Research Advances in Chronic Fatigue Syndrome

1. Research Advances in Chronic Fatigue Syndrome Nancy Klimas, MD University of Miami CFS and GWI Research Center Miami VA Medical Center

2. Definition - CFS • >6 mo. debilitating fatigue, unexplained by preexisting illness or psychiatric co morbidity, and at least 4 of eight symptom criteria: • post exertional relapse • concentration and cognitive complaints • myalgia • arthralgia • sore throat • painful lymph nodes • new headaches • unrefreshing sleep

3. CFS/ME Clinical Case Definition • 1. Substantial reduction in activity level due to new onset, persistent fatigue • 2. Post exertional malaise • 3. Sleep dysfunction • 4. Pain – myalgia, headaches • 5. Neurologic/Cognitive Manifestations • 6. At least one symptom from 2 of the following: - Autonomic manifestations eg. OI, IBS - Neuroendocrine manifestations eg. temperature intolerance, weight change - Immune manifestations eg. tender lymph nodes, sore throat, flu-like symptoms • Link to full report www.iacfs.net

4. Subpopulations CFS “Caseness” CFS Autonomic Immune HPA

5. CFS “Caseness” • Subpopulations HPA Autonomic Immune

6. Other overlapping conditions • Fibromyalgia • Gulf War Illness (VA CSP 16 fold increased risk of CFS in Gulf War veterans) Eisen et al Ann Int Med 2005 7;142(11):881 • Multiple Chemical Sensitivity

7. Epidemiology: DePaul University • Latinos: 726 per 100,000 • African Americans: 337 per 100,000 • Caucasians: 224 per 100,000 • Women: 522 per 100,000 • Men: 291 per 100,000 CDC Wichita study: 85% undiagnosed, 50% reduction in household income – 9 billion/yr US loss productivity Jason et al Psychosom Med. 2000 Sep-Oct;62(5):655-63 Reeves et al Biomed Central 2005

8. Attitudes of Physicians • Of 811 GP’s 44% did not feel confident making the diagnosis, 41% did not feel confident treating • More likely to have confidence if they had a friend or family member with CFS, having more patients with CFS. • Concludes that education emphasizing acceptance of CFS as a real entity results in improved confidence in treatment Bowen J et al Family Pract 2005 April 1

9. Model of CFS Pathogenesis Genetic Predisposition Triggering event / infection Mediators (Immune, endocrine, neuroendocrine, psychosocial) Health Outcome/Persistence

10. Genetic Predisposition - CFS • HLA DR haplotypes in 112 South Florida CFS patients, compared to 5,000 regional and national controls • 4 to 6 fold increased relative risk for DR4, DR3 and DQ3. (Keller et al, 1992) • Seattle CFS Cooperative Research Center Twin study - genetic predisposition, hereditability estimate of 51% (2nd World Conf)

11. Evidence for Triggering event/ infection - CFS • 60 to 80% of CFS subjects date the onset of their illness to an acute viral-like illness (Komaroff, Buchwald) Less so in population based studies. (Reeves, Jason) • Andrew Lloyd and colleagues in Australia performed a prospective study during and after acute EBV, Q fever or Ross River Virus -Anergy during acute infection predicted persistent CFS like symptoms • Peter White described post EBV illness in a prospective trial

12. Model of CFS Pathogenesis Genetic Predisposition Triggering event / infection Mediators (Immune, endocrine, neuroendocrine, psychosocial) Health Outcome/Persistence

13. CRF • CNS Symptoms • Altered perceptions • fatigue • pain • Cognitive changes • concentration • memory • Mood alterations • depression • anxiety • Sleep disturbances • unrefreshing sleep • altered sleep-wake cycle Physical stress activates immune system and HPA axis Emotional stress activates immune system and HPA axis • Hypothalamic-Pituitary-Adrenal Axis • Relative Hypocortisolemia • Musculoskeletal System • Myalgia & Arthralgia • Gastrointestinal Tract • Altered bowel habits • Abdominal pain • Heart and Blood Vessels • Altered blood pressure • responses • Dizziness • Immune System • Lymph node tenderness • Sore throat • Enhanced Cytokines

