1. Miami UNIVERSITY OF SCHOOL OF MEDICINE Immunology and Infection in Chronic Fatigue Syndrome Nancy Klimas, MD

2. Model of CFS Pathogenesis Genetic Predisposition Triggering event / infection Mediators (Immune, endocrine, neuroendocrine, psychosocial, viral reactivation or persistence) CFS/ME

3. Genetic Predisposition - CFS HLA DR haplotypes in 112 South Florida CFS patients, compared to 5,000 regional and national controls 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) 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); similar results in Sweden, Australian studies Seattle CFS Cooperative Research Center Twin study - genetic predisposition, hereditability estimate of 51% (2nd World Conf); similar results in Sweden, Australian studies

4. Evidence for Triggering event/ infection - CFS 60 to 80% of CFS subjects onset an acute viral- like illness (Komaroff, Buchwald) Less so in population based studies. (Reeves, Jason) 60 to 80% of CFS subjects onset 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 - anergy during acute infection predicted persistent CFS like symptoms Andrew Lloyd and colleagues in Australia performed a prospective study - anergy during acute infection predicted persistent CFS like symptoms Severity of initial infection single best predictor Severity of initial infection single best predictor

5. Model of CFS Pathogenesis Genetic Predisposition Triggering event / infection Mediators (Immune, endocrine, neuroendocrine, psychosocial, viral reactivation or persistence) CFS/ME

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

10. Video link: The leukocyte. http://www.studiodaily.com/main/searchlist/6850.html http://multimedia.mcb.harvard.edu/media.html

11. .

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

13. Photo by Leventhal, Karnovsky and Martz

14. 0 5 10 15 20 25 30 35 40 Subjects GWI CFS Controls NK Cytotoxicity: % K562 Cells Killed at Target to Effector Cell Ratio of 1:1

15. Intracellular Cytolytic Granules: * Perforin * Granzyme A * Granzyme B Cell Surface Antigen: CD56 Perforin is a molecule in cytotoxic lymphocytes necessary for killing of virus infected and tumor cells. Natural Killer Cell

16. Natural killer (NK) cell cytolytic capacity was measured by quantitative flow cytometry of intracellular content of perforin, with data expressed as relative number of molecules of perforin per CD3- CD56+ lymphocyte (rMolPer/NK cell).

17. 0 1000 2000 3000 4000 5000 6000 7000 Subjects GWI CFS Controls Intracellular Perforin

20. CD26 (dipeptidyl peptidase IV) is involved in the activation of T cells, and is expressed on antigen-reactive memory T cells. As reported by the Miami CFS research group, the percentage and number of CD26+ lymphocytes is elevated in CFS.

21. 0 10 20 30 40 50 60 70 Subjects GWI CFS Controls Fig. 3. Lymphocyte Activation in GWI and CFS: Percent of CD2+CD26+ Lymphocytes Lymphocyte Activation in GWI and CFS: Percent of CD2+CD26+ Lymphocytes

22. Qualitative flow cytometry showed fewer numbers of molecules of DPPIV/CD26 on T and NK cells in CFS patients.

23. NPY is stored in sympathetic nerve terminals and is released along with catecholamines during stress- induced activation. Only a few peptidases are capable of cleaving NPY due to its unique 3-diminsional structure. DPPIV/CD26 is one such peptidase. Neuropeptide-Y (NPY) is peptide,which participates in the regulation of a large number of physiological and pathophysiological processes in the cardiorespiratory system, immune system, nervous system and endocrine system.

24. BIOMARKERGWICFSHC NPY (pmol/L) 37*4152 *Significantly different from HC (p<.05) In the GWI patients, we found a reduced amount of NPY in plasma.

25. ....

26. ...

