Genomic hsd-Mu(lac) operon fusion mutants of Escherichia coli K-12.

Genomic (chromosomal)hsd-Mu(lac) operon fusions have been constructed in two strains of Escherichia coli K-12 for the three hsd genes, hsdRK, hsdMK and hsdSK, using MudX and lambda placMu53. Expression of hsdK mutants ranged from 16 to 74 units (u) (with a mean of 52 u) for fusions to promoter pres and ranged from 26-75 u (also with a mean of 52 u) for fusions to promoter pmod. The expression of the two hsdK promoters was measured in different stages of growth. The pres fusion mutant showed a lag in beta-galactosidase (beta Gal) production, as compared to the pmod fusion mutant. One r-Km-K mutant (JR205) showed more than ten times the beta Gal activity of other insertion mutants. The activity of this mutant decreased by 20-fold upon the transfer of F101-102, which includes the wild-type hsd region. Positive gene-dosage effect was observed using F' plasmids containing the hsd-lacZ region.

A new restriction-modification system, KpnBI, recognized in Klebsiella pneumoniae.

A unique DNA restriction-modification (R-M) system has been identified in the GM236 strain of Klebsiella pneumoniae using the newly isolated phage, SBS. The system was designated KpnBI. The gene (hsdRKpnBI) complementing the restriction activity of the KpnBI system was cloned in pBR322. The nucleotide sequence of the cloned DNA revealed one open reading frame (ORF) of 3035 bp. Analysis of the deduced amino-acid sequence shows seven helicase motifs which are common to the restriction (R) subunit of both type-I and type-III R-M systems. Computer analysis (Dendrogram) of the R polypeptide of KpnBI suggests a closer relationship to EcoR124/3I, a member of type-IC family, than to other representative type-I and type-III systems.

Clonal origin of hepatitis B virus-associated hepatocellular carcinoma.

The clonal origin of tumors was studied in two multifocal hepatocellular carcinomas arising from hepatitis B viral cirrhosis. DNA extracted from several tumor nodules was analyzed for the presence of hepatitis B virus genome by the Southern blot technique. Unique clonal integration of the viral DNA sequences occurred in both cases. In each case, identical integration bands occurred among multiple nodules of liver tumor. These results are strong evidence of a unicentric origin of these tumors, although the tumors' gross appearance is suggestive of multifocal origin.

Detection of hepatitis delta virus in serum and liver tissue by molecular hybridization. Validation of a rapid spot-hybridization technique.

Serologic tests for detection of delta hepatitis virus (HDV) antigen and antibody have recently been supplemented with a Northern blot hybridization assay for HDV RNA. However, this technique is cumbersome for analysis of multiple samples. In order to simplify detection of HDV RNA, the authors have tested a spot-hybridization method with a new HDV cDNA probe. Their method has proved to be rapid, sensitive, and specific for HDV RNA even when less ultracentrifugation was used for recovering serum RNA. Results for HDV RNA were concordant by both spot and Northern blot hybridization in 12 serum samples from patients with known delta hepatitis, whereas in seven cases spot-hybridization was superior in detecting liver HDV RNA. The concordance between HDV RNA by spot hybridization and delta antigen was complete, whereas that between delta IgM (44% overall) or IgG (67% overall) was less strong. The authors' observations indicate that this new technology permits detection of HDV RNA with relative ease and could be applicable to the evaluation of large numbers of cases with delta hepatitis.

Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus.

Anti-hepatitis delta virus (anti-HDV) antibodies were measured by solid phase IgG and IgM capture radioimmunoassays (RIA) as well as by a competitive binding enzyme immunoassay (EIA) in both acute and chronic HDV infections. EIA anti-delta test measures total delta antibody without discriminating IgM from IgG anti-delta. Low titer IgG antibodies were detected by both techniques with equal sensitivity. High titer IgG antibodies reached the end point sooner with EIA than with RIA (10(-3) versus greater than 10(-6)). When IgM anti-HDV was present without accompanying IgG anti-HDV, EIA failed to identify the antibody. Presence of high titer rheumatoid factor in the serum and lipemic samples produced false-positive results by EIA. Usage of undiluted serum samples for EIA probably exaggerates the factors contributing to false-positive reaction.

Serum hepatitis B virus DNA in acute hepatitis B.

