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S Mihm

Publications and source records attributed to S Mihm.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of gene expression profile in rat Kupffer cells stimulated with IFN-alpha or IFN-gamma.

BACKGROUND AND AIM: Kupffer cells are intrasinusoidal space located macrophages with phagocytic capacity. Interferons are cytokines with antiviral, antiproliferative and immunomodulatory activities which may influence the activity of Kupffer cells. Aim of this study was to evaluate Kupffer cell gene expression after interferon-alpha or interferon-gamma stimulation in order to investigate a link between these cytokines and macrophage activation. METHODS: Rat Kupffer cells were cultured for 24 h and divided into three groups: unstimulated; stimulated with interferon-alpha and stimulated with interferon-gamma. After 8 h stimulation total RNA was extracted and processed according to Affymetrix protocols and hybridised on R34A microarray gene set. Data analyses was performed using Microarray Analysis Suite 5.0 software. Genes showing remarkably different expression in microarray analysis were confirmed by real-time PCR. RESULTS: Nearly 4000 out of the 8800 genes represented in the array were expressed by Kupffer cells. Among these, interferon-alpha up-regulates 91 genes by over two-fold (antiviral, antigen processing and presentation, and tumour suppressor/proapoptotic genes) and down-regulates 72 genes by 50% or more. Interferon-gamma up-regulates 70 genes by over two-fold and down-regulates 78 genes by 50% or more. Most of the genes induced by interferon-alpha are also induced by interferon-gamma. Down-regulated genes include growth factors and genes involved in cell cycle/proliferation. Real-time PCR confirms the results of the array. CONCLUSION: Interferons directly target rat Kupffer cells and are involved in the regulation of a wide variety of genes. Their expression profile shed light onto molecular mechanism of Kupffer cells activation in specific pathways such as antiviral and antitumour processes.

Animals↗
PubMed2001

Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization.

Hepatitis C virus (HCV) infection causes acute and often also chronic liver disease. Worldwide, prevalence of infection is estimated to exceed that of human immunodeficiency virus infection fourfold. Because of the lack of appropriate animal models, knowledge of interactions between virus and host is still limited. Assumptions regarding pathogenesis or the activation status of innate antiviral host responses, for instance, derive mainly from clinical observations and from expression analyses of selected genes. To obtain a more objective insight into virus-host interrelationships, we used suppression-subtractive hybridization to compare gene expression in HCV-infected and non-HCV-infected liver tissues samples. Four differentially expressed genes were found: (i) the gamma interferon (IFN-gamma)-inducible chemokine IP-10 gene; (ii) the IFN-alpha/beta-inducible antiviral MxA gene; (iii) the gene encoding IFN-alpha/beta-inducible p44, shown to be associated with ultrastructural cytoplasmic entities within hepatocytes of non-A, non-B hepatitis-infected chimpanzees; and (iv) the gene encoding IFN-alpha/beta/gamma-inducible IFI-56K, a protein recently shown to interact with the eukaryotic translation initiation factor eIF-3. Compared to hepatic gene expression in patients with liver diseases unrelated to viral infections, expression in patients with chronic HCV infection was up to 50-fold higher. While in patients with chronic HBV infection IP-10 was slightly activated as well, the IFN-alpha/beta-regulated genes were not. Revealing a dominance of hepatic interferon-regulated processes in chronic HCV infection, data on the enhanced expression of the IFN-gamma regulated IP-10 support earlier findings and may explain the composition of the hepatic cellular infiltrate. The data on enhanced expression of IFN-alpha/beta inducible genes might be germane to therapeutic considerations.

Chemokine CXCL10↗
PubMed2001

HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

Adult↗
PubMed2000

Liver infiltrating T lymphocytes express interferon gamma and inducible nitric oxide synthase in chronic hepatitis C virus infection.

