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Biomedical subjects

G Buchan

Publications and source records attributed to G Buchan.

13 recordsLinked to original sources
PubMed1995

The cloning and sequencing of cervine interleukin 10.

We report the cloning and sequencing of the cervine interleukin-10 gene. Specific cDNA was amplified by PCR using primers based on the bovine sequence. This was cloned into pGEM 5Zf and several clones were sequenced. The 762 nucleotide product coded for a 179 amino acid protein which was 86% homologous with its bovine and 77% homologous with its human counterparts. There is a strongly hydrophobic signal sequence consisting of the first 20 amino acids and a potential glycosylation site at amino acids 134-136. There are three regions, comprising 34% of the protein, which show complete homology between the cervine, bovine and human sequences. The transcription of the gene was shown by Northern Blotting where a single, 1.8kb, mRNA transcript was detected 4-8 hours after activation of peripheral blood mononuclear cells with mitogen.

Amino Acid Sequence↗
PubMed1991

Cytokine assays: role in evaluation of the pathogenesis of autoimmunity.

Cytokines are protein mediators involved in inflammation, the immune response, cell growth, repair and fibrosis. All of these processes are ongoing in active autoimmune diseases such as rheumatoid arthritis (RA), and so it would be expected that many cytokines would be actively produced in RA joints or Graves' disease (GD) thyroid glands. The cDNA cloning of cytokines has permitted the generation of pure recombinant molecules, and of newer more sensitive assays, and spurred the rapid development of knowledge in this field. Here we review the molecular strategies devised to study the possible role of cytokines in the pathogenesis of RA and GD, and describe some of the initial results. After 'cataloguing' the relative abundance of various cytokines, we sought to discover which cytokines are of major importance in pathogenesis. For that purpose we used neutralizing anti-cytokine antibodies and found that TNF alpha is one of the major signals regulating the production of IL-1 in the RA but not in the osteoarthritic (OA) joint. In order to further understand the dynamics of the cytokine network, the localization of the cytokine-producing cells by immunostaining and in situ hybridization has also been performed. The latter techniques are particularly valuable for attempting to establish the role of the target cell, such as thyroid epithelium, in the pathogenesis of disease. Cytokines act on cells via binding to high-affinity receptors. The last two years has been the cDNA cloning of many molecules encoding cytokine receptor chains, and it is now possible to begin to evaluate the other half of the cytokine pathway. Taken together, there are now exciting opportunities for the molecular dissection of the cytokine events occurring in auto-immune tissues.

Animals↗
PubMed1990

Cytokine production in the rheumatoid joint: implications for treatment.

Cytokines are protein mediators that play a part in inflammation, the immune response, cell growth, repair, and fibrosis. All of these are continuing processes in active rheumatoid arthritis (RA), and so it would be expected that many cytokines would be actively produced in RA joints. Here, the molecular strategies devised to study the possible role of cytokines in the pathogenesis of RA, are reviewed and some of the initial results described. The relative abundance of various cytokines is 'catalogued' and then attention is turned to an attempt to discover which cytokines are of major importance in the pathogenesis. Neutralising antibodies to cytokines were used for that purpose, and it was found that tumour necrosis factor alpha (TNF alpha) is one of the major signals regulating the production of interleukin-1 in the RA, but not in the osteoarthritic joint. To understand further the dynamics of the cytokine network localisation of the cytokine producing cells by immunostaining--for example, TNF alpha, is currently being established.

Arthritis, Rheumatoid↗
PubMed1989

Regulation of expression of human IL-1 alpha and IL-1 beta genes.

IL-1 gene expression was investigated in human blood mononuclear cells. IL-1 alpha and IL-1 beta mRNA were induced with LPS or TNF. Kinetic measurements on Northern blots revealed that these stimuli elicited qualitatively similar changes in IL-1 mRNA levels, and that expression of IL-1 mRNA was transient. IL-1 beta mRNA was the predominant mRNA species and remained elevated for somewhat longer than IL-1 alpha mRNA. TNF and IFN-gamma synergized to induce both species of IL-1 mRNA and IL-1 bioactivity. Transcriptional control, as measured by nuclear run on assays, partly determines the greater levels of IL-1 beta mRNA because the rate of IL-1 beta transcription was greater than that of IL-1 alpha. Cycloheximide (CHX) was able to induce IL-1 mRNA but did not induce transcription of either IL-1 gene. When added to cultures pretreated with TNF or LPS, CHX superinduced IL-1 mRNA, but IL-1 transcription was not increased. If added simultaneously CHX blocked TNF-induced IL-1 gene transcription, suggesting that TNF may induce factors required for IL-1 gene transcription. CHX increased the stability of both IL-1 alpha and IL-1 beta mRNA, demonstrating the existence of a post-transcriptional form of control. In half-life experiments IL-1 beta mRNA was more stable than IL-1 alpha mRNA, indicating that post-transcriptional control also contributes to the greater steady state levels of IL-1 beta. Taken together, the available evidence suggests IL-1 alpha and IL-1 beta mRNA are regulated differentially at both the transcriptional and post-transcriptional level.

