INK4A/Rb rather than on regulation of p19ARF/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16 Bmi1 is part of the polycomb repressive complex 1 (PRC1), a transcriptional repressive complex that silences genes during embryonic development. PRC1 complexes act through recognition of H3K27Me3 epigenetic tags on histone 3 INK4A and p19ArfINK4A and p19Arf suggested that p16INK4A/p19Arf independent targets are responsible for the severe phenotype in mice Among the Bmi1 target genes are regulators of stem cell self-renewal, partially by repressing the senescence and apoptosis-regulating genes p16INK4A and p19Arf dependent as well as independent oncogenic functions of Bmi1 have been shown In cancer, both p16INK4A/Rb mediated effects rather then through p19ARF/p53 effects.Many reports have shown over expression of Bmi1 in tumors of astroglial, neural or neuroendocrine origin Table 1.All animal experiments have been conducted with approval of the ethical committee of the Netherlands Cancer Institute under references 04.003 B21/04.003 B38ext and 04.003 B23/04.003 B39ext. Genetically engineered mice with predispositions leading to cancer were checked twice every week for the occurrence of tumors. Animals were immediately sacrificed when a tumor was detected. All procedures have been conducted according to the standard operating procedures of the institute.Figure S1A, C and results not shown). The first mouse model is referred to as Bmi1LSL and the latter as Bmi1Lox66/Lox71. Detailed information on the transgenes is available at MGI . Reference numbers are: Bmi1LSL, MGI:4398910, Gt(ROSA)26Sor<tm1Nki>, also named Bmi-CTS, when inbred on a FVB background: NKI strain# 1353, when inbred on a C57BL/6 background: NKI strain# 1656. Bmi1Lox66/Lox71, MGI:4398914, Gt(ROSA)26Sor<tm2Nki>, also named Bmi-CTI, when inbred on a FVB background: NKI strain# 1148, when inbred on a C57BL/6 background: NKI strain# 1657.The cDNA encoding mouse Bmi1 with a downstream internal ribosome entry site (IRES)-eGFP cassette was cloned in the Rosa 26 targeting construct CEB11. This construct contains three transcriptional stop (poly A) signals flanked by LoxP sites to enable removal of this stop cassette upon LoxP-recombination (LSL transgenic strain was subsequently crossed with Actin-Cre (Acre) strain to analyze the phenotype when Bmi1 is activated constitutively throughout the mouse. In addition, transgenic mice were crossed to Glial fibrillary acidic protein promoter driven Cre 2Brn), LoxP, LSL to analyze the contribution of Bmi1 over expression on top of this background. All breeding was done on an FVB background. All tumors were analyzed by the pathologists of the NKI and in case of doubt, dr. Annemiek Rosemöller, of the VU Medical Center, Amsterdam was consulted.The Bmi1LSL locus by LoxP recombination in the liver by intravenous injecting adenovirus which expressed Cre . For this, 2×109 virus particles per mouse (estimated 2% infection of hepatocytes) in a total volume of 100 µl was injected. Wild type mice were treated simultaneously as a reference. One week before the experiment, the mice were given cyclosporine  to reduce immunosupression of adenovirally infected cells. Rosa reporter mice (R26R) which have a Cre inducible beta galactosidase gene targeted to the Rosa26 locus were used as a control for the viral infection  or 1∶200 (in mouse tumors unless otherwise stated)), ACTH (Organon 11150), hGH (DAKO A570), LH , Prolactine (DAKO A569), TSH (DAKO A574), NCAM-1 , CAM 5.2 (B&D 349205), Chormogranine A (DAKO A430), anti-human Ki 67 , anti-mouse Ki 67 , Phospho-Histone H3 (Upstate 06-570), glial fibrillary acidic protein , S-100 protein , synaptophysin , p75 NTR, also named low affinity nerve growth factor (NGF) receptor , F4/80 antigen, a 160 kD glycoprotein expressed by murine macrophages , neurofilament , Keratin-8 . Antibodies were detected by peroxidase staining using the Powervision system (Immunologics) followed by visualization on a Zeiss Axiovert microscope.For electron microscopy, a selected area from paraffin-embedded material was dissolved in 1% osmiumtetroxide in toluene and embedded in epoxyresin LX-112. Light microscopy sections were stained with toluidine blue. EM sections were stained with tannic acid, uranyl acetate and lead citrate and examined in a Philips CM10 transmission electron microscope .Detection of eGFP in recombined transgenic ES cells was done on a Facs scan (BD).bona fide oncogene in non-lymphoid compartments, we analyzed the effects of over expression of Bmi1 in mice. For this, we developed a conditional lox-stop-lox Bmi1 transgene model, further on referred to as Bmi1LSL. In this model, Bmi1 is conditionally over expressed from the ROSA26 locus, under control of a combined CMV and beta-Actin (CAG) promoter. This enables constitutive expression of Bmi1 in the cellular compartment of interest independent of the transcriptional signals that normally regulate Bmi1 . All subsequent steps described below were performed with the Bmi1LSL mice.Bmi1 is a proto-oncogene which was identified by its ability to initiate lymphoid tumors Proper gene targeting was confirmed by Southern blot  with an expected frequency of 25%, p>0.1) and mice that were alive were observed until day E15.5-E18.5 suggesting that a neonatal defect is the reason for the death of the mice. Since Bmi1 deficient mice have strong growth defects, we analyzed growth during embryonic development ; Bmi1LSL mice allowing transgenic expression of Bmi1 in GFAP positive cells, which encompass different types of mature astrocytes as well as neural progenitors. Interestingly, these mice generated tumors with a latency of about one year, which shows that transgenic over expression of Bmi1 is sufficient to generate solid tumors  of which 6 tumors showed intracranial localization. In addition, a mammary tumor and a primitive neuroectodermal tumor (PNET) were found. The intracranial tumors consisted of cells of uniform size with round to oval nuclei with a fine chromatin patterning and a moderate to abundant amount of pale cytoplasm. These tumors are referred to as typical and this was observed in 5 out of 6 cases. In one case we observed an atypical form with anaplastic cells with a pleiomorphic nuclei and a thin rim of pale cytoplasm. This latter form was sometimes observed in combination with the typical form as well. The highly vascularised tumors localized to the pituitary gland/hypothalamus as observed from their caudal localization relative to the brain . To confirm that the tumors were derived from endocrine cells of the pituitary gland, the tumors were subjected to immunohistochemistry using a panel of pituitary hormones. This showed that the typical tumors were positive for adrenocorticotropic hormone  as are commonly observed in hormone producing pituitary adenomas (M and N).The number of Ki67 positive cells  ranged from less than 1% to up to 8.4% comparable to human pituitary specimens that typically show between 1% to 3.8% positivity (MIB index). Cytokeratin 8/18 as stained by the CAM5.2 antibody was negative (1). Additional analysis of neuroendocrine markers (Table S1) showed that the tumors stained positive for the intermediate lobe pituitary marker beta-endorphin (B-END) and synaptophysin (SYN), which are commonly observed in pituitary adenomas NGFR (not shown) and the glandular epithelial marker KER8. The tumor that showed the atypical cytomorphology had a different marker profile consisting of a mosaic pattern of expression of the neuroendocrine markers  mice Human pituitary adenomas are derived from the anterior lobe LSL potentially represses p19ARF/p53 function, we anticipated that GFAP-Cre; Bmi1LSL; RbLox/Lox mice might form medulloblastomas. To test this hypothesis, we crossed GFAP-Cre;Bmi1LSL on a RbLox/Lox background (n = 12). We analyzed the incidence of tumors and these were histologically classified by two independent pathologists  and these formed medulloblastomas with an early onset as expected . These data indicate that the oncogenic function of Bmi1 in GFAP positive cells depends on p16INK4A/Rb regulation and less on p19ARF/p53 regulation.Combined loss of p53 and Rb mediated by GFAP-Cre in the cerebellum leads to medulloblastoma Lox/Lox;Bmi1LSL n = 12, GCre;RbLox/Lox n = 20). Although we did observe transgenic Bmi1 expression  is therefore dominant over inactivation the locus by Bmi1 over expression (which happens in in 40% of the cases).Earlier reports describing pituitary tumors because of Rb loss described a similar localization and histology LSL; Rbox/LoxL mice, shows that the transgenic Bmi1 over expression is insufficient to functionally inactivate the p19ARF/p53 pathway and indicates that the oncogenic role of Bmi1 is primarily dependent on repression of p16INK4A/Rb.Together, our results show that transgenic over expression of Bmi1 is sufficient to generate adrenocorticotropic pituitary tumors and a subset of clinical specimens of pituitary adenomas show high expression of Bmi1. The phenotype of Bmi1 over expression overlaps with the phenotype observed in Rb deficient mice and absence of medulloblastoma in GFAP-Cre; Bmi1Bmi1 is a proto oncogene and it’s over expression has been observed in many tumors of neural and astroglial origin Table 1.INK4A expression by hypermethylation is a common mechanism in human pituitary tumors INK4A locus or, alternatively, points to an incomplete transcriptional repression of the p16INK4A pathway or could simply reflect the efficiency of Cre-mediated recombination events for the alleles used. Since no higher incidence of pituitary tumors was found upon combined Rb loss and Bmi1 over expression, this indicates that the oncogenic function of Bmi1 is dependent on the presence of a functional