author: Fumihiko UranoCorresponding author: Fumihiko Urano, <email@example.com>.
date: 2014-03
references:
title: Wolfram Syndrome iPS Cells: The First Human Cell Model of Endoplasmic Reticulum Disease

Wolfram syndrome is a rare autosomal recessive genetic disorder with clinical signs apparent in early childhood. This condition is characterized by childhood-onset diabetes, optic nerve atrophy, deafness, diabetes insipidus, and neurodegeneration, and it results in death in middle adulthood (1–3). Genetic and experimental evidence strongly suggest that endoplasmic reticulum (ER) dysfunction is a critical pathogenic component of Wolfram syndrome (4,5). However, there is a lack of complete understanding of the pathways and biomarkers involved in the disease process due to the limitations of animal models that do not accurately reflect human patients. As a result, despite the underlying importance of ER dysfunction in Wolfram syndrome, there are currently no therapies that target the ER, a deficiency that points to the urgent need to develop a human cell model of this condition. In this issue, Shang et al. (6) report that this has been successfully accomplished.

ER is a membrane network within the cytoplasm of cells that is involved in protein synthesis, calcium storage, redox regulation, steroid synthesis, and cell death. Recent clinical and genetic evidence indicate that acquired or inherited ER dysfunction can cause rare genetic diseases such as Wolfram syndrome, as well as many common diseases, including type 1 and type 2 diabetes, atherosclerosis, and neurodegenerative diseases (7–11). Thus, ER is an emerging target for both rare and common chronic conditions. However, it has been challenging to study the efficacy of pharmacological agents that could potentially reverse ER dysfunction due to the lack of a human cell model of ER disorders.

In Wolfram syndrome, pancreatic β-cells and neuronal cells are selectively destroyed as a consequence of mutations in the *WFS1* gene. This gene encodes a transmembrane protein localized to the ER, suggesting that ER dysfunction is a major pathogenic component of Wolfram syndrome. In animal and cell models of Wolfram syndrome, WFS1 mutations lead to elevated ER stress levels, pancreatic β-cell dysfunction, and initiation of ER stress–associated cell death (5,12,13). Shang et al. showed that Wolfram syndrome patient induced pluripotent stem (iPS) cell–derived β-cells also showed increased levels of ER stress molecules, as well as decreased insulin content. Upon exposure to experimental ER stress, Wolfram syndrome patient iPS cell–derived β-cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. These observations were all consistent with previous findings and could validate the roles of WFS1 in insulin production, secretion, and protection against ER stress (14). Moreover, Shang et al. showed that 4-phenyl butyric acid (4PBA), a chemical protein folding and trafficking chaperone, could restore normal insulin synthesis and the ability to upregulate insulin secretion in Wolfram syndrome patient iPS cell–derived β-cells, a finding that suggested 4PBA is a candidate drug for treating patients with Wolfram syndrome.

We can expect Wolfram syndrome patient iPS cell lines and Wolfram iPS cell–derived β-cells to be cornerstones for developing novel therapeutic modalities for Wolfram syndrome and other diseases involving ER dysfunction. We can use these cells to screen and identify drugs for treating patients with Wolfram syndrome and other ER-associated diseases. Chemical chaperones such as 4PBA and tauroursodeoxycholic acid (TUDCA) are good candidates. It has been shown that these compounds can mitigate ER stress in peripheral tissues, such as liver, in mouse models of diabetes (15). Glucagon-like peptide 1 agonists and rapamycin are encouraging compounds because they have been shown to prevent ER stress–mediated β-cell death in mouse and cell models (16,17) (Fig. 1). Clinical trials evaluating the efficacy of these drugs should be seriously considered after we carefully test these compounds in β-cells and neurons derived from Wolfram iPS cells. It has been shown that WFS1 polymorphisms and mutations are associated with type 2 diabetes and adult-onset diabetes, suggesting that drugs effective for Wolfram syndrome may also be useful for the treatment of more prevalent forms of diabetes, type 1 and type 2 diabetes (18,19). Thus, the potential public health implications of the new findings are considerable.

In the future, we can use these cells to regenerate damaged tissues including pancreatic β-cells, retinal ganglion cells, and neurons in patients with Wolfram syndrome (Fig. 1). Rapid progress in genetic editing technologies and regenerative medicine will make it possible to correct WFS1 mutations in patient-specific iPS cell lines and regenerate patients' damaged cells. Shang et al. (6) have taken us a step closer toward these goals. As a physician and scientist who is working on Wolfram syndrome, a devastating form of juvenile diabetes, I truly appreciate their important accomplishment. We should make the best use of these cells to develop treatments for Wolfram syndrome—efforts that may lead to breakthroughs in diabetes treatment.

## Article Information

**Acknowledgments.** The author thanks all of the participants in the Wolfram Syndrome International Registry (<http://wolframsyndrome.dom.wustl.edu/>) and the Washington University Wolfram Clinic for their time and effort. The author also thanks the Washington University Wolfram Study Group members and the study staff for advice and support.

The author dedicates this article to M. Alan Permutt, MD, whose untimely death from cancer was a great loss for us all.

**Funding.** This work was supported by grants from National Institutes of Health (DK-067493, DK-016746, DK-020579, and UL1-TR-000448), JDRF (47-2012-760 and 17-2013-512), American Diabetes Association (1-12-CT-61), Samuel E. Schechter Professorship, the Ellie White Foundation for Rare Genetic Disorders, and the Jack and J.T. Snow Scientific Research Foundation to F.U.

**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.

# References


abstract: In March, 2012 we held the first Mideast conference on "Depression and Anxiety Spectrum disorders: from basic science to the clinic and back", at the University of Amman, Jordan. This event brought together both clinical and basic scientists with expertise in depression and anxiety spectrum disorders. The meeting took place in a large lecture hall at the University of Jordan Medical School. The audience included faculty, residents, and students. The Dean of the Medical School opened the meeting, welcoming the guest speakers and participants.
author: Suzanne N Haber; Ziad Safadi; Mohammad R Milad
date: 2013
institute: 1Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
references:
title: Meeting report: "Depression and Anxiety Spectrum disorders: from basic science to the clinic and back"

Anxiety disorders are generally characterized by irrational and excessive fear along with chronically elevated levels of anxiety. The trigger for the inappropriate fear and anxiety may be clear and well-defined in some of the disorders, such as specific phobias, posttraumatic stress disorder, or social anxiety disorder, or may be unspecific or not defined in other disorders such as panic disorder and generalized anxiety disorder. Depression is commonly characterized by lost of interest in engaging in daily activities, feelings of hopelessness, lack of energy and concentration, and feelings of incompetence. Depression and anxiety share common clinical features. Between 1/3 and 2/3 of patients who suffer from major depression experience some form of anxiety as part of their illness \[1\]. Moreover, the prevalence of major depression in patients with anxiety disorders is significantly higher than in the general population. Co-morbidity between anxiety and depression is common \[2,3\]. This relationship extends to family studies. Twin studies suggest shared genetic vulnerability to depression and anxiety. Effective treatments for depression and anxiety also notably overlap: medications acting on monoaminergic systems, and psychotherapies based on fear extinction and other methods geared toward reducing avoidance and increasing goal-directed, rewarding activities. Importantly, there is also a common neural network associated with these diseases. This network is central to the pharmacological and behavioral approaches for treatment and the hypotheses that drive the research of the underlying pathophysiology. The network includes the prefrontal cortex, specifically the orbital and anterior cingulate cortices, the ventral striatum, amygdala, and the brainstem catacholaminergic systems, particularly dopamine and serotonin \[4\].

Although there are no 'hard' data available for most Middle East countries \[5\], the population afflicted with these diseases is growing according to our colleagues practicing in there. Moreover, these diseases are massively under diagnosed and under treated. This meeting provided a unique opportunity to discuss the basic and clinical science that focus on the biological underpinnings of these mental health disorders.

The goal of the meeting was three-fold: first, to bring together experts on the pathophysiology and treatment of depression and anxiety spectrum disorders in a culturally diverse environment, where such disorders are prevalent, but exposure to cutting-edge therapies and research is limited; second, to explore the directions in which science is driving this field by presenting data on experimental and therapeutic approaches to understanding and treating these syndromes; and finally, to address the possibilities for future training and research in the region. The last goal was particularly important, as this meeting provided a forum to explore needs of the region and possible future collaborations.

