Abstracts from IACFS/ME International Conference, 9/23/2011-9/24/2011, Ottawa, Canada

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IACFS/ME Biennial International Conference Ottawa, Ontario, Canada

Abstracts from General Session September 23, 2011

Gammaretroviruses of Mice and Their Links to Prostate Cancer and CFS/ME Christine Kozak, Ph.D.

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD, 20892-0460, USA

Gammaretroviruses of three distinct host range tropisms have been isolated from the laboratory mouse. These viruses differ in receptor usage, distribution among wild mouse species and strains of laboratory mice,pathogenicity and sensitivity to host restriction factors. Two of these three host range groups, the xenotropic and polytropic mouse leukemia viruses (together termed XP-MLVs) are widely distributed in house mouse species, mice that live in closest contact with humans. XP-MLVs rely on the XPR1 receptor for entry into cells as does the xenotropic murine leukemia virus-related virus (XMRV) initially identified in human patient samples. Despite their initial description as viruses incapable of infecting mouse cells, the xenotropic viruses have the broadest host range of the MLVs. Nearly all nonrodent mammals are susceptible to X-MLVs, as are all wild mouse species and some inbred strains of laboratory mice. Their XPR1 receptor is highly polymorphic, and there are 5 functional variants of Xpr1 in Mus species and laboratory mouse strains that differ in their ability to support entry of XMRV and various isolates of XP-MLVs. The distribution of XP-MLVs and Xpr1 variants in wild mouse populations provides a good example of how diversifying selection can be driven by genetic conflicts. Restrictive receptor variants evolved in Eurasian house mouse populations exposed to XP-MLV infection suggesting that positive selection favors antiviral alleles in virus-infected species. The ecotropic and polytropic MLVs have long been linked to disease induction in mice, and the discovery that all wild mice and some laboratory strains are also susceptible to X-MLV has made it possible to examine the disease inducing potential of these viruses in mice as well as in other model systems. X-MLVs are capable of establishing infection in mice carrying permissive XPR1 alleles, but X-MLV does not induce or accelerate disease in mice with permissive receptors inoculated as adults or neonates, and X-MLVs do not readily establish productive infection in monkeys. Host factors that restrict retroviruses effectively limit virus spread and disease induction in mice and other species.

a good example of how diversifying selection can be driven by genetic conflicts. Restrictive receptor variants evolved in Eurasian house mouse populations exposed to XP-MLV infection suggesting that positive selection favors antiviral alleles in virus-infected species. The ecotropic and polytropic MLVs have long been linked to disease induction in mice, and the discovery that all wild mice and some laboratory strains are also susceptible to X-MLV has made it possible to examine the disease inducing potential of these viruses in mice as well as in other model systems. X-MLVs are capable of establishing infection in mice carrying permissive XPR1 alleles, but X-MLV does not induce or accelerate disease in mice with permissive receptors inoculated as adults or neonates, and X-MLVs do not readily establish productive infection in monkeys. Host factors that restrict retroviruses effectively limit virus spread and disease induction in mice and other species.

Session: VIROLOGY RESEARCH

Session Chair: Jose Montoya, M.D. Multi-laboratory Evaluations of XMRV Detection Assays Graham Simmons, Ph.D. John M. Coffin2, Indira K. Hewlett3, Shyh-Ching Lo4, Judy A. Mikovits5, William H. Switzer6, Jeffrey M. Linnen7, Francis Ruscetti8, Simone A. Glynn9 and Michael P. Busch1 1Blood Systems Research Institute and Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94118, USA 2National Cancer Institute and Department of Molecular Biology & Microbiology and Program in Genetics, Tufts University, Boston, MA 02111, USA. 3Office of Blood Research and Review, FDA, Bethesda, MD 20892, USA 4Division of Cellular and Gene Therapies and Division of Human Tissues, FDA, Bethesda, MD 20892, USA 5Whittemore Peterson Institute and University of Nevada, Reno, NV 89557, USA

Session: VIROLOGY RESEARCH

Session Chair: Jose Montoya, M.D.

Multi-laboratory Evaluations of XMRV Detection Assays Graham Simmons, Ph.D., John M. Coffin2, Indira K. Hewlett3, Shyh-Ching Lo4, Judy A. Mikovits5, William H. Switzer6, Jeffrey M. Linnen7, Francis Ruscetti8, Simone A. Glynn9 and Michael P. Busch1 1Blood Systems Research Institute and Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94118, USA 2National Cancer Institute and Department of Molecular Biology & Microbiology and Program in Genetics, Tufts University, Boston, MA 02111, USA. 3Office of Blood Research and Review, FDA, Bethesda, MD 20892, USA 4Division of Cellular and Gene Therapies and Division of Human Tissues, FDA, Bethesda, MD 20892, USA 5Whittemore Peterson Institute and University of Nevada, Reno, NV 89557, USA

6Division of HIV/AIDS Prevention, CDC, Atlanta, GA 30333, USA 7Gen-Probe Incorporated, San Diego, CA, USA 8Laboratory of Experimental Immunology, National Cancer Institute- Frederick, Frederick, MD 21701, USA 9Transfusion Medicine and Cellular Therapeutics Branch, NHLBI, Bethesda, MD 20892, USA

Background:

The Blood XMRV Scientific Research Working Group was established to design and coordinate collaborative studies to investigate the prevalence of XMRV in blood donors using standardized XMRV assays.

Materials And Methods:

A multi-phase study has been designed to evaluate XMRV nucleic acid and serological detection assays in terms of sensitivity, specificity and reproducibility; assess assay performance on various specimen types represented in existing blood donor/recipient repositories, and determine the prevalence of XMRV in blood donors. Phase I involved production of whole blood (WB) and plasma analytical performance panels spiked with XMRV infected cells or virus, respectively. These panels were tested in a blinded fashion using XMRV nucleic acid amplification testing (NAT) developed by seven participating laboratories. Phase II represented pilot studies to compare XMRV detection using frozen PBMCs, WB and plasma derived from individuals identified as XMRV viremic in a previous study. Additionally, serology was performed on plasma by two laboratories. Phase III involves further evaluation of the clinical sensitivity and specificity of candidate NAT, serology and culture assays by using a blinded panel of 15 pedigreed positive samples, together with pedigreed negative samples and spiked positive controls.

Results:

In phase I, all laboratories detected at least 136 proviral copies/ml and 5/7 assays demonstrated even more sensitive limits of detection. 5/7 plasma RNA assays performed similarly, with limits of detection of 80 RNA copies/ml or less. The initial unblinded pilot study in phase II resulted in two laboratories detecting MLV-like sequences in the plasma, but not PBMCs or WB, from all four subjects. A third laboratory detected no viral sequences. A second, blinded, pilot study using the same four subjects and two validated negative controls was less conclusive, with three laboratories detecting no viral sequences with any of the samples. A FACS-based serological assay detected antibodies in 3/4 XMRV-positive individuals, but also in 1/2 negative controls. A western-based assay found no evidence of serology in any sample. Results from Phase III are expected soon.

Conclusions:

The Blood XMRV SRWG has established a collaboration between many of the laboratories conducting research into XMRV and its detection in blood and has initiated steps to compare performance of XMRV assays using analytical and clinical panels comprised of blood samples from XMRV-positive and negative pedigreed subjects.

Detection Of Anti-XMRV Antibodies In Serum of CFS Patients and Healthy Blood Donors in Belgium

Kenny De Meirleir, M.D.

Marc Frémont2, Svetlana Khaliboulina3, Vincent C. Lombardi3, Cassandra Puccinelli3, Kristine Metzger2, Judy A. Mikovits3 1. Department of Human Physiology, Vrije Universiteit Brussel, Brussels, Belgium 2. RED Laboratories, Zellik, Belgium 3. Whittemore Peterson Institute, Reno, Nevada, USA

Objectives:

Xenotropic murine leukemia virus–related virus (XMRV) is a new human gammaretrovirus originally identified in prostate cancer patients with a deficiency in the antiviral enzyme RNase L. An association has been made between XMRV and Chronic Fatigue Syndrome (CFS), with a 2009 study reporting the presence of XMRV DNA in the blood of 67% of CFS patients, whereas only 3,7% of healthy controls tested positive. In 2010 another study detected murine leukemia virus (MLV)-like GAG sequences in 86,5% of CFS patients, versus only 6,8% of healthy blood donors. A number of other studies, however, have failed to detect XMRV DNA in the blood of CFS patients. The objectives of this study were to investigate the association between CFS and XMRV in a Belgian population of patients, and to estimate the prevalence of XMRV infections in the general population in Belgium.

Methods:

A flow cytometry-based assay was used to detect the presence of circulating anti-XMRV antibodies in the serum of 84 Belgian CFS patients. A subgroup of these patients (21) have developed CFS after receiving a blood transfusion.

Serum obtained from 44 Red Cross healthy blood donors was also tested. Samples were collected in Belgium and sent, blinded, to the Whittemore Peterson Institute in Reno for analysis.

Results:

48 out of 84 patients (57%) presented circulating antibodies against XMRV (10 out of the 21 patients who received a transfusion). In contrast, only 7 out of 44 controls had anti-XMRV antibodies (16%).

Conclusions:

The higher prevalence of serology positives in the patient population, compared to the controls, supports the idea that XMRV is involved in the pathogenesis of CFS. The finding that 16% of healthy blood donors present evidence of infection with XMRV or a related virus raises questions regarding the need to screen blood donors for asymptomatic XMRV infections.

Prof. Kenny De Meirleir, M.D., Ph.D., Department of Human Physiology, Vrije Universiteit Brussel, Pleinlaan 2, B- 1051 Brussels Belgium, Email: de.meirleir@telenet.be

Detection of MLV-like gag Sequences in Blood and Cell Lines Incubated With Plasma From CFS Patients and Controls

Maureen Hanson, Ph.D.

L.L. Lee1, L. Lin1, D.E. Bell2, D. Ruppert3, S. Levine4, D.S. Bell5. 1Cornell University, Molecular Biology and Genetics, Ithaca NY, 2State University of New York, Dept. of Medical Anthropology, Buffalo NY, 3Cornell University, School of Operations Research and Information Engineering, Ithaca NY, 4Private Practice, New York City, 5State University of New York, Dept. of Pediatrics, Buffalo NY

Objectives:

To determine whether viruses related to XMRV could be detected in peripheral blood from adult subjects who are either ill with CFS, are recovered from CFS, or have no history of a CFS diagnosis.

Methods:

Subjects were divided into five groups. Ten subjects were severely ill with CFS, ten met Fukuda criteria at one time but now considered themselves recovered, and ten subjects from the same geographic area in Western New York were healthy and had never been diagnosed with CFS. Standard instruments were administered to assess the health status of the subjects in these three groups. An additional ten ill subjects and ten control subjects lacking any CFS history were recruited from a physician’s practice in New York City and a different region of upstate New York, respectively. Blood was collected in EDTA tubes and nucleic acids made from PBMCs or whole blood. Plasma was incubated with human cells in culture. Nested PCR with USB Hot-Start IT FideliTaq was performed with gag primers. Any PCR products of expected sizes were sequenced. Samples were tested for mouse contamination with primers to IAP and/or mouse mitochondrial DNA. Control experiments in which human nucleicacid samples were spiked with mouse DNA were performed to determine the sensitivity of the assays for mouse contamination.

Results:

The SF-36 scores of the ten individuals who considered themselves recovered were significantly lower than ten members of the healthy control group from the same Western New York area, according to Hotelling’s T2 test. Tukey's multiple comparison of means indicates that there are highly significant differences between the scores of the Western New York "severe" and controls on all 7 instruments. gag sequences were detected in CFS subjects’ blood as well as in some healthy controls. gag sequences were detected that were more similar to the MLV-like sequences reported by Lo et al. (2010) than to the XMRV sequences reported by Lombardi et al. (2009). MLV-like gag sequences could be detected in nucleic acids prepared from whole and fractionated blood that were negative for the presence of mouse DNA when sensitive assays were performed. Possible reasons for false positive and false negative results when performing highly sensitive PCR assays will be presented.

Conclusion:

gag sequences were detected by PCR in whole blood genomic DNAs that were negative for mouse IAP and mitochondrial DNA. gag sequences similar to polytropic MLVs were obtained. The sensitivity of the PCR assays used requires extreme caution in interpreting results. Maureen R. Hanson, Ph.D., Liberty Hyde Bailey Professor, Dept. of Molecular Biology and Genetics, Cornell University, Biotech. Bldg., Ithaca, NY 14853 USA. mrh5@cornell.edu

Chronic Widespread Musculoskeletal Pain, Fatigue, Depression and Disordered Sleep in Chronic Post- SARS Syndrome; A Case-Controlled Study

Harvey Moldofsky, M.D., Dip.Psych., FRCPC, FAPA

John Patcai

Background:

The long term adverse effects of Severe Acute Respiratory Syndrome (SARS), a viral disease, are poorly understood.

Methods:

Sleep physiology, somatic and mood symptoms of 22 Toronto subjects, 21 of whom were healthcare workers, (19 females, 3 males, mean age 46.29 yrs.+/- 11.02) who remained unable to return to their former occupation (mean 19.8 months, range: 13 to 36 months following SARS) were compared to 7 healthy female subjects. Because of their clinical similarities to patients with fibromyalgia syndrome (FMS) these post-SARS subjects were similarly compared to 21 drug free female patients, (mean age 42.4 +/- 11.8 yrs.) who fulfilled criteria for fibromyalgia.

