abstract: Detailed comprehensive molecular analysis using families and multiple matched tissues is essential to determine whether imprinted genes have a functional role in humans.
  .
  See research article: <http://genomebiology.com/2011/12/3/R25>
author: Gudrun Moore; Rebecca Oakey
date: 2011
institute: 1Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK; 2Department of Medical and Molecular Genetics, Kings College London, 8th Floor Tower Wing, London SE1 9RT, UK
references:
title: The role of imprinted genes in humans

# Research highlight

Imprinted or parent-of-origin-dependent gene expression has over the past 25 years developed into an exciting and dynamic research field. Its functional or even evolutionary importance is considered most relevant in mammals and in flowering plants \[1\]. In mammals the link to the existence of the placenta and the differences between the two parental sexes in terms of resources and evolutionary drive through imprinting has been the focus of much debate. One fundamental question remains: has parent-of-origin gene expression evolved and been maintained because of the different needs of the mother and father in producing viable, strong offspring? The mother needs to survive the pregnancy but the father's drive is focused on the offspring being the fittest. Much of the functional relevance of the research in the imprinting field, particularly with its application to the human, has grown out of this 'resources for fittest' debate. A study in this issue of *Genome Biology* \[2\] starts to analyze more thoroughly which genes are truly imprinted in humans using genome-wide assessment.

Imprinting in the mouse is well understood. It was discovered separately by the Surani \[3\] and McGrath \[4\] groups in the early 1980 s, who found that gynogenetic embryos (which contain only maternal genomes) developed differently *in utero* and with emphasis on different tissues to the androgenotes (only paternal genomes). Interestingly, the androgenotes had a more developed placenta and the gynogenotes had a better developed embryo. Links were soon made between imprinted gene models in the mouse and human diseases, imprinted genes were implicated in many fetal growth syndromes, and they were shown to regulate maternal-fetal interactions, postnatal feeding behaviors and neurological development. Disturbance of the apparently rigorous mono-allelic imprinted gene expression was also linked to cancer, and alterations in imprinting methylation patterns or expression in peripheral blood leukocytes were considered as biomarkers for cancer \[5\].

The study by Morcos *et al.* \[2\] extends this human analysis comparison further. Here the authors \[2\] make a genome-wide assessment of imprinted expression in paired sets of samples of adult human tissue, comparing lymphoblastoid cell lines with primary fibroblasts. These two cell lines are both relatively easy to obtain from humans with ethical approval. Using families they could track parental-allele-specific expression, and using paired tissue samples they could study tissue-specific variation between lymphoblastoid cells and fibroblasts. To truly confirm whether a gene is imprinted, differential methylation, tissue-specific expression and parental allele origin must all be tracked in the same family. Observing differentiated methylated patterns in isolation, however, does not always totally reflect monoallelic expression \[6\]. These all-inclusive experiments can be done relatively easily in mouse but are ethically impossible to copy in humans. These authors \[2\] have achieved the best compromise by using matched tissues and by studying families. Their results are both interesting and intriguing.

Previous careful comparative analysis between the imprinted genes in mouse and humans showed that roughly half of the mouse imprinted genes are either not or never have been imprinted in humans. Of the about 140 imprinted genes identified so far in the mouse, only 60 are imprinted in humans and several are specific to humans. In addition, some have different tissue-specific expression profiles; for example, growth factor receptor binding protein 10 (*GRB10*) in humans is imprinted only in invasive trophoblasts (maternally expressed) and brain (paternally expressed) \[7\], whereas in mouse it is maternally expressed in most embryonic tissues and predominantly paternally expressed in brain \[8\]. If the regulation of gene dosage is so important, why is there not greater conservation of imprinted expression? Or maybe the genes still imprinted in humans have been selected and/or maintained for important reasons. Epigenetics provides the mechanisms through which imprinting influences gene expression. These mechanisms affect the processes of cell differentiation and embryonic growth, although they are as yet not completely understood. When epigenetic mechanisms go awry, transcriptional activity may be perturbed and result in disorders and syndromes. This underscores the rationale for studies such as these \[2\], particularly on a genome-wide scale, for identifying imprinted genes and classifying their conservation across mammalian species.

In the study by Morcos *et al.* \[2\], of the 44 informative imprinted genes from the literature that were analyzed, 19 were validated as imprinted using this rigorous assessment. More importantly, only 1 in 13 candidate imprinted genes were confirmed. This demonstrates again that only over 50% of mouse imprinted genes are truly imprinted in humans in the adult tissues assayed and only 10% of candidates can be verified.

One caveat of this approach stems from the fact that human embryonic tissues are extremely difficult to access and thus the authors \[2\] used lymphoblastoid cells and fibroblasts instead; this has some limitations. It is known that imprinting is important in the developing embryo and fetus and typically occurs in a tissue-specific manner. So the use of transformed lymphoblastoid cells as a human tissue resource does not necessarily reflect the *in situ* state. It could be argued that the true role of imprinted genes is in fetal development but, even so, analysis of fetal tissues and placenta has also revealed much lower numbers of imprinted genes in humans than in mouse \[9\]. In humans there are fewer imprinted genes and these may be the ones that are most relevant for the 'resources for fittest' needs that are most important in human fetal growth.

This study \[2\] plus other work on human tissues in this dynamic field are all helping to clarify the numbers of imprinted genes in humans and lead towards an understanding of the role of imprinting in humans. There remains no doubt that gene dosage control in the developmental period is exquisitely sensitive and needs accurate control mechanisms. The future focus in humans needs to be on careful dissection of the function of those genes that are confirmed to be imprinted using methods similar to those in this study \[2\].


date: 2014-01-10
title: Findings of research misconduct.

# Findings of Research Misconduct

**Notice Number:** NOT-OD-14-037

**Key Dates
Release Date:** January 10, 2014

**Related Announcements
** None

None

**Issued by
** Department of Health and Human Services ([DHHS](http://www.hhs.gov/))

Purpose

Notice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:

Dong-Pyou Han, Ph.D., Iowa State University of Science and Technology: Based on the report of an inquiry conducted by the Iowa State University of Science and Technology (ISU), a detailed admission by the Respondent, and additional analysis conducted by ORI, ORI and ISU found that Dr. Dong-Pyou Han, former Research Assistant Professor, Department of Biomedical Services, ISU, engaged in research misconduct in research supported by National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), grants P01 AI074286, R33 AI076083, and U19 AI091031.

ORI and ISU found that the Respondent falsified results in research to develop a vaccine against human immunodeficiency virus-1 (HIV-1) by intentionally spiking samples of rabbit sera with antibodies to provide the desired results. The falsification made it appear that rabbits immunized with the gp41-54 moiety of the HIV gp41 glycoprotein induced antibodies capable of neutralizing a broad range of HIV-1 strains, when the original sera were weakly or non-reactive in neutralization assays. Falsified neutralization assay results were widely reported in laboratory meetings, seven (7) national and international symposia between 2010 and 2012, and in grant applications and progress reports P01 AI074286-03, -04, -05, and -06; R33 AI076083-04; U19 AI091031-01 and -03; and R01 AI090921-01. Specifically:

a\. Respondent falsified research materials when he provided collaborators with sera for neutralization assays from (i) rabbits immunized with peptides from HIV gp41-54Q (and related antigens HR1-54Q, gp41-54Q-OG, gp41-54Q-GHC, gp41-54Q-Cys and Cys-gp41-54Q) to assay HIV neutralizing activity, when Respondent had spiked the samples with human IgG known to contain broadly neutralizing antibodies to HIV-1; and (ii) rabbits immunized with HIV gp41-54Q to assay HIV neutralizing activity, when Respondent had spiked the samples with sera from rabbits immunized with HIV-1 gp120 that neutralized HIV.

b\. Respondent falsified data files for neutralization assays, and provided false data to his laboratory colleagues, to make it appear that rabbits immunized with gp41-54Q and recombinant Lactobacillus expressing gp41-64 (LAB gp41-64) produced broadly reactive neutralizing antibodies, by changing the numbers to show that samples with little or no neutralizing activity had high activity.

Dr. Han has entered into a Voluntary Exclusion Agreement and has voluntarily agreed for a period of three (3) years, beginning on November 25, 2013:

\(1\) To exclude himself from any contracting or subcontracting with any agency of the United States Government and from eligibility or involvement in nonprocurement programs of the United States Government referred to as \`\`covered transactions' pursuant to HHS' Implementation (2 CFR Part 376 et seq) of OMB Guidelines to Agencies on Governmentwide Debarment and Suspension, 2 CFR Part 180 (collectively the \`\`Debarment Regulations'); and

\(2\) To exclude himself voluntarily from serving in any advisory capacity to the U.S. Public Health Service (PHS) including, but not limited to, service on any PHS advisory committee, board, and/or peer review committee, or as a consultant.

Inquiries

Please direct all inquiries to:**

** David E. Wright, Ph.D.**
** Director**
** Office of Research Integrity**
** 1101 Wootton Parkway, Suite 750**
** Rockville, MD 20852**
** Telephone: 240-453-8800


abstract: For the first time in history, psychiatrists during the Nazi era sought to systematically exterminate their patients. However, little has been published from this dark period analyzing what may be learned for clinical and research psychiatry. At each stage in the murderous process lay a series of unethical and heinous practices, with many psychiatrists demonstrating a profound commitment to the atrocities, playing central, pivotal roles critical to the success of Nazi policy. Several misconceptions led to this misconduct, including allowing philosophical constructs to define clinical practice, focusing exclusively on preventative medicine, allowing political pressures to influence practice, blurring the roles of clinicians and researchers, and falsely believing that good science and good ethics always co-exist. Psychiatry during this period provides a most horrifying example of how science may be perverted by external forces. It thus becomes crucial to include the Nazi era psychiatry experience in ethics training as an example of proper practice gone awry.
author: Rael D Strous
date: 2007
institute: 1Department of Psychiatry, Beer Yaakov Mental Health Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
references:
title: Psychiatry during the Nazi era: ethical lessons for the modern professional

# Background

During the Nazi era, for the first time in history, psychiatrists sought to systematically exterminate their patients. It has been acknowledged that the medical profession was profoundly involved in crimes against humanity during this period, with various publications describing this malevolent period of medical history. It is less known, however, that psychiatrists were among the worst transgressors. At each stage of the descent of the profession into the depths of criminal and genocidal clinical practice lay a series of unethical decisions and immoral professional judgments. Furthermore, very little has been published on lessons that may be learned from this dark period in the history of psychiatry and on ethical principles that may be extrapolated for the future practice of clinical and research psychiatry and for inclusion in educational programs. This paper reviews the role of psychiatrists in the Nazi era and analyzes the underlying misconceptions that led to the aberrant behavior. Finally, some recommendations for inclusion of the study of this period in ethics training are presented \[26\].

