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Biomedical subjects

K A Fleming

Publications and source records attributed to K A Fleming.

At least 73 records · Page 4Linked to original sources
PubMed1993

Primary biliary cirrhosis: contribution of HLA class II allele DR8.

Primary biliary cirrhosis is a chronic cholestatic disease of unknown aetiology which predominantly affects middle-aged women. It is thought to be autoimmune in nature, but unlike many autoimmune diseases no clear HLA association has been described. Several studies have suggested conflicting associations with HLA class II, although a DR8 association is most frequently described. To test the hypothesis that primary biliary cirrhosis is associated with a certain HLA class II locus we genotyped 130 patients with the disease from the north-east region of England and 363 local healthy controls. HLA-DRB1 and confirmatory DQA and DQB genotypes were determined by TaqI restriction fragment DNA length polymorphism analysis. In addition, a polymerase chain reaction technique (double ARMS) was used to investigate the DRB3 locus (DR52) in 98 primary biliary cirrhosis patients and 107 local controls. We found an increased frequency of HLA-DR8 (18.5% vs 9.2%, p < 0.005, relative risk of 2.0 [1.3-3.1]) in the primary biliary cirrhosis group. HLA-DR8-positive primary biliary cirrhosis patients had a higher serum bilirubin level (p = 0.03) than DR8-negative patients. There was no difference in the DR52 frequencies and no association with markers of disease severity. These results support earlier serological findings, although the association between primary biliary cirrhosis and DR8 is weaker than previously described. In addition, DR8-positivity may identify a clinical subgroup with a worse prognosis.

Adult↗
PubMed1992

Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping.

We have developed a new method for analysis of complex genetic systems by a combination of the Amplification Refractory Mutation System (ARMS) and Single-Strand Conformation Polymorphism (SSCP) analysis: ARMS-SSCP. Thus, a complex allelic series is subdivided into a number of groups by ARMS followed by the identification of specific alleles using SSCP analysis. We have shown that the HLA alleles at the DRB3 and DQB1 loci were distinguishable from each other using ARMS-SSCP. In 36 individuals typed for the DRB3 and 48 individuals typed for the DQB1 loci, ARMS-SSCP results were in complete agreement with those obtained using the established method of sequence-specific oligonucleotide (SSO) hybridisation. With silver staining, ARMS-SSCP is a rapid, non-radioactive and reliable method which also offers the possibility for detecting new HLA alleles. We have demonstrated that ARMS-SSCP can be performed using fluorescent PCR primers, a feature which gives the method potential for automation.

Base Sequence↗
PubMed1992

Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues.

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.

Antibodies, Viral↗
PubMed1992

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

Animals↗
PubMed1992

A survey of cytomegalovirus (CMV) DNA in primary sclerosing cholangitis (PSC) liver tissues using a sensitive polymerase chain reaction (PCR) based assay.

Reactivation of cytomegalovirus (CMV) has been implicated as a possible etiological agent in primary sclerosing cholangitis (PSC) partly because of the ability of CMV infection to cause hepatobiliary damage, and further because of the recent recognition of a PSC-like syndrome in AIDS patients, many of whom have hepatobiliary infection with CMV. Direct evidence of CMV infection in PSC has come from a study detecting CMV DNA in 7/7 PSC livers, but only 5/20 controls. We have developed an assay for CMV-DNA by amplification of the immediate early region of CMV using the polymerase chain reaction, followed by Southern blotting and 32P oligoprobing of the amplification product. This system has an average sensitivity of at least 25 copies of CMV-DNA per 5000 formalin-fixed paraffin-embedded cells. 37 PSC and 19 control samples of formalin-fixed paraffin-embedded hepatobiliary tissues were studied. Amplification for the beta-globin in each sample was used as an amplification control, and fetal lung with known CMV infection as the CMV-positive control. 37/37 PSC tissues amplified for beta-globin, and one of these was positive for CMV-DNA. All 19 controls amplified for beta-globin, with none being positive for CMV. The lack of CMV-DNA in 35/36 PSC samples at a level of 25 copies per 5000 cells, we believe, rules out any significant CMV reactivation in these tissues, and suggests that CMV replication and re-activation is not responsible for the progression of PSC.

