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Biomedical subjects

A R Poole

Publications and source records attributed to A R Poole.

At least 163 records · Page 9Linked to original sources
PubMed1983

Lymphocyte transformation to connective tissue antigens in adult and juvenile rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus, and a nonarthritic control population.

Transformation of peripheral blood lymphocytes after exposure to connective tissue antigens was measured in patients with adult (n = 35) and juvenile rheumatoid arthritis (n = 34), osteoarthritis (n = 21), ankylosing spondylitis (n = 15), and systemic lupus erythematosus (n = 26) and in control subjects (n = 36). The connective tissue antigens included homologous cartilage-type proteoglycan, cyanogen bromide-derived peptides of type I, II, and III collagens, and type I and II helical collagens. Lymphocyte transformation was not detected in the osteoarthritic and control groups, with one exception. Sensitization to at least one connective tissue antigen was detected in approximately one-third of the rheumatoid arthritic and lupus patients and in one-quarter of the juvenile rheumatoid patients. In ankylosing spondylitis, positive responses occurred to proteoglycan in 20% of patients tested but never to collagens or peptides. Sensitivity to proteoglycan was detected only in ankylosing spondylitis except for one patient with juvenile rheumatoid arthritis. In patients with systemic lupus erythematosus and both forms of rheumatoid arthritis, lymphocyte transformation was usually more frequently detected to peptides than to the helical collagens. In adult rheumatoid arthritis, type II peptides elicited an elevated number of responses (14%) as did type I (9%) and III (8%) peptides to lesser degrees. Responses to type I (4%) and II (4%) helical collagens were infrequent. Rheumatoid arthritic patients usually exhibited sensitivity to only one antigen and lymphocyte transformation was often detected when the arthritis was improving. In juvenile rheumatoid arthritis, lymphocyte transformation was detected to peptides of type I (16%), II (9%), and III (29%) collagens and to helical type I (12%) and II (8%) collagens. In systemic lupus erythematosus, sensitization was detected to peptides of type I (13%), II (20%), and III (14%) collagens and to helical type I collagen (18%) but not type II collagen. Simultaneous sensitivity to several antigens often occurred in both systemic lupus erythematosus and juvenile rheumatoid arthritis. Examination of individual patients in all three rheumatic disease groups revealed that immune sensitivity developed to collagen peptides rather than to the helical molecules, particularly in the case of type II collagen. Thus, some patients with inflammatory arthritis exhibit immune responses to connective tissue components which are, as a group, characteristic for each type of arthritis. These responses, which were not obviously associated with disease activity, may develop as a result of inflammation or trauma which destroys connective tissue and exposes molecules, in either a native or degraded state, to cells of the immune system. Expression of sensitivity to these tissue antigens may contribute to the chronicity of the inflammatory arthritides.

Adult↗
PubMed1983

Immune regulation of collagenase secretion in rheumatoid and osteoarthritic synovial cell cultures.

Primary cultures of synovial cells were obtained by proteolytic dispersion of synovial tissue from patients with rheumatoid arthritis (n = 19), psoriatic arthritis (n = 2), osteoarthritis (n = 13) and other joint problems (n = 3). The levels of endogenously secreted collagenase were variable from patient to patient but did not differ significantly between rheumatoid arthritis and osteoarthritis. The endogenous collagenase secretion was likely a consequence of mononuclear cell factor (MCF) release from monocytes/macrophages which have been shown to be present among the heterogeneous primary rheumatoid synovial cell population (Dayer et al., 1976). As also demonstrated by these investigators, medium containing MCF can be generated from peripheral blood mononuclear cells in a T lymphocyte-dependent process by the addition of phytohemagglutinin (PHA). The addition of such medium stimulated collagenase secretion from all our synovial cell cultures regardless of the endogenous level. Protein synthesis but not synovial cell proliferation was required for MCF stimulation of collagenase secretion. The direct addition of PHA to primary synovial cell cultures stimulated collagenase secretion in some but not all cases indicating the presence of T lymphocytes in these positively-responding cultures. In some of these primary synovial cell cultures in which the addition of PHA stimulated collagenase secretion, secretion was also stimulated by the addition of collagen peptides, native collagen, proteoglycan or purified protein derivative of tuberculin. We propose that, in these instances, MCF release is mediated by antigen-sensitized lymphocytes. Antigen-responsive cultures were not restricted to the rheumatoid population. Our data are compatible with the idea that infiltrated lymphocytes in inflamed synovial tissue become sensitized to cartilage and joint capsule components released during tissue degradation and contribute to matrix destruction by mediating MCF release with consequent stimulation of collagenase synthesis and secretion from synovial cells.

