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Biomedical subjects

M J Arthur

Publications and source records attributed to M J Arthur.

At least 37 records · Page 2Linked to original sources
PubMed1998

Immunogenetic analysis of a panel of monoclonal IgG and IgM anti-PDC-E2/X antibodies derived from patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Autoantibodies with specificity for the E2 component of the pyruvate dehydrogenase complex (PDC-E2) are commonly present in primary biliary cirrhosis. The aim of this study was to generate and characterise human anti-PDC-E2 monoclonal antibodies and analyse immunoglobulin gene usage and mutation for clues to pathogenesis. METHODS: Peripheral B-lymphocytes from two patients with primary biliary cirrhosis were used to generate heterohybridomas secreting PDC-E2 specific monoclonal antibodies. The antibodies were characterised by ELISA, immunoblotting, indirect immunofluorescence and enzyme inhibition techniques, and their encoding immunoglobulin genes were amplified, cloned and sequenced. RESULTS: Four IgGlambda and one IgMlambda monoclonal antibodies specific for PDC-E2 were generated: all gave bands at 74 kD and 52 kD on PDC immunoblots, two clones were specific for the lipoylated inner lipoyl domain, and all inhibited target enzyme function. Sequence analysis suggested unrestricted VH gene usage, but a strong preference for lambda light chains. The extent of somatic mutation was high (3-20%), with evidence for antigen selection in 3/5 VH sequences. CONCLUSIONS: These monoclonal antibodies closely resemble the hallmark autoantibodies of primary biliary cirrhosis. Their specificities demonstrate true cross reactivity between an epitope on PDC-E2 and Protein X, and the existence of a subset of B cells that recognise only the lipoylated form of the antigen. The pattern of immunoglobulin gene mutations suggests an antigen-driven selection of high affinity IgG autoantibodies, supporting a possible role for exogenous antigen in the pathogenesis of primary biliary cirrhosis.

Amino Acid Sequence↗
PubMed1998

Macromolecular aspartate aminotransferase.

Macroenzymes are serum enzymes that have a greater molecular mass than the corresponding enzyme normally found in serum (Klonoff. West J Med 1980; 133: 392-407). Serum AST (aspartate aminotransferase) has rarely been reported to complex with immunoglobulins, resulting in an elevation in serum AST activity. Physicians should be aware of this condition so that patients are not subjected to invasive and costly procedures unnecessarily (Litin et al. Mayo Clin Proc 1987; 62: 681-687). We report on such a patient with a raised AST due to macroenzyme formation.

Adult↗
PubMed1998

Rat hepatic stellate cell expression of alpha2-macroglobulin is a feature of cellular activation: implications for matrix remodelling in hepatic fibrosis.

1. Hepatic stellate cells are key mediators of hepatic fibrosis. We have studied hepatic stellate cell expression of the collagenase and general protease inhibitor alpha2-macroglobulin after activation in tissue culture and in response to certain cytokines. 2. Hepatic stellate cells isolated by Pronase-collagenase digestion were activated by culture on uncoated plastic. By Northern analysis hepatic stellate cells undergoing activation (5 days) expressed alpha2-macroglobulin mRNA and alpha2-macroglobulin could be immunolocalized to hepatic stellate cells from 5 to 15 days of culture. 3. By ELISA of cell culture supernatants hepatic stellate cell secretion of alpha2-macroglobulin was found to increase from 2. 78+/-1.13 ng x ml-1 x microgram-1 DNA per 24 h at 5 days of culture (n=8) to 13.55+/-4.64 ng x ml-1 x microgram-1 DNA per 24 h at 15 days of culture (n=7). Stimulation of hepatic stellate cells with interleukin-6 at 5 days caused a significant increase in alpha2-macroglobulin expression as did exposure to Kupffer-cell conditioned medium. However, exposure of hepatic stellate cells to interleukin-1, transforming growth factor-beta1 and tumour necrosis factor-alpha had no significant effect. 4. During profibrotic liver injury plasma alpha2-macroglobulin levels were found to increase to between 850% and 250% of the control value (100%) after bile duct ligation (72 h to 13 days respectively), and to 1166% and 1106% of the control value during progressive CCl4-induced fibrosis (24 h to 4 weeks respectively). 5. These data suggest that hepatic stellate cells are a potential source of the potent protease inhibitor alpha2-macroglobulin, expression of which may inhibit matrix remodelling during progressive fibrosis.