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15. Immune Activation DR, CD26 expression TH2 cytokine shift Proinflammatory cytokines expression TNF-a, IL-1, IL6 Functional defects NK Cell dysfunction CD8 abnormalities perforins, granzymes Macrophage abnormalities Antibody production Immune abnormalities in CFS

16. Immunology What’s New? • Exercise induced complement activation1 • NK phenotypes predict risk 2 • Lower mRNA and TGF-beta1 production 3 • Increased neutrophil apoptosis 4 • Autoantibodies - anti-microtubule-associated protein 2, sDNA5 • CTL defect equals that of NK cells 1 Sorensen, Jones et al J Allergy Clin Immun 2003 112(2):397-403 2 Stewart et al Cytometry 2003 53(1)2633 3 Tomoda A et al Psychiatry Res 2005 134(1):101 4 Kennedy et al J Clin Parhol 2004 57(8) 891 5 Vernon and Reeves, J Autoimmune Dis 2005 25:2:5 6 Maher Fletcher and Klimas 2006

17. Viral Persistence/Reactivation HHV6 virus is present in 22 to 54% of patients in cross sectional studies (Ablashi, Krueger, Knox), HHV6 virus is present in 79% of CFS patients in longitudinal studies (HHV6 PCR assay, Knox) HHV6 virus is present in the spinal fluid of 28 of 120 CFS patients (Peterson), and 7 of 35 CFS samples (Knox). Enterovirus is present in 13% of CFS muscle samples (Douche-Aourik, 2003) EBV – dUTPase as a immune modulator, up regulating inflammatory cytokines (Glaser, 2005) (Glaser et al Brain Behavior and Immunity 2005 19(2):91-103)

18. HPA Axis dysregulation • Demitrack low basal cortisols in CFS subjects, hypothalamic dysfunction- • Dinan and colleagues - evidence of deficiency of hypothalamus, pituitary, and adrenal hypofunction. • Small adrenal gland in depressed and non depressed CFS subjects, enlarged adrenal in depressed control group. • Bennett et al studied 500 FM patients with basal IGF-I levels which were significantly lower than controls.

19. Endocrinology Reduced Cortisol output via several mechanisms A) heightened negative feedback B) heightened receptor function C) impaired ACTH and cortisol responses to challenge DHEA functional abnormality (early data) Abnormal seritonin function IL-6 increase associates with low cortisol CRH mediated Many confounding factors (deconditioning, sleep, comorbid depression, stress, medication) Cleare AJ Endocr Rev 2003 24(2):236-52 Papanicolau Neuroimmunomodulation 2004 11(2)65-74 Maes M Neuro Endocrinol Lett 2005 Oct 30;26(5)

20. Renin Aldosterone Axis • High Prevalence of Renin-Aldosterone Axis Abnormalities in Patients with Chronic Fatigue Syndrome (CFS) • 30 of the 33 patients had at least one value of supine or upright PRA or supine or upright serum AL outside of the 95% Tolerance Interval (TI) for normal volunteers • 16 patients had low serum AL and low or normal PRA, consistent with Hyporeninemic Hypoaldosteronism (HH) • The underlying defect may be autonomic nervous system dysfunction and/or a primary adrenal defect. Zuckerbraun, E, Kim, HS, Daigle, K, Lee, ML, Friedman, TC, Charles R. Drew University

21. Autonomic Dysfunction • Neurally mediated hypotension (Rowe) • Orthostatic hypotension (Streeten) • Parasympathetic dysfunction(Sisto) • Sympathetic over activation (Pagini, De Becker) • Study in adolescents mirrored that of adults

22. Balancing Act sympathetic parasympathetic

23. Autonomic Nervous System • Haemodynamic Instability Score taken during tilt table testing predicts CFS with 90% sensitivity. 1 • Heart Rate variability as a predictor of CFS 2 • Gastric emptying delayed in 23/32 CFS subjects 3 • 1 Naschitz QJ Med 2003 96(133-142) • 2 YamamotoExp Biol Med 2003 228(2):167-74 • 3 Burnet BMC Gastrenterol 2004 (1):32

24. Autonomic Dysfunction • Drops in BP followed by CFS relapse • Exhaustive treadmill testing results in cognitive function decline (LaManca et al) • Perfusion abnormalities of brain stem, cerebellum (Costa et al) • Mid cerebral reduced perfusion (Schwartz et al)

