Markus Reiser

Comprehensive Analysis of CD8+-T-Cell Responses against Hepatitis C Virus Reveals Multiple Unpredicted Specificities

ABSTRACT The hepatitis C virus (HCV)-specific CD8+-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8+-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8+-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8+-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

Full-Breadth Analysis of CD8+ T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy

ABSTRACT Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

Serum interleukin 4 and interleukin 10 levels in patients with chronic hepatitis C virus infection

BACKGROUND/AIMS Immune-mediated mechanisms are believed to play an important pathogenetic role in chronic hepatitis C virus infection. Interleukin 4 (IL-4) and IL-10 are secreted by T helper-2 type cells (Th2) which may downregulate cell-mediated immune effector mechanisms important in the host defense against intracellular pathogens. This study aimed to determine Th2 cytokine levels in chronic hepatitis C virus infection. METHODS Serum IL-4 and IL-10 levels were measured in 74 patients with chronic hepatitis C virus infection and 20 healthy controls. The expression of CD30 in liver, a marker that is preferentially expressed in Th2 cells, was also determined by immunohistochemical staining in 37 patients. RESULTS Serum IL-4 and IL-10 were below the detection limit (5 pg/ml) in all 20 healthy controls. However, 36 patients (49%) had elevated serum IL-4 levels (range 5-106 pg/ml, p<0.001) and 23 patients (31%) had elevated serum IL-10 levels (range 5-37 pg/ml, p<0.05). There was no correlation between serum IL-4 and IL-10 levels. There was also no correlation between serum IL-4 and IL-10 levels and any of the clinical (age, gender, mode of acquisition), biochemical (serum alanine transaminase levels), virologic (viremia level, genotype), and histological parameters examined. Twenty of 37 liver biopsy specimens from patients with chronic hepatitis C virus infection showed occasional CD30+ lymphocytes, suggestive of Th2 phenotype. However, in 20 of the 37 patients with paired cryostat liver sections, IL-4 was not detected in any of these patients, suggesting that IL-4 was not produced in the liver in patients with chronic hepatitis C virus infection. CONCLUSIONS This study showed that serum Th2 cytokines are elevated (but at a low level) in a proportion of patients with chronic hepatitis C virus infection. However, the elevated Th2 cytokine levels may represent a systemic response and not a result of increased local production within the liver.

Analysis of the Evolutionary Forces in an Immunodominant CD8 Epitope in Hepatitis C Virus at a Population Level

ABSTRACT Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.

Degree of cross‐genotype reactivity of hepatitis C virus–specific CD8+ T cells directed against NS3

The inherent sequence diversity of the hepatitis C virus (HCV) with the existence of multiple genotypes that differ up to 20% at the amino acid level represents one of the major obstacles for immune control. Accordingly, immune control of a heterologous virus challenge, particularly across genotypes, is difficult to achieve; however, the overall role of genotype‐specific sequence differences has not yet been defined at the epitope level. The aim of this study was to determine the role of genotype‐specific sequence differences for the CD8+ T cell response against HCV. We analyzed a cohort of anti‐HCV–positive injection drug users infected with HCV genotype 1 (n = 17) or genotype 3 (n = 22) or undetectable HCV‐RNA (n = 14) with overlapping peptides covering consensus sequences of NS3 from both genotypes. Importantly, the majority of HCV‐specific CD8 T cells were specific for one genotype only indicating that sequence differences between genotypes are relevant at the epitope level. Interestingly, T cells active against both genotypes were significantly more frequent in HCV‐RNA–negative subjects. Of note, we identified five subjects with undetectable viremia and coexistence of two T cell populations—one for each genotype—suggesting immune control of two different genotypes. Conclusion: We systematically analyzed the degree of cross‐genotype reactivity of HCV‐specific T cells and have shown that CD8 responses targeting different HCV genotypes can be primed in the same individual and that such responses potentially characterize a subgroup among injection drug users being protected from chronic HCV infection. (HEPATOLOGY 2009.)

