Biomedical subjects

A R Jilbert

Publications and source records attributed to A R Jilbert.

34 records · Page 2Linked to original sources

PubMed1990

Use of recombinant hepatitis delta antigen in diagnostic assays for HDV antibody.

The gene encoding the hepatitis delta virus (HDV) structural antigen (HD Ag) was inserted into a Rous sarcoma virus expression vector and the recombinant plasmid used to direct the synthesis of recombinant HD Ag (rHD Ag) in a continuous hepatoma cell line. A competitive radioimmunoassay for serum antibody to HDV using rHD Ag was developed and was found to be equally suitable for diagnostic purposes to a radioimmunoassay using infected liver-derived HD Ag. Similarly, rHD Ag was shown to be serologically equivalent to liver-derived HD Ag within the limit of the blocking titrations performed. The rHD Ag-positive cell line was also used in an indirect immunofluorescence assay to detect anti-HD. Similar titres of anti-HD were detected by both radioimmunoassay and immunofluorescence and identical samples were positive for anti-HD by either assay. In a sample of prison inmates with high prevalence of both HBV and HDV, anti-HD was confined almost exclusively to those with persistent HBV infection and not to those in whom HBV infection had cleared. The availability of rHD Ag will permit wider development of diagnostic anti-HD assays, and the use of two such assays is presented in this study.

Antigens, Viral↗

PubMed1990

Serological analysis of duck hepatitis B virus infection.

A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen and antibody; viraemia (DHBV DNA or DHBsAg) was detected in all ducks inoculated within 3 weeks post-hatch, and persistent infection developed in 93% of birds in this group. In contrast, only 80% and 60% of ducks inoculated 4- and 6-weeks post-hatch respectively developed viraemia, and approximately 70% of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated 8 weeks post-hatch and in uninfected controls. A typical anti-DHBs seroconversion developed subsequently in 2 of 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection develops with increasing age that may be related to several factors including host immunity. This model may help elucidate similar age-related features of human hepatitis B virus infections.

Animals↗

PubMed1990

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

Antigens, Viral↗

PubMed1988

Virus-liver cell interactions in duck hepatitis B virus infection. A study of virus dissemination within the liver.

Thirty-five 1-day-old Pekin-Aylesbury ducks were inoculated intravenously or intraperitoneally with duck hepatitis B virus, and the time-course of infection was examined by Southern-blot, dot-blot, and in situ hybridization and by immunohistochemistry. Randomly scattered single infected hepatocytes were first seen on days 1 and 2 after inoculation and by day 3 occurred as single cells, pairs, and groups of 5-10 adjoining cells. From day 4 after inoculation all hepatocytes were positive for duck hepatitis B surface antigen and deoxyribonucleic acid. Duck hepatitis B virus deoxyribonucleic acid levels in liver extracts and serum increased logarithmically from days 2 to 3 to a plateau by days 4 to 5 after inoculation. Infected and control birds showed no significant differences during the first 7 days in terms of liver histology, hepatocyte morphology, or mitotic activity. It was concluded that (a) virus gains access to randomly distributed hepatocytes without first replicating in other cell types, and then begins disseminating to adjacent cells following anatomic boundaries; (b) markers of infection in liver and serum show reproducible kinetics, thus making this in vivo system amenable to further quantitative study; and (c) hepatocytes in this system are highly permissive to virus replication without the development of significant cytopathology.

Animals↗

PubMed1987

Duck hepatitis B virus DNA in liver, spleen, and pancreas: analysis by in situ and Southern blot hybridization.

Tissues from a 10-week-old Pekin duck, experimentally infected at 1 day of age with duck hepatitis B virus (DHBV), were examined for the presence of replicative levels of DHBV DNA by in situ and Southern blot hybridization. Hepatocytes, pancreatic lymphoid follicle, exocrine, and endocrine cells, and splenic mononuclear cells all contained DHBV DNA localized predominantly to the cytoplasm of infected cells. Duck hepatitis B surface antigen distribution in the same tissues correlated well with the presence of DHBV DNA in many of these cells. In hepatocytes and pancreatic islet cells, 60% of DHBV DNA was present as single-stranded DNA, indicating the likelihood of ongoing virus replication in these cell types and providing further evidence that hepadnavirus DNA replication occurs largely within the cell cytoplasm. In contrast, DHBV in mononuclear cells within splenic germinal centers was wholly double-stranded, suggesting that limited, if any, DHBV DNA replication was occurring in this cell type. These data provide further information about the pathogenesis and cell-specific sites of DHBV infection.

