Biomedical subjects

P J Townsend

Publications and source records attributed to P J Townsend.

At least 19 recordsLinked to original sources

PubMed1999

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T.

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.

Blotting, Northern↗

PubMed1998

Genomic organisation, alternative splicing and polymorphisms of the human cardiac troponin T gene.

Troponin T (TnT) is a component of the troponin complex which regulates muscle contraction in response to alterations in intracellular calcium ion concentration. In human heart, multiple isoforms of cardiac TnT have been described on the basis of antibody studies and molecular cloning of corresponding cDNAs. These isoforms are all derived from the transcription of a single gene, TNNT2, located on chromosome 1q32, and generated by alternative splicing. We show here that isoform diversity is achieved by the use of both alternative exons and alternative acceptor sites and present the organisation of the human TNNT2 gene, which is composed of 17 exons spread over 17 kb. A potential structure of the promoter region is also presented. Several polymorphisms in both the exonic and intronic regions were identified, some of which may act as modulators of the expression of this gene.

Alternative Splicing↗

PubMed1997

Assignment of the human cardiac/slow skeletal muscle troponin C gene (TNNC1) between D3S3118 and GCT4B10 on the short arm of chromosome 3 by somatic cell hybrid analysis.

We have determined the chromosomal location of the human cardiac/slow skeletal muscle troponin C gene (TNNC1) to the short arm of chromosome 3 using somatic cell hybrids. PCR-based analysis of a 'monochromosomal' hybrid panel identified the presence of the TNNC1 gene on human chromosome 3 and subsequent analysis of the Genebridge4 radiation hybrid panel located the gene between D3S3118 (7.3cR) and GCT4B10 (4.2cR) with a lod score of > 3.0.

Chromosome Mapping↗

PubMed1997

Assignment of the human fast skeletal muscle troponin C gene (TNNC2) between D20S721 and GCT10F11 on chromosome 20 by somatic cell hybrid analysis.

The chromosomal location of the human fast skeletal muscle troponin C (TNNC2) gene was determined using somatic cell hybrids. PCR-based analysis of a 'monochromosomal' hybrid panel identified the presence of the TNNC2 gene on human chromosome 20 and subsequent analysis of the Genebridge4 radiation hybrid panel located the gene between D20S721 and GCT10F11 with a lod score of > 3.

Chromosome Mapping↗

PubMed1997

Localization of the fast skeletal muscle troponin I gene (TNNI2) to 11p15.5: genes for troponin I and T are organized in pairs.

We have localized the gene encoding the fast skeletal muscle isoform of troponin I (TNNI2) to 11p15.5 by PCR-based analysis of somatic cell hybrid panels: based on the Genebridge4 radiation hybrid panel, TNNI2 is coincident with the marker D11S922. The gene encoding the fast skeletal muscle troponin T gene (TNNT3) has been previously assigned to 11p15.5 suggesting that TNNI2 and TNNT3 may be closely linked. The overall location of genes encoding troponin I and T isoforms now reveals that they are organized at three loci each containing a troponin I/troponin T gene pair. This organization contrasts with all other sarcomeric protein genes and has implications for the evolution of these two gene families, for their regulation and for the analysis of mutations suspected to result in cardiomyopathy.

