Distribution of MICA alleles and haplotypes associated with HLA-B in Greek population.

INTRODUCTION: The Major Histocompatibility Complex Class I-related chain A gene (MICA) is a highly polymorphic functional gene located close to the HLA-B locus. Certain MICA alleles have been related to inflammatory and autoimmune diseases while MICA antibodies have been implicated in organ allograft rejection or graft-versus-host disease (GVHD). AIM: The aim of this study was to identify the frequencies of MICA alleles and MICA ~ HLA-B haplotypes in the Greek population since, as far as we know, these data are still limited. METHODS: DNA was obtained from 277 unrelated healthy Greek individuals of Caucasian origin, volunteer donors of blood stem cells. HLA-B* and MICA* genotyping was performed by reverse PCR-SSOP. RESULTS: A total of 18 MICA alleles were defined in the present study. The five most frequent alleles in the Greek population were MICA*008 (24.6%), MICA*009 (22.36%), MICA*018 (16.03%), MICA*002 (8.02%) and MICA*004 (7.17%) which altogether account for 77.8% of all alleles. The most common MICA ~ HLA-B haplotypes were MICA*018 ~ B*18 (12.5%) and MICA*009 ~ B*51(11.5%). CONCLUSIONS: The five most frequent MICA alleles in the Greek population were *008, *009, *018, *002, *004. In other Caucasian populations, two of these alleles (*008, and *004) were observed in similar frequencies. MICA*002 was observed less frequently (8.02%) in the Greek population compared to other Caucasian groups (frequencies > 15%). Also, MICA*009 and MICA*018 had elevated frequencies (above 15%) whereas in other Caucasian populations they were found around 10% or less. These data may be important for the elucidation of the role that MICA polymorphisms play in organ and stem cell transplantation and to identify the relation of certain MICA with susceptibility to specific diseases.

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity.

AIM: To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity. METHODS: There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 degrees C. Dimethyl sulphoxide (Me2SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks' balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me2SO in multi-well tissue culture plates at 1 degrees C/min to -80 degrees C. After storage overnight, the valve leaflets were warmed at approximately 11 degrees C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann's solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth. RESULTS: After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me2SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me2SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks' solution (Group 3) (chi2, p=0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (chi2, p=0.007). The rates of cell growth in Group 2 (two-step addition of Me2SO) and Group 3 (one-step addition of Me2SO with additional Hanks' solution rinse) were similar and faster than the Group 4 (one-step addition of Me2SO without the additional Hanks' rinse). CONCLUSION: Single-step addition of Me2SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks' solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me2SO, and the 5% Me2SO step in the two-step protocol, merely served to reduce this carryover.