p16INK4A locus, this is substantiated by the observation that in the latter case, 40% of the tumors are positive in IHC for Bmi1, indicating that the remaining 60% of the tumors have inactivated the Rb allele independent of Bmi1 over expression.The incomplete penetrance and long latency period (1 year) of tumor formation upon Bmi1 over expression suggested that additional genetic or epigenetic defects might facilitate the generation of tumors. Loss of Rb itself has been shown to induce pituitary tumors in mouse models LSL mice in non transformed cells of the pituitary gland of postnatal day 8 mice indicates that that transgenic activation has occured before this time point. The occurrence of pituitary tumors using GFAP-Cre mediated recombination of Rb has been shown before in vitro effects, however, in vivo effects were moderate, reflecting the fact that high activity of p16INK4 and p19ARF are seen in cultures of neural stem cells, thereby increasing the reliance of cells upon Bmi1. In contrast, p16INK4 and p19ARF are not detectably expressed by neural stem/progenitor cells in developing or young adult mice We do not know at which stage GFAP-Cre mediated transgene activation is accomplished in our transgenic model although enhanced expression of Bmi1 in Gcre;Bmi1We show that the tumors in the transgenic mice were localized to the pituitary gland, although this could be shown for large intracranial tumors only because of lack material of earlier neoplastic stages. The majority (n = 5 out of 6) of the tumors showed a typical cytomorphology and these tumors expressed ACTH. One tumor expressed multiple markers in a mosaic pattern and was referred to as an atypical type Table 3.ARF/p53. Hence, our data point to a Rb mediated function of Bmi1 in our model. The p16INK4A or the p53 pathways are frequently mutated in glioblastoma Transgenic expression of Bmi1 failed to generate medulloblastoma even in the presence of the predisposing deletion of Rb. In contrast, the p53/Rb double deficient mice control mice developed medulloblastoma after half a year as expected In our mouse model, we identified both anterior and intermediate lobe tumors. In contrast, human pituitary adenomas are considered to be derived from the anterior lobe only since all tumors resemble cell types of the anterior lobe hormone producing cell types In conclusion, we show here that Bmi1 transgene over expression is sufficient to drive pituitary tumors and we also show that more than 50% of clinical pituitary adenomas have high expression of Bmi1. Furthermore, Bmi1 initiated tumorigenesis is not enhanced by Rb loss which shows that the oncogenic function is largely dependent on Rb function and not on p53 function, which is further substantiated by the observation that Bmi1 overexpression is not sufficient to repress p53 sufficiently to generate medulloblastomas.Figure S1LSL construct contains a transcriptional stop sequence that can be removed by LoxP recombination and the Bmi1Lox66/Lox71 construct contains two partially mutated LoxP sites that recombine upon Cre expression resulting reversion of the DNA that is flanked by the LoxP sites which results in the formation of one recombined LoxP site that has a low chance of reversal of the recombination process. ES cells that were targeted with the Bmi1LSL construct (B) or Bmi1LOX66/LOX71 construct (C) show eGFP expression after Cre mediated activation of the transgene.(A) Comparison of the two targeting constructs for conditional over expression of Bmi1. Two LoxP recombination methods are used, the Bmi1(TIF)Click here for additional data file.Figure S2LSL mice (middle panel) when compared to a wild type controls (lower panel).Bmi1 immunohistochemistry of postnatal day 8 pituitary glands (upper panel) shows enhanced expression of Bmi1 in Gcre;Bmi1 The Bmi1 antibody was used at a concentration of 1∶50. Bar is 10 µm.(TIF)Click here for additional data file.Figure S3Immunohistochemistry shows that Bmi1 transgenic mice generate pituitary tumors that stain positive for (A) beta-endorphin (B-END) and (B) synaptophysin (SYN). The tumor is negative for (C) the neural marker neurofilament (NF) as well as for the astroglial and schwann cell markers (D) GFAP and (E) S100, respectively. No Keratin 8 (KER8) staining was observed (F).(TIF)Click here for additional data file.Figure S4LSL transgenic mice shows expression of PCNA , Ki67  and phosphorylated Histone H3 , respectively.Immunohistochemistry of a typical and an atypical tumor that were generated in GCre;Bmi1(TIF)Click here for additional data file.Table S1LSLP induced tumors.IHC analysis of additional markers in Gcre; BmiP(DOC)Click here for additional data file.


The utility of the WHO-defined stage 1 and 2 clinical factors used in WHO HIV/AIDS clinical staging in predicting low CD4 cell count has not been established in Uganda. Although the WHO staging has shown low sensitivity for predicting CD4<200cells/mmTo validate the World Health Organisation HIV/AIDS clinical staging in predicting initiation of antiretroviral therapy in a low-resource setting and to determine the clinical predictors of low CD4 cell count in Uganda.3 and 350cells/mm3 was 53.5% and 49.1% respectively. Angular cheilitis, papular pruritic eruptions and recurrent upper respiratory tract infections were found to be significant predictors of low CD4 cell count among participants in WHO stage 1 and 2.Data was collected on 395 participants from the Joint Clinical Research Centre, of whom 242 (61.3%) were classified as in stages 1 and 2 and 262 (68%) were females. Participants had a mean age of 36.8 years (SD 8.5). We found a significant inverse correlation between the CD4 lymphocyte count and WHO clinical stages. The sensitivity the WHO clinical staging at CD4 cell count of 250 cells/mmThe WHO HIV/AIDS clinical staging guidelines have a low sensitivity and about half of the participants in stages 1 and 2 would be eligible for ART initiation if they had been tested for CD4 count. Angular cheilitis and papular pruritic eruptions and recurrent upper respiratory tract infections may be used, in addition to the WHO staging, to improve sensitivity in the interim, as access to CD4 machines increases in Uganda. There have been significant declines in HIV-related morbidity and mortality since the advent of anti-retroviral therapy (ART) Some previous studies have explored the utility of using other predictors of low CD4 cell count to guide initiation of antiretroviral therapy. Other clinical factors, such as anaemia and body mass index have low sensitivities in detecting eligibility for HAART initiation In Uganda, although the availability of CD4 testing is increasing, its access is limited due to cost and a lack of laboratory infrastructure, particularly in rural health facilities. Specifically, CD4 testing is not available in decentralized health units that include district hospitals and health centre IV facilitiesthat serve the largest population of HIV-infected Ugandans.The WHO clinical staging system for HIV/AIDS was developed in 1990 and revised in 2007. It uses clinical parameters to classify subjects into any one of four categories i.e. stage 1 to IV, progressing from primary HIV infection to advanced HIV/AIDS. It is these categories that are used to guide decision making for the management of HIV/AIDS patients where there is limited access to laboratory services 3 to make decisions on ART initiation. However, in Uganda the CD4 cut-off for ART initiation changed from 200 cells/mm3 to 250 cells/mm3 or 350 cells/mm3 for the World health organisation. Previous studies of the utility of the WHO HIV/AIDS clinical guidelines for determining ART eligibility, using the cut-off of <200 cells/mm3 have found the sensitivity to be in the range of 51–52% and the specificity to be in the range 68–88% Initially the WHO clinical staging for HIV/AIDS was used in line with CD4 cut-offs of 200 cells/mm3 and <350 cells/mm3. Evaluation of the WHO HIV/AIDS staging guidelines in comparison to the newer CD4 cell count cut-offs for ART eligibility is needed to determine the potential level of misclassification. In addition, examination of the individual clinical components of the WHO HIV disease staging is needed because some clinical factors associated with stage I or II illness may be predictive of more advanced immune suppression in Uganda. We postulate that if there are significant clinical factors in stages I and II that are predictive of low CD4 counts, they could improve identification of ART-eligible subjects, thereby reducing the missed opportunities for timely ART initiation. The goal of this study was therefore to determine the diagnostic properties  of the WHO HIV/AIDS clinical staging guidelines at CD4<250cells/mm3 and <350cells/mm3 and to determine the WHO HIV/AIDS Stage I and II clinical factors (symptoms and signs) that are predictive of CD4<250cells/mm3 and CD4<350 cells/mm3.Despite the wide use of the WHO HIV/AIDS clinical guidelines in Ugandan primary health care facilities, they have not yet been evaluated in Uganda against the new CD4 cut-offs for ART eligibility of <250cells/mmTo address the study objectives, we conducted a multi-centre, cross-sectional study from January to April 2007 at three JCRC sites located at Mengo , Jinja  and Kasana . All the three sites offered free HIV/AIDS treatment and care at the time of study recruitment. The study sites were selected for convenience. The number of subjects enrolled from each site was proportional to the number of ARV treatment naïve HIV patients seen at each study site in the previous 3 months. Subjects aged ≥18, known HIV positive, ART treatment naïve, and consented to participate in the study were consecutively enrolled to participate in the study.