[The analysis of concentration of cyclosporine A in blood using liquid chromatographic mass spectrometry].

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, hemosiderosis, and hepatic fibrosis in a renal graft recipient].

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.

[Chronic hepatitis B and C in renal graft recipients].

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.

Lipopolysaccharide-dependent and lipopolysaccharide-independent pathways of monocyte desensitisation to lipopolysaccharides.

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.

Evidence for antibody and immune-complex deposition in heart transplantation: a report of three cases.

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.

Virus-induced blast transformation of lymphocytes and its inhibition by F(ab1)2 fragments of human IgG.

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.

[Distribution and features of infection with hepatitis viruses B and C during hemodialysis treatment]. / Rasprostranennost' i osobennosti infitsirovaniia virusami gepatitov B i C v usloviiakh lecheniia gemodializom.

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.

[Use of fluorescein isothiocyanate to label plasma membrane of lymphocytes]. / Ispol'zovanie fluorestseinizotiotsianata dlia mecheniia plazmaticheskikh membran limfoidnykh kletok.

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.

[Immunopathologic study of the myocardium in dilated cardiomyopathy]. / Immunopatologicheskoe issledovanie miokarda pri diliatatsionnoi kardiomiopatii.

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.

[Humoral immunopathologic changes in the myocardium after clinical allotransplantation of the heart]. / Gumoral'nye immunopatologicheskie izmeneniia v miokarde pri allotransplantatsii serdtsa v klinike.

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.

[Prednisolone sensitivity of peripheral blood mononuclear cells of patients with systemic lupus erythematosus clinically resistant to glucocorticoids and the effects of carbon hemoperfusion]. / Chuvstvitel'nost' k prednizolonu mononuklearnykh kletok perifericheskoi krovi bol'nykh sistemnoi krasnoi volchankoi, klinicheski rezistentnykh k deiistviiu gliukokortikoidov, i vliianie gemokarboperfuzii.

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.

[Neoislets obtained from rat pancreas do not contain passenger leukocytes]. / Neoostrovki, poluchennye iz podzheludochnoi zhelezy krysy, ne soderzhat leikotsitov-passazhirov.

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.

Alterations in mononuclear cell tumour necrosis factor-alpha (TNF-alpha) response in patients on long term cuprophane haemodialysis.

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.

[Interactions of mononuclear cells of peripheral blood with islet cells in vitro]. / Vzaimodeistvie mononuklearov perifericheskoi krovi s ostrovkovymi kletkami in vitro.

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.

[Effect of the preliminary incubation of human peripheral blood lymphocytes on the intensity of transformation in phytohemagglutin stimulation]. / Vliianie predvaritel'noi inkubatsii limfotsitov perifericheskoi krovi cheloveka na intensivnost' transformatsii pri stimuliatsii fitogemaggliutininom.

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.

[Effect of alpha 1-protease inhibitor (alpha 1-antitrypsin) on the intensity of phytohemagglutinin stimulated lymphocyte transformation]. / Vliianie al'fa l-proteaznogo ingibitora (al'fa 1-antiripsina) na intensivnost' transformatsii limfotsitov, stimulirovannykh fitogemaggliutininom.

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.

[The cytology of the urinary sediment in the differential diagnosis of the complications in patients with kidney allografts]. / Tsitologiia osadka mochi v differentsial'noi diagnostike oslozhnenii u bol'nykh s allotransplantatami pochek.

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.

[Preliminary lymphocyte activation by a mitogen as a condition for demonstrating the ability of the Fab-fragment of normal IgG to inhibit lymphocyte transformation]. / Predvaritel'naia aktivatsiia limfotsitov mitogenom kak uslovie dlia proiavleniia sposobnosti Fab-fragmentov normal'nogo IgG ingibirovat' transformatsiiu limfotsitov.

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.