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Extrahepatic manifestations of HCV

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  • Hepatitis C is associated with many extrahepatic manifestations, including nonspecific antibody production, essential mixed cryoglobulinemia, glomerular nephritis, porphyria cutanea tarda (PCT), leukoclastic vasculitis, non-Hodgkin’s lymphoma, autoimmune thyroiditis, diabetes, and Sjögren’s syndrome.
  • Many individuals with hepatitis C have circulating autoantibodies and are sometimes incorrectly diagnosed with other disorders. For example, 70% of patients with hepatitis C have circulating rheumatoid factor. Approximately one third have cryoglobulins, and anywhere from 13% to 21% have low-titer or high-titer antinuclear antibodies (ANA). A slightly lower percentage have smooth muscle antibodies—about 5% have antibodies to liver or kidney microsomes—and about 7% have antithyroid antibodies. These antibodies are all significantly higher than we see in the control population without hepatitis C.
  • Cryoglobulinemia is classified into 3 types. Type 2 cryoglobulinemia is found almost exclusively in individuals with hepatitis C and is associated with polyclonal IgG, monoclonal IgM, and rheumatoid factor.
  • Cryoglobulinemia can cause several manifestations. In addition to the dermatitis, it can also cause vasculitis and myalgias. Clearly some fibromyalgia patients have myalgias from hepatitis C and cryoglobulinemia. Cryoglobulinemia can also cause arthralgias. Many of these patients are rheumatoid factor and/or ANA positive. Cryoglobulinemia can also cause membranal proliferative glomerular nephritis, neuropathy, and in some cases, chronic fatigue syndrome.
  • This slide illustrates the dermatitis of cryoglobulinemia—a patchy discoloration in the lower extremities resulting from deposition of cryoglobulins in small capillaries. Ulcerations may develop, and these areas can be very pruritic.
  • Why do individuals with hepatitis C develop so many autoantibodies? The schematic in this slide illustrates one of the proposed mechanisms. Because hepatitis C is a rapidly reproducing virus that is constantly changing because of the high mutation frequency, it is able to evade the immune response. Because the immune system wants to constantly attack this virus, there is a constant immune stimulation, causing clonal expansion of B cells. Under genetic and environmental factors which are poorly defined, the immune system produces polyclonal IgG, monoclonal IgM, and rheumatoid factor. These antibodies bind to HCV causing large aggregates called cryoglobulins, which trap hepatitis C in dependent areas and blood vessels causing the symptoms of cryoglobulinemia.
  • Figures used with permission from Karsten Wursthorn, MD.
  • It is important to realize that there is no specific relationship between the presence of these autoantibodies and the severity of HCV infection or the genotype of hepatitis C. There is a correlation between rheumatoid factor titer and cryoglobulinemia, but not symptomatic cryoglobulinemia. An important point to realize is that circulating autoantibodies from true autoimmune disorders can result in false-positive hepatitis C reactions, as we mentioned earlier.
  • Another extrahepatic manifestation that is well recognized now by dermatologists is PCT, a genetic disease of bilirubin metabolism. Individuals who develop the skin manifestations of PCT usually have an underlying liver disease. In the past this liver disease was attributed to alcohol, but it now seems clear that one of the primary drivers for PCT is hepatitis C. In this series of 2 case series and 3 uncontrolled series including more than 250 patients, prevalence of hepatitis C was about 70% in patients with PCT.
  • Another extrahepatic manifestation of hepatitis C is sialadenitis, or inflammation of the salivary glands. Sialadenitis is caused by inflammation or migration of lymphocytes into the salivary glands. It can mimic primary Sjögren’s syndrome, but there are several factors that differentiate it from Sjogren’s. Hepatitis C-induced sialadenitis is negative for the antibodies that are positive in Sjögren’s syndrome: Sjogren’s-specific antibody A and B. On biopsy of the salivary glands, it is evident that the way the lymphocytes infiltrate the salivary gland is different between the 2 disorders. In hepatitis C, the infiltration is milder: it is pericapillary and involves mostly CD8 cells. In primary Sjögren’s syndrome, the lymphocyte involvement is severe, periductal, and involves mostly CD4 cells. In HCV-induced sialadenitis, patients experience dry mouth, which is often why they will seek clinical attention. Patients with primary Sjögren’s syndrome also have dry mouth, but in contrast to HCV sialadenitis, they will have also have xerophthalmia.
