Coinfection with GBV-C is associated with a reduced mortality rate in HIV-infected patients. GBV-C is not known to cause any disease, but it is possible that its presence leads to an inhibition of HIV replication. However, GBV-C infection could also be a marker for the presence of other factors that lead to a favorable HIV response.

To assess the significance of cell-mediated immunity, T cells were derived from the peripheral blood and liver tissue of hepatitis B virus (HBV)-infected patients and controls. The analysis of the 3H-thymidine-uptake in response to a panel of recombinant HBV antigens revealed that peripheral blood mononuclear cells (PBMC) of the 25 viremic patients with inflammatory active, chronic hepatitis B, 16 with wild-type and nine with HBe-minus HBV mutant infection, showed stronger proliferative responses to HBc and HBe antigens than 16 asymptomatic nonviremic HBsAg carriers with normal aminotransferase levels (HBc: SI 19.3 +/- 3.9 vs. 13.0 +/- 3.2 vs. 8.0 +/- 1.2; P < .01 and HBe: SI 16.6 +/- 4.0 vs. 10.7 +/- 3.5 vs. 6.9 +/- 1.5; P < .05). In 15 patients with acute self-limited hepatitis B, however, significantly stronger HBc antigen-specific T-cell responses were observed during HBV clearance and HBe/anti-HBe seroconversion, whereas in nine completely HBV-immunized patients only minor proliferative responses to HBV antigens were observed. Six HBe/HBcAg- and two HBeAg-specific CD4+ T-cell lines could be expanded from liver tissue and peripheral blood of six viremic patients with chronic hepatitis B. Irrespectively of HBV mutations the HBV-specific activation of the T-cell lines was restricted by the presence of HLA-DR molecules and resulted in the release of Th1-like cytokine patterns. Follow-up of interferon (IFN) recipients showed simultaneous short-term increase of HBc/HBe-specific T-cell reactivities in responder patients during HBV clearance and HBe/anti-HBe seroconversion, whereas in nonresponders high virus load and HBV-specific immune responses were in imbalance.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the results of a multicenter evaluation of a new assay for the detection of hepatitis C virus (HCV) RNA in human serum or plasma based on transcription-mediated amplification (HCV TMA). Analysis of combined data obtained from 15 independent sites, including 4 sites in the United States and 11 in Europe, by using preproduction kits showed a limit of detection of 9.8 IU/ml and an overall mean specificity of 97.9%. In addition, assay runs and samples were valid consistently (97.8% of assay runs and 98.0% of specimen results). Of the 15 sites that participated in the multicenter evaluation, 2 subsequently carried out additional performance studies with production kits in support of the assay's registration in France. Comparison of the findings from these two sites during the multicenter evaluation and during the registration studies showed an overall improvement in assay performance. A statistically significant (P < 0.001) improvement was achieved for both specificity and specimen validity in the registration studies, which were 99.4 and 98.1%, respectively. Combined data from the two sites showed a lower limit of detection of approximately 2.4 IU/ml and an improved assay validity of 100%, although the sample size was too small to show statistical significance at the 0.05 level. In summary, the performance characteristics of HCV TMA indicate that this assay is a reliable tool for the detection of HCV RNA in serum or plasma. Improvement in assay performance has been demonstrated with refinement of assay reagents, instrumentation, and operator experience.Qualitative hepatitis C virus (HCV) RNA assays play a key role in both the diagnosis and monitoring of HCV infection. The enzyme immunoassays (EIAs) that detect antibodies to HCV cannot differentiate between active and resolved infection. Qualitative HCV RNA assays detect viral genomes and therefore can both confirm the presence of active infection and demonstrate its presence 4 to 6 weeks before antibody seroconversion takes place (1). Most qualitative HCV RNA tests are 10-to 100-fold more sensitive than are quantitative HCV RNA tests and up to 1,000-fold more sensitive than is the HCV core antigen assay, rendering them the method of choice both for confirming active infection and for assessing viral clearance in response to therapy (4, 7). Indeed, according to the recent National Institutes of Health consensus statement on management of hepatitis C (http://consensus.nih-gov/cons/116 /116cdc_intro.htm), a qualitative HCV RNA assay with a lower limit of detection of 50 IU/ml or less should be used to confirm acute or chronic HCV infection in a patient with a positive EIA result as well as to indicate sustained virologic response, defined as the absence of detectable HCV RNA in serum at 24 weeks after the end of treatment.A new assay for the detection of HCV RNA in human serum or plasma based on transcription-mediated amplification (HCV TMA) has recently been introduced. Developed and manufactured by Gen-Probe Inc. (San Diego, Calif.) and marketed by Bayer Co...

