I and IIb 2018 Intravenous Interferon α/β receptor Anrukinzumab (=IMA-638) Humanized monoclonal antibody IgG1κ Disease modifying Asthma phase II ?results UC phase II no benefit IL-13 Apolizumab Humanized monoclonal antibody Antineoplastic non-Hodgkin's lymphoma abandoned 2009 toxic effects 2009 CLL phase I/II HLA-DRβ Arcitumomab CEA-Scan FDA 1996 EU 1996 Withdrawn EU market 2005 Murine monoclonal antibody IgG1 Fab' Diagnostic imaging Gastrointestinal cancers Colorectal cancers CEA Ascrinvacumab Human monoclonal antibody Antineoplastic mesothelioma Nothing in pub med or web search Activin receptor-like kinase 1 Aselizumab Humanized monoclonal antibody Disease modifying Severe injured patients phase II 2004, no benefit L-selectin (CD62L) Atezolizumab MPDL3280A Tecentriq FDA 2016 Fc engineered, humanized monoclonal antibody IgG1κ Antineoplastic agent, treat metastatic urothelial carcinoma , non-small cell lung cancer Phase III Bladder/urothelial cancer phase I Breast cancer phase Ib triple marker neg breast cancer Intravenous Binds to PD-L1 and blocks interactions with the PD-1 and B7.1 receptors FDA-approved atezolizumab (TECENTRIQ, Genentech, Inc.), in combination with bevacizumab, paclitaxel, and carboplatin for the first-line treatment of patients with metastatic nonsquamous, nonsmall cell lung cancer (NSq NSCLC) with no EGFR or ALK genomic tumor aberrations Atidortoxumab Human monoclonal antibody IgG1κ Limited information on use and development Negative search PubMed-internet Staph aureus alpha toxin Atinumab Human monoclonal antibody IgG4κ Disease modifying Acute spinal cord injury RTN4 Atorolimumab Developed?? Human monoclonal antibody IgG3 Disease modifying hemolytic disease of the newborn Rhesus factor Avelumab Bavencio FDA 2017 Human monoclonal antibody IgG1λ Antineoplastic Cancers, ovarian, gastric, nonsmall cell lung (NSCLC), metastatic, solid tumors phase II Studies completed metastatic Merkel cell carcinoma Intravenous PD-L1 Azintuxizumab vedotin Chimeric/humanized monoclonal antibody IgG1 Antineoplastic Nothing in PubMed CD319 BAN-2401 Humanized monoclonal antibody IgG1 Disease modifying Alzheimer A phase IIb study ongoing started 2013 Intravenous Soluble Aβ amyloid protofibrils Bapineuzumab Humanized IgG1 monoclonal antibody Disease modifying Alzheimer's disease Phase III no more studies discontinued research 2012 ARIA-E, amyloid-related imaging abnormalities–edema Intravenous Beta amyloid Fibrillary and soluble β amyloid Basiliximab Simulect FDA 1998 EU 1998 Chimeric monoclonal antibody IgG1κ Immunosuppressive agents Prophylaxis of acute rejection in allogeneic renal transplantation Intravenous CD25 (α chain of IL-2 receptor) Bavituximab Chimeric monoclonal antibody IgG1κ IgG3 (SUNRISE trial) Cancer, viral infections (Hep C) phase III NSCLC failed to improve survival Sunrise trial stopped Feb 2016, phase II/III breast cancer, phase II pancreatic cancer, phase I/II trial hepatocellular carcinoma, phase I malignant melanoma + rectal cancer good response rectal; not for prostate cancer, phase II hepatitis C not resulted? Phosphatidylserine BAY-103356 CDP 571 Humicade Senlizumab Humanized monoclonal antibody IgG4κ For research only Phase II 1995 TNF α BCD-100 Human monoclonal antibody Antineoplastic Phase II/III melanoma NCT03269565 (complete Dec 2019) Intravenous Programmed cell death-1 (PD1) Bectumomab LymphoScan Fab'-IgG2κ Antineoplastic Non-Hodgkin's lymphoma (detection) CD22 Begelomab Begedina Murine IgG2b Disease modifying GvHD phase II/III DPP4 binds CD26 on T lymphocytes Belantamab mafodotin Humanized monoclonal antibodymab Antineoplastic No studies or info on clinical trial, PubMed, FDA substance BCMA Belatacept Nulojix FDA 2011 Soluble fusion Protein consisting of the modified extracellular domain of CTLA-4 fused to Fc domain of a recombinant human monoclonal antibody IgG1 Immunosuppressive agents Prophylaxis renal transplant rejection in adults Phase III FDA approved Intravenous Selectively inhibits T-cell activation through costimulation blockade binds to both CD80 and CD86 blocking CD28 Belimumab Benlysta FDA 2011 EU 2011 LymphoStat-B Human monoclonal antibody IgG1λ Disease modifying Kidney transplant phase II Treat SLE (testing phase III for renal involvement) Phase II Rheum arthritis failure Phase II Srogren ± GVHD ongoing Intravenous Subcutaneous B-cell activating factor (BAFF), B-lymphocyte stimulator Bemarituzumab Humanized monoclonal antibody Antineoplastic FGFR2 Benralizumab Fasenra FDA 2017 EU 2017 Humanized monoclonal antibody IgG1κ Disease-Modifying Asthma phase III completed Severe asthma eosinophilic subtype Subcutaneous Interleukin-5 (IL-5α) receptor alpha subunit-directed cytolytic (CD125) Berlimatoxumab Human monoclonal antibody Staph aureus bicomponent leukocidin No studies clinical, no find creative, zero pub med Bermekimab MABp1 T2-18C3 CA-18C3 Xilonix Human monoclonal antibody IgG1κ Disease modifying psoriasis phase III x 2 2020 Ank spond II 2022 Psor arth II 2020 III 2020 Subcutaneous Intravenous IL17A Bersanlimab Human monoclonal antibody ICAM-1 Bertilimumab CAT-214 Human monoclonal antibody IgG4κ Disease modifying Severe allergic disorders phase II atopic dermititis Ongoing studies bullous pemphigoid and ulcerative colitis phase II Intravenous CCL11 (eotaxin-1) Besilesomab Scintimun EU 2010 Not FDA approved Murine monoclonal antibody IgG1κ Diagnostic use Inflammatory lesions and metastases (detection) CEA-CAM8-related antigen Bevacizumab Avastin FDA 2004 EU 2005 Humanized monoclonal antibody IgG1κ BiTE Antineoplastic agent Antiangiogenesis inhibitor Colorectal cancer 2004, NSCLC 2006, RCC 2009, GBM phase III, ovarian cancer, metastatic cervical cancer, fallopian 2014 Breast cancer (FDA removed approval for breast cancer 2010) Recurrent glioblastoma multiform Non squamous nonsmall cell lung cancer Intravenous solution or ophthalmic injection May not be so good for GBM or ovarian VEGF-A anti-angiogenesis inhibitor Bezlotoxumab Zinplava FDA 2016 EU 2017 Human monoclonal antibody IgG1 Disease modifying phase III studies done MODIFY I and II Modify III ongoing Pseudomembranous colitis Intravenous Clostridium difficile colitis anti-B toxin Biciromab FibriScint Murine monoclonal fragment Fab' IgG1κ Detect cardiovascular thromboembolism (diagnosis) Fibrin II, beta chain Bimagrumab BYM338 Human monoclonal antibody IgG1λ Disease modifying Myostatin inhibitor DM II decrease BMI phase II Sporadic inclusion body myositis phase III not meet endpoint Treat sarcopenia in older adults phase II Intravenous Activin A receptor type IIB (ACVR2B) Bimekizumab Humanized monoclonal antibody IgG1κ Disease modifying Ankylosing spondylitis (2018 II, 2022 II, +), plaque psoriasis (2021 III, 2020 III, 2019 III, +), psoriatic arthritis (2020), RA (2017 II), UC phase II Subcutaneous IL 17A and IL 17F Bivatuzumab mertansine Humanized monoclonal antibody IgG1 Antineoplastic squamous cell carcinoma, breast phase I fail x 2, head/neck or esophagus phase I fail, toxicity CD44 v6 Bleselumab Human monoclonal antibody IgG4κ Disease modifying organ transplant rejection phase II 2020 to prevent FSGS in kidney transplant patients Phase II psoriasis- medication tolerated with minimal reaction, no benefit to disease process Intravenous CD40 Blinatumomab Blincyto FDA 2014 EU 2015 Murine(scFv - kappa - heavy) - (scFv - heavy - kappa) BiTE Antineoplastic Ph chrom neg pre-B ALL (CD19+) phase II B- cell precursor acute lymphoblastic leukemia (ALL) initial or relapsed/refractory Intravenous Bispecific T-cell engager monoclonal antibody construct that directs CD-3 positive effector memory T cells to CD19-positive target cells Blontuvetmab Blontress Canine monoclonal antibody IgG2 κ/λ Veterinary treat canine B-cell lymphoma CD20 Blosozumab Humanized IgG4κ Disease modifying Osteoporosis 3 phase I and one phase 2 injection site reaction and antibodies to antibody Intravenous Subcutaneous SOST Antisclerostin Bococizumab RN316 PF-04950615 Humanized IgG2κ Disease modifying Dyslipidemia Phase III 2019 Discontinued secondary to antidrug antibodies, no primary endpoint achieved Subcutaneous intravenous Neural apoptosis-regulated proteinase 1 PCSK9 (proprotein convertase subtilisin/kexin type 9, neural apoptosis-regulated convertase 1, NARC1, NARC-1, proproteine convertase 9, PC9) Brazikumab Human monoclonal antibody IgG2λ Disease modifying Ulcerative colitis phase II 2021 Phase I/II completed Crohn. Phase III ongoing Subcutaneous IL23 Brentuximab vedotin Adcetris FDA 2013 Breakthrough therapy status by FDA 2018 Chimeric humanized monoclonal antibody IgG1κ Antineoplastic Hodgkin lymphoma Anaplastic large-cell lymphoma Intravenous CD30 (TNFRSF8) an antibody-drug conjugate (ADC) 3 parts: anti-CD30 (cAC10, a cell membrane protein of the tumor necrosis factor receptor), a microtubule disrupting agent monomethyl auristatin E (MMAE) and a protease-cleavable linker that attaches MMAE covalently to cAC10. The combination disrupts the intracellular microtubule network causing cell-cycle arrest and apoptotic cellular death Briakinumab Human monoclonal antibody Disease modifying psoriasis, Drug development stopped for psoriasis, phase IIb study in Crohn's Intravenous IL-12, IL-23 Brodalumab AMG827 Siliz FDA 2016 Human monoclonal antibody IgG2κ Disease modifying Plaque psoriasis Completed phase III Subcutaneous Receptor IL-17RA Brolucizumab RTH258 ESBA1008 FDA review 2018 Humanized single chain antibody fragment (scFv κ) Disease modifying Wet or age-related macular degeneration phase III to be completed Sept 2018, 2020 HAWK (NCT02307682) and HARRIER (NCT02434328) phase III trials good results Intravitreal https://www.novartis.com/news/media-releases/new-novartis-phase-iii-data-brolucizumab-demonstrate-reliability-12-week-treatment-interval VEGFA Brontictuzumab Humanized IgG2λ Antineoplastic Phase I Colorectal Lymphoid Adenoid cystic Solid tumors Intravenous Notch 1 Burosumab KRN23 Crysvita FDA 2018 Human monoclonal antibody IgG1κ Disease modifying X-linked hypophosphatemia Phase III completed Subcutaneous https://www.creativebiolabs.net/burosumab-overview.htm FGF 23 phosphaturic hormone fibroblast growth factor 23 Cabiralizumab Humanized monoclonal antibody IgG4κ Antineoplastic metastatic pancreatic cancer phase II 2020 Many other cancers phase I Intravenous CSF1R Camidanlumab tesirine ADCT-21 Human monoclonal antibody Antineoplastic B-cell Hodgkin's lymphoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia 2018 phase I Advanced solid tumors with literature evidence of CD25(+) treg content Head and neck Nonsmall cell lung Gastric, esophageal, Pancreas, bladder, Renal cell, melanoma, Triple-negative breast, ovarian phase I 2021 Intravenous CD25 Sinilimab Camrelizumab IBI308 China pending approval Humanized monoclonal antibody IgG4κ Antineoplastic Phase III nasopharyngeal cancer 2021 Phase III esophageal cancer 2021 Programmed cell death 1 (PDCD1) Canakinumab ACZ885 Ilaris FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Cryopyrin- associated periodic syndromes Including familial cold auto-inflammatory syndrome and Muckle –Wells syndrome ; tumor necrosis factor receptor-associated periodic syndrome (TRAPS); hyperimmunoglobulin D syndrome (HIDS)/mevalonate kinase deficiency (MKD) and familial Mediterranean fever (FMF) Systemic Juvenile idiopathic arthritis Treat Juvenile idiopathic arthritis phase III NSCLC 2025 phase III CVD rejected by FDA Behcet Subcutaneous IL-1β Cantuzumab mertansine Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase I 2007 Intravenous Mucin CanAg Cantuzumab ravtansine Humanized monoclonal antibody IgG1κ Antineoplastic Cancers MUC1 Caplacizumab-yhdp Cablivi (Nanobody program) FDA 2019 EU 2018 Humanized single variable domain antibody (bivalent nanobody) Disease modifying Inhibits interaction vWF and platelets Treat acquired TTP Phase III Hercules study completed Intravenous Subcutaneous VWF Capromab pendetide Prostascint FDA 1996 Murine monoclonal antibody Diagnostic imaging Prostatic carcinoma cells detection Intravenous Tumor surface antigen PSMA Carlumab Human monoclonal antibody IgG1κ Antineoplastic Prostate phase II no long term benefit Pulm fibrosis phase II no benefit Intravenous hMCAF/MCP-1 (human macrophage/monocyte chemotactic protein-1) Carotuximab TRC105 Chimeric monoclonal antibody IgG1κ Antineoplastic angiosarcoma Hepatocellular car phase I/II 2020 Glioblastoma multi-phase II 2014 terminated poor accrual ?results Angiosarcoma phase III 2019 TAPPAS trial Prostate ca phase II 2021 NSCLC phase I 2019 Intravenous Endoglin (CD105) Catumaxomab Removab FDA approved pend 2017) EU approved 2009 Removab: A trifunctional rat/murine hybrid antibody IgG2a/IgG2b Antineoplastic Removab Ovarian cancer phase II, malignant ascites phase II, gastric cancer phase II (ovarian, gastric, colon, pancreatic, breast, endometrial) Proxinium Head and neck cancer Intraperitoneal EpCAM, CD3 Catumaxomab consists of one "half" (one heavy chain and one light chain) of an anti-EpCAM antibody and one half of an anti-CD3 antibody, so that each molecule of catumaxomab can bind both EpCAM and CD3. In addition, the Fc-region can bind to an Fc receptor on accessory cells like other antibodies, which has led to calling the drug a trifunctional antibody. cBR96-doxorubicin immunoconjugate aka SGN-15 Humanized monoclonal antibody IgG1κ Antineoplastic Cancer Sponsorship ceased 2005 Cedelizumab CIMZIA Humanized monoclonal antibody IgG4κ Prevent organ transplant rejection CD4 Cemiplimab Libtayo FDA 2018 Human monoclonal antibody IgG4 Antineoplastic Nonsmall cell lung cancer (NSCLC) phase I 2021, phase III 2022 × 3 Oropharynx phase II 2022 Multiple myeloma phase II 2022 Ovarian ca phase II 2022 Head neck squamous cell carcinoma phase II 2020 Cutaneous squamous cell Glioblastoma multiforme phase II 2021 Lung ca phase II 2022 Cervical cancer phase III 2023 Intravenous Programmed cell death receptor PCDC1 Cergutuzumab amunaleukin Aka RO6895882, CEA-IL2v Humanized monoclonal antibody Antineoplastic phase I Dec 2018 Intravenous IL2 Certolizumab pegol CDP870 Cimzia FDA 2008 EU 2009 Recombinant, humanized antibody Fab' fragment Disease-Modifying Crohns Rheumatoid arthritis (phase IIIs completed) Psoriatic arthritis phase III Ankylosing spondylitis Subcutaneous Tumor necrosis factor α blocker Cetrelimab Relatimab Human monoclonal antibody IgG4κ Antineoplastic Nothing on PubMed or creative lab Substance is registered with FDA Programmed cell death 1 Cetuximab IMC-225 Leukeran Erbitux FDA 2004 EU 2004 Recombinant chimeric monoclonal antibody IgG1κ Antineoplastic agent Metastatic colorectal cancer and head and neck cancer NSCLC Intravenous solution EGFR Citatuzumab bogatox Humanized Fab IgG1κ Antineoplastic ovarian cancer and other solid tumors Study phase I terminated 2008 EpCAM Cixutumumab Human monoclonal antibody IgG1κ Antineoplastic Solid tumors Sarcoma phase II Esophageal cancer phase II Rhabdomyosarcoma phase II no benefit Liver cancer phase I Low antitumor effect Pancreas no benefit 2012 Intravenous IGF-1 receptor (CD221) Clazakizumab ALD−518 Humanized monoclonal antibody Disease modifying rheumatoid arthritis phase II 2015 × 3 Crohn disease phase II 2013 Highly sensitized renal transplant candidates phase II 2020 Treat post-tx rejection kidney phase II 2020 Antibody-mediated rejection phase III 2027 Subcutaneous IL6 Clenoliximab Chimeric monoclonal antibody Disease modifying Rheum Arth No study since 2003 CD4 Clivatuzumab tetraxetan (90)Y-clivatuzumab tetraxetan hPAM4-Cide Humanized monoclonal antibody IgG1κ Antineoplastic Pancreatic cancer Phase III 2017 PANCRIT-1 study. Study terminated no increase improvement of overall survival MUC1 Codrituzumab Humanized monoclonal antibody IgG1κ Antineoplastic HCC Phase Ib no response Phase II no response Glypican 3 Cofetuzumab pelidotin Humanized monoclonal antibody IgG1κ Antineoplastic Nothing on PubMed or creative lab Substance is not registered with FDA Protein tyrosine kinase 7 (PTK7) Coltuximab ravtansine SAR3419 Chimeric monoclonal antibody IgG1 conjugated to DM4 (N2′-(4-((3-carboxypropyl)dithio)-4-methyl-1-oxopentyl)-N2′-deacetylmaytansine) Antineoplastic Relapse/refractory ALL phase II 2015 low clinical response Phase II moderate response CD19 Conatumumab AMG655 Human monoclonal antibody IgG1κ Antineoplastic Phase II 2019: Advanced solid tumors Carcinoid Colorectal cancer Locally advanced Lymphoma Metastatic cancer Nonsmall cell lung cancer Sarcoma Solid tumors Colon cancer phase Ib/II no benefit Intravenous TRAIL-R2 Concizumab Humanized IgG4κ Disease modifying Hemophilia A and B phase II 2020 Subcutaneous Kunitz-type protease inhibitor 2 domain of tissue factor pathway inhibitor (TFPI) Cosfroviximab ZMapp Chimeric monoclonal antibody IgG1κ Triple monoclonal antibody cocktail Disease modifying Ebola virus Ongoing studies show benefit but not enough enrolled to power study Ebola virus glycoprotein Crenezumab RG7412 MABT5102A Humanized monoclonal antibody IgG4 Disease modifying Alzheimer's disease phase III study ongoing prodromal/mild AD 2021 Phase III 2022 Intravenous 1-40-β-amyloid Crizanlizumab SelG1 FDA review possible 2019 Humanized monoclonal antibody IgG2κ Disease modifying Sickle cell disease phase II 2022 children Phase II adults decrease pain crisis Intravenous P Selectin Crotedumab Human monoclonal antibody IgG4κ Disease modifying DM type II No results GCGR Cusatuzumab ARGX-110 Humanized monoclonal antibody IgG1 Antineoplastic Phase I completed safe Phase I/II CTCL dec 2018 Nasopharyngeal carcinoma 2018 Intravenous CD70 Dacetuzumab HU-S2C6 ASKP1240 SGN-40 Humanized monoclonal antibody IgG1 Antineoplastic Hematologic cancers Multiple myeloma phase I 2007 Large B-cell lymphoma phase II 2009 enrollment stopped no benefit CLL phase II 2006 NHL phase I Renal transplant (CIRRUS I) phase II 2022 SLE nephritis phase II 2020 Intravenous CD40 Daclizumab Zenapax Zinbryta FDA 1997 EU 1999 Zenapax withdrawn from market Apr 2009 for commercial reasons Zinbryta withdrawal 2018 secondary to risk/benefit profile Humanized monoclonal antibody IgG1κ Disease modifying Prevention of organ transplant rejections Phase IV kidney transplants, multiple sclerosis phase III 2018 pulled from market secondary inflammatory brain disorders Biogen Heart transplant phase IV 108 studies Zanapax discontinued from market by Roche (basiliximab replace) CD25 (α chain of IL-2 receptor) Dalotuzumab Humanized monoclonal antibody IgG1κ Antineoplastic Phase I multiple Phase II breast no improvement × 2 Phase III colon no improvement Ped solid phase I Intravenous IGF-1 receptor (CD221) Dapirolizumab pegol Humanized monoclonal antibody IgG1κ SLE phase II Nov 2018 Phase I safe Intravenous CD154 (CD40L) Daratumumab Darzalex FDA 2015 EU 2016 Human IgG1κ Antineoplastic agent Multiple myeloma relapse/refractory Phase III completed Intravenous solution CD38 Induces CDC, ADCC, ADCP, and apoptosis Dectrekumab QAX576 Human monoclonal antibody IgG1κ Cancers, asthma phase II, idiopathic pulmonary fibrosis, eosinophilic Esophagitis phase II some benefit but primary endpoint not achieved, Keloids, Crohn's disease phase II trials 2013 Nothing on PubMed Substance is registered with FDA, creative lab IL-13 Demcizumab Humanized monoclonal antibody IgG2κ Antineoplastic NSCLC phase II 2018 Phase I safety established with 50% tumor regression response Intravenous Delta-like ligand 4DLL4 DLL4 and Notch1, signaling stimulated by DLL4 plays a role in development of blood vessels throughout life Denintuzumab mafodotin HBU-12 SGN-CD19A Humanized monoclonal antibody IgG1κ Antibody-drug conjugate (ADC) composed of a humanized anti-CD19 monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) Antineoplastic LBCL phase II terminated study by company Acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma Phase I 2017 Phase II 2018 terminated by sponsor Intravenous CD19 Denosumab AMG162 Prolia FDA 2010 EU 2010 Xgeva FDA 2011 EU 2011 Human monoclonal antibody IgG2 Disease modifying Osteoporosis FREEDOM trial, bone metastases, etc. 186 studies Phase III completed Melanoma phase II 2022 Bone giant cell tumor phase II 2025 Subcutaneous Receptor activator of nuclear factor kappa-B ligand (RANKL) Xgeva: Prevention of skeletal-related events (SREs) in adults with bone metastases from breast and castration-resistant prostate cancer. Prolia: Osteoporosis Depatuxizumab mafodotin ABT 414 Chimeric humanized monoclonal antibody IgG1κ CONJUGATED TO AURISTATIN F Glioblastoma Phase III Nov 2019 Children phase III 2020 Intravenous EGFR Derlotuximab biotin Iodine (131 I) derlotuximab biotin Chimeric monoclonal antibody IgG1κ Immunoassays Potential for glioblastoma multiforme Histone complex Detumomab Murine monoclonal antibody IgG1 Antineoplastic B-lymphoma cell Nothing on PubMed or clinical trials Substance is not registered with FDA, is on creative lab CD3E Dezamizumab GSK-2398852 Humanized monoclonal antibody IgG1κ Disease modifying Treat amyloidosis Transthyretin cardiomyopathy amyloidosis (ATTR-CM), suspended pending data review Aug 2018 phase I x 4 Intravenous Serum amyloid P component Dinutuximab APN311 Unituxin FDA 2015 EU 2015 then withdrawn EU Chimeric monoclonal antibody IgG1κ Antineoplastic Neuroblastoma phase I 2022 SCLC phase III Nov 2019 Osteosarcoma phase II Dec 2018 Neuroblastoma phase II 2020 Intravenous GD2 ganglioside Diridavumab CR6261 Human monoclonal antibody IgG1λ Disease modifying Infectious disease/influenza A Very good response in animal study mice Phase II 2019 Intravenous Influenza A hemagglutinin Domagrozumab PF-06252616 Humanized monoclonal antibody IgG1κ Disease modifying Duchenne muscular dystrophy phase II 2018 Phase I completed GDF-8 Dorlimomab aritox F(ab') 2 Murine Nothing on PubMed or clinical trials Substance is not registered with FDA, or creative lab Dostarlimab TSR042 WBP285 FDA review pending 2019 Humanized monoclonal antibody IgG4κ Antineoplastic Solid tumor Phase I, II, III studies ongoing Ovarian CA (first study) phase III 2023 Programmed cell death protein-1 (CD279) PCDP1 Drozitumab PRO95780 rhuMAB DR5 Human monoclonal antibody IgG1λ Antineoplastic Colorectal cancer Ib 2012 Preclinical rhabdomyosarcoma 2018 Chondrosarcoma not efficacious NHL results? Intravenous Death receptor 5 (DR5) Duligotuzumab MEHD7945A Human monoclonal antibody IgG1κ Antineoplastic squamous head and neck phase II no benefit Colon ca phase II no benefit Anti-EGFR × Anti-HER3 bispecific antibody Dupilumab Dupixent FDA 2017 Human monoclonal antibody IgG4 Disease modifying asthma, atopic dermatitis Ongoing studies Subcutaneous IL4 Durvalumab Imfinzi FDA 2017 Human monoclonal antibody IgG1κ Antineoplastic agent Treat NSCLC stage III phase I, urothelial carcinoma Intravenous PD-L1 (CD274) and CD80—inhibit binding of programmed death ligand 1 to PD-1 and CD80 allowing T cell to recognize and kill tumor cells Dusigitumab MEDI 573 Human monoclonal antibody IgG2λ Antineoplastic Breast cancer phase II results? HCC phase II results? Intravenous ILGF2 Duvortuxizumab MGD011 Chimeric/humanized monoclonal antibody Antineoplastic B-cell malignancy Phase I/II Jul 2018/2020 Intravenous CD19, CD3E Ecromeximab KW2871 Chimeric monoclonal antibody IgG1κ Antineoplastic Metastatic melanoma Phase I/II clinical activity limited Intravenous GD3 ganglioside Eculizumab Soliris PNH FDA 2007 EU 2007 aHUS, and myasthenia gravis FDA 2018 EU 2018 Japan 2018 Humanized monoclonal antibody IgG1/4 Immuno-regulation Paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (HUS ) Generalized myasthenia gravis (MG) Phase II CAD Intravenous C5 Edobacomab E5 Murine monoclonal antibody No improved survival Endotoxin Edrecolomab Panorex Murine monoclonal antibody IgG2κ Antineoplastic Colorectal carcinoma phase III 2003 no improvement Intravenous Glycoprotein EpCAM/17-1A Efalizumab Raptiva FDA 2003 EU 2004 Withdrawn both markets 2009 Recombinant humanized monoclonal antibody IgG1κ Disease modifying (2003 approved) psoriasis Subcutaneous Voluntary withdrawal 2009 Human CD11a Increase risk progressive multifocal leukoencephalopathy (PML) Efungumab MYC123 Mycograb Mycograb C28Y Human scFv Antiinfectious agent Invasive Candida infection Intravenous Heat shock protein 90 (Hsp90) Eldelumab Mdx 1100 Human monoclonal antibody IgG1κ Crohn's disease phase IIa no significant response, ulcerative colitis phase IIb prim endpoint not achieved Rheum arthritis phase II Intravenous Interferon γ-induced protein CXCL 10 Elezanumab PR-1432051 ABT-555 Human monoclonal antibody IgG1λ Spinal cord injury and multiple sclerosis phase II 2021 Intravenous REPULSIVE GUIDANCE MOLECULE FAMILY MEMBER A (RGMA) Elgemtumab LJM716 Human IgG1κ Antineoplastic Breast gastric phase I Intravenous ERBB3 (HER3) Elotuzumab PDL063 Empliciti FDA 2015 EU 2016 Human IgG1κ Antineoplastic Multiple myeloma Phase III completed and ongoing Intravenous SLAMF7 Elsilimomab B-E8 Humanized monoclonal antibody IgG1 Antineoplastic multiple myeloma Not effective in mice IL-6 Emactuzumab RG7155 Humanized monoclonal antibody IgG1 ANTINEOPLASTIC Phase I 2019 solid tumors Phase II 2025 REDIRECT study ovarian, fallopian tube cancer Pancreatic phase II 2020 HUMAN MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR (CSF1R, CD115) Emapalumab NI-0501 Gamifant FDA 2018 EU pending Human monoclonal antibody IgG1λ Hemophagocytic lymphohistiocytosis Phase III 2021 Intravenous Interferon γ Emibetuzumab LA480 LY2875358 Humanized monoclonal antibody IgG4κ Bivalent antibody Antineoplastic NSCLC phase II 2020 Advanced cancer Gastric safe ?effective adenocarcinoma Gastroesophageal junction adenocarcinoma Hepatocellular cancer Renal cell carcinoma Nonsmall cell lung cancer phase II Jan 2018 Phase I safe with tumor response Intravenous Hepatocyte growth factor receptor (HHGFR) and MET signaling Emicizumab ACE910 Hemlibra FDA 2018 Humanized monoclonal antibody IgG4κ Bispecific Disease modifying Hemophilia A phase III 2020 With or without inhibitors Subcutaneous Activated F9, F10 Enapotamab vedotin Human monoclonal antibody IgG1κ Antineoplastic Nothing on PubMed or clinical trials Substance is not registered with FDA, or creative lab Human growth factor receptor AXL Enavatuzumab PDL192 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2011 No responses and liver pancreatic toxicity Intravenous TWEAK receptor Enfortumab vedotin FDA review pending 2019 Human monoclonal antibody Antineoplastic bladder cancer phase I Phase II ongoing Nectin-4 Anti-Nectin-4 Monoclonal antibody attached to a microtubule-disrupting agent, monomethyl auristatin E (MMAE) Enlimomab pegol Murine monoclonal antibodyIgG2a Disease modifying Stroke Nothing on PubMed or clinical trials Substance is not registered with FDA ICAM-1 (CD54) Enoblituzumab MGA 271 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2022 children Neuroblastoma Rhabdomyosarcoma Osteosarcoma Ewing sarcoma Wilms tumor Desmoplastic small round cell tumor Phase I melanoma, NSCLC 2018 Phase II prostate 2021 CD276 (B7–H3) Enokizumab MEDI528 Humanized monoclonal antibody IgG1κ Asthma phase II No improvement Intravenous IL9 Enoticumab REGN421 Human monoclonal antibody IgG1κ Antineoplastic Phase 1 2014 ovarian cancer + Delta-like canonical notch ligand 4 (DLL4) Ensituximab NEO-201 NPC-1C Chimeric monoclonal antibody IgG1κ Antineoplastic Phase II pancreatic and colorectal cancer 2017 Intravenous 5AC Enterecept RHU-TNFR:FC Enbril FDA 2003 1-235-Tumor necrosis factor receptor fusion protein attached to recombinant human IgG1 Fc fragment Disease modifying Antirheumatic drug Not effective for inflammatory bowel disease Subcutaneous TNFα Epitumomab cituxetan AS-1402 HuHMFG-1 Sontuzumab Humanized monoclonal