Nucleic acid-based cross-linking assay for detection and quantification of hepatitis B virus DNA

Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects of ribosome biogenesis in eukaryotic cells. Scores of snoRNAs have been discovered in recent years from various eukaryotes, and the total number is predicted to be up to 200 different snoRNA species per individual organism. We have created a comprehensive database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured). The database consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual snoRNAs. The utilities section provides an introduction to the database. The master table lists all known S. cerevisiae snoRNAs and their major properties. Information in the individual tables includes: alternate names, size, family classification, genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene disruption phenotypes, functional properties and associated RNAs and proteins. All information is accompanied with appropriate literature references. The database is available on the World Wide Web (http://www.bio.umass. edu/biochem/rna-sequence/Yeast_snoRNA_Database/snoRNA_ DataBase.html), and should be useful for a wide range of snoRNA studies.     

Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay

BACKGROUND: Previous studies have suggested a variety of factors that may be associated with the presence of hippocampal formation (HF) atrophy in patients with complex partial seizures (CPS), including a history of complex or prolonged febrile seizures (FS), age at seizure onset, and epilepsy duration. OBJECTIVE: To determine whether epilepsy duration is related to HF atrophy. Methods: We performed MRIs on 35 patients with uncontrolled CPS who had temporal lobe ictal onset on video-EEG. None had evidence for an alien tissue lesion or extra-hippocampal seizure onset. All had a history of secondary generalization. Brain structures were drawn on consecutive images and pixel points summed from successive pictures to calculate volumes. RESULTS: Nine patients with a history of complex or prolonged FS had smaller ipsilateral HF volume and ipsilateral/contralateral ratio than did patients without a history of FS. Epilepsy duration had a significant relation to ipsilateral HF volume and ipsilateral/contralateral ratio. In a multivariate analysis, the effect of duration, but not age at onset or scan, was significant. Patients with a history of FS did not have earlier age at epilepsy onset or longer duration. Conclusions: A history of FS predicted the severity of HF atrophy in our patients. Age at onset or study was not a significant factor. Epilepsy duration, however, did have a significant effect, suggesting that, after an initial insult, progressive HF damage may occur in patients with persistent seizures.     

Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system

OBJECTIVE: Amylin, a secretory peptide of beta-cells, is the constituent peptide of islet amyloid, which is characteristic of NIDDM, and changes in amylin secretion in response to therapies may influence the rate of production of islet amyloid. The primary objective of this study was to determine whether therapy with sulfonylurea or basal insulin in NIDDM would alter amylin secretion in a way that might affect the formation of islet amyloid. RESEARCH DESIGN AND METHODS: In a randomized crossover design, eight subjects with NIDDM underwent three 8-week periods of therapy with diet alone, sulfonylurea, or exogenous basal insulin, with evaluation of amylin, amylin-like peptide (ALP), and glucose and C-peptide concentrations, both during fasting and after a standard breakfast. Changes in beta-cell function (% beta) were assessed, in the basal state by homeostasis model assessment (HOMA) and in the stimulated state by hyperglycemic clamps. Seven nondiabetic control subjects each underwent a meal profile and hyperglycemic clamp. RESULTS: Both sulfonylurea and insulin therapy reduced basal glucose concentrations compared with diet alone, but neither reduced the increased postprandial glucose increments. Both sulfonylurea and insulin therapy increased basal % beta, assessed by HOMA, but only sulfonylurea increased the second-phase C-peptide responses to the hyperglycemic clamp. Sulfonylurea increased time-averaged mean postprandial amylin and ALP concentrations compared with diet alone (geometric mean [1-SD range] for amylin, 4.9 [2.0-11.8] vs. 3.0 [1.4-6.2] pmol/l, P = 0.003; for ALP, 16.4 [8.5-31.7] vs. 10.1 [4.9-20.8] pmol/l, P = 0.001). Insulin therapy reduced basal ALP concentrations compared with diet alone (2.9 [1.5-5.6] vs. 6.0 [2.6-13.6] pmol/l, P = 0.03), but had no effect on postprandial concentrations of amylin (3.0 [1.3-6.5] pmol/l) or ALP (10.0 [5.5-18.1] pmol/l). CONCLUSIONS: By increasing postprandial concentrations of the constituent peptides of islet amyloid, sulfonylurea therapy might increase the rate of deposition of islet amyloid and thereby accelerate the decline of % beta in NIDDM, compared with diet therapy alone.     

