Biomedical subjects

J H Pringle

Publications and source records attributed to J H Pringle.

At least 55 records · Page 3Linked to original sources

PubMed1994

Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy.

Glomerular IgA in IgA nephropathy (IgAN) is at least in part polymeric, and is thought to derive from the mucosal IgA system in view of the association between mucosal infection and haematuria in this condition. To investigate this hypothesis, an in situ hybridization (ISH) technique was developed for the detection of J chain mRNA, the expression of which has been correlated with the secretion of high level polymeric immunoglobulin (pIg). Endoscopic duodenal biopsies from ten patients and matched controls were examined by: (i) two color immunofluorescence (IF); (ii) ISH; and (iii) combined ISH and IF, to permit simultaneous identification of plasma cell type. IF revealed a reduction in the percentage of IgA plasma cells (P < 0.02) and increased absolute numbers of IgG cells (P < 0.02) in patient biopsies. ISH demonstrated fewer J chain mRNA expressing plasma cells (P < 0.005) with lower signal intensity (P < 0.002) in patients' biopsies compared with controls. Combined ISH and IF confirmed a reduction in J chain mRNA-positive IgA plasma cells in the patient biopsies (P < 0.02). The reduction in J chain mRNA expression in duodenal IgA plasma cells in IgAN argues against the gastrointestinal lamina propria as the source of glomerular pIgA.

Adult↗

PubMed1994

Absence of Coxsackie viruses in idiopathic inflammatory muscle disease by in situ hybridization.

The role of coxsackie virus infection in the pathogenesis of idiopathic inflammatory muscle disease (IIMD) has been investigated by many workers with conflicting results. This study uses in situ hybridization, with digoxigenin-labelled oligonucleotide probes complementary to the coxsackie B virus genome, to investigate the presence of virus RNA in muscle biopsies from 26 patients with IIMD. In the five cases of inclusion body myositis studied, there was focal probe-binding to nuclei and cytoplasm, and in nine cases probe-binding to mast cells was seen. In both of these instances probe-binding was non-specific and not due to hybridization. None of the cases showed the presence of coxsackie virus RNA within muscle and it is concluded that lytic infection of myocytes by coxsackie virus does not occur in IIMD.

Adolescent↗

PubMed1994

Demonstration of mitochondrial ribosomal RNA in frozen and paraffin-embedded sections of skeletal muscle by in situ hybridization.

We have used in situ hybridization with synthetic oligonucleotide probes to the 16S sub-unit of mitochondrial ribosomal RNA to detect mitochondrial accumulations in biopsies of skeletal muscle from 16 patients with mitochondrial myopathies. In all cases, the distribution of the hybridization signal matched the pattern of succinate dehydrogenase activity in adjacent cryostat sections. In situ hybridization also allowed visualization of mitochondrial accumulations in paraffin-embedded sections of skeletal muscle that had been fixed in either De Castro's solution or formalin. DNase pre-treatment did not significantly diminish the intensity of hybridization signal, suggesting that the hybridization is predominantly to RNA. The advantages of this technique are that it allows the investigation of mitochondrial disorders when only paraffin-embedded tissue is available and should also facilitate the demonstration of mitochondrial accumulations in tissues other than skeletal muscle.

Biopsy↗

PubMed1994

Absence of Epstein-Barr virus RNA in multiple sclerosis as assessed by in situ hybridisation.

Epidemiological and serological evidence has suggested a role for Epstein-Barr virus infection in the aetiology of multiple sclerosis. Epstein-Barr virus-specific RNA was looked for in the brains of 10 patients with multiple sclerosis by in situ hybridisation. A total of 21 plaques was examined. In all of these preservation of RNA was shown by hybridisation of control probes to mitochondrial rRNA but no signal was detected with the Epstein-Barr virus probes. It is unlikely that persistent or latent Epstein-Barr virus infection of the CNS occurs in multiple sclerosis, although present findings do not exclude a role for Epstein-Barr virus in the initiation of this disorder.

Aged↗

PubMed1993

Absence of Epstein-Barr virus in carcinoma of the cervix.