BACKGROUND: Pathogenesis of hepatitis C virus (HCV) associated liver injury is thought to be due to the host antiviral immune response. Using a quantitative, competitive RT-PCR technique, we recently showed that expression of interferon gamma (IFN-gamma) and IFN-gamma inducible type of nitric oxide synthase (iNOS) is increased in homogenised liver tissue of patients with chronic HCV infection. AIMS: To determine the cellular origin of IFN-gamma and iNOS expression and to examine the hypothesis that T cell derived IFN-gamma secretion induces iNOS in hepatocytes in chronic HCV infection. METHODS: By applying a non-radioactive in situ hybridisation method combined with indirect immunofluorescence, 33 liver biopsy specimens from patients with chronic HCV infection were studied for cellular expression of IFN-gamma and iNOS mRNA. RESULTS: In chronic HCV infection, both IFN-gamma and iNOS gene expression were significantly increased. IFN-gamma and iNOS mRNA were observed in CD3+ lymphocytes infiltrating portal tracts and hepatic lobules, but not in hepatocytes. CONCLUSIONS: Results are consistent with previous reports that IFN-gamma and iNOS transcripts are elevated in chronic HCV infection. In contrast to the hypothesis, IFN-gamma expressing T cells do not induce iNOS in hepatocytes, but probably in T cells. T lymphocytes expressing IFN-gamma and/or iNOS have the potential to participate in autocrine and paracrine pathways that may contribute to the pathobiology of chronic hepatitis C.

Adult↗
PubMed2000

Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase-mediated cellular antiviral responses.

The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system. Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR. The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver. ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material. As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique. Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression. The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability.

Adolescent↗
PubMed2000

MxA gene expression in peripheral blood mononuclear cells from patients infected chronically with hepatitis C virus treated with interferon-alpha.

Hepatitis C virus (HCV) infection causes acute and often chronic liver disease. The treatment of choice is interferon-alpha (IFN-alpha). The proportion of patients responding to therapy in terms of a sustained virological response, however, is relatively low. One possible reason for the lack of effectiveness might be neutralization of the drug by host's inhibitory factors. Recent kinetic studies suggested that high doses of IFN-alpha-, especially during the initial phase of therapy, might improve the virological response rates. Eighteen patients infected chronically with HCV were treated with IFN-alpha either at a standard dose (3 x 10(6) to 6 x 10(6) IU IFN-alpha three times weekly) for 6 to 12 months or with an intensified therapy (6 x 10(6) IU IFN-alpha daily) for at least one month. As surrogate parameter for the intracellular effect of the drug, MxA gene expression was quantified in RNA preparations from peripheral blood mononuclear cells. Beta-2-microglobulin (beta2M) concentrations were measured in serum. Serum HCV RNA titers were monitored in parallel. When compared to healthy individuals, untreated patients infected chronically with HCV were found to express 2.8-fold higher amounts of MxA specific transcripts. MxA gene expression and serum beta2M concentrations were found to be induced after administration of IFN-alpha, independent of the virological response not only during the initial phase of the intensified therapy but also over several months during standard therapy. It is concluded from these results that both early non-effectiveness of high dose IFN-alpha therapy as well as long-term non-effectiveness of standard therapy are not due to IFN-alpha inhibitory or neutralizing elements in serum.

Adult↗
PubMed1999

Clearance of HCV RNA in a chronic hepatitis C virus-infected patient during acute hepatitis B virus superinfection.

The routes of hepatitis B virus and hepatitis C virus transmission are quite similar and coexistence of both viruses in one patient is not a rare phenomenon. Until now, the natural course of liver diseases induced by coinfections has not been well documented and the mechanisms of interaction between the two viruses and the human host have not been fully clarified. We report the case of a patient suffering from chronic hepatitis due to hepatitis C virus who developed an acute hepatitis B virus superinfection. Serum hepatitis C virus ribonucleic acid became undetectable by reverse transcriptase/polymerase chain reaction at diagnosis of acute hepatitis B virus infection. At the same time, there was a striking increase in the serum concentrations of the antibodies against C22 and C33c hepatitis C virus antigens. Four months after clinical resolution of the acute hepatitis, hepatitis B surface antigen was undetectable in serum and three months later antibodies against hepatitis B surface antigen appeared. Two years after acute hepatitis B virus infection, the patient has had no relapse of markers for viral replication of hepatitis B virus. Transaminases are within the reference range and hepatitis C virus ribonucleic acid is undetectable in both serum and liver tissue. We hypothesize that acute hepatitis B virus infection stimulated a specific humoral response against hepatitis C virus as well as triggering non-specific defense mechanisms which finally eliminated both viruses.

