Heteroduplex formation as a means to exclude contamination in virus detection using PCR.
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Hepatitis B virus↗
Biomedical subjects
K A Fleming
Publications and source records attributed to K A Fleming.
Mannan-binding protein in human liver.
Mannan-binding protein (MBP) is a Ca(2+)-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles C1q, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies.
Adolescent↗
Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications.
We have developed a novel double Amplification Refractory Mutation System (double ARMS) using a highly polymorphic region 5' to the human delta-globin gene as a model system. The double ARMS approach involves using two allele-specific ARMS primers simultaneously during DNA amplification by the polymerase chain reaction (PCR). The resulting system is highly sensitive and more specific than single ARMS. In addition, this approach enables the elucidation of the relationship of polymorphic sites on the same chromosome and thus allows the direct determination of haplotypes. We have also demonstrated that this system can be used in conjunction with inverse PCR, the resulting double ARMS inverse PCR (DARMSI-PCR) may allow haplotype determination on polymorphic sites which are separated further apart than the length limit imposed by PCR. The double ARMS approach has numerous other applications in molecular biology including HLA typing, virology, forensic pathology and the investigation of the phenomenon of chimerism following bone marrow transplantation.
Alleles↗
HLA typing by double ARMS.
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Base Sequence↗
Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.
A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.
Base Sequence↗
Detection of single-copy fetal DNA sequence from maternal blood.
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Amino Acid Sequence↗
Human papillomaviruses in anogenital warts in children: typing by in situ hybridisation.
OBJECTIVE: To identify the types of human papillomaviruses found in anogenital warts in children and to relate these to clinical and social information. DESIGN: In situ hybridisation using biotin labelled DNA probes to 11 types of human papillomavirus was performed on biopsy specimens from 17 children with anogenital warts. SETTING: Nuffield department of pathology and the department of dermatology, Oxford. PATIENTS: Children in one group were referred by general practitioners or paediatricians to the dermatology department, where biopsies were performed. The other children were seen in four different hospitals, and biopsy specimens were submitted to the laboratory at the physician's or pathologist's request. RESULTS: Of the 17 biopsy specimens, 10 contained cells positive with a probe to a genital human papillomavirus type (types 6 or 11), while six were positive with a skin virus type (types 2 or 3). One was negative. The virus type present bore no relation to the site or appearance of the warts. The virus type did, however, appear to correlate with groups of children. Skin types were commoner in older children (over 4 years), in those with a relative who had skin warts, and in children with warts elsewhere; there was no relation with the child's sex and no suspicion of sexual abuse in these children. These circumstances suggested non-sexual transmission, such as autoinoculation. In contrast, genital types were commoner in girls, in children under 3 years, in children with relatives with genital warts, and in those with no warts elsewhere. Nevertheless, there was suspicion or evidence of sexual abuse in only half these children, suggesting that other routes of transmission--for example, perinatal--might have been implicated. CONCLUSION: Anogenital warts in children may contain either skin or genital wart virus type. Although the type of human papillomavirus present may give some indication of the likely mode of transmission, this can be interpreted only in conjunction with all available clinical and social information. The type of virus does not provide proof of the presence or absence of sexual transmission.
Anus Diseases↗
Liver abnormality in ovarian hyperstimulation syndrome.
Ovarian hyperstimulation syndrome (OHSS) is a potentially fatal condition associated with the therapeutic induction of ovulation in infertility. Liver function abnormality has been previously reported in four patients, one of whom had ultrastructural abnormalities on liver biopsy. This paper describes a patient presenting with severe OHSS 16 days after ovulation had been induced. Liver function abnormality was apparent 11 days later, with a sustained rise in alkaline phosphatase and aspartate aminotransferase (AST) which lasted up to 2 months. A liver biopsy performed during the second month of her protracted hospital admission showed marked zonal fatty change (acinar zone 1) and associated inflammation, with mitochondrial crystalline inclusions and rough endoplasmic reticulum dilatation on electron microscopy. This report discusses the clinical features and possible aetiological factors.
Adult↗
Combined non-isotopic in situ hybridisation and immunohistochemistry on routine paraffin wax embedded tissue: identification of cell type infected by human parvovirus and demonstration of cytomegalovirus DNA and antigen in renal infection.
A method for combined immunohistochemistry using alkaline phosphatase antialkaline phosphatase (APAAP) and in situ hybridisation using biotinylated probes was developed. The method requires no change to either technique and no additional procedures between them. The procedure was able to show the cell types involved in parvovirus infection of the fetus. The efficiency of immunohistochemistry and in situ hybridisation for the detection of cytomegalovirus in kidney were also compared: occasional cells contained cytomegalovirus DNA but not antigen. The method is rapid, straightforward, and has wide applications in the study of viral infections, genes, and gene products in tissue sections because it permits the combined demonstration of antigen and nucleic acid on the same section in routine clinical material.
Antigens, Viral↗
Gastric endocrine cells share a clonal origin with other gut cell lineages.