Antigens↗
PubMed1983

Complexity of proteoglycan organization in articular cartilage: recent observations.

Antibodies to aggregating high buoyant density cartilage proteoglycans (PG) and to link proteins have been used to study the distribution of these molecules in adult bovine and human articular cartilages. The work on bovine cartilages has been described in detail elsewhere. In human articular cartilage there was a striking deficiency of PG in the interterritorial region of the deep zone and of link protein in territorial sites in this and the mid zone. Thus aggregated PG stabilized in their interaction with link protein, are probably mainly present in the interterritorial matrix of the mid zones of human cartilage and in the deep zone of bovine cartilage. This apparent deficiency of PG in the interterritorial matrix of the deep zone in adult human cartilage may produce greater weaknesses in this older cartilage.

Animals↗
PubMed1983

Immunity to homologous collagens and cartilage proteoglycans in rabbits.

The in vitro incorporation of [3H]-thymidine into spleen cells was used to show that rabbits with experimentally-induced inflammatory arthritis of 1-4 months duration often develop cellular immunity to purified homologous cartilage proteoglycans, type I and III collagens and, less frequently, to type II collagen. Responses to collagens were primarily directed to antigenic determinants exposed on peptides in degraded molecules, whereas responses to proteoglycans were seen with both native and degraded molecules. These in vitro blastogenic responses were shown to be completely dependent on the presence of T lymphocytes in the cultures. This expression of immunity was not demonstrable in rabbits with long duration arthritis (7-12 months) with any of the antigens tested. Some rabbits injected with homologous proteoglycans demonstrated T-cell-dependent cellular, but not humoral, immunity to the injected antigens. In contrast, rabbits injected with heterologous human proteoglycans developed cellular and humoral immunity to the immunizing proteoglycan, but failed to develop cellular immunity to rabbit proteoglycan. Some of these rabbits did, however, produce circulating antibodies which reacted with rabbit proteoglycans. Moreover, antibodies produced by immunizing rabbits with proteoglycans from bovine, chicken and rat cartilages and the hyaluronic acid binding region from rat cartilage proteoglycan often cross-reacted with rabbit proteoglycan, indicating that there are species-common antigenic determinants present in these proteoglycans and in the hyaluronic acid binding region and that these are recognizable in rabbit proteoglycans by rabbit antibodies. This ability to induce selectively cellular or humoral immunity to proteoglycans should be useful for future investigations of the role of such immunity in the pathogenesis of arthritis, although induction of immunity to proteoglycan was not accompanied by any demonstrable synovitis in these rabbits as has been observed in some strains of rats and mice for type II collagen.

Animals↗
PubMed1982

Enzyme-linked immunosorbent assay analyses of the hyaluronate-binding region and the link protein of proteoglycan aggregate.

An enzyme-linked immunosorbent assay, in combination with an independent inhibition step, was established to quantitate two components of the proteoglycan aggregate, namely link protein and hyaluronate-binding region, at concentrations below 100 ng/ml. The presence of other components of the aggregate in the samples to be tested influenced quantitation in a specific manner. The apparent antigenicity of link protein increased 2-5 times when either purified proteoglycan monomer, purified hyaluronate-binding region, or purified hyaluronate (macromolecular or oligomers) were present in the link protein samples. These findings are interpreted as showing different states of conformation or degree of association of the link protein with other components of aggregate in solution. In separate experiments, a 2-4-fold increase in the apparent antigenicity of purified hyaluronate-binding region was observed when hyaluronate molecules with at least 20 disaccharides were present in the samples. Co-incubation of the hyaluronate-binding region or proteoglycan monomer with either purified link protein or with smaller hyaluronate oligomers did not change its antigenicity in the assay. However, when hyaluronate oligomers with 8 disaccharides were included in a mixture of macromolecular hyaluronate with hyaluronate-binding region, the increase in apparent antigenicity was blocked. The results illustrate the inherent difficulties in using the enzyme-linked immunosorbent assay for the quantitation of link protein or proteoglycan monomers in samples where these macromolecules can associate with themselves or other components of proteoglycan aggregates.

