bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of two human IgM monoclonal antibodies reactive with HLA‐B27
Authors: Wölpl A. , Toubert A. , Siemoneit K. , Eiermann T. H. , Neumayer H. , Goldmann S. F.
Published: December 2008

Abstract: We describe here the generation and characterization of two human monoclonal IgM antibodies (UL‐4F11 and UL‐F6) reactive with HLA‐B27. The monoclonal antibody (mAb) UL‐4F11 is cytotoxic for peripheral mononuclear cells and, therefore, useful as typing reagent for HLA‐B27 and HLA‐B38. Protein chemistry showed that the mAb UL‐4F11 precipitates HLA‐B27 molecules. Epitope mapping analysis suggests that the amino acids 45, 67, 82 and 83 (alpha‐1 domain) of the HLA‐B27 sequence are necessary for mAb UL‐4F11 reactivity. The mAb UL‐F6 is suitable for complement dependent lysis of lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells with HLA‐B27 (B*2701, B*2702, B*2703, B*2705, B*2707), B13, B40 (60, 61), B47 and B48 specificities. Its reactivity indicates that the amino acid valine in position 152 and glutamic acid in position 163 of the alpha‐2 domain are crucial for the binding epitope.
Expression of an unusual Bw4 epitope by a subtype of HLA‐B8 [B*0802]
Authors: Arnett K. L. , Adams E. J. , Gumperz J. E. , Darke C. , Marsh S. G. E. , Gelsthorpe K. , Parham P.
Published: December 2008

Abstract: The primary structure of a variant HLA‐B8 antigen has been determined by cDNA cloning and sequencing. The variant, B*0802 differs, from the common B*0801 subtype at positions 77–83 of the α1 helix that determine the Bw4 and Bw6 public epitopes. Whereas B*080l has the common Bw6 motif, B*0802 has the Bw4 motif found in B*13 and B*44 allotypes. Serological analysis of B cell lines expressing B*0802 and of a B*0802 transfectant made with the HLA‐A, B negative cell line 721.221 shows that B*0802 reacts with Bw4‐specific antibodies, but at a level much lower than expected for Bw4 positive HLA‐B allotypes.
Allelic heterogeneity of HLA‐B35 subtypes in different populations as assessed by DNA typing
Authors: Satz M. L. , Fernández‐Viña M. , Theiler G. del C. , Marcos Y. C. , Lindel N. , Capucchio M. , Gorodezky C. , Fainboim L. , Stastny P.
Published: December 2008

Abstract: HLA‐B35, a class I antigen differentially associated to several diseases in different ethnic groups, comprises at least eight alleles which differ among them by one to six amino acids. In the present work a rapid DNA typing procedure was used to investigate the distribution of the various HLA‐B35 alleles in different populations. The approach is based on a group‐specific PCR amplification of a set of closely related HLA‐B alleles sharing a Thr in position 45 of the alpha‐1 domain. The amplified DNA was then hybridized to a panel of sequence‐specific oligonucleotide (SSO) probes designed to recognize the polymorphic residues in previously reported HLA‐B35 subtypes. This methodology was successfully tested in 100 individuals of four different populations, previously typed by serology as HLA‐B35, and in six reference panel cells of the 10th International Histocompatibility Workshop. HLA‐B*3501 was the predominant subtype in all populations. B*3502, B*3503 and, to a lesser extent B*3508, were also found. Among Mexican Mestizos, thirteen individuals had patterns of SSO hybridization suggestive of new B35 alleles. The evolutionary considerations on the different B35 alleles and their extended B35, Cw4 haplotypes are discussed.
Phototyping: comprehensive DNA typing for HLA‐A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence‐specific primers (PCR‐SSP)
Authors: Bunce M. , O'Neill C. M. , Barnardo M. C. N. M. , Krausa P. , Browning M. J. , Morris P. J. , Welsh K. I.
Published: December 2008

Abstract: We have developed a single DNA typing method which uses 144 sequence‐specific primer (SSP) reactions to simultaneously detect all known HLA‐A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as “Phototyping”. Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA‐B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem‐free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.
C4 polymorphism and extended HLA haplotypes in Namibian San and Khoi and in South African Xhosa
Authors: Creemers P. C. , du Toit E. D.
Published: December 2008

We studied C4A and C4B polymorphisms and HLA‐B and ‐DR associations in the San, Khoi and Xhosa. C4A and C4B alleles were determined using conventional protein allotyping methods. The C4A*3, C4B*1 haplotype had a high frequency (30–55%) in all populations. The frequency of C4A*3, C4B*Q0 was 7–19%. The C4A*Q0, C4B*1 haplotype was frequent (15%) in the Khoi but very rare in the San (P0.001). C4A*12 A*91, C4B*Q0 was frequent in the Xhosa (15%) but rare in the San and Khoi (P0.001). Alleles C4A*5 and C4A*6, and the C4B*2 B*92 duplication were only found in the Xhosa. C4A alleles A*4, A*45, A*58, A*12, A*14, A*19 and the C4A*3 A*91 duplication were only found in the San/Khoi population group. In the San, fourteen extended haplotypes were found in a relatively high frequency (2–7%). In the Xhosa, one extended haplotype (B42, C4A*12 A*91, C4B*Q0, DR18) was found in a very high frequency (13%) and was characteristic for this group; five other extended haplotypes were found with a low frequency (3%).
The human leukocyte antigen TAP2 gene defines the centromeric limit of melanoma susceptibility on chromosome 6p
Authors: Lee J. E. , Loflin P. T. , Laud P. R. , Lu M. , Reveille J. D. , Lawlor D. A.
Published: December 2008

