Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis

Although several reports concerning the detection of hepatitis C virus (HCV) by in situ hybridization have been published, there are no data concerning the relative viral load in infected hepatocytes or about its relation with serum viremia levels. To address these issues, liver biopsies from 10 patients with chronic hepatitis C were analyzed by in situ hybridization and digital image analysis of hybridization signals. Serum HCV RNA levels were measured using the Amplicor Monitor test. HCV RNA was detected by in situ hybridization in the hepatocytes of the ten liver samples. The hybridization signals were mainly found in the cytoplasm. The relative viral load per infected cell fit the second order polynomial curves in all cases. The minimum and maximum relative viral load per infected hepatocyte differed in the ten cases; however, large differences were not observed in the mean relative viral load among the samples, especially when compared with the increasing values detected for copy number per milliliter in serum. The percentage of infected cells ranged from 4.8% to 87.6% in the ten cases. The percentage of positive cells correlated with the serum viremia levels. Our data suggest that HCV viremia does not depend on the relative viral load per infected cell but on the number of infected hepatocytes.     

Clinical implications of GBV-C/HGV infection in patients with "HCV-related" chronic hepatitis

BACKGROUND/AIMS: To evaluate the clinical, biochemical and histological implications of a concomitant HGV infection in "HCV-related" chronic liver disease. METHODS: Eighty-three HCV-RNA positive patients with chronic liver disease were tested for GBV-C/HGV coinfection by heminested PCR. RESULTS: Twenty-two (26.5%) patients were found to be positive for GBV-C/HGV RNA. GBV-C/HGV+ patients differed significantly from GBV-C/HGV- ones for younger age, higher frequency of history of drug addiction, which in turn might favor coinfection with interferon-sensitive HCV genotypes (3a), and increased probability of long-term response to interferon. GBV-C/HGV infection appears to have no responsibility for specific aspects of HCV infection such as biochemical or histological cholestatic features, lymphoid follicles, symptomatic cryoglobulinemia or presence of serum autoantibodies, including LKM1. It does not worsen the HCV-related disease (ALT levels and histological activity) and does not significantly interfere with HCV infection, as explored by the number of hepatocytes positive for HCV antigens. The amount of steatosis (mean score) was shown to be higher in GBV-C/HGV+ patients. A virological follow up was performed in 17 interferon-treated GBV-C/HGV+ patients On the whole, GBV-C/HGV seems to be as sensitive to IFN treatment as HCV, but recurrence after withdrawal is more frequent. In spite of this, ALT levels often remain normal after treatment withdrawal. CONCLUSIONS: The present data suggest that GBV-C/HGV infection, apart from more marked liver steatosis, does not modify the overall picture of chronic hepatitis due to HCV infection.     

Hepatitis C virus infection of salivary gland epithelial cells. Lack of evidence

BACKGROUND: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports. AIMS: To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease. METHODS: Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing. RESULTS: HCV-RNA was detected in all sera with titers ranging from 5.42 x 10(5) genome equivalents/ml to 123.2 x 10(5) genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2 x 10(5) genome equivalents/ml) was detected in 3/20 saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples. CONCLUSIONS: There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.     

Childhood Lead Poisoning in Russia: A Site-specific Pediatric Blood Lead Evaluation

The association between the actual status of cryoglobulins (CGs) expression in hepatitis C-related chronic liver disease (C-CLD) and different types of liver pathology, HCV-RNA titers and genotypes was investigated. Sixteen out of 1340 ordinary clinical specimens were CGs-positive (1%), and in 8 of them (50%) the patients blood was HCV-RNA positive. CGs was detected in 63% of C-CLD patients as a whole, but when compared with the histological findings in the liver, it was 88% in F3, and 92% in A3, and thus high percentages were detected in patients with progression of liver fibrosis and patients with strong activity. Serum IgG, IgM, transaminase and gamma-GTP levels were significantly higher in the patients with CGs, and their RA test and C3dCIC levels tended to be higher, but the differences were not significant, and no association was found with the anti-nuclear antibody positive level, or the HCV-RNA titers or genotypes. Based on the above, there was a clear involvement of HCV infection, especially the activity and histological progression of hepatitis in CGs formation in Japan as well, but there were few extrahepatic manifestations, suggesting differences in CGS levels and immune response to CGs from cases in Western countries.     

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.     

