Biomedical subjects

J H Pringle

Publications and source records attributed to J H Pringle.

At least 73 records · Page 4Linked to original sources

PubMed1991

Epstein-Barr viral DNA in Hodgkin's disease: amplification and detection using the polymerase chain reaction.

The presence of Epstein-Barr virus (EBV) DNA in biopsy tissues from patients with Hodgkin's disease (HD) was investigated by the polymerase chain reaction (PCR) using primers specific to a sequence within the EBV Bam H1W region. EBV genome was detected in 33 of 57 (58 per cent) cases of HD. Viral DNA was, however, also demonstrated in nine of 24 non-Hodgkin's lymphomas, in three of nine non-neoplastic lymph nodes and in seven of 12 normal peripheral blood samples used as controls. In all cases, the band obtained following PCR was verified using Southern blotting and hybridization with highly specific Bam H1W probes. The results suggest that the technique is sufficiently sensitive to detect EBV in persistent latent infection in B-lymphocytes. Distinction between virus present as a possible aetiological agent of malignancy or as a latent infection is not possible when PCR is used under these conditions. The possible role of EBV as an aetiological agent of HD remains unresolved.

Adolescent↗

PubMed1991

Demonstration of insulin and glucagon mRNA in routinely fixed and processed pancreatic tissue by in-situ hybridization.

Human insulin and glucagon mRNA were identified in routinely processed pancreatic tissue by non-radioactive in-situ hybridization using digoxigenin-labelled oligonucleotide probes. Cocktails of synthetic oligonucleotides complementary to human insulin and glucagon mRNA were labelled with digoxigenin using terminal deoxynucleotidyl transferase (Tdt). Specific hybrids were detected with alkaline phosphatase-labelled anti-digoxigenin antibody and visualized by BCIP-nitroblue tetrazolium indicator substrate. The results showed highly sensitive and specific staining of islet cells on a range of routinely formalin-fixed and paraffin-embedded tissues. Post-mortem pancreatic tissue from adults and stillborn neonates yielded acceptable signals as long as tissue morphology was well preserved. Preliminary investigations using pancreatic endocrine cell tumours gave clear easily interpretable signals which were comparable to conventional immunostaining. The application of this technique promises to be of value in the investigation of pancreatic disease.

Adult↗

PubMed1991

The expression of parathyroid hormone messenger RNA in normal and abnormal parathyroid tissue.

The distribution and expression of preproparathyroid hormone (PTH) mRNA were investigated in parathyroid tissue from 57 parathyroidectomy specimens. PTH mRNA was detected by in situ hybridization using digoxigenin-labelled oligonucleotide probes. Cell morphology was seen to correlate with PTH mRNA expression. Strong expression of PTH mRNA was confined to cells which on haematoxylin and eosin staining had large vesicular nuclei. These included both vacuolated and non-vacuolated cells. Chief cells with small dark nuclei and scanty cytoplasm had little or no expression. In both adenoma and chief cell hyperplasia, the striking difference from normal was the greatly increased proportion of cells expressing PTH mRNA. In adenomas, the rim of uninvolved parathyroid tissue showed PTH mRNA expression similar to that of normal parathyroid. In hyperplasia, there was frequently concordance of staining within individual nodules. The findings establish morphological criteria for activity of parathyroid tissue and support current concepts of the different pathogenesis of hyperplasia and adenoma. The expression of PTH mRNA in oxyphil change and parathyroid carcinoma was also investigated.

Humans↗

PubMed1991

High-grade T-cell lymphoma following treatment with cyclosporin A.

A 47-year-old man with persistent severe oropharyngeal ulceration developed a high-grade T-cell lymphoma soon after commencing treatment with cyclosporin A. Using Southern blotting to identify T-cell beta-chain gene rearrangements, evidence of clonal restriction was found both in blood and lymph node DNA samples. Two BamH1 rearranged bands were demonstrated in both samples. In the blood a 16 Kb band predominated, with a weaker 28 kb band. In the lymph node sample this pattern was reversed. The findings suggest that a bi-clonal population of T-lymphocytes or clonal evolution of an existing T-cell monoclone had developed, and that cyclosporin contributed to the emergence of a high-grade T-cell lymphoma.

Antigens, CD↗

PubMed1991

Cervical intraepithelial neoplasia and squamous cell carcinoma of the anus in sexually active women.

Twenty-five patients with squamous cell carcinoma (SCC) of the anus have presented over an 8 year period; 18 were female. Six of 9 patients aged under 50 years were female. Five of these women had been treated for a previous cervical malignancy (2 invasive) and 4 practised anal intercourse; human papillomavirus (HPV) type 16 DNAs were isolated from their arcival anal/cervical paraffin sections. Signals were confined to the nuclei of the invasive anal SCC cells and the transformation zone of the cervix. HPV 6, 11 and 18 DNAs were not identified. Young women with cervical intraepithelial (CIN) III or invasive cervical SCC found in association with HPV infection are at risk of developing anal SCC (P less than 0.001; Fisher's exact).