# Role of psychiatrists in Nazi atrocities

The professional status of psychiatrists did not place any obstacle to their participation in Nazi crimes, and many demonstrated a profound commitment to the atrocities. Psychiatrists were instrumental in instituting a system of identifying, notifying, transporting, and killing hundreds of thousands of mentally ill and "racially and cognitively compromised" individuals in settings ranging from centralized psychiatric hospitals to prisons and death camps. Their role was *central* and *critical* to the success of Nazi policy, plans, and principles. Psychiatrists, along with many other physicians, facilitated the resolution of many of the regime's ideological and practical challenges, rather than taking a passive or even active stance of resistance \[1\]. Psychiatrists played a prominent and central role in two categories of the crimes against humanity, namely sterilization and euthanasia \[2\]. It was psychiatrists (many of whom were senior professors in academia) who sat on planning committees for both processes and who provided the theoretical backing for what transpired. It was psychiatrists who reported their patients to the authorities and coordinated their transfer from all over Germany to gas chambers situated on the premises of the six psychiatric institutions: Brandenburg, Grafeneck, Hartheim, Sonnenstein, Bernburg, and Hadamar \[2,3\]. It was psychiatrists who coordinated the "channeling" of patients on arrival into specially modified rooms where gassing took place. It was psychiatrists who saw to the killing of the patients (initially using carbon monoxide and later, starvation and injection). Finally, it was psychiatrists who faked causes of death on certificates sent to these patients' next of kin. It has been estimated that over 200,000 individuals with mental disorders of all subtypes were put to death in this manner \[4-7\]. Much of this process took place before the plan to annihilate the Jews, Gypsies and homosexuals of Europe. Hitler never gave the order to kill patients with mental illness. He only permitted it in a letter written in October 1939 and backdated to September 1, 1939 \[2,6\]. Psychiatrists were therefore never ordered to facilitate the process or carry out the murder of mentally ill...they were empowered to do so. Activity by psychiatrists and psychiatric institutions thus constituted the connection between euthanasia and the larger scale annihilation of Jews and other "undesirables" such as homosexuals in what came to be known as the Holocaust. Parenthetically, only one physician ever came to command an extermination camp. His name was Dr Imfried Eberl, a psychiatrist, who established Treblinka based on his experience as the Brandenburg Psychiatry Facility medical superintendent. He managed the camp for six months until he was fired for inefficiency in disposing of the thousands of bodies he succeeded in accumulating \[2\].

# Attitude of mainstream psychiatry to Nazi psychiatry practice following the war

While it would be expected that the involvement of psychiatrists in such a profound manner would be well-known in the field, this is not the case. Little has been published on the subject in mainstream psychiatry journals and even less is part of the formal education process for medical students and psychiatry residents. Several reasons may be proposed for this. First, it remains an embarrassment for the field that so many senior members – professors, department heads and internationally known figures – were so intimately involved. Second, many of those involved continued to practice and conduct research long after the war and were protected by colleagues. Third, and arguably most important, what psychiatrists did was based upon a paradigm shift in how patients and mental illness were viewed. Activities of psychiatrists became much of a value judgment in how they "read" the community and principles of neo-Darwinism with subsequent consideration of racial hygiene. In the absence of firm and unbending timeless ethical underpinnings to the practice of psychiatry, many felt that what they were doing was correct from a moral and scientific standpoint; therefore, they were not the demons and "paradigms of evil" that we perceive them to be. Their actions were a colossal misjudgment based on what today we may term "pseudoscience", but which at the time was deemed correct by many. Although actions based on "scientific theories" of mental illness in the past have led to patient deaths – one example being Henry Cotton and his belief that mental illness results from focal infection or chronic sepsis \[8\] – the extent and scale of the German psychiatrists' actions during the Nazi era remains unprecedented. These rationalizations based on faulty scientific theory and unethical medical practice were difficult to accept and therefore the nature and extent of these activities remained on the backbenches of the academic literature until more recently, when these issues have begun to be faced in an era of openness and transparency.

# Common assumptions leading to gross ethical misconduct

In addition to resting on poor science, the atrocities of the German psychiatric establishment were based upon several fundamental errors of ethical, professional, and scientific conduct. While many may simply brush off any deeper consideration of the issues with the stance that "they were just evil", such an approach only deepens the risk that such events will be repeated. The truth cannot be more different: perversion of ethical medical practice due to theoretical misjudgment and fundamental error in approach to the patient are what led to these atrocities of catastrophic proportions. So where did they go wrong? Several misconceptions lay at the source:

## 1) Medical ethics is ethnic, cultural, and time sensitive

The theory behind such a proposition is that much of medical ethics is time and culture bound \[9\]. Therefore what may be unethical now may not necessarily have been unethical then. This approach inculcates a relative attitude to the atrocities, minimizing the severity of the injustice and gross professional negligence so inherent in what transpired. Certain aspects of medical ethics transcend time and culture. Except under very specific and precise circumstances, such as when there is a serious and immediate risk to others, a physician should always respect autonomy, beneficence, and patients' confidentiality and dignity. Although it may be suggested that there is a major leap between disregard of these time-honored factors and the genocide of euthanasia, this is how it all began – it may even define the central thread of the atrocities to the mentally ill. While the form of ethical medical practice may depend on resources and cultural nuances (Tarasoff etc.), the basis for ethical behavior should remain constant, irrespective of time and place. Thus, while some maintain that for one generation a practice may be considered unethical but not for another \[9\], this is a misconception, as certain practices and concepts do not change with context. It is therefore never appropriate to kill one's patients *en masse* based on diagnosis and economic and racial-hygiene considerations for the community at large.

## 2) Philosophical constructs and ideas should define clinical practice

During the period of the Nazi regime, psychiatry supported compulsory sterilization and euthanasia of the physically and mentally ill, and subsequently, the killing of "inferior" races. They did this by applying scientifically invalid conclusions from evolutionary biology \[10\]. Aside from the fact that these philosophical constructs and scientific paradigms of evolutionary theory were flawed, they were also immoral and contravened basic tenets of medical ethics and clinical practice. Much of this approach was based on theories of neo-Darwinism. Furthermore, ever since Francis Galton in 1865 first published the idea of eugenics (a term rooted in the Greek "good in birth" or "noble in heredity"), individuals with mental illness had been targeted by eugenics programs, with psychiatrists intimately involved in the theoretical debate. The eugenics movement was not limited to Germany, and proponents of eugenics were prominent in several other countries including most notably Britain and the USA \[11\]. Interestingly, during the period in which euthanasia of the mentally ill was taking place in Germany, a fascinating debate transpired between two prominent American academics and was published in the *American Journal of Psychiatry* in 1942. Foster Kennedy, professor of neurology at Cornell University in New York, argued that all children with proven mental retardation ("feeblemindedness") over the age of five should be put to death. Leo Kanner, however, maintained that such individuals might still serve a purpose to society – garbage collection, postmen, etc. – as well as give meaning to their parents by virtue of having to care for them. Astoundingly, no one emphasized the unethical nature of putting individuals with disability to death. Instead, the editorial, published anonymously, appeared to side with Kennedy, and advised help for the parents in coming to terms with such a reality for their children and for the need for "enabling legislation" in order to facilitate the process legally (apparently in contrast to that of the German experience) \[12\]. The Nazi experience, which took much of the concept to fruition, was an extreme perversion of this movement, which existed already (at least at the conceptual level) in the minds of many psychiatrists supporting the idea.

## 3) Preventative medicine is more important than curative medicine

In the interests of preserving the future quality and purity of the Aryan race, racial hygiene became the battle cry of the German nation with Nazi medicine attempting to prevent the proliferation of illness. Within this context, it became the role of physicians in general, and psychiatrists in particular, to define who should be eliminated in order to best preserve the German nation's uniqueness and "higher-being". Thus in place of managing mental illness with the available tools (which were minimal) or investing resources in research for more appropriate treatment, it became important for physicians and psychiatrists to prevent such forms of illness or defects through euthanasia \[7\]. A particular focus was placed on psychiatric patients in the racial-hygiene program because they were perceived as weakening the "master-race" with no known cure. Therefore these "lives not worth living" were deemed useless and dangerous to German society and, in order to prevent their dissemination, the process of eliminating them in the context of the sterilization and euthanasia program came about. This procedure of trying to prevent illness, while a noble concept, should never be instituted at the expense of (and complete exclusion of) treating illness, as the disastrous Nazi program proved \[7\]. Even if one accepts their reasoning – and in this case they were wrong – selective sterilization of the mentally-ill would never significantly reduce the frequency of mental illness based on the Hardy-Weinberg law of preservation of rare recessive genes in a population of phenotypically normal carriers \[10\]. The Nazis embraced an exclusionary biological and racial determinism that removed any reparative function from clinical psychiatry. What remained was prevention of mental illness. Psychiatrists lived up to the challenge.