Abstract: A single human leukocyte antigen (HLA) class II allele, DQB1*0301, is strongly associated with melanoma, and the HLA‐DR locus provides the telomeric boundary for melanoma susceptibility in the HLA class II region of chromosome 6. However, the centromeric boundary is unknown. This study was designed to determine whether the adjacent upstream transporter associated with antigen processing (TAP) locus, TAP2, constitutes the centromeric boundary of disease susceptibility in melanoma. Molecular oligotyping of TAP2 genes was performed for 36 Caucasian patients with melanoma and for 32 Caucasian control individuals by both amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) and PCR‐sequence‐specific oligonucleotide (SSO) typing. TAP2 allele frequencies in the melanoma patients were compared to those in non‐melanoma Caucasian control populations, and to HLA‐DQ allele frequencies determined by molecular oligotyping. While HLA‐DQB1*0301 was more common in this group of 36 melanoma patients compared to a group of 200 controls (56 percent vs. 27 percent, Bonferoni‐corrected chi‐square p=0.01), no significant differences were observed in TAP2 allele frequencies between melanoma patients and controls. The TAP2 locus represents the centromeric boundary of disease susceptibility for melanoma in the class II region of chromosome 6p. These results support an etiologic role for HLA‐DQB1*0301 in melanoma susceptibility.
Validation of large data sets, an essential prerequisite for data analysis: an analytical survey of the Bone Marrow Donors Worldwide
Authors: Schipper R.F. , Oudshoorn M. , D'Amaro J. , van der Zanden H. G. M. , de Lange P. , Bakker J. T. , Bakker J. , van Rood J. J.
Published: December 2008

Large data sets like the Bone Marrow Donors Worldwide (BMDW) data set can be used for population genetic analyses. The qualities of such data sets are unique. To be able to use the BMDW data for analyses, several problems, like limited size and selective DR typing, of the data have to be solved and the quality of the registry data subsets has to be examined. We describe these problems and methods to overcome them. Also, we give an overview of the qualities of the different registry subsets. Sixteen of the twenty‐nine examined subsets contain data that can be used for population genetic analysis. We will deal with these analyses in the future. Additionally, we present a method to calculate the minimum number of individuals required for reliable haplotype frequency estimation.
Complement C4A, C4B and BF haplotypes in Koreans
Authors: Park K. S. , Park M. H. , Juji T. , Tokunaga K.
Published: December 2008

Specific alleles at C4A, C4B and BF loci occur in populations and are inherited in complotypes, which are linked with particular HLA haplotypes. Considerable differences in complement allele and complotype frequencies have been observed among various ethnic groups. In the present study, 109 Korean families were analyzed for complement and complotype polymorphism. Thirty‐four different complotypes were detected: the most common was BF*S‐C4A*3‐C4B*1 (S31) with a frequency of 42.2%, followed by S42 (14.3%) and F31 (13.8%). Three complotypes, S42, F31, and FQ01, showed positive linkage disequilibrium. Some of the complotypes were linked with characteristic HLA haplotypes. Two complotypes carrying duplicated C4A genes, S3+31 (BF*S‐C4A*3‐C4A*3‐C4B*1) and S3+2Q0(BF*S‐C4A*3‐C4A*2‐C4B*Q0), were exclusively associated with HLA‐A24‐Cw7‐B7‐DR1‐DQ1 and A24‐CBL‐B52‐DR15‐DQ1 haplotypes, respectively. Twelve families showed recombinant haplotypes, nine in the class I region, three between the HLA‐B and HLA‐DR loci, and none in the class III region. Maternal recombination occurred twice as frequently as paternal. The results obtained in this study represent the frequencies of complotypes and extended HLA haplotypes of well‐defined Koreans, based on a family study.
A rapid HLA‐DRB1*04 subtyping method using PCR and DNA heteroduplex generators
Authors: Savage D. A. , Tang J. P. , Wood N. A. P. , Evans J. , Bidwell J. L. , Wee J. L. K. , Oei A. A. , Hui K. M.
Published: December 2008

We describe here a rapid polymerase chain reaction (PCR)‐based method for the identification of HLA‐DRB1*0401‐*0412 alleles. The method is based on the generation of specific DNA heteroduplex patterns between PCR products derived from selective group‐specific amplification of the various DRB1*04 alleles and PCR products derived from two synthetic DNA heteroduplex generator (DHG) molecules following non‐denaturing polyacrylamide minigel electrophoresis. One DHG was designed to detect DRB1*0401, *0405, *0407, *0408, and *0409 alleles, whilst the other was designed to detect DRB1*0402, *0403, *0404, *0406, *0410, *0411, and *0412 alleles. Characteristic heteroduplex patterns were obtained for all DRB1*04 alleles tested both in homozygous and heterozygous situations. Both DHG and PCR‐SSP (sequence‐specific primer) typing were performed on 41 DRB1*04 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA‐DRB1*0403, *0405, and *0406 were found to account for 95% of the DRB1*04 alleles in the population studied. The DHG technique described is technically simple and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for resolving DRB1*04 combinations which are indistinguishable by PCR‐SSO (sequence‐specific oligonucleotide) or PCR‐SSP subtyping.
Loss of an HLA haplotype in pancreas cancer tissue and its corresponding tumor derived cell line
Authors: Torres M. J. , Ruiz‐Cabello F. , Skoudy A. , Berrozpe G. , Jimenez P. , Serrano A. , Real F. X. , Garrido F.
Published: December 2008

