Donor non-specific MICA antibodies in renal transplant recipients.

Despite recent advances in solid organ transplantations, an antibody mediated rejection caused by donor specific antibodies is still a major problem in kidney graft survival. Besides HLA-induced humoral response, antibodies against MICA antigens have recently attracted attention because of their possible role in graft rejection. The aim of our study was to establish whether renal recipients produce antibodies against MICA molecules due to the transplantation and if they are specific for MICA antigens of the donors. MICA antibody screening was performed in 124 kidney recipient sera. 22 sera, that were found to be MICA antibody positive, were further examined for MICA antibody profiles and compared with donor MICA alleles. The analysis of MICA antibody positive sera showed mostly more complex reactivity patterns. A significant fraction of patient sera (59%) reacted not only with the donor MICA antigens, but also with other MICA patterns. A match between antibody specificities and MICA antigens was observed in 41% of renal recipients only. On the other hand, as much as in 36% of recipient sera were detected antibodies against their own MICA molecules. We did not prove a complete correlation between the recipient MICA antibody specificities and MICA antigens of the donor. We assume that MICA antibody induction occurs not only due to the allogeneic stimulation itself but also due to other factors that need to be elucidated.

Circulating TGF-ß1, glycation, and oxidation in children with diabetes mellitus type 1.

The present study investigates the relationship between diabetes metabolic control represented by levels of HbA1c, early glycation products-(fructosamine (FAM)), serum-advanced glycation end products (s-AGEs), lipoperoxidation products (LPO), advanced oxidation protein products (AOPP) and circulating TGF-ß in young patients with DM1. The study group consisted of 79 patients with DM1 (8-18 years). 31 healthy children were used as control (1-16 years). Baseline characteristics of patients were compared by Student's t-test and nonparametric Mann-Whitney test (Statdirect), respectively. The correlations between the measured parameters were examined using Pearson correlation coefficient r and Spearman's rank test, respectively. A P value < 0.05 was considered as statistically significant. HbA1c was measured by LPLC, s-AGEs spectrofluorimetrically, LPO and AOPP spectrophotometrically and TGF-ß by ELISA. Our results showed that parameters of glycation and oxidation are significantly higher in patients with DM1 than in healthy control. The level of serum TGF-ß was significantly higher in diabetics in comparison with control: 7.1(3.6; 12.6) versus 1.6(0.8; 3.9) ng/mL. TGF-ß significantly correlated with age and duration of DM1. There was not found any significant relation between TGF-ß and parameres of glycation and oxidation. However, these results do not exclude the association between TGF-ß and the onset of diabetic complications.

Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays.

Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive (51)chromium ((51)Cr) release assay is a "gold standard" for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90 min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland-Altman statistical method was applied to measure true agreement for all CAM-(51)Cr, CFSE-(51)Cr, DiO-(51)Cr and MTG-(51)Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard (51)Cr release assay. Considering linear relationships between data obtained with four fluorochromes and (51)Cr release assay as well as linear regression analysis with R(2)=0.9393 value for CAM-(51)Cr pair, we found the CAM assay to be the most closely related to the (51)Cr assay.