Results:

Chronic post-SARS is characterized by persistent fatigue, diffuse myalgia, weakness, depression, and nonrestorative sleep with associated REM-related apneas/hypopneas, an elevated sleep EEG cyclical alternating pattern, and alpha EEG sleep anomaly. Post- SARS patients had symptoms of pre and post-sleep fatigue and post sleep sleepiness that were similar to the symptoms of patients with FMS, and similar to symptoms of patients with chronic fatigue syndrome. Both post-SARS and FMS groups had sleep instability as indicated by the high sleep EEG cyclical alternating pattern rate. The post-SARS group had a lower rating of the alpha EEG sleep anomaly as compared to the FMS patients. The post-SARS group also reported less pre-sleep and post-sleep musculoskeletal pain symptoms.

Conclusions:

The clinical and sleep features of chronic post-SARS form a syndrome of chronic fatigue, pain, weakness, depression and sleep disturbance, which overlaps with the clinical and sleep features of FMS and chronic fatigue syndrome.

Publication: Moldofsky and Patcai: Chronic widespread musculoskeletal pain, fatigue, depression and disordered sleep in chronic post-SARS syndrome; a case-controlled study.

BMC Neurology 2011 11:37. See: http://www.biomedcentral.com/1471-2377/11/37

Session: VIROLOGY RESEARCH AND REVIEW

Session Chair: Jose Montoya, M.D.

Role of the Immune Response in CFS

Jose Montoya, M.D.

Abstract Unavailable

The Case FOR XMRV/Human Gammaretroviruses (HGRVs) in ME/CFS Judy Mikovits, Ph.D.

In 2009 using a classical virology approach of viral isolation and transmission, electron microscopy, serology and PCR, Lombardi et al demonstrated the first isolation of XMRV from blood from patients with chronic fatigue syndrome (CFS) predominately from the west coast of the United States. In 2010, Lo et al. extended these studies by detecting nucleic acids of MLV-related variants in the peripheral blood mononuclear cells of CFS from the northeastern United States suggesting additional strains capable of infecting humans exist. In a study of 300 CFS patients, 13 developed lymphoproliferative disorders. Of those tested, 11/11 were positive for XMRV and 9/9 positive for clonal TCR gamma rearrangements. Spontaneous development of four immortalized B cells lines occurred during culture of cells from CFS patients. Three developed from B cells isolated from the peripheral blood (two of whom had B cell lymphoma) and one from a bone marrow biopsy. The B cell lines have a mature CD20+, CD23+ phenotype and produce infectious XMRV. Virus production occurred despite extensive hypermutation of the proviruses in these cells by APOBEC3G. Therefore, XMRV infection may accelerate the development of B cell malignancies by either indirect chronic stimulation of the immune system and/or by direct infection of the B-cell lineage. Since viral load in peripheral blood is low, these data suggest that B cells in tissues such as spleen and lymph nodes could be anin vivo reservoir for XMRV. We have also identified an inflammatory cytokine and chemokine signature that distinguishes XMRV infected CFS patients from healthy controls with 94% sensitivity and specificity. Monitoring immune dysfunction affords the opportunity to begin to understand the pathogenesis of XMRVs. In addition to these data, recent advances in developing tests for detection and characterization of variants of XMRV will be also be discussed

Judy A Mikovits, PhD, Research Director, Whittemore Peterson Institute, University of Nevada, Reno MS 0552, 1664 N Virginia St, Reno NV 89557-0552, phone: 775-682-8264, fax 775-996-7159

The Case AGAINST Human Gamma Retroviruses (HGRV) in CFS/ME John Coffin, Ph.D.

Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111,USA

Xenotropic MLV-related retrovirus (XMRV) was first reported about 5 in a few cases of prostate cancer, but did not attract much attention until its reported association with a large fraction of chronic fatigue syndrome cases about 2 years ago. The publication of the XMRV-CFS connection created a ripple of excitement and interest in the scientific, medical, and patient communities reminiscent of the reports of the association of another retrovirus—HIV--with AIDS some 25 years previously. However, most of the results of the XMRV paper – isolation of infectious virus from patients, frequent detection of virus in plasma and PBMCs by PCR, detection of antiviral antibodies - remain to be replicated outside of the laboratories that authored the original report despite considerable effort worldwide. Indeed, XMRV is now considered by most virologists to be the consequence of a collection of artifacts originating from endogenous murine leukemia viruses prevalent in laboratory and wild mice.

There are several related, but distinct, issues that need to be considered. First, various mouse (Mus musculus) subspecies carry over a hundred different endogenous proviruses closely (>90%) related to XMRV in their DNA. Second, mice are extremely widespread, as is their DNA, which can be found sporadically on laboratory surfaces, as well as contaminants of common reagents and materials. Sensitive PCR assays can detect “XMRV” related sequences in DNA from tiny fractions of one cell. To detect such contamination, we developed a more sensitive assay based on mouse IAP sequences present in thousands of copies per cell. Third, although only a few of the endogenous MLV proviruses encode infectious virus, it has been known since the 1970s that some of them can give rise to virus that can infect human tumor lines when passaged through nude mice. Indeed, A virus identical to XMRV is produced by the 22RV1 prostate cancer line that was derived in just this way. In initial reports, however, XMRV did not appear to be sufficiently similar to known proviruses to have been derived this way. However, we have recently shown that this is exactly how it did arise, but not from infection of the precursor CWR22 xenograft with a single virus, but rather with a recombinant between the progeny of two previously undescribed proviruses found in the nude mice used for passage. Since the predicted recombinant is ancestral to all XMRV isolates, and cannot have arisen more than once, it must have found its way into many laboratories as the 22Rv1 cell line was distributed worldwide and, by means that remain to be worked out, into clinical samples from CFS patients.

Session: TREATMENT ADVANCES

Chair: Eleanor Stein, M.D.

Health/Performance and Response Status of XMRV/pMRV Antibody Positive vs. Negative Chronic FatigueSyndrome (CFS) Subjects in a Phase III Clinical Trial

David R. Strayer, M.D.

Judy A. Mikovits2, Vamsidhar Vurimindi1, William A. Carter1 1Hemispherx Biopharma, Inc., Philadelphia, PA; 2Whittemore Peterson Institute, Reno, NV

Background:

CFS is a severe disorder consisting of profound fatigue and a variety of other debilitating symptoms that affects up to 4 million Americans. Recently, one of us (JAM) identified DNA from a human gamma retrovirus (XMRV) in 67% of CFS subjects. Evidence also suggested that approximately 50% of the CFS infected subjects mounted a specific antibody response against XMRV (Science 326, 585-589 (2009)). The objective of this study was to compare demographic parameters, health/performance status and response of XMRV/pMRV antibody positive vs. negative CFS subjects enrolled in a Phase III clinical trial evaluating the safety and efficacy of a toll-like receptor 3 (TLR3) agonist, rintatolimod (PolyI:PolyC12U, Ampligen®).

Materials and Methods:

Two-hundred-eight (208) evaluable subjects, who met the 1988/1994 Case Definitions for CFS, participated in this randomized, placebo-controlled, double-blinded, multicenter study. Only severely debilitated patients were selected for this study. The primary endpoint was exercise treadmill duration. Subjects received rintatolimod (200-400 mg) or an equivalent volume of placebo twice weekly by IV infusion for 40 weeks. Baseline (or earliest available specimen) serum samples from all 208 subjects were analyzed for antibodies directed against XMRV/pMRV.

Results:

Seventy (33.7%) of the 208 CFS subjects were positive for antibodies directed against XMRV/pMRV, while 138 (66.3%) were negative. There was no significant difference in the number of CFS subjects positive or negative for antibody with regard to age, gender, duration of CFS, cognitive dimension (SCL90-R), exercise treadmill duration, or SF-36 vitality score (p>0.3). However, the subjects negative for antibody had a lower Activity of Daily Living score (66.9 vs. 71.2, p=0.010, ANOVA) and a lower overall activity level based upon a lower activity monitor score (183K vs. 210K, p=0.033, ANOVA). The percent of subjects with a >25% increase in exercise treadmill tolerance (ETT) at Week 40 compared to Baseline was significantly greater for subjects receiving rintatolimod (39%) vs. placebo (23%), p=0.016 (2 tailed Fisher’s Exact Test). Although, there was a trend for greater improvement in exercise duration with rintatolimod treatment for both the XMRV/pMRV antibody cohorts receiving rintatolimod, the antibody positive subgroup had a greater relative percent of subjects showing a >25% increase in ETT with rintatolimod compared to placebo than the antibody negative cohort. An analysis of concomitant medications utilized by CFS subjects to help treat symptoms of CFS showed that, when compared to placebo, the rintatolimod treated cohort positive for antibodies had a greater percentage of subjects with a decrease in CFS-related medication use at the end of the study (24%), p=0.039 vs. the antibody negative subjects (13%), p>0.10.

Conclusions:

These results indicate that approximately 1/3 of the CFS subjects have a detectable immune response directed against XMRV/pMRV and that this antibody positive group may respond more favorably to rintatolimod, an antiviral and immune modulator, than the antibody negative cohort. Additional studies to further evaluate XMRV/pMRV in this CFS population are underway.

Rifampin Augments the Effects of Oxymatrine/Equilibrant (oxm/equi) In Patients with Myalgic Encephalomyelitis/CFS

John K. Chia, M.D. Andrew Chia. EV Med Research

Rifampin augments the effects of oxymatrine/Equilibrant (oxm/equi) in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). John Chia, Andrew Chia. EV Med Research

Objectives:

Chronic enterovirus infection has been implicated in the immunopathogenesis of ME/CFS. Previously, we demonstrated the benefit of oxm/equi, an herbal immune booster, in 50% of ME/CFS patients. Concomitant administration of rifampin in one patient resulted in flu-like symptoms and ulceration of infected pharyngeal tissues, which was followed by symptomatic improvement and decrease of chronically elevated Coxsackievirus B3, 4 antibodies. We evaluated the adjunctive effect of rifampin in patients who were taking oxm/equi.

Method:

46 patients who fulfilled the CDC criteria for ME/CFS were treated with rifampin 300 mg po bid for 7 days while taking oxm/equi (32 responders and 14 non-responders, duration 1.32±0.86 years). 45 patients treated with oxm/equi without rifampin, and 45 outpatients treated with doxycycline and rifampin for MRSA (methicillinresistant Staphylococcus aureus) infections served as controls. Laboratory studies including CBC, chemistry panel, CPK were obtained before and during treatment if patient had flu-like symptoms. Cytokine gene expression of peripheral blood was performed before and during rifampin treatment for 10 ME/CFS treatments.

Results:

31/46 (67%) patients developed significant flu-like symptoms lasting few days during or after the one-week rifampin treatment. 23/33 (70%) of responders and 0/13 non-responders had additional improvement of fatigue and other symptoms ( p <0.01, X2 test). 21/33 (64%) responders who had taken oxm/equi ≥ 1-2 years were able to discontinue the herbs within weeks or months of flu-like symptoms and remained in remission. 0/45 ME/CFS patients on oxm/equi alone and 0/45 MRSA-infected patients on doxycycline and rifampin developed flu-like symptoms. Laboratory studies showed no significant changes, and gene expression study of 12 cytokines demonstrated increase of TNF-α and IL-1α,β mRNA while on rifampin and oxm/equi.

Conclusion:

Flu-like symptoms were commonly observed in patients who took oxm/equi concomitantly with rifampin, as compared to controls. Subsequent symptomatic improvement was observed in > 60% of oxm/equi responders. Short course of rifampin may be beneficial in ME/CFS patients who are responding to oxm/equi. The possible mechanism of enhanced immune response will be discussed and further investigated.

Brief Self-Management of UCF/CFS in Primary Care: A Randomized Trial Fred Friedberg, Ph.D. Janna Coronel, MA

Objective:

The objective of this study was to test a brief self-management protocol in a primary care setting, in people with medically unexplained chronic fatigue (UCF) and chronic fatigue syndrome (CFS). An effective self-management plan has the potential (1) to improve the generally poor outcomes for UCF and CFS patients in primary care, (2) to greatly expand the availability of behavioral health care for UCF and CFS, and (3) to reduce medical and behavioral utilization for UCF and CFS. The proposed study is an extension of an efficacious two-session selfmanagement clinical trial for CFS in secondary care (Powell et al., 2001). The hypothesis was tested that a brief self-management- focused cognitive-behavioral intervention will yield improvements in fatigue, physical and role functioning, and psychological distress in comparison to the two control conditions: standard medical care alone or standard medical care plus an attention control symptom monitoring condition.

Methods:

We tested the efficacy of a two - session self-management-focused cognitive-behavioral intervention in a target sample of 108 persons with UCF or CFS. Participants were randomly assigned to one of three study conditions: (1) standard medical care alone; (2) standard medical care plus a nurse-delivered attention control condition of symptom monitoring; or (3) standard medical care plus a nurse-delivered self-management cognitive-behavioral treatment delivered by a nurse.

