Holger-Andreas Elsner

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients.

Background and objective: Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. Methods: From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg−1 over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. Results: A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 ± 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 ± 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. Conclusions: Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.

Aberrant expression of HLA-B*3565Q is associated with a disrupted disulfide bond

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw−; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C→G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques.

The nature of introns 4–7 largely reflects the lineage specificity of HLA–A alleles

Abstract For most HLA-A alleles the phylogeny of the 3′ non-coding regions has not yet been studied systematically. In this study, we have determined the sequences of introns 4–7 in 50 HLA-A variants, and have computed nucleotide substitution rates and phylogenetic relationships. The A2/A28,A9, and A10 groups were characterized by clear lineage specificity. For the A19 group, lineage specificity was weaker. A*3001 clustered together with the alleles of the A1/A3/A11/A36 serological family, but not with the A19 group alleles. Reduced lineage specificity was also observed for the alleles of the A1/A3/A11/A36 groups. The 3′ intron sequences of A*8001 were clearly distinct from all other alleles studied. In several cases two allelic groups shared identical intron sequences, whereby the patterns varied with the introns. A similar situation has been previously described for the 5′ introns. Since recombination is the major mechanism of HLA diversification, the intronic lineage specificity corresponds to the comparatively lower recombination rate of the HLA-A 3′ exons. The low level of recombination within the 3′ region of HLA-A is supported by the low CpG content with a maximum of 3.0% in this region compared with up to 10.7% in the 5′ region. Apart from phylogenetic studies of HLA diversity and diversification, the sequence data obtained in our study may prove valuable for the development of a haplotype-specific sequencing strategy for the HLA-A 3′ exons and for the explanation of recombination events in newly described HLA class I alleles.

Peptide-binding motif of HLA-A*6603

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.

Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

BackgroundThe effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated.MethodsWhole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay.ResultsThere were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol.ConclusionsAccordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.

Impact of tobacco smoking on platelet function in apheresis products in vitro.

Background and Objectives  The aim of this study was to analyse the platelet function, over a 5‐day time‐period, of apheresis‐derived platelet concentrates obtained from smokers and non‐smokers.

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients

MICA and MICB, as members of the major histocompatibility complex (MHC) class I-chain-related genes (MIC), encode stress-inducible glycoproteins that act as activating ligands for NKG2D and gammadelta T-cell receptor-bearing cells. We here describe the identification of eight novel MICB variants, including a null allele, which were identified in peripheral blood leukocytes of gastric MALT lymphoma patients. Only two of the novel alleles are characterized by point mutations, whereas the other variants display a recombination of known exonic MICB sequences that may be best explained by intragenic conversions. The novel MICB null allele is characterized by a Cytosin (C) deletion in a stretch of four Cs beginning from nucleotide 135 of exon 2 that leads to a premature stop codon (TGA) at codon 66.