  • Lichen planus is another dermatologic disorder commonly seen in individuals with hepatitis C. Lichen planus is seen in less than 1% of the general population. In total, HCV infection will be seen in 10% to 30% of patients with lichen planus. Lichen planus is characterized by pruritic papules that look like flat-topped, raised lesions on the skin. The lesions can occur anywhere on the body but are most commonly seen in the oral mucosa. Histologically, the affected skin is densely packed with T lymphocytes, which is another manifestation of hepatitis C.
  • Another extrahepatic manifestation of hepatitis C is B-cell lymphoma. In 8 case series that evaluated more than 1700 patients, you can see that individuals with B-cell lymphoma had a prevalence of hepatitis C of approximately 25% overall. In the control population of other cancers the incidence of hepatitis C was much lower and similar to what we see in the general population. Clearly this constant immune proliferation and clonal expansion of B cells driven by hepatitis C can at times transfer or progress to B-cell lymphoma.
  • It is important to realize that there is no specific relationship between the presence of these autoantibodies and the severity of HCV infection or the genotype of hepatitis C. There is a correlation between rheumatoid factor titer and cryoglobulinemia, but not symptomatic cryoglobulinemia. An important point to realize is that circulating autoantibodies from true autoimmune disorders can result in false-positive hepatitis C reactions, as we mentioned earlier.
  • Diabetes has recently been recognized to be associated with hepatitis C. In this study prevalence of diabetes mellitus and insulin resistance was much higher in patients with hepatitis C than in healthy individuals from the National Health and Nutrition Examination Survey study when adjusted for age, race, and sex.
  • Transcript

    • 1. 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
    • 2. Chronic Hepatitis C Virus Extrahepatic 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)
    • 3. Chronic Hepatitis C Virus Autoantibodies 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
    • 4. Autoantibodies Are Frequent in Both Hepatitis B and C SMA ANA
    • 5. LKM1 Is Seen Only in Hepatitis C Virus Infection Liver Kidney
    • 6. Molecular Mimicry Similarity with E1 region of HCV PGHITGHRMAWDMMM HCV310-24 DELLTEHRMTWDPAQ CYP2D6252-66 Vergani D, 2006
    • 7. Molecular Mimicry Similarity with E1 region of HCV PGHITGHRMAWDMMM HCV310-24 DELLTEHRMTWDPAQ CYP2D6252-66 Vergani D, 2006
    • 8. CRYOGLOBULIN • Immunoglobulin which undergoes reversible temperature-induced insolubilization
    • 9. Cryoglobulinaemias Classification Immunoglobulin Classification I II Monoclonal No rheumatoid factor Polyclonal IgG Monoclonal IgMκ Rheumatoid factor Polyclonal IgG III Polyclonal IgM Cacoub P, et al. Curr Opin Rheumatol. 2002;14:29-35. Primary Secondary mixed HCV infection Secondary mixed Infections Autoimmune disorders Lymphoproliferative diseases
    • 10. HCV and Mixed Cryoglobulinaemic Syndrome (MCS) Clinical Manifestations • Minor: – – – – Purpura Fatigue Arthralgias Sensitive neuropathy • Major: – – – – Glomerulonephritis Motor neuropathy Hyperviscosity syndrome Systemic vasculitis
    • 11. HCV and Cryoglobulinaemia Purpura, Vasculitis • Occurs in dependent areas • Deposition of cryoglobulins in small capillaries • Pruritic • Ulcerations may develop
    • 12. Diagnostic Criteria of Mixed Cryoglobulinaemia Criteria Major Minor Serological • 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.
