Upregulation of OX40 ligand on monocytes contributes to early virological control in patients with chronic hepatitis C

Dysfunctional hepatitis C virus (HCV) specific CD4⁺ T cells are known to contribute to inadequate adaptive immunity in chronic hepatitis C (CHC), although the underlying mechanisms remain largely undefined. In this study, OX40 ligand (OX40L) expression was investigated in 41 treatment‐naïve CHC patients, 20 sustained virological responders and 36 healthy subjects. We observed that OX40L expression was significantly upregulated in peripheral monocytes in CHC patients compared with sustained virological responders and healthy subjects. OX40L upregulation correlated significantly with plasma viral load rather than serum alanine aminotransaminase levels. Furthermore, longitudinal analyses indicated that upregulated OX40L expression on monocytes is closely associated with rapid or early virological responses in patients receiving pegylated IFN‐α/ribavirin treatment. In vitro, HCV core antigen strongly stimulated monocyte expression of OX40L and blockade of TLR2 signaling significantly downregulated OX40L expression. More importantly, elevated OX40L expression was also shown to be closely associated with elevation of the HCV‐specific CD4⁺ T‐cell response and in vitro blockade of OX40L expressed on monocytes led to impaired CD4⁺ T‐cell function. These findings, therefore, implicate OX40L expression can be used as a marker to evaluate antiviral treatment efficacy and extend the notion that enhancement of OX40L expression could be a good way for immunotherapy in CHC patients.     

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 × 10³ copies/μg RNA; range: 0 to 3.6 × 10⁴ copies/μg RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001) but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden. J. Med. Virol. 54:265–270, 1998. © 1998 Wiley-Liss, Inc.     

Detection and genotyping of the hepatitis C RNA in tear fluid from patients with chronic hepatitis C

Tear fluid from 51 patients with chronic hepatitis C virus (HCV) infection was analyzed for the presence of the hepatitis C RNA to assess the potential role of this fluid in virus transmission. HCV sequences were amplified from sera and tear fluids by nested polymerase chain reaction using primers from the 5′ non coding region of the virus genome. Positive samples were genotyped by the LiPA^TM procedure. HCV RNA was detected in 76.5% (39/51) of the sera and in 9.8% (5/51) of the tear fluid samples. The presence of the RNA in the tear fluid was independent of the severity of the hepatitis and of the viral load as measured by the branched DNA^TM assay. The genotypes of the tears and serum isolates were different for two patients. For another patient, the HCV RNA was positive in the tear sample but negative in the serum sample. These findings suggest that tear fluid may transmit HCV but the source of HCV RNA in this fluid needs to be better understood. J. Med. Virol. 51:231–233, 1997. © Wiley-Liss, Inc.     

Changes in antibody titers to hepatitis C virus following interferon therapy for chronic infection

The use of quantitative assays for hepatitis C virus specific antibodies (anti-HCV) as a prognostic marker was evaluated in 31 patients with chronic hepatitis C treated with interferon (IFN). Changes in titers of serum HCV-RNA and anti-HCV antibodies; anti-C11 (anti-core), anti-C100 (anti-NS3), and anti-C7 (anti-NS3) were investigated. Recombinant IFN-α 2a was administered and the patients were followed for more than 1 year. The patients were classified into three groups according to their responses to IFN: 11 sustained responders with continuous normalizations of serum alanine aminotransferase (ALT) levels; 14 transient responders with transient decreases in ALT; and six nonresponders who had no changes in ALT levels. Ten of 11 sustained responders had a continuous decrease in anti-C11 titers after completion of treatment, decreasing to less than half of pretreatment titers. No patients in the other two groups had a continuous decrease in anti-C11 titers. Although sustained responders had decreases in anti-C100 and anti-C7 titers after IFN therapy, these titers also decreased in some patients in the other two groups. HCV-RNA was not detected in the sera of 10 of 11 sustained responders following IFN therapy. In contrast, while 9 of 10 transient and nonresponders had a decrease or disappearance of HCV-RNA at the completion of therapy, they had increased levels thereafter. These results indicate that anti-HCV-core (anti-C11) titers most closely reflect the status of HCV replication. A quantitative assay for anti-HCV-core antibody can be used as a predictive marker of remission in IFN-treated patients with chronic hepatitis C. © 1994 Wiley-Liss, Inc.     

