MiamiUNIVERSITY OF

SCHOOL OF MEDICINE

Immunology and Infection in Chronic Fatigue Syndrome

Nancy Klimas, MD

Model of CFS Model of CFS PathogenesisPathogenesisGenetic PredispositionGenetic Predisposition

Triggering event / infectionTriggering event / infection

Mediators (Immune, endocrine, Mediators (Immune, endocrine, neuroendocrine, psychosocial, viral neuroendocrine, psychosocial, viral

reactivation or persistence)reactivation or persistence)

CFS/MECFS/ME

Genetic Predisposition - Genetic Predisposition - CFSCFS

HLA DR haplotypes in 112 South HLA DR haplotypes in 112 South Florida CFS patients, compared to Florida CFS patients, compared to 5,000 regional and national controls5,000 regional and national controls

4 to 6 fold increased relative risk for 4 to 6 fold increased relative risk for DR4, DR3 and DQ3. (Keller et al, 1992)DR4, DR3 and DQ3. (Keller et al, 1992)

Seattle CFS Cooperative Research Seattle CFS Cooperative Research Center Twin study - genetic Center Twin study - genetic predisposition, hereditability estimate predisposition, hereditability estimate of 51% (2nd World Conf); similar of 51% (2nd World Conf); similar results in Sweden, Australian studiesresults in Sweden, Australian studies

Evidence for Evidence for Triggering event/ Triggering event/ infection - CFS infection - CFS 60 to 80% of CFS subjects onset an 60 to 80% of CFS subjects onset an

acute viral-like illness (Komaroff, acute viral-like illness (Komaroff, Buchwald) Less so in population based Buchwald) Less so in population based studies. (Reeves, Jason)studies. (Reeves, Jason)

Andrew Lloyd and colleagues in Andrew Lloyd and colleagues in Australia performed a prospective Australia performed a prospective study - anergy during acute infection study - anergy during acute infection predicted persistent CFS like symptomspredicted persistent CFS like symptoms

Severity of initial infection single best Severity of initial infection single best predictorpredictor

Model of CFS Model of CFS PathogenesisPathogenesisGenetic PredispositionGenetic Predisposition

Triggering event / infectionTriggering event / infection

Mediators (Immune, endocrine, Mediators (Immune, endocrine, neuroendocrine, psychosocial, viral neuroendocrine, psychosocial, viral

reactivation or persistence)reactivation or persistence)

CFS/MECFS/ME

CRFCRF

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 axisEmotional stress activates immune system and HPA axis

Video link: The Video link: The leukocyteleukocyte

..http://www.studiodaily.com/main/searchlist/6850.html

http://multimedia.mcb.harvard.edu/media.html

Immune cascade

N atura l K illerC ells (T h1)

C D 8 ce llskill virus

H elper C D 4 cellT h1 cytokines

IL-2, IN F ac tivates C D 8

B ce llsm ake antibody

prevent and helpc lear in fec tion

H elper C D 4 cellT h2 cytokines

IL-6, IL-10ac tivates B ce lls

Macrophage presents antigen

..

Immune abnormalities in Immune abnormalities in CFSCFS

Immune ActivationImmune Activation

DR, CD26 DR, CD26 expressionexpression

TH2 cytokine shiftTH2 cytokine shift Proinflammatory Proinflammatory

cytokines cytokines expression TNF-a, expression TNF-a, IL-1, IL6IL-1, IL6

Functional defectsFunctional defects

NK Cell dysfunctionNK Cell dysfunctionCD8 abnormalitiesCD8 abnormalities perforins, granzymesperforins, granzymesMacrophage Macrophage

abnormalitiesabnormalitiesAntibody productionAntibody production

Photo by Leventhal, Karnovsky and Martz

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Subjects

GWI

CFS

Controls

NK Cytotoxicity: % K562 Cells Killed at Target to Effector Cell Ratio of 1:1

IntracellularCytolytic 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

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).

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Subjects

GWI

CFS

Controls

Intracellular Perforin

Hi NKCCLo NKCC

Subject Groups

8000

6000

4000

2000

0

rMol

ecul

es /

Perforin

CFS

NK

Cel

lp = 0.05

p = 0.01

Controls

Hi NKCCLo NKCC

CFS

2000

1600

1200

800

400

0

rMol

ecul

es /

Subject Groups

CD

3+C

D8+

Cel

l

Controls

p = 0.01

p = 0.01

Perforin

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.

