High predictability of a sustained virological response (87%) in chronic hepatitis C virus genotype 1 infection treatment by combined IL28B genotype analysis and γ-glutamyltransferase/alanine aminotransferase ratio: a retrospective single-center study.

BACKGROUND: Chronic hepatitis C virus genotype 1 (HCV-G1) infection is treated with pegylated interferon-α and ribavirin. Predictive factors for treatment success are even more important now as direct-acting antiviral agents are available. METHODS: Clinical and laboratory parameters were analyzed by uni- and multivariate statistical means in 264 patients with HCV-G1 infections with regard to treatment outcome. RESULTS: The overall sustained virological response (SVR) rate was 44%. Univariate analyses revealed SVRs to be associated with age, high alanine aminotransferase (ALT) and low γ-glutamyltransferase (γ-GT) serum activities, a low pretreatment γ-GT/ALT ratio, rapid virological response (RVR), and absence of steatosis. Multivariate analyses unveiled IL28B rs12979860 genotype (CC vs. CT: OR = 2.8, CI: 1.5-4.9, p = 0.001; CC vs. TT: OR = 7.1, CI: 3.1-16.7, p < 0.001), low pretreatment γ-GT/ALT ratio (OR = 2.5, CI: 1.7-3.3, p < 0.001), age (OR = 0.96, CI: 0.94-0.98, p = 0.001) and RVR (OR = 4.18, CI: 2.85-8.65, p < 0.001) to be significantly related to treatment outcome. Patients with the IL28B rs12979860 CC genotype and a low pretreatment γ-GT/ALT ratio achieved the highest rate of a SVR with the highest predictive values (OR = 26.7, 95% CI: 10-71.1, p < 0.0001). CONCLUSION: The pretreatment γ-GT/ALT ratio significantly enhances the predictability of the IL28B genotype. Employing this combination will help to identify patients who will most likely benefit from an interferon-α-based combination therapy in a nontriaged ordinary setting.

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.

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives.

Mitogenically stimulated human peripheral blood lymphocytes and T cell clones were found to have weak membrane transport activity for the disulfide cystine but strong membrane transport activity for the thiol amino acid cysteine. Cysteine, however, is represented at the lowest concentration among all protein-forming amino acids in the blood plasma. Complementary laboratory experiments have shown that the cysteine supply is indeed limiting for important lymphocyte functions. Proliferative responses of mitogenically stimulated lymphocytes and T-cell clones and the activation of cytotoxic T cells in allogeneic mixed lymphocyte cultures are strongly influenced by small variations in the extracellular cysteine concentration even in the presence of relatively high and approximately physiologic concentrations of cystine. Cysteine can be substituted by N-acetylcysteine but not by cystine. The more detailed analysis revealed that the extracellular supply of cysteine influences strongly the intracellular level of glutathione (GSH) and also the activity of the transcription factor NF kappa B that regulates the expression of several immunologically relevant genes. In vitro experiments including double-chamber experiments with macrophages and lymphocytes revealed, moreover, that cysteine plays an important role as a regulatory mediator between these cell types. The cysteine supply is impaired directly or indirectly in several pathologic conditions that are associated with immunodeficiencies, including the acquired immune deficiency syndrome (AIDS). Cysteine or cysteine derivatives may therefore be considered for the treatment of patients with HIV-1 infection.

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.

Immunomodulatory action of eicosanoids and other small molecular weight products of macrophages.

There is an increasing body of evidence that T cell-mediated immune response is regulated by a variety of small molecular weight products of macrophages. One of the best known immunoregulatory products in this context is prostaglandin E2 (PGE2) which is known to regulate both T cell and macrophage functions. Recently, ornithine, cysteine, and lactate have also been recognized as immunoregulatory mediators. In analogy to the hormone-like cytokines and lymphokines, all these substances are produced by immunologically relevant cells (macrophages) at a variable and regulated rate; and they have been shown to regulate the functional activities of other cells (T cells and macrophages). This brief review describes the key observations that underscore the important regulatory role of these metabolites in physiological and pathological conditions.

Suppression of cytotoxic T-lymphocyte activation by pyruvate and L-alanine.

The activation of cytotoxic T lymphocytes (CTL) in allogeneic mixed-lymphocyte cultures was found to be strongly inhibited if 1-3 X 10(-2) M L-alanine or the structurally and biochemically related substance pyruvate was present in the period from 7 to 19 or from 19 to 120 hr. The cytotoxic response was not inhibited when L-alanine or pyruvate was present during the first 7 hr of the culture period. L-Alanine produced also little or no suppression, if added on Day 3 of the culture. L-Lactate or D-alanine at similar concentrations was not suppressive during the entire culture period. The suppression by pyruvate and L-alanine was strongly reduced by 1 X 10(-4) M adenosine. Adenosine in combination with an interleukin-2 (IL-2)-containing EL-4-cell supernatant was even more effective. Pyruvate and alanine (1-3 X 10(-2) M) also inhibited the DNA synthesis in mixed-lymphocyte cultures on Day 5 by about 50%, but both substances had practically no effect on DNA synthesis in cultures that had been supplemented with an IL-2-containing EL-4 supernatant. They had also no effect on the IL-2-dependent proliferation of several T-cell clones or of concanavalin A-activated thymocytes. These relatively selective regulatory effects of pyruvate and L-alanine may be useful for the analysis of the biochemical pathways during lymphocyte activation and/or for a selective manipulation of the immune response.