Animals↗
PubMed1982

The heterogeneity of link proteins isolated from human articular cartilage proteoglycan aggregates.

Link proteins, isolated from proteoglycan aggregates prepared from human articular cartilage, were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. When subjected to the former technique the native link proteins resolve into three components of molecular weights 48,000, 44,000, and 41,000. Link protein, isolated following clostripain digestion of proteoglycan aggregate, is present as a single component of molecular weight 41,000. Under all conditions of isoelectric focusing tested, the native link proteins resolve into at least nine subcomponents having pI values between 6.0 and 7.0. The clostripain-treated link protein resolves into at least five subcomponents which have pI values similar to those of the more basic subcomponents observed in the native molecules. One source of heterogeneity contributing to both isoelectric focusing profiles is variation in sialic acid content, since neuraminidase treatment of the link protein preparations produces a shift to subcomponents with more basic pI. The electrophoretic data are consistent with the two larger link protein components representing the same protein core, but being substituted to different degrees with oligosaccharide chains that may have variable sialic acid contents. The smallest link protein component, which may be derived from either of the larger moieties by limited proteolytic cleavage, is also substituted with sialic acid-containing oligosaccharides.

Cartilage, Articular↗
PubMed1982

Immunoferritin binding to proteoglycan monomers. An electron microscopic study.

Antibody to native bovine nasal cartilage proteoglycan monomer was shown by enzyme-linked immunosorbent assay to react with the purified hyaluronic acid binding region of the monomer. Antibody was digested with pepsin to produce F(ab')2 and labeled with glutaraldehyde-activated ferritin. F(ab')2 and F(ab')2-ferritin were reduced and alkylated to render them monovalent (Fab'). Antibody Fab' binding to native proteoglycan monomer was studied by electron microscopy of monomer reacted with ferritin-labeled antibody Fab' spread in a cytochrome c film. Ferritin-labeled antibody Fab' bound primarily at one end of the proteoglycan monomer. This binding was partly inhibitable by unlabeled monovalent antibody Fab', demonstrating immunospecificity. The end of the monomer with bound ferritin sometimes appeared as the thin segment, previously observed to bind to hyaluronic acid. These observations indicate that the hyaluronic acid binding region is at only one end of each proteoglycan monomer and that ferritin-labeled antibody Fab' selectively attaches to this part of native proteoglycan monomers. This methodology should be useful for future structural studies of isolated proteoglycans.

Animals↗
PubMed1982

Immunity to homologous type III collagen after partial meniscectomy and sham surgery in rabbits.

Cellular immunity to rabbit types I, II, and III collagen was detected after both partial medial meniscectomy and sham operation in rabbits when an in vitro 3H-thymidine incorporation assay was used. The blastogenic responses were commonly directed towards peptides derived from collagens by cyanogen bromide cleavage used to mimic proteinase degradation products. The responses to type III collagen peptides were by far the strongest and were seen in most rabbits that were operated on. We suggest that the responses to types I and II collagen were caused by a cross-reaction with type III collagen peptides. This conclusion was supported by the observation that spleen cells from rabbits directly immunized with homologous type III collagen peptides in Freund's complete adjuvant responded strongly to the immunizing peptides and also cross-reacted with types I and II collagen peptides. Immunity to cartilage proteoglycans was observed primarily in rabbits that had undergone meniscectomy and had severe cartilage degeneration. These results indicated that immunity to collagens will develop merely as a result of joint surgery, whereas immunity to proteoglycans is largely dependent upon an osteoarthritic lesion.

Animals↗
PubMed1982

Age-related changes in the antigenicity of human articular cartilage proteoglycans.

Rabbits were immunized with adult or foetal human articular cartilage proteoglycans and cell-mediated and antibody immunity assessed in vitro. Cellular immunity to proteoglycans of different ages was measured by the stimulation of 3H-thymidine incorporation into cultured spleen cells. Antibodies to proteoglycans in the rabbit sera were detected with an enzyme-linked immunosorbent assay. The ability of adult and foetal proteoglycans to preabsorb antibody activity in this assay was compared. The results from both the cellular and antibody studies support the same conclusion, that adult proteoglycans express all foetal antigenic determinants and have additional determinants not seen on foetal proteoglycans. The reactivity of rabbit spleen cells to proteoglycans of different ages indicated that these determinants appear during maturation when the major changes in proteoglycan structure occur. The results did not appear to be due to differences in chondroitin sulphate content of the preparations, since the same observations were made with chondroitinase ABC-digested proteoglycans. The observations might be explained by other differences in glycosylation between the preparations or by postulating the synthesis during maturation of an additional proteoglycan species with a different core-protein. Such changes in the antigenicity of articular cartilage proteoglycans with age could account for the development of autoimmunity to proteoglycans in inflammatory joint diseases and may play a role in their pathogenesis.