    • 13. 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
    • 14. Immune Manifestations of HCV Pathogenesis HCV evades the immune response Chronic B-cell stimulation by HCV antigen (E2 ?) Y Y Y Y Polyclonal IgG Y Y Y Y Y Y Y Cryoglobulin traps HCV Monoclonal IgM RF Genetic and environmental factors
    • 15. Questions • 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 ?
    • 16. 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/CD81binding 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)
    • 17. AA Insertions within HVR1 in Patients with and without Cryoglobulins Patient ID No. of clones with changes 117 (1b) TR 387 1/20 ?? ?? ?? 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) Controls (9.1%) Position 171 (1b) Cryo + (6.2%) Insertion GAG GTV 385 385 9/10 1/10 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 .
    • 18. 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. Cryo pos + 384 386 388 391 395 396396 397 398 405 386 388 391 395 397 398 399 405 Controls neg 384 386 388 391 395 396396 397 398 386 388 391 395 397 398 399 399 405 405 Bianchettin G et al., J Virol 2007;81:4564-71 .
    • 19. Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HCV Infection and Healthy Controls p=0.0002 p=0.0007 100 % CD86 B cells % CD69 B cells 100 80 60 40 20 p=0.0002 80 60 40 20 0 0 100 p=0.0001 p<0.0001 p=0.03 2 80 60 40 20 100 % CD71 B cells % CD183 B cells p=0.0031 p=0.0004 p=0.021 p=0.0019 80 60 40 20 0 0 Total CD27+ CD27- Healthy controls (n=36) Total CD27+ HCV+ patients (n=50) CD27-
    • 20. MW 1 2 3 4 5 6 7 8 9 10 11 12 100bp Genomic HCV RNA MW 1 2 3 4 5 6 7 8 9 10 11 12 1/4 160bp Full 160bp Minus-strand HCV RNA MW. 50bp DNA Ladder, 10 water, 11 Neg. ctrl, 12 Pos. ctrl 1. B.A. 2. C.E. 3. F.R. 4. M.T. 5. F.I. CD19+/CD69+ 6. B.A 7. C.E 8. M.T. 9. F.I. CD19+/CD69Pugnale, Negro, Mondelli, unpublished
    • 21. One Signal is Sufficient to Activate Human Memory B Cells Bystander T cell help / Cytokines Proliferation Differentiation CD40 OR TLR Innate immunity derived signals Bernasconi et al Science 2002
    • 22. N. of IgG-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls after Stimulation with CD40L ± IL10 or CpG ± IL2 p = 0.0227 SFC/105 PBMC 1500 750 0 Media CpG Healthy Controls (n=44) HCV Patients (n=56) 900 SFC/105 PBMC CpG+IL2 p = 0.0006 450 p = 0.0128 0 Media CD40L CD40L+IL10
    • 23. Putative Mechanisms of B-Cell Activation in HCV Infection B Cell CD 81 E2 BCR (Ig) CD21 ↓ Activation threshold CD19 Polyclonal B cell activation B cell clonal expansion Lymphoproliferative Disease Autoimmunity
    • 24. Cryoglobulinaemia Therapeutic Strategies Rationale Strategy Eradicate aetiological agent • PEG-IFNα + Ribavirin Reduce inflammation • Steroids Remove CIC • Cyclophosphamide • Plasmapheresis Promote CIC clearance by RES • Lac diet Eliminate B lymphocytes • Rituximab
    • 25. Rituximab • Murine IgG1κ 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
    • 26. Rituximab: Clinical Studies beyond NHL • • • • • • • • RA (only FDA approved condition) SLE ITP Autoimmune haemolytic anaemia Pemphigus Waldenstrom’s macroglobulinaemia Wegener’s granulomatosis MCS
    • 27. Clinical Response to Rituximab 375 mg/m 2 for 4 Weeks in HCV+ Patients with MCS A 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
    • 28. 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)