BACKGROUND: Cervical epithelium is known to contain receptors for Epstein-Barr virus (EBV) and is a recognized site of viral shedding. Recent cases of nasopharyngeal carcinoma have been associated with cervical carcinoma, leading to speculation of a possible etiologic link between EBV and cervical carcinoma. METHODS: This study investigates the presence of EBV in 24 cases of invasive cervical carcinoma (9 adenocarcinomas and 15 squamous cell carcinomas) and 10 cases of cervical intraepithelial neoplasia (4 adenocarcinomas in situ and 6 squamous: 3 CIN II, 3 CIN III), using in situ hybridization. RESULTS: In one case, EBV was demonstrated in infiltrating lymphocytes adjacent to tumor epithelium, but in no cases did the malignant epithelium show the presence of EBV. CONCLUSIONS: These results suggest that EBV does not play a role in the etiology of cervical carcinoma, although further studies are needed.

Adenocarcinoma↗

PubMed1993

In situ correlation of synthesis and storage of parathormone in parathyroid gland disease.

The distribution and expression of parathyroid hormone (PTH) were investigated in normal and abnormal parathyroid tissue. PTH was detected using a monoclonal antibody with specificity for the 44-68 region of the PTH molecule. Prominent reactivity for PTH was seen in normal parathyroid with a granular pattern of staining. Active parathyroid tissue (adenoma and hyperplasia) showed much less reactivity for PTH, although there was prominent reactivity in the normal tissue around adenomas. Comparison of expression of PTH with that of parathormone mRNA showed a reciprocal pattern in normal tissue and, to a less marked extent, in abnormal tissue. Parathyroid carcinoma in particular had coinciding areas of PTH and PTH mRNA expression. Oxyphil cells had little or no PTH expression, except in the associated 'colloid' in some cases. The findings indicate an inverse relationship between storage and cellular synthesis of PTH, this being more marked in physiological than in pathological conditions of the parathyroid.

Adenoma↗

PubMed1993

Demonstration of Coxsackie virus RNA in formalin-fixed tissue sections from childhood myocarditis cases by in situ hybridization and the polymerase chain reaction.

This is a combined study using in situ hybridization and the polymerase chain reaction to investigate the presence of Coxsackie virus RNA in formalin-fixed tissue from cases of childhood myocarditis. Of the ten cases studied, two were positive by both methods. The virus RNA was predominantly located in areas showing an inflammatory cell infiltrate and myofibre necrosis. These findings suggest that direct lytic infection of myocytes by virus is responsible for myocarditis in these cases, rather than an autoimmune process, which has been suggested previously. The findings in one case, where the virus showed a marked sub-endocardial distribution, may have implications for the aetiology of endocardial fibroelastosis by confirming a viral tropism for this location. The techniques used in this study are easily repeatable and can be directly applied to look for viruses in a number of other diseases where a viral aetiology is suspected.

Base Sequence↗

PubMed1993

Demonstration of light chain mRNA in Hodgkin's disease.

The lineage of Hodgkin and Reed-Sternberg cells is still unclear. Detection of both immunoglobulin light chains in Hodgkin and Reed-Sternberg cells by immunohistochemistry is a well-known phenomenon. However, up to now, in situ hybridization techniques have failed to demonstrate light chain messenger(m) RNA in Hodgkin and Reed-Sternberg cells. In this investigation, we have analysed 26 cases of Hodgkin's disease (nodular lymphocyte predominant Hodgkin's disease, mixed cellularity, and nodular sclerosis type) using digoxigenin-labelled oligonucleotide probes for kappa and lambda light chains by in situ hybridization. In nearly half of the cases of nodular lymphocyte predominant Hodgkin's disease and in one case of mixed cellularity type, mRNA for only one light chain could be clearly demonstrated in the lymphocytic and histiocytic cells, Hodgkin, and Reed-Sternberg cells. These results support the idea that at least some cases of Hodgkin's disease are B-cell neoplasms.

Hodgkin Disease↗

PubMed1993

Flow cytometric quantitation of sequence-specific mRNA in hemopoietic cell suspensions by primer-induced in situ (PRINS) fluorescent nucleotide labeling.

We modified a technique of RNA detection based upon primed in situ labeling of RNA (PRINS) to quantitate poly(A) and histone mRNA in HL60 cell suspensions by flow cytometry. The direct PRINS technique was used to label poly(A) mRNA, based upon the incorporation of fluoresceinated nucleotides into cDNA chains generated by reverse transcription from the site of oligo(dT) primer-specific annealing on the poly(A) template. By this method, poly(A) fluorescent signals of up to 20-fold above background were obtained when primer concentrations were saturating. Nonspecific binding of the fluorochrome-conjugated nucleotide proved to be the greatest source of background fluorescence in cell suspensions. This was considerably reduced by preincubation of the cells in lysine and an excess of cold nucleotides prior to the PRINS reaction. The application of indirectly fluoresceinated antibodies to the FITC moiety of the conjugated nucleotide (indirect PRINS) enhanced detection of a fluorescent signal generated from cells by the direct PRINS technique after stringent histone priming. Cell cycle analysis, based upon DNA histograms obtained after PRINS labeling, showed the histone fluorescence to be primarily in S-phase cells of an exponentially growing population. Confocal laser scanning microscopy revealed good retention of cellular architecture, with cytoplasmic localization of poly(A) and histone mRNA fluorescence and heterogeneous mRNA expression among cells. Our modified PRINS labeling of RNA represents a useful advance in the precise quantitation of RNA species in cell suspension by flow cytometry.

Flow Cytometry↗

PubMed1993

Detection of immunoglobulin heavy chain gene rearrangements in Hodgkin's disease using PCR.

AIM: To detect clonal rearrangements of the immunoglobulin (Ig) heavy chain gene in Hodgkin's disease tissue using the polymerase chain reaction (PCR). METHODS: DNA extracted from 36 samples of Hodgkin's disease was analysed using PCR and primers from conserved sequences in the variable (VH) and joining (JH) regions. RESULTS: Clonal rearrangement was detected only in one case. Evidence of clonal immunoglobulin gene rearrangement had been detected previously in this case using conventional Southern blot analysis. CONCLUSIONS: The sensitivity of the two techniques is equivalent and clonal Ig heavy chain gene rearrangements are rare in Hodgkin's disease.

Base Sequence↗

PubMed1993

Analysis of antigen receptor genes in Hodgkin's disease.

AIM: To analyse the configuration of the antigen receptor genes in Hodgkin's disease. METHODS: DNA extracted from 45 samples of Hodgkin's disease was analysed using Southern blotting and DNA hybridisation, using probes to the joining region of the immunoglobulin heavy chain gene, the constant region of kappa immunoglobulin light chain gene, and the constant region of the beta chain of the T cell receptor gene. RESULTS: A single case of nodular sclerosing disease showed clonal rearrangement of the immunoglobulin heavy and light chain genes, all other samples having germline immunoglobulin genes. The nature of the clonal population in the diseased tissue is uncertain, because the intensity of the rearranged bands did not correlate with the percentage of Reed-Sternberg cells present. The T cell receptor genes were in germline configuration in all the samples. CONCLUSIONS: Antigen receptor gene rearrangement is a rare finding in unselected cases of Hodgkin's disease.

Adult↗

PubMed1992

The demonstration of a subset of carcinoid tumours of the appendix by in situ hybridization using synthetic probes to proglucagon mRNA.

Previous studies using immunohistochemistry have shown variable hormone production by carcinoid tumours of the appendix. In order to confirm the existence of a specific subset of these tumours, in situ hybridization using synthetic oligonucleotide probes to detect pre-proglucagon and pre-proinsulin mRNA was performed in formalin-fixed, paraffin-embedded material from eight tubular carcinoids, 12 insulin carcinoids, and two mucinous carcinoids. The results were correlated with standard silver and mucin stains. All tubular carcinoids but none of the insular or mucinous carcinoids contained proglucagon mRNA. Proinsulin mRNA was not detected in any of the tumours. Tubular carcinoids of the appendix constitute a definable subset of appendiceal carcinoids which have a similar distribution and prognosis to typical insular carcinoids and can be diagnosed on haematoxylin and eosin-stained sections confirmed by routine special stains. The main need for recognition is to avoid confusion with mucinous carcinoids, which have a worse prognosis and may require more aggressive treatment.

Appendiceal Neoplasms↗

PubMed1992

Vimentin expression by Reed-Sternberg cells in Hodgkin's disease.

The expression of vimentin in Reed-Sternberg cells in 61 samples of Hodgkin's disease (HD) was examined using an avidin-biotin-peroxidase complex technique. Forty biopsies (66%) expressed vimentin, and expression was seen in all subtypes of HD. No immunophenotypic differences between vimentin-positive and vimentin-negative cases were noted. The significance of such expression is unclear, but may be related to the alterations in growth and differentiation that are typical of neoplastic cells.

Hodgkin Disease↗

PubMed1992

Demonstration of the distribution of coxsackie virus RNA in neonatal mice by non-isotopic in situ hybridization.

