bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA typing of human platelet antigen systems 1, 2, 3 and 5 in B‐lymphoblastoid cell lines of the International Histocompatibility Workshop
Authors: Bein G. , Hackstein H. , Klüter H.
Published: December 2008

Alloimmunization against human platelet antigens (HPA) can cause thrombocytopenia in different clinical settings, including neonatal alloimmune thrombocytopenia, post transfusion purpura, and refractoriness to platelet transfusion. Recently, DNA based methods have been described for analysis of the polymorphism of the human platelet antigens. However, until now, no reference material for standardization purposes is available. We thus determined the DNA polymorphism of the human platelet antigen (HPA) systems 1, 2, 3 and 5 in B‐lymphoblastoid cell lines collected for the International Histocompatibility Workshops. DNA typing of the HPA systems was done by PCR amplification with sequence‐specific primers (PCR‐SSP). A new enzyme (AmpliTaq Gold) was introduced to enhance the specificity of the PCR. We present a panel of five B‐lymphoblastoid cell lines, representing all heterozygous and homozygous genotypes of the tested HPA systems. This work thus provides the reference material required for DNA based diagnosis of human platelet antigens.
Trinucleotide repeat polymorphism within exon 5 of the MICA gene (MHC class I chain‐related gene A): allele frequency data in the nine population groups Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian
Authors: Ota M. , Katsuyama Y. , Mizuki N. , Ando H. , Furihata K. , Ono S. , Pivetti‐Pezzi P. , Tabbara K. F. , Palimeris G. D. , Nikbin B. , Davatchi F. , Chams H. , Geng Z. , Bahram S. , Inoko H.
Published: December 2008

We recently identified a trinucleotide repeat polymorphism, (GCT)n, within the transmembrane (TM) segment of the human MHC class I MICA gene (MHC class I chain‐related gene A). Five distinct alleles (A4, A5, A5.1, A6, A9) corresponding to 4, 5, 5 with one nucleotide insertion, 6 and 9 repetitions, respectively, have been detected in various HLA‐homozygous B cell lines. Here we present allele frequencies for this trimeric short tandem repeat (STR) in 604 unrelated individuals collected from nine human populations (Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian) determined using the polymerase chain reaction (PCR) combined with fluorescent‐based automated fragment detection technology. All alleles were present in each population, but allelic distributions varied from one population to another. No new alleles (such as A7 or A8) were identified. The evolutionary and structural significance of these data as well as the potential application to forensic medicine is discussed.
Nomenclature for factors of the HLA system, update January/February 1997
Authors: Marsh Steven G. E.
Published: December 2008

The following sequences have been submitted to the Nomenclature Committee in the period following the publication of the last report “Nomenclature for factors of the HLA system, 1996” and, following agreed policy, have been assigned official allele designations. Full details of all sequences will be published in the forthcoming report “Nomenclature for factors of the HLA system, 1997”.

Below are listed the newly assigned sequences, confirmations of previously reported sequences and some sequences which are corrections of those originally reported. The accession number of each sequence is given and these can be used to retrieve the sequence files from either the EMBL, GenBank or DDBJ data libraries. Although accession numbers have been assigned by the data libraries and most sequences are already available, there is still the possibility that an author may not yet have allowed the sequence to be released; in such a case, you will have to contact the submitting author directly.
HLA‐B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket
Authors: García F. , Galocha B. , Villadanps J. A. , Lamas J. R. , AIbar J. P. , Marina A. , de Castro J. A. López
Published: December 2008

B*2701 differs from B*2705 by three amino acid changes: DY74, DN77, LA81, and from B*2702 only by two: DY74 and T180. Tyr74 is located in the C/F cavity of the peptide‐binding site, and is unique to B*2701 among HLA‐B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed. In addition, sequencing of the peptides bound in vivo by B*2701 and the Y74 mutant was carried out. The main distinctive feature of B*2701 was its presentation of peptides with Gln2. Synthetic analogs bound in vitro similarly as the corresponding ligands with Arg2. Moreover, both Gln2 and Arg2 were dominant upon pool sequencing of B*2701‐ bound peptides, and 2 of 8 natural ligands contained Gln2. Suitability of Gln2 was largely determined by the Y74 change, as indicated by: 1) binding of Gln2 analogs to this mutant, and 2) detection of Gln2 by pool sequencing of Y74‐bound peptides. B*2701 bound peptides with C‐terminal aromatic or Leu residues, and interacted with these motifs more strongly than B*2702. The Y74 mutation alone was not responsible for poor binding of peptides with C‐terminal basic residues to B*2701, since they bound efficiently and at least one was presented in vivo by this mutant. Most peptides bound to the A81 mutant worse than to B*2705, but frequently better than to B*2701 or B*2702, suggesting that other subtype changes were compensatory. The peptide specificity of B*2701 suggests that this subtype may determine susceptibility to spondyloarthropathy.
Association between polymorphisms at the human IL‐10 locus and systemic lupus erythematosus
Authors: Eskdale J. , Wordsworth P. , Bowman S. , Field M. , Gallagher G.
Published: December 2008

