Specific cellular immune response and cytokine patterns in patients coinfected with hepatitis C viru

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Specific cellular immune response and cytokine
patterns in patients coinfected with hepatitis C
virus and Schistosoma mansoni
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SANAA M. KAMAL, LEONARDO BIANCHI, AHMED AL
TAWIL, MARGARET KOZIEL, KHALIFA EL SAYED KHALIFA,
THOMAS PETER, and JENS W. RASENACK,
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• Schistosomiasis and HCV coinfection is common in
  Egypt
• Patients coinfected with HCV and schistosomiasis
  exhibit a unique clinical virological and
  histological pattern manifested by viral
  persistence with high HCV RNA titers, as well as
  higher necro-inflammatory and fibrosis scores in
  their liver biopsy samples.

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AIM OF THE WORK
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• The study was done to investigate the influence
  of S. mansoni on the HCV-specific cellular
  immune response by
• Proliferative response of peripheral blood
  mononuclear cells (PBMCs) to HCV and
  schistosomal. antigens.
• Determination of proliferative response cells
  subtypes.
• Cytokines (TNF-?, INF-?, IL-4, IL-10) production
  by PBMCs in response to HCV and schistosomal.
  antigens.
• Cytokines levels in the patients sera.
• Correlations between the proliferative response
  and cytokines production and the
  necroinflammatory changes in the liver.

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• MATERIALS AND METHODS

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PATINETS

• The study was done on 85 patients and 10 health
  control subjects
• Group A 30 patients chronically infected with
  HCV
• Seropositive for HCV antibody.
• Positive for HCV RNA by PCR.
• Elevated ALT and AST for more than 6 months.
• Liver biopsy showing evidence of chronic
  hepatitis.

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PATINETS

• Group B included 20 patients infected with
  schistosomiasis alone.
• History of schistosomiasis.
• Detection of S. mansoni ova in stool or rectal
  biopsy.
• Seropositivty for schistosomal antibodies by
  Indirect Haemagglution Assay (IHA)
• Group C included 35 patients with chronic HCV
  and S. mansoni coinfection. All the patients
  acquired schistosomiasis before HCV.

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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 control subjects were
  isolated and cultured in triplicates 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 for 5 days 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

• 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.
• Cells cultured with the respective antigen or
  mitogen were incubated for 30 minutes with 2
  µg/ml of the monoclonal antibodies, washed twice
  with cold PBS and then analyzed by flow cytometry
  in a fluorescent activated cell sorter.

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Cytokines Measurement

• 48 hours supernatants of mitogen or antigen-
  stimulated PBMCs were collected.
• Sera form the three groups under study were
  collected.
• The supernatants and sera were stored at 85?C
  until assayed for TNF-?, INF-?, IL-4, IL-10 using
  commercial ELISA Kits according to the
  manufacturers instructions.

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Histological Assessment

• Liver biopsy samples were stained with H and E
  and a connective tissue stain.
• The samples were read in a blinded fashion.
  Necroinflammatory changes were graded with a
  staging score of 0 - 18. Fibrosis was evaluated
  with a staging score of 0 - 6.
• The correlation between the proliferative
  response and cytokines production, and the
  necroinflammatory changes and fibrosis of the
  liver were studied.

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RESULTS
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Demographic and baseline characteristics of
patients with HCV or/and S. mansoni
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Percentages of proliferative PBMCs, from the
groups under study, in response to HCV proteins,
schistosomal antigens and PHA
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Stimulation indices of proliferative PBMCs, from
the groups under study, in response to HCV
proteins, schistosomal antigens and PHA
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Relation of HCV specific CD4 proliferative
response to HCV RNA virus load
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INF-? production by PBMCs, from the groups under
study, in response to HCV proteins, schistosomal
antigens and PHA
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TNF-? production by PBMCs, from the groups under
study, in response to HCV proteins, schistosomal
antigens and PHA
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IL-4 production by PBMCs, from the groups under
study, in response to HCV proteins, schistosomal
antigens and PHA
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IL-10 production by PBMCs, from the groups under
study, in response to HCV proteins, schistosomal
antigens and PHA
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Cytokines levels in the sera of the patients
groups (A, B, C) and control subjects (N)
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Correlation between cytokines, and
necroinflammatory and fibrosis scores
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CONCLUSION
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• Coinfected patients showed significant
  qualitative and quantitative suppressed cellular
  response to HCV antigens, when compared to
  patients with HCV alone.
• Coinfected patients mounted strong qualitative
  and quantitative cellular response to SEA and
  SWAP that was not significantly different from
  the responses in patients infected with S.
  mansoni alone.
• Study of the cell subtypes showed that the
  proliferative response was of CD4 subtype

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• Patients showing positive HCV-specific cellular
  responses had lower mean virus load.
• PBMCs stimulation with HCV antigen produced a
  type 1 cytokines (INF-? and TNF-?) profiles in
  patinets infected with HCV alone, compared with a
  type 2 (IL-4 and IL-10) predominance in patients
  coinfected with HCV and S. mansoni.
• On the other hand, stimulation of PBMCs with SEA
  and SWAP resulted in a predominant type 2
  cytokines profile (IL-4 and IL-10) in coinfected
  and isolated S. mansoni infected patinets.

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• Sera of patients with isolated HCV infection
  showed type 1 cytokines profile compared with a
  type 2 predominance in patients coinfected with
  HCV and S. mansoni.
• A significant correlation was found between
  TNF-? and the necroinflammatory scores in
  patients with isolated HCV infection, such
  correlation was not apparent among Coinfected and
  Isolated S. mansoni infected patients.
• On the other hand, there was a significant direct
  correlation between IL-4 levels and the
  fibrosis/cirrhosis score and an inverse
  correlation between INF-? levels and
  fibrosis/cirrhosis score.

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• These findings demonstrate that the inability to
  mount an HCV-specific CD4/Th1 cell response
  plays a role in the persistence and severity of
  HCV infection in patients with S. mansoni
  coinfection

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THANK YOU