Acute Disease↗
PubMed1999

Ratio of serum gamma-GT/ALT rather than ISDR variability is predictive for initial virological response to IFN-alpha in chronic HCV infection.

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-alpha. Because the proportion of patients responding to therapy with sustained or even just with transient elimination of viral RNA is low, several potential prognostic parameters have been evaluated to predict the outcome of the therapy. The present study aimed to prove the validity of a predictive parameter described previously for initial virological response, namely the ratio of serum gamma-glutamyltransferase/alanine transaminase (gamma-GT/ALT) activity in connection with virus genotypes 1a, 1b, and 3a, prospectively and to compare the predictive value of these combined parameters with amino acid variability within the interferon sensitivity determining region (ISDR). The prospective analysis confirmed previous data on the predictive value of the serum gamma-GT/ALT ratio. Concerning ISDR variability, the majority of ISDR sequences obtained from the mostly nonresponding type 1b-infected individuals (23/28) resembled nonmutant types (27/ 28). Isolates from type 3a-infected patients responding to therapy in the majority of cases (13/ 20) exclusively resembled nonmutant types when compared with databank type 3a sequences, but were mutant when compared with the prototype sequence HCV-J. However, the initial virological responsiveness among both type 1b- and type 3a-infected patients did not correlate to ISDR variability. In contrast, virological responsiveness was closely related to serum gamma-GT/ALT ratio. The data are not necessarily contrary to the concept that the number of amino acid exchanges within the ISDR compared with the prototype HCV-J sequence is related to some extent to IFN-alpha sensitivity. The ratio of serum gamma-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter.

Adult↗
PubMed1997

Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype.

Hepatitis C virus (HCV) causes acute and often chronic hepatitis. On the basis of variations in nucleotide sequence, at least six genotypes and several subtypes have been identified. Histopathologically, chronic HCV infection is characterized by relatively mild hepatic inflammatory activity and a low degree of fibrosis, but hepatic lesions might be accompanied by bile duct damage, intraportal lymphoid aggregates, steatosis, or a combination of these manifestations. The histopathological lesions thus appear quite heterogeneous. To address the question of whether distinct histopathological manifestations are related to particular genotypes of HCV, 90 patients with chronic HCV infection were analyzed regarding histopathological features, biochemical liver parameters, demographic data, and virus genotype. The results revealed a significantly higher prevalence of both steatosis and bile duct lesions among patients infected by HCV type 3a compared to patients infected by types 1a or 1b. Furthermore, the data suggest interrelationships between virus genotype, patient's age, and a history of intravenous drug abuse. However, none of the histopathological manifestations were found to be related to a history of drug abuse. The data further corroborate the relationship of HCV type 1b infection to age, duration of disease, and the degree of fibrosis, respectively. Irrespective of HCV genotype, elevated serum ALT activity was shown to be associated with pronounced inflammatory activity or pronounced steatosis as well. Thus, the current data support the hypothesis that distinct genotypes of HCV appear to be associated with distinct manifestations of disease.

Adult↗
PubMed1997

Hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis C virus infection: relation to hepatic viral load and liver injury.