There were two general topics, with sub-topics within each. The first general topic, "Biological basis of mood and anxiety disorders: A clinical and basic science overview", focused on an overview of where the field is now, including a brief history. This session started with 2 general overviews, one on clinical aspects of mood and anxiety disorders (Dr. Greenberg) and a general discussion of the reward circuit that underlies these disorders (Dr. Haber). These general introductions were followed by talks focused specifically on state of the art clinical and basic science approaches. Dr. Eskandar gave an overview of neurosurgical and physiological studies in both animal and human studies; Dr. Akil gave an overview of the underlying biology associated with stress and depression; and Dr. Dougherty discussed using neuroimaging approaches to understanding these diseases. The second topic, "Biological basis of mood and anxiety disorders: New advances", focused on ongoing research. Dr. Moghaddam discussed reward and anxiety processing in adolescents, their vulnerability to disease, and goals for prevention; Dr. Aston-Jones talked about ongoing studies focused on the locus coeruleus; Dr. Greenberg discussed the clinical and basic science perspective of obsessive-compulsive disorder while Dr. Grace focused on animal models of the disease. Finally, the last afternoon of the meeting was dedicated to trauma and fear, with Dr. Pitman discussing stress, anxiety, and trauma, Dr. Milad focusing in fear and fear extinction, and Dr. Quirk discussing the neurobiology of fear learning. In addition there were some specialized talks, giving a perspective from the regions. These included a presentation from Dr. Jadiry on the prevalence of anxiety and mood disorders. Dr. Sarhan summarized cognitive behavioral therapy of Islamic religious OCD, and Dr. BaniMustafa on depression in primary care patients. We summarize the talks below.

The meeting was very well received and prompted numerous thoughtful questions and discussions. Students made comments such as "This meeting has completely changed my thinking and now I want to do research in neuroscience." Faculty comments were equally positive; "The meeting was a success by all standards this is not personal view alone but the feedback I got from meeting people who attended." Several also indicated how important it was to have this meeting. "The meeting about anxiety and depression was a great opportunity for clinicians and neuroscientists to meet to discuss the latest research in the field, which is not given the attention it deserves in undergraduate and postgraduate medical education taking in consideration that more than half of those who suffer from anxiety and depression go unrecognized and never receive treatment." This impact on medical and graduate students is perhaps the most important legacy of the symposium.

Topic 1: Biological basis of mood and anxiety disorders: A clinical and basic science overview.

## 1A. Clinical overview and the reward circuit

Dr. Benjamin D. Greenberg from the Departments of Psychiatry and Human Behavior in the Alpert Medical School of Brown University gave the first talk, entitled "*Overview of mood & anxiety disorders".* Dr. Greenberg emphasized several main points: that patients with the same categorical diagnosis as defined in the DSM system are in fact heterogeneous; that longitudinal stability of most diagnoses is poor; and that co-occurrence ("comorbidity") of diagnoses tends to be the rule. For example, one- to two-thirds of individuals with an anxiety disorder will have a lifetime mood disorder; conversely, two-thirds of individuals who develop major depression or dysthymia will also have an anxiety disorder during their lifetimes.

In this context, he noted that the development of medication treatments in psychiatry was initially focused on how medications affected core clinical features of affected individuals as much as (or more than) than on treating illnesses as sepble entities. Such core features include an impaired ability to respond to reward after motivated behaviors, or positive emotional and social experiences (anhedonia). He proposed that an essential step towards making it possible for clinicians to target specific treatments to those individuals most likely to benefit from them, was to seek to understand behavioral and brain mechanisms underlying how effective behavioral and medication treatments may change such clinical features. The same approach should allow refinement of existing therapies, and development of new ones. Moreover, progress in this domain should enhance public understanding of psychiatric illnesses as disorders of the brain and behavior, which, in time, should reduce stigma and encourage treatment seeking, across cultures.

Dr. Suzanne Haber Professor at the Department of Pharmacology and physiology at the University of Rochester Medical School gave a presentation entitled, "*Understanding the Reward Circuit: Implications for Depression and Anxiety Spectrum Disorders*". The presentation began with a brief overview of the background and the history of research of the reward system, followed by describing the reward network. She noted that while many brain regions respond to reward, central to the network are structures embedded within the cortico-basal ganglia system and include, the orbital, and anterior cingulate cortices, the ventral striatum, the ventral tegmental, midbrain dopamine neurons. In addition, other structures including the dorsal prefrontal cortex, amygdala, hippocampus, thalamus, lateral habenular n., and specific brainstem structures, such as the pedunculopontine n., and the raphe n., are key components in regulating the reward circuit. Connectivity between these areas forms a complex neural network. This network mediates different aspects of reward processing, including value, magnitude, and anticipation. Dr. Haber discussed how delineating these neurocircuits are key elements for understanding anatomical substrates of psychiatric diseases.

Classically, the cortico-basal ganglia system has been functionally divided into septe and segregated circuits. However, while the topographic organization of cortico-striatal projections are well documented, there is increasing evidence that there are also regions of interface between terminals from different cortical areas, suggesting functional integration to link motivation to behavioral output, critical for developing appropriate action plans. Dr. Haber demonstrated two ways in which cortico-basal ganglia circuits can integrate this information across functional circuits, through cortico-striatal projections, and through striatal-midbrain-striatal connections.

Projections from the cortical regions associated with motivation terminate in specific striatal regions, but also converge with terminals from associative areas. This complex interweaving and convergence provides an anatomical substrate for modulation between these motivation and cognition. This creates activation sites that may be particularly important for incentive-based learning. In addition, there is an interface between different functional striatal regions via the midbrain DA cells. Dr. Haber demonstrated how this interface is organized in an ascending spiral interconnecting different functional regions of the striatum. This anatomical arrangement creates a hierarchy of information flow, and provides an anatomical basis for the limbic/cognitive/motor interface via the midbrain dopamine neurons.

Dr. Haber ended her talk by describing translational studies, linking animal anatomical studies with neuroimaging. Diffusion tensor imaging (DTI) is increasingly used to develop models of prefrontal connections, to examine chances in white matter (WM) integrity associated with disease, and to target surgical interventions, including deep brain stimulation for psychiatric disease. Dr, Haber described how her lab uses nonhuman primate tracing studies coupled with 3-D reconstructions of WM bundles to delineate the rules frontal cortex axons follow to reach their targets. She then demonstrated that human prefrontal WM pathways follow these organizational rules. Taken together, these data provide the foundation for dividing white matter bundles into functional components and for predicting what is likely to be carried at different points through each bundle. Understanding this organization is key for determining specific connections that are likely to be captured at different neurosurgical targets.

## 1B. What we can learn from clinical and basic science

Dr. Emad Eskandar, MD, from the Department of Neurosurgery Massachusetts General Hospital presented two talks at the symposium. The first talk was entitled "Physiological Mechanisms of Basal Ganglia Disorders". In this presentation, Dr. Eskandar presented data on the role of the basal ganglia and movement psychiatric disorders. First, Dr Eskandar gave a description of the relevant anatomy and physiology, followed by a reviewed surgical treatment options such as deep brain stimulation. This portion of talk discussed the rational for treatment and the key technical aspects of the surgery. Finally, Dr. Eskandar reviewed a number of prospective randomized clinical trials illustrating the benefits and limitations of the surgical treatments. Later in the symposium, Dr. Eskandar gave a talk entitled "Insights from Human and Primate Neurophysiology" which focused on the important role played by basic physiologic research in translating scientific findings into useful treatments for human disorders.

Dr. Huda Akil Professor of Neuroscience & Psychiatry and Director of the Molecular and Behavioral Neuroscience Institute, at the University of Michigan. She began her talk entitled "The Neurobiology of Stress and Depression: Animal Models and Human Studies" by noting that her late father, Dr. Fakher Akil (Akel) was one of the founders of the University of Jordan almost 50 years ago, and a member of the faculty of its Psychology Department when it was first established. She shared photographs of Dr. Akil with His Majesty the late King Hussein and other founding members at the dedication of the University of Jordan, and she noted that this gave this meeting a very special meaning for her.

Dr. Akil emphasized the burden of depression around the world, and the underlying complexity of the illness, as it has many genetic and environmental causes, converging to disturb the functioning of brain circuits that control our responses to stress and our short term and long-term moods. But currently available treatments for depression only work for a subset of patients, and at least a third of depressed individuals do not respond to any of the current treatments and become increasingly more depressed, some of them ending their lives tragically. So, how can science discover the biological causes of depression and identify new molecules that may become targets for new types of antidepressant drugs?

Dr. Akil described how her laboratory has created animal models of increased vulnerability to depression. One model uses a genetic approach in mice, and shows that increasing the activity of a gene that responds to stress only in the brain, and only during early development, is sufficient to produce an anxious and emotionally labile animal that serves as an excellent model for bipolar (manic-depressive) disorder. Thus, the stress the glucocorticoid receptor gene, GR, may be a good target for treatment in some patients.

Another approach was to create models of "temperament" by developing genetically bred lines of rats that are either prone to anxiety and depression-like behaviors or resistant to stress. These animals show characteristics of "internalizing disorders" versus "externalizing disorders". Studies of developmental, molecular and neural bases of these differences have already yielded many surprising discoveries.