With the use of the dot blot hybridization technic, sera from 30 consecutive patients with acute hepatitis B were examined for the presence of hepatitis B virus (HBV)-DNA. In an additional five patients with acute hepatitis B, serial serum samples, beginning before the serum alanine amino transferase elevation to the clearance of hepatitis B surface antigen, also were tested for various hepatitis B virus markers, including HBV-DNA. Thirteen of the 30 patients (43%) had circulating HBV-DNA and HBeAg at the time of their first clinic visit. However, 11 additional patients with HBeAg did not have HBV-DNA in their sera. In the 13 patients with HBV-DNA, there was no correlation between the titers of HBeAg and the levels of HBV-DNA. Examination of the serial serum samples from the additional five patients showed HBV-DNA and HBeAg to be present several days before the peak of serum alanine amino transferase. In all five patients, HBV-DNA was present in the serum before the appearance of IgM anti-HBc. However, HBsAg was present in all these patients' sera at the time of HBV-DNA positivity. None of the patients in this study became chronic carriers of hepatitis B virus.

Markers of viral replication in patients with chronic hepatitis B virus infection.

Serum samples from 56 patients with biopsy-proven chronic B viral hepatitis without superimposed delta hepatitis were analyzed for the various markers of viral replication, including serum hepatitis B e Ag (HBeAg), hepatitis B virus deoxyribonucleic acid (HBV-DNA), and hepatitis B core antigen (HBcAg) in the liver tissues. Twenty-seven patients had persistent viral hepatitis (PH) and 29 patients had chronic active hepatitis (CAH) with or without cirrhosis. HBV-DNA was identified in the sera of 81% of patients with PH and 60% of patients with CAH. Significantly higher levels of HBV-DNA were found in patients with PH than in those with CAH. Both HBeAg in serum and HBcAg in liver correlated positively with serum HBV-DNA. Nine patients had serum HBV-DNA in the absence of HBeAg (four had anti-HBe), and seven of these nine patients had stainable HBcAg in the liver (two did not have staining). None of these patients had hepatic HBcAg in the absence of serum HBV-DNA. When these patients were stratified according to their epidemiologic background, serum HBV-DNA was present in a significantly higher number of male homosexuals than in any other groups. This was unrelated to their status of human immunodeficiency viral serology.

Serum hepatitis B virus-DNA in chronic hepatitis B and delta infection.

Hepatitis B-associated delta agent, a defective RNA virus requiring helper functions of hepatitis B virus (HBV), has been shown to interfere with HBV replication. Low titers of serum hepatitis B surface antigen, absence of hepatitis B e antigen, and low levels of stainable hepatitis B core antigen in liver cells usually seen in chronic delta infection are indirect evidences of such an interference. Measurement of serum HBV-DNA by hybridization with phosphorus 32-labeled HBV-clone DNA is the most sensitive method currently available to detect HBV replication. Using this method, we found that only two of 13 patients with chronic delta infection showed serum HBV-DNA positivity in comparison with seven of 14 patients who had chronic hepatitis B without delta infection. These two groups were matched for hepatitis B e antigen status and liver histopathology. Thus, we report direct evidence of delta agent interfering with the replication of the helper (HBV) virus.

The stability of serum hepatitis C viral RNA in various handling and storage conditions.

To investigate whether certain handling and storage conditions of serum samples could alter the sensitivity and specificity of the hepatitis C virus (HCV) RNA assay, we studied serum samples obtained from five patients known to be positive for HCV RNA and two patients with autoimmune chronic active hepatitis. Samples were subjected to one of the following conditions: (1) immediate storage at -20 degrees C, (2) five freeze-thaw cycles, (3) storage at 4 degrees C for 5 days, and (4) storage at room temperature for 5 days. Detection of HCV RNA was performed by polymerase chain reaction. Titers of HCV RNA were determined by serial end point dilutions. We found that the titer of HCV RNA was reduced by only one logfold in samples subjected to conditions 2 through 4 in two of the five patients. False-positive results were not seen with the serum samples that were subjected to similar conditions from the two negative control patients. We conclude that serum HCV RNA is resistant to degradation under routine laboratory handling and storage conditions.

Study of preneoplastic changes of liver cells by immunohistochemical and molecular hybridization techniques.