Animals↗

PubMed1986

Histological aspects of in situ hybridization. Detection of poly(A) nucleotide sequences in mouse liver sections as a model system.

This study examined the detection of cellular poly(A) sequences in mouse liver sections by in situ hybridization using a 3H-labelled poly(dT) probe. Parameters examined included possible losses of target poly(A) sequences from sectioned cells, access of probe to target sequences, section thickness, hybridization conditions, autoradiographic efficiency, specific activity of probes and specificity of reaction. An improved protocol was devised that resulted in good preservation of histological detail in sectioned tissue blocks, and a calculated hybridization efficiency of 50%-100%. With the use of probes of defined sequence, the protocol should allow detection of unique mRNA sequences within single cells with an estimated sensitivity of 6-12 unique mRNA molecules per sectioned cell.

Animals↗

PubMed1986

Production of a monoclonal antibody to human interferon-alpha (IFN-alpha) and its use to identify IFN-alpha-producing cells in virus infection in vivo.

A monoclonal antibody to human interferon-alpha (IFN-alpha) was produced using affinity-purified IFN-alpha, that reacted with recombinant human IFN-alpha 2, but not with IFN-alpha 1, IFN-alpha M1 or IFN-beta. Indirect immunofluorescence using this monoclonal (designated 6C3) and anti-IFN-alpha polyclonal antibodies identified cells expressing IFN-alpha. After Sendai virus induction of normal human buffy-coat cells the proportion of monocytes and lymphocytes expressing IFN-alpha rose progressively from 0% to 50% and 34% respectively, preceding peak IFN-alpha titres in the culture supernatants. Around 80-90% of polymorphs were IFN-alpha-positive using both antisera, with or without IFN induction, although very little IFN bioactivity was released to the supernatant of polymorph cultures after IFN induction. Sections of hepatitis B virus infected human liver tissue showed foci of IFN-alpha-positive infiltrating mononuclear cells and (to a lesser extent) fibroblasts in patients who had active cirrhosis and evidence of virus replication. These findings suggest that polymorphs constitutively express IFN-alpha 2 related antigenic activity, whose biological activity is at present unknown; and demonstrates the identification of IFN-alpha-expressing cells in sections of tissue undergoing natural virus infection.

Antibodies, Monoclonal↗

PubMed1985

Cytoplasmic (but not nuclear) hepatitis B virus (HBV) core antigen reflects HBV DNA synthesis at the level of the infected hepatocyte.

Hepatitis B virus (HBV) core antigen (HBcAg) detected by the peroxidase-antiperoxidase technique was present in the nucleus and cytoplasm of some infected hepatocytes but only in the cytoplasm of other hepatocytes. When cells expressing HBcAg were examined by in situ hybridization for the presence of HBV DNA, the intracellular level of cytoplasmic HBV replicative intermediate DNA correlated with the level of cytoplasmic HBcAg, but not with the presence or absence of nuclear HBcAg. This suggests that nuclear HBcAg may not be directly involved in hepadnavirus replication.

Cell Nucleus↗

PubMed1983

Patterns of single- and double-stranded hepatitis B virus DNA and viral antigen accumulation in infected liver cells.

Liver sections from five patients with persistent hepatitis B virus (HBV) infection and active cirrhosis were shown to contain intracellular HBV DNA by in situ hybridization using cloned 3H-labelled HBV DNA probes. Two classes of infected cells, with different distributions throughout the liver, were distinguished: (i) cells containing a low copy number of double-stranded HBV nucleotide sequences, confined to the cell nucleus and thought to represent HBV DNA, and (ii) cells containing large amounts (estimated to be greater than 10 or 15 genome copies per cell) of HBV DNA, much of it in a single-stranded form and largely confined to the cell cytoplasm; these single-stranded regions represented widely separated regions of the HBV genome, in contrast to the structure of the DNA in mature virions. It is likely that these latter cells may be supporting viral DNA synthesis. Cells with large amounts of cytoplasmic HBV DNA invariably contained hepatitis B surface antigen (HBsAg) and in addition contained either no detectable hepatitis B core antigen (HBcAg), or cytoplasmic HBcAg or nuclear HBcAg in that order of frequency. Cytoplasmic HBcAg was highly predictive of the presence of large amounts of cytoplasmic HBV DNA in the same cell, while either nuclear HBcAg, or cytoplasmic HBsAg, were often seen both in cells with and without such levels of DNA. These patterns, relating HBV DNA and antigen content in naturally occurring asynchronous infection in a heterogeneous cell population, should provide a background to further studies of the virus replication cycle with a defined experimental system, when such a system becomes available.