Dr. Akil then spoke about a complementary approach, where she and her collaborators begin their search in the brains of patients who had died when severely depressed compared to non-depressed controls. Using broad genetic and gene expression strategies, they identify molecules in specific brain regions that differentiate the severely depressed brains. Then, they return to the animal models to examine the function of these molecules. Using this strategy, they discovered that a family of growth factors, the FGF system, is severely disrupted in major depression. Studies in the animal models show that FGF2, which is low in depressed human brains, serves as a natural anti-depressant and anti-anxiety agent that is present in all brains, but differences in its activity can modify mood. Thus, animals that are prone to anxiety and depression, like depressed humans, have low levels of FGF2. Social stress also lowers the levels of FGF2. Importantly, replacing FGF2 in the brains of anxiety-prone animals reverses these tendencies, acts as an antidepressant and increases their activity and exploration. Thus FGF family has become a new target for studies of depression and as a potential target for developing new types of antidepressants.

In sum, combining basic science research and human clinical studies, and using genetic, molecular, anatomical and behavioral approaches are yielding exciting new insights into the causes of depression and hold promise for developing new treatment strategies for these stress-related brain disorders.

Dr. Darin D. Dougherty from the Department of Psychiatry at Massachusetts General Hospital and Harvard Medical School gave a presentation entitled, *"Neuroimaging, what we know and what we need to know"*. The presentation began with a brief overview of studies utilizing modern neuroimaging technology that have helped to elucidate regarding the pathophysiology of major depressive disorder (MDD) and obsessive-compulsive disorder (OCD). In brief, when compared to healthy volunteers, MDD appears to be associated with decreased resting activity in more dorsal neocortical brain regions associated with cognitive processes and increased resting activity in more ventral limbic and plimbic brain regions associated with the neurovegetative symptoms common to MDD. In addition, during neuroimaging studies that use a variety of activation pdigms in subjects with MDD and healthy volunteers, these dorsal regions are hyporeactive during cognitive tasks and these ventral regions are hyperreactive during affective tasks in subjects with MDD. Lastly, most of these abnormalities are attenuated following successful treatment (with either pharmacotherapy or psychotherapy) of the major depressive episode. In contrast, when compared to healthy volunteers, subjects with OCD exhibit greater resting activity in a specific cortico-thalamo-striato-cortical pathway that includes the orbitofrontal cortex (OFC), thalamus, and head of the caudate nucleus and these abnormalities can be amplified during pdigms that temporarily worsen OCD symptomatology. As is the case with MDD, these abnormalities are attenuated following successful treatment (with either pharmacotherapy or psychotherapy) of OCD.

After describing the rudimentary pathophysiology of both MDD and OCD, Dr. Dougherty described initial attempts to understand the effects of deep brain stimulation (DBS) at the ventral capsule/ventral striatum (VC/VS) target on these circuits using functional neuroimaging techniques. Because of the presence of metal in the DBS leads, most studies to date have used positron emission tomography (PET). The use of ^15^O isotopes (either in CO~2~ in room air or in water) allows for the study of acute changes in blood flow. Interestingly, studies using PET and ^15^O in subjects with VC/VS DBS for OCD have found acute blood flow changes in all nodes (OFC, thalamus, caudate) of the very circuit implicated in the pathophysiology of OCD. Initial data from studies using ^18^FDG and PET in subjects with VC/VS DBS for OCD to identify chronic (e.g., following 3-6 months of DBS) changes are suggesting that the same brain regions that exhibit these acute changes in blood flow continue to demonstrate changes from baseline following chronic treatment with VC/VS DBS. Compble studies in subjects receiving VC/VS DBS for MDD are ongoing. Dr. Dougherty concluded with a review of potential future directions, including using baseline neuroimaging data to identify individuals who are more likely to respond following DBS and to possibly tailor electrode targeting.

Topic 2: Biological basis of mood and anxiety disorders: New advances.

## 2A. Depression and Stress

Dr. Bita Moghaddam from the department of neuroscience at University of Pittsburgh gave a talk entitled " Neuronal mechanisms for behavioral and psychiatric vulnerability in adolescents". Dr. Moghaddam discussed how adolescents often respond differently than adults to the same salient motivating contexts, like peer interactions and stressors. Adolescence to early adulthood is also a period of psychiatric vulnerability and the typical time of symptom onset for several psychopathologies, including depression, addiction and schizophrenia. Thus, while the great majority of animal and human research related to these illnesses has focused on adults, research findings on the adolescent to early adulthood developmental age range may be more relevant to understanding the biological mechanisms that may contribute to the disease process and prevention. Adolescence encompasses puberty, a time of reproductive development, but also characterized by a myriad of non-reproductive socio-behavioral changes. Concomitant with these behavioral changes, adolescents undergo significant neurodevelopmental changes, including axonal growth, myelination and synaptic pruning, and changes in modulatory regulation of prefrontal cortex activity. Despite its importance, this stage of development remains understudied at mechanistic levels that are relevant to motivated behavior. This has been in part due to technical limitations associated with adolescent human and animal models. We believe that age-related changes in the ways salient stimuli are processed in key brain regions could underlie the unique predilections and vulnerabilities of adolescence. Because motivated behavior is the central issue, it is critical that age-related comparisons of brain activity be undertaken within motivational contexts. Dr. Moghaddam presented single neuron and large-scale electrophysiological data showing that the pattern of activation of cortico-striatal circuits, and the inhibitory-excitatory balance in these circuits, is different during motivated behavior in adolescent rats. These data indicate that there is reduced efficiency in the processing of cortical neural activity and related behaviors in adolescents. Furthermore, during this developmental period, regions critically involved in learning and habit formation are highly responsive to reward. This work highlights neuronal processing differences in adolescence that suggest possible mechanisms contributing to the vulnerability that this period presents for psychiatric illnesses.

Dr. Gary Aston-Jones, from the Medical University of South Carolina gave a talk entitled "The cortex in context: Locus coeruleus, optimal performance, and maximal utility" Optimal performance on a specific task requires selective attention to filter out extraneous stimuli and behaviors. It is also important, however, to disengage from the current task and find a new behavioral goal if motivation or the stimulus context changes. This tension between selective attention and the search for a new task in an altered context has been characterized as the exploitation-exploration tradeoff, and is critical for adaptive behavior. LC neurons exhibit two patterns or modes of activity in monkeys performing cognitive tasks: a *phasic mode* in which these cells are transiently activated selectively following decisions that were made, and a *tonic mode* when these cells have elevated tonic levels of discharge but no phasic responses. We hypothesize that the phasic mode of LC activity promotes selective behavioral responding to task-defined stimuli (selective attention), whereas the tonic mode produces a broader sampling of stimuli in the environment (search for new task, high behavioral flexibility). Both of these modes serve to increase the utility of behavior: The phasic mode increases utility on a short time-scale by facilitating selective responding in the task at hand, whereas the tonic mode increases long time-scale utility by promoting the identification of a new task that is adaptive in a changed context. In view of this functional framework, impairments of LC activity and ability to fluidly and adaptively change activity modes can produce deficits involved in several mental disorders, including attention deficit-hyperactivity disorder (persistent tonic mode), autism (persistent phasic mode), or sleep disorders and depression (persistently low activity).

## 2B. OCD and Anxiety

Dr. Benjamin D. Greenberg from the Departments of Psychiatry and Human Behavior in the Alpert Medical School of Brown University gave second talk entitled OCD*, clinical and basic science perspectives".* He discussed one specific diagnosis, obsessive-compulsive disorder (OCD). He presented data on the population prevalence, course, clinical features (including subtypes based on differing symptom profiles) and the full range of current treatment approaches from behavior therapy to neurosurgery. The presentation of the range of clinical features was intended to give clinicians and others in the audience assistance in diagnosing OCD, the prevalence of which has historically been under-appreciated throughout the world. While current epidemiological information suggests that OCD, which typically has its onset in childhood or adolescence, affects about one percent of adults in any given year, many affected individuals fail to seek treatment, or may do so only after a long delay. As for other psychiatric illnesses, accurate diagnosis is often best accomplished via obtaining information from multiple sources, including potentially family members.

Then, after describing conventional behavioral and medication treatments, Dr. Greenberg turned to the subset of OCD sufferers with disabling, "intractable" illness, in whom surgical approaches might be considered. Determining if the strict selection criteria for, e.g., deep brain stimulation (DBS) are met requires exhaustive presurgical evaluations in multiple domains of symptoms, comorbidities, cognition and daily functioning. In the main stimulation approach, DBS leads implanted in the ventral anterior limb of the internal capsule and adjacent ventral striatum (VC/VS) are intended to modulate the same prefrontal cortex-basal ganglia-thalamic circuitry targeted by lesion procedures. Related approaches include targeting VS subterritories (nucleus accumbens or ventral caudate) using DBS.

Outcomes data are mainly open-label, though small studies have used sham-controlled DBS. In one case a sham gamma knife lesion was compared to actual ablations in the VC/VS, in the first controlled trial of a lesion procedure for any psychiatric illness. Across procedures, meaningful benefit appeared in 30-60% when assessed after varying delays. The longstanding clinical impression that exposure-based behavioral therapies (exposure and response prevention, or ERP) can become more effective after vs. before neurosurgery has continued. This has led to explicit incorporation of ERP early in the post-surgery course in at least one study.