Treatment of chronic hepatitis C in patients with HIV/HCV coinfection

Hepatitis C virus (HCV) infection is one of the most frequent causes of comorbidity and mortality in the human immunodeficiency virus (HIV) population, and liver-related mortality is now the second highest cause of death in HIV-positive patients, so HCV infection should be countered with adequate antiviral therapy. In 2011 began the era of directly acting antivirals (DAAs) and the HCV NS3/4A protease inhibitors telaprevir and boceprevir were approved to treat HCV-genotype-1 infection, each one in combination with pegylated interferon alfa (Peg-IFN) + ribavirin (RBV). The addition of the first generation DAAs, strongly improved the efficacy of antiviral therapy in patients with HCV-genotype 1, both for the HCV-monoinfected and HIV/HCV coinfected, and the poor response to Peg-IFN + RBV in HCV/HIV coinfection was enhanced. These treatments showed higher rates of sustained virological response than Peg-IFN + RBV but reduced tolerability and adherence due to the high pill burden and the several pharmacokinetic interactions between HCV NS3/4A protease inhibitors and antiretroviral drugs. Then in 2013 a new wave of DAAs arrived, characterized by high efficacy, good tolerability, a low pill burden and shortened treatment duration. The second and third generation DAAs also comprised IFN-free regimens, which in small recent trials on HIV-positive patients have shown comforting preliminary results in terms of efficacy, tolerability and adherence.     

Longitudinal study of hepatitis C viremia in chronic hepatitis C

Serial serum samples from 20 untreated patients with chronic hepatitis C virus (HCV) infection were tested for HCV RNA by a nested polymerase chain reaction assay using primers from the highly conserved 5′ noncoding region to determine the relationship between hepatitis C viremia and the activity of liver disease during the natural course of chronic HCV infection. Semiquantitation of serum HCV RNA level was achieved by testing serial 10-fold dilutions of RNA extracts to determine the end-point titer. All the patients were HCV RNA positive at presentation. There was a poor correlation between the initial HCV RNA titer and serum transaminase levels. All patients except one were persistently HCV RNA positive during a follow-up period of 1.5-15 years, although 17 (85%) had periods of normal or near-normal transaminase levels. There was no correlation between changes in the serum transaminase levels and HCV RNA titer. Patients with chronic HCV infection have persistent viremia despite fluctuations in ALT levels.     

The effects of direct-acting antiviral agents on the frequency of myeloid-derived suppressor cells and natural killer cells in patients with chronic hepatitis C

Currently, hepatitis C antiviral therapy is entering a new era with the use of direct-acting antiviral (DAA) agents. However, the precise immunological influences of DAA therapy in patients with chronic hepatitis C (CHC) are insufficiently understood. This study aimed to investigate the effects of DAA therapy on the frequency of myeloid-derived suppressor cells (MDSCs), T lymphocytes, and natural killer (NK) cells in patients with CHC. Thirty-two treatment-naive CHC patients were treated with DAA therapy, and the frequency of immune cells was analyzed by flow cytometry at various time points during and after therapy. Sixteen healthy donors were recruited for comparison. DAA therapy decreased the frequency of MDSCs and monocytic MDSCs in patients with CHC to a normal level. DAA therapy also increased the CD8⁺ T and NK cell levels in patients with CHC. In addition, activation (NKp30 and NKp46) and inhibitory (NKG2A) receptors on NK cells were downregulated to yield an NK cell phenotype resembling that observed in the healthy controls. This study provides insight into the normalization of immune cell levels under DAA therapy and indicates that restoration of the immune system in patients with CHC strongly supports long-term curative hepatitis C virus eradication.     

Proapoptotic IL-18 in patients with chronic hepatitis C treated with pegylated interferon-alpha

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. In mainland of China, It was estimated the population of 1.3 billion infected with HCV. HCV is not cytopathic. Immune response that is essentially conducted by cytokines may play an important role in the pathogenesis of HCV infection. Interleukin (IL)-18, mainly produced by monocytes/macrophages, plays an important role in the immune system by enhancing T cell responses, regulating interferon-gamma (IFN-γ) production and promoting the development of T helper cell Th1 immune responses. Raised serum levels of IL-18 have recently been reported in patients with chronic hepatitis C before antiviral therapy. Herein we report the IL-18 sequential changes in patients with hepatitis C during the period of pegylated interferon (PEG-IFN) alpha treatment for 48 weeks. We established the correlation of plasma IL-18 level and alanine aminotransferase (r = 0.77, P < 0.05). Hepatic inflammatory activity in chronic hepatitis C was shown to be closely associated with an increased amount of IL-18. HCV-infected patients had raised IL-18 levels (93.67 ± 23.58 pg/ml versus 59.73 ± 24.06 pg/ml; P < 0.001) comparing donor negativity for HCV. PEG-IFN alpha-2a treatment induces a marked decline in IL-18 and remission of hepatic inflammatory in responders at week 24 and week 48 follow-up time point, while increased levels persist in those in whom the HCV infection was not eliminated by the therapy. We proposed declined IL-18 levels favor for virus solution, while persistent raised IL-18 associated with PEG-IFN treatment failure.     