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Subjects

GWI

CFS

Controls

Lymphocyte Activation in GWI and CFS:Percent of CD2+CD26+ Lymphocytes

CFS Controls

8000

6000

4000

2000

0

p < 0.0002

Molecules of CD26 on T Cells

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

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.

BIOMARKERBIOMARKER GWIGWI CFSCFS HCHCNPY (pmol/L)NPY (pmol/L) 37*37* 4141 5252

*Significantly different from HC (p<.05)

In the GWI patients, we found a reduced amount of NPY in plasma.

LYMPHOCYTE PROLIFERATION IN RESPONSE TO PHA: MEAN (+/- S.D.) NET CPMIN CFS PATIENTS ABOVE OR BELOW THE MEAN ON COGNITIVE DIFFICULTIES SCALE

210000

180000

150000

120000

90000

60000

30000 LOW CDS HIGH CDS

P = .O3

.... ....

80

65

50

35

20

5

Gro

up M

eans

(+/-

SD)

Medical Outcomes Survey Short Form-36 ( SF-36) Women with CFS

SF-36 measures the impact of illness including limitations due to physical health, pain, vitality, social functioning, emotional problems and general mental health.

LOW NKCC NORMAL NKCCN = 22 N = 19

p = .003

.

• . • .

55

41

28

14

0

Group M

eans (+/- SD)

Paced Auditory Serial Addition Task (PASAT)Women with CFS

The PASAT is an objective measure of:

Low NKCC Normal NKCCn = 22 n = 19

p = <.001

rate of information processing, sustained attention and divided attention

Immune –Endocrine LinkImmune –Endocrine Link

IL-6 increase associates with low IL-6 increase associates with low cortisol, CRH mediatedcortisol, CRH mediated

Papanicolau Neuroimmunomodulation 2004 11(2)65-74Papanicolau Neuroimmunomodulation 2004 11(2)65-74Maes M Neuro Endocrinol Lett 2005 Oct 30;26(5)Maes M Neuro Endocrinol Lett 2005 Oct 30;26(5)

Viral Viral Persistence/ReactivatioPersistence/Reactivatio

n n HHV6 virus is present in 22 to 54% of patients in HHV6 virus is present in 22 to 54% of patients in

cross sectional studies (Ablashi, Krueger, Knox), cross sectional studies (Ablashi, Krueger, Knox), HHV6 virus is present in 79% of CFS patients in HHV6 virus is present in 79% of CFS patients in longitudinal studies (HHV6 PCR assay, Knox)longitudinal studies (HHV6 PCR assay, Knox)

HHV6 virus is present in the spinal fluid of 28 of HHV6 virus is present in the spinal fluid of 28 of 120 CFS patients (Peterson), and 7 of 35 CFS 120 CFS patients (Peterson), and 7 of 35 CFS samples (Knox). samples (Knox).

Enterovirus is present in 13% of CFS muscle Enterovirus is present in 13% of CFS muscle samples (Douche-Aourik, 2003); samples (Douche-Aourik, 2003);

EBV – dUTPase as a immune modulator, up EBV – dUTPase as a immune modulator, up regulating inflammatory cytokines (Glaser, 2005)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)

Viral and Immune Viral and Immune Interactions and HealthInteractions and Health

J Chia showed GI biopsies with J Chia showed GI biopsies with enterovirus inclusions, found in enterovirus inclusions, found in patients with CFS and abdominal patients with CFS and abdominal complaintscomplaints

Novel mechanisms of Novel mechanisms of virus mediated chronicityvirus mediated chronicity

Glaser et al found evidence of regulatory Glaser et al found evidence of regulatory peptides encoded by EBV expressed in CFS peptides encoded by EBV expressed in CFS despite the absence of replicative virusdespite the absence of replicative virus

These peptides are known to modulate These peptides are known to modulate immune function, inducing pro-inflammatory immune function, inducing pro-inflammatory and Type 2 cytokinesand Type 2 cytokines

Lerner’s group found evidence of a subgroup Lerner’s group found evidence of a subgroup of CFS patients with incomplete viral of CFS patients with incomplete viral expression and cardiac motility expression and cardiac motility abnormalities; subset of CFS with IgM EBVabnormalities; subset of CFS with IgM EBV