Recent studies have shown elevated IL‐10 levels in several rheumatic autoimmune diseases, and particularly in systemic lupus erythematosus (SLE). Such changes may have a genetic basis. We studied two novel polymorphic dinucleotide repeats in the IL‐10 promoter region (IL 10.G and IL 10.R) in order to investigate their possible significance in association with this condition in a group of 56 Caucasian SLE patients compared with 102 ethnically matched controls. The results show that there is an allelic imbalance between SLE patients and controls at the IL 10.G microsatellite; this observation is supported by a significant difference in genotype distribution. The nature of autoantibody production and the presence or absence of renal involvement also appeared to be associated with certain IL 10.G microsatellite alleles, although the small size of individual clinical sub‐groupings may have influenced this result. No association with the IL 10.R microsatellite was observed. Overall, the differences observed at the IL 10.G microsatellite between SLE patients and controls suggest that the IL‐10 locus contributes to the genetic background important for the development of this disease. Although the moderate sample size described in this study requires that the results be interpreted carefully, they provide an interesting and useful framework for future study.
A strong association between HLA‐B*5101 and Behçet's disease in Greek patients
Authors: Mizukl N. , Ohno S. , Ando H. , Chen L. , Palimeris G. D. , Stavropoulos‐Ghiokas E. , Ishihara M. , Goto K. , Nakamura S. , Shindo Y. , Isobe K. , Ito N. , Inoko H.
Published: December 2008

Behçet's disease is known to be associated with HLA‐B51, one of the split antigens of HLA‐B5, among many different ethnic groups. In a Greek population, an increased incidence of HLA‐B5 in the patient group also been reported. Because the B51 antigen has been recently identified to comprise seven alleles, B*5101‐B*5107, we performed HLA‐B51 allele genotyping by the PCR‐SSP method as well as serological HLA‐A and ‐B typing among 31 Greek patients with Behçet's disease to investigate whether there is any correlation between one particular B51‐associated allele and Behçet's disease. The frequency of B51 was remarkably high (80.6%) in the patient group as compared to the ethnically matched control group (26.7%). In addition, HLA‐A26 was also increased in the patients (29.0%) as compared with the healthy controls (3.3%). B51 allele genotyping revealed that all these B51‐positive patients carried B*5101. This study revealed a strong association of Behçet's disease in Greeks with one of B51 subantigens, providing insight into the molecular mechanism underlying an HLA association with Behçet's disease.
Dimorphic primers derived from intron 1 for use in the molecular typing of HLA‐B alleles
Authors: Cereb N. , Yang S. Y.
Published: December 2008

We have identified a dimorphic site in intron 1 of the HLA‐B gene. Oligotyping was performed on about 3000 samples using primers derived from this dimorphic site in combination with a locus‐specific primer derived from intron 3. The distribution of B‐alleles bearing each of the dimorphic sequences was approximately equal. These primers were mutually exclusive and yielded approximately 50% of the heterozygous samples as apparently homozygous in PCR products. Intermediate and almost high‐resolution oligotyping of HLA‐B alleles was achieved using 35 and 63 hybridization probes, respectively. This dimorphic site will provide a useful tool for otherPCR‐based HLA‐B typing approaches
Maternal DRB1*1501, DQA1*0102, DQB1*0602 haplotype in fetomaternal alloimmunization against human platelet alloantigen HPA‐6b (GPIIIa‐G1n489)
Authors: Westman P. , Hashemi‐Tavoularis S. , Blanchette V. , Kekomäki S. , Laes M. , Porcelijn L. , Kekomäki R.
Published: December 2008

Fetomaternal incompatibility of platelet alloantigens may lead to alloimmunization and neonatal alloimmune thrombocytopenia (NAIT). Human platelet alloantigen (HPA) 6b, which associates with residue Gln 489 of platelet membrane glycoprotein IIIa, has been described as a cause of NAIT. We have studied the MHC genes of all available family members in the six thus far reported families with a thrombocytopenic newborn and fetomaternal HPA‐6b incompatibility. Maternal HPA‐6b antibodies could be detected in five mothers to the altogether seven thrombocytopenic male infants. The MHC genes HLA‐DRB, ‐DQA1, ‐DQB1, ‐DPB1, TAP1,2 and HSP70‐Hom were studied by using polymerase chain reaction (PCR)‐based DNA analysis methods. All five mothers with detectable circulating HPA‐6b antibodies at the time of delivery shared an identical DRB1 *1501, DQA1 *0102, DQB1 *0602 haplotype. The sixth, HPA antibody‐negative mother and a HPA‐6b‐negative mother to a healthy HPA‐6b+ child were negative for this haplotype. The frequency of DRB1*15‐positive haplotype was increased in immunized mothers (100%) as compared with the general Finnish population (27%), but the association was not statistically significant after correction. We conclude that there is a potential association between the MHC haplotype DRB1 *1501, DQA1 *0102, DQB1 *0602 and alloimmunization to the HPA‐6b antigen and that this alloimmunization probably involves different HLA class II molecules from immunization to HPA‐la.
Evaluation of trophoblast HLA‐G antigen with a specific monoclonal antibody
Authors: Loke Y. W. , King A. , Burrows T. , Gardner L. , Bowen M. , Hiby S. , Howlett S. , Holmes N. , Jacobs D.
Published: December 2008

A monoclonal antibody to HLA‐G has been generated by immunizing HLA‐A2.1/human β2‐microglobulin (β2m) double transgenic mice with murine L cells transfected with both human β2m and HLA‐G. This monoclonal antibody, designated as G233, has been found not to cross‐react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA‐G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA‐G in the fetal thymus raises the question as to how T‐cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA‐G expressed on the surface of trophoblast is complexed with p2m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA‐G may also occur as free heavy chains not associated with p2m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several iso‐forms of HLA‐G from trophoblast similar to the characteristic spot array previously described for HLA‐G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA‐G.
The pattern of expression of CD147/neurothelin during human T‐cell ontogeny as defined by the monoclonal antibody 8D6
Authors: Kirsch A. H. , Jr. L. A. Diaz , Bonish B. , Antony P. A. , Fox D. A.
Published: December 2008