    • 29. 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 Patient 1 100 80 60 40 20 250 200 150 100 50 0 100 200 300 Days Metzler F, et al. AASLD 2008. Abstract 848. 400 300 200 100 0 -200 Patient 2 Patient 3 108 30 106 20 106 104 10 104 102 0 200 400 Days 0 102 0 500 1000 Days 108 HBV DNA (IU/mL) 1st rituximab dose Anti-HBs (IU/mL) Serum ALT (U/L) Days 92, 146, 320 after 2-3 doses of rituximab
    • 30. Phase 2 Single-Arm Study on Low-Dose Rituximab for Symptomatic, Refractory Mixed Cryoglobulinaemia (AIFA Protocol FARM6KMZFY) 5 or less responders STOP Patients Wk 12 interim analysis Rituximab 250 mg/m2 × 2 one week apart (n = 16) 50% reduction in BVAS & cryocrit Wk 52 Follow-up Rituximab 250 mg/m2 × 2 one week apart (n = 36) Size of initial group 16 patients Size of second group 36 patients Criterion for stopping on a negative finding at 1st stage 5 or less responders Criterion for positive finding at 2nd stage: 27 or more responders Significance level 0.03 Expected number of patients under null hypothesis 46 Power 0.85 (expected response rate 60 ± 15%) Expected number of patients under alternative hypothesis 50 (Fiorilli, Mondelli, Zignego, Pozzato, Co-PI’s)
    • 31. BASELINE RITUXIMAB (after 2 infusions) 5.29 0.16 CD19 FITC CD19 FITC 4 WEEKS 8 WEEKS 12 WEEKS 0.03 0.13 3.14 CD19 FITC 16 WEEKS 6.81
    • 32. B Lymphocyte Count after RITUXIMAB Treatment #1 #2 % B Lymphocytes (CD19+) Rituximab Rituximab #3 #4 Rituximab Rituximab Time (weeks)
    • 33. Rituximab • Excellent results on vasculitic ulcers and severe renal involvement. • No effect on peripheral neuropathy
    • 34. Clinical, Immunological, and Virological Efficacy of Rituximab ± PEG-IFNα2b 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.
    • 35. Cryoglobulinaemia Therapeutic 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
    • 36. Acknowledgements Area di Ricerca Infettivologica Fondazione IRCCS Policlinico San Matteo and University of Pavia Lab Team: • Gabriella Bianchettin • Antonella Cerino • Stefania Varchetta • Barbara Oliviero • Enrica Paudice Clinical Team: • Serena Ludovisi • Giuseppe Michelone • Marco Zaramella 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
    • 37. 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
    • 38. 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
    • 39. ANTI-THYROID ANTIBODIES IN PATIENTS WITH VIRAL CHRONIC HEPATITIS (Preziati et al, 1995) Anti-Thyroid AutoAb TPO-Ab Tg-Ab CH-HCV (n=78) positive Titer (KU/l) CH-HBV (n=49) positive Titer (KU/l) 36% 16% (17 – 1190) (57 – 836) 0 − 10% (64 – 282)
    • 40. Extrahepatic Effects of HCV Porphyria Cutanea Tarda Fargion (1992) De Castro (1993) Criber (1995) 2 case series 3 uncontrolled series 280 patients Alcohol: 36%-77% Stolzel (1995) Kondo (1997) 100 80 60 40 PCT 20 0 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.)
    • 41. Extrahepatic Effects of HCV Lymphocytic Sialadenitis HCV Sialadenitis Primary Sjögren’s Syndrome SS-A, SS-B Negative Positive Lymphocytic capillaritis Mild Pericapillary Mostly CD8 cells Severe Periductal Mostly CD4 cells Absent 8%-36% Present Present Characteristic Sicca syndrome: • Xerophthalmia • Xerostomia
    • 42. Extrahepatic Effects of HCV Lichen 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 Nagao Y, et al. J Gastroenterol Hepatol. 2004;19:1101-1113.
