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Biomedical subjects

B Ryffel

Publications and source records attributed to B Ryffel.

At least 127 records · Page 7Linked to original sources
PubMed1989

Identification of interleukin 4 receptor-associated proteins and expression of both high- and low-affinity binding on human lymphoid cells.

Interleukin 4 (IL4) produced by activated T cells expresses its biological effects on T and B lymphocytes by binding to specific membrane receptors. Cross-linking of human recombinant 125I-IL4 to peripheral blood mononuclear cells identifies a trimolecular complex consisting of a 65/70-kDa doublet and a 110-kDa protein. Scatchard analysis reveals about 300 IL4 binding sites/cell on resting cells with an equilibrium binding constant (Kd) of approximately 100 pM. Stimulation by anti-CD3 antibodies causes an up-regulation of IL4 receptors by a factor of 2 to 3 without any change in binding affinity. In addition to this high-affinity binding site a second class of a previously unidentified, low-affinity receptor (Kd approximately 30 nM, approximately 9000 sites/cell) is expressed on resting lymphocytes. The number of low-affinity binding sites for IL 4 also increases twofold upon cell activation. Exogenous IL 4 enhances the expression of its receptor on resting lymphocytes and this effect is further increased by anti-CD3 activation. Binding of IL4 to its receptor is specific, being only inhibited by IL 4 and not by IL2. By contrast, the gibbon leukemia cell line MLA 144 expresses only high-affinity receptors for IL4. Cross-linking studies reveal a 45/50-kDa IL 4 receptor-associated doublet in addition to the three proteins identified in human peripheral blood mononuclear cells. The functional significance of the different proteins composing the receptor for IL4 is discussed.

Animals↗
PubMed1989

Cyclosporin A: action and side-effects.

Cyclosporin A (CSA) is a new, potent immunosuppressive agent which is of proven value in organ transplantation. The use of CSA is associated with numerous side-effects, of which a dose-dependent nephrotoxicity is the most serious. CSA nephrotoxicity can be divided into two major groups: (i) functional toxicity without significant morphological lesions and (ii) morphological forms of toxicity with tubular and/or vascular-interstitial lesions. Vascular-interstitial toxicity is the most serious form because the renal lesions are irreversible.

Acute Kidney Injury↗
PubMed1989

Identification of the multidrug resistance-related P-glycoprotein as a cyclosporine binding protein.

The immunosuppressive agent cyclosporine A has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a 3H-cyclosporine diazirine analogue was used to photolabel viable MDR Chinese hamster ovary cells. The 170-kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled. The binding of 3H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H. These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative. The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Biotinylated recombinant interleukin-2. A tool for research on the interleukin-2 receptor.

Recombinant interleukin-2 was biotinylated using a N-hydroxyl-succinimidyl [3H]biotin ester. The biotinylated lymphokine retained full binding and growth-promoting activities when assayed on the interleukin-2-dependent murine cell line HT-2. In preliminary studies, biotinylated interleukin-2 was used in conjunction with immunogold staining to demonstrate cell surface interleukin-2 receptors using both light and electron microscopy techniques. In addition, with rabbit anti-biotin antibodies, biotin-interleukin-2 was able to precipitate the 55 kDa IL-2 receptor from murine HT-2 cells. Thus, biotin-interleukin-2 represents a useful, non-radioactive tool for studying the structure and function of the interleukin-2 receptor.

Animals↗
PubMed1988

Identification of cyclophilin as the erythrocyte ciclosporin-binding protein.

