Management of Extrahepatic Manifestations of Chronic Hepatitis C

Mario U. Mondelli

Department of Infectious Diseases,University of Pavia, Fondazione IRCCS Policlinico San Matteo

Kasr Al-Aini International Post Graduate Course of Hepatology, Cairo University, 10-12 October 2009

Chronic Hepatitis C VirusExtrahepatic Manifestations

• Non organ-specific antibodies• Mixed cryoglobulinaemia (Types II & III):

– Purpura (Leukocytoclastic vasculitis)– Glomerulonephritis– Peripheral neuropathy

• Non-Hodgkin’s lymphoma: low grade, MZ• Autoimmune thyroiditis (up to 36% anti-TPO prevalence,

CD81 on thyrocytes, ↑ IL-8)

• Porphyria cutanea tarda (Metanalysis showed OR 274.78)

• Diabetes mellitus (defects in insulin signalling)

• Lichen planus• Sicca (non-Sjögren’s syndrome) (SS-A, SS-B neg.; no xerophtalmia)

Chronic Hepatitis C VirusAutoantibodies

HCV % Control %

Rheumatoid factor 70 8

Cryoglobulins >50 <1

ANA

• > 1:40

• > 1:180

21

13

10

2

Antismooth muscle (SMA)

• > 1:40

• > 1:180

21

7

2

<1

Anti–liver-kidney microsome (LKM) 5 <1

Autoantibodies Are Frequent in Both Hepatitis B and C

SMA ANA

Liver Kidney

LKM1 Is Seen Only in Hepatitis C Virus Infection

DELLTEHRMTWDPAQ CYP2D6252-66

HCV310-24PGHITGHRMAWDMMM

Similarity with E1 region of HCV

Molecular Mimicry

Vergani D, 2006

DELLTEHRMTWDPAQ CYP2D6252-66

HCV310-24PGHITGHRMAWDMMM

Similarity with E1 region of HCV

Molecular Mimicry

Vergani D, 2006

CRYOGLOBULIN

• Immunoglobulin which undergoes reversible temperature-induced insolubilization

Cacoub P, et al. Curr Opin Rheumatol. 2002;14:29-35.

CryoglobulinaemiasClassification

Immunoglobulin Classification

IMonoclonalNo rheumatoid factor

Primary

IIPolyclonal IgGMonoclonal IgMRheumatoid factor

Secondary mixedHCV infection

IIIPolyclonal IgGPolyclonal IgM

Secondary mixedInfectionsAutoimmune disordersLymphoproliferative diseases

HCV and Mixed Cryoglobulinaemic Syndrome (MCS) Clinical Manifestations

• Minor:– Purpura– Fatigue– Arthralgias– Sensitive neuropathy

• Major:– Glomerulonephritis– Motor neuropathy– Hyperviscosity syndrome– Systemic vasculitis

HCV and CryoglobulinaemiaPurpura, Vasculitis

• Occurs in dependent areas

• Deposition of cryoglobulins in small capillaries

• Pruritic

• Ulcerations may develop

Criteria Major MinorSerological • Mixed cryoglobulinaemia

• Low C4• Rheumatoid factor• HCV or HBV infection

Pathological • Leukocytoclastic vasculitis • Mono-oligoclonal B-cell infiltrate in liver or BM

Clinical • Purpura • Membranous-proliferative glomerulonephritis• Peripheral neuropathy• Cutaneous ulcers

Ferri C, Zignego AL, Pileri SA. J Clin Pathol 2002; 55: 4 – 13.

Diagnostic Criteria of Mixed Cryoglobulinaemia

How to Combine Diagnostic Criteria

• Confirmed MCS:– Serological MCS (± low C4) + purpura + leukocytoclastic vasculitis.– Serological MCS (± low C4) + 2 minor symptoms + 2 minor

serological/pathological findings.

• Incomplete or possible MCS: – Serological MCS or low C4 + 1 minor symptom + 1 minor

serological finding ± compatible pathological findings.– Purpura and/or leukocytoclastic vasculitis + 1 minor symptom +

1 minor serological finding ± compatible pathological findings– 2 minor symptoms + 2 serological findings ± compatible

pathological findings

Immune Manifestations of HCVPathogenesis

Monoclonal IgM RF

Cryoglobulin traps HCV

HCV evades theimmune response

Chronic B-cell stimulation by

HCV antigen (E2 ?)