A combination of immunohistochemical, biochemical, and recombinant DNA techniques were used to investigate class I expression in 26 pancreatic adenocarcinomas and 6 autologous tumor‐derived cells. The prevalence of HLA losses was found to be comparable to that observed in other tumor types (>35%), using monomorphic and locus‐specific antibodies. In one patient, the original tumor tissue, a tumor derived cell line (IMIM‐PC‐2), and EBV‐transformed lymphocytes were available for study. The patient's phenotype was A25, A30, B18, B18. However, A30 allele product could not be detected in the original tumor not in the cultured tumor cells. In addition, A30 allele could not be isolated from cDNA or genomic clones from the cultured tumor cells whereas it was isolated from the autologous lymphoblastoid cell line. Using isoelectric focusing analysis a significant reduction in the B18 heavy chain product was also observed in the tumor cell line, IMIM‐PC‐2, suggesting the absence of expression of one allele. Further studies revealed loss of heterozygosity at DR and other loci of chromosome 6 and cytogenetic data strongly suggested deletion of a full chromosome 6. This work indicates for the first time that loss of a full HLA haplotype occurs in tumor tissue and suggests that this mechanism may contribute to the progression of human cancer.
HLA‐G transcription studies during the different stages of normal and malignant hematopoiesis
Authors: Amiot L. , Onno M. , Renard I. , Drénou B. , Guillaudeux T. , Bouteiller P. Le , Fauchet R.
Published: December 2008

Specific expression of the non classical class I HLA‐G gene on trophoblasts, the only fetal tissue in contact with maternal cells which lack MHC class I antigens, may indicate a role of this gene in fetal‐maternal tolerance. We recently reported HLA‐G transcription in peripheral blood leukocytes. In this work, we have investigated HLA‐G transcription in hematopoietic stem cells, in different hematopoietic lineages and in malignant cells by using a RT‐PCR technique. PCR amplification with primers specific to the exon 2 and the 3′ untranslated region has enabled to detect HLA‐G transcription in B and T cell populations. No transcription was found in CD34+ cells, in thymocytes, in polynuclear cells, in monocytes and in natural killer cells. Among the malignancies analyzed, HLA‐G is transcribed in 2 of 13 cases of acute leukemia characterized by a monocytic contingent, in 3 of 6 CLL and in all the cases of B‐NHL (n=6). No HLA‐G transcription was detected in myeloma (n=2). The splicing type does not seem to be linked to a lymphocyte subpopulation nor to a malignant proliferation stage. These results suggest that HLA‐G is a marker of mature lymphoid cells and may play an immunological function as a peptide presenting molecule. HLA‐G transcription in some cases of malignancy might indicate a contribution to the tumoral progression by blocking natural killing reaction.
Nucleotide sequences of MHC class I introns 1, 2, and 3 in humans and intron 2 in nonhuman primates
Authors: Cereb N. , Kong Y. , Lee S. , Maye P. , Yang S. Y.
Published: December 2008

HLA‐class I genes are the most polymorphic genetic system yet known. The polymorphic substitutions are mostly located in exon 2 and 3, encoding α1 and α2 domains, respectively, which are involved in peptide binding and T cell receptor interaction. In this study, we present the sequences of the introns neighboring the polymorphic exons in humans with few examples from non‐human primates. In general, intron sequences are found to be less polymorphic than the adjacent exons, displaying numerous locus‐specific and group‐specific sites. These sequences will provide important information for developing DNA based typing strategies for HLA‐class I alleles.
Brief communication The primate MHC contains sequences related to the fibroblast growth factor receptor gene family
Authors: Leelayuwat C. , Abraham L. J. , Pinelli M. , Townend D. C. , Wilks A. F. , Dawkins R. L.
Published: December 2008

We have cloned and sequenced a genomic region centromeric of the HLA‐B locus from different MHC ancestral haplotypes. These haplotypes are associated with several diseases. The sequences were analyzed for coding potential and their relevance to disease associations were assessed with respect to the level of polymorphism. Analysis of sequences located approximately 25kb centromeric of HLA‐B reveals the existence of fibroblast growth factor receptor related sequences. These sequences designated PERB1 (FGFR6) reveal 80% homology, at both nucleic acid and amino acid level, to the immunoglobulin domain 1 (Ig‐1) of the human fibroblast growth factor receptor 3 (FGFR3) gene. Amino acid comparison of the Ig‐1 domain of PERB1 to those of other FGFR molecules indicates that PERB1 is more closely related to FGFR3 and FGFR5 than to FGFR1, FGFR2 or FGFR4. Genomic sequence analysis, however, reveals no consensus splice sites and indicates the existence of inframe premature stop codons in the putative coding sequences. The results suggest that these sequences may represent FGFR gene fragments existing within the central MHC. Sequence analysis of the Mhc in 6 chimpanzee and one orangutan indicates that the existence of PERB 1 predates the spe‐ciation of the three species. The fact that the MHC contains a mixture of functional and nonfunctional (pseudo) genes suggests that a functional copy of PERB1 (FGFR6) may exist within or in close proximity to the MHC.
Frequency of platelet‐specific alloantigens in a Danish population
Authors: Steffensen R. , Kaczan E. , Varming K. , Jersild C.
Published: December 2008