Mean severity decreases from "very severe" to "moderate" illness were usually but not always plleled by functional gains. Given that the chronically severe symptoms in these patients are profoundly disruptive to the life course, capturing possible changes in functioning might require a year-long period of observation, even if symptom lessening appears earlier. The prognostic significance of differences in OCD clinical phenomenology to surgical outcomes is beginning to be addressed. Ego-dystonic OCD symptoms primarily based in harm avoidance might improve most, although symptoms of "incompleteness" (motivated by an intense desire to achieve a "just right" feeling) have lessened in some patients as well.

Translational research mapping ventral prefrontal-basal circuitry in primates has delineated potential pathways of DBS effect. Stimulation-related changes in behavioral phenotypes related to fear conditioning and extinction are the focus of ongoing collaborative work, in concert with our controlled multicenter trial of VC/VS DBS for intractable OCD. For OCD sufferers as a whole, the major promise of this line of research is that better delineation of the neurocircuit and behavioral mechanisms necessary to therapeutic benefit will have application using less invasive treatment strategies.

Dr. Anthony A. Grace from the Departments of Neuroscience, Psychiatry and Psychology at the University of Pittsburgh presented a talk entitled "Animal Models of OCD: Frontal Cortical Disruption and Modulation by DBS." In this talk Dr. Grace covered what we have found about the mechanisms of deep brain stimulation (DBS) action and how it can relate to therapy in humans. An issue in understanding DBS is a better understanding of its mechanism of action. DBS has been carried out in the treatment of neurological disorders by stimulating regions that previous studies have targeted for lesions to achieve a therapeutic action. This gave rise to the "functional lesion" hypothesis of DBS action. However, the stimulation frequency typically used for DBs is 130 Hz, which for neurophysiologists typically is associated with long-term potentiation rather than inhibition. Therefore, we examined the effects of DBS using the ventral striatal circuit that is typically used for the treatment of Obsessive Compulsive Disorder (OCD) in humans.

OCD is typically associated with hyperactivity in frontal cortical structures, notably the orbitofrontal cortex. We found that DBS of the ventral striatum for 90 minutes caused a decrease in the firing rate of orbitofrontal neurons, along with firing through antidromic activation of a small percentage of the pyramidal output neurons in this area. In contrast, when we presented single stimuli, we found that there was a classical NMDA-dependent long-term potentiation of the evoked field potential, which is a recording of the total electrical signal originating in the orbitofrontal cortex caused by the stimulation. Moreover, interneurons in the cortex were found to be more responsive to stimulation. This led to the conclusion that DBS caused antidromic activation of a small number of pyramidal neurons and, via local axon collaterals, long-term potentiation of the excitation of interneurons. Therefore, the DBS was not causing a "functional lesion," but instead caused activation of interneurons to increase inhibition within the orbitofrontal cortex. This is consistent with the concept that the overactivity in the orbitofrontal cortex may have been due to deficits in interneuron inhibition.

Besides inhibiting pyramidal neurons, interneurons have another important function within the cortex and other structures - they control the generation of rhythmic activity within these structures. Rhythmic activity is believed to represent the functional state of a region, with beta and gamma oscillations representing neuronal activation that correlates with increases in metabolism seen with functional imaging. In awake animals, the effects of DBS are transient in nature, wearing off minutes after turning off the stimulation; this suggests that the brain is organized in a manner to "push back" from the effects of DBS. However, when the stimulation is done for at least 5 days, there are significant changes in these frequencies. First, the short-term increases in power seem to give way to changes in coherence, or the organization of activity between regions. In particular, the orbitofrontal cortex showed increased coherence between itself and other regions, suggesting that the orbitofrontal cortex was now more responsive to inputs. This is consistent with the functional pathology of OCD - i.e., the orbitofrontal cortex is responsible for devaluing a stimulus; something that OCD patients fail to do. Therefore, when an OCD patient finds that a door is locked, instead of devaluing its significance in the face of data, their orbitofrontal cortex keeps "reminding" them that the door may indeed be unlocked. By restoring the ability of the orbitofrontal cortex to respond to this information, DBS may enable the patient to "let go" of inappropriate stimuli in the face of evidence to the contrary.

## 2C. Trauma and fear

Dr. Roger Pitman a Professor of Psychiatry at Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA delivered a lecture entitled, "Stress, Anxiety, and Trauma." Because of the breadth of this topic, Dr. Pitman focused on his group's research into biological aspects of posttraumatic stress disorder (PTSD). He noted that although PTSD is still largely regarded as a psychological phenomenon, ultimately the impact of an environmental event, such as a psychological trauma, must be understood at organic, cellular, and molecular levels. Dr. Pitman reviewed research in the psychophysiology laboratory beginning in the 1980's indicating that trauma victims diagnosed with PTSD showed larger heart rate, skin conductance, and facial electromyogram responses during personal, script-driven imagery of their traumatic events. When the script-driven imagery method was taken into the positron emission tomography (PET) scanner, results indicated that PTSD subjects also showed greater concurrent activation of amygdalocentric anterior plimbic brain areas thought to underlie emotional experience. However, one brain region, the ventromedial prefrontal cortex (vmPFC), showed pdoxically reduced activation in PTSD subjects during traumatic imagery. Further work with functional magnetic resonance imaging (fMRI) revealed an inverse association between vmPFC and amygdala activation, suggesting that vmPFC activation acts to inhibit amygdala and thereby confers resilience.

Pitman also presented results of a 15-year ongoing study of identical twins discordant for combat exposure in Vietnam, which has addressed the origin of biological abnormalities in PTSD. If his combat-unexposed co-twin shares an abnormality in a PTSD veteran, it is unlikely to have been acquired as a result of combat trauma but rather likely represents a familial vulnerability factor. If it is not shared, it is likely to have been acquired. This research has suggested that greater heart rate response to loud (startling) tones, lower electroencephalographic P300 event-related potential (ERP) response, increased P2 intensity dependence, lower vmPFC volume according to voxel-based morphometry, and impaired retention of extinction of a de novo conditioned fear response are acquired PTSD biomarkers, whereas more neurological soft signs (suggestive of subtle central nervous system impairment), diminished volume of the hippocampus, and both hypermetabolism and hyperreactivity of dorsal anterior cingulate cortex (a brain region found to promote the fear response) are familial vulnerability factors for PTSD.

Dr. Mohammed R Milad from the Department of Psychiatry at the Department of Psychiatry and Massachusetts General Hospital presented a talk entitled "Neurobiology of learning not to fear: implications to PTSD and beyond". In his presentation, Dr. Milad presented data on the neural correlates of fear extinction using a translational and multi-modal approach. After a brief definition and description of the classical fear conditioning pdigms used to study fear extinction, Dr. Milad presented data describing the basic mechanisms of how fear extinction learning is formed and stored with a network of brain regions including the ventromedial prefrontal cortex (vmPFC), the amygdala and the hippocampus. Dr. Milad presented data regarding the apparently apposing effects of two prefrontal regions on fear expression: the activation of the dorsal anterior cingulate cortex (dACC) seems to enhance the expression of conditioned fear, and the vmPFC seems to quell the expression of fear. The function of these brain regions within the context of fear learning and extinction seems to be impaired in patients diagnosed with PTSD, and that such functional impairment seem to be associated with exaggerated fear expression in PTSD patients.

In the second part of his talk, Dr. Milad described the potential role of gonadal hormones, such as estrogen, on fear extinction and how these hormones may contribute to sex differences in fear learning and extinction and may also contribute to the differences in the epidemiology of PTSD between men and women. In brief, Dr. Milad presented data showing that an increased estrogen level in both female rats and in women is associated with enhanced consolidation of extinction memory. The elevated levels of estrogen seem to interact with the functional activation of the fear extinction network, specifically, the higher the estrogen levels, the more activation the vmPFC exhibits during extinction recall in women. The importance of assessing hormonal status in women during treatment for PTSD and other anxiety disorders was highlighted in the presentation.

Dr. Gregory Quirk from the University of Puerto Rico presented a talk entitled "Neurobiological Basis of Conditioned Fear and Extinction" in which he talked about the neural mechanism of fear conditioning, using rats as a model organism. Fear conditioning has become a popular model of emotional learning, in which neurons in the amygdala, hippocampus, and prefrontal cortex acquire information about stimuli (such as tones) that predict danger (such as shocks). Disruption of such circuits leads to excessive fear seen in anxiety disorders such as PTSD and OCD. Quirk showed that expression of fear in the amygdala is under bidirectional control by the prelimbic and infralimbic subdivisions of prefrontal cortex. Modulation of activity in these circuits can increase fear either by facilitating expression or by impairing extinction of fear. In addition to modulating fear (such as freezing), these areas also modulate avoidance behaviors, in which rats escape a shock by moving to a safe place. Deep brain stimulation (DBS) of the ventral striatum has been shown to help some patients suffering from OCD, and Quirk showed rodent data suggesting that DSB may act by augmenting plasticity in extinction circuits located in amygdala and prefrontal areas. In collaboration with Dr. Haber, Quirk is characterizing the striatal-cortical circuits by which DBS enhances extinction of fear.