Base Sequence↗
PubMed1992

Generation of digoxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction.

As the polymerase chain reaction (PCR) can be used for the generation of vector-free probes, the optimum conditions for incorporation of digoxigenin-11-dUTP into hepatitis B virus (HBV) probes have been investigated. High yields of double-stranded or single-stranded probes can be obtained by utilizing a pair of primers or one primer alone. The probes were tested by dot-blot hybridization on HBV plasmid DNA, slot-blot hybridization on total cellular RNA of Alexander cells and Southern blot hybridization on cellular DNA of Alexander cells and HBV plasmid DNA. They were also tested by in situ hybridization (ISH) on HBV-positive biopsy liver tissue. A ratio of dig-dUTP:dTTP of 1:3 gave highest sensitivity in DNA hybridization. No loss of amplification efficiency and sensitivity was observed when the final concentration of dig-11-dUTP and dTTP was reduced to 20 microM and 60 microM respectively, compared to 200 microM each of dATP, dCTP, dGTP. Several different sizes of double-strand probes were compared by dot-blot hybridization. Longer probes were more sensitive. Strong signal could also be obtained by combination of two or three small probes, which have overlapping sequences. Single-stranded DNA probes had advantages of simplicity of use, high sensitivity and strand specificity.

Base Sequence↗
PubMed1992

In vitro culture for the detection of infectious human parvovirus B19 and B19-specific antibodies using foetal haematopoietic precursor cells.

The inability to culture human parvovirus B19 in standard cell lines has rendered investigation of clinical samples for the presence of infectious virus problematic. Using haematopoietic precursors derived from first trimester foetal liver as targets for infection, and non-isotopic in situ hybridization to detect intracellular viral DNA, we have assessed infectivity in stored serum samples taken from nine volunteers at different stages following intranasal inoculation with parvovirus B19. Infectious virus was detected as early as 3 days after inoculation, the cessation of infectivity correlating with the rise in specific IgM. In all but two samples, infectivity correlated with the detection of B19 DNA by dot-blot hybridization, although in vitro culture was 10-fold more sensitive than dot-blot hybridization. B19 DNA was detected by the polymerase chain reaction in serum from one volunteer up to 36 days after inoculation, although samples containing specific antibody were non-infectious. Infection of erythroid precursors was completely inhibited by preincubation of virus with serum containing high titre B19-specific IgM and IgG. Unexpectedly, this was associated with a strong B19 DNA hybridization signal within the cytoplasm of phagocytic macrophages. This culture and detection system is a rapid and sensitive means of detecting infectious virus in serum samples, and of assessing the neutralizing ability of B19-specific antibodies.

Antibodies, Viral↗
PubMed1992

Immunophenotyping of fetal haemopoietic cells permissive for human parvovirus B19 replication in vitro.

Human parvovirus B19 is known to inhibit erythroid colony formation in vitro, but the precise stage of differentiation at which erythroid precursors become capable of supporting viral replication has not been accurately determined. In order to address this issue, haemopoietic cells derived from first trimester fetal liver were cultured in medium containing B19 antigen-positive serum. Infected cells were phenotyped by combining immunohistology for cell-type specific antigens with non-isotopic in situ hybridization for B19 nucleic acid. Strong nuclear hybridization signal was detected as early as 8 h after infection in erythroid precursors labelling with antibodies to glycophorin A, glycophorin C, CD43, CD36 and HLA-ABC (pronormoblast or normoblast phenotype). Giant erythroid precursors labelling with the same five antibodies were a pathognomonic feature of infected cultures, but contained relatively little B19 nucleic acid. Hybridization signal was not detected in progenitor cells of more primitive erythroid phenotype or in nuclei of cells of other lineages, though B19 DNA was occasionally localized within the cytoplasm of macrophages. Double-labelling with antibody Ki-67 confirmed that proliferating cells were targets for B19 infection. Co-detection of cell-type specific antigens and viral nucleic acid is a powerful tool for investigating host cell specificity, and suggests that proliferating late erythroid precursors are the only haemopoietic cells fully permissive for B19 infection.