DNA, Single-Stranded↗

PubMed1981

Detection of hepatitis B virus DNA sequences in infected hepatocytes by in situ cytohybridisation.

Plasmid pHBV 114 DNA, which contains 73% of the genome of hepatitis B virus (HBV), was radiolabelled with tritium to 1-2 X 10(8) dpm/microgram by nick translation and used as a radioactive probe to detect HBV DNA present in sections of infected liver tissue by in situ hybridisation followed by autoradiography. Factors affecting the sensitivity of the reaction were examined, including different methods of fixation, hybridisation time, temperature, and buffers. The specificity of the reaction for detecting viral DNA was carefully established by the use of unrelated DNA probes, pretreatment of sections with DNAase, and comparing the stability of the binding of DNA probe at different temperatures, with the melting curve of double-stranded DNA in solution. In the one liver studied in detail, cells containing large amounts of viral DNA were distributed in foci corresponding to areas containing morphologically damaged hepatocytes. This observation suggested a relationship between active viral replication and cell damage. Viral DNA was found mainly in the cytoplasm, although a minority of nuclei in these foci were also positive.

Autoradiography↗

PubMedDate not supplied

Correlation between liver histology and markers of hepatitis B virus replication in infected patients: a study by in situ hybridization.

Liver sections from 18 patients positive for hepatitis B surface antigen (HBsAg), and from 12 negative patients, were examined for the presence of hepatitis B virus (HBV) DNA using an in situ hybridization assay that would identify only those hepatocytes containing more than 10 to 15 HBV genome equivalents per cell. Such cells are likely to be undergoing active viral replication, rather than latent infection. The findings were correlated with results of tissue immunofluorescence for HBV antigens and the presence of serum hepatitis B e antigen (HBeAg), together with histologic assessment of each liver. HBV DNA detected in the above assay was predominantly cytoplasmic; it was associated with the presence of hepatitis B core antigen (HBcAg) in hepatocytes and HBeAg in serum, and to a lesser extent with cirrhosis and immunosuppression, but not with the presence of HBsAg in hepatocytes, nor with histological evidence of disease activity judged by the presence of piece-meal necrosis and lobular and portal tract inflammation. These findings support the view that liver HBcAg and serum HBeAg are markers of virus replication, and demonstrate that active liver disease in HBsAg-positive patients may occur with or without such markers of replication. It is proposed that alternative mechanisms for hepatocyte injury may apply in different chronic HBV patients, one related to virus replication and one dependent on immunological factors.

Adolescent↗

PubMedDate not supplied

Cellular localization of alpha-interferon in hepatitis B virus-infected liver tissue.

Cells expressing alpha 2-interferon were identified by indirect immunofluorescence using both a polyclonal and a monoclonal anti-alpha-interferon antibody reagent. In hepatitis B or delta virus infection, focal clusters of alpha-interferon-positive infiltrating mononuclear cells and (to a lesser extent) fibroblasts were regularly seen in liver sections from patients who had chronic active hepatitis and cirrhosis and evidence of virus replication, but in a minority of patients with chronic persistent hepatitis B and not in nonvirally infected livers. This report provides evidence for local alpha-interferon production near the site of virus replication in hepatitis B infection, identifies mononuclear cells and fibroblasts (but not hepatocytes) as the main cell types producing interferon in this infection and suggests that locally produced alpha-interferon may be a natural regulator of virus replication in HBsAg-positive chronic active hepatitis. Furthermore, serological characterization of the interferon species produced locally may predict which particular interferon species could be of the greatest therapeutic benefit in specific disease states or individual patients.

Antigens, Viral↗

PubMedDate not supplied

Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection.

