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Biomedical subjects

R M Hembry

Publications and source records attributed to R M Hembry.

At least 73 records · Page 4Linked to original sources
PubMed1989

Immunolocalization of metalloproteinases and their inhibitor in the rabbit growth plate.

UNLABELLED: The synthesis and distribution of three metalloproteinases, collagenase, stromelysin, and gelatinase, and of the tissue inhibitor of metalloproteinase were examined in the distal femoral growth plate, the secondary center of ossification, and the perichondral ossification groove of Ranvier in newborn to six-week-old rabbits. Specific antisera to each of the enzymes and to the tissue inhibitor of metalloproteinase were used to identify their distribution in the extracellular matrix of the growth plate and to determine the associated tissues and cells that are responsible for their synthesis. Immunolocalization using tissue that was cultured in the presence of monensin to augment accumulation of intracellular antigen revealed that the growth-plate chondrocyte is responsible for the synthesis of the metalloproteinases and the tissue inhibitor of metalloproteinase, and that there is a unique pattern of synthesis in each zone. Chondrocytes of the resting and proximal proliferative zones were shown to synthesize and secrete all of the metalloproteinases and the tissue inhibitor of metalloproteinase. Synthesis of collagenase also was demonstrated in the remainder of the proliferative zone and in the most distal cells of the hypertrophic zone. The presence of collagenase in the distal cells suggests their involvement in vascular invasion. By culturing tissues in the presence of antibodies, we were able to demonstrate collagenase and the tissue inhibitor of metalloproteinase throughout the growth-plate matrix. Staining of the extracellular matrix, implying active collagenase, was also found in the matrix of the proliferative and hypertrophic zones, suggesting that degradation of tissue may occur at a distance from the cells that synthesize the enzyme. Simultaneous localization with two different antibodies demonstrated that growth-plate chondrocytes are capable of synthesizing collagenase and the tissue inhibitor of metalloproteinase, both independently of one another and coordinately. Stromelysin was found to be synthesized in all zones, implying that it plays an important role in degradation. Monocyte-conditioned media stimulates synthesis of collagenase in growth-plate cells, principally through the action of interleukin-1. All chondrocytes throughout the growth plate, including hypertrophic cells, can be stimulated to produce collagenase. These changes in metalloproteinase and in the tissue inhibitory of metalloproteinase in the growth plate are crucial to remodelling of the matrix during development, and the appearance of metalloproteinases and the tissue inhibitory of metalloproteinase in the secondary ossification center and groove of Ranvier indicates that changes at these sites are similar to remodeling in the growth plate. CLINICAL RELEVANCE: The life cycle of the chondrocyte in the growth plate is central to the process of endochondral ossification, bone growth, and development. Our new data on the zonal synthesis of metalloproteinase and of th

Animals↗
PubMed1988

Role of collagenase in colonic anastomoses: a reappraisal.

Increased collagenolysis, with reduction in collagen concentration, has been incriminated in the breakdown of colonic anastomoses but previous studies have measured only collagen levels and non-specific collagenolytic activity. Collagenase, the initiating enzyme in collagen degradation, is synthesized on demand and controlled by tissue inhibitor of metalloproteinases (TIMP). Antibodies to collagenase and TIMP were applied to colonic anastomoses in rabbits to investigate the role of the enzyme during healing. Within 12 h of operation, secreting cells and extracellular collagenase were identified at the everted edges of the bowel wall. After 24 h, collagenase activity was accompanied by TIMP secretion in the same localized regions, and by the third postoperative day very few cells were still synthesizing enzyme in these areas, although extracellular activity remained visible. TIMP-secreting cells, however, were seen in a layer of connective tissue sealing the serosal surface of the anastomosis. At 7 days, both enzyme and inhibitor were found only in small aggregates of secreting cells in the deeper layers. The localization and extent of collagenase and TIMP activity accorded well with a normal healing response as, at all times, the enzyme was confined to the immediate vicinity of the suture line.

Anastomosis, Surgical↗
PubMed1987

Bacterial antigens induce collagenase and prostaglandin E2 synthesis in human gingival fibroblasts through a primary effect on circulating mononuclear cells.