Antigens, Surface↗
PubMed1992

Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver.

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.

Adult↗
PubMed1992

Clinical and histopathological features of parvovirus B19 infection in the human fetus.

OBJECTIVE: To provide a comprehensive description of the clinical and histopathological features associated with parvovirus B19 infection of the human fetus. SUBJECTS: All cases of parvovirus B19-related fetal death presenting to the John Radcliffe Hospital, Oxford, over a 16 year period. Diagnosis was confirmed retrospectively by non-isotopic in situ hybridization for parvovirus B19 DNA. RESULTS: The ten cases occurred in two clusters (1979-80 and 1988-89) and presented between 15 and 29 weeks gestation. In at least three cases maternal infection was asymptomatic. Nine fetuses were grossly hydropic at necropsy. Histological features common to all cases included the presence of typical intranuclear inclusions in erythroid precursor cells and evidence of vasculitis within placental villi. Inflammatory changes were also present in the myocardium of four cases, with evidence of subendocardial fibroelastosis in three. CONCLUSIONS: Histological features of fetal parvovirus B19 infection are similar across a range of gestational ages. The heart failure and hydropic state associated with fetal parvovirus infection may be of multifactorial aetiology, and not due to fetal anaemia alone.

Adult↗
PubMed1992

Prevalence of anti-neutrophil antibody in primary sclerosing cholangitis and ulcerative colitis using an alkaline phosphatase technique.

The detection of a nuclear anti-neutrophil antibody in patients with primary sclerosing cholangitis (PSC), using an immunoperoxidase technique, was recently reported by us. Subsequently, detection of a cytoplasmic anti-neutrophil antibody was reported by others, using a two stage procedure of enzyme linked immunosorbent assay followed by an immunofluorescent method. Detection of cytoplasmic anti-neutrophil antibody in PSC, which, in contrast to that two stage procedure, uses a simple one step immuno-alkaline phosphatase method is now reported. Normal human neutrophils were cytocentrifuged, ethanol fixed, and then incubated with coded patients' sera. Rabbit anti-human immunoglobulin conjugated with alkaline phosphatase was used to detect the bound antibody. Fast red was used to visualise the reaction. Twenty three of 30 (77%) PSC patients showed positive granular cytoplasmic staining (with some perinuclear accentuation) with a network of cytoplasmic filaments. Fifteen of 45 (33%) ulcerative colitis patients and 1 of 3 chronic active hepatitis patients showed similar staining. Thirty five of 152 patients with ulcerative colitis, chronic active hepatitis, and a variety of other liver diseases showed a different pattern of cytoplasmic labelling, with no surrounding filaments. Seventy nine patients, including seven PSC patients and 33 normal subjects, were negative. In comparison, 86% of PSC patients, 57% of patients with primary biliary cirrhosis, 50% of normal subjects, and well over 60% of patients with ulcerative colitis, Crohn's disease, alcoholic liver disease, and chronic active hepatitis were positive using the one step immunofluorescent method. This method is more specific for PSC than those described in recent reports and may be of diagnostic importance.

Adult↗
PubMed1992

Non-isotopic in situ hybridisation and immunophenotyping of infected cells in the investigation of human fetal parvovirus infection.