Polyclonal IgG

Y

Y

Y

YGenetic and

environmentalfactors

Y Y

Y

YY

Y

Y

• Are specific viral protein sequence changes or recurrent amino acid motifs responsible for aberrant polyclonal B cell stimulation in chronic HCV infection ?

• Is polyclonal B cell activation a general feature of chronic HCV infection and what are the mechanisms responsible for initiation and maintenance of this phenomenon ?

Questions

HCV Sequence Changes Found in Cryoglobulinaemic patients

• Insertion at position 385 (HVR1) detected in 5 (24%) of 21 patients with cryoglobulinaemia and in none of controls (Gerotto et al., Blood 2001;98:2657-63).

• Two HVR1 positions (389 and 398) and 3 HVR2/CD81-binding site positions (474, 493, 497) associated with cryoglobulinaemia (Hofmann et al., Blood 2004;104:1228-9).

• No specific HVR1 motifs associated with cryoglobulinaemia (Rigolet et al., Leukemia 2005; 19:1070-1076)

AA Insertions within HVR1 in Patients with and without Cryoglobulins

Patient ID Insertion Position No. of clones with changes

Cryo + (6.2%) 117 (1b) TR 387 1/20

171 (1b) ?? ?? ??

28 (2a/c) GLSL

GLTL

404

404

9/11

1/11

169 (2a/c) ASSSM

SSPTA

SSPMA

384

385

385

8/12

3/12

1/12

193 (2a/c) GAG

GTV

385

385

9/10

1/10

Controls (9.1%) 17 (1b) GPG

ELG

385

385

4/12

2/12

191 (2a/c) ARY

TRQ

ARQ

TRR

*

385

385

385

385

6/11

1/11

3/11

1/11

183 (2a/c) RTV

RKT

RTA

384

384

384

2/10

1/10

Bianchettin G et al., J Virol 2007;81:4564-71 .

Weblogo of the Positions Highlighted by the PCA Analysis of 548 HVR1 Sequences.

The higher the letter, the higher the frequency of the amino acid in that position.

384 386 388 391 395 396 397 398 399 405

384 386 388 391 395 396 397 398 399 405

pos

neg

384 386 388 391 395 396 397 398 399 405

384 386 388 391 395 396 397 398 399 405

384 386 388 391 395 396 397 398 399 405384 386 388 391 395 396 397 398 399 405

384 386 388 391 395 396 397 398 399 405384 386 388 391 395 396 397 398 399 405

Cryo +

Controls

Bianchettin G et al., J Virol 2007;81:4564-71 .

% C

D69 B

cells

% C

D183 B

cells

% C

D86 B

cells

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

CD27-Total CD27+ CD27-Total CD27+

% C

D71 B

cells

Healthy controls (n=36) HCV+ patients (n=50)

p=0.0002 p=0.0007 p=0.0031 p=0.0002

p=0.0001 p<0.0001 p=0.032 p=0.0004 p=0.021 p=0.0019

Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HCV Infection and Healthy Controls

MW. 50bp DNA Ladder, 10 water, 11 Neg. ctrl, 12 Pos. ctrl 1. B.A. 2. C.E. 6. B.A 3. F.R. 7. C.E 4. M.T. 8. M.T. 5. F.I. 9. F.I.

CD19+/CD69+ CD19+/CD69-

Genomic HCV RNA

1 2 3 4 5 6 7 128 9 10 11MW

100bp

Minus-strand HCV RNA

160bp

1 2 3 4 5 6 7 128 9 10 11MW

160bp

1/4

Full

Pugnale, Negro, Mondelli, unpublished

One Signal is Sufficient to Activate

Human Memory B Cells

CD40

Innate immunity derived signalsTLR

Bystander T cell help / CytokinesProliferation

Differentiation

Bernasconi et al Science 2002

OR

SF

C/1

05 P

BM

CS

FC

/105

PB

MC

Media CD40L CD40L+IL10

Healthy Controls (n=44)

HCV Patients (n=56)

p = 0.0227

Media CpG CpG+IL2

p = 0.0128

p = 0.0006

N. of IgG-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls after Stimulation with CD40L ± IL10 or CpG ± IL2

0

450

900

0

750

1500

CD81

CD19

CD21

E2E2B CellBCR (Ig)

