Cell surface enzymes of brain cortex cells or lymphocytes during early allogeneic reaction.

The aim was to study the role of major histocompatibility complex (MHC), in mice named H-2, during early allogeneic reactions (AR) of brain cortex cells or lymphocytes. We used neuronal and glial enriched perikarya, spleen and thymus lymphocytes or their subpopulations. Rat AR was also assayed between C-6 astrocytoma cells and spleen lymphocytes. We demonstrated that: 1) H-2 dependent stimulation of Na+,K+-ATPase and ouabain-sensitive K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activities represented specific response in both AR of unseparated brain cells or lymphocytes. On the other hand, non-specific AR-induced stimulation of Ca2+-ATPase activity was observed. 2) Allogeneic enriched glial fractions reacted similarly by the same enzyme activation in contrast to no change in AR between enriched neuronal fractions. Allorecognition ability of glial cells was confirmed by AR between C-6 astrocytoma cells and lymphocytes. 3) Mature thymus lymphocytes exerted alloreactivity by specific activation of Na+,K+-ATPase or K+-pNPPase, in contrast to no change in AR between immature lymphocyte subpopulations. 4) MHC Class II monoclonal antibody inhibited Na+,K+-ATPase and K+-pNPPase activities in brain cells as well as in thymus and spleen lymphocytes in a dose-dependent manner. Results support former studies about alloantigen-induced uncoupling in brain oxidative cortex metabolism (Kováru Med. Biol. 58: 273, 1980) via Na+,K+-ATPase and K+-pNPPase inhibition by mechanism which can mimic MHC restriction.

Immunolocalization of cyclophilin in normal and cyclosporin A-treated human lymphocytes.

A polyclonal rabbit antibody against a protein fraction (10-30 kDa) of human thymuses with a high CsA-binding activity of dominant protein cyclophilin (CPH) was prepared and characterized. In immunoblotting with the cell lysate from JURKAT T cell line, this antibody specifically reacted with 18-kDa protein corresponding to CPH. In indirect immunofluorescence the antibody visualized granular structures in JURKAT cells and human peripheral blood lymphocytes. In JURKAT cells cultivated with 1-2 micrograms of CsA per ml for 7 days a much weaker reaction of the antibody was found, compared with non-treated cells. In some CsA-treated cells the antibody visualized various 'star-like' or filamentous structures. A similar staining pattern has also been obtained in lymphocytes of the patients receiving CsA therapy. Complementary staining with rhodamine-tagged phalloidin revealed changes in F-actin distribution of CsA-treated JURKAT cells. In conclusion, the treatment with CsA induces dramatic changes of CPH cellular distribution, which may take part in the final therapeutic effect of the drug.

A natural specific intracellular ligand as a reagent for cyclosporine blood level determination.

We isolated and characterized a non-immunoglobulin fraction from calf thymuses which was found very active in binding [3H]labelled cyclosporine (CsA). It contains the natural, intracellular receptor cyclophilin, specifically binding to the active site of the CsA molecule, which may also play a part in the immunosuppression of human immunocompetent cells. This material with a high selectivity could be used, instead of specific monoclonal or polyclonal antibodies, to determine CsA levels in patient's blood.

[Antibodies against deoxyribonucleoproteins in circulating immune complexes in the blood of patients with autoimmune diseases]. / Protilátky proti deoxyribonukleoproteinu v cirkulujících imunitních komplexech a sérech nemocných s nekterými autoimunitními chorobami.

In 40 patients with various autoimmune diseases antibodies against desoxyribonucleoprotein (DNP) were assessed by enzyme immunoanalysis (ELISA) in serum and after dissociation of the isolated precipitate of circulating immune complexes (CIC) in the presence of polyethylene glycol. The results indicate that antibodies against DNP are not specific for systemic lupus erythematosus (SLE) and can be detected in serum and in particular in CIC in various autoimmune conditions. In SLE they may be important for evaluation of the activity of the disease, in particular if estimated concurrently in serum and in CIC.

HLA molecule--cytoskeleton associations induced by cross-linking with specific antibodies.

Sera of patients with antibodies against HLA-typed panel, control human sera, anti-beta 2-microglobulin monoclonal antibody recognizing HLA class I molecules, anti-HLA class II monoclonal antibodies and control antibodies were used in indirect immunofluorescence study of living human peripheral blood lymphocytes and their cytoskeletons. Lymphocytes immobilized on concanavalin A-precoated glass slides enabling extraction procedures and repeated washings were found useful for such a study. If the lymphocytes were treated with human polyclonal anti-HLA antibodies only, or with two antibodies (i.e. with a specific monoclonal anti-HLA antibody and an anti-mouse Ig polyclonal antibody labelled with fluorescein isothiocyanate (FITC), the cross-linking of the surface molecules caused their resistance to the detergent effect. Murine monoclonal anti-HLA antibodies alone, bound to the cell surface molecules, formed immune complexes easily extracted with Triton solution. In such cases, the second anti-mouse Ig antibody labelled with FITC did not visualize specific molecules in the remaining matrix. No significant changes in the microtubules of cells treated with anti-HLA antibodies were observed.

CMV infection in patients with chronic renal failure and in those following transplantation.

Serum anti-CMV IgG was found in 62% of healthy blood donors. Patients in a chronic haemodialysis programme were seropositive in 80%, and kidney graft recipients in 93 to 100%, depending on the post-transplantation period. Median of the IgG titre was 1:10 in healthy persons, 1:80 in dialysed patients, and 1:40 in graft recipients. Anti-CMV IgG was never detected in healthy blood donors. IgM positivity was found in one of 30 dialysed patients, and in four of 27 kidney graft recipients. Compared with healthy blood donors, a higher percentage of CD 8 lymphocytes and LGL was found in dialysed patients exhibiting an anti-CMV IgG above 1:40. NK activity was impaired in all patients in the chronic haemodialysis programme.

Changes in the microtubular skeleton of human lymphocytes produced by immunosuppressive substances.

The technique of immunofluorescent visualization of the microtubular skeleton of human lymphocytes isolated from the peripheral blood of healthy donors and immobilized on Concanavalin A--precoated slides was used. These lymphocyte monolayers are suitable for extraction and employment of indirect immunofluorescence. Using a monoclonal antitubulin antibody, one can visualize the MT apparatus of control human lymphocytes as an organizing centre with attached beamlike structures. Lymphocytes treated with ATG or monoclonal OKT 3 antibody in vitro manifest changes in the shape of cells, inhibition of the blast-like cell formation, and an atypical MT system: disturbance or even disappearance of the beamlike structure, formation of bundles and "caps" resulting, in some cases, in shedding of a part of cytoplasm. Rabbit ATG binds to all the cells of the monolayer, and can be shown even after cell extraction. Control experiments with nonspecific rabbit or murine IgG did not induce any of the above changes. Reorganization of the MT apparatus of human lymphocytes affected by ATG or OKT 3 in vitro can be explained either by a direct association of antigenic determinant with MT, or by the persistence or/and internalization and further processing of immune complexes of antigenic membrane determinants with specific antibodies.

Contribution to rapid diagnosis of infectious mononucleosis.

By indirect immunofluorescence (IF) technique humoral antibodies to Epstein-Barr virus capsid antigen (EB-VCA) and to cytomegalovirus (CMV) were detected in 47% and 9% of persons with infectious mononucleosis (IM), respectively. In 23% of the patients examined, IgM antibodies to both viruses were detected, while in 8% of them high titres of IgG only were found in the absence of IgM class antibodies to EB-VCA or to CMV. The finding of IgM antibody to EB-VCA was in good correlation with the persisting symptoms of the disease. Discrepancy between the presence of specific IgM and the absence of heterophilic antibodies was observed in some children and in all persons with persistent or recurrent signs of IM. In the latter, specific IgM was found only during exacerbation of the disease, but during remissions IgG antibodies persisted in high levels. Antibodies to Epstein-Barr virus nuclear antigen (EBNA) were detected in all chronically ill persons and antibodies to the R-component of Epstein-Barr virus early antigen (EA) were present in the majority of them.