AIMS: To compare the use of biotinylated and digoxigenin labelled probes for diagnosis of human fetal parvovirus B19 infection in formalin fixed, paraffin wax embedded tissues; and to assess the cellular distribution of the virus in positive cases. METHODS: Sections of lung tissue from 23 cases of anatomically normal non-immune fetal hydrops presenting between 1984 and 1989, and from 13 control cases of hydrops due to chromosomal abnormality were probed for B19 DNA by in situ hybridisation using both biotinylated and digoxigenin labelled probes. The distribution of the virus was then investigated in all cases of fetal B19 infection confirmed in this laboratory to date (n = 11) by combining in situ hybridisation for viral DNA (using the digoxigenin system) with immunohistological labelling for a range of cellular antigens. RESULTS: Five unequivocal cases of B19 infection were identified among the 23 fetuses with unexplained hydrops using both probe labels. When combined with data from previous studies of the period 1974-1983, the results indicate that B19 infection was responsible for 27% of cases of anatomically normal non-immune hydrops and 8% of all cases, of non-immune hydrops presenting to this hospital over 15 years. False positive signal was seen in an additional three cases, using biotinylated probes. Digoxigenin labelled probes gave greater specificity and permitted detailed investigation of tissues high in endogenous biotin. Though most cells containing B19 DNA colabelled as erythroid precursors, viral DNA was frequently detected within mononuclear-phagocytic cells. In three cases viral signal was also found within occasional myocardial cells labelled by antibody to desmin. CONCLUSIONS: A relatively high proportion of cases of anatomically normal, non-immune hydrops are caused by B19 infection. Digoxigenin is a more reliable probe label than biotin for in situ hybridisation in archival fetal tissues. Double labelling for cellular antigens and viral nucleic acid is a powerful technique for investigating virus-host cell interactions, and provides evidence that cell types other than those of erythroid lineage may have a role in human fetal parvovirus infection.

DNA Probes↗
PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗
PubMed1992

A nude mouse xenograft model of fetal intestine development and differentiation.

This report describes a novel in vivo model of intestinal differentiation. Fourteen day, undifferentiated fetal rat small intestine, stripped of the major part of its mesenchyme, suspended in a type I collagen gel and then xenografted into a nude mouse, undergoes small intestinal morphogenesis and cytodifferentiation. All four major epithelial lineages, namely Paneth, goblet, columnar and endocrine are present. Double-label nonisotopic in situ hybridization, employing biotinylated and digoxigenin-labelled whole rat DNA and whole mouse DNA probes, was performed to distinguish donor cells from host cell types. The outer longitudinal smooth muscle layer, and the major part of the lamina propria, including pericryptal fibroblasts, are of host mouse origin; the inner circular smooth muscle layer is of donor rat origin. Cells of the muscularis propria and lamina propria acquired smooth muscle alpha-actin, presumably under the influence of the donor endoderm. Furthermore, this xenograft develops a host vascular network, and cells with the morphological appearance of lymphocytes are present within the intestinal epithelium. The production of chemotactic factors by the endoderm is postulated because grafting of collagen gel alone results in a minimal invasion by stromal cells which do not express smooth muscle alpha-actin.

Animals↗
PubMed1992

Viral respiratory infection and SIDS.

One of the many factors which have been implicated in the aetiology of SIDS is infection of the respiratory tract, particularly viral infection. This applies particularly to those infants who die from SIDS who are more than 3 months old. The evidence for this belief is based on both epidemiological and pathological factors. Among the epidemiological factors are the pronounced seasonal variation of SIDS (it being commoner in winter); the increased incidence of pre-existing illness, particularly upper respiratory infections, in the two weeks before death; and the increased occurrence of SIDS during epidemics of viral infection in the community. Not all of these factors are universally accepted, however, particularly when appropriate controls are investigated. The necropsy evidence includes the presence of lymphoid inflammatory infiltrates in the respiratory tract, particularly the upper respiratory tract. While these are present in many cases of SIDS, they are not present in all. Postmortem isolation of respiratory viruses has also given conflicting results: some authors show an apparent increase compared with controls, while others do not. No specific virus has been implicated. Part of the reason for these conflicting epidemiological and pathological results is failure to use proper controls. An additional explanation may be the technical difficulties involved in isolating viruses. Apart from the problems resulting from postmortem effects, culture, immunofluorescence, and ELISA tests are known to give significant false negative rates. Accordingly, newer, potentially more sensitive and robust techniques, such as molecular hybridisation, are being applied to cases of SIDS to determine whether viral infection is more common than is currently recognised. Whatever the outcome of these investigations, it is highly unlikely that viral infection per se is the cause of SIDS. One or more additional factors are also involved which may include an abnormal immune response, generation of thermal stress, precipitation of respiratory obstruction, bacterial overgrowth with toxin release, or suppression of the arousal response.

Humans↗