## 4) Psychiatrists have a particular role in channeling societal issues and public discussion

Many psychiatrists maintain that they have an inherent responsibility more than other medical professions to be involved in community affairs. This is because psychiatry by nature advocates a holistic approach to the patient, which often includes taking into account societal factors and contemporary ideology. Thus while the unique role of psychiatry in the genocide may be overstated, since other areas of medicine were also involved, psychiatrists fitted in particularly well. The dangers inherent in such involvement, while not obvious, are, however, prominent when important boundaries become blurred. Clinical practice and political machinations need to be kept separate. Many psychiatrists during the Nazi era were state-controlled and this further facilitated their conforming to the program. The rights of individuals cannot be totally ignored in the interests of society. The dangers become particularly acute in psychiatry compared to other subspecialties in medicine since it may be suggested that the field of psychiatry is often used in order to remove undesirables from society and place them in asylums. It may be argued that labeling of mental disease and its classification is a means of controlling members of the community who do not comply with accepted norms; therefore their freedom should be taken away and replaced with hospitalization. However, while at times there may be a fine line separating mental health and illness, it becomes very clear that the extent to which Nazi psychiatry allowed the political and community atmosphere to influence and govern clinical practice was grossly unethical, murderous, and unacceptable to an extreme extent.

## 5) Political and economic pressures may influence clinical practice

The management of patients must be dictated primarily by the patient's best interests and not by virtue of any ideology that may be prevalent at the time in society. This may include economic "ideological" considerations. Thus while pressures may exist "encouraging" the physician to make decisions one way or another based on the prevailing mood or tendency of the community at any time or place, this should be resisted and medical management should continue, unaffected by external considerations. The patient has to receive individual management and not be treated according to what is in vogue at the time. Psychiatrists should be wary of political and economic pressures that impinge upon medical decisions and health service provision. Nazism was supposed to be "applied biology" \[13\]. Science in general and psychiatry in particular needs to be independent from contemporary sociological and political contexts as well as protected from political abuse, even when embraced by the medical establishment. It has been proposed that the primary downfall of Nazi medicine was the failure of physicians to challenge the substantive core of Nazi values "Too many physicians were willing to go with the political flow; too many were unwilling to resist, to 'deviate' from 'commonly accepted' practices" \[14\]. Sound medical practice should be protected from the movement of political forces.

## 6) Psychiatrists/scientists have a responsibility to "enhance" mankind

Much of the early involvement by psychiatric clinicians and researchers in the process of "racial purification" arose from a genuine desire to improve mankind and not necessarily from the perspective of racist genocide. While no direct parallel can be drawn, today many continue in a sincere scientific effort towards the "enhancement" of man through molecular biology and genetic engineering \[15\]. Appropriate dialogue is required in order to ensure that the desire for "improving man", creating a "better human", does not come at the expense of the individual patient.

## 7) The interests of science take priority over the interests of the individual patient

Clinical management and research participation may appear to be equivalent, but they are not. A clear distinction must be made between the two and the patient must be aware of this. Research is critically important for the future of good medical practice and is fundamental to the philosophy of medical ethics in psychiatry which would be reflected in the long-term striving for excellence in clinical management. However, it should always be made clear to the patient that participation is voluntary and that more conventional treatment regimes exist and are available if preferred. Particular issues such as scientific validity, favorable risk-benefit ratio, voluntaryism, and decisional capacity, while important in all aspects of clinical practice, become of acute importance with respect to individuals with mental illness \[16,17\]. The Nazi experience, which completely disregarded such factors in the interests of "science" and racial-hygiene, is a prime example of the dangers inherent when such factors are not respected. Ethical commitment to research safeguards needs to be reflected in appropriate standards, guaranteeing appropriate study participation \[16\]. Refusal to participate in a study should likewise never interfere with the doctor-patient relationship and in the case of a patient agreeing to participate in research, it remains the duty of the physician to protect the health of the individual.

## 8) High-quality science and high-quality ethics always co-exist

It has become easy for those in the West to dismiss the depths of unethical medical practice of the Nazi physicians by categorizing it as bad science. This is easier to accept than the possibility that even within the context of good science, ethical behavior by physicians may go astray. In fact, the Nazi era in Germany was a time of remarkable scientific advances in several areas including cancer research and treatment, biochemistry, and quantum mechanics to name a few. In addition, the Nazis were pioneers of jet-propelled air flight, guided missiles, electronic computers, electron microscopes, and atomic fission \[14\]. Thus, scientific advancement does not necessarily go hand in hand with ethical advancement. It would be incorrect to brush off the ethical challenges that true scientific advancement in medicine may present, since the connection with true ethical practice is not necessarily a natural one.

# Ethics in the training of future psychiatrists

The theory of "medical ethics" has become a requirement in residency training programs in several countries around the world \[18\]. This has become a particularly pressing issue considering the need to understand principles of research ethics and roles of psychiatrists as investigators and researchers. The teaching of medical ethics during residency is particularly well-timed because professional identity and ethical practices are in their formative stages. Such ethics training is important in order to define the critical role that the four principles of autonomy, beneficence, non-maleficence, and justice play in clinical practice \[19\]. Several reports have been published that extol the virtues of medical ethics training in psychiatry residency training programs \[20\].

However, while the importance of such training programs is well recognized, so was the importance of medical ethics acknowledged in Germany in the 1930's. In fact, Germany possessed one of the most advanced and sophisticated codes of medical ethics in the world in existence from 1931. Some have even suggested that in certain aspects it was stricter than the subsequent Nuremberg Code or Helsinki Accord \[14,21\]. Doctors in general and psychiatrists in particular who were involved in the euthanasia program were not morally blind or devoid of the power of moral reflection. This belief would render the guilty parties not responsible for their actions. However, such codes did not help and when it came to bringing to fruition the ideology and plans of the wider society's beliefs, psychiatrists cooperated fully. They even took enthusiastic initiative in the process, allowing societal politics and ideas to interfere with clinical practice. Furthermore, although a broader consideration of potential abuse and malpractice in other totalitarian regimes would further strengthen the importance of the subject, a focus on Nazi psychiatric practice in particular brings to the fore clearly a most apt and recent example of how such interference can go awry. The example of Nazi psychiatry is a prime illustration of how ethics training without a focus on history is useless; where policy, even though existing, can be disregarded in the most grotesque fashion.

Furthermore, Cowley \[19\] has argued that although many medical schools have now given medical ethics a secure place in the curriculum, they err in treating it like a scientific body of knowledge. Ethics is a unique subject precisely because of its widespread relevance in all areas of life, and any teaching has to start from this shared understanding and from the familiar ethical concepts of ordinary language. Ethical jargon obscures the essential integration of ethics with the personal and "drives a wedge between ethical concepts and ethical conduct". This may have accounted for some of the unethical conduct of "Hitler's psychiatrists" in their disregard of basic principles despite the existence of strong ethical policy. "Ethical mantras" have little value when they exist away from a context of a mature understanding and self-reflection that needs to precede good ethical judgment and professionalism \[19\].

The question remains: why did so many psychiatrists willingly participate in the process of mass murder of the mentally ill? Perhaps some light may be shed on this issue by consideration of similar behavior as reported by Browning \[22\] in the history of an "ordinary" and unremarkable battalion of the Order Police that participated in mass shootings and deportations. Browning describes how these ordinary men were not coerced to kill, but rather participated in a willing fashion due to peer pressure, government sanction and following of orders, and in order to advance their own interests (careerism). This is in contrast to Goldhagen \[23\], who suggested that the average German citizen either participated in or ignored genocidal actions during the Nazi Era due to ingrained anti-Semitism, which was an intrinsic part of German society and had built up over centuries.

Yet another approach proposes that psychiatrists during the Nazi era were at particularly high risk for moral and ethical breaches because of how society and they themselves defined their role and power. Inherent in their work lay the risk of dehumanizing the patients with whom they had daily involvement, individuals at the extremes of human behavior. Moreover, psychiatry by nature incorporates contemporary ideology in its approach to the individual and society, and psychiatrists during that period were in essence state-controlled. All of these factors may have led to their tendency to objectify patients \[reviewed in \[1\]\]. Thus, these psychiatrists were primed to become involved in furthering Nazi ideology.

These differing approaches and considerations need to be conveyed to students of psychiatry, emphasizing that merely explaining the actions of psychiatrists and other physicians during this period by saying "they were evil" is misleading and reductionistic \[24\]. Danger exists in such an approach since it would preclude consideration of one's own risk for involvement in such a process.

Despite the wealth of ethics literature and the requirement for medical ethics in training programs, the experience of Nazi German psychiatry receives minimal mention, if at all, in contemporary medical student and resident ethics training courses. This is a serious oversight, since well-developed ethical principles did not stop the trespassing of political ideology into clinical practice and research in the 1930's. The result was equally devastating to the patients and to the practice of the profession. Every psychiatry resident needs to know about this. A knowledge and appreciation of Nazi psychiatry practices should become an important component of an integrated program of psychiatry ethics, with a focus on the human aspects of the psychiatric clinician and researcher, and a warning against the influences of political and community ideology impinging upon professional practice.

The content of ethics training for medical students and residents will remain a creative exercise for educators. Such a program should include an informative historical review of this period, including information on the unethical medical activities that transpired, case studies of psychiatrists at all levels of involvement, a consideration of various ethical frameworks for psychiatric care that were violated, as well as a consideration of why psychiatrists came to be so inextricably involved. Such a course would pave the way for a discussion about creating the most optimal framework of ethics for psychiatric practice. Several approaches may be considered, including the recent one by Bloch and Green \[25\], who proposed a model based on a "complementarity of principalism" (pragmatic focus on respect for autonomy, beneficence, non-maleficence and justice) and care ethics (highlighting character traits pertinent to caring for vulnerable psychiatric patients).

Thus, in addition to the usual formal medical ethics training in which the importance of autonomy, beneficence, confidentiality, and professionalism is emphasized, an in-depth appreciation of the actions of German psychiatrists during the Nazi years should be imparted. Since medical students and residents today are learning in an ethical environment that is unprecedented in its complexity \[20\], it becomes crucial to include the Nazi era medical experience as an example of proper practice gone awry, despite its being in the interest of science, and despite receiving the support of many of the foremost world leaders of the profession. The responsibility of psychiatrists to act as moral agents in the interests of patients, especially in the area of psychiatry, is thus of paramount value.