Recently, it was demonstrated in chronic hepatitis C that the release of IgG and IgM anti-HCV antibodies by mononuclear cells (PBMCs) correlated with inflammatory activity, HCV persistence in serum, and negative outcome from antiviral therapy. Thus, persistent antigenic stimulation of the antibody-secreting B cells has been suggested. In this study, PBMCs were derived from 13 patients with chronic hepatitis C. Nucleic acids were extracted by the guanidine-thiocyanate-method, and plus- and minus-stranded HCV-RNAs were determined using primers from the 5'-untranslated region of HCV. Simultaneously, unstimulated PBMCs were cultured for 8 days and anti-HCV antibodies were detected in the supernatants by EIA and RIBA. Seven patients (53.8%) had both plus- and minus-stranded HCV-RNA in PBMCs, while anti-HCV antibodies were secreted in vitro. One of 2 patients with plus- but not minus-stranded HCV-RNA in PBMCs was anti-HCV positive in vitro, whereas 4 patients without HCV-infected PBMCs were anti-HCV negative in vitro. Eight patients received antiviral therapy with interferon-alpha 2b. Four nonresponders and 1 partial responder had plus- and minus-stranded HCV-RNA in PBMCs and anti-HCV secretion in vitro. On the other hand, 2 complete responders and another partial responder showed neither HCV infection of PBMCs nor anti-HCV secretion in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the T-cell receptors (TCR) used by synovial cytotoxic T lymphocytes (CTL) of patients with reactive arthritis (ReA) may be crucial to better understanding the pathogenetic mechanism underlying the HLA-B27 association of spondylarthropathies. The authors, therefore, sequenced 25 TCRB chains from HLA-B27-restricted CD8+ CTL clones and two clonal lines specific for self- or Yersinia enterocolitica antigen isolated from synovial fluids of 3 HLA-B27+ patients with ReA and PBL of one healthy HLA-B27+ individual. Fourteen non-HLA-B27-restricted CTL served as controls. Both autoreactive and Y. enterocolitica specific HLA-B27-restricted CTL used a highly limited set of VB genes with preferential rearrangement of three closely related VB families (VB 13, 14, 17), suggesting that these families contain a preferred site for contact with the HLA-B27 molecule. In addition, the presence of limited TCRBJ usage, limited heterogeneity in CDR3 sequences and dominant clones from individual donors among these CTL indicate that TCRB chain usage is further restricted by a limited set of peptides bound to the HLA-B27 molecule. Limited TCR usage by SF CTL of ReA patients may lay a basis for therapeutical manipulation of the T-cell response in the spondylarthropathies.

A number of naturally occurring hepatitis B virus (HBV) mutants unable to synthesize the hepatitis B e antigen (HBeAg) have been identified in patients characterized by HBV DNA and anti-HBe in their serum. Because the analysis of the HBV-associated DNA and antigens in the liver tissue is still not complete, we investigated the precore sequence of HBV DNA and its encoded proteins in the liver tissue of 32 patients positive for HBV DNA and anti-HBe in their serum. Three different groups of patients were identified. Group I (n = 14) was characterized by viral DNA sequences with a G-A transition in the distal precore gene region, thus creating a termination codon (TAG). Liver tissue from this group was negative for HBeAg but positive for hepatitis B core antigen (HBcAg) and a peptide containing the last 10 aminoacids of the precore and the first four aminoacids of the c region. Group II (n = 6) showed variable mutations in base sequences further upstream and negative for HBeAg and the precore peptide. Group III (n = 12) contained wild-type HBV DNA sequences in the tissue and positivity for precore- and core-encoded proteins. We conclude from our study that the group of patients with HBV DNA and anti-HBe in the serum is rather heterogeneous, including HBV DNA mutations in the precore region as well as infection with the wild-type virus being negative for HBeAg in the serum. The precore peptide is produced and expressed in the tissue even when the formation of a stop codon at base 1896 prevents the complete translation of the entire HBe protein.

Orthotopic liver transplantation (OLT) is a possible treatment for acute or chronic liver failure due to hepatitis B virus (HBV) infection, but reinfection of the graft can be a serious complication. The aim of this study was to monitor HBV markers, to analyse pre-core-/core-mutations as well as to identify the viral population causing reinfection after OLT, and to investigate the emergence or disappearance of these mutants in patients receiving immunosuppressive treatment. Fifty-four pre-and posttransplant serum samples of 17 patients were analysed. All patients underwent OLT for HBV-related liver disease and had HBV-DNA before and after OLT. Total DNA was extracted from all sera and a 240 bp fragment comprising the pre-core region of HBV was amplified by polymerase chain reaction (PCR). Pre-core mutants of HBV were determined by direct sequencing of these PCR products and by sequencing of PCR clones. Eight of 17 patients were infected with pre-core wildtype HBV before OLT (group A). Seven of eight patients of group A were reinfected by pre-core wildtype HBV after OLT. In one of eight patients in addition to wildtype HBV a mutant strain (nt. 1899 G-->A) was detected. Nine of 17 patients were infected with pre-core mutant HBV before OLT (group B). Six of nine patients of group B were reinfected with the same mutant population; in one, an additional pre-core mutation emerged; two patients lost pre-core mutant HBV (nt. 1896 and 1899 G-->A). In one of the latter two, a pre-core start-codon mutant (nt. 1816 G-->T), not detectable before OLT, emerged, in the other a nt. 1897 G-->A stop-codon mutant persisted. Five patients of each group were followed-up for more than 24 (25 to 58) months on immunosuppressive therapy. In all five patients of group A, pre-core wildtype of HBV persisted during long-term follow up. Two of five patients of group B were infected stably with a stop-codon HBV-mutant nt. 1896. In three patients, the nt. 1896 stop-codon mutant disappeared during immunosuppressive therapy. However, in one of the latter three, an HBV stop-codon mutant nt. 1897 persisted. In conclusion, most patients who underwent OLT for HBV-related disease were reinfected with the same virus population that existed before OLT. In rare cases, new mutants emerged after OLT or preexisting mutants were lost. During long-term follow-up on immunosuppressive therapy, in the majority of patients pre-core mutants disappeared and wildtype HBV became the predominant virus strain.

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