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Biomedical subjects

C R Parish

Publications and source records attributed to C R Parish.

At least 73 records · Page 4Linked to original sources
PubMed1991

Isolation and characterization of cell adhesion molecules from the marine sponge, Ophlitaspongia tenuis.

Previous studies suggested that cell adhesion in the marine sponge, Ophlitaspongia tenuis, is mediated by a 35 kDa cell surface protein which interacts with an extracellular sulfated polysaccharide. This paper describes a simple and efficient procedure for isolating both putative cell adhesion molecules from detergent lysates of O. tenuis cells, the procedure being based on the fortuitous affinity of the sponge polysaccharide for heparin. The purified polysaccharide inhibits O. tenuis sponge cell aggregation, is highly sulfated and represents a glycosaminoglycan containing glucuronic acid. N-sulfated glucosamine and, possibly, glucose. The purified 35 kDa protein has a high affinity for the sponge polysaccharide and also, selectively interacts with dextran sulfate, a polysaccharide that has been shown previously to both bind to the sponge cell surface and inhibit aggregation of O. tenuis cells. Collectively, the data supports the hypothesis that the 35 kDa molecule is the major cell adhesion protein in O. tenuis. Preliminary data also suggests that the sponge contains an endogenous glycan hydrolase which can cleave the sponge polysaccharide.

Animals↗
PubMed1991

Dextran sulfate induces changes in the free intracellular calcium ion concentration of a subpopulation of immature thymocytes.

Previous studies have shown that certain sulfated polysaccharides, such as dextran sulfate (DxS), can induce a rapid and sustained increase in the free intracellular calcium ion concentration ([Ca2+]i) of thymocytes, whereas they cannot induce a change in the [Ca2+]i of peripheral lymphocytes. This study examined this phenomenon in more detail and clearly demonstrated that only immature thymocytes can respond to DxS. Cortisone-resistant thymocytes failed to exhibit an increase in [Ca2+]i when exposed to DxS. Furthermore, analysis of [Ca2+]i changes at the single cell level using a flow cytometer demonstrated that only a subpopulation of immature thymocytes responded to DxS. Additional flow cytometry studies revealed that DxS and Con-A induce a [Ca2+]i response in different subpopulations of thymocytes, approximately 30% of thymocytes responding to Con-A and 15-20% to DxS. Thus, responsiveness to Con-A and DxS, in terms of increases in [Ca2+]i, may represent a novel marker for two different thymocyte subpopulations. Such metabolic markers could be used to complement the conventional serological methods currently employed to define stages in thymocyte differentiation.

Animals↗
PubMed1991

Conservation of a polyanion binding site in mammalian and avian CD4.

A polyanion binding site was identified recently on human CD4 which is distinct from the human immunodeficiency virus (HIV)-gp120 binding region but which incorporates the first two immunoglobulin (Ig)-like domains of the molecule. To determine if this site is conserved in other species, several polyanions that blocked monoclonal antibody (mAb) binding to human CD4 were examined for their ability to inhibit the binding of mAb to mouse, rat, pig, sheep and chicken CD4. It was found that aurintricarboxylic acid (ATA) was a particularly effective inhibitor, blocking mAb binding to human, mouse, pig, sheep and rat CD4 by greater than 90% and to chicken CD4 by 80-90%. The polyanions dextran sulphate (DxS), polyvinyl sulphate (PVS) and polyanethole sulphonate (PAS) were also effective inhibitors of anti-CD4 mAb binding in most species, although there were clear species differences in the effects obtained. The polyanions did not inhibit mAb binding to a variety of other cell-surface antigens in the different species, with the exception of sheep CD8, suggesting that the inhibitory effects observed were essentially CD4 specific. Collectively these data indicate that a polyanion binding site is conserved in mammalian and avian CD4. Comparison of the amino acid sequences of human, mouse and rat CD4 revealed that basic residues in human CD4 which could participate in a polyanion binding site are conserved in mouse and rat CD4. It is proposed that this conserved polyanion binding site of CD4 interacts with a sulphated glycosaminoglycan chain which is associated with class II major histocompatibility complex (MHC) molecules containing recently processed antigen.

Amino Acid Sequence↗
PubMed1991

Characterization of lymphocyte receptors for glycosaminoglycans.