The status of hepatitis B virus DNA was investigated by in situ hybridization in multifocal areas of a noncancerous hepatitis B virus-associated cirrhosis. This liver exhibited a marked degree of dysplasia and adenomatous hyperplasia. The results of these studies were correlated with the histopathology and immunohistochemical stains for hepatitis B core and surface antigens. There was clear evidence of a marked reduction to absence of hepatitis B viral DNA by in situ hybridization and absence of HBc and HBsAg in the foci of liver cell dysplasia and adenomatous hyperplasia. These results support the hypothesis that liver cell dysplasia and adenomatous hyperplasia are preneoplastic in nature.

Relapse of acute B viral hepatitis--role of delta agent.

Serologic markers for delta agent were evaluated in 39 patients with acute hepatitis B and clinical relapse within 30 days from the initial episode. Eighteen of the 39 patients (46%) had evidence of acute delta infection. Delta antigenaemia preceded the appearance of antibodies in seven of these 18 patients; delta antigenaemia occurred during the initial episode of illness and the appearance of the antibody coincided with the relapse. Eight of these patients developed severe relapse with fulminant course which resulted in two deaths. This study reveals that delta infection is one of the important causes of severe relapse in cases of acute B viral hepatitis.

Host dependent modulation of hepatitis delta virus replication in chronic delta hepatitis.

To determine whether host dependent differences modulated hepatitis delta virus replication in chronic delta hepatitis, we tested HDV RNA in homosexual and intravenous drug abuser populations. Overall, the seroprevalence of HDV RNA in the two groups with matching clinical characteristics was 72% (76/106 patient visits). A trend for greater prevalence of HDV RNA was noted at initial presentation in homosexuals (82%) compared to intravenous drug abusers (60%, P less than 0.05) and this trend appeared to be maintained during two years of sequential follow-up. The seroprevalence of co-appearing IgM and IgG anti-HD antibodies was similar in the two groups of patients. However, in HDV RNA positive homosexuals IgG anti-HD antibody was more prevalent, and additionally, assumed concordance with HDV RNA of 92% although the significance of this observation is unclear. The difference in prevalence of HIV in the two groups did not reach statistical significance. Prospective studies are required to confirm differences in HDV replication in various patient groups and to define underlying mechanisms.

Correlation of IgM anti-hepatitis D virus (HDV) to HDV RNA in sera of chronic HDV.

One hundred forty-four serum samples from 52 patients with chronic hepatitis D virus infection were analyzed for hepatitis D virus RNA by dot-blot hybridization using hepatitis D virus cDNA probe labeled with 32P. The results were correlated with the presence of serum IgM anti-hepatitis D virus and hepatitis D antigen in liver biopsy specimens when available. Although there was a trend of positive correlation between serum hepatitis D virus RNA and IgM anti-hepatitis D virus, no statistical significance could be found. In the serum samples with hepatitis D virus RNA, 32% were found to be negative for IgM anti-hepatitis D virus. Therefore, in chronic hepatitis D virus, absence of IgM anti-hepatitis D virus does not rule out active viral infection, as suggested by previous studies. There was a strong correlation between serum hepatitis D virus RNA and hepatic hepatitis D virus antigen. These data indicate that detection of hepatitis D virus RNA in serum samples is a reliable noninvasive marker of active viral infection.

Prevalence of hepatitis G virus RNA in the sera of patients with HIV infection.

OBJECTIVE: The routes of transmission of the hepatitis G virus (HGV) are similar to those responsible for infection with HIV. We sought to evaluate the prevalence of HGV RNA in the sera of HIV-infected patients. METHODS: The sera of 157 HIV-infected patients were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) using established primers for HGV. Patients were divided into group 1 (positive circulating hepatitis B surface antigen [HBsAg]), group 2 (positive anti-hepatitis C virus [HCV] antibody) and group 3 (without markers for HBV or HCV). RESULTS: The overall prevalence of HGV RNA was 22%; prevalence was higher in group 1 (49%) than in groups 2 (16%) or 3 (7%). Patients with positive HGV RNA had laboratory values similar to HGV RNA-negative patients except for higher CD4 counts. Patients with an estimated risk duration of < or = 14 years were more likely to be HGV RNA-positive than patients at risk for >15 years. HGV RNA was found as frequently in patients with a homosexual lifestyle as in injection drug users (IDU). Multivariable analysis showed that the presence of HBsAg was the strongest factor associated with the presence of HGV RNA in serum. CONCLUSIONS: Patients with HIV and HBV coinfection are significantly more likely to be HGV RNA-positive. Patients with a risk factor duration for >15 years were less likely to be HGV RNA-positive, pointing to a decrease in HGV RNA prevalence over time. This study supports the notion that homosexual lifestyle, in addition to injection drug usage and blood product transfusion, is a risk factor for HGV infection.