The professional burden of the memory of what transpired during the Nazi Era by the hands of members of the psychiatry profession is great. Those that were inextricably involved were colleagues, and this requires us to grapple with the intrinsic guilt of the profession, and to take responsibility to fix fundamental flaws in how we view patients and their management. A dark side to medicine exists: psychiatry, academia, and science played a key role in the establishment of National Socialism and all that ensued. The experience of psychiatry during the Nazi era provides an example of how science can be perverted by politics and therefore can become vulnerable to misuse and abuse. An exclusive focus on the monstrous aspects of Nazi medicine enables us to dismiss such events as aberrant and deviant, with a subsequent failure to internalize the inherent and very real dangers of the perversion of science and clinical management by outside political influences. Psychiatry cannot afford to turn a blind eye to such a past.

# Competing interests

The author declares that he has no competing interests.


abstract: # Background
  .
  Alternate day modified fasting (ADMF) is an effective strategy for weight loss in obese adults.
  .
  # Objective
  .
  The objective of this study was to examine the dietary and physical activity adaptations that occur during short-term ADMF, and to determine how these modulations affect rate of weight loss.
  .
  # Methods
  .
  Sixteen obese subjects (12 women/4 men) completed a 10-week trial consisting of 3 phases: 1) 2-week control phase, 2) 4-week ADMF controlled feeding phase, and 3) 4-week ADMF self-selected feeding phase.
  .
  # Results
  .
  Body weight decreased (*P* \< 0.001) by 5.6 ± 1.0 kg post-treatment. Energy intake on the fast day was 26 ± 3% of baseline needs (501 ± 28 kcal/d). No hyperphagic response occurred on the feed day (95 ± 6% of baseline needs consumed, 1801 ± 226 kcal/d). Daily energy restriction (37 ± 7%) was correlated to rate of weight loss (*r* = 0.42, *P* = 0.01). Dietary fat intake decreased (36% to 33% of kcal, *P* \< 0.05) with dietary counseling, and was related to rate of weight loss (*r* = 0.38, *P* = 0.03). Hunger on the fast day decreased (*P* \< 0.05) by week 2, and remained low. Habitual physical activity was maintained throughout the study (fast day: 6416 ± 851 steps/d; feed day: 6569 ± 910 steps/d).
  .
  # Conclusion
  .
  These findings indicate that obese subjects quickly adapt to ADMF, and that changes in energy/macronutrient intake, hunger, and maintenance of physical activity play a role in influencing rate of weight loss by ADMF.
author: Monica C Klempel; Surabhi Bhutani; Marian Fitzgibbon; Sally Freels; Krista A Varady
date: 2010
institute: 1Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, USA; 2Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA; 3Department of Biostatistics, University of Illinois at Chicago, Chicago, IL, USA
references:
title: Dietary and physical activity adaptations to alternate day modified fasting: implications for optimal weight loss

# Introduction

Rates of obesity have dramatically increased over the past three decades. At present, 34% of adults in the United States are obese (body mass index (BMI) \> 30 kg/m^2^) \[1\]. According to the National Heart Blood and Lung Institute (NHLBI) Obesity Guidelines \[2\], dietary interventions should be implemented as the first line of treatment to help obese individuals lose weight. The most common diet therapy prescribed by practitioners is daily calorie restriction (CR). CR involves decreasing energy intake by 15 to 40% of baseline needs everyday. Evidence from short-term CR trials (8 to 24 weeks) demonstrate that CR is an effective means of decreasing body weight by 5 to 10% from baseline in obese patients \[3-6\].

Although CR is the most frequent diet strategy implemented to facilitate weight loss \[7\], many obese patients find it difficult to adhere to CR since food intake must be limited *every day*\[8-10\]. Alternate day modified fasting (ADMF) was created as an alternative to CR to improve compliance with dietary restriction regimens \[11\]. ADMF includes a "feed day" where food is consumed ad-libitum over a 24-h period, alternated with a "fast day", where food intake is partially reduced for 24-h. ADMF only requires an individual to restrict food intake on *every other day*, and as such, greatly increases adherence to these protocols. To date, four ADMF human trials have been performed \[12-15\], two of which were weight loss studies \[13,14\]. In the first trial by Johnson et al. \[13\], 8 weeks of modified ADMF, which allowed for 20% of energy needs to be consumed on the fast day, decreased body weight by 8% from baseline in overweight adults. In the second study conducted by our group \[14\], 8 weeks of modified ADMF (i.e. 25% energy intake on the fast day, alternated with an ad libitum feed day) resulted in a 6% weight loss in obese individuals. Although these preliminary findings suggest that ADMF may be an effective weight loss strategy, \[13,14,16\] what has yet to be examined is the dietary and physical activity adaptations that contributed to this pronounced weight loss by ADMF. Key questions that remain unresolved include: Are obese subjects able to dramatically change their meal pattern and limit their energy intake to 25% of needs on the fast day? If this is the case, what degree of hyperphagia occurs on the feed day in response to this lack of food on the fast day, and how does this affect net energy restriction and rate of weight loss? Moreover, how long does it take for obese subjects to become habituated to ADMF (i.e. no longer feel hungry on the fast day)? Furthermore, what changes in habitual physical activity occur during ADMF, and how do these changes affect rate of weight loss?

Accordingly, the objective of the present study was to examine the dietary and physical activity adaptations that occur during short-term ADMF, and to determine how these modulations affect rate of weight loss.

# Methods

## Subject selection

This study was approved by the Office for the Protection of Research Subjects at the University of Illinois, Chicago, and all volunteers gave their written informed consent prior to participation in the trial. As reported previously \[14\], participants were recruited by means of advertisements placed in community centers in the Chicago metropolitan area. Inclusion and exclusion criteria were assessed by an in-person interview. Participants meeting the following criteria were included in the study: age 35 to 65 y; body mass index between 30 and 39.9 kg/m^2^; weight stable for 3 months prior to the beginning of the study (i.e. less than 5 kg weight loss or weight gain); non-diabetic; no history of cardiovascular disease; lightly active (i.e. \< 3 h/week of light intensity exercise at 2.5 to 4.0 metabolic equivalents (METs) for 3 months prior to the study); not participating in an exercise class; non-smoker; and not taking weight loss, lipid or glucose lowering medications. Peri-menopausal women were excluded from the study, and post-menopausal women (absence of menses for more than 2 y) were required to maintain their current hormone replacement therapy regimen for the duration of the study.

## Experimental design

Obese participants were enrolled in the study as a single cohort. Subjects participated in a 10-week trial consisting of three consecutive dietary intervention phases: (1) 2-week pre-loss control phase, (2) 4-week weight loss/ADMF controlled feeding phase, and (3) 4-week weight loss/ADMF self-selected feeding phase. During *Phase 1*, each subject maintained their usual eating and exercise habits in order to maintain a stable body weight. During *Phase 2*, subjects participated in a 4-week controlled-feeding ADMF period. All subjects consumed 25% of their baseline energy needs on the fast day (24 h), and then ate ad libitum on each alternating feed day (24 h). Individual baseline energy requirement was determined by the Mifflin equation \[17\]. Subjects were provided with a calorie-restricted meal on each fast day, and ate ad libitum at home on the feed day. The provided fast day meal was formulated for each subject using Nutritionist Pro Software (version 4.3, Axxya Systems, Stafford, TX). All diets were prepared in the metabolic kitchen at the Human Nutrition Research Unit (HNRU) at the University of Illinois, Chicago, and were provided as a 3-day rotating menu consisting of typical American foods. Meals provided on fast days during the controlled feeding phase are displayed in Table 1. Each feed/fast day began at midnight, and all fast day meals were consumed between 12.00 pm and 2.00 pm to ensure that each subject was undergoing the same duration of fasting. During *Phase 3*, all subjects participated in a self-selected feeding ADMF period in conjunction with weekly dietary counseling. This phase was put in place to determine if subjects could maintain the ADMF regimen on their own at home. During this phase, subjects still consumed 25% of their baseline energy needs on the fast day (between 12.00 pm and 2.00 pm), and ate ad libitum on the feed day. No food was provided to the subjects during this phase. Instead, a Registered Dietician met with each subject each week (for approximately 30 min per session) to develop individualized fast day meal plans. These plans included menus, portion sizes, and food lists that were consistent with each subject's food preferences and prescribed calorie levels for the fast day. Subjects were also instructed how to make healthy food choices on the ad libitum feed days, by choosing low fat meat and dairy options, and increasing fruit and vegetable intake.

Meal components of provided fast day meals during controlled feeding phase

| Foods           | Fast day 1         | Fast day 2       | Fast day 3        |
|-----------------|--------------------|------------------|-------------------|
| Entree          | Chicken fettuccini | Vegetarian pizza | Chicken enchilada |
| Fruit/vegetable | Carrot sticks      | Apple            | Orange            |
| Snack           | Cookie             | Peanuts          | Crackers          |

## Weight loss assessment

Body weight was measured weekly to the nearest 0.25 kg in the fasted state, without shoes, and in light clothing using a balance beam scale (HealthOMeter, Sunbeam Products, Boca Raton, FL).

## Reported food intake on feed days

Each participant completed a 3-day food record on 2 feed days during the week, and on 1 feed day during the weekend, at each week of the 10-week trial. Thus, a total of 30 feed day food records were collected for each subject. At baseline, the Research Dietician provided 15 min of instruction to each participant on how to complete the food records. These instructions included verbal information and detailed reference guides on how to estimate portion sizes and record food items in sufficient detail to obtain an accurate estimate of dietary intake. Subjects were instructed to record food items, in as much detail as possible, in the blank food diary provided. Any mixed foods were broken down to individual food items to be recorded one per line. Participants were not required to weigh foods but were asked to measure the volume of foods consumed with household measures (i.e. measuring cups and measuring spoons). When a commercial product was consumed, subjects were asked to indicate the weight of the product to assess portion size. Food records were collected at the weigh-in each week, and were reviewed by the Dietician for accuracy and completeness. All dietary information from the food records was entered into the food analysis program, Nutritionist Pro (Axxya Systems) by a single trained operator to alleviate inter-investigator bias. The program was used to calculate the total daily intake of energy, fat, protein, carbohydrate, cholesterol, and fiber.