A novel monoclonal antibody termed 8D6 was generated against the human leukemic T‐cell line Molt 13 and was found to recognize the neurothelin structure, recently termed CD147. UM8D6 precipitated a single band from HSB2 cells of molecular weight 35 kDa nonreduced and 40 kDa reduced. CD 147 is broadly expressed on human hematopoietic cells and is expressed more intensely on thymocytes than on mature peripheral blood T cells. A clear distinction was found by three‐color flow cytometry between antigen density on single positive mature thymocytes subsets compared with the corresponding subset from peripheral blood. The 8D6 antibody did not have direct effects on T‐cell activation or apoptosis, and the function of CD147 in development and activation of T cells is not yet clear. Nevertheless, its highly regulated expression during T‐cell differentiation and the structural characteristics of this antigen suggest that it has an interesting and important role in T‐cell biology.
Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing‐based typing of the HLA‐A gene
Authors: Kotsch K. , Wehllng J. , Köhler S. , Blasczyk R.
Published: December 2008

We present a sequencing‐based typing strategy for the HLA‐A locus that is generally applicable to all HLA class I genes. Sequencing‐based typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5′ flanking region, 5′ untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR‐based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene‐specific 5′ and 3′ universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time‐consuming and less laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.
Three newly identified A*24 alleles: A*2406, A*2413 and A*2414
Authors: Gao X. , Lester S. , Matheson B. , Boettcher B. , McCluskey J.
Published: December 2008

Three previously unknown A24‐related alleles were identified by PCR‐SSO typing and confirmed by DNA sequencing in Australian Aboriginal populations (A*2406, 2413) and in individuals of South American descent (A*2414). A*2406 and A*2413 both have two adjacent (but different) nucleotide substitutions in codon 156 in exon 3 compared to A*2402, resulting in a single amino acid replacement in each allele. The South American A*2414 is apparently a hybrid between A2 and A24 with a segment of the A*24 sequence between codons 95 and 107 in exon 3 replaced with the A*02 sequence. Interallelic sequence exchange is the most likely mechanism in the generation of all three novel alleles. Compared to A*2402, the four amino acid substitutions in the A*2414 molecule would be expected to significantly change the shape of the peptide binding cleft, leading to selection of different peptide ligands. The single amino acid replacements in position 156 of the two Australian Aboriginal A*24 alleles may also have significant functional effects. In particular, Trp replacing Gin in position 156 (A*2406) is predicted to markedly reduce the volume of the peptide binding cleft, influence the interaction of HLA pockets with peptide side chains, and therefore, cause major changes in peptide presentation. These newly defined alleles may reflect the adaptive process of HLA genes to local environments.
Interactions of HLA‐B*4801 with peptide and CD8
Authors: Martinez‐Naves E. , Barber L.D. , Madrigal J.A. , Vullo C.M. , Clayberger C. , Lyu S.‐C. , Williams R.C. , Gorodezky C. , Markow T. , Petzl‐Erler M.L. , Parham P.
Published: December 2008

Functional properties of the B*4801 allotype were investigated using HLA class I‐deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl‐terminus. In anin vitrocell‐cell binding assay, B*4801 binds CD8α homodimers weakly due to the presence of a threonine residue at position 245 in the α3domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8α homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8α, alloreactive T‐cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti‐CD8 monoclonal antibodies. Analysis of 25 B*48‐expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.
A nucleotide insertion in exon 4 is responsible for the absence of expression of an HLA‐A*01 allele
Authors: Laforet M. , Froelich N. , Parissiadis A. , Pfeiffer B. , Schell A. , Faller B. , Woehl‐Jaegle M.‐L. , Cazenave J.‐P. , Tongio M.‐M.
Published: December 2008

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified whereas the corresponding antigen was not detected on the cell surface. In the present report, we describe four members of a family in whom an HLA‐A 1 allele identified at the molecular level was typed as A “blank” by lymphocytotoxicity. This serologically blank antigen was undetectable by isoelectric focusing (IEF). Sequencing of the HLA‐A*01 allele from the promoter region to the eighth exonic region revealed insertion of a “C” nucleotide at the beginning of the fourth exon as compared to the common HLA‐A*0101 allele. This mutation causes a frame shift, giving rise to an early stop codon in the fourth exon.
An HLA‐B null allele (B*1526N) with a stop codon in exon 3 generated by a point mutation
Authors: Mine H. , Ishikawa Y. , Hojo M. , Imashuku S. , Tokunaga K. , Juji T. , Saji H. , Yokoyama S.
Published: December 2008