Biomarkers↗

PubMed1996

Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy is a phenotypically and genetically heterogeneous disease. In some families, the disease is linked to the CMH2 locus on chromosome 1q3, in which the cardiac troponin T gene (TNNT2) has been identified as the disease gene. The mutations found in this gene appear to be associated with incomplete penetrance and poor prognosis. Because mutational hot spots offer unique possibilities for analysis of genotype-phenotype correlations, new missense mutations that could define such hot spots in TNNT2 were looked for in unrelated French families with familial hypertrophic cardiomyopathy. METHODS AND RESULTS: Family members were genotyped with microsatellite markers to detect linkage to the four known disease loci. In family 715, analyses showed linkage to CMH2 only. To accurately position potential mutations on TNNT2, its partial genomic organization was established. Screening for mutations was performed by single-strand conformation polymorphism analysis and sequencing. A new missense mutation, Arg102Leu, was identified in affected members of family 715 because of a G-->T transversion located in the 10th exon of the gene. Penetrance of this new mutation is complete; echocardiographic data show a wide range of hypertrophy; and there was no sudden cardiac death in this family. CONCLUSIONS: The codon 102 of the TNNT2 gene is a putative mutational hot spot in familial hypertrophic cardiomyopathy and is associated with phenotypic variability. Analysis of more pedigrees carrying mutations in this codon is necessary to better characterize the clinical and prognostic implications of TNNT2 mutations.

Adult↗

PubMed1995

Molecular cloning of human cardiac troponin T isoforms: expression in developing and failing heart.

Troponin T, which links the troponin complex to tropomyosin, is found as multiple isoforms in the hearts of many animal species. Changes in isoform composition have been correlated with variation in myofilament sensitivity to calcium. In order to determine the origin of diversity of the cardiac troponin T (cTnT) isoforms indicated by existing protein data, we have determined the sequences and patterns of expression of mRNAs encoding troponin T in fetal and adult heart and those present in adult heart in end-stage failure. Three main regions of alternative splicing within the cTnT coding region were identified using reverse-transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNAs are developmentally regulated and some of the fetal forms are expressed in adult failing heart. The molecular structure of the spliced regions was determined from cloned cDNAs and RT-PCR products. In the 5' region of the mRNA, isoforms are generated by the inclusion or exclusion of 15-, 3- and 27-nucleotide (nt) sequences and by the inclusion or exclusion of a separate 3-nt sequence. In the 3' region of the mRNA, alternative splicing involves a 9-nt sequence which can be present in full, in part or not at all. A further splicing site was identified in the central region involving a 234-nt sequence and resulting in rare but detectable mRNAs. This work demonstrates the complexity of cTnT RNA composition in human heart and provides the information necessary to address the function of cTnT isoforms in contraction.

Adult↗

PubMed1994

Human cardiac troponin T: identification of fetal isoforms and assignment of the TNNT2 locus to chromosome 1q.

The troponin complex is located on the thin filament of striated muscle and is composed of three component polypeptides: troponin T, troponin I, and troponin C. Three troponin T genes have been described on the basis of molecular cloning in humans and other vertebrates. These are expressed in a tissue-specific manner and encode the troponin T isoforms expressed in cardiac muscle, slow skeletal muscle, and fast skeletal muscle, respectively. Each of these genes is subject to alternative splicing, resulting in the production of multiple tissue-specific isoforms. We have cloned cDNAs encoding human cardiac troponin T from adult heart and have used these to demonstrate that multiple cardiac troponin T mRNAs are present in the human fetal heart, resulting from alternative splicing in the 5' coding region of the gene. Hybridization of the cloned cDNAs to genomic DNA identifies a single-copy gene, and using somatic cell hybrid analysis, we have mapped the corresponding gene locus (designated TNNT2) to the long arm of chromosome 1 (1cen-qter).

Amino Acid Sequence↗

PubMed1992

Enhancement of the antigen-presenting function of monocytes by cholesterol: possible relevance to inflammatory mechanisms in extrinsic allergic alveolitis and atherosclerosis.