Animals↗
PubMed1998

Tissue inhibitors of metalloproteinases, hepatic stellate cells and liver fibrosis.

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. Following liver injury, these cells proliferate and are activated to a profibrogenic myofibroblastic phenotype. In addition to increased matrix protein synthesis, there is evidence to indicate that these cells are able to regulate matrix degradation. In the early phases of their cellular activation, HSC release matrix metalloproteinases with the ability to degrade the normal liver matrix. When HSC are fully activated, there is a net down-regulation of matrix degradation mediated by increased synthesis and extracellular release of tissue inhibitors of metalloproteinase (TIMP)-1 and -2. These studies in cell culture have been complemented by in vivo studies of hepatic TIMP-1 and TIMP-2 gene expression. In advanced human liver disease of various aetiologies, there is increased TIMP-1-mRNA and protein and increased TIMP-2-mRNA in fibrotic liver compared with control liver. Temporal studies of progressive rat liver fibrosis caused by bile duct ligation or by carbon tetrachloride, indicate an important role for increased TIMP-1 and TIMP-2 expression in pathogenesis. Moreover, in a rat model of reversible liver fibrosis, matrix remodelling and resolution of liver fibrosis is closely associated, temporally, with a marked decrease in TIMP-1 and TIMP-2 expression. These combined cell culture and in vivo findings have led us to investigate the mechanisms of regulation of TIMP-1 gene expression in hepatic stellate cells. Our recent data indicate that transcriptional regulation of TIMP-1 gene expression in HSC is mediated via a mechanism which differs considerably from that previously identified in skin fibroblasts. We conclude that increased TIMP-1 and TIMP-2 expression by HSC plays an important role in the pathogenesis of liver fibrosis. This may represent an important therapeutic target in the design of anti-fibrotic strategies for chronic liver disease.

Animals↗
PubMed1997

Pathogenesis of liver fibrosis.

1. Liver fibrosis is a common sequel to diverse liver injuries. It is characterized by an accumulation of interstitial collagens and other matrix components. The hepatic stellate cell is pivotal to the pathogenic process. Fibrotic liver injury results in activation of the hepatic stellate cell which undergoes a phenotypic change to a proliferative myofibroblast-like cell which synthesizes excess interstitial collagens and other matrix components. 2. The process of initiation of activation and its perpetuation result from complex, often interrelated series of signalling mechanisms which converge on this effector cell. Such mechanisms include alterations in matrix resulting in changed cell-matrix interactions and stimulation by cytokines released from damaged hepatocytes, infiltrating inflammatory cells, Kupffer cells and matrix. Foremost among the profibrotic cytokines is transforming growth factor beta 1. 3. Once the hepatic stellate cell is activated the preceding matrix changes and recurrent injurious stimuli will perpetuate the activated state. 4. Despite the accumulation of excess collagens, the liver retains a capacity for matrix degradation. This capacity may be overwhelmed and any secreted matrix remodelling enzymes may be inhibited by the concurrently secreted tissue inhibitors of metallo-proteinase-1 and alpha 2-macroglobulin. 5. Our understanding of the molecular pathogenesis of liver fibrosis is increasing. It is anticipated that this knowledge will provide novel therapeutic avenues to treat this disease process.

Cell Differentiation↗
PubMed1997

A multicentre randomised trial comparing octreotide and injection sclerotherapy in the management and outcome of acute variceal haemorrhage.

