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Biomedical subjects

K A Fleming

Publications and source records attributed to K A Fleming.

At least 37 records · Page 2Linked to original sources
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

Conventional patterns of human intestinal proliferation in a severe-combined immunodeficient xenograft model.

The present work describes the pattern of human intestinal proliferation in an immunodeficient murine xenograft model, which we have shown to closely mimic cell division in normal paediatric gut. Cellular proliferation was measured using a double-label technique combining MIB-1 immunohistochemistry and [3H]thymidine autoradiography, to critically compare values for the tissue growth fraction (G1, G2, S- and M-phase cells) and DNA synthesizing (S-phase) cells in xenograft epithelium, lamina propria, muscularis externa and intraepithelial lymphocytes. The MIB-1 monoclonal antibody (which recognises the cell-cycle dependent nuclear antigen Ki-67) specifically labelled proliferating human cells within the xenografts and did not cross-react with dividing murine cells. This was confirmed using ultrastructural in situ hybridisation with human- and mouse-specific DNA probes to identify the genetic origin of proliferating cells. In general, we found a good tissue correlation between MIB-1 and [3H]thymidine labelling, the only exception being an apparent dysregulation of Ki-67 antigen expression in regenerating xenograft epithelium. In developed xenograft intestine, the highest levels of proliferation were consistently recorded within the crypt epithelium, where 15.7%-26.7% of cells were actively cycling and S-phase occupied approximately half of the cell cycle. The frequency distribution of proliferating epithelial cells within small and large intestinal xenograft crypts was clearly tissue-specific, showing typical patterns of cell division. Therefore, the presence of functional pluripotent epithelial stem cells and conventional spatio-temporal patterns in cellular proliferation, migration, de-cycling, lineage commitment and cytodifferentiation now makes this an attractive experimental model with which to study human intestinal crypt responses to various types of tissue manipulation, e.g. cytotoxic, radiotherapeutic, dietary, endocrine and gene-targeting therapy.

Animals↗
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↗
PubMed1995

Focal destructive cholangiopathy associated with amoxycillin/clavulanic acid (Augmentin).

Amoxycillin/clavulanic acid (Augmentin) has been widely used as a broad spectrum antibiotic since its introduction in 1981, since which time a number of reports of adverse hepatic reaction to the drug combination have been published. This paper describes five patients presenting with cholestatic illness within 8 weeks of a course of amoxycillin/clavulanic acid. The clinical picture indicated a direct link between the illness and the drug combination. Hepatic histology revealed a distinctive focal destructive cholangiopathy in all five patients, which has not previously been reported. Two also showed a granulomatous reaction, which has only previously been reported in one patient. Parallels are drawn with other diseases displaying bile duct destruction, and it is suggested that immunologically mediated drug-induced biliary damage may be involved. One of the five patients developed chronic liver disease with persistence of cholestatic liver biochemical tests, which has not previously been reported. The severity of the reaction and its prolonged course merit wider recognition of the possible adverse hepatic reaction to amoxycillin/clavulanic acid.

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

Superficial thrombophlebitis, dysplasia, and cholangiocarcinoma in primary sclerosing cholangitis.

Cholangiocarcinoma occurs in approximately 10% of patients with primary sclerosing cholangitis. Usually, liver failure, rapidly progressing jaundice, and an increase in alkaline phosphatase levels are suggestive diagnostic features. We report two cases of patients with primary sclerosing cholangitis who developed cholangiocarcinoma without jaundice and with no changes in their serum biochemistry. Both patients were taking ursodeoxycholic acid at the time of tumor diagnosis. Initial suspicion of malignancy was based on the development of superficial thrombophlebitis. Liver histology showed evidence of bile duct epithelial dysplasia in areas free from tumor in one patient, and in the other, bile duct epithelial dysplasia preceded the appearance of cholangiocarcinoma by at least 18 months. In one of the cases, the dysplastic epithelium stained positively for carcinoembryonic antigen. The histological finding of bile duct epithelial dysplasia in patients with primary sclerosing cholangitis may suggest either imminent or actual development of cholangiocarcinoma and may thus affect consideration of orthotopic liver transplantation. In addition, the development of superficial thrombophlebitis in patients with primary sclerosing cholangitis should arouse suspicion of the presence of cholangiocarcinoma even if there is no evidence of deterioration of the liver function or a dominant stricture on endoscopic retrograde cholangiography.

Adult↗
PubMed1994

HLA DR4 is a marker for rapid disease progression in primary sclerosing cholangitis.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is an inflammatory disease of the biliary tree associated with an increase in the HLA alleles DR3, DR52a, DR2, Dw2, and a decrease in DR4. However, it is not certain which of these alleles provides the primary associations. Our aim was to establish the primary HLA associations with PSC and to assess the ability of HLA alleles to mark for disease progression. METHODS: By applying molecular techniques to archival tissue, we have genotyped 83 PSC patients from two populations and 131 controls for the alleles HLA DR2, DR3, DR4, DRw12, DR52a, and Dw2. RESULTS: HLA DR3, DR52a, DR2, and Dw2 were all significantly increased in PSC, with the relative risk for DR52a and Dw2 being greater than for DR3 and DR2, respectively. HLA DR4 was significantly decreased, but this may be artifactual to the DR3, DR2 increase. HLA DR4 and not DR52a marks for rapid disease progression in both our PSC populations. CONCLUSIONS: HLA DR52a and Dw2 are the best candidate alleles for providing the known HLA association with PSC. HLA DR4 and not DR52a marks for rapid disease progression in our two PSC populations.

Amino Acid Sequence↗
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↗