This paper describes attempts to isolate and characterize glycosaminoglycan (GAG)-binding molecules on the surface of lymphocytes and lymphoma cell lines and relate their expression to splenic and lymph node homing capacity. Initial binding studies with radiolabelled GAG and rosetting studies with GAG-coupled erythrocytes revealed that there are receptors on lymphocytes for the major classes of GAG (i.e. hyaluronic acid, chrondroitin sulfates, heparin), but lymphocytes bind heparin much more avidly than other GAG species. Analysis of the binding of solubilized radiolabelled cell-surface molecules to immobilized GAG revealed cell-type specific expression of GAG-binding molecules. Thus, each of four lymphoma cell lines tested gave a characteristic pattern of GAG-binding molecules, some molecules being unique to a particular cell line and others being shared by some of the lines. Similarly, splenocytes expressed at least 10 distinct GAG-binding molecules with molecular weights (MW) ranging from 10,000 to 100,000, whereas thymocytes expressed additional GAG-binding proteins of 190,000 and 250,000 MW. Furthermore, splenocytes differed from thymocytes by possessing a unique family of cell-surface molecules which reacted with each GAG. Immunoprecipitation studies demonstrated that the GAG-binding molecules on splenocytes did not correspond to any of the cell-surface antigens tested, notably the cell adhesion molecules MEL-14, CD11/CD18 and CD44, although CD8 bound weakly to heparin. Four lymphoma cell lines with well-characterized migration properties were examined for GAG-binding molecules which may control lymphocyte migration. It was found that no one GAG-binding protein could be correlated with the entry of cells into a particular lymphoid organ. Nevertheless, the role of GAG-binding molecules in the subsequent positioning of lymphocytes within lymphoid organs requires further investigation.

Animals↗
PubMed1990

New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.

16 fluorochromes were examined for their ability to label viable lymphocytes in vitro and yield fluorescence detectable by fluorescence microscopy and flow cytometry. Of these fluorochromes, four intracellular dyes were found to be suitable for in vivo migration studies. They were H33342, the well known DNA-binding dye which excites and emits in the UV range, and three fluorescein based cytoplasmic dyes, namely BCECF-AM, Calcein-AM and CFSE which excite and emit in the visible range. Lymphocytes labelled with H33342, BCECF-AM and Calcein-AM were suitable for short term in vivo migration experiments with detection by flow cytometry 2-3 days post injection. In contrast lymphocytes labelled with CFSE, a fluorochrome which can covalently couple with intracellular macromolecules, were detected by flow cytometry up to 8 weeks post injection and thus this fluorochrome is ideal for long term migration experiments. Due to marked differences in fluorescence profiles, BCECF-AM and Calcein-AM could be used for short term double labelling experiments using the flow cytometer in which entry of injected lymphocytes into lymphoid organs was quantified. Similarly, in vivo localization of lymphocyte subpopulations could be examined by fluorescence microscopy utilizing differences in fluorescence excitation and emission spectra of lymphocytes labelled with H33342 and one of the fluorescein based dyes.

Animals↗
PubMed1990

A polyanion binding site on the CD4 molecule. Proximity to the HIV-gp120 binding region.

Recent studies have demonstrated that sulfated polyanions (SP) are potent inhibitors of HIV infection in vitro, appearing to inhibit virus attachment. To understand the mode of action of these compounds a large panel of SP were examined for their ability to inhibit HIV infection, block anti-CD4 mAb binding and, when immobilized, bind soluble CD4 and virion gp120. Based on anti-CD4 mAb binding-inhibition studies a SP binding site was identified on the CD4 molecule. Dextran sulfate (DXS)-500 kDa, polyvinylsulfate (PVS), and polyanethole sulfonate were particularly potent SP inhibitors, blocking the binding of 11 of the 12 anti-CD4 mAb tested. These 11 mAb are known to interact with the two amino-terminal Ig-like domains of CD4. In fact, DXS-500 kDa exhibited an hierarchy of inhibition of anti-CD4 mAb which suggests that SP bind to a conformational site incorporating the first two Ig-like domains of CD4. This SP binding site is clearly distinct but closely associated with the gp120 binding region of CD4. In terms of anti-HIV activity there was no evidence that SP act at the virion level as rgp120 did not bind to immobilized SP and preincubation of virions with SP did not affect infectivity. In contrast, many of the SP tested showed some affinity for CD4 based on anti-CD4 mAb blocking studies and binding of soluble CD4 to immobilized SP. The most active in this regard were DXS-500 kDa and PVS, whose anti-HIV activity could be entirely due to disruption of the CD4-gp120 interaction. However, with SP such as heparin, fucoidan, the carrageenans, and polyanethole sulfonate, although CD4 blocking may contribute to anti-HIV activity, some other anti-viral mechanism is also operating. Finally, pentosan sulfate, a SP with anti-HIV activity comparable to DXS-500 kDa and PVS, showed little or no reactivity with CD4 and must inhibit HIV infection by a totally CD4-independent mechanism.