This study reports the first data on gene frequencies of platelet alloantigens HPA‐1, HPA‐2, HPA‐3, HPA‐4 and HPA‐5 in a population of unrelated Danish blood donors using PCR‐techniques. The observed gene frequencies fit the Hardy‐Weinberg equilibrium, and the calculated phenotype frequencies are similar to those obtained in other Caucasian populations: HPA‐la and ‐lb occur in 96.6% and 30.3% of 557 unrelated respectively. HPA‐2a and ‐2b in 99.4% and 15.9% of 163 tested, HPA‐3a and ‐3b in 88.3% and 63.2% of 163 tested, HPA‐4a and ‐4b in 100% and 0% of 131 tested, and finally HPA‐5a and ‐5b in 100% and 15.7% of 427 tested. It is a major technical improvement to use PCR techniques for genomic typing of HPA. Not only is it possible to perform HPA typings in severely thrombocytopenic patient and on amniotic fluid cells of the fetus of alloimunized mothers, but it must be expected that accuracy of the HPA typing will increase considerably, as has been the case with genomic HLA class II typing. Finally, use of PCR technique combined with allele‐specific primers is suitable for accurate large scale typing of platelet donors, which may be useful in special clinical settings.
Six new DR52‐associated DRB1 alleles, three of DR8, two of DR11, and one of DR6, reflect a variety of mechanisms which generate polymorphism in the MHC
Authors: Smith A. G. , Nelson J. L. , Regen L. , Guthrie L. A. , Donadi E. , Mickelson E. M. , Hansen J. A.
Published: December 2008

We have sequenced DNA from six new DR52‐associated DRB1 alleles initially detected by PCR/SSOP analysis. Three DR8 associated alleles differed from previously known alleles by single nucleotide substitutions. DRB1*0807 and DRB1*0811 both vary from DRB1*08021 at codon 57 resulting in two different amino acids at this residue. DRB1 *0807 was identified in samples of Brazilian origin while *0811 was identified among samples from the Tlingit Native American population of Southeast Alaska. DRB1*0814, identified in a family of Chinese origin, differed from DRB1*08032 at codon 12 at both the nucleotide and the amino acid level. In addition, two alleles of DR11, DRB1*1113 and *1119, were each detected in Caucasian individuals. DRB1*1113 differs from other DR11 alleles at codons 37, 67, 70 and 74, while DRB1*1119 differs from *1101 by a single nucleotide substitution at codon 67. Finally, DRB1*1418 was detected in a sample from an Asian or Pacific Islander and shares sequences with several other DR52‐associated DRB 1 alleles. These six DRB 1 alleles appear to have been generated by either gene conversion events, DRB1*1113 and * 1418, or by point mutations, DRB1*0814, *0807, *0811 and *1119, although the single nucleotide substitutions found in the latter three alleles are also present in at least one other DRB1 allele and, therefore, could have been the product of gene conversions.
HLA‐DPA1typing by PCR amplification with sequence‐specific primers (PCR‐SSP) and distribution ofDPA1alleles in Caucasian, African and Oriental populations
Authors: Aldener‐Cannavá A. , Olerup O.
Published: December 2008

In the present study PCR primers were designed for detecting all knownDPA1variability, i.e. the presently recognized sixDPA1alleles 0103 to 0401, and also for separation of the fourDPA1*02alleles, by PCR amplification with sequence‐specific primers (PCR‐SSP). For each sample seven different PCR reactions were performed which allowed the identification of all DPAl alleles and the resolution of allDPA1genotypes. Forty‐eight cell lines and 100 donor spleen cells were investigated by theDPA1PCR‐SSP technique. In the forty‐eight known workshop cell‐lines no false positive or false negative results were obtained. The 100 donor spleen cells were only typed by the PCR‐SSP technique and in their DNAs only one or twoDPA1alleles were found. Twenty cell lines and twenty donor spleen cells were typed on two separate occasions and interpreted blindly. The reproducibility between the repeated typings was 100%. The length of the specific products ranged from 103 to 258 base pairs and the amplification patterns obtained were easy to interpret. In conclusion,DPA1typing by the PCR‐SSP method is an accurate typing technique with high sensitivity, specificity and reproducibility. Analysis of the distribution ofDPA1alleles was performed in 100 Caucasian samples, 100 African samples and 80 Oriental samples, including separation of the fourDPA1*02alleles. The population study showed a characteristic distribution ofHLA‐DPA1alleles. Each ethnic group appeared to have one (Caucasians), or two (Africans and Orientals), frequentDPA1allele(s) and a high frequency ofDPA1homozygotes, suggesting that, like for theDPB1locus, balancing selection does not appear to be affecting the evolution of theDPA1locus.
Stable inheritance of an HLA‐“blank' phenotype associated with a structural mutation in the HLA‐A*0301 gene
Authors: Lienert K. , Russ G. , Lester S. , Bennett G. , Gao X. , McCluskey J.
Published: December 2008

A serological family study identified an HLA‐A “blank” segregating through three generations of apparently healthy individuals. The HLA‐A*0301 allele was assigned by DNA genotyping in each of the three individuals. Complete absence of cellular expression of the HLA‐A3 antigen was associated with a 6 nucleotide deletion in exon 3 of the A*0301 gene. The in‐frame deletion of nucleotides 373–378 results in the absence of residues C101 and D102 from the mature HLA‐A heavy chain. Cysteine 101 is involved in the formation of the highly conserved disulfide bridge in the α2 domain of the class I molecule, and deletion of this residue is believed to be highly disruptive to proper folding and function of the class I molecule.
Expression of intercellular adhesion molecule‐3 (ICAM‐3/CD50) in malignant lymphoproliferative disorders and solid tumors
Authors: Terol M. J. , Cid M. C. , Lóipez‐Guillermo A. , Juan M. , Yagüe J. , Miralles A. , Vilella R. , Vives J. , Cardesa A. , Montserrat E. , Campo E.
Published: December 2008