Extrinsic allergic alveolitis (EAA) (synonym: hypersensitivity pneumonitis) is a hypersensitivity lung disease characterized by lymphocytic infiltrates in the pulmonary interstitial tissues. We have previously reported that the numbers of lymphocytes in bronchoalveolar lavage (BAL) samples in this disease correlate with levels of cholesterol and neutral lipid-laden 'foamy' macrophages. We have also reported that the macrophages express an increased density of MHC class II antigens (in particular HLA-DQ) which are known to be essential for antigen recognition by T lymphocytes. The aim of the present study was to explore whether cholesterol is capable of enhancing the antigen-presenting function of mononuclear phagocytes by modulating the expression of HLA-D region products. Incubation of purified monocytes from healthy volunteers with cholesterol in serum-free medium induced a significant increase in both the percentages of monocytes expressing HLA-DQ (P less than 0.02) and in the intensity of expression of the three HLA-D sub-region products, HLA-DQ, -DP and -DR (P less than 0.02, less than 0.01, less than 0.05, respectively). The cholesterol pre-incubated monocytes also exhibited enhanced antigen-presenting function (P less than 0.05), compared with controls pre-incubated without cholesterol. These findings indicate that increases in cholesterol in the extracellular milieu may augment antigen presentation by modulating the expression of HLA-D region products on antigen-presenting cells. Apart from EAA, this observation may also have relevance to inflammatory mechanisms in atherosclerosis, where 'foamy' macrophages also occur in association with hypercholesterolaemia.

Alveolitis, Extrinsic Allergic↗

PubMed1992

Induction of lymphocyte proliferation by antigen-pulsed human neutrophils.

We have investigated whether purified antigen-pulsed human neutrophils can induce a proliferative response in purified resting blood lymphocytes. Neutrophils were pulsed with soluble tetanus toxoid (dose range 25-250 Lf/ml) and co-cultured with autologous lymphocytes that had been depleted of MHC class II expressing cells. The antigen-pulsed neutrophils induced an increase of lymphocyte proliferation which was dependent on the antigen dose and the neutrophil/lymphocyte ratios. Neutrophils were less potent than autologous monocytes in stimulating lymphocyte proliferation. Blocking with a monoclonal antibody to a common determinant of the human MHC class II complex failed to reduce the lymphoproliferative effects and allogenic antigen-pulsed neutrophils were also able to elicit lymphocyte proliferation similar to autologous neutrophils. We conclude that antigen-pulsed neutrophils are able to induce lymphocyte proliferation in a non-MHC-restricted fashion.

Antigen-Presenting Cells↗

PubMed1992

Gene expression during cardiac development.

The vertebrate heart forms as two concentric epithelial cylinders of myocardium and endocardium separated by an extended basement membrane matrix commonly referred to as cardiac jelly. Subsequent maturation involves a complex series of events including asymmetric changes in cell shape and division which contribute to bending and the formation of the bulboventricular loop, the formation of specialised tissues including endocardial cushion tissue of the atrioventricular (AV) and outflow tract regions, the development of conductive tissue and myocyte maturation leading to the overall pattern of expression characteristic of mature heart muscle. These processes depend on a precise spatial and temporal control of gene expression both of genes encoding regulatory molecules and those encoding structural components of the heart. In this chapter we address three aspects of cardiac development, namely, the determination of cell fate during formation of endocardial cushion tissue in the embryonic heart, transitions in troponin gene expression during fetal myocyte maturation, and the use of cloning techniques based on the polymerase chain reaction for identifying transcription factors present in the heart.

Animals↗

PubMed1990

Increases in HLA-DQ, DP, DR, and transferrin receptors on alveolar macrophages in sarcoidosis and allergic alveolitis compared with fibrosing alveolitis.

We have used flow cytometric methods to detect and quantify HLA-DR, DQ, and DP antigens and transferrin receptors on alveolar macrophages in lavage samples from 36 patients with granulomatous lung diseases (extrinsic allergic alveolitis [EAA], n = 13; sarcoidosis, n = 23), and 12 patients having fibrosing alveolitis (FA) (cryptogenic fibrosing alveolitis, n = 3; FA and scleroderma, n = 8; FA and primary biliary cirrhosis, n = 1). HLA-DR, DQ, and DP antigens were expressed on the majority of alveolar macrophages in all the patients, and the percentages of positive cells were similar to those in control subjects without lung disease. However, the amounts expressed were higher in those with EAA and sarcoidosis than in the FA group or control subjects, the most significant differences being in HLA-DQ and HLA-DP expression. Transferrin receptor expression was also higher in the granulomatous lung diseases. In sarcoidosis, higher levels of HLA-DQ correlated with lower lung function measurements (Dco p less than 0.025, FVC p less than 0.025, FEV1 p less than 0.005), suggesting this may be a marker of disease activity. HLA-DP levels also showed a trend (p less than 0.1) of inverse correlation with lung function. Levels of HLA-DQ (p less than 0.005) and HLA-DP (p less than 0.001) correlated more closely than HLA-DR with numbers of lymphocytes in the lavage fluids, and HLA-DQ levels correlated with increasing proportions of lymphocytes in proliferation (p less than 0.05). We suggest that high levels of HLA-DQ and DP on alveolar macrophages may be more relevant than HLA-DR to the enhanced antigen-presenting function of these cells in sarcoidosis, and possibly also in EAA.

Adult↗

PubMed1990

Lavage versus serum measurements of lysozyme, angiotensin converting enzyme and other inflammatory markers in pulmonary sarcoidosis.

The aim of this study was to explore whether amounts of angiotensin converting enzyme (ACE) and lysozyme produced within the lungs correlate more closely than serum levels of these enzymes, or other inflammatory markers, with chest radiographic profusion scores, lung function and therapy response in patients with pulmonary sarcoidosis. We have studied 25 patients, and levels in bronchoalveolar lavage (BAL) were used to determine "local" enzyme production by reference to serum and lavage albumin. Before treatment, serum lysozyme levels were elevated in more patients (80%) than serum ACE levels (40%). They also gave the best overall correlation with clinical measurements prior to treatment and falls in serum lysozyme closely parallelled improvement in lung function (transfer factor for carbon monoxide (DLCO)) on therapy. The only other markers showing significant correlations with disease severity were lavage neutrophil counts per ml and "local" ACE measurements prior to treatment. The value of pre-treatment levels of the different inflammatory markers in predicting response to corticosteroid therapy was explored and the only significant finding was that BAL lymphocyte percentages and numbers.ml-1 were initially higher in patients with lower post-treatment chest X-ray scores (p less than 0.01 and p less than 0.05, respectively). We conclude that serum lysozyme levels appear to be a more useful marker of overall disease activity in sarcoidosis than measurements of other inflammatory markers. However, BAL lymphocyte counts were the best predictive marker of radiographic response to corticosteroids.

Adult↗

PubMed1986

Bronchoalveolar lavage in patients with interstitial lung diseases: side effects and factors affecting fluid recovery.

One hundred and seventy patients with interstitial lung diseases undergoing bronchoalveolar lavage (BAL), were contrasted with 51 patients undergoing fibreoptic bronchoscopy alone to define the factors which predispose to post-lavage side-effects. Transient post-bronchoscopy fall in the peak expired flow (PEF) greater than or equal to 20% occurred in both groups (24% and 23% respectively), and thus was probably related to the bronchoscopy procedure. Post-lavage pyrexia (greater than or equal to 1 degree C) occurred only in the patients undergoing BAL (26%), p less than 0.001. Only 4% with pyrexia required antibiotics, and only 2% with falls in PEF needed bronchodilator therapy. The only significant clinical association was more frequent pyrexia in patients on treatment with prednisolone, particularly in women (p less than 0.01). Pyrexia was also associated with higher lavage fluid introduction volumes (greater than 240 ml). Side effects did not relate to the percentages of lavage fluid recovered, although smokers had lower recoveries and, recoveries tended to be higher in sarcoidosis than cryptogenic fibrosing alveolitis. Serial lavages in 25 patients caused no significant increase in side effects.

Adult↗

PubMed1985

Numerical and functional alterations in circulatory lymphocytes in cigarette smokers.

