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The method of analysis of concentration of immunosuppressant of cyclosporine A in whole blood was developed. The highly effective liquid chromatography with mass spectrometric detection was applied using device of "ionic trap" type. The optimal conditions of analysis are established. The tryout of method was carried out using blood samples of healthy donors and patients underwent allotransplantation of organs. The comparison was made of the developed method with method of fluorescence polarized immunoassay Abbott TDX applied in clinical diagnostic. The higher selectivity of the proposed method to cyclosporine A as compared with Abbott TDX was established.

Chronic hepatitis B is a frequent concomitant disease in recipients of a renal graft that worsens results of kidney transplantation due to renal and extrarenal complications. Much rarer hemochromatosis either has genetic roots (hereditary hemochromatosis) or results from multiple blood transfusions and hemolysis during treatment by hemodialysis (secondary hemochromatosis). Combination of chronic hepatitis B and hemochromatosis increases the risk of chronic liver disease leading to cirrhosis and hepatocellular carcinoma. Success of antiviral therapy combined with massive phlebotomy is illustrated by a case of kidney transplantation to a patient with chronic hepatitis B of large duration and iron overload syndrome.

Clinical and morphological features of chronic hepatitis B (CHB), C (CHC), and B+C (CHB+C) were studied in 283 renal graft recipients. High total bilirubin serum levels were detected significantly more often in CHB and CHB+C patients vs. CHC patients. High ALT activity was noted in 65% of CHB patients and only in 45% of CHC patients (p = 0.003). Stable low activity of hepatitis prevailed in renal recipients; it was noted in 56.7% of CHB patients, 66.2% of CHC patients, and 62% of CHB+C patients. The character of pathomorphological liver changes in chronic viral hepatitis was studied in 53 renal graft recipients using puncture biopsy. Histopathological activity index (HAI, Knodell R.G. et al., 1981) witnessed a more severe liver lesion in CHB vs. CHC and CHB+C. Thus, inflammatory activity in CHB was found to be minimal or low in 13 patients, and moderate or high in 11 patients, whilst a minimal or low activity in CHC or CHB+C was found in 16 and 10 patients, respectively, and a moderate activity was detected only in two and one, respectively (p = 0.016 and 0.024 compared with CHB). Advanced hepatic fibrosis or cirrhosis was significantly more frequent (p = 0.006) in CHB patients (eight out of 24) than in CHC ones (none out of 18). The rate of advanced sclerotic changes in CHB+C was lower (one out of 10 patients) than that in CHB, and similar to CHC. Thus, clinico-morphological manifestations were more prominent in renal graft recipients with CHB vs. CHC.

What is a contribution of the humoral (vascular) and mixed type of the rejection episodes to all the episodes of heart allograft rejection is not quite clear, though this factor is of considerable importance for the choice of the treatment methods. The hearts from recipients, as well as endomyocardial biopsies of the heart allografts and postmortem material were investigated with the aim to determine the immunopathological process. Overall, 420 samples from 80 patients were analyzed. Immunofluorescence examination of endomyocardial biopsy showed that in 8 from 44 patients with heart allograft in postoperative period for the first six weeks there were revealed the immunomorphological signs of the acute humoral rejection, manifested as fixation of immunoglobulins and complement in capillaries. Six of them exhibited rejection of mixed type. Most patients in the later postoperative period exhibited a discrete local fixation of immunoglobulins and complement in myocardium, that can be assessed as one of the compartments of the chronic rejection process. In cases of the secondary administration of serum preparations, the fixation of immune complexes was shown in sarcolemma and capillaries, and can be proposed as a sign of serum disease. Repeated acute rejection episodes of humoral or mixed types raised at the first six weeks after transplantation. In the period from 1-5 years after operation, patients displayed discrete deposits of the immunoglobulins and complement as part of the chronic rejection process.

The prevalence of hepatitides B and C was evaluated in 140 patients treated by hemodialysis. Almost half of patients (48%) had acute hepatitis B which completely resolved. Acute hepatitis B was detected in 6% in the course of observation. In 6% chronic hepatitis B was diagnosed, and in 24% chronic hepatitis C. A combination of hepatitides B and C was diagnosed in 2% patients. Only 12% patients were not infected with hepatitis. Genotype 1b predominated in patients with HCV infection (73%); genotypes 1a, 21, and 3a were equally incident (9%). Replication of HBV and HCV in patients with uremia under conditions of hemodialysis was detected in 83 and 86% patients, respectively. Relationship between HBV and HCV infection and the duration of hemodialysis treatment was analyzed. The percentage of non-infected patients persistently decreased, and the time course of HBV and HCV infection was different. Infection with HBV after the beginning of hemodialysis occurred sooner (16.0 +/- 4.0 months) than with HCV (30.2 +/- 4.6 months, p < 0.04). The levels of SGPT and SGOT in patients with various manifestations of HBV and HCV infection treated by hemodialysis were followed up.

Endomyocardial diagnostic biopsies, recipient heart removed at operation, endomyocardial biopsies of allotransplants and postmortem material were studied using immunofluorescence to specify immunopathological process and to detect humoral rejection. Altogether 306 samples from 55 patients were studied. In the early postoperative period (one year) 8 out of 18 patients with heart transplants repeatedly showed immunopathologic picture of acute humoral (vascular) rejection which was characterized by a widespread immunoglobulin G and complement fixation in the capillary walls accompanied by enhanced capillary permeability and fibrin deposition in intestitial tissue. Such patients often had graft dysfunction. 1 to 5 years after transplantation in 24 out of 37 patients discrete focal immunoglobulin and complement fixation was observed as one of chronic rejection component.

Urine sediment from 56 renal transplant recipients was tested cytologically. The recipients had 7 episodes of acute rejection (AR), 8 episodes of cyclosporin nephrotoxicity (CNT), acute tubular necrosis (ATN), combined complications, bacterial infection in 11, 6 and 16 cases, respectively. 12 patients had stable renal function. Cytospin preparations were made for Pappenheim staining. FACScan cytograms were obtained based on light scattering (cell size and granularity). Low cell count in patients with stable renal function and higher levels of mononuclear cells in all cases of a complicated postoperative period were seen. In acute tubular necrosis the sediment had a high scatter pattern, i.e. high density and large particles, while in cyclosporin nephrotoxicity renal epithelial cells had a low scatter pattern. Polymorphs were typical of the bacterial infection and acute tubular necrosis. Acute rejections were associated with elevated lymphocyte number. Microscopic examination revealed predominance of collect duct cells, lymphocytes with morphological signs of activation in acute rejection and proximal tubular cells in cyclosporin nephrotoxicity. Episodes of combined complications were difficult for diagnosis due to their mixed cellularity.

Reaggregation of pancreatic islet cells during their cultivation in stationary culture leads to formation of neoislets--compact insulin secreting clusters. By the means of indirect immunofluorescence it has been shown that neoislets have no leukocytes-passengers, common for normal islets and responsible for allograft rejection.

Immunofluorescence microscopy of endomyocardial biopsy specimens from heart allograft recipients identified immunopathologic changes in three of 17 patients. These changes included immunoglobulin G and complement C3 deposition in tissue structures such as capillary endothelium and basal membranes, cardiomyocyte sarcolemma, and interstitial tissue. Moreover, the immunopathologic changes could be correlated with acute cellular rejection episodes evidenced by endomyocardial biopsy criteria.

We have investigated TNF-alpha secretory response of peripheral blood mononuclear cells (PBMC) from 13 uraemic patients undergoing regular haemodialysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells of haemodialysis patients exhibited increased TNF-alpha release in vitro in the absence of activating stimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-alpha from dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its independence of trace amount of lipopolysaccharide (LPS) present in the medium as contaminant. Furthermore, predialysis PBMC were considerably more sensitive to stimulation with 10(7) pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF-alpha release from monocytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-alpha secretion from PBMC above the level of cells cultured on tissue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-alpha release. Furthermore, PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-alpha in response to a subsequent LPS stimulation, while a 2-day treatment of cells with LPS was followed by LPS refractory state. Therefore, direct contact with CM induces in PBMC a long-lasting TNF-alpha response which is not down-regulated by the acquisition of refractoriness in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation of the observation that predialysis PBMC exhibit elevated TNF-alpha secretory capacity.

Myocardial tissues of patients with dilated cardiomyopathy were studied by immunofluorescence. While immunoglobulin A fixation was observed in myocardial capillary wall and cardiomyocyte sarcolemma in the majority of patients (11 of 12), immunoglobulin G and C3 complement component were a rare finding. In the vessel wall of heart allografts immunoglobulin A fixation occurred 3-6 days after transplantation. As a result of the intensive immunosuppressive therapy which was used after the operation immunoglobulin A disappeared from heart allografts within 4-5 weeks. Immunoglobulin A fixation in the heart of patients with dilated cardiomyopathy is attributed to the anti-tissue or antivirus antibodies and probably is involved in the development of this disease.

The present study demonstrates that with time in culture blood monocytes (MO) lose their ability to express procoagulant activity (PCA) and secrete tumor necrosis factor-alpha (TNF alpha) in culture medium in response to lipopolysaccharide (LPS) stimulation. Thus, upon 10 micrograms/ml LPS stimulation for 4 hours 2-day-old MO produced lower levels of PCA and TNF alpha than fresh MO. The decrease in responsiveness was not caused by cell death, since in the case of TNF alpha it was fully reversible by interferon-gamma (IFN-gamma). Compared with cells pre-incubated in medium alone, the responsiveness of MO pre-incubated in LPS was further decreased. Thus, in MO LPS pre-incubation was followed by an LPS refractory state. It was expected that the decrease in responsiveness induced by cultivation in medium alone was mediated by LPS contamination of culture medium. However, as we were unable to prevent this decrease by neutralizing LPS contamination of the culture medium with polymyxin B, the loss in LPS-induced activities of cultured MO is likely to be mediated by culture conditions other than LPS contamination. Taken together the present data demonstrate that LPS-dependent as well as LPS-independent pathways of MO desensitization to LPS exist.

Peripheral blood mononuclear cells from Type I diabetic patients health donors or Wistar rats were cultured with rat islet cells for 18 h. Then TNF content of the medium and basal and stimulated insulin release were determined. Mononuclear cells from both healthy donors and diabetic patients could inhibit the insulin release with no correlation to TNF content. Addition of lipopolysaccharide resulted in a 5-7 times increase of TNF content of the medium followed by a more pronounced inhibition of insulin release. Rat mononuclear cells inhibited the beta-cell function almost completely and initially produced large amounts of TNF. The data indicate that inhibition of insulin release by blood mononuclear cells in vitro does not reflect anti beta-cell specific cellular immunity, involves cytokines and, probably depends upon the initial properties of the cells.

Purified rat islets were dissociated into single-cell suspension with an EDTA-Trypsin treatment. During a stationary culture in vitro the islet cells reassociated forming aggregates (neoislets). Electron microscopy revealed that the aggregates consisted mostly of beta-cells and not numerous alpha-cells. They showed a good insulin-secreting capacity and were able to increase insulin release in response to glucose plus theophylline. The lack of passenger leukocytes makes the neoislets particularly suited for experimental and clinical transplantation.

Peculiarities attending inhibition of the PHA-induced blast-cell transformation of human lymphocytes by F(ab')2 fragment of rabbit IgG were studied. It was shown that the fragment did not affect the intensity of blast-cell transformation if the lymphocytes were preliminarily incubated with the fragment for 24 h at 37 degrees or 4 degrees C and then transferred to the fresh medium containing PHA. However, if the fragment was added to the cells 24 or 48 h following PHA it produced a significant inhibition of the blast-cell transformation. These data may indicate that F(ab')2 fragment interferes with the lymphocyte transformation only when the cells are already activated with PHA.

A study was made of the sensitivity to prednisolone (PS) of peripheral blood mononuclear cells (MS) of patients with systemic lupus erythematosus (SLE) with renal lesion showing resistance to glucocorticoids (GC). It was shown that in most of the patients (60%) MC were poorly inhibited by PS whereas the rest of the patients (40%) demonstrated in vitro sensitivity compared to that of the donors and even higher in some cases. Thus, the group of SLE patients showing clinical resistance to GC action was inhomogeneous in sensitivity of their MC to PS; only some patients showed resistance associated with the properties of the MC system. Hemocarboperfusion was shown to produce a considerable effect on MC sensitivity to PS: higher sensitivity was noted in all the cases, it was particularly noticeable in the group of patients with low basal sensitivity to PS. An analysis of the data obtained led to a conclusion that hemocarboperfusion raised MC sensitivity to PS. This mechanism was shown to determine the efficacy of the method of therapy of resistance to GC.

The effect of neutral SH-dependent proteinase of the spleen on transformation of human peripheral blood lymphocytes has been studied. Proteinase (1--10 micrograms/ml) inhibits phytohemagglutinin-stimulated (PHA) lymphocyte transformation and has no effect on spontaneous transformation. Proteinase does not alter the mitogenic properties of PHA and possesses no cytotoxicity. Therefore, inhibition of stimulated lymphocyte transformation is either resultant of proteinase direct action on the cell or associated with the inhibitor factor formation.

Thermo- and acid-stable serine proteases inhibitor from the rabbit blood serum (TASPI) was shown to inhibit the human peripheral blood lymphocytes transformation stimulated by phytohemagglutinin (PHA) or concanavalin A. The extent of inhibition depended on the concentration of the preparation and its specific activity. The maximal inhibition of lymphocytes proliferation constituted 50 to 70%. TASPI displayed no cytotoxic activity. Considerably more effective inhibition was demonstrated by TASPI addition to the culture medium 24 hours after the addition of PHA. The antiprotease activity of crude human serum and that inactivated under different conditions is described. The results obtained suggest the participation of TASPI in the control of biological activity of the lymphoid tissue cells.

alpha 1-Antitrypsin (alpha 1-AT) decreases the intensity of human peripheral blood lymphocyte transformation stimulated by phytohemagglutinin. The degree of inhibition is influenced by the antiprotease activity of alpha 1-AT. It is shown that maximal inhibition of transplantation is 50%. Participation of alpha 1-AT in the control of biological activity of lymphoid tissue cells is suggested.

Monovalent and bivalent Fab-fragments of normal human or rabbit gamma-globulin suppressed blasttransformation of human lymphocytes induced by phytohemagglutinin and concanavalin A. Peptic F(ab)2-fragments from highly-purified rabbit anti-DNP antibody displayed suppressing activity similar to that of the fragments of normal gamma-globulin. Fab-fragments affected blasttransformation when added to lymphocytes either simultaneously with the PHA or 24 and 48h after the mitogen. The data obtained may indicate that the inhibiting of lymphocyte blasttransformation produced by the gamma-globulin fragments was not caused by their competing with mitogens for the receptors on the target-cell; the Fab-fragment activity was probably determined by the structures located outside the antibody active centre.

Preincubation of lymphocytes in leukocytic suspension or those separated on verografin-Ficoll gradient at 37 degrees C leads to enhancement of blast transformation in response to phytohemagglutinin (PHA) stimulation. This effect is reversed by preincubation in the presence of soybean trypsin inhibitor (500 micrograms/ml). The results obtained suggest the participation of neutral proteinases from neutrophils or monocytes in the modification of PHA response.

Blast transformation of lymphocytes from persons immunized by vaccines against tick-borne Japanese encephalitides, in response to stimulation by homologous viral antigens was studied. 3H-Thymidine incorporation into lymphocytes was completely inhibited by F(ab1)2 fragments of normal human IgG containing no antibody to the viruses examined. A correlation of the inhibitory action of F(ab1)2 fragments on lymphocyte transformation induced by viruses and phytohaemagglutinin was observed.

The possibility of using fluoresceine isothiocyanate to label plasmatic membranes of lymphocytes was explored. Membrane labeling can be done under physiological conditions. The label interacted with the surface located membrane component, and the binding centers became saturated with the label concentration which left the membrane undamaged. Modification of the membrane surface resulting from fluoresceine isothiocyanate treatment influenced significantly intercellular interactions mediating mitogen-induced blast transformation.

It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.