Previous studies on the distribution of circulating ciclosporin have shown that the majority of the drug is associated with erythrocytes. In order to investigate the nature of ciclosporin-erythrocyte binding, binding studies were performed on isolated erythrocytes. At therapeutic concentrations (approx. 0.5 microgram/ml in whole blood) greater than 90% of the erythrocyte associated ciclosporin was found in the cytosol. The cytosolic binding capacity was approximately (2-2.5).10(5) molecules of ciclosporin per cell. A lower affinity binding of the drug to the plasma membrane occurred only at higher ciclosporin concentrations. The ciclosporin-binding species was purified from erythrocyte cytosol using ciclosporin-Affigel affinity chromatography. This revealed a 16 kDa protein, similar in size to the ciclosporin-binding protein, cyclophilin, previously identified in lymphocyte cytosol. Immunochemical analysis using rabbit anti-bovine spleen cyclophilin antisera revealed that the erythrocyte ciclosporin-binding protein was either cyclophilin or a closely related protein. It is concluded that intracellular ciclosporin-binding within erythrocytes is mostly attributable to the presence of a single protein or protein family represented by cyclophilin. The presence of (2-2.5).10(5) copies of this binding protein within each erythrocyte is responsible for the ciclosporin found associated with erythrocytes.

Binding, Competitive↗
PubMed1988

Comparison of the structure of the murine interleukin 2 (IL 2) receptor on cytotoxic and helper T cell lines by chemical cross-linking of 125I-labeled IL 2.

The structure of the murine interleukin 2 receptor (IL 2R), on a cytotoxic (CTLL) and a helper (HT2) cell line, has been studied by a combination of chemical cross-linking with 125I-labeled IL 2 and immunoprecipitation with an anti-receptor monoclonal antibody (7D4). In CTLL cells both methods detected the major 57-kDa IL 2-binding protein and in addition the cross-linking studies revealed the presence of a 70-75-kDa protein associated with the high-affinity receptor. In the HT2 cell line, however, immunoprecipitation studies revealed three additional proteins of 18, 22 and 37 kDa to the expected 50-kDa receptor protein. Again cross-linking studies demonstrated the presence of a 70-75-kDa protein, which was not immunoprecipitable with the 7D4 antibody. The low molecular polypeptides in HT2 cell were associated with the low-affinity receptor and represented most likely breakdown products of the 50-kDa protein. Whereas the 18- and 22-kDa proteins were involved in ligand binding, the 37-kDa fragment carried the epitope recognized by the 7D4 antibody. Comparative studies with two IL 2R antibodies, PC61 and 7D4, revealed that only PC61 inhibited the formation of the IL 2 alpha/beta chain complex, although both antibodies reportedly prevent the biological response to IL 2. It is speculated that the 37-kDa fragment, which reacts with the 7D4 antibody, might be involved in IL 2 signal transduction. Finally there was no evidence for the existence of a high molecular weight component of the IL 2R, previously described as gamma chain. In summary, the two-chain structure of the IL 2R has been confirmed for both murine cell lines with some heterogeneity of the alpha chain. The possibility was raised that a 37-kDa fragment of the alpha chain plays a role of in signal transduction.

Animals↗
PubMed1988

A comparison of cyclosporine binding by cyclophilin and calmodulin and the identification of a novel 45 KD cyclosporine-binding phosphoprotein in Jurkat cells.

Cyclosporine mediates its immunosuppressive effect by preventing the synthesis of lymphokine mRNA during the process of T lymphocyte activation. Although the detailed molecular mechanism by which CsA achieves this effect is unknown, two proteins have been identified as putative intracellular CsA-receptor proteins. One of these, calmodulin, is an important Ca++-binding protein and enzyme cofactor and the other, cyclophilin, is a novel protein that is reported to have protein kinase activity. In this study the CsA-binding capacity of both these proteins has been assessed using CsA-coated ELISA plates and CsA-affinity gel matrices. CsA binding was shown by cyclophilin whereas no CsA-calmodulin binding could be detected under identical conditions. However, it was not possible to demonstrate any cyclophilin-associated protein kinase activity. Jurkat cells were probed for the presence of CsA-binding proteins using the CsA-affinity gel matrix; a 17 KD protein, most probably cyclophilin, was identified as the major CsA-binding protein. In addition, a previously unidentified CsA-binding 45 KD phosphoprotein was precipitated from 32P-labeled Jurkat cells. These results would support cyclophilin as the major, if not only, intracellular receptor protein for CsA. However, the relationship between binding of CsA to cyclophilin and/or the 45 KD phosphoprotein and the immunosuppressive effects of CsA is still unknown.

Calmodulin↗
PubMed1988

Cyclosporine--relationship of side effects to mode of action.

Although cyclosporine has high specificity for the immune system, immunosuppressive therapy with CsA is often complicated by nephrotoxicity. The main morphologic targets of CsA nephrotoxicity include the tubular epithelial and endothelial cells. These cells were investigated in vitro. CsA caused a dose- and time-dependent inhibition of cell growth, vacuolization and fatty change in adherent cells, detachment, and cell death. Inhibition of 3H-TdR incorporation in cells of both tubular epithelial and endothelial origin occurred between 3 microM and 10 microM. Electron microscopy studies revealed cellular swelling, dilatation of the endoplasmic reticulum, and the presence of lipid droplets and giant mitochondria. The content of the main CsA-binding protein, cyclophilin, in these cell-lines was 5-10 micrograms/mg protein and did not differ in various cell lines, including T cells. Immunohistochemistry using rabbit anticyclophilin antibody revealed diffuse distribution of cyclophilin in the cytosol, nuclear membrane, and nucleolus. Whereas lymphoid cell functions are inhibited at 10-100 nM, CsA had no effect on tubular epithelial and endothelial cells at these concentrations. At concentrations of 3-10 microM, CsA caused growth inhibition and cytotoxicity on cells of lymphoid and nonlymphoid origin. Present evidence shows little, if any, relationship of side-effects to the mode of action of CsA.

Animals↗
PubMed1988

Syngeneic graft-versus-host disease induced by cyclosporine--a reappraisal.

Cyclosporine-induced graft-versus-host disease has been described in lethally irradiated rats and mice following syngeneic bone marrow reconstitution. To further study this apparently CsA-restricted phenomenon, we followed reported protocols by administering CsA orally at 50 or 100 mg/kg/day to irradiated, syngeneically reconstituted C57B1/6 mice. No clinical evidence of GVHD was observed for more than 8 weeks after CsA discontinuation. Ear biopsies and circulating immunoglobulin levels 2-4 weeks after stopping CsA failed to demonstrate histological or serological evidence of GVHD, respectively, compared with mice allogeneically reconstituted with Balb/c marrow. To further follow a previous report, CsA 50 mg/kg/day orally or 10 mg/kg/day intraperitoneally was given to normal C57B1/6 mice prior to using their spleen or bone marrow cells for reconstitution of lethally irradiated syngeneic mice. Clinical monitoring and histological examination of recipients 2-5 weeks after reconstitution again failed to confirm GVHD. Thus our results were uniformly negative in attempting to reproduce syngeneic GVHD in mice. Existing data on rats and humans are reviewed, showing that syngeneic or autologous GVHD is not CsA-restricted and that the syndrome could be equated to the chronic form of GVHD found in rats and patients after allogeneic bone marrow transplantation.

Animals↗
PubMed1987

Light and electron microscopic changes in the kidney of Wistar rats following treatment with cyclosporine A.

The morphological changes found in kidneys of patients undergoing chronic Cyclosporine A (CSA) treatment could, despite of numerous attempts, not yet be reproduced in reliable experimental models. The purpose of the present investigation was to systematically scan the in vivo fixed kidneys in CSA treated Wistar rats at the light and electron microscopic level. The study revealed changes in the proximal tubules, i.e., inclusion bodies, vacuolization, microcalcifications and regeneration. Some glomeruli displayed a thickening of the parietal sheet of Bowman's capsule. At the electron microscopic level, individual necroses of myocytes in the tunica media of afferent arterioles were observed. Thus, at the ultrastructural level, the kidneys of CSA treated normotensive Wistar rats do reveal morphological changes similar to those occurring in man.

Animals↗