antibody IgG1 Antineoplastic Breast cancer phase II 2012 no benefit Episialin MS4A1 (membrane-spanning 4-domains subfamily A member 1, CD20 (HMFG-1) Epratuzumab HLL2 AMG412 Humanized monoclonal antibody IgG1κ ADCC/CDC Antineoplastic B-ALL phase III ongoing 2018 Disease modifying SLE phase III no improvement Intravenous CD22 Eptinezumab ALD403 FDA review possible 2019 Monoclonal antibody IgG1κ Disease modifying Migraine phase III Calcitonin gene-related peptide Erenumab Aimovig FDA May 2018 Human monoclonal antibody IgG2λ Disease modifying Migraine phase III Calcitonin gene-related peptide (CGRP) Erlizumab Rhumab CD18 Humanized IgG1 F(ab') 2 fragment Antineoplastic (lab tests) Immunosuppressive drug phase I study cough up blood and phase II did not meet goals Heart attack, stroke, traumatic shock ??no successful CD18 drug to date LGL type leukemia ITGB2 (CD18) and LFA-1 block growth factor of blood vessels stop lymphocytes from moving into inflamed tissue Ertumaxomab Rexomun Rat/murine hybrid triomab, murine IgG2a HET2 target, RAT IgG2bλ CD3 target Antineoplastic Breast Gastric, esophageal Phase II studies terminated company to focus on other plans not safety concerns concentrate on catumaxomab Phase I found safe 2016 Intravenous HER2/neu, CD3 Etaracizumab or etaratuzumab MEDI-522 Abegrin Vitaxin Humanized monoclonal antibody IgG2κ Antineoplastic Melanoma phase II 2010 not beneficial, prostate cancer, ovarian cancer small and large bowel cancer phase I and II completed results unreported 2017 Intravenous Integrin αvβ3 Etigilimab Humanized monoclonal antibody IgG1κ Nothing on pubmed or clinical trials Substance is registered with FDA, or is not in creative lab TIGIT T-cell immunoreceptor with Ig and ITIM domains Etrolizumab PRO145223 RHUMAB BETA7 Humanized monoclonal antibody IgG1κ Disease modifying Inflammatory bowel disease UC phase III 2020 × 4/2023/2024/2025 Crohn phase III 2021 Subcutaneous Integrin β7 Inhibits binding of αEβ7 to E-cadherin Evinacumab REGN1500 Human monoclonal antibody IgG4κ Disease modifying Dyslipidemia Phase II 2020 Phase III 2020/2022 Angiopoietin 3 Evolocumab Repatha FDA 2015 EU 2015 Human monoclonal antibody IgG2λ Disease modifying hypercholesterolemia Completed phase III Heterozygous familial hypercholesterolemia, CVD Subcutaneous Proprotein convertase subtilisin kexin type 9 (PCSK9) Exbivirumab Humanized monoclonal antibody IgG1λ Disease modifying prevent disease Hep B Oral therapy Abstract of randomized study of 50 patients Hepatitis B surface antigen Fanolesomab RB5-IGM NeutroSpec Murine monoclonal antibody Diagnostic imaging Appendicitis (diagnosis only) CD15 Faralimomab Murine monoclonal antibody IgG1 Nothing on PubMed or clinical trials Substance is not registered with FDA, or is not in creative lab Interferon receptor Faricimab RG7716 RO6867461 Humanized monoclonal antibody IgG1mab Disease modifying angiogenesis, ocular vascular diseases STAIRWAY, BOULEVARD, RHINE, Yosemite phase II and III studies phase III 2022 for diabetes maculae edema AMD LUCERNE phase III 2022 TENAYA phase III 2022 Intravitreous ANTIVASCULAR ENDOTHELIAL GROWTH FACTOR/ANTIANGIOPOIETIN 2 BISPECIFIC ANTIBODY (VEGF-A and Ang-2) Farletuzumab MORAB-003 Humanized monoclonal antibody IgG1κ Antineoplastic Ovarian cancer phase III subgroup may benefit Intravenous Folate receptor 1 Fasinumab REGN475 SAR164877 MT5547 Human monoclonal antibody IgG4κ Disease modifying acute sciatic pain phase III Knee arthritis pain phase III 2021 Subcutaneous (auto injector) Human nerve growth factor (HNGF) FBTA05 Bi20 Lymphomun Rat IgG2b (CD3)/murine IgG2a (CD20) hybrid trifunct Antineoplastic Chronic lymphocytic leukemia trial terminated recruitment too slow Intravenous? CD20/CD3 Felvizumab Humanized monoclonal antibody IgG1κ Antiinfectious agent Respiratory syncytial virus infection Nothing on PubMed or clinical trials Substance is not registered with FDA, but is in creative lab Respiratory syncytial virus Fezakinumab Human monoclonal antibody IgG1λ Disease modifying Rheumatoid arthritis, psoriasis (not good for) Atopic dermatitis phase IIb good results Intravenous IL-22 Fibatuzumab Ifabotuzumab Humanized monoclonal antibody IgG1κ Disease modifying Myelodysplastic syndrome Research in Australia for GBM phase I Ephrin receptor A3 Ficlatuzumab SCH 900105 AV 299 Humanized monoclonal antibody IgG1κ Antineoplastic Head and neck cancer phase I 2020 Pancreatic phase I 2023 NSCLC phase I/II 2013 AML phase I 2020 Intravenous Hepatocyte growth factor (HGF) hepapoietin A Figitumumab CP751871 Human monoclonal antibody IgG2κ Ceased development in 2011 by pfizer Antineoplastic Adrenocortical carcinoma, nonsmall cell lung carcinoma etc. ?additional benefit in phase I Insulin-like growth factor receptor IGF-1 receptor (CD221) Firivumab Human monoclonal antibody IgG1κ Disease modifying Influenza A virus hemagglutinin Nothing on PubMed or clinical trials Substance is registered with FDA, and is in creative lab INFLUENZA A VIRUS HEMAGGLUTININ HA Flanvotumab IMC20D7S Human monoclonal antibody IgG1κ Antineoplastic Melanoma phase I 2012 Intravenous No published data TYRP1 (glycoprotein 75) Fletikumab Human monoclonal antibody IgG4 Disease modifying Rheumatoid arthritis phase IIa good, phase IIb no results IL 20 Flotetuzumab MGD006 S80880 Humanized di-scFv dual affinity retargeting (DART) to CD123 and CD3 Antineoplastic Hematologic malignancies (ALL, NHL) Phase II not yet recruiting 2018 Intravenous? IL 3 receptor Fontolizumab HuZAF Humanized monoclonal antibody IgG Disease modifying Treat Crohn's clinical development stopped despite some benefit phase II ustekinumab is better IFN-γ FOR46 Antibody drug conjugate Antineoplastic Phase I for multiple myeloma failed remission or relapse Phase I prostate cancer Intravenous CD46 Foralumab Human monoclonal antibody IgG1κ Disease modifying NASH phase II 2019 Oral CD3 epsilon Foravirumab Human monoclonal antibody IgG1κ Disease modifying rabies (prophylaxis) Nothing in pub med or clinical trials Rabies virus glycoprotein Fremanezumab LBR-101 RN307 Ajovy FDA 2018 Humanized monoclonal antibody IgG2κ Disease modifying migraine and cluster headache phase III 2019 Subcutaneous α-Calcitonin gene-related peptide Fresolimumab Humanized monoclonal antibody IgG4 Disease modifying Idiopathic pulmonary fibrosis (IPF), scleroderma, focal segmental glomerulosclerosis (phase 2), cancer (kidney cancer and melanoma) Need larger study for FSGS Good response scleroderma https://newdrugapprovals.org/2016/01/30/fresolimumab/ TGF β 1 Frovocimab LY3015014 Humanized monoclonal antibody IgG4κ Disease modifying hypercholesterolemia Completed phase II trials 2014 good response and safe Subcutaneous PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9 (PCSK9) Frunevetmab https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies - cite_note-WHOList 116-17 NV-02 Veterinary monoclonal antibody IgG1κ Veterinary Feline muscle nerve growth factor Fulranumab AMG403 Human monoclonal antibody IgG2κ Disease modifying Pain osteoarthritis pain phase III 2017 Subcutaneous Nerve growth factor Galcanezumab LY2951742 Emgality FDA 2018 Humanized monoclonal antibody IgG4κ Disease modifying Migraine Phase III completed Cluster HA phase III 2020 Subcutaneous Calcitonin gene-related polypeptides (CGRPs) α and β Galiximab IDEC-114 Chimeric monoclonal antibody IgG1λ ADCC/CDC Antineoplastic lymphoma phase II 2015 minimal response ORR 10.3% Intravenous CD80 Gancotamab MM-302 Human cFv Single chain fragment Antineoplastic Breast cancer phase I–III Intravenous HER2/neu Ganitumab AMG479 Human monoclonal antibody IgG1κ Antineoplastic Pancreatic phase III—no increase benefit Phase III for rhabdomyosarcoma and Ewings 2021 Not beneficial in NSCLC Intravenous IGF-1 receptor (CD221) Gantenerumab R04909832 R1450 Human monoclonal antibody IgG1κ Disease modifying Alzheimers Phase III stopped for potential futility additional studies at higher dosing (DIAN in a phase II/III trial in individuals at risk For and with early-stage autosomal-dominant AD phase III 2021 Subcutaneous Beta amyloid Gatipotuzumab PankoMab-GEX Humanized monoclonal antibody IgG1κ Antineoplastic Ovarian, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), gynecological cancers (GYN) phase I used for diag/prog now Intravenous Musculus, antimucin (MUC1) Gavilimomab ABX-CBL Murine monoclonal antibody IgM Disease modifying Graft versus host disease phase III completed 2005 less effective than antithymocyte antibody CD147 (basigin) Gedivumab RG7745 RO6876802 Human monoclonal antibody IgG1κ Disease modifying Influenza virus A No studies PubMed/clinical trials Influenza virus hemagglutinin HA Gemtuzumab ozogamicin Mylotarg AML FDA 2000 Voluntary withdrawal 2010 VOD now black box warning Returned to market with FDA approval 2017 Humanized monoclonal antibody IgG4/toxin conjugate Antineoplastic Acute myelogenous leukemia Many ongoing and completed studies Intravenous CD33 Gevokizumab XOMA 052 Humanized monoclonal antibody IgG2κ Disease modifying DM phase II (late stage) no results Other dz too 24 studies behcet uveitis failed primary end point phase III (Eyeguard _B) 2015 Subcutaneous IL-1β Gilvetmab PD1 Veterinary monoclonal antibody IgG2κ Antineoplastic No studies clinical trial, pub med CANIS FAMILIARIS PROGRAMMED CELL DEATH PROTEIN 1 (PCDC1) Gimsilumab MORAb-022 Human monoclonal antibody IgG1 Disease modifying Rheumatoid arthritis Asthma Phase I poster presentation of safety results good 2016 Intravenous HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (CSF2) Girentuximab WX-G250 CG250 Rencarex Reductane Chimeric monoclonal antibodyIgG1κ Radioactive labeled ab Antineoplastic Clear cell renal cell carcinoma for treatment and imaging Intravenous Carbonic anhydrase 9 (CA-IX) Glembatumumab vedotin CR011 Human monoclonal antibody IgG2κ Antibody drug complex Antineoplastic Melanoma phase II, breast cancer Intravenous Human glycoprotein NMB extracellular domain (GPNMB) Golimumab CNTO 148 Simponi FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis, psoriatic arthritis , juvenile rheum arth, ankylosing spondylitis many studies, UC, DM1 phase I (2020, 2021) Subcutaneous, intravenous TNF-α Gomiliximab IDEC-152 Lumiliximab ST-152 Chimeric monoclonal antibody IgG1κ ADCC/CDC Allergic asthma ? Antineoplastic CLL Phase I, phase 2/3 2014 Failed efficacy CD23 (IgE receptor) Gosuranemab BIIB092 IPN-007 FDA orphan drug status Humanized monoclonal antibody IgG4κ Progressive supranuclear palsy Phase I 2020 Alzheimer 2021 Intravenous τ protein Guselkumab CNTO 1959 Tremfya FDA ? Human monoclonal antibody IgG1λ Disease modifying Psoriasis Adenomatous polyposis Subcutaneous IL23A Hu3F8 Humanized monoclonal antibody IgG3 Antineoplastic Phase I For neuroblastoma mod tox and substantial effect on tumor Phase II ongoing Intravenous GD2 ganglioside Ianalumab Human monoclonal antibody IgG1κ Immunomodulation Autoimmune hepatitis Human cytokine receptor BAFF-R Ibalizumab Trogarzo FDA/EU approved Humanized monoclonal antibody IgG4 Disease modifying anti-HIV Phase III CD4 Ibritumomab tiuxetan IDEC-129 IDEC-IN2B8 IDEC-Y2B8 Zevalin FDA 2002 EU 2004 Murine monoclonal antibody IgG1κ YT 77 or In 98 bound Antineoplastic Follicular non-Hodgkin's lymphoma , B-cell NHL , multiple myeloma conditioning for BMT, B-cell DLCL, mantle cell Many studies CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35) Icrucumab IMC 18F1 Human monoclonal antibody IgG1κ Antineoplastic No benefit breast phase II No benefit colon phase II No benefit urothelial phase II Intravenous Vascular endothelial growth factor receptor (VEGFR-1) Idarucizumab Praxbind FDA 2015 EU 2015 Humanized monoclonal antibody Fab fragment Antidotes Drug reversal agent Reversal of anticoagulant effects of dabigatran Phase III trial RE-VERSE AD Intravenous Dabigatran etexilate Igovomab Indimacis-125 Murine F(ab') 2 Diagnostic imaging Ovarian cancer (diagnosis) Iladatuzumab vedotin RG7986 Humanized monoclonal antibody IgG1κ Antineoplastic Nothing in PubMed or clinical trials Human gene B29 protein (CD97B) Imalumab BAX69 Human monoclonal antibody IgG1κ Antineoplastic Intraperitoneal infusion, intravenous Macrophage migration inhibitory factor (MIF) Imaprelimab Humanized IgG1κ Antineoplastic Nothing in PubMed or clinical trials Melanoma cell adhesion molecule (MCAM) Imciromab pentetate Myoscint FDA approved 1996 Withdrawn from market Murine monoclonal antibody fragment Fab IgG2aκ Diagnostic Cardiac imaging Cardiac myosin Imgatuzumab RG7160 RO5083945 GA201 HUMA-B Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal 2013 Head and neck 2017 NSCLC 2017 Epidermal growth factor receptor (EGFR, HER1) IMGN632 Monoclonal antibody with antibody drug conjugate Antineoplastic AML, ALL phase I 2021 NCT03386513 Intravenous CD123 Inclacumab RG1512 RO4905417 Human monoclonal antibody IgG4κ Disease modifying Cardiovascular disease phase II Intravenous Selectin P Indatuiximab ravtansine Chimeric monoclonal antibody IgG4κ Antineoplastic Preclinical breast cancer CD138 (syndecan-1) SDC1 Indusatumab vedotin TAK-264 MLN0264 Human monoclonal antibody IgG1κ conjuagated via a mc-val-cit-PABC linker to monomethyl auristatin E {MMAE (5F9-mc-val-cit-PABC-MMAE)} Antineoplastic Gastrointestinal, pancreatic, gastroesophageal Safe phase I, phase II pancreatic min response, three studies terminated by company business Intravenous Guanylate cyclase C (GUCY2C) Inebilizumab MEDI-551 Humanized monoclonal antibody IgG2κ ADCC Antineoplastic Refractory DLBCL phase II 2016 Disease modifying systemic sclerosis, multiple sclerosis Neuromyelitis optica Intravenous CD19 Infliximab Remicade FDA 1998 EU 1999 Inflectra FDA 2016 EU 2013 Remsima EU 2013 Human-murine chimera IgG1κ Human constant, murine variable region Disease modifying Remicade Crohn's disease; ulcerative colitis; rheumatoid arthritis; ankylosing spondylitis; psoriatic arthrits; plaque psoriasis Inflectra Spondylitis; ankylosing; arthritis; rheumatoid colitis; ulcerative arthritis; psoriatic Crohn's disease; psoriasis Remsima Spondylitis; ankylosing arthritis; rheumatoid colitis; ulcerative Crohn's disease; arthritis; psoriatic psoriasis Intravenous solution TNF-α Inolimomab Murine monoclonal antibody Disease modifying GVHD phase III No better than ATG in 3 studies Abandoned not in 2017 Cochrane review CD25 (α chain of IL-2 receptor) Inotuzumab ozogamicin G544 Besponsa FDA 2017 Humanized monoclonal antibody IgG4κ ADCC/CDC Antineoplastic ALL phase II 2023 Multiple other studies Intravenous CD22 Intetumumab CNTO095 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors (prostate cancer, melanoma) Melanoma phase II possible benefit 2011 Prostate cancer no additional benefit 2013 phase II Intravenous CD51 Ipilimumab Yervoy FDA 2011 EU 2011 Human monoclonal antibody IgG1κ Antineoplastic agent Bladder carcinoma (trials ongoing) Melanoma Ipilimumab MDX010 MDX101 BMS-734016 Yervoy FDA 2011 melanoma Metastatic renal cancer/colorectal cancer 2018 Human monoclonal antibody IgG1κ Antineoplastic Melanoma (checkmate 067) Renal cell carcinoma (checkmate 214) Colorectal cancer Pancreatic? Intravenous CD152 cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and blocks interaction with its ligands CD80/CD86 Iratumumab MDX060 Human monoclonal antibody IgG1κ Antineoplastic Hodgkin's lymphoma phase II completed Clinical research discontinued 2009 Intravenous CD30 (tumor necrosis factor receptor superfamily, Member 8; TNFRSF8) aka Ki-1 Ag Isatuximab SAR650984 FDA review possible 2019 Chimeric monoclonal antibody IgG1κ Antineoplastic multiple myeloma Phase I 2019 Phase II 2022 Phase III 2025 Intravenous CD 38 Iscalimab CFZ533 Human monoclonal antibody IgG1κ Disease modifying Potential treat autoimmune disease Lupus nephritis phase II 2020 Myasthenia gravis GVHD Kidney transplant 2022 phase II Preclinicals Intravenous CD40 Istiratumab MM-005 MM-141 Human monoclonal antibody IgG1 Antineoplastic Advanced solid tumors Pancreatic cancer phase II 2018 Insulin-like growth factor I receptor/neuregulin receptor HER3 (IGF1R, CD221) Itolizumab Alzumab FDA Humanized monoclonal antibody IgG1κ Disease modifying Psoriasis GVHS phase II 2022 CD6 Ixekizumab Taltz Humanized monoclonal antibody Disease modifying Phase III radiographic axial spondyloarthritis Psoriatic arthritis Subcutaneous IL 17A Keliximab Became clenoliximab IgG4 Chimeric monoclonal antibody IgG1λ Disease modifying Chronic asthma Rheumatoid arthritis CD4 Labetuzumab hMN14 CEA-Cide Humanized monoclonal antibody IgG1 Antineoplastic Colorectal cancer Gastrointestinal phase II 2021imagin Phase II completed therapy 2003 Intravenous CEA Lacnotuzumab MCS110 Humanized monoclonal antibody IgG1κ Antineoplastic Breast, pigmented villonodular synovitis (PVNS) Squam ESOP phase II 2024 Gastric 2019 CSF1, MCSF Ladiratuzumab vedotin Humanized monoclonal antibody IgG1κ Antibody drug conjugate Antineoplastic Phase I triple-negative breast cancer 2017 ongoing study LIV-1 Lampalizumab RG7417 Humanized monoclonal fragment IgG1κ Disease modifying Geographic atrophy secondary to age-related macular degeneration phase III Jan 2018 ineffective Intravitreous Complement factor D (CFD) Lanadelumab SHP643 DX-2930 Takhzyro FDA 2018 Human monoclonal antibody IgG1κ Disease modifying Angioedema phase III 2019 Subcutaneous Kallikrein (KLKB1) Landogrozumab LY2495655 Humanized monoclonal antibody IgG4κ Disease modifying Muscle wasting disorders, i.e., after hip surgery phase II Pancreatic cancer phase II no benefit cancer, some muscle improvement but primary objective not met Intravenous Subcutaneous Human growth differentiating factor 8 (GDF-8) aka myostatin (MSTN) Laprituximab emtansine IMGN289 Chimeric monoclonal antibody No trials or PubMed or creative lab only in FDA registry ?new EGFR Larcaviximab ZMAPP Chimeric monoclonal antibody IgG1κ Disease modifying Ebola virus No studies clinical trial or PubMed Ebolavirus glycoprotein Lebrikizumab MILR1444A TNX-650 RG-3637 PRO301444 Humanized monoclonal antibody IgG4κ Disease modifying Asthma phase III Atopic dermatitis HL phase II 2007 Subcutaneous injection Interleukin-13 (IL-13) Lemalesomab Murine monoclonal antibody IgG1κ Diagnostic agent NCA-90 (granulocyte antigen) Lendalizumab Olendalizumab ALXN-1007 Humanized monoclonal antibody IgGκ Disease modifying Antiphospholipid syndrome GI GVHD Intravenous Anticomplement 5A Lenvervimab Humanized IgG1κ Disease modifying Hepatitis B No studies in clinical trials or PubMed Hepatitis B surface antigen Lenzilumab KB-003 Human monoclonal antibody Antineoplastic chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia phase I Intravenous GRANULOCYTEMACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) Lerdelimumab CAT-152 Trabio Human monoclonal antibody IgG4 Disease modifying Phase I studies ?Cancer and fibrosis Trials stopped for fibrosis after glaucoma surgery Transforming growth factor β 2 Leronlimab PRO-140 FDA review 2018 Humanized IgG4κ Disease modifying HIV phase III ongoing no results published good results phase II Subcutaneous Chemokine receptor 5 (CCR5) Lesofavumab RG70026 Human monoclonal antibody IgG1κ Disease modifying Influenza A No studies clinical trials or PubMed Hemagglutinin HA Letolizumab Humanized synthetic light chain variable region (scFv) Disease modifying inflammatory diseases No studies clinical trials or PubMed or creative labs TRAP Lexatumumab HGS1018 HGS-ETR2 Human monoclonal antibody IgG1λ Antineoplastic Breast Pancreatic Intravenous Tumor necrosis factor receptor superfamily member 10B/death receptor 5 (TRAIL-R2) Libivirumab Humanized monoclonal antibody IgG1κ Antiinfectious Prevent disease Hep B Oral therapy Abstract of randomized study of 50 patients Hepatitis B surface antigen Lifastuzumab vedotin DBNIB0600A Humanized monoclonal antibody Antineoplastic Ovarian cancer Phase 2 Intravenous Phosphate-sodium cotransporter Ligelizumab QGE031 Humanized monoclonal antibody IgG1κ Disease modifying SSevere asthma and chronic spontaneous urticaria phase II and III ongoing trial 2021 Subcutaneous Immunoglobulin E (IGHE) Lilotomab satetraxetan Betalutin Murine monoclonal antibody IgG1 Antineoplastic NHL 2020/phase II 2025 Diffuse lg B-cell lymphoma 2019 CD37 Lintuzumab SGN33 Humanized monoclonal antibody IgG1κ Antineoplastic AMLphase I/II 2022 Mult myeloma phase I 2020 Myelodysplastic phae II 2011 Intravenous CD33 Lirilumab IPH2102 Human monoclonal antibody IgG4 Antineoplastic Solid and hematological cancers No good aml, squam cell head neck no good, bladder cancer ongoing? May benefit MDS Intravenous Killer cell immunoglobulin like (KIR2D) Block the interaction between KIR2DL-1,-2,-3 inhibitory receptors and their ligands Lodelcizumab LFU720 Humanized monoclonal antibody IgG1κ Disease modifying Hypercholesterolemia Unknown studies in clinical trials and PubMed Proprotein convertase subtilisin/kexin type 9 (PCSK9) Lokivetmab Cytopoint FDA approved for dogs only Canis monoclonal antibody IgG2κ Disease modifying Veterinary Clinical signs of atopic dermatitis in dogs Canis lupus familiaris IL31 Loncastuximab tesirine ADCT-402 Chimeric monoclonal antibody IgG1κ Antineoplastic Diffuse large B-cell lymphoma phase II 2020 Intravenous CD19 Lorvotuzumab mertansine BB-10901 IMGN901 Humanized monoclonal antibody IgG1κ Antineoplastic SCLC Ovarian AML phase II Wilm, rhabdomyosarcoma, Neuroblast, MPNST, Synovial sarcoma 2018 phase II Intravenous CD56 Losatuxizumab vedotin ABBV-221 Chimeric/humanized monoclonal antibody IgG1 Antineoplastic Epidermal growth factor (EGRF, ERBB1 HER1) Lucatumumab HCD122 Discontinued development by Novartis 2013 Human monoclonal antibody IgG1κ Antineoplastic Multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma Intravenous CD40 Lulizumab pegol Humanized monoclonal antibody Disease modifying SLE Phase I safe Phase II no response Intravenous Subcutaneous CD28 Lumretuzumab RG7116 RO5479599 Humanized monoclonal antibody IgG1κ Antineoplastic Intravenous CD28; receptor for tyrosine-protein kinase(erbB-3, HER3) Lupartumab amadotin BAY-1129980 Human monoclonal antibody IgG Antineoplastic Phase I terminated Why? Intravenous GPI- anchored cell surface-associated protein C4.4A (LYPD3) Lutikizumab ABT981 Humanized monoclonal antibody Disease modifying Osteoarthritis Phase IIa no effect Subcutaneous Interleukin 1 alpha/interleukin 1 beta Mapatumumab HGS1012 Human monoclonal antibody IgG4λ Antineoplastic Hepatocellular no benefit Multiple myeloma Cervical cancer NSCLC no benefit NHL Bladder cancer may be beneficial Tumor necrosis factor receptor superfamily member 10A; cytokine receptor DR4 (death receptor 4 tumor necrosis receptor apoptosis-induced ligand (TRAIL-R1) Margetuximab MGAH22 Chimeric/Humanized monoclonal antibody IgG1κ Antineoplastic Breast cancer Gastric cancer/GEC phase Ib/II trial Intravenous erbB2/HER2 Marstacimab PF-06741086 Human monoclonal antibody IgG1λ Disease modifying Bleeding with hemophilia phase II 2020 Subcutaneous Tissue pathway factor inhibitor (TFPI) Maslimomab Murine monoclonal antibody Immunosuppressive Unknown no studies and not listed in creative lab or FDA T-cell receptor Matuzumab EMD 72000 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal, lung and stomach cancer weakly beneficial Intravenous Epidermal growth factor receptor (EGFR) Mavrilimumab CAM3001 Human monoclonal antibody IgG4λ Disease modifying rheumatoid arthritis phase IIb good Subcutaneous GMCSF receptor α-chain MEDI565 MT111 AMG211 Fab IgG1 BiTE Antineoplastic Gastrointestinal adenocarcinoma phase I 2018 Intravenous CD3 and CEA Mepolizumab SB-240563 Bosatria Nucala FDA 2015 EU 2015 Human monoclonal IgG1κ Disease modifying No benefit in eosinophilic esophagitis Beneficial allergic severe asthma Subcutaneous Interleukin-5 (IL-5) antagonist Metelimumab CAT 192 Humanized monoclonal antibody IgG4 Disease modifying Scleroderma Dropped from further development TGF β 1 Milatuzumab HLL1 IMMU-115 Humanized monoclonal antibody IgG1κ Antineoplastic Multiple myeloma Lupus Leukemia Intravenous CD74 Minretumomab MOAB CC49 Murine monoclonal antibody IgG1 Diagnostic Tumor detection/diagnostic/prognostic Failed phase I clinical trials Tumor-associated glycoprotein 72 (TAG-72) Mirikizumab LY3074828 Humanized monoclonal antibody Disease modifying Psoriasis phase III 2020 UC phase III 2023 LUCENT 1 2021 phase III LUCENT 2 2022 Intravenous IL23A Mirvetuximab soravtansine M9346A IMGN853 Chimeric monoclonal antibody IgG1 Antineoplastic Ovarian phase III 2019 Breast ca phase II 2020 Intravenous Folate receptor alpha Mitumomab BEC-2 Murine monoclonal antibody Antineoplastic SCLC phase III no benefit 2005 GD3 ganglioside Modotuximab 1024 DS Zatuximab Futuximab SYM004 Chimeric monoclonal antibody IgG1κ Antineoplastic Antineoplastic Colorectal Phase 2019 Phase III 2025 Subcutaneous EGFR extracellular domain III/HER1 Mogamulizumab AMG761 KM8761 Poteligeo FDA 2018 Humanized monoclonal antibody IgG1κ Antineoplastic Adult T-cell leukemia/lymphoma Solid tumors Many studies ongoing Intravenous CC chemokine receptor CCR4 MOR202 MOR03087 Human monoclonal antibody IgG1 Antineoplastic multiple myeloma phase I 2018 Intravenous CD38 Monalizumab NN8765 IPH2201 Humanized monoclonal antibody IgG4κ Disease modifying Rheumatoid arthritis, antineoplastic gynecologic malignancies, and other cancers phase II 2021 NSCLC phase II 2022 s/p stem cell transplant phase I 2020 CLL phase II 2019 Intravenous Killer cell lectin-like receptor subfamily C member1 (NKG2A, CD159A, CD94) that recognize nonclassical HLA (i.e., HLA-E) Morolimumab Human monoclonal antibody IgG1 ?Diagnostic No studies in pub med, creative lab or FDA substance Rhesus factor Mosunetuzumab RG7828 BTCT4465A Humanized monoclonal antibody IgG1κ bispecific Antineoplastic NHL phase II 2023 DLBCL phase II 2023 Intravenous Subcutaneous CD3E, MS4A1, CD20 Motavizumab MEDI-524 Numax FDA not approved 2010 Older drug just as effective with less side effects Humanized monoclonal antibody IgG1κ Disease modifying Respiratory syncytial virus phase III completed Safety concerns hives and allergic reactions Intramuscular Respiratory syncytial virus glycoprotein F Moxetumomab pasudotox Lumoxiti FDA 2018 Recombinant immunotoxin comprised of a variable fragment (Fv) of a Murine IgG4 anti-CD22 monoclonal antibody genetically Fused to a truncated fragment of Pseudomonas exotoxin A Antineoplastic Hairy cell leukemia Phase I ALL peds Intravenous CD22 Muromonab-CD3 Muromab Aka teplizimab/MGA031 Orthoclone OKT3 FDA 1986 EU 1986 (country specific approval) Humanized monoclonal antibody IgG2aκ Disease modifying Prevention of kidney transplant rejection Many trials GVHD, NASH and T2DM, giant cell myocarditis AbATE Intravenous Oral CD3 Nacolomab tafenatox Murine monoclonal fragment Fab Antineoplastic ?Colorectal cancer No studies in clinical trial or PubMed C242 antigen Namilumab MT203 Human monoclonal antibody IgG1κ Disease modifying Ank spond psoriasis, RA phase II Subcutaneous Colony-stimulating factor 2 (CSF2) Naptumomab estafenatox TTS-CD3 ANYARA ABR-217620 Murine monoclanl antibody fragment Fab Antineoplastic Nonsmall cell lung carcinoma, renal cell carcinoma phase III completed primary endpoint not achieved Intravenous Tumor-associated antigen 5T4 Naratuximab emtansine IMGN529 Chimeric monoclonal antibody IgG1κ Antineoplastic B-Cell lymphoma NHL Intravenous Tetraspanin-26 (CD37) Narnatumab IMC-RON-8 Ron8 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors phase I Intravenous Human cell surface receptor RON (CD 135) macrophage-stimulating 1 receptor Natalizumab Antegran Antegren Tysabri FDA 2004 EU 2006 Humanized monoclonal antibody IgG4κ Disease modifying Relapsing multiple sclerosis, Crohn's disease Intravenous L selectin (CD62L) α4-subunit of α4β1 and α4β7 integrins of leukocytes (except neutrophils) (VLA-4) Navicixizumab OMP 305B83 Humanized/chimeric monoclonal antibody IgG2κ Antineoplastic Phase I study colorectal gyn tumors Intravenous Delta-like 4 (DLL4) Vascular endothelial growth factor A (VEGF-A) Navivumab CT-P27 Human monoclonal antibody IgG1κ Disease modifying Influenza A No studies PubMed Influenza A virus hemagglutinin HA Naxitamab HU3F8 Humanized monoclonal antibody IgG3 Antineoplastic High-risk neuroblastoma and refractory osteomedullary disease study 2023 ?Intravenous c-Met Ganglioside anti-GD2 Nebacumab Humanized monoclonal antibody IgM Withdrawn for safety, Efficacy and commercial reasons Endotoxin Necitumumab IMC-11F8 Portrazza FDA 2015 EU 2016 Human monoclonal antibody IgG1κ Antineoplastic Nonsmall cell lung carcinoma Intravenous EGFR Nemolizumab CIM331 CD14152 Humanized monoclonal antibody IgG2κ Disease modifying Eczema phase I and II Subcutaneous Interleukin-31 receptor A (IL31RA) NEOD001 Birtamimab ELT1-01 HU2A4 Humanized monoclonal antibody IgG1κ Disease modifying Primary systemic amyloidosis lack clinical benefit Intravenous Amyloid A protein/amyloid light chain Nesvacumab REGN910 SAR307746 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors not as beneficial as other agents in breast cancer Disease modifying Macular degeneration Intravenous Angiopoietin 2 Netakimab Chimeric monoclonal antibody Disease modifying Psoriasis PLANETA study (Russia, future EU and China) Interleukin 17A Nimotuzumab Theracim Theraloc Humanized monoclonal antibodyIgG1κ Antineoplastic Squamous cell carcinoma, head and neck cancer, nasopharyngeal cancer, glioma Intravenous EGFR Nirsevimab MEDI8897 Human monoclonal antibody IgG1κ Disease modifying Respiratory syncytial virus phase II 2018 Intramuscular Respiratory syncytial virus fusion protein (RSVFR) Nivolumab Opdivo FDA 2015 EU 2015 Human monoclonal antibody IgG4κ immunoglobulin Antineoplastic agent Programmed death receptor-1 (PD-1) blocking antibody NSCLC, bladder cancer, renal cell cancer phase III 2021 Hodgkin lymphoma Melanoma Small cell lung cancer Squamous carcinoma head and neck Colorectal cancer GBM no added benefit 2017 Intravenous Blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2 Nofetumomab merpentan Verluma FDA 1996 No longer marketed in USA Murine monoclonal fragment IgG2bκ Fab Cancer diagnostic imaging SCLC Antitumor Membrane-spanning 4-domains, subfamily A, member 1 Obiltoxaximab ETI-204 Anthim FDA 2016 Chimeric monoclonal antibody IgG1κ Disease modifying Bacillus anthracis anthrax phase IV 2021 Intravenous Intramuscular Bacillus anthracis spores PA component of B. anthracis toxin Obinutuzumab GA101HUMAB RG7159 RO5072759 Afutuzumab Gazyvaro FDA 2013 Humanized monoclonal antibody IgG1κ Antineoplastic lymphoma phase II (MCL, DLBCL) Chronic lymphocytic leukemia Phase II 2021 Intravenous CD20 Induces B-cell apoptosis Ocaratuzumab LY2469298 AME-133V Humanized monoclonal antibody IgG1κ Antineoplastic NHL Pemphigus phase III Intravenous CD20 Ocrelizumab Ocrevus FDA 2017 Humanized monoclonal antibody IgG1κ Disease modifying Multiple sclerosis Intravenous CD20 Odulimomab Murine monoclonal antibody Disease modifying Transplant rejection Only studied in mice Lymphocyte function-associated antigen-1 (LFA-1 (CD11a)) Ofatumumab Arzerra FDA 2009 EU 2010 Human monoclonal antibody IgG1κ Complement-dependent cytotoxicity (CDC) Antineoplastic CLL Phase III 10% ORR after ritux Phase II as first line 86% ORR With CHOP 100% ORR with 62% CR Intravenous CD20 Olaratumab IMC3G3 Lartruvo FDA 2016 EU 2016 Human monoclonal antibody IgG1κ Antineoplastic Sarcoma phase II 2023 Ovarian not beneficial Intravenous Platelet derived growth factor receptor alpha (PDGF-R α) Oleclumab MEDI9447 Human monoclonal antibody IgG1λ Antineoplastic pancreatic phase II 2021 Colorectal cancer Bladder cancer phase I 2020 Breast cancer phase II 2022 NSCLC phase II 2022 Intravenous? 