Results:

At the three month follow-up, sample sizes were as follows: fatigue self-management (FSM) = 21; Symptom Monitoring (SM) = 26; and Usual care (UC) = 21. Forty percent met Fukuda criteria for CFS. Controlling for age, sex and illness duration at the three-month follow-up assessment, a significant reduction was found on the fatigue severity scale (p<.05). No significant changes were found on diary fatigue ratings, the SF-36PF, Beck Anxiety Inventory or the Beck Depression Inventory. Actigraphy significantly declined across all conditions (p<.05). Patient global impression of change (PGIC) ratings were as follows for the three conditions (FSM/SM/UC): Improved (13/ 6/4); Unchanged (5/9/11); Worse (2/5/2). Despite little change on our standard measures, brief interviews with study participants revealed that both worsened and unchanged patients across conditions attributed their PGIC ratings to external negative events or lack of healthy activities, whereas improved patients reported increased awareness of their behaviors and affirmative steps to pursue healthy activities.

Conclusion:

A brief, standardized illness management service for UCF/CFS showed modest improvement in fatigue severity and PGIC ratings. PGIC ratings of improved, unchanged, and worsened overall appeared to reflect different attitudes toward the illness and/or differential exposures to negative major life events. These findings indicate a role for self-management activities in generating improved outcomes.

Fred Friedberg, PhD, Research Associate Professor, Department of Psychiatry, Putnam Hall/South Campus, Stony Brook University, Stony Brook, NY 11794-8790. fred.friedberg@stonybrook.edu

Session: FIBROMYALGIA: ARE TENDER POINTS NECESSARY? A DEBATE Chair: Lucinda Bateman, M.D.

Tender Points are Important

Roland Staud, M.D.

Population studies have demonstrated moderately strong associations (odds ratios range 1.3-3.1) between the presence of pain in a body segment and the presence of tender points within that segment. Further, there is evidence of increasing number of tender points with increasing number of painful segments. The reporting of nonspecific pain, aching, or stiffness, is also associated with high tender point counts. Importantly, there is no unique cut off at which local pains and tender points occur concurrently in a widespread form. This is consistent with the observation that fibromyalgia (FM) represents one end of a spectrum of musculoskeletal pain and tender points, and that both traits are continuous in the general population. Tender points have been successfully used for the definition of study populations like fibromyalgia. For clinical purposes, however, tender points seem to provide little mechanistic information about individuals’ pain and associated symptoms.

Tender Points are Unnecessary Daniel J. Clauw, M.D.

Abstract Unavailable

Session: DIAGNOSING CFS/ME; DIFFICULT CLINICAL CASES

Session Chair: Nancy Klimas, M.D.

Case Presentations by: Charles Lapp, M.D., Lucinda Bateman, MD, Rosamund Vallings, MNZM, MB BS, Derek Enlander, M.D.

Nancy Klimas M.D.

Abstract

In this workshop, experts will present difficult cases, and discuss the diagnostic and management implications. Cases may include “look alike cases” that presented with the signs and symptoms of ME, but in fact were found to be caused by another disorder; complex management issues; medication use and medication intolerance and other issues of interest to the practicing clinician. Related conditions such as Gulf War Illness will be included in the case discussion. The workshop will welcome interchange with the clinicians attending the session. Basic scientists may also hear in this discussion some of the issues that trigger further research ideas. Charles Lapp, M.D., Lucinda Bateman, MD, Rosamund Vallings, MNZM, MB BS, and Derek Enlander, M.D. will present cases that will serve as a platform for discussion.

Rosamund Vallings MB BS

Case study - A Cautionary Tale

This is a case study of a female with a long history of Chronic Fatigue Syndrome, which followed a typical relapsing and remitting course over many years. She was often very ill interspersed with bouts of reasonably good health. She was admitted to hospital aged 60 during a very serious “relapse” and died a week after admission. This study serves to remind us of the importance of ongoing surveillance and the need to focus on other diagnostic possibilities over time.

Lucinda Bateman MD

Case Study Patient 1: 39 year old male attorney who was ill for 15 years with mild CFS, then became unable to work. Healthy and very physically active in youth. Fitness conscious. Married at age 23 in 1993.

1994. First child born. Ankle fracture required surgery. Completed college while working in family business. Family business turmoil. Brother commits suicide. Gradually developed unusual fatigue, frequent sore throat and swollen glands, low grade fevers, then pain in the shoulders and sternum. Became less able to exercise vigorously.

1995-2001: Completed college and law school (PCP said he was "just depressed" and thought the challenge would cheer him up). Two more kids born. Marital discord. Low function at times due to severe fatigue, achiness, sore throats, low grade fevers, hot flashes, night sweats and cognitive difficulties. Missed class often to stay in bed and stayed in bed on weekends. Often too ill to mow the small lawn and other times he could play basketball but end up in bed afterward.

2001-2007: Worked as an attorney. More stable but continued chronic symptoms and reduced activity tolerance. Managed basic work hours, could weights but not aerobic exercise, and continued to have fatigue, achiness, brain fog and night sweats. Diagnosed with hyperthyroidism, post surgical hypothyroidism, hypogonadism, hyperlipidemia and obstructive sleep apnea. All were treated. Compliant with CPAP.

2008: Busy period of work. Wife in hospital. Vestibular neuronitis with severe vertigo. Resolved in a few weeks. After this he was progressively less able to maintain a normal work schedule. Cognitive dysfunction made analytical work difficult. After another negative workup to rule out causes of fatigue, the firm partners encouraged him to apply for medical disability leave. 2 year LTD awarded for depression/anxiety.

2009: Full evaluation was done by a CFS specialist. CFS diagnosis confirmed.

MEDS: Synthroid, Lipitor, Androgel, CPAP Physical Exam: BMI 35 (muscular habitus with mild obesity) BP 138/95, pulse 80, supine; BP 140/95, Pulse 100, standing at 3 min (later BPs 140/90- 150/100) 0/18 fibromyalgia tender points. Lab: Normal except Vitamin D 22 (subsequently supplemented without improvement) Does not meet criteria for MDD or any other exclusionary mental health condition. Symptoms: fatigue and cognitive problems, unrefreshing sleep, headaches, mild myalgia and arthralgia, marked activity intolerance with post-exertion relapse, few infection/immune symptoms. Able to complete ADLs, do light household activities, run light errands 2-3 times in a week, and walk on a treadmill for 5-7 minutes, 3 mph, 2-3 times per week on better days. He was able to drive the truck to 3 day family hunting trip, but unable to participate [stayed in the campground] and went home early. Vigorous, sustained physical activity, or intense cognitive tasks result in relapse symptoms of 1-3 days duration. Sad about losing his career and anxious about the financial support of his family. Does he meet CFS criteria? Does he have OI/POTS? If so, how should it be treated? Should his blood pressure be treated with a medication? Should he be on Lipitor? How should his metabolic syndrome be addressed? He is maximizing testosterone to maintain muscle mass. Testosterone levels top normal. Is this OK? What else can be done for him? Lucinda Bateman MD Case Study Patient 2: 25 year old woman with severe CFS/FM. Married 1 year. No children. Childhood: Born with cord around neck but seemed to recover. Dyslexia in elementary school. Bitten by a monkey in Brazil and got rabies shots. Youth: Soccer and swim teams. Played violin. Diagnosed with auditory processing disorder. College: Age 17 in 2003. ROTC Air Force 40 hours/week. Major in aerospace engineering 20 cr hr/ semester. Worked 10 hours/week. 4 hours sleep/night not uncommon. 2004-2005: Gradual onset sleep disturbance, generalized weakness and fatigue, concentration difficulties, abdominal pain. After ROTC boot camp (32 days) she never recovered and developed total body aching. Spring 2006: Failed many courses due to inability to function. Quit ROTC. Quit school. Tried to go on a mission to Hong Kong. Worsened and came home due to "seizures"[shaking tremor attacks] and worsening pain. June 2007: CFS/FM Evaluation Primary symptoms: Abdominal pain, nausea/dizziness (vertiginous and OI), fatigue/weakness, seizure-like episodes when too tired. Additional symptoms: constant headache, attention/concentration, disturbed sleep, night sweats, sore throats, numbness and tingling (shoulders, arms, feet) MEDS: Armour thyroid 15 mg bid (for "fatigue"---later d/c), B12 po, fish oil 1 gm bid, multivitamin EXAM: BMI 25. BP 90/64 Pulse 72 supine. BP 88/58 Pulse 88 standing at 3 min. Pharynx mildly erythematous with moderately large tonsils. Toes are cool with delayed capillary refill. 11/18 TP Testing: SF36 scores very low except for emotional well being. Pain diagram: whole body sparing lower legs. Symptom scores: 9-10 for pain, fatigue, cognition. 7-8 headache, sleep. 4-6 mood. 2007-2011: Interval diagnoses and treatment Interstitial cystitis. Dysmenorrhea and chronic pelvic pain. GERD/IBS. Eczema, allergies, reflux related asthma. Mild. Neurology, urology, gyn consults. Brain MRI, EEG, Tilt table test, PSG, echo all normal. CURRENT MEDS: amitriptyline 50 mg qhs (for IC), Savella 100 mg bid (mildly improved pain), zolpidem 10 mg q hs, Lortab 7.5/325 bid PRN, alprazolam 1 mg qd, Vit D, Vit C, Mag, Calcium, multiple vitamin. Push oral fluids and sodium. OCP

MEDICATIONS that were not tolerated or not helpful: Fatigue: Adderall, Nuvigil, Ritalin Pain: cyclobenzaprine, desipramine, Lyrica, gabapentin, zonisamide, Cymbalta, tramadol, NSAIDS, APAP Sleep: melatonin, Lunesta, temazepam, Seroquel, trazodone Currently able to spend about 1-2 hours out of reclining position daily. Makes jewelry at home. The only intervention that controls pain is activity limitation. How can pain be improved? Can function be improved? Doesn’t get along with in-laws. Should she try to convince them she is a good choice for their son? Should she pursue her dream of having a family…i.e. get pregnant? What should she be advised about medications during pregnancy? Charles W. Lapp, M.D. Case Study In this workshop, experts will present difficult cases, and discuss the diagnostic and management implications. Cases may include “look alike cases” that presented with the signs and symptoms of ME, but in fact were found to be caused by another disorder; complex management issues; medication use and medication intolerance and other issues of interest to the practicing clinician. Related conditions such as Gulf War Illness will be included in the case discussion. The workshop will welcome interchange with the clinicians attending the session. Basic scientists may also hear in this discussion some of the issues that trigger further research ideas. Charles Lapp, M.D., Lucinda Bateman, MD, Rosamund Vallings, MNZM, MB BS, and Derek Enlander, M.D. will present cases that will serve as a platform for discussion. Abstracts from General Session Saturday, September 24, 2011 Session: CASE DEFINITIONS FOR RESEARCH AND PRACTICE Chair: Kenneth J. Friedman, PhD The New International Consensus Criteria for ME: Content and Context Bruce M. Carruthers, M.D., CM. FRCP(C) Contents- the general thrust of the 2003 Canadian Consensus Criteria was retained and developed further. Several changes were made- e.g. the 6 month waiting period was no longer required, but left to clinical judgment. The symptom pattern of Post-Exertional- Neuroimmune-Exhaustion (PENE) was kept criterial and further articulated. Symptoms and symptom interactive patterns arising from the following subsystems- neurocognitive, pain processing, sleep disturbances, neurosensory and motor, immune, gastrointestinal, genitourinary and endocrine subsystems as well as energy transport impairments (cardiovascular, microvascular, respiratory, thermostatic homeostasis, intolerance of temperature extremes and stress intolerance) are noted if present. Interactive dynamical pattern matches between PENE symptom patterns and those from pathophysiologial subsystems for individuals and groups of patients are noted for causal projectability over time will be mutually confirmative as real kinds. Modifications for paediatric cases were added. The past historical context is described as well as future implications of this case definition plus any descendents are discussed regarding future research directions, case segregation, and treatments. In conclusion, it is hoped that this case definition and its descendents will continue to emphasize both the clinical/epidemiological/research realms of observation and challenge all participants to integrate them into a mutual confirmation/disconfirmation process that characterizes both clinical medicine, epidemiology and science in general.

