Plasma glutathione concentration in patients with chronic hepatitis C virus infection

Summary. It has recently been proposed that a depletion of glutathione (GSH) may be a contributing factor to viral persistence and resistance to interferon-α (IFN-α) therapy in chronic hepatitis C virus (HCV) infection. The aim of this study was: (1) to compare plasma GSH levels in patients with chronic HCV infection and normal healthy controls; and (2) to correlate GSH levels with liver histology and serum HCV RNA levels. Twenty-four patients with compensated chronic hepatitis C and 2 7 healthy subjects were studied. Serum and heparinized plasma were prospectively prepared and frozen within 1 h of collection. Plasma glutathione and glutathione peroxidase (GP) levels were measured spectrophotometrically. The serum HCV RNA level was quantitated by the branched chain DNA signal-amplification assay. Plasma GSH levels were not decreased in patients with chronic HCV infection but were actually greater than in controls (control 1.2 7 ± 0.12 μg ml^-1, HCV 1.62 ± 0.11 μg ml^-1, P < 0.05). There was also no difference in plasma GP activity between these two groups (control 0.233 ± 0.007 U ml^-1, HCV 0.230 ± 0.007 U ml^-1). Among the patients with chronic HCV infection, there was no correlation between either plasma GSH or GP levels and the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST), serum HCV RNA level, or liver histology. This study demonstrates that chronic HCV infection does not decrease the plasma GSH and GP levels.     

Assessment of hepatitis C virus RNA and genotype from 6807 patients with chronic hepatitis C in the United States

Hepatitis C virus (HCV) RNA status and HCV genotype have become important tools in the diagnosis and monitoring of therapy in chronic HCV infection. To establish a database with respect to HCV genotype and serum HCV RNA concentrations in chronic hepatitis C patients in the United States, we analysed 6807 chronic hepatitis C patients who had HCV RNA and HCV genotype tests conducted at a central laboratory. The HCV RNA concentration cut-off for the lower 25th percentile of this population (low titre) was 0.9 × 10⁶ copies ml^–1. The median HCV RNA concentration was 3.5 × 10⁶ copies ml^–1 and the cut-off for the upper 25th percentile (high titre) was 5 × 10⁶ copies ml^–1. Male patients had a median HCV RNA concentration of 3.9 × 10⁶ copies ml^–1, which was significantly higher than the median HCV RNA level for females (2.75 × 10⁶ copies ml^–1; P  < 0.001). HCV genotype 1 was detected in 73% of patients; genotype 2 in 14%; genotype 3 in 8%; mixed genotype in 4%; and genotypes 4, 5 and 6 with a frequency of < 1%. Patients from the Northeast, Southeast and Midwest had significantly ( P  < 0.001) more infections with genotype 1 than patients from the Western and Southern regions. African–American patients were more likely to be infected with genotype 1 when compared with Caucasian, Hispanic or Asian Pacific Islanders ( P  < 0.001). Patients infected with HCV genotype 1 and mixed HCV genotypes had significantly higher serum HCV RNA concentrations when compared with HCV genotypes 2 and 3 ( P  < 0.001 for all comparisons).     

Increased soluble IL-2 receptor levels in HCV-infected haemophiliacs: a possible indicator of liver disease severity

SUMMARY. We have measured sIL-2R in 60 haemophiliacs and 20 male control subjects. Haemophiliacs were grouped according to their HIV/HCV antibody status. Group 1 ( n = 20) comprised HIV + ve/HCV + ve, group 2 ( n = 27) HIV - ve/HCV + ve and group 3 ( n = 13) HIV - ve/HCV - ve. Group 4 comprised the normal control subjects. We also examined, retrospectively, the relationship between the severity of chronic liver disease, assessed histologically, and sIL-2R levels in selected patients. There was no significant difference between sIL-2R levels of the group 1 and group 2 patients, and the levels for both were significantly greater than those of either the group 3 patients or the control subjects. sIL-2 levels were also higher in selected patients with cirrhosis than in those with chronic active hepatitis (CAH) or chronic persistent hepatitis (CPH). We conclude that in haemophiliacs, chronic HCV-related liver disease is associated with increased plasma levels of sIL-2R and that the degree of elevation may reflect the severity of the associated chronic liver disease.     

Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection

CD8⁺ T lymphocyte responses are important in the clearance of viral infections. In chronic infections they may contribute to pathogenesis. To investigate the role of CD8⁺ T lymphocyte responses in viral clearance and chronic hepatitis C we have compared hepatitis C virus (HCV) specific cytotoxicity and interferon-gamma (IFN-γ) production in patients with resolved-acute, and chronic HCV infection. CD8⁺ T cell responses to a panel of 13 HCV T cell peptide epitopes were studied using Elispot assays of IFN-γ production and chromium release cytotoxicity assays. Responses of seven patients with resolved acute HCV infection were compared with those of 14 chronically infected patients. HCV-specific cytotoxicity differentiated the two populations of patients. The majority (71%) of patients with resolved acute infection tested positive to 42% of relevant peptides compared with the minority (28%) of patients with chronic hepatitis C ( P =0.03) who responded to only 8% of relevant peptides ( P =0.0009). In contrast, HCV-specific IFN-γ production was detected in 86% of patients with either resolved or chronic infection in response to 42% and 35%, respectively, of relevant peptides tested (not significant). In patients with chronic infection the magnitude of the HCV-specific IFN-γ production was inversely correlated to viral load ( R ²=0.52; P =0.042). Failure to clear HCV infection may be attributable to the presence of noncytolytic IFN-γ producing CD8⁺ T lymphocytes in chronically infected patients. However these CD8⁺ T cells may play a beneficial role in contributing to the control of viral load in chronic hepatitis C.     

Stability of hepatitis C virus RNA in serum from samples collected in a closed-tube system for serum separation and transport, as measured by a quantitative competitive PCR assay

SUMMARY. Recovery of hepatitis C virus (HCV) RNA, after variable time intervals from collection, was assessed using a closed-tube system for collection, separation and transport (SST^(tm) tubes). Blood from four hepatitis C-infected patients was collected in 12 SST^(tm) tubes and centrifuged within 1 to 3 h of collection. Tubes were then left 0, 8, 12, 24. 48 and 72 h at room temperature and at 4°C before removing serum. Hepatitis C virus RNA levels were measured by quantitative polymerase chain reaction (PCR) using the AMPLICOR HCV MONITOR^(tm) assay. Hepatitis C virus RNA levels in these samples were stable for at least three days at both temperatures. Polymerase chain reaction signals never decreased by more than 0.5 log. The reproducibility of the assays showed that the quantitative PCR method can be used with the storage conditions tested here. Our data suggests that processing blood in SST^(tm) tubes may be very useful in following hepatitis C virus RNA titres in infected patients, especially those receiving treatment.     

Procollagen-III Peptide and Chronic Viral C Hepatitis

Chronic hepatitis develops in at least half of persons acutely infected with hepatitis C virus (HCV). Ten to 25% of these patients will develop cirrhosis. Serum pro-collagen-III peptide (PIIIP) may be of value in predicting the development of chronic active fibrogenic liver disease. It has been reported that in chronic viral C hepatitis, the levels of hepatitis C virus-RNA (HCV-RNA) correlate directly with the severity of hepatic histology and inversely with response to interferon therapy. Objectives: The aims of this study were to correlate the level of PIIIP with HCV-RNA concentrations, ALT values, and histological severity in patients with chronic viral C hepatitis. Methods: Eighty-six patients with chronic C hepatitis were divided into three groups: group I (n = 34), mild chronic active hepatitis, group II (n = 25), moderate to severe chronic active hepatitis, and group III (n = 27), cirrhosis. HCV-RNA was measured by Quantiplex, and PIIIP was measured by radioimmunoassay-gnostic assay. Results: Mean ± SD level of ALT in group I was 114 ± 48 U/L, group II was 169 ± 115 U/L, and group III was 160 ± 94 U/L. The mean ± SD level of HCV-RNA in group I was 110 ± 130 × 10⁵ Eq/ml, in group II was 140 ± 140 × 10⁵ Eq/ml, and in group III was 70 ± 80 × 105 Eq/ml. The mean ± SD level of PIIIP in group I was 0.6 ± 0.2 U/ml, in group II was 0.9 ± 0.4 U/ml, and in group III was 1.2 ± 0.6. There was a significant difference in the levels of PIIIP among the three groups ( p = 0.0001). There was no correlation among ALT, HCV-RNA, and PIIIP in any of the three groups. Conclusions: PIIIP peptide determinations in patients with chronic viral C hepatitis are reflective of histological severity and may provide relatively nonin-vasive means of following disease progression.     