25. Neuropeptides • Seritonin and its precursers: abnormalities of CSF tryptophan levels Badawy et al J Psychopharm 2005 19(4):385 • PET scan – 5HT1A receptor binding reduced in CFS, particularly in the hippocampus • Cleare AJ et al Biol Psychiatry 2005 1;57(3):239-46 • Reduction of seritonin transporters (5HTTs) most pronounced in the anterior cingulate by PET scan. Yamamoto S et al Neuroreport 2004 15(17):2571

26. Neuroimaging • Reductions in global grey matter associated with reductions in physical activity (28 subjects, 28 matched controls).deLange et al Neuroimage 200526(3):777 • Utilization of more extensive regions of the brain to process tasks using fMRI and mPASAT. Lange G et al Neuroimage 2005 26(2):513

27. Sleep Physiology • Circadian Sleep - Wake neuroendocrine and immune functions in CFS (Modolfsky) • altered diurnal patterns in cortisol, prolactin • altered diurnal patterns of NK cell function • alpha wave intrusion on sleep EEG , reduced stage III and IV • Higher %REM (Twin study, 22 discordant twins)1 1 Watsonet al Sleep 2003 26(3):32-8

28. Muscle • Oxidative stress study, measuring protein carbonyls suggested higher levels of protein oxidation than controls 1 • Exercise testing in 189 CFS subjects resulted in clinically significant subgroups 50% showing moderate to severe functional impairment. Unexpected blunted HR and BP responses noted. 2 • Sarcoplasmic reticulum defect – conduction and calcium transport abnormalities3 1 Smirnova et al Mol Chem Biochem 2003 248(1-2):93-5 2Vaness Med Sci Sports Exerc 2003 35(6):908-13 3Fulle et al NeuromusculDisord 2003 13(6):479-84

29. Muscle • Cardiac muscle – cardiac output related to severity,and predicted exercise induced relapse1 • Subset of CFS patients with IgM EBV or CMV Ab at risk for cardiac motility abnormalities and occassionally true cardiomyopathy 2 • Raises the issue of incomplete viral replication, activating immune responses as suggested by Glaser et al 3 1 Peckerman et al AJ Med Sci 2003 326(2)55 2 Lerner M et al In Vivo 2004(18) 4:417 3 Glaser R Brain Behavior Immun 2005 19(2):91

30. Exercise – new studies • No association between pain related fear of movement and exercise capacity and disability Nijs J et al Phys Ther 2004 84(8):696 • Exercise induced pain thresholds increase in controls and decrease in CFS subjects Whiteside A et al Pain 109(3):497

31. Exercise – new studies • A subset of fit healthy controls deprived of exercise develop symptoms similar to CFS/FM. The subset is predicted by baseline abnormalities of autonomic, immune and HPA axis abnormalities.1 • Patients with Rnase L abnormalities have abnormal exercise physiology which is mediated in part by immune dysfunction 2 • Response to exercise shows accentuated oxidative stress, and marked alterations of muscle membrane excitablility, sufficient to explain muscle pain and post exertional malaise. 3 1Glass JM et al J Psychosom res 2004 57(4):391 2 Snell CR In Vivo 2005 19(2)387, Nijs J et al Med Sci Sports Exerc 2005 Oct 37(10):1647 3 Jammes Y et al J Intern Med 2005 257(3):229

32. Model of CFS Pathogenesis Genetic Predisposition Triggering event / infection Mediators (Immune, endocrine, neuroendocrine, psychosocial) Health Outcome/Persistence

33. Molecular Epidemiology Laboratory Strategy DNA Protein Abundance Protein Function mRNA Genomics Proteomics Activity-based Protein Profiling

34. Microarray Production Sample Preparation Plasma DNA clones oligonucleotides Blood PBMCs PBMCs Lymphoprep Erythrocytes + Granulocytes PCR amplification Purification Total RNA Robotic printing Labelled cDNA Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarray Technology

35. Gradual onset Sudden onset • 23 women with CFS from Wichita • Measure expression of 3,800 genes • Question: • Could gene expression profiles and differentially expressed genes distinguish subtypes of CFS? Of the 3,800 genes, 117 were significantly • Different between gradual vs sudden onset