Contrasting Case Definitions Leonard Jason, Ph.D. Abigail Brown, Erin Clyne, Lindsey Bartgis, Meredyth Evans, Molly Brown DePaul University Objectives: There has been considerable debate about what case definition to use with the illness commonly known as CFS. For example, some have speculated that the initial definitions of ME ((Dowsett et al., 1994; Goudsmit et al., 2009; Ramsay, 1988)) and later on the Canadian criteria of ME/CFS (Carruthers et al., 2003) select a group of patients that have more severe functional impairments than the Fukuda et al. (1994) criteria. This presentation contrasts individuals diagnosed with the Myalgic Encephalomyelitis/chronic fatigue syndrome (ME/CFS) Canadian case definition (Carruthers et al., 2003) with those that did not meet these criteria (Non-ME/CFS) but met the Fukuda et al. (1994) chronic fatigue syndrome (CFS) criteria. The study also compared individuals diagnosed with another case definition involving Myalgic Encephalomyelitis (ME) (based on criteria from Dowset et al., 1994; Goudsmit et al., 2009; Hyde, 2007; Ramsay, 1988) with those that did not meet these criteria (Non-ME), but met the Fukuda et al. criteria. Methods: The sample of patients had been diagnosed with CFS by the Fukuda et al. criteria and were later categorized as meeting ME/CFS and/or ME criteria. Results: In general, the ME/CFS criteria identified a group of patients with more functional impairments and physical, mental and cognitive problems than the Non-ME/CFS criteria. The ME criteria identified patients with more functional impairments, and more severe physical and cognitive symptoms than the Non-ME condition. Katon and Russo (1992) have argued that a requirement of more symptoms to meet criteria could inadvertently select for individuals with psychiatric problems. Similarly, Kroenke (2003) found similar results examining 15 variables within a fatigued sample. It is certainly possible that the differences on so many measures between the ME/CFS and the Non-ME/CFS groups was due to the larger number of symptoms of higher frequency and severity who met the ME/CFS criteria. Conclusion: The current CFS case definition of Fukuda et al. (1994) has been used internationally by researchers for over 15 years. It is possible that some patients meeting these criteria do not have core symptoms such as post-exertional malaise or memory/concentration problems. By specifying 7 symptoms as with the ME/CFS criteria or by specifying 4 symptoms with the ME criteria, it may be possible to identify a more homogenous and impaired group of patients. The current study suggests that these other ME and ME/CFS criteria might be used to identify patients with possibly more homogenous and severe symptomatology and functional impairment. Both ME/CFS and ME criteria appear to select a more severe group of patients than those that only meet the Fukuda et al. criteria. Leonard A. Jason, Ph.D., Director, Center for Community Research, DePaul University, 990 W. Fullerton Ave., Suite 3100, Chicago, Il. 60614. Ljason@depaul.edu Data Mining Leonard Jason, Ph.D. Beth Skendrovic, Jacob Furst, Molly Brown, Meredyth Evans, Abby Brown DePaul University Objectives: Data mining may be a useful tool in aiding in the diagnosis of ME/CFS. There are many challenges in diagnosing ME/CFS. Some symptoms associated with it are common of other illnesses, and there are competing definitions that investigators may use. More work with data mining in ME/CFS research could aid in further identification of cardinal symptoms, leading to better diagnostic ability. This would also combine an objective, computer driven decision with a physician’s medically influenced decision to come up with a better and more reliable way to diagnose and treat ME/CFS. This article contrasts two case definitions for Myalgic Encephalomyelitis/chronic fatigue syndrome (ME/CFS). We compared the empiric CFS case definition (Reeves et al., 2005) and the Canadian ME/CFS Clinical case definition (Carruthers et al., 2003) with a sample of individuals with CFS as defined by the Fukuda et al. (1994) criteria versus those without CFS from a community-based sample. Methods: Data mining with decision trees was used to identify the best items to identify patients with CFS. Data mining is a statistical technique that was used to help determine which of the survey questions were most effective for accurately classifying cases as defined by the two case definitions versus others contained by the study.

Results: The empiric criteria identified 79% of patients with CFS and the Canadian criteria identified 87% of patients with CFS. Items identified by the Canadian criteria had more construct validity. ME/CFS is often thought to include post-exertional malaise and neurocognitive disorders, and both did emerge as predictive factors for the Canadian criteria, but not when using the Reeves et al. (2005) empiric case criteria. In addition, sleeping disorders and pain symptoms, other key symptoms of ME/CFS, did emerge for the Canadian criteria as well as in the immune areas (i.e., sore throat and multiple chemical sensitivities), and this supports evidence for the Canadian criteria. In contrast, the empiric criteria tended to identify more general areas, including less activity, social and role functioning problems, and some pain issues. However, critical symptoms such as post-exertional malaise, neurocognitive symptoms and sleep disorders were not identified as discriminating symptoms with the Reeves et al. (2005) criteria. Conclusion: The study’s overall findings were that the Reeves et al. (2005) criteria were not as capable of identifying cases from non-cases as the Canadian criteria (Carruthers et al., 2003). The Reeves criteria have been criticized as being more general and broader than the Fukuda et al. (1994) criteria, and the results of this study suggest that these criteria are only able to discriminate 79% of cases from others, whereas the Canadian criteria were able to 87% of cases. In addition, when examining the items selected in both analyses, it is apparent that the Canadian criteria appear to select cardinal and central features of the illness. Leonard A. Jason, Ph.D., Director, Center for Community Research, DePaul University, 990 W. Fullerton Ave., Suite 3100, Chicago, Il. 60614. Ljason@depaul.edu Pathways to Pathogenesis: Standardized Measures of CFS/ME Illness Domains Elizabeth R.Unger, M.D., Ph.D. Case definitions are used in at least two different circumstances. First, a case definition, when simply and easily applied, may be used as a substitute for a specific diagnostic test to measure disease in a population. This would be done when use of the diagnostic test is impractical for delivering information in a timely and cost-effective manner. An example of this situation is use of “flu-like illness” determined by telephone interview as a surrogate to monitor seasonal or pandemic influenza. A second circumstance occurs when there is no diagnostic test, and case identification requires the use of specific descriptive measures. This is the current situation with CFS/ME, a complex and heterogeneous disorder likely to involve multiple pathways to pathogenesis. Case definitions are essential for public health agencies to determine burden of illness, for clinicians to appropriately diagnose and manage patients, and for researchers to identify risk factors and the underlying biologic basis for illnesses. Limitations in the ability of case definitions to identify homogenous patient populations could be addressed by standardizing how the definitions are applied, or by narrowing the definitions through increased criteria needed to meet the definition, or both. An alternative approach is to improve measures of illness domains (questionnaires and biologic) to allow patients identified by any case definition to be phenotypically sub-grouped in a way that allows the underlying biology to be discovered. Presenting author: Elizabeth R. Unger PhD, MD, Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, NCEZID, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G41, Atlanta, GA 20222, USA. eunger@cdc.gov The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency. Session: IDENTIFYING ABNORMALITIES IN CFS/ME: THE IMPORTANCE OF EXERCISE CHALLENGE Chair Staci R. Stevens, M.A. Exercise Testing to Quantify Effects of Fatigue on Functional Capacity in Patients With CFS Betsy A. Keller, B.A. Micale, FG Ithaca College Objective: The purpose of this study was to assess the effects of post-exertional malaise (PEM) on functional capacity and anaerobic threshold in subjects diagnosed with chronic fatigue syndrome (CFS). Methods: Subjects were 10 females and 2 males (41.3+1.11 yrs) diagnosed with CFS by a physician experienced in the diagnosis of CFS. To induce PEM, each subject completed a maximum exercise test on a cycle ergometer. A second maximum exercise test was

performed 24 hrs later to assess the effects of exercise-induced PEM on functional capacity. Maximum oxygen consumption (VO2max), maximum heart rate (HRmax), anaerobic threshold (AT), maximum workload (Wmax), workload at AT (ATwork), and respiratory exchange ratio (RER) were measured. RER is an objective indicator of substrate utilization and subject effort during exercise. Results: Significant decreases from test 1 to test 2 were 13.5% for VO2max (21.5 to 18.6 ml.kg-1.min-1; p<0.01), 8 bpm for HRmax (p<0.01), 18.8% for AT (12.0 to 9.7 ml.kg-1.min-1; p<0.05), 9.4% for Wmax (121 to 109 W, p<0.05), and 17.3% for ATwork (58.3 to 48.2 W; p<0.05). However, there was no change in maximum RER indicating that subject effort was maximum and also comparable during both tests. Conclusion: Results indicate that PEM decreased maximum functional capacity by more than 13% to below 5 METS; a level at or below that which is required by many job-related activities and IADLs. To compare, VO2max in healthy individuals is highly reproducible over days and even months (r>.95)1, with a SEM of < 6%1,2. Thus, for subjects in this study, an expected variation between tests would be ±1.29 ml.kg-1.min-1 in contrast to the observed decrease of 2.9 ml.kg-1.min-1. Furthermore, PEM decreased AT to below 3 METS (e.g., light-moderate speed walking), which is a level of many activities considered to be sedentary in nature. Thus, completion of sedentary ADLs and IADLs for those with CFS requires production of energy via anaerobic processes that will further contribute to PEM and exacerbate symptoms of CFS. Since many daily activities fall into the 3-5 MET range, individuals with CFS will exacerbate symptoms associated with PEM simply by completing normal daily activities. 1Taylor, HL, Buskirk, E & Henschel, A. (1955). Maximal oxygen intake as an objective measure of cardiorespiratory performance. J Appl Physiol, 8, 73-80. 2Katch, VL, Sady, SS & Freedson, P. (1982). Biological variability in maximum aerobic power. Med Sci Sports & Ex, 14(1), 21-25. Betsy A. Keller, PhD, Professor, Exercise & Sport Sciences, Ithaca College, 318 Center for Health Sciences, Ithaca, NY 14850 keller@ithaca.edu Frank G. Micale, M.S., Clinical Associate Professor, Exercise & Sport Sciences, Ithaca College, 317 Center for Health Sciences, Ithaca, NY 14850 fmicale@ithaca.edu The Importance of Exercise Challenge Christopher Snell, Ph.D. The absence of reliable diagnostic laboratory tests or biomarkers presents significant problems for persons with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), treating physicians, and the ME/CFS research community alike. Typically ME/CFS diagnoses rely on self-report measures. An alternative to this approach is to employ direct, objective multi-system, measures of physical function that may also provide insights to the underlying pathophysiology of ME/CFS. One such methodology is cardiopulmonary exercise testing (CPET). The principles underlying CPET are simple. Physical exertion requires that the cardiovascular system supply oxygen (O2) to active muscles and the pulmonary system remove carbon dioxide (CO2) from the blood. Taxing these systems has the capacity to reveal abnormalities that may not be apparent at rest and thus elucidate the mechanisms underlying exercise intolerance in ME/CFS. Some key measures available from CPET include: maximal aerobic capacity (Peak VO2 or VO2 max ); ventilatory or anaerobic threshold (VT); and peak respiratory exchange ratio (RER). CPET permits accurate comparison of subjects across serial exercise tests and should be of prime consideration for any clinical intervention trial with functional endpoints. CPET data also allow for the more reliable interpretation of results when an exercise challenge is used to elicit ME/CFS symptoms. As a quantifiable measure of both physiological stress and effort, CPET enables direct comparison between patients and controls on these measures. CPET also has the capacity to objectively document PEM in ME/CFS patients. A significant change in exercise capacity over consecutive tests, it could be argued, is clear evidence of PEM. Session: THE LATEST RESEARCH IN IMMUNOLOGY Chair: Nancy Klimas, M.D. Natural Killer Cell Number and Function in a Prospective Cohort of Adolescents with Chronic Fatigue Syndrome and Controls Following Mononucleosis Benjamin Katz, M.D. Maurice O’Gorman2, Deli Wang3, Cynthia Mears1, Yukiko Shiraishi4, Renee Taylor4 1Pediatrics, Northwestern University Feinberg School of Medicine 2Pathology, Northwestern University Feinberg School of Medicine 3Biostatistics Research Core, Children’s Memorial Research Center 4Occupational Health, University of Illinois at Chicago

Introduction: Chronic fatigue syndrome (CFS) is a complex condition involving severe fatigue and disabling musculoskeletal and cognitive symptoms. Whether immunologic dysfunction accompanies CFS is controversial. Arguably the most consistent immunologic disturbance associated with CFS is reduced function of natural killer (NK) cells. Objectives: We examined NK function in our cohort of adolescents following infectious mononucleosis (IM) and recovered controls matched for age, sex and Tanner stage. Methods: Nine adolescents with CFS and 9 matched, recovered controls had blood drawn for NK cell quantitation and functional analysis that was performed blinded at 6, 12 and 24 months following IM. At each time point, NK cell quantification was ascertained by flow cytometry as %CD56+ cells, and NK cell function was determined using K562 cells and 3 different dilutions of patient lymphocytes. NK cell numbers were scored as high, normal or low. NK cell function was scored as normal, low or borderline by pre-determined parameters by an investigator blinded as to the patient’s diagnosis. Statistical analysis was conducted using generalized linear mixed model with repeated measurements and linear mixed model with SAS 9.2. Results: There were 27 evaluable time points for the CFS patients and 25 for the controls. There was no difference in NK numbers between cases and recovered controls. NK function was significantly higher in case patients with CFS 6 months following IM than in recovered controls (p=0.02). Conclusion: We could not confirm decreased NK cell function in adolescents with CFS following IM. Acknowledgement: Funded by R01HD4330101AI from the National Institute of Child Health and Human Development Presenting author: Ben Z. Katz, MD, Professor of Pediatrics, Northwestern University Feinberg School of Medicine Attending Physician, Division of Infectious Diseases, Children’s Memorial Hospital 2300 Children’s Plaza, Chicago, IL 60614, bkatz@northwestern.edu Disparities In Innate and Adaptive Immune Cell Activities in Chronic Fatigue Syndrome Ekua W. Brenu, PhD candidate M. Van Driel1,2, K.J. Ashton2, S.B. Ramos2, J. Keane2, D.R. Staines1,3 , S. Marshall-Gradisnik1,2 1. Faculty of Health Science and Medicine, Population Health and Neuroimmunology Unit, Bond University, Robina, Queensland, Australia. 2. Faculty of Health Science and Medicine, Bond University, Robina, Queensland, Australia. 3. Queensland Health, Gold Coast Public Health Unit, Southport, Robina, Gold Coast, Queensland, Australia. Objective: Cell specific immune investigations have demonstrated a possible link between Chronic Fatigue Syndrome (CFS) and failure to maintain immunological homeostasis. The most common immune cells with known dysfunction in CFS are cytotoxic cells, Natural killer cells and CD8+T cells. This study examined cytotoxic function and markers in CFS patients at 6 months intervals to determine the stability of these observations over time. Methods: 90 CFS patients (mean age 46.5yrs ±11.7) and 50 healthy controls (mean age 41.9yrs ± 9.6) participated in the study. Flow cytometeric protocols were used in the assessment of cytotoxic activity and cell phenotypes and RTqPCR analysis in screening for levels of cytoxic molecules that depict the various cytotoxic pathways. These molecules include, granzymes, perforin, interferon (IFN)-ã and tumour necreosis (TNF-á). Results: Preliminary results indicate that compared to the healthy controls, CFS patients demonstrate significant decreases in cytotoxic activity at baseline, at 6 and at 12 months. Additionally, NK CD56 bright cells remained decreased in the CFS participants. Cytotoxic, molecules were also differentially expressed in these cells in comparison to the healthy group. Conclusion: This study demonstrates and confirms reduced immune function in patients with CFS. These findings substantiate the use of NK cell cytotoxic function as a potential biomarker for CFS. Ekua Weba Brenu HBSc, Grad Dip BMed, Faculty of Health Science and Medicine, Bond University, Gold Coast, Queensland 4229, Australia, Email: ebrenu@student.bond.edu.au Longitudinal Assessment of Adaptive Immune Regulation in Chronic Fatigue Syndrome Ekua W. Brenu, PhD candidate K.J. Ashton2, M. Van Driel1,2, S. Hardcastle1,2, D.R. Staines1,2, S. Marshall-Gradisnik1,2