## Reported food intake on fast days

During the ADMF controlled feeding phase, subjects were asked to report any additional food item consumed that was not included in the provided meal. During the ADMF self-selected feeding phase, each participant was asked to record his or her food intake on each fast day. At the beginning of this phase, the Research Dietician went over the food record instructions once again with each subject. Fast day food records were collected at the weigh-in each week, and all records were reviewed for accuracy and completeness by the Dietician. Dietary information from the fast day food records was analyzed by a single trained operator using Nutritionist Pro (Axxya Systems).

## Hunger, satisfaction with diet, and fullness assessment

Subjects completed a validated visual analog scale (VAS) on each fast day, in the evening, approximately 5 min before going to bed (reported bedtime ranged from 8.20 pm to 1.40 am) \[18\]. In brief, the VAS consisted of 100-mm lines, and subjects were asked to make a vertical mark across the line corresponding to their feelings from 0 (not at all) to 100 (extremely) for hunger, satisfaction with diet, or fullness. The VAS was collected at the weigh-in each week and reviewed for completeness. Quantification was performed by measuring the distance from the left end of the line to the vertical mark.

## Physical activity assessment

Habitual, free-living physical activity was assessed by a pedometer (Digiwalker SW-200, Yamax Corporation, Tokyo, Japan SW). Subjects wore the pedometer each day throughout the 10-week trial. The pedometer was worn attached to the participant's waistband during waking hours (except while bathing or swimming), and reset to zero each morning. Number of daily steps were recorded in a pedometer log provided, and the log was collected by study personnel at the weigh-in each week. No subjects were enrolled in an exercise class, and all participants were asked to refrain from joining any exercise programs during the course of the study. In this way, any changes in physical activity during the study could be estimated by the use of the pedometer.

## Statistics

Results are presented as means ± standard error of the mean (SEM). Tests for normality were included in the model. One-factor repeated measures analysis of variance was performed to determine an overall *P* value over time. The main variables tested included body weight, energy intake, nutrient intake, hunger, satisfaction and fullness. The Bonferroni correction was used to assess significance. Relations between continuous variables (i.e. body weight, energy intake, nutrient intake, hunger, satisfaction and fullness) were assessed by simple regression analyses as appropriate. Data were analyzed by using SPSS software (version 18.0 for Mac OS X; SPSS Inc., Chicago, IL).

# Results

## Subject characteristics at baseline

Of the 52 participants screened, 20 were deemed eligible to participate in the study, and 16 (4 men/12 women) completed the entire 10-week trial. Subjects who completed the study were middle age (46 ± 3 y, 35-65 y), obese (BMI 34 ± 1 kg/m^2^, 30.2-39.9 kg/m^2^), sedentary (2.4 ± 0.3 h/week of physical activity), and borderline hypercholesterolemic (LDL cholesterol level 106 ± 10 mg/dl). Eight participants were African-American, 2 were Caucasian, and 6 were Hispanic.

## Changes in body weight in response to ADMF

During the control phase, body weight remained stable (week 1: 96.4 ± 5.3 kg, week 2: 96.5 ± 5.2 kg). At the end of the ADMF controlled feeding phase (week 6), body weight decreased (*P* \< 0.001) to 93.8 ± 5.0 kg (feed day measurement) and 93.7 ± 5.0 kg (fast day measurement). By the end of the ADMF self-selected feeding phase (week 10), body weight was further reduced (*P* \< 0.001) to 92.8 ± 4.8 kg (feed day measurement) and 90.8 ± 5.0 kg (fast day measurement). Thus, a total weight loss of 5.6 ± 1.0 kg (-0.7 ± 1.0 kg per week) was attained after 8 weeks of ADMF.

## Degree of energy restriction achieved with ADMF and relation to body weight changes

Energy intake and percent energy restriction were determined from food record data collected on feed and fast days. Mean completion rate of feed and fast day food records was 83 ± 5%, and 86 ± 4%, respectively. Energy intake on feed and fast days during each week of the trial is displayed in Figure 1A. During the control phase, mean energy intake was 1937 ± 180 kcal. Mean feed day energy intake (1801 ± 226 kcal) at each week of the trial was similar to that of the control phase, and did not differ between ADMF controlled-feeding and self-selected feeding phases. Mean energy intake on the fast day (501 ± 28 kcal, 26 ± 3% of baseline needs consumed) was lower (*P* \< 0.001) than that of the feed day at each week of the trial. The ratio of energy consumed on the fast day versus the feed day during the controlled feeding phase (0.28 ± 0.03) did not differ from that of the self-selected feeding phase (0.30 ± 0.05). Percent energy restriction is reported in Figure 1B. Over the course of the trial, percent daily energy restriction remained high and stable (37 ± 7%), and did not differ between the ADMF controlled-feeding and self-selected feeding phases. Degree of energy restriction achieved by ADMF was correlated to rate of weight loss (*r* = 0.42, *P* = 0.01) and absolute post-treatment weight loss (*r* = 0.48, *P* = 0.008).

## Hyperphagic response

Hyperphagia on the feed day in response to the lack of food on the fast day is reported in Figure 2. We hypothesized that the participants would increase their energy intake on the feed day by approximately 125% of their baseline needs. However, no such hyperphagic response was observed, as mean feed day energy intake (1801 ± 226 kcal) was similar to calculated requirements (1896 ± 160 kcal) at each week of the trial. Thus, on average, subjects were only consuming 95 ± 6% of their calculated energy needs on the feed day.

## Changes in nutrient intake during ADMF and relation to body weight changes

The nutrient composition of feed and fast day meals during each phase of the trial is displayed in Table 2. During the control phase, subjects were consuming a high fat (\> 35% of kcal), high saturated fat (\> 7% of kcal), high cholesterol (\> 200 mg/d), and low fiber diet (\< 25 g/d), as per the National Cholesterol Education Program (NCEP) dietary guidelines \[19\]. During the ADMF controlled feeding phase, the nutrient composition of feed day diet was similar to that of the control phase (i.e. high total fat, high saturated fat, high cholesterol and low fiber). During the ADMF self-selected feeding phase, total fat (33 ± 4% kcal) and saturated fat (7 ± 1% kcal) intake on the feed day decreased (*P* \< 0.05), relative to the control phase. Dietary cholesterol, however, was still above the recommended daily allowance (223 ± 27 mg/d), and dietary fiber (15 ± 1 g) was still below the recommended intake level on the feed day. Decrease in total fat intake was related to rate of weight loss (*r* = 0.38, *P* = 0.03).

Nutrient composition of feed day and fast day meals during each phase of the trial^1^

|  | Pre-loss control phase^2^ | Weight loss/ADMF controlled feeding phase |  | Weight loss/ADMF self-selected feeding phase |  |
|----|----|----|----|----|----|
|  |  | **Feed day^2^** | **Fast day^3^** | **Feed day^2^** | **Fast day^2^** |
|  |  |  |  |  |  |
| Energy (kcal) | 1937 ± 180 | 1792 ± 228 | 413 ± 20 | 1645 ± 187 | 588 ± 46 |
| Protein (% kcal) | 18 ± 1 | 18 ± 1 | 23 ± 1 | 19 ± 1 | 20 ± 1 |
| Carbohydrate (% kcal) | 46 ± 3 | 47 ± 3 | 52 ± 0 | 46 ± 2 | 51 ± 3 |
| Total fat (% kcal) | 36 ± 5^a^ | 36 ± 6^a^ | 25 ± 1^b^ | 33 ± 4^b^ | 29 ± 1^b^ |
|  Saturated fat (% kcal) | 11 ± 1^a^ | 10 ± 1^a^ | 6 ± 1^b^ | 7 ± 1^b^ | 9 ± 1^a^ |
|  Monounsaturated fat (% kcal) | 11 ± 1 | 12 ± 1 | 11 ± 1 | 13 ± 1 | 8 ± 1 |
|  Polyunsaturated fat (% kcal) | 10 ± 2 | 11 ± 1 | 8 ± 1 | 10 ± 1 | 9 ± 1 |
|  Trans fat (% kcal) | 4 ± 1^a^ | 3 ± 1^a^ | 0^b^ | 3 ± 1^a^ | 3 ± 1^a^ |
| Cholesterol (mg) | 249 ± 46^a^ | 239 ± 24^a^ | 68 ± 3^b^ | 223 ± 27^a^ | 73 ± 9^b^ |
| Cholesterol (mg/kcal) | 0.13 ± 0 | 0.13 ± 0 | 0.17 ± 0 | 0.14 ± 0 | 0.12 ± 0 |
| Fiber (g) | 16 ± 2^a^ | 12 ± 2^a^ | 10 ± 1^b^ | 15 ± 1^a^ | 7 ± 1^b^ |
| Fiber (g/kcal) | 0.008 ± 0 | 0.008 ± 0 | 0.02 ± 0 | 0.009 ± 0 | 0.01 ± 0 |

^1^ Values reported as mean ± SEM. Values in the same row with different superscript letters are significantly different, *P* \<0.05 (One-factor ANOVA with Bonferroni analysis).

^2^ Food intake self-reported each week using 3-d food record.

^3^ Food was provided on the fast day during the controlled feeding phase.

## Hunger, satisfaction with diet, and fullness

Changes in hunger, satisfaction, and fullness during the trial are displayed in Figure 3. During the first week of ADMF, hunger scores were elevated. However, after two weeks of ADMF, hunger scores decreased (*P* \< 0.05) and remained low throughout the rest of the trial. Satisfaction with the ADMF diet was low during the first 4 weeks of the intervention, but gradually increased (*P* \< 0.05) during the last 4 weeks of the study. Fullness scores remained low during the entire 8-week ADMF intervention.

## Changes in physical activity habits

All subjects wore a pedometer each day throughout the entire trial to assess changes in physical activity habits. On average, subjects were very compliant with pedometer use, and steps were recorded on 87 ± 4% of study days. We hypothesized that subjects would feel less energetic on the fast days, and would therefore take less steps/d on fast days than feed days. Interestingly, no difference was noted when fast day values (6416 ± 851 steps/d) were compared to feed day values (6569 ± 910 steps/d) (Figure 4). Moreover, physical activity remained constant throughout the 10-week study, as steps/d taken during the control phase was similar to that of the ADMF phases.