Adult↗

PubMed1990

Demonstration of Epstein-Barr viral DNA in formalin-fixed, paraffin-embedded samples of Hodgkin's disease.

The presence of Epstein-Barr virus (EBV) DNA in formalin-fixed, paraffin-embedded samples of Hodgkin's disease (HD) was investigated by Southern blot hybridization using a specific EBV Bam H1W fragment probe. DNA from 16 samples of HD in children and young adults was compared with that from ten samples in adults older than 45 years. A further eight cases of DNA extracted from fresh samples of HD were also studied, in order to compare the results obtained with fresh and fixed tissue samples. Hybridization was demonstrated in 15 of the 34 cases of HD studied in contrast to 3 or the 34 control specimens of non-Hodgkin's lymphoma, non-neoplastic lymph nodes, and carcinomas. No differences between the two age groups compared were found. The results of this study suggest that there is a definite association between EBV and HD, although the exact nature of this association remains to be established. It has also been shown that DNA of sufficient quality for the detection of EBV DNA can be extracted from formalin-fixed, paraffin-embedded material, and that comparable results can be obtained using DNA extracted from fresh and fixed tissue.

Adolescent↗

PubMed1990

In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes.

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.

Biotin↗

PubMed1990

Zonal distribution of immunoglobulin-synthesizing cells within the germinal centre: an in situ hybridization and immunohistochemical study.

Immunohistologic studies have shown that synthesis of cytoplasmic immunoglobulin (cIg) is a normal function of some follicle centre cells (FCCs). The mechanisms regulating this synthesis of immunoglobulin and its function within the germinal centre are still poorly understood. In this study we applied a recently developed in situ hybridization method for the detection of kappa and lambda light chain mRNA to reactive lymph nodes and tonsils in order to investigate further the immunoglobulin-synthesizing cells of the germinal centre. FCCs containing detectable levels of light chain mRNA corresponded closely to cells containing cIg. The detection of light chain mRNA rather than its immunoglobulin product was found to be an advantage in that problems associated with the detection of extracellular immunoglobulin were eliminated. This was most apparent in germinal centres where the absence of 'network' immunoglobulin led to the observations that immunoglobulin-synthesizing FCCs are predominantly small centrocytes and that in a proportion of germinal centres they localize in that part of the light zone closest to the dark zone. This zonal distribution of immunoglobulin-synthesizing FCCs raises the possibility of further functional and micro-environmental subcompartments within the light zone.

Antibody-Producing Cells↗

PubMed1989

Identification of a human ribosomal protein mRNA with increased expression in colorectal tumours.

A human ribosomal protein cDNA was selected from a normal colon cDNA library on the basis of overexpression in familial adenomatous polyposis. Nucleotide sequence analysis was used to identify this cDNA as corresponding to the human equivalent of the rat ribosomal protein L31 (HL31). We have quantified the expression of HL31 mRNA in colorectal tumours and found overexpression in 23 out of 23 cases. Our results indicate that HL31 is associated with a malfunction of normal growth regulatory mechanisms in these tumours, and suggest a role for HL31 in proliferation and neoplasia.

Adenocarcinoma↗

PubMed1989

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.

Base Sequence↗

PubMed1989

Functional activity of intestinal epithelium demonstrated by mRNA in situ hybridization.

To elucidate the synthetic activity in relation to the morphology of the epithelial cells of the small and large intestine, in situ hybridization with a poly-deoxyribothymidine (poly d(T)) probe was applied to paraffin sections of formalin-fixed blocks. This effectively displays poly-adenylated RNA and, by implication, messenger RNA (mRNA). By minimizing proteinase K pretreatment, the relative concentrations of cellular mRNA were visualized. This revealed minimal mRNA in crypt columnar cells, and maximal mRNA in proliferating cells and in cells showing terminal differentiation. The latter include surface epithelial cells, endocrine cells, Paneth cells, and maturing, but not mature, goblet cells. The goblet cells showing positive hybridization can be regarded as active cells and show characteristic morphology. Such cells are particularly evident in untreated coeliac disease, remitting ulcerative colitis, and transitional mucosa. The proliferating cells showing increased hybridization include normal mitotically active crypt epithelium, regenerating epithelium in ulcerative colitis, adenomatous epithelium, and adenocarcinomatous epithelium. The similarity of hybridization between metaplastic polyp epithelium and surface colonocytes is consistent with the concept that metaplastic polyps are formed of cells showing premature terminal differentiation.

Adenocarcinoma↗

PubMed1989

The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: in vivo experiments.

The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains.

Bacterial Proteins↗

PubMed1989

Rapid identification of donor and recipient cells after allogeneic bone marrow transplantation using specific genetic markers.