Our previous work suggests that one mechanism through which connective tissue breakdown might occur in periodontal diseases is the production of metalloproteinases, including collagenase, by gingival fibroblasts. In this study we investigated whether highly purified preparations of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from a number of putative periodontal pathogens could induce monolayer cultures of human gingival fibroblasts to synthesize collagenase and prostaglandin E2. Using both biochemical assays and immunocytochemical techniques, we found that cells synthesized only very small amounts of collagenase in direct response to LPS or LTA (0.1 to 20.0 micrograms/ml). At the highest dose of both antigens, prostaglandin E2 production was increased. We then studied whether LPS and LTA could signal collagenase production by interacting primarily with a different cell type. Our results show that LPS and LTA were each able to stimulate cultures of human blood mononuclear cells (greater than 95% monocytes) to release collagenase-inducing cytokines. By indirect immunocytochemistry, we found that a large proportion of human gingival fibroblasts was activated to produce collagenase by supernatants from LPS- and LTA-stimulated mononuclear cells, whereas gingival fibroblasts cultured with supernatants from unstimulated mononuclear cells were not. Furthermore, in a population of activated fibroblasts we demonstrated, using a double-labeling technique, that some cells made collagenase and the specific tissue inhibitor of metalloproteinases (TIMP) simultaneously. As yet, the collagenase-inducing signals remain poorly characterized but the interleukins-1 and tumor necrosis factors seem likely candidates.

Actinobacillus↗
PubMed1987

Tissue inhibitor of metalloproteinases (TIMP) regulates extracellular type I collagen degradation by chondrocytes and endothelial cells.

A specific antiserum to purified rabbit tissue inhibitor of metalloproteinases (TIMP) was raised in sheep, characterized and used to investigate the role of TIMP in a model system. Chondrocytes and endothelial cells cultured on 14C-labelled type I collagen films and stimulated to produce collagenase were unable to degrade the films unless the anti-TIMP antibody was added. The degradation induced was inhibited by a specific anti-rabbit collagenase antibody. It was concluded that TIMP is a major regulatory factor in cell-mediated collagen degradation.

Animals↗
PubMed1987

Actin filaments in normal dermis and during wound healing.

During wound healing, it has been suggested, modified fibroblasts rich in actin filaments are responsible for wound contraction. With the use of specific fluorescent probe (NBD-phallacidin), the distribution of actin filaments are compared in normal dermis and in several wound contraction models, including open and burn wounds and full and thin-thickness skin autografts. Fibroblasts of normal dermis are slightly stained with NBD-phallacidin. Fibroblasts with actin filaments are increased in autografts, particularly at Days 15 and 21 after grafting, and are prominent in open and burn wounds. The wound contraction rate is not directly related to the presence of actin-staining fibroblasts. After stabilization of the contraction of open or burn wounds, fibroblasts rich in actin filaments remain. The superficial layer of full-thickness skin graft contains a similar actin distribution without concomitant contraction. It is concluded that the distribution of actin-rich fibroblasts corresponds morphologically to previous areas of necrosis or injury.

Actin Cytoskeleton↗
PubMed1986

Characterization of a specific antiserum to rabbit stromelysin and demonstration of the synthesis of collagenase and stromelysin by stimulated rabbit articular chondrocytes.

An antiserum to rabbit bone stromelysin (proteoglycanase) was raised in sheep and characterized as specific, recognizing the enzyme from both different tissue sources and different species. This antiserum and a specific antiserum to rabbit bone collagenase were used in the study of metalloproteinase production by rabbit articular chondrocytes stimulated with either interleukin 1 or mononuclear cell-conditioned medium. It was shown by electroimmunoblotting that the apparently co-ordinate (mole:mole) induction of collagenase and stromelysin activity with time correlated in either case with an increase in enzyme protein. The stimulated production of both enzymes could be modified in parallel by a variety of compounds. Immunohistochemical studies confirmed that although most cells were producing both metalloproteinases simultaneously, some chondrocytes produced detectable levels of only one. The data are discussed in relation to the mechanisms of breakdown in connective tissues.

Animals↗
PubMed1986

Commitment to expression of the metalloendopeptidases, collagenase and stromelysin: relationship of inducing events to changes in cytoskeletal architecture.

Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-O-tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 micrograms/ml CB for 3-24 h, or with various concentrations of CB (0-2 micrograms/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 micrograms/ml CB for only 3 h, greater than 85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immunofluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (less than 0.5 micrograms/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for collagenase and stromelysin gene expression.

Actins↗
PubMed1986

Immunolocalization of collagenase and tissue inhibitor of metalloproteinases (TIMP) in hypertrophic scar tissue.

Normal, mature and hypertrophic dermal scars were examined by indirect immunofluorescence for the presence of collagenase, tissue inhibitor of metalloproteinases (TIMP) and cathepsin D. Significant extracellular immunoprecipitation of both collagenase and TIMP were found in areas of all scars judged to be actively remodelling, whereas inactive areas were predominantly negative. TIMP was also present in endothelial cells of patent blood vessels, but found not to be present in the enlarged endothelial cells of occluded vessels. Negligible amounts of extracellular cathepsin D were found.

Adolescent↗
PubMed1986

Characterization of a specific antiserum for mammalian collagenase from several species: immunolocalization of collagenase in rabbit chondrocytes and uterus.

A specific antiserum to rabbit bone collagenase was raised in a sheep and shown to react with collagenase from several mammalian species. This antiserum was used to demonstrate that only active collagenase binds to and can be immunolocalized on collagen fibrils and, by indirect immunofluorescence, the secretion of latent collagenase by stimulated rabbit chondrocytes and cells of the post-partum involuting rabbit uterus. The ionophore monensin was used to demonstrate intracellular accumulation of collagenase in the Golgi apparatus of both stimulated chondrocytes and involuting uterine cells. Collagenase was not detectable in either normal cartilage or non-gravid uterus. These results are discussed in relation to other studies of collagenase immunolocalization reported in the literature.

Animals↗
PubMed1986

Morphologic examination of mesenchymal cells in healing wounds of normal and tight skin mice.

The healing process of an open wound as effected by wound contraction is complete by 3 weeks in the normal mouse. In contrast, its onset is delayed by 3 weeks and complete healing requires 6 weeks in the tight skin mouse (TSM), a mutant mouse strain with the autosomal dominant gene for tight skin. Possible mechanisms for this delay were evaluated. The frequency and distribution of myofibroblasts were studied during the 3-week delay in wound contraction by actin staining and electron microscopy. It was determined, by electron microscopy and phalloidin staining, that myofibroblasts were found in high density in noncontracting TSM wounds. Electron microscopy showed, however, that these myofibroblasts were surrounded by a pericellular matrix that separated their surface from adjacent collagen fibers. No pericellular matrix was found around cells in granulation tissue of normal mice. At 3 weeks, as TSM wounds began to contract, the number and intensity of cells stained by phalloidin in this tissue was less than that seen earlier. The pericellular matrix was fragmented at this time, and cell surface and collagen fiber associations were apparent. Finally, at 5 weeks, when wound contraction was well developed in the TSM, only a small area in the center of the healing wound beneath the epidermis contained phalloidin-positive myofibroblasts. Electron-microscopic examination of the residual granulation tissue at this time revealed the complete absence of the pericellular matrix. It is postulated that during the 3-week delay in wound closure, the presence of a localized pericellular matrix prevents the interaction between cells and collagen fibers necessary for the reorganization of collagen. It is also thought that the tightly adherent uninjured skin surrounding the healing wound may cause delayed wound closure. There was no evidence that the absence of myofibroblasts is responsible for delayed wound contraction.

Animals↗
PubMed1985

Immunolocalization of tissue inhibitor of metalloproteinases (TIMP) in human cells. Characterization and use of a specific antiserum.

A specific antiserum to pure human amniotic fluid metalloproteinase inhibitor (TIMP) was raised in a sheep. This antiserum was used to demonstrate: firstly, the immunological identity of the TIMP activities from amniotic fluid and culture medium of human foetal lung fibroblasts; and secondly, by indirect immunofluorescence, the secretion of TIMP by human foetal lung fibroblasts, chondrocytes, epithelial cells and smooth muscle cells. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was used to stimulate secretion of TIMP by human foetal lung fibroblasts and the ionophore monensin was used to demonstrate intracellular accumulation of TIMP in the Golgi apparatus of these cells. These results are discussed in relation to other inhibitors of collagenase reported in the literature, which are probably identical to TIMP.