36. Differentially Expressed Genes in Numerous Pathways

37. Genetic studies • Differential expression of 35 genes of 9522 tested. T Cell activation, neuronal and mitochondrial regulatory abnormalities Kaushik J Clin pathol 2005 58(8):826 • Abnormalities of Immune response genes in post-infection fatigue suggest genetic variations in susceptibility to persistent fatigue. Helbig QJM 2005 98(8):565 • Pre-post exercise challenge gene studies saw differences in genes that regulate ion transport, intracellular cell functions. Challenge studies such as these may be more useful than single cross sectional studies. Whistler et al BMC Physiol 2005 24;5(1):5

38. CFS is a Complex Illness • Illness represents alterations in complex systems of homeostasis • Not a result of a single mutation or single environmental factor • Arise from a combined action of many genes, environmental factors and risk-conferring behavior

39. Treatment • Pathogenesis based approaches

40. Pathogenesis Directed Interventions • Immune - Ampligen, future immunomodulators • HPA axis interventions - Growth hormone, cortisol • NMH treatments (plasma expansion, sympathetic and parasympathetic stimulants/inhibitors); • Sleep - pharmacologic and nonpharmacologic

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42. Immune modulatory approaches • Ampligen, a immune modulator and antiviral (Phase 3 recently completed) • Allergy immunotherapy to down regulate allergic drive • Future immunomodulators (trials underway): Isoprinosine, thalidomide, anti-TNFa monoclonal Ab • Proof of concept: Autologous lymphocyte study

43. Treating HHV6a?Association vs. causation • Blood PCR HHV6 a did not predict HHV6 virus is present in the spinal fluid • CSF did not predict blood • Of 120 CSF samples, 44 had abnormalities of protein, glucose or cells. Of the 44 , 28 were positive for HHV6(26), EBV (1), or CMV(1). • 5 of 8 CSF PCR positive treated until CSF cleared returned to full time employment (Peterson); in his experience TK inhibitors did not clear CSF, patients required foscarnet or cidofovir • Open label valgancyclovir 9 of 12 responders in high titer EBV plus HHV6 selected cohort , (Jose Montoya of Stanford) • Placebo control trials have not been completed

44. Autonomic Dysfunction Neurally mediated hypotension (Rowe) Orthostatic hypotension (Streeten) Parasympathetic dysfunction (Sisto) Sympathetic over activation (Pagini, De Becker) Balancing Act sympathetic parasympathetic

45. Implications for treatment - NMH • “Pipes and a pump”, wired by the autonomic nervous system • Fill the space - fluid vs. cells • compress the space - alpha 1 agonists (e.g. midodrine), • anti-phlebitic stockings • regulate the pump - beta blockers

46. HPA axis interventions - • Growth hormone – phase 1 (Antwerp study) • Cortisol – conflicting phase 2 study results (London, NIH) • Restoration of sleep cycle (circadian rhythm)

47. Sleep • Re-establish circadian rhythm Conditioned response to bed - avoid bed for resting, reading, use bed for sleeping. Establish “bedtime”. • Avoid short acting hypnotics (alpha trappers) • tricyclics, doxepan are longer acting, and don’t trap in alpha wave • mirtazapine (Remeron), sodium oxybate or gamma hydroxybutyrate, (Xyrem) act as stage 4 inducers

48. Pain and Sleep • pregabulin (Lyrica) 529 subjects, placebo control trial in fibromyalgia • Significant improvement in pain, sleep and fatigue (48% improved vs 27% placebo), 450 mg dose had greater reduction in pain scores.

49. Nutritional interventions • Oxidative stress studies suggest interventions such as glutathione, N-acytylcysteine, alpha lipoic acid, NADH • Vitamin studies suggest B vitamins, Vitamin C, magnesium, sodium, zinc, l-tryptophan, L carnitine, co-Q10, and essential fatty acids • highlighted interventions have phase 2 studies published

50. Nutritional interventions • Dangers: • Licorice root – potassium deficiencies • “supplements” that are actually hormones • “supplements” that have iffy contents – eg. St John’s wort, melatonin • Products that make unsubstantiated claims • Under and over hydration