Topic 3: Anxiety and depression: perspective from the region.

Dr. Abdul-Monaf Al-Jadiry, Professor and chair of Psychiatry, Department of Medicine, Medical Faculty, The University of Jordan, Amman, Jordan gave a presentation entitled, "Prevalence of Anxiety and Mood Disorders and the link to Trauma: An Iraqi Perspective". The presentation initiated with a general conclusion, based on literature review, about the impact of trauma on mental health in general, and the prevalence of anxiety and mood disorders in particular. Dr Al-Jadiry presented data from Iraqi Mental Health Survey (IMHS) to demonstrate the link between the two. IMHS is the first nationwide mental health epidemiological survey in Iraq and the second in the Middle East after Lebanon. The IMHS was undertaken in the years 2006-207, amidst circumstances of extreme instability and minimal security following the second gulf war. IMHS used World Mental Health Survey (WMHS) methodology and diagnostic instrument to provide data on the prevalence of mental health disorders, their impact and treatment, and their relationship to violence in the population. Trained interviewers used the "Composite International Diagnostic Interview (CIDI), Version 3" as a diagnostic tool for the assessment of mental disorders. The IMHS for the first time provides evidence-based data about the prevalence of mental health problems and mental health of Iraqi people. The survey provides an initial indicator of lifetime prevalence, and 12 months and 30 days prevalence rates alongside the experience of trauma. The importance of the Iraqi example springs from the fact that the state of Iraqis has been unique, as they have suffered more than any other people from decades of war trauma, sanctions and violence, which are still going on.

The IMHS demonstrated that 11.58% of population suffered from any anxiety disorder, including stress disorder, lifetime, and 7.82% from any affective disorder lifetime, with a significant difference between women and men, age groups, and several other socio-demographic variables. Moreover, the overall lifetime exposure to traumatic events, according to the survey was 60.02%. In the lifetime prevalence rate of these disorders cases had significant higher exposure to trauma. The most striking finding of the survey is the very high exposure of the population to various types of trauma and the relative low prevalence rates of anxiety and mood disorders though in line with prevalence rates reported in Lebanon and Afghanistan. Dr. Al-Jadiry attributed this discrepancy to the biological nature of people vulnerability for anxiety and mood disorders in addition to inherent protective sociocultural characteristics of Iraqis; these include: development of resilience, high community support, rituals to canalize distress and bereavement in a socially acceptable manner, and viewing losses as divine states. Moreover, the effect of stigma of mental illness is to make some patients hide their symptoms. Dr Al–Jadiry concluded, despite massive exposure to traumatic events, the prevalence of anxiety and mood disorders amongst Iraqis is relatively lower than expected, though in accordance with the literature from post conflict zones.

Dr. Walid Sarhan a consultant Psychiatrist from Amman Jordan gave a talk entitled "Cognitive behavioral therapy of Islamic religious OCD". The talk started by brief background of Islamic religion with emphases on how its comprehensive application to people's daily lives and its rules to be followed in praying, washing, cleaning may relate to OCD symptoms expressed in some of the patients in the region. In managing those patients, Dr. Sarhan discussed how therapists may need to be aware of the compatibility of the cognitive behavioral therapy with the Islamic religion. The clinical experience of dealing with cleaning, abolition, praying, divorce and doubts in beliefs were discussed in light of the literature, while at the same time addressing the common clinical challenges presented by Arab Muslim patients suffering from OCD. Dr. Sarhan discussed how religious individuals with authority to declare legal rulings or decrees (Fatwa) on some aspects of the Islamic religion may also influence patient's symptoms. The clinician's knowledge of some of these rulings or "fatwas" may be necessary to understanding how some aspects of the OCD symptoms (especially those related to cleansing) came to exist.

In obsessions related to one's faith, Dr. Sarhan suggested that clinician's knowledge of the Islamic religion might be used as an additional tool to help aid the treatment; a clinician could make use of the teachings of the prophet to his followers as the key element in cognitive therapy.

Dr. Radwan A. BaniMustafa from the University of Jordan Medical School & Hospital gave a talk entitled "Depression in Primary patients with chronic medical diseases" he described a study which included a group of 407 primary care patients with chronic medical diseases. These patients were seen in the primary care clinics in Jordan University hospital in 2011. Prevalence of depression in this sample was 45.5%. Depression was significantly associated with young age, low level of education, chronic headache and chronic musculoskeletal diseases. In addition, it was associated but not significantly so with having more than three medical problems, chronic cardiopulmonary disorders, chronic Skin and thyroid disorders, female sex and single status.

## Acknowledgements

Funding for this meeting was made possible by NIMH (PI: Haber) and the International Brain Organization (IBRO). The meeting was hosted and supported by the University of Jordan, School of Medicine and the University of Rochester.


abstract: There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto-immune reaction; Well's syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. In contrast, the United States Centers for Disease Control and Prevention states that Thimerosal is safe and there is "no relationship between \[T\]himerosal\[-\]containing vaccines and autism rates in children." This is puzzling because, in a study conducted directly by CDC epidemiologists, a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy was found. The CDC's current stance that Thimerosal is safe and that there is no relationship between Thimerosal and autism is based on six specific published epidemiological studies coauthored and sponsored by the CDC. The purpose of this review is to examine these six publications and analyze possible reasons why their published outcomes are so different from the results of investigations by multiple independent research groups over the past 75+ years.
author: Brian Hooker; Janet Kern; David Geier; Boyd Haley; Lisa Sykes; Paul King; Mark Geier\*Brian Hooker: <email@example.com>[^1]
date: 2014
institute: ^1^Simpson University, 2211 College View Drive, Redding, CA 96001, USA; ^2^Institute of Chronic Illness, Inc., 14 Redgate Court, Silver Spring, MD 20905, USA; ^3^University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA; ^4^University of Kentucky, Lexington, KY 40506, USA; ^5^CoMeD, Inc., Silver Spring, MD, USA
references:
title: Methodological Issues and Evidence of Malfeasance in Research Purporting to Show Thimerosal in Vaccines Is Safe

# 1. Introduction

Thimerosal is an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, in the past and present. To date, there have been over 165 studies that focused on Thimerosal and found it to be harmful \[1, 2\]. (A comprehensive list of these studies is shown at <http://mercury-freedrugs.org/docs/20140329_Kern_JK_ExcelFile_TM_sHarm_ReferenceList_v33.xlsx>.) Of these studies, 16 were conducted to specifically examine the effects of Thimerosal on human infants and/or children \[3–18\]. Within these studies, which focused on human infants and/or children, the reported outcomes following Thimerosal exposure were (1) death \[3\]; (2) acrodynia \[4\]; (3) poisoning \[5\]; (4) allergic reaction \[6\]; (5) malformations \[7\]; (6) autoimmune reaction \[8\]; (7) Well's syndrome \[9\]; (8) developmental delay \[10–13\]; and (9) neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism \[10, 11, 14–18\].

However, the United States (US) Centers for Disease Control and Prevention (CDC) still insists that there is "no relationship between \[T\]himerosal\[-\]containing vaccines and autism rates in children" \[19\]. This is a puzzling conclusion because, in a study conducted directly by the CDC, epidemiologists assessed the risk for neurologic and renal impairment associated with past exposure to Thimerosal-containing vaccine (TCV) using automated data from the Vaccine Safety Datalink (VSD) and found a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy \[20\]. The database for that study was "from four health maintenance organizations \[HMOs\] in Washington, Oregon, and California, containing immunization, medical visit, and demographic data on over 400,000 infants born between 1991 and 1997." In that initial study, Verstraeten et al. \[20\] "categorized the cumulative ethyl-Hg exposure from \[T\]himerosal\[-\]containing vaccines after one month of life and assessed the subsequent risk of degenerative and developmental neurologic disorders and renal disorders before the age of six." They "applied proportional hazard models adjusting for HMO, year of birth, and gender, and excluded premature babies." The reported results showed that "the relative risk (RR) of developing a neurologic development disorder was 1.8 (95% confidence intervals \[CI\] 1.1–2.8) when comparing the highest exposure group at 1 month of age (cumulative dose \> 25 *μ*g) to the unexposed group." Similarly, they "also found an elevated risk for the following disorders: autism (RR 7.6, 95% CI = 1.8–31.5), nonorganic sleep disorders (RR 5.0, 95% CI = 1.6–15.9), and speech disorders (RR 2.1, 95% CI = 1.1–4.0)" in the highest exposure group.