Amniotic Fluid↗
PubMed1984

A comparative study of fibroblasts in healing freeze and burn injuries in rats.

In rats, the healing process of a full-thickness dermal freeze injury differs from that of a burn wound. Whereas burn wounds heal by wound contraction, the movement of surrounding normal skin over the defect, freeze wounds heal without wound contraction. That absence of contraction may be due to the freeze wound's lack of myofibroblasts, the cells reportedly associated with wound contraction. Myofibroblasts can be demonstrated histologically by staining the F-actin filaments of the stress fibers with NBD-phallacidin, a fluorescent reagent specific to F-actin filaments. Fibroblasts in normal dermis have no staining stress fibers. However, staining myofibroblasts are uniformly distributed in the granulation tissue of the healing burn and in the islands of granulation tissue between residual connective tissue fibers in the healing freeze wound. These residual dermal fibers were identified by their patterns of birefringence. Residual connective tissue matrix persists following cold trauma and acts like an internal splint. Burn trauma destroys cells and the connective tissue matrix, which is completely replaced with granulation tissue which undergoes wound contraction. Freeze trauma kills the cellular components of dermis, while some residual connective tissue fibers endure. This study shows that the connective tissue matrix can play an important role in the control of wound contraction.

Animals↗
PubMed1982

Evidence that extracellular cathepsin D is not responsible for the resorption of cartilage matrix in culture.

Cathepsin D, the major lysosomal aspartic proteinase, is responsible for the autolysis of cartilage at slightly acidic pH, and it has been suspected of making a significant contribution to the breakdown of the living tissue, such as in stimulated by retinol. Our finding, however, has been that neither inhibitory antibodies against cathepsin D, nor chemical inhibition with pepstatin, significantly decreases the rate of degradation of proteoglycan in the organ culture system. Most of the other proteinase inhibitors tested were similarly ineffective, although the EDTA and 1,10-phenanthroline inhibited the resorption by a cytotoxic effect. We conclude that although cartilage matrix degradation has clear characteristics of proteolytic process, the identity of the enzyme(s) responsible remains obscure.

Animals↗
PubMed1982

Rabbit cranial suture fibroblasts under tension express a different collagen phenotype.

We present evidence that cells in fibrous joints respond to tensile mechanical stress by synthesizing significant quantities of type III collagen. Under normal conditions sutural fibroblasts synthesize type I collagen. Type III comprised some 20 per cent of the newly synthesized collagen in stressed joints, a level that was achieved within 24 h of the onset of tension and remained unchanged throughout a 4-day experimental period. These findings suggest that the biomechanical environment of a connective tissue cell is an important determinant of the type of collagen synthesized. However, we propose that the effect is likely to be an indirect one by virtue of its influence on the metabolic activity of the cells.

Animals↗
PubMed1980

Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury.

Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.

Antibody Affinity↗
PubMed1979

Experimental model for scleritis.

Rabbits were sensitized over a prolonged period to ovalbumin by intradermal injection. Ovalbumin was then injected into the limbus of the eye. A corneoscleral lesion, similar to necrotizing scleritis in humans, was produced. The clinical progression of the lesion is described and its histologic appearance discussed in relation to the human disease.

Animals↗
PubMed1978

Preparation of antibody fragments: conditions for proteolysis compared by SDS slab-gel electrophoresis and quantitation of antibody yield.

Monovalent antibody fragments are now recognized to have major advantages over intact immunoglobulin G in immunohistochemistry, but the methods commonly used for their preparation do not necessarily give a maximal yield of active antibody fragments and take little account of species differences. Sodium dodecyl sulphate (SDS) slab-gel electrophoresis demonstrated large differences in the susceptibility to digestion of antibodies of different species and was found to be valuable in the selection of optimal digestion conditions. Promising results were obtained with sheep antibodies by use of low concentrations of trypsin, and an immunoadsorbent technique showed a high yield of antibody fragments.

Binding Sites↗