Detection of hepatitis E virus genome and gene products in two patients with fulminant hepatitis E

Non-isotopic in situ hybridization (digoxigenin-labeled probe directed towards hepatitis E virus ORF1) and immunohistochemistry (against hepatitis E virus ORF2 and ORF3) were applied to detect hepatitis E virus genome and gene product in the liver tissue of two patients with fulminant hepatitis E seropositive for hepatitis E virus RNA. Both hepatitis E virus RNA and hepatitis E virus antigens were detected exclusively in the cytoplasm of hepatocytes and not detected in other cell types. In both patients, more than 50% of the hepatocytes were positive for both hepatitis E virus RNA and hepatitis E virus antigens, most of which showed degenerative changes. This is consistent with the histological appearance of marked loss of hepatocytes with acinar collapse. Interestingly, denaturation of the RNA before in situ hybridization was found to enhance hepatitis E virus RNA detection. We conclude that: (1) hepatitis E virus RNA and hepatitis E virus antigens can be demonstrated in the liver in hepatitis E virus-related fulminant hepatitic failure, (2) hepatitis E virus is hepatocyte-tropic within the liver, (3) cytoplasmic localization of hepatitis E virus RNA and hepatitis E virus antigens is consistent with cytoplasmic replication, and (4) the presence of degenerative changes in hepatitis E virus positive cells, together with the histological appearance of hepatocyte loss in the absence of significant inflammatory infiltrate, suggests that hepatitis E virus-related fulminant hepatitic failure is mediated by a cytopathic mechanism.     

On the experimental distinction between ssbs and dsbs in circular DNA

Positive serum anti-nuclear antibody (ANA) and anti-smooth muscle antibody (SMA) have been reported in 10-66% of patients with chronic hepatitis C virus (HCV) infection from Western countries. However, the mechanism involved in this immunological disorder is still unknown. This study was carried out to evaluate the prevalence and clinical significance of positive serum auto-antibodies in Chinese patients with chronic hepatitis C and to assess the role of serum HCV-RNA titre and HCV genotype in the presence of serum auto-antibodies. Serum ANA, SMA and anti-mitochondrial antibody (AMA) were measured in 122 patients with chronic hepatitis C. Clinical, biochemical and virological data (serum HCV-RNA titre and HCV genotype) were compared between patients with and without serum auto-antibodies. Fifty-eight (48%) patients were associated with positive serum auto-antibodies: 42 (34%) positive for ANA, six (5%) positive for SMA, nine (7%) positive for both ANA and SMA and one (1%) positive for AMA. Clinical parameters (age, sex, blood transfusion history), liver biochemical tests, the presence of cryoglobulinaemia or cirrhosis, and the response to interferon treatment were not significantly different between patients with and without positive serum auto-antibodies. Serum HCV-RNA levels and HCV genotypes were also not significantly different between the two groups. Logistic regression analysis showed that none of the previously mentioned parameters were significant predictors to associate with serum auto-antibodies in chronic hepatitis C. We concluded that 48% of Chinese patients with chronic hepatitis C were associated with positive serum auto-antibodies. Hepatitis C virus genotypes and serum HCV-RNA levels were not correlated to the presence of serum auto-antibodies. The clinical significance and actual pathogenesis of this phenomenon remain to be clarified.     

Detection by a solid phase independent enzyme immunoassay of monoclonal anti-idiotypic antibodies against monoclonal antibodies neutralizing Semliki Forest virus and encephalomyocarditis virus

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the rate limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Inhibitors of HMG-CoA reductase are teratogenic in vivo and induce neural tube defects in rat embryo culture, effects which appear unrelated to cholesterol deficiency. This study is the first to localize HMG-CoA reductase mRNA by in situ hybridization (ISH). Expression of reductase mRNA was examined in post-implantation rat embryos, and for control purposes in rat liver and UT-1 cells, using a digoxigenin-11 (dig-11) labelled cRNA probe. Eighteen-day fetal liver showed heavy but patchy hybridization, and adult rat liver showed strong hybridization only on some periportal hepatocytes, which was absent in livers of fasted animals. UT-1 cells stimulated to overexpress HMG-CoA reductase mRNA were strongly positive with the same probe. Control hybridizations with sense strand RNA probe, or with cRNA probe on pre-RNased tissue were negative. Strong hybridization signal for HMG-CoA reductase mRNA was observed in all tissues of the post-implantation rat embryo, from egg cylinder to 30 somite stages (7 to 12 days). Heavy signal was noted in primitive ectoderm and neural tube. The wide embryonic and extraembryonic distribution and abundance of HMG-CoA reductase mRNA may reflect developmental requirements for products of the mevalonate pathway, e.g., isoprenoids for post-translational farnesylation of p21ras.     

Absence of nonpercutaneous transmission of hepatitis C virus in a colony of chimpanzees

Transmission of hepatitis C virus (HCV) was studied in a colony of 85 chimpanzees using assays for anti-HCV and HCV-RNA. Thirteen of the 85 sera were positive for anti-HCV, and 12 of the 13 were also positive for HCV-RNA. All of the anti-HCV positive sera except one were obtained from chimpanzees which had been inoculated with non-A, non-B hepatitis virus. On the other hand, only one of 63 sera of chimpanzees without history of experimental infection of the virus was positive for anti-HCV. Transmission to this chimpanzee was thought to be a needle contaminated with HCV. All 39 samples of chimpanzees born in the center were negative for both anti-HCV and HCV-RNA. Sixteen of their mothers had undergone experimental infection, and 6 of them were positive for both anti-HCV and HCV-RNA. These results suggest that nonpercutaneous transmission, including sexual and mother-to-infant transmissions, is not an important mode of transmission. If these findings apply to humans, definition of inapparent sources of the infection is needed.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Shoulder joint instability after primary arthroplasty

Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, co-amplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.     