Hepatitis C virus (HCV) causes acute and often also chronic liver disease. Hepatocellular injury might result from both a host response directed to inhibit viral spread and from processes initiated by the virus itself. To study mechanisms involved in hepatocellular injury, liver tissue from chronically HCV-infected patients was analyzed for the expression of the inducible isoform of nitric oxide synthase (iNOS) and for interferon gamma (IFN-gamma) by a quantitative, competitive reverse-transcription-polymerase chain reaction (RT-PCR) technique. Moreover, hepatic viral load was determined by two independent techniques, and liver tissue was evaluated histopathologically in detail. Liver tissue from HCV-infected patients was shown to express elevated levels of iNOS transcripts compared with non-HCV-infected patients. Increased iNOS transcript expression, however, was not accompanied by significantly elevated serum nitrite plus nitrate (NOx) concentration, although some of the chronic HCV-infected patients reached markedly higher serum NOx levels than the control group or healthy individuals, respectively. Hepatic iNOS expression was found to be positively correlated to hepatic HCV-RNA content on the one hand, and weakly to hepatic IFN-gamma expression, previously shown to be solely associated with hepatic necro-inflammatory activity among the histopathological parameters studied, on the other hand. In contrast to IFN-gamma transcript expression, neither hepatic iNOS expression nor hepatic HCV-RNA content were related to hepatic inflammatory activity. The presented data are compatible with the assumption that HCV might be able to stimulate iNOS expression both directly and indirectly via its capacity to induce IFN-gamma.

Adult↗
PubMed1997

Interferon-alpha treatment of hepatitis C virus-associated mixed cryoglobulinemia.

BACKGROUND/AIMS: Chronic hepatitis C virus infection is frequently associated with mixed cryoglobulinemia. The efficacy of interferon-alpha treatment in the presence of cryoglobulinemia, particularly the rate of sustained responders, has not yet been well defined. METHODS: Fifty-nine consecutive patients with chronic HCV infection were studied prospectively with regard to the presence of cryoglobulinemia and their biochemical and virological response to interferon-alpha2a therapy. RESULTS: Cryoglobulins were detected in sera of 23 patients. For this latter group of patients, significant differences were found compared to the 36 patients without cryoglobulinemia, i.e. the prevalence of female sex was higher, the duration of liver disease was longer and distinctive laboratory abnormalities, e.g. higher rheumatoid factor activity, were noted as well as a higher prevalence of cirrhosis. The distribution of HCV genotypes and serum HCV RNA titers was similar in the two groups. Interferon-alpha treatment regimens were not different regarding mean cumulative dose and mean duration of therapy. The response to therapy was almost identical, i.e. 35% of patients with cryoglobulinemia showed a sustained response compared to 22% of patients without cryoglobulinemia. The percentages of patients showing a relapse or breakthrough were similar in both groups. Pre-treatment viremia levels were higher in non-responders compared to sustained responders. Non-responders appeared to be more frequent among patients infected with genotypes 1a and 1b, especially among male patients without cryoglobulinemia. CONCLUSIONS: The presence of cryoglobulinemia per se in chronic HCV-infected patients does not adversely affect the outcome of interferon-alpha therapy, including the rate of sustained response.

Adult↗
PubMed1997

Clinical, biochemical, and histological changes in hepatitis C virus infection-associated cryoglobulinemia.

The most common extrahepatic manifestation of HCV infection is mixed cryoglobulinemia (MC). 62 unselected patients with chronic HCV infection were prospectively evaluated for the presence of cryoglobulinemia and associated clinical and biochemical parameters. Furthermore, a putative relationship between the HCV genotypes and cryoglobulinemia was tested. Histological features typical for HCV infection were comparatively analyzed in cases with and without cryoglobulinemia. Whether an intrahepatic Th2-response is responsible for the strong antibody production causing cryoglobulinemia was also examined. Cryoglobulins were detected in sera of 30 patients (approximately equal to 48%). Patients with cryoglobulinemia were on the average elder, showed an apparent longer duration of infection, and suffered more frequently from arthralgia, accompanied by a significant increase of total serum IgM concentration and rheumatoid factor activity. The HCV genotype distribution among patients with cryoglobulinemia was not different from that found in patients without cryoglobulinemia. In cases with cryoglobulinemia, an increased activity of chronic hepatitis and a higher grade of liver fibrosis was noted. The prevalence of HCV-typical histological lesions among all patients were: Portal lymphocytic aggregates (40%), bile duct damage (35%), steatosis (47%), and intracellular acidophilic bodies (29%). A significant correlation, however, could not be found between cryoglobulinemia and the presence of HCV-typical histological lesions. An intrahepatic Th2-response causing an increased antibody production could not be observed in cases with cryoglobulinemia.

