Characterization of MICA gene polymorphism of HLA complex in the Slovak population.

BACKGROUND: The function of the MHC class I polypeptide-related sequence A (MICA) gene, which belongs to the MHC class I chain-related genes, is to trigger cytolysis of target cells mediated by NKG2D receptor recognition in NK (Natural Killer) cells and CD8 T-lymphocytes. The MICA gene has a high degree of polymorphism, especially observed in exons 2-5. MICA allelic diversity has been reported in association with some autoimmune diseases such Behcet's disease, psoriasis and diabetes, as well as with organ rejections. AIM: The aim of this study was to analyse MICA gene polymorphism in the Slovak population, to establish frequencies of MICA alleles and to compare the results with those found in other Western Eurasian populations. No such study has been performed previously in the Slovak population. SUBJECTS AND METHODS: This study examined DNA samples from 124 unrelated Slovak individuals (51 women and 73 men with an average age of 40.3 years) using direct sequencing of MICA exons 2-5. Allele and genotype frequencies were calculated by direct counting and statistical analysis was carried out using Arlequin software. RESULTS: This study identified 15 out of 71 MICA alleles. The most frequent allele was MICA(*)008 (37.1%) followed by alleles MICA(*)002 (16.5%) and MICA(*)009 (11.3%). The rarest alleles were MICA(*)027, MICA(*)006 (both 0.8%) and MICA(*)057 (0.4%), respectively. The most frequent genotypes were 008/008 and 008/002, both with a frequency of 13.7%. Exon 5 microsatellite polymorphism screening revealed five MICA alleles, namely A4, A5, A5.1, A6 and A9. The most frequent was allele A5.1 (37.1%) and the rarest A5 (8.1%). Finally it was found that haplotype MICA*008 A5.1 was the most frequent (37.1%). CONCLUSION: A comparison of these results with those reported in the literature revealed similarity in MICA polymorphism to that found in other Western Eurasian populations. The data will be useful for further association studies on MICA polymorphism and its function.

Biochemical evaluation of the antiplatelet effect of aspirin in patients at different levels of cardiovascular risk.

BACKGROUND: The phenomenon called aspirin resistance is being intensively discussed. METHODS: To evaluate the biochemical aspirin response, the method of urinary 11-dehydro TXB2 levels measurement was used. Quantitative detection of TXB2 in urine was determined by competitive enzyme immunoassay, using human Thromboxane B2 ELISA-kit. We investigated the urine samples from 69 patients. RESULTS: The mean urinary levels of 11-dehydro TXB2 were significantly lower in patients in the primary and secondary types of aspirin prevention comparing with the control group of patients not taking aspirin. The difference in thromboxane concentrations between the two groups of patients taking aspirin did not reach statistical significance. Our results did not show significant differences in the biochemically measured aspirin response when comparing diabetics with non-diabetics. Similarly, the observed tendency to higher thromboxane levels in women did not show to be significantly different from men. CONCLUSION: Our pilot study did not show any significant differences among patients at different cardiovascular risk. Since there is currently no standard laboratory method to detect aspirin non-responders available, the term aspirin resistance remains controversial and requires further research. Every effort should be done to improve patients' compliance and to prevent clinically relevant interactions of aspirin with ibuprofen. The elimination of these two factors as was the case in our study may provide better efficacy of the antithrombotic prevention by aspirin (Fig. 2, Tab. 4, Ref. 19). Full Text (Free, PDF) www.bmj.sk.

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines.

Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 microg per 0.1 mL dose than at the 1 microg per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-gamma and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both T(H)1 (TNF) as well as T(H)2 (IL-4) cytokines (7-10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.

Efficacy of recombinant herpes simplex virus 1 glycoprotein D candidate vaccines in mice.

To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.

HLA-G1-transfected K562 cells do not inhibit NK-cell-mediated lysis in europium release cytotoxicity assay.

The class Ia of HLA molecules are recognised by NK-cells either by inhibitory or stimulatory NK-receptors. When inhibitory signals prevail over stimulatoryones, the target cells expressing the class Ia of HLA molecules are not lysed by NK-cells. Similarly, class Ib of HLA molecules have been reported to induce the inhibitory signal in NK-cells. The cell line K562 is deprived of both class Ia and class Ib of HLA molecules, respectively, the fact of which enhances the lysis of K562 cells when they are co-cultivated with NK-cells. To study the role of HLA-G molecules in NK-cell cytotoxic activity, HLA-G tranfected K562 cells were used as target cells. NK-cells were isolated from the peripheral blood of 4 unrelated persons using Miltenyi's Biotec isolation system. The purity of directly isolated NK cells (CD56 Multisort kit) was 74.1%, and that of indirectly isolated NK-cells (NK-cell isolation kit) 69.4%. The europium release cytotoxicity assay was used in all experiments. The percentage of cytotoxicity ranged from 19% to 24% when K562 target cells were used. Similar results were obtained with the HLA-G1-transfected target cells: the percentage of cytotoxicity ranged from 17% to 29%. Our preliminary results indicate that NK-cells are able to lyse both, K562 cells and the HLA-G1-transfected K562 cells. (Tab. 1, Fig. 8, Ref. 21.).