Considering the many peer-reviewed published research studies that have shown harm from Thimerosal, including studies in which Thimerosal exposure is associated with the subsequent diagnosis of neurodevelopmental disorders (16 studies) such as autism, and the just-described evidence from the CDCs own research, which found evidence of a relationship between the level of Thimerosal exposure and the risk of a subsequent autism diagnosis, how does the CDC conclude that there is no evidence of that relationship? The foundation for the CDC's current stance apparently is based primarily on six specific published epidemiological studies that the CDC has completed, funded, and/or cosponsored, starting in the late 1990s. These studies include (1) the Madsen et al. \[21\] ecological study of autism incidence versus Thimerosal exposure in Denmark, (2) the Stehr-Green et al. \[22\] ecological study of autism incidence versus Thimerosal exposure in Denmark, Sweden, and California, (3) the Hviid et al. \[23\] study of autism incidence versus Thimerosal exposure in Denmark (also ecological), (4) the Andrews et al. \[24\] cohort study of autism incidence and Thimerosal exposure in the United Kingdom, (5) the published Verstraeten et al. \[25\] CDC cohort study of autism incidence and Thimerosal exposure in the United States, and (6) the more recent Price et al. \[26\] case-control study of autism incidence and Thimerosal exposure in the United States. Although the CDC cites several other publications to purport the safety of Thimerosal, only these six specifically consider its putative relationship to autism.

The purpose of this review is to examine these six publications \[21–26\] which were "overseen" by the CDC and which claim that prenatal and early childhood vaccine-derived Thimerosal exposures are not related to the risk of a subsequent diagnosis of autism or autism spectrum disorder (ASD). This review analyzes possible reasons why their published outcomes are so different from the results of investigations by multiple independent research groups over the past 75+ years. The review begins with an examination of the Madsen et al. \[21\] study.

# 2. The Madsen et al. 2003 Study

The CDC-sponsored Madsen et al. \[21\] study examined whether discontinuing the use of TCVs in Denmark led to a decrease in the incidence of autism. Data were obtained from the Danish Psychiatric Central Research Register, which contains all psychiatric admissions since 1971 and all outpatient contacts in psychiatric departments in Denmark since 1995. The study authors examined the data from 1971 to 2000 and reported that rate of autism increased with the removal of Thimerosal from vaccines (starting in 1992, the year that Thimerosal-containing early childhood vaccines were phased out).

Although there are several concerns about the methodology used, the most serious concern involves diagnosis. As described in the paper, estimates of total autism cases in Denmark were only based on diagnoses occurring during inpatient visits from 1971 to 1994 and then during both inpatient and outpatient visits from 1995 to the last year of the study in 2000. Thus, the inclusion criteria are greatly expanded two years after the phaseout of Thimerosal from infant vaccines in Denmark, creating an "artificial increase" in autism prevalence. The authors conceded that "the proportion of outpatient to inpatient activities was about 4 to 6 times as many outpatients as inpatients with variations across time and age bands." However, in an earlier publication by Madsen et al. \[27\], the same authors had stated regarding this same data, "in our cohort, 93.1% of the children were treated only as outpatients…" Unlike the statement in the Madsen et al. \[21\] study, the 2002 paper indicates that the ratio between outpatients and inpatients in the 1971–2000 dataset was 13.5 : 1, which would account for an even greater increase in cases diagnosed starting in 1995 (i.e., after the probable completion of the phaseout of TCVs that started in 1992).

In addition, the authors stated that the Danish registry which was used to count cases did not include a large Copenhagen clinic before 1993. This clinic accounted for as many as 20% of the autism cases nationwide, which would again artificially inflate the autism incidence observed in Denmark after the phaseout of TCVs was initiated in 1992. The authors do not mention this change in inclusion criteria (i.e., the addition of a new clinic in the registry) neither do they attempt to adjust their analysis in accordance with the anomaly. It was revealed, instead, in a similar paper by Stehr-Green et al. \[22\] where the authors state regarding the Denmark registry of autistic patients, "Prior to 1992, the data in the national register did not include cases diagnosed in one large clinic in Copenhagen (which accounts for approximately 20% of cases occurring nationwide)."

Also, the diagnosis criteria for "autism" changed within the course of the study. From 1971 to 1993, the ICD-8 standards for diagnosis (psychosis protoinfantilis 299.00 or psychosis infantilis 299.01) were used to measure autism incidence. However, from 1994 to 2000, the ICD-10 standard (infantile autism, F84.1) was used. Although the authors did not address the impact of the change in diagnostic criteria, this could result in as much as a 25-fold increase in cases as the instantaneous change in autism prevalence in Denmark, due to this change, went from a low of 1.2/10,000 to a high of 30.8/10,000 \[28\].

Another disconcerting methodological issue was that the 2001 data, which showed a strong downward trend in autism rates in at least two of the three age groups (continuing from 1999 through 2001), was not included in the final publication. This was apparent because when the paper was initially submitted for publication, it included the 2001 data. After the paper was rejected for publication by the Journal of the American Medical Association (JAMA) and the Lancet, it was submitted to the journal Pediatrics again including the 2001 data. As stated by one of the peer-reviewers of the Pediatrics submission, "The drop of incidence shown for the most recent years is perhaps the most dramatic feature of the figure, and is seen in the oldest age group as well as the youngest. The authors do not discuss whether incomplete ascertainment in the youngest children or delay in recording of data in the most recent years might play a role in this decline, or the possibility that this decrease might have come about through elimination of \[T\]himerosal" (January 23, 2003, communication between Dr. Poul Thorsen, Aarhus University, and Dr. Coleen Boyle, CDC scientist). In response to this criticism, the authors removed the 2001 incidence numbers. The authors' decision to withhold these data resembles scientific malfeasance, especially when coupled with the previously discussed problematic methods for counting autism cases. If the scientists believed that downward trend between 1999 and 2001 was caused by some phenomenon unrelated to the phaseout of the TCVs, these scientists should have included those data and then explained the trend within the discussion of the data.

If the 2001 data had been included in the final publication, the results would have been consistent with a more recent CDC study \[29\] where a decreasing trend of autism prevalence in Denmark after the removal of Thimerosal in 1992 was reported. Instead of large increases in autism prevalence after 1992, the recent Danish study revealed that the autism spectrum disorder prevalence in Denmark fell steadily from a high of 1.5% in 1994-95 (when children receiving Thimerosal-free formulations were too young to receive an autism diagnosis and, because of the known offset in diagnosis, most of those being diagnosed had been born 4 to 8 years earlier \[from 1985 to 1990\]) to a low of 1.0% in 2002–2004 (more than 10 years after the phasein of the use of Thimerosal-free vaccine formulations was started in 1992).

# 3. The Stehr-Green et al. 2003 Study

The CDC's Stehr-Green et al. \[22\] study compared the prevalence/incidence of autism in California, Sweden, and Denmark with average exposures to TCVs. Graph-based ecologic analyses were used to examine population data from the state of California (national immunization coverage surveys and counts of children diagnosed with autism-like disorders seeking special education services in California); Sweden (national inpatient data on autism cases, national vaccination coverage levels, and information on use of all vaccines and vaccine-specific amounts of Thimerosal); and Denmark (national registry of inpatient/outpatient-diagnosed autism cases, national vaccination coverage levels, and information on use of all vaccines and vaccine-specific amounts of Thimerosal).

The study followed and appeared to be conducted in response to California study data \[30\], which was presented to the Institute of Medicine's Immunization Safety Review Committee. The California data showed that increased uptake of Thimerosal-containing vaccines in California during the 1990s correlated with a corresponding increase in autism diagnoses. In the Stehr-Green et al. \[22\] study, the researchers stated that the reliability of the autism prevalence data, citing that the California data included autism spectrum disorder diagnoses such as pervasive development disorder (PDD), could account for the increase. However, in a published response to this paper, Blaxill and Stehr-Green \[31\] stated that the California prevalence rates were reported based solely on autism cases.

In the Stehr-Green paper, the Sweden autism prevalence data showed an increase in autism rates from 5- 6 cases per 100,000 in 1980–82 to a peak of 9.2 cases per 100,000 in 1993. In Sweden, TCVs were phased out starting in 1987. Denmark's autism prevalence data was identical to that reported in the Madsen et al. \[21\] study critiqued previously. For Denmark, the authors reported an astounding 20-fold increase in autism prevalence between 1990 and 1999, despite the phaseout of TCVs that started in 1992.

In addition, the data from Sweden were based on inpatient (hospital) visits only. This limitation (counting a small fraction of the total number of cases) likely accounted for the erratic swings in the annual numbers of autism cases reported in that country. Also, the Thimerosal exposure level based on the Swedish vaccination schedule during this time period was much less (a nominal maximum of 75 *μ*g of Hg by two years of age) than that possible in California (and the United States as a whole) where developing children nominally received up to 237.5 *μ*g of Hg by 18 months of age through the standard immunization schedule. In conclusion, the Stehr-Green et al. study was problematic in its attempt to combine ecological data from three different countries that, relative to each other, demonstrated different vaccination policies and widely different Thimerosal exposure levels.

# 4. The Hviid et al. (2003) Study

The Hviid et al. \[23\] population-based cohort study, widely cited by the CDC, compared rates of autism prevalence among individuals who received Thimerosal-free vaccines to those receiving TCVs. The authors report that there was no evidence of increased autism prevalence with Thimerosal exposure.