2 Lerner M et al In Vivo 2004(18) 4:417; (18)2:1012 Lerner M et al In Vivo 2004(18) 4:417; (18)2:101

3 Glaser R Brain Behavior Immun 2005 19(2):913 Glaser R Brain Behavior Immun 2005 19(2):91

Treating HHV6a?Treating HHV6a?Association vs. causationAssociation vs. causation

Blood PCR HHV6 a did not predict HHV6 virus Blood PCR HHV6 a did not predict HHV6 virus is present in the spinal fluid is present in the spinal fluid

CSF did not predict bloodCSF did not predict blood Of 120 CSF samples, 44 had abnormalities of Of 120 CSF samples, 44 had abnormalities of

protein, glucose or cells. Of the 44 , 28 were protein, glucose or cells. Of the 44 , 28 were positive for HHV6(26), EBV (1), or CMV(1).positive for HHV6(26), EBV (1), or CMV(1).

5 of 8 CSF PCR positive treated until CSF 5 of 8 CSF PCR positive treated until CSF cleared returned to full time employment cleared returned to full time employment (Peterson); in his experience TK inhibitors did (Peterson); in his experience TK inhibitors did not clear CSF, patients required foscarnet or not clear CSF, patients required foscarnet or cidofovircidofovir

Open label valgancyclovir 20 of 23 responders Open label valgancyclovir 20 of 23 responders in high titer EBV plus HHV6 selected cohort , in high titer EBV plus HHV6 selected cohort , (Jose Montoya) (Jose Montoya)

Placebo control trials have not been completedPlacebo control trials have not been completed

Clinical TrialsClinical Trials M Lerner reported a phase 1 trial M Lerner reported a phase 1 trial

of valgangciclovir in CFS patients of valgangciclovir in CFS patients with evidence of cardiac with evidence of cardiac dysfunction. 37 patients were dysfunction. 37 patients were treated in an open label study, treated in an open label study, with improvement in all 37. He with improvement in all 37. He emphasized the need to hydrate, emphasized the need to hydrate, monitor renal and liver function.monitor renal and liver function.

Genomics, Proteomics, and Genomics, Proteomics, and Viral ChipsViral Chips

IACFS Conf 2007 :IACFS Conf 2007 : Inflammation pathways, IL-6, TNFa Inflammation pathways, IL-6, TNFa

upregulated in a subgroup, suggesting upregulated in a subgroup, suggesting monoconal ab blockers as a treatment monoconal ab blockers as a treatment (Kerr, Vernon, Olano)(Kerr, Vernon, Olano)

CFS proteome (Baruniuk)CFS proteome (Baruniuk) Serum analysis using infrared spectroscopy Serum analysis using infrared spectroscopy

(Sakudo, Watanabe, Ikuta, Kuratsune)(Sakudo, Watanabe, Ikuta, Kuratsune) 28 potential microbes under study (Kerr)28 potential microbes under study (Kerr) Reno research group using viral chip Reno research group using viral chip

technology in CFS and CFS associated technology in CFS and CFS associated malignancymalignancy

ConclusionConclusion Immune dysfunction in CFS contributes Immune dysfunction in CFS contributes

to the overall symptom complex, and to the overall symptom complex, and contributes to the persistence of the contributes to the persistence of the illness, both directly and through illness, both directly and through interaction with neuropeptides and interaction with neuropeptides and hormones.hormones.

There is increasing evidence of viral There is increasing evidence of viral reactivation in at least a subset of the reactivation in at least a subset of the patient population. Interventive trials are patient population. Interventive trials are currently focused on HHV6 virus.currently focused on HHV6 virus.

This work is helping to identify subgroups This work is helping to identify subgroups of CFS patients, identifying biomarkers, of CFS patients, identifying biomarkers, and potential treatment options.and potential treatment options.

Thank You!Thank You!Immunology/Virology:Immunology/Virology:Drs. Mary Ann Fletcher, Kevin Maher, Roberto PatarcaDrs. Mary Ann Fletcher, Kevin Maher, Roberto Patarca

Autonomic:Autonomic:Dr. Barry HurwitzDr. Barry Hurwitz

Health Assessment:Health Assessment:Drs. Michael Antoni, Mary Catherine Segota, Jackie JuncoDrs. Michael Antoni, Mary Catherine Segota, Jackie Junco

Professional links:Professional links:IACFS/ME on line: IACFS/ME on line: www.iacfs.netCDC on line: CDC on line: www.cdc.govNIH on line: NIH on line: www.nih.gov

Photo by Leventhal, Karnovsky and Martz