Virological characteristics of HCV infection in Japanese haemophiliacs

It has been found that almost all haemophiliacs treated with pooled concentrates of clotting factor VIII or IX before 1985/6 have been infected with hepatitis C virus (HCV). In order to clarify the characteristics of HCV infection in Japanese haemophiliacs, we investigated the HCV genotype and HCV-RNA level in 80 patients with haemophilia who had been confirmed to be positive by a second-generation HCV antibody test. HCV-RNA was detected in 60 (75.0%) individuals and various HCV genotypes were found. Although 80% (48/60) of the patients had genotype 1b, the frequency of each genotype was quite different from that in HCV-infected non haemophiliac Japanese. Particularly, multiple HCV genotypes were observed in 27 (46.7%) patients. The mean (± SD) level of HCV-RNA was 5.3 × 10⁵ ±  1.1 × 10⁶ copies mL⁻¹. The viral load in patients with genotype 2a was significantly less common than those with genotype 1a ( P = 0.0007), genotype 1b ( P = 0.0009) and combined genotype 1a/1b ( P = 0.0019). In patients co-infected with human immunodeficiency virus (HIV), the HCV-RNA level was significantly higher ( P = 0.05) than in those without co-infection. However, there was no significant difference ( P = 0.25) in the HCV-RNA level with HCV/HIV co-infection among the 40 patients with group 1 genotypes. We conclude that this biased distribution of HCV genotypes in Japanese haemophiliacs reflects their specific mode of HCV infection. Moreover, these results suggest that super-infection with HIV does not greatly influence the HCV load in patients with no marked immunological deterioration.     

Serum levels of soluble interleukin-2 receptors and effects of interferon-α for patients with chronic hepatitis C virus

To characterize the role of serum soluble interleukin-2 receptor (sIL-2R) in hepatitis C virus (HCV) infection, the level of sIL-2R was measured by ELISA in 117 subjects with chronic HCV infection and in 23 healthy controls. HCV RNA was detected by polymerase chain reaction in all subjects with HCV infection. Forty-seven patients with chronic hepatitis and 10 with liver cirrhosis were treated for six months with natural interferon-. The sIL-2R levels of 40 asymptomatic HCV carriers (632±340 units/ml), 47 patients with chronic hepatitis (547±204 units/ml), 10 with cirrhosis (679±239 units/ml), and 20 with hepatocellular carcinoma (1145±487 units/ml) were significantly higher than those of healthy controls (380±191 units/ml) (P<0.05, respectively). The levels of sIL-2R increased, as did the histological activity index scores (r=0.348,P<0.01). The level of sIL-2R rose after the initial administration of interferon in all 57 patients. In patients in whom HCV RNA was eliminated from the sera within a six-month follow-up after cessation of treatment, the level of sIL-2R reverted to basal values, but in patients in whom HCV RNA was not eliminated the value was significantly higher than that before treatment. These results suggest that monitoring serum sIL-2R in patients with chronic HCV infection treated with interferon may provide information concerning the possibility of the elimination of HCV RNA.     