Seventy, 1-day-old ducklings inoculated intraperitoneally with duck hepatitis B virus and 30 controls have been studied over a 2-year period. Infection with duck hepatitis B virus occurred in all inoculated ducks, although this was not associated with clinical morbidity. Duck hepatitis B virus DNA was first detected in liver on Day 3, in pancreatic acinar cells on Day 4, serum on Day 6, splenic red and white pulp on Day 7 and in the renal glomurulus on Day 14, using a combination of dot, Southern blot and in situ hybridization techniques. Peak levels of circulating virus, as determined by DNA polymerase levels, occurred 1 to 4 weeks postinoculation. Mild degrees of portal inflammation were seen in sections of liver tissue in both infected and control ducks. However, moderately severe inflammatory changes were present in 8 of 22 infected birds compared with 0 of 18 controls (p less than 0.025). Appearance of this inflammatory infiltrate 6 weeks postinoculation coincided with a decrease in levels of duck hepatitis B virus DNA in hepatocytes and within the pancreatic acinar cells. At the same time, duck hepatitis B virus DNA became increasingly localized to the splenic germinal centers, and viral DNA was first detected in pancreatic islet cells. No histological changes accompanied the extra-hepatic tissue infection. The sequence and significance of duck hepatitis B virus infection in liver and extra-hepatic tissues is discussed in relation to the pathogenesis of hepatitis B virus infection in man.

Animals↗

PubMedDate not supplied

Characterisation of duck thrombocytes.

Duck thrombocytes were initially identified in peripheral blood mononuclear cells (PBMCs) purified from whole blood on Ficoll-Paque density gradients by examining stained smears of these cells. These thrombocytes could be readily purified from lymphocytes on the FACStar cell sorter by their increased side-scatter. They were similar to chicken thrombocytes in both appearance and function; they had a diameter of 4.5-6 microm and contained large vacuoles and were able to phagocytose carbon and Staphylococcus aureus. A monoclonal antibody (mAb) BA3 was generated which binds specifically to duck thrombocytes and has facilitated the characterisation of these cells which comprise up to 50-60 per cent of cells in Ficoll-Paque purified duck PBMCs.

Animals↗

PubMedDate not supplied

Optimization of an in vitro assay which measures the proliferation of duck T lymphocytes from peripheral blood in response to stimulation with PHA and ConA.

The in vitro proliferative responses of duck PBMCs purified from Ficoll-Paque density gradients to the mitogens PHA and ConA show a great deal of duck-to-duck variation. Better responses were consistently obtained by using nylon wool fractionation to increase the proportion of duck T lymphocytes in PBMC preparations and then culturing these preparations with homologous monocytes, purified from PBMC preparations by their adherence properties. We have also established that the addition of homologous red blood cells enhances the in vitro proliferative responses of duck T lymphocytes, especially when limiting doses of PHA and ConA are used. Duck T lymphocytes showed greater and more consistent proliferation when cultures were incubated at 37 degrees C as compared to incubation at 41.6 degrees C. The improved consistency of higher proliferative responses with this assay should make it more suitable for detecting in vitro proliferative responses of antigen-specific T lymphocytes, as a measure of in vivo induced cell mediated immune responses.

Animals↗

PubMedDate not supplied

Immune responses to duck hepatitis B virus infection.

The duck hepatitis B virus (DHBV) was the first hepatitis B virus identified from an avian host. It is a member of the Hepadnaviridae family of viruses. All members of this family display similar genomic organization and replication strategies and cause species-specific infections that result in either transient (acute) or persistent infection. Hepadnavirus infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious 'empty' surface antigen particles into the bloodstream. Hepadnavirus replication is non-cytopathic and immune responses to viral antigens are thought to be responsible for the liver damage seen in both transient and persistent infection and for the clearance of virus from infected cells. This has provided the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human hepatitis B virus (HBV) infection. It follows that detailed understanding of the immune responses induced during transient and persistent infection may well facilitate the development of more effective approaches to immunotherapy in patients with persistent infection and may also provide a means of reducing the liver damage associated with this infection, without reducing the effectiveness of the immunity required to eliminate the virus. Immune responses to hepadnavirus infection have been studied primarily in humans, following natural infection with HBV, but studies have also been performed with the woodchuck hepatitis virus (WHV) and the DHBV models. This manuscript reviews the recent studies of immune responses to DHBV infection.

Animals↗