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Fifty-four chimpanzee Patr-DRB and five human HLA-DRB second exons were cloned and sequenced from thirty-five chimpanzees and four human B-cell lines and compared with known Mhc-DRB sequences of these two species. Equivalents of the HLA-DRB1*02, -DRB1*03, -DRB1*07 allelic lineages and the HLA-DRB3, -DRB4, -DRB5, -DRB6, and -DRB7 loci were all found in the chimpanzee. In addition, two chimpanzee Patr-DRB lineages (Patr-DRBX and -DRBY) were found for which no human counterparts have been described. None of the Patr-DRB sequences is identical to known HLA-DRB sequences. The Patr-DRB1*0702 and HLA-DRB1*0701 alleles are the most similar sequences in a comparison between the two species and differ by only two nucleotides out of 246 sequenced. Equivalents of the HLA-DRB1*01, -DRB1*04, and -DRB1*09 alleles were not found in our sample of chimpanzees. A per locus comparison of the number of Patr-DRB alleles with the HLA-DRB alleles shows that the Patr-DRB3, -DRB4, -DRB5, and -DRB6 locus are, thus far, more polymorphic than their human homologs. The polymorphism of the Patr-DRB1 locus seems to be less extensive than that reported for the HLA-DRB1 locus. Nevertheless, the Patr-DRB1 locus seems to be the most polymorphic of the Patr-DRB loci. Phylogenetic analyses indicate that the HLA-DRB1*09 allele may have originated from a recombination between a Mhc-DRB5 allele and the DRB1 allele of a Mhc-DR7 haplotype. Although recombination seems to increase the diversity of the Patr-DRB alleles, its contribution to the generation of Patr-DRB variation is probably low. Hence, most Patr-DRB diversity presumably accumulated via recurrent point mutations. Finally, two distinct Patr-DRB haplotypes are deduced, one of which (the chimpanzee equivalent of the HLA-DR7 haplotype) is probably older than 6-8 million years.

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.