Sexual behaviour and multiple infections in drug abusers

We have studied the prevalence and the serological profile of HBV, HCV, HDV and HIV infections in 137 Italian subjects addicted to the intravenous use of heroine and correlated the virological findings with sexual behaviour. HBV and HCV viremia were also measured in 114 patients. Anti-HCV was detected in 81% of the addicts, and one or more markers of HBV infection were detected in 62.8% (4.4% were carriers of HBsAg, 58.4% had evidence of past HBV infection and 13.1% of the latter also had HDV markers). Anti-HIV was positive in 23.4%; 26% of those positive for anti-HCV and 4.6% of those positive for HBV markers had no other viral marker: none had only anti-HIV. HBV-DNA was negative in the carriers of HBsAg, and HCV-RNA was not detected in any of the HBsAg carriers who also had circulating anti-HCV. Overall, 34% of the anti-HCV positive addicts had HCV-RNA in their blood. The prevalence of the virus infection correlated with the duration of drug addiction but not with sexual behaviour, and sexual behaviour did not influence the acquisition of any virus. HCV infection was most frequent and probably the first infection to occur, but exposure to HBV was also common despite a low rate of HBsAg carriage. The prevalence of HDV infection was high (50%) in the HBsAg carriers, while the overall prevalence of HIV was lower (23%) than expected. Lack of HBV-DNA and HCV-RNA in carriers of HBV with anti-HCV in serum may indicate that HBV and HCV mutually inhibit their own replication.     

An investigation of the viral pathogenesis of Kikuchi-Fujimoto disease. Lack of evidence for Epstein-Barr virus or human herpesvirus type 6 as the causative agents

Histiocytic necrotizing lymphadenitis of Kikuchi and Fujimoto is a well-defined clinicopathologic entity of unknown cause. Both the Epstein-Barr virus (EBV) and human herpesvirus type 6 (HHV-6) have been suggested as potential etiologic agents. Twenty cases of Kikuchi-Fujimoto disease were studied for the presence of EBV DNA and HHV-6 DNA by the polymerase chain reaction (PCR), and in situ hybridization in the case of EBV. Cellular DNA from sections of formalin-fixed, paraffin-embedded lymph node tissue was amplified using the PCR technique and oligonucleotide primers to the EBV BamH1 W, lymphocyte-determined membrane antigen, or the EBNA-1 region. These studies were performed in three separate laboratories. In addition, 12 cases were examined by in situ hybridization, eight of which had shown at least one positive PCR signal for EBV. The presence of HHV-6 was assessed by PCR using primers to part of the pZVH14 sequence. Biopsy specimens from eight patients (40%) showed a strong positive signal for EBV in at least one laboratory, while an additional three specimens (15%) showed a weaker positive signal. Five cases studied showed rare positive cells by in situ hybridization, and one case had scattered positive cells. All samples lacked HHV-6 genomic templates. These findings indicate that HHV-6 does not play a role in the pathogenesis of Kikuchi-Fujimoto disease and do not implicate EBV as a causal agent for Kikuchi-Fujimoto disease, since EBV was detected in only a fraction of cases with a low number of positive cells detected by in situ hybridization. Further, some discrepancies were identified in the positive results for EBV in samples studied by multiple laboratories. These results indicate that inconsistent results by PCR may occur with very low levels of viral genomes and that different laboratories perform DNA amplification at different efficiencies. Alternatively, laboratory contamination may give rise to false-positive results. Therefore, a positive result for EBV should be interpreted with caution and should be confirmed by repeated study (PCR) or by independent methodology (in situ hybridization).     

The significance of choroid plexus cysts in an unselected population: results of a multicenter study

BACKGROUND: Cytotoxic T lymphocytes and Kupffer cells are essential components of the immune response during liver diseases. Recent studies have highlighted the role of cytotoxic T lymphocytes using Fas and its ligand in induced hepatocyte death during acute and chronic hepatitis. METHODS: In the present work, the main purpose was to investigate perforin and granzyme B expression in liver biopsies of patients with chronic hepatitis (10 HBV, 14 HCV and 10 autoimmune hepatitis) using immunohistochemistry. The liver biopsies of two normal individuals were also studied in the same conditions. RESULTS: Few intrahepatic T lymphocytes expressed perforin and granzyme B, while a large number of Kupffer cells were positive for both proteins in all the patients tested. The co-localization of perforin and granzyme B, and CD3 or CD68 antigens was visualized, respectively, in T cells and Kupffer cells, using confocal microscopy. In situ hybridization assays confirmed that perforin and granzyme B mRNAs were present in the liver during chronic hepatitis. The results were similar among the three groups of patients and whatever the activity of the disease. Perforin and granzyme B expression was lacking in liver samples from normal individuals. Conclusions: These data suggest a minor role for the T cell-mediated perforin/granzyme B death pathway, and a putative role for Kuppfer cells via lytic protein release, during chronic hepatitis.