An HLA‐B null allele was identified in a Japanese family during histocompatibility testing for bone marrow transplantation. The propositus was a healthy Japanese woman with three children, and her parents were cousins. Serological HLA typing of the family members indicated that the propositus was homozygous for the A24‐Cw4‐B blank (B null)‐DR4.2‐DQ3 haplotype. Total RNA was extracted from peripheral blood of the propositus was converted to first‐strand cDNA using reverse transcriptase. The cDNA was amplified by the polymerasee chain reaction (PCR) using HLA‐B locus‐specific primers. The PCR product showed no change in size upon polyacrylamide gel electrophoresis (PAGE) compared to that of normal controls, suggesting that HLA‐B gene mRNA was normally expressed. The nucleotide sequence of the cDNA was the same as that of B*1501 except at nucleotide 369 or codon 123, where C was replaced with A; TAC encodes Tyr whereas TAA is a stop codon. This point mutation may have truncated the HLA‐B molecule of the propositus, resulting in the negative results we obtained with anti‐HLA‐B sera.
The tetramer model: a new view of class II MHC molecules in antigenic presentation to T cells
Authors: Pareja E. , Tobes R. , Martin J. , Nieto A.
Published: December 2008

Crystallographic studies suggest a plausible divalent interaction between T‐cell receptor (TCR) and MHC class II molecules. In addition, biochemical data suggest that these divalent MHC molecules are preformed at the membrane of the antigen‐presenting cell. The tetramer model is based on these preformed tetrameric class II molecules that can be loaded with identical or different peptides in their two grooves. This enables divalent class II molecules to deliver two different messages to T cell: 1) a two‐peptide message, in which the tetramer with two identical peptides is able to cross‐link two TCRs triggering full activation of a T cell. At the thymic level we propose that this message induces negative selection; or 2) a one‐peptide message: only one of the peptides loaded in the class II tetramer is able to interact with that TCR. This message would be involved in triggering partial activation phenomena in mature lymphocytes, whereas in thymocytes this message would mediate positive selection. Since high concentrations of a peptide would favor the load of tetramers with identical peptides, the tetramer could therefore be viewed as a quantitative‐qualitative transducer that would trigger different responses depending on the concentration of antigenic peptides.
A rapid molecular method (polymerase chain reaction with sequence‐specific primers) to genotype for ABO blood group and secretor status and its potential for organ transplants
Authors: Procter J. , Crawford J. , Bunce M. , Welsh K.I.
Published: December 2008

We hypothesize that kidneys from non‐secretor blood group A2 donors may be used for transplant into non‐A recipients. In addition, we believe that organs from A2 donors may be used in non‐A recipients where the anti‐A titer is low. In order to reliably identify non‐secretor A2 kidneys from cadaver donors, we have developed a rapid molecular method. The PCR‐SSP‐based method was developed to genotype ABO blood group and secretor status. Samples of known blood group ABO and Lewis phenotype determined by standard serological methods were used to appraise the method. A retrospective renal cadaver donor study was conducted to assess the potential of using A2 non‐secretor organs for transplantation into non‐A recipients. Phenotype frequencies of blood group A donors were 76% and 24% for A1 and A2 subgroups respectively, whereas 27% of the donor sample population were non‐secretors. Three donors were identified as A2 non‐secretors, and analysis was performed to theoretically place the organs by considering them as blood group O. These results coupled with a detailed analysis of HLA type and antibody status of our panel suggests that using A2 donors would be a useful adjunct to strategies for transplanting highly sensitized patients and redressing the donor‐recipient imbalance in terms of blood group.
DQ microsatellite association studies in three ethnic groups
Authors: Lin L. , Jin L. , Kimura A. , Carrington M. , Mignot E.
Published: December 2008

Polymorphism at the level of three microsatellite markers (DQCAR, DQCARII, G51152) located in the HLA‐DQ region was characterized in 78 10th International Histocompatibility Workshop B‐cell lines, 718 random Japanese Asians, 99 Norwegian Caucasians and 95 New Guinean Aborigines with established HLA‐DRB1, ‐DQA1 and ‐DQB1 typing. DQCAR, DQCARII, and G51152 result in 13, 13, and 11 alleles respectively. All three markers were in tight linkage disequilibrium with HLA‐DRB1, ‐DQA1 and ‐DQB1. DRB1, DQA1, DQCARII, DQCAR, DQB1, and G51152 haplotypes could be defined for all subjects. In fact, DQ microsatellite typing data could predict DQA1 and DQB1 genotypes with high accuracy and may be used as a simple first pass HLA‐DQ typing method. The haplotype data was also used to determine recombination in the DRB1‐DQA1 (about 80 kb), DQA1‐DQCARII (about 4.5 kb), DQCARII‐DQCAR (about 7.5 kb), DQCAR‐DQB1 (about 1‐1.5 kb) and DQB1‐G51152 (about 20–25 kb) genomic segments and the relative rate of slippage microsatellite mutations for DQCAR, DQCARII, and G51152. This led us to conclude that recombination is more frequent in the DRB1‐DQA1 and DQCAR‐DQCARII segments, thus suggesting cross‐overs within small genomic segments are not proportional to genetic distance. We also observed that DQCAR had a higher mutation rate than DQCARII or G51152 and that 1 or 2 CA slippage mutations were arising more frequently from large size microsatellite alleles.
HLA DRB1‐DQA1‐DQB1 haplotype diversity in two African populations
Authors: Schnirtger L. , May J. , Loeliger C.‐C. , Gallin M. Y. , Erttmann K. D. , Bienzle U. , Kremsner P. G. , Meyer C. G.
Published: December 2008

HLA class II DRB1‐DQA1‐DQB1 haplotypic polymorphism was determined in 120 Liberian and 230 Gabonese individuals. In our study groups, the number of allelic variants observed for each locus was similar to that found in non‐African populations. However, 39 novel haplotypes and several yet unrecognized DRB1‐DQA1 and DQA1‐DQB1 combinations were identified. The extent of HLA‐haplotypic variability in Africans appears to result from the high degree of allele combinations rather than from allelic polymorphism.
Cell surface phenotype and ultramicroscopic analysis of purified human enterocytes: a possible antigen‐presenting cell in the intestine
Authors: Martín‐Villa J.M. , Ferre‐López S. , López‐Suárez J.C. , Corell A. , Pérez‐Blas M. , Arnaiz‐Villena A.
Published: December 2008