    • 43. 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.
    • 44. Extrahepatic Effects of HCV B-Cell Lymphoma Ferri (1994) 8 case series 1754 pts evaluated Mazzaro (1996) Silvestri (1996) Izumi (1996) McColl (1996) Zignego (1997) DeRosa (1997) Zuckerman (1997) 30 20 10 0 B Cell Lymphoma 10 20 Controls 30
    • 45. Three Signals Are Required to Activate Human Naïve B Cells (1) Ag (1’) Costimulation p-MHC Ag tlr (2) Cognate T cell help CD40 Proliferation Survival (3) Innate immunity derived signals Ruprecht & Lanzavecchia, Eur J Immunol 2006;36:810-6
    • 46. Molecular Mimicry CYP2D6252-71: Major epitope of LKM1 DELLTEHRMTWDPAQPPRDL 252 271
    • 47. Multiple Hits Hypothesis Protein Database Search DRLSPRPPAQPPRRR IE 175 HSV-1 EHRMTWDPAQPPRDL CYP2D6257-271 E1 HCV EBNA-2 Epstein-Barr GHRMAWDMMMNWSPT QLPPPAAPAQPPPGV PMIAAAPPAQPPSQP AARTAPAPAQPPSPA IE63 H. Cytomegalovirus J1L H. Cytomegalovirus
    • 48. Background  Chronic HCV infection is associated with extrahepatic manifestations including autoantibody production and abnormal B-cell proliferation
    • 49. Functional Studies on Memory B-Cells in Chronic HCV Infection PBMC 500-3,000/w Anti-CD40+IL10 CpG 2006 7 d ± IL2 6 d Ig producing-cells (ELISPOT)
    • 50. Molecular Mimicry  LKM-1 targets CYP2D6  CYP2D6 shares an amino acid sequence with HCV  LKM-1 may arise through a mechanism of molecular mimicry
    • 51. 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 2/17 (11.8%) genotype 2a/c 2/34 (5.9%) genotype 1b 1/16 (6.25%) genotype 1b Bianchettin G et al., J Virol 2007;81:4564-71 .
    • 52. Human Mature B Cell Phenotype B cell areas of lymph nodes and spleen IgMhi,IgDhi CD19,CD20,CD21hi CD38lo, ABCB1(?) Blood Naïve IgM, IgG, IgA, IgDlo CD19,CD20,CD27 CD38 CD126 CD138 Memory Plasmacell CD19lo,CD20lo Plasmablast IgG,CD69, CD86… Ab Bone marrow and inflamed tissues
    • 53. % CD27+ & CD27B cells Proportions of Total and Memory (CD27+) and Naïve B Cells in Healthy Controls and Patients with Chronic HCV or HBV Infection 100 % CD19+ cells 30 20 10 0 Controls HCV Healthy controls (n=36) 75 50 25 0 CD27+ HBV HCV+ patients (n=50) CD27- HBV+ patients (n=22)
    • 54. Eradication of HCV Infection Associates with a Decreased Expression of Activation Markers and CXCR3 Rosa et al., PNAS 2005;102:18544-
    • 55. Stimulation with CpG Oligonucleotides Is Sufficient for Optimal Expansion of Memory B Cells CpG Media CD19+ CD19+/CD27+ CD19+/CD27-
    • 56. 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.