27. 55 41 28 14 0 Group Means (+/- SD) Paced Auditory Serial Addition Task (PASAT) Women with CFS The PASAT is an objective measure of: Low NKCCNormal NKCC n = 22 n = 19 p = <.001 rate of information processing, sustained attention and divided attention

28. Immune –Endocrine Link IL-6 increase associates with low cortisol, CRH mediated Papanicolau Neuroimmunomodulation 2004 11(2)65-74 Maes M Neuro Endocrinol Lett 2005 Oct 30;26(5)

29. 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) (Glaser et al Brain Behavior and Immunity 2005 19(2):91-103)

30. Viral and Immune Interactions and Health J Chia showed GI biopsies with enterovirus inclusions, found in patients with CFS and abdominal complaints J Chia showed GI biopsies with enterovirus inclusions, found in patients with CFS and abdominal complaints

31. Novel mechanisms of virus mediated chronicity Glaser et al found evidence of regulatory peptides encoded by EBV expressed in CFS despite the absence of replicative virus Glaser et al found evidence of regulatory peptides encoded by EBV expressed in CFS despite the absence of replicative virus These peptides are known to modulate immune function, inducing pro-inflammatory and Type 2 cytokines These peptides are known to modulate immune function, inducing pro-inflammatory and Type 2 cytokines Lerner’s group found evidence of a subgroup of CFS patients with incomplete viral expression and cardiac motility abnormalities; subset of CFS with IgM EBV Lerner’s group found evidence of a subgroup of CFS patients with incomplete viral expression and cardiac motility abnormalities; subset of CFS with IgM EBV 2 Lerner M et al In Vivo 2004(18) 4:417; (18)2:101 3 Glaser R Brain Behavior Immun 2005 19(2):91

32. Treating HHV6a? Association vs. causation Blood PCR HHV6 a did not predict HHV6 virus is present in the spinal fluid Blood PCR HHV6 a did not predict HHV6 virus is present in the spinal fluid CSF did not predict blood 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). 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 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 20 of 23 responders in high titer EBV plus HHV6 selected cohort, (Jose Montoya) Open label valgancyclovir 20 of 23 responders in high titer EBV plus HHV6 selected cohort, (Jose Montoya) Placebo control trials have not been completed Placebo control trials have not been completed

33. Clinical Trials M Lerner reported a phase 1 trial of valgangciclovir in CFS patients with evidence of cardiac dysfunction. 37 patients were treated in an open label study, with improvement in all 37. He emphasized the need to hydrate, monitor renal and liver function. M Lerner reported a phase 1 trial of valgangciclovir in CFS patients with evidence of cardiac dysfunction. 37 patients were treated in an open label study, with improvement in all 37. He emphasized the need to hydrate, monitor renal and liver function.

34. Genomics, Proteomics, and Viral Chips IACFS Conf 2007 : IACFS Conf 2007 : Inflammation pathways, IL-6, TNFa upregulated in a subgroup, suggesting monoconal ab blockers as a treatment (Kerr, Vernon, Olano) Inflammation pathways, IL-6, TNFa upregulated in a subgroup, suggesting monoconal ab blockers as a treatment (Kerr, Vernon, Olano) CFS proteome (Baruniuk) CFS proteome (Baruniuk) Serum analysis using infrared spectroscopy (Sakudo, Watanabe, Ikuta, Kuratsune) Serum analysis using infrared spectroscopy (Sakudo, Watanabe, Ikuta, Kuratsune) 28 potential microbes under study (Kerr) 28 potential microbes under study (Kerr) Reno research group using viral chip technology in CFS and CFS associated malignancy Reno research group using viral chip technology in CFS and CFS associated malignancy

35. Conclusion Immune dysfunction in CFS contributes to the overall symptom complex, and contributes to the persistence of the illness, both directly and through interaction with neuropeptides and hormones. Immune dysfunction in CFS contributes to the overall symptom complex, and contributes to the persistence of the illness, both directly and through interaction with neuropeptides and hormones. There is increasing evidence of viral reactivation in at least a subset of the patient population. Interventive trials are currently focused on HHV6 virus. There is increasing evidence of viral reactivation in at least a subset of the patient population. Interventive trials are currently focused on HHV6 virus. This work is helping to identify subgroups of CFS patients, identifying biomarkers, and potential treatment options. This work is helping to identify subgroups of CFS patients, identifying biomarkers, and potential treatment options.

36. Thank You! Immunology/Virology: Drs. Mary Ann Fletcher, Kevin Maher, Roberto Patarca Autonomic: Dr. Barry Hurwitz Health Assessment: Drs. Michael Antoni, Mary Catherine Segota, Jackie Junco Professional links: IACFS/ME on line: www.iacfs.net www.iacfs.net CDC on line: www.cdc.gov www.cdc.gov NIH on line: www.nih.gov www.nih.gov

37. Photo by Leventhal, Karnovsky and Martz