5′-nucleotidase CD73 Olokizumab Humanized monoclonal antibody IgG4κ Disease modifying rheumatoid arthritis Phase I 2014 phase IIb mod results IL6 Omalizumab IGE25 RG3648 Xolair Humanized monoclonal antibody IgG1κ Disease modifying allergic asthma Urticaria Subcutaneous IgE Fc region Omburtamab Murine monoclonal antibody IgG1κ Antineoplastic Neuroblastoma Phase III 2022 Intracerebroventricular treatment CD276 OMS721 Human monoclonal antibody Disease modifying Atypical hemolytic uremic syndrome phase III 2020 Lupus nephritis phase II 2018 Intravenous Mannan-binding lectin-associated serine protease-2 (MASP-2) Onartuzumab PRO143966 RO5490258 METMAB Humanized monoclonal antibody IgG Antineoplastic Intravenous Human scatter factor receptor kinase Ontuxizumab MORAB-004 Chimeric/humanized monoclonal antibody Antineoplastic No clinical response Intravenous Endosialin tumor endothelial marker-1 (TEM1) Onvatilimab Human monoclonal antibody IgG1κ Nothing in PubMed Vista (V-domain immunoglobulin suppression of T activation (VSIR) Opicinumab BIIB033 Human monoclonal antibody IgG1 Disease modifying multiple sclerosis Phase II 2020 Leucine-rich repeat and immunoglobulin domain containing neurite outgrowth inhibitor receptor interacting protein-1 (LINGO-1) LINGO-1 Oportuzumab monatox VB4-845 Vicinium Proxinium FDA 2005 EU 2005 Additional approval pending 2019 Humanized monoclonal antibody fragment scFv Antineoplastic Bladder phase III Head and neck cancer Intravescical Epithelial cell adhesion molecule (EPCAM) and tumor-associated calcium signal transducer 1 (TACSTD1) and pseudomonas exotoxin A immunotoxin fusion protein (anti-EPCAM antibody fragment-Pseudomonas exotoxin fusion protein) Oregovomab MAB-B43.13 OvaRex Murine monoclonal antibody IgG1κ Antiidiopathic antibody to ovarian antigen CA-125 Antineoplastic Ovarian cancer Not effective in achieving increase RFS or OS Ovarian phase I 2021 Phase II 2019 Subcutaneous Intravenous CA-125 Orticumab RG7418 Human monoclonal antibody fragment Fab Disease modifying Antiinflammatory Oxidized low-density lipoprotein oxLDL Otelixizumab Chimeric humanized monoclonal antibody IgG1 Disease modifying Diabetes mellitus type 1 TTEDD phase II DEFEND-1 phase III failed DEFEND-2 phase III- no real benefit Subcutaneous CD3 Otilimab MOR103 GSK3196165 Human monoclonal antibody IgG1λ Disease modifying Osteoarthritis, rheumatoid arthritis phase II 2012 Multiple sclerosis phase II 2014 Intravenous Granulocyte-macrophage colony-stimulating factor (GMCSF) Otlertuzumab TRU-016 Humanized monoclonal antibody IgG fragment Antineoplastic CLL phase I and II 2014 and 2019 Intravenous CD37 Oxelumab OX40L R4930 HUMAB OX40L Human monoclonal antibody IgG1κ Disease modifying Asthma mainly preclinical mice Many clinical studies ongoing leukemia and asthma Intravenous OX-40 (CD252) Ozanezumab GSK1223249 Humanized IgG1 Disease modifying ALS phase II 2015 ALS no good Intravenous Neurite outgrowth inhibitor (NOGO-A) Ozoralizumab ATN 103 Humanized monoclonal antibody Disease modifying Rheumatoid arthritis phase II 2012 Subcutaneous TNF-α Pagibaximab Chimeric monoclonal antibody Disease modifying Staph sepsis low birth weight infants Phase II/III studies 2010 Intravenous Lipoteichoic acid Palivizumab Synagis, Abbosynagis FDA 1998 EU 1999 Humanized monoclonal antibody IgG1κ Disease modifying RSV many phase III studies Intramuscular F protein of respiratory syncytial virus Pamrevlumab FG-3019 Human monoclonal antibody IgG1κ Disease modifying Idiopathic pulmonary fibrosis (IPF), Antineoplastic Pancreatic cancer Muscular dystrophy phase II 2021 Diabetes nephropathy Connective tissue growth factor (CTGF) Insulin-like growth factor binding protein 8 (IGFBP-8) Panitumumab ABENIX ABX-EGF https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antibodies - cite_note-WHOList91-38 Vectibix FDA 2006 EU 2007 Human monoclonal antibody IgG2κ Antineoplastic Metastatic colorectal cancer Intravenous EGFR/erbB-1/HER1 PankoMab-GEX Gatipotuzumab Humanized monoclonal antibody IgG1κ Antineoplastic Phase IIb 2017 Phase I solid tumors 2019 Intravenous Tumor-specific glycosylation of MUC1 Panobacumab Aerumab 11 AR-101 KBPA-101 Human monoclonal antibody Antimicrobial Pseudomonas aeruginosa infection Intravenous Pseudomonas aeruginosa serotype O11 Parsatuzumab MEGF0444A RG-7414 Human monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase II 2014 no benefit Epidermal growth factor-like domain 7 (EGFL7) Pascolizumab Humanized monoclonal antibody Disease modifying Not effective trails aborted IL-4 Pasotuxizumab Chimeric/humanized monoclonal antibody fragment Antineoplastic No studies Folate hydrolase/prostate-specific membrane antigen (PSMA) Pateclizumab RG7415 PRO283698 MLTA3698A Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis Phase II not as efficacious as adalimumab but had response Subcutaneous Lymphotoxin-α Patritumab AMG888 U3-1287 Human monoclonal antibody IgG1κ Antineoplastic May not be beneficial head/neck NSCLC CT site ErbB3 (HER3) Spartalizumab PDR001 FDA review possible 2019 Humanized monoclonal antibody Antineoplastic Breast cancer phase II 2021 NSCLC 2021 Melanoma phase II 2022 Phase III 2020 Intravenous PD1, PDCD1, CD279 Pembrolizumab MK-3475 Keytruda FDA 2014 EU 2015 FDA 2018 for metastatic Merkel cell carcinoma, HCC, NSCLC Humanized monoclonal antibody IgG4κ Antineoplastic Squamous carcinoma trachea, NSCLC , urothelial (HCC phase II) Melanoma cHL, LgB cell lymph Gastric cancer Cervical cancer Hepatocellular carcinoma Intravenous Trials for multiple myeloma discontinued by FDA PD-1 Pemtumomab HMFG1 antibody labeled with 90Yttrium Theragyn Murine monoclonal antibody Antineoplastic Phase III Europe 2009/US 2013 no benefit after 3.5 years follow-up MUC1/human milk fat globule antigen 1 (HMFG1) Perakizumab Humanized monoclonal antibody IgG1κ Disease modifying psoriatic arthritis Phase I discontinued IL 17A Pertuzumab Perjeta FDA2012 EU 2013 Omnitarg Humanized monoclonal antibody IgG1 Antineoplastic agent HER2-positive metastatic breast cancer Gastric/breast cancer Phase III gastric Intravenous Extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 protein (HER2/neu) Pexelizumab Humanized scFv Disease modifying acute myocardial infarctions APEX-AMI trial negative results PRIMO-CABG I and II trials no significant benefit C5 Pidilizumab CT-011 Humanized monoclonal antibody IgG1κ Antineoplastic Mult myeloma DLBCL Pontine glioma Pancreas Melenaoma HCC Antiinfection Intravenous PD-1 Pinatuzumab vedotin Humanized monoclonal antibody ADC consisting of the microtubule-disrupting agent, monomethyl auristatin E (MMAE), conjugated to an anti-CD22 mAbvia the protease-cleavable peptide linker maleimidocaproylvaline-citrulline(vc)-p-aminobenzoyloxycarbonyl Antineoplastic B-cell NHL phase I study good response Phase II completion 2019 Intravenous CD22 Pintumomab Murine monoclonal antibody Not therapeutic Diagnostic imaging adenocarcinoma antigen Adenocarcinoma (imaging) Placulumab Human monoclonal antibody V-kappa)2 FC Disease modifying pain and inflammatory diseases Development discontinued 2012 Human TNF Plozalizumab MLN1202 HU1D9 Withdrawn by company Humanized monoclonal antibody IgG1κ Disease modifying Diabetic nephropathy and arteriovenous graft patency RA no benefit Intravenous CC chemokine receptor 2 (CCR2) Pogalizumab MOXR0916 R07021608 Vonlerolizumab Humanized monoclonal antibody IgG1κ Antineoplastic Solid tumors phase I 2019 may be safe but may not be effective No formal manuscripts yet Intravenous Tumor necrosis factor receptor superfamily member 4 (ACT35, OX40, CD134) Polatuzumab vedotin FCU2711 RO5541077-000 FDA review 2018 Humanized monoclonal antibody IgG1κ Antineoplastic NHL phase II 2019 DLBCL phase III 2023 Intravenous CD79B Ponezumab RN1219 PF-04360365 Humanized monoclonal antibody IgG2 Disease modifying Alzheimer's disease Safe but no clinical efficacy 2013 Intravenous Human beta-40-amyloid Aβ40 Porgaviximab C2G4 Chimeric monoclonal IgG1κ Antiinfectious Ebola virus disease No known ongoing studies Zaire ebolavirus glycoprotein Prasinezumab PRX002 RG7935 RO7046015 Humanized monoclonal antibody IgG1κ Disease modifying Parkinson's disease Phase II 2021 Intravenous Anti-alpha-synuclein (NACP) Prezalizumab AMG-557 MEDI5872 Humanized monoclonal antibody IgG2 Disease modifying SLE phase II 2018 Sjogren's Subcutaneous B7-related protein inducible T-cell costimulator ligand (ICOSL) Priliximab cMT 412 CEN 000029 Chimeric monoclonal antibody Disease modifying Crohn's disease, multiple sclerosis IN FDA no known studies CD4 Pritoxaximab Chimeric monoclonal antibody IgG1κ Antiinfectious E. coli shiga toxin type-1 Pritumumab Human monoclonal antibody IgG1κ Antineoplastic Brain cancer Phase II studies in Japan, could not find literature reportedly increase survivability 10 fold Vimentin Quilizumab MEMP1972A RG-7449 Anti-M1 Humanized monoclonal antibody IgG1κ Disease modifying Asthma phase II 2014 no great benefit Urticaria phase II 2014 no great benefit Allergic rhinitis Subcutaneous Intravenous M1 prime segment of membrane bound IgE (IGHE) Racotumomab Vaxira Murine monoclonal antibody IgG1κ Antineoplastic Nonsmall cell lung cancer phase III 2016 cimavax better (recombinant EGF injection) 2 more months survival over placebo Neuroblastoma phase II 2020 Intradermal Subcutaneous N-glycolylneuraminic acid gangliosides (NGNA ganglioside) Radretumab F16SIP L19SIP radiolabeled with I 331 Human monoclonal antibody Imaging study PET Antineoplastic Lymphoma brain mets 2012 phae I Stage III NSclC Fibronectin extra domain-B Rafivirumab CR57 Human monoclonal antibody IgG1λ Used in cocktail and with vaccination Antiinfectious Rabies (prophylaxis) No known studies Rabies virus glycoprotein Ralpancizumab RN317 PF-05335810 Humanized monoclonal antibody IgG2κ Disease modifying Dyslipidemia phase I 2017 PCSK9 (proprotein convertase subtilisin/kexin type 9, neural apoptosis-regulated convertase 1, NARC1, NARC-1, proprotein convertase 9, PC9) Ramucirumab LY3009806 IMC-1121B Cyramza FDA 2014 EU 2014 Human monoclonal antibody IgG1κ Antineoplastic Urothelial phase III done Adenocarcinoma stomach and GE junction phase II 2023 Colorectal cancer NSCLC HCC phase III 2017 no additional benefit Intravenous VEGFR2 Ranevetmab NV-01 Veterinary monoclonal antibody IgG1κ canine Disease modifying Osteoarthritis in dogs Nerve growth factor-β (NGF-β) Ranibizumab RBZ RG-3645 RHuFAb Lucentis FDA 2006 EU 2007 Humanized monoclonal fragment IgG1κ Fab Disease modifying Macular degeneration (wet form) post market studies phase II Intravitreal Vascular endothelial growth factor A (VEGF-A) Ravagalimab PR-1629977 ABBV-323 Humanized monoclonal antibody IgG1κ Disease modifying UC phase II 2023 Intravenous Subcutaneous CD40 Ravulizumab ALXN1210 Ultomiris FDA 2019 EU pending Humanized monoclonal antibody IgG2/IgG4κ Disease modifying Paroxysmal nocturnal hemoglobinuria (PNH) Phase III 2021 similar to eculizumab, atypical hemolytic uremic syndrome phase III 2021 Intravenous Complement C5 (C5) Raxibacumab ABthrax FDA 2012 Human monoclonal antibody IgG1λ Antiinfectious Treat inhalation anthrax Intravenous Bacillus anthracis protective antigen Refanezumab GSK249320 Humanized monoclonal antibody IgG1κ Disease modifying recovery of motor function after stroke Phase II completed 2011 no benefit Intravenous Myelin-associated glycoprotein Regavirumab MCA C23 TI-23 Human monoclonal antibody Antiinfectious Cytomegalovirus glycoprotein B ONLY STUDIES IN RATS 1994 Cytomegalovirus infection Relatlimab BMS-986016 Human monoclonal antibody IgG4κ Antineoplastic Melanoma phase II 2022 Colon cancer phase II 2022 Chordoma phase II 2020 Cannot find manuscripts but company website phase II good results Glioblastoma phase I 2020 Intravenous Lymphocyte activation gene 3 (LAG3) CD223 Remtolumab ABT-122 Human monoclonal antibody Disease modifying RA Phase II 2016 no increased benefit over adalimumab Subcutaneous Interleukin 17 alpha, TNF-α Reslizumab DCP 835 Scheme 55700 CEP-38072 Cinqair FDA 2016 EU 2016 Humanized monoclonal antibody IgG4κ Disease modifying Inflammations of the airways asthma completed and ongoing, skin and gastrointestinal tract, polyarteritis stage II 2018 Rhino sinusitis 2020 Intravenous Subcutaneous IL-5 Rilotumumab AMG-102 Human monoclonal antibody IgG2κ Antineoplastic Gastric completed phase III 2015 not effective NSCLC phase II 2014 no benefit Glioma phase II no response Intravenous Hepatocyte growth factor (HGF) Rinucumab REGN2176 Human monoclonal antibody IgG4κ Disease modifying neovascular age-related macular degeneration phase II 2014 Intravitreal Platelet-derived growth factor receptor beta Risankizumab ABBV-066 BI-655066 FDA/EU pending approval Humanized monoclonal antibody IgG1κ Disease modifying Crohn's disease phase II good, phase III ongoing psoriasis phase II response better than ustekinumab, psoriatic arthritis, and asthma Subcutaneous IL23A Rituximab GP2013 IDEC-102 RG-105 MabThera, Rituxan FDA 1997 EU 1998 Chimeric monoclonal antibody IgG1κ Antineoplastic Non-Hodgkin lymphomas, chronic lymphocytic leukemias, some autoimmune disorders, i.e., rheumatoid arthritis, >2K studies ongoing Subcutaneous CD20 Rivabazumab pegol Humanized monoclonal antibody fragment Fab' IgG1κ Antiinfectious No studies found Pseudomonas aeruginosa type III secretion system Rmab Rabishield Made in India Human monoclonal antibody Antiinfectious Postexposure prophylaxis of rabies Rabies virus G glycoprotein Robatumumab 19D12 SCH 717454 MK-7454 P04722 Human monoclonal antibody IgG1κ Antineoplastic Colorectal phase II 2009 little benefit Ewings no response 2016 Intravenous Insulin-like growth factor I (IGF-1 receptor) (CD221) Roledumab Human monoclonal antibody IgG1κ Immunomodulation Rh disease Phase III 2017 Intravenous RHD Romilkimab SAR156597 HUBTI3_2_1 Humanized chimeric monoclonal antibody IgG4 bispecific Disease modifying Systemic sclerosis phase II 2019 Pulm fibrosis phase II 2017 no benefit Subcutaneous Interleukin 13 and IL4 Romosozumab Evenity FDA pending EU pending Japan 2018 Humanized monoclonal antibody IgG2κ Disease modifying Postmenopausal osteoporosis phase III study FRAME Men phase III BRIDGE phase III Intravenous Sclerostin/scleroscin SOST Rontalizumab rhuMAb IFNalpha Humanized monoclonal antibody Disease modifying Systemic lupus erythematosus phase II 2013 end points not met Subcutaneous IFN-α Rosmantuzumab OMP-131R10 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase I 2018 Intravenous Root plate-specific spondin r-spondin-3 WNT? (wingless/integrated) Rovalpituzumab tesirine SC0002 SC16LD6.5 ABBV-181 Humanized monoclonal antibody IgG1κ Antineoplastic Small cell lung cancer phase I 2018 Phase II 2024 Intravenous Delta-like ligand-3 (DLL3) Rovelizumab Hu23F2G LeukArrest Humanized monoclonal antibody IgG1κ Disease modifying Hemorrhagic shock, MI stroke phase III goals not met 2000 CD11, CD18 Rozanolixizumab UCB7665 Chimeric/humanized monoclonal antibody IgG4κ Thrombocytopenia ITP phase II 2019 Myasthenia gravis phase II 2018 Subcutaneous Intravenous Neonatal Fc receptor (FCGRT) Ruplizumab Antova Humanized monoclonal antibody Disease modifying lupus and lupus nephritis not effective Life-threatening thromboembolism BioDrugs. 2004; 18(2):95–102. Costimulation blockade in the treatment of rheumatic diseases CD154 (CD40L) Sacituzumab govitecan IMMU-132 FDA/EU pending approval Humanized monoclonal antibody IgG1κ Antineoplastic agent Prostate cancer phase II 2021 Urothelial phase II 2020 Trip neg breast ca phase III 2020 NSCLC SCLC UC Intravenous Tumor-associated calcium signal transducer 2 (TROP-2) inhibits topoisomerase I Samalizumab ALXN6000 BAML-16-001-S1 Humanized monoclonal antibody IgG2/G4κ Antineoplastic CLL MM phase I 2010 (terminated by sponsor) AML phase II 2021 Intravenous OX-2 membrane glycoprotein (CD200) Samrotamab vedotin Chimeric/humanized monoclonal antibody IgG1κ Antineoplastic No studies found Leucine-rich repeat-containing protein 15 (LRRC15) Sarilumab REGN88 SAR153191 Kevzara FDA?EU/Japan under review approved in Canada Human monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis phase III 2015/2020/2027(preg exposure), ankylosing spondylitis Juvenile idiopathic arthritis phase II 2022 Subcutaneous IL6 Satralizumab SA237 Sapelizumab FDA review possible 2019 Humanized monoclonal antibody IgG2κ Disease modifying Neuromyelitis optica phase III 2019/2020 Subcutaneous? IL6 receptor Satumomab pendetide OncoScint CR103 FDA 1992 Murine monoclonal antibody IgGκ fragment Fab' Diagnostic imaging Detection colorectal and ovarian cancer Intravenous Tumor-associated glycoprotein (TAG-72) Secukinumab AIN457 Cosentyx FDA 2015 EU 2015 Human monoclonal antibody IgG1κ Disease modifying Uveitis, rheumatoid arthritis psoriasis phase II 2019 over 100 other studies arthritis ; psoriatic psoriasis; spondylitis; ankylosing Subcutaneous IL 17A Selicrelumab CP 870.893 RG7876 RO-7009789 Human monoclonal antibody IgG2κ Antineoplastic Solid tumors phase I 2020 Pancreatic cancer phase II 2020 Colon cancer phase II 2021 Mesothelioma phase ib 2015 Subcutaneous Intravenous Tumor necrosis factor receptor superfamily member 5 (CD40) Seribantumab MM121 SAR256212 Human monoclonal antibody IgG2λ Antineoplastic Breast phase II 2020 Ovarian phase I 2014 Intravenous Receptor tyrosine-protein kinase erbB-3 (HER3) Setoxaximab Chimeric monoclonal antibody IgG1κ Antiinfection E. coli No known studies or clinical use E. coli shiga toxin type-2 Setrusumab BPS804 MOR05813 Human monoclonal antibody IgG2 Disease modifying Osteogenesis imperfecta phase II 2020 Intravenous Sclerostin (SOST) Sevirumab MSL-109 Antiinfectious CMV retinitis early termination trial secondary to safety Phase II 2003 Cytomegalovirus infection SHP647 Ontamalimab PF-00547659 Human monoclonalantibodyIgG2κ Disease modifying Crohn's/UC phase III 2020–2025 × 7 Phase II study 2007 better response in UC than in Crohn (?more time needed to evaluate clinical significance Subcutaneous Mucosal addressin cell adhesion molecule (MADCAM) Sibrotuzumab BIBH1 F19 Humanized monoclonal antibody IgG1κ Antineoplastic Colorectal cancer phase II 2003 failed Lung cancer2001 Intravenous FAP Sifalimumab MDX-1103 MEDI-545 CP145 Humanized monoclonal antibody IgG1κ Disease modifying SLE phase II 2015 dermatomyositis, polymyositis Intravenous Subcutaneous IFN-α Siltuximab CLLB8 CNTO-328 Sylvant FDA 2014 EU 2014 Chimeric monoclonal antibody IgG1κ Antineoplastic Multiple myeloma phase II 2019 DM type I phase I 2017 Schizophrenia adjunct 2020 phase II Multicentric Castleman's disease (MCD) with HIV negative and HHV-8 negative Intravenous IL-6 Simtuzumab AB0024 GS-6624 Humanized monoclonal antibody IgG4κ Disease modifying Hepatic fibrosis Phase II 2016 no benefit Pulm fibroses phase II 2017 no benefit Myelo fibr 2017 phase II Subcutaneous Intravenous Lysyl oxidase homolog 2 (LOXL2) Siplizumab MEDI-507 Humanized monoclonal antibody IgG1κ Antineoplastic CD2 T Or NK cells Sirtratumab vedotin Human monoclonal antibody Antineoplastic Nothing in PubMed or clinical trials SLITRK6 Sirukumab Human monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis Phase III done good results Subcutaneous IL-6 Sofituzumab vedotin Humanized monoclonal antibody Antineoplastic Ovarian pancreatic Phase I (2014) CA-125 Solanezumab LY2062430 Humanized monoclonal antibody IgG1 Disease modifying Alzheimer's Phase III study discontinued no effect In preclinical trial for secondary prevention 2022 Hereditary AD phase III 2021 Intravenous Beta amyloid Solitomab MT110-011 AMG110 Murine monoclonal antibody bispecific T-cell engager (BiTE) Antineoplastic Gastrointestinal, lung, and other cancers Phase I 2015 Intravenous Epithelial cell adhesion molecule (EpCAM) CD3 Sonepcizumab LT1009 iSONEP Humanized monoclonal antibody Disease modifying Choroidal and retinal neovascularization phase II 2015 not so good Antineoplastic phase II renal cancer 2017 potential Intravenous Intravitreous Sphingosine-1-phosphate (S1P) Stamulumab Humanized monoclonal antibody Disease modifying muscular dystrophy Animal studies, minimal efficacy Phase I/II studies ongoing (no improvement) Intravenous Myostatin Sulesomab IMMU-MN3 LeukoScan EU 1997 Murine monoclonal IgG1 fragment Fab′ Diagnostic Osteomyelitis (imaging) NCA-90 (granulocyte antigen) Suptavumab REGN2222 SAR438584 Human monoclonal antibody IgG1κ Antiinfectious Medically attended lower respiratory disease phase III 2017 not meet primary endpoint Another study no data yet at 30 mg/kg dose Intramuscular Resp sync virus fusion protein (RSVFR) Sutimlimab BIVV009 Chimeric/humanized monoclonal antibody IgG4κ Disease modifying cold agglutinin disease phase III 2020 Intravenous Complement C1s (C1s) Suvizumab KD-247 Humanized monoclonal antibody IgG1κ Antiinfectious HIV Phase I KD-247 2007 Intravenous Human immunodeficiency virus glycoprotein 120 third variable loop Suvratoxumab MEDI4893 Human monoclonal antibody IgG1κ Disease modifying Nosocomial pneumonia phase II 2018 Intravenous Staphylococcus aureus alpha toxin Tabalumab LY2127399 Human monoclonal lantibodyIgG4κ Antineoplastic Rheum arthr phase III 2013 no signif response SLE phase III 2015 endpoints not met Mult myelo phase I 2014 may not treat but be prognostic Subcutaneous Cytokine B-cell activating factor (BAFF) Tacatuzumab tetraxetan HAFP-31 AFP-Cide Humanized monoclonal antibody yttrium 77 Antineoplastic No studies in clinical trial or PubMed Alpha-fetoprotein Tadocizumab C4G1 YM337 Humanized monoclonal antibody fragment IgG1κ Fab' Disease modifying Percutaneous coronary intervention phase I 1999 ?not further developed Integrin αIIbβ3 Talacotuzumab CSL362 JNJ-56022473 Humanized monoclonal antibody IgG1-2κ Antineoplastic AML phase III 2018 MDS phase II 2019 SLE 2019 phase I Intravenous Interleukin 3 receptor subunit-α (IL3Rα, CD123) Talizumab C21/AL-90 TNX-901 Humanized monoclonal antibody IgG1κ Disease modifying Peanut allergy Allergic reaction Phase II 2003 good results legal issues shelved the drug Subcutaneous IgE Tamtuvetmab AT-005 Tactress Canine monoclonal antibody IgG2λ CD52 Tanezumab RN624 PF-4383119 FDA review possible 2019 Humanized monoclonal antibody IgG2 Disease modifying Pain Osteoarthritis Back pain Metastatic cancer pain Phase II ∼2008 Nerve growth factor (NGF) Tanibirumab Olinvacimab TTAC-0001 Human monoclonal antibody IgG1 Disease modifying Antineoplastic Phase I glioblastoma 2020 Breast cancer phase I 2020 AMD murine no human studies found Intravenous VEFR-2 Taplitumomab paptox Murine monoclonal antibody IgG1κ Antineoplastic No studies pub med or clinical trials CD19 Tarextumab OMP-59R5 Human monoclonal antibody IgG2 Antineoplastic Phase II trial NSCLC no benefit 2017 Pancreatic phase II 2017 Intravenous Notch2/3, Notch receptor Tavolimab MEDI0562 Chimeric/humanized monoclonal antibody IgG1κ Antineoplastic Head and neck phase I 2024 Ovarian cancer phase II 2023 Tumor necrosis factor receptor superfamily member 4 (TNFRS4) OX40L receptor (CD134) Technetium (99 mTc) acritumomab Rabbit monoclonal IgG Not for use in humans- research purpose only CEA Technicium (99 mTc) Fanolesomab NeutroSpec FDA2004 Murine monoclonal IgM radiolabeled Disease modifying osteomyelitis Sales and marketing suspended (2005) Diagnostic scans for acute appendicitis Intravenous CD15 Tefibazumab INH–H2002 Aurexis Humanized monoclonal antibody IgG1κ Antiinfectious Staphylococcus aureus infection Phase II 2006 Intravenous Clumping factor A Telimomab aritox (TAB-885) Recombinant murine monoclonal antibody Fab with ricin Antineoplastic T Cell lymphoma/leukemia No studies in pub med or clinical trials CD5 Telisotuzumab vedotin ABT-700 Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2017 