Adult↗
PubMed1996

Preferential virological response to interferon-alpha 2a in patients with chronic hepatitis C infected by virus genotype 3a and exhibiting a low gamma-GT/ALT ratio.

Hepatitis C virus infection causes acute and often chronic hepatitis. Therapy with interferon-alpha has been shown to induce remission of the inflammatory process within the liver and also elimination of the virus. However, only about 50% of treated patients respond in terms of at least a transient disappearance of viral RNA from the circulation below the limit of detection. In order to find prognostic factors for responsiveness, patients with chronic hepatitis C virus infection were analyzed for virus genotype and pretreatment biochemical liver parameters including serum AST, ALT, and gamma-GT activities. Whereas the initial biochemical response to interferon-alpha 2a was found not to be related to virus genotype, the initial virological response was found to be closely related to infection by genotype 3a and to a low pretreatment ratio of serum gamma-GT/ALT activity. These data confirm and extend the importance of virus genotype for responsiveness to interferon-alpha therapy and introduce an additional, host-specific parameter with a potential predictive value, namely the pretreatment ratio of serum gamma-GT/ALT activity.

Adult↗
PubMed1996

High inflammatory activity is associated with an increased amount of IFN-gamma transcripts in peripheral blood cells of patients with chronic hepatitis C virus infection.

The mechanisms underlying the chronic hepatic inflammatory process in hepatitis C virus (HCV) infection are not well understood. Some models of experimentally induced hepatitis point to a role of interferon-gamma (IFN-gamma) secreted by liver-infiltrating peripheral blood lymphocytes (PBMC) in mediating hepatocellular injury. In the present study, IFN-gamma gene expression was analysed in PBMC and in liver biopsy specimens from patients with chronic HCV infection using a quantitative reverse transcriptase polymerase chain reaction technique. IFN-gamma gene expression by PBMC from HCV-infected patients exhibiting elevated serum transaminase activities was found to be increased up to ninefold when compared with (1) healthy individuals, (2) HCV-infected patients exhibiting normal or only slightly elevated serum enzyme activities, or (3) patients with drug-induced elevated serum transaminase activity. A histo-pathological evaluation of liver biopsy sections revealed further that high IFN-gamma gene expression by PBMC was associated with a more pronounced degree of inflammatory activity. In individual patients, the expression of IFN-gamma by PBMC was shown to parallel closely serum transaminase activities during IFN-alpha 2a therapy. Moreover, liver biopsy material from patients chronically infected with HCV contained higher amounts of IFN-gamma transcripts than liver tissue from patients with liver disorders unrelated to HCV infection or without any liver disease. These data thus demonstrate a close association between the amount of IFN-gamma transcripts in PBMC and in liver tissue and the inflammatory activity in chronic HCV infection in man.

Adult↗
PubMed1996

A reevaluation of the association of hepatitis C virus replicative intermediates with peripheral blood cells including granulocytes by a tagged reverse transcription/polymerase chain reaction technique.

BACKGROUND/AIMS: Persistence of hepatitis C virus at extrahepatic sites is of both basic and clinical interest. The clinical interest arises mainly from the occurrence of reinfections of the hepatic allograft following transplantation. Therefore, any extrahepatic association of virus, e.g. with peripheral blood cells, appears relevant. METHODS: In this study we employed for the first time the recently developed tagged reverse transcription/polymerase chain reaction procedure to determine the presence of genomic HCV RNA and antigenomic replicative intermediates in RNA preparations from sera, peripheral blood mononuclear cells, and polymorphonuclear granulocytes of 29 patients with chronic hepatitis C virus infection. RESULTS: All sera were found to contain both genomic and antigenomic HCV RNA. In addition to peripheral blood mononuclear cells, viral nucleic acids were found to be associated with polymorphonuclear granulocytes, too. CONCLUSIONS: In individual patients different patterns were observed for the distribution of hepatitis C virus genomes and antigenomes among peripheral blood mononuclear cells and polymorphonuclear granulocytes, apparently neither related to pretreatment biochemical parameters, nor to response following interferon-alpha 2a treatment, nor to hepatitis C virus genotype.

Base Sequence↗