Histological improvement of chronic liver disease after spontaneous serum hepatitis C virus clearance

The long-term histological and virological outcomes of spontaneous circulating hepatitis C virus (HCV) clearance were studied in chronic liver disease. Between 1979 and 1984, three patients underwent laparoscopy for chronic non-A, non-B liver disease, and two were found to have cirrhosis and one with chronic active hepatitis. After HCV assays became available in 1990, they were positive persistently for HCV antibody without serum HCV RNA. Reductions of antibody levels to HCV core and/or nonstructural proteins were observed, and liver biopsies were undertaken between 1995 and 2000. Liver biopsies at 11-19 years after laparoscopy disclosed marked alleviation of liver inflammation and fibrosis in each case although a low grade of inflammation remained. The two patients with cirrhosis no longer showed histological features of cirrhosis, and the poor liver function in one patient had been ameliorated. Liver specimens from two patients were subjected to polymerase chain reaction to detect positive and negative HCV RNA strands and hepatitis B virus DNA. Only the positive HCV RNA strand was detected for one patient who had previously cirrhosis. Liver specimens were examined from another six nonviremic HCV-seropositive individuals without chronic liver disease. Five patients displayed low-grade liver inflammation without evident fibrosis, but none had any viral genome in the liver. These findings suggest that spontaneous circulating HCV clearance in chronic liver disease confers favorable liver histological outcome, although occult HCV infection persists. J. Med. Virol. 69:41-49, 2003.     

Sofosbuvir-based therapy for patients with chronic hepatitis C: Early experience of its efficacy and safety in Korea

Background/Aims

The previous standard treatment for chronic hepatitis C (CHC) patients, comprising a combination of pegylated interferon (IFN) and ribavirin, was associated with suboptimal efficacy and severe adverse reactions. A new era of direct-acting antivirals is now dawning in Korea. Early experience of applying sofosbuvir-based therapy to CHC patients in Korea is reported herein.

Methods

Data on efficacy and safety were collected for CHC patients treated with a combination of sofosbuvir plus ribavirin or sofosbuvir/ledipasvir with or without ribavirin.

Results

This retrospective study included 25 consecutive patients who received sofosbuvir-based therapy (19 with genotype 1b and 6 with genotype 2) at Seoul National University Hospital from May 2014 to April 2015. A virologic response was achieved at week 4 by 85.7% and 80% of the patients with genotypes 1b and 2, respectively. The HCV-RNA level decreased more slowly in IFN-experienced than in treatment-naïve patients with genotype 1b. However, the sustained virologic response at week 12 (SVR12) rate did not differ among these patients, and was as high as 100%. The presence of cirrhosis significantly increased the risk of a virologic response failure at week 4 (OR, 11.0; P=0.011) among patients with HCV genotype 1b. Only five patients (20%) experienced minor adverse events, including grade 1 fatigue and headache. The hemoglobin level decreased slightly after sofosbuvir-based therapy, but there was no case of premature discontinuation of this therapy.

Conclusions

In a real clinical practice, sofosbuvir-based therapy for CHC patients in Korea achieved optimal antiviral efficacy with insignificant adverse events. Long-term follow-up data are warranted to ensure the sustained antiviral efficacy and long-term safety of sofosbuvir-based IFN-free therapy.     

High seroprevalence against hepatitis E virus in patients with chronic hepatitis C virus infection

BackgroundHepatitis E virus (HEV) genotype 3 is endemic in Europe. Superinfection with HEV in patients with underlying chronic liver disease can cause hepatic decompensation leading to increased morbidity and mortality.ObjectivesThe prevalence of anti-HEV antibodies was investigated in 204 patients with chronic hepatitis C virus (HCV) infection and different stages of fibrosis.Study designSera were analyzed for anti-HEV IgG, IgM and HEV RNA.ResultsThe median age of the patients was 55 years (IQR 40–62 years); 126 (62%) were men. Ninety-eight (48%) patients had a METAVIR fibrosis stage F2 or higher. The prevalence of anti-HEV IgG was 30% (62/204), which was significantly higher than among Swedish blood donors (17%, p < 0.01). The prevalence of anti-HEV antibodies was associated with higher age (OR 1.08 (1.05–1.11); p < 0.01). It was also higher for patients with a prior history of blood transfusion (48%) as compared to intravenous drug use (IDU; 26%) as the risk factor for acquisition of the HCV infection (OR 2.72 (1.2–6.19); p < 0.02). The prevalence of anti-HEV IgG was also significantly higher in patients with significant fibrosis, i.e. ≥F2 (38%; OR 2.04 (1.11–3.76); p = 0.02) and/or neoplasm (72%; OR 7.27 (2.46–21.44); p < 0.01).ConclusionsWhen adjusted for age, the prevalence of anti-HEV antibodies was significantly higher in patients with previous or current malignant liver disease compared to blood donors. The lack of significant correlation between HCV and HEV infections indicate low level of transmission of HEV by IDU. HEV infections warrant more attention, especially in patients with preexisting liver disease.     