Rarity of microsatellite genomic instability in B-cell non-Hodgkin's lymphomas in hepatitis C virus-infected patients

Several groups have emphasized the likely impli-cation of the hepatitis C virus (HCV) in a fraction of B-cell non-Hodgkin's lymphomas. Since only a minority of patients with HCV infection and monoclonal mixed cryoglobulinaemia develop overt lymphoma, the identification of predisposing factors has relevant clinical implications. The replication error phenotype (RER⁺), as revealed by widespread microsatellite instability, is caused by defects in DNA mismatch repair genes, and has been frequently disclosed in subsets of B-cell lymphomas with underlying infection and chronic inflammation. We therefore investigated the occurrence of the RER⁺ phenotype in a series of eight consecutive B-cell NHLs in patients with chronic infection by HCV. A polymerase chain reaction-based assay was used to analyse an extended panel of 15 microsatellite loci.
Microsatellite instability was not observed in six tumour samples in any locus; the two remaining cases showed instability at only one locus.
Therefore genetic instability by defects in DNA mis-match repair genes should not represent the general mechanism predisposing to overt lymphoma in HCV-infected patients. Although a clearer definition of HCV-related B-cell disorders should better address future studies on genetic instability in larger series, we recommend additional oncogenetic pathways as the target of further research.     

Oral prostaglandin (PGE2) therapy for chronic viral hepatitis B and C

The cytoprotective effects of prostaglandins have been utilized in the prevention of hepatitis B virus reactivation after liver transplantation. This pilot study evaluated the effects of oral prostaglandin E₂ (PGE₂) in chronic viral hepatitis B and C. Twenty patients with chronic hepatitis B and 20 patients with chronic hepatitis C received 4mgday^–1 PGE₂ for 6 months. The lymphocyte antiviral enzyme 2',5'-oligoadenylate synthetase (2',5'-OAS) and peripheral blood monocyte procoagulant activity (PCA) were measured before, during and after the treatment. Three of 20 hepatitis B and five of 20 hepatitis C patients withdrew from the study. Eight of 17 hepatitis B patients responded: in seven of these eight patients, serum alanine aminotransferase (ALT) levels normalized; loss of viral replication was sustained in all eight patients; and seroconversion from hepatitis Be antigen (HBeAg) to hepatitis Be antibody (HBeAb) positivity occurred in seven patients over the 48-week duration of this study. In 14 of the 15 hepatitis C patients, hepatitis C virus (HCV) RNA remained detectable and the serum ALT levels remained elevated. 2',5'-OAS levels and PCA values did not correlate with other markers of response to PGE₂ therapy in either chronic hepatitis B or C. In summary, PGE₂ was associated with sustained loss of viral replication in 47% of chronic hepatitis B patients; no beneficial effects were apparent in chronic hepatitis C.     

Sustained response to interferon-alpha2a monotherapy of young blood donors with minimal-to-mild chronic hepatitis C. The Donor Surveillance Study Group

Subjects with minimal-to-mild chronic hepatitis C may suffer long-term consequences of hepatitis C virus (HCV) infection. Nonetheless, they are not candidates for antiviral treatment, mainly because little data are available concerning the efficacy and safety of therapy. Thirty-two HCV RNA positive individuals aged 18–45 years, who had a histological activity index score ≤ 8 and alanine aminotransferase (ALT) levels ≤ 1.5 times lower than the normal limit for at least 1 year, were prospectively enrolled among a cohort of 35358 candidate blood donors, and treated with 4.5 mega units (MU) of recombinant interferon-α2a (IFN-α2a) thrice weekly for 6 months, and for an additional 6 months if a virological response was observed. Twelve months after the completion of treatment, 13 of 31 evaluable patients were HCV RNA negative, accounting for a sustained response rate of 42%. Patients without fibrosis had a lower response rate than those with mild fibrosis (two of 14 vs 11 of 17; P =0.012). In responders, median aminotransferase levels were significantly lower after therapy than before (11.04 ± 3.98 vs 27.3 ± 12.32 U l⁻¹, respectively; P  < 0.005). When the analysis was limited to the six responders whose pretreatment aminotransferase levels were consistently normal, this difference was still significant (9.33 ± 4.12 vs 20.58 ± 6.73 U l⁻¹; P =0.002). In conclusion, a durable suppression of viraemia can be obtained by IFN monotherapy in a relatively high proportion of young subjects with minimal-to-mild chronic hepatitis C, especially when portal fibrosis is found on liver biopsy. The disappearance of viraemia always leads to a reduction in the degree of hepatocellular necrosis.     