BACKGROUND: Few studies have compared vasoactive drugs with endoscopic sclerotherapy in the control of acute variceal haemorrhage. Octreotide is widely used for this purpose, but its value remains undetermined. AIMS: To compare octreotide with endoscopic sclerotherapy for acute variceal haemorrhage. PATIENTS: Consecutive patients with acute variceal haemorrhage. METHODS: Patients were randomised at endoscopy to receive either a 48 hour intravenous infusion of 50 pg/h octreotide (n = 73), or emergency sclerotherapy (n = 77). RESULTS: Overall control of bleeding and mortality was not significantly different between octreotide (85%, 62 patients) and sclerotherapy (82%, 63 patients) over the 48 hour trial period (relative risk of rebleeding 0.83; 95% confidence interval (CI) 0.38 to 1.82), irrespective of Child's grading or active bleeding at endoscopy. One major complication was observed in the sclerotherapy group (aspiration) and two in the octreotide group (pulmonary oedema, severe paralytic ileus). During 60 days of follow up there was an overall trend towards an increased mortality in the octreotide group which was not statistically significant (relative risk of dying at 60 days 1.91, 95% CI 0.97 to 3.78, p = 0.06). CONCLUSIONS: The results of this study indicate that intravenous octreotide is as effective as injection sclerotherapy in the control of acute variceal bleeding, but further controlled trials are necessary to evaluate the safety of this treatment.

Acute Disease↗
PubMed1996

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis.

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.

Amino Acid Sequence↗
PubMed1996

The plasminogen-activating system in hepatic stellate cells.

Urokinase plasminogen activator (uPA) generates plasmin, a process inhibited by plasminogen-activator inhibitor (PAI)-1 and localized to the cell surface by binding of uPA to a specific receptor. Plasmin degrades extracellular matrix (ECM) both directly and by activation of matrix metalloproteinases (MMPs). Because stellate cells play a central role in the pathogenesis of liver fibrosis both via production of ECM proteins and through secretion of MMPs, their contribution to plasmin generation was assessed. Stellate cells were prepared from rat liver and cultured on plastic. Northern analysis showed cellular expression of messenger RNA (mRNA) for PAI-1, uPA, and uPA receptor. Zymography/reverse zymography identified cell-surface-associated uPA activity and uPA and PAI-1 in culture media. Net uPA activity in culture media was maximal after 7 days in culture and then declined, whereas PAI-1 antigen levels remained consistently elevated between 7 and 21 days in culture. Stellate cell-mediated plasmin generation was also seen in in vitro cultures supplemented with plasminogen. Because hepatic stellate cells (HSCs) contain retinoids and release them on activation, the effect of retinoic acid on the plasminogen-activating system was also assessed. Treatment of cultured HSCs with retinoic acid (1 micromol/L) increased uPA secretion 2.6-fold but did not alter PAI-1. We conclude that stellate cells synthesize key components of the plasminogen-activating system and generate plasmin and therefore have the ability to regulate MMP activation. Upregulation of uPA synthesis by retinoic acid may have implications in matrix remodeling in sites of stellate cell activation in which high concentrations of retinoids may be achieved.

Adipocytes↗
PubMed1996

Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver.

BACKGROUND & AIMS: Tissue inhibitors of metalloproteinases may contribute to liver fibrosis by preventing remodeling of fibrillar collagens by interstitial collagenase. This hypothesis was investigated by comparing the relative expression of messenger RNA for interstitial collagenase, gelatinase A, and tissue inhibitor of metalloproteinases 1 and 2 in fibrotic and normal human liver. METHODS: Hepatic expression of metalloproteinases and their inhibitors was examined using ribonuclease protection assay, immunocytochemistry, and immunoassay. Lipocyte expression of tissue inhibitors of metalloproteinases was examined using Northern blotting and reverse zymography. RESULTS: Messenger RNA levels for tissue inhibitors of metalloproteinase 1 and 2 were elevated to 260%-526% of levels in normal liver in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. In fibrotic livers, tissue inhibitor of metalloproteinase 1 protein was 367%-724% of normal. Gelatinase A messenger RNA level increased to 324%-430% of normal values in fibrotic liver, but interstitial collagenase messenger RNA level was not significantly altered. Normal human liver lipocytes activated by culture on plastic expressed messenger RNA and protein for tissue inhibitor of metalloproteinase 1 and 2. CONCLUSIONS: Increased expression of tissue inhibitors of metalloproteinases relative to interstitial collagenase may promote deposition of interstitial collagens in liver fibrosis. Our studies further suggest that lipocytes are an important source of tissue inhibitors of metalloproteinases in progressive liver fibrosis.