# Discussion

Preliminary reports indicate that ADMF may be an effective strategy to help obese individuals lose weight \[13,14\]. However, the dietary and physical activity adaptations that contributed to this pronounced weight loss by ADMF were not tested previously. We show here, for the first time, that weight loss by ADMF occurred due to change in meal pattern, i.e. obese subjects limited their energy intake to 25% of needs on the fast day with no hyperphagic response on the feed day. This change in meal pattern helped these subjects to achieve a marked degree of energy restriction (37% net daily) which was related to the pronounced weight loss attained (5.6 kg in 8 weeks). This study is also the first to demonstrate that subjects become habituated to the ADMF diet (i.e. feel very little hunger on the fast day) after approximately 2 weeks, and that physical activity habits are not affected by fasting on alternate days.

A key objective of the present study was to examine the degree of energy restriction achieved by ADMF and to investigate how this relates to rate of weight loss. In order to measure energy intake and percent energy restriction, we asked obese participants to complete food records on feed and fast days throughout the trial. Results from the food record analysis reveal that obese subjects were able to consistently limit their energy intake to approximately 25% of needs (500 kcal) on the fast day. Our data also show that the ratio of energy consumed on the fast day versus the feed day did not differ between phases. However, it should be noted that there was a trend towards consuming less energy on the feed days, and more energy on the fast days over the course of the trial. It will therefore be of interest in long-term ADMF studies to examine whether restriction gradually diminishes on the fast day after several months of diet. The degree of hyperphagia that occurred on the feed day in response to the lack of food on the fast day was also assessed. Our data indicate that no hyperphagic response took place as subjects only consumed approximately 95% of their calculated energy needs on each feed day throughout the trial. These findings therefore suggest that obese subjects are able to drastically change their meal pattern in a way that conforms to the ADMF protocol. Nevertheless, there are several limitations to these data that must be discussed. First and foremost, it is well known that obese subjects underreport energy intake by 20 to 40% when completing food records \[20,21\]. The extent to which these subjects underreported energy intake became apparent when we tried to relate reported energy intake to the weight loss achieved. From the food record data, we calculated that, on average, subjects were restricted by 37% of calculated needs every day. If the subjects were indeed restricted by this amount, this would have resulted in a rate if weight loss of 1.2 kg/week. In actuality, the rate of weight loss was 0.7 kg/week. This disparity between reported intake and weight loss can be observed when examining the limited amount of weight lost during the 4-week self-selected feeding phase (body weight reduction of 93.8 kg to 92.8 on the feed day, equivalent to 1 kg of weight loss). The incongruity between self-reported energy intake and rate of weight loss therefore suggests that subjects were underreporting energy intake. In view of this, it will be important for future ADMF trials to assess energy intake and energy restriction by more robust methods, such as the doubly labeled water technique \[22,23\]. It should also be mentioned that assessing body weight changes by ADMF is difficult as weight measurements are drastically different from feed to fast day. This discrepancy in body weight is most likely due to the additional weight of food present in the gastrointestinal tract, and not changes in fat mass from day to day. As a potential solution, future trials of ADMF should average body weight measurements taken from consecutive feed and fast days to attain a more accurate assessment of weight.

In addition to energy intake, we also examined changes in dietary macronutrient composition throughout the course of the trial. We hypothesized that during the ADMF controlled feeding phase (weeks 3-6), when dietary counseling was not provided, subjects would instinctively choose higher fat/more energy dense foods on the feed day to make up for the lack of energy consumed on the fast day. Interestingly, fat intake did not increase from the baseline period (36% of kcal) to the ADMF-controlled feeding period (36% of kcal). These preliminary data suggest that subjects are not likely to consume higher fat diets on the feed day when partaking in an ADMF regimen. We also hypothesized that the dietary counseling provided during the self-selected feeding phase (weeks 7-10), would help subjects decrease total fat, saturated fat, and cholesterol intake, while increasing fiber intake. Results reveal that counseling assisted these individuals in lowering their total fat and saturated fat intakes to levels that conform with NCEP dietary recommendations \[19\], and that these changes in fat intake were related to rate of weight loss. On the other hand, dietary counseling appeared to have no effect on cholesterol or fiber intake. This lack of effect of dietary counseling on the intakes of these nutrients has been reported previously \[24\]. It should also be noted that fiber intake on the fast day was particularly low (7-10 g/d). Since quantity of food consumed on the fast day is limited, it would be difficult for individuals to meet fiber requirements \[25\]. As such, it is recommended that future trials in the ADMF field provide a fiber supplement on the fast day to help individuals meet recommendations \[19,26\].

Changes in perceived hunger, satisfaction with diet, and fullness were also evaluated on each fast day throughout the trial. This study is the first to show that obese subjects become habituated with ADMF after approximately 2 weeks of diet (i.e. feel very little hunger on the fast day). Our data also demonstrate that subjects become more satisfied with ADMF after approximately 4 weeks of diet. Feelings of fullness, however, remained low across the course of the trial suggesting that subjects never felt "full" at any point while undergoing 8-weeks of ADMF. These findings may have important implications for long-term adherence to ADMF by obese men and women \[27-29\]. More specifically, since hunger virtually diminishes, and since satisfaction with diet considerably increases within a short amount of time (2-4 weeks), it is likely that obese participants would be able to follow the diet for longer periods of time. It is important to note, however, that the subjects only completed the VAS scales pre-bedtime. Thus, the data only reflects their feelings immediately before going to bed, and is not indicative of their feelings of hunger and satisfaction throughout the day. Future trials in this area should administer these VAS scales throughout the day to obtain a more complete data set for these variables. It should also be noted that hunger spiked at week 8. We speculate that this may have occurred because this study week corresponded to Memorial Day weekend, and subjects may have felt hungrier while attending food-related celebrations. Moreover, trials examining the ability of obese subjects to comply with ADMF for longer durations (i.e. 24 to 52 weeks), and in consequence, lose larger amounts of weight, will be an important focus of future research in this field.

The effects of ADMF on habitual physical activity was also assessed by having the subjects wear a pedometer on everyday of the study. We hypothesized that subjects would feel less energetic on the fast days, and would therefore be less physically active (i.e. take less steps/d) on fast days than feed days. Surprisingly, physical activity level did not differ between feed and fast days. Moreover, there was no difference in activity level when steps/d taken during the ADMF phase were compared to steps/d taken during the control phase. Similar results have also been reported in normal weight individuals undergoing ADMF \[12\]. These data suggest that obese individuals are able to maintain their level of habitual physical activity despite decreases in energy intake on the fast day. This maintenance of physical activity while undergoing ADMF would thus allow obese individuals to lose weight consistently on feed and fast days as energy expenditure would stay constant.

A key limitation of this study is that there was no true control group. Having a control arm run parallel to the treatment (ADMF) arm would have strengthened the study by allowing us to: 1) compare changes in the ADMF group to that of a non-restricted control group at each time point, and 2) identify events (such as holidays) that may have resulted in deviations from the prescribed diet. Future studies aiming to test similar objectives should employ a control group where possible.

In summary, these findings indicate that obese subjects quickly adapt to ADMF, and that changes in energy/macronutrient intake, hunger level, and maintenance of physical activity play a role in influencing rate of weight loss by ADMF. These preliminarily data offer promise for the implementation of ADMF as a long-term weight loss strategy in obese populations.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

MCK performed all the diet analyses and assisted with trial coordination and manuscript preparation. SB coordinated the human clinical trial. MF and SF assisted with data analysis and manuscript preparation. KAV designed the study and wrote the manuscript. All authors read and approved the final manuscript.

## Acknowledgements

We are grateful for the help of Kathryn Tomaszewski and Kristin Hoody during the analysis phase of the trial. Funding source: University of Illinois at Chicago, Departmental funding.


abstract: The 'action' in genome-level evolution lies not in the large gene-containing segments that are conserved among related species, but in the breakpoint regions between these segments. Two recent papers in *BMC Genomics* detail the pattern of repetitive elements associated with breakpoints and the epigenetic conditions under which breakage occurs.
author: David Sankoff
date: 2009
institute: 1Department of Mathematics and Statistics, University of Ottawa, 585 King Edward Avenue, Ottawa K1N 6N5, Canada
references:
title: The where and wherefore of evolutionary breakpoints

For many years, dating back to well before the genomics era, there have been numerous observations and hypotheses of associations between the presence or absence of breakpoints of chromosomal evolution and prominent features of the genomic landscape: telomeres, centromeres, recombination hotspots, gene deserts or gene-rich regions, isochores, cytogenetically fragile sites, oncological rearrangements, segmental duplications, transposons and other repetitive elements. Two recent papers in *BMC Genomics* take somewhat different tacks on this subject. Longo *et al.* \[1\] capitalize on new sequencing resources for the tammar wallaby, *Macropus eugenii*, to substantiate the links between the rapid and complex patterns of evolution of centromeric sequence and recurrent rearrangement activity in marsupials, and to discover one evolutionary breakpoint region in humans that has repetitive element similarity to corresponding regions in marsupials. Lemaitre *et al.* \[2\] combine a high-resolution breakpoint localization procedure with specialized data that they have calculated or obtained on DNAse sensitivity, CG content, hypomethylation and replication origins \[3\] to dispel some of the most widespread folklore in the field. They show that propensity to breakage is not favored in gene deserts but, on the contrary, is closely related to transcriptional activity and DNA accessibility in a region, a conclusion that lends a decidedly epigenetic flavor to our understanding of rearrangement.

# The ephemeral breakpoint

A breakpoint or breakpoint region is not a tangible physical entity in a genome; it is an analytical construct arising only in the comparison of two genomes and, as such, exists or not, and has one set of characteristics or another, depending on the assumptions and methodology of this comparison. When we can identify two contiguous chromosomal segments in one genome, each of which seems orthologous to a different segment in another genome, and these latter segments are not contiguous, we can say that there is a breakpoint. When one of the segments is small (according to a threshold of anywhere from 10^2^ to 10^6^ base pairs), we might wish to consider the two breakpoints delimiting the segment as reflecting a single breakpoint. If the two segments are actually contiguous in the second genome but one is inverted compared with its orientation in the first genome, we might want to count the breakpoint or not. Normally, the DNA alignment of the two genomes will not be such that the breakpoint can be pinpointed as separating two specific adjacent base pairs, but rather there will be a more or less lengthy region in the middle of the segment on the first genome that does not align well to either of the two segments of the second genome or their flanking sequences. Instead of break 'point', we have a break 'region' with its own particular characteristics \[4\].