4. Faculty of Health Science and Medicine, Population Health and Neuroimmunology Unit, Bond University, Robina, Queensland, Australia. 5. Faculty of Health Science and Medicine, Bond University, Robina, Queensland, Australia. 6. Queensland Health, Gold Coast Population Health Unit, Southport, Gold Coast, Queensland, Australia. Objective: Chronic Fatigue Syndrome (CFS) is known to persist for more than 6 months with a very slow recovery rate. It is not known whether immunological abnormalities in CFS remain stable over time or change during the course of the disease. Additionally cytokine measurements have not been consistent across studies which may be associated with the fluctuating pattern of the disease. This longitudinal study assesses proteins and receptors secreted and expressed by CD4+T lymphocytes in CFS patients over time, at baseline, 6 and 12 months. Method: 50 CFS meeting the CDC case definition and 30 non-CFS control participants were recruited from two states in Australia. Peripheral blood mononuclear cells were preferentially isolated from whole blood samples collected from participants. The samples were then assessed for the expression of Th1, Th2, Th17 cytokines, IL-1α, IL-1β and TGF-β using cytometric bead array and flex set kits. Result: At baseline there was an increase in IL-10, TNF-α and IFN-γ in the CFS group compared to the healthy control group. However, after 6 months IL-2 was significantly increased and IL-10 and IL-17A were significantly decreased in the CFS group while after 12 months only IL-2 was observed to be significantly increased in the CFS group. Conclusion: These results suggest that the cytokine profile in CFS changes during disease progression. This may be associated with disease severity and/or concurrent environmental stressors. Hence there is a need to match experimental findings with data on clinical disease progression. Ekua Weba Brenu HBSc, Grad Dip BMed, Faculty of Health Science and Medicine, Bond University, Gold Coast, Queensland 4229, Australia, Email: ebrenu@student.bond.edu.au Promoter DNA Methylation and Expression of Perforin in CFS and Controls Virginia R. Falkenberg, Ph.D. Toni Whistler, Janna Murray, Elizabeth R Unger, Mangalathu S. Rajeevan. Chronic Viral Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Objectives: Perforin plays a key role in immune surveillance and several studies report decreased perforin protein and mRNA in peripheral blood of patients with chronic fatigue syndrome (CFS). Factors that modulate gene-environmental interaction and thus the pathophysiology of disease include gene silencing by DNA methylation. The objectives of this study were to determine the pattern of perforin gene methylation in conjunction with perforin gene expression and whether these features were altered in CFS. Methods: Subjects (34 CFS and 47 non-fatigued, NF) selected from a population based study underwent the Trier Social Stress Test (TSST), a standardized psychosocial test that induces stress, and is known to influence cortisol secretion. Blood samples were collected prior to (10:30am, T1), and after the TSST (3:05 pm, T2). DNA extracted from peripheral blood mononuclear cells (PBMC) was used to examine site-specific CpG methylation levels in the methylation sensitive region (MSR) of the promoter (sites -876, -776, -744, -720, -691, -670 and -650). This was quantified by bisulfite treatment of DNA followed by pyrosequencing. RNA from PBMCs collected at the same time points was used to quantify perforin mRNA expression by LightCycler real-time RT-PCR. Total RNA from peripheral blood collected at the same time points was used in the Affymetrix Human Exon Array 1.0 platform. Results: Methylation of the MSR ranged from 38%-79% and no differences in CpG site-specific methylation of perforin was detected between CFS and NF at T1 or T2. In PBMC, there was no difference in the perforin expression between CFS and NF at T1 but expression was significantly higher in CFS than NF (1.4 fold, p=0.02) at T2. NF subjects had reduced perforin expression (0.8 fold, p=0.008) and methylation levels were increased by 4% (range 2.6-4.3, p= 0.01-0.05) at four CpG sites (- 876, -744, -691, and -670) at T2 compared to T1. However in CFS subjects, methylation levels were increased by

6% (range 4.7-6.8, p=0.02-0.03) at T2 compared to T1 at two positions (-776 and -744) without a corresponding change in expression. Expression results by real-time RT-PCR and exon arrays were concordant. Conclusion: While increased promoter DNA methylation correlated with reduced perforin expression in NF, this relationship was not seen in CFS. The small but statistically significant differences in methylation were detected over the course of the day were different for the NF and CFS groups. Further studies are needed to confirm these results and to evaluate explanations (changes in cell population, circadian rhythm or stress) for the observed dynamics in perforin DNA methylation and expression. Presenting author: Virginia R. Falkenberg PhD, Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, NCEZID, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-41, Atlanta, GA 30333, USA fse9@cdc.gov Session: NEW DEVELOPMENTS IN PEDIATRIC ME/CFS Chair: Teruhisa Miike, M.D, Ph.D. Linking Lymphocyte Metabolites with Clinical Course in Post-Infectious Fatigue Gordon Broderick, Ph.D. R. Ben Hamo2, S. Efroni2, B.Z. Katz3, M.R.G. O’Gorman3, L. Nathanson4, M.A. Fletcher4, S.D. Vernon5, R. Taylor6. 1Dept. of Medicine, University of Alberta, Edmonton, AB, Canada; 2 Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel; 3Dept. Pediatrics, Northwestern University, Chicago, IL, USA; 4Dept. of Medicine, University of Miami, Miami, FL, USA; 5CFIDS Association of America, Charlotte, NC, USA, 6Dept. of Occupational Therapy, University of Illinois at Chicago, Chicago, IL, USA. Objectives: Chronic Fatigue Syndrome (CFS) affects between 1 and 4 million individuals and costs an estimated $35 billion per year in lost productivity and health care. As CFS can follow Epstein-Bar virus (EBV) and other systemic infections, our objective was to describe differences in immune activation in post-infective CFS (PI-CFS) patients compared to recovered controls. Methods: We studied 301 Chicago-area adolescents prospectively over 24 months following diagnosis of monospot-positive infectious mononucleosis (IM). Cluster analysis of subjects chronically fatigued at 24 months (4.3% of cohort) revealed 3 clinical courses: i) a sustained increase in fatigue after a early partial remission (C1), ii) a monotonic decrease in fatigue (C2), or iii) a slow decrease in fatigue after a peak at 12 months (C3). Cryopreserved samples of peripheral blood mononuclear cells (PBMC) were also recovered from 7 PI-CFS subjects and matched recovered controls. Duplicate gene expression profiles were obtained in these samples using the GeneChip Human Gene 1.0 ST microarray (Affymetrix, Santa Clara, CA). A novel computational method was used to assign probabilities of discrete up and down-expressed states for each gene in every individual sample. These probabilities were then combined to identify consistent representation of known molecular interactions and quantify the activity level of close to 600 cellular pathways catalogued in the National Cancer Institute (NCI)/Nature Pathway Interaction Database (PID) and the KEGG database. Patients and patient groups were then compared statistically on the basis of the estimated activity levels of these pathways. Results: Previous analysis of plasma cytokines in this cohort indicated immune signaling anomalies specific to PI-CFS subjects and present to different extents in each fatigue sub-group. Consistent with this, 20% of expressed genes (of 92 with fold change>2, p<0.05) supported cell signaling and/or immune function. Close to half however (47%) supported cell metabolic function. Derivation of pathway activity levels in individual subjects greatly reduced the false discovery rates (FDRs) isolating 5 pathways with significantly altered activity in PI-CFS (FDR<0.10, p< 0.005). Phenylalanine metabolism and Trk neuronal receptor signaling were significantly suppressed in PI-CFS (p=0.002, 0.004; FDR=0.08, 0.09) while starch metabolism, glycolysis and pentose phosphate metabolism were up-regulated (p=0.000, 0.001, 0.003; FDR=0.04, 0.07, 0.09). Of these, phenylalanine metabolic activity also supported the separation of fatigue sub-groups, with higher activity being linked to a more favorable prognosis. Conclusion: These preliminary results suggest that observed differences in cytokine expression are consistent with altered metabolic activity in circulating lymphocytes in PI-CFS patients. These differences may also inform on the course and underlying causes of this illness.

• Gordon Broderick, Ph.D., Associate Professor, Department of Medicine, University of Alberta, Suite 225B College

Plaza, 8215 112 Street NW, Edmonton, AB, T6G 2C8, Canada; gordon.broderick@ualberta.ca A Trial for Prevention of CCFS Onset from The View Point of Sleep Issue Terusha Miike, M.D., Ph.D. Nobuyuki Ymamashita2) (a teacher's consultant) 1) Hyogo Childrenfs Sleep and Development Medical Research Center, Hyogo, Japan 2) A board in charge of local public schools. in Yawata City,Kyoto, Japan. Objectives: We have been considering that CCFS has been completed with the following order. 1) In spite of night active modern type daily life, people should keep classical morning active daily life, 2) which causes chronic sleep deprivation, 3) developed failure of neuronal function maintenance, 4) induced the derangement of the biological clock which is important and necessary for a human social life and life itself, 5) resulted neuronal fatigue and loss of neurons,6) these conditions connected with each other, finally complete the failure of the everyday life, 7) resulted in school non-attendance and/or so called CCFS. Therefore we think that we will be able to prevent CCFS by reading sleep deficient sign from everyday life of children. Methods: Then we investigated the habit of the sleep-wake rhythm for 5,100 students (ranged 6-15 years of age) in Yawatacity, Kyoto to get actual information to contribute to our purpose. We asked all students to record the consecutive 14 days sleep-wake log and studied following subjects, 1) total sleep time, 2) sleep onset time, 3) wake-up time and 4) sleep-wake pattern. The sleep log was classified into the 8 kinds of patterns, which was made beforehand. (1: holiday sleep supply, 2:short sleep, 3:long sleep, 4:irregular sleep, 5:sleep after get home, 6:sleep fragmentation, 7:overlapping, 8:normal sleep) .. Results and Conclusion.. Average bed time (First grader: 9:14, Second 9:15, Third 9:16, Fourth 9:33, Fifth 10:03, Sixth 10:21, Seventh 10:49, Eighth 11:15, Ninth 11:51 pm).. Average rise time (First grader: 6:58, Second 7:01, Third 7:01, Fourth 7:00, Fifth 7:06, Sixth 7:10, Seventh 7:03, Eighth 7:14, Ninth 7:31am). Average total sleep time (First grader: 9.8, Second 9.7, Third 9.7, Fourth 9.5, Fifth 9.0, Sixth 8.8, Seventh 8.2, Eighth 8.0, Ninth 7.7 hrs). In addition we found that prolonged short sleep and irregular sleep pattern are the strong risk factors that suggest a difficulty of performing daily school life, in this study. It is considered that sleep deprivation and irregular life style has a direct connection to the CCFS. According to these data we conceived that it may be possible to prevent CCFS by observing a lifestyle, especially a sleep-wake rhythm of children. (Teruhisa Miike, M.D. Ph.D. Chief of Hyogo Childrenfs Sleep and Development Medical Research Center, 1070, Akebono-Cho Nishi-Ku Kobe, Hyogo, 651-2181, Japan E-mail: t_miike@hwc.or.jp) Therapeutic Outcome by Two-months Intensive Sleep-Wake Circadian Rhythm Treatments in Japanese Children and Adolescents with Chronic Fatigue Seiki Tajima, M.D. Shigeyuki Matsuzawa, Kazumi Takai, Teruhisa Miike. Hyogo childrenfs sleep and development medical research center Objectives: In the last two decades, we have reported relationship between biological clock system and childhood chronic fatigue (CCF). Biological clock dysfunction is associated with energy metabolism, immune system and frontal lobe dysfunction. Based on these evidences, we have treated patients with CCF at new medical research center for CCF and developmental disorder since April 2009. Here we show the therapeutic outcome of our first year trial for translating evidence into practice. Methods: 30 patients (15 boys and 15 girls, age 11 to 25) with chronic fatigue caused by sleep deprivation were admitted in the first year of our center. All patients were treated for 8 weeks with bright light therapy, thermal therapy, medication, cognitive behavioral therapy and lifestyle teaching. Self-sleep-logs (S-log) have been recorded during hospitalization. 48hr core body temperature (CBT) monitoring was performed at the beginning and the end of therapy. Delay of circadian rhythm (.60min.), poor daily variation (ƒ¢CBT<1.0‹C) and totally high CBT (1.0‹C higher than control) were detected in CBT recordings. Long total sleep time (.10hr), delayed sleep phase (sleep onset later than 24:00), irregularity of sleep onset and offset (larger variation than 90min.) and sleep segmentation (segmented more than 7 days per 2 weeks) were also detected in S-log recordings. With or without these factors were compared between the beginning and the end of therapy using fisherfs exact test.