The fate of the donor graft in allogeneic bone marrow transplantation, the presence of chimaerism and early relapse can be monitored by identification of the donor or recipient origin of haemopoietic cells in peripheral blood and bone marrow. We have done this by the use of highly informative locus-specific hypervariable DNA probes in two sex and blood group matched transplants. In four sex mismatched transplants the Y chromosome was identified by in situ hybridization using a biotinylated Y probe. Early engraftment by day 13 was detected in five of the six patients. Transient chimaerism occurred in half of the cases and was accompanied by the temporary appearance of the Philadelphia chromosome in one patient with chronic myeloid leukaemia without subsequent relapse. Persistence of the graft in the face of falling peripheral counts was documented in four of the six patients studied. The morphology as well as origin of the haemopoietic cells could be characterized by the Y probe analysis. In one patient, recipient lymphocytes were shown to co-exist with myeloid series of donor origin. We conclude that both techniques are highly specific, sensitive and can provide information within 24-48 h. Thus they are of value in guiding early therapeutic intervention in allogeneic transplantation.

Blotting, Southern↗

PubMed1989

In situ hybridization detection of light chain mRNA in routine bone marrow trephines from patients with suspected myeloma.

A method for the detection of immunoglobulin light chain mRNA by situ hybridization has been applied to routine bone marrow trephines, from patients suspected of having multiple myeloma. The principle aim was to define monoclonal populations even in cases lacking a paraprotein or an obvious atypical plasma cell proliferation. The expression of light chain mRNA in plasma cells in routine bone marrow trephines from 14 patients with suspected myeloma was studied. Plasma cells in the marrow ranged from 4% to 99%. Clonal populations based on light chain restriction were delineated in 12 patients, using biotinylated probes to kappa and lambda mRNA and visualized using an alkaline phosphatase/fast red naphthol capture method. Normal tonsil and bone marrow trephines were used as controls. In situ hybridization was found to be a specific, safe and rapid technique. It could be applied to both fixed and fresh tissue. It was found to lack the background staining of interstitial immunoglobulin, particularly in sclerotic cases, which is so often encountered with immunocytochemistry. Additionally, this technique will type plasma cell neoplasms (undetected by routine immunocytochemistry) in post-transcriptional block or non-secretory myeloma.

Bone Marrow↗

PubMed1988

Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue.

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.

DNA↗

PubMed1988

How do we teach pathology? The laser videodisc and computer-assisted learning.

A laser videodisc, holding 54,000 still frames per side, can be used to provide visual material of excellent quality for pathology teaching. In a collaborative project between ten pathology departments, the first pathology videodisc has recently been completed and the production of the disc is described. When used as illustrative material for computer-assisted learning programs, the videodisc offers accurate random access of still frames and allows the overlay of computer-generated graphics onto the video picture. This can be used to pre-identify every individual feature of a particular frame, and an example tutorial program is described to show how this may be used to improve the students' understanding of what they see. Details and costs are given for a workstation suitable for this application and potential future developments for this teaching method are discussed. Although objective assessment of the cost-effectiveness of interactive video in teaching pathology has yet to be undertaken, one suitably designed program can give individual tuition to many students, making this a most efficient use of teaching time.

Computer-Assisted Instruction↗

PubMed1988

Large granular lymphocytosis associated with rheumatoid arthritis.

A 74 year old woman with rheumatoid arthritis, hepatosplenomegaly, neutropenia, and peripheral blood lymphocytosis is described. The lymphocytes had a large granular morphology and expressed a CD3+ CD8+ Leu7+ surface antigen phenotype. They did not have natural killer cell function. Southern analysis of the lymphocyte DNA using two restriction enzymes showed a rearranged pattern for the T cell receptor beta chain gene, indicating a monoclonal lymphoproliferation. Large granular lymphocytosis is a rare and heterogeneous phenomenon, which has become more clearly characterised through the application of molecular biology techniques. Most cases appear to be forms of T cell leukaemia with a chronic benign course. The association between rheumatoid arthritis and large granular lymphocytosis is emphasised.

Aged↗

PubMed1987

The immunohistology of Hodgkin's disease--Reed-Sternberg cells and their variants.

Thirty-three cases of Hodgkin's disease were analysed by immunoalkaline phosphatase and immunoperoxidase techniques, using a monoclonal antibody panel, including markers of B-cells, T-cells, macrophages, granulocytes, and the antibody Ki-1. Hodgkin's cells were found to express markers generally regarded as T-cell, B-cell, myeloid, or monocyte associated. Furthermore, heterogeneity of marker expression was also seen within the Hodgkin's cell population in any single case. Morphological and immunohistological analogies between cells involved in antigen presentation and Hodgkin's cells are described, suggesting possible relationships between these cell types. Anti-Leu M1 was not found to be a particularly sensitive marker of Hodgkin's disease under the conditions used in this study.

Antibodies, Monoclonal↗