*T 5 Diff CP, while the CD4 cell count was assessed using TriTEST CD4 FITC/CD8PE/CD3 PerCP (TRUCOUNT) reagent method. Quality assurance procedures are routinely conducted to compare the results to that of external laboratories .Clinicians with training in HIV/AIDS clinical staging administered structured questionnaires to study participants. They recorded the socio-demographic data , and clinical factors used for the WHO HIV/AIDS staging guidelines. The latter were used these to determine the patients' clinical stage α2P(1−P)÷W2 where W is the width of confidence intervals was 1%, Zα at 95% confidence interval = 1.96, with an estimate of 50% of subjects that were expected to be eligible for ART The minimum sample size of 385 subjects for the study was determined using the cross sectional studies with 4Z3 and <350 cells/mm3 and calculated 95% confidence intervals for proportions, using STATA version 10.0 . We estimated the association between CD4<250cells/mm3 and <350 cells/mm3 and stages I and II clinical features using odds ratios at 95% confidence intervals. Variables with odds ratios and p≤0.2 at bi-variate analysis were included in a multivariate logistic regression model to determine the adjusted odds ratios for the association between clinical stages I & II clinical factors and low CD4 counts.We determined the diagnostic properties of the WHO clinical staging compared to CD4<250cells/mmEthical approval was obtained from the Faculty of Medicine Research and Ethics Committee of Makerere University, the Joint Clinical Research Centre, and the Uganda National Council of Science and Technology. Written informed consent was obtained from each subject before enrolment into the study.Between January and April 2007 a total of 417 known HIV patients were screened. Twenty two (5.3%) participants were excluded from the study due to; age <18 years (n = 1), prior ART experience (n = 2) and inaccessible CD4 results (n = 19). A total of 395 participants were enrolled into the study. The subjects were enrolled from JCRC Mengo (75%), Jinja (14%) and Kasana (11%). The majority (61%) of the 395 subjects were classified as in clinical stages 1 and 2. Over two thirds, (68%) of all enrolled subjects were females. . Three h3 (SD = 270.93) as compared to male participants with 216.77 cells/mm3 (SD = 226.08), (p<0.0012), while the mean CD4 counts among the urban-based population was higher at 301.62 cells/mm3 (SD = 260.57) compared to the rural-based population with CD4 cell count of 206.03 cells/mm3 (248.12), p<0.0016.The median CD4 cell count of those in WHO HIV/AIDS clinical stages 1 and 2 was significantly higher than that of participants in clinical stages 3 and 4.  We found3 and 350 cells/mm3 were 53.5% and 49.1%, while the specificities were 81.1% and 86.8% at CD4 counts of 250cells/mm3 and 350 cells/mm3. The positive predictive values at CD4 cell count of 250cells/mm3 and 350cells/mm3 was 79.1% and 90.2%, and the negative predictive values were 56.6% and 40.9%, respectively. (3 and CD4<350cells/mm3).The sensitivities of the WHO clinical staging at CD4 cell count cut-offs of 250 cells/mmctively.  There we3 while angular cheilitis and recurrent upper respiratory tract infections  that had either angular cheilitis or pruritic eruption had a CD4 cell count of <250cells/mm3. Thus 44 subjects would have been correctly considered eligible for ART if any of the two clinical features had been used, while only 19 of the 63 subjects would have been started on ART earlier than the required time if they were considered. The identification of 44 eligible patients using the supplemental clinical symptoms of angular cheilitis and papular pruritic eruption would have improved the sensitivity of the clinical staging from 53.5% to 73.0% and subsequently reduced the false negative rate from 46.5% to 27%.To assess the effect of the use of angular cheilitis and papular pruritic eruption on the sensitivity, specificity and false negative rate to predict CD4<250cells/mm3 among patients in Stage I and II, a variable “clinical factor present” was generated if any of the patients in Stage II disease had one or both clinical factors of angular cheilitis and recurrent upper respiratory tract infection present.To assess the effect of the use of angular cheilitis and recurrent upper respiratory tract infections on the sensitivity, specificity and false negative rate to predict CD4<350cells/mm3 and therefore would have been eligible for ART initiation, while only 18 of the 81 subjects would have been treated earlier than the required time if it they were considered. The identification of63 eligible subjects using only angular cheilitis and recurrent upper respiratory tract infections would have improved the sensitivity of the clinical staging from 49.1% to 71.5% and subsequently reduced the false negative rate from 50.9% to 28.5%.Sixty three (77.8%) of the 81 subjects that had either angular cheilitis or upper respiratory tract infections on both were found to have CD4 counts <350cells/mm3 and 350cells/mm3, respectively. Our findings replicate the low sensitivities that have been previously found in studies of the WHO staging guidelines conducted in Africa Although the WHO HIV/AIDS clinical staging plays a critical role in guiding primary health workers in initiating antiretroviral therapy 3 and 350cells/mm3 respectively, which was also similar to previous studies using various CD4 cell count cut-offs 3 and at CD4<350cells/mm3 of 79.1% and at 90.2%, respectively; and negative predictive values of 56.6% and 40.9% respectively.We found that the specificity of the WHO HIV/AIDS clinical staging guidelines were at 81.1% and 86.8% at CD4 counts of 250cells/mmThe finding that females had a higher mean CD4 cell count than the males is similar to previous findings that demonstrated that women in stage 1 were less likely to have a lower CD4 cell count compared to the men The urban based population in this study may have had a higher mean CD4 cell count compared to the rural population because of better access to the HIV counselling and testing services leading to earlier HIV diagnosis.3 while angular cheilitis and recurrent upper respiratory tract infections predicted a CD4 cell count <350 cells/mm3. These results are in agreement with a Tanzanian study that found that HIV associated mucocutaeous manifestations may improve the sensitivity of the WHO staging guidelines In our study, we found that angular cheilitis and papular pruritic eruptions were significant predictors of CD4 cell count <250cells/mmWhile the WHO HIV/AIDS clinical guidelines are an affordable method to determine ART eligibility, routine or low cost CD4 T-cell count, as compared with WHO HIV/AIDS clinical staging in a resource limited setting is very cost-effective for sub Saharan Africa However, the availability of the tests and the laboratory personnel is still limited in Uganda. Therefore, determining other clinical factors that are predictive of low CD4 cell count in clinical stages I and II, will be very useful in improving the identification of subjects eligible for ART initiation until low cost CD4 cell count testing is widely available in Uganda. We recommend that similar studies with larger sample sizes be conducted in other developing countries to assess the role of clinical predictors, in addition to WHO staging guidelines in determining equivalents to CD4 counts.3 and 350cells/mm3. Further studies are recommended to assess the association between rare clinical features and CD4 cell counts.Our study had much strength; there was minimal referral bias since this was a multi centre study. Random error was minimised by having an adequate sample size for the common clinical predictors. The study was limited by a lack of sufficient sample size to detect the association between rare clinical conditions and CD4 counts below 250cells/mmWHO HIV/AIDS clinical stage misclassification was minimised by use of experienced clinicians trained in the study protocols and blinding of the clinicians to the CD4 results.Based on the findings of this study, we recommend increased access to CD4 cell count testing machines in resource limited settings. In the interim, more study is needed to confirm the clinical features that we found to be predictive of low CD4 counts. If these results are confirmed, then ART initiation among persons with these clinical features should be started regardless of WHO HIV/AIDS clinical stage. This will go a long way in identifying more patients eligible for ART.


Studies investigating the outcome of conservative scoliosis treatment differ widely with respect to the inclusion criteria used. Prospective cohort studies are available using the SRS inclusion criteria for studies on bracing ,2. This 34 patients (of 152) fulfilled the SRS inclusion criteria with an average age of 12.06 years (10 – 13 years), average Cobb angle of 31 degrees (25 – 40°), an average Risser stage of 0,35, average in-brace Cobb angele of 13° (= 59% of in-brace correction). There were 17 thoracic, 10 double major, 6 lumbar and 2 thoracolumbar curve patterns. After change of workplace of the second author the patients could not be followed up as planned. Therefore a telephone interview was performed by the first author.28 patients (average age 16.5 years) have been reached, 9 of them were still under treatment. No patient has been operated (Rate of surgery 0%) and only one was not satisfied with cosmetic outcome of treatment.Rate of surgery was far less than reported in recent studies using the same inclusion criteria even when all drop outs where rated as failures ,2.Rate of surgery can be reduced with the help of Chêneau braces of the latest standard and satisfactory in-brace correction.


We discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined.125I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver.We generated mutant mice with a novel Cthrc1 null allele by homologues recombination. Cthrc1 null mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 null mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 null mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with Cthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology. We originally discovered collagen triple helix repeat containing 1 (Cthrc1) in a screen for novel sequences induced in rat carotid arteries upon balloon catheter injury Targeted replacement of the first exon of the Cthrc1 gene by a LacZ reporter gene in mice was reported to demonstrate expression of Cthrc1 in inner ear hair cells Expression analyses at the RNA level using in situ hybridization have identified the sites of Cthrc1 expression during embryonic development. In addition, our studies have also shown that Cthrc1 is expressed by the activated fibroblast of remodeling tissues following injury The pituitary gland is the master endocrine gland, with the anterior pituitary expressing and secreting a variety of hormones and the posterior pituitary releasing oxytocin as well as vasopressin expressed by neurosecretory cells of the hypothalamus. Colloid-filled follicles of the anterior pituitary containing PAS (periodic-acid Schiff reaction) positive material have been reported in several vertebrates including humans Here we generated mutant mice with a novel targeted Cthrc1 null allele and focused on the analysis of their phenotype in adulthood. Monoclonal antibodies were generated against C terminal and N terminal epitopes of Cthrc1, which allowed us to localize Cthrc1 in tissues of a variety of species including pig. Our results demonstrate circulating levels of Cthrc1 in human plasma including expression in the anterior pituitary as well as neurosecretory nuclei of the hypothalamus.Guide for Care and Use of Laboratory Animals. All surgical interventions were performed under general anesthesia with tribromoethanol/tert. amyl alcohol. Human plasma samples were obtained under a protocol approved by the Institutional Review Board of the Maine Medical Center (protocol number 3657).All protocols involving animals were approved by the Institutional Animal Care and Use Committee of the Maine Medical Center (protocol numbers 0905 and 1112) and were in compliance with all applicable regulations and guidelines including the National Institutes of Health 5′-CCACTGGAAACCTCTGGAGTTG-3′ and 5′-AAGTTCACACAAAGGAAGCCCCGC-3′) and the mutant allele (primers: 5′-GTGTGTTTTGAGGTGTGGTCCC-3′ and 5′TGGATGTGGAATGTGTGCGAGG-3). These animals have been backcrossed on the 129S6/SvEv (Taconic) or C57BL/6J background for 11 generations. Cthrc1 null mice for experiments were derived from matings of homozygous mutants. The mutant allele was backcrossed regularly with 129S6/SvEv or C57BL/6J breeder stock to prevent genetic drift. The age of the mice used for specific experiments is indicated in the figure legends.We generated a novel Cthrc1 null allele by replacing exons 2, 3 and 4 with a neomycin cassette using the targeting vector pKO Scrambler NTKV-1905 (Stratagene) . Exon 1 Mice were fed a standard rodent diet  and water ad libitum, and housed with dry cellulose bedding  under a 14 hour daylight-10 hour night cycle. Litter size was determined as the number of pups weaned at the age of 21 days after birth. Growth curves were obtained for Cthrc1 null and wild type mice on the 129S6/SvEv (n = 5–33 per time point).Livers of seven month old mixed gender Cthrc1 null and wild type mice on the C57Bl/6J background were examined to assess histology and lipid content. Lipid storage was determined by image analysis of oil red O stained sections using the same approach as described previously 5′- GACCCCTTCATTGACCTCCACTAC-3′ and 5′-ACATACTCAGCACCAGCATCGC-3′ for Gapdh and 5′-GAATGCCTGAGGGAAATCTTTGAG-3′ and 5′-CGTCTCCTTTTGGGTAATCTGCG-3′ for Cthrc1. The annealing temperature was 55.9°C and 35 cycles of amplification were performed.Pig tissues were obtained from euthanized animals (6 to 12 months of age) of the Advanced Trauma Operative Management (ATOM) courses conducted at our facility or from a local slaughterhouse. Primer sequences used for RT-PCR of pig cDNA were Rat tissues from three month old male Sprague Dawley rats were kindly provided by Dr. Renée LeClair .Mixed gender wild type and Cthrc1 null mice on the 129S6/SvEv background were used for this assay . Samples were obtained from identical sites of the liver and the right gastrocnemius muscle. Glycogen from 10 mg fresh-frozen tissue was hydrolyzed to glucose in 500 µl of 2 N HCl for 2 h at 100°C with vortexing every 30 minutes. Samples were neutralized with 500 µl 2 N NaOH and 50 µl 1 M TRIS pH = 7.6. The glucose hexokinase reagent (ThermoFisher) was used as directed. A standard curve was established with glucose standards covering the range of 0.00312 to 0.1 µmole. Using GraphPad Prism software, concentrations of glucose in the samples were calculated based on the standard curve. Three different samples from each organ were assayed in duplicates and mean values were calculated and compared using Student’s t-test. The assay was performed four times with separate sets of samples from different mice and similar results were obtained each time. The results of a representative experiment are shown.Body composition was determined as described using a Lunar PIXImus II Mouse Densitometer  For glucose tolerance testing, mixed gender wild type and Cthrc1 null mice on the 129S6/SvEv background  were injected intraperitoneally with 1 g of glucose per kg of body weight after 16 hours of fasting. For insulin stress testing, mixed gender, non-fasted wild type and Cthrc1 null mice on the 129S6/SvEv background  were injected intraperitoneally with insulin . Blood was obtained for glucose level measurements using the One Touch Ultra glucometer at the indicated times.A rabbit monoclonal antibody was raised against a synthetic peptide of the conserved C terminus of Cthrc1 (GDASTGWNSVSRIIIEELP) using the services of Epitomics, Inc. . Clone Vli-55 was suitable for Western blotting and immunohistochemistry on paraffin-embedded, paraformaldehyde-fixed tissues. Rabbit monoclonal Vli-55 was used at 20 ng/ml for immunohistochemistry on paraffin-embedded, formalin-fixed tissue section following antigen retrieval with citrate buffer . Subsequent steps of the immunostaining procedure were executed as previously published For detection of human Cthrc1 in plasma, mouse monoclonal antibodies were raised against a synthetic peptide with sequence of the N terminus of human Cthrc1 (SEIPKGKQKAQLRQRE) using the hybridoma services of Maine Biotechnology Services . Anti-Cthrc1 clones 10G7 and 19C7 detected human Cthrc1 expressed in CHO-K1 cells by indirect ELISA without amplification in the low picogram range. Protein A-purified antibodies were conjugated to magnetic beads  following the manufacturer’s protocol. EDTA plasma was obtained from healthy volunteers. 15 ml of plasma were incubated overnight at 1°C with anti-Cthrc1 conjugated magnetic beads, and washed extensively with phosphate buffer prior to elution with 0.1 M glycine, pH = 2.6. The eluate was immunoblotted with HRP-conjugated monoclonal anti-Cthrc1 antibodies following SDS-PAGE under reducing conditions.125I (Perkin Elmer) using iodination tubes (Pierce). Six µg of radioactive labeled Cthrc1 were infused into adult anesthetized Cthrc1 null mice via the left carotid artery (n = 3 mice). Blood samples were obtained at indicated times and Cthrc1 levels were determined in a gamma counter. The half-life in circulation was calculated from the clearance curve. SDS-PAGE analysis followed by autoradiography was performed on 1 µl of plasma obtained thirty minutes after injection of 125I-Cthrc1 to verify its integrity. All tissues were harvested six hours after 125I-Cthrc1 injection following extensive perfusion with lactated Ringer’s solution to remove as much blood from organs as possible. 125I-Cthrc1 per mg wet weight of tissue was measured by gamma counting.An adenovirus was generated expressing rat Cthrc1 with a C terminal myc/6×His tag. CHO-K1 cells were transduced with this adenovirus and Cthrc1 protein was purified from the conditioned medium with HIS-Select affinity gel (Sigma) following the supplier’s instructions. Silver-stained SDS-PAGE gels demonstrated >95% purity of the purified protein (not shown). A BCA protein assay (Pierce) was used to determine the concentration of the purified protein. Purified Cthrc1 was labeled with HEK293-T and CHO-K1 cells were grown as described and transfected with an expression vector for human Cthrc1 using Fugene6 HD (Roche) Data are expressed as means ± standard deviation. Student’s t-test was used for all calculations. P≤0.05 was considered significant.in vivo, we generated a novel Cthrc1 null allele by replacing three of the four exons (exons 2–4) with a neomycin cassette (tm1VliCthrc1) (To characterize Cthrc1 function 1tm1Vli) . This mu1tm1Vli) . Mice weTo verify that our Cthrc1 allele is a null mutation, we generated and characterized anti-Cthrc1 antibodies. We succeeded in developing highly sensitive and specific rabbit monoclonal antibodies against the conserved C terminus of Cthrc1. These antibodies allowed us to detect Cthrc1 by immunoblotting and in formalin fixed, paraffin embedded tissue sections by immunohistochemistry .The average litter size was slightly lower for Cthrc1 null mice, however, this did not reach a statistically significant level . Growth Liver histology with staining of sections for carbohydrates with the Periodic Acid Schiff (PAS) stain suggested increased carbohydrate storage in hepatocytes of Cthrc1 null mice on the 129S6/SvEv background. Quantification of the glycogen content in liver tissue with biochemical methods confirmed increased glycogen content in livers from Cthrc1 null mice . MeasureRoutine histology of livers from seven month old Cthrc1 null mice on the C57BL/6J background revealed strikingly larger hepatocytes compared to wild type controls. Overall this resulted in a significantly lower cell density . The livWe undertook a comprehensive immunoblot-based and immunohistochemistry-based survey of Cthrc1 expression in tissues from adult mice, rats and pigs. Expression of Cthrc1 in two month old C57BL/6J mice was detected in the supraoptic nucleus son,  and paraIn wild-type mice older than six months of age, we occasionally observed small accumulations of Cthrc1 immunoreactivity in the anterior pituitary . In all As shown above, certain areas of the brain constitutively express Cthrc1 but we currently have no evidence that Cthrc1 from those sites enters the circulation. In brains from pigs, we found foci of paraventricular cells of the lateral ventricles that expressed Cthrc1  and thesIn the anterior pituitary immunoreactive Cthrc1 was observed in close association with chromophobe cells  suggesti125I-labeled Cthrc1 into the carotid artery of Cthrc1 null mice and obtained the clearance curve shown in Immunoblotting of plasma from Cthrc1 transgenic mice We generated mouse monoclonal antibodies suitable for detection of Cthrc1 by ELISA. These antibodies conjugated to magnetic beads were used to isolate Cthrc1 from 15 ml of plasma. For one sample (#1)  the eluaWe originally discovered Cthrc1 in a screen for genes upregulated in balloon-injured arteries, where it was highly induced in activated adventitial cells. Database searches and sequence alignments revealed that Cthrc1 is a highly conserved and unique gene in chordates with no homologues found in lower species such as flies or worms Yamamoto et al. Here we generated a novel Cthrc1 null mutant mouse and focused on the characterization of its phenotype in adulthood. Unlike the two other reported targeted Cthrc1 mutants In summary, our study identifies Cthrc1 as a novel circulating hormone with metabolic effects. Expression in the hypothalamus, pituitary gland and remodeling tissues are likely to contribute to Cthrc1 plasma levels.125I-Cthrc1 to the liver supports this concept. The Cthrc1 null mutant mice examined here were derived from matings of homozygous null mice. Therefore, we cannot rule out the possibility that some of the metabolic abnormalities seen in the null mutants were due to maternal influences. However, litter sizes of the wild type and the homozygous null matings were comparable and we did not see any evidence of early postnatal failure to thrive on any genotype.While Cthrc1 expression was not detectable in the liver and skeletal muscle of the wild type, examination of these organs in our Cthrc1 mutant mice on the C57BL/6J background revealed excessively fatty livers and increased glycogen levels in skeletal muscle and livers of Cthrc1 null mice on the 129S6/SvEv background. This ultimately led us to consider the possibility that Cthrc1 functions as a hormone. We have currently no data related to the mechanism how Cthrc1 affects these organs but our findings do suggest that hepatocytes and myocytes in vivo may express a receptor for Cthrc1 and the binding of With a sensitive monoclonal anti-Cthrc1 antibody suitable for detection by ELISA we succeeded in demonstrating the presence of Cthrc1 in plasma. Working with plasma we deliberately avoided the use of secondary anti-IgG antibodies because even minimal cross-reactivity with human immunoglobulins could make discrimination between Cthrc1 and the similarly migrating band of the immunoglobulin light chain difficult on a Western blot. As shown in The current study also sheds light on the identity of colloid-filled follicles and the anterior pituitary as a source of Cthrc1. In guinea pigs, the first few colloid follicles of the anterior pituitary are detected at the age of 6 months with an average of just over 4 µm in size