Results: Delay of CBT circadian rhythm (p<0.01), long total sleep time (p<0.0001), delayed sleep phase (p<0.05) and irregularity of sleep onset and offset (p<0.0001) were significantly improved at the end of therapy. Conclusion: Intensive sleep-wake circadian rhythm treatments were effective to improve circadian rhythm. However, recoveries of other chronic fatigue related symptoms and poor performances were insufficient at the time of discharge. Therefore, just a part of patients has resumed normal activity yet. Recovery from sleep disturbance is not the goal but the first stage of improvement for the patients with chronic fatigue. From this point of view, more clinical trials will be needed. Presenting author: Seiki Tajima, MD., Address: 1070 Akebono-cho, Nishi-ku, Kobe, 651-2181, Japan. E-mail address: s_tajima@hwc.or.jp What is the Natural History of Chronic Fatigue Syndrome in Young People? Dr Katherine Rowe, Judith Moon. Royal Children’s Hospital Melbourne, Australia. Objectives: To follow up consecutive patients referred to the CFS clinic at the Royal Children’s Hospital between 1991 and 2009 regarding their level of functioning, self-reported perception of recovery, duration of illness and the usefulness of management strategies. Methods: Seven hundred and eighty eight young people age 6-18 years (mean 15 years) (M:F 1:3), were referred from family doctors or consultant pediatricians for diagnosis and management or secondary consultation. Diagnosis required a defined onset over hours or days of persistent or relapsing, debilitating fatigue, which was exacerbated with exercise and did not resolve with bed rest, duration of illness greater than 6 months, and fulfilling the criteria of Holmes et al (1988) and Fukuda et al., (1994). Standardised historical, symptom and psychological data were obtained from 398 and standardised history only, from an additional 390. 398 were followed up prospectively with questionnaires approximately each 2 years, while the second group were contacted by phone during 2010 and 2011, and a questionnaire sent if consent was obtained. The follow up questionnaire recorded functional outcomes, demographics, duration of illness, use of alternative health practitioners and reported usefulness of management strategies. Results: Questionnaire follow up data were obtained on at least one occasion for 342 of the 398 (86%). Six occasions between 1996 and 2008 provided 804 returns allowing more accurate timing of reported recovery with multiple data points. 78% of the additional 390 were able to be traced and provided information. The follow up for both groups ranged from 1.7 years to 21 years. The average duration of illness was reported as 5 years with range 1-15 years. By 5 years 60% reported recovery. By 12 years, 88% reported recovery (n=256), although in approximately 1/3 there was an indication that they were conscious of monitoring their workload. Less than 5% were not either studying or working part or full time, often due to other factors than CFS. Many had married (n=38) and those with children (n=15) reported being well. 90% completed or intended to complete post-secondary training. The only alternative practitioners that were deemed helpful were those that provided some relief for muscle pain with massage, or who provided good dietary advice. Restrictive diets and supplements did not reach placebo levels of response. Symptom management and the strategy of balancing social contact, physical activity, educational input and a commitment to regularly attend at least one activity each week as the most useful assistance. Every young person devised a different balance of activities and program depending on severity of illness, stage of education, family circumstances and life interests. Engagement in education was best predictor of functional outcome. Conclusion: The outcomes for young people in Australia are generally positive although prolonged. Ongoing support particularly in navigating the education system was highlighted by them as an essential contributor to the quality of their life and their ability to cope. Session: New DEVELOPMENTS IN EPIDEMIOLOGY Chair: Kenneth J. Friedman, Ph.D. Natural History Leonard Jason, Ph.D. DePaul University Nicole Porter, Jessica Hunnell, DePaul University, Alfred Rademaker, Northwestern University, Judith A. Richman, University of Illinois, Chicago.

Objectives: Despite growing knowledge about long-term predictors of chronic fatigue syndrome (CFS) outcomes, many followup studies are not prospective in that they either rely on retrospective self-report at a single point in time or they consist of longitudinal data that are analyzed in a cross-sectional manner without taking into account the influence of baseline findings. Clearly, there is a need for more research on the incidence and course of CFS in ethnically and socioeconomically diverse, community populations. Methods: The present project was carried out in two stages. In Stage 1, we attempted to re-contact the 213 adults who were medically and psychiatrically evaluated from 1995-1997. These adults were previously evaluated in our original Wave 1 CFS epidemiology project (Jason, Jordan et al., 1999). Stage 2 of the study encompasses a structured psychiatric assessment and a complete physical examination and a structured medical history. The original Wave 1 sample is a stratified random sample of several neighborhoods in Chicago specifically selected to contain individuals from different ethnic and socioeconomic profiles. Although the CFS group had a high rate of follow-up, those in the other groups were much more difficult to track over time. Fortunately, we did not find significant sociodemographic differences at Wave 1 between those we retained in the sample versus those that we were not able to re-contact, and this provides support for the generalizability of the outcomes to the larger sample. Results: The study’s major finding was that rates of CFS appear to have been relatively stable over the period of time from Wave 1 to 2. As rates of CFS were .42% in Wave 1 (Jason, Richman et al., 1999), estimates from our current natural history study suggest that these rates have stayed relatively constant over the past decade. Sixty-seven percent of participants with CFS at Wave 1 continued to have CFS in our sample at Wave 2. Of the new cases of CFS over time, 75% came from the ICF group, suggesting that this group is at higher risk of developing CFS. In addition, 50% of the remitters went from a CFS diagnosis to the ICF group, indicating that while remitters no longer met CFS case definition, half were still suffering from chronic severe fatigue. Among all the variables in this study, only for post-exertional malaise did the CFS group significantly differ from the other conditions. This reaffirms the importance of this being a cardinal and critical symptom for CFS, and all of the individuals in the CFS group had this symptom either at Waves 1 or 2. Finally, a high level of mortality was found (18% of those with medical or psychiatric exclusions group, 12.5% for the CFS group). Conclusion: There are few studies that have been able to provide estimates of long term CFS outcomes, particularly in culturally diverse, community-based samples. In the present 10 year natural history study, the CFS group for the most part remained rather ill over time. Leonard A. Jason, Ph.D., Director, Center for Community Research, DePaul University, 990 W. Fullerton Ave., Suite 3100, Chicago, Il. 60614. Ljason@depaul.edu CFS Knowledge And Illness Management Behavior Among U.S. Healthcare Providers and The Public Elizabeth Unger, M.D., Ph.D. Dana J. Brimmer, Ph.D., MPH, Roumiana S. Boneva, James F. Jones, , Centers for Disease Control and Prevention Objectives: Chronic fatigue syndrome (CFS) is a challenge because of unknown etiology and diagnostic biomarkers, and treatment relies on symptom management. Previous research has shown positive CFS knowledge, attitudes and beliefs among physicians and awareness among the public. We compared CFS knowledge and illness management behavior between healthcare providers (HCP) and the public in order to identify gaps and need for educational interventions. Methods: We used DocStyles, a 2009 web-based panel survey of primary care physicians, OB/GYNs, pediatricians, dermatologists, and nurse practitioners, and HealthStyles, a 2010 public consumer mail survey to ask questions about CFS knowledge and illness management behavior. The HCP sample is drawn from an opt-in, verified Epocrates Honors panel. HealthStyles used stratified random sampling to match the national population on region, income, age, and household size. Both surveys asked about CFS awareness, CFS symptoms, and if CFS is a medical or psychiatric condition. HCP were also asked if they have ever made a CFS diagnosis and how they treat and manage CFS. HealthStyles respondents were asked if they knew someone diagnosed with CFS, and then also if they or someone they knew thought they had CFS, what they would do to find out more about the illness.

Results: The response rate was 46% for DocStyles and 67% for HealthStyles with a sample size of 2,000 and 4,184 participants, respectively. Males comprised 65% of DocStyles physicians and 48% of the HealthStyles sample. Among HCP, 94% heard of CFS compared to 57% of the public. HCP had two to three times higher recognition rates on eight CFS symptom criteria as compared to the general public. When asked if CFS were both medical and psychiatric, 71% of HCP agreed as compared to 30% of the public. Two percent of the public considered CFS a psychiatric condition vs 14% of HCP. A higher proportion of the public considered CFS a medical condition: 27% vs 8% of HCP. Uncertainty whether CFS was either a medical or a psychiatric condition was higher among the public (42%) compared to HCP (8%). Thirty-seven percent of HCP reported ever making a diagnosis of CFS and nearly 10% of the public knew someone diagnosed with CFS. HCP who reported ever diagnosing CFS were more likely to categorize CFS as a medical condition (Chi-square = 98.6, p<0.01). Top three ways in which HCP manage CFS were: referring to a medical specialist (35%), prescribing medications (29%), and referring to a psychologist (26%) or prescribing graded exercise therapy (26%). If diagnosed with CFS, the public would seek information by talking to a family doctor (72%), searching the Internet (54%), and talking to medical specialist (25%). Only 7% would join a support group. Conclusion: Our results are consistent with findings that HCP have high awareness and knowledge of CFS, and nearly 80% of HCP classified CFS as a medical or as both medical and psychiatric condition. However, HCP and public view of medical or psychiatric conditions differed. History of CFS diagnosis for HCP may influence this classification. More than 1/3 of HCP had ever diagnosed CFS, which is notable considering the relatively low prevalence of CFS. Study results support development and maintenance of CFS educational materials for the Internet and providing family doctors with tools to engage patients in conversations about CFS. Corresponding author: Dana J. Brimmer, Ph.D., M.P.H., Behavioral Scientist, McKing and CDC, 1600 Clifton Road, MS-A15, Atlanta, Georgia, 30333, dyv4@cdc.gov. Disclaimer: The findings and views in this report are those of the authors and do not necessarily reflect the views of the funding agency. Profile of the Patient with Chronic Fatigue Syndrome; Experience with a Population- Based Registry Jose Alegre, M.D. Ruiz E, Garcia Quintana AM, Karaki M, Aliste E, Montaner L, Saez N, Fernandez de Sevilla T. Unidades del CFS. Hospital Vall d´Hebrón y Centro Médico Delfos. Barcelona. (Spain). Objectives: In Spain, there are no epidemiologic studies analyzing the characteristics of patients diagnosed with chronic fatigue syndrome (CFS) according to the criteria of Fukuda. Thus, the prevalence and incidence of this nosologic condition, which causes considerable disability in personal, social, and work-related activity, is currently unknown. This study determines the sociodemographic, clinical, and therapeutic characteristics of a large series of CFS patients in our setting. Patients and Method: All patients who consulted for disabling chronic fatigue and met the diagnostic criteria of Fukuda were included. Patients underwent a diagnostic protocol that included complete laboratory analyses, chest x-ray, abdominal ultrasound, and psychiatric assessment. Sociodemographic data, symptoms, work situation, and treatments prescribed at the time of the diagnosis were recorded. . Results: The study included 981 patients with CFS (91 men and 890 women), with a mean age of 47,9 years, 66% were married, 60% carried out specialized work, and 7% were housewives. Among the total, 60% had a secondary school or university education. There was a family background of CFS in 12%, fibromyalgia in 10%, and other immunological diseases in 26.4%. The mean age at the onset of symptoms was 37,5 years and the mean interval from the onset of fatigue to the diagnosis was 116,5 months. The onset was sudden in 20% and gradual in 61%. An evident trigger was documented in 60% (infection, delivery, and a stressful life event). At the time of the diagnosis, 62.5% of patients were not working (sick leave 34% and work disability 37%). The treatment received at diagnosis included medication for the symptoms (analgesic, anxiolytic, and antidepressive agents) in 78,3%, alternative treatments in 3%, and programmed physical exercise and/or cognitive behavioral therapy in 5%. Conclusions: When evaluating a patient with incapacitating chronic fatigue, it is essential to identify cases that meet the criteria for CFS. In our setting, this condition predominantly affects middle-aged women who have a secondary or university education and work at specialized jobs. The onset of symptoms often occurs following an identifiable trigger. The condition leads to severe dysfunction in the personal, social, and work-related activities of daily life.