Polyclonal B cell activation

LymphoproliferaLymphoproliferative Diseasetive Disease

B cell clonal expansion

Putative Mechanisms of B-Cell Activation

in HCV Infection

AutoimmunityAutoimmunity

Activation threshold

CryoglobulinaemiaTherapeutic Strategies

Rationale Strategy

Eradicate aetiological agent

• PEG-IFN + Ribavirin

Reduce inflammation • Steroids

Remove CIC • Cyclophosphamide• Plasmapheresis

Promote CIC clearance by RES

• Lac diet

Eliminate B lymphocytes • Rituximab

Rituximab

• Murine IgG1 humanised mAb • Binds CD20 on mature B cells• Acts by:

– Complement-mediated cytotoxicity– ADCC– Opsonization and clearance by RES– Pro-apoptotic ?

• Approved for treatment of NHL

Rituximab: Clinical Studies beyond NHL

• RA (only FDA approved condition)• SLE• ITP• Autoimmune haemolytic anaemia• Pemphigus• Waldenstrom’s macroglobulinaemia• Wegener’s granulomatosis• MCS

Clinical Response to Rituximab 375 mg/m2

for 4 Weeks in HCV+ Patients with MCSA Systematic Review of 13 Studies

Clinical Features

N. of Cases Complete Response N. (%)

Purpura 33 24 (73)

Arthralgia 30 16 (53)

Neuropathy 25 9 (36)

Glomerulonephritis 13 9 (70)

Cryocrit 15 11(73)

Cacoub, P et al. Ann Rheum Dis 2008;67:283-7

Phase 2 Single-Arm Study on Low-Dose Rituximab for Symptomatic, Refractory Mixed Cryoglobulinaemia

(AIFA Protocol FARM6KMZFY)

• 250 mg/m2 x 2

• 1-year follow-up

• Objectives:– Reduce treatment costs– Reduce side effects ( HCV RNA, infections)

(Fiorilli, Mondelli, Zignego, Pozzato, Co-PI’s)

Reactivation of HBV with Rituximab Administration

• Anti-HBs titers dropped precipitously after rituximab administration in vaccinated patients – Median titers 80 IU/mL before administration dropped to 38 IU/mL after

administration (P < .05)

• 3 of 20 patients with inactive HBV had detectable HBV DNA at Days 92, 146, 320 after 2-3 doses of rituximab

Metzler F, et al. AASLD 2008. Abstract 848.

25020015010050

10080604020

0 100 200 300

400

300

200

100

0

108

106

104

102

-200 0 200 400

30

20

10

00 500 1000

108

106

104

102

Patient 1 Patient 2 Patient 3

Ant

i-HB

s (I

U/m

L)S

erum

ALT

(U

/L)

HB

V D

NA

(IU

/mL)

1st r

ituxi

mab

dos

e

Days Days Days

Patients

Rituximab 250 mg/m2 2 one week apart (n = 36)

Wk 12interim analysis Wk 52

Phase 2 Single-Arm Study on Low-Dose Rituximab for Symptomatic, Refractory Mixed Cryoglobulinaemia

(AIFA Protocol FARM6KMZFY)

Follow-upRituximab 250 mg/mRituximab 250 mg/m22 2 2 one week apart one week apart (n = 16)(n = 16)

50% reduction in BVAS & cryocrit

Size of initial group 16 patientsSize of second group 36 patientsCriterion for stopping on a negative finding at 1st stage 5 or less respondersCriterion for positive finding at 2nd stage: 27 or more respondersSignificance level 0.03Expected number of patients under null hypothesis 46Power 0.85 (expected response rate 60 ± 15%)Expected number of patients under alternative hypothesis 50

(Fiorilli, Mondelli, Zignego, Pozzato, Co-PI’s)

5 or less responders5 or less respondersSTOPSTOP

BASELINE RITUXIMAB (after 2 infusions)

0.16

0.03 0.13 3.14

4 WEEKS 8 WEEKS 12 WEEKS

6.81

16 WEEKS

CD19 FITC

5.29

CD19 FITC CD19 FITC

% B

Lym

ph

ocy

tes

(CD

19+

)

Rituximab

Rituximab

Rituximab

Rituximab

#1 #2

#3 #4

Time (weeks)

B Lymphocyte Count after RITUXIMAB Treatment

Rituximab

• Excellent results on vasculitic ulcers and severe renal involvement.