To complete the deconstruction of the breakpoint terminology, we can naïvely imagine the free ends of two or more double-stranded breaks in DNA molecules flailing around inside the nucleus until they are repaired (incorrectly), resulting in a rearrangement within a chromo some or involving two chromosomes. This does indeed happen as a result of radiation, toxic or mechanical stress or, as is clearly demonstrated by Lemaitre *et al.* \[2\], following normal cellular activity that requires regions of open chromatin. It should be emphasized, however, especially where breakpoints are associated with repetitive elements, rearrangements do not derive from any actual DNA breakage, but from nonhomologous recombination caused by faulty alignment of repetitive elements during meiosis.

The Longo *et al.* article \[1\] contains a carefully executed and controlled analysis of the distribution of different kinds of repetitive elements in selected segments from three kinds of genomic region in the tammar wallaby: centromeric regions, breakpoint regions (actually three locations in one breakpoint region) and euchromatic regions not containing a breakpoint. They showed a dramatic enrichment in the breakpoint region of sequence characteristic of endogenous retroviruses (ERVs) and LINE1 transposable elements, and a deficiency of SINE and CR1 transposable element sequences, when compared with the euchromatic regions, with the centromeric regions falling in between the two other patterns. In addition, in a human genomic region containing parts homologous to the marsupial breakpoint region and parts homologous to one of the euchromatin selections, the pattern of repetitive elements makes a transition from ERVs and LINE1s to SINEs. This is suggestive of an association between neocentromeric tendencies, regional instabilities around evolutionary breakpoints and the incorporation of specific kinds of repetitive elements. Although the authors' \[1\] longstanding interest in marsupial evolution and the role of centromeres in genomic rearrangements, as well as the availability of new sequence resources on the tammar wallaby, are certainly sufficient motivation for the study of repetitive elements in this context, and given the very different patterns known for human and primate pericentromeric evolution, it will now be important to generalize this work to genomes for which sequencing is essentially complete and to undertake a more comprehensive survey of repetitive elements in regions of each kind.

# Reuse and recurrence

The term 'breakpoint reuse' is used in the rearrangements literature to cover two rather different concepts. In its original algorithmic use \[5\], it denoted the excess of the number of rearrangements necessary to transform one genome into another compared with half the number of breakpoints induced by the comparison of two genomes (given that inversions and reciprocal translocations normally create two breakpoints each). This was accounted for by assuming that some breakpoints (without specifying which ones) were used more than once in the transformation. Soon afterwards, its most frequent meaning became the recurrence of the same breakpoint in two lineages but not their common ancestor with respect to an outgroup lineage \[6\]. Despite the attractiveness of these concepts to many authors (such as Longo *et al.* \[1\]), neither breakpoint reuse nor breakpoint recurrence is solidly established as a major evolutionary phenomenon, in contrast to well-known disease-causing somatic cell rearrangements. The original concept of reuse, which did not pertain to particular breakpoints but only their aggregates, has rarely if ever been systematically and quantitatively documented at the level of all the individual breakpoints induced by a pair of genomes. Indeed, the algorithmic results suggesting breakpoint reuse are not only wildly variable depending on how telomeric breakpoints are weighted \[7\], but are in any case predictable artifacts of highly constrained models of evolution through rearrangement \[8\] (models that permit no deletion of chromosome segments, no chromosome or chromosomal arm duplication, no segmental duplication, no transpositions, no jumping translocations and no deletion of paralogous syntenic blocks or interleaving deletions of duplicated blocks), and of the levels of resolution used in defining synteny blocks and breakpoint regions \[9,10\]. In the breakpoint definition above, if two breakpoints are collapsed when the small segment between them is below threshold size (a common practice), this mistakenly shows up as an increase in breakpoint reuse. As for the phylogenetic recurrence of breakpoints, the major source in this field \[6\] actually shows that 80% of the breakpoints in their mammalian phylogeny are not recurrent, and that almost all of the remaining ones affect the syntenically unstable rodent lineage. The tiny proportion of apparently recurrent breakpoints in the rest of the phylogeny would be hard to distinguish from coincidence, given the resolution of the synteny block construction.

The connection between the 'fragile sites' in traditional cyto genetics and evolutionary breakpoints is exceedingly weak \[11\] and, indeed, statistically insignificant except through a heuristically contrived categorization of the data. The same may be said for the oft-cited attempt \[6\] to associate cancer breakpoints with evolutionary breakpoints by selectively comparing only two of the reported frequency categories of neoplastic breakpoints.

# Accident and selection

An e volutionary breakpoint is the product not only of some meiotic accident at a site predisposed to breakage or nonhomologous recombination. It is also a configuration that has managed to do all of the following: make it through steps of abnormal chromosome alignment and segregation to the gamete stage; participate in creating a viable heterokaryotypic zygote that eventually develops into reproductive maturity; endure generations of likely negative selection; and emerge through genetic drift as a homokaryotypic feature of some presumably small bottleneck population. Predisposition to breakage at the cellular level is just the first step on the road to fixation, and phenotypic selection operating at the meiotic, embryonic, adult and population levels has a more important role. Somatic cells presumably have many of the same predispositions to physical breakage, although not of course to nonhomologous recombination, but cancer cells do not have to survive meiosis or life outside the affected individual, and that may be a large part of the reason why the repertoire and quantitative distribution of rearrangements in tumor genomes are very different from those in evolution \[12\].

Genetic deduction appealing to selection-based arguments at the gene expression level, together with indirect and anecdotal evidence, has recently prompted speculation about prohibition of rearrangement breakage in short inter genic regions in mammals \[13\]. These claims, however, have effectively been demolished by Lemaitre *et al.* \[2\], who measured directly and systematically, at a high level of resolution, the connections between both high rate of breakage and short intergenic distances and four strong correlates of transcriptional activity: GC content, proximity to origins of replication (as inferred from 'N-domains' \[3\]), hypomethylation (based on CpG ratios) and DNase sensitivity. This innovative and convincing work, to which the authors added support ranging from the classic Bernardi theory of isochores \[14\] to the more recent mammalian replicon model, overturns the conventional genetic wisdom and reopens evolutionary questions about mechanisms promoting neutral variation at the karyotypic level. It adds a weighty contribution to the accumulating body of results, such as those on the gibbon *Nomascus leucogenys leucogenys* \[15\] and those previously produced by the O'Neills-Graves collaboration on marsupials, cited in the Longo *et al.* article \[1\], on the epigenetic conditioning of evolutionary chromosome rearrangement.

### Acknowledgements

This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). DS holds the Canada Research Chair in Mathematical Genomics.


abstract: The authors describe the development, implementation, and evaluation of their innovative social marketing campaign.
author: Katherine Ahrens; Charlotte K Kent; Jorge A Montoya; Harlan Rotblatt; Jacque McCright; Peter Kerndt; Jeffrey D Klausner\* To whom correspondence should be addressed. E-mail:<firstname.lastname@example.com>
date: 2006-12
references:
title: Healthy Penis: San Francisco's Social Marketing Campaign to Increase Syphilis Testing among Gay and Bisexual Men

# The Problem: Sharp Increase in Syphilis among Men Who Have Sex with Men

San Francisco experienced a sharp rise in early syphilis between 1999 and 2002, with the number of cases rising from 44 to 494 per year (Figure 1). Rates continued to rise through 2004. Since 1999, most syphilis cases have been among men who identified as gay or bisexual (88%), were white (60%), and were infected with HIV (61%) (Figure 1). In June 2002, the San Francisco Department of Public Health (SFDPH), STD Prevention and Control Services launched a social marketing campaign, called Healthy Penis, designed to increase syphilis testing and awareness among gay and bisexual men.

Social marketing, like marketing in the private sector, is a research-driven approach to behavior change \[1\]. Unlike commercial marketing, however, it is truly consumer-centered and designed to increase the health of the consumer \[2\]. The success of any social marketing campaign can be attributed to the net effect of its five main components: branding, segmentation, price, placement, and promotion \[1–3\]. The first component, *branding*, focuses on the health behavior message so that the desired behavior, or product, appeals to the needs and values of the consumer through functional and emotional attributes.

The second component is concerned with the *segmentation* of the intended audience—developing a campaign message that emphasizes the target population's values, attitudes, and beliefs and/or capitalizes on their current stage of behavior change. Messages that are customized in this manner will ensure that the health behavior (product) is appealing and applicable to each subgroup.

The third component is *price*. In social marketing, price is the social, psychological, or physical cost the consumer associates with performing the health behavior. The fourth component is product *placement*. In public health, this involves delivering the resources that make the desired health behavior possible at a time when it will most likely be sought out. The fifth component is the *promotion* of the health behavior through communication media like print, television, radio, outdoor advertising, or face-to-face techniques that are determined by the information-consumption habits of the consumer. Notable successful social marketing campaigns include North Carolina's "Click It or Ticket" campaign aimed at increasing seatbelt use, Florida's "truth" anti-smoking campaign, and the United States National WIC (Women, Infants and Children) Breastfeeding Promotion project \[4\].

The impact of a social marketing campaign is evaluated based on the measurement of change in the targeted behavior, i.e., increased syphilis testing in the case of Healthy Penis, and the extent to which this change is associated with people who received the campaign messages. To that end, we initially evaluated the Healthy Penis campaign in 2002–2003 and found a higher rate of syphilis testing among those aware of the campaign than those unaware of the campaign \[5\]. In addition, syphilis knowledge was higher among those aware, a secondary goal of the campaign.

In this article we describe the development, implementation, and evaluations of the Healthy Penis campaign. We present a simple method to evaluate the immediate and long-term effectiveness of a public health media campaign designed to change behavior. We include our most recent evaluation, conducted in 2004–2005, which assessed the long-term effectiveness of the campaign \[6\] and determined if respondents aware of the campaign 2.5 years after it began displayed the same syphilis knowledge and testing behavior as those initially aware of the campaign in the first evaluation.