Quantification of infectious duck hepatitis B virus by radioimmunofocus assay

We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate hamster arterioles in vivo and which endothelial mediators are potentially involved. To this end the plasma viscosity was elevated by exchanging blood for dextran-erythrocyte solution thereby augmenting WSS. Diameters of small and large arterioles as well as red blood cell velocities were measured before and after exchange of blood for solutions of identical haematocrit containing either high- (HMWD) or low-molecular weight dextran (LMWD). The potential role of endothelial autacoids was investigated by local application of the NO-synthase inhibitor NG-nitro-L-arginine (L-NNA), the inhibitor of cyclooxygenase, indomethacin (3 microM), or the K+-channel blocker, tetrabutylammonium (TBA, 0.1 mM) to assess the potential effects of EDHF. HMWD (n = 11 animals) increased plasma viscosity by 64 +/- 3% and dilated arterioles of all branching orders (A1-A4) significantly [by 24 +/- 3% (A1-A2) and 32 +/- 3% (A3-A4)]. This dilation compensated fully for the calculated initial increase of WSS. LMWD (n = 6) did not affect plasma viscosity or arteriolar diameters. Tissue treatment with L-NNA (30-300 microM, n = 12) substantially diminished the HMWD-induced dilation in small arterioles (A3-A4; to 13 +/- 3%; P<0.05) and virtually abolished it in large ones (A1-A2). Consequently, the calculated WSS increased significantly in these arterioles (by 31 +/- 5%). TBA combined with L-NNA (n = 4) did not reduce further the remaining dilation. Indomethacin (n = 6) had no effect on HMWD-induced dilation. We conclude that an increase of WSS induces a mainly NO-mediated arteriolar dilation. This dilation occurs in all arteriolar branching orders and is of sufficient magnitude to compensate for the initial WSS-increase. Thus, any elevations of WSS fulfil the requirement for a signal to change diameter along the arteriolar tree in a coordinated manner. The fully compensating dilation which we observed indicates that WSS is a controlled variable. It does, however, raise questions as to its role as a continuous endothelial stimulus.     

Frequent detection of hepatitis B virus X-gene DNA in hepatocellular carcinoma and adjacent liver tissue in hepatitis B surface antigen-negative patients

Hepatitis B virus is associated with human hepatocellular carcinoma. We performed polymerase chain reaction for the X, C, S, and preS2/S regions of the viral genome in 23 hepatitis B surface antigen-negative hepatocellular carcinomas and adjacent liver. Hepatitis B viral genomes were detected in 17 of 23 tumors and adjacent tissues (73.9%). Among recognized transactivators, the X gene was present in 16 (69.6%) cases of hepatocellular carcinoma, but preS2/S was detected in only 7 (30.4%). Hepatitis B virus C and S regions were detected in 3 (13.0%) and 9 (39.1%) hepatocellular carcinomas, respectively. Serologic study revealed antibodies to hepatitis B surface antigen, hepatitis B core antigen, and hepatitis B e antigen in 14 patients; among these, X-gene DNA was detected in 12 of 14 tumors (85.7%). The X gene was also detected in 4 of 9 tumors of seronegative patients. The X gene, present in many hepatocellular carcinomas, may promote hepatocellular carcinoma in hepatitis B surface antigen-negative patients.     

In vitro transformation by hepatitis B virus DNA

There is strong epidemiological evidence that the hepatitis B virus (HBV) contributes to the development of hepatocellular carcinoma (HCC). In several immortalized cell lines, an in vitro transforming activity of HBV DNA and expression vectors for the viral protein X (HBx) has now been demonstrated. Furthermore, it appears as if still unknown parts of the HBV genome other than HBx contribute to the transforming activity of HBV DNA in vitro. Only one of several studies found that HBx-transgenic mouse lines develop HCC. A mouse line transgenic for the large surface protein of HBV develops HCC due to concomitant necroinflammatory infection. Growing evidence shows the importance of recombination of integrated viral DNA and cellular DNA for HCC development. A direct transforming potential of one of these viral integrates has been demonstrated. Chemical carcinogens are more effective in HBV-containing cell lines or transgenic mice.     

Evaluation of enzyme immunoassay for hepatitis B virus DNA based on anti-double-stranded DNA

We have evaluated a new enzyme immunoassay technology to detect the products of PCR-based amplification that may be applicable to routine testing of hepatitis B virus (HBV) DNA. Two hundred eight serum samples were studied: 73 were basal samples and 135 were sequential serum samples from patients with chronic hepatitis, some of whom were being treated with alpha interferon. We compared the new detection method (PCR-DNA enzyme immunoassay [DEIA]) with dot blot hybridization performed without prior PCR amplification and with two other methods for detection of PCR products: agarose gel electrophoresis with ethidium bromide staining (PCR-EB) and dot blot (PCR-dot blot). For hepatitis B-antigen-positive basal samples, HBV DNA was detected in 70.4% by dot blot, 74.1% by PCR-EB, and 100% by PCR-DEIA and PCR-dot blot; for anti-hepatitis B e-antigen basal samples, HBV DNA was found in 10.5% by dot blot and PCR-EB and in 42.1% by PCR-DEIA and PCR-dot blot. Chi-square tests showed a strong association between dot blot and PCR-EB and between PCR-DEIA and PCR dot blot. Using PCR-dot blot as the reference, dot blot shows a 56.9% sensitivity and a 100% specificity, PCR-EB shows a 55.0% sensitivity and a 100% specificity, and PCR-DEIA shows a 95.4% sensitivity and a 97% specificity. We conclude that the technical advantages of the DEIA method and its high sensitivity and specificity may facilitate the use of PCR in routine testing for HBV DNA in clinical microbiology laboratories.     

Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.     

Colourimetric point mutation assay: for detection of precore mutants of hepatitis B

Rats with unilateral dopamine (DA) depletions (hemiParkinson analogue rats) produced by intracerebral 6-hydroxydopamine injection are impaired in using the contralateral (bad) limbs for postural adjustments. This article examines whether the bad limbs are impaired in applying the forces required to initiate postural adjustments that anticipate and accompany voluntary movements. The rats were trained to reach for food using their good paw while standing on small platforms, each of which measured force changes produced by an individual limb. In one condition the force platforms were aligned to support the limb placement of normal rats and in the second they were aligned to permit the DA-depleted rats to use a compensatory reaching stance. It was found that the bad limbs of the DA-depleted rats produced normal supporting reactions but did not initiate adjustments in posture. Postural adjustments were initiated with the good limbs and preceded rather than accompanied the reaching movements. When constrained to use the posture of normal rats, the DA-deplete rats could not reach successfully, but when allowed to adjust their stance to increase reliance on the good limbs, reaching performance improved. Measures of ground reaction forces confirm that DA-depleted rats can support posture but cannot initiate postural adjustments with their impaired limbs.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-alpha therapy (n = 37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Selective inhibition of the duck hepatitis B virus by a new class of tetraazamacrocycles

The antiviral activity of a new class of N,N,N',N",NA'-pentakis (omega-aminoalkyl) tetraazamacrocycles was evaluated in primary duck hepatocyte cultures infected with the duck hepatitis B virus (DHBV). Three of the four tested compounds were able to selectively inhibit DHBV replication by acting at an early step of the hepadnavirus infection but were associated with significant toxicity.     

Experience with lamivudine against hepatitis B virus

After several nucleoside analogues have been tested against chronic hepatitis B virus (HBV) infection with minimal success, lamivudine seems to be a highly effective new therapeutic option. This review focuses on nucleoside metabolism and on the molecular action of lamivudine as well as on results of clinical studies for several indications. We report on results of trials on the use of lamivudine for chronic HBV infection, chronic HBV under immunosuppression and prophylaxis or treatment of HBV reinfection before or after orthotopic liver transplantation. Aspects of combination therapy of different nucleoside analogues as well as on combination of lamivudine with interferon are also highlighted. Although lamivudine seems to be highly effective in most patients at the start of therapy, development of resistance by mutations in the viral polymerase is a significant clinical problem. The mode of resistance development is compared with the situation in HIV infection. Possible cross-resistance with other nucleoside analogues and the perspectives of lamivudine therapy are also considered.     

A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia

The abnormal lymphocytes in adult T cell leukemia (ATL) reveal a peculiar morphology that is characterized by indented or lobulated nuclei. While human T lymphotropic virus type I (HTLV-I) is thought to be integrated in ATL cells, the correlation between the nuclear irregularities and HTLV-I infection is obscure. We have devised a novel single cell polymerase chain reaction (PCR) technique to examine the integration of HTLV-I provirus genome in cells from two patients with ATL. To isolate single cells, peripheral blood smears were prepared on thin polyester slides and stained with May-Grünwald-Giemsa. Morphologically defined single cells were cut out after light microscopy. The HTLV-I DNA sequences were detected not only in ATL cells but also in normal-looking lymphocytes. This novel PCR method may provide a valuable tool for understanding the molecular events associated with HTLV-I infection at the single cell level.     

Multi-measurement method comparison of three commercial hepatitis B virus DNA quantification assays

Serum specimens (327) from patients with chronic hepatitis B were evaluated for hepatitis B virus (HBV) DNA using three commercial assays--Chiron Quantiplex (CA), Digene Hybrid Capture (DA) and Abbott HBV DNA assay (AA). The HBV DNA values obtained following evaluation were used to compare the linearity, responsiveness and precision of each assay and to determine the conversion between the three different assay values. The comparison was accomplished using a new statistical approach termed the multi-measurement method (MMM). MMM is a minimal bias, non-linear regression technique that allows simultaneous multiassay performance evaluation as well as assay value interconversion. MMM analysis demonstrated that the CA was more sensitive and responsive than either the DA or the AA. Both the CA and DA were more precise than the AA. Validation of the MMM results was performed using two additional data sets of 551 and 100 specimens, respectively. MMM is a novel statistical tool that has a broad application for the generation of statistically normalized laboratory data and for interassay standardization.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.