• No effect on peripheral neuropathy

Clinical, Immunological, and Virological Efficacy of Rituximab ± PEG-IFN2b and Ribavirin

Complete Response Rituximab/PEG–IFN/RBV

(n=20)

Rituximab only

(n=12)

Clinical 80% 58%

Immunological 67% 46%

Virological 55% 0%

Terrier et al. Arthritis Rheum 2009;60:2531-40.

CryoglobulinaemiaTherapeutic Strategies

Clinical Symptoms Treatment

Asymptomatic Monitor

Mild-moderate Low-dose steroids ± Lac diet

Moderate-Severe PEG-IFN + RBV, Low-dose steroids

Severe, rapidly progressive Plasmapheresis + steroids + Cyclophosphamide;

Rituximab + PEG-IFN + RBV

Chronic hepatitis +

mild cryoglobulinaemia

Peg-IFN + RBV ± Rituximab

Acknowledgements

Lab Team:• Gabriella Bianchettin • Antonella Cerino• Stefania Varchetta• Barbara Oliviero• Enrica Paudice

Clinical Team:• Serena Ludovisi• Giuseppe Michelone • Marco Zaramella

Area di Ricerca Infettivologica Fondazione IRCCS Policlinico San Matteo and

University of Pavia

Collaborators:• Franco Negro, Dept.of Gastroenterology, University of Geneva• Anna Tramontano, Dept. of Biochemistry, University of Rome La Sapienza• Milvia Casato, Dept. of Medicine, University of Rome La Sapienza• Giampaolo Merlini, Centre for Amyloidosis, University of Pavia

Chronic Hepatitis C Virus Autoantibodies (cont’d)

• No relationship between presence of autoantibodies and

– Severity of chronic HCV

– HCV genotype

• Correlation between rheumatoid factor titre and

– Cryoglobulinaemia

– But not symptomatic cryoglobulinaemia

• Circulating autoantibodies from autoimmune disorders may result in

– False positive anti-HCV

HCV Prevalence and Crude and Adjusted O.R. in Patients with B-NHL vs. Controls

Total # HCV+ % HCV+ Crude O.R. (95% C.I.)

Adjusted O.R. (95% C.I.)

Controls 396 22 5.6 1 1

Patients with B-NHL

400 70 17.5 3.6 (2.2-6.0) 3.1 (1.8-5.2)

Mele A, et al. Blood. 2003;102:996-999

ANTI-THYROID ANTIBODIES IN PATIENTS WITH VIRAL CHRONIC HEPATITIS

(Preziati et al, 1995)

CH-HCV (n=78)

positiveTiter (KU/l)

CH-HBV (n=49)

positiveTiter (KU/l)

36%(17 – 1190)

0

16%(57 – 836)

10%(64 – 282)

TPO-Ab Tg-Ab

Anti-Thyroid AutoAb

Extrahepatic Effects of HCVPorphyria Cutanea Tarda

2 case series3 uncontrolled series280 patientsAlcohol: 36%-77%

Fargion (1992)

De Castro (1993)

Criber (1995)

Stolzel (1995)

Kondo (1997)

0

20406080100

PCT

0 5 10 15 20

Control

Combined meta-analysis of 7 studies: OR 274.78, CI 104.12-725.13

(Gisbert et al., J Hepatol 2003;39:620-7.)

CharacteristicHCV

Sialadenitis

Primary

Sjögren’s Syndrome

SS-A, SS-B Negative Positive

Lymphocytic capillaritis

Mild

Pericapillary

Mostly CD8 cells

Severe

Periductal

Mostly CD4 cells

Sicca syndrome:• Xerophthalmia• Xerostomia

Absent

8%-36%

Present

Present

Extrahepatic Effects of HCVLymphocytic Sialadenitis

Nagao Y, et al. J Gastroenterol Hepatol. 2004;19:1101-1113.

Extrahepatic Effects of HCVLichen Planus

• Occurs in < 1% of the general population• 10%-30% of patients with chronic HCV• Appearance

– Flat topped, violaceous, pruritic papules– Throughout body – Oral mucosa

• Histology– Dense infiltration of dermis with T lymphocytes

Meta-Analysis of Case-Control Studies Comparing the Prevalence of HCV Infection in Patients with PCT and in Healthy Controls.

Gisbert et al., J Hepatol 2003;39:620-7.