Status of hepatitis B virus DNA in alcoholic liver disease: a study of a large urban population in the United States.

Two reports have shown hepatitis B virus DNA in serum and liver tissue in alcoholic liver disease with negative serum HBsAg, suggesting a pathogenetic role for hepatitis B virus. We studied hepatitis B virus DNA in serum and liver from three groups of alcoholic patients; (Group 1) 50 patients without liver disease, (Group 2) 108 patients with alcoholic liver disease and (Group 3) five patients with alcoholic liver disease and hepatocellular carcinoma. Serum was tested for HBsAg, anti-hepatitis B core and anti-hepatitis B surface by radioimmunoassay and hepatitis B virus DNA by direct spot hybridization. Liver tissue from Groups 2 and 3 (113 patients) was examined by Southern blot analysis using 32P-labeled hepatitis B virus DNA clone from pBR322. Controls were 21 patients with chronic hepatitis B virus (14 patients with chronic active hepatitis, seven patients with cirrhosis and hepatocellular carcinoma). Serum and tissue were analyzed for hepatitis B virus DNA. Hepatitis B virus DNA was not detected in either serum or liver tissue in any of the 163 patients (Groups 1 to 3). In contrast, among the controls, hepatitis B virus DNA was present in the serum of 15 of the 21. Tissue DNA in those with chronic active hepatitis revealed 10/14 with free hepatitis B virus DNA, two with integrated sequences and two with no viral sequences. All seven patients with hepatocellular carcinoma had integrated viral DNA sequences in the tumor tissues. From these results, it appears that hepatitis B virus does not play a role in the pathogenesis of alcoholic liver disease.

Acute delta hepatitis: serological diagnosis with particular reference to hepatitis delta virus RNA.

To evaluate serologic diagnosis of hepatitis delta virus, we tested HDV RNA in stored sera from 48 patients with acute delta hepatitis who were identified with anti-HD antibodies. Initial sera were positive for HDV RNA in 27 of 48 (56%) patients. In comparison, isolated IgM anti-HD was present in 18 (38%) patients, although IgM and IgG anti-HD were present concurrently in 16 (33%) additional patients. Overall, either HDV RNA or IgM anti-HD was present in 69% of the initial sera. The HDV infection was self-limiting in all except two patients who died of fulminant hepatitis and nine others in whom chronic delta hepatitis ensued. Patterns of HDV seropositivity during progression to chronicity induced variable persistence, disappearance or recrudescence of either HDV RNA or IgM and IgG anti-HD. Results of HDV RNA and IgM anti-HD tests were concordant in only 40-50% of instances. Our results indicate that serological testing for HDV RNA is direct and will demonstrate HDV replication in a large number of cases with acute delta hepatitis. Testing for IgM anti-HD could provide supplemental evidence for HDV infection. Sequential testing for these markers will facilitate assessment of the outcome of acute HDV infection.

Spontaneous exacerbation of disease activity in patients with chronic delta hepatitis infection: the role of hepatitis B, C or D?

Forty-six patients with chronic hepatitis delta virus infection were followed between 6 and 116 mo (mean = 32.8 mo; median = 24 mo). Nineteen patients (41%) demonstrated clinical courses with episodes of biochemical reactivation (ALT levels greater than or equal to 10 times baseline values [group A]). Twenty-seven patients (59%) had stable clinical courses without biochemical reactivation (group B). Patients in group A were younger than those in group B (30.5 vs. 35.3 yr; p = 0.03), were less likely to be intravenous drug abusers (16% vs. 52%; p = 0.01) and were more likely to be homosexual (58% vs. 22%; p = 0.01). Serum hepatitis B virus DNA, hepatitis delta virus RNA, IgM antibody to HBc, HBeAg, antibody to HBe and IgG and IgM antibody to hepatitis delta virus were measured in all patients. In group A, these markers were studied before and during reactivation and during remission. In group B, these parameters were studied in a random fashion at 7- to 10-mo intervals. The presence of antibodies to human immunodeficiency virus and hepatitis C virus was assessed in all patients. A total of 38 biochemical reactivation episodes was noted among the 19 patients in group A. Eleven had sequential changes in hepatitis delta virus markers, suggesting that the exacerbations were due to hepatitis delta virus. In three, the sequential changes of viral markers were consistent with the exacerbations due to hepatitis B virus. In five other patients, no sequential changes in viral markers could be demonstrated to correlate with the biochemical exacerbations.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of HDV, HBV and HIV in chronic B and D infections and in reactivation of chronic D infection.