Cycloheximide↗
PubMed1989

Intrathyroidal cytokine production in thyroid disease.

The intrathyroidal production of Interferon gamma, tumour necrosis factor alpha and beta, Interleukin-1 alpha and beta, Interleukin-6, platelet-derived growth factor A and of transforming growth factor-beta was analysed in patients with autoimmune and non-autoimmune thyroid disease. Cytokines were assessed indirectly by slot blot mRNA analysis in fresh tissue samples (unpurified cells, infiltrating mononuclear cells and thyroid follicular cells), in thyroid follicular cells in primary culture, as well as in thyroid-derived T-cell clones. The production of Interleukin-1 alpha and beta, Interleukin-6 and transforming growth factor beta was additionally measured by bioassay. Cytokine production by thyroid-infiltrating mononuclear cells generally did not differ between autoimmune and non-autoimmune samples, the whole panel of all cytokines being found in freshly purified cells as well as in thyroid-derived T-cell clones from patient with Graves' disease, as well as with multinodular non-toxic goitre. Thyroid follicular cells produced Interleukin-1 alpha, Interleukin-6 and transforming growth factor beta. Interleukin-1 and Interleukin-6 production did not differ between thyroid follicular cells from autoimmune and non-autoimmune thyroids. Transforming growth factor beta was, however, lower in non-toxic goitre than in Graves' disease and in normal thyroid tissue, but could be increased by exposure of the cells to micromolar concentrations of iodide. This seemed of special interest, as transforming growth factor beta proved to inhibit thyroid follicular cell growth in response to known growth stimuli, such as insulin-like growth factor I or epidermal growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune Diseases↗
PubMed1989

Transforming growth factor beta regulates thyroid growth. Role in the pathogenesis of nontoxic goiter.

The production and growth regulatory activity of transforming growth factor beta were studied in human thyroid tissue. As estimated by its mRNA expression in fresh tissue samples, transforming growth factor beta was produced in normal and in diseased thyroid glands. Transforming growth factor beta mRNA was mainly produced by thyroid follicular cells and in lesser quantities by thyroid infiltrating mononuclear cells. The concentrations of transforming growth factor beta mRNA were lower in iodine-deficient nontoxic goiter than in Graves' disease and normal thyroid tissue. Transforming growth factor beta protein secretion by cultured thyroid follicular cells was also low in nontoxic goiter, but could be increased by addition of sodium iodide (10 microM) to the culture medium. Recombinant transforming growth factor beta did not affect basal tritiated thymidine incorporation in cultured thyroid follicular cells, but inhibited, at a concentration of 10 ng/ml, the growth stimulatory influence of insulin-like growth factor I, epidermal growth factor, transforming growth factor alpha, TSH, and partly that of normal human serum on cultured thyroid follicular cells. This inhibition was greater in Graves' disease than in nontoxic goiter. These results suggest that transforming growth factor beta may act as an autocrine growth inhibitor on thyroid follicular cells. Decreased transforming growth factor beta production and decreased responsiveness to transforming growth factor beta may be cofactors in the pathogenesis of iodine-deficient nontoxic goiter.

Adult↗
PubMed1989

Analysis of intrathyroidal cytokine production in thyroid autoimmune disease: thyroid follicular cells produce interleukin-1 alpha and interleukin-6.

Cytokine production was studied in thyroid tissue from patients with Graves' disease, Hashimoto's thyroiditis and non-toxic goitre. The expression of interferon gamma, tumour necrosis factor alpha and beta, interleukin-1 alpha and beta, interleukin-6 and platelet-derived growth factor A chain was assessed by slot-blot analysis of the respective mRNA in freshly isolated tissue samples. All seven cytokines were detected in patients of all groups. Although the respective mRNA levels were, in general, higher in thyroid autoimmune disorders, this appeared to relate to the degree of the lymphocytic infiltration of the thyroid gland at the time of surgery. Purified thyroid follicular cells expressed high levels of interleukin-1 alpha and interleukin-6 mRNA and when established in primary culture, purified thyroid follicular cells from Graves' disease as well as non-toxic goitre produced interleukin-1 alpha and interleukin-6 bioactivity spontaneously. In the case of interleukin-1 this could be further augmented by addition of lipopolysaccharide to the thyroid follicular cell cultures. These results demonstrate that the lymphocytic infiltrate found in autoimmune and non-autoimmune thyroid disorders is associated with cytokine production. Additionally we have shown that intrathyroidal cytokine production is not restricted to thyroid-infiltrating mononuclear cells, but may also involve thyroid follicular cells both in vivo and in vitro. The cytokines produced by thyroid follicular cells may have an important role in stimulating autoantigen specific T cells in vivo as both interleukin-1 and interleukin-6 facilitate T cell activation.

