Hepatitis C virus (HCV) serotype in the asymptomatic HCV-infected patients from selected groups

In asymptomatic hepatitis C virus (HCV)-infected blood donors and persons from high risk groups (intravenous drug users and hemodialyzed patients) HCV serotypes 1, 2, 3, 4 and 6 have been detected. The most frequent was serotype 1. In the group of intravenous drug users (IDU) a coexistence of different serotypes was observed.     

Hepatitis C virus diagnostics: technology, clinical applications and impacts

To determine the basis of the therapeutic efficacy of Psoralens and UVA (PUVA) in inflammatory skin diseases, the effect of PUVA on the expression of cell adhesion molecules of keratinocytes was investigated in vitro. The addition of IFN-gamma and TNF-alpha to human keratinocytes in culture up-regulated the expression of ICAM-1 and HLA-DR on the cell surface. The cultured human keratinocytes were exposed to UVA light in the presence of 8-methoxypsoralen (PUVA). The ICAM-1 and HLA-DR surface molecules were stained by monoclonal antibodies, and the intensity of the resultant fluorescence was analyzed by FACS-can. PUVA treatment suppressed the expression of these cell surface molecules, with increasing UVA fluence. Moreover, PHA-blasts failed to adhere to PUVA treated keratinocytes. When keratinocytes were treated with PUVA prior to the addition of IFN-gamma and TNF-alpha, ICAM-1 and HLA-DR expression was suppressed. These results suggest that one of the therapeutic mechanisms of PUVA in inflammatory skin diseases is by inhibition of the adhesion of activated lymphocytes to keratinocytes due to suppression of cell surface molecules. It also suggests that PUVA may be useful as maintenance therapy for inflammatory skin diseases.     

Hepatitis C virus infection in the elderly. Epidemiology, prophylaxis and optimal treatment

Chronic infection with the hepatitis C virus (HCV) occurs throughout the world and appears to be the main cause of hepatocellular carcinoma. Studies have shown that, in areas of high endemicity, the prevalence of HCV infection is low in children but high in people aged > 60 years. Medical interventions were found to play an important role in the spread of HCV infection, because elderly patients became infected via contaminated blood transfusions or when contaminated syringes and needles were used. Maternal and sexual transmission do not appear to be the main routes of HCV infection. Interferon treatment eliminates HCV in 20 to 30% of patients with chronic HCV infection. The response to interferon therapy is usually complete in 70 to 80% of people with low levels of HCV RNA, HCV of genotype 2 and young women, but poor in elderly patients. Because liver disease can be severe in elderly patients, more effective therapies are clearly needed.     

Hepatitis C virus infection, mixed cryoglobulinemia, and non-Hodgkin's lymphoma: an emerging picture

Hepatitis C virus (HCV) is a single-stranded RNA agent which expresses its genetic informations in the form of a single, large polyprotein encoded by an open reading frame (ORF) that extends through most of its genomic RNA. Proteolytic cleavage of the ORF product is essential for the virogenesis and the production of viral progeny. HCV is responsible for chronic liver disease, cirrhosis and possibly hepatocellular carcinoma. Viral persistence is considered the greatest problem in the management of HCV infection. It may result from several mechanisms, two of which are established. In the first, the high rate of genetic variations during viral replication results in the production of mutants capable of escaping the immune attack. In the second, the virus infects cells of the immune system itself, which represent a privileged site that cannot be reached by virus-specific T cell response. Involvement of lymphoid cells in the early stages of HCV infection may provide insight into the pathobiologic patterns of extrahepatic dissemination (lymph nodes, major salivary glands, kidneys, blood vessels). Dissemination of HCV-infected lymphoid cells throughout the organism is likely to maintain a mobile and extensive reservoir of the virus. In this respect, extrahepatic sites may act as a source of continuous reinfection of hepatocytes. Studies of intrahepatic B lymphocytes indicate that they are infected with HCV, clonally expanded and activated to secrete IgM molecules with rheumatoid factor activity. This strongly suggests that HCV directly stimulates B cell expansion, which may result in an indolent stage of lymphoproliferation (i.e., mixed cryoglobulinemia) or in frank B cell non-Hodgkin's lymphoma (NHL). The frequency of NHL, however, is much lower than that of HCV infection, suggesting that HCV alone is not able to induce tumors and that cellular events, in addition to the presence of virus and virus-encoded products, are necessary in order to obtain a malignant B cell phenotype. The demonstration of HCV productive infection in bone marrow-recruited and circulating pluripotent hematopoietic CD34+ stem cells indicates that HCV replication occurs in the early differentiation stages of hematopoietic progenitors. These are stable cell populations and are likely to represent the initial site of infection and a continuous source of virus production.     