ICAM‐3/CD50 is a recently described LFA‐1 counter receptor that seems to play an important role in the initiation of immune responses. In this study we have examined the expression of ICAM‐3/CD50 in a large series of human neoplasms including 101 Non‐Hodgkin's lymphomas (NHL), 26 Hodgkin's disease, and 38 solid tumors to define the distribution patterns of this molecule in malignant neoplasms and their possible correlation with clinical and pathological characteristics of the patients. In NHL, ICAM‐3/CD50 was expressed in almost all the tumors with a tendency to be lost in high grade lymphomas. Reed‐Sternberg cells and their variants in Hodgkin's disease were always negative independently of the histological subtype of the disease. No expression was observed in tumor epithelial cells of the 38 solid tumors examined. Strong endothelial cell staining was observed in 31% of the NHL and 31% of Hodgkin's disease. ICAM‐3 expression in these cases was restricted to small tumor vessels. ICAM‐3 expression in endothelial cells of NHL was significantly more frequent in high grade (40%) than in low grade lymphomas (14%) (p=0.012). In addition, tumor vessels were also positive in 29% of solid rumors independently of the histological type. No correlation was observed between ICAM‐3 expression in tumor or endothelial cells and other clinical and pathological characteristics of the patients. These findings indicate that ICAM‐3 expression in human tumors is restricted to hematological neoplasms with a tendency to be lost in high grade lymphomas and Hodgkin's disease. ICAM‐3 is also expressed by endothelial cells from tumor‐associated neovascularization in both lymphoid and solid tumors.
The renal cell carcinoma lysis by a specific cytotoxic T cell clone is independent of the Fas/Fas‐L cytotoxic pathway
Authors: Caignard A. , Guillard M. , Cai Z. , Asselin‐Paturel C. , Carayol G. , Chouaib S.
Published: December 2008

The expression of Fas antigen at the surface of renal cell carcinoma and the susceptibility to Fas‐mediated lysis by a tumor specific CTL clone were investigated. Renal cell carcinoma cell lines expressed Fas antigen and were susceptible to apoptosis mediated by antibodies to Fas/APO1. Using RT‐PCR, we further showed that these cell lines expressed mRNA for Fas deleted transmembrane region, corresponding to a soluble form of Fas/APO‐1. To investigate the role of the Fas/FasL pathway in the cytotoxic response against RCC cells, we analyzed the induction of Fas‐L on a tumor specific T cell clone (CTL 8C2), previously generated against one RCC cell line. Fas‐L expression on CTL 8C2 was detected by RT‐PCR after stimulation with autologous tumor cells. However, the cytotoxic activity of CTL 8C2 was completely abolished when EGTA was added, suggesting that the cytolysis was mainly mediated by a Ca++‐dependent pathway, perforin/granzyme‐based.
Peptides bound endogenously by HLA‐Cw*0304 expressed in LCL 721. 221 cells include a peptide derived from HLA‐E
Authors: Tzeng C.‐M. , Adams E. J. , Gumperz J. E. , Percival L. , Wells R. S. , Parham P. , Barber L. D.
Published: December 2008

The peptide‐binding specificity of HLA–Cw*0304 was determined. Sequence analysis of endogenously‐bound peptides isolated from Cw*0304 expressed by LCL 721. 221 (221 for short) cells transfected with Cw*0304 cDNA revealed this class I allotype preferentially binds peptides possessing alanine at position 2 and leucine or methionine at the C‐terminus. One peptide isolated from Cw*0304 expressed by 221 cells has sequence identity to residues 116–126 of HLA‐E. Expression of HLA‐E by 221 cells was confirmed by isolation of mRNA transcripts for HLA‐E*0101 and detection of β2‐microglobu‐lin (β2‐m)‐associated HLA‐E protein.
New HLA–DR haplotypes containing the DRB6 pseudogene
Authors: Salazar M. , Granja C. B. , Selvakumar A. , Fresia B. , Alosco S. , Dupont B. , Yunis E. J.
Published: December 2008

An unexpected probe reaction pattern was observed in two samples during HLA‐DR typing by PCR‐Sequence Specific Oligonucleotide Probes. In order to confirm the unusual typings, samples were analyzed by PCR‐Sequence Specific Primers, cloning, and nucleotide sequencing of the second exon of the HLA‐DRB‐genes. The confirmed DR, DQ phenotype for one sample was DRB1*0701, DRB4*01, DRB5*0101, DRB6*0201, DQB*0602, DQB1*0202. The phenotype of other sample was DRB1*1602, DRB1*1302, DRB3*0301, DRB6*0101, DQB1*0501, DQB1*0502. The first sample has the novel combination of DRB1*0701 with DRB5*0101 and DRB6*0201. The second sample has either DRB6*0101 together with DRB 1*1602 in absence of any DRB5 allele or DRB6*0101 together with DRB1*1302, DRB 3*0301. We postulate that the most likely haplotype in sample #1 is DRB1*0701, DRB5*0101, DRB1*0602 which could have arisen from gene conversion. The most likely haplotype in sample #2, DRB 1*1602, DRB6*0101, DQB1*0502 would have arisen from an homologous recombination event.
African‐American HLA class II allele and haplotype diversity
Authors: Just J. J. , King M.‐C. , Thomson G. , Klitz W.
Published: December 2008