Background. Hepatitis C virus (HCV) genotype 3 is known to cause steatosis (fatty liver) that is more frequent and severe than other genotypes. We previously identified sequence elements within genotype 3 HCV Core domain 3 that were sufficient for lipid accumulation. Aims. We examined various genotype 3 Core domains for lipid droplet localization and compared the lipid droplet binding regions of domain 2 with a genotype 1 isolate. Methods. We generated HCV Core domain constructs fused with green fluorescent protein and performed immunofluorescence to visualize lipid droplets. Results. Constructs containing HCV Core domain 2 are appropriately localized to lipid droplets with varying degrees of efficiency. When compared to genotype 1, there are polymorphisms within domain 2 that do not appear to alter lipid droplet localization. Conclusions. In summary, the differences in a steatosis-associated HCV Core genotype 3 isolate do not appear to involve altered lipid droplet localization. The significant differences with genotype 3 core protein that have been highlighted are the phenylalanine at position 164 and sequence polymorphisms within domain 3. Our results here demonstrate that neither of these differences contribute to significant differences in lipid droplet localization between genotype 3 and genotype 1 isolates. Deletion constructs from genotype 3 Core required domain 2 to localize to lipid droplets and sequence differences between genotype 3 and genotype 1 isolates within domain 2 did not occur within the critical regions for lipid droplet localization.In this study, we sought to further define the differences with HCV Core genotype 3 that contribute to clinical steatosis and These results further separate the phenomena of lipid droplet localization and intracellular lipid accumulation when examining the differences in HCV genotype 3 isolates. Previous studies have focused on regions within domain 2 being important for lipid droplet localization –33. In aIn the initial work describing regions within domain 2 that were required for lipid droplet binding, Hope and McLauchlan used deletion mutants of domain 2 and Boulant et al. constructed GFP fusion constructs , 31. OurWhen analyzing the sequence differences in domain 2 between genotype 3 and genotype 1 isolates, several things are notable. One is that all of the sequence differences are within the interhelical region or helix 2. Based on the work of Boulant et al., these 2 regions were absolutely essential for association with lipid droplets . HoweverOur results should be analyzed in the light of certain limitations. First, we used GFP fusion constructs which raises concern about the context of these results. However, our full-length GFP fusion and our domain 2 alone fusion both localized to lipid droplets, which is as predicted based on previously published work. The second limitation is this system lacks the context of other viral proteins and the entire viral life cycle. Again, these results are consistent with previous results using the JFH cell culture system.In conclusion, we have demonstrated that HCV Core genotype 3 exhibits the same requirements for lipid droplet localization as genotype 1 isolates and that sequence differences observed in genotype 3 do not alter the critical residues involved in this process. These results further separate lipid droplet localization from intracellular lipid accumulation in the mechanisms of steatosis formation in HCV genotype 3 infections.


This reagent reduction strategy can be used for reducing the cost of enumerating CD4 lymphocytes in high-volume laboratories from resource-limited settings.Enumeration of CD4 lymphocytes is essential for the clinical management of HIV-infected patients, but it can be difficult to afford in developing countries. In this study we evaluated a reagent reduction strategy for reducing the cost of enumerating CD4 cell absolute count and percentage using the FACSCalibur flow cytometer (Becton Dickinson). We compared the protocol recommended by the manufacturer with a protocol that used half of the usual amount of CD3/CD4/CD45 monoclonal antibody reagent in 100 samples from HIV-infected patients in a rural hospital in India. The concordance correlation coefficient between the two protocols was 0.976 for CD4 cell count and 0.984 for CD4 cell percentage. We did not find significant bias when performing Deming regression or Bland-Altman analysis. Sensitivity and specificity were 97% and 98.5% for identifying patients with less than 200 CD4 cells/ In 2010, it was estimated that 34 million people were living with HIV/AIDS and more than 90% were living in low and middle-income countries  + b[slope]x). If the value of the y-intercept is significantly different from 0 indicates a constant bias and if the value of the slope is significantly different from 1 indicates a proportional bias. The agreement between the two protocols was assessed by using the Altman-Bland method and concordance correlation coefficients (CCCs) [Statistical analysis was performed using Stata Statistical Software . Bias was estimated using Deming regression . Deming s (CCCs) . μL  and the mean CD4 lymphocyte count with protocol B was 349 cells/μL . The mean CD4 lymphocyte percentage with protocol A was 18.4%  and the mean CD4 lymphocyte percentage with protocol B was 18.4% . The study included 100 samples from HIV-infected patients. The mean CD4 lymphocyte count with protocol A was 366 cells/y-intercepts included 0 and the confidence intervals for slopes included 1. The concordance correlation coefficient was slightly higher for CD4 cell percentage than for CD4 cell count. Correlation and Bland-Altman plots are shown in Estimation of bias and agreement between the two protocols are presented in μL need to initiate prophylaxis against Pneumocystis jirovecii pneumonia and this cutoff is useful for identifying patients in risk of opportunistic infections [μL is an indication for initiating antiretroviral therapy in patients older than 5 years and having less than 25% CD4 lymphocytes is an indication for initiating antiretroviral therapy in children aged 2 to 5 years [Sensitivities and specificities of protocol B for three clinically relevant cutoffs are presented in fections . In HIV- 5 years .Although the reagent reduction strategy has been successfully evaluated in previous studies , to our Protocol B is able to provide reliable results of CD4 cell count and percentage with half of usual amount of monoclonal antibody reagent. This reagent reduction strategy can be used for reducing the cost of enumerating CD4 lymphocytes with the FACSCalibur system.


Two of these five loci have been suggested by previous association studies , and this study provides further support for these findings. Using a dataset of post-mortem brain samples assayed for gene expression (n = 399) and methylation (n = 292), we identified methylation and expression changes associated with PD risk variants in PARK16/1q32, GPNMB/7p15, and STX1B/16p11 loci, hence suggesting potential molecular mechanisms and candidate genes at these risk loci.A previous genome-wide association (GWA) meta-analysis of 12,386 PD cases and 21,026 controls conducted by the International Parkinson's Disease Genomics Consortium (IPDGC) discovered or confirmed 11 Parkinson's disease (PD) loci. This first analysis of the two-stage IPDGC study focused on the set of loci that passed genome-wide significance in the first stage GWA scan. However, the second stage genotyping array, the ImmunoChip, included a larger set of 1,920 SNPs selected on the basis of the GWA analysis. Here, we analyzed this set of 1,920 SNPs, and we identified five additional PD risk loci (combined  This paper describes the largest case-control analysis of Parkinson's disease to date, with a combined sample set of over 12,000 cases and 21,000 controls. After combining our findings with an independent replication dataset of more than 3,000 cases and 29,000 controls, we found five additional PD risk loci in addition to the 11 loci previously identified in earlier consortium efforts. This successful study further demonstrates the power of the GWA scan experimental design to find new loci contributing to disease risk, even in the context of complex disorders like Parkinson's disease. These new findings provide insights into the etiology of PD and will promote a better understanding of its pathogenesis. LRKK2PINK1SNCAPARK2PARK7SNCA and MAPT as unequivocal risk loci BST1GAKHLA-DRACMSD, STK39, MCCC1/LAMP3, SYT11, and CCDC62/HIP1RSNCA, LRRK2, MAPT, BST1, GAK and HLA-DRUntil the recent developments of high throughput genotyping and genome-wide association (GWA) studies, little was known of the genetics of typical Parkinson's disease (PD). Studies of the genetic basis of familial forms of PD first identified rare highly penetrant mutations in We conducted a two-stage association study. Combining stage 1 and stage 2, the data consist of 12,386 PD cases and 21,026 controls genotyped using a variety of platforms . Stage 1Here, we report the combined analysis for this full set of 1,920 SNPs. This step1+2 analysis identified seven new loci that passed genome-wide significance in the meta-analysis. During the process of analyzing these data and preparing for publication, we became aware that another group was also preparing a large independent GWA scan in PD for publication . Following discussion with this group we agreed to cross validate the top hits from each study by exchanging summary statistics for this small number of loci.To provide further insights into the molecular function of these associated variants, we tested risk alleles at these loci for correlation with the expression of physically close gene  and the methylation status (methQTL) of proximal DNA CpG sites in a dataset of 399 control frontal cortex and cerebellar tissue samples extracted post-mortem from individuals without a history of neurological disorders.p<10−3. We submitted the most associated SNP in each region for probe design and follow-up genotyping using the ImmunoChip platform. For each region of interest, we also added four SNPs in high level of linkage disequilibrium (LD) to provide redundancy where the most associated SNP would not pass the Illumina probe design step or the assay for that SNP would fail. To complete the array design we also added all non-synonymous dbSNPs located in known PD associated regions In addition to eleven loci that passed genome-wide significance in stage 1 p<5×10−8, −10 overall). Taking either the nearest gene (or the strongest candidate when available) to designate these regions, these five loci are 1q32/PARK16STBD1, 7p15/GPNMB, 8p22/FGF20STX1B.On the basis of stage 1+2 results, seven new SNPs passed our defined genome-wide significance threshold . To understand the potential biological consequences of risk variation at this locus we tested whether rs708723 was correlated with either gene expression or DNA methylation status of proximal transcripts or CpG sites respectively (NUCKS1 (p = 1.8×10−7) and RAB7L1 (p = 7.2×10−4). We also found correlations with the methylation state of CpG sites located in the FLJ3269 gene (p = 3.9×10−22).rs708723/1q32 has been previously reported as PD associated PARK16,  but