A simple method for the demonstration of Coxsackie virus RNA by in situ hybridization is described. Oligonucleotides complimentary to conserved sequences of Coxsackie B genome were synthesised and labelled with digoxigenin using commercially available reagents. In addition to detecting all five Coxsackie B strains examined, six strains of Coxsackie A were also demonstrated by these probes. Using one of the oligonucleotides separately it was possible to distinguish Coxsackie A strains from the strains of Coxsackie B virus examined. This study demonstrates the presence of viral RNA in mice tissues showing morphological evidence of damage, confirming the suspected tropisms of these viruses. The method described is directly applicable to the study of the presence of these viruses in human tissue from diseases where a viral aetiology is suspected.

Animals↗

PubMed1992

Proteins encoded by the Escherichia coli replication terminus region.

The replication terminus region (31 to 35 min) of the Escherichia coli chromosome contains very few mapped genes (two per min) compared with the remainder of the chromosome, and much of the DNA appears dispensable. In order to determine whether, despite this, the terminus region consists of protein-coding sequences, we cloned 44 kb (1 min) of terminus region DNA (that surrounding trg at 31.4 min) and examined its ability to catalyze protein synthesis in vitro or in minicells. We were able to account for more than half the coding capacity of the cloned DNA with proteins synthesized in these systems, indicating that the sparsity of mapped genes in the terminus region does not result from a lack of identifiable coding sequences. We can therefore conclude that the terminus region is composed mainly of expressable, albeit inessential, protein-encoding genes.

Bacterial Proteins↗

PubMed1992

Simultaneous in situ hybridisation of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin wax embedded sections.

AIMS: The development of a technique for simultaneous in situ hybridisation for native mRNA and conventional immunofluorescence for cytoplasmic antigens in routine pathology specimens. METHODS: Cocktails of synthetic deoxyoligonucleotides coding for immunoglobulin J chain and kappa light chain were 3' end labelled enzymatically with digoxigenin using terminal deoxynucleotidyl transferase. Native mRNA sequences were "unmasked" using proteolytic digestion with proteinase K and hybrid detection was achieved with an alkaline phosphatase labelled anti-digoxigenin antibody. Alkaline phosphatase was visualised with Fast red/naphthol AS-MX phosphate. Fluorescein isothiocyanate (FITC) conjugated anti-isotype antibodies were used simultaneously at the detection stage to identify the isotype production by individual plasma cells in endoscopic duodenal biopsy specimens. RESULTS: The IgA plasma cells of the lamina propria were identified by immunofluorescence and hybrids were detected in the anticipated plasma cell population by Fast red visualisation. The reaction product was visible in bright field or ultraviolet illumination which allowed FITC and Fast red labels to be visualised together under ultraviolet light at 490 nm. Dual labelled cells were clearly visible. Morphology was well preserved throughout. CONCLUSIONS: This technique permits the demonstration of specific mRNA species in cells expressing immunoglobulin. It combines all the advantages of non-radioactive synthetic oligonucleotide probes and conventional immunofluorescence techniques in routine formol-saline fixed and paraffin wax embedded sections with good retention of morphology.

Antigens↗

PubMed1992

Oncogene expression in primary myelodysplasia: correlation with haematological, karyotypic, and clinical progression.

AIMS: To see if the relative expressions of proto-oncogenes that are increased in acute myeloid leukaemia are raised in patients with myelodysplastic syndromes (MDS), and to see if they increase with progression to leukaemia. To note if there is a correlation between morphology, karyotype, and these proto-oncogene expressions and if any one proto-oncogene can predict prognosis. METHOD: Bone marrow from 130 patients was analysed at six monthly intervals over two years for relative mRNA expression of seven oncogenes, karyotype, and morphology. The technique used slot blot hybridisation and densitometric analysis. The results were compared with 14 surgical controls and 30 people with vitamin deficiency anaemia. RESULTS: Six of seven oncogenes showed increased expression which progressed with time, but did not correlate with morphological or karyotypic changes. Expression of four of the seven oncogenes was increased in megaloblastic and iron deficiency anaemia. C-mos showed differences among the five morphological subgroups; it correlated with abnormal location (p = 0.025) and seemed to influence prognosis. CONCLUSION: Increased proto-oncogenes reflect the overall marrow perturbation in MDS. C-mos may reflect persistence of monocyte pathway which confirms marrow stability.

Aged↗

PubMed1991

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization.

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.

Hodgkin Disease↗