Release Of 35S Research Articles

Murine B-cell leukemia lymphoma (BCL1) cells as a target for NK cell-mediated immunotherapy

Natural killer (NK) cells are important for their ability to recognize and lyse tumor cells and virus infected cells. NK cells express triggering receptors that are specific for non-MHC ligands. This article describes the 35S release cytotoxic assay, which measures the ability of NK cells derived from spleen cells taken from polyIC-treated mice to lyse B-cell leukemia (BCL1) cells. BCL1 cells express ligands for NKp46 on the cell surface membrane and they are sensitive to allogeneic but not syngeneic IL-2 activated natural killer cells.

Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz sand.

Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer,followed bythe slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and 35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is approximately 3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

Anti-interleukin-1 and anti-CD44 interventions producing significant inhibition of cartilage destruction in an in vitro model of cartilage invasion by rheumatoid arthritis synovial fibroblasts.

To establish an in vitro model for the investigation of destructive processes in rheumatoid arthritis (RA), to study the interaction between fibroblasts, macrophages, and chondrocytes, and to evaluate strategies to inhibit joint destruction in RA. Human and bovine chondrocytes cultured in sponges pretreated with native bovine embryonic extracellular matrix produced a cartilaginous matrix reflected by the incorporation of 35S into proteoglycans. The 3-dimensional culture system was optimized for the number of chondrocytes (10(5) cells/sponge), the timing of 35S incorporation (day 21 after chondrocyte isolation), and the medium (20% fetal calf serum). RA synovial fibroblasts (RASF; 10(5)) were added, and the matrix destruction mediated by these RASF was monitored by the release of 35S. The system was modulated by the addition of monocytes (U937 cells), cytokines (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]), interleukin-1 receptor antagonist (IL-1Ra), and monoclonal antibodies against IL-1beta and CD44. RASF destroyed the bovine cartilaginous matrix within 2 weeks (days 5-12) and the human cartilaginous matrix within 3 weeks (days 10-18). Compared with the effect of RASF alone (mean +/- SD 948 +/-180 counts per minute/week), the addition of U937 cells (a monocytic cell line), IL-1beta, or TNFalpha to the incubation medium increased the destruction of human cartilaginous matrix by at least 71% up to 90% (ranging from 1,618+/-204 cpm/week to 1,802+/-307 cpm/week). IL-1Ra and anti-IL-1beta monoclonal antibodies reduced the destruction of human matrix by 45% and 35%, respectively; this was partially reversed by the addition of U937 cells. The pretreatment of RASF with anti-CD44 monoclonal antibody (an adhesion molecule and receptor for hyaluronic acid) inhibited the destruction of the cartilaginous matrix by an average of 41% over 3 weeks. This model is envisioned to study distinct aspects of human destructive joint diseases under in vitro conditions and to replace and/or supplement animal experiments in basic research and drug testing. Based on the fact that proinflammatory cytokines enhance destruction whereas IL-1Ra and antibodies against IL-1beta and CD44 inhibit the process, it is concluded that anti-IL-1- and anti-CD44-directed therapies may help prevent cartilage destruction in RA.

Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes.

Cartilage degradation, a hallmark of rheumatoid arthritis, is attributed to serine and metalloproteases secreted by neutrophils, synovial lining cells, macrophages, and chondrocytes. A large proportion of synovial fluid lymphocytes contains the granule-associated serine proteases granzymes A and B. We report that lysates of IL-2-stimulated lymphocytes contain an enzymatic activity (ECMase; cartilage extracellular matrix 35S release assay; extracellular matrix degrading activity) that solubilizes matrix synthesized by chondrocyte monolayers. ECMase activity is inactivated by the serine protease inhibitor diisopropylfluorophosphate, is stored in dense granules and cleaves aggrecan proteoglycans but not free glycosaminoglycans, hyaluronic acid, or type II collagen. ECMase is mediated by a cationic protein with biochemical properties identical to granzyme B, inasmuch as it preferentially hydrolyzes the substrate Boc-Ala-Ala-Asp-SBzl, immunochemically cross-reacts with an antibody that binds to a conserved amino-terminal region of lymphoid-myeloid serine proteases, and has amino-terminal sequence identity with human Q31 granzyme B. Using an agarose gel electrophoresis technique to assess cleavage of the rat sarcoma aggrecan, the catalytic efficiency of granzyme B for the digestion of aggrecan (catalytic efficiency = 1.7 x 10(7) M-1 s-1) was 425-fold faster than the catalytic efficiency reported for human stromelysin-1 at pH 7.5 (catalytic efficiency 4000 M-1 s-1) and 3200-fold faster than granzyme A. Based on these observations, we propose that granzyme B, secreted from cytotoxic lymphocytes within the rheumatoid joint, may contribute to cartilage loss by degrading resident aggrecan.

Effect of hylan on cartilage and chondrocyte cultures

The protective role of hylan, a hyaluronan [hyaluronic acid (HA)] derivative, was studied in explanted bovine cartilage and isolated chondrocytes. Cartilage and chondrocytes were exposed to degradative enzymes (lysate from activated polymorphonuclear leukocytes), oxygen-derived free radicals (ODFR), conditioned media from mononuclear cells (MCCM), and interleukin-1 (IL-1), in the presence and absence of hylan. The effect of HA was also studied. In cartilage explants susceptibility to pertubation was evaluated in terms of 35S release and proteoglycan depletion and was compared to control cultures; high viscosity hylan was found to reduce 35S release in cartilage explants caused by degradative enzymes, ODFR, MCCM, and IL-1. The hylan effect was reversible and viscosity-dependent. In chondrocyte cultures, high viscosity hylan was effective in reducing cell injury caused by degradative enzymes and ODFR. The data suggest that the glycosaminoglycan hylan, as well as native HA, may mediate exposure to and/or response to stimuli associated with initiation of degenerative processes in cartilage tissues.

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO42− reduction rates in the sediment, (2) depletion of SO42− in the overlying water column and (3) release of35S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO42− in the overlying water.

Mechanical stimulation by intermittent compression stimulates sulfate incorporation and matrix mineralization in fetal mouse long-bone rudiments under serum-free conditions

Mechanical stimulation evoked by intermittent hydrostatic compression (IC) in a closed culture system has been shown to stimulate calcification of fetal long-bone rudiments in the presence of serum [6]. We have studied effects of IC on sulfate metabolism and matrix mineralization under serum-free conditions, in short-term (24 hours) cultures of mineralizing long-bone rudiments in alpha minimum essential medium (MEM) + 0.2% bovine serum albumen (BSA). Exposure to IC for 24 hours stimulated radiosulfate incorporation into the papain-digestible pool in the noncalcifying epiphyses and, to a larger extent, in the calcifying diaphysis. The percentage release of 35S from prelabeled rudiments was stimulated in the epiphyses, but inhibited in the diaphyses. The changes in sulfate metabolism of matrix mineralization, in hypertrophic cartilage, and the diaphyseal bone collar, were judged from the increase in length of the diaphysis. This study shows that under serum-free conditions, mechanical stimulation by IC increases sulfate content while stimulating mineralization in calcifying cartilage of fetal long-bone rudiments. Mechanical stimulation seems to be an important regulator of cartilage calcification.

The functional significance of the sympathetic innervation of mucous glands in the bronchi of man.

1. Pieces of human bronchi, from lung resected for carcinoma of the bronchus, were mounted in Ussing chambers and given [35S]sulphate as radiolabelled precursor of mucous glycoproteins (mucins). The release of 35S, bound to macromolecules, into the luminal half-chamber was used as an index of mucin secretion. 2. Noradrenaline, at concentrations of 1, 10 and 100 microM, was given into both halves of the Ussing chamber. At the lowest concentration, noradrenaline failed to change mucin output, but at the two higher concentrations it stimulated output. 3. In other experiments the sympathetic nerves in the bronchial wall were labelled with 5-hydroxydopamine and examined under the electron microscope. The distances between adrenergic nerve varicosities and submucosal glands were measured; some sympathetic nerve varicosities were seen within 1 microns of gland cells. 4. A simple mathematical model for the diffusion of noradrenaline was used to predict the concentrations of the transmitter likely to result at different distances from a nerve if one or more vesicles of noradrenaline were released. 5. The model predicts that the release of a single large vesicle of noradrenaline is likely to generate an effective concentration of transmitter provided that the nerve is within 1 micron of the target cell.

Autoradiographic studies of the distribution of 35sulfate label in ferret trachea: effects of stimulation.

Na2(35)SO4 is among the common isotopic mucin precursors used to label airway secretory cells and monitor their active discharge in response to drugs. Previous work has established that 35S is taken up by multiple cell types in the trachea, yet there is no direct evidence linking 35S release with secretory activity by any specific cell type. In this study, we have used autoradiography to identify the sites of uptake and release of 35S in ferret trachea. Confirming work performed in other species, we found uptake sites include surface epithelium (ciliated and goblet cells), submucosal glands (serous and mucous cells) and cartilage. Extending these findings using a "pulse-chase" protocol, we found that 35S turns over very rapidly in ciliated but not submucosal gland cells or cartilage. Specific grain density over epithelium declined from 0.12 +/-0.006 grains/micron 2 immediately after the pulse to 0.05 +/- 0.004 grains/micron 2 at 4 h. In contrast, corresponding figures for the glands and cartilage showed no spontaneous loss of label during the same period. At 4 h, when the epithelium contained very little 35S label, we exposed tracheal rings in vitro to neurotransmitter receptor agonists (bethanechol, phenylephrine, and isoproterenol; all at 10(-5) M). Counts in the medium (determined by scintillation spectrometry) increased 2-3 times in response to each agonist. These increases were prevented by preincubating tracheal rings with appropriate antagonists. Autoradiography showed that stimulated glands contained many fewer silver grains than untreated or antagonist-blocked glands. In contrast, neither cartilage nor epithelium showed decreased labeling after stimulation. These results indicate that (a) sulfated glycoconjugates turn over rapidly in the tracheal epithelium and may account for much sulfated material spontaneously released into organ culture medium and into the tracheal lumen, and (b) 4 h after Na2(35)SO4 incubation, the major source of 35S-labeled macromolecules released from ferret trachea by neural agonists is the submucosal glands.

Control of mucoglycoprotein output from the rabbit nose.

A method for the collection of rabbit nasal washings to analyse outputs of mucous glycoproteins is described. The radiolabels sodium [35S]sulphate and [3H]glucose are bound to the glycoproteins. The release of bound 35S and 3H was enhanced by cervical sympathetic nerve stimulation. Adrenoceptor agonists (phenylephrine, dobutamine and salbutamol) given I.V. increased the output of 35S, and the last two drugs increased the output of 3H. The blocking effects of thymoxamine and propranolol on these responses are described. Pilocarpine (given I.V. or intranasally) produced large increases in 35S release; histamine had little effect. Irritants (ammonia and cigarette smoke) and diluted serum or plasma, given intranasally, produced large increases in 3H output, and sometimes enhanced 35S release. Radiolabelled nasal washings fractionated on Sepharose CL-4B gel chromatography columns formed two peaks, with most of the radioactivity in the high molecular weight (mucous glycoprotein) peak.

Quantitative relationship between histamine and 35S release from isolated rat mast cells due to the beta-adrenoceptor blocking drug Kö 1124

Quantitative relationship between histamine and 35S release from isolated rat mast cells due to the beta-adrenoceptor blocking drug Kö 1124

Quantitative correlation between histamine and 35S release from isolated rat mast cells due to the beta-adrenergic blocking drug Kö 1124.

An attempt was made to explain the mechanism of histamine release from isolated rat mast cells induced by the beta-adrenergic blocking drug Kö 1124. This drug at the highest concentration used released 12 times more histamine than most other investigated beta-blockers. The release of histamine with Kö 1124 was dose and temperature dependent. The maximal histamine release was at pH 8 and in the absence of calcium ions. Increased calcium concentration decreased histamine release significantly. The effect of Kö 1124 on histamine release from mast cells was inhibited only by cocaine and 2,4-dinitrophenol; other metabolic inhibitors were ineffective. The histamine release due to Kö 1124 was not followed by an equal release of 35S. Isoprenaline in equimolar concentration decreased histamine release induced by Kö 1124 significantly. The release of 35S-labelled granules was decreased or blocked by isoprenaline.

Distribution of 35S labeled sulfated mucopolysaccharids in the recipient eye after corneal allografting

The release of 35S labeled corneal glycoprotein from an allogenic interlamellar corneal graft was studied in randomly selected rabbits. Simultaneously, the uptake of 35S by the recipient animal was investigated. These changes followed for a period of 80 h indicate that there is a constant 35S release fromthe donor cornea and that it is the recipient cornea which takes up the major part of the 35S from the donor. The iris, choroid, and sclera of the recipient also take up a certain percentage of the donor's 35S while the aqueous humor, the optic nerve, and the local lymph nodes take up only minimal amounts.

Breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates from guinea pig, rabbit, and human

Degradation of the noncollagenous protein-mucopolysaccharide matrix of rabbit ear cartilage in vitro by various enzyme preparations is reported. Cartilage matrix degradation was quantitated by measuring the release of soluble, 35S-labeled, anionic material from cartilage that was previously labeled in vivo with Na 2 35SO 4. The release of 35S was specific for chondromucoprotein degradation, as evidenced by the parallel release of mucopolysaccharide split products, precipitation of 35S with cetyltrimethyl ammonium bromide and enzyme specificity. Autolytic breakdown of chondromucoprotein proceeded predominantly at pH 5.0 and to a very limited extent at pH 7.4 during an 18-hr incubation at 37°C. Trypsin, chymotrypsin, and elastase provoked chondromucoprotein degradation at pH 7.4, whereas hyaluronidase was more active at pH 5.0. Purified bacterial collagenase did not promote significant breakdown of the noncollagenous portion of cartilate matrix at neutral or acid pH. Lysosome granule lysates of guinea pig and rabbit peritoneal polymor-phonuclear leukocytes were capable of degrading the chondromucoprotein of cartilage matrix at pH 5.0 but not at pH 7.4. Conversely, lysates of granules from human leukocytes damaged cartilage matrix at pH 7.4 but not at pH 5.0. Gold sodium thiomalate (10 −4 M) and human serum (10%) inhibited the degradation of guinea pig, matrix provoked by autolysis and by the leukocyte granuleslysates of guinea pig, rabbit and human. The demonstration in human leukocyte granules of neutral protease(s) that damage the noncollagenous chondromucoprotein matrix of cartilage and the finding that gold, an antirheumatic drug, inhibits this deleterious effect, may have a significant bearing on the pathology of connective tissue disease.

Altered mucopolysaccharide metabolism in organ cultures of fetal hamster bones infected by the H-1 strain of parovirus.

AbstractInfection with the H‐1 strain of parvovirus results in death and deformities in hamster embryos, retardation of skeletal growth and development in immature hamsters, and delayed repair of osseous wounds in older animals. Several aspects of this interference with bone growth can be reproduced as a laboratory model by infection of organ cultures of fetal hamster tibiae with H‐1 virus. To investigate the pathogenesis of this virus‐induced retardation of skeletal growth, the incorporation and release of 35S by infected and control tibiae was measured in this organ culture model to determine the effect of H‐1 virus infection on the rates of synthesis and loss of mucopolysaccharides by fetal bone. Ash weights and mineral‐free dry weights of infected and control bones were also compared to detect differences in degrees of mineralization and cartilage formation. By these methods, growth retardation was found to be associated with decreased cartilage formation and increased synthesis and loss of mucopolysaccharides or their components. No difference was found between the mineral contents of infected and control bones. It is proposed that H‐1 virus infection interferes with formation of the matrix of cartilage and that intensified 35S incorporation reflects a compensatory but ineffective effort to reverse this inhibition by increasing mucopolysaccharide synthesis. Although no quantitative difference was detected between the mineral contents of infected and control bones, it is suggested that disordered matrix formation may contribute to the delayed or incomplete mineralization of osseous tissues that occurs during later stages of maturation in this animal model of virus‐induced dwarfism.

RELEASE OF CARTILAGE MUCOPOLYSACCHARIDE-DEGRADING NEUTRAL PROTEASE FROM HUMAN LEUKOCYTES

The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of (35)S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of (35)S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 10(6) cells provoked two to four times more (35)S and beta-glucuronidase (beta-G) release from cartilage than phagocytic enzyme release conditions. beta-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and beta-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10(-5)M colchicine, whereas the protease activity, but not the release process, was inhibited by 10(-6)M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis.

Quantitative correlation between degranulation and histamine release following exposure of rat mast cells to compound 48-80 in vitro.

AbstractThe relationship between the release of histamine and 35S‐labelled heparin from mast cells exposed to compound 48/80 in vitro has been studied in order to elucidate the mechanism of histamine release induced. The cells were taken from rats injected 3, 6, 12 or 20 days earlier with Na235SO4. A good correlation was found between the two release curves. From studies of the 35S/protein ratio in the extruded granules and granules obtained by water lysis of the washed compound 48/80‐treated cells it appears that the 35S‐labelled heparin was first evenly distributed with respect to the granule protein 12 to 20 days after injection. Using cells taken after such an interval the ratio percentage histamine release/percentage 35S release was found to be 1.1–1.4. We conclude from these results and from the similarity between the time courses of histamine and 35S release that the mechanism of histamine release induced by compound 48/80 involves an initial extrusion of histamine‐cotnaining granules, followed by an exchange of histamine in the extruded granules and cations in the extracellular medium.