Molecular genetic techniques were used to type nine loci in the HLA class II region in 241 unrelated African‐Americans from New York City (NYC). Several effects attributable to recent genetic admixture were evident: the number of distinct class II alleles and haplotypes was larger in the African‐Americans than in people of African or European origin, the allele frequencies were more consistently even, and linkage disequilibrium was present across the entire class II region. The African‐American DRB1 allele frequencies almost always fell between frequencies among samples from northern Europe and the Gambia, two possible founding populations. The exceptions are attributed to the contribution of other genetically dissimilar African groups to the African‐American gene pool. DRB1 allele frequencies (specifically DRB 1*1501) and some haplotypes of DRB 1‐DPB1 were different in our NYC and the 11th International Histocompatibility Workshop (IHW) samples of African‐Americans. The high level of allele and haplotype diversity found in African‐Americans has important implications for the construction of pools of unrelated potential donors for tissue transplantation.
Molecular cloning of two new HLA‐C alleles: Cw*1801 and Cw*0706
Authors: Vilches C. , Bunce M. , Sanz L. , de Pablo R. , Puente S. , Kreisler M.
Published: December 2008

Nucleotide sequence analysis of the HLA‐C alleles of the GB92 cell line, heterozygous for B*8101 and B*4407, revealed the existence of two new allelic variants: Cw*1801 and Cw*0706. The former allele, initially detected as a PCR‐SSP variant, displays a hybrid aspect, sharing sequence motifs with Cw*07 at exons 1 and 2, and with'Cw*04 at distal exons. In serological assays, Cw*1801 is only recognized by some cross‐reactive sera. Cw*0706 shows a primary structure closely related to previously known Cw7 alleles, but carries new sequence motifs at its 3′‐end. Preliminary data indicate that Cw*1801 is associated to B*8101 and that Cw*0706, B*4407 could account for a part of the Cw7, B44 haplotypes observed in African populations.
Unique HLA antigen frequencies in the Finnish population
Authors: Sirén M. K. , Sareneva H. , Lokki M. L. , Koskimies S.
Published: December 2008

The Finnish Bone Marrow Donor Registry was established 1992 to serve Finnish patients in urgent need of bone marrow transplantation. This study details the HLA antigen frequencies, including those of the A 19 subtypes, in the Finnish population. Large regional variations were found in antigen frequencies between the different geographical areas of Finland. In particular, antigens A9, B12, B35, Cw4 and DR3 display regional frequency deviations, but B7, B8 and B15 also exhibit regional variations. The present population is the largest (n= 10,000) ever HLA‐typed in Finland. 97% of the donors were HLA‐A‐B‐DR typed. Confirmation of the serological HLA type was performed by DNA typing on 3% of the donors in the registry. A potential donor was found for 52% of Finnish patients in need of a matched unrelated donor for a bone marrow transplantation. Due to the ethnic origin of the Finns, it is not easy to find suitable bone marrow donors for Finnish patients in worldwide registries. It is thus important to maintain a national bone marrow donor registry which recruits donors from all over the country.
Polymorphisms of TAP 1 and TAP2 genes in Graves' disease
Authors: Rau H. , Nicolay A. , Usadel K. H. , Finke R. , Donner H. , Walfish P. G. , Badenhoop K.
Published: December 2008

Graves' disease is an autoimmune disorder in which HLA DQA1*0501 and DQB1*0201 confer predisposition. The genes for transporters associated with antigen processing (TAP1 and TAP2) locate near to HLA DQ coding regions and display only a limited degree of polymorphism. Since polymorphisms of TAP might influence susceptibility to Graves' disease by a possibly different selection of antigenic peptides, we investigated sequence variants of TAP1 and TAP2 genes in 235 patients with Graves' disease and 218 random healthy controls by polymerase chain reaction (PCR) followed by sequence specific oligonucleotide analysis (SSO), single strand conformational polymorphism (SSCP) analysis and amplification refractory mutation system (ARMS). TAP1*0301 (Val‐333/Asp‐637: 71% vs. 55% in controls. p< .0008, RR=2.05) and TAP2*0101 (Val‐379/Ala‐565/Thr‐665/stop‐687: 83% vs. 69% in controls, p< .003, RR=2.20) showed a positive association with Graves' disease whereas TAP1*0401 a negative (Ile‐333/Gly‐637: 4% vs. 13% in controls, p< .001, RR=0.25). After selection of patients and controls for HLA DQA1*0501 a similar association was found for TAP1*0301 (72% vs. 50% in controls, p< .002, RR=2.63) and TAP1*0401 (4% vs. 16% in controls, p< .004, RR=0.22), when matching for HLA DQB1*0201 as well as for TAP1*0401 (3% vs. 16% in controls, p< .005, RR=0.18). Our findings indicate that the positive association of TAP1*0301 and the negative of TAP1*0401 with Graves' disease cannot only be explained by linkage disequilibrium between TAP alleles and HLA DQ. Therefore, these TAP alleles contribute to genetic susceptibility in Graves' disease as additional permissive and protective factors.
Haplotypic diversity of DQA1*03 and *05 subtypes differing at amino acid residue 160 encoded in the third exon in 2215 Japanese individuals
Authors: Kaneshige T. , Hashimoto M. , Kinoshita T. , Moribe T. , Inagawa A. , Itou Y. , Fukunishi T. , Teraoka H. , Inoko H.
Published: December 2008