The study authors stated that the mean age of autism diagnosis within their population was 4.7 years with a standard deviation of 1.7 years. However, cases and controls as young as 1 year of age were included within the analysis. Accordingly, controls that were less than the mean age of diagnosis minus two standard deviations (1.3 years) from that age had a 97.5% probability of actually being individuals who will later develop autism and are therefore possibly misclassified. Similarly, in this study, the mean age for an ASD diagnosis was 6.0 years with a standard deviation of 1.9 years. Thus, the study methodology is questionable because it appears to have underascertained the number of cases diagnosed with autism and ASD.

In addition, rather than counting persons within the cohort, the authors counted "person-years of follow up." With this technique, each age group (one-year-olds, two-year-olds, etc.) was considered equally, despite the fact that younger age groups were much less likely to receive an autism diagnosis. This again contributed to the undercounting of the cases with a diagnosis of autism and ASD and biased the study towards the null hypothesis (that there is no statistically significant Thimerosal exposure effect on the outcomes observed).

# 5. The Andrews et al. (2004) Study

The Andrews et al. \[24\] study was a retrospective cohort study completed using records from a database in the United Kingdom, where autism prevalence rates were compared for children receiving Thimerosal-containing DTaP and DT vaccines. In the Andrews et al. \[24\] study, Cox's proportional-hazards ratios were used to evaluate periods of followup in the cohort examined by the investigators using the records in the general practitioner research database (GPRD), a database that was known to have a significant level of errors. These investigators reported that increased organic-Hg exposure from TCVs was associated with a significantly reduced risk for diagnosed general developmental disorders and for unspecified developmental delay (although there was a significantly higher risk for diagnosed tics).

Considering that there are several studies conducted by independent investigators that have found that exposure to Thimerosal is a risk factor for neurodevelopmental delay and disorders \[10, 11, 16\], the reduced rate of neurodevelopmental delay and disorders with Thimerosal exposure found in the Andrews et al. \[24\] study suggests possible methodological issues.

This result may have occurred, in part, because other studies examined cohorts with significantly different childhood vaccine schedules and with different diagnostic criteria for outcomes. This difference may also exist because these other studies that found Thimerosal to be a risk factor for neurodevelopmental delay and disorders employed different epidemiological methods, especially with respect to the issue of follow-up period for individuals in the cohorts examined. The method used to measure follow-up period for individuals is a critical issue in all studies examining the relationship between exposures and the subsequent risk of a neurodevelopmental disorder diagnosis, especially in those instances where the postexposure periods for all of the participants in the study are essentially the same. This is because the risk of an individual being diagnosed with a neurodevelopmental disorder is not uniform throughout his/her lifetime. As observed in the present study, the initial mean age for any neurodevelopmental disorder diagnosis was 2.62 years old, and the standard deviation of mean age of the initial diagnosis of neurodevelopmental disorder was 1.58 years old. These findings are highly problematic because (1) any follow-up method that fails to consider the lag time between birth and age of initial neurodevelopmental disorder diagnosis will likely not be able to observe the true relationship between exposure and the subsequent risk of a neurodevelopmental disorder diagnosis and (2) statistically, the mean and standard deviation age of diagnosis as reported lead to the nonsensical result that a significant portion (2.5%) of the children in this study were diagosed with a neurodevelopmental disorder more than six months before they were born (i.e., the mean age minus two standard deviations, 2.62 − \[2 × 1.58\] = −0.54 years of age).

Another issue with this study is that the authors used a nontransparent, multivariate regression technique to analyze vaccine uptake and autism prevalence data. The study included one dependent variable (autism) and multiple independent variables, including two independent variables (Thimerosal exposure levels and year of birth) that were "correlated" with each other, since Thimerosal exposures increased with time. Thus, the researchers did not report a statistical analysis of the effect of Thimerosal exposure on autism incidence, despite the fact that the authors stated that no such effect was observed. Moreover, the methods used in this study can create a problem in regression known as "multicolinearity." In this case, since the time variable and the vaccine exposure variable are correlated, they actually compete to explain the outcome effect. Inclusion of the time variable reduces the significance of the exposure variable. Yet, the authors did not explain why they included a time variable that competes with the exposure variable. Unfortunately, the authors of this study never released the raw data so that a valid single-variable analysis could be conducted to ascertain the probability of an association between Thimerosal exposure and the risk of autism.

It is also important to note that the UK Thimerosal exposure (a maximum of 75 *μ*g of Hg by 4 months of age) was not comparable to that in the United States (a maximum of 75 *μ*g of Hg by 2 months of age and 187.5 *μ*g of Hg by 6 months of age). Thus, this study should not be extrapolated to the probability of an autism-Thimerosal association based on the US vaccination schedule.

# 6. The Verstraeten et al. (2003) Study

The CDC's published Verstraeten et al. \[25\] study consists of a cohort analysis of a subset of records from the medical records databases for several of the HMOs whose records were maintained in a central data repository, the Vaccine Safety Datalink (VSD). This study was conducted in at least five separate phases. In the final phase (i.e., the results reported in the publication), the authors stated that there was no relationship between Thimerosal exposure in vaccines and autism incidence. However, no data are reported in the published study to support this conclusion.

Results from the first phase of the study released in an internal presentation abstract by Verstraeten et al. \[20\] (mentioned earlier) using records from four (4) HMOs showed that infants who were exposed to greater than 25 *μ*g of Hg in vaccines and immunoglobulins at the age of one month were 7.6 times more likely to have an autism diagnosis than those not exposed to any vaccine-derived organic Hg. Within the same abstract, Verstraeten reports that the risk for any neurodevelopmental disorder was 1.8, the risk for speech disorder was 2.1, and the risk for nonorganic sleep disorder was 5.0. All relative risks were statistically significant.

In the second phase of the study, a different approach was taken: exposure was compared at 3 months of age, rather than one month. Results of this phase showed that children exposed to the maximum amount of organic Hg in infant vaccines (62.5 *μ*g) were 2.48 times more likely to have autism diagnosis compared to those exposed to less than 37.5 *μ*g of Hg in vaccines. These results were also statistically significant. No assessment against a "no exposure" control was apparently completed in this study phase.

In the third phase of the study, in which more data stratification methods and different inclusion/exclusion criteria were applied to the analysis, the relative risk of autism for children at three months of Thimerosal exposure dropped to 1.69. At this point, evidence in an email from Verstraeten, the lead investigator, written to a colleague outside of the CDC (obtained by the authors via the US Freedom of Information Act of 1950 as amended), suggests that Verstraeten could have been receiving pressure within the CDC to apply unsound statistical methods to deny a causal relationship between Thimerosal and autism. In this email, Verstraeten states (<a href="#fig1" data-ref-type="fig">Figure 1</a>), "I do not wish to be the advocate of the anti-vaccine lobby and sound like being convinced that thimerosal is or was harmful, but at least I feel we should use sound scientific argumentation and not let our standards be dictated by our desire to disprove an unpleasant theory."

The fourth and fifth phase of the study used records from only two of the original HMOs and incorporated a third HMO, Harvard Pilgrim, into the analysis. Some critics of the study questioned the use of Harvard Pilgrim, as this HMO appeared to be riddled with uncertain record keeping practices, and the state of Massachusetts had been forced to take it over after it declared bankruptcy. In addition, the HMO used different diagnostic codes than the other two HMOs used in phases 2 and 3. Other criticisms include that the study used younger children, from 0 to 3 years of age, even though the average age for an autism diagnosis at the time was 4.4 years. Since half of the children receiving an autism diagnosis would be over 4.4 years of age, far greater than the maximum age in the study at 3 years, this analysis excluded more than 50% of all autism cases from this HMO. Also, the cohort from this HMO contained 7 times fewer individuals than the main cohort from the previous study (i.e., HMO B), and there was no apparent attempt to assess the power of this HMO to show any statistically significant effect.

Also of note is the lack of variability within strata among the different HMOs in the Verstraeten et al. \[25\] study. By design, a cohort study seeking to assess the effect of some treatment on a subsequent outcome should be designed to maximize the range of the independent "treatment" variable (Thimerosal exposure in this instance) in order to determine if there is indeed an "effect" in the dependent postexposure outcome variable (neurological disorders in this study). However, the authors knowingly stratified the analysis based on the participants' gender, year of birth, month of birth, and clinic most often visited. This effectively reduced the variability of Thimerosal exposure within the strata to the point that it reduced the capability of the final analysis to find any but the "strongest" Thimerosal exposure-related outcome effects. The problems with such "overmatching" practices have been discussed in detail in peer-reviewed scientific literature and will be treated in greater detail in the forthcoming review of the CDC's Price et al. \[26\] paper.