Antibodies, Monoclonal↗
PubMed1990

Mapping the dextran sulfate binding site on CD2.

This study has analysed the binding of a series of anti-CD2 monoclonal antibodies (MoAbs) to T cells in the presence of the sulfated polysaccharide dextran sulfate (2.3 sulfates/monosaccharide, 500 kDa) (DXS) to define the DXS binding site on CD2. The results show that DXS interacts primarily at the T11(2) epitope. Thus five anti-CD2 MoAbs which bound to the T11(2) epitope were inhibited in their binding by DXS. In contrast, seven anti-CD2 MoAbs that totally inhibited sheep red blood cells (SRBC) rosetting (identifying the T11(1) epitope) were unaffected in their binding to T cells in the presence of DXS. Three MoAbs which partially inhibited SRBC rosetting and thereby defining only part of the T11(1) epitope, were also inhibited in their binding by DXS. Consistent with the conclusion that the DXS binding site on CD2 is associated with the T11(2) epitope was the observation that interaction of DXS with CD2 resulted in augmented binding of the four MoAbs defining the T11(3) epitope, possibly reflecting an increased expression of the T11(3) (activation, CD2R) epitope of CD2. Collectively, the data presented support the notion that a natural ligand for the T11(2) epitope of CD2 will be identified as a sulphated carbohydrate structure.

Antibodies, Monoclonal↗
PubMed1989

Inhibition of experimental allergic encephalomyelitis by the alpha-glucosidase inhibitor castanospermine.

The alkaloid castanospermine is a potent inhibitor of oligosaccharide processing in vitro. Our recent findings indicating the importance of carbohydrate moieties in some critical step of the neuro-immunologic inflammatory process of allergic encephalomyelitis prompted us to investigate the effect of castanospermine on this disease process. The alkaloid inhibited passively induced allergic encephalomyelitis in a dose-dependent manner when administered continuously for 7 days beginning at the time of lymphocyte transfer. Although clinical disease was totally inhibited, treated animals did have inflammatory lesions in the central nervous system. These lesions were qualitatively different from those seen in untreated animals in that the inflammatory cells were tightly packed around the vessels and showed little migration into surrounding tissues. Castanospermine also effectively inhibited clinical disease in recipient animals which had had a previous episode of allergic encephalomyelitis. Castanospermine did not alter the disease when treatment was started after the onset of clinical symptoms.

Alkaloids↗
PubMed1989

Phosphosugars are potent inhibitors of central nervous system inflammation.

Adoptively transferred allergic encephalomyelitis can be inhibited by various phosphosugars, particularly mannose-6-phosphate. The sugar specificity suggests that inhibition may be due to depletion of lymphocyte cell-surface lysosomal enzymes, which are essential for the passage of lymphocytes across the vascular endothelium and the entry of lymphocytes into the central nervous system parenchyma.

Animals↗
PubMed1989

Receptors on lymphocytes for endogenous splenic glycosaminoglycans.

Previous studies have shown that lymphocytes carry cell surface receptors for sulphated polysaccharides (SPS), and SPS recognition may play a role in lymphocyte migration and positioning in vivo. This paper describes attempts to isolate and characterize the endogenous glycosaminoglycans (GAGs) of murine spleen and determine whether splenic lymphocytes carry cell surface receptors for these GAGs. A procedure was devised for isolating GAGs from murine spleen in good yield and high purity and the GAG preparation was then radiolabelled for subsequent binding studies. It was found that the splenic GAGs bound to murine splenocytes in a saturable, rapid and reversible manner with only a small subpopulation of the splenic GAG preparation being involved in binding. This reactive species was chondroitinase ABC-resistant and nitrous acid-sensitive, indicative of a heparan sulphate/heparin-like molecule. Furthermore, using immunofluorescent flow cytometry studies it was demonstrated that the majority of spleen cells have receptors for these GAGs. Subsequent ion-exchange fractionation and SDS-PAGE analysis of chondroitinase ABC-resistant GAGs confirmed that the splenic GAG recognized by splenocytes was a heparan sulphate/heparin molecule of approximately 20,000 MW with a binding affinity to splenocytes of approximately 5 X 10(-8) M. Additional binding inhibition studies indicated two possible binding sites for splenic GAGs on the splenocyte surface, one being fully inhibited by a range of SPS such as heparin (both coagulant and anticoagulant forms), pentosan sulphate, fucoidan, dextran sulphate, lambda- and iota-carrageenan, and the second being partially inhibited by kappa-carrageenan. The possible relevance of these heparan sulphate/heparin receptors on splenocytes to lymphocyte positioning in vivo is discussed.