thisectively . We founSTX1B, the proximal gene to the most associated SNP rs4889603 is SETD1A. However, STX1B is located 18 kb upstream of rs4889603 and is a more plausible PD candidate gene STX1B (In the case of 16p11/ne STX1B .FGF20 gene (NCBI build 36.3), for which association with PD has been suggested previously in familial PD samples FGF20 or not remains unclear.The SNP rs591323 in the 8p22 region is located ∼150 kb downstream of the STBD1 and 7p15/GPNMD have not been previously implicated in PD etiology. We found that the risk allele of rs156429, the most associated SNP in the 7p15 region, is associated in our eQTL dataset with decreased expression of the proximal transcript encoded by NUPL2 (GPNMB itself (The regions 4q21/by NUPL2 . The samB itself . NeitherNMD3 and 8q21/MMP16) showed strong evidence of association in stage 1 and 2 but were not disease associated in the Do et al dataset. Further replication is required to clarify the role of variation at these loci in risk for PD.Two additional loci (3q26/LRRK2 gene The strongly associated G2019S variant in the SNCA locus p>0.01) after conditioning on the main SNP in the region. In contrast, after conditioning on the most associated SNPs rs356182 in the SNCA region, several SNPs remained convincingly associated (p = 9.7×10−8 for rs2245801 being the most significant).The ImmunoChip array design provides some power to detect whether multiple distinct association signals exist at individual loci. Indeed, if a SNP showed an independent and sufficiently strong association in stage 1, it would have been included in stage 2 provided that it was not located in the same 10 kb window as the primary SNP in the region. There is precedent for this in PD, with the previous identification of independent risk signals at the LRKK2Lastly, we performed a risk profile analysis to investigate the power to discriminate cases and controls on the basis of the 16 confirmed common associated variants . For eacPARK16, 8p22/FGF20) implicate regions that had been previously associated with PD risk PARK16 showed convincing evidence of association in the Japanese population FGF20 locus had been previously reported in a study of familial PD STX1B/16p11, STBD1/4q21 and GPNMB/7p15) are new.The combination of GWA scans and imputation methods in large cohorts of PD cases and controls has enabled us to identify five PD associated loci in addition to the 11 previously reported by us. Two of these loci (1q32/p = 2.2×10−3) improvement of our ability to discriminate PD cases from controls.Adding the eleven previously reported common variants PARK16 region with the RAB7L1 and NUCKS1 genes  is in low LD (r2 = 0.21) with rs2301134, the SNP reported in SNCA/4q22. A more exhaustive fine-mapping analysis using either sequencing of large cohorts or targeted genotyping arrays will also be required to fully explore this locus.The ImmunoChip data provide limited resolution for the detection of multiple independent association signals in these regions. A previous study As yet, we do not know which of the variants and which genes within each region are exerting the pathogenic effect. We cannot exclude that some of the currently reported variants are in fact tagging high penetrance, but rare, mutations −7, minor allele frequencies at a minimum of 1%, exclusion of first degree relatives, and the exclusion of ancestry outliers based on either principal components or multidimensional scaling analyses using either PLINK Participating studies were either genotyped using the ImmunoChip as part of a collaborative agreement with the ImmunoChip Consortium, or as part of previous GWA studies provided by members of the IPDGC or freely available from dbGaP Single SNP test statistics were combined across datasets using a score test methodology, essentially assuming equal odds ratio across cohorts. In addition, fixed and random effects meta-analyses were implemented in R (version 2.11) to confirm that the score test approximation does not affect the interpretation of the results. We also tested the relevant SNPs heterogeneity across cohorts and no significant heterogeneity was detected .2 value on their genotyping platform and provided us with the following summary statistics: odds ratio, direction of effect, standard error for the estimated odds ratio and one degree-of-freedom trend test P-value.We communicated to our colleagues in charge of the independent study  the seven SNPs listed in Quantitative trait analyses were conducted to infer effects of risk SNPs on proximal CpG methylation and gene expression. For the five replicated SNP associations , all avaTable S1Summary of results for fixed and random effects meta-analysis, as estimates of effect heterogeneity across cohorts and SNP used at the Do et al replication stage.(XLSX)Click here for additional data file.Table S2Summary of the quality control parameters applied to the GWA datasets included in this study.(XLSX)Click here for additional data file.Table S3Complete list of tested QTL associations (expression and methylation).(XLSX)Click here for additional data file.Text S1Membership of the Wellcome Trust Case Control Consortium 2.(DOC)Click here for additional data file.


LAMA2 gene that encodes the laminin α2 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected based on homozygosity mapping and laminin α2 chain deficiency.Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases.We investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2 gene, a homozygous T > C substitution at position +2 of the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients from the 4 families revealed variable disease severity particularly as regards motor achievement and disease progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases indicating a founder effect of the mutation in all 4 families.A previously described mutation in the LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical  LAMA2 gene encoding the laminin α2 chain , α-dystroglycan , α-dystroglycan , perlecan  and collagen VI  were investigated in patient 3. Dystrophin, α-, β- and γ-sarcoglycan  were tested in patients 6 and 8.Cryosections were incubated in primary antibodies diluted in PBS for 1-2 h at room temperature or overnight at 4°C. Slides were washed 5 min ×2 in PBS, then secondary antibodies were applied in PBS for 30 min-1 h at room temperature. After washing again 5 min ×2 in PBS, coverslips were mounted using ProLongGold with DAPI . Secondary antibodies were either goat-anti-mouse tagged with AlexaFluor488 or goat-anti-rat tagged with AlexaFluor594 (both from Molecular Probes).LAMA2 exons (exon numbering according to the Leiden muscular dystrophy database) [Genomic DNA was extracted from peripheral blood and analyzed by the polymerase chain reaction touchdown method using oligonucleotide primers flanking the intron-exon junctions of all 65 atabase) . AberranTotal RNA was prepared from skeletal muscle using TRI Reagent . The isolated RNA was reverse transcribed using a First-Strand cDNA Synthesis Kit , the resulting cDNA was amplified by reverse transcriptase PCR (RT-PCR) and sequenced using appropriate primers.Haplotype analysis was performed on genomic DNA using microsatellite markers provided by Genethon human genetic linkage map  and flanClinical findings are summarized in Additional file Electrocardiogram (performed in 5 patients) and echocardiogram (performed in 4 patients) were normal; cardiac signs were reported in none of the patients. Respiratory support was necessary in patient 3 who died of respiratory failure at 7 years and in patient 5 who also died of respiratory failure at 16 years. No mental retardation, epilepsy or eye abnormalities were observed. MRI or CT, performed in 8 patients, revealed white matter changes in all cases  showed mild dystrophic features; whereas muscle biopsies from patients 5 (taken at 3 years and 2 months), 6 (taken at 5 years) and 8 (taken at 4 years) showed marked dystrophic features. Immunofluorescence analysis of the laminin α2 chain was only performed in patient 3 (with 3 antibodies) and in patient 8 (1 antibody); this revealed greatly reduced staining intensity with the 3 antibodies in patient 3  revealed abnormal conformers in exon 26. The exon was sequenced and showed a T > C substitution at position +2 of the consensus donor splice site. This mutation has been described previously in two siblings from a consanguineous Saudi family unrelated to ours . Direct Because of the consanguinity in the Saudi families and presumed consanguinity in the Sudanese family, it is likely that all affected siblings are homozygous for this mutation.LAMA2 gene in the 4 index cases indicated an identical mutation-associated haplotype (containing the mutation) between marker D6S407 and intragenic polymorphism G6286A (data not shown), suggesting remote consanguinity and a founder effect in all 4 families. By contrast, the downstream markers  were not identical in the 4 cases.Investigation of flanking and intragenic microsatellite markers of the LAMA2 gene associated with variable clinical phenotype. The main phenotypic differences among our 9 cases regard motor achievement. Patients 1 and 2 from the Sudanese family walked at 4 years; patients 7, 8 and 9 from a Saudi family walked at 3, 3.5 and 4 years, respectively. These 5 patients were still walking at 8, 11, 12 and 13 years when last seen, while patients 3, 4, 5, and 6 from the 2 other Saudi Arabian families never walked.In 3 families from Saudi Arabia and 1 from Sudan we have identified a previously reported homozygous c.3924 + 2 T > C mutation in the CK levels were also variable, being very high  in patients 3, 6, 7, and 8; high  in patients 1, 2, 4 and 5; and normal in patient 9.Features common to all were floppiness in infancy, delayed motor milestones or failure to achieve walking, brain white matter attenuation on MRI or CT (not done in 1 patient) and development of joint contractures/foot deformities. Eye and cardiac abnormalities were not observed. In patients 3 and 5 respiratory compromise was present, and both died of respiratory failure at 7 and 16 years, respectively.LAMA2 mutation as found in our patients (but characterized as 3973 +2 T > C according to the previous LAMA2 nucleotide sequence numbering). Both were mildly affected: the boy at 3.5 years had muscle hypotonia and inadequate head control, but walked at 26 months; his younger sister also had hypotonia from early infancy and poor head control and achieved walking at 3 years 8 months. Both had slightly reduced laminin α2 chain expression as investigated by 2 antibodies recognizing the G domain, and highly reduced expression using an antibody recognizing the N terminal. These results demonstrated for the first time that use of more than one antibody can provide valuable indications as to what domain(s) of the laminin α2 chain may be affected in CMD patients. Thereafter, this procedure became the standard method for staining muscle of patients with CMD and has also been used for prenatal diagnosis.In 1997 Allamand et al.  reportedBy contrast, in our patient 3, analyzed with 3 different antibodies, laminin α2 chain expression was markedly reduced: clearly more so than the cases described in Allamand et al. . He was It is noteworthy that our cases show a much wider clinical spectrum than suggested by the siblings described by Allamand et al. : from thLAMA2 gene, which was unrelated to laminin α2 chain expression (completely undetectable in both cases). Such clinical variability implies the presence of other genetic or epigenetic factors able to influence disease phenotype .LAMA2- associated haplotype. Modifier genes, such as those coding for proteins that interact with laminin α2, and/or epigenetic factors, for instance those involved in regulatory signaling functions, are likely to contribute to the observed phenotypic variability. A drawback of the present study is that muscle biopsies were stained for laminin α2 in only 2 cases and extensive immunohistological characterization was only possible in one case.The data of the present study provide further evidence that the clinical spectrum of MDC1A is more heterogeneous than previously thought; motor achievement and disease progression are particularly variable, making it difficult to formulate prognoses even in patients with an identical LAMA2 gene in our patients, strongly suggest a founder effect. The mutation probably originated in Saudi Arabia since studies on severe childhood autosomal recessive muscular dystrophy (SCARMD), the common form of muscular dystrophy in North Africa and the Arabian Peninsula [http://www.dmd.nl/. The clinical implication is that a laminin α2 chain deficiency in Middle Eastern or Sudanese patients should initially prompt a search for the c.3924 + 2 T > C mutation in the LAMA2 gene.We emphasize, finally, that the identical intragenic polymorphisms and upstream microsatellite markers of the eninsula -21 indiceninsula . FurtherLAMA2-associated haplotype.Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.The authors declare that they have no competing interests.CDB carried out the molecular and microsatellite analysis and drafted the manuscript; EB performed molecular analysis; MAMS evaluated clinical and neurologic features of the patients, collected DNA and muscle biopsies and revised the manuscript critically for important intellectual content; MCM carried out homozygosity mapping and linkage analysis; SAM performed immunochemical evaluation of muscle biopsies and prepared the figures; MZS, MMM and ZAK evaluated neurologic, neurophysiologic and MRI features; CAW and KPC supervised the study and critically revised the manuscript; LM and RM contributed to writing the clinical reports, revised the manuscript critically and provided financial support; MM was responsible for study design, supervised the study and manuscript completion. All authors read and approved the final manuscript.Table S1. Clinical findings.Click here for fileFigure S1. Sequencing of genomic DNA from control (A) and patient (B) showing the T > C transition at position +2 of the consensus donor splice site of exon 26. Direct sequencing of the cDNA revealing a 189 bp in-frame deletion, corresponding to aberrant skipping of the whole of exon 26: (C) control and (D) patient.Click here for file