There has been considerable debate about the ontological origin of gut endocrine cells as being either from the neural crest (or primitive epiblast) or from the endodermal stem cell. We have attempted to define the ontological origin of endocrine cells by applying an experimental system that uses a marker to identify one of the two phenotypes present in chimaeric mice as suggested by Ponder et al. (1985). This study involved two separate experiments. The first made use of the unique staining properties of Dolichos biflorus agglutinin (DBA), a lectin that binds to the N-acetyl galactosamine sugar residues present on the surface of C57Bl mouse gut, but absent from RoRIII mouse gut, in C57Bl----RoIII mouse chimaeras at the ultrastructural level. A four-stage procedure for staining at the EM level was developed. Although mature villous endocrine cells stained for DBA, immature endocrine cells did not, either in the positive crypts of chimaeric mouse gut or in gut from C57Bl positive controls. Thus a second marker was chosen. This experiment combined immunocytochemistry (to identify gastric antral gastrin cells chosen as a representative neuroendocrine cell) with in situ DNA hybridization for the mouse male chromosome repeat sequence PY 353 (to identify XY cells) in XX----XY chimaeric mice. This study showed that the sex chromosomal pattern in the gastrin cells parallels that of other cells in the same gastric gland and therefore are clonal with them. This suggests that gut endocrine cells share a common stem cell with other epithelial cell lineages in the antrum and are endodermally derived.
Animals↗
Prenatal sex determination by DNA amplification from maternal peripheral blood.
The polymerase chain reaction was used to amplify a Y-specific repeat sequence in peripheral blood DNA samples from 19 pregnant women who had a gestational age of 9 to 41 weeks. Y-specific sequences were amplified from all 12 women who bore a male fetus but in none of 7 women who bore a female fetus. With stringent precautions against contamination, this technique may assist prenatal diagnosis of sex-linked genetic disorders.
Blood↗
Oral epithelial dysplasia in vitamin B12 deficiency.
Widespread soreness and focal ulceration of the oral mucosa developed in a woman with vitamin B12 deficiency shortly after hydroxocobalamin therapy had commenced. An oral mucosal biopsy showed disordered epithelial maturation with marked cytologic atypia. However, during the next month of treatment, the patient's oral mucosa became clinically and histologically normal. Pathologists should be aware that vitamin B12 deficiency may be associated with epithelial changes similar to those associated with premalignancy.
Aged↗
Peripheral blood and portal tract lymphocyte populations in primary sclerosing cholangitis.
The relative distribution of lymphocyte subpopulations in the blood and liver of patients with primary sclerosing cholangitis (PSC) and related diseases has been studied using immunoenzyme techniques. The peripheral blood CD4/CD8 T lymphocyte ratio was significantly higher in active ulcerative colitis (UC) and in PSC with inactive UC than in inactive UC alone. In contrast, no relationship with disease activity was seen in Crohn's disease. The portal tract t lymphocyte count per high power field (mean +/- S.D.) was higher in pre-cirrhotic PSC (173 +/- 105) and primary biliary cirrhosis (PBC: 210 +/- 110) than in histologically normal liver (42 +/- 27). However, the overall portal tract CD4/CD8 ratio was similar in PSC (1.49), PBC (1.89) and normal controls (1.63). The results are consistent with immunological involvement in the pathogenesis of PSC, but argue against the hypothesis that changes in the peripheral blood T cell subsets are due to sequestration at the site of tissue inflammation.
Adolescent↗
Hemangiosarcoma of the rectum after chronic anorectal ulceration.
A 37-year-old man with a 30-year history of recurrent anorectal ulceration developed a hemangiosarcoma of the rectum. This diagnosis was made based on histology and immunocytochemistry. We believe this is the fifth reported case of hemangiosarcoma of the rectum and is another example of a rare granulation tissue sarcoma.
Adult↗
Identification of the origin of cells in human basal cell carcinoma xenografts in mice using in situ hybridization.
In an attempt to identify more clearly the origin of cells in human basal cell carcinoma xenografts in mice, paraffin and frozen sections were subjected to in situ DNA hybridization with biotin labelled human and murine DNA probes. Human skin and mouse skin sections were used as controls. As expected, the implanted epithelium reacted with the human DNA probe and the surface epithelium and most of the stromal cells reacted with the murine probe. However, the stroma immediately surrounding the implanted epithelium contained cells of human origin mixed with murine cells. Occasional murine cells (presumed inflammatory) were present in the human implanted epithelium. Assessment showed no correlation between the degree of differentiation of the implanted epithelium and the ratio of human/murine cells in the contiguous stroma. This technique provides a sensitive test for identifying human cells in xenografts and may be useful in assessing the role of stromal cells in the differentiation of a variety of carcinomas.
Animals↗
The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras.
Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.
Animals↗
In vitro amplification of hepatitis B virus sequences from liver tumour DNA and from paraffin wax embedded tissues using the polymerase chain reaction.
A 185 base pair fragment from the core-polymerase overlap region of the hepatitis B virus (HBV) genome was amplified using the polymerase chain reaction (PCR). The results were compared with those of Southern blotting on extracted DNA from eight hepatocellular carcinomata. The data agreed with those of Southern blotting in six cases (two positive, four negative) but in two other positive cases PCR failed to amplify HBV sequences. This suggests deletion or mutation, or both, of this viral region in these cases. PCR was also used to amplify HBV sequences from formalin fixed, paraffin wax embedded tissue. Tissue inhibition of PCR occurred which increased with the number of tissue sections. It was present in tissues from different organs and species and fixed by different procedures, thus highlighting the need for a positive control during amplification. Use of formalin fixed Alexander cells, however, showed a sensitivity of one viral copy per 5000 cells. Confirmation of the identity of the PCR products was carried out using PCR-generated biotinylated probes, and suggested the insertion of extra nucleotide sequences or infection with an HBV variant in one case.
Base Sequence↗