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Biomedical subjects

B Ryffel

Publications and source records attributed to B Ryffel.

At least 19 recordsLinked to original sources
PubMed1992

Covalent binding of cyclosporine inhibits irreversibly T-lymphocyte activation.

A diazirine derivative of cyclosporine (PL-CS) was used to photolabel recombinant human cyclophilin (rhCyp), the cytosolic receptor for the immunosuppressant cyclosporine. The affinity of PL-CS for rhCyp and the immunosuppressive activity were 10-fold reduced as compared to cyclosporine A. Whereas cyclosporine immunosuppression was fully reversible, UV cross-linking of PL-CS resulted in permanent inhibition of lymphocyte activation as shown by proliferation of anti-CD3 stimulated human peripheral lymphocyte, interleukin (IL)-2 gene transcription and IL-2 synthesis in the human T-leukemia cell line Jurkat. In vivo photolabeling of viable Jurkat cells revealed that a 21-kDa complex was the major radiolabeled product which was identified as a cyclophilin-cyclosporine complex. In addition, cyclophilin B (25 kDa) and proteins of an unidentified nature at 40, 46 and 60 kDa were observed in Jurkat cells. The cyclosporine-resistant human fibroblast cell line MRC5 displayed a different labeling pattern: cyclophilin B (25 kDa) and a 65-kDa protein were the major labeled products, while the 46- and 60-kDa components were not detectable and cyclophilin was only faintly labeled. In summary, covalent cyclosporine binding caused irreversible lymphocyte inactivation and revealed in addition to cyclophilin other specifically labeled proteins in lymphoid cells. The role and identity of these proteins is presently unknown.

Affinity Labels↗
PubMed1992

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

Amino Acid Isomerases↗
PubMed1992

The carcinogenicity of ciclosporin.

Experimental data relevant for the evaluation of the carcinogenic potential of the immunosuppressant ciclosporin are reviewed: Firstly, the mode of action of ciclosporin at the level of lymphocyte gene transcription, secondly, the main adverse effects especially nephrotoxicity and thirdly, the results of the chronic bioassays. The experimental data are discussed together with the clinical evidence of increased incidence of tumors, especially lymphoproliferative disorders under ciclosporin immunosuppression. Conventional immunosuppression (azathioprine, anti-lymphocyte globulin, prednisone) also demonstrates comparable risks to develop tumors. Lympho-proliferative lesions regress after dose reduction or cessation of treatment. Furthermore, combinations of various immunosuppressants may result in a higher incidence of viral infection and malignancy. In summary, chemical immunosuppression carries the intrinsic risk of tumor growth. In the case of ciclosporin, which has no direct genotoxic effect, tumor promotion is probably dose-dependent. Thus, the risk may be reduced by low dosage and by avoiding combination therapies with additional immunosuppressants.

Animals↗
PubMed1992

Pathology induced by interleukin-6.

Interleukin 6 (IL-6) is a multi-functional cytokine which plays an important role in the immune response, hemopoiesis and host defense. Recombinant human IL-6 (rhIL-6) was administered at high doses to mice, rats and non-human primates. In all species IL-6 had an immunostimulatory and hemopoietic (especially on megakaryocytes) effect. An acute phase response was most pronounced in non-human primates, which was not, however, associated with any major histopathological liver change. Finally, no evidence of glomerular pathology was found. Neutralising antibodies were detected within 10 days of rhIL-6 administration in all species.

Animals↗
PubMed1992

SRI 62-834, a cyclic ether analogue of the phospholipid ET-18-OCH3, displays long-lasting beneficial effect in chronic relapsing experimental allergic encephalomyelitis in the Lewis rat. Comparison with cyclosporin and (Val2)-dihydrocyclosporin effects in clinical, functional and histological studies.

The therapeutic effect of the ether phospholipid SRI 62-834, which lacks the characteristics of an immunosuppressive agent, was compared with those of two immunosuppressive drugs, cyclosporin and valine2-dihydrocyclosporin, in a rat model of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). Drug treatment was initiated at the beginning of the first spontaneous remission on day 15 and was discontinued on day 31. Whereas the untreated rats experienced two paralytic relapses around days 21 and 31, the progression of CR-EAE was prevented during the period of drug administration. Protection with both cyclosporin and its derivative was complete, but SRI 62-834 only attenuated the clinical disease. The absence of paralytic symptoms was reflected by a distinct reduction in mononuclear cell infiltration in the central nervous system at days 21 and 31 in treated animals. The main difference between the two drug classes became apparent after withdrawal of therapy. Discontinuation of SRI 62-834 resulted in a long-lasting beneficial effect, with the rats remaining clinically normal and showing no histopathological changes. However, cyclosporin only delayed the clinical symptoms which reappeared after cessation of treatment. The exacerbated paralytic relapse, which followed about 1 week later and was associated with severe perivascular cell infiltrates and tissue destruction, subsequently became chronic in several animals. By contrast, withdrawal of valine2-dihydrocyclosporin partially prevented disease relapse and markedly reduced severity of symptoms without progression of a chronic disease. These results demonstrate the clear differences in the mode of action of these compounds in CR-EAE and suggest that SRI 62-834 could be an interesting candidate for the treatment of multiple sclerosis.

Animals↗
PubMed1992

Immunosuppression and cancer: the ciclosporin case.

Experimental and clinical data relevant for the evaluation of the carcinogenic potential of the immunosuppressant ciclosporin are reviewed. Ciclosporin binds reversibly to the cytosolic receptor protein ciclophilin. Ciclophilin is likely involved in the blockade of lymphocyte activation-induced gene transcription of various growth factors, especially interleukin-2. The drug has no effect on the transcription of housekeeping genes nor does it activate any gene. Ciclosporin may inhibit tumor cell growth, notably those which are growth factor dependent. At high concentration virus-transformed cells, especially Epstein-Barr-infected B-lymphocytes, may escape the control of specific cytotoxic T-lymphocytes. Ciclosporin has no genotoxic activity, and has no DNA-binding property. In experimental studies ciclosporin did not cause cancer in the absence of an initiating event (e.g. chemical mutagen). However, by its immunosuppressive property, the drug may allow the growth of initiated tumor cells in vivo, an effect which is dose-dependent. In clinical use ciclosporin immunosuppression is associated with an increased incidence of lymphoproliferative disorders and other malignancies particularly of the skin when compared with a normal, not immunosuppressed population. Conventional immunosuppression (azathioprine, antilymphocyte globulin, prednisone) also demonstrates comparable risks to develop tumors. Lymphoproliferative lesions regress after dose reduction or cessation of treatment. Furthermore, combinations of various immunosuppressants with associated 'over-immunosuppression' may result in a higher incidence of viral infection and malignancy. In summary, chemical immunosuppression carries the intrinsic risk of tumor growth. In the case of ciclosporin this effect is dose dependent. Thus, the risk may be reduced by low dosage and by avoiding combination therapies with additional immunosuppressants.

Animals↗
PubMed1991

A brief review of modern toxicologic pathology in regulatory and explanatory toxicity studies of chemicals.

Macroscopic and histologic evaluation of animal studies for general toxicity and carcinogenicity are cornerstones of the risk assessment of new chemical entities. Standard toxicopathologic evaluation is mainly based on the study of paraffin sections stained with hematoxylin and eosin. There are, however, a number of new approaches and techniques which have improved the objectivity of evaluation and the accuracy of cell identification, and provided deeper insight into the molecular biological mechanisms of toxicity and carcinogenicity. Such approaches include the standardization of the nomenclature, the creation of data banks for morphological alterations, the use of computers to register pathological findings in toxicity studies and to statistically evaluate incidences, and the use of morphometry. Other modern techniques are immunohistochemistry, in situ hybridization, and the assessment of cell kinetics.

Animals↗
PubMed1991

Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for cyclosporine.

The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a 3H-cyclosporine diazirine analogue (3H-PL-CS) was used to photolabel viable MDR 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↗
PubMed1991

Tumor necrosis factor receptors in lymphoid tissues and lymphomas. Source and site of action of tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF alpha), which is produced by germinal center dendritic reticulum cells (DRC) in lymphoid tissue, plays a regulatory role in a local immune response. However no information is available on the nature and location of cells responding to this cytokine. Thus TNF receptor distribution was investigated in situ by immunohistochemistry using monoclonal antibodies directed against the p75 and p55 receptor proteins. Receptor expression was unique and restricted to the lymphoreticular tissue. The p75 receptor was found on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal center DRCs, which are the main site of TNF alpha production. The two receptor proteins were expressed on distinct cell populations of the lymphoid system and no coexpression was observed. Preliminary results indicate that TNF receptor (TNFR) expression is regulated; Upregulation of TNFR proteins was found in reactive hyperplasia together with increased TNF alpha expression. In lymphoproliferative disorders, expression of the p75 receptor and TNF alpha was found mainly in high-grade malignant non-Hodgkin lymphomas. In summary, TNF alpha produced by germinal center DRCs might regulate an in vivo immune response through autocrine and paracrine pathways. Thus TNF alpha might signal, through the distinct TNFR proteins, the p55 and p75 receptor, which are expressed on different cell types in lymphoid tissue.

Humans↗
PubMed1991

Tumour necrosis factor receptor distribution in human lymphoid tissue.

The nature and location of cells responding to tumour necrosis factor-alpha (TNF-alpha) were investigated in situ by immunohistochemistry using monoclonal antibodies (mAb) directed against the p75 and p55 proteins of the TNF receptor. Receptor expression was found in the thymus and secondary lymphoid tissues. In the thymus the p75 receptor was confined to medullary lymphoblasts and dendritic cells, which co-stain with the Tac protein of the interleukin-2 (IL-2) receptor. In lymph nodes and other secondary lymphoid tissues, the p75 receptor was expressed on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal centre dendritic reticulum cells (DRC), which is the main site of TNF-alpha production. TNF receptor (TNFR) proteins were up-regulated in reactive hyperplasia together with increased TNF-alpha expression. Surprisingly, no TNFR was detectable on non-lymphoid tissues. The species specificity of these TNFR antibodies was high: whereas the antibodies cross-reacted with epitopes in non-human primates, no immunoreactivity was detected in lower animal species, e.g. dog, rabbit and rodents. The data presented suggest that TNF-alpha, which is produced by germinal centre DRC, might regulate an in vivo immune response through autocrine and paracrine pathways, e.g. through the p55 and p75 receptor proteins, which are expressed at different sites of the lymphoid tissue.

Animals↗
PubMed1991

Distribution of the cyclosporine binding protein cyclophilin in human tissues.

Cyclophilin (CYP) is the major intracellular binding protein for the immunosuppressive drug cyclosporine (CS). CYP distribution was investigated in human tissues by solid-phase immunoassay, Western and Northern blot analysis as well as immunohistochemistry. CYP was found in all tissues examined at concentrations in the range of 1 microgram/mg protein. Furthermore, mRNA specific for CYP was found in every tissue, indicating local production of the protein. Immunohistochemical investigations revealed preferential parenchymal and only little stromal localization. Within certain organs, e.g. kidneys, regional differences of immunoreactive CYP was evident. The presence of CYP was also investigated in several lymphoid and non-lymphoid cell lines and was found at comparable concentrations. Immunogold staining confirmed cytosolic, but revealed also nuclear localization of CYP. The possible role of this abundant CS binding protein is discussed.

Amino Acid Isomerases↗
PubMed1990

Inhibition of interleukin 4 receptor expression on human lymphoid cells by cyclosporin.

The effect of the immunosuppressant cyclosporin (CsA) on the expression of interleukin (IL) 4 membrane receptors on human peripheral blood mononuclear cells (PBMC) was investigated after cell activation by anti-CD3 antibody, IL 2 or IL 4. Previous studies with 125I-labeled IL 4 identified on resting lymphocytes a trimolecular complex consisting of a 65/70-kDa doublet and a 110-kDa protein with approximately 300 high-affinity binding sites (Kd 100 pM) and approximately 9000 low-affinity binding sites (Kd 30 nM). Upon cell activation by anti-CD3 antibody both low- and high-affinity binding sites increased about threefold concomitant with up-regulation of all the cross-linked proteins. CsA inhibited anti-CD3 antibody-induced up-regulation of IL 4 receptor (IL 4R)-associated proteins as well as the expression of high-affinity binding sites. However, up-regulation of IL 4R by its own ligand or IL 2 and the growth-promoting effect of IL 4 on activated, IL 4R+ T cells were CsA resistant. Since CsA inhibits the synthesis of IL 4, exogenous IL 4 was added to the cultures and it partially reversed the inhibitory effect of CsA on cell proliferation as well as on IL 4R expression. It is concluded that the inhibitory effect of CsA on IL 4R expression may contribute to the immunosuppressive effect of the drug.

Affinity Labels↗
PubMed1990

Renal side-effects of cyclosporin A with special reference to autoimmune diseases.

At therapeutic drug levels, the functional changes which occur are a reduction of glomerular filtration rate and renal plasma flow. At higher doses, morphological changes develop which may result, particularly in severe cases, in acute or chronic renal failure. The threshold for the development of irreversible vascular-interstitial lesions mainly depends on the increment of serum creatinine, age and drug dosage or drug blood level. Based on the experience with cyclosporin A (CyA), the following recommendations have been made for its clinical use, especially in patients with autoimmune diseases. The initial dose should not exceed 5 mg/kg body weight and the dose should be reduced if blood CyA levels are over 250 ng/ml; in addition, a dose reduction is recommended if serum creatinine values exceed 30% of pre-treatment values or if other signs of CyA toxicity, such as hepatotoxicity or hypertension, are found. Strict adherence to these suggestions should allow treatment of patients for prolonged periods without irreversible morphological lesions.

Autoimmune Diseases↗
PubMed1990

Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant.

Antibodies, Monoclonal↗