Adolescent↗
PubMed1982

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate.

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.

Animals↗
PubMed1982

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein.

Monospecific antibodies to bovine nasal cartilage proteoglycan monomer and link protein were used to demonstrate that immunologically related molecules are present in the bovine eye and associated tissues. With immunofluorescence microscopy, reactions for both proteoglycan and link protein were observed in the sclera, the anterior uveal tract, and the endoneurium of the optic nerve of the central nervous system. Antibody to bovine nasal cartilage proteoglycan also reacted with some connective tissue sheaths of rectus muscle and the perineurium of the optic nerve of the central nervous system. Antibody to proteoglycan purified from rat brain cross-reacted with bovine nasal cartilage proteoglycan, indicating structural similarities between these proteoglycans. ELISA studies and crossed immunoelectrophoresis demonstrated that purified dermatan sulphate proteoglycans isolated from bovine sclera did not react with these antibodies but that the antibody to cartilage proteoglycan reacted with other molecules extracted from sclera. Two molecular species resembling bovine nasal link protein in size and reactivity with antibody were also demonstrated in scleral extracts: the larger molecule was more common. Antibody to link protein reacted with the media of arterial vessels demonstrating the localization of arterial link protein described earlier. Tissues that were unstained for either molecule included the connective tissue stroma of the iris, retina, vitreous body, cornea, and the remainder of the uveal tract. These observations clearly demonstrate that tissues other than cartilage contain molecules that are immunologically related to cartilage-derived proteoglycans and link proteins.

Animals↗
PubMed1982

An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage.

Monospecific antibodies to bovine cartilage proteoglycan monomer (PG) and link protein (LP) have been used with immunoperoxidase electron microscopy to study the distribution and organization of these molecules in bovine articular cartilage. The following observations were made: (a) The interterritorial matrix of the deep zone contained discrete interfibrillar particulate staining for PG and LP. This particulate staining, which was linked by faint bands of staining (for PG) or filaments (for LP), was spaced at 75- to 80-nm intervals. On collagen fibrils PG was also detected as particulate staining spaced at regular intervals (72 nm), corresponding to the periodicity of collagen cross-banding. The interfibrillar PG staining was often linked to the fibrillar PG staining by the same bands or filaments. The latter were cleaved by a proteinase-free Streptomyces hyaluronidase with the removal of much of the interfibrillar lattice. Since this enzyme has a specificity for hyaluronic acid, the observations indicate that the lattice contains a backbone of hyaluronic acid (which appeared as banded or filamentous staining) to which is attached LP and PG, the latter collapsing when the tissue is fixed, reacted with antibodies, and prepared for electron microscopy. Thishyaluronic acid is anchored to collagen fibrils at regular intervals where PG is detected on collagen. PG and LP detected by antibody in the interterritorial zones are essentially fully extractible with 4 M guanidine hydrochloride. These observations indicated that interfibrillar PG and LP is aggregated with HA in this zone. (b) The remainder of the cartilage matrix had a completely different organization of PG and LP. There was no evidence of a similar latticework based on hyaluronic acid. Instead, smaller more closely packed particulate staining for PG was seen everywhere irregularly distributed over and close to collagen fibrils. LP was almost undetectable in the territorial matrix of the deep zone, as observed previously. In the middle and superficial zones, stronger semiparticulate staining for LP was distributed over collagen fibrils. (c) In the superficial zone, reaction product for PG was distributed evenly on collagen fibrils as diffuse staining and also irregularly as particulate staining. LP was observed as semiparticulate staining over collagen fibrils. The diffuse staining for PG remained after extraction with 4 M guanidine hydrochloride. (d) In pericellular matrix, most clearly identified in middle and deep zones, the nature and organization of reaction product for PG and LP were similar to those observed in the territorial matrix, except that LP and PG were more strongly stained and amorphous staining for both components was also observed. (e) This study demonstrates striking regional variations of ultrastructural organization of PG and LP in articular cartilage...

Animals↗
PubMed1982

Stimulation of collagenase secretion from rheumatoid synovial tissue by human collagen peptides.

Dayer et al. demonstrated that mononuclear cells from human peripheral blood produce a mononuclear-cell factor that stimulates collagenase secretion from adherent rheumatoid synovial cells. The production of this mononuclear-cell factor can be stimulated by phytohemagglutinin, a T-cell mitogen. We have examined immune mechanisms by which collagenase secretion may be stimulated from explants of rheumatoid synovial tissue and from primary monolayer cultures of rheumatoid synovial cells. Conditioned media from cultures of normal peripheral-blood mononuclear cells that had been activated by phytohemagglutinin stimulated collagenase secretion from most explants and from all monolayer cultures that were examined. The direct addition of phytohemagglutinin stimulated collagenase secretion in explants from eight of fourteen patients and in monolayer cultures from three of six patients. These observations indicate the presence of responsive T lymphocytes in rheumatoid synovial tissue and implicate them in the stimulation of collagenase secretion from synovial cells. The direct addition of type-II collagen peptides to rheumatoid explants also stimulated collagenase secretion in explants from six to fifteen patients. Four of five of these patients displayed moderate or severe disease activity. Other patients showed a marginal stimulation of collagenase secretion on addition of type-II collagen peptides (four of fifteen patients) and type-I collagen peptides (three of twelve patients). None of the patients with mild or inactive disease responded to type-II collagen. With one exception, the addition of collagen peptides to monolayer cultures did not stimulate collagenase secretion. An increased production of factors that enhance or inhibit the migration of polymorphonuclear leukocytes was also observed in explant cultures in response to the addition of phytohemagglutinin (in seven of twelve patients), of type-I collagen peptides (in five of ten patients), and of type-II collagen peptides (in five of eleven patients). There was no correlation between production of these factors and stimulation of collagenase secretion in rheumatoid synovial tissue.

Arthritis, Rheumatoid↗
PubMed1982

Secretion of a thiol proteinase from mouse mammary carcinomas and its characterization.

Spontaneous mammary tumors from C3H/HeJ mice and transplants established from mammary tumors were investigated for their capacity to secrete thiol-dependent proteinase activity in organ culture explants. More activity was detected in culture media from spontaneous tumors than from transplanted spontaneous tumors. The accumulation of thiol proteinase in the culture medium was inhibited by cycloheximide, hydrocortisone, and aldosterone, but not by estradiol or the peptide hormones insulin or prolactin. The thiol proteinase is similar in enzymic properties to lysosomal cathepsin B, but its physical properties are different. It is stable to alkaline pH, has a larger molecular size on gel filtration (relative M.W. 39,000) and shows a different isoenzyme pattern to liver cathepsin B on analytical isoelectric focusing. The characteristics of this thiol proteinase are very similar to an enzyme secreted from malignant human breast tumors.

2-Naphthylamine↗
PubMed1981

Identification of core protein, an intermediate in proteoglycan biosynthesis in cultured chondrocytes from the Swarm rat chondrosarcoma.

After incubating cultured chondrocytes from the Swarm rat chondrosarcoma for 30 min with [3H]serine, a labeled macromolecule was found predominantly as Mr = approximately 370,000 species which was subsequently identified as a core protein precursor to cartilage proteoglycan from the following properties: (a) it was immunoprecipitated along with completed proteoglycan from cell extracts by an antiserum to the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid. Its immunoprecipitation could be inhibited completely by the addition of purified hyaluronic acid-binding region to the extracts, indicating the presence of common antigenic determinants with this region of the proteoglycan core protein. (b) the core protein precursor was able to interact with the hyaluronic acid and link protein in proteoglycan aggregates added as carrier to extracts to form mixed aggregates of high buoyant density in associative CsCl density gradients. Labeled core protein precursor and link protein were subsequently isolated from the mixed aggregates from the top of dissociative CsCl density gradients. (c) radioactivity in core protein precursor after a 30-min pulse of [3H]serine disappeared after inhibiting further protein synthesis with cycloheximide concurrent with the appearance of label in completed proteoglycan molecules.

Animals↗