We analyzed the frequencies and haplotypes of DQA1*03 and *05 subtypes, DQA1*03011 or DQA1*0302 and DQA1 *0501 or DQA1*0503, respectively, differing only at codon 160 in the non‐polymorphic third exon of the DQA1 gene. Of these, 1,862 and 337 individuals selected as DQA1*03‐ and DQA1**05‐positive samples, respectively among 2,215 unrelated Japanese were typed for their nucleotide variation at residue 160 using PCR‐SSP. As observed in other populations, all the samples carrying DQA1*03011 (Gene Frequency, GF: 7.8%) were found to share DQB1*0302, whereas those carrying DQA1*0302 (GF: 44.3%) were associated with a variety of DQB1 alleles including DQB1*0302. Both of the DQA1‐DQB1 haplotypes with DQA1*03011 and DQA1*0302 carrying DRB1*0406, DQA1*03011‐DQB1*0302 and DQA1*0302‐DQB1*0302, showed a strong linkage disequilibrium with B62 (p< .0001, p< .005). These results suggested that DQA1*03011 was generated from a single amino acid change at residue 160 in the DQA1*0302‐DQB1*0302 haplotype. However, none of the haplotypes with two different DQA1*03 subtypes carrying DRB1*0403, *0405, *0802 and *0901 showed a linkage disequilibrium with any common B‐locus antigens, revealing extensive haplotypic diversity of the DQA1*03 group. For example, DRB1*0802 haplotypes showed linkage disequilibria with two different B‐locus antigens, B35 and B61 depending on the presence of DQA1*03011 and DQA1*0302, respectively. The GFs of DQA1*0501 and *0503 were 5.1% and 2.7%, respectively. The DQA1*05 associated haplotypes in the DR52‐antigen group with DQB1*0301 were divided into two groups, depending on the bimorphism at residue 160. Such a high degree of haplotypic diversity in association with DRB1 and B alleles observed in the DQA1*03 and *05 groups related to amino acid variation at residue 160, which may affect biological function such as the interaction between CD4 and HLA‐DQ molecules, seems to reflect selective pressure in the evolutionary process of HLA antigens
An unusual DRB1*1503 haplotype without a detectable DRB5 locus in a black African family
Authors: Nesci S. , Talevi N. , Andreani M. , Manna M. , Iliescu A. , Lucarelli G.
Published: December 2008

A DRB1*1503 allele not associated with DRB5 locus has been detected in an African family during routine HLA typing for bone marrow transplantation. PCR/SSOP analysis showed the DR2‐associated alleles in all the family members but the DRB5 locus appeared to be absent in the patient and his brother. The samples were then analyzed for the presence of DRB6 pseudogenes and we found that the unusual haplotype was associated with DRB6*0101 allele. This finding strengthen the hypothesis of a recombination hot spot between DRB1 and DRB6 genes.
Possible protective role of HLA‐B*2706 for ankylosing spondylitis
Authors: Ren E. C. , Koh W. H. , Sim D. , Boey M. L. , Wee G. B. , Chan S. H.
Published: December 2008

HLA‐B27 is strongly associated with ankylosing spondylitis (AS) but the role of the HLA molecule itself is still unclear. In this study on Singapore Chinese, we have subtyped 50 B27 positive AS patients and 45 B27 positive normals and found that the B*2706 allele has a significant negative association with disease (p=0.047). Together with recent data indicating the existence of AS “protective” B27 alleles, our data shows that the HLA molecule itself plays a crucial role in disease development.
Review article Microsatellites in the HLA region: an overview
Authors: Foissac A. , Crouau‐Roy B. , Fauré S. , Thomsen M. , Cambon‐Thomsen A.
Published: December 2008

Microsatellites are repeats of a DNA base motif (1–6 bp, mostly CA repeats) up to 100 times; they are distributed regularly all over the genome. Many of them are polymorphic and their high polymorphism is based upon a variable number of repeats. They are widely used for genetic mapping, linkage analysis, population genetics, evolutionary studies and in forensic medicine. Such markers have also been described in the HLA region since 1991, and a growing interest in their potential applications is being expressed. The aims of this review are: 1) to outline the presently available information from literature and molecular databases concerning 53 microsatellites in the HLA region (localization, type of repeat, number of alleles, heterozygosity, primers used for amplification); 2) to address the question of technical pitfalls when using such markers; 3) to discuss specific features such as their mutation rate (10‐3 to 10‐6), which is higher than that reported for HLA genes, and their linkage disequilibrium with HLA alleles; 4) to present an integrated map of microsatellites and genes of this region; and 5) to provide a synopsis of their different applications in HLA‐related fields (disease studies, population genetics, recombination point studies, HLA region mapping, transplantation) along with perspectives for future use. Although some HLA region microsatellites have already been applied to the analysis of more than 10 diseases, it is now evident that their use in population genetics and the determination of genomic compatibility in bone marrow transplantation represent growing areas of application.
Interleukin 2 induces a transient downregulation of protein phosphatase 1 and 2A activity in human T cells
Authors: Brockdorff J. , Nielsen M. , Dobson P. , Geisler C. , Röpke C. , Svejgaard A. , Ødum N.
Published: December 2008