    • 57. CIDENCE OF THYROID DYSFUNCTION IN HCV PATIENT TREATED WITH IFNα Author Treated Thyroid dysfunction Marcellin ’95 Okanoue ’96 Koh ’97 Kakizaki ‘’99 Dumolin ’99 Wong ’02 248 677 1043 439 598 246 21 (8.5%) 18 (2.7%) 69 (6.6%) 17 (3.9%) 60 (10%) 9 (3.6%) Total 3251 194 (6.0%)
    • 58. RISK FACTORS FOR DEVELOPMENT OF THYROID DYSFUNCTION DURING IFNα THERAPY Risk factors Treated Thyroid dysf. R.R. (C.I.) Sex Females Males 1176 1774 153 53 4.4 (3.2 – 5.9) TPO-Ab Positive Negative 38 428 8 23 3.9 (1.9 – 8.1) Marazuela ’96; Fernandez-Soto ’98; Hsieh ’00; Vial ’95;Okanoue ’96; Koh ’97; Kakizaki ’99; Deutsch ’97; Roti ‘96
    • 59. 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
    • 60. Chronic HCV and Diabetes Mellitus Case Prevalence 20 Observed Expected • Number of Cases 16 • • 12 8 4 • 0 Females Males Zein CO, et al. Am J Gastroenterol. 2005;100:48-55. 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
    • 61. 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
    • 62. Enlarged Pericapsular Lymph Node in a Patient with Chronic HCV Infection >CD20+ B cells
    • 63. Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HBV or HCV Infection and Healthy Controls p=0.0013 p=0.0002 80 60 40 20 0 p=0.0152 p=0.0031 80 p=0.0002 60 40 20 0 p=0.001 p<0.0001 p<0.0001 p=0.032 100 % CD71 B cells % CD183 B cells 100 p=0.0001 p=0.012 p=0.0078 100 p=0.0007 % CD86 B cells % CD69 B cells 100 p=0.0009 80 60 40 20 80 p=0.0004 p=0.021 p=0.0019 60 40 20 0 0 Total CD27+ Healthy controls (n=36) CD27HCV+ patients (n=50) Total CD27+ HBV+ patients (n=22) CD27-
    • 64. Proportions of CXCR3+ Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HCV Infection and Healthy Controls p < 0.0001 p < 0.0001 %CD183 B cells 100 80 60 40 20 0 TOT TOT CD27+ CD27+ CD27- CTRLS HCV+ PTS n=33 CD27- n=29 Cerino et al., in preparation
    • 65. % CD183/CD19+ cells Correlation between Serum HCV RNA and CD183 (CXCR3) Expression on B cells 100 Speaman r=0.408 75 p=0.012 50 25 0 0 1 2 3 4 15 5 16 % CD183/CD19+27+ cells 100 Speaman r=0.59 75 p=0.0001 50 25 0 0 1 2 3 4 5 HCV RNA (IU/ml x 10 6 ) 15 16
    • 66. N. of IPC-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls Following Stimulation with CD40L ± IL10 900 IgG SFC/105 PBC p=0.0193 450 p=0.0128 0 Media CD40L+IL10 900 IgA SFC/105 PBC SFC/105 PBC 900 CD40L 450 IgM 450 0 0 Media CD40L CD40L+IL10 Healthy Controls (n=31) Media HCV Patients (n=33) CD40L CD40L+IL10
    • 67. N. of IPC-Producing Cells in Patients with Chronic HCV Infection and Healthy Controls Following Stimulation with CpG ± IL2 2000 IgG SFC/105 PBC p=0.0202 1000 0 Media 1000 0 Media CpG+IL2 2000 IgA SFC/105 PBC SFC/105 PBC 2000 CpG CpG CpG+IL2 Healthy Controls (n=41) IgM 1000 0 Media HCV Patients (n=49) CpG CpG+IL2
    • 68. Proportions of Activated Memory (CD27+) and Naïve (CD27-) B-Cells in Patients with Chronic HCV Infection and Healthy Controls p= 0.0049 p= 0.0004 %CD69 B cells 100 80 60 40 20 0 TOT TOT CD27+ CTRLS n=33 CD27+ CD27- CD27- HCV+ PTS n=29 Cerino et al., in preparation
    • 69. …an autoimmune disease is a viral disease in which the virus is unknown. Rolf Zinkernagel - Immunol Rev. 1996;152:21-45