An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family.

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58-77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38-57 and codons 78-88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.

Immunogenetics of HLA null alleles: implications for blood stem cell transplantation.

The transplantation of haematopoietic stem cells is a potentially curative therapy for a variety of haematological and non-haematological diseases. Matching of donor and recipient for human leucocyte antigens (HLA) is pivotal for the success of blood stem cell transplantation. HLA null alleles are characterized by the lack of a serologically detectable product. Because serological HLA diagnostics are increasingly replaced by DNA-based typing methods considering only small regions of the genes, null alleles may be misdiagnosed as normally expressed variants. The failure to identify an HLA null allele as a non-expressed variant in the stem cell transplantation setting may result in an HLA mismatch that is highly likely to stimulate allogeneic T cells and to trigger graft-vs-host disease. For some HLA null alleles, the translation into a truncated polypeptide chain seems possible, which thus might act as minor histocompatibility antigens. Because the prevalence of HLA null alleles may be around 0.3% or even higher, a screening strategy for HLA null alleles should, therefore, be implemented in the clinical laboratory. It may consist of the combination of serology and standard molecular typing techniques. As the standard molecular techniques are sometimes troublesome especially for characterizing the cytosine island at the 5' end of HLA class I exon 4 and need continuously be updated, an alternative approach may consist of sequencing all samples from genomic DNA for exons 2-3 or 4 (class I) or exon 2 (class II), including the adjacent intron splicing sites. This approach will detect 36/40 so far known non-expressed variants and has the potential to easily uncover novel variants, thus essentially minimizing the risk of overlooking these challenging variants.

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.

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. MATERIALS AND METHODS: Smoker and non-smoker plateletpheresis products were investigated on days 1, 3 and 5 of storage. Receptor expression (as evaluated by flow cytometry) and the platelet aggregation response were measured. RESULTS: There was only a slight loss of platelet function in apheresis products from smokers compared to non-smokers. CONCLUSIONS: Smoking does not significantly change the quality of platelet preparations. The current practice, of not to exclude smokers from platelet donation, can be continued.

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.

The phylogenies of introns 4-7 demonstrate an inconsistent pattern between human leukocyte antigen-C group topologies.

In this study, we have sequenced introns 4-7 in 31 human leukocyte antigen-C (HLA-C) alleles representing all allelic groups. Intron sequences show a patchwork pattern of polymorphism. Bootstrap support for phylogenetic lineages and for differentiation between groups is limited due to the high homology of intron sequences, where the substitution of a single nucleotide may lead to the assignment to different clusters. The intron data suggest the idea of a Cw*06/Cw*12 family, which is closely related to a hypothetical Cw*05/Cw*08 family. Moreover, a third family consisting of Cw*01/Cw*04/Cw*18 may exist. The intron data compiled in our study may be the basis for further sequencing studies. The detection of three novel alleles (Cw*0401new, Cw*140201new, and Cw*1701new) suggests that the HLA-C polymorphism might have been strongly underestimated to date.

HistoCheck: rating of HLA class I and II mismatches by an internet-based software tool.

HLA polymorphism is a major barrier for hematopoietic stem cell and solid organ transplantation. To estimate the allogeneic potential between HLA-mismatched stem cell donor/recipient pairs, we recently proposed a matching score (dissimilarity index) that is based on the structural data of HLA class I molecules, and on the functional similarity of amino acids (AA). This first approach revealed new features about presumptive subtype allogenicities within the HLA-A*23 and A*24 groups. We have now developed an internet-based software tool ("HistoCheck") that is capable to assess the allogenicity (matching score) between any pair of clinically relevant HLA class I, and also class II, alleles. Newly described HLA sequences will be regularly integrated into the database according to the nomenclature for factors of the HLA system updates. The software is intended to be a first step for estimating the allogenicity of HLA mismatches in peculiar clinical settings, as long as there are no reliable in vitro or clinical studies available. The algorithm can later be modified according to functional data, for example, peptide-binding specificities. With the extension of the sequence similarity concept to all clinically relevant HLA class I and II loci, HistoCheck may contribute to prevent HLA mismatching being a matter of chance.

The nature of introns 4-6 suggests reduced lineage specificity in HLA-B alleles.

For most HLA-B alleles, coding sequences of the 3' part of the genes still need to be determined, and sequences of the 3' noncoding regions have yet to be studied systematically. In this study, we have determined the sequences of introns 4-6 in all HLA-B allelic groups, and computed nucleotide substitution rates and phylogenetic relationships. These sequences demonstrated an inconsistent pattern of intralineage specificity, intralineage diversity, and interlineage diversity that is best characterized by a patchwork pattern. Apart from phylogenetic studies about HLA diversity and diversification, the sequence data obtained in our study may prove valuable for haplotype-specific sequencing of the 3' part of HLA-B and for the explanation of recombination events in newly described HLA-B alleles.

Lack of toxic side effects in neutrophils following hyperbaric oxygen.

Conflicting data have been reported about the impact of repeated HBO2 exposure on the production of superoxide radicals during the neutrophil respiratory burst (RB) and on phagocytosis. In this study we wanted to see if exposure to hyperoxia would affect human neutrophil RB and phagocytosis. Short- and long-term effects after single or repetitive HBO2 exposure of 2.5 atmospheres absolute over a period of 90 min were studied in 40 healthy volunteers. The RB was measured by the intracellular oxidation of dihydrorhodamine after induction by Escherichia coli (E. coli), or priming with recombinant tumour necrosis factor alpha (TNF-alpha), followed by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. The phagocytic activity was determined by the intake of FITC-labelled opsonized E. coli. No differences could be found between RB and phagocytic activity before and after HBO2 therapy, regardless of short- or long-term exposure. These findings indicate that exposure to hyperoxia does not impair these two important functions of the human innate host defense.

HLA-B*4431: a new HLA-B variant with a complex ancestral history involving HLA-B*44, B*40 and B*07 alleles.

We here describe the identification of the new allele HLA-B*4431, which was found in three members of a Turkish family. Sequencing of the new allele following haplotype-specific PCR amplification revealed that exon 2 is identical to HLA-B*4402, whereas exon 3 resembles a HLA-B*40 variant with the exception of position 572, where a single nucleotide transversion (C > G) leads to an amino acid exchange (Trp162Ser). The generation of the 3' part of B*4431 may be best explained by a separate recombination between B*40 and B*07. Although B*4431 consists of B44 in its alpha1 domain and of B60(40) in its alpha2 domain; the new allele only displayed B44 seroreactivity, which demonstrates that epitopes crucial for B60(40) specificity must be located in the alpha1 domain.

HLA-B*3531, a hybrid of B35 and B61, implications for diagnostic approaches to alleles with complex ancestral compositions.

The serological characterization of allelic variants that have been generated by large-scale interallelic recombination events indicates which residues may be involved in the formation of epitopes crucial for serological recognition. The allelic product of HLA-B*3531 is composed of B35 in its alpha1 domain and of B61(40) in its alpha2 domain. Both specificities are only weakly detectable with available sera. Allelic products with 'mixed' serology also represent a challenge to DNA-based HLA typing methods, as only the sequence motif of one ancestral allele may be recognized. In this case the hidden specificity would not be considered in the matching process and might not be recognized as an antigen 'unacceptable' to the recipient.

Sequence similarity matching: proposal of a structure-based rating system for bone marrow transplantation.

Recent advances in DNA-based typing have led to the detection of a continuously growing number of HLA alleles. For this reason, HLA matching in transplantation of hematopoietic stem cells from unrelated donors has become increasingly complicated. When there is no genotypically identical sibling and there are several alternative potential donors that all have a mismatch at a relevant HLA locus, until now no rating system has existed indicating different levels of allogenicity. In order to find a theoretical approach to this problem we propose a rating system ('dissimilarity index') based on structural data of HLA class I molecules, and on published data about frequencies of naturally occurring amino acid exchanges. For demonstration we employ our rating system for the comparison of the HLA-A*23 and A*24 groups, both of which allelic products are subdivisions of the serological HLA-A9 family. Remarkable differences between the subtypes were revealed, which were superior to a simple sequence comparison. More surprisingly, it was uncovered that some alleles of the A*24 group showed fewer differences to A*2301 than to alleles within their own subtype group. Sequence similarity matching may serve as a starting point for the clinical evaluation of acceptable mismatches within the HLA-A9 family and serve as a model for other HLA class I groups.

Non-expression of HLA-A*2901102 N is caused by a nucleotide exchange in the mRNA splicing site at the beginning of intron 4.

We describe the identification of the novel human leukocyte antigen (HLA) blank allele A*2901102 N which was detected in an individual of mixed race. The serological HLA class I typing was A1; B7,44 whereas PCR-SSP indicated the presence of an additional A*29 allele. The pedigree analysis demonstrated that the new blank allele segregated with the haplotype A*29null B*07, inherited from the individual's Vietnamese father. A single G-->Tau transversion was detected at position 1 of intron 4, which is a highly conserved nucleotide position in vertebrate splice donor sites. Accordingly, it is very likely that this nucleotide exchange inhibits the splicing of intron 4, resulting in a premature stop codon further downstream. Despite this alteration, transcription into mRNA was demonstrated.

Identification of the novel allele HLA-DRB1*1137 which probably originated from DRB1*11011: implications for mismatch with its ancestor allele at bone marrow transplantation.

The identification of the new allele HLA-DRB1*1137, which was found in a Caucasian individual, is described. In the sequence analysis the new allele differs from DRB1*11011 by position 227 (T>A) which is located in exon 2. At the protein level, the new allele has one amino acid difference compared to DRB1*1101 (Phe47Tyr). Residue 47 is likely to contribute to the peptide binding site of HLA-DR11 and thus to be important for peptide binding. However, as phenylalanine and tyrosine have very similar physical and chemical features allogenicity in case of mismatch at bone marrow transplantation may be weak.

Attenuation of sepsis-related immunoparalysis by continuous veno-venous hemofiltration in experimental porcine pancreatitis.

OBJECTIVES: In light of evidence suggesting that hemofiltration favorably influences septic diseases by removing sepsis mediators, the impact of different modalities of continuous veno-venous hemofiltration (CVVH) on outcome and immunologic derangements in porcine pancreatogenic sepsis was evaluated. DESIGN: Randomized, controlled intervention trial. SUBJECTS: Forty-eight minipigs of either sex. INTERVENTIONS: Pancreatitis was induced by intraductal injection of sodium taurocholate (4%, 1 mL/kg body weight [BW]) and enterokinase (2 U/kg BW). Animals were allocated either to untreated controls-group 1-or to one of three treatment groups-group 2: low-volume CVVH (20 mL/kg BW), no change of hemofilters; group 3: low-volume CVVH, filters changed every 12 hrs; and group 4: high-volume CVVH (100 mL/kg BW), filters changed every 12 hrs. Survival represented the major parameter of the study. Serum cytokine levels, sepsis-related down-regulation of major histocompatibility complex II and CD14 expression on leukocytes, bacterial translocation, and endotoxemia were further parameters evaluated in the study. MEASUREMENTS AND MAIN RESULTS: High-volume CVVH combined with periodic filter change was significantly superior compared with less intensive treatment modalities (low-volume CVVH, no filter change) in sepsis protection. Long-term survival (>60 hrs) was found in 67% of group 4 and 33% of group 3 animals (p <.05), whereas in controls and group 2 no animal survived. CVVH ameliorated the initial serum tumor necrosis factor-alpha response and prevented sepsis-induced in vitro endotoxin hyporesponsiveness. Down-regulation of major histocompatibility complex II and CD14 expression on monocytes was significantly improved by CVVH. Improved oxidative burst and phagocytosis capacity in polymorphonuclear leukocytes suggested that leukocyte function was stabilized by CVVH. Also, CVVH significantly reduced bacterial translocation and endotoxemia. CONCLUSIONS: Hemofiltration reversed sepsis-induced immunoparalysis in a porcine model of bile acid-induced pancreatitis. Implications for human pancreatitis must be validated in prospective, clinical protocols.

Non-expression of HLA-B*5111N is caused by an insertion into the cytosine island at exon 4 creating a frameshift stop codon.

The identification of the "blank" allele HLA-B*5111N, which was detected in German and Czech individuals, is described. In the pedigree analysis this new allele segregates with the serological haplotype HLA-A2; B-; DR4 which is frequent in Czech population. The non-expression of B*5111N is caused by the insertion of an additional cytosine molecule at the cytosine island between the nucleotides 621-626 (codons 183-185, first three codons of exon 4) leading to a frame shift that creates a stop codon at codon 196. This insertion may be explained either by conversion with the pseudogene HLA-J or by slipped-strand mispairing. In order not to overlook the presence of alleles with altered expression in case of hematopoietic stem cell transplantation, both serological and DNA-based typing should be performed (Note).

HLA-DRB fluorotyping by dark quenching and automated analysis.

In fluorescence-based sequence-specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA-DRB1,3,4,5 low-resolution fluorotyping method, we have constructed two probes quenched by the non-fluorescent acceptor dye DABCYL. The HLA-DRB-specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA-DRB-specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan system 7700 was supplemented by an EXCEL-based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA-DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.

The nucleotide diversity of MICA and MICB suggests the effect of overdominant selection.

The major histocompatibility complex (MHC) class I-related molecules A and B (MICA and MICB) are stress-inducible cell surface antigens that are recognized by immunocytes bearing the receptor NKG2D. In our study we estimated the average number of synonymous (pis) and nonsynonymous nucleotide substitutions (pia) per site in exons 2-4 of MICA and MICB. In exons 2 and 3 of MICB only nonsynonymous substitutions were found, and in exon 3 of MICA pia clearly exceeded pis. This finding is in contrast to the evolutionary parameters found in most other genes, and is reminiscent of the elevated pia values caused by overdominant selection in the peptide-binding region of conventional MHC class I molecules. It may be explained by the hypothetical interaction with nonpeptide antigens, or by resistance against pathogens.

Increased diversity within the HLA-B*07 group: identification of the two novel alleles B*0709 and B*0710.

The identification of the two novel alleles, HLA-B*0709 and B*0710, is described. B*0709 differs from B*07021 by a nucleotide exchange at position 419 (A>C) which is located in exon 3. At the protein level the nucleotide exchange results in an amino acid residue difference (Tyr116Ser). The other newly detected allele, B*0710, differs from B*07021 by a nucleotide exchange at position 272 (A>G) which is located in exon 2. This nucleotide exchange also leads to an amino acid substitution (Tyr67Cys). The allogeneic potential in case of mismatch with other alleles of the B*07 group at bone marrow transplantation was assessed. The peptide motifs between B*0709 and B*0711 may be very similar and thus the alloreactive potential in case of mismatch may be low. In contrast, mismatches of B*0709 and B*0710 with other B*07 alleles are likely to stimulate alloreactive T cells.

Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German university pediatric hospital.

Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children's hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms.