Liver-infiltrating CD56 positive T lymphocytes in hepatitis C virus infection

AIM: Hepatitis C virus (HCV) is a major cause of post-transfusional and sporadic hepatitis, and leads to chronic liver disease. It has been suggested that virus-specific cytotoxic T lymphocytes are responsible for liver injuries that occur in HCV-infected patients. However, the detailed characteristics of these lymphocytes have not yet been defined. We have previously reported that CD56+ T lymphocytes, as intermediates between natural killer cell and T lymphocytes, predominantly infiltrated the liver and were increased in patients with chronic hepatitis related to HCV (CH-C). MATERIAL AND METHODS: We obtained peripheral blood and liver tissues from 32 patients diagnosed as having CH-C, and 10 other liver disease patients (5 chronic hepatitis related to HBV, 5 alcoholics), and analyzed peripheral blood and liver-infiltrating lymphocytes using flow cytometric and immunohistochemical techniques. RESULTS: The CD56+ T lymphocyte ratio in the liver of patients with a high histology activity index (HAI) score for chronic hepatitis was higher than that of patients with a low HAI score and patients with other liver diseases. In addition, T lymphocytes from patients with chronic hepatitis with a high HAI score carried mostly gamma delta-TCR. There was a correlation between the ratio of CH-C and serum alanine aminotransferase, category I (periportal inflammation and necrosis), and IV (fibrosis) of the HAI scoring system. The ratio was highest in zone 1 of the hepatic lobules. CONCLUSION: The correlation between CD56+ T lymphocyte ratios and hepatocellular damage was examined. These findings suggest strongly that liver-infiltrating CD56+ T lymphocytes play an important pathologic role in hepatocellular injury in CH-C.     

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV‐coinfected patients

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV‐infected and HIV/HCV‐coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy‐derived liver‐infiltrating lymphocytes from 26 HIV/HCV‐coinfected, 10 chronic HCV‐infected and 10 HIV‐infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T‐cell frequency by flow cytometry. CD8⁺ T cells expressing the exhaustion marker PD‐1 were increased in HCV‐infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4⁺CD25⁺Foxp3⁺ T cells, as well as CD4⁺CD25⁺PD1⁺ T cells, were more frequent in HIV/HCV‐coinfected than in HCV‐monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD‐1⁺ T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8⁺ expression was observed only in PD‐1⁺CD8⁺ T cells from HCV‐infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8⁺ T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.     

Compartmentalization and its implication for peripheral immunologically‐competent cells to the liver in patients with HBV‐related acute‐on‐chronic liver failure

Aims: This study attempts to characterize the feature of immunologically competent cells (ICCs) and evaluate its clinical implication in patients with acute‐on‐chronic liver failure (ACLF) in relation to chronic hepatitis B virus (HBV) infection. Methods: Circulating ICCs were examined in ACLF patients (n = 75), as well as in patients with hepatitis B (CHB, n = 31), CHB‐related liver cirrhosis (LC, n = 36), and normal controls (NC, n = 30). Intrahepatic ICCs in some patients were further analyzed via immunohistochemical and flow cytometric assays. Results: Total lymphocytes, CD4⁺ T cells, CD8⁺ T cells, and NK cells in circulation were numerically lower in the ACLF and LC groups compared to the CHB and NC groups. Importantly, the number of these cells was significantly lower in non‐surviving ACLF patients compared with surviving ACLF patients. In comparison to NC, ACLF patients displayed a significantly higher ratio of liver‐infiltrating CD4⁺ T‐cell frequency than its circulating counterpart, suggesting that the possiblility of the ICCs compartmentalization from the peripheral blood into the liver in ACLF. Immunohistochemical analysis showed that intrahepatic CD4⁺ cells, CD8⁺ cells, and CD56⁺ cells were significantly higher in the ACLF group compared with the other three groups, suggesting a stronger cellular immune response‐mediated inflammation in ACLF group than other patient groups. Conclusions: The abnormal prevalence of circulating and intrahepatic ICCs possibly acts as an important factor that may drive the progression of HBV‐related ACLF.     

Hepatitis C viraemia reversibly maintains subset of antigen‐specific T‐bet+ tissue‐like memory B cells

Background: Chronic antigen exposure and/or ageing increases the frequency of T‐box expressed in T cells (T‐bet)‐expressing B‐lymphocytes in mice. The frequency and significance of B‐cell T‐bet expression during chronic hepatitis C (HCV) infection in human subjects has never been described. Methods: Healthy controls, cirrhotic and noncirrhotic HCV‐infected patients, and non‐HCV patients with cirrhosis were recruited. Peripheral blood mononuclear cells were phenotyped for expression of T‐bet and related markers by flow cytometry. In a subset of patients who underwent antiviral therapy and were cured of HCV infection (sustained virological response), the dynamics of T‐bet expression in B cells was monitored. After cure, convalescent B cells were tested for T‐bet expression after re‐exposure to infected plasma or recombinant HCV proteins. Results: Forty‐nine patients including 11 healthy donors, 30 hepatitis C‐infected individuals (nine with liver cancer, 13 with cirrhosis, eight without cirrhosis) and eight patients with cirrhosis due to non‐HCV‐related cause were recruited. We found that B cells in patients with chronic HCV exhibited increased frequency of T‐bet+ B cells relative to noninfected individuals (median 11.5% v. 2.2%, P<.0001) but that there were no significant differences between noncirrhotic, cirrhotic and cancer‐bearing infected individuals. T‐Bet⁺ B cells expressed higher levels of CD95, CXCR3, CD11c, CD267 and FcRL5 compared to T‐bet^? B cells and predominantly exhibit a tissue‐like memory CD27^?CD21^? phenotype independent of HCV infection. T‐bet⁺ B cells in HCV‐infected patients were more frequently class‐switched IgD^?IgG⁺ (40.4% vs. 26.4%, P=.012). Resolution of HCV infection with direct‐acting antiviral (DAA) therapy leads to a marked reduction in the frequency of T‐bet⁺ B cells (median 14.1% pretreatment v. 6.7% end of treatment v. 6.1% SVR12, P≤.01). Re‐exposure of convalescent (cured) B cells to viremic plasma and recombinant HCV E2 protein led to re‐expression of T‐bet. Conclusion: Chronic antigenemia in chronic HCV infection induces and maintains an antigen‐specific T‐bet⁺ B cell. These B cells share markers with tissue‐like memory B cells. Antigen‐driven T‐bet expression may be a critical suppressor of B‐cell activation in chronic HCV infection.     

Impact of oral silymarin on virus‐ and non‐virus‐specific T‐cell responses in chronic hepatitis C infection

Silymarin displays anti‐inflammatory effects on T lymphocytes in vitro. The immunomodulatory properties of oral silymarin in vivo in humans with chronic hepatitis C have not previously been characterized. We hypothesized that silymarin would suppress T‐cell proliferation and pro‐inflammatory cytokine production of virus‐ and non‐virus‐specific T cells while increasing anti‐inflammatory IL‐10 production in vivo. Patients from one site of the SyNCH‐HCV double‐masked, placebo‐controlled study of oral silymarin in prior interferon nonresponders with chronic hepatitis C provided blood samples at baseline and treatment week 20. Mononuclear cells were stimulated with recombinant HCV proteins and controls in ³H‐thymidine proliferation assays, IFNγ ELISPOT and IL‐10 ELISPOT. The frequency of CD4⁺CD25^hi and CD4⁺foxp3⁺ regulatory T cells, serum cytokine levels, serum IP‐10 and lymphocyte interferon‐stimulated gene expression were also quantified at baseline and week 20. Thirty‐two patients were recruited (10; placebo, 11; 420 mg three times a day, 11; 700 mg three times a day). Serum ALT and HCV RNA titres did not change in any group. HCV‐specific CD4⁺ T‐cell proliferation and the frequency of IFNγ‐ and IL‐10‐producing T cells were not significantly changed in silymarin‐treated subjects. However, C. albicans‐induced T‐cell IFNγ and phytohaemagglutinin‐induced T‐cell proliferation were suppressed by silymarin therapy. A trend towards augmentation of interferon‐induced ISG15 expression was present in the high‐dose silymarin group. While no effect on HCV‐specific T cells was identified, these data confirm that high‐dose oral silymarin exerts modest nonspecific immunomodulatory effects in vivo. The impact of this anti‐inflammatory effect on long‐term liver health in chronic hepatitis C merits future clinical investigation.     

Expansion of CD4+CD25+FoxP3+ regulatory T cells in hepatitis C virus‐related chronic hepatitis,cirrhosis and hepatocellular carcinoma

Aim: Regulatory T (Treg) cells may play a pivotal role in the persistence of hepatitis C virus (HCV) infection and the development of hepatocellular carcinoma (HCC). Therefore, we examined their frequency in peripheral blood from patients with HCV‐positive chronic hepatitis (CH), cirrhosis (LC) and HCC. Methods: Treg cells were identified as CD4⁺, CD25⁺ and FoxP3⁺ T lymphocytes using three‐color FACS. The frequency of Treg cells was expressed as a percentage of the total CD4⁺ T lymphocytes, and the phenotype of Treg cells was examined using CD45RA. Results: Treg cells were significantly increased in CH (5.88 ± 0.19%, n = 76; P < 0.01), LC (6.10 ± 0.28%, n = 40; P < 0.001) and HCC (6.80 ± 0.30%, n = 57; P < 0.0001) compared to healthy control (5.13 ± 0.25%, n = 31). However, Treg cells were not increased with the progression of fibrosis or the grade of inflammations. Treg cells were slightly increased in early‐stage HCC (6.91 ± 0.40%) compared with advanced‐stage HCC (6.58 ± 0.39%), but these results were not statistically significant. In a serial examination, a distinct increase in Treg cells after local therapy for early‐stage HCC was a hallmark of early recurrence. Most expanded Treg cells in HCC were CD45RA^‐, suggesting that a memory‐type Treg population had differentiated in the periphery and not in the thymus. Conclusion: We observed an increase in Treg cells in HCV‐related chronic liver disease, particularly in HCC, and these cells were shown to be memory‐type Treg cells.     

IL28B genetic variation and hepatitis C virus–specific CD4+ T‐cell responses in anti‐HCV‐positive blood donors

Summary. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome‐wide association studies have shown a strong correlation between single‐nucleotide polymorphisms (SNPs) near the interleukin‐28B (IL28B) gene and spontaneous or treatment‐induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV‐specific T‐cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti‐HCV‐positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV‐specific CD4⁺ T‐cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon‐γ ELISpot assay. The rs12979860‐CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV‐specific CD4⁺ T‐cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T‐cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T‐cell responses were only observed among those with non‐CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4⁺ T‐cell responses towards NS3 were only evident among those with non‐CC haplotypes.     

Study of liver‐targeted regulatory T cells in hepatitis B and C virus in chronically infected patients

Introduction: Regulatory T cells (Tregs) play a critical role in chronic viral infections. The role of Tregs in chronic hepatitis B (CHB) and chronic hepatitis C (CHC) is unknown. This study examined the distribution and frequency of forkhead box p3⁺ (Foxp3⁺) Tregs in the liver tissue and compared the clinicopathological characteristics of CHB and CHC patients. Methods: Liver needle biopsies were obtained from 26 patients who were hepatitis B surface antigen positive and 27 patients who were hepatitis C virus antibody positive. Results: The ratio of Foxp3⁺ Tregs in CD3⁺ T cells was similar in HBV and in HCV cases. In HBV cases, the variables that were positively associated with the ratio of Foxp3⁺ Tregs in CD3⁺ T cells included the serum alanine aminotransferase level (R=0.402, P=0.025) and the ratio of CD8⁺ T cell plus CD56⁺ NK cell against CD4⁺ T cell (R=0.53, P=0.005). The ratio of Foxp3⁺ Tregs in CD3⁺ T cells increased more in the severe activity group than in the mild activity group (P=0.04). In HCV cases, the ratio of Foxp3⁺ Tregs in CD3⁺ T cells increased significantly in terms of the genotype2 (P=0.0002) and male gender (P=0.04). In addition, the ratio of Foxp3⁺ Tregs in CD3⁺ T cells showed a negative correlation with the ratio of CD8⁺ T cell plus CD56⁺ NK cell against CD4⁺ T cell (R=?0.508, P=0.005) and HCV viral load (R=?0.482, P=0.001). Conclusions: Liver‐targeted regulatory T cells present similarly in CHB and CHC, but their relationship with the effector cell population, the inflammation grade or the viral load is different between CHB and CHC.     

Oral anti‐CD3 immunotherapy for HCV‐nonresponders is safe,promotes regulatory T cells and decreases viral load and liver enzyme levels: results of a phase‐2a placebo‐controlled trial

Orally administered anti‐CD3 antibodies are biologically active in the gut through induction of regulatory T cells, exert an immune‐modulatory effect, and alleviate insulin resistance and liver damage in patients with NASH. Aims: To determine the safety of oral anti‐CD3 monoclonal antibody (MAb) immunotherapy in chronic HCV patients with associated immune dysfunction. Methods: Four groups (n = 9) of chronic HCV patients who were nonresponders to interferon plus ribavirin therapy received oral placebo (group A) or anti‐CD3 MAb at one of three dosage levels for 30 days. Patients were followed for safety parameters and serum levels of liver enzymes, virus, cytokines and regulatory T cells. Results: Oral anti‐CD3 immunotherapy was safe and well tolerated; no treatment‐related adverse events were noted. The following improvements were noted relative to pretreatment levels: HCV viral load and AST and ALT levels decreased in the low‐ and high‐dose groups following 30 days of therapy. In two of the treated groups, an increase in regulatory T cells (CD4⁺ CD25⁺) was noted. The positive effects were somewhat more apparent in subjects with initially elevated liver enzyme levels. Conclusions: Oral anti‐CD3 MAb immunotherapy for nonresponder HCV patients was safe and well tolerated. Trends and statistically significant improvements were observed as reductions in viral load and liver enzyme levels, along with an increase in regulatory T‐cell levels. These data support a role for the immune system in the pathogenesis of HCV infection and suggest that this immunotherapy is worthy of evaluation in combination with HCV antiviral drugs.     

In situ expression of Granzyme B and Fas‐ligand in the liver of viral hepatitis

Abstract: Background/Aims: The molecular mechanism involved in hepatocellular injury in viral hepatitis remains to be clarified. Methods: We investigated the in situ expression of effector molecules of cytotoxic T lymphocytes such as Fas‐ligand (Fas‐L), perforin and Granzyme B (Gr‐B) immunohistochemically in liver tissues from 20 patients with chronic hepatitis B (CHB) and C (CHC). The degree of cell infiltration was analysed semi‐quantitatively and compared with the histological activity index (HAI). Fas‐L was expressed in both CD4 and CD8 T‐cells in the portal tract as well as in the parenchyma. Results: Immunostaining of serial sections demonstrated that mononuclear cells at interface hepatitis and focal necrosis were mainly Fas‐L positive CD8 T‐cells. On the other hand, the expression of perforin or Gr‐B was limited to a few mononuclear cells in the portal tract and parenchyma. Semi‐quantitative analysis showed a positive correlation between HAI and the grade of infiltration of CD8 T‐cells or Fas‐L‐positive cells, while the correlation was not apparent between HAI and the number of Gr‐B positive cells. The expression of these molecules was not different between types of viruses. Conclusions: These results suggest that Fas‐L‐positive CD8 T‐cells play a major role in the pathogenesis of liver cell injury in chronic hepatitis.     

Serum HCV RNA levels correlate with histological liver damage and concur with steatosis in progression of chronic hepatitis C

The role of HCV RNA levels and host factors in the severity of liver injury was studied. Enrolled were 298 consecutive liver biopsy-proven chronic hepatitis (CH) C patients (179 men; median age: 52 years, range 19–68; CH, 198; cirrhosis, 100) and 18 chronic hepatitis C with normal ALT. HCV genotypes were: 1a, 4.3%; 1b, 53%; 2a/c, 28%; 3a, 7%; 4, 1.3%, and mixed 6.4%. Serum HCV RNA levels were similar for all genotypes (median: 2.8 × 10⁶ eq/ml; range <0.2–69). In patients with chronic hepatitis without cirrhosis, the serum HCV RNA levels reflected the grade of liver necroinflammatory activity (R = 0.45; P < 0.001) and the stage of fibrosis (R = 0.51; P < 0.001), regardless of age, gender, HCV genotype, hepatic steatosis, and hepatic iron overload. Patients with high serum HCV RNA levels (3 × 10⁶ eq/ml) had higher ALT values (P < 0.002) than those with lower HCV RNA levels. Patients with normal ALT showed low HCV RNA levels (median: 0.82 × 10⁶ eq/ml) and histological features of minimal or mild chronic hepatitis. Cirrhotic patients showed significantly lower levels of viremia than those with chronic hepatitis with a similar HAI. The data of a subgroup of 62 patients with an established time of infection showed that for a similar duration of disease, patients with serum HCV RNA levels 3 × 10⁶ eq/ml had a significantly higher fibrosis score than those with lower levels. HAI and fibrosis score were significantly higher in patients with HCV RNA levels 3 × 10⁶ eq/ml and grade 3–4 steatosis than those with lower HCV RNA levels and steatosis grades. The data indicate that the liver damage is correlated with the HCV RNA levels and that a high viral load acts together with steatosis in accelerating the progression of liver injury.     

Ribavirin downmodulates inducible costimulator on CD4+ T cells and their interleukin-10 secretion to assist in hepatitis C virus clearance

Background and Aim: The immunological mechanism by which ribavirin (RBV) polarizes the T‐helper (Th) 1/2 balance toward Th1 predominancy is not fully understood. We therefore examined whether RBV affects costimulatory signaling, which is known to be essential for regulating the Th1/2 balance. Methods: The expression of costimulatory molecules and their ligands, and levels of various cytokines, released from CD4⁺ T cells obtained from healthy individuals or patients with chronic hepatitis C virus (HCV) infection were analyzed. Results: In CD4⁺ T cells, RBV selectively downmodulates the expression of inducible costimulator (ICOS), a ligand for B7–H2 on dendritic cells, which mainly differentiates Th0 into Th2 cells. Moreover, the levels of interleukin‐10 (IL‐10) released from RBV‐stimulated CD4⁺ T cells also decreased, indicating that the downmodulation of ICOS induced by RBV might be correlated with the decrease in IL‐10 released from Th cells, leading to the inhibition of Th2 activity. An analysis of the association between ICOS kinetics and hepatitis C virus (HCV) elimination in hepatitis C patients receiving combined pegylated interferon and RBV indicated that HCV elimination tended to occur more frequently in patients showing ICOS downmodulation with RBV treatment. A decrease in IL‐10 production by CD4⁺ T cells was also observed in association with ICOS downregulation in patients who succeeded in HCV elimination. Conclusions: The downmodulation of ICOS in correlation with a reduction in IL‐10 produced by CD4⁺ T cells is possibly the immunological mechanism of action of RBV, which polarizes the Th1/2 balance toward a Th1 cytokine profile, thus contributing to the elimination of cells chronically infected with HCV.     

Heterozygosity for the hemochromatosis gene in liver diseases – prevalence and effects on liver histology

Abstract: Background/Aims: The effect of heterozygosity for the C282Y mutation in the HFE hemochromatosis gene on iron accumulation and disease progression in liver disease patients is unclear. Methods: We investigated the prevalence of this mutation in 531 patients and 205 healthy controls. In addition, we assessed the influence of the mutation on liver histology in 34 C282Y heterozygous and 124 age‐, sex‐ and disease‐matched controls without the mutation using the modified HAI and Chevallier score. Results: The highest prevalence of the C282Y mutation was observed in patients with autoimmune hepatitis (17.2%, p<0.01) compared to 6.4% in healthy controls. Heterozygotes with hepatitis C and B virus infection showed higher ferritin and hepatic iron concentrations than patients without the mutation. However, we did not detect significant differences in necroinflammatory or fibrosis scores between carriers of the mutation and controls. Conclusions: There are marked differences in the prevalence of the C282Y mutation in patients with different liver diseases, with the highest prevalence rates in autoimmune hepatitis and PBC. However, the C282Y mutation alone only leads to a mild increase in iron accumulation in the majority of the patients, with the exception of H63D/C282Y compound heterozygotes. We found no evidence for more pronounced fibrosis in C282Y heterozygotes.     

Circulating CD56dim natural killer cells and CD56+ T cells that produce interferon‐γ or interleukin‐10 are expanded in asymptomatic,E antigen‐negative patients with persistent hepatitis B virus infection

Infection with hepatitis B virus (HBV) can result in spontaneous resolution or chronic infection, which can remain asymptomatic or can progress to cirrhosis and/or hepatocellular carcinoma. The host immune response is thought to be a major determinant of the outcome of HBV infection and virus‐specific cytotoxic T lymphocytes (CTL) can mediate immunity against the virus and cause liver pathology. Antigen‐nonspecific innate lymphocytes may also contribute to HBV infection and liver disease, therefore, we examined the frequencies, phenotypes, cytolytic activities and cytokine profiles of circulating natural killer (NK) cells, CD1d‐restricted invariant natural killer T (iNKT) cells and CD56⁺ T cells in 102 asymptomatic HBV‐infected patients and compared them with those in 66 uninfected control subjects. NK cells expressing low levels of CD56 (CD56^dim) and CD56⁺ T cells were significantly expanded in the circulation of HBV‐infected patients compared with control subjects. CD1d expression and iNKT cell frequencies were similar in both groups. Despite these expansions, we did not detect augmented natural or cytokine‐induced cytotoxicity in the HBV‐infected subjects. All lymphocyte populations studied produced interferon‐γ (IFN‐γ) significantly more frequently when taken from HBV‐infected patients compared with when taken from healthy controls. Additionally, NK cells from the patients more frequently produced interleukin‐10. As our HBV‐infected cohort consisted of asymptomatic patients with low viral loads, we propose that CD56^dim NK cells and CD56⁺ T cells control HBV infection by noncytolytic mechanisms.     

Clinical,virological and histopathological features: long‐term follow‐up in patients with chronic hepatitis C co‐infected with S. mansoni

Abstract: Background/Aims: Infection with Schistosoma mansoni is endemic in Egypt leading to hepatic schistosomiasis and eventually portal hypertension. The prevalence of antibodies against hepatitis virus C among Egyptians is 14–51%. The aim of the present study was to investigate the influence of schistosomiasis on chronic hepatitis C with respect to the natural course of the disease, immunology, virology and histology. Patients and Methods: One hundred and twenty‐six Egyptian patients classified into three groups: group A: chronic hepatitis C (n=33); group B: chronic schistosomiasis (n=30) and group C: chronic hepatitis C and chronic schistosomiasis (n=63) were enrolled and prospectively followed for 62.7±22 months. Patients infected with other hepatic viruses and/or parasites were excluded. Detailed history, clinical examination, CD4⁺ and CD8⁺ lymphocyte counts in blood, hematological and blood chemical values, abdominal ultrasonography, upper endoscopy, HCV RNA titer by RT/PCR, genotype and histological activity index in the liver biopsy were determined. Results: Thirty patients (48%) with HCV and schistosomiasis had liver cirrhosis and Child‐Pugh class C vs. five (15%) in HCV patients and none in the schistosomal group. HCV RNA levels ranged between 0.07 and 13×10⁵ copies/ml in group A, and between 1 and 25×10⁵ copies/ml in group C. HCV genotype 4 was detected in 58 patients with co‐infection (92%) and 21 patients with HCV alone (64%). Patients with coinfection showed higher grading and staging scores in their liver biopsies. Hepatocellular carcinoma was detected only in patients with coinfection. During follow‐up, the mortality rate was 12%, 3% and 48% in group A, B and C, respectively. Conclusions: Patients with concomitant HCV and schistosomiasis infection were characterized by more advanced liver disease, higher HCV RNA titers, predominance of HCV genotype 4, higher histologic activity, higher incidence of cirrhosis and hepatocellular carcinoma as well as a much higher mortality rate.     

Persistent hepatitis C virus (HCV) infection impairs HCV‐specific cytotoxic T cell reactivity through Mcl‐1/Bim imbalance due to CD127 down‐regulation

Summary. In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2⁺ HCV⁺ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8⁺/pentamer⁺ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127^low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127^high correlated with undetectable viral loads (P < 0.001). Directly ex vivo, pentamer⁺ cell frequency was similar according to CD127 expression level. Nevertheless, CD127^low pentamer⁺ cell proliferation after specific in vitro challenge was impaired (P < 0.05), although this was corrected by z‐VAD‐fmk treatment (P < 0.05). Mcl‐1 expression was low directly ex vivo (P < 0.01), and Bim was up‐regulated after antigen encounter (P < 0.05) of CD127^low pentamer⁺ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer⁺ cells correlated positively with CD127 expression level (P < 0.001) and with pentamer⁺ cell reactivity (P < 0.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127^low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.     

Polarization of granulocytic myeloid‐derived suppressor cells by hepatitis C core protein is mediated via IL‐10/STAT3 signalling

Myeloid‐derived suppressor cells (MDSCs) have been described as suppressors of T‐cell function in many malignancies. Impaired T‐cell responses have been observed in patients with chronic hepatitis C virus infection (CHC), which is reportedly associated with the establishment of persistent HCV infection. Therefore, we hypothesized that MDSCs also play a role in chronic HCV infection. MDSCs in the peripheral blood of 206 patients with CHC and 20 healthy donors were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) of healthy donors cultured with hepatitis C virus core protein (HCVc) were stimulated with or without interleukin 10 (IL‐10). Compared to healthy donors and certain CHC patients with sustained viral response (SVR), CHC patients without SVR presented with a dramatic elevation of G‐MDSCs with the HLA‐DR^?/lowCD33⁺CD14^?CD11b⁺ phenotype in peripheral blood. The frequency of G‐MDSCs in CHC patients was positively correlated with serum HCVc, and G‐MDSCs were induced from healthy PBMCs by adding exogenous HCVc. Furthermore, we revealed a potential mechanism by which HCVc mediates G‐MDSC polarization; activation of ERK1/2 resulting in IL‐10 production and IL‐10‐activated STAT3 signalling. Finally, we confirmed that HCVc‐induced G‐MDSCs suppress the proliferation and production of IFN‐γ in autologous T‐cells. We also found that the frequency of G‐MDSCs in serum was associated with CHC prognosis. HCVc maintains immunosuppression by promoting IL‐10/STAT3‐dependent differentiation of G‐MDSCs from PBMCs, resulting in the impaired functioning of T‐cells. G‐MDSCs may thus be a promising biomarker for predicting prognosis of CHC patients.     