Extrahepatic Effects of HCVB-Cell Lymphoma

8 case series1754 pts evaluated

Ferri (1994)

Mazzaro (1996)

Silvestri (1996)

Izumi (1996)

McColl (1996)

Zignego (1997)

DeRosa (1997)

Zuckerman (1997)

0102030

B Cell Lymphoma

0 10 20 30

Controls

Three Signals Are Required to Activate Human Naïve B Cells

CD40

p-MHC

(1) Ag

(3) Innate immunity derived signals

tlr(2) Cognate T cell help

(1’) Costimulation

Ag

ProliferationSurvival

Ruprecht & Lanzavecchia, Eur J Immunol 2006;36:810-6

DELLTEHRMTWDPAQPPRDL

CYP2D6252-71: Major epitope of LKM1

271252

Molecular Mimicry

Protein Database Search

EHRMTWDPAQPPRDL

DRLSPRPPAQPPRRR IE 175 HSV-1

CYP2D6257-271

E1 HCVGHRMAWDMMMNWSPT

QLPPPAAPAQPPPGV EBNA-2 Epstein-Barr

PMIAAAPPAQPPSQP IE63 H. Cytomegalovirus

AARTAPAPAQPPSPA J1L H. Cytomegalovirus

Multiple Hits Hypothesis

Chronic HCV infection is associated with extrahepatic manifestations including autoantibody production and abnormal B-cell proliferation

Background

Functional Studies on Memory B-Cells in Chronic HCV Infection

PBMC500-3,000/w

Anti-CD40+IL10

7 d 6 d

Ig producing-cells (ELISPOT)

CpG 2006 IL2

CYP2D6 shares an amino acid

sequence with HCV

LKM-1 targets CYP2D6

LKM-1 may arise through a

mechanism of molecular mimicry

Molecular Mimicry

Prevalence of HVR1 Insertions in Patients with and without

Cryoglobulinaemia

113 Patients

80 Cryo + 33 Controls

5 (6.25%) with insertions

3 (9.1%)with insertions

3/46 (6.5%) genotype 2a/c

2/34 (5.9%) genotype 1b

2/17 (11.8%)genotype 2a/c

1/16 (6.25%)genotype 1b

Bianchettin G et al., J Virol 2007;81:4564-71 .

Human Mature B Cell Phenotype

B cell areas of lymph nodes and spleen

IgMhi,IgDhi

CD19,CD20,CD21hi

CD38lo, ABCB1(?)

IgM, IgG, IgA, IgDlo

CD19,CD20,CD27

CD38CD126CD138

Bone marrow andinflamed tissues

MemoryBlood Naïve Plasmacell

AbPlasmablast

CD19lo,CD20lo

IgG,CD69,CD86…

0

10

20

30

0

25

50

75

100

Healthy controls (n=36) HCV+ patients (n=50) HBV+ patients (n=22)

% C

D1

9+

cells

Controls HCV HBV

% C

D2

7+

& C

D2

7-

B c

ells

CD27+ CD27-

Proportions of Total and Memory (CD27+) and Naïve B Cells in Healthy Controls and

Patients with Chronic HCV or HBV Infection

Eradication of HCV Infection Associates with a Decreased Expression of Activation Markers and

CXCR3

Rosa et al., PNAS 2005;102:18544-9

Stimulation with CpG Oligonucleotides Is Sufficient for Optimal Expansion of

Memory B Cells

CD19+/CD27+ CD19+/CD27-CD19+

Me

dia

Cp

G

Reactivation of HBV With Rituximab Administration

• Retrospective analysis of patients who received 1 course of rituximab at single center: 2005-2007

• 180 of 258 patients (70%) treated with rituximab tested for HBV – Vaccinated: 46% – Negative: 39% – Anti-HBc/anti-HBs positive: 11% – HBV DNA positive: < 1%

Metzler F, et al. AASLD 2008. Abstract 848.

INCIDENCE OF THYROID DYSFUNCTION IN HCV PATIENTSTREATED WITH IFN

248677

1043439598246

3251

21 (8.5%)18 (2.7%)69 (6.6%)17 (3.9%)60 (10%)

9 (3.6%)

194 (6.0%)

Marcellin ’95Okanoue ’96Koh ’97Kakizaki ‘’99Dumolin ’99Wong ’02

Total

Treated Thyroid dysfunctionAuthor

RISK FACTORS FOR DEVELOPMENT OF THYROIDDYSFUNCTION DURING IFN THERAPY

11761774

38428

15353

823

SexFemalesMales

TPO-AbPositiveNegative

Treated Thyroid dysf.Risk factors

4.4 (3.2 – 5.9)

3.9 (1.9 – 8.1)

R.R. (C.I.)

Marazuela ’96; Fernandez-Soto ’98; Hsieh ’00; Vial ’95;Okanoue ’96; Koh ’97;Kakizaki ’99; Deutsch ’97; Roti ‘96

Chronic Hepatitis C Virus Autoantibodies (cont’d)

• No relationship between presence of autoantibodies and

– Severity of chronic HCV

– HCV genotype

• Correlation between rheumatoid factor titre and

– Cryoglobulinaemia

– But not symptomatic cryoglobulinaemia

• Circulating autoantibodies from autoimmune disorders may result in

– False positive anti-HCV

Zein CO, et al. Am J Gastroenterol. 2005;100:48-55.

Chronic HCV and Diabetes MellitusCase Prevalence

• N = 179 with chronic HCV• Prevalence of diabetes

mellitus and insulin resistance noted

• Compared with expected rate based on NHANES III study after adjusting for– Age– Sex– Race

• Prevalence of DM or insulin resistance higher in those with chronic HCV

0

4

8

12

16

20

Females Males

Nu

mb

er o

f C

ases

ObservedExpected

Pathogenetic Mechanisms Responsible for Development of Type 2 Diabetes in HCV Infection

Early Defects in Upstream Insulin Signalling

Components (IRS-1, PI3-kinase, Akt)

Increased Insulin Resistance

Type 2 Diabetes Mellitus

Aytung et al., Hepatology 2003;38:1384-92

Enlarged Pericapsular Lymph Node in a Patient with Chronic HCV Infection

>CD20+ B cells

CD27-

% C

D69 B

cells

% C

D86 B

cells

% C

D71 B

cells

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

% C

D183 B

cells

Total TotalCD27+ CD27+ CD27-

Healthy controls (n=36) HCV+ patients (n=50) HBV+ patients (n=22)

p=0.0013

p=0.0002

p=0.0009

p=0.0007p=0.0031

p=0.0078

p=0.0002

p=0.0152p=0.001

p=0.0001 p=0.012p<0.0001 p<0.0001 p=0.032

p=0.0004 p=0.021 p=0.0019

Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HBV or HCV Infection and Healthy Controls

TOT TOT CD27+ CD27+ CD27- CD27-0

20

40

60

80

100

CTRLS HCV+ PTS

%C

D18

3 B

cel

ls

p < 0.0001p < 0.0001

Proportions of CXCR3+ Memory (CD27+) and Naïve (CD27-)

B-Cells in Patients with Chronic HCV Infection and Healthy Controls

n=33 n=29

Cerino et al., in preparation

Correlation between Serum HCV RNA and CD183 (CXCR3) Expression on B cells

0

25

50

75

100

0

25

50

75

100

% C

D183/C

D19+

cells

% C

D183/C

D19+

27+

cells

HCV RNA (IU/ml x 106)

0 1 2 3 4 5

15 16

0 1 2 3 4 5

15 16

Speaman r=0.408

p=0.012

Speaman r=0.59

p=0.0001

N. of IPC-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls Following Stimulation with CD40L ± IL10

HCV Patients (n=33)Healthy Controls (n=31)

0

450

900

CD40L+IL10

p=0.0128

p=0.0193

IgG

CD40L

0

450

900

SF

C/1

05 P

BC

IgA

Media

Media0

450

900

Media CD40L CD40L+IL10

IgM

CD40L CD40L+IL10

SF

C/1

05 P

BC

SF

C/1

05 P

BC

N. of IPC-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls Following Stimulation with CpG ± IL2

0

1000

2000

Healthy Controls (n=41) HCV Patients (n=49)

Media CpG CpG+IL2

IgA

0

1000

2000 IgG

Media CpG CpG+IL2

p=0.0202

0

1000

2000

Media CpG CpG+IL2

IgM

SF

C/1

05 P

BC

SF

C/1

05 P

BC

SF

C/1

05 P

BC

TOT TOT CD27+ CD27+ CD27- CD27-0

20

40

60

80

100

CTRLS HCV+ PTS

%C

D69

B c

ells

p= 0.0004p= 0.0049

Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HCV Infection and

Healthy Controls

n=33 n=29

Cerino et al., in preparation

…an autoimmune disease is a viral disease

in which the virus is unknown.

Rolf Zinkernagel - Immunol Rev. 1996;152:21-45