• This study is supported by a research grant (Beca Mutua Madrileña, 2007)

Abstracts from General Session Sunday, September 25, 2011 Session: RESEARCH DEVELOPMENTS IN GENOMICS AND GENETICS Chair: Christine Kozak, Ph.D. Expression Patterns of miRNAs in Lymphocytes In Patients with Chronic Fatigue Syndrome Ekua W. Brenu, PhD candidate K.J. Ashton2, G. Atkinson2, M. Van Driel1,2, D.R. Staines1,3, S. Marshall-Gradisnik1,3 7. Faculty of Health Science and Medicine, Population Health and Neuroimmunology Unit, Bond University, Robina, Queensland, Australia. 8. Faculty of Health Science and Medicine, Bond University, Robina, Queensland, Australia. 9. Queensland Health, Gold Coast Population Health Unit, Southport, Gold Coast, Queensland, Australia. Objective: MicroRNAs (miRNAs) and transcription factors regulate gene expression and thus are important in modulating the immune responses. Changes in these molecules may be implicated in diseases such as Chronic Fatigue Syndrome (CFS). A number of transcription factors have been shown to be upregulated in CFS patients. However, the role of miRNAs remains to be determined. As cytotoxic activity is decreased in Natural Killer (NK) cells and CD8+T cells, this study assesses the role of miRNAs molecules in CD8+T cells and NK cells in CFS patients. Methods: 30 CFS patients meeting the CDC case definition (45.3±11.7 yrs) and 30 healthy controls (41.8±9.6 yrs) were recruited into the study. Blood samples were collected from all participants following which lymphocytes were preferentially isolated via a negative isolation system to yield a pure sample of NK and CD8+T cells. RNA was extracted and converted into cDNA and miRNAs of interest were assessed using RT-qPCR. Statistical analysis was performed using the t-test. Results: Of the fifteen miRNAs investigated six were found to be down regulated in both the NK and CD8+T cells in CFS patients compared with healthy controls. Most of these miRNAs target genes that are involved in cell cycle regulation, apoptosis and toll like receptor expression. Conclusion: This study confirms changes in miRNA expression in cytotoxic cells that may be related to the poor function of these cells in CFS patients. Ekua Weba Brenu (PhD Candidate) HBSc, Grad Dip BMed, Faculty of Health Science and Medicine, Bond University Gold Coast, Queensland 4229, Australia, Email: ekbrenu@bond.edu.au Pathway-Focused Genetic Evaluation of Immune and Inflammation Related Genes in CFS Mangalathu S. Rajeevan, Ph.D. Irina Dimulescu, Janna Murray, Maung M. Khin, Virginia Falkenberg, Elizabeth R Unger. Chronic Viral Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Objectives: There is evidence that immune and inflammatory alterations are important in CFS. The objective of this study was to determine if genetic variants in inflammation and immune pathways could be linked to CFS as well as to quantitative measures of functional impairment, fatigue and symptom inventory. Methods: Participants were identified from a population-based study. This analysis included 362 Caucasian subjects: 121 non-fatigued (NF); 50 CFS with no medical/psychiatric exclusions (CFS); 129 fatigued but insufficient symptoms or fatigue for CFS (ISF) with no medical or psychiatric conditions; and 62 CFS except for medical/psychiatric exclusions (CFS-exclusions). We used a pathway-focused genetic analysis of immune and inflammation related genes with the Affymetrix Immune and Inflammation Chip that covers 11K single nucleotide polymorphisms (SNP) in 1000 genes representing 38 sub-pathways in immune response and inflammation. The manufacturer’s protocol was followed for genotyping and accuracy was validated by pyrosequencing. Golden Helix SVS software was used for genetic analysis. SNP functional annotation was done using SPOT and GenomePipe programs.

Results: Compared to NF controls, CFS was associated with 34 functionally relevant SNPs (p=2.68 x10-2 – 1.31 x10-5). Twelve of these SNPs are in genes playing a role in pathways related to complement cascade (SERPINA5, CFB, CFH, MASP1 and C6), chemokines (CXCL16, CCR4, CCL27), cytokines/cytokine signaling (IL18, IL17B, IL2RB), and Toll-like receptor signaling (TIRAP, IRAK4). While 11 out of 34 SNPs remained associated with ISF compared to NF, only 4 of the 34 SNPs remained associated with CFS-exclusions. A polymorphism (rs11214105) in the 5′upstream regulatory region of IL18 was associated with both CFS and ISF (CFS, p=1.52 x 10-2; ISF, p=2.03 x 10-2). In CFS, this SNP associated with MFI subscale of physical fatigue (p=7.1 x10-3), SF-36 subscale of body pain (p=9.7 x10-3) and summary score for CFS case defining symptoms (2.6 x 10-5). With all these associations, the minor allele increased the risk of the associated phenotype. Similarly, the minor allele of rs7616342 in KCNH8, representing the p38/MAPK signaling pathway, increased the risk for CFS (p=1.31 x 10-5), and was also associated with MFI mental fatigue subscale (p=9.0 x 10-3). Conclusion: This study identified a number of novel and functionally relevant genetic variants in complement cascade, chemokine and cytokine signaling pathways associated with CFS. Differences in these associations found for subjects with exclusionary conditions otherwise meeting criteria for CFS (CFS-exclusions) suggests important differences between these groups. Further replication and functional studies are needed to support the results of this study. Presenting author: Mangalathu S. Rajeevan PhD, Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, NCEZID, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-41, Atlanta, GA 30333, USA mor4@cdc.gov The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency Gene Expression Of Sensory Ion Channels, Adrenegic Receptors and Cytokines: Potential Biomarkers for CFS and Fibromyalgia Lucinda Bateman, M.D. A.R. Light, A.T. White, R.W. Hughen, T.A. VanHaitsma, and K.C. Light. University of Utah and Fatigue Consultation Clinic, Salt Lake City UT Objectives: To determine whether baseline and/or post-exercise expression of genes involved in signaling and modulating sensory fatigue and muscle pain are potential biomarkers for distinguishing patients with Chronic Fatigue Syndrome (CFS) and Fibromyalgia Syndrome (FM) from healthy controls. Methods: Forty eight Patients with CFS-only or CFS with comorbid FM, 18 Patients with FM that did not meet criteria for CFS, and 49 healthy Controls underwent moderate exercise (25 min at 70% of age-predicted maximum heart rate on Air- Dyne). Blood samples were taken before and 0.5, 8, 24, and 48 hours after exercise. Leukocytes were immediately isolated in buffer, number coded for blind processing, and flash frozen. Using real-time, quantitative PCR, the amount of mRNA for 13 genes (relative to control gene) involved in sensory ion channel, adrenergic, and immune functions was compared between groups at baseline and following exercise. Visual-analogue measures of fatigue and pain were taken before, during, and after exercise, including concurrently with all blood samples. Changes in amounts of mRNA were correlated with these measures, with history of orthostatic intolerance and with blinded ratings of disorder severity by the treating physician derived from multiple clinics. Results: No gene expression changes occurred following exercise in Controls except for inconsistent increases in β-1 adrenergic receptor. In 71% of CFS patients, moderate exercise increased most sensory ion channels and adrenergic receptors and one cytokine gene for 48 hours. These post-exercise increases correlated with numerical ratings of fatigue and pain, and greater increases were shown by patients with higher physician ratings of disorder severity. In contrast, for the other 29% of CFS patients, adrenergic α-2A receptor expression was decreased at all time points after exercise; other genes were not altered. History of orthostatic intolerance was significantly more common in the α-2A decrease subgroup. FM only patients showed no post-exercise alterations in gene expression, but their pre-exercise baseline mRNA for two sensory ion channels and one cytokine were significantly higher than Controls. Conclusions: At least two subgroups of CFS patients can be identified by gene expression changes following exercise. The larger subgroup showed increases in mRNA for sensory ion channels and adrenergic receptors and a cytokine. Both self

rated and physician-rated symptom severity was associated with greater post-exercise increases in these genes. The smaller subgroup contained most of the CFS patients with orthostatic intolerance, showed no post-exercise increases in any gene, and was defined by decreases in mRNA for α-2A adrenergic receptor. FM only patients can be identified by baseline increases in 3 genes. Post-exercise increases for 4 genes meet published criteria as an objective biomarker for CFS, and could be useful in guiding treatment selection for different subgroups. Presenting author: Lucinda Bateman, M.D., Adj. Assist. Professor, Depts. of Anesthesiology, Fam and Prev. Medicine, Internal Medicine, University of Utah; and Director, Fatigue Consultation Clinic, 1002 East South Temple, Suite 408, Salt Lake City UT 84102 USA. Email: fcclinic@xmission.com Gene-Exposure Interactions In The Etiology Of Gulf War Illness: Evidence Of Increased Vulnerability to Neurotoxicants in Identifiable Veteran Subgroups Lea Steele, Ph.D. Objectives: Epidemiologic and clinical studies have implicated wartime exposures to neurotoxicants (pyridostigmine anti-nerve gas pills, pesticides, and low-level nerve agents) as risk factors for Gulf War illness (GWI), but it is unclear why some troops developed GWI after the 1991 Gulf War (GW) while others, with similar experiences and exposures, remained healthy. This study investigated whether genotype or activity of paraoxonase (PON1), a circulating enzyme whose isoforms differentially hydrolyze pesticides and nerve agents, is associated with variable risk for GWI in veterans of the 1991 Gulf War. Methods: Case-control study of a population-based sample of 91 veterans who served in the Army as enlisted personnel during the 1991 Gulf War: 49 Gulf War veterans with GWI, 19 GW veteran controls, and 23 nondeployed veteran controls. Veterans provided information on wartime experiences and exposures, and blood samples for determining PON1 genotype at position 192, and PON1 activity in three substrates: paraoxon, phenyl acetate, and diazoxon. In addition to general case-control comparisons, exploratory analyses evaluated interactions between genotype and exposures in the risk for GWI. Results: Overall, a somewhat higher proportion of GW cases (22%) than GW controls (6%) were PON1192 RR homozygotes (p=0.09). PON1 activity in paraoxon was significantly lower in GW controls than in GWI cases (p=0.04) and nondeployed controls (p=0.05), with no significant differences noted in other substrates. In exploratory evaluation of GWI risk in PON1192 genetic subgroups, QQ homozygotes were at significantly increased risk for GWI if they reported wearing pesticide-treated uniforms (OR = 21.0, exact p<0.01) or prolonged use of pyridostigmine bromide (OR=11.2, exact p<0.01), although these exposures were not associated with GWI in veterans with QR and RR genotypes. Among veterans with QR and RR genotypes, the only significant GWI risk factor was hearing chemical alarms in theater (OR=7.6, exact p=0.04), while hearing alarms was not a risk factor for QQ homozygotes (OR= 0.75, exact p=0.34). Conclusions: Findings support earlier indicators that GWI may be associated with PON1 genotype and activity levels, with GW PON1192RR homozygotes at somewhat increased risk for GWI overall. Detailed investigations from our small sample provided significant results in support of the following hypotheses: 1) GW veterans whose PON1 genotype (QQ) is known to provide slower hydrolysis of some organophosphate pesticides were at increased risk for GWI in relation to reported use of pesticides and prolonged use of pyridostigmine bromide during deployment, and 2) GW veterans who carry the R allele at PON1192, which is known to provide inefficient hydrolysis of sarin, were at increased GWI risk if they heard chemical alarms in theater. These preliminary findings identify significant geneexposure interactions in the directions expected for substrates preferentially hydrolyzed by PON1192 Q and R isozymes, and warrant further evaluation in a larger sample. Lea Steele, Ph.D., Research Professor of Biomedical Studies, Baylor University, Director, Baylor Complex Illness Research Initiative, One Bear Place, Box # 97224, Waco, TX 76798, Tel. 254-710-2635, Email: Lea_Steele@baylor.edu Comparing Gene Expression Patterns in CFS and GWI Using the Kerr ME/CFS Platform Lina Garcia, M.D. Jonathan Kerr, MD^, Mary Ann Fletcher, PhD*, Connie Sol, MA*, and Nancy Klimas, MD** *University of Mimi Miller School of Medicine, , ^St George's University of London, United Kingdom,**corresponding author nklimas@miami.edu, University of Miami Miller School of Medicine and Miami VA Medical Center

There have been a number of studies utilizing genomics to better understand and define CFS/ME. Jonathan Kerr’s group published a series of studies that defined 79 genes associated with this illness, then used the same method to develop biologically defined subgroups (1). The Miami group has been studying both GWI and CFS/ME using gene activation patterns and proteomics, before during and after an exercise challenge to better understand the mediators of persistence and relapse. In this study we collaborated with Dr Kerr, comparing CFS/ME (n=25), control (n=53) and GWI samples (n=25), the GWI samples studied were drawn prior to the exercise challenge. The data from the CFS/ME cohort confirmed the findings from Dr. Kerr’s earlier studies. There were significant differences when compared to controls in expression of genes that regulate intracellular pathways mitochondrial function, cell wall and signaling pathways. Genes which regulate cytokine regulation were also significantly different than controls, particularly the pro-inflammatory cytokines TNFa and IL6; antiviral pathways Interferon alpha, beta and omega, and the anti-inflammatory cytokine IL10. When compared to Gulf War Illness there are some important overlaps: EB12, an EBV induction gene is 6 fold higher than controls in CFS/ME, 2 fold higher in GWI, both significant differences (p<.005) ETS1, a viral oncogene was also upregulated in both groups. (p<.-0005) Transcription factor 3, which regulates immunoglobulin production, was markedly elevated in GWI, less so though significantly elevated in CFS/ME. (p<.005) Apoptosis genes were markedly upregulated in both groups though GWI saw elevations were 400 fold higher than CFS/ME. (p<.0005). However, the overall trend was that most of the gene regulation abnormalities that are associated with CFS in the Kerr platform were not significantly different in GWI than in controls , and often moved in the opposite direction down regulating intracellular processes in GWI that were upregulated in CFS (73 of 87 genes studied). Using a comprehensive platform, additional genes specific for GWI have been identified by the Miami group (presented separately). 1. Kerr JR, et al. 2008. Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis. J Infect Dis 197(8):1171-84. Session: ADVANCES IN BRAIN AND NEUROENDOCRINE FUNCTIONING Chair: Andrew H. Miller, M.D. Regional Grey and White Matter Volumetric Changes in Chronic Fatigue Syndrome (Myalgic Encephalomyelitis): A Voxel-Based Morphometry 3T MRI Study I. H. Treasaden, M.B., B.S., LRCP, MRCS, FRCPsych, LLM BK Puri 1 Department of Imaging, Hammersmith Hospital, London, UK, 2 Three Bridges Unit, WLMHT, Middlesex, UK, 3 Department of Physical Education and Sport Sciences, University of Limerick, Republic of Ireland Objectives: It is not established whether or not myalgic encephalomyelitis/chronic fatigue syndrome (CFS) is associated with structural brain changes. The aim of this study was to investigate this by conducting the largest voxel-based morphometry study to date in CFS. Methods: High-resolution structural 3-T cerebral MRI scanning was carried out in 26 CFS patients and 26 age- and gendermatched healthy volunteers. Voxel-wise generalized linear modeling was applied to the processed MR data using permutation-based non-parametric testing, forming clusters at t > 2.3 and testing clusters for significance at p < 0.05, corrected for multiple comparisons across space. Results: Significant voxels (p < 0.05, corrected for multiple comparisons), depicting reduced grey matter volume in the CFS group, were noted in the occipital lobes (right and left occipital poles; left lateral occipital cortex, superior division; and left supracalcrine cortex); the right angular gyrus; and the posterior division of the left parahippocampal gyrus. Significant voxels (p < 0.05, corrected for multiple comparisons), depicting reduced white matter volume in the CFS group, were also noted in the left occipital lobe. Conclusion: These data support the hypothesis that significant neuroanatomical changes occur in CFS, and are consistent with the complaint of impaired memory that is common in this illness; they also suggest that subtle abnormalities in visual processing, and discrepancies between intended actions and consequent movements, may occur in CFS.