Serum levels of soluble interleukin-2 receptors in acute and chronic viral hepatitis

To analyze interleukin-2-dependent immunoregulatory function in hepatitis B virus infection and in other forms of viral hepatitis, levels of soluble interleukin-2 receptors (sIL-2R) were measured by an enzyme-linked assay in sera from patients with acute and chronic viral hepatitis of different etiology. Increased sIL-2R levels were detected in the early phase of acute hepatitis type A and type B, but not during acute non-A, non-B hepatitis. Among 46 patients with chronic hepatitis B virus infection, levels of sIL-2R were significantly increased only in cases with chronic active hepatitis, while they were about normal in chronic persistent hepatitis or in healthy carriers of the infection. These differences were independent of virus replication, being maintained when patients were stratified according to HB_eAg/anti-HB_e status and to serum HBV-DNA. Nine patients with chronic active hepatitis type B and high sIL-2R levels at presentation were followed prospectively for two to eight years, and in HB_eAg -positive patients, the behavior of receptor levels closely paralleled disease activity. These results, which may reflect increased shedding of IL-2R by activated T lymphocytes in patients with active destruction of HBV infected hepatocytes, indicate the usefulness and potential prognostic importance of serum slL-2R determination in patients with chronic viral hepatitis. Patients with chronic non-A, non-B hepatitis had much lower sIL-2R levels, although their liver disease was similar to hepatitis B cases, suggesting that different pathogenetic mechanisms operate in these patients.     

慢性HCV感染与机体细胞免疫功能关系的探讨

目的:探讨慢性丙型肝炎病毒感染患者HCV RNA增殖状态与T细胞亚群功能及血浆中IL-2和SIL-2R活性的关系。方法:以间接免疫荧光法、ELISA法和RT-PCR分别检测75例慢性HCV感染患者(其中HCV RNA阳性47例,HCV RNA阴性28例)外周血T细胞亚群、IL-2、sIL-2R的水平和HCV RNA。结果:慢性HCV感染患者周围血CD_3~ 、CD_4~ 淋巴细胞亚群,CD_4~ /CD_3~ 比值及IL-2水平均显著低于正常对照组(P<0.01),而sIL-2R明显升高(P<0.01);血清HCV RNA阳性患者T细胞亚群,CD_4~ /CD_3~ 比值及IL-2水平显著低于HCV RNA阴性患者(P<0.01)。结论:慢性HCV感染患者存在机体细胞免疫功能低下,HCV RNA阳性患者较阴性患者更甚,提示细胞免疫功能受抑可能是HCV持续增殖的原因。     

Hepatic inflammatory cytokine mRNA expression in hepatitis C virus-human immunodeficiency virus co-infection

Summary.  Although epidemiologic studies have documented that hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients have accelerated fibrogenesis, especially those with CD4⁺ cell counts <200 cells/mm³, the pathogenic mechanisms are poorly understood. We investigated whether severe immunodeficiency in co-infection is associated with changes in intrahepatic inflammatory cytokine mRNA levels. We measured interferon (IFN)-γ, tumour necrosis factor-α, transforming growth factor (TGF)-β₁, interleukin (IL)-4, IL-10, IL-12p35 and IL-12p40 mRNA levels by real-time PCR performed on liver samples from HCV mono-infected ( n  = 19) and HCV/HIV co-infected ( n  = 24) patients. Co-infected patients had decreased intrahepatic mRNA levels of IFN-γ ( P  = 0.09), IL-4 ( P  = 0.05) and IL-12p35 ( P  = 0.04) compared with mono-infected patients, while IL-10 was increased ( P  = 0.07). In co-infected patients, IFN-γ mRNA levels increased linearly with increasing peripheral CD4⁺ cell counts by 1.23 times relative to the calibrator for every 100 CD4⁺ cells/mm³ increase ( P  = 0.02). No other cytokines were significantly associated with CD4⁺ cell counts. In conclusion, HIV-induced lymphopenia may result in hepatic inflammatory cytokine suppression in HCV/HIV co-infection. Intrahepatic IFN-γ levels are significantly reduced in patients with advanced immunodeficiency. Further studies are needed to assess whether decreased IFN-γ secretion by HCV-specific CD4⁺ cells may account for accelerated fibrogenesis in these patients.     

Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C

We evaluated the synergistic effect of zinc supplementation on the response to interferon (IFN) therapy in patients with intractable chronic hepatitis C in a pilot study using natural IFN-α with or without zinc. No clinical differences were observed between patients treated with IFN alone ( n =40) and IFN with polaprezinc (IFN + Zn, n =35). All patients were positive for HCV genotype Ib and had more than 10⁵ copies of the virus/mL serum. Ten million units of natural IFN-α was administered daily for 4 weeks followed by the same dose every other day for 20 weeks. In the IFN + Zn group, patients received an additional dose of 150 mg/day polaprezinc orally throughout the 24-week IFN course. No additional side-effects of polaprezinc were noted but four out of 40 IFN alone treatment and three out of 35 IFN + Zn group withdrew because of side-effects. Complete response (CR) was defined as negative HCV RNA in the serum on PCR and normal aminotransferase level 6 months after therapy. Incomplete response (IR) was normal liver enzyme and positive serum HCV RNA. Both of them were evaluated at the 6 months after the completion of the treatment. Patients with higher levels of serum HCV (more than 5 × 10⁵ copies/mL) had little response in both treatment groups. Patients with moderate amount of HCV (10⁵ to 4.99 × 10⁵/mL) showed high response rates in combination group (CR: 11/27, 40.7%; CR + IR 15/27, 64.3%), better than IFN alone (CR: 2/15, 18.2%; CR + IR: 2/15, 18.2%). Serum zinc levels were higher in patients with IFN + Zn group than in the IFN group. Our results indicate that zinc supplementation enhances the response to interferon therapy in patients with intractable chronic hepatitis C.     

Hepatitis G virus infection in Japanese haemophiliacs

The recently identified hepatitis G virus (HGV) (also known as GB virus-C) has been considered as a blood-transmissible agent. As many haemophiliacs have risk factors for infectious agents, to clarify the frequency of HGV infection is important. HGV-RNA was investigated in 77 Japanese haemophiliacs who had been treated with nonvirus-inactivated concentrates derived from pooled plasma. Detection of HGV-RNA was performed with a nested RT-PCR that recognizes the 5'-NCR of the HGV genome. HGV-RNA was detected in 19 (24.7%), including four (21.0%) infected with HGV alone, 12 (63.2%) co-infected with HCV and three (15.8%) who were HBV carriers. The patients infected with HGV alone showed a normal ALT level of 18.7 ± 4.1 IU L⁻¹. Most (36/37, 97.3%) of the patients with abnormal ALT levels had HCV-RNA. Patients infected with HCV alone or co-infected with HCV and HGV showed higher ALT levels of 108.8 ± 90.2 IU L⁻¹ ( n = 39) and 67.6 ± 62.6 IU L⁻¹ ( n = 11), respectively. However, there was no significant difference ( P = 0.16) in ALT levels between HCV infection alone and HCV/HGV co-infection. On the other hand, four of the patients who could be followed over 10 years showed HGV-RNA persistently. In two who underwent liver biopsy, the histological evidence showed no definitive fibrotic and necro-inflammatory changes. These results indicate that HGV infection has frequently occurred in haemophiliacs. It is possible that HGV infection does not cause aggressive hepatitis with elevated ALT levels, and that co-infection with HGV may not aggravate hepatitis caused by HCV.