Epithelial cells of the intestine seem to act as antigen‐presenting cells to surrounding lymphoid tissue and may be crucial to maintain the pool of peripheral T lymphocytes. The scope of this study was to carry out an immunophenotypic and ultramicroscopic analysis of purified human enterocytes to elucidate their role as antigen‐presenting cells, in the immune responses in the gut‐associated lymphoid tissue. A method has been developed to obtain purified and viable human enterocyte populations, later labeled with relevant monoclonal antibodies directed to leukocyte antigens and subjected to cytofluorometric analysis. Phenotypic analysis revealed the presence of markers common to “classical” antigen‐presenting cells (CD14, CD35, CD39, CD43, CD63 and CD64), reinforcing the idea that enterocytes may act as such. Moreover, several integrins (CD11b, CD11c, CD18, CD41a, CD61 and CD29) were also found. CD25 (IL‐2 receptor a chain) and CD28, characteristic of T cells, were detected on the surface of these cells; this latter finding rises the possibility that enterocytes could be activated by IL‐2 and/or via CD28 through binding to its ligands CD80 or CD86. Finally, the presence of CD21, CD32, CD35 and CD64 that may bind immune complexes via Fc or C3, suggests their participation in the metabolism of immune complexes. Furthermore, the finding of a Birbeck'slike granule in the cytoplasm of the cells, shows that enterocytes contain an ultramicroscopic feature previously thought to be characteristic of Langerhans' cells, an antigen‐presenting cell. The phenotype detected on the surface of enterocytes, along with their ultramicroscopic characteristics, suggests that they may play an important role in the immune responses elicited in the gut, presenting antigens to surrounding lymphoid cells, and establishing cognate interactions with them.
A high frequency of Mamu‐A *01 in the rhesus macaque detected by polymerase chain reaction with sequence‐specific primers and direct sequencing
Authors: Knapp L.A. , Lehmann E. , Piekarczyk M.S. , Urvater J.A. , Watkins D.I.
Published: December 2008

SIV infection of rhesus macaques is an excellent model for HIV infection of humans. Unfortunately, it is has been difficult to identify macaques expressing particular MHC class I alleles. Here we describe the use of PCR‐SSP for Mamu‐A *01 typing of rhesus macaques. The Mamu‐A *01 allele was amplified from genomic DNA using Mamu‐A *07‐specific primers and positive PCR products were directly sequenced. Our technique identified 15 Mamu‐A*01‐positive animals of 68 tested. We validated our molecular analysis by showing that lymphocytes from 8 Mamu‐A *01‐positive animals expressed Mamu‐A*01 as determined by immunoprecipitation and 1‐D IEF. The technical simplicity and accuracy of this typing method should facilitate selection of Mamu‐A *01‐positive rhesus macaques for AIDS virus pathogenesis and vaccine studies.
Identification of the DRB1*1313 allele in a sibling pair
Authors: Noreen H. , Lin Y.‐S. , Davidson M. , Johnson S. , Segall M.
Published: December 2008

Sequence‐specific oligonucleotide probe hybridization (PCR/SSOP), which is now routinely used in HLA laboratories to type for alleles of the class II loci, can also detect the presence of previously unknown alleles. In the course of clinical typing for DRB1, we identified two samples with probe hybridization patterns appropriate for DRB1*1313, a previously submitted sequence that had been withdrawn. Direct sequencing showed that the two individuals carried an allele with an exon 2 sequence identical to that previously submitted for DRB1*1313.
Clarification of HLA‐B serologically ambiguous types by automated DNA sequencing
Authors: Lee K.W. , Steiner N. , Hurley C.K.
Published: December 2008

Abstract: Assignment of HLA‐B types can be hampered by ambiguous reactivity of the typing sera resulting in inaccurate HLA‐B assignments. In this study, 19 Korean samples exhibiting ambiguous serologic reactivities were characterized by DNA sequencing. Alleles identified from 7 samples were previously undetected in this population (B*1517, B*4101, B*4701, B*5001, and B*5106) and from 9 samples were common alleles in this population (B*4002, B*4003, B*4006, B*1501, B*1401, B*67012, and B*5401). Three samples were putative HLA‐B homozygotes. Three major factors causing serologic ambiguity were identified: weak or false negative reactivity of typing sera (52.4%); cross or false positive reactivity of the sera (38.1%); and absence of information on the reaction patterns due to the lack of appropriate sera in the typing kit (e.g. B*4101 encoded molecule) or to the presence of recently characterized molecules (e.g. B*5106 encoded molecule) (9.5%). Overall, sequencing was helpful in clarifying ambiguous serologic reaction patterns improving the HLA typing for the Korean population.
DPB1*7601, a novel DPB1 variant in the Caucasoid population
Authors: Rozemuller E.H. , Allebes W.A. , van den Berg‐Loonen E.P.M , Hidajat M. , van der Zwan A.‐W. , Tilanus M.G.J. , Joosten I.
Published: December 2008