Regulation of cytotoxic T-lymphocyte activation by L-lactate and pyruvate.

The activation of cytotoxic T lymphocytes (CTL) in vivo was found to be strongly augmented by two injections of 0.2 ml 1 X 10(-1) M pyruvate in spite of the relatively high concentration of glucose (approximately 10(-2) M) in the blood. The repeated injection of 1 X 10(-1) M L-lactate, in contrast, was found to suppress cytotoxic responses in vivo. The activation of CTL and DNA synthesis in mixed lymphocyte cultures, on the other hand, was found to be suppressed by pyruvate (1 X 10(-2) M), and was substantially augmented by 1 X 10(-2) M L-lactate. The glucose concentration in the culture medium was also approximately 10(-2) M. Taken together, these results suggest that the utilization of glucose is relatively ineffective and that the respiratory metabolism is a more effective source of energy during the early T cell activation. The results suggest also that the aerobic glycolysis of macrophages and their release of L-lactate may contribute to their function as accessory cells in immune responses. The differences between the in vivo and in vitro results are discussed.

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.

Modulation of transcription factor NF kappa B activity by intracellular glutathione levels and by variations of the extracellular cysteine supply.

HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We now show that a depletion of intracellular glutathione in a human T cell line (Molt-4) inhibits the activation and nuclear translocation of the transcription factor NF kappa B, whereas incubation with increasing extracellular concentrations of cysteine inhibits the DNA-binding and transactivating activity of NF kappa B. Because inhibition of DNA-binding activity is associated with increasing intracellular glutathione disulfide levels and GSSG can be shown to inhibit the DNA-binding activity directly in cell-free systems, our studies suggest that GSSG is a physiologically relevant inhibitor in intact cells also. NF kappa B controls many immunologically important genes, so our studies suggest that the immune system may be sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.

Distinct effects of glutathione disulphide on the nuclear transcription factor kappa B and the activator protein-1.

Oxidative conditions potentiate the activation of the nuclear transcription factor kappa B (NF kappa B) and the activator protein-1 (AP-1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NF kappa B and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NF kappa B activation in human T lineage cells (Molt-4) by 12-O-tetradecanoyl-phorbol 13-acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NF kappa B activation. These effects of BCNU and hydrogen peroxide were not seen in glutathione-depleted cells. However, NF kappa B and AP-1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NF kappa B but increased that dependent on AP-1. This selective suppression of NF kappa B was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NF kappa B more effectively than that of AP-1, while AP-1 was inhibited more effectively by oxidized thioredoxin.

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.

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.

Release of L-alanine by tumor cells.

Culture supernatants from the weakly immunogenic T cell lymphoma L5178Y ESb were found to contain substantial amounts of alanine and lactate at a ratio of about 1:10. Supernatants from cells of the highly immunogenic mutant line ESb-D also contained lactate but only minute amounts of alanine. Moreover, ESb cells converted 14C-labeled glucose or pyruvate into labeled alanine and lactate at a ratio of about 1:10, whereas ESb-D cells yielded only labeled lactate and no detectable alanine. The injection of L-alanine in combination with L-lactate into mice strongly suppressed the capacity of their spleen cells to generate cytotoxic responses. The injection of L-alanine also suppressed the immunogenicity of ESb-D cells, as demonstrated by the generation of cytotoxic activity in vivo and by the in vivo immunization (priming) for secondary cytotoxic responses against ESb-D cells in vitro. Taken together, these experiments suggest the possibility i) that the ESb cells prevent the induction of cytotoxic responses by releasing immunosuppressive alanine, and ii) that the immunogenic mutant ESb-D may have gained immunogenicity by losing this immunosuppressive property.

HIV-induced cysteine deficiency and T-cell dysfunction--a rationale for treatment with N-acetylcysteine.

Markedly decreased plasma cystine and cysteine concentrations have been found in HIV-infected patients at all stages of the disease and in SIV-infected rhesus macaques. The elevated glutamate levels found in the same patients aggravate the cysteine deficiency by inhibiting the membrane transport activity for cystine. The intact immune system appears to require a delicate balance between pro-oxidant and antioxidant conditions, maintained by a limited and well-regulated supply of cysteine. This balance is obviously disturbed in HIV infection and may contribute to the pathogenesis of AIDS.