Absence of extensive genetic heterogeneity of hepatitis C virus in antibody-negative chronic hepatitis C

Hepatitis C virus (HCV) carriers usually have antibodies to HCV; however, there are viremic individuals without these antibodies. To investigate whether variations of the viral genome are responsible for this discrepancy, the nucleotide and deduced amino acid sequences of HCV capsid and nonstructural regions obtained from 15 viremic patients were examined. These 15 patients were infected with type 1b HCV, and 10 did not have antibody to HCV assayed with second-generation tests. The nucleotide homology of the 5 seropositive and 10 seronegative patients with the HCV prototype sequence were 91.6% and 91.9%, respectively, in the capsid region. There was no apparent difference in the deduced amino acid sequences between the two groups of patients studied (94% vs. 95%). The nucleotide and amino acid sequences of a part of the nonstructural region 3 also showed similar results. These findings suggest that absence of antibodies against both capsid and nonstructural peptides in HCV carriers is not caused by genetic heterogeneity of the viral epitopes. © 1996 Wiley-Liss, Inc.     

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Pathology of hepatitis C virus infection

This paper reviews our current knowledge of hepatitis C virus, its structure, epidemiology and pathological effects in man. The histopathological features of acute and chronic hepatitis due to hepatitis C virus are detailed and illustrated. The possible pathogenetic mechanisms involved in the liver injury are outlined and the significance of genotypic subtypes of the virus and of host genetic predisposition in relation to the liver injury and the response to therapy are summarized.     

Identification of a new autoantibody in patients with chronic hepatitis

To comprehensively study autoantibodies in patients with chronic hepatitis (CH), especially those with hepatitis C virus (HCV) infection, proteins extracted from HepG2 cells were separated by two-dimensional electrophoresis. Spots reacting with sera from 15 patients with CH-C were detected by Western blotting. Proteins extracted from the spots were subjected to mass spectroscopy for identification by mass fingerprinting. Antigenicity of the proteins identified was confirmed by Western blotting and enzyme-linked immunoabsorbent assay. The localization of the autoantigens so detected was investigated by immunohistochemistry. Among 20 protein spots detected, four were identified as actin, heat shock protein (HSP) 70, HSP60, and a novel protein (hepalaminin). Hepalaminin consists of two domains of laminin β-2 and a specific domain. Autoantibodies against the specific domain were detected in 60.8% of patients with CH-C, 37.7% of those with CH-B, 42.3% of those with autoimmune CH, 28.6% of nonalcoholic steatohepatitis, 10.0% of asymptomatic HCV carriers, but in no healthy volunteers. Antihepalaminin positivity in CH-C and CH-B was related to histologic grading. Immunohistochemical staining demonstrated that hepalaminin is present in the cytoplasm of hepatocytes and cholangiocytes but not of fibroblasts or the vascular epithelium. Hepalaminin is a novel protein expressed in hepatocytes and cholangiocytes. Autoimmunity to this protein may exacerbate inflammation in chronic viral hepatitis.     

Quantitative measurement of hepatitis C virus core antigen is affected by the presence of cryoglobulins

Mixed cryoglobulinaemia is associated strikingly with HCV infection. The aim of this study was to assess whether the adherence to proper methods of collecting samples for cryoglobulin detection was critical or not on virological parameters in hepatitis C virus (HCV) patients. We studied 56 consecutive patients. Blood samples were collected using a conventional method and a blood collection method at 37 degrees C adapted to cryoglobulin detection. HCV core antigen and HCV RNA were measured in sera and cryoglobulins issued from both blood collection methods. In cryoglobulin-positive patients, serum concentrations of HCV core antigen, but not that of HCV RNA, were significantly higher when a conventional method was used, compared to a blood collection method at 37 degrees C (P = 0.001). In the cryoprecipitates, concentration of HCV core antigen was optimum when the blood collection method at 37 degrees C, rather than the conventional method, was applied for cryoglobulin detection (P < 10(-4)). The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37 degrees C rather than the conventional method (P < 0.001). HCV parameter measurements and cryoglobulin study should not be performed on the same serum samples due to the potential impact of blood collection methods on results.