Serine protease inhibitors as anti-hepatitis C virus agents

Approximately 3% of the worldwide population (i.e., more than 170 million people) are chronically infected with the hepatitis C virus (HCV). An estimated 20% of these patients will develop liver cirrhosis within a mean of 20 years, and 2–5% of cirrhotic patients will die of end-stage liver disease or hepatocellular carcinoma. The currently approved antiviral therapy with pegylated interferon (pegIFN) and ribavirin induces a sustained virological response (SVR) in 40–50% of patients infected with genotype 1, the most prevalent HCV type. In this review, we focus on the development and clinical application of serine protease inhibitors as anti-HCV agents. Although highly active in inducing a significant decline of serum HCV RNA, the rapid development of resistance must be counteracted in combination with other antiviral agents, currently pegIFN-α and ribavirin. Two serine protease inhibitors have reached clinical Phase III trials, increasing SVR rates and shortening treatment duration when combined with pegIFN and ribavirin. Trials of interferon-free targeted combination therapies are currently underway.

Induction of cell proliferation arrest and apoptosis in hepatoma cells through adenoviral-mediated transfer of p53 gene.

BACKGROUND/AIM Loss of p53 function is common in hepatocellular carcinoma and is associated with an extremely poor prognosis. The aim of the study was to evaluate the biologic effect of adenoviral-mediated gene transfer of wild-type p53 gene in four hepatoma cell lines with different p53 genetic makeup. METHODS Recombinant adenovirus expressing wild-type p53 was used. Recombinant adenoviruses with either an empty expression cassette or expressing beta-galactosidase gene served as controls. RESULTS High-level expression of wild-type p53 was achieved with adenoviral-mediated gene transfer. The expressed p53 protein showed nuclear localization and its expression was associated with an induction of p21 and bax expression. Expression of the p53 gene was associated with inhibition of tumor cell proliferation and induction of apoptosis. Expression of p53 was also associated with an upregulation of CD95 (Apo-1/Fas) gene expression, which may predispose the tumor cells to undergo apoptosis induced by the Fas Ligand/Fas cytolytic pathway. An additional anti-tumor effect, in terms of allowing the replication-defective adenovirus to replicate, was observed in hepatoma cells with homozygous deletion of p53 genes and to a lesser extent, hepatoma cells with mutated p53 genes. CONCLUSIONS These data showed that adenoviral-mediated gene transfer is effective in delivering p53 gene to tumor cells, and the multiple pathways involved in their antitumor activities.

Erkrankungen des Magen-Darm-Traktes

Eine gastroosophageale Refluxkrankheit (GERD) liegt vor, wenn ein Risiko fur organische Komplikationen durch einen gesteigerten gastroosophagealen Reflux (GER) und/oder eine signifikante Storung der Lebensqualitat infolge der Refluxbeschwerden besteht (C). Unter GERD werden die verschiedenen Manifestationen NERD („non erosive reflux disease“), erosive Osophagitis (ERD), Barrett-Osophagus und extraosophageale Manifestationen subsumiert (C).

Krankheiten des Magen-Darm-Traktes und der Bauchspeicheldrüse

Traumen durch stumpfe oder penetrierende Verletzungen der gastrointestinalen Hohlorgane (Magen, Duodenum, Dunndarm, Pankreas, Kolon) konnen zur Organperforation und/oder Blutung mit der Notwendigkeit der zeitnahen chirurgischen Intervention fuhren. Bei Ingestion starker Sauren oder Laugen stehen Verletzungen des Osophagus im Vordergrund; Nekrosen bis hin zur Perforation des Magens sind jedoch moglich. Traumatische Verletzungen des Pankreas bedurfen eines interdisziplinaren Vorgehens (Endoskopie, Chirurgie). Der Grad der Schadigung orientiert sich am dauerhaften Funktionsverlust (Kurzdarmsyndrom, Anus praeter, endokrine/exokrine Pankreasinsuffizienz).

Krankheiten der Leber und Gallenwege

Die Mehrzahl der Gallenwegsverletzungen ereignet sich bei Operationen. Als Folge konnen sich Gallenfisteln in die freie Bauchhohle, den Magen-Darm-Trakt oder das Bronchialsystem oder Strikturen der Gallenwege entwickeln. Bei daraus entstehenden chronischen Cholangitiden und Cholestasen ist die Entwicklung einer biliaren Leberzirrhose moglich. Verletzungen der Leber konnen akut zu lebensbedrohlichen Blutungen und Schockzustanden fuhren. Die Bestimmung von GdB/GdS/MdE orientiert sich an sekundaren Funktionsstorungen. Wegen der guten Regenerationsfahigkeit des Leberparenchyms sind anhaltende Storungen der Organfunktion nach Lebertrauma die Ausnahme.