# Healthy Penis Campaign Development

The Healthy Penis campaign was launched during the annual San Francisco Lesbian Gay Bisexual Transgender Parade in late June 2002 and continued through December 2005. A San Francisco–based social marketing firm, Better World Advertising, created the campaign in collaboration with the SFDPH to target the gay and bisexual community. This campaign was part of a broad multifaceted effort begun in 1999 by the SFDPH in response to the syphilis epidemic \[7\].

The campaign was developed in collaboration with the Los Angeles–based Stop the Sores syphilis prevention campaign, which allowed us to lower our start-up costs to \$75,000 in 2002. The campaign was continued through the end of 2005 at an additional cost of \$295,000 \[7\]. Three-quarters of the campaign's first-year funds were spent on campaign development, with the remaining quarter spent on displaying campaign materials \[8\].

Syphilis is often initially asymptomatic and testing is readily available in San Francisco for easy diagnosis. Based on these characteristics and input from the community partners group, the SFDPH determined that the primary campaign health behavior message should be "get tested," with the main objective of changing community norms around syphilis testing. Secondary objectives were to increase awareness about the syphilis epidemic in gay and bisexual men and to increase knowledge about syphilis (symptoms, routes of transmission, the link between syphilis and HIV transmission, and so on). The complete methodology for selection of campaign concepts and themes for this campaign has been described in a previous manuscript \[5\].

The Healthy Penis campaign was promoted in neighborhoods where the greatest concentration of gay or bisexual men lived and where there were businesses that catered to this population. The campaign incorporated the use of humorous cartoon strips that featured characters like Healthy Penis and Phil the Sore to: (1) promote syphilis testing, (2) publicize the rise of syphilis among gay and bisexual men, (3) provide information on syphilis transmission, symptoms, and prevention, and (4) delineate the connection between syphilis and HIV (Figure 2). These cartoon strips were initially published semi-monthly in a popular gay Bay Area publication. After publication, poster-size reproductions were posted on the streets; in bars and commercial sex venues; on bus shelters and bus advertising; on palm cards; and on banner advertisements on one of the most popular Internet sites for meeting sex partners among gay men.

Topics addressed in campaign cartoons initially emphasized all four areas described above and then after 1.5 years focused primarily on the message "get tested." In addition to messages contained within the cartoon strip, brief text boxes were displayed on the bottom of the cartoon images describing modes of syphilis transmission (skin-to-skin contact, oral, anal, and vaginal sex), symptoms (painless sore and/or rash), and explaining that syphilis is curable. These text boxes were displayed on cartoons from June 2002 to October 2003 with decreasing frequency.

# Evaluation of the Campaign: What Impact Has It Had?

## Survey methodology.

To evaluate the effectiveness of the campaign, we conducted two waves of surveys using the same instrument: one six months after the campaign began (December 2002–February 2003; Evaluation I), and a second 2.5 years after the campaign began (September 2004–March 2005; Evaluation II). For each survey, men were intercepted at coffee shops, bars, markets, laundromats, sex clubs, a clean and sober community center, on sidewalks, and in other venues located in campaign-targeted neighborhoods.

Respondents were asked about basic demographic information; unaided (e.g., spontaneous mention) and aided (e.g., prompted response) awareness of the Healthy Penis campaign; perceived key messages of the campaign; syphilis knowledge (via open-ended questions); sexual practices in the past month; HIV status; and how many times they were tested for syphilis in the past six months.

Comparisons were made among those aware of the campaign between the first and second sample of respondents. Recent history of syphilis testing was compared with campaign awareness level for each evaluation separately. Analysis included Fisher's Exact and Cochran-Armitage tests, and logistic regression.

## Survey population.

Two hundred and forty-four interviews were conducted with San Francisco residents and included in the first evaluation; 150 interviews were included in the second evaluation. For each evaluation, all respondents were men who have sex with men (MSM) and most were white and HIV negative. Our respondents were similar to the MSM population in San Francisco. More detailed characteristics of the two samples are described in Table 1. There were no significant demographic differences between Evaluation I and II participants.

###### Demographic Characteristics of Respondents from First and Second Evaluations of the Healthy Penis Campaign

![](pmed.0030474.t001)

## Campaign awareness.

Campaign awareness was high with 80% (*n* = 194/244) and 85% (*n* = 127/150) of respondents aware of the campaign in Evaluation I and II, respectively. Unaided and aided awareness was similar between the two evaluations; 33% versus 41% spontaneously mentioned the Healthy Penis campaign (unaided awareness) when asked to recall recent advertisements or public events about sexual health issues, and an additional 47% versus 44% recognized the campaign (aided awareness) when shown a campaign image (Evaluation I and II, respectively). There was no difference in overall campaign awareness, unaided awareness, or aided awareness between the two evaluations (*p* = 0.23, *p* = 0.13, *p* = 0.60, respectively). Among respondents aware of the campaign, campaign exposure was greater in the first evaluation; Evaluation I respondents reported a median of six exposures (mean, 18) over the previous three months, while Evaluation II respondents reported a median of five times (mean, six) in the previous three months (Wilcoxon two-sample test, *p* \< 0.0001). Perceptions of key messages among respondents who were aware of the campaign did not change markedly between Evaluations I and II, with the most common message identified being "get tested" (53% versus 55%, respectively, *p* = 0.73).

## Syphilis testing in the last 6 months.

Both evaluations found a positive association between campaign awareness and recent syphilis testing—the primary objective of the social marketing campaign. An increasing proportion of respondents reported syphilis testing in the previous six months by campaign awareness level (none, aided awareness, and unaided awareness): Evaluation I: 26%, 40%, 55% (Cochran-Armitage trend test z = −3.303, *p* = 0.001); Evaluation II: 35%, 42%, 60% (Cochran-Armitage trend test z = −2.304, *p* = 0.02) (Figure 4). After controlling for potential confounders (age, HIV status, casual sex partners in the last month) in a multivariable logistic regression model, each increase in campaign awareness level during Evaluation I was associated with a 90% increase in likelihood for having tested for syphilis in the past six months (odds ratio \[OR\] 1.9 \[95% confidence interval (CI), 1.3–2.9\]). Other variables significantly related to syphilis testing in Evaluation I included being HIV infected (OR 4.9 \[95% CI, 2.3–10.5\]), and having casual sex partners in the last month (OR 2.0 \[95% CI, 1.1–3.9\]). The same multivariable model applied to Evaluation II respondents found each increase in campaign awareness level to be associated with a 76% increase in likelihood for syphilis testing (OR 1.76 \[95% CI, 1.01–3.1\]), and no other factors were significant in the model, although HIV-infected respondents showed an increased likelihood of syphilis testing (OR 2.1 \[95% CI, 0.9–5.2\], *p* = 0.10). In both evaluations, a higher proportion of HIV-positive respondents reported syphilis testing than did HIV-negative respondents (Evaluation I 71% versus 34%, p \< 0.05, Evaluation II 63% versus 44%, *p* = 0.07).

Overall, reported syphilis testing in the previous six months did not change from the first evaluation to the second evaluation (42% versus 49%, *p* = 0.25).

## Syphilis knowledge.

There were few significant differences between evaluations among respondents aware of the campaign in terms of knowledge of syphilis, including knowledge of symptoms; groups most affected by syphilis; how to find out if you have syphilis; health consequences of untreated syphilis; and the relationship between syphilis and HIV acquisition and transmission (Figure 3).

## Syphilis incidence.

In 2005, incidence of early syphilis was lower than in the previous three years, with decreases in cases in gay/bisexual men accounting for the drop \[9\]. Although our campaign evaluations were cross-sectional and causality cannot be determined, it does appear that the Healthy Penis campaign, along with other SFDPH syphilis elimination efforts \[7\], may have led to this decrease in syphilis incidence.

# Conclusions from Evaluations

MSM who were aware of the Healthy Penis campaign were more likely than those unaware to have recently tested for syphilis and to have greater knowledge about syphilis. This effect was sustained for almost three years.

# Next Steps for Healthy Penis

After initial evaluation of the Healthy Penis campaign, elements have been used in Philadelphia, Seattle, Palm Springs, and Santa Clara County, California ([www.healthypenis.org](http://www.healthypenis.org)). Thus the campaign can be translated to other jurisdictions. Because much of the material is already developed, other jurisdictions may adapt and use the campaign at a lower cost than was originally incurred in San Francisco, although they will need to conduct evaluations to ensure the campaign is effective for their population. In addition, the principles used in the development, implementation, and evaluation of Healthy Penis are universally applicable for any social marketing campaign.

Based on our experience with the Healthy Penis campaign, we believe spending more start-up resources on campaign development rather than campaign placement \[8\] was probably what led to our high level of campaign awareness, demonstrating that our campaign resonated with our target population. We also learned that partnering with another jurisdiction (Los Angeles) lowered start-up costs and creating a Community Partners Group helped us tailor our campaign messages to be most effective. And most importantly, we learned that careful, well-planned evaluations are critical in assessing the impact of a campaign, which then supported continuing the campaign and implementing it elsewhere. Our evaluations strongly suggest that the Healthy Penis social marketing campaign was effective in augmenting syphilis testing and increasing syphilis awareness and knowledge in the San Francisco gay and bisexual community. This effect might have contributed to decreased syphilis incidence in 2005.

The authors would like to thank Les Pappas from Better World Advertising, the STD Community Advisory Group for Syphilis Elimination, and the SFDPH employees who administered the evaluation surveys.

**Author contributions.** CKK, JAM, HR, JM, PK, and JDK conceived and designed the experiments. KA, JAM, and HR analyzed the data. KA, CKK, JAM, HR, JM, PK, and JDK wrote the paper. JM worked closely with Better World Advertising in the planning and development of the campaign in 1999.

# References

### Abbreviations

CI

:   confidence interval

MSM

:   men who have sex with men

OR

:   odds ratio

SFDPH

:   San Francisco Department of Public Health