Simultaneous numerical and functional studies of circulatory lymphocytes were undertaken in healthy non-smoking and cigarette-smoking volunteers. The smokers all had light to moderate histories of less than 50 pack years. By contrast with non-smokers (n = 32), the smokers (n = 14) had a significant increase in the total number of lymphocytes, surface immunoglobulin bearing (sIg+) cells, total T-cells (T3+) and T helper-inducer cells (T4+), and a trend of increase in T suppressor-cytotoxic cells (8+). These changes differ from those in heavy smokers who have been reported to show significantly increased T suppressor-cytotoxic but significantly decreased T helper-inducer cells. Although the proportions of T-cell subsets did not differ significantly in the light to moderate smokers compared with non-smokers, in vitro T-suppressor function against the Ig-secreting response of allogeneic B-cells to pokeweed mitogen (PWM) stimulation was significantly impaired. The proliferative response of T-cells to phytohaemagglutinin (PHA) was, however, similar in both groups. This suggests that smoking may exert a selective influence upon a subset of T suppressor cells. In cytotoxicity assays, smokers showed a significant decrease in natural killer cell (NK) activity but not in antibody dependent cellular cytotoxicity (ADCC). It appears that these alterations are reversible since a group of ex-smokers (n = 10) were indistinguishable from our non-smoking group in all studies. The implications regarding the link between smoking and increased susceptibility to infection and malignancy are discussed; and these findings should be borne in mind in basic studies of lymphocytes.

Adult↗

PubMed1981

Bronchoalveolar macrophages in sarcoidosis and cryptogenic fibrosing alveolitis.

Previous reports suggest that blood monocytes and tissue epithelioid cells in patients with sarcoidosis are "activated', but few studies have been undertaken on human alveolar macrophages. In the present study, bronchoalveolar lavage samples have been obtained from eighteen patients with sarcoidosis and these have been compared with twenty controls and twenty-nine patients with non-granulomatous chronic inflammatory interstitial lung disease (cryptogenic fibrosing alveolitis). Assessment of the functional state of the macrophages was made by measurements of C3b receptor sites on the cell membrane, intra and extracellular lysosomal enzyme (beta-D-glucosamidase) and the degree of spreading of macrophages on glass. C3b receptor sites and intracellular levels of lysosomal enzyme were significantly reduced in sarcoidosis compared to controls; levels of extracellular enzyme in the lavage supernatant fluid and macrophage spreading were similar to controls. These features suggest that alveolar macrophages from patients with sarcoidosis are not "activated'. By contrast, in cryptogenic fibrosing alveolitis, macrophages show a greater extracellular:intracellular ratio of lysosomal enzyme and more spreading, suggestive of "activation'.

Adult↗

PubMed1980

Complement activation during cardiopulmonary bypass.

Conversion of C3, the third component of complement, has been studied using a two-dimensional immunoelectrophoretic method. Evidence of C3 conversion has been obtained in stored donor blood, in patients during and after cardiac surgery involving cardiopulmonary bypass, and occasionally during thoractomy for pulmonary resection. It is suggested that complement activation is one factor contributing to tissue injury after cardiopulmonary bypass.

Adult↗

PubMed1980

Alveolar macrophage lysosomal enzyme and C3b receptors in cryptogenic fibrosing alveolitis.

Levels of the lysosomal enzyme N-acetyl-beta-glucosaminidase were measured in alveolar macrophage extracts and lung lavage fluid obtained from patients with cryptogenic fibrosing alveolitis and a control group. Evidence is presented which suggests that macrophages from patients with cryptogenic fibrosing alveolitis secrete this enzyme in vivo. To investigate one of the possible mechanisms of stimulating this secretion, an assessment was made of the C3b receptor sites on alveolar macrophages. While there were fewer receptor sites on the macrophages from patients with cryptogenic fibrosing alveolitis, which might indicate that they were occupied in vivo, this difference did not reach statistical significance. It is suggested that the alveolar macrophage is not merely a phagocyte but that it also has a more active role in the pathogenesis of cryptogenic fibrosing alveolitis.

Acetylglucosaminidase↗