Animals↗
PubMed1988

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.

Antibodies, Monoclonal↗
PubMed1988

Automated fluorometric assay for T cell cytotoxicity.

A fluorometric assay avoiding the use of radioactivity has been developed for detecting cytotoxic T lymphocytes (Tc cells). The method involves labelling targets with Hoechst dye no. 33342 (H33342) which becomes brightly fluorescent on binding to DNA. Lysis of target cells by Tc cells is quantified by measuring the release of fluorescent H33342 into the supernatant of culture wells. The fluorescence is measured using an automated Microfluor reader which allows results to be obtained rapidly. The assay has been used to detect alloreactive Tc cells and H-2 restricted Tc cells against influenza virus in a short-term 6 h assay using P815 and L929 as targets with comparable results to those obtained with 51Cr labelling. In contrast, lymphocyte blasts were found to be less sensitive in 6 h fluorometric assays when compared with the 51Cr assay. In long-term overnight assays (possible because of the low spontaneous release of H33342 from targets) lymphocyte blasts gave high specific lysis and some anti-self reactivity. The cause of the anti-self reactivity may reflect fundamental differences between the H33342 and 51Cr release assays.

Animals↗
PubMed1988

Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides.

A number of sulfated polysaccharides were tested for their ability to inhibit passively induced experimental allergic encephalomyelitis (EAE) in rats. Heparin and fucoidan both completely inhibited passive EAE even when treatment was begun 3 days after transfer of cells. Pentosan sulfate was partially inhibitory whereas chondroitin-4-sulfate had no effect. Inhibition was not merely due to killing of the cells since active sensitization 14 days after cell transfer resulted in an early onset of disease indicating the persistence of transferred cells as memory cells. Although all the inhibitory polysaccharides are anticoagulants, it would appear that this function alone is not the reason for inhibition since a heparin preparation devoid of anticoagulant activity also partially inhibited EAE. Actively induced EAE was also significantly delayed by treatment with heparin. The results are discussed in terms of the polysaccharides inhibiting the enzymatic dependent movement of lymphocytes across central nervous system vascular endothelium.

Animals↗
PubMed1988

Evidence that Thy-1 and Ly-5 (T-200) antigens interact with sulphated carbohydrates.

Recent studies have demonstrated that lymphocytes express an array of cell surface receptors for sulphated polysaccharides (SP). Experiments were undertaken to determine the binding characteristics of these receptors and establish whether any known lymphocyte cell surface antigens interact with sulphated carbohydrates. It was found that murine thymocytes lack receptors for chondroitin-4-sulphate but express saturable, high affinity binding sites for heparin, fucoidan and dextran sulphate, with an apparent affinity constant range of 0.03-2.6 x 10(-9) mol/l. Binding inhibition experiments revealed one class of binding sites on murine thymocytes that is shared by heparin, fucoidan and dextran sulphate and another class of sites that is dextran sulphate-specific. The cell surface receptors for the SP were affinity-purified by applying detergent lysates of 125I-labelled thymocyte membranes to SP-coupled solid supports. It was found that the Thy-1 and Ly-5 (T-200 or leucocyte common antigen) molecules of murine thymocytes bind to sulphated carbohydrates, although the two molecules differed substantially in their reactivity with the four different SP tested. Furthermore, only subpopulations of the Thy-1 and Ly-5 molecules interacted with sulphated sugars. Four additional sulphated carbohydrate-binding molecules were also detected. It is suggested that the SP-binding molecules are involved in the interaction of lymphocytes with glycosaminoglycans on other cells and in the interstitial space.

Animals↗
PubMed1988

The T11(3) epitope of the CD2 molecule, sheep erythrocytes, and the alternative T-cell activation pathway.

In this study, results are presented showing that the T11(3) epitope of the CD2 molecule is expressed on unfractionated peripheral blood mononuclear cells, resting T cells and activated T cells. T11(3) is not therefore, an activation-specific CD2 epitope. Sheep erythrocytes (SRC) which present a cell-surface ligand for CD2 could not effectively replace anti-T11(2) in initiating proliferation of resting T cells, stimulated by anti-T11(3). SRC with either of these anti-CD2 monoclonal antibodies did activate resting T cells, but proliferation required the presence of added IL-2.

Animals↗