PubMed Health⌕ Search

Biomedical subjects

C J Handley

Publications and source records attributed to C J Handley.

At least 37 records · Page 2Linked to original sources
PubMed1995

Antibody reactivity to mycobacterial 65 kDa heat shock protein: relevance to autoimmunity.

Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule.

Amino Acid Sequence↗
PubMed1994

Polymorphonuclear neutrophils release 35S-labelled proteoglycans into cartilage during frustrated phagocytosis.

Rabbit peritoneal polymorphonuclear neutrophils (PMN), incubated in medium containing [35S]sulphate, incorporated 35S into proteoglycan and protein fractions. Approximately 46% of the 35S-labelled macromolecules associated with the PMN cells after 1 h of incubation were recovered in a cytoplasmic granule extract, the majority being present in azurophil granules. Analysis of the azurophil granule fraction showed that approximately 90% of the 35S-labelled macromolecules were proteoglycans. When challenged with heat-aggregated rabbit gamma-globulin in the presence of cytochalasin B and cGMP, PMN were induced to release granular enzymes but did not release 35S-labelled proteoglycans into the incubation medium. When incubated with articular cartilage slices, PMN released their granule 35S-labelled proteoglycan into the medium and into the cartilage matrix. Granule enzymes and 35S-labelled granule proteoglycan were extracted from the cartilage tissue after incubation and 35S-labelled macromolecules were detected in the cartilage tissue by autoradiography.

Animals↗
PubMed1994

Characterization of extracellular matrix macromolecules from bovine synovial capsule.

Synovial capsule from the metacarpophalangeal joints of cattle was shown to be a highly collagenous tissue, with a hydroxyproline content of 100 +/- 1 micrograms/mg dry weight and a water content of 70 +/- 3.6%. Type-I collagen made up 83% of the collagen present, and the remainder was type III. When incubated in explant culture, synovial capsule incorporated [3H]acetate into both glycoproteins and hyaluronan and [3H]acetate and [35S]sulfate into proteoglycans. The rate of synthesis of proteoglycans by synovial tissue was shown to be similar to that measured for collateral ligament from the same joint. Two populations of proteoglycans were observed to be synthesized by synovial capsule. More than 90% of the 35S-labelled proteoglycans eluted with a K(av) of 0.7 on Sepharose CL-4B, and the remainder of the radiolabelled macromolecules eluted from the column with a K(av) of less than 0.5. Analysis of the major population of proteoglycans showed it to consist of a dermatan sulfate-containing proteoglycan with a core protein of 45,000 Da that had the same N-terminal amino acid sequence as decorin.

Animals↗
PubMed1994

Immunochemical studies of opsonic epitopes of the lipopolysaccharide of Leptospira interrogans serovar hardjo.

Leptospiral lipopolysaccharides (LPS) are the main antigens responsible for immunity in leptospirosis. In this investigation we studied the nature of the antigenic determinants of LPS extracted from Leptospira interrogans serovar hardjo (reference strain Hardjoprajitno). The reactions of anti-LPS monoclonal antibodies (mAbs) MUM/F1-4/hardjo (IgM) and MUM/F1-6/hardjo (IgG) with whole cell lysates in Western immunoblotting analysis were unaffected by proteinase K treatment. Periodate treatment of the LPS destroyed the binding of MUM/F1-6/hardjo but preserved that of MUM/F1-4/hardjo. Alkaline phosphatase decreased significantly the binding of MUM/F1-4/hardjo to the LPS but only slightly that of MUM/F1-6/hardjo. On the other hand, phosphodiesterase totally destroyed the binding capacity of both monoclonal antibodies in enzyme immunoassays (EIA). A number of mono- and oligosaccharides was used in EIA inhibition studies. Mannose-6-phosphate and galactose-6-phosphate inhibited the binding of MUM/F1-4/hardjo (50% inhibition at a concentration of 5 mM) to the antigen, but glucose-6-phosphate did not. Galactosamine and mannosamine inhibited the binding of MUM/F1-6/hardjo (50% inhibition at a concentration of 3-4 mM), whereas only a weak inhibition was observed with glucosamine. In contrast, N-acetylated amino sugars did not show any inhibition. An O-acetyl group also appears to be involved in the antigen-antibody binding process.

Antibodies, Bacterial↗
PubMed1993

Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin B and an inhibitor of matrix metalloproteinases. Evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation.

OBJECTIVE: To investigate mechanisms of cartilage matrix destruction by a study of the effects of a specific inactivator of cathepsin B and an inhibitor of several matrix metalloproteinases (MMP) on cartilage proteoglycan release. METHODS: Cartilage explants were treated with either recombinant human interleukin-1 alpha (rHuIL-1 alpha) or retinoic acid in the presence or absence of the inhibitors, and proteoglycan release was quantitated. Tests for nonspecific effects of the inhibitors included reversibility, rates of protein synthesis and glycolysis, and effects on other rHuIL-1 alpha-mediated events. RESULTS: The cathepsin B inactivator inhibited rHuIL-1 alpha-stimulated proteoglycan release at nanomolar concentrations, but failed to significantly inhibit retinoic acid-stimulated proteoglycan release. An inhibitor of MMP was inhibitory to both rHuIL-1 alpha-stimulated release and retinoic acid-stimulated release. CONCLUSION: Cathepsin B is implicated in rHuIL-1 alpha-stimulated loss of cartilage proteoglycan. Its lack of involvement in retinoic acid-stimulated proteoglycan release suggests the existence of at least 2 pathways of cartilage proteoglycan breakdown, which may converge at the activation of a matrix prometalloproteinase.

Aggrecans↗
PubMed1993

Intrasynovial levels of sulphated glycosaminoglycans and autoantibodies to type II collagen in rheumatoid arthritis: a correlative analysis.

It is uncertain whether the autoantibodies to type II collagen that occur frequently in the serum and synovial fluid of patients with rheumatoid arthritis (RA), but rarely in other articular diseases, are primary or secondary to cartilage damage. Hence, we measured antibodies in synovial fluid from patients with RA and other articular diseases and related these to the concentration of sulphated glycosaminoglycans, as a measure of ongoing cartilage catabolism. Synovial fluids from 42 patients with RA and 30 patients with other articular diseases were studied. We found that levels of antibodies to native and denatured collagen were significantly higher in RA than in all other articular diseases, whereas concentrations of sulphated glycosaminoglycans were similar. The absence of any correlation between levels of sulphated glycosaminoglycans and antibodies to collagen weighs against the occurrence of such antibodies in RA as a secondary effect of cartilage damage.

Adult↗
PubMed1992

Modulation of aggrecan and link-protein synthesis in articular cartilage.

The addition of serum or insulin-like growth factor-I (IGF-I) to the medium of explant cultures of bovine articular cartilage is known to stimulate the synthesis of aggrecan in a dose-dependent manner. The half-life of the pool of proteoglycan core protein was measured in adult articular cartilage cultured for 6 days in the presence and absence of 20 ng of IGF-I/ml and shown to be 24 min under both sets of conditions. The half-life of the mRNA pool coding for aggrecan was also determined and shown to be approx. 4 h in cartilage maintained in culture with or without IGF-I. The pool size of mRNA coding for aggrecan core protein increased 5-6-fold in cartilage explants maintained in culture in medium containing 20% (v/v) fetal-calf serum; however, in tissue maintained with medium containing IGF-I there was no increase in the cellular levels of this mRNA. This suggests that aggrecan synthesis is stimulated by IGF-I at the level of translation of mRNA coding for the core protein of this proteoglycan and that other growth factors are present in serum that stimulate aggrecan synthesis at the level of transcription of the core-protein gene. Inclusion of serum or IGF-I in the medium of cartilage explant cultures induced increases in the amounts of mRNA coding for type II collagen and link protein, whereas only serum enhanced the amount of mRNA for the core protein of decorin.

Aggrecans↗
PubMed1992

Synthesis of 35S-labelled macromolecules by polymorphonuclear neutrophils. Evidence for the production of [35S]sulphite which can modify both endogenous and exogenous proteins.

The incorporation of [35S]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of 35S-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80% of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that 35S-labelled macromolecules isolated from the cell extract migrated with Kav. of 0.36, while corresponding material isolated from the medium migrated with Kav. of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise 35S-labelled proteins of various molecular masses. The 35S label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [35S]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [35S]sulphite from [35S]sulphate. The release of [35S]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [35S]sulphite reacted with these proteins to form a stable 35S-labelled conjugate.

Animals↗
PubMed1992

Effect of insulin-like growth factor-I on the synthesis and distribution of link protein and hyaluronan in explant cultures of articular cartilage.

Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of explant cultures of adult articular cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate-namely link protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix proteins by articular cartilage compared with tissue maintained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of cartilage explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of link protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized link protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled link protein was lost from the matrix of the explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.

Aggrecans↗
PubMed1992

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.

Amino Acid Sequence↗
PubMed1992

Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants.

This article describes the effects of bound forms of insulin-like growth factors (IGFs) on proteoglycan metabolism by bovine articular cartilage in explant culture. When these growth factors were added to articular cartilage explants complexed with their native serum binding proteins (BPs), both IGF-I-BP complex and IGF-II-BP complex stimulated proteoglycan synthesis to different degrees over a 3-day period. When added to the medium of cultures of articular cartilage over 5 days, IGF-II-BP complex induced high rates of synthesis and low rates of catabolism of proteoglycans, giving rise to tissue levels of proteoglycan similar to those observed in fresh tissue. When articular cartilage was maintained in culture with the same concentration of IGF-I-BP complex, tissue levels of proteoglycans fell over the culture period because of lower rates of proteoglycan synthesis. Analysis of the proteoglycans synthesized by articular cartilage in the presence of free or bound IGF-I or IGF-II showed that these growth factors stimulated the rate of synthesis of the large proteoglycan species present in cartilage but did not affect the synthesis of the small proteoglycans.

Animals↗
PubMed1992

The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants.

Hyaluronan was shown to have the same turnover time as aggrecan in explant cultures of adult bovine articular cartilage. Inclusion of fetal calf serum in the culture medium resulted in a similar decrease in the rate of catabolism of both hyaluronan and proteoglycan. Less than 9% of the hyaluronan lost from the explants in the course of the experiment was recovered from the culture medium as hyaluronan, suggesting that the catabolism of hyaluronan involves the uptake of this glycosaminoglycan by the chondrocytes. Analysis of the molecular size of the newly synthesized hyaluronan in these cultures showed that the hyaluronan was initially synthesized as large macromolecules that were gradually depolymerized with time within the extracellular matrix. The resulting size distribution of newly synthesized hyaluronan molecules after 12 days in culture was similar to that determined for the endogenous hyaluronan. The kinetics of depolymerization of the newly synthesized hyaluronan was consistent with a random fragmentation of the macromolecule. The rate constants for the depolymerization of hyaluronan indicate that oxygen-derived radicals may be involved in the fragmentation of this macromolecule. Inclusion of either cycloheximide or proteinase inhibitors in the medium of the explant cultures resulted in a marked decrease in the rate of loss of hyaluronan from the tissue and in the inhibition of the depolymerization of the newly synthesized macromolecule. This suggests that both the catabolism and the depolymerization of hyaluronan are cell mediated and depend on metabolically active cells.

Age Factors↗
PubMed1992

Mechanism of catabolism of aggrecan by articular cartilage.

Characterization of aggrecan core protein peptides appearing in the medium of adult articular cartilage maintained in tissue culture showed that eight major peptides could be detected. The two largest peptides had the same N-terminal sequence as bovine aggrecan core protein and probably represent partly degraded aggrecan lost to the medium in the form of the proteoglycan aggregate. The three next smallest peptides were all shown to have another N-terminal sequence which corresponded to a sequence in the interglobular domain starting at alanine residue 393 of the human aggrecan core protein (K. Doege et al., 1991, J. Biol. Chem. 266, 894-902). Two other peptides were isolated and shown to have two different N-terminal amino sequences corresponding to sequences in the chondroitin sulfate attachment domain 2 of the core protein starting at alanine residue 1839 and leucine residue 1939 of human aggrecan. This suggests that the catabolism of aggrecan by adult articular cartilage occurs by the proteolytic cleavage of the core protein of this proteoglycan at three separate sites. Examination of the amino acid sequences around each of these cleavage sites showed a similar pattern TEGE decreases ARGS, TAQE decreases AGEG, and VSQE decreases LGQR, suggesting that a single proteinase may be involved in the catabolism of aggrecan. Analysis of synovial fluids and serum of age-matched animals revealed the presence of aggrecan core protein peptides corresponding in size to those detected in vitro, thus indicating the cleavage observed in explant culture is the same as that which occurs in vivo.

Aggrecans↗
PubMed1991

The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate.

The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains.

Animals↗
PubMed1990

Characterization and synthesis of macromolecules by adult collateral ligament.

Bovine collateral ligament was found to have a water content of 67.5 +/- 2.5%, the tissue was highly collagenous containing 100.3 +/- 15.1 micrograms hydroxyproline/mg dry weight. Type I collagen was the major collagen present with small amounts of Type III and V. The hexuronate content of the tissue was found to be 2.62 +/- 0.40 micrograms hexuronate/mg dry weight of tissue. On incubation in vitro collateral ligament incorporated [35S]sulfate and [3H]acetate into proteoglycans and [3H]acetate into hyaluronate and glycoproteins. The rate of synthesis of proteoglycans by collateral ligament was shown on a weight basis to be greater than that of tendon but lower than that of articular cartilage. Analysis of the proteoglycans present in collateral ligament showed two populations of proteoglycans to be present. Approx. 20% of the total proteoglycans present were large chondroitin- and keratan sulfate-containing proteoglycans capable of forming aggregates with hyaluronate. The major species of proteoglycan present were small dermatan sulfate proteoglycans made up of a core protein with a molecular mass of 45,000 daltons with one dermatan/chondroitin sulfate glycosaminoglycan chain of 30,000 daltons attached. The N-terminal amino acid sequence of the core protein of this proteoglycan showed it to be analogous to the core protein of dermatan sulfate proteoglycan II.

Amino Acid Sequence↗
PubMed1990

The N-terminal sequence of the large proteoglycan of articular cartilage.

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.

Amino Acid Sequence↗