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Biomedical subjects

R W Moskowitz

Publications and source records attributed to R W Moskowitz.

At least 91 records · Page 5Linked to original sources
PubMed1984

Biosynthesis of sulfated proteoglycan in vitro by cells derived from human osteochondrophytic spurs of the femoral head.

Cells derived from organ-explant culture of the cartilaginous component of osteochondrophytic spurs of human femoral heads were incubated with [35S]-sulfate in order to study sulfated proteoglycan biosynthesis in vitro. Secondary monolayer cultures incorporated [35S]-sulfate into macromolecules which were recovered in the bottom fraction (dA1) of a CsCl gradient after ultracentrifugation in associative buffer (0.5 M guanidine X HCl). The incorporated [35S]-sulfate in fraction dA1 from the culture medium eluted in two peaks with average partition coefficients (Kav) of 0.14 and 0.45 respectively, on Sepharose CL-2B eluted with dissociative buffer (4 M guanidine X HCl). A significant percentage of incorporated [35S]-sulfate was found in the medium dA4 fraction (44%). The Kav of this fraction on Sepharose CL-2B was 0.66 with a shoulder of incorporated [35S]-sulfate at Kav, 0.22. In contrast to the culture medium, cellular CsCl gradient fractions dA1-dA3 showed Kav's on Sepharose CL-2B ranging from 0.63-0.75. Cellular fraction dA4 was even more polydisperse. A dD1 fraction (proteoglycan monomer) prepared by CsCl ultracentrifugation in dissociative buffer of [35S]-sulfate labelled culture medium eluted with a Kav of 0.25 on Sepharose CL-2B identical to the Kav of bovine nasal cartilage A1D1 and human tissue osteophyte A1D1 chromatographed under identical conditions. Glycosaminoglycan analysis demonstrated significant amounts of chondroitin 6- and 4-sulfate in unfractionated culture medium and in those proteoglycan fractions generated from culture medium (dA1, dA2 and dD1). In contrast, cellular fractions dA1-dA3 and medium fraction dA4 were enriched in dermatan sulfate. The size of the [35S]-sulfated glycosaminoglycan chains analyzed by Sepharose CL-6B chromatography showed considerable polydispersity (Kav range, 0.29-0.52). The results of this study indicated that cells derived from the cartilaginous component of human osteophyte synthesized several distinct populations of sulfated proteoglycans. These results may reflect the heterogeneity of cells which grow out from osteophyte organ explants and become established in monolayer culture.

Cells, Cultured↗
PubMed1984

Application of new techniques to separation of proteoglycan aggregates from normal and destabilized rabbit articular cartilages.

Proteoglycans were prepared from rabbit articular cartilages by classical techniques employing 4.0 M guanidine. HCl by transport ultracentrifugation techniques on the purified proteoglycans, present. A new method of extracting the cartilage with 0.4 M guanidine. HCl in the presence of highly purified collagenase is presented. The same yield of proteoglycans on extraction of normal cartilage was obtained as with the classical technique, but a larger proportion of intermediate and large aggregates was obtained with the new than with the classical methodologies. The osteoarthritic cartilage was obtained from 6 month old animals, 3 months after a partial medial meniscectomy had been performed. The profile of proteoglycans from osteoarthritic cartilage consisted predominately of monomers, and a small content of aggregates spread over intermediate and large size ranges. It is postulated that by the methods of extraction, the profile of proteoglycan aggregates present in vivo is more faithfully reproduced than obtained by the classical methodologies.

Animals↗
PubMed1984

IgG-induced experimental immune synovitis: hormonal modulation of in vitro splenic immune responses to homologous antigens.

The effect of oestrogen or anti-oestrogen administration on gross pathology and in vitro cell-mediated immune responses to homologous IgG, native and denatured interstitial collagens and PPD was studied in an IgG-induced rabbit model of immune synovitis. During induction of synovitis, rabbits were administered oestradiol valerate (0.075 mg/kg/day) or tamoxifen, an anti-oestrogen (2.0 mg/kg/day, high dose or 0.5 mg/kg/day, low dose) or placebo injections. Low dose tamoxifen administration was associated with significant improvement P less than 0.05 in immune synovitis with regard to gross pathology, when compared to placebo and the oestradiol treatment group. High dose tamoxifen treatment was not associated with significant improvement in observed synovitis. With regard to cell-mediated immune responses, spleen cells derived from immune synovitis rabbits were observed to increase 3H-thymidine uptake on incubation with native or denatured homologous collagens. Modulation of these immune responses to antigens was observed in anti-oestrogen treated rabbits with immune synovitis. In vitro cell-mediated immune responses to denatured type I, II and III collagens, PPD, as well as native type II and III collagens were not observed in the low dose tamoxifen treatment group. However, in vitro immune responses to these antigens were observed in spleen cell cultures from immune synovitis rabbits treated with either high dose tamoxifen or oestradiol valerate. The data suggest that in vivo anti-oestrogen administration can modulate the in vitro cell-mediated immune response to connective tissue constituents observed in immune synovitis. Concomitant with reduced immune responses is a significant reduction in the observed lesions of the inflammatory response.

Animals↗
PubMed1983

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways.

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.

Animals↗
PubMed1983

Phospholipase C activity in plasma membranes isolated from lapine synovial cells in monolayer culture.

Plasma membranes were isolated from lapine synovial cells grown in monolayer culture using discontinuous sucrose gradient centrifugation techniques. 5'nucleotidase was detected in great abundance while glucose-6 phosphate dehydrogenase and cytochrome oxidase were present at low to undetectable levels. Plasma membranes incubated at 37 degrees C for 60 min with [3H]-arachidonyl-phosphatidylinositol/phosphatidylserine synthesized [3H]-diacylglycerides. Little if any [3H]-diacylglyceride synthesis was measured when [3H]-arachidonyl-phosphatidylcholine or [3H]-arachidonyl-phosphatidylethanolamine were used as substrates. These results are consistent with a plasma membrane-associated phosphatidylinositol-specific phospholipase C from lapine synovial cells in culture.

Animals↗
PubMed1983

'Gelatinase-like' activity from articular chondrocytes in monolayer culture.

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TCA and TCB fragments of human Type II collagen into smaller peptides at 37 degrees C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1.10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.

Animals↗
PubMed1983

Arthritis of hemochromatosis. Clinical spectrum, relation to histocompatibility antigens, and effectiveness of early phlebotomy.

Five patients who presented with arthritis as the sole manifestation of hereditary hemochromatosis and 51 family members were studied. Studies included clinical evaluation for the presence of arthritis and hemochromatosis, roentgenography of hands, knees, and pelvis, serum iron and serum ferritin measurements, complete HLA typing for 50 of the A and B loci, and, when indicated, liver biopsy. Arthritis occurred in 45 percent of persons with hemochromatosis. Although typical involvement of second and third metacarpophalangeal joints was observed in all five patients and some family members, two with typical arthritis did not have characteristic radiographic changes, two had constitutional symptoms without arthropathy, and one had unilateral hand changes. A specific HLA haplotype (A2/B17 in Family 1 and A29/B15 in Family 2) correlated with hereditary hemochromatosis but not with the arthropathy. Phlebotomy alleviated the early constitutional symptoms but did not help advanced arthritis. Anti-inflammatory drugs, intraarticular injections of glucocorticoids, and resection osteotomies of metacarpal heads were other treatment modalities.

Anti-Inflammatory Agents↗
PubMed1983

Estradiol and tamoxifen stimulation of lapine articular chondrocyte prostaglandin synthesis.

The effect of estradiol and tamoxifen on prostaglandin (PG) synthesis by rabbit articular chondrocytes in secondary monolayer cultures was investigated. Radioimmunoassay for PGE2, PGF2 alpha, 6-oxo-PGF1 alpha and thromboxane B2 was performed on media from cultures containing estradiol and tamoxifen (10-12M-10-7M). Radiometric thin-layer chromatography was also carried out. The time course of estradiol/tamoxifen effect on chondrocyte PG synthesis was evaluated and its relationship to cell density in culture examined. Estradiol stimulated the synthesis of PGs by chondrocytes. Stimulation was noted at picomolar concentrations of estradiol without further stimulation at markedly higher concentrations. In time studies, after a lag, the effect of estradiol was present fully by 5 hrs, remained steady for 24 hrs and then declined by 48 hrs. Estradiol stimulation of PG synthesis was dependent upon chondrocyte culture plating density. Tamoxifen stimulated chondrocyte PG synthesis to relatively lower levels than estradiol. The characteristics of estradiol/tamoxifen stimulation of chondrocyte PG synthesis suggest a mechanism involving estradiol cytoplasmic receptors.

Animals↗
PubMed1983

Intraarticular hyaluronic acid injection and synovial prostaglandins in experimental immune synovitis.

In vitro studies suggest that synovial fluid hyaluronic acid may have a role in reducing joint inflammation. The effect of intraarticular injection of sodium hyaluronate in a model of experimental immune synovitis was assessed. In addition, tissue prostaglandin content of synovia with and without immune synovitis was compared. Intraarticular hyaluronic acid administered at 2 doses was not effective in reducing the induced inflammation. With immune synovitis there was an increase in the total synovial prostaglandins. When related to total prostaglandins, prostacyclin was decreased, prostaglandin F2 alpha and thromboxane were increased and prostaglandin E2 was the same in synovitis as compared to controls.

Animals↗
PubMed1982

Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs.

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [(35)S]sulphate or [(14)C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [(35)S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [(35)S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60-80% of the [(35)S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (K(av.)) of the proteoglycan monomer on Sepharose CL-2B was 0.28-0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2-D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [(35)S]sulphate and [(14)C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60-70%), with 9-11% keratan sulphate in the monomer proteoglycan; (3) K(av.) values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH(4)-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

Cartilage↗
PubMed1982

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes.

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.

6-Ketoprostaglandin F1 alpha↗
PubMed1982

Response of articular chondrocytes to pituitary fibroblast growth factor (FGF).

Rabbit chondrocytes from pooled articular joints have been delineated by their time of attachment to culture flasks after initiation of primary monolayer culture, either attached (48-AT) or floating (48-F) after 48 hours. A general population of chondrocytes (attached after 72 hours, 72-AT) was also studied. The growth-promoting activity of pituitary fibroblast growth factor (FGF) and its effect on sulfated-proteoglycan synthesis was studied on each chondrocyte population in secondary monolayer culture. 3H-thymidine incorporation during a 1-hour pulse was stimulated by FGF (100 ng/ml) in each chondrocyte population. The response of AT-72 chondrocytes to FGF required an additional fetal bovine serum supplement, while 48-F cells responded independent of serum. The response of 48-AT chondrocytes to FGF (100 ng/ml) during a 1-hour pulse with 3H-thymidine was increased in low serum (0.5-2.0%) rather than when high serum (8-10%) was present in the culture medium. FGF reduced 35SO4 incorporation into sulfated-proteoglycans in the 48-AT and 48-F chondrocyte populations, but not in the 72-AT population. The reduction in 35SO4 incorporation in the 48-AT and 48-F chondrocytes was not characterized by alterations in the hydrodynamic size of the sulfated-proteoglycans as measured by Sepharose CL-2B chromatography nor by changes in the types of sulfated-glycosaminoglycans produced. These results indicated that FGF produced quantitative rather than qualitative alterations in chondrocyte sulfated-proteoglycan synthesis. The latter appears uncoupled from the growth-promoting activity of FGF on chondrocytes.

Animals↗
PubMed1981

Neutral proteinases from articular chondrocytes in culture. 2. Metal-dependent latent neutral proteoglycanase, and inhibitory activity.

Monolayer and spinner cultured rabbit articular chondrocytes released into the medium latent metal-dependent enzyme with activity against bovine proteoglycan. Pretreatment of medium with p-aminophenylmercuric acetate or trypsin followed by soybean trypsin inhibitor significantly increased enzyme activity. The monolayer-cultured chondrocytes released more of this activity than spinner cultures. The neutral proteoglycanase activity increased with medium concentration and incubation time. Like the human cartilage proteoglycanase, its pH optimum on proteoglycan subunit was 7.25. Gel filtration on BioGel P-30 indicated that the proteoglycanase occurred in two molecular weight forms: 20 000--30 000 and 13 000. The latent enzyme was about 30 000--40 000. The metal-chelators, o-phenanthroline (5 mM) and EDTA (10 mM) inhibited the activated proteoglycanase almost completely, but trypsin and chymotrypsin inhibitors had little effect. The cultured chondrocytes also released into the media a heat-labile inhibitor against the proteoglycanase. The inhibitory activity was present in the nonactivated media and eluted on Sephadex G-100 chiefly at a position corresponding to molecular weights of 10 000--13 000.

Animals↗
PubMed1981

Prostaglandin biosynthesis by lapine articular chondrocytes in culture.

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostacyclin) and prostaglandin F2 alpha. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B2 (the stable metabolite of thromboxane A2). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [3H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E2 relative to 6-oxo-prostaglandin F1 alpha than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prostaglandins found in articular cartilage. Minimal quantities of thromboxane B2 were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F1 alpha relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prostaglandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.

6-Ketoprostaglandin F1 alpha↗
PubMed1981

Neutral proteinases from articular chondrocytes in culture. I. A latent collagenase that degrades human cartilage type II collagen.

Culture media collected from secondary monolayer and spinner cultures of rabbit articular chondrocytes showed evidence of collagenolytic activity by the following criteria: (1) Amicon PM-10 concentrates of culture medium released [14C] glycine from reconstituted rabbit skin collagen fibrils at 37 degrees C; (2) medium concentrated by lyophilization decreased the relative viscosity of human cartilage collagen in solution. The loss in viscosity was partially inhibited if medium was preincubated with o-phenanthroline, and (3) degradation of human cartilage collagen after 60 h incubation at 24 degrees C was characterized primarily by the appearance of 75 000 dalton (TCA) and 25 000 dalton ((TCB) products. The majority of the collagenase (EC 3.4.24.3) from cultured chondrocytes was secreted in latent form, since preincubation with either trypsin or p-aminophenylmercuric acetate significantly increased activity against human cartilage collagen. Chondrocyte collagenase may be important in mediating the normal slow turnover of cartilage collagen and may be particularly active in collagen destruction associated with early stages of synovial joint arthritides, before attack by non-cartilage cells or extra-articular soft tissues.

Animals↗