Hepatitis C virus c100 antigen in liver tissue from patients with acute and chronic infection

A pool of murine monoclonal antibodies developed against c100 antigen, a hepatitis C virus-associated protein encoded by the NS3/NS4 virus genome, was used to detect hepatitis C virus in liver biopsy specimens from patients with acute and chronic hepatitis C virus infection. The antigen was present in the cytoplasm of liver cells only. The immunoreactive signal appeared as large, distinct, brilliant fluorescent granules with no clear relationship to cellular structures. No obvious membrane c100 antigen accumulation was observed. Distribution of c100-containing hepatocytes was directly correlated with viral replication in acute hepatitis. All three acute-hepatitis patients were positive for hepatitis C virus RNA (as detected on polymerase chain reaction) in serum and displayed c100 antigen in 50% to 70% of hepatocytes, with a distinct topographical relationship with necrotic areas and inflammatory cell accumulation. Conversely, very low numbers of infected cells and no relationship between tissue c100 antigen expression and sites of liver cell necrosis and inflammation were found in 14 chronic hepatitis C virus infection patients. Furthermore, though all patients had measurable levels of serum hepatitis C virus RNA, only eight (57%) had detectable c100 antigen in liver sections. Indeed, these two distinct immunopathological patterns were inversely related to the development of c100 antibody in serum. Specificity of hepatocellular c100 antigen deposits was established through extensive absorption experiments using structural and nonstructural hepatitis C virus recombinant proteins. However, tissue processing was found to be a crucial step in the demonstration of hepatitis C virus antigen in fresh frozen liver tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution

In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation.     

Hepatitis C virus genotypes in liver transplant recipients: impact on posttransplant recurrence, infections, response to interferon-alpha therapy and outcome

BACKGROUND: End-stage liver disease due to hepatitis C virus (HCV) is the most common indication for liver transplantation in U.S. veterans. We investigated the influence of HCV genotypes on the incidence and timing of recurrent HCV hepatitis, survival, infectious morbidity, and response to interferon-alpha therapy in this unique patient population. METHODS: HCV genotype was determined by direct sequencing of the NS5 region of HCV with type-specific primers. RESULTS: Genotype 1a (66%, 32/47) was the predominant genotype. Type 1b was found in 25% (12/47) of patients and type 2b was found in 9% (4/47). Histopathologically recurrent HCV hepatitis developed in 53% (25/47) of the patients after transplantation. This group included 45% (14/31) of the patients with type 1a, 67% (8/12) of the patients with type 1b, and 25% (1/4) of the patients with type 2b (P>0.5). The time to recurrence and the severity of HCV recurrence as defined by aminotransferase levels or Knodell scores were not different among the three genotypes. There was a trend toward a higher incidence of major infections in patients with type 1b (75%) versus type 1a (48%) and type 2b (50%) (P=0.11). The response to interferon-a therapy did not differ significantly among the genotypes. Mortality at 5 years was 16% (5/31) in patients with genotype 1a, 42% (5/12) in patients with genotype 1b, and 50% (2/4) in patients with genotype 2b (P=0.06). CONCLUSIONS: The incidence, time to recurrence, and response to interferon-alpha therapy did not differ between the various genotypes in our liver transplant recipients. However, there was a trend toward higher infectious morbidity and overall mortality in patients with genotype 1b after transplantation.     

Two major proteins from locust plasma are involved in coagulation and are specifically precipitated by laminarin, a beta-1,3-glucan

Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260,000 (P260) and 85,000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260-P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260-P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260-P85 complex was added to a fully activated proPO system.     

Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi.     

Hepatitis C virus serotype-specific core and NS4 antibodies in injecting drug users participating in the Amsterdam cohort studies

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75. 5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5' untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.     

GB virus C RNA in serum, liver, and peripheral blood mononuclear cells from patients with chronic hepatitis B, C, and D

We examined fiber density, compound muscle action potential (CMAP) amplitude, and motor unit number estimate (MUNE) of the abductor digiti minimi and grip strength longitudinally. We sought to determine the effects of ALS on these measurements and to evaluate which of these tests may be more sensitive in evaluating progression of ALS and possibly predicting survival. Ten patients were examined at months 0, 3, and 6. A significant decrease in MUNE and increase in fiber density were observed at months 3 and 6 (p < 0.02) compared with baseline (month 0). Mean CMAP and grip strength declined, but not significantly. The decrease in MUNE over 6 months was significantly greater than that of CMAP and grip strength (p < 0.025). The significant changes in MUNE and fiber density over time suggest that they are more sensitive in measuring the rate of progression of ALS. To evaluate further the utility of these tests, we arbitrarily divided the patients into equal groups based on length of survival. MUNE declined significantly in the group with shorter survival (p < 0.01). Conversely, fiber density increased significantly in patients with longer survival (p < 0.01). With similar statistical analysis there were no significant differences in decline of CMAP or grip strength in either subgroup over 6 months. Our study suggests that MUNE and fiber density are more sensitive than CMAP and grip strength in detecting progression of ALS. Furthermore, we raise the hypotheses that a greater increase in fiber density identifies a group of patients with ALS who will have longer survival, and that a greater decline in MUNE identifies a group with a worse prognosis.     

Stevens-Johnson syndrome and toxic epidermal necrolysis are severity variants of the same disease which differs from erythema multiforme

A new classification, based on the pattern and distribution of cutaneous lesions, separates erythema multiforme major from Stevens-Johnson syndrome. A retrospective re-classification of 76 cases supported the validity of that separation by demonstrating differing causes and pathology. Another prospective international case-control study found differing demographic characteristics and risk factors between erythema multiforme major on the one hand and Stevens-Johnson syndrome or toxic epidermal necrolysis on the other. Erythema multiforme major was mainly related to Herpes virus infection, while Stevens-Johnson syndrome and toxic epidermal necrolysis were associated with drug reactions.