Stimulation of human CD4* T cell lines with interleukin 2 (IL‐2) induces tyrosine, serine and threonine phosphorylation of a series of proteins involved in the IL‐2 receptor (IL‐2R) signaling pathway. Here, we examined whether IL‐2 induces changes in the activity of protein serine/threonine phosphatases in antigen specific, CD4* human T cell lines. Using inhibitors of protein phosphatases 1 (PP1), PP2A, and PP2B, we provide evidence, that IL‐2 induces a downregulation of PP activity in the cytoplasmic/membrane fraction. Thus, IL‐2R ligation for 30 min triggers a 16 percent decrease in total PP2A activity (p<0.00005, n=17) and a seven percent decrease in PP1 activity (p<0.00005, n=17). Cytokine‐induced downregulation of PP2A activity reaches a maximum 60 min after IL‐2R ligation, and returns to baseline levels within two hours. Downregulation of PP1 activity reaches a maximum after 30 min and is largely reversed one hour after IL‐2 stimulation. As determined from immunoblotting experiments using a specific anti‐PP1 or anti‐PP2A antibody, the amount of PP1 and PP2A recovered from cytosolic/membrane fraction remains unchanged after IL‐2 treatment suggesting that the drop in PP1/PP2A activity might be due to a regulatory change rather than to a change in the amount of PP1 and PP2A. In conclusion, we provide evidence, for the first time, that IL‐2 induces a transient downregulation of PP2A activity in T cells. In addition, our findings indicate that cytoplasmic PPl activity is transiently downregulated following IL‐2R ligation in antigen‐specific, human CD4* T cells.
A feline homologue of CD1 is defined using a feline‐specific monoclonal antibody
Authors: Woo J. C. , Moore P. F.
Published: December 2008

The characteristics of a feline homologue of CD1, defined by a murine monoclonal antibody, Fel.5F4 (IgG1), are described. This antibody precipitated a 49 kDa protein from biotinylated feline thymocyte extract in conjunction with a 14 kDa protein, consistent with β2 microglobulin subunit. The tissue distribution of this antigen was restricted to cortical thymocytes and antigen presenting cells of the thymic medulla, epidermis (Langerhans cells), dermis and occasional dendritic cells in the mantle and periarteriolar lymphoid areas of the spleen. Although flow cytometry demonstrated a continuous distribution of antigen expression on thymocytes, antigen density was found to decrease with age, consistent with physiological thymic involution. Thymocytes with high density expression of this antigen were predominantly restricted to cells with dual expression of CD4 and CD8 as defined by feline specific murine monoclonal antibodies Fel.7B12 (IgG1) and Fel.10E9 (IgG1) respectively. The tissue distribution of this CD1 homologue indicates that it is a member of the classic thymic CD1 family. This feline homologue of CD1 was distinct from CD1c by virtue of its lack of expression in peripheral blood and splenic mantle zone B cells. An unequivocal distinction could not be made between CD la and CD 1b based on tissue distribution due to species variation in expression of these CD1 molecules. Although, the biochemical characteristics of this feline CD1 homologue more closely match with CD 1a. The pattern of tissue expression and biochemical characteristics of the feline CD1 antigen appear largely similar to those described for human and other species.
Sequencing‐based typing of HLA‐A locus using mRNA and a single locus‐specific PCR followed by cycle‐sequencing with AmpliTaq DNA polymerase, FS
Authors: Norgaard L. , Fugger L. , Jakobsen B. K. , Svejgaard A.
Published: December 2008

The large number (59) of alleles now known at the HLA‐A locus is a serious challenge to the existing methods for HLA typing, including many of the DNA based methods. Here, we describe a sequencing‐based typing (SBT) protocol for typing of HLA‐A alleles using a single A‐locus‐specific PCR. This reaction amplifies an 824 base pair product from cDNA, prepared from mRNA, covering exons 1–3 and most of exon 4. This product allows identification of all possible combinations of two alleles from this locus. The sequencing strategy used for allele assignment contains several improvements compared to those previously published. The enzyme AmpliTaq® DNA Polymerase, FS, used, combines high‐quality sequencing, i.e. long reads, low background, and uniform peak heights making the identification of heterozygous positions very reliable in a fast and easy protocol developed by determining the optima for a number of variables. Thus, this strategy meets most of the requirements for the use of sequencing in HLA typing. Furthermore, this method is very flexible. The use of a PCR primerpair tailed with the recognition sites for two different sequencing primers allows the application of the same sets of fluorescent‐labelled sequencing primers regardless of the amplified locus. Thus, the protocol can very easily be extended to cover the B‐ and C‐locus too, simply by adding PCR reactions specific for these loci to the protocol. Using this protocol, we investigated a total of 65 cell lines and clinical samples, many of the latter chosen from samples difficult to type by serology. Our method gave in all cases unambiguous results and proved functional for work requiring the highest resolution.
Nucleotide sequence of HLA‐B*5505, which expresses a unique HLA class I polymorphism
Authors: Szmania S. , Seurynck K. , Baxter‐Lowe L. A.
Published: December 2008

The nucleotide sequence reported in this article will appear in the GenBank database under the accession number U63653. The name HLA‐B*5505 was officially assigned by the WHO Nomenclature Committee in 1996. This follows the agreed policy that, subject to the conditions stated in the most recent nomenclature report (1), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO nomenclature report.
Expression of human leukocyte antigens class I and class II on cultured biliary epithelial cells after cytomegalovirus infection
Authors: Scholz M. , Cinatl J. , Blaheta R. A. , Kornhuber B. , Markus B. H. , Doerr H. W.
Published: December 2008

The influence of cytomegalovirus (CM V) infection on the HLA expression on cultured biliary epithelial cells (BEC) was investigated. CMV‐infection augmented expression of HLA class I but not of HLA class II. CMV reduced the IFN‐gamma‐mediated induction of the de novo expression of HLA class II while the stimulated expression of HLA class I was not impaired. Autologous but not allogeneic PBL responded to CMV‐infected BEC. This response resulted in upregulation of HLA class I on BEC which was significantly higher compared with the expression on infected BEC alone or on uninfected BEