Biomedical subjects

C R Parish

Publications and source records attributed to C R Parish.

At least 91 records · Page 5Linked to original sources

PubMed1988

Monosaccharide inhibition of cytotoxic T-cell function: demonstration of clone-specific effects.

A range of monosaccharides has been tested for their capacity to influence the induction and effector function of alloreactive cytotoxic T (Tc) cells. Strain-specific differences in the capacity of monosaccharides to inhibit Tc cell induction have been demonstrated. Monosaccharides can also inhibit effector function of target cell lysis, but this could only be demonstrated by assessing the effect of sugars added to limiting dilution cultures of alloantigen-stimulated T cells. B10.A(4R) anti-BALB/c Tc cells have been reproducibly inhibited by D-glucosamine and D-galactosamine, as well as D-galacturonic acid, at both the induction and effector phases of the Tc cell response. Analysis of monosaccharide inhibition of cytotoxicity in limiting dilution cultures has confirmed that D-glucosamine is the most effective inhibitor of B10.A(4R) anti-BALB/c Tc cells, while D-galactosamine and D-galacturonic acid inhibit cytotoxicity in only some limiting dilution wells. Analysis of several B10.A(4R) anti-BALB/c Tc cell clones has revealed at least two different 'clone-specific' patterns of inhibition by D-glucose, D-glucuronic acid and D-galacturonic acid. Since Tc cell recognition of antigen is generally specific for class I major histocompatibility complex (MHC) antigens, this data implicates a role for MHC-associated carbohydrate structures expressed by target cells in T-lymphocyte interactions with antigen.

Animals↗

PubMed1987

Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases.

Recent studies in this laboratory demonstrated that several sulphated polysaccharides can inhibit metastasis of the rat mammary adenocarcinoma 13762 MAT, probably by preventing the passage of tumour cells through the walls of blood vessels. In order to directly test this possibility, 13762 MAT cells were cultured with (35S)O4(=)-labelled subendothelial extracellular matrices (ECM) and ECM degradation was monitored in either the presence or absence of different sulphated polysaccharides. Degradation products were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent autoradiography. The 5 sulphated polysaccharides that had previously been shown to possess anti-metastatic activity were potent inhibitors of the degradation of subendothelial ECM by 13762 MAT cells. In contrast, of the 4 polysaccharides tested that failed to inhibit metastasis, 3 had no effect on ECM breakdown and one (carrageenan-kappa) was substantially less effective at inhibiting ECM degradation than the anti-metastatic preparations. It was also shown that 13762 MAT cells produce a heparan sulphate-specific glycosidase (heparanase) that degrades the heparan sulphate side-chains of the ECM, the action of this enzyme rather than that of other ECM-solubilizing enzymes being inhibited by the antimetastatic sulphated polysaccharides. Additional experiments indicated that the anti-coagulant activity of the polysaccharides probably plays a minor role in their anti-metastatic effects since heparin, almost completely depleted (98-99.5%) of heparin molecules with anti-coagulant activity by passage over an anti-thrombin III column, retained its ability to inhibit 13762 MAT heparanases and was almost as effective as unfractionated heparin at inhibiting tumour-cell metastasis. Collectively, these data suggest that sulphated polysaccharides inhibit the metastasis of 13762 MAT cells by inhibiting tumour-cell-derived heparanases involved in the penetration of the vascular endothelium and its underlying basement membrane by tumour cells.

Animals↗

PubMed1987

The effect of sulfated polysaccharides on the free intracellular calcium ion concentration of lymphocytes.

Recent studies have demonstrated that murine lymphocytes express specific cell-surface receptors for a range of sulfated polysaccharides. In order to determine whether polysaccharide binding induces transmembrane signaling, the effects of sulfated polysaccharides on the free intracellular calcium ion concentration [( Ca2+]i) of mouse thymocytes and spleen cells were determined. Cells were loaded with Indo-I, a fluorescent indicator of calcium ion concentration. The validity and limitations in the use of this indicator in the determination of [Ca2+]i are documented. Dextran sulfate (Mn = 500,000), iota-carrageenan, lambda-carrageenan and kappa-carrageenan all cause relatively large changes in the [Ca2+]i of thymocytes (change in [Ca2+]i greater than 50 nM). Of these, dextran sulfate (Mn = 500,000) always had the greatest effect on [Ca2+]i. Smaller responses were obtained with heparin and dextran sulfate (Mn = 5000), while no response was obtained with chondroitin 4-sulfate, chondroitin 6-sulfate, pentosan sulfate or fucoidin. This response pattern (with the exception of fucoidin and pentosan sulfate) corresponds with the expression of thymocyte receptors for these polysaccharides. The increase in [Ca2+]i caused by the sulfated polysaccharides requires extracellular Ca2+ ions however, it is unlikely that voltage-dependent ion channels are involved in these responses. In contrast to thymocytes, although spleen cells express receptors for sulfated polysaccharides, they were unresponsive to all of the sulfated polysaccharides tested, suggesting a basic difference between thymocytes and peripheral T and B lymphocytes in their response to the binding of sulfated polysaccharides.

Animals↗

PubMed1987

Analysis of the inhibition of tumour metastasis by sulphated polysaccharides.

Lung metastases resulting from the intravenous (i.v.) injection of cells from the rat mammary adenocarcinoma 13762 MAT were significantly reduced by a variety of sulphated polysaccharides, the most effective being heparin, fucoidan and Carrageenan lambda. Although all the inhibitory polysaccharides were anticoagulants, it is unlikely that anticoagulation is the total explanation of their antimetastatic effect because: (i) heparin preparations from 2 different suppliers, although exhibiting comparable anticoagulant activities, differed 10-fold in their antimetastatic capability; (ii) certain sulphated polysaccharides consistently gave a 30% difference in the number of metastatic lesions, yet exhibited identical anticoagulant activity; and (iii) the entrapment of 13762 MAT cells in the lung was not impaired by heparin or fucoidan. It was more probable that the sulphated polysaccharides were interfering with the passaging of tumour cells across the capillary wall as heparin significantly inhibited metastasis when injected up to 3 hr after lodgement, and heparin and fucoidan caused a gradual loss of tumour cells from the lung which only became apparent greater than 1 hr following cell lodgement. The data did not eliminate the possibility that tumour cell adhesion to the endothelium occurred via sulphated polysaccharide recognition. A negative correlation existed between the sulphated polysaccharides that bound to the surface of the tumour cells and those that inhibited metastasis.

Animals↗

PubMed1987

A role for sulfated polysaccharide recognition in sponge cell aggregation.

Molecules binding sulfated polysaccharides were detected as lectins in cholate lysates of cells from twelve sponge species. Each species exhibited a unique binding profile. The pattern of binding indicated that the specificity was most probably determined by the orientation of the sulfate groups on the polysaccharide chains. Cells from each of the three species examined in more detail were found to express sulfated polysaccharide-binding molecules at their surface and at least one of the polysaccharides recognized was found to inhibit the reaggregation of cells from each species. Moreover, in all but one instance, lectins for the inhibitory polysaccharide were both detected in cell lysates and shown to be expressed at the cell surface. Sulfated polysaccharides, therefore, appeared to be involved in cell interaction events in the Porifera. This conclusion was confirmed by the isolation via ion exchange chromatography of an endogenous polysaccharide from an O. tenuis cell extract. This molecule contained uronic acid and hexose units in a ratio of 2:1, 11.9% sulfur and less than 0.5% protein. It inhibited the aggregation of O. tenuis cells and the agglutination of dextran-sulfate- and polyvinyl-sulfate-coupled erythrocytes by O. tenuis cell lysates. O. tenuis cell aggregation was also inhibited by polyvinyl sulfate and dextran sulfate and molecules binding these compounds were expressed on the surface of O. tenuis cells. Thus, is was probable that the cell surface receptor for polyvinyl sulfate and dextran sulfate and isolated sponge sulfated polysaccharide are one and the same. Finally, using a dextran sulfate affinity procedure, a 35 kD dextran-sulfate-binding protein was isolated from the surface of O. tenuis cells. The possibility that the polysaccharide isolated from O. tenuis cell extracts in the absence of calcium is the monomeric form of a cell aggregation-enhancing factor is discussed.

Animals↗

PubMed1987

Evidence that the cytoskeleton plays a key role in cell adhesion.

A range of pharmacological agents with defined effects on cell metabolism was used to determine the metabolic requirements of three cell adhesion systems: aggregation of cells from the sponge, Ophlitaspongia tenuis; fibronectin-induced adhesion of fibroblasts to substrata and an in vitro murine thymocyte-macrophage interaction. Cell adhesion in all three systems was found to have similar metabolic requirements, implying that the mechanism of cell adhesion has been conserved through evolution. In fact, based on analysis of F-actin organization in fibroblasts, all of the pharmacological agents that inhibited cell adhesion were found to disrupt the cytoskeleton, suggesting that the cytoskeleton plays a central role in the adhesion process, presumably via redistribution of cell surface molecules. This concept was supported by the finding that the same drugs that inhibited cell adhesion inhibited anti-Ig-induced redistribution of surface Ig on B lymphocytes. The drug inhibition studies also revealed that two drugs, bromophenacyl bromide (BPB) and nordihydroguaiaretic acid (NDGA), that were previously believed to be selective inhibitors of arachidonic acid synthesis and metabolism, are also potent disruptors of the cytoskeleton.

Acetophenones↗

PubMed1987

Analysis of the genetic control of lymphocyte positioning.

A possible role for the major histocompatibility complex (MHC) in the localization of lymphocytes in different lymphoid organs was investigated using inbred mouse strains. Lymphocytes labelled with the intracellular fluorochrome Hoechst 33342 (H33342) were transfused intravenously (IV) into unimmunized mice and the distribution of these labelled lymphocytes examined. In some combinations (e.g. C57BL/6----CBA) 2 h after injection allogeneic lymphocytes accumulated in the region between the marginal zones and outer aspects of the white pulp of the spleen. In contrast, in syngeneic controls (e.g. CBA----CBA) the lymphocytes migrated normally into the while pulp. Similar results were obtained in Peyer's patches. Mapping studies in the spleen indicated that the failure to migrate normally is predominantly controlled by the MHC complex, although some non-MHC genes may play a role. In the case of the MHC the most definitive combination was BALB/c-H-2dm2 (H-2L deletion mutant) lymphocytes transfused into BALB/c recipients, the mutant lymphocytes failing to migrate normally and, therefore, implicating the H-2L region in the phenomenon. No differences in the viability of labelled lymphocytes at 6 and 24 h after injection into either syngeneic or allogeneic recipients suggests that the inability of cells to passage through lymphoid organs may represent inappropriate receptors rather than elimination of the allogeneic lymphocytes by natural killer cells (NK) as previously proposed.

Animals↗

PubMed1986

Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation.

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH4)2SO4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH4)2SO4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly-2/3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH4)2SO4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH4)2SO4 concentrations, precipitation of cellular proteins at two different (NH4)2SO4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.

Ammonium Sulfate↗

PubMed1986

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.

Animals↗

PubMed1986

Binding sites for glycosaminoglycans on developing sympathetic neurones.

Recent studies have suggested that cell to cell communication in the immune system is mediated by cell surface receptors for glycosaminoglycans (GAGs) [Parish et al, 1984]. The intention of this study was to see whether similar recognition molecules for GAGs are present on sympathetic neurones. A mechanical dissociation technique was used to isolate neurones from superior cervical ganglia (SCG) of rats aged between gestational day 19 and postnatal day 21. Receptors for GAGs on sympathetic neurones were detected by the ability of neurones to form rosettes with sheep red blood cells coupled with one of 12 different GAGs. It was found that SCG cells bind to all the GAGs tested. In addition, a range of developmental binding patterns for the various GAGs was found.

Animals↗

PubMed1986

Cell surface receptors for sulphated polysaccharides: a potential marker for macrophage subsets.

The expression of a diverse array of receptors for sulphated polysaccharides on lymphocytes has been demonstrated by Parish & Snowden (1985). This paper presents evidence to suggest that other cell types, namely macrophages, polymorphonuclear leucocytes, mast cells and fibroblasts, can bind similar polysaccharides. Using a rosetting assay and eleven structurally unique polysaccharides, each cell type was observed to bind a characteristic array of these polysaccharides. Analysis of the polysaccharide reactivity of macrophages revealed that BCG-activated and thioglycollate-elicited macrophages express an expanded repertoire of reactivity compared to resident peritoneal macrophages. For example, only thioglycollate-elicited macrophages, but not resident and BCG-activated peritoneal macrophages, reacted with the glycosaminoglycans, chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate, while both BCG- and thioglycolate-activated, but not resident peritoneal macrophages, bound pentosan polysulphate-coupled sheep erythrocytes. The expression of the receptors for chondroitin-4 and -6-sulphate was observed to be cyclic and peaked at 2 and 5-6 days after thioglycollate treatment. Preliminary analyses of the functional significance of the observed binding of polysaccharides to macrophages revealed that heparin, fucoidan and kappa-carrageenen were specifically endocytosed. However, endocytosis of all other test polysaccharides was not observed. Finally, polysaccharide-coupled sheep erythrocytes were not phagocytosed, even though they interacted strongly with the macrophage surface. The possible relevance of these observations to an inflammatory response and as a means of identifying cellular subsets is discussed.

Animals↗

PubMed1985

Rapid screening of monoclonal antibodies by spin adherence double immunosorbent test (SADIST).

The spin adherence double immunosorbent test (SADIST) is a simple, rapid immunoassay with sensitivity similar to the enzyme-linked immunosorbent assay (ELISA). A 1-step SADIST has been found suitable for rapid screening of hybridomas for antigen-specific monoclonal antibodies (MAb). In this procedure hybridoma supernatants are added to antigen coated microplates followed by commercially available antiglobulin beads. The microplate is immediately centrifuged. Wells containing antigen-specific MAb produce a mat of beads whilst wells without antigen-specific MAb produce a button of beads. No washing or incubation steps are necessary and results are read within minutes of adding beads to test supernatants. By comparison, ELISA tests require several hours to perform with multiple wash steps and further reagent additions. A 2-step SADIST was also assessed. Supernatants are incubated in the microplate as for an ELISA and a wash step precedes the addition of antiglobulin beads. A panel of 117 hybridoma supernatants was selected to assess the suitability of the SADIST techniques for hybridoma screening. The supernatants were added to antigen-coated microplates and SADIST and ELISA tests performed. The SADIST correctly discriminated most hybridoma supernatants that were clearly positive or negative by ELISA. It was also found possible to perform SADIST followed by ELISA tests on the same microplate well without significantly affecting ELISA values.

Antibodies, Monoclonal↗

PubMed1985

Topographical studies of lymphocyte localization using an intracellular fluorochrome.

A procedure for analysing the topographical localization in tissue sections or whole-organ mounts of lymphocytes labelled with an intracellular DNA-binding fluorochrome, Hoechst dye No. 33342, is described. The localization of intravenously injected lymphocytes in spleen, popliteal lymph nodes, and Peyer's patches was followed up to 7 days. In the case of spleen, both B and T lymphocytes initially localised in the marginal zone. Subsequently, B cells appeared to exit via the red pulp, while T cells aggregated around vessels in the white pulp. In Peyer's patches, B and T lymphocytes localized to different lymphoid areas. The advantages and potential applications of this technique are discussed.

Animals↗

PubMed1985

Nonimmune lymphocyte-macrophage interaction. I. Quantification by an automated colorimetric assay.

Previous studies have demonstrated a spontaneous, nonimmune interaction between lymphocytes and macrophages. This paper describes an automated colorimetric assay based on the dye, rose bengal, to quantify this interaction. The procedure entails allowing lymphocytes to adhere to preformed macrophage monolayers in the wells of microplates and then staining bound lymphocytes with rose bengal. Dye uptake and the consequent number of lymphocytes bound were quantified using an automated spectrophotometer developed for reading microplates. This procedure was used to confirm and extend the basic parameters of the system. The interaction was found to be temperature dependent but the kinetics and percentage of cells binding varied with the source of lymphocytes. However, all lymphocyte populations tested, namely, mature and immature thymocytes, T and B lymphocytes, and a range of thymoma cell lines, bound to macrophages. Furthermore, all macrophage populations examined had the ability to bind lymphocytes. The interaction also showed no strain specificity and generally lacked species specificity. It is proposed that the interaction is a highly dynamic process that enables lymphocytes to scan the surface of macrophages for self and/or foreign antigens.

Age Factors↗

PubMed1985

Nonimmune lymphocyte-macrophage interaction. II. Evidence that the interaction involves sulfated polysaccharide recognition.

This paper describes attempts to determine the molecular basis of the nonimmune interaction between lymphocytes and macrophages. Initial studies revealed that the interaction could be inhibited by simple sugars, six out of the thirty-five tested being inhibitory. Furthermore, the majority of the inhibitory sugars were charged and subsequent studies revealed that some sulfated polysaccharides, notably kappa-carrageenan, were potent inhibitors of the interaction. Further experiments revealed that the lymphocyte-macrophage interaction was indeed mediated by kappa-carrageenan-specific receptors on lymphocytes. The results supporting such a conclusion were as follows: When the interacting cells were preincubated with kappa-carrageenan, it was found that kappa-carrageenan exerted its inhibitory effect at the lymphocyte rather than the macrophage level. Separation of splenocytes into kappa-carrageenan-binding and -nonbinding subpopulations resulted in a corresponding enrichment and depletion of lymphocytes that reacted with macrophages. Lymphocytes were found to express kappa-carrageenan-reactive molecules, these molecules being detected on the surface of lymphocytes by rosetting and in detergent lysates as hemagglutinins. Furthermore, the polyanion specificity of these kappa-carrageenan-specific receptors/hemagglutinins closely resembled the specificity of inhibition of the lymphocyte-macrophage interaction. Pronase-resistant material in macrophage, but not lymphocyte lysates, effectively inhibited both the lymphocyte-macrophage interaction and the recognition of kappa-carrageenan by lymphocytes, suggesting that a kappa-carrageenan-like structure is expressed by macrophages.

Animals↗

PubMed1985

Lymphocytes express a diverse array of specific receptors for sulfated polysaccharides.

Lymphocyte receptors for sulfated polysaccharides were detected in two ways, namely, by the ability of lymphocytes to form rosettes with sheep red blood cells (SRBC) coupled with one of fourteen different sulfated polysaccharides, and by the ability of cholate extracts of lymphocytes to hemagglutinate the same sulfated polysaccharide-coupled SRBC. It was found that murine lymphocytes lacked receptors for a number of glycosaminoglycans, such as hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate, but reacted strongly with heparin, arteparon, and a number of sulfated polysaccharides of plant and bacterial origin. In each case receptor activity was demonstrated by rosetting and by the ability of lymphocyte lysates to strongly agglutinate sulfated polysaccharide-coupled SRBC. The receptors exhibited a high degree of diversity as evidenced by (a) only subpopulations of lymphocytes, particularly splenic B cells, expressing receptors for some of the sulfated polysaccharides and (b) hemagglutination-inhibition analyses revealing numerous subsets of receptors with different binding specificities. Receptor diversity was further highlighted by a 48% difference in the hemagglutination-inhibiton results between thymus and spleen. It is proposed that these receptors are involved in cell-cell communication and lymphocyte homing and recirculation. The likely target structures for the receptors in vivo are the heparan sulfates, a ubiquitous and structurally diverse family of sulfated glycosaminoglycans.

Animals↗

PubMed1984

Intracellular fluorescent labelling of cells for analysis of lymphocyte migration.

A procedure for analysing in vivo migration of lymphocytes labelled in vitro using intracellular fluorochromes is described. Comparison of carboxyfluorescein diacetate, a cytoplasmic label, with Hoechst dye No. 33342 (H33342), a DNA-binding fluorochrome, indicated that H33342 is superior. The concentration of H33342 used for labelling does not significantly affect viability or lymphocyte migration and permits long-term visualization. H33342 allows quantitation of in vivo migration in cell suspension and histological localization in frozen sections. Fluorescence is retained in fixed frozen sections for at least 3 months. This method can be used for analyses of lymphocyte migration and maturation.

Adenocarcinoma↗

PubMed1984

An improved colorimetric assay for T cell cytotoxicity in vitro.

An improved colorimetric assay for estimation of cytotoxic T (Tc) cells is described. The method involves staining thioglycollate-induced macrophage targets with the dye neutral red prior to addition of cytotoxic T cells and estimating macrophage survival at the end of the assay by measuring dye remaining in viable targets. The method using macrophage targets is more sensitive than the 51Cr release assay employing macrophages or a variety of other targets. It may be used to detect alloreactive and H-2 restricted Tc cells in both short-term (4 h) and long-term (24 h) assays and overcomes some variability encountered with a previously described colorimetric procedure. Furthermore, the method is cheap, fast, reliable and avoids the use of radioactivity.

Animals↗