Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

BACKGROUND: Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS: CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS: A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION: These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

The V3 loop of human immunodeficiency virus type-1 envelope protein is a high-affinity ligand for immunophilins present in human blood.

Human immunodeficiency virus type-1 (HIV-1) infection requires binding of the envelope protein gp120 to host CD4 receptors and the action of the chemokine receptors CXCR4 or CCR5, which define cell tropism. The proline-containing V3 loop of gp120 determines the selection of the chemokine receptor and participates in conformational changes on binding of gp120 to CD4. In this study, we show that macrophage-tropic and T-cell-tropic V3 loop peptides bind specifically to the active site of the immunophilins FK506-binding protein (FKBP12), and cyclophilins A and B. Macrophage-tropic and T-cell-tropic V3 loop peptides inhibited the peptidyl-prolyl cis-trans isomerase (PPIase) activities of the immunophilins. Kd values in the range 0.036-4.1 microM were determined with V3 loop peptides labeled with an environmentally sensitive fluorophore. The observed binding properties of the V3 loop peptides reveal structural motifs of linear water-soluble peptidic substrates for tight interaction with immunophilins. FKBP12, and cyclophilins A and B were found to be present in normal human blood in the ranges 0.8-1.7, 1.4-2.3 and 2.4-3.1 nM, respectively, as demonstrated by PPIase activity measurements and western blot analysis. Cyclophilins A and B levels in serum of HIV-1-infected individuals were increased 3.6-fold and 1.6-fold. Due to the interaction of immunophilins with V3 loop peptides and with the envelope protein gp120, a role of immunophilins in HIV pathogenesis as conformases or docking mediators seems possible, since immunophilin receptors on cell membranes and immunophilin-related virulence factors of pathogens have been identified.

The human erythrocyte membrane contains a novel 12-kDa inositolphosphate-binding protein that is an immunophilin.

A 12-kDa inositolphosphate-binding protein has been identified as a component of the human erythrocyte membrane. Its robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by the immunosuppressant drugs FK506 and rapamycin indicates that it is an immunophilin belonging to the FKBP class. The finding that its peptidylprolyl cis-trans isomerase activity is also strongly inhibited by nanomolar concentrations of the second messengers inositol 1,4,5-trisphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) suggests that IP3 and IP4 could be physiological ligands for this membrane-associated immunophilin.

Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies.

Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against peptides copying the most divergent parts of the two sequences. These antibodies allowed us to develop an ELISA capture assay selective for either isotype. Thus, we showed that leukocyte CyPB concentration was almost ten times lower than that of CyPA, and that in contrast to the results described in the literature, only CyPB was released in plasma. Moreover, CyPB levels in leukocytes and plasma were found to correlate for the same donor, but no relationship was found with CyPA level.

The effect of cyclosporine administration on the cellular distribution and content of cyclophilin.

Cyclophilin (CYP), an intracellular protein sharing amino acid sequence identity with the enzyme peptidyl-prolyl cis-trans isomerase has become the leading candidate for the receptor responsible for cyclosporine biological effects. Avid binding of CYP to cyclosporine and immunosuppressive cyclosporine metabolites has been demonstrated, while nonimmunosuppressive cyclosporine metabolites have tended not to bind to cyclophilin. A previous immunohistochemical analysis documented that CYP localized principally to the cytoplasmic cellular compartment, but nuclear staining was observed among some cells. This study was undertaken to more precisely define the ultrastructural distribution of CYP, and to determine whether CYP cellular content was affected by CsA therapy. Untreated Wistar rats or those receiving 7 days of CsA (15 mg/kg/day, i.p.) were anesthetized, perfusion-fixed in situ, and sacrificed. Analyses of lymph node, spleen, thymus, kidney, liver, heart, brain, and ileum used an affinity purified, rabbit anticyclophilin IgG. Transmission electron microscopy was performed after staining with anti-CYP using a horseradish peroxidase/biotin/avidin technique. Quantitative immunofluorescence was measured by confocal microscopy using anti-CYP, with a biotin/avidin/phycoerythrin technique. Cyclophilin localized to the cytoplasmic compartment--however, association with mitochondria endoplasmic reticulum, Golgi, and with the nuclear membrane among lymphocytes, as well as cells from kidney, liver and ileum--was documented. Cyclophilin was not identified within the nucleus proper. Tissues obtained from animals receiving CsA exhibited a generalized increase in CYP content compared with tissues from untreated controls, suggesting the possibility that CsA may exert a regulatory influence upon CYP gene activation. Collectively, the data were consistent with the hypothesis that CYP exerts a central role in cellular metabolism, and that CsA-mediated biologic effects result from the CsA/CYP interaction.

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.

[Natural resistance of the body in appendicular peritonitis in children]. / Sostoianie estestvennoi rezistentnosti organizma pri appendikuliarnom peritonite u detei.

It is found that in appendicular peritonitis, as the inflammatory process progresses, the protective functions of the organism are lowered, and the intensity of inflammatory changes is increased. The determination of the natural resistance status of the body in peritonitis in children may be used for an objective estimation of the process course, the efficacy of therapeutic measures and prognostication.

[Immunologic reactivity of patients with acute leukemia]. / Ob immunologicheskoi reaktivnosti bol'nykh ostrym leikozom.

In sera of patients with acute leucosis the authors have determined antibodies to alpha-toxin, streptolysin-O, the complement, lysosyme. B-lysins and C-reactive protein. It was found that the indices of immune reactivity in patients with acute leucosis are dependent on a morphological variant of the disease, the duration of the conducted therapy and presence of complications. The most high immunity indices in patients with acute leucosis were observed in a primary active stage of the disease and during the period of remission. Considerable reduction of the immunity was noted in the terminal stage of the disease.