Adipocytes↗
PubMed1996

Effect of gonadotrophin on cell and matrix retention and expression of metalloproteinases and their inhibitor in cultured human granulosa cells modelling corpus luteum function.

Granulosa cells were prepared from follicular aspirates obtained at oocyte collection for in-vitro fertilization (IVF) and maintained in culture. Substantial loss of cells from the culture surface occurred in the absence of gonadotrophin when cells were maintained on a thin layer of extracellular matrix (ECM) using a defined, serum-free medium. This cell loss was clearly and significantly reduced in the presence of human chorionic gonadotrophin (HCG) by days 4-6 of culture, and occurred in conjunction with loss of ECM. Analysis of culture medium by zymography using gelatin as substrate demonstrated the presence of metalloproteinases (MMP), MMP-9 (gelatinase B) appearing as the predominant band. Measurement of overall gelatinase activity in culture media revealed a progressive fall in gelatinase expressed on a per cell basis in media from HCG-treated cultures and this was less marked in controls. This suppression of gelatinase activity was consistent with an observed increase in production of tissue inhibitor of metalloproteinase-1 (TIMP-1) by HCG-treated cells, which was significant by days 6-8 of culture. We speculate that stabilization of the ECM may be an important aspect of HCG action in the corpus luteum.

Cells, Cultured↗
PubMed1996

Iron overload and liver fibrosis.

The pathogenesis of liver fibrosis in genetic haemochromatosis and other iron overload states remains enigmatic. Recent advances in the cellular and molecular pathogenesis of liver fibrosis have determined a central role for hepatic stellate cells. These become activated to a myofibroblastic phenotype following most forms of liver injury and are the major cellular source of collagens and other matrix proteins laid down in fibrotic liver. Similar changes have now been reported in the liver in genetic haemochromatosis, with activation of stellate cells becoming more prominent with increasing hepatic iron concentration. In contrast to other liver diseases, this apparently occurs in the absence of significant necroinflammatory change. Unravelling the mechanism of liver fibrogenesis in iron overload states may, therefore, provide important general insights into the pathogenesis of liver fibrosis. The present article reviews current knowledge of this field with emphasis on the role of lipid peroxidation, sideronecrosis of hepatocytes and spillover of iron to Kupffer cells. An attempt is made to draw these observations together with previous studies of the mechanisms of stellate cell activation in other models and diseases. A unifying hypothesis emerges that helps to define some of the next research questions in the pathogenic mechanisms of liver fibrosis in iron overload.

Humans↗
PubMed1996

Augmentation by eosinophils of gelatinase activity in the airway mucosa: comparative effects as a putative mediator of epithelial injury.

1. We have studied the release of gelatin-degrading enzymes from isolated sheets of bronchial mucosa in the presence and absence of eosinophils. 2. Isolated sheets of bovine bronchial mucosa released gelatin-degrading activity in similar amounts from both the apical and basolateral aspects of the tissue. Gelatinolytic activity could not be increased by treatment of the mucosal sheets with calcium ionophore, A23187. 3. The activity of the released gelatinases could be inhibited by chelation of divalent cations or by the matrix metalloproteinase inhibitors, BB-94 and BB-250. However, inhibitors of serine proteinases, or of cysteine proteinases were without effect. In zymography, major bands of gelatin-degrading activity consistent with gelatinases A and B were identified. 4. Addition of guinea-pig eosinophils to the basolateral aspect of bronchial mucosa for 60 min resulted in an increase in the gelatinolytic activity of the conditioned medium, irrespective of whether the eosinophils were stimulated with ionophore A23187 or not. However, only ionophore-stimulated eosinophils reacted to produce sufficient tissue damage to increase the transepithelial flux of serum albumin. 5. Purified eosinophils were a poor source of gelatinolytic activity, indicating that when interacting with the bronchial mucosa their effect is to increase the apparent release and/or activation of gelatinases derived from the airway mucosa. 6. After organomercurial activation, recombinant human progelatinase A increased the permeability of the bronchial mucosa to mannitol. However, the activity of enzyme and duration of exposure required to do this were greater than the amounts of gelatinase activity detected during eosinophil-mediated injury. Sheets of airway mucosa were also resistant to injury evoked by high concentrations of hydrogen peroxide or plasmin. 7. Collectively, these results suggest that if gelatinases are involved in eosinophil-mediated injury and repair of the bronchial mucosa, they require other mediators to act in concert to bring about outright epithelial cell detachment. This does not preclude the possibility that gelatinases are crucial in rendering the airway mucosa hyperfragile.

Animals↗
PubMed1995

Rat hepatic lipocytes synthesize and secrete transin (stromelysin) in early primary culture.

BACKGROUND & AIMS: Hepatic lipocyte proliferation and activation are pivotal in liver fibrosis. Disruption of normal lipocyte-matrix interactions may contribute to this process. The synthesis of transin, which degrades normal liver matrix, by culture-activated hepatic lipocytes was investigated. METHODS: Lipocytes were isolated by pronase/collagenase perfusion, density gradient centrifugation, and centrifugal elutriation. Transin messenger RNA in lipocytes was analyzed by Northern blotting. Transin activity was analyzed by zymography, Western blotting, immunocytochemistry, and quantitative [14C]beta-casein degradation assay. RESULTS: Transin messenger RNA was detected in early primary culture (3-5 days) but not in freshly isolated lipocytes or late primary culture. Zymography of lipocyte medium showed caseinolytic activity (relative molecular weight, 57 kilodaltons and 60 kilodaltons) inhibited by ethyl-enediaminetetraacetic acid but not thiol or serine protease inhibitors. Immunoblotting and immunocytochemistry confirmed the presence of transin in media and cells. Quantitative transin activity decreased progressively with increasing duration of primary lipocyte culture and myofibroblastic transformation. CONCLUSION: Rat hepatic lipocytes express the transin gene and secrete its product during the early phase of lipocyte activation in primary culture. Because this enzyme degrades a wide spectrum of normal basement membrane proteins and activates progelatinase B and interstitial collagenase, it may have an important role in liver injury and fibrosis.

Adipocytes↗
PubMed1995

Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes.

1. Activated hepatic lipocytes are central to the pathogenesis of liver fibrosis as the principal source of both interstitial collagens and matrix-degrading metalloproteinases. In progressive fibrosis there is a failure to degrade interstitial collagens with a reported decrease in collagenase activity. In these studies we investigate expression of the potent collagenase inhibitor, tissue inhibitor of metalloproteinase-1, and interstitial collagenase in end-stage autoimmune chronic active hepatitis and activated human hepatic lipocytes in culture. 2. Messenger RNA transcripts for interstitial collagenase and tissue inhibitor of metalloproteinase-1 in explanted human liver were quantified by ribonuclease protection assay and densitometric analysis. This indicated that tissue inhibitor of metalloproteinase-1 and interstitial collagenase expression in autoimmune chronic active hepatitis were also coordinately up-regulated. 3. Using Northern analysis of RNA from human lipocytes in primary culture on plastic, mRNA for interstitial collagenase could not be detected in unstimulated cells but was present after stimulation with tumour necrosis factor alpha. Tissue inhibitor of metalloproteinase-1 mRNA was present in unstimulated lipocytes and up-regulated fivefold in response to tumour necrosis factor alpha. Using activity assay of serum-free conditioned media, interstitial collagenase could not be detected in unstimulated primary cultures, primary cultures stimulated with tumour necrosis factor alpha or transforming growth factor beta-1 (n = 3 and n = 4 respectively) or in passaged lipocytes (n = 6). In contrast, free tissue inhibitor of metalloproteinase-1 activity was present in unstimulated and passaged cultures and this was increased in response to tumour necrosis factor alpha and transforming growth factor beta-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune Diseases↗