From this study, we can conclude that there is significant influence of HIV infection on the clinical course of chronic HDV as follows: In these patients, there is simultaneous replication of both HBV and HDV and the suppression of HBV by HDV is modified. There is decreased antibody response to HDV, however, the degree of liver injury is not altered. Although these patients tend to have "reactivation episodes" as frequently as the HIV negative group, no correlation of serum ALT to HDV-RNA could be found. The possibility of these episodes resulting from injury due to other viruses such as non-A, non-B cannot be excluded.

Short-term prednisone therapy affects aminotransferase activity and hepatitis C virus RNA levels in chronic hepatitis C.

BACKGROUND/AIMS: The effects of corticosteroids on chronic hepatitis B have provided insight into the mechanism of liver cell injury caused by hepatitis B. In this study, this model was applied to investigate the effects of prednisone on alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels in chronic hepatitis C. METHODS: Ten patients with chronic hepatitis C who had increased levels of ALT and HCV RNA detectable in serum were given a 7-week course of a tapering dose of prednisone. Quantitation of serum HCV RNA was determined by polymerase chain reaction (PCR) and by branched-chain DNA amplification. RESULTS: ALT levels decreased in 8 of 10 patients during therapy. Mean ALT levels of all 10 patients decreased from 184 to 84 U/L (P = 0.002) and then rebounded in 7 of the 8 patients after discontinuation of prednisone. HCV RNA was detectable by the branched DNA technique in 9 of 10 patients. These values increased in all 9 patients during prednisone therapy. The mean serum HCV RNA levels increased from 40.9 before treatment to 414.3 Eq/mL x 10(5) during treatment (P = 0.043). Using PCR, HCV RNA titers increased one log-fold in 8 of 10 patients (geometric mean of 1:4420 to 1:23410). HCV RNA levels decreased to pretreatment values within a mean of 2.8 weeks (range, 1-5) after discontinuation of prednisone. CONCLUSIONS: These responses in ALT and HCV RNA suggest the participation of an immune-mediated mechanism in the liver cell injury in chronic hepatitis C.

Clinical significance of concomitant hepatitis C infection in patients with alcoholic liver disease.

The significance of antibodies to hepatitis C virus in patients with chronic alcoholic liver disease is unclear. Prior studies have utilized the first-generation enzyme-linked immunosorbent assay, which is limited by problems with sensitivity and specificity. Hepatitis C virus infection in 137 patients with biopsy-proven alcoholic liver disease was assessed with second-generation hepatitis C virus antibody assays and reverse transcription-polymerase chain reaction for detection of hepatitis C virus RNA in the serum. The patients were categorized into three groups according to results of serological testing. Discriminant-function analysis was used to determine which factors (risk, biochemical and histological) could best differentiate the three groups. Thirty-three patients were reactive on second-generation enzyme-linked immunosorbent assay/second-generation recombinant immunoblot assay and RNA positive (group 1). Twelve were reactive on second-generation enzyme-linked immunosorbent assay/second-generation recombinant immunoblot assay but RNA negative (group 2). Eighty-six were nonreactive on second-generation enzyme-linked immunosorbent assay, and six were reactive on second-generation enzyme-linked immunosorbent assay but negative on second-generation recombinant immunoblot assay and negative for hepatitis C virus RNA (group 3). Seventy-six percent of patients in group 1 and 58% in group 2 had parenteral risk factors, compared with only 1% in group 3 (p < 0.00001). The mean ALT level was higher in group 1 patients (p < 0.05). The mean histologic activity index was significantly higher in group 1 (p = 0.0007).(ABSTRACT TRUNCATED AT 250 WORDS)