Histone tails play an important role in nucleosome structure and dynamics. Here we investigate the effect of truncation of histone tails H3, H4, H2A and H2B on nucleosome structure with 100 ns all-atom molecular dynamics simulations. Tail domains of H3 and H2B show propensity of  Histone tails are the most common sites of post-translational modifications. Tail modifications alter both inter and intra nucleosomal interactions to disrupt the condensed chromatin structure, thereby playing crucial role in gene access. Here we investigated histone tail functions on the stability of a single nucleosome in atomic detail by selectively truncating tail domains in molecular dynamics simulations. Our study revealed that truncation of H3 or H2A tail results in structural alterations in the nucleosome core whereas truncation of H4 or H2B tail does not. A potential role of H2A C terminal tail in regulating nucleosome stability is discussed. Finally, an  Eukaryotic DNA is organized into nucleosomes The The nucleosomal organization of eukaryotic chromatin presents a physical barrier to DNA access and also acts as a repository of epigenetic marks controlling chromosomal behavior during different periods of the cell cycle Post-translational modifications of histones play a key role in the regulation of gene access in eukaryotes A major challenge in chromatin research is to characterize the effect of tail modifications on nucleosome mobility and stability. Evidence suggests that the modifications may recruit chromatin-binding proteins Truncating the end of the H2A C terminal domain results in a 2.4 fold increase in the nucleosome sliding rate Although progress has been made Simulations were performed on the intact nucleosome and on tail truncated nucleosomes with each of the four types of histone tail  truncated, one at a time. The N-terminal tail domains of H3, H4, H2A and H2B were removed up to residues 26, 17, 11 and 20, respectively, and residues 118–128 were also removed from the C-terminal tail domain of H2A . SimulatThe starting structure of all molecular dynamics (MD) simulations was taken from the For each of the simulations, the water molecules and ions were first energy minimized and equilibrated at 300 K for 160 ps with the solute kept fixed. The whole system was then energy minimized for 10000 steps using the conjugate gradient method and keeping the positions of the protein backbone atoms fixed. Harmonic restraints on the backbone atoms were then relaxed stepwise during 30 ps heating and 160 ps equilibration of the whole system.http://www.nics.tennessee.edu/computing-resources/kraken) generating 1 ns of trajectory took approximately 1700 CPU hours on 504 cores generating about 50 gigabyte of raw data.It was observed that H3 and H2A tail truncation destabilizes the nucleosome structure. To verify these results 2 additional independent simulations of H3 tail-truncated nucleosome and 1 additional simulation of H2A tail-truncated nucleosome were performed. Each of the simulations was 100 ns long, making a total of 8 simulations and 800 ns of combined trajectory. On the Kraken Cray XT5 machine  histone tails adopt disordered conformations with many amino acids having occupancy zero. In the simulation these tail domains primarily remain bound to DNA with a low degree of ordering. The overall secondary structure conservation in histones is plotted in To examine the overall stability of the histones during the 100 ns intact nucleosome trajectory we calculated the RMSD of each histone (excluding tails) from the equilibrated structure . The ploTo quantify the overall effect of tail truncation on the nucleosome structure, we define an order parameter, upper panel).To illustrate how tail truncation affects nucleosome structure ulations . Tail trulations . We callTo locate the structural domain of the H2A monomer that is responsible for the increased RMSD, we computed ajectory . The RMSajectory .The simulations reported here are non-equilibrium simulations each of which can follow a different pathway. However, we found that certain changes involving the H2A(2)Realistic parametrization of force fields for the nucleic acids has been a long-standing problem While analyzing the DNA dihedral parameters we did find that one nucleotide base (Cyt49(J)) is unstacked and the neighboring bases (Gua98(I)-Cyt50(J)) show unusual (non Watson-Crick) base-pairing in the intact nucleosome simulation. However, this did not lead to any unusual fluctuation in the neighboring amino-acid residues. Furthermore, since the DNA backbone and helical parameter values derived from all other base pairs are in agreement with x-ray and simulation data, this may not be artefactual.DNA major and minor groove characteristics are plotted in In the intact nucleosome simulation the DNA RMSD stabilizes around 3.2 Å  Analysis of the trajectories with truncated H3 tails revealed an active role of histone arginines in structural alterations in the H2A(2)Concomitant with the above Arg81 interaction change, there is also a change of interaction of the nearby Arg88 of H2A(2) . WhereasThe change of interaction of arginines upon truncation of the H3 tails is likely a result of local change of electrostatic environment . In the Interestingly, truncation of the H2A tails (simulation 1) causes interaction changes in the same H2A(2)The crystal structure of the nucleosome core particle shows that the histone H2A C terminus lies close to the H3 Truncation of the H3 tail destabilizes contact of the H2A docking domain with the surrounding amino acids . In TablDestabilization of the H2A C terminus upon H3 tail truncation affects the C terminus-DNA contact. In the intact nucleosome simulation the H2A C terminus (Lys118 and Lys119) makes stable contact with the DNA through hydrogen bonding. Upon H3 tail truncation the C terminus switches between states ‘in contact’ with DNA and ‘detached’ .Truncation of H2A tails removes H2A C terminus-DNA contacts. This affects the remaining part of the H2A tail differently in the independent simulations : in simulation 1 the H2A docking domain breaks contact with Lys44 and Ile51, in the other only minimal loss of contact with Lys44 is observed . FurtherThe aim of the present study was to provide atomic-level information on interactions within the nucleosome that are altered upon tail truncation. This was accomplished by multiple all-atom MD simulations of intact and tail-truncated nucleosomes, with each trajectory covering a time frame 5 times longer than previously-reported simulations comparing intact and tail-truncated nucleosomes The formation of ractions . The staHyperacetylation of lysine residues neutralizes its positive charge by transferring an acetyl moiety onto the The reason for observing interaction changes for one H2A monomer in H3 tail-truncated simulations is not clearly understood (a comparison of interaction changes between histone monomers is provided in Supplementary in vitro studies The H2A docking domain provides the interaction surface between the histone H3–H4 tetramer and the H2A–H2B dimer . DestabiIn the simulations we observe a correlation between the breaking of contacts of the H2A docking domain with close by amino acid residues and the change of interaction of Arg88 of the H2A(2)e formed . When the formed .We also found that certain changes involving the alteration of sidechain hydrogen bonding of Arg88 are common to all the H3 and H2A tail-truncated simulations Click here for additional data file.Text S2Finding H2A docking domain contacts. Through the contact map analysis we want to find contact residues between the H2A docking domain and its surrounding which also form an interacting pair. To do this it is necessary to verify the contact map based information with visualization in 3D. This is achieved by the Molsurfer program which, in addition to the 2D map, has an interface for viewing in 3D (WebMol) as shown in Fig. S19. In the Figure the 2D contact map is shown the left panel whereas in the right panel the docking domain and its surrounding are shown in 3D in backbone representation. In the program when the cursor is pointed to a grid location on the 2D map the corresponding position is shown on the 3D interface by a red dot. One can then ‘focus’ (or zoom in) on this red dot to see which residues are forming an interacting pair and are in contact. Furthermore, the residue contacts found using Molsurfer are validated by visualizing the trajectory in VMD.(PDF)Click here for additional data file.Figure S1DNA phosphorous atom B-factors obtained from X-ray crystallography (dotted line) and those computed from the last 50 ns of intact and tail-truncated nucleosome simulations (continuous lines). The B-factors are shown for the two chains of DNA: I and J. The labels under the curves indicate the histone chains and secondary structure elements that make intermolecular contacts with the DNA.(PDF)Click here for additional data file.Figure S2DNA helical parameter fluctuations during intact nucleosome simulation. Average helical parameters with fluctuations (standard deviation) indicated as error bars are compared with those obtained from the crystal structure (1KX5.pdb).(PDF)Click here for additional data file.Figure S3DNA phosphorous atom RMSD versus simulation time for intact nucleosome.(PDF)Click here for additional data file.Figure S4Number of hydrogen bonds between Arg88 of H2A(2) and Glu105 of H3(1) or Ala135 of H3(2) as a function of time for tail-truncated nucleosome simulations. In the H2A tail-truncated nucleosome simulation number 2 no hydrogen bond was formed between Arg88 and Glu105.(PDF)Click here for additional data file.Figure S5The 2D contact map of the H2A docking domain with the WebMol interface for viewing the docking domain and its surrounding in 3D. In the WebMol interface atoms are shown in backbone representation. The interface between the H2A docking domain and its surrounding appears as a mesh.(PDF)Click here for additional data file.Table S1Comparison between 20 nsand 100 ns nucleosome trajectories.(PDF)Click here for additional data file.Table S2Interaction change in histone monomers with respect to key findings.(PDF)Click here for additional data file.Video S1Arg81 interacting with Gln55 and Lys56 of H3(1) and Val107 of H2A(2) in the intact nuclesome simulation.(MPG)Click here for additional data file.Video S2Time course of interaction changes in Arg81 during the H3 tail-truncated simulation.(MPG)Click here for additional data file.Video S3Arg88 making stable hydrogen bonds to Asn94, Gly98 and Val100 of H2A(2) in the intact nucleosome simulation.(MPG)Click here for additional data file.Video S4In the H3 tail-truncated simulation Arg88 sidechain moves towards the DNA and then makes stable hydrogen bonds to Glu105 of H3(1) and Ala135 of H3(2).(MPG)Click here for additional data file.


Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factors of STEC are Shiga toxin type 1 and/or 2 . We previously reported that intraperitoneal (i.p.) injection of rats in the late stage of pregnancy with culture supernatant from recombinant E. coli expressing Stx2 and containing lipopolysaccharide (LPS) induces premature delivery of dead fetuses. It has been reported that LPS may combine with Stx2 to facilitate vascular injury, which may in turn lead to an overproduction of nitric oxide (NO). The aim of this study was to evaluate whether NO is involved in the effects of Stx2 on pregnancy. Pregnant rats were i.p. injected with culture supernatant from recombinant E. coli containing Stx2 and LPS (sStx2) on day 15 of gestation. In addition, some rats were injected with aminoguanidine (AG), an inducible isoform inhibitor of NO synthase (iNOS), 24 h before and 4 h after sStx2 injection. NO production was measured by NOS activity and iNOS expression by Western blot analysis. A significant increase in NO production and a high iNOS expression was observed in placental tissues from rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body weight and killed 12 h after injection. AG caused a significant reduction of sStx2 effects on the feto-maternal unit, but did not prevent premature delivery. Placental tissues from rats treated with AG and sStx2 presented normal histology that was indistinguishable from the controls. Our results reveal that Stx2-induced placental damage and fetus mortality is mediated by an increase in NO production and that AG is able to completely reverse the Stx2 damages in placental tissues, but not to prevent premature delivery, thus suggesting other mechanisms not yet determined could be involved.Shiga toxin-producing  Escherichia coli (STEC) cause a significant public health risk due to contamination of food and water supplies. Gastrointestinal infection with STEC causes diarrhea and hemorrhagic colitis, and is the leading cause of hemolytic uremic syndrome (HUS), a systemic complication that is attributed to expression of Shiga toxins (Stx) In vivo and in vitro studies have demonstrated that LPS may combine with Stx to facilitate vascular injury Shiga toxin-producing To obtain timed pregnant females, both male and virgin female Sprague-Dawley rats (250–300 g of body weight) from the School of Veterinary Medicine animal facility of the University of Buenos Aires were used.ad libitum and were housed under controlled conditions of light  and temperature (23–25°C). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals of the University of Buenos Aires .Mating was performed by placing females in the cages of males for several days. Day 1 of gestation was determined when sperm was observed in the vaginal smear. Animals received food and water E. coli DH5α expressing Stx2 were incubated overnight at 37°C with shaking at 200 rpm in Luria-Bertani broth (Difco Laboratories) supplemented with 100 µg/ml ampicillin (Sigma Aldrich Co. USA). Bacterial cells were then removed by centrifugation, and the resultant supernatant (sStx2) was filtered through 0.22 µm pore size filter units  and assayed for toxicity to Vero cells as previously described 50) corresponds to the dilution required to kill 50% of Vero cells. Stx2 cytotoxicity in sStx2 was approximately 1×104 CD50/ml corresponding to 400 ng/ml when compared with the cytotoxicity of pure Stx2 on Vero cells. Supernatant from E. coli DH5α containing only the plasmid was used as control (sControl). Lipopolysaccharide (LPS) content in the culture supernatant was determined using the HEK-Blue LPS Detection Kit . A sample of 1 ml of supernatant contained 30 ng of LPS (75 ng LPS/µg of Stx2 protein).The stx2 gene was cloned into pGEM-T-Easy  and recombinant ad libitum.In order to examine the effects of sStx2 on the delivery time and fetal status, pregnant rats were intraperitoneally (i.p.) injected with 0.05–1.5 ml of sStx2 containing approximately 0.07–2 ng Stx2/g body weight (wt) on day 14–16 of pregnancy (late stage). Control rats were injected with the same volume of sControl. The rats were individually housed under controlled conditions of light, humidity, and temperature, with food and water available Animals were observed every 30 minutes for any signs of morbidity , vaginal bleeding, and/or preterm delivery (pups present in the cage) after sStx2 or sControl administration. The beginning of preterm delivery was defined as the delivery of the first pup.Pregnant rats on days 15 of gestation were randomly divided into three groups of five rats each. One group was i.p. injected with 0.5 ml of sStx2 containing 0.7 ng Stx2/g body wt, another with 1.5 ml of sStx2 containing 2 ng Stx2/g body wt, and the last one with 0.5 or 1.5 ml of sControl. All rats were anesthetized and killed by cervical dislocation 12 h after treatment, and the kidneys and placentas were removed.14C]arginine into L-[14C]citrulline was used to quantify NOS activity according to a method previously described 2, and 1 mM DTT (dithiothreitol), and incubated at 37°C with 10 µM L-[14C] arginine  and 0.5 mM NADPH (nicotinamide adenine dinucleotide phosphate). After 15 min, samples were centrifuged for 10 min at 10,000 g and applied to a Dowex AG50-X8 column , and L-[14C] citrulline was eluted. L-[14C]Citrulline radioactivity was measured by liquid scintillation counting. Enzyme activity is reported as fmoles of L-[14C]citrulline per mg of protein per 15 min.The conversion of L-arginine into L-[14C]citrulline. We observed a significant increase in NOS activity in placental tissues of rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body wt and killed 12 h after injection  protein was detected as a 130 kDa band by western blot in placental tissues from both control and experimental rats killed 12 h after injection . The expThe increase in NOS activity detected in placental tissues after injection of sStx2 containing 0.7 ng Stx2/g body wt decreased significantly when pregnant rats were treated with 100 µg AG/g body wt 24 h before and 4 h after Stx2 injection .The treatment with AG also decreased the iNOS protein expression observed in placentas from rats injected with 0.7 ng or 2 ng Stx2/g body wt. Values returned to the same levels as those observed in placentas from control rats . Both NOUteri and fetuses from pregnant rats injected with sStx2 (2 ng Stx2/g body wt) showed intrauterine hemorrhage and fetal death 48 h post injection . TreatmePlacentas from rats injected with 0.7 or 2 ng Stx2/g body wt and killed 12 h post injection presented necrotic areas . In contTreatment with 100 µg AG/g body wt 24 h before and 4 h after sStx2 injection containing 2 ng Stx2/g body wt did not prevent the premature delivery of dead fetuses. In contrast, all the pregnant rats treated only with AG or sControl delivered normal live pups on days 22 or 23 of gestation. After Stx2-treatment, all rats were able to become pregnant and deliver normal pups at term.Our data show that sStx2 (a combination of Stx2 and LPS) induces a significant increase in NO production and levels of iNOS protein in placental tissues from pregnant rats, suggesting overproduction of NO plays an important role in sStx2-induced placental toxicity and fetal mortality NO is produced from L-arginine under the catalytic control of nitric oxide synthase (NOS). Three NOS isoforms have been identified: the endothelial (eNOS) and neuronal (nNOS) isoforms, which are responsible for basal, i.e. constitutive, NO production, and iNOS, which is activated by a variety of agonists including LPS and tumor necrosis factor (TNF). In contrast to the constitutive NOS isoforms, iNOS is thought to account for the increased NO production seen in pathological states Although there are several clinical studies on the enhanced NO production in patients with HUS Here we found that AG did not prevent premature delivery, thus indicating that other mechanisms not yet determined could be involved. It is well known that Stx may act in concert with LPS to elicit cellular dysfunction In summary, the present results allow us to conclude that the Stx2-induced premature delivery in rats in the late gestational stage is, at least in part, mediated by an increase in NO production resulting from the output of iNOS.