Dr. Ian H. Treasaden, M.B., B.S., MRCS, LRCP, FRCPsych, LL.M., Consultant Psychiatrist and Honorary Clinical Senior Lecturer, West London MHT and Imperial College London; Head of Forensic Neuroscience, Department of Imaging, Imperial College, UK. Three Bridges Unit, West London Mental Health NHS Trust, Uxbridge Road, Southall, Middlesex UB1 3EU, England, UK. E-mail: ian.treasaden@wlmht.nhs.uk Evidence For Reduced Aldosterone in Persons with Chronic Fatigue Syndrome Roumiana S. Boneva, M.D., Ph.D. James F. Jones, Elizabeth R. Unger Chronic Viral Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA. Objectives: Aldosterone, a mineralocorticoid hormone of the adrenal gland, controls the absorption of salt and water in the kidneys and the intestine and thus the overall blood volume. Mineralocorticoid receptors in the brain are involved in regulation of blood volume and sympathetic outflow regulation. A few studies have suggested lower blood volume and lower heart stroke volume in persons with chronic fatigue syndrome (CFS) and some persons with CFS experience postural hypotension. Because altered cortisol secretion has been found in a number of CFS studies, adrenal dysfunction has been suggested. Surprisingly, only one prior study examined aldosterone in CFS, finding lower aldosterone in CFS compared to controls. The current study was performed to explore the hypothesis that aldosterone levels in persons with CFS may differ from those in controls of similar age, sex, and race. Methods: Participants were identified from a population-based study including 70 CFS cases who met the international 1994 CFS case definition and 212 controls of similar age and race. A morning blood sample, collected after participants rested 30 minutes in supine position, was used to test for serum aldosterone. Testing was performed at Quest Diagnostics using liquid chromatography tandem mass spectrometry (analytical sensitivity 1 ng/dL). The Wilcoxon nonparametric test was used for comparison of non-normally distributed numeric variables (aldosterone). Chi square test and logistic regression were used to assess magnitude of associations; for these tests aldosterone was dichotomized at its median value 4 ng/dL in controls, which also equaled the lowest normal lab reference value. Results: Cases and controls did not differ significantly in mean age (47.8 and 47.7, respectively) or race distribution (78.3% and 80.7% Whites, respectively). The CFS group had a higher proportion of women (91.3% vs 67.5%) and a higher mean body mass index than the control group (BMI, 28.9 vs 26.9), p<0.005 for both. The CFS group had lower aldosterone levels compared to controls (mean 4.46, median 3, range 1 to 19 ng/dL vs mean 6.05, median 4, range 1to 76 ng/dL, respectively), p<0.0001 (Wilcoxon non-parametric test). Persons with CFS were 65% more likely to have aldosterone level of <4 ng/dL, OR=1.65 (95% CI, 0.95-2.85), p=0.07. The OR changed slightly after adjusting for BMI, OR=1.58 (0.93-2.74), p=0.11, but minimally after adjusting for sex, OR= 1.69 (95% CI, 0.96- 2.97), p=0.07. A limitation of the study is that aldosterone was measured only in supine position (less sensitive for identifying alterations in aldosterone secretion) and information on dietary salt intake was not available. Conclusions: These results support a previous study’s finding of relatively lower aldosterone levels in CFS subjects compared to controls. Further studies of aldosterone in CFS should measure its response to challenge such as salt restriction and changes in aldosterone levels from recumbent to upright position. Presenting author: Roumiana S. Boneva MD, PhD, Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, NCEZID, Centers for Disease Control and Prevention, 1600 Clifton Road, MS A15, Atlanta, GA 30333, USA rboneva@cdc.gov The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency Interaction of Self-And Illness-Related Cognitive Processing In The Right Anterior Insula of CFS Patients: An fMRI Study Andrew H. Miller, M.D. Jones JF1, Rajendra J2, Drake D2, Miller A2, Unger ER1, Tian H2, Pagnoni, G3. 1Chronic Viral Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta, GA; Emory 2University School of Medicine, Atlanta, GA; 3University of Modena and Reggio Emilia, Modena University, Modena, Italy Objectives: Based on the core clinical complaints of CFS patients several groups have suggested that CFS symptoms may be at least partially linked to altered cognitive or pre-cognitive processing in the central nervous system. Targeting the identification of the neural substrates of alterations of internal body signals and self-related information seems warranted. This study investigates brain responses to self- and illness-related semantic information in a sample of

CFS patients compared to matched control subjects, using functional magnetic resonance imaging (fMRI). We focus on the right anterior insula (rAIC), for its purported role in interoceptive processing and awareness (Craig 2002, 2009, Nat Rev Neurosci), and on the interaction of the self-related and illness-related semantic processing. Methods: Twenty-one subjects meeting the 1994 International criteria for CFS, and 42 non-fatigued (NF) subjects performed a semantic processing task while undergoing an fMRI scan. The stimuli consisted of visually presented short sentences requiring the participants to provide a “true” or “false” answer. The semantic content was arranged according to a 2 x 2 x 2 factorial design, where the three factors were (a) Self-related: yes/no, (b) Illness-related: yes/no, and (c) Valence: negative/positive. We assessed the effect of processing Self-related versus Non selfrelated semantic information, across the two groups of CFS and NF subjects in a single acquisition run of functional images by examining a set of regions of interest. Results: In both CFS and NF subjects: 1) the insular response to self-related sentences tended to decrease compared to response to non self-related sentences, in both CFS and NF subjects; 2) the insular response to non-self sentences did not differ with respect to illness-related material or not; 3) the insular response to self-related/illness-related sentences was greater than that for self-related/non illness-related only in CFS subjects. A qualitatively similar pattern was observed for the response time data. The statistical significance for the group difference in the interaction effect (3-way ANOVA: Group x Self-related x Illness-related) was p=0.0034 for the rAIC activation data and p=0.0022 for the reaction time data. Conclusions: Self is a multifaceted construct that relies in part on interoception (monitoring of internal physiology and consequences of external stimuli) via the anterior insular cortex through recognition of subjective feelings (Craig). The changes observed here in CFS subjects indicate responses to an increased mental load or to a cognitive conflict within the semantic dimensions of self and illness. This is evidence that (1) there is a real alteration of body physiology underlying the CFS symptoms, and the observed altered rAIC response reflects the (normal) cognitive and pre-cognitive acquisition of an abnormal physiological landscape in the body; or (2) the actual interoceptive landscape is acquired cognitively and pre-cognitively in an altered way in CFS subjects, enhancing the prominence of normal bodily signals related to fatigue. Corresponding Author: James F. Jones, MD, CVDB, CDC, 1600 Clifton Rd, MS-A15, Atlanta, GA, 30333, USA, jaj9@cdc.gov. Disclaimer: The findings and views in this report are those of the authors and do not necessarily reflect the views of the funding agency. Decreased Basal Ganglia Activation in CFS Subjects is Associated With Increased Fatigue Andrew H. Miller, M.D. Miller, A.H.1, Jones, J.F.2, Drake, D.F.1, Tian, H.2, Unger, E.R.2, Pagnoni, G.3 1Emory University School of Medicine, Atlanta, GA, 2Chronic Viral Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta, GA, 3University of Modena and Reggio Emilia, Modena University, Modena, Italy Objectives: Altered basal ganglia function has been associated with fatigue in a number of neurologic disorders, as well as in patients exposed to chronic immune stimulation. Patients with chronic fatigue syndrome (CFS) have been shown to exhibit symptoms suggestive of decreased basal ganglia function as reflected by psychomotor slowing on neurocognitive testing, which in turn correlated with fatigue. In addition, CFS patients have been found to have increased markers of immune activation. In order to directly test the hypothesis of decreased basal ganglia function in CFS, we conducted a functional magnetic resonance (fMRI) study on a sample of CFS patients and matched controls, using a reward-processing experimental protocol. Methods: A community-derived sample of 59 male and female subjects, including 18 patients diagnosed with CFS according to 1994 CDC criteria and 41 non-fatigued healthy controls, participated in the study. All subjects were free of psychotropic medications as well as significant depressive symptoms, as determined by a Zung Depression score <60. Groups were similar in age, sex, and race. While undergoing fMRI scanning, subjects performed a monetary gambling task previously shown to strongly activate basal ganglia in the win versus lose condition. To focus our analysis on the specific basal ganglia regions activated by the task, the following procedure was employed: (1) a whole-brain group analysis revealing the general activation pattern for the win-lose contrast across all subjects was performed; (2) the resultant statistical parametric brain map thresholded at p<0.05, corrected for multiple

was intersected with a set of basal ganglia regions of interest (ROIs: caudate nucleus, putamen, and globus pallidus), obtained from a probabilistic cytoarchitectonic brain atlas included in the SPM Anatomy Toolbox; (3) for each subject, the average value of win-lose activation contrast in each ROI was extracted for group comparisons and correlational analyses. Results: Compared to non-fatigued controls, patients with CFS exhibited significantly decreased activation in the right caudate (p=0.01) and right globus pallidus (p=0.02). Decreased activation in the right globus pallidus was significantly correlated with increased mental fatigue (r2=0.49, p=0.001), general fatigue (r2=0.34, p=0.01) and reduced activity (r2=0.29, p=0.02), as measured by the Multidimensional Fatigue Inventory. No such relationships were found in control subjects. Conclusions: These data suggest that reduced basal ganglia activation may contribute to symptoms of fatigue in CFS subjects. Given the central role of dopamine in basal ganglia regulation, these data also indicate that alterations in dopamine metabolism may be involved. Further understanding of potential alterations of dopamine transmission and metabolism in basal ganglia, due to activated immune pathways or other causes, may lead to new pharmacologic strategies targeting dopamine and the basal ganglia for the treatment of CFS symptoms. The findings and views in this report are those of the authors and do not necessarily reflect the views of the funding agency. Corresponding Author: Andrew H. Miller, M.D., Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 1365-B Clifton Rd., 5th Floor, Room B5101, Atlanta, GA, 30322, USA, amill02@emory.edu Assessment of Regional Cerebral Blood Flow in CFS Using Arterial Spin Labeling MRI Jonathan P. Dyke, Ph.D. Dyke JP1, Weiduschat N1, Mao X1, Pillemer S2, Murrough JW2, Natelson B3, Mathew SJ2,4, Shungu DC1 1Dept of Radiology Weill Cornell Medical College, NY; 2Dept of Psychiatry, Mount Sinai School of Medicine, NY; 3Beth Israel Medical Center, NY; 4Dept of Psychiatry, Baylor College of Medicine, TX Objectives: Chronic Fatigue Syndrome (CFS) is an unexplained illness characterized by debilitating fatigue that is not ameliorated by sleep[1,2]. In two previous independent samples of CFS, we found increased ventricular lactate, which we had postulated to be due to oxidative stress, a secondary mitochondrial dysfunction and/or decreased regional cerebral blood flow (rCBF). To investigate the latter possibility, we used arterial spin labeling MRI[3] to compare rCBF in patients with CFS, and in age-and sex-matched patients with major depressive disorder (MDD) and healthy volunteers (HV). Methods: Fourteen CFS [31.9±8.6 yrs, 3M/11F], 13 MDD [31.4±9.9 yrs, 5M/8F] and 13 HV [27.6±7.4 yrs, 6M/7F] subjects were recruited for this study. The two patient groups were psychotropic medication-free for at least 1 week prior to scanning. rCBF was measured in each participant using ASL on a 3.0T GE MRI system. The resulting rCBF images were reconstructed on-line and normalized to the Montreal Neurological Institute (MNI) PET template. Groupwise voxel-based analysis of the ASL data was performed using SPM version 5, followed by between-group ANCOVA comparisons in which age and gender were covariates. Results: Significantly decreased rCBF values were found in the left anterior cingulate cortex (ACC) [p=0.039] and the right lingual region [p=0.016] in CFS compared to HV, while a trend toward significantly lower rCBF was found in the left ACC region in MDD subjects compared to HV [p = 0.08]. rCBF values for CFS and MDD did not differ significantly [p>0.05].

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