SCLC phase II 2022 NSCLC phase II 2021 Intravenous Hepatocyte growth factor receptor HGFR Tenatumomab Murine monoclonal antibody IgG2b Antineoplastic Phase I 2017 Phase II brain tumors 2010 Intravenous P24821, tenascin C Teneliximab Chimeric monoclonal antibody IgG1 Not in clinical trials 2009 CD40 (TNF receptor superfamily member 5) Teplizumab MGA031 PRV-031 hOKT3g1(Ala-Ala) Humanized monoclonal antibody IgG1κ Disease modifying type I DM phase II completion AbATE trial 2019 Psoriasis phase I and II completed 2010 study stopped secondary to injection reaction severe allergy Intravenous Subcutaneous CD3 Tepoditamab Human monoclonal antibody IgG1κ bispecific Antineoplastic No studies on PubMed or clinical trials c-type dendritic cell-associated lectin 2 (CLEC-2A, MCLA-117) and CD3 Teprotumumab RV001 R-1507 RO4858696 HZN-001 FDA review possible 2019 Human monoclonal antibody Disease modifying Thyroid eye disease phase II 2017 Graves phase III 2020 Intravenous Insulin-like growth factor receptor type I (IGF-1 receptor) (CD221) Tesidolumab LFG316 NOV-4 Human monoclonal antibody Phase I 2017 PNH phase II 2020 AMD phase II 2015 not beneficial Intravenous Intravitreous C5 Tetulomab tetraxetan LU-177 Betalutin Humanized monoclonal antibody Antineoplastic Animal studies 2013 CD37 Tezepelumab MEDI9929 AMG-157 Human monoclonal antibody IgG2λ Disease modifying Asthma, atopic dermatitis Phase II 2017 Subcutaneous Thymic stromal lymphopoietin (TSLP) Theralizumab TGN1412 TAB08 Humanized monoclonal antibody Antineoplastic Solid tumors phase I 2020 Disease modifying Rheum arth, SLE phase II Intravenous CD28 History of cytokine storm at higher doses 2006 Tibulizumab LY3090106 Humanized monoclonal antibody bispecific tetravalent Disease modifying Autoimmune disorder Phase I 2020 Subcutaneous Intravenous No manuscripts found specific to this antibody Human B-cell activating factor of the tumor necrosis factor family interleukin 17 (BAFF) Tigatuzumab CS-1008 TRA-8 Humanized monoclonal antibody IgG1κ Antineoplastic Colon phase II 2011no added benefit Colon phase I 2013 NSCLC phase II 2011 no benefit Pancreatic phase II 2008 benefit TN breast canc 2015 phase II no added benefit Cytokine receptor DR5 (death receptor 5) TRAIL-R2 Tildrakizumab MK-3222 SCH-900222 Ilumya Ilumetri FDA 2018 Humanized monoclonal antibody IgG1κ Immunologically mediated inflammatory disorders Mod/severe psoriasis phase III 2018-20 Subcutaneous IL23 Timigutuzumab Humanized monoclonal antibody IgG1κ Antineoplastic No studies in clinical trial or PubMed erbB2/HER2 Timolumab BTT-1023 Human monoclonal antibody Disease modifying Scler cholang phase II 2019 Intravenous AOC3 Tiragotumab MTIG-7192A RG6058 RO7092284 Human monoclonal antibody IgG1κ Antineoplastic Phase I 2020 NSCLC phase II 2021 HL phase II 2019 Intravenous Nothing published yet T-cell IG and immune-receptor tyrosine-based inhibitory motif (TIGIT) Tislelizumab China pending approval Humanized monoclonal antibody Antineoplastic NSCLC phase III 2020 Gastric phase III 2022 Esophageal cancer phase III 2021 NHL phase II 2020 Intravenous Nothing published yet 2019 PCDC1, CD279 Tisotumab vedotin Human monoclonal antibody IgG1κ Antineoplastic Ovary cancer Cervix cancer Endometrium cancer Bladder cancer Prostate cancer Esophagus cancer Lung cancer, NSCLC Squamous cell carcinoma of the head and neck Pancreatic phase II 2022/3 Intravenous Coagulation factor III Tocilizumab MRA R-1569 RG-1569 RHPM-1 RO-4877533 Atlizumab Actemra, RoActemra FDA 2010 EU 2009 Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis >100 studies Behcet syndrome Intravenous Subcutaneous IL-6 receptor Tomuzotuximabfibri Humanized monoclonal antibody IgG1κ Antineoplastic Phase I 2019 EGFR, HER1 Toralizumab E−6040 IDEC-131 Humanized monoclonal antibody IgG1κ Disease modifying rheumatoid arthritis, lupus nephritis etc. Phase II trials failed with TE CD154 (CD40L) Tosatoxumab Human monoclonal antibody IgG1λ Antiinfectious No studies PubMed or Clin trials Staphylococcus aureus α-hemolysin Tositumomab and iodine 131 Tositumomab Bexxar FDA 2003 Murine monoclonal antibody IgG2aλ Antineoplastic Follicular lymphoma ( NHL ) >100 studies Intravenous CD20 Tovetumab MEDI-575 Human monoclonal antibody IgG2κ Antineoplastic Phase I/II 2012 Glioblastoma limited clin activity Intravenous Platelet-derived growth factor receptor α (CD140a) Tralokinumab CAT-354 Human monoclonal antibody IgG4 Disease modifying asthma phase IIb +/−, atopic dermatitis phase II 2016 Intravenous Subcutaneous IL-13 Trastuzumab 4D5v8 R-597 SYD977 Herceptin FDA 1998 EU 2000 Herceptin Hylecta FDA 2019 –trastuzumab/hyaluronidase Herzuma 2018 Humanized monoclonal antibody IgG1κ Antineoplastic Breast cancer Gastric and gastro-esophageal junction cancer HER2-positive phase III Subcutaneous Intravenous HER2/neu Trastuzumab Deruxtecan DS-8201 Hercion FDA breakthrough therapy Antibody drug conjugate humanized antibody IgG1κ with topoisomerase I inhibitor (DXd) Antineoplastic breast cancer phase I study breast, gastric, colorectal, salivary, and nonsmall cell lung cancer participated in part 2 2020 phase II DESTINY-Breast01 HER2 Trastuzumab emtansine RG-3502 PRO132365 Kadcyla FDA2013 EU 2013 Humanized monoclonal antibody IgG1κ as ADC Antineoplastic Breast cancer Intravenous HER2/neu TRBS07 Ektomab 3funct Antineoplastic Melanoma GD2 ganglioside Tribbles-related protein (TRB) family members are the mammalian orthologs of Drosophila tribbles. Tribbles was originally identified as a cell cycle regulator during Drosophila development. Tribbles genes are evolutionary conserved, and three TRB genes (TRB1, TRB2 and TRB3) have been identified in mammals. TRBs are considered pseudokinases because they lack an ATP binding site or one of the conserved catalytic motifs essential for kinase activity. Instead, TRBs play important roles in various cellular processes as scaffolds or adaptors to promote the degradation of target proteins and to regulate several key signaling pathways. Recent research has focused on the role of TRBs in tumorigenesis and neoplastic progression. In this review, we focus on the physiological roles of TRB family members in tumorigenesis through the regulation of the ubiquitin-proteasome system and discuss TRBs as biomarkers or potential therapeutic targets in cancer Tregalizumab BT-061 Humanized monoclonal antibody IgG1κ Disease modifying Rheumatoid arthritis Phase IIb no benefit Subcutaneous CD4 Tremelimumab (aka ticilimumab) ∗CP-675,206 Human monoclonal antibody IgG2 Antineoplastic agent NSCLC, small cell lung cancer, urethelial cancer phase II 2020, head and neck cancer and colon phase I 2021 Mesothelial phase IIb DETERMINE not beneficial >100 studies CTLA4 (cytotoxic T lymphocyte-associated antigen 4, CD152 Trevogrumab REGN1033 SAR391786 Human monoclonal antibody IgG4κ Disease modifying Muscle atrophy due to orthopedic disuse and sarcopenia phase II 2020 Myostatin, growth differentiation factor 8 (GDF8) TRL3d3 3D3 IgG Studies only in mice to this point Ati-G protein antibody (RSVGV) Tucotuzumab celmoleukin EMD-273066 HUKS-IL2 Humanized monoclonal antibody IgG1 Antineoplastic Ovarian phase II 2008 Lung, kidney, bladder phase I 2000 no benefit Intravenous Interleukin2 (EpCAM) Tuvirumab Humanized monoclonal antibody Antiinfectious Not effective in achieving primary efficacy as assessed by neutralization of circulating HBsAg Intravenous Hepatitis B virus surface antigen Ublituximab TG-1101 FDA review pending 2019 Chimeric monoclonal antibodyIgG1κ Antineoplastic Chronic lymphocytic leukemia, follicular cell lymphoma phase II 2020 Disease modifying Multiple sclerosis phase II 2019, phase III 2021 Awaiting result looks good prelim Intravenous CD20 MS4A1 Ulocuplumab Human monoclonal antibody IgG4 Antineoplastic CLL phase I 2014 Phase I/II Waldenstrom macroglobulinemia 2025 Phase I/II AML 2021 Intravenous CXCR4 (CD184) Urelumab BMS-663513 Human monoclonal antibody IgG4κ Antineoplastic CLL phase II 2020 Solid tumors phase II 2023 Intravenous Human receptor 4-1BB (CD137) Urtoxazumab TMA-15 Humanized monoclonal antibody IgG1κ Disease modifying EHEC animal studies Escherichia coli (EHEC) shiga toxin 2 Ustekinumab Stelara FDA 2009 EU 2009 Human monoclonal antibody IgG1κ Disease modifying Crohn disease Plaque psoriasis Psoriatic arthritis Subcutaneous Intravenous p40 subunit of interleukin 12 (IL-12p40), IL-23 Utomilumab PF-05082566 Human monoclonal antibody IgG2 Antineoplastic Diffuse large B-cell lymphoma Phase I 2021 phase II 2020 Breast phase II 2025 Intravenous 4-1BB (CD137) Vadastuximab talirine H2H12EC Chimeric monoclonal antibody IgG1κ Antineoplastic Acute myeloid leukemia phase II 2017 phase III 2017 MDS phase II 2017 Intravenous CD33 Vanalimab Mitazalimab Humanized monoclonal antibody IgG1λ Antineoplastic? No studies clinical trial or PubMed Immune checkpoint receptor, tumor necrosis receptor family CD40, (TNFRSF5) Vandortuzumab vedotin Humanized monoclonal antibody Antineoplastic Prostate cancer STEAP1 Vantictumab OMP-18R5 Human monoclonal antibody IgG2mab Antineoplastic NSCLC, breast phase I 2017 Intravenous Frizzled receptor Vanucizumab RG-7221 RO5520985 Humanized monoclonal antibody IgG1κ bispecific ANG-2/VEGF-Amab Antineoplastic Phase I 2018 Intravenous Angiopoietin 2/vascular endothelial growth factor A Vapaliximab BTT-1002 HUVAP Chimeric monoclonal antibody IgG2κ No studies in PubMed or clinical trials Vascular adhesion protein AOC3 (VAP-1) Varisacumab GNR-011 R-84 Human monoclonal antibody IgG1κ No studies in PubMed or clinical trials VEGF-A Varlilumab CDX-1127 Human monoclonal antibody IgG1κ Antineoplastic Solid tumors and hematologic malignancies Phase I 2017, phase II 2019/20 Melanoma phase II 2018/21 Intravenous CD27 Vatelizumab GBR500 SAR339658 Humanized monoclonal antibody IgG4 Disease modifying UC phase II 2016 MS phase II 2016 withdrawn lack of efficacy Α2β1 integrin I domain ITGA2 (CD49b) Vedolizumab LDP02 MLN02 Entyvio FDA 2014 EU 2014 Humanized monoclonal antibody IgG1κ Disease modifying Crohn disease Ulcerative colitis In CD resolution extraintestinal manifestations Intravenous Integrin α4β7 Selectively blocks trafficking of Memory T cells to inflamed gut tissue by inhibiting a4b7-mucosal addressin cell adhesion molecule-1 (MAd-CAM-1) interaction Veltuzumab IMMU-106 HA20 Humanized monoclonal antibody IgG1κ Antineoplastic Non-Hodgkin's lymphoma phase II 2013 ITP phase II 2016 Subcutaneous CD20 Vepalimomab Murine monoclonal antibody AOC3 (vascular adhesion protein-1) Vesencumab MNRP1685A Human monoclonal antibody IgG1mab Antineoplastic Solid malignancies Phase I 2011 proteinuria Neuropilin1 (NRP1) Visilizumab Nuvion Humanized monoclonal antibody IgG2 Disease modifying Prevent GVHD Not effective in UC CD3 Vobarilizumab Humanize monoclonal scFv Disease modifying inflammatory autoimmune diseases Nothing in PubMed IL6R Volociximab M200 Chimeric monoclonal antibody IgG4κ Antineoplastic Solid tumors NSCLC phase I/II 2010 Disease modifying phase I AMD terminated no results Integrin α5β1 Vopratelimab JTX-2011 Humanized monoclonal antibody IgG1κ Antineoplastic Solid tumors phase II 2022 Intravenous Inducible T-cell costimulator (ICOS) Vorsetuzumab mafodotin H1F6 SGN-70 Humanized monoclonal antibody Antineoplastic Phase I 2017 Intravenous CD70 Votumumab HumaSPECT Diagnostic EU 1998 Withdrawn from market 2003 Human monoclonal antibody Diagnostic Human colon cancer imaging Tumor antigen Cytokeratin tumor-associated antigen (CTAA16.88) Vunakizumab SHR-1314 Humanized monoclonal antibody IgG1 Disease modifying Psoriasis phase II 2019 Subcutaneous Nothing published Interleukin 17 alpha Xentuzumab BI-836845 Humanized monoclonal antibody Antineoplastic Breast, prostate, solid phase I 2019 Intravenous No clinical studies published Insulin-like growth factor (IGF1, IGF2) XMAB-5574 Tafasitamab MOR00208 FDA review possible 2019 Humanized monoclonal immunoglobulin fragment κ Fc Antineoplastic Diffuse large B-cell lymphoma phase II 2015/18/19/22 phase III 2022 Intravenous CD19 Zalutumumab 2F8 HUMAX-EGFR HuMax-EGFr Suspended for commercialization Human monoclonal antibody Antineoplastic Squamous cell carcinoma of the head and neck phase II 2011 phase III 2016 Intravenous EGFR Zanolimumab HuMax-CD4 (trade name) Humanized monoclonal antibody IgG1κ Antineoplastic CTCL Phase II good results Phase III suspended by company? Intravenous CD4 Zenocutuzumab Humanized monoclonal antibodyIgG1 bispecific epidermal growth factor receptors her2,her3 Antineoplastic ERBB3, HER3 Ziralimumab Human monoclonal antibody IgM Disease modifying immunosuppressive No studies clinical trials or PubMed CD147 (basigin) Zolbetuximab IMAB362 Claudiximab Chimeric monoclonal antibody IgG1κ ADCC enhance antibody Antineoplastic gastric cancer phase I, IIb Phase III 2023 Gastrointestinal adenocarcinomas and pancreatic tumor Intravenous Claudin protein (CLDN18.2) Zolimomab aritox H65-RTA ZX-CD5 Orthozyme CD 5 plus Human monoclonal antibody IgG1 Disease modifying Not effective in preventing GVHD 1994 CD5 Auristatins are water-soluble dolastatin analogs of dolastatin 10. Dolastatin 10 belongs to dolastatin family and it can powerfully bind to tubulin, thus inhibiting polymerization mediated through the binding to the vinca alkaloid-binding domain, and causes cell to accumulate in metaphase arrest. Ulcerative Colitis Mabs being studied for UC but not yet approved by the FDA include bimekizumab, etrolizumab, golimumab, mirikizumab, ravagalimab, sacituzumab govitecan, ontamalimab, and vatelizumab. Refer to Table 16.1 . Autoimmune Diseases Autoimmune diseases affect many organs and tissues including liver, gall bladder, pancreas (β islet cells in diabetes mellitus), nerve junctions (myasthenia gravis), thyroid, bone and joints, blood vessels, and multiorgan systems, systemic lupus erythematosus (SLE). Autoimmune arthritis is of multiple types including psoriatic, sclerosis, rheumatoid arthritis (RA), and SLE. Many of these diseases are mediated by antibody or cellular autoimmunity but ultimately appear to be secondary to an underlying abnormality in T-cell immune-regulatory control. These disease processes are historically controlled with antiinflammatory agents, immunosuppressive/immunomodulatory agents, or low-dose chemotherapy. Those with resultant hormone deficiencies are supplemented with hormones depleted by the disease process. It is hoped that passive antibody therapy will mitigate the sequelae of these inflammatory processes. Plaque psoriasis/psoriatic arthritis Psoriasis affects 2%–3% of the world population and is an inflammatory skin disease. Brodalumab (Siliz) is a human Mab (IgG2κ) with specificity to IL-17 receptor A (IL-17RA). It is FDA approved for treatment of plaque psoriasis, and its mechanism of action is by inhibiting IL-17A, IL-17F, IL-17C, IL-25, and IL-17A/F heterodimer cytokine-induced responses including release of proinflammatory cytokines. When compared to ustekinumab, response rates nearly doubled with brodalumab in phase II and phase III trials during induction and maintenance therapies. 275 , 276 Other therapies currently also approved or being studied for treatment of this disease include bermekimab (MABp1,T2-18C3, CA-18C3, Xilonix), bimekizumab, briakinumab, certolizumab pegol (Cimzia), etanercept (Enbrel), infliximab (Remicade, Inflectra, Remsima), itolizumab (Alzumab), adalimumab (Humira, Amjevita), ustekinumab (Stelara), secukinumab (AIN457, Cosentyx), guselkumab (Tremfya), tildrakizumab (MK-3222, SCH-900,222, Ilumya, Ilumetri), risankizumab (ABBV-066, BI-655,066), mirikizumab (LY3074828), namilumab (MT203), netakimab, and vunakizumab. Refer to Table 16.1 . Withdrawn from market or ineffective for treating psoriasis include efalizumab (Raptiva), fezakinumab, bleselumab, and teplizumab (MGA031, PRV-031, hOKT3g1(Ala-Ala)) Refer to Table 16.1 . Systemic juvenile idiopathic arthritis Abatacept (Orencia) is a recombinant soluble fusion protein of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human IgG1. Its mechanism of action is as selective costimulation modulator as it inhibits T lymphocyte activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. This medication is FDA approved for both juvenile idiopathic arthritis (JIA) and adult RA. 277 , 278 , 279 Plaque psoriasis/psoriatic arthritis Psoriasis affects 2%–3% of the world population and is an inflammatory skin disease. Brodalumab (Siliz) is a human Mab (IgG2κ) with specificity to IL-17 receptor A (IL-17RA). It is FDA approved for treatment of plaque psoriasis, and its mechanism of action is by inhibiting IL-17A, IL-17F, IL-17C, IL-25, and IL-17A/F heterodimer cytokine-induced responses including release of proinflammatory cytokines. When compared to ustekinumab, response rates nearly doubled with brodalumab in phase II and phase III trials during induction and maintenance therapies. 275 , 276 Other therapies currently also approved or being studied for treatment of this disease include bermekimab (MABp1,T2-18C3, CA-18C3, Xilonix), bimekizumab, briakinumab, certolizumab pegol (Cimzia), etanercept (Enbrel), infliximab (Remicade, Inflectra, Remsima), itolizumab (Alzumab), adalimumab (Humira, Amjevita), ustekinumab (Stelara), secukinumab (AIN457, Cosentyx), guselkumab (Tremfya), tildrakizumab (MK-3222, SCH-900,222, Ilumya, Ilumetri), risankizumab (ABBV-066, BI-655,066), mirikizumab (LY3074828), namilumab (MT203), netakimab, and vunakizumab. Refer to Table 16.1 . Withdrawn from market or ineffective for treating psoriasis include efalizumab (Raptiva), fezakinumab, bleselumab, and teplizumab (MGA031, PRV-031, hOKT3g1(Ala-Ala)) Refer to Table 16.1 . Systemic juvenile idiopathic arthritis Abatacept (Orencia) is a recombinant soluble fusion protein of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human IgG1. Its mechanism of action is as selective costimulation modulator as it inhibits T lymphocyte activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. This medication is FDA approved for both juvenile idiopathic arthritis (JIA) and adult RA. 277 , 278 , 279 Rheumatoid Arthritis Certolizumab pegol alone or with methotrexate improves quality of life in RA and may cause disease remission and reduce joint damage. 280 Ankylosing Spondylitis Certolizumab pegolis is also approved for use with ankylosing spondylitis. Systemic Lupus Erythematosus Belimumab (Benlysta) is a human Mab (IgG1λ) that binds to B-cell activating factor and acts as a B-lymphocyte stimulator-specific inhibitor. It was approved by the FDA in 2011 for treatment of adult patients with active, autoantibody-positive SLE receiving standard therapy. This medication also decreases episodic frequency of lupus nephritis. 281 , 282 , 283 Cardiovascular Disease Despite marked improvement in survival from cardiovascular disease, this illness remains the number one cause of mortality in the US. This process causes injury to the endothelium of blood vessels of the heart secondary to toxins, accumulation of cholesterol, or chronic low-grade inflammation. Treatment has been preventive, primarily during actual injury or following injury. Therapies involve changes in behavior (diet, exercise, and cessation of tobacco use), pharmacologic to control contributing underlying illness (hypercholesterolemia, hypertension, diabetes type I and II), to diminish injury through thrombolytics, stents, vasodilators, supplemental oxygen, or to control sequelae of infarctions (cardiac dysfunction/failure). Passive antibody therapies are being tried to decrease the effects of some of the contributing factors of atherosclerotic plaque formation. Abciximab (ReoPro) is a chimeric recombinant monoclonal fragment (IgG1 Fab') with specificity to platelet glycoprotein IIb/IIIa receptor (CD41 7E3)/Intergrin α-IIb that prevents platelets from binding to fibrinogen. This Mab also prevents coagulation factor XIII from binding to platelets allowing stabilization of clots and are more easily lysed. The Fc portion of the antibody is removed to decrease thrombocytopenias. This antibody is used during high-risk coronary intervention to prevent clot formation and cardiac ischemia. 284 Alirocumab (Praluent) is a human Mab (IgG1) with specificity to proprotein convertase subtilisin/kexin type 9. This medication is used to control cholesterol levels in patients at high risk for cardiovascular events and in patients with familial hypercholesterolemia who are not controlled by other agents. 285 , 286 , 287 Evolocumab (Repatha) is a human Mab (IgG2λ) FDA approved for the treatment of hypercholesterolemia in patients with familial hypercholesterolemia or history of cardiovascular disease. This Mab has specificity to PCSK9. This medication reduced low-density lipoprotein (LDL) and cholesterol levels by 60% even after statin therapy. Hazard ratios for primary and secondary endpoints were less than one (∼0.80–0.85) with fewer cardiovascular-related death or infarction and stroke. 288 , 289 Under future watch is frovocimab (LY3015014) a humanized Mab (IgG4κ) with specificity to PCSK9 that completed phase I and II trials. There was up to 50% reduction in LDL cholesterol levels. Phase III studies have yet to be performed. 290 An additional antibody is lodelcizumab a humanized Mab (IgG1κ); however, no studies were found in clinicaltrials.gov or in Pubmed searches. Bococizumab is a humanized Mab (IgG2κ) that was in phase III trial, which was discontinued secondary to primary endpoints not being achieved. 291 Neurologic Diseases Besides autoimmune and malignant diseases of the neurologic system, there are also diseases of the central nervous system classified as degenerative. Such diseases include supranuclear palsy (SNP), Alzheimer's, and Parkinson's. Alzheimer's is likely the most common cause of dementia first described in 1907. This disease may be depicted as presenile or senile dementia and progresses at a similar rate no matter age of onset. This disease has a genetic predisposition causing it to occur in younger age groups. Histological changes include diffuse plaques (containing amyloid), neurofibrillary plaques, and neuronal loss especially in the hippocampus and temporal regions. Medical management may reverse some of the symptoms but does not prevent disease progression. Parkinson's is a mainly sporadic degenerative disease with a gradual progressive course mainly affecting motor function more than memory. It was first described in 1817. This is a disease of the substantia nigra characterized by loss of melanin containing nerve cells and eosinophilic intracytoplasmic inclusions. Aside from emotional support and physical therapy, medical therapy is used to decrease tremors including anticholinergic drugs for tremors at onset, beta blockers for intention tremors, and levodopa for postural imbalance and akinesia. Deep brain stimulation is also used to treat symptoms later on as disease progresses. SNP starts in the same age range as Parkinson's (middle to later in life) that was first described in 1963 with disturbances in gait and balance secondary to rigidity of trunk muscles. Loss of neurons and gliosis is seen in the midbrain. Medical treatment is relatively unsuccessful. Multiple sclerosis is a demyelinating disease most often seen in young adults. The clinical manifestations are diverse and the progression can be chronic, acute, or remitting and relapsing. Medications and therapeutic plasma exchange have been used to treat this debilitating disease with limited efficacy. Clinical trials are ongoing looking at Mab therapies for treatment of these four neurologic degenerative diseases. Multiple Sclerosis Alemtuzumab (Lemtrada) is a humanized Mab (IgG1κ) targeting CD52 that depletes lymphocytes (B and T cell) as reported earlier and is FDA approved for treatment of acute relapsing and remitting multiple sclerosis. 292 Ocrelizumab (Ocrevus) is a humanized Mab (IgG1κ) with specificity to CD20 (a B-cell membrane protein). In phase II trials, there were decreases in brain lesions on imaging, and decrease rate of disability decline in primary progressive multiple sclerosis. 293 Natalizumab (Tysabri) is a monoclonal IgG4κ humanized antibody with specificity to cell adhesion molecule (CD62L) that is FDA approved for relapsing multiple sclerosis. 294 , 295 The mabs to watch out for in the future and are in clinical trials include anifrolumab a human monoclonal antibody in phase I trials; elezanumab is a human Mab (IgG1λ) with specificity to repulsive guidance molecule family member-A that is in phase II trials to be completed 2021; and finally inebilizumab (MEDI-551) is a humanized monoclonal antibody (IgG2κ) with specificity to CD19 (a B-cell lymphocyte protein). This Mab mechanism of action is via ADCC and has completed phase I trials with good safety profile and response in decreasing lesions seen on contrast enhanced magnetic resonance imaging. Otilimabis (MOR103) is a human Mab (IgG1λ) completing phase I studies with good safety profile that targets granulocyte-macrophage colony-stimulating factor. Ublituximab is in phase II clinical studies to be completed in 2019, and phase III studies are scheduled to be completed in 2021. This Mab is a chimeric Mab (IgG1κ) with specificity to CD20 MS2A1. 296 , 297 , 298 , 299 , 300 Additional Mab have serious adverse effects such as daclizumab a humanized monoclonal (IgG1κ) with specificity to (CD25 {IL-2Rα}); or are ineffective as is opicinumab a human Mab IgG1 with specificity to Leucine-rich repeat and immunoglobulin domain containing neurite outgrowth inhibitor receptor interacting protein-1 which in a phase II trial was no more beneficial than placebo in treating optic neuritis in multiple sclerosis patients. 301 , 302 Alzheimer's Disease Aducanumab is a human Mab IgG1 with specificity to β-amyloid (N-terminus 3–6) soluble oligomers and insoluble fibers. Phase III clinical trials are ongoing since 2015. BAN-2401 is a humanized Mab IgG1 with specificity to β-amyloid fibrillary and soluble β amyloid and is in phase IIb clinical studies since 2013. Gosuranemab (BIIB092, IPN-007) is a humanized Mab IgG4κ with specificity to the tau protein and is in clinical trials to treat Alzheimer's disease scheduled to be completed in 2021. Gosuranemab is also in phase I studies to treat progressive suranuclear palsy and will be completed in 2020. Crenezumab (RG7412, MABT5102A) is a humanized Mab IgG4 with specificity to 1–40 β-amyloid and is on phase III studies scheduled to be completed in 2021 and 2022. Gantenerumab (R04909832, R1450) is a human Mab IgG1κ with targets β-amyloid. This Mab on initial phase III studies was found to be ineffective. Ongoing phase II/III trials are currently in place at higher dosing in a clinical population of people with autosomal dominant form of Alzheimer's disease. Solanezumab (LY2062430) is a humanized Mab IgG1 with specificity to beta amyloid. Initial phase III trials discontinued for lack of efficacy in preventing Alzheimer's disease. Ongoing phase III trials are now in place for secondary prevention of this disease and will be completed in 2021 and 2022. Mab antibodies studied and were ineffective include bapineuzumab, gantenerumab (R04909832, R1450), and ponezumab (RN1219, PF-04,360,365). Parkinson's Disease Prasinezumab (PRX002, RG7935, RO7046015) is a humanized Mab IgG1κ with specificity to α-synuclein. This Mab is in phase II clinical trials to treat Parkinson's and will be completed in 2021. Multiple Sclerosis Alemtuzumab (Lemtrada) is a humanized Mab (IgG1κ) targeting CD52 that depletes lymphocytes (B and T cell) as reported earlier and is FDA approved for treatment of acute relapsing and remitting multiple sclerosis. 292 Ocrelizumab (Ocrevus) is a humanized Mab (IgG1κ) with specificity to CD20 (a B-cell membrane protein). In phase II trials, there were decreases in brain lesions on imaging, and decrease rate of disability decline in primary progressive multiple sclerosis. 293 Natalizumab (Tysabri) is a monoclonal IgG4κ humanized antibody with specificity to cell adhesion molecule (CD62L) that is FDA approved for relapsing multiple sclerosis. 294 , 295 The mabs to watch out for in the future and are in clinical trials include anifrolumab a human monoclonal antibody in phase I trials; elezanumab is a human Mab (IgG1λ) with specificity to repulsive guidance molecule family member-A that is in phase II trials to be completed 2021; and finally inebilizumab (MEDI-551) is a humanized monoclonal antibody (IgG2κ) with specificity to CD19 (a B-cell lymphocyte protein). This Mab mechanism of action is via ADCC and has completed phase I trials with good safety profile and response in decreasing lesions seen on contrast enhanced magnetic resonance imaging. Otilimabis (MOR103) is a human Mab (IgG1λ) completing phase I studies with good safety profile that targets granulocyte-macrophage colony-stimulating factor. Ublituximab is in phase II clinical studies to be completed in 2019, and phase III studies are scheduled to be completed in 2021. This Mab is a chimeric Mab (IgG1κ) with specificity to CD20 MS2A1. 296 , 297 , 298 , 299 , 300 Additional Mab have serious adverse effects such as daclizumab a humanized monoclonal (IgG1κ) with specificity to (CD25 {IL-2Rα}); or are ineffective as is opicinumab a human Mab IgG1 with specificity to Leucine-rich repeat and immunoglobulin domain containing neurite outgrowth inhibitor receptor interacting protein-1 which in a phase II trial was no more beneficial than placebo in treating optic neuritis in multiple sclerosis patients. 301 , 302 Alzheimer's Disease Aducanumab is a human Mab IgG1 with specificity to β-amyloid (N-terminus 3–6) soluble oligomers and insoluble fibers. Phase III clinical trials are ongoing since 2015. BAN-2401 is a humanized Mab IgG1 with specificity to β-amyloid fibrillary and soluble β amyloid and is in phase IIb clinical studies since 2013. Gosuranemab (BIIB092, IPN-007) is a humanized Mab IgG4κ with specificity to the tau protein and is in clinical trials to treat Alzheimer's disease scheduled to be completed in 2021. Gosuranemab is also in phase I studies to treat progressive suranuclear palsy and will be completed in 2020. Crenezumab (RG7412, MABT5102A) is a humanized Mab IgG4 with specificity to 1–40 β-amyloid and is on phase III studies scheduled to be completed in 2021 and 2022. Gantenerumab (R04909832, R1450) is a human Mab IgG1κ with targets β-amyloid. This Mab on initial phase III studies was found to be ineffective. Ongoing phase II/III trials are currently in place at higher dosing in a clinical population of people with autosomal dominant form of Alzheimer's disease. Solanezumab (LY2062430) is a humanized Mab IgG1 with specificity to beta amyloid. Initial phase III trials discontinued for lack of efficacy in preventing Alzheimer's disease. Ongoing phase III trials are now in place for secondary prevention of this disease and will be completed in 2021 and 2022. Mab antibodies studied and were ineffective include bapineuzumab, gantenerumab (R04909832, R1450), and ponezumab (RN1219, PF-04,360,365). Parkinson's Disease Prasinezumab (PRX002, RG7935, RO7046015) is a humanized Mab IgG1κ with specificity to α-synuclein. This Mab is in phase II clinical trials to treat Parkinson's and will be completed in 2021. Allergic Diseases Allergic reactions develop because of immunologic stimulation of IgE antibodies followed by their interaction with allergens and mast cells. Effects can be local (dermatitis) or systemic (respiratory, cardiovascular, and gastrointestinal). Treatment is either avoidance of the allergens or supportive therapy in acute allergic reactions including pharmacologic treatment with type 1 and 2 histamine blockers, glucocorticosteroids, and if life-threatening epinephrine. Passive antibody therapies are being studied and approved to curtail severe reactions. Asthma Asthma affects 24 million individuals in the US, and up to 10% of asthma patients have severe disease that may be uncontrolled despite high doses of standard-of-care asthma medications requiring additional use of chronic oral corticosteroids. Benralizumab (Fensenra) is a humanized Mab (IgG1κ) with specificity to CD125 (IL-5Rα). This Mab is approved to treat severe asthma of the eosinophilic subtype in ages 12 and older. Its mechanism of action is to decrease the number of eosinophils via ADCC. Basophils are also depleted. 303 Atopic Dermatitis Dupilumab (Dupixent) is a human monoclonal gG4 antibody with specificity to interleukin-4 receptor subunit-alpha (IL-4Rα) that is approved to treat severe atopic dermatitis in adults. 304 Asthma Asthma affects 24 million individuals in the US, and up to 10% of asthma patients have severe disease that may be uncontrolled despite high doses of standard-of-care asthma medications requiring additional use of chronic oral corticosteroids. Benralizumab (Fensenra) is a humanized Mab (IgG1κ) with specificity to CD125 (IL-5Rα). This Mab is approved to treat severe asthma of the eosinophilic subtype in ages 12 and older. Its mechanism of action is to decrease the number of eosinophils via ADCC. Basophils are also depleted. 303 Atopic Dermatitis Dupilumab (Dupixent) is a human monoclonal gG4 antibody with specificity to interleukin-4 receptor subunit-alpha (IL-4Rα) that is approved to treat severe atopic dermatitis in adults. 304 Coagulopathy and Other Benign Hematologic Diseases Coagulopathies are usually either autoimmune or genetic. In factor VIII deficiency, recombinant factor VIII is used to replace lack of this protein. However, patients may develop antibodies to factor VIII leading to high titers of inhibitors. Furthermore, patients without deficiency may also develop autoantibodies to factor VIII de novo leading to coagulopathies. Other factor combinations as well as recombinant active factors have been created to overcome these inhibitory antibodies. Mabs with bispecific binding are also being researched as another avenue for treatment. ITP can lead to critical low platelet levels increasing risk for severe bleeding. ITP can occur in both adult and pediatric settings as it is considered an autoimmune disease. Typically, this is treated with steroids and IVIG. In addition, as mentioned earlier, RhD + patients have benefitted from polyclonal medications directed against the D antigen. Recently, Mab to treat this disease have been developed and will be discussed next. Thrombotic thrombocytopenic purpura (TTP) is a blood disorder that does not lead to bleeding but to development of diffuse thrombi in small blood vessels. More often, this disorder is secondary to an autoimmune inhibitory antibody to the disintegrin and metalloproteinase with thrombospondin type 1 motif member-13 (ADAMTS-13), known as acquired TTP. Inhibiting this zinc containing metalloprotease leads to lack of cleavage of large multimers of von Willebrand Factor (vWF). The large vWF multimers then more easily bind to platelets resulting in platelet clots in small blood vessels. More rarely, this disorder is secondary to an inherited deficiency of ADAMTS-13. This patient population with congenital deficiency is managed with transfusion of FFP to replace the deficient enzyme. Acquired TTP is typically treated with therapeutic plasma exchange (TPE). This treatment modality removes the inhibitory antibody and ultralarge vWF multimers. Similarly, TPE will replete the missing enzyme. Immunosuppressive agents may be added if only TPE is not effective. A Mab preventing interaction of vWF and platelets was recently approved for use in treating this disorder. 305 , 306 Caplacizumab-yhdp (Cablivi) is a humanized single-variable-domain immunoglobulin (Nanobody) that inhibits the interaction between ultralarge vWF multimers and platelets and is directed against vWF. It induces a faster response to therapy with TPE and decreases relapse with continued use during TPE. This medication is then used post-TPE treatment until immunological evidence of disease is controlled to prevent relapse. 305 , 307 , 308 This medication was FDA approved for use in TTP in 2019. Atypical hemolytic uremic syndrome (aHUS) is a disorder of the complement system due to uncontrolled activation. This disorder presents with thrombocytopenia, thrombi, and renal dysfunction. Historically, this illness was treated with TPE; however, end-stage renal failure occurred in 30% of patients and about 65% mortality in subsequent relapses with increasing incidence of renal failure. There are now two monoclonal antibodies approved for the treatment of aHUS. Refer to Table 16.1 . Sickle Pain Crisis In sickle cell disease, one of the frequent complications is pain crises. This is usually treated with analgesics, oxygen, hydration, and transfusions (simple or exchange). Monoclonal antibodies are being developed to treat pain crises in sickle cell patients in both adult and pediatric populations. Crizanlizumab is a humanized Mab (IgG2κ) with specificity to selectin P. One phase II trial was completed in 2016 and three additional phase II studies will be completed between 2021 and 27 to treat vasoocclusive pain crisis. This medication may be under FDA review as early as 2019. 309 , 310 Sickle Pain Crisis In sickle cell disease, one of the frequent complications is pain crises. This is usually treated with analgesics, oxygen, hydration, and transfusions (simple or exchange). Monoclonal antibodies are being developed to treat pain crises in sickle cell patients in both adult and pediatric populations. Crizanlizumab is a humanized Mab (IgG2κ) with specificity to selectin P. One phase II trial was completed in 2016 and three additional phase II studies will be completed between 2021 and 27 to treat vasoocclusive pain crisis. This medication may be under FDA review as early as 2019. 309 , 310 Infections Antimicrobials have historically been developed against a variety of viral, bacterial, fungal, and parasitic infections. These pharmaceuticals target differences from human cells of these particular organisms such as cell wall or membrane structure, genetic make-up, transcription/translation of genetic material, or metabolic pathways. Often organisms develop resistance to entire categories of these medications. Earlier in the chapter, passive polyclonal antibodies were discussed in the treatment of some of these infectious agents and we will now discuss research in monoclonal therapies to pathogenic microorganisms. Clostridium difficile Enterocolitis from Clostridium difficile is a community or hospital acquired infection increasing morbidity and mortality in those that acquire it. Treatment is supportive or with fecal transplants or antibiotics. Bezlotoxumab (Zinplava) is a human Mab (IgG1) with specificity to Clostridium difficile 's B toxin. It is used to treat pseudomembranous colitis and prevent C. difficile reinfection. 311 , 312 Actoxumab, a monoclonal antibody against C. difficile toxin A, has shown not to be clinically significant. Respiratory Syncytial Virus Respiratory syncytial virus (RSV) infects almost all children by 2 years old and poses extra risk in preterm infants. Supportive therapy, RSV-IG or IVIG, and antiviral therapy have been used to mitigate the sequela of this infection with optimal response yet to be seen. No vaccines have yet to be developed for this infection. Recently, monoclonal antibodies have been FDA approved or are undergoing pre/clinical trials to treat this infectious process and include palivizumab, Nirsevimab (MEDI8897), TRL3d3 (3D3), and ALX-0171. 313 , 314 Not beneficial or safe in use for RSV: motavizumab, Suptavumab (REGN2222, SAR438584). Influenza virus Influenza is a worldwide respiratory infectious problem with cyclic epidemics yearly. Supportive therapy, yearly vaccinations, and antivirals are used to decrease the morbidity and mortality caused by this sometimes virulent pathogen. Both polyclonal and monoclonal therapies are being evaluated to better treat these infections. Mabs in pre/clinical trials include diridavumab (CR6262), firivumab, gedivumab (RG7745, RO6876802), lesofavumab (RG70026), and Navivumab (CT-P27). Rabies Rabies is a devastating viral infection with swift mortality if not treated quickly after initial exposure. Vaccines usually react too slowly and have to be combined with polyclonal IVIG infusions. Monoclonal therapy was previously studied but usually the virus mutates quickly and the infection is not controlled. More recently, in clinical trials, cocktails of Mabs are being tried to more closely mimic the benefits of polyclonal therapies. These Mabs include foravirumab, rafivirumab (CR57), and Rmab. Hepatitis B virus HBV is one of if not the most common infections in the world. Even though antivirals are available and effective, only recently they have they been widely used in the infant population and not just "high"-risk individuals. Mabs to treat this infection that are being investigated include libivirumab. Mab that is not found to be effective is tuvirumab. Ebola Ebola is a relatively rare but devastating hemorrhagic infection. Most care is supportive with various studies being performed to prevent/mitigate this disease. Vaccines are under development as well as passive polyclonal therapies. Mab therapies being developed or studied include porgaviximab (C2G4), cosfroviximab, and larcaviximab. For these and other bacterial, fungal, and viral antiinfectious agents, information may be found in Table 16.1 . Clostridium difficile Enterocolitis from Clostridium difficile is a community or hospital acquired infection increasing morbidity and mortality in those that acquire it. Treatment is supportive or with fecal transplants or antibiotics. Bezlotoxumab (Zinplava) is a human Mab (IgG1) with specificity to Clostridium difficile 's B toxin. It is used to treat pseudomembranous colitis and prevent C. difficile reinfection. 311 , 312 Actoxumab, a monoclonal antibody against C. difficile toxin A, has shown not to be clinically significant. Respiratory Syncytial Virus Respiratory syncytial virus (RSV) infects almost all children by 2 years old and poses extra risk in preterm infants. Supportive therapy, RSV-IG or IVIG, and antiviral therapy have been used to mitigate the sequela of this infection with optimal response yet to be seen. No vaccines have yet to be developed for this infection. Recently, monoclonal antibodies have been FDA approved or are undergoing pre/clinical trials to treat this infectious process and include palivizumab, Nirsevimab (MEDI8897), TRL3d3 (3D3), and ALX-0171. 313 , 314 Not beneficial or safe in use for RSV: motavizumab, Suptavumab (REGN2222, SAR438584). Influenza virus Influenza is a worldwide respiratory infectious problem with cyclic epidemics yearly. Supportive therapy, yearly vaccinations, and antivirals are used to decrease the morbidity and mortality caused by this sometimes virulent pathogen. Both polyclonal and monoclonal therapies are being evaluated to better treat these infections. Mabs in pre/clinical trials include diridavumab (CR6262), firivumab, gedivumab (RG7745, RO6876802), lesofavumab (RG70026), and Navivumab (CT-P27). Rabies Rabies is a devastating viral infection with swift mortality if not treated quickly after initial exposure. Vaccines usually react too slowly and have to be combined with polyclonal IVIG infusions. Monoclonal therapy was previously studied but usually the virus mutates quickly and the infection is not controlled. More recently, in clinical trials, cocktails of Mabs are being tried to more closely mimic the benefits of polyclonal therapies. These Mabs include foravirumab, rafivirumab (CR57), and Rmab. Hepatitis B virus HBV is one of if not the most common infections in the world. Even though antivirals are available and effective, only recently they have they been widely used in the infant population and not just "high"-risk individuals. Mabs to treat this infection that are being investigated include libivirumab. Mab that is not found to be effective is tuvirumab. Ebola Ebola is a relatively rare but devastating hemorrhagic infection. Most care is supportive with various studies being performed to prevent/mitigate this disease. Vaccines are under development as well as passive polyclonal therapies. Mab therapies being developed or studied include porgaviximab (C2G4), cosfroviximab, and larcaviximab. For these and other bacterial, fungal, and viral antiinfectious agents, information may be found in Table 16.1 . Influenza virus Influenza is a worldwide respiratory infectious problem with cyclic epidemics yearly. Supportive therapy, yearly vaccinations, and antivirals are used to decrease the morbidity and mortality caused by this sometimes virulent pathogen. Both polyclonal and monoclonal therapies are being evaluated to better treat these infections. Mabs in pre/clinical trials include diridavumab (CR6262), firivumab, gedivumab (RG7745, RO6876802), lesofavumab (RG70026), and Navivumab (CT-P27). Rabies Rabies is a devastating viral infection with swift mortality if not treated quickly after initial exposure. Vaccines usually react too slowly and have to be combined with polyclonal IVIG infusions. Monoclonal therapy was previously studied but usually the virus mutates quickly and the infection is not controlled. More recently, in clinical trials, cocktails of Mabs are being tried to more closely mimic the benefits of polyclonal therapies. These Mabs include foravirumab, rafivirumab (CR57), and Rmab. Hepatitis B virus HBV is one of if not the most common infections in the world. Even though antivirals are available and effective, only recently they have they been widely used in the infant population and not just "high"-risk individuals. Mabs to treat this infection that are being investigated include libivirumab. Mab that is not found to be effective is tuvirumab. Ebola Ebola is a relatively rare but devastating hemorrhagic infection. Most care is supportive with various studies being performed to prevent/mitigate this disease. Vaccines are under development as well as passive polyclonal therapies. Mab therapies being developed or studied include porgaviximab (C2G4), cosfroviximab, and larcaviximab. For these and other bacterial, fungal, and viral antiinfectious agents, information may be found in Table 16.1 . Immunomodulation In solid organ transplants, cellular or humoral immunity can develop against the transplant leading to acute or chronic rejection. An additional complication with these and stem cell transplants is severe GVHD. In the past, these transplant complications were treated with high-dose glucocorticosteroids, immunosuppressive medication, chemotherapeutic agents, IVIG, or T-cell lymphocytic specific immunoglobulins. Recently, Mabs have been added to this armamentarium to better control these adverse reactions to transplantations. Basiliximab (Simulect) is a chimeric Mab (IgG1κ) with specificity to CD25 IL-2α. The only FDA-approved indication for this medication is prophylaxis of acute rejection in renal transplant patients. There are multiple ongoing studies of this biological for other organ transplants including liver, lung, and heart as well as for inflammatory/immunologic diseases such as GVHD following stem cell transplantation, ulcerative colitis, and uveitis. 315 , 316 , 317 , 318 , 319 Belatacept (Nulojix) is a soluble fusion protein consisting of the modified extracellular domain of CTLA-4 fused to the Fc domain of a recombinant human Mab IgG1. This Mab selectively inhibits T-cell activation through costimulation blockade binding to both CD80 and CD86 while blocking CD28 via tighter binding than its parent antibody abatacept. Refer to Table 16.1 . Metabolic Syndromes Hypercholesterolemia is associated with increased risk for cardiovascular disease/atherosclerosis secondary to inherited or dietary etiologies. Diet and exercise are used to treat mild forms of these disorders. Medications such as nicotinic acid, fibrates, bile acid binding resins, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors are used for more severe forms of these disorders. Phase III studies have been completed with monoclonal antibodies for patients' refractory to the previously mentioned forms of therapy. Hypophosphatemia Burosumab (KRN23, Crysvita) is a human Mab IgG1κ with specificity to phosphaturic hormone fibroblast growth factor 23 (FGF 23). This hormone is a regulator of phosphate and vitamin D homeostasis. FGF23 inhibits the enzyme CYP27B1 and stimulates CYP24A1, thereby reducing circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D. This medication is FDA approved for the treatment of X-linked hypophosphatemic rickets. 320 , 321 Osteoporosis Denosumab (Prolia) is an FDA-approved human Mab (IgG2) that is a receptor activator of nuclear factor κB ligand that inhibits development and activity of osteoclasts. As Prolia, this medication is used to prevent or treat osteoporosis in women. 322 , 323 , 324 This medication under the trade name Xgeva is also used to prevent skeletal-related events in adults with bone metastasis from breast, prostate cancers, and multiple myeloma. 325 , 326 Hypophosphatemia Burosumab (KRN23, Crysvita) is a human Mab IgG1κ with specificity to phosphaturic hormone fibroblast growth factor 23 (FGF 23). This hormone is a regulator of phosphate and vitamin D homeostasis. FGF23 inhibits the enzyme CYP27B1 and stimulates CYP24A1, thereby reducing circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D. This medication is FDA approved for the treatment of X-linked hypophosphatemic rickets. 320 , 321 Osteoporosis Denosumab (Prolia) is an FDA-approved human Mab (IgG2) that is a receptor activator of nuclear factor κB ligand that inhibits development and activity of osteoclasts. As Prolia, this medication is used to prevent or treat osteoporosis in women. 322 , 323 , 324 This medication under the trade name Xgeva is also used to prevent skeletal-related events in adults with bone metastasis from breast, prostate cancers, and multiple myeloma. 325 , 326 Endocrine Disorders Diabetes may be classified as primary or secondary. In this chapter, we will be mainly interested in both insulin-dependent (Type I) and insulin-independent types (Type II). Type I diabetes mellitus is generally secondary to loss of β cells in the islets of Langerhans and subsequent loss of insulin production. Type II typically is secondary to decreased sensitivity to the effects of insulin. In type I, insulin is replaced exogenously depending on glucose levels. In type II, medications are given to stimulate islet cells to produce more insulin. Mabs are being developed to potentially mitigate the autoimmune process leading to Type I diabetes mellitus or the sequela of renal failure often seen with this disease. For type II, Mabs are being investigated to potentially decrease body mass index and thus decrease disease severity. Refer to Table 16.1 . Other Clinical Disorders Age-related macular degeneration (AMD) is the leading irreversible cause of visual loss affecting the elderly. Two forms include a dry form with deposits in the macula or a wet form involving abnormal growth of blood vessels. The wet form, even though less frequent, is associated with more severe visual acuity loss. Antiangiogenesic drugs or laser treatments are used to slow the progression or even partially reverse visual loss. Some trials have been completed while others are ongoing using Mab to treat the wet form of AMD. Brolucizumab was found as good as if not better than aflibercept in a phase III clinical trial. 327 Cryopyrin-associated periodic syndromes (including familial cold auto-inflammatory syndrome and Muckle-Wells syndrome); tumor necrosis factor receptor-associated periodic syndrome (TRAPS); hyperimmunoglobulin D Syndrome (HIDS)/mevalonate kinase deficiency and familial Mediterranean fever (FMF) may also respond to canakinumab. 328 Potential Future Uses of Monoclonal Antibodies and Their Targets Passive antibody therapy continues to be useful clinically whether polyclonal or monoclonal therapy is implemented. Increased utilization of the classic polyclonal antibody preparations continue especially in the realm of infections. In the past 3 years, monoclonal therapy has evolved and revolutionized treatment in many areas. As targets are identified to modify disease pathology no matter its genre we continue to get a better handle on morbidity and mortality. We are learning that not only is the target important put the portion of the target mediating the effect we intend to modify is also important. Importantly, modification of antibodies to be more compatible with the immune system while decreasing rapidity of clearance also allows for more consistent therapy. There are also many targets yet to be discovered or only now being developed as in the canonical wingless/integrated (WNT) signaling. This receptor family is important in a multitude of diseases not limited to: hereditary colorectal cancer, various types of sporadic cancers, intellectual disability syndrome, Alzheimer's disease, bipolar disorder, bone diseases, and vascular diseases. One monoclonal antibody rosmantuzumab (OMP-131R10), a humanized Mab (IgG1κ), is in phase I trials to treat colorectal cancer. 329 , 330 Other disease processes have yet to find their optimal therapy (Alzheimer's) or are advancing to fuller therapeutic benefit. The future is wide open for this newer class of pharmaceuticals as they continue to develop to full fruition. Table 16.2 Summary of Polyclonal Antibody Therapies. Generic Drug Name Brand Name Additional Brand Names AHFS Classification Dosage Form(s) Restricted Medication Antithymocyte globulin (equine) Atgam Immunosuppressive agent Intravenous solution Antithymocyte globulin (rabbit) Thymoglobulin Immunosuppressive agent Intravenous solution Antivenin Latrodectus mactans Black widow Antivenin Serums Intravenous solution Antivenin micrurus Eastern and Texas coral Snake Antivenin Serums Intravenous solution Botulism immune globulin BabyBIG Crotalidae polyvalent immune Fab Crofab Serums Intravenous solution Cytomegalovirus immune globulin Cytogam Serums Intravenous solution Yes Digoxin immune Fab Digibind Serums Intravenous solution Hepatitis B immune globulin Hepagam-B Serums Intramuscular solution, Intravenous solution Hepatitis B immune globulin BayHepB HepaGam B, Hyper Hep B, Nabi-HB High antibody titer Ebola FFP High antibody titer influenza FFP Immunoglobulin (generic) Gamunex Vivaglobin, Cuvitru, Privigen, gammagard, octagam, gamunex, hizentra, Bivigam, Carimune, Flebogamma, Gamastan, Gamimune, Gammaplex, gammar, Panglobulin, Panzyga, Sandoglobulin Intravenous, Subcutaneous Treat XLA, CVID, Hyper IgM syndromes, Wiskott Aldrich syndrome Rabies immune globulin Bayrab HyperRAB, Imogam rabies, KedRAB Respiratory syncytial virus immune globulin RespiGam Rho (D) immune globulin WhinRho RhoGam Rhophylac, MicRhoGAM, BatRhoD, HyperRho Serums Intravenous, intramuscular solutions Rimabotulinumtoxin B Myobloc Other Miscellaneous Therapeutic agents Injection solution Yes Rozrolimupab Anti-RhD Prevent isoimmunization ITP Tetanus immune globulin Baytet Hypertet Varicella zoster immune globulin VariZIG Searched sites for table information. Monoclonal. https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm279174.htm . https://fdasis.nlm.nih.gov/srs/ . https://clinicaltrials.gov/ct2/ . https://www.ncbi.nlm.nih.gov/pubmed/ . https://chem.nlm.nih.gov/chemidplus/rn . https://druginfo.nlm.nih.gov/drugportal/ . https://www.creativebiolabs.net/ .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800162/

Food Safety Knowledge, Attitudes and Behavior among Dairy Plant Workers in Beijing, Northern China

The safety of milk and dairy products has always been one of the focuses of consumers, the food industry and regulatory agencies. The purpose of this study was to gain insight into the food safety knowledge, attitudes and behavior of dairy plant workers. A cross-sectional survey was performed between May and August 2015 in three dairy plants in Beijing, northern China. A total of 194 dairy plant workers were interviewed to collect information on food safety knowledge, attitudes and self-reported behavior. The 194 dairy plant workers interviewed showed a sufficient level of knowledge (mean score 34 on a scale from 0–58), perfect attitudes (mean score 17 on a scale from 0–18), and perfect behavior (mean score 38 on a scale from 8–40). Only 39% of workers correctly determined specific pathogens or diseases that could be conveyed through milk and dairy products. 24% of workers knew the correct method of washing hands. A significant positive association was observed between attitudes and knowledge ( p 0.05). The mean specific pathogen/disease knowledge score was 11 (range 0–20 points; SD 4.8) with 39% above threshold level of 12 points. About 60% of the respondents knew that foodborne diseases may lead to miscarriage in pregnant women. Most of the respondents (60–90%) recognized that preschoolers, pregnant women and older people, and HIV-positive persons and cancer patients are at high risk of becoming ill due to the pathogenic microorganisms in food. A low proportion of the respondents knew that people who frequently eat outside (7%) and teenagers (28%) are not the population at high risk. In total, 52% and 62% of the respondents correctly answered that anthrax and brucellosis could be conveyed through milk and dairy products, respectively. However, the corresponding figure was only 4% for bovine viral diarrhea and 6% for Johne's disease, both of these diseases could not be conveyed through milk and dairy products. The percentages of correct responses of E. coli O157:H7, Salmonella spp., Staphylococcus aureas , L. monocytogens , Campylobacter spp. and noroviruses were 88%, 74%, 73%, 56%, 53% and 48%, respectively. However, correct responses to Clostridium botulinum (8%) and Toxoplasma gondii (7%) were generally low. Nearly 80% of respondents knew the optimal method to clean fresh vegetables and fruits (with cool running water). However, only 24% knew the best way to wash hands, which includes wetting, applying soap, rubbing for 20 s, rinsing and drying hands. Approximately 60% of the respondents could not specify the recommended freezer temperature (−18 °C). However, 68% of the respondents knew the maximum refrigeration temperature (4 °C) to maintain the safety of foods. 3.3. Attitudes The mean attitude score was 17 (SD = 1.8) with 99% above 9 ( Table 3 ). There was no statistical difference in mean attitude scores between age and gender groups ( p > 0.05). More than 96% of the respondents agreed that use of work uniform, masks and caps, raw foods stored separately from cooked food, food safety training for workers and monitoring of refrigerator temperatures have a great influence on decreasing risk of food contamination. 8% of the respondents disagreed with the statement that workers with wounds on their hands should not handle food with their bare hands. 3.4. Behavior All respondents had a total self-reported behavior score above 20. The mean behavior score was 38 (SD = 3.2), ranging from 24 to 40 points ( Table 4 ). There was no statistical difference in mean behavior scores between age and gender groups ( p > 0.05). More than 85% of the respondents always wore work uniform and cap, never wore watch and decoration, always washed hands after handling waste/garbage or after using the toilet, never smoke or ate/drank during their work except for breaks. However, among the 193 dairy plant workers, only 64% (123/193) of them always wore masks, whereas 7% (14/193) seldom or never wore masks while working. 3.5. Correlation Results Significant weak associations were observed between attitudes with knowledge (rho = 0.261, p 0.05). The mean specific pathogen/disease knowledge score was 11 (range 0–20 points; SD 4.8) with 39% above threshold level of 12 points. About 60% of the respondents knew that foodborne diseases may lead to miscarriage in pregnant women. Most of the respondents (60–90%) recognized that preschoolers, pregnant women and older people, and HIV-positive persons and cancer patients are at high risk of becoming ill due to the pathogenic microorganisms in food. A low proportion of the respondents knew that people who frequently eat outside (7%) and teenagers (28%) are not the population at high risk. In total, 52% and 62% of the respondents correctly answered that anthrax and brucellosis could be conveyed through milk and dairy products, respectively. However, the corresponding figure was only 4% for bovine viral diarrhea and 6% for Johne's disease, both of these diseases could not be conveyed through milk and dairy products. The percentages of correct responses of E. coli O157:H7, Salmonella spp., Staphylococcus aureas , L. monocytogens , Campylobacter spp. and noroviruses were 88%, 74%, 73%, 56%, 53% and 48%, respectively. However, correct responses to Clostridium botulinum (8%) and Toxoplasma gondii (7%) were generally low. Nearly 80% of respondents knew the optimal method to clean fresh vegetables and fruits (with cool running water). However, only 24% knew the best way to wash hands, which includes wetting, applying soap, rubbing for 20 s, rinsing and drying hands. Approximately 60% of the respondents could not specify the recommended freezer temperature (−18 °C). However, 68% of the respondents knew the maximum refrigeration temperature (4 °C) to maintain the safety of foods. 3.3. Attitudes The mean attitude score was 17 (SD = 1.8) with 99% above 9 ( Table 3 ). There was no statistical difference in mean attitude scores between age and gender groups ( p > 0.05). More than 96% of the respondents agreed that use of work uniform, masks and caps, raw foods stored separately from cooked food, food safety training for workers and monitoring of refrigerator temperatures have a great influence on decreasing risk of food contamination. 8% of the respondents disagreed with the statement that workers with wounds on their hands should not handle food with their bare hands. 3.4. Behavior All respondents had a total self-reported behavior score above 20. The mean behavior score was 38 (SD = 3.2), ranging from 24 to 40 points ( Table 4 ). There was no statistical difference in mean behavior scores between age and gender groups ( p > 0.05). More than 85% of the respondents always wore work uniform and cap, never wore watch and decoration, always washed hands after handling waste/garbage or after using the toilet, never smoke or ate/drank during their work except for breaks. However, among the 193 dairy plant workers, only 64% (123/193) of them always wore masks, whereas 7% (14/193) seldom or never wore masks while working. 3.5. Correlation Results Significant weak associations were observed between attitudes with knowledge (rho = 0.261, p < 0.001) and behavior (rho = 0.200, p < 0.01). There was no significant association between knowledge and behavior. Education level was significantly positively related with knowledge (rho = 0.221, p < 0.05), attitudes (rho = 0.159, p < 0.05), and behavior (rho = 0.173, p < 0.05), though the associations were weak. There was no significant association between duration of employment with knowledge, attitudes, and behavior. 4. Discussion This is the first study to assess knowledge, attitudes and behavior toward food safety among dairy plant workers in China. Our findings demonstrated that workers employed in the three dairy enterprises in northern China have relatively lower-level of knowledge, yet satisfactory attitudes and behavior. The majority (78%) of the dairy plant workers answered correctly to more than 50% of the questions in the knowledge section. However, their knowledge of the specific diseases or pathogens that could be conveyed through milk and dairy products, was relatively low. Previous studies had showed that dairy farm workers did not have enough knowledge of foodborne diseases or pathogens [ 10 ]. About 50% of the respondents in this study were unaware that norovirus could spread from milk and dairy product to humans, while C. botulinum and T. gondii could not. More than 40% of workers were unsure whether anthrax, Campylobacter or L. monocytogens could convey through milk and dairy products to humans. This is surprising as the majority of dairy plant workers participated in food safety training courses. There is a need to develop more tailored training programs for dairy plant workers. Future research should assess the effectiveness of mandatory continued education activities in food safety for dairy plant workers. E. coli O157:H7 is one of the most virulent pathogens responsible for foodborne disease globally. Cattle are the main host for E. coli O157:H7 and outbreaks are frequently attributed to the ingestion of contaminated milk and dairy products of cattle origin [ 14 ]. In 1999, a large-scale E. coli O157:H7 outbreak in China had resulted in 195 hospital admissions due to hemolytic uremic syndrome and 177 deaths [ 15 ]. In the present study, the proportion of respondents who identified E. coli O157:H7 as a milk-borne pathogen was greater than those who identified other pathogens as milk-borne. This difference could be due to the fact that the issue of E. coli O157:H7 has been highly publicized through media in China. About 40% of the respondents in this study were unaware that foodborne diseases might cause miscarriage in pregnant women. Meanwhile, 44% of the respondents were unaware that L. monocytogenes could spread from milk and dairy products to humans. Although affected pregnant women usually have mild symptoms, L. monocytogenes can lead to severe outcomes for fetus or newborn infant; infection in utero can cause preterm labor or spontaneous miscarriage and stillbirth [ 16 ]. Southwestern Ontario artisan cheese makers perceived L. monocytogenes to be the most harmful biological hazard to the health of consumers and to their businesses [ 8 ]. The proportions of correct answers to optimal refrigeration temperature for food storage (68%) and freezing of food (38%) varied greatly in our study. The figures were lower than found in other studies in Lebanon (78% and 55%) [ 17 ] and in Romania (76%, respectively) [ 18 ]. It is worth noting that there may not be a single "maximum refrigeration temperature in order to maintain the safety of food" ( Table 2 , Question 28). As L. monocytogenes can grow in permissive foods even when these foods are stored at 4 °C. Correspondingly, the "monitoring of refrigeration temperatures" may have no effect on "decreasing the risk of food contamination" ( Table 3 , Question 7). In several studies on foodborne outbreaks, the hands of workers have been determined as the origin of pathogens in the involved food vehicles. Hand washing is the easiest and most efficient manner to remove pathogens on the hands [ 19 ]. In line with findings from previous studies in Lebanon and Romania [ 17 , 18 ], almost all respondents (nearly 90%) in our study always wash hands after handling waste/garbage, or after toilet visits. However, only 24% of respondents in the present study knew the most hygienic hand-washing steps, lower than previous studies [ 18 , 20 ]. The study highlights the correlation between the level of education and the level of food safety knowledge, attitudes and behavior. Previous studies showed that workers with higher educational levels have a significantly higher food safety knowledge [ 13 ]. Compared with the perfect attitudes and behavior, the relatively lower-level knowledge presented in the study suggests the inefficiency of the food safety training programs, indicating the Beijing dairy enterprises need to further improve training on employee food safety knowledge. An efficient training program on food safety knowledge should at least include materials on major foodborne pathogens, high-risk population, safe storing temperature, and appropriate cleaning and hygiene procedures. Without well-trained workers who are aware of the importance of safety procedures in the food processing, it is difficult to implement and maintain a functional food safety system. The limitations of this study are similar to other research on knowledge, attitudes and behaviors. The inherent limitation of such research is that the response may be influenced by social expectations. The sample size regarding the number of participants is sufficient, but they were recruited from only three plants. As the study is not anonymous, there may be an influence between the involvements of company management upon the collection of the questionnaires. Another serious limitation is that the sampling of participants was "convenience sampling". While the survey may not be representative of all dairy plant workers, it does indicate problems of professional knowledge and behaviors in a high percentage of those surveyed and the work does suggest problems that need to be addressed. The focus of this study is on dairy plant workers in Beijing, China, so the findings should not be extended to all dairy plant workers in the country. Besides, dairy farming is also an important part of the dairy industry, therefore, similar assessment of food safety knowledge, attitudes and behavior is required for dairy farm workers in the future. 5. Conclusions We conclude that workers employed in dairy enterprises in northern China have a relatively lower level of knowledge, yet satisfactory attitudes and behavior. The knowledge of microbial food hazards and hand hygiene remains an issue that needs to be emphasized in food safety training programs in the Chinese dairy enterprises. Education level is a determinant of attitudes and behavior with regard to the proper handling of milk and dairy products.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836467/

Staphylococcal Enterotoxin B–Specific Monoclonal Antibody 20B1 Successfully Treats Diverse Staphylococcus aureus Infections

Background. Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. Methods. Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. Results. Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. Conclusions. mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562545/

Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel

Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest–host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest–host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest–host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with L-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,L-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10877574/

Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra- ortho -fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis -azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein. Introduction Toxins are poisonous substances produced by living organisms, which are present in all kingdoms of life. 1 − 6 A number of known protein-based toxins belong to the pore-forming toxins (PFTs) family. 7 , 8 PFTs are usually produced as water-soluble monomers, with the ability to bind to target membranes and assemble into an oligomeric pore complex, thereby disrupting or perforating the lipid bilayer. 7 , 8 Typically, organisms employ PFTs to gain entry into host cells to acquire nutrition or use PFTs as a defense mechanism. 9 − 11 Of note, the human immune system involves pore-forming proteins in its defense machinery. 12 , 13 Consequently, PFTs are known to play a crucial role in the virulence mechanism of various pathogens. 14 − 17 While some of these inherently toxic proteins have targeting capabilities, such as the latrotoxin of the deadly black widow spider that selectively punctures neurons, 18 , 19 others, like actinoporins, lyse indiscriminately all sphingomyelin-containing membranes. 20 − 22 Since all mammalian cell surfaces contain sphingomyelin, the cytolytic activity of actinoporin PFTs can potentially result in the destruction of human cells. However, to precisely and safely utilize PFTs as novel chemotherapeutics, it is necessary to establish external control over their pore-formation ability. Under physiological conditions, the pore formation of PFTs is triggered by the lipid composition of the target cell membrane, 23 − 25 the presence of specific cell surface receptors, 26 , 27 proteolysis, 28 or pH changes in the intracellular microenvironment. 29 Several PFTs with nonspecific cytolytic activity have been modified with antibody fragments for targeting toward specific cell lines, 30 − 32 with some requiring additional protease activation to initiate the pore-forming process. 30 , 33 , 34 However, none of the aforementioned strategies allow for external control without substantial modification of the PFT by incorporating additional protein domains. 35 Taken together, to allow for future therapeutic application, it is key to establish external, spatiotemporal control over the nonspecific cytolytic activity of PFTs. In this context, light is an excellent stimulus that can be easily externally delivered at specific time points and locations. 36 − 39 Organic chemistry provides artificial actuators that can reversibly respond to different wavelengths of light: molecular photoswitches. 39 − 42 Photoswitches are small organic molecules that change their shape and properties in a reversible manner upon irradiation with light. 40 They have been applied, inter alia , in photopharmacological approaches to control the action of small molecules, DNA, peptides, and, most importantly in this context, proteins. 36 , 38 , 41 − 47 The structure and activity of numerous proteins have been manipulated by light with the help of covalently attached photoswitches via single attachment or two-point attached cross-linkers. 42 , 44 , 48 To the best of our knowledge, there are only two reported examples of using light for external control over the activity of PFTs. In 1995, the Bayley group reported a photocaged β-hemolysin PFT whose cytolytic activity could be irreversibly triggered by UV-light irradiation. 49 More recently, our group has published the first cell-lysing fragaceatoxin C (FraC) construct which reversibly performed controlled nanopore assembly with application of UV and white light. 50 FraC labeled with a water-soluble azobenzene at strategic locations lysed both blood and cancer cells upon light activation ( cis isomer), while the inactivated trans isomer did not show cytotoxicity at the same concentrations. However, UV-light irradiation was required for both of the described systems, which effectively prohibited their use in a biological system. UV light is not biocompatible as it can damage tissue and mutate DNA and has a low penetration depth. 51 − 56 Therefore, it is necessary to develop a PFT-based system that can be fully operated with visible light, thus allowing external spatiotemporal manipulation, to bring this concept closer to a photocontrolled PFT-based therapeutic approach. The major challenge of shifting the absorbance of photoswitches, in particular the most often applied azobenzenes, to the visible light region, has attracted much attention in the recent years. 57 − 63 Visible-light-responsive and water-soluble systems often feature many drawbacks, such as difficult synthesis, insolubility in water, susceptibility to glutathione reduction, and short half-lives. 58 , 65 − 67 Therefore, there are only a few examples of fully visible-light-controlled manipulation of proteins. In the majority of cases, freely diffused photoswitches, namely tetra- ortho -fluoro substituted 68 − 75 and tetra- ortho -chloro substituted azobenzene, 61 , 76 diazocines, 77 or other photoswitches, 78 were used to modulate the activity of proteins. However, there are only a few examples in which the photoswitch is covalently attached to a protein. As such, tetra- ortho -fluorinated azobenzene-based amino acids were encoded into mammalian cells via genetic code expansion enabling incorporation of the switch in vivo . 79 , 80 Furthermore, both fluorinated and chlorinated azobenzenes were tethered to ionotropic receptors to modulate their activity. 81 , 82 Visible light-responsive photoswitches with two attachment points, so-called cross-linkers, have been introduced as a staple to control the secondary structure of bioactive short peptides. 58 , 75 , 83 − 85 However, to the best of our knowledge, there is only one example in which the visible-light responsive switch (based on the tetra- ortho -methoxy azobenzene core) was utilized to control a protein function through cross-linking the binding site of a guanine-N7 methyltransferase to achieve visible-light mediated methylation of the 5′ cap of mRNA. 86 Nevertheless, none of the described examples exhibited pharmacological (cytolytic or cytotoxic) activity, specifically, when targeting cancer cells. Therefore, in this study, we report for the first time the fully visible-light-mediated control of protein toxicity with a water-soluble molecular photoswitch. Results and Disscussion Molecular Design The ability to be operated with light that features low toxicity and deep tissue penetration is crucial for advancing the light-responsive PFT design toward developing a potential therapeutic. Therefore, we aimed to design a photoswitchable pendant for covalent attachment to the PFT that can be fully controlled with visible light ( Figure 1 c). Due to its synthetic accessibility and generally favorable photophysical properties, 41 , 48 , 55 the azobenzene photoswitch was chosen. It has been shown in literature that introduction of four substituents in the ortho positions of the azobenzene core results in n−π* band separation for the two isomers allowing manipulation by visible light. 56 − 58 , 62 , 87 Specifically, we selected the tetra-fluorinated core for three reasons: first, due to its optimal photophysical properties, such as generally long half-lives of the metastable cis state and high photostationary state distribution (PSD) ratios; 87 − 90 second, known stability toward glutathione reduction; and third, because of the electron-poor nature of the ortho -fluoro azobenzene, which allows for later stage functionalization via nucleophilic aromatic substitution (S N Ar). 91 Figure 1 (a) Schematic representation of a FraC monomer (PDB 4TSY ( 64 )) labeled with an azobenzene photoswitch; the trans isomer inhibits binding of the monomer to the lipid bilayer, while upon irradiation with green light the formed cis isomer allows binding to the membrane and further assembly into the oligomeric pore complex in the target membrane (yellow), (b) Schematic representation of a FraC monomer, inserted into the target membrane, where the residues that were herein mutated into cysteine are highlighted (gray spheres), as well as sphingomyelin molecules stabilizing the pore (orange). (c) Design elements of a visible-light-responsive photoswitch A with a water-solubilizing group and an attachment handle for covalent protein modification. Next to visible light, the issue of water solubility of inherently aromatic photoswitches poses a great challenge for their applications in biological systems. 42 The main limitations for the introduction of water-solubilizing groups into the photoswitch structure include challenging synthesis and purification and the often detrimental effects of the solubilizing group and the aqueous medium on the photophysical properties of the switch. Therefore, the possibility to modify the electron-poor fluorinated azobenzene via S N Ar to introduce a water-solubilizing group at a later stage in the synthetic route, instead of starting with water-soluble compounds, would enable working with standard organic solvents during most of the synthetic route and minimizes repeated tedious purification under aqueous conditions. Furthermore, utilizing S N Ar to functionalize the azobenzene core provides an orthogonal and selective method that accommodates other functionalization approaches, such as cross-coupling reactions. The PFT chosen for photoswitch modification was FraC, a relatively small protein (21 kDa) with a known crystal structure that is relatively easy to purify. 64 , 92 − 94 FraC monomers assemble into octameric pores upon encountering cell membranes containing sphingomyelin, a lipid which is present in all mammalian cell surfaces. 64 , 95 Since wild-type FraC does not contain a cysteine residue, the visible-light-responsive photoswitch furnished with a reactive chloroacetyl group was attached to a free cysteine genetically introduced by site-specific mutation, providing an extra level of precision in the design. The mutation sites, namely, W112 and Y138, were selected for their location at the interface of the formed oligomeric pore and the lipid bilayer of the target cell ( Figure 1 a). 64 Furthermore, the selected positions are located in the sphingomyelin binding pockets. Sphingomyelin naturally stabilizes the assembled wild-type FraC pore, 64 therefore we anticipated that modification of FraC at the selected locations would substitute sphingomyelin with the photoswitch and thus have a larger effect on the nanopore assembly process. After purification of the labeled FraC mutants, the hemolytic activity was evaluated using defibrinated red blood cells and human hypopharyngeal squamous cell carcinoma. Synthesis For the synthesis of the water-soluble visible-light-responsive switch A , we utilized the unique susceptibility of the tetra- ortho -fluoro-azobenzene core to S N Ar for installing the water solubilizing group ( Scheme 1 ). Aniline 1 was oxidized to the nitroso derivative 3 and subsequently submitted to the Baeyer–Mills reaction with aniline 2 in a mixture of solvents (toluene, acetic acid, and TFA) to yield azobenzene 4 in good yield (83%) ( Scheme 1 ). The sulfonation step was performed with azobenzene 4 and a weak nucleophile, sodium sulfite, in a 1/1 mixture of water and EtOH to ensure solubilization of both substrates. The water-soluble azobenzene 5 was purified by reverse phase chromatography using a mildly basic ammonium bicarbonate buffer and ACN mixture as eluents. After isolation of the pure sulfonated product 5 , Buchwald–Hartwig coupling with acetamide was performed at 90 °C in degassed DMF. Pure azobenzene 7 was isolated after reversed phase purification in a high yield (73%). The acetamide was hydrolyzed in the presence of concentrated hydrochloric acid and the isolated amine 8 underwent reaction with chloroacetic anhydride (neat) to obtain final photoswitch A . Azobenzene A was purified from the excess anhydride by several centrifuge washes with ether and fully characterized by NMR spectroscopy, HRMS, and LCMS analysis (See Supporting Information sections 1.2 and 3.1). Scheme 1 Synthesis of Water-Soluble Visible Light-Responsive Switch A Photophysical Properties First, the photophysical properties of A were investigated in DMSO as a standard solvent for validating the photophysical properties of switches for biological applications. The UV–vis spectrum of azobenzene A clearly shows separation of the n−π* bands of both isomers thus allowing efficient switching exclusively with visible light ( Figure 2 b,c,h). The band separation is sufficient to ensure good isomer distribution at the photostationary state (PSS), as determined by NMR spectroscopy, revealing that high amounts of the respective isomer were formed upon irradiation with green (530 nm) and blue (430 nm) light (PSD 530nm = 81% cis , PSD 430nm = 79% trans ). The photophysical properties in DMSO, including fatigue resistance over multiple switching cycles, were accompanied by a half-life over 9 h at 20 °C. Figure 2 (a) Scheme depicting photoswitching of A (R = CH 2 Cl) and 7 (R = CH 3 ) with visible light. (b) UV–vis spectrum of A at PSS 530nm and PSS 430nm (80 μM, DMSO). (c) PSD determination of A with 1 H NMR spectroscopy (2 mM, DMSO- d 6 ) by relative integration of the shifted signals shown in bold. (d) UV–vis spectrum of 7 at PSS 530nm and PSS 430nm (50 μM, 15 mM Tris, 150 mM NaCl, pH = 7.5). (e) PSD determination of 7 in Tris buffer with 19 F NMR spectroscopy (0.88 mM, 15 mM Tris, 150 mM NaCl, pH = 7.5 with 10% DMSO- d 6 ) by relative integration of the shifted signals shown in bold. The signal between −117.8 and 118.6 in the 19 F-NMR is likely due to a fluorinated impurity which was impossible to be removed, despite several purification attempts. The signal does not react to irradiation. (f) Stability of 7 (50 μM) toward reduction by GSH (10 mM) in 15 mM Tris buffer, 150 mM NaCl, pH = 7.5, at 20 °C. Fatigue resistance in the presence of GSH with marked time points at which the UV–vis spectra are shown in panel g.(h) Half-life determination of A at 80 μM in DMSO. (i) Half-life determination of 7 at 50 μM in 15 mM Tris, 150 mM NaCl, pH = 7.5. All at 20 °C. Furthermore, we tested the photophysical properties in an aqueous environment as similar as possible to the buffer used to elute the FraC monomer. This is important, since many azobenzenes show a substantially shorter half-life of the metastable cis isomer in water. 42 , 96 While photoswitch A completely dissolved in Tris buffer at a concentration of 10 mM, it also contains a reactive chloroacetyl group highly susceptible to reaction with nucleophiles, such as Tris, which is the main component of the buffer used for the purification of FraC. Therefore, analogue 7 not bearing the chloroacetyl moiety ( Figure 2 d,e,i) was used as a model compound for studying the photophysical properties in Tris buffer. To our delight, azobenzene 7 also exhibited favorable photophysical properties in the aqueous medium with similar PSS ratios (PSD 530nm = 83% cis , PSD 430nm = 76% trans ), showing no fatigue upon numerous photoswitching cycles and, most importantly, a half-life of over 12 h at 20 °C that is sufficient for the PFT activity assays. Furthermore, azobenzene 7 was used to assess the stability of the switch in the presence of glutathione (GSH). Resistance to GSH reduction is often tested for photoswitches used in biological applications since the environment in living cells is naturally highly reducing. 97 − 99 Azobenzene 7 underwent several switching cycles in the presence of 10 mM GSH solution in Tris buffer indicating resistance to GSH reduction ( Figure 2 f,g). After investigating the photophysical properties of the water-soluble, visible-light-responsive molecular photoswitches in both DMSO and Tris buffer, it was concluded that molecule A can be reversibly and fully controlled with visible light while remaining stable in the presence of GSH and having a half-life sufficiently long for the cell lysis assays used to assess the reactivity of PFTs. Protein Labeling, PFT Activity Assays, and Cell Lysis Two mutants of FraC with cysteine located at positions 138 or 112 were chosen for attachment of visible-light-responsive molecule A ( Figure 1 b). In the wild type both cysteines are located at the interface of the formed pore and the lipid bilayer, in the locations where naturally the sphingomyelin binding pocket is positioned. 64 Sphingomyelin stabilizes the formation of the full pore complex and, in the wild-type pore, its presence in the target membrane is crucial for nanopore assembly. 64 Therefore, we anticipated that introduction of an aromatic photoswitch in the sphingomyelin pocket locations could have the largest effect on the pore-forming activity. Covalent attachment of switch A to the FraC monomers containing a freely accessible cysteine was performed in a degassed phosphate buffer solution in order to prevent oxidation of the free thiol. The presence of a mild reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), was avoided during the labeling reaction since the reactive chloroacetyl group of switch A can react with the reagents. After labeling, the excess azobenzene was removed via size exclusion chromatography, while the content of any remaining unmodified monomer is the same across samples, possibly causing some background activity. For details of protein expression, labeling, and purification, see SI 1.4 . The cytolytic activity of the labeled FraC constructs was evaluated by performing a hemolytic activity assay on defibrinated red blood cells. In the initial studies, it was observed that ambient light, even stray light from computer screens, significantly affected the hemolytic activity of the constructs. This observation was in accordance with the finding that the free switch A and its derivatives, being responsive to visible light, very efficiently formed a mixture of both isomers under any ambient light. Therefore, all the hemolytic activity assays, as well as handling of the samples after labeling with switch A , were performed strictly under dim red light. Before addition to the red blood cells, the freshly prepared labeled FraC mutants were preirradiated with either green (530 nm, "ON") or blue (430 nm, "OFF") light. Both FraC(Y138C)– A and FraC(W112C)– A exhibited hemolytic activity within a short time span upon addition at the tested concentrations and showed a significant difference in the activity of the ON and OFF samples ( Figure 3 a,b). The analysis of the dose–response curves confirmed that FraC(W112C)– A was generally more active (EC50 530nm (ON) = 0.7 μM, EC50 430nm (OFF) = 1.1 μM) compared to FraC(Y138C)– A , while FraC(Y138C)– A (EC50 530nm (ON) = 2.2 μM, EC50 430nm (OFF) = 5.2 μM) showed a larger difference in activity of the ON and OFF solutions ( Figure 3 a,b). For both labeled mutants, the OFF samples, containing predominantly the trans isomer of switch A , were still relatively active, which is likely a consequence of the presence of the cis isomer (the PSD 430nm of molecule 7 in Tris buffer is only 76% trans , Figure 3 e). Besides the possibility that the trans - A labeled FraC is also somewhat active, the OFF sample contains 24% cis - A labeled FraC, which might be causing the observed activity. Nevertheless, both mutants exhibited a significant difference in activity of the labeled FraC constructs upon irradiation with two different wavelengths of visible light. Furthermore, the steep dose–response curves (Hill coefficient n (FraC112C– trans - A ) = 5.3 and n (FraC112C– cis - A ) = 6.5, n (FraC138C– trans - A and − cis - A ) = 3.6), which are inherent to the cooperative nature of the FraC assembly mechanism involving a multimeric pore, are working in favor for this photopharmacological system as a small difference in concentration results in a large response difference. In addition, to confirm the reversibility upon irradiation, a sample of FraC(Y138C)– A was subjected to subsequent irradiation cycles with green and blue light, while an aliquot was taken after each irradiation step and its activity was tested on blood ( Figure 3 c). Consecutive activation of the construct upon λ = 530 nm light irradiation and deactivation upon application of λ = 430 nm light was observed. However, some loss of activity was observed for each of the performed cycles, suggesting slow degradation of the construct upon successive photoswitching cycles. Figure 3 Hemolytic activity of labeled FraC– A upon irradiation with 530 (in green) and 430 (in blue) nm LED where the EC50 values are expressed in μM: (a) EC50 curves for FraC(W112C)– A ; (b) EC50 curves for FraC(Y138C)– A . (c) Reversibility test of hemolytic activity of mutant FraC(Y138C) where the P values were determined by one-way ANOVA analysis between the highlighted conditions, A after 10 min upon subsequent irradiation cycles (approximately 2 μM). (d) Cytolytic activity of FraC(Y138C)– A on FaDu cancer cells where the P values were determined by one-way ANOVA analysis between the ON (green) or OFF (blue) and the control and between ON and OFF (highlighted with bracket) conditions. The asterisks represent P values as follows: **** P ≤ 0.0001; *** P ≤ 0.001; ** P ≤ 0.01, and * P ≤ 0.05. Experiments a, b and c were performed as four independent replicates, while d was performed in duplicate. Finally, we evaluated the cytolytic activity of the visible-light responsive FraC proteins toward human cancer cells. Since both the wild-type FraC and the Y138C mutant exhibited the highest cytolytic activity toward FaDu (hypopharyngeal squamous carcinoma) cells, this cell line was selected for evaluating the cytolytic activity of the visible light-responsive FraC(Y138C)– A . As in the hemolytic assays, the samples were preirradiated with the corresponding light (530 nm ON, and 430 nm OFF). At higher concentrations, the activated ON sample of FraC(Y138C)– A exhibited higher cytolytic activity compared to the OFF sample ( Figure 3 d). This result serves as a proof of principle illustrating that the modified FraC nanopore labeled with a water-soluble photoswitch can selectively destroy cancer cells upon activation with visible light. Molecular Design The ability to be operated with light that features low toxicity and deep tissue penetration is crucial for advancing the light-responsive PFT design toward developing a potential therapeutic. Therefore, we aimed to design a photoswitchable pendant for covalent attachment to the PFT that can be fully controlled with visible light ( Figure 1 c). Due to its synthetic accessibility and generally favorable photophysical properties, 41 , 48 , 55 the azobenzene photoswitch was chosen. It has been shown in literature that introduction of four substituents in the ortho positions of the azobenzene core results in n−π* band separation for the two isomers allowing manipulation by visible light. 56 − 58 , 62 , 87 Specifically, we selected the tetra-fluorinated core for three reasons: first, due to its optimal photophysical properties, such as generally long half-lives of the metastable cis state and high photostationary state distribution (PSD) ratios; 87 − 90 second, known stability toward glutathione reduction; and third, because of the electron-poor nature of the ortho -fluoro azobenzene, which allows for later stage functionalization via nucleophilic aromatic substitution (S N Ar). 91 Figure 1 (a) Schematic representation of a FraC monomer (PDB 4TSY ( 64 )) labeled with an azobenzene photoswitch; the trans isomer inhibits binding of the monomer to the lipid bilayer, while upon irradiation with green light the formed cis isomer allows binding to the membrane and further assembly into the oligomeric pore complex in the target membrane (yellow), (b) Schematic representation of a FraC monomer, inserted into the target membrane, where the residues that were herein mutated into cysteine are highlighted (gray spheres), as well as sphingomyelin molecules stabilizing the pore (orange). (c) Design elements of a visible-light-responsive photoswitch A with a water-solubilizing group and an attachment handle for covalent protein modification. Next to visible light, the issue of water solubility of inherently aromatic photoswitches poses a great challenge for their applications in biological systems. 42 The main limitations for the introduction of water-solubilizing groups into the photoswitch structure include challenging synthesis and purification and the often detrimental effects of the solubilizing group and the aqueous medium on the photophysical properties of the switch. Therefore, the possibility to modify the electron-poor fluorinated azobenzene via S N Ar to introduce a water-solubilizing group at a later stage in the synthetic route, instead of starting with water-soluble compounds, would enable working with standard organic solvents during most of the synthetic route and minimizes repeated tedious purification under aqueous conditions. Furthermore, utilizing S N Ar to functionalize the azobenzene core provides an orthogonal and selective method that accommodates other functionalization approaches, such as cross-coupling reactions. The PFT chosen for photoswitch modification was FraC, a relatively small protein (21 kDa) with a known crystal structure that is relatively easy to purify. 64 , 92 − 94 FraC monomers assemble into octameric pores upon encountering cell membranes containing sphingomyelin, a lipid which is present in all mammalian cell surfaces. 64 , 95 Since wild-type FraC does not contain a cysteine residue, the visible-light-responsive photoswitch furnished with a reactive chloroacetyl group was attached to a free cysteine genetically introduced by site-specific mutation, providing an extra level of precision in the design. The mutation sites, namely, W112 and Y138, were selected for their location at the interface of the formed oligomeric pore and the lipid bilayer of the target cell ( Figure 1 a). 64 Furthermore, the selected positions are located in the sphingomyelin binding pockets. Sphingomyelin naturally stabilizes the assembled wild-type FraC pore, 64 therefore we anticipated that modification of FraC at the selected locations would substitute sphingomyelin with the photoswitch and thus have a larger effect on the nanopore assembly process. After purification of the labeled FraC mutants, the hemolytic activity was evaluated using defibrinated red blood cells and human hypopharyngeal squamous cell carcinoma. Synthesis For the synthesis of the water-soluble visible-light-responsive switch A , we utilized the unique susceptibility of the tetra- ortho -fluoro-azobenzene core to S N Ar for installing the water solubilizing group ( Scheme 1 ). Aniline 1 was oxidized to the nitroso derivative 3 and subsequently submitted to the Baeyer–Mills reaction with aniline 2 in a mixture of solvents (toluene, acetic acid, and TFA) to yield azobenzene 4 in good yield (83%) ( Scheme 1 ). The sulfonation step was performed with azobenzene 4 and a weak nucleophile, sodium sulfite, in a 1/1 mixture of water and EtOH to ensure solubilization of both substrates. The water-soluble azobenzene 5 was purified by reverse phase chromatography using a mildly basic ammonium bicarbonate buffer and ACN mixture as eluents. After isolation of the pure sulfonated product 5 , Buchwald–Hartwig coupling with acetamide was performed at 90 °C in degassed DMF. Pure azobenzene 7 was isolated after reversed phase purification in a high yield (73%). The acetamide was hydrolyzed in the presence of concentrated hydrochloric acid and the isolated amine 8 underwent reaction with chloroacetic anhydride (neat) to obtain final photoswitch A . Azobenzene A was purified from the excess anhydride by several centrifuge washes with ether and fully characterized by NMR spectroscopy, HRMS, and LCMS analysis (See Supporting Information sections 1.2 and 3.1). Scheme 1 Synthesis of Water-Soluble Visible Light-Responsive Switch A Photophysical Properties First, the photophysical properties of A were investigated in DMSO as a standard solvent for validating the photophysical properties of switches for biological applications. The UV–vis spectrum of azobenzene A clearly shows separation of the n−π* bands of both isomers thus allowing efficient switching exclusively with visible light ( Figure 2 b,c,h). The band separation is sufficient to ensure good isomer distribution at the photostationary state (PSS), as determined by NMR spectroscopy, revealing that high amounts of the respective isomer were formed upon irradiation with green (530 nm) and blue (430 nm) light (PSD 530nm = 81% cis , PSD 430nm = 79% trans ). The photophysical properties in DMSO, including fatigue resistance over multiple switching cycles, were accompanied by a half-life over 9 h at 20 °C. Figure 2 (a) Scheme depicting photoswitching of A (R = CH 2 Cl) and 7 (R = CH 3 ) with visible light. (b) UV–vis spectrum of A at PSS 530nm and PSS 430nm (80 μM, DMSO). (c) PSD determination of A with 1 H NMR spectroscopy (2 mM, DMSO- d 6 ) by relative integration of the shifted signals shown in bold. (d) UV–vis spectrum of 7 at PSS 530nm and PSS 430nm (50 μM, 15 mM Tris, 150 mM NaCl, pH = 7.5). (e) PSD determination of 7 in Tris buffer with 19 F NMR spectroscopy (0.88 mM, 15 mM Tris, 150 mM NaCl, pH = 7.5 with 10% DMSO- d 6 ) by relative integration of the shifted signals shown in bold. The signal between −117.8 and 118.6 in the 19 F-NMR is likely due to a fluorinated impurity which was impossible to be removed, despite several purification attempts. The signal does not react to irradiation. (f) Stability of 7 (50 μM) toward reduction by GSH (10 mM) in 15 mM Tris buffer, 150 mM NaCl, pH = 7.5, at 20 °C. Fatigue resistance in the presence of GSH with marked time points at which the UV–vis spectra are shown in panel g.(h) Half-life determination of A at 80 μM in DMSO. (i) Half-life determination of 7 at 50 μM in 15 mM Tris, 150 mM NaCl, pH = 7.5. All at 20 °C. Furthermore, we tested the photophysical properties in an aqueous environment as similar as possible to the buffer used to elute the FraC monomer. This is important, since many azobenzenes show a substantially shorter half-life of the metastable cis isomer in water. 42 , 96 While photoswitch A completely dissolved in Tris buffer at a concentration of 10 mM, it also contains a reactive chloroacetyl group highly susceptible to reaction with nucleophiles, such as Tris, which is the main component of the buffer used for the purification of FraC. Therefore, analogue 7 not bearing the chloroacetyl moiety ( Figure 2 d,e,i) was used as a model compound for studying the photophysical properties in Tris buffer. To our delight, azobenzene 7 also exhibited favorable photophysical properties in the aqueous medium with similar PSS ratios (PSD 530nm = 83% cis , PSD 430nm = 76% trans ), showing no fatigue upon numerous photoswitching cycles and, most importantly, a half-life of over 12 h at 20 °C that is sufficient for the PFT activity assays. Furthermore, azobenzene 7 was used to assess the stability of the switch in the presence of glutathione (GSH). Resistance to GSH reduction is often tested for photoswitches used in biological applications since the environment in living cells is naturally highly reducing. 97 − 99 Azobenzene 7 underwent several switching cycles in the presence of 10 mM GSH solution in Tris buffer indicating resistance to GSH reduction ( Figure 2 f,g). After investigating the photophysical properties of the water-soluble, visible-light-responsive molecular photoswitches in both DMSO and Tris buffer, it was concluded that molecule A can be reversibly and fully controlled with visible light while remaining stable in the presence of GSH and having a half-life sufficiently long for the cell lysis assays used to assess the reactivity of PFTs. Protein Labeling, PFT Activity Assays, and Cell Lysis Two mutants of FraC with cysteine located at positions 138 or 112 were chosen for attachment of visible-light-responsive molecule A ( Figure 1 b). In the wild type both cysteines are located at the interface of the formed pore and the lipid bilayer, in the locations where naturally the sphingomyelin binding pocket is positioned. 64 Sphingomyelin stabilizes the formation of the full pore complex and, in the wild-type pore, its presence in the target membrane is crucial for nanopore assembly. 64 Therefore, we anticipated that introduction of an aromatic photoswitch in the sphingomyelin pocket locations could have the largest effect on the pore-forming activity. Covalent attachment of switch A to the FraC monomers containing a freely accessible cysteine was performed in a degassed phosphate buffer solution in order to prevent oxidation of the free thiol. The presence of a mild reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), was avoided during the labeling reaction since the reactive chloroacetyl group of switch A can react with the reagents. After labeling, the excess azobenzene was removed via size exclusion chromatography, while the content of any remaining unmodified monomer is the same across samples, possibly causing some background activity. For details of protein expression, labeling, and purification, see SI 1.4 . The cytolytic activity of the labeled FraC constructs was evaluated by performing a hemolytic activity assay on defibrinated red blood cells. In the initial studies, it was observed that ambient light, even stray light from computer screens, significantly affected the hemolytic activity of the constructs. This observation was in accordance with the finding that the free switch A and its derivatives, being responsive to visible light, very efficiently formed a mixture of both isomers under any ambient light. Therefore, all the hemolytic activity assays, as well as handling of the samples after labeling with switch A , were performed strictly under dim red light. Before addition to the red blood cells, the freshly prepared labeled FraC mutants were preirradiated with either green (530 nm, "ON") or blue (430 nm, "OFF") light. Both FraC(Y138C)– A and FraC(W112C)– A exhibited hemolytic activity within a short time span upon addition at the tested concentrations and showed a significant difference in the activity of the ON and OFF samples ( Figure 3 a,b). The analysis of the dose–response curves confirmed that FraC(W112C)– A was generally more active (EC50 530nm (ON) = 0.7 μM, EC50 430nm (OFF) = 1.1 μM) compared to FraC(Y138C)– A , while FraC(Y138C)– A (EC50 530nm (ON) = 2.2 μM, EC50 430nm (OFF) = 5.2 μM) showed a larger difference in activity of the ON and OFF solutions ( Figure 3 a,b). For both labeled mutants, the OFF samples, containing predominantly the trans isomer of switch A , were still relatively active, which is likely a consequence of the presence of the cis isomer (the PSD 430nm of molecule 7 in Tris buffer is only 76% trans , Figure 3 e). Besides the possibility that the trans - A labeled FraC is also somewhat active, the OFF sample contains 24% cis - A labeled FraC, which might be causing the observed activity. Nevertheless, both mutants exhibited a significant difference in activity of the labeled FraC constructs upon irradiation with two different wavelengths of visible light. Furthermore, the steep dose–response curves (Hill coefficient n (FraC112C– trans - A ) = 5.3 and n (FraC112C– cis - A ) = 6.5, n (FraC138C– trans - A and − cis - A ) = 3.6), which are inherent to the cooperative nature of the FraC assembly mechanism involving a multimeric pore, are working in favor for this photopharmacological system as a small difference in concentration results in a large response difference. In addition, to confirm the reversibility upon irradiation, a sample of FraC(Y138C)– A was subjected to subsequent irradiation cycles with green and blue light, while an aliquot was taken after each irradiation step and its activity was tested on blood ( Figure 3 c). Consecutive activation of the construct upon λ = 530 nm light irradiation and deactivation upon application of λ = 430 nm light was observed. However, some loss of activity was observed for each of the performed cycles, suggesting slow degradation of the construct upon successive photoswitching cycles. Figure 3 Hemolytic activity of labeled FraC– A upon irradiation with 530 (in green) and 430 (in blue) nm LED where the EC50 values are expressed in μM: (a) EC50 curves for FraC(W112C)– A ; (b) EC50 curves for FraC(Y138C)– A . (c) Reversibility test of hemolytic activity of mutant FraC(Y138C) where the P values were determined by one-way ANOVA analysis between the highlighted conditions, A after 10 min upon subsequent irradiation cycles (approximately 2 μM). (d) Cytolytic activity of FraC(Y138C)– A on FaDu cancer cells where the P values were determined by one-way ANOVA analysis between the ON (green) or OFF (blue) and the control and between ON and OFF (highlighted with bracket) conditions. The asterisks represent P values as follows: **** P ≤ 0.0001; *** P ≤ 0.001; ** P ≤ 0.01, and * P ≤ 0.05. Experiments a, b and c were performed as four independent replicates, while d was performed in duplicate. Finally, we evaluated the cytolytic activity of the visible-light responsive FraC proteins toward human cancer cells. Since both the wild-type FraC and the Y138C mutant exhibited the highest cytolytic activity toward FaDu (hypopharyngeal squamous carcinoma) cells, this cell line was selected for evaluating the cytolytic activity of the visible light-responsive FraC(Y138C)– A . As in the hemolytic assays, the samples were preirradiated with the corresponding light (530 nm ON, and 430 nm OFF). At higher concentrations, the activated ON sample of FraC(Y138C)– A exhibited higher cytolytic activity compared to the OFF sample ( Figure 3 d). This result serves as a proof of principle illustrating that the modified FraC nanopore labeled with a water-soluble photoswitch can selectively destroy cancer cells upon activation with visible light. Conclusion PFTs hold great potential for future therapeutic applications, especially due to their high potency in destroying cancer cells. However, the main limitation is their nondiscriminative cytotoxic activity toward normal mammalian cells, which would result in severe off-target side effects of the treatment. To develop PFT-based therapeutics, it is crucial to enable the local activation of their lytic activity with an external, biocompatible trigger, such as visible light. 33 Here, we aimed to design a fully visible-light responsive PFT by labeling the mutated FraC protein with a water-soluble, green- and blue-light-responsive azobenzene photoswitch in strategically selected locations for manipulating nanopore assembly. The selected azobenzene photoswitch features tetra- ortho -fluoro groups which result in favorable photophysical properties and stability, as well as an electron-deficient nature thus enabling functionalization of the molecule via S N Ar reaction. This unique synthetic strategy allowed for the later stage introduction of the water-solubilizing sulfonate group. While the water-soluble molecules still proved to require challenging reversed phase purification, the final photoswitch A was successfully obtained in sufficient amounts. Both switch A and its model analogue 7 exhibited favorable photophysical properties also under aqueous conditions. Molecule A was covalently attached to the chosen cysteine mutants of the FraC monomers. The fully visible-light-responsive FraC construct demonstrated a difference in hemolytic activity where the cis isomer of A (ON sample) was the more active species. Furthermore, FraC(Y138C)– cis - A was also more active than the blue light-irradiated construct toward FaDu cancer cells, confirming the first report of a fully visible-light-controlled cytolytic protein which destroys cancer cells. Taken together, our strategy paves the way for the development of photocontrolled PFT-based cancer therapeutics. Methods The Supporting Information contains synthetic schemes and procedures, 1 H, 13 C and 19 F NMR spectra, HRMS analysis of compounds, sulfonation reaction optimization, protocol description of protein expression, labeling, purification, and the hemolytic activity assay. Safety Statement No unexpected or unusually high safety hazards were encountered in this study. Chemical Synthesis trans -4-((4-Bromo-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 5 ) Azobenzene 4 (2.0 g, 5.7 mmol) and sodium sulfite (1.0 equiv, 0.72 g, 5.7 mmol) were weighed in a round-bottom flask and were subjected to three vacuum–dry nitrogen cycles. The solids were dissolved in 150 mL of the solvent mixture (water/EtOH, 1:1, v/v), and the solvent was degassed by purging with dry N 2 for 10 min. The reaction mixture was heated to 50 °C and stirred vigorously overnight. The solvent was partially removed in vacuo , and the resulting mixture was extracted with DCM (3 × 100 mL) to remove the remaining starting material which is not soluble in water. The aqueous layer was loaded on Celite and freeze-dried to remove water. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in CAN). The obtained fractions were freeze-dried, and the pure product 5 (440 mg, 19%) was isolated as a yellow solid. Due to limited solubility in CAN and formation of aggregates at higher concentrations, the 13 C spectrum was performed at relatively low concentration. Therefore, we also collected 2D NMR spectra to add to the characterization. Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 7.57–7.49 (m, 1H), 7.45 (d, J = 8.7 Hz, 1H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −120.44 (d, J = 10.6 Hz), −120.49 (d, J = 9.9 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ: 156.2, 155.7, 154.5, 154.0, 149.4, 131.9, 130.3, 124.7, 117.1, 117.0, 110.8. HR-MS (ESI−) calculated for C 12 H 4 BrF 4 N 2 O 3 S – [M – ] 410.9057 found: 410.9060. 1 H NMR (500 MHz, CD 3 CN, drop DCl) δ 7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H). 19 F NMR (376 MHz CD 3 CN, drop DCl) δ −120.31 (d, J = 9.5 Hz), −120.40 (d, J = 9.0 Hz). 13 C NMR (101 MHz, CD 3 CN, drop DCl) δ 157.4 (d, J = 5.1 Hz), 156.9 (d, J = 3.8 Hz), 154.8 (d, J = 5.1 Hz), 154.3 (d, J = 3.7 Hz), 148.6, 132.96 (m), 131.0 (t, J = 9.4 Hz), 125.7 (t, J = 12.4 Hz), 117.9 (d, J = 3.9 Hz), 111.5 (dd, J = 23.2, 3.3 Hz). trans -4-((4-Acetamido-2,6-difluorophenyl)diazinyl)-3,5-difluorobenzenesulfonate ( 7 ) Compound 5 (300 mg, 0.726 mmol), acetamide (1.20 eq, 51.5 mg, 0.871 mmol), Cs 2 CO 3 (3.5 eq, 828 mg, 2.54 mmol), Pd 2 dba 3 (5 mol %, 33.2 mg, 0.0360 mmol), and XantPhos (20 mol %, 84.0 mg, 0.145 mmol) were weighed into a dry sealable vial equipped with a magnetic stirrer. The solids were subjected to three vacuum–dry nitrogen cycles. Peptide grade DMF (8 mL) was added via a syringe, and the mixture was degassed via five freeze–thaw–pump cycles. (The mixture was frozen by submerging the vial into liquid nitrogen. The frozen solid was put under vacuum, and the valve was closed, leaving a residual vacuum. The reaction mixture was thawed by submerging it into lukewarm water while observing some bubbling. The mixture was again frozen, and the process was repeated.) The reaction mixture was placed under nitrogen and left to stir at 90 °C for 4 h. The crude product mixture was diluted with water/ACN and most of the solvent was removed by rotary evaporation. The remaining solution was transferred with additional water, loaded on Celite, and freeze-dried. The crude product on Celite was dissolved in water and freeze-dried another two times to remove all the remaining traces of DMF. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in ACN). The product elutes at 30% ACN. The obtained fractions were freeze-dried, and the pure product was isolated as an orange solid (208 mg, yield = 73%). Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 8.94 (s, 1H), 7.46 (d, J = 9.7 Hz, 2H), 7.43 (d, J = 12.3 Hz, 2H), 2.12 (s, 3H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −119.68 (d, J = 12.5 Hz), −122.25 (d, J = 9.8 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ 170.6, 156.6 (dd, J = 7.8, 4.2 Hz), 154.9 (d, J = 6.3 Hz), 152.8 (t, J = 7.7 Hz), 144.3 (t, J = 14.4 Hz), 132.3, 127.7, 111.3 (d, J = 3.8 Hz), 111.2 (d, J = 3.8 Hz), 103.7 (d, J = 3.1 Hz), 103.5 (d, J = 3.0 Hz), 24.6. HRMS (ESI−) calcd for C 14 H 8 F 4 N 3 O 4 S – : 390.0166, found 390.0179. trans -4-((4-Amino-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 8 ) Compound 7 (170 mg, 0.43 mmol) was dissolved in concentrated aq. HCl (10 mL, 37%) in an oven-dried sealable vial equipped with a magnetic stirrer. The solution was purged with dry nitrogen for 5 min. The dark purple reaction mixture was heated to 40 °C and left to stir overnight. The reaction mixture was transferred to a flask, and the solvent was removed by rotary evaporation with base (aqueous NaHCO 3 solution) in the rotary evaporator collection flask. The remaining black solid was dissolved in water and freeze-dried in Corning Falcon conical tubes. The product was used for the next step, as obtained by freeze-drying in a sealable vial equipped with a magnetic stirrer. trans -4-((4-(2-Chloroacetamido)-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( A ) Crude aniline 8 (150 mg, 0.4 mmol) was transferred to a sealable vial, and chloroacetic anhydride (2.5 g) was added. The mixture was subjected to three vacuum/dry nitrogen cycles and left under N 2 to stir at 87 °C for 4 h. The dark purple reaction mixture was dripped into cold ether to precipitate the pure product. The mixture was centrifuged (4000 rpm) and washed multiple times with cold ether. The pure product was obtained as a dark purple solid (200 mg, yield >95%). Mp > 250 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.20 (s, 1H), 7.57 (d, J = 12.0 Hz, 2H), 7.40 (d, J = 12.0 Hz, 2H), 4.37 (s, 2H). 19 F NMR (565 MHz, DMSO- d 6 ) δ −118.06 (d, J = 12.6 Hz), −120.82 (d, J = 10.1 Hz). 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.0, 156.6 (d, J = 6.4 Hz), 155.4–154.4 (m), 153.2 (d, J = 3.8 Hz), 152.1 (d, J = 7.0 Hz), 142.9 (t, J = 14.0 Hz), 130.7, 126.4, 110.2 (d, J = 3.6 Hz), 110.0 (d, J = 3.4 Hz), 103.1 (d, J = 3.0 Hz), 103.0–102.9 (m), 43.5. HRMS (ESI−) calcd for C 14 H 7 ClF 4 N 3 O 4 S – : 423.9787, found 423.9790. Protein Expression, Labeling, and Purification The FraC(W112C) and FraC(Y138C) constructs were created and expressed as described previously. 50 In brief, the constructs were transformed into the electrocompetent E. cloni EXPRESS BL21(DE3) cells and cultured in 1 L of 2×YT medium supplemented with 100 μg/mL ampicillin at 37 °C while shaking at 200 rpm, until reaching an OD 600 of ∼0.8. Protein expression was induced by addition of 0.5 mM isopropyl β- d -thiogalactopyranoside (IPTG) and subsequent culturing overnight at 25 °C while shaking at 200 rpm. The cells were harvested by centrifugation at 8000 g for 10 min at 4 °C and subjected to one freeze–thaw cycle at −80 °C to increase susceptibility to cell lysis. Cell pellets, from 50 mL of cell culture, were resuspended in 10 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 M urea, 1 mM MgCl 2 , 2.5 mM TCEP, 10 μg/mL lysozyme, 0.1 U/mL DNase) and incubated for 20 min at RT while rotating at 25 rpm. The cells were further disrupted via probe sonication (Brandson) for 2 min at 25% output power. Cell debris were removed via centrifugation at 8000 g for 30 min at 4 °C. The supernatant was incubated with 300 μL (bead volume) of Ni-NTA resin (Qiagen) for 10 min while rotating at 10 rpm. The incubated Ni-NTA resin was loaded onto a gravity flow column (Bio-Rad) and washed with 15 mL of wash buffer 1 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM imidazole, and 2.5 mM TCEP) and 20 mL of nitrogen degassed wash buffer 2 (50 mM NaH 2 PO 4 pH 9.5, 150 mM NaCl). Wash buffer 2 is used to remove Tris-HCl, imidazole, and TCEP that can inhibit the labeling of the light switch. The FraC constructs were eluted from the Ni-NTA resin with 3 times 250 μL of nitrogen degassed elution buffer (50 mM NaH 2 PO 4 pH 9.5, 150 mM NaCl, 200 mM EDTA) in a darkroom in light protective microtubes. After each elution step, the protein concentration was immediately determined spectroscopically (ε 280nm (FraC)= 43.9 × 10 3 M –1 cm –1 ) and the azobenzene A was added in a 1:50 ratio (protein/azobenzene). The stock solution of switch A was previously freshly prepared in 1/1 solution of water/DMSO. The labeling mixture was incubated with the protein for 2 h while shaking and preventing any ambient light exposure ( Figure S1 ). The excess photoswitch was separated from the labeled protein with two desalting columns (HiTrap Desalting column, Cytiva) connected to an Äkta pure chromatography system. The sample was applied to the HiTrap desalting columns equilibrated with running buffer (15 mM Tris-HCl pH 9.5). Protein elution was monitored by measuring absorbance at 280, 365, and 420 nm wavelengths. The first eluted peak corresponds to the FraC monomers labeled with the visible light switch ( Figure S2 ). Collected elution fractions of the first peak were combined and concentrated by using the rotary evaporator at 35–40 °C. Protein concentration was determined by UV-spectroscopy while approximating the same extinction coefficient as unlabeled FraC (ε 280nm (FraC)= 43.9 × 10 3 M –1 cm –1 ). Labeled FraC monomers were stored at 4 °C until further use. Cancer Cell Cytolytic Activity Assay Cancer Cells The human hypopharyngeal squamous carcinoma cell line FaDu was obtained from the American Type Culture Collection (HTB-43, ATCC). The adherently growing FaDu cancer cells were cultured in DMEM medium (Lonza) supplemented with 10% fetal calf serum (FCS, ThermoFisher) at 37 °C in a humidified 5% CO 2 atmosphere. Assessment of Anticancer Activity of FraC Variants The capacity of the various FraC constructs to eliminate FaDu cancer cells was assessed by flow cytometry using the annexin-V-FITC/propidium iodide staining method. In short, FaDu cancer cells were cultured in 48-well culture plates (1.5 × 10 4 cells/well) and treated (or not) with the indicated concentrations of control buffer, FraC-138C-ON, or FraC-138C-OFF for 2 h at 37 °C. Subsequently, cancer cells were harvested from the respective wells and evaluated for the percentage cells Annexin-V/PI positive by flow cytometry. Safety Statement No unexpected or unusually high safety hazards were encountered in this study. Chemical Synthesis trans -4-((4-Bromo-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 5 ) Azobenzene 4 (2.0 g, 5.7 mmol) and sodium sulfite (1.0 equiv, 0.72 g, 5.7 mmol) were weighed in a round-bottom flask and were subjected to three vacuum–dry nitrogen cycles. The solids were dissolved in 150 mL of the solvent mixture (water/EtOH, 1:1, v/v), and the solvent was degassed by purging with dry N 2 for 10 min. The reaction mixture was heated to 50 °C and stirred vigorously overnight. The solvent was partially removed in vacuo , and the resulting mixture was extracted with DCM (3 × 100 mL) to remove the remaining starting material which is not soluble in water. The aqueous layer was loaded on Celite and freeze-dried to remove water. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in CAN). The obtained fractions were freeze-dried, and the pure product 5 (440 mg, 19%) was isolated as a yellow solid. Due to limited solubility in CAN and formation of aggregates at higher concentrations, the 13 C spectrum was performed at relatively low concentration. Therefore, we also collected 2D NMR spectra to add to the characterization. Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 7.57–7.49 (m, 1H), 7.45 (d, J = 8.7 Hz, 1H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −120.44 (d, J = 10.6 Hz), −120.49 (d, J = 9.9 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ: 156.2, 155.7, 154.5, 154.0, 149.4, 131.9, 130.3, 124.7, 117.1, 117.0, 110.8. HR-MS (ESI−) calculated for C 12 H 4 BrF 4 N 2 O 3 S – [M – ] 410.9057 found: 410.9060. 1 H NMR (500 MHz, CD 3 CN, drop DCl) δ 7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H). 19 F NMR (376 MHz CD 3 CN, drop DCl) δ −120.31 (d, J = 9.5 Hz), −120.40 (d, J = 9.0 Hz). 13 C NMR (101 MHz, CD 3 CN, drop DCl) δ 157.4 (d, J = 5.1 Hz), 156.9 (d, J = 3.8 Hz), 154.8 (d, J = 5.1 Hz), 154.3 (d, J = 3.7 Hz), 148.6, 132.96 (m), 131.0 (t, J = 9.4 Hz), 125.7 (t, J = 12.4 Hz), 117.9 (d, J = 3.9 Hz), 111.5 (dd, J = 23.2, 3.3 Hz). trans -4-((4-Acetamido-2,6-difluorophenyl)diazinyl)-3,5-difluorobenzenesulfonate ( 7 ) Compound 5 (300 mg, 0.726 mmol), acetamide (1.20 eq, 51.5 mg, 0.871 mmol), Cs 2 CO 3 (3.5 eq, 828 mg, 2.54 mmol), Pd 2 dba 3 (5 mol %, 33.2 mg, 0.0360 mmol), and XantPhos (20 mol %, 84.0 mg, 0.145 mmol) were weighed into a dry sealable vial equipped with a magnetic stirrer. The solids were subjected to three vacuum–dry nitrogen cycles. Peptide grade DMF (8 mL) was added via a syringe, and the mixture was degassed via five freeze–thaw–pump cycles. (The mixture was frozen by submerging the vial into liquid nitrogen. The frozen solid was put under vacuum, and the valve was closed, leaving a residual vacuum. The reaction mixture was thawed by submerging it into lukewarm water while observing some bubbling. The mixture was again frozen, and the process was repeated.) The reaction mixture was placed under nitrogen and left to stir at 90 °C for 4 h. The crude product mixture was diluted with water/ACN and most of the solvent was removed by rotary evaporation. The remaining solution was transferred with additional water, loaded on Celite, and freeze-dried. The crude product on Celite was dissolved in water and freeze-dried another two times to remove all the remaining traces of DMF. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in ACN). The product elutes at 30% ACN. The obtained fractions were freeze-dried, and the pure product was isolated as an orange solid (208 mg, yield = 73%). Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 8.94 (s, 1H), 7.46 (d, J = 9.7 Hz, 2H), 7.43 (d, J = 12.3 Hz, 2H), 2.12 (s, 3H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −119.68 (d, J = 12.5 Hz), −122.25 (d, J = 9.8 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ 170.6, 156.6 (dd, J = 7.8, 4.2 Hz), 154.9 (d, J = 6.3 Hz), 152.8 (t, J = 7.7 Hz), 144.3 (t, J = 14.4 Hz), 132.3, 127.7, 111.3 (d, J = 3.8 Hz), 111.2 (d, J = 3.8 Hz), 103.7 (d, J = 3.1 Hz), 103.5 (d, J = 3.0 Hz), 24.6. HRMS (ESI−) calcd for C 14 H 8 F 4 N 3 O 4 S – : 390.0166, found 390.0179. trans -4-((4-Amino-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 8 ) Compound 7 (170 mg, 0.43 mmol) was dissolved in concentrated aq. HCl (10 mL, 37%) in an oven-dried sealable vial equipped with a magnetic stirrer. The solution was purged with dry nitrogen for 5 min. The dark purple reaction mixture was heated to 40 °C and left to stir overnight. The reaction mixture was transferred to a flask, and the solvent was removed by rotary evaporation with base (aqueous NaHCO 3 solution) in the rotary evaporator collection flask. The remaining black solid was dissolved in water and freeze-dried in Corning Falcon conical tubes. The product was used for the next step, as obtained by freeze-drying in a sealable vial equipped with a magnetic stirrer. trans -4-((4-(2-Chloroacetamido)-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( A ) Crude aniline 8 (150 mg, 0.4 mmol) was transferred to a sealable vial, and chloroacetic anhydride (2.5 g) was added. The mixture was subjected to three vacuum/dry nitrogen cycles and left under N 2 to stir at 87 °C for 4 h. The dark purple reaction mixture was dripped into cold ether to precipitate the pure product. The mixture was centrifuged (4000 rpm) and washed multiple times with cold ether. The pure product was obtained as a dark purple solid (200 mg, yield >95%). Mp > 250 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.20 (s, 1H), 7.57 (d, J = 12.0 Hz, 2H), 7.40 (d, J = 12.0 Hz, 2H), 4.37 (s, 2H). 19 F NMR (565 MHz, DMSO- d 6 ) δ −118.06 (d, J = 12.6 Hz), −120.82 (d, J = 10.1 Hz). 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.0, 156.6 (d, J = 6.4 Hz), 155.4–154.4 (m), 153.2 (d, J = 3.8 Hz), 152.1 (d, J = 7.0 Hz), 142.9 (t, J = 14.0 Hz), 130.7, 126.4, 110.2 (d, J = 3.6 Hz), 110.0 (d, J = 3.4 Hz), 103.1 (d, J = 3.0 Hz), 103.0–102.9 (m), 43.5. HRMS (ESI−) calcd for C 14 H 7 ClF 4 N 3 O 4 S – : 423.9787, found 423.9790. trans -4-((4-Bromo-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 5 ) Azobenzene 4 (2.0 g, 5.7 mmol) and sodium sulfite (1.0 equiv, 0.72 g, 5.7 mmol) were weighed in a round-bottom flask and were subjected to three vacuum–dry nitrogen cycles. The solids were dissolved in 150 mL of the solvent mixture (water/EtOH, 1:1, v/v), and the solvent was degassed by purging with dry N 2 for 10 min. The reaction mixture was heated to 50 °C and stirred vigorously overnight. The solvent was partially removed in vacuo , and the resulting mixture was extracted with DCM (3 × 100 mL) to remove the remaining starting material which is not soluble in water. The aqueous layer was loaded on Celite and freeze-dried to remove water. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in CAN). The obtained fractions were freeze-dried, and the pure product 5 (440 mg, 19%) was isolated as a yellow solid. Due to limited solubility in CAN and formation of aggregates at higher concentrations, the 13 C spectrum was performed at relatively low concentration. Therefore, we also collected 2D NMR spectra to add to the characterization. Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 7.57–7.49 (m, 1H), 7.45 (d, J = 8.7 Hz, 1H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −120.44 (d, J = 10.6 Hz), −120.49 (d, J = 9.9 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ: 156.2, 155.7, 154.5, 154.0, 149.4, 131.9, 130.3, 124.7, 117.1, 117.0, 110.8. HR-MS (ESI−) calculated for C 12 H 4 BrF 4 N 2 O 3 S – [M – ] 410.9057 found: 410.9060. 1 H NMR (500 MHz, CD 3 CN, drop DCl) δ 7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H). 19 F NMR (376 MHz CD 3 CN, drop DCl) δ −120.31 (d, J = 9.5 Hz), −120.40 (d, J = 9.0 Hz). 13 C NMR (101 MHz, CD 3 CN, drop DCl) δ 157.4 (d, J = 5.1 Hz), 156.9 (d, J = 3.8 Hz), 154.8 (d, J = 5.1 Hz), 154.3 (d, J = 3.7 Hz), 148.6, 132.96 (m), 131.0 (t, J = 9.4 Hz), 125.7 (t, J = 12.4 Hz), 117.9 (d, J = 3.9 Hz), 111.5 (dd, J = 23.2, 3.3 Hz). trans -4-((4-Acetamido-2,6-difluorophenyl)diazinyl)-3,5-difluorobenzenesulfonate ( 7 ) Compound 5 (300 mg, 0.726 mmol), acetamide (1.20 eq, 51.5 mg, 0.871 mmol), Cs 2 CO 3 (3.5 eq, 828 mg, 2.54 mmol), Pd 2 dba 3 (5 mol %, 33.2 mg, 0.0360 mmol), and XantPhos (20 mol %, 84.0 mg, 0.145 mmol) were weighed into a dry sealable vial equipped with a magnetic stirrer. The solids were subjected to three vacuum–dry nitrogen cycles. Peptide grade DMF (8 mL) was added via a syringe, and the mixture was degassed via five freeze–thaw–pump cycles. (The mixture was frozen by submerging the vial into liquid nitrogen. The frozen solid was put under vacuum, and the valve was closed, leaving a residual vacuum. The reaction mixture was thawed by submerging it into lukewarm water while observing some bubbling. The mixture was again frozen, and the process was repeated.) The reaction mixture was placed under nitrogen and left to stir at 90 °C for 4 h. The crude product mixture was diluted with water/ACN and most of the solvent was removed by rotary evaporation. The remaining solution was transferred with additional water, loaded on Celite, and freeze-dried. The crude product on Celite was dissolved in water and freeze-dried another two times to remove all the remaining traces of DMF. The crude product was purified by automatic chromatography on reversed-phase silica gel (C18) with a gradient of the solvent mixture (0–100% 10 mM NH 4 HCO 3 (aq) in ACN). The product elutes at 30% ACN. The obtained fractions were freeze-dried, and the pure product was isolated as an orange solid (208 mg, yield = 73%). Mp > 250 °C. 1 H NMR (600 MHz, CD 3 CN, drop D 2 O) δ 8.94 (s, 1H), 7.46 (d, J = 9.7 Hz, 2H), 7.43 (d, J = 12.3 Hz, 2H), 2.12 (s, 3H). 19 F NMR (565 MHz, CD 3 CN, drop D 2 O) δ −119.68 (d, J = 12.5 Hz), −122.25 (d, J = 9.8 Hz). 13 C NMR (151 MHz, CD 3 CN, drop D 2 O) δ 170.6, 156.6 (dd, J = 7.8, 4.2 Hz), 154.9 (d, J = 6.3 Hz), 152.8 (t, J = 7.7 Hz), 144.3 (t, J = 14.4 Hz), 132.3, 127.7, 111.3 (d, J = 3.8 Hz), 111.2 (d, J = 3.8 Hz), 103.7 (d, J = 3.1 Hz), 103.5 (d, J = 3.0 Hz), 24.6. HRMS (ESI−) calcd for C 14 H 8 F 4 N 3 O 4 S – : 390.0166, found 390.0179. trans -4-((4-Amino-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( 8 ) Compound 7 (170 mg, 0.43 mmol) was dissolved in concentrated aq. HCl (10 mL, 37%) in an oven-dried sealable vial equipped with a magnetic stirrer. The solution was purged with dry nitrogen for 5 min. The dark purple reaction mixture was heated to 40 °C and left to stir overnight. The reaction mixture was transferred to a flask, and the solvent was removed by rotary evaporation with base (aqueous NaHCO 3 solution) in the rotary evaporator collection flask. The remaining black solid was dissolved in water and freeze-dried in Corning Falcon conical tubes. The product was used for the next step, as obtained by freeze-drying in a sealable vial equipped with a magnetic stirrer. trans -4-((4-(2-Chloroacetamido)-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzenesulfonate ( A ) Crude aniline 8 (150 mg, 0.4 mmol) was transferred to a sealable vial, and chloroacetic anhydride (2.5 g) was added. The mixture was subjected to three vacuum/dry nitrogen cycles and left under N 2 to stir at 87 °C for 4 h. The dark purple reaction mixture was dripped into cold ether to precipitate the pure product. The mixture was centrifuged (4000 rpm) and washed multiple times with cold ether. The pure product was obtained as a dark purple solid (200 mg, yield >95%). Mp > 250 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.20 (s, 1H), 7.57 (d, J = 12.0 Hz, 2H), 7.40 (d, J = 12.0 Hz, 2H), 4.37 (s, 2H). 19 F NMR (565 MHz, DMSO- d 6 ) δ −118.06 (d, J = 12.6 Hz), −120.82 (d, J = 10.1 Hz). 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.0, 156.6 (d, J = 6.4 Hz), 155.4–154.4 (m), 153.2 (d, J = 3.8 Hz), 152.1 (d, J = 7.0 Hz), 142.9 (t, J = 14.0 Hz), 130.7, 126.4, 110.2 (d, J = 3.6 Hz), 110.0 (d, J = 3.4 Hz), 103.1 (d, J = 3.0 Hz), 103.0–102.9 (m), 43.5. HRMS (ESI−) calcd for C 14 H 7 ClF 4 N 3 O 4 S – : 423.9787, found 423.9790. Protein Expression, Labeling, and Purification The FraC(W112C) and FraC(Y138C) constructs were created and expressed as described previously. 50 In brief, the constructs were transformed into the electrocompetent E. cloni EXPRESS BL21(DE3) cells and cultured in 1 L of 2×YT medium supplemented with 100 μg/mL ampicillin at 37 °C while shaking at 200 rpm, until reaching an OD 600 of ∼0.8. Protein expression was induced by addition of 0.5 mM isopropyl β- d -thiogalactopyranoside (IPTG) and subsequent culturing overnight at 25 °C while shaking at 200 rpm. The cells were harvested by centrifugation at 8000 g for 10 min at 4 °C and subjected to one freeze–thaw cycle at −80 °C to increase susceptibility to cell lysis. Cell pellets, from 50 mL of cell culture, were resuspended in 10 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 M urea, 1 mM MgCl 2 , 2.5 mM TCEP, 10 μg/mL lysozyme, 0.1 U/mL DNase) and incubated for 20 min at RT while rotating at 25 rpm. The cells were further disrupted via probe sonication (Brandson) for 2 min at 25% output power. Cell debris were removed via centrifugation at 8000 g for 30 min at 4 °C. The supernatant was incubated with 300 μL (bead volume) of Ni-NTA resin (Qiagen) for 10 min while rotating at 10 rpm. The incubated Ni-NTA resin was loaded onto a gravity flow column (Bio-Rad) and washed with 15 mL of wash buffer 1 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM imidazole, and 2.5 mM TCEP) and 20 mL of nitrogen degassed wash buffer 2 (50 mM NaH 2 PO 4 pH 9.5, 150 mM NaCl). Wash buffer 2 is used to remove Tris-HCl, imidazole, and TCEP that can inhibit the labeling of the light switch. The FraC constructs were eluted from the Ni-NTA resin with 3 times 250 μL of nitrogen degassed elution buffer (50 mM NaH 2 PO 4 pH 9.5, 150 mM NaCl, 200 mM EDTA) in a darkroom in light protective microtubes. After each elution step, the protein concentration was immediately determined spectroscopically (ε 280nm (FraC)= 43.9 × 10 3 M –1 cm –1 ) and the azobenzene A was added in a 1:50 ratio (protein/azobenzene). The stock solution of switch A was previously freshly prepared in 1/1 solution of water/DMSO. The labeling mixture was incubated with the protein for 2 h while shaking and preventing any ambient light exposure ( Figure S1 ). The excess photoswitch was separated from the labeled protein with two desalting columns (HiTrap Desalting column, Cytiva) connected to an Äkta pure chromatography system. The sample was applied to the HiTrap desalting columns equilibrated with running buffer (15 mM Tris-HCl pH 9.5). Protein elution was monitored by measuring absorbance at 280, 365, and 420 nm wavelengths. The first eluted peak corresponds to the FraC monomers labeled with the visible light switch ( Figure S2 ). Collected elution fractions of the first peak were combined and concentrated by using the rotary evaporator at 35–40 °C. Protein concentration was determined by UV-spectroscopy while approximating the same extinction coefficient as unlabeled FraC (ε 280nm (FraC)= 43.9 × 10 3 M –1 cm –1 ). Labeled FraC monomers were stored at 4 °C until further use. Cancer Cell Cytolytic Activity Assay Cancer Cells The human hypopharyngeal squamous carcinoma cell line FaDu was obtained from the American Type Culture Collection (HTB-43, ATCC). The adherently growing FaDu cancer cells were cultured in DMEM medium (Lonza) supplemented with 10% fetal calf serum (FCS, ThermoFisher) at 37 °C in a humidified 5% CO 2 atmosphere. Assessment of Anticancer Activity of FraC Variants The capacity of the various FraC constructs to eliminate FaDu cancer cells was assessed by flow cytometry using the annexin-V-FITC/propidium iodide staining method. In short, FaDu cancer cells were cultured in 48-well culture plates (1.5 × 10 4 cells/well) and treated (or not) with the indicated concentrations of control buffer, FraC-138C-ON, or FraC-138C-OFF for 2 h at 37 °C. Subsequently, cancer cells were harvested from the respective wells and evaluated for the percentage cells Annexin-V/PI positive by flow cytometry. Cancer Cells The human hypopharyngeal squamous carcinoma cell line FaDu was obtained from the American Type Culture Collection (HTB-43, ATCC). The adherently growing FaDu cancer cells were cultured in DMEM medium (Lonza) supplemented with 10% fetal calf serum (FCS, ThermoFisher) at 37 °C in a humidified 5% CO 2 atmosphere. Assessment of Anticancer Activity of FraC Variants The capacity of the various FraC constructs to eliminate FaDu cancer cells was assessed by flow cytometry using the annexin-V-FITC/propidium iodide staining method. In short, FaDu cancer cells were cultured in 48-well culture plates (1.5 × 10 4 cells/well) and treated (or not) with the indicated concentrations of control buffer, FraC-138C-ON, or FraC-138C-OFF for 2 h at 37 °C. Subsequently, cancer cells were harvested from the respective wells and evaluated for the percentage cells Annexin-V/PI positive by flow cytometry. Supplementary Material cb3c00640_si_001.pdf

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