Another methodological concern about the Verstraeten et al. \[25\] study is related to the issue of the minimum follow-up period required for individuals in the cohorts examined to ensure that all the cases in the cohort will have been identified with a high degree of certainty. This issue has been mentioned as a problem in the previous studies. As mentioned earlier, the method used to determine the minimum follow-up period for individuals is a critical issue in all studies examining the relationship between exposures and the subsequent risk of a neurodevelopmental disorder diagnosis, especially in those instances where the exposures to all participants in the study are the same or essentially the same. This is the case because the risk of an individual being diagnosed with a neurodevelopmental disorder is not uniform throughout his/her lifetime. Any follow-up method that fails to consider the lag time between birth and age of initial neurodevelopmental disorder diagnosis will likely not be able to observe the true relationship between exposure and the subsequent risk of a neurodevelopmental disorder diagnosis. Verstraeten et al. \[25\] included children in the control group who were too young (down to "0" years of age) to receive a neurodevelopmental disorder diagnosis.

Within this study, Verstraeten et al. \[25\] still found significantly increased risk ratios for tics and language delay. However, the authors stated that, because these results were not consistent between the HMOs tested, these significantly increased risk ratios could not be used to make a determination of the potential adverse consequences of organic-Hg exposure from TCVs.

# 7. The Price et al. 2010 Study

In 2010, the CDC published another epidemiology study on Thimerosal and autism \[26\]. This case-control study was conducted using the records from three managed care organizations (MCOs) consisting of 256 children with an ASD diagnosis and 752 controls that were matched by birth year, gender, and MCO to the children with an ASD diagnosis. Exposure to Thimerosal in vaccines and immunoglobulin preparations was determined from electronic immunization registries, medical charts, and parent interviews. Conditional logistic regression was used to assess associations between ASD, autistic disorder (AD), and ASD with regression and exposure to ethyl-Hg during prenatal, birth-to-1-month, birth-to-7-month, and birth-to-20-month periods. Their published finding was that prenatal and infant Thimerosal exposure from TCVs and Thimerosal-containing immunoglobulin posed no statistically significant risk of autism.

As mentioned earlier, in case-control studies, the main methodological concern is the phenomenon called "overmatching." This concern for overmatching in the Price et al. \[26\] study was voiced previously by DeSoto and Hitlan \[32\]. In their comprehensive analysis of overmatching errors specific to the Price paper, DeSoto and Hitlan \[32\] stated that "Matching cannot—or should not—be done in a way that artificially increases the chance that within\[-\] strata exposure is the same; this happens when a matching variable is a significant predictor of exposure and is called overmatching."

Cases were matched with controls of the same age and sex, within the same HMO and essentially the same vaccination schedule, using the same vaccine manufacturers. DeSoto and Hitlan then state further, regarding the lack of variability of Thimerosal exposure in the Price study, "Across the different years, the average cumulative exposure varies from 42.3 *μ*g to 125.46 *μ*g; while within the birth year stratas (sic), the mean exposures do not vary by more than 15 micrograms." In other words, the maximum level of variation in Thimerosal exposure in the cases and controls being compared was 15 *μ*g of Hg, as compared to the "83" *μ*g of Hg range for the average cumulative exposures in the cohort studies. Moreover, this range is much less than the range of Thimerosal exposures that could have been used to determine risk including (a) 0 to 50 *μ*g of Hg for one-month exposures, (b) 0 to 190 *μ*g of Hg for seven-month exposures, and (c) 0 to 300 *μ*g of Hg for 20-month exposures. Finally, regarding the Price study, DeSoto and Hitlan \[32\] concluded, "this paper is flawed. Unfortunately, there is not an analytic fix for overmatching: it is \[a\] design flaw."

Prenatal Thimerosal exposure for the children within the study arose from the Thimerosal-preserved inactivated-influenza vaccine given during pregnancy and the Rho immunoglobulin administered to pregnant women to prevent Rh-factor incompatibility injury to the developing child. Unlike postnatal exposure from TCVs in the recommended childhood vaccination schedule, prenatal exposures would not be overmatched in a study design that stratified the participants based on their birth year or HMO. Evidence from the background CDC report regarding the Price study showed a significant risk of regressive autism due to prenatal Thimerosal exposure levels, at exposure levels as low as 16 *μ*g of Hg \[33\]. However, the risk of regressive autism due to prenatal Thimerosal exposure reported in that paper was 1.86 and yielded a *P* value of 0.072 which was deemed as insignificant based on the authors' "cut-off" value of *P* \< 0.05. However, *P* values between 0.05 and 0.10 are "marginally significant" and should merit further study. In addition, upon further analysis, it was found that the 2009 background report \[33\] to the Price et al. \[26\] study showed that the prenatal Thimerosal exposure model was run in six different ways and that the most reliable methods (those that factored out the postnatal Thimerosal exposure effects) found highly statistically significant relative risks of up to 8.73 (*P* = 0.009) for regressive ASD due to prenatal Thimerosal exposures from Thimerosal-containing influenza vaccines and Rho immunoglobulin products relative to no such prenatal Thimerosal exposures. Curiously, these more compelling results were not reported in the paper. Withholding these data from the publication and, instead, reporting a significantly lower value could appear to constitute scientific malfeasance on the part of the authors of this study.

# 8. Conclusion

As seen in this review, the studies upon which the CDC relies and over which it exerted some level of control report that there is no increased risk of autism from exposure to organic Hg in vaccines, and some of these studies even reported that exposure to Thimerosal appeared to decrease the risk of autism. These six studies are in sharp contrast to research conducted by independent researchers over the past 75+ years that have consistently found Thimerosal to be harmful. As mentioned in the Introduction section, many studies conducted by independent investigators have found Thimerosal to be associated with neurodevelopmental disorders. Several studies, for example, including three of the six studies covered in this review, have found Thimerosal to be a risk factor for tics \[10, 17, 24, 25, 34, 35\]. In addition, Thimerosal has been found to be a risk factor in speech delay, language delay, attention deficit disorder, and autism \[10, 11, 15–17, 24, 25, 34\].

Considering that there are many studies conducted by independent researchers which show a relationship between Thimerosal and neurodevelopmental disorders, the results of the six studies examined in this review, particularly those showing the protective effects of Thimerosal, should bring into question the validity of the methodology used in the studies. A list of the most common methodological issues with these six studies is shown in <a href="#tab1" data-ref-type="table">Table 1</a>. Importantly, other than the Hviid et al. \[23\] study, five of the publications examined in this review were directly commissioned by the CDC, raising the possible issue of conflict of interests or research bias, since vaccine promotion is a central mission of the CDC. Conceivably, if serious neurological disorders are found to be related to Thimerosal in vaccines, such findings could possibly be viewed as damaging to the vaccine program.

## Acknowledgment

Funding was provided by the nonprofit Institute of Chronic Illnesses, Inc. and CoMeD, Inc.

## Conflict of Interests

All of the investigators on the present study have been involved in vaccine/biologic litigation.

Methodological issues most common in each of the six reviewed studies.

<table>
<thead>
<tr>
<th style="text-align: left;">Study reviewed</th>
<th style="text-align: left;">Methodological issues</th>
</tr>
</thead>
<tbody>
<tr>
<td style="text-align: left;">Madsen et al. [<span class="citation" data-cites="B21">21</span>]</td>
<td style="text-align: left;">(i) Changing entrance criteria in ecological studies.<br />
(ii) Withholding important results from the final publication.<br />
(iii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.</td>
</tr>
<tr>
<td colspan="2" style="text-align: left;"></td>
</tr>
<tr>
<td style="text-align: left;">Stehr-Green et al. [<span class="citation" data-cites="B22">22</span>]</td>
<td style="text-align: left;">(i) Changing entrance criteria in ecological studies.<br />
(ii) Withholding important results from the final publication.<br />
(iii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.</td>
</tr>
<tr>
<td colspan="2" style="text-align: left;"></td>
</tr>
<tr>
<td style="text-align: left;">Hviid et al. [<span class="citation" data-cites="B23">23</span>]</td>
<td style="text-align: left;">(i) Accounting for "person-years" regarding exposure rather than actual exposure levels.<br />
(ii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.</td>
</tr>
<tr>
<td colspan="2" style="text-align: left;"></td>
</tr>
<tr>
<td style="text-align: left;">Andrews et al. [<span class="citation" data-cites="B24">24</span>]</td>
<td style="text-align: left;">(i) Accounting for "person-years" regarding exposure rather than actual exposure levels.<br />
(ii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.</td>
</tr>
<tr>
<td colspan="2" style="text-align: left;"></td>
</tr>
<tr>
<td style="text-align: left;">Verstraeten et al. [<span class="citation" data-cites="B25">25</span>]</td>
<td style="text-align: left;">(i) Cohort of children too young for followup for an autism diagnosis.<br />
(ii) "Overmatching" phenomena due to too closely matched cases and controls.<br />
(iii) Withholding important results from the final publication.</td>
</tr>
<tr>
<td colspan="2" style="text-align: left;"></td>
</tr>
<tr>
<td style="text-align: left;">Price et al. [<span class="citation" data-cites="B26">26</span>]</td>
<td style="text-align: left;">(i) "Overmatching" phenomena due to too closely matched cases and controls.<br />
(ii) Withholding important results from the final publication.</td>
</tr>
</tbody>
</table>

[^1]: Academic Editor: Jyutika Mehta