A prospective study of T‐ and B‐lymphocyte subpopulations,CD81 expression levels on B cells and regulatory CD4+CD25+CD127low/−FoxP3+ T cells in patients with chronic HCV infection during pegylated interferon‐alpha2a plus ribavirin treatment

Summary. Resolution of hepatitis C virus (HCV) infection requires a complex interplay between innate and adaptative immune responses. The role of lymphocyte subpopulations during combined antiviral treatment remains to be defined. This study was conducted to assess the effect of pegylated interferon‐alpha2a (pegIFN‐α2a) and ribavirin treatment on peripheral blood lymphocytes, mainly on CD81 expression on B cells and CD4⁺CD25⁺CD127^low/?FoxP3⁺ regulatory T cells (Tregs) in patients with chronic HCV infection. Thirty‐five patients with chronic HCV infection who started pegIFN‐α2a and ribavirin treatment were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained at baseline before treatment (BT), mid‐treatment (MT), the end of treatment (ET) and 24 weeks post‐treatment (PT). During combined antiviral treatment, a significant decrease in the percentage of CD3⁺, CD8⁺, CD3⁺gamma/delta (γδ)⁺, CD19⁺ lymphocyte subpopulations and Tregs was observed. There was also a significant increase in the percentage of the CD4⁺ lymphocyte subpopulation and in CD81 expression levels on CD19⁺ B cells when BT was compared with ET (all P < 0.05). Seventeen patients were nonresponders (NR) and 18 had a sustained virological response (SVR). At baseline, NR patients had higher CD81 expression levels on CD19⁺ B cells (P = 0.017) and a higher Tregs percentage (P = 0.025) than SVR patients. Our results suggest that immunomodulation fluctuates during antiviral treatment and that percentage CD81 expression levels on B cells and Tregs might be useful as an immunological prognostic factor for pegIFN‐α2a and ribavirin treatment response in chronic HCV infection.     

Expresssion of bcl-2 oncoprotein in cases of acute and chronic viral hepatitis type B and type C: a clinicopathologic study

The bcl-2 gene is involved in the regulation of programmed cell death by providing a survival advantage to rapidly proliferating cells. This study investigates bcl-2 protein expression in liver biopsies with acute or chronic viral hepatitis B (HBV) or C (HCV). The study comprised 60 liver biopsies with hepatitis B or C. These included 10 biopsies from cases with acute lobular hepatitis (ALH), and 50 with chronic hepatitis (CH). In addition, 10 liver biopsies were used as controls. In CH cases, HAI grade ranged from 3/18 to 15/18, and stage from 1 to 6 (6HBV/8HCV). Immunohistochemical bcl-2 expression was assessed using the streptavidin-biotin method and the presence of apoptotic cells was evaluated by TUNEL method. Immunohistochemical and in situ hybridization (TUNEL) results were expressed following morphometric analysis. In CH cases, bcl-2 was detected in portal and intralobular lymphocytes, and in cholangiolar epithelial cells of the interface area. In ALH and control cases, bcl-2 was expressed only in lymphocytes. Lymphocytic bcl-2 expression was correlated directly with categories A, C and D of HAI (P < 0.001), whereas the degree of fibrosis was reversibly correlated to bcl-2 expression. In conclusion, in cases of chronic hepatitis, bcl-2 expression in cholangioles of interface area suggests that this oncoprotein may be involved in regulation of growth of these epithelial cells.     

Eradication of hepatitis C virus could improve immunological status and pyoderma gangrenosum‐like lesions

Hepatitis C virus (HCV) can affect immune cells and induce various kinds of immune‐related diseases including pyoderma gangrenosum. We experienced a difficult‐to‐treat case of pyoderma gangrenosum‐like lesions in a patient with HCV infection. The patient was treated with pegylated interferon (PEG IFN)‐α‐2b and ribavirin (RBV) therapy and achieved a sustained virological response. Before the eradication of HCV, the frequency of T‐helper 17 cells was remarkably high in comparison to chronic hepatitis C patients without extrahepatic immune‐related diseases. Moreover, we could detect negative and positive strand‐specific HCV RNA in the CD19⁺ B lymphocytes and CD4⁺ T lymphocytes. However, after the eradication of HCV, the immunological status became normal and the pyoderma gangrenosum‐like lesions became stable without immunosuppressive therapy. Here, we report a sequential immunological analysis during PEG IFN/RBV therapy and the beneficial effect of HCV eradication in difficult‐to‐treat pyoderma gangrenosum‐like lesions.