Abstract: A new DPB1 allele has been identified in a Caucasoid individual, DPB1*7601. The sequence of the complete second exon has been confirmed by cloning and subsequent sequencing. This allele differs by one amino acid, at codon 36, from DPB1*1401, as indicated by SBT and PCR‐SSP analysis. The amino‐acid motif introduced by the change is shared by DPB1*0401 and some rare alleles. It remains unclear whether the change is due to interallelic microgen conversion or a single point mutation.
Molecular, serological and population studies on a novel HLA‐B allele — HLA‐B*5002
Authors: Darke C. , Guttridge M.G. , Thompson J. , Street J. , Thomas M.
Published: December 2008

Abstract:A novel HLA‐B allele (B*5002), detected as a discrepancy between serological and PCR‐SSP HLA‐A and B phenotyping of bone marrow panel donors, was identified by nucleotide sequencing of exons 2 and 3. Titration studies on 39 HLA‐B12/B21 cross‐reactive antisera showed that the serological specificity of HLA‐B*5002 was HLA‐B45. PCR‐SSP testing of 287 serologically defined HLA‐B45‐positive subjects from a panel of 12,411 donors, together with HLA‐B*45 and B*5002 frequency data on 4,342 PCR‐SSP typed subjects, indicated that 4.53% of serologically defined HLA‐B45‐positive subjects possess HLA‐B*5002 and not HLA‐B*4501. The pheno‐type frequency of HLA‐B*5002 was 0.08954%; gene frequency was 0.00045 (n=16,753). In 73.3% of instances B*5002 appeared to be present on a haplotype with DRBl*0406 and DQB1*0402, 54.6% of which possessed A*2301. The B*5002, DRBl*0406, DQB1*0402 haplotype represents 52.4% of all haplotypes with DRB1*04 and DQB1*04 and 78.6% of haplotypes possessing DRBl*0406 and DQBl*0402.
Characterization of the HLA class I genotypes of a Venezuelan Amerindian group by molecular methods
Authors: Martfnez‐Arends A. , Layrisse Z. , Argüello R. , Herrera F. , Montagnani S. , Matos M. , Ross J. , Dunn P. , Marsh S.G.E. , Madrigal J.A.
Published: December 2008

Abstract: We have characterized the HLA class I genotypes of the Yucpa, a tribe of Venezuelan Amerindians, using molecular methods. The study was carried out on DNA extracted from unrelated individuals using low resolution ARMS‐SSP typing with sequence‐specific primers, high resolution typing using reference strand conformation analysis (RSCA), and for some samples sequence‐based typing (SBT). The following class I alleles were found to be present in this tribe: for the HLA‐A locus A*0204, A*0212, A*0213, A *2402, A*3101 and A*6801; for the B locus B *1522, B *3512, B*3905, B *3909, B*4004 and B*52012, and for C locus Cw *0102, Cw*0302/4, Cw*0401, Cw*0702 and Cw*1503. This is the first rime these alleles have been described in this group, although all of them have previously been reported as being present in other Amerindian tribes. The study confirmed the high frequency of HLA‐B39 which was previously observed in serologi‐cal analysis of this tribe, and indicated that two different B*39 alleles were present in this population. The identification of the class I alleles by molecular methods for this ethnic group confirms the restricted polymorphism of the MHC molecules previously obtained by serology and has allowed a more accurate definition of the different alleles present in this population.
HLA region microsatellite polymorphisms in juvenile arthritis
Authors: Feichtlbauer P. , Gomolka M. , Brünnler G. , Eisenhut T. , Truckenbrodt H. , Albert E.D.
Published: December 2008

Abstract: A number of microsatellite polymorphisms located in the MHC region of the human chromosome 6 have been analysed in a large group of patients with juvenile arthritis (JA) (n=177) and in 157 controls. There have been no significant associations for the alleles of the microsatellite polymorphisms D6S‐105, D6S‐510, TNFA, TNFC, TNFD, TNFE, HSP. Allele frequencies and HLA associations were listed for the non‐associated micro‐satellite loci. The microsatellite locus DQ CAR, which is localized between DQA1 and DQB1, shows a significant positive association with JA for the allele DQ CAR 121 and a negative association for the allele DQ CAR 111. The allele DQ CAR 121 is strongly associated with DQA1*0501 and with DQB1*0301 both in the normal controls and in the patient population. This pair of DQA/DQB alleles corresponds to the DQ molecule DQ7 on the cell surface, which has been described to be strongly associated with JA. Investigations of the two and three‐point haplotypes of DQ CAR with alleles of its neighboring loci have shown that the association with DQ CAR 121 is secondary to the association with DQ7 previously observed. Thus, using eight HLA linked microsatellite polymorphisms in the region from HLA‐A to HLA‐DQ, we have not found any evidence for further loci which might be involved in the coding for susceptibility for JA.
Typing of HLA‐B*15 alleles using sequence‐specific primers
Authors: Yu N. , Ohashi M. , Alosco S. , Salazar M. , Cao K. , Vina M. Fernandez , Yunis E.J.
Published: December 2008

Abstract:We have developed a DNA based typing method to detect 38 knownB*15alleles using sequence‐specific primers (PCR‐SSP). This method involves 38 primers and 39 PCR‐SSP reactions with results that can be obtained in 3 hours. The method is easy, fast and suitable for clinical typing for bone marrow and organ transplantation. We have typed 106 HLA‐B15 samples using this method. For homozygous HLA‐B15 samples, someB*15allele combinations need to be resolved by additional PCR reactions not included in this article. The method allows the detection of potential new alleles requiring sequencing for confirmation, and it is useful to resolve unusual serological reaction patterns for different HLA‐B15 serological specificities. In addition, it could be used to resolve ambiguous PCR‐SSOP typing results and for recognition of mismatches in serologically matched unrelated individuals.
Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor‐α gene promoter
Authors: Uglialoro A.M. , Turbay D. , Pesavento P.A. , Delgado J.C. , McKenzie F. E. , Gribben J.G. , Hartl D. , Yunis E.J. , Goldfeld A.E.
Published: December 2008

Abstract: We have identified three new human tumor necrosis factor‐α (TNF‐α) promoter polymorphisms with single nucleotide (nt) substitutions at ‐862, ‐856, and ‐574 nt relative to the TNF‐α transcription start site. The ‐862 and ‐856 nt TNF‐α promoter polymorphisms occur with high frequency in Caucasian and Cambodian individuals and are each non‐ran‐domly associated with three extended HLA haplotypes. This study, in which 61 independent TNF‐α promoters were analyzed spanning from ‐977 to +93 nt relative to the TNF‐α mRNA cap site, establishes a new canonical TNF‐α promoter sequence. Furthermore, we show that none of the three novel polymorphisms at ‐862, ‐856 and ‐574 nt or polymorphisms previously described at positions ‐238, ‐308 and +70 have an effect upon TNF‐α gene expression in activated lymphocytes. Thus, these TNF‐α promoter polymorphisms likely serve as markers for neighboring genes encoding HLA or other undefined molecules in the MHC that may influence disease susceptibility.
Haplotypes comprising subtypes of the DQB1*06 allele direct the antibody response after immunisation with hepatitis B surface antigen
Authors: Langö‐Warensjö A. , Cardell K. , Lindblom B.
Published: December 2008

Abstract: Two HLA class II haplotypes, HLA‐{DQB1*0602; DQAl*0102; DR15} and HLA‐{DQB1*0603; DQA1*0103; DRB1*1301} were found to be less common in 52 nonresponders compared with 68 responders, P<0.025 and P<0.05 respectively, after vaccination with hepatitis B surface antigen (HBsAg). Another haplotype, HLA‐(DQBl*0604; DQA1*0102; DRB1*1302), had a significantly higher frequency in the nonresponders (P <0.005). The nonresponders and responders were nonsmoking, healthy individuals with an antibody concentration of <10 IU/1 and >100 IU/1 respectively. The three haplotypes comprise either of three different DQB1*06 subtypes. Two of the seven amino acids that differ between the two responder alleles DQBl*0602 and *0603 and the nonresponder allele *0604 are located in the peptide‐binding groove of the DQB1 molecule. In addition to this finding, amino acid 86 in the DRB1 molecule seems to determine the response against HBsAg. DRB1*1301 and DR15 in the responder haplotypes have a Val at this position while the nonresponder haplotype has a Gly. These results suggest a role for both the DQB1* 06 alleles and the DRB1 alleles *1301, *1302 and DR15 to direct either a response or a nonresponse against HBsAg. Sixteen HLA class II genotypes were found to be shared by 25 nonresponders and 32 responders. This finding of HLA‐identical nonresponders and responders indicates an influence of other genetic factors in addition to the HLA system in the response to HBsAg.
HLA class I and class II of the Nivkhi, an indigenous population carrying HTLV‐I in Sakhalin, Far Eastern Russia
Authors: Lou H. , Li H.‐C. , Kuwayama M. , Yashiki S. , Fujiyoshi T. , Suehara M. , Osame M. , Yamashita M. , Hayami M. , Gurtsevich V. , Ballas M. , Imanishi T. , Sonoda S.
Published: December 2008

Abstract: The Nivkhi are a native people isolated in the Nogliki region of Sakhalin, Far East Russia, where our group recently recognized human T‐cell lymphoma virus type I (HTLV‐I) infection. In order to trace the Nivkhi's ethnic background and the HTLV‐I carriers, we investigated HLA class I and II allele types of 53 Nivkhi (including four HTLV‐I carriers). Major HLA class I alleles of the Nivkhi were A*24, A*02, B*40, B*48, B*27, B*35 with allele frequencies similar to the Orochon, a native people isolated in Northeast China. Major Nivkhi class II alleles were DRBl*0901, DRB1*1401, DRB1*1201, DRB1*1106 with allele frequencies similar to the Ainu in Hokkaido, Japan, but dissimilar to other Asian Mongoloids, including the general Japanese population. The same HLA class I and II allele frequencies are found in both Nivkhi HTLV‐I carriers and the background population. A dendrogram of HLA class I alleles showed that the Nivkhi were closely related to the Orochon and Yakut, and remotely related to the Japanese, Ainu and other Asian Mongoloids. Interestingly, the Nivkhi were intermediately related to the Amerindians (Inuit, Tlingit and Andeans), a relationship closer than to the Japanese and Asian Mongoloids. These results suggested the Nivkhi might be related to some genetic group of Northeast Asian Mongoloids like the Orochon and Yakut, being infected with HTLV‐I in the distant past before diverging into the current major Mongoloid ethnic groups.
High incidence of the Cys 282 Tyr mutation in the HFE gene in the Irish population ‐implications for haemochromatosis
Authors: Murphy S. , Curran M.D. , McDougall N. , Callender M.E. , O'Brien C.J. , Middleton D.
Published: December 2008

Abstract: A simple PCR‐SSOP approach based on a single PCR product has been developed to screen the HFE gene for the haemochromatosis‐associated mutations Cys 282 Tyr and His 63 Asp. Using this approach the prevalence of these mutations in a cohort (30) of haemochromatosis patients and normal controls (404) was determined. Ninety percent of the haemochromatosis patients were homozygous for the Cys 282 Tyr mutation. In the normal population we found an increased incidence of the Cys 282 Tyr mutation (17.3%; 95% confidence limits 0.136–0.209) which was also reflected in the higher frequency of Cys 282 Tyr homozygotes (1.24%; 95% confidence limits 0.0015–0.0232). Linkage disequilbrium analysis confirmed the association between A*03 and Cys 282 Tyr. However, strong linkage disequilibrium occurred with the HLA‐A*03‐associated allele HLA‐B*14 but not the HLA‐A*03‐associated allele HLA‐B*07. The His 63 Asp was found to be in linkage disquilibrium with HLA‐A*29.
Staphylococcal enterotoxin‐A directly stimulates signal transduction and interferon‐γ production in psoriatic T‐cell lines
Authors: Nielsen M.‐B. , Ødum N. , Gerwien J. , Svejgaard A. , Bendtzen K. , Bregentholt S. , Rōpke C. , Geisler C. , Dohlsten M. , Kaltoft K.
Published: December 2008

Abstract: Bacterial superantigens such as staphylococcal enterotoxin‐A (SEA) have been implicated in the pathogenesis of psoriasis vulgaris. Major histocompatibility complex (MHC) class II molecules are high affinity receptors for SEA, and T cells found in psoriatic skin lesions express high levels of MHC class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen‐presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T‐cell receptor (TCR), and iii) production of interferon‐γ (IFN‐γ), but not autocrine mitogenesis in CD8‐posi‐tive T clones obtained from skin lesions of a patient with psoriasis vulgaris. Psoriatic T cells do not respond to SEA molecules if mutations are introduced in the TCRβ‐ or in both the two MHC class II α‐ and β‐binding sites of SEA. Mutations in only one of the two MHC class II binding sites of SEA has different effects on T‐cell activation. Thus, SEA molecules with a mutation in the MHC class LT β‐binding site induce protein tyrosine phosphorylation, but not LFN‐γ production or co‐stimulation of cytokine‐mediated proliferation. In contrast, SEA with a mutation in the MHC class II α‐binding site induces IFN‐γ and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co‐stimulatory effect on cytokine‐mediated proliferation. This suggests that both MHC class II binding sites are involved in the autopresentation of SEA by psoriatic T cells. In conclusion, we provide evidence that SEA directly activates MHC class II‐posi‐tive psoriatic T‐cell lines to produce IFN‐γ, a key cytokine in the pathogenesis of psoriasis vulgaris.;
A reliable and efficient high resolution typing method for HLA‐C using sequence‐based typing
Authors: van der Vlies S.A. , Voorter C.E.M. , van den Berg‐Loonen E.M.
Published: December 2008

Abstract: Serological typing of HLA‐C has been poor and almost half of its alleles are serologically undetectable blanks in most populations. Therefore, DNA typing techniques have been used to identify and type for the HLA‐C gene. Sequence‐based typing (SBT) has proven a major typing strategy for highly polymorphic HLA genes. The technique enables direct identification of all sequence motifs without the need to continuously adjust primers. Here we describe a reliable solid‐phase SBT strategy for HLA‐C which can be used to distinguish all currently known HLA‐C alleles without prior knowledge gained by low resolution typing. Exons 2 and 3 were amplified and sequenced and if necessary sequences of exons 1 and 5 were determined. A total of 257 individuals were typed for HLA‐C using this protocol and 30 of the 42 known HLA‐C alleles were detected. All heterozygous combinations found in this study were unambiguously discriminated.

One hundred and forty‐four individuals from the Dutch population were typed randomly. In this group Cw*0701 and *0702 were the most frequently detected alleles. Of the serological Cw blank alleles Cw*1203 was found to have the highest frequency (16%). From the total group 212 individuals were typed serologically and 106 were retyped with 97 selected antisera to further compare serological and molecular defined phenorypes. Discrepancies between serological typing and SBT are mainly attributable to the serologically Cw blank alleles Cw*12–18.

The high resolution SBT protocol described will be a valuable tool for the identification of HLA‐C alleles and the determination of the role of HLA‐C in marrow and organ transplantation.
HLA‐E polymorphism in patients with recurrent spontaneous abortion
Authors: Steffensen R. , Christiansen O.B. , Bennett E.P. , Jersild C.
Published: December 2008

Abstract: The aim of this study was to compare the frequencies of five HLA‐E alleles in 82 women with recurrent spontaneous abortions with that of 150 random Danish controls. PCR sequence‐specific oligonucleotide typing was carried out to detect polymorphism in exons 2 and 3 of the HLA‐E gene. In informative samples sequencing of these two exons was also undertaken to confirm the presence of the HLA‐E*01031 allele. HLA‐E*0101, HLA‐ E*01032 and HLA‐E*01031 were detected with frequencies of 56.7%, 33.6% and 9.6% in controls and 58,5%, 32.9% and 8.5% in patients with recurrent abortion, respectively. No HLA‐E*0102 and E*0104 alleles could be detected. Since the HLA‐E allele distribution was similar in women with recurrent spontaneous abortion and controls, it is suggested that maternal HLA‐E polymorphism per se does not play any role in the pathogenesis of this disorder of pregnancy.