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Biomedical subjects

K A Fleming

Publications and source records attributed to K A Fleming.

At least 19 recordsLinked to original sources
PubMed1995

High prevalence of pancreatic islet amyloid in patients with end-stage renal failure on dialysis treatment.

Islet amyloid polypeptide (IAPP) is the main proteinaceous component of pancreatic islet amyloid, which is a characteristic feature of type 2 diabetes. The factors responsible for amyloid deposition are unclear. Patients with end-stage renal failure (ESRF) on dialysis treatment have increased insulin resistance which is associated with hypersecretion of beta-cell products. Furthermore, elevated concentrations of circulating IAPP are found in these patients due to reduced renal clearance of IAPP. To determine the prevalence of islet amyloid in this group of patients, pancreas was examined from 23 non-diabetic [aged 62 (29-79) years, median and range] and four type 2 diabetic [aged 67 (56-72) years] patients with ESRF on dialysis treatment. Pancreatic specimens from 30 non-diabetic control subjects [aged 67.5 (56-86) years] and 14 type 2 diabetic subjects without renal disease [aged 69 (48-86) years] were used as control groups. Islet amyloid was present in all type 2 diabetic patients with ESRF and in 12 out of 14 type 2 diabetic control subjects (86 per cent). Amyloid deposits were found in 8 out of 23 non-diabetic patients with ESRF (35 per cent), which was a higher prevalence than that found in non-diabetic control subjects (3 per cent) (P < 0.01). This may be related to undiagnosed (pre)diabetes. Elevated secretion rates of IAPP due to insulin resistance and high circulating IAPP concentrations as a result of severely reduced renal clearance of IAPP will cause high pericellular concentrations of IAPP. This condition is likely to enhance amyloid fibril formation in pancreatic islets similar to that observed in type 2 diabetes.

Adult↗
PubMed1995

Combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus B19 infection.

Parvovirus B19 is a single-stranded DNA virus with a specific tropism for human erythroid precursor cells. The virus codes for two overlapping structural (capsid) proteins and one non-structural protein which is thought to perform essential functions in viral replication, transcription and packaging. The ultrastructural localization of these proteins was achieved in cultured haemopoietic cells derived from fetal liver which had been infected in vitro and subsequently embedded in LR White acrylic resin. Postembedding immunogold detection of B19 structural and non-structural proteins was combined with localization of viral nucleic acid by in situ hybridization using a digoxigenin-labelled probe and different sized gold labels. The majority of the B19 capsid protein and DNA present in cells harvested 48 hours post-infection co-localized within the centri-nuclear region of erythroid cells demonstrating characteristic chromatin margination. Relatively little DNA hybridization signal was present over paracrystalline inclusions strongly labelled with anti-capsid protein monoclonal antibody R92F6. Viral DNA and capsid protein were co-localized in apparent egress from the nucleus through nuclear pores. B19 non-structural protein was detected in association with both nuclear and cytoplasmic arrays of capsids, supporting the view that this protein plays an important role in viral packaging and remains associated with the complete viral particle until its release from the cell. Co-localization of viral nucleic acid and proteins at the ultrastructural level is a flexible, rapid and highly specific tool for examination of viral life-cycles within cells.

Capsid↗
PubMed1995

Human intestinal development in a severe-combined immunodeficient xenograft model.

The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed clear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a 'selection' process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types.

Absorption↗
PubMed1995

Application of a polymorphic Y microsatellite to the detection of post bone marrow transplantation chimaerism.

A highly sensitive Y-microsatellite amplification system was developed for the detection of post bone marrow transplantation (BMT) chimaerism. The system is able to detect the DNA equivalent of a single male cell in a background of 10(5) female cells. For clinical applications it has the distinct advantage that the polymorphic nature of the PCR product allows the checking of the genuineness of a positive PCR signal by reference to its allelic type. This extra degree of precision enhances the accuracy of Y-PCR for clinical diagnosis.

Alleles↗
PubMed1995

Plasminogen activator inhibitor-2 expression in inflamed appendix.

Plasminogen activator inhibitors are thought to be responsible for the abolition of fibrinolytic activity in inflamed peritoneum. This reduction in the fibrin clearing capacity of the peritoneum promotes the formation of intra-abdominal adhesions. High concentrations of plasminogen activator inhibitor-2 (PAI-2) have been previously found in inflamed peritoneal tissue using immunoassays, but it is undetectable in normal peritoneum. The aim of this study was to localize plasminogen activator inhibitor-2 production in tissue by in situ mRNA hybridisation. Sections of normal and inflamed human appendix were hybridised with a digoxigenin labelled cDNA probe. In normal appendix staining was confined to macrophages in the mucosa. Macrophage staining was also seen in inflamed tissue but with a wider distribution throughout the appendix wall. PAI-2 was also localized to mesothelial cells of inflamed but not normal appendix. Cell identities were confirmed using immunohistochemistry directed against cell specific markers. Staining was absent from control slides incubated with plasmid DNA or PAI-2 probe following ribonuclease digestion. The identification of the cells expressing the PAI-2 gene in peritoneum increases our understanding of the pathophysiological process leading to fibrin deposition within the abdomen during peritonitis.

Appendicitis↗
PubMed1995

Acute hepatitis C infection after sexual exposure.

A case is described of a woman with acute hepatitis C infection whose partner had chronic hepatitis C infection and where heterosexual contact was the only major risk factor. Infection of both partners was confirmed serologically and by the finding of virus RNA by reverse transcription and polymerase chain reaction amplification. Nucleotide sequence analysis of the NS5 region (RNA polymerase) was used to show that both partners were infected with virus of the same genotype (1a). The nucleotide sequence of virus RNA found in the female patient is closest to variants cocirculating in the male contact, consistent with transmission having occurred between the two.

Acute Disease↗
PubMed1995

Liver histology in hepatitis C infection: a comparison between patients with persistently normal or abnormal transaminases.

Forty two cases of confirmed hepatitis C virus (HCV) infection with available liver histology were studied. Most patients, 23 of 42 (55%) had abnormal liver function tests but 19 of 42 (45%) had persistently normal liver transaminases (mean aspartate transaminase (AST) 24.1 IU/l, mean follow up 10 months). Histological examinations in the group with normal AST activities were normal in two of 19 (11%), showed non-specific reactive hepatitis in eight of 19 (42%), chronic persistent hepatitis in six of 19 (31%), and chronic active hepatitis in three of 19 (16%). Twenty three of 42 (55%) had either persistently or temporary raised liver transaminases (mean AST 96.2 IU/l, mean follow up 16 months). Histological examinations in this second group with abnormal liver biochemistry showed reactive hepatitis in five of 23 (22%), chronic persistent hepatitis in six of 23 (26%), chronic active hepatitis in 10 of 23 (43%), and cirrhosis in two (9%). Average alcohol intake was significantly higher in the group within abnormal liver function (17.8 v 6.4 units, p = 0.01). Although serious pathology was more frequent in the abnormal transaminase group, significant liver pathology (chronic persistent hepatitis or chronic active hepatitis) was found in nine of 19 (47%) of cases with repeatedly normal transaminases. Liver biopsy is advised in all cases of chronic hepatitis C infection to accurately assess both the degree of fibrosis and the current activity of the disease.

Adult↗
PubMed1994

Prenatal determination of fetal rhesus D status by DNA amplification of peripheral blood of rhesus-negative mothers.

We have developed a sensitive PCR-based assay for the RhD gene and used it to detect circulating fetal cells from RhD-positive fetuses from peripheral blood of RhD-negative mothers. With further improvement in diagnostic accuracy, this assay may have implications in the management of RhD-sensitized pregnancies in women whose partners are heterozygous for the RhD gene. Further studies are required to determine the relationship between maternal anti-D levels and circulating fetal cell numbers.

Amniotic Fluid↗
PubMed1994

A 2-year follow-up study of anti-neutrophil antibody in primary sclerosing cholangitis: relationship to clinical activity, liver biochemistry and ursodeoxycholic acid treatment.

A specific anti-neutrophil antibody was recently described in primary sclerosing cholangitis and was detected using an indirect immunoalkaline phosphatase method. The present study examined the relationship of this antibody with respect to time, clinical activity, liver biochemistry, histology and ursodeoxycholic acid treatment. Fourteen patients with primary sclerosing cholangitis were followed up at regular intervals for 2 years. Clinical activity, liver biochemistry, histology and anti-neutrophil antibody titre were determined during the study period. A fluctuating course of the anti-neutrophil antibody was noted in both untreated patients and in those who were on ursodeoxycholic acid. This finding will have clinical implications if anti-neutrophil antibody is used to screen for potential primary sclerosing cholangitis in the future. The results suggest that several determinations of the antibody would need to be carried out over a period of time. No correlation was found between the antibody titre, clinical activity, liver biochemistry or histology. Anti-neutrophil antibody in primary sclerosing cholangitis probably has no pathogenic significance and is an epiphenomenon.

Adult↗
PubMed1994

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

AIMS: To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS: Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS: Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS: There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.

Base Sequence↗
PubMed1994

Defining the immunogenetic susceptibility to primary biliary cirrhosis.

Primary biliary cirrhosis is a chronic cholestatic disease, thought to be immune-mediated with genetic susceptibility encoded in the major histocompatibility complex. In northern Europeans, the best established associations are with HLA-DR8 and the complement allele, C4B2. These associations could be due to a single susceptibility locus on an extended haplotype linking HLA-DR8 and C4B2 or to both HLA-DR8 and C4B2 independently conferring disease susceptibility. C4B2 genotyping was performed on 64 patients with primary biliary cirrhosis and 61 controls matched for ethnic background and frequency of HLA-DR8. C4B2 was associated with HLA-DR8 (p < 0.05) in PBC. No difference in the frequency of C4B2 was detected between control and disease populations, suggesting that HLA-DR8 and C4B2 are in linkage disequilibrium and that C4B2 is not a susceptibility locus for PBC. Taq I polymorphisms were screened in the disease and control populations with the cosmid probe G91, located midway between the HLA-DR and complement loci. One G91 restriction fragment (G91A) was found to be associated with both HLA-DR8 and C4B2, at equal frequency in health and disease, providing evidence of an HLA-DR8-G91A-C4B2 extended haplotype. The frequency of G91A was the same in the disease and control populations, suggesting that G91A does not confer disease susceptibility. These findings establish G91 as the telomeric boundary for disease susceptibility associated with HLA-DR8, encoded on chromosome six. These studies help define the immunogenetic susceptibility locus for primary biliary cirrhosis.

Alleles↗