Acute hepatitis C without and with schistosomiasis: correlation with hepatitis Cspecific CD4 T cell

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Acute hepatitis C without and with
schistosomiasis correlation with hepatitis
C-specific CD4 T -cell and cytokine response
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SANAA M. KAMAL, JENS W. RASENACK, LEONARDO
BIANCHI, AHMED AL TAWIL, KHALIFA EL SAYED
KHALIFA, THOMAS PETER, HODA MANSOUR, WAFAA EZZAT,
and MARGARET KOZIEL
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• Schistosomiasis and HCV coinfection is common in
  Egypt
• Patients coinfected with HCV and schistosomiasis
  have higher incidence of cirrhosis and
  hepatocellular carcinoma than patients with
  chronic HCV monoinfection matched for age,
  disease duration, and HCV genotype.
• Several studies in individuals who experienced
  complete virologic recovery have found a
  significant association between a strong and
  maintained HCV-specific CD4 T-cell response and
  viral clearance in acute hepatitis C.

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• Morbidity in humans infected with S. mansoni and
  in experimental schistosomiasis results primarily
  from deposition of parasite ova in the portal
  areas with granuloma formation.
• Irreversible fibrosis and severe portal
  hypertension occurs in more than 60 of cases.
• The severity of disease appears to be regulated
  by the balance of Th1- versus Th2-type cytokines.
  Established S. mansoni infection is characterized
  by a strong Th2 immunologic bias.

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AIM OF THE WORK
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• To characterize the kinetics of HCV-specific CD4
  responses and cytokine patterns in patients with
• Isolated acute hepatitis C
• Acute hepatitis C and S. mansoni coinfection in
  whom the immune response biased towards the Th2
  immune response.
• Correlation of these CD4 responses and cytokine
  patterns to the clinical outcome and progression
  of acute hepatitis C.

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• MATERIALS AND METHODS

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PATINETS

• The cohort (follow up study) was done on 32
  patients in whom acute HCV infection was
  diagnosed based on the following criteria
• Sudden onset of malaise, jaundice, fever, and
  other symptoms related to liver disease in a
  previously healthy individual
• Elevated values of ALT 20 times above the upper
  limit of normal (40 U/L)
• Positive anti-HCV antibody
• Positive for HCV RNA by PCR

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PATINETS

• Patients were further classified according to S.
  mansoni status into
• Group A 15 patients with hepatitis C
  monoinfection
• Group B 17 patients with hepatitis C and
  schistosomiasis coinfection, in whom the disease
  was diagnosed.
• History of schistosomiasis.
• Detection of S. mansoni ova in stool or rectal
  biopsy.
• Seropositivty for schistosomal antibodies by
  Indirect Haemagglution Assay (IHA)

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PATINETS FOLLOW UP

• Patients were examined monthly during the first
  6 months, then semiannually until the end of the
  study (72 4.6 months).
• Clinical examination
• Liver enzymes, bilirubin, albumin, PT
• PCR for detection of HCV RNA
• Proliferative response and cytokine production by
  PBMCs on in vitro stimulation with HCV and
  schistosomal antigens
• Liver biopsies for patinets who did not clear the
  infection, 6-8 months after clinical onset and at
  the end of follow up.

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Antigens and Mitogens

• HCV antigens
• purified recombinant core antigen, nonstructural
  antigen (NS) 3, 4 and 5
• Soluble egg antigen (SEA)
• Soluble adult worm antigen protein (SWAP)
• Non-specific control positive antigens
  Phytohemagglutinin (PHA) and tetanus toxoid.
• Negative control antigen
• superoxide dismutase (SOD))

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Proliferation Assay

• PBMCs from patients and 20 control subjects were
  isolated and cultured in triplicates for 5 days
  in 96well plates in the presence of HCV proteins
  (10 µg/ml), schistosomal antigens (15-50 µg/ml),
  PHA (1/200 dilution) and SOD as a negative or no
  stimulation control. Cultures were incubated at
  37?C in a humidified atmosphere with 5 Co2.
• The amount of 3H-thymidine incorporated by
  cells was determined by liquid scintillation
  counting. Results were expressed as the
  stimulation index
• SI counts per minute incorporated (cpm) in
  response to antigen/cpm incorporated in the
  absence of the antigen.
• SI exceeding 3 SD above the mean SI of healthy
  control subjects was considered positive.

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Determination of cell subtypes

• CD4 and CD8 Cell depletion proliferation assay
  was performed before and after CD4 and CD8
  depletion on para magenetic microbeads conjugated
  to a monoclonal mouse antihuman-CD4 and
  antihuman-CD8.
• Determination of CD4 and CD8 subtypes was
  performed by using flow cytometry after staining
  of the cells with isothiocynate-labeled anti-CD4
  and anti-CD8 antibodies.

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Cytokines Measurement

• 48 hours supernatants of mitogen or
  antigen-stimulated PBMCs were collected and
  stored at 85?C until assayed for INF-?, and
  IL-10 using commercial ELISA Kits according to
  the manufacturers instructions.

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Histological Assessment

• Liver biopsies were performed only for patients
  who did not clear the infection (PCR).
• Samples were taken within 6-8 months after
  clinical onset and at the end of follow up period
• Samples were stained with H and E and a
  connective tissue stain and examined in a blinded
  fashion by 2 pathologist using the grading and
  scoring system.
• Biopsy specimens were also assessed for
  morphologic features of schistosomiasis.

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RESULTS
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Demographic baseline characteristics of patients

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Characteristics of patients with self limited or
chronic monoinfection and chronic coinfection
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Relation of HCV specific CD4 proliferative
response to clinical, virologic and cytokines
profiles in patients with self limited HCV
infection
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Relation of HCV specific CD4 proliferative
response to clinical, virologic and cytokines
profiles in HCV patients with chronic evolution
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Relation of HCV specific CD4 proliferative
response to clinical, virologic and cytokines
profiles in coinfected patients
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Relation of HCV specific CD4 proliferative
response to clinical, virologic and cytokines
profiles in patients of groups A, B and C
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Relation of HCV specific CD4 proliferative
responses to viral load and fibrosis progression
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INF-? production by PBMCs, from the groups under
study, in response to HCV proteins during the
acute and at 48 weeks
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IL-10 production by PBMCs, from the groups under
study, in response to HCV proteins during the
acute and at 48 weeks
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Histological Assessment
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CONCLUSION
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• 30 (5 out of 15) with acute HCV monoinfection c
  leared the virus
• None of the coinfected patients achieved viral
  clearance
• Monoinfected patients who cleared the infection
  mounted early and strong CD4 proliferative
  response with early production of Th1 cytokine
  INF-? that peaked 6-12 weeks after infection and
  maintained throughout the follow up.
• Monoinfected patients with chronic evolution
  mounted less vigorous initial and transient CD4
  proliferative response that failed to clear the
  infection with production of lower amounts of the
  type 1 cytokine INF-? that switched to Th0 (INF-
  ?, IL10) or to type Th2(IL-10)

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• Few coinfected patients showed weak transient
  proliferative response, but most failed to mount
  any response to HCV antigens
• Coinfected patients mounted strong proliferative
  response to SEA and SWAP.
• Coinfected patients who mounted weak
  proliferative response to HCV antigens produced
  Th0 (INF-?, IL-10) that switched gradually toTh2.
• Most coinfected patients produced predominant Th2
  cytokines in response to HCV and schistosomal
  antigens.

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• Patients showing positive HCV-specific cellular
  responses had lower mean virus load and lower
  fibrosis progression rates.
• Both parameters showed significant inverse
  correlation with CD4 proliferative responses.
• At baseline biopsy both mono- and coinfected
  patients had no fibrosis with no significant
  difference in the necroinflammatory score.
• At the end of the follow up coinfected patients
  had significant higher fibrosis scores than
  monoinfected (4.3 0.9, 0.8 0.5, respectively)
  and higher fibrosis progression rates (0.53
  0.13, 0.1 0.06, respectively).

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• These findings demonstrate that patients with
  acute HCV and schistosomiasis coinfection cannot
  clear viremia and show rapid progression to
  fibrosis once chronic infection is established.
  This rapid progression is due to the strong
  schistosoma-induced Th2 response in peripheral
  immune responses.

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