Adult↗
PubMed1988

Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis.

High levels of interleukin 6 (IL 6/B cell stimulatory factor-2) were detected in synovial fluids from the joints of patients with active rheumatoid arthritis (RA). The cells found in freshly isolated synovial fluid constitutively expressed IL 6 mRNA. The synovial tissues obtained by joint biopsy were also found to produce IL 6 in vitro. Immunohistochemical analysis demonstrated that CD2+ T cells as well as CD20+ blastoid B cells in the synovial tissues produce IL 6. The data indicate that IL 6 is generated constitutively in RA and its overproduction may explain the local as well as the generalized symptoms of RA, since IL 6 can function as B cell growth and differentiation factor as well as hepatocyte-stimulating factor.

Antigens, CD20↗
PubMed1988

Role of HLA class II and cytokine expression in rheumatoid arthritis.

HLA class II expression is notable in rheumatoid arthritis. We have investigated the mechanism of HLA class II regulation in the joints and found local synthesis, as judged by mRNA levels to be high. The role of antigen presentation in maintaining class II mRNA was explored, and blocking presentation by using monoclonal antibodies to HLA class II inhibited synthesis of mRNA for HLA-DR alpha chain. HLA class II expression is maintained by cytokines and so cytokine production in rheumatoid joints was investigated. It was chosen to use mRNA analysis by slot blotting as a screening assay, and the expression of many cytokines was detected. Levels of these were maintained in culture in the absence of extrinsic stimulation.

Antibody Formation↗
PubMed1988

Detection of activated T cell products in the rheumatoid joint using cDNA probes to Interleukin-2 (IL-2) IL-2 receptor and IFN-gamma.

Attempts to detect immune mediators in RA synovial fluids by bioassay or radioimmunoassay have yielded conflicting results, and so we have begun to analyse the complex immunological reactions occurring within the rheumatoid joint using recombinant DNA technology. High levels of Interleukin-2 (IL-2) and IL-2 receptor transcripts were found in the mononuclear cells of the rheumatoid lesions. Interferon gamma (IFN gamma) mRNA was also detected, although at lower level than IL-2. To investigate the possible relevance of IL-2 and IL-2 receptor mRNA expression to the chronicity of the disease, RA joint cells were cultured in the absence of any stimulus, and the duration of mRNA expression compared to that of blood mononuclear cells (PBM), optimally stimulated. IL-2 mRNA was found to persist in culture for many days, in contrast to its transient (less than 24 h) presence in stimulated PBM. IL-2 receptor expression was also prolonged. In contrast IFN gamma mRNA, present at biopsy in 10/12 RA samples, was found to increase significantly in vitro. These results suggest that persistent T cell activation is of importance in the pathogenesis of RA, and suggests that prolonged mediator production (IL-2 and IFN gamma) may be of importance. The elevation of IFN gamma mRNA in culture and its lower relative expression suggests that there are inhibitory immunoregulatory influences within the RA joint. To determine whether abnormal IL-2 mRNA expression may be due to a genetic defect in the region controlling IL-2 gene expression, Southern blotting analysis of genomic DNA was performed with a 5' flanking probe using normal, RA and systemic lupus erythematosis patients. No abnormalities were detected.

Arthritis, Rheumatoid↗
PubMed1988

Interleukin-1 and tumour necrosis factor mRNA expression in rheumatoid arthritis: prolonged production of IL-1 alpha.

In rheumatoid arthritis there is a chronic immune and inflammatory reaction which can lead to the destruction of the diseased joint. Cytokine gene expression was studied in synovial cells using cDNA probes specific for human interleukin 1 (IL-1), -alpha and IL-1 beta, tumour necrosis factor (TNF), -alpha and TNF beta (lymphotoxin); protein molecules which induce cartilage degradation and bone resorption. In all cases studied, IL-1 mRNA was present in freshly isolated synovial cells from fluid or membrane. Compared to levels of IL-1 mRNA found in optimally activated normal blood mononuclear cells, the levels of IL-1 alpha mRNA were high in seven of the nine patients studied, whereas IL-1 beta mRNA, the dominant form in blood, was relatively lower. TNF alpha and TNF beta mRNA were also detected. Rheumatoid synovial cells, cultured without any stimulus, continued to express high levels of IL-1 alpha mRNA for up to 5 days, compared to the 24 h response of activated blood cells; IL-1 beta mRNA in culture was also prolonged. Cultures of rheumatoid joint cells produced IL-1 bioactivity, with roughly equal amounts of IL-1 alpha and beta, as assessed using neutralizing antibodies. TNF bioactivity was also detected which may be of importance as TNF induces the production of IL-1. The finding of these mediators produced in large amounts in active rheumatoid synovial cells suggests that mutually stimulatory cell interactions, mediated by these molecules, may be important in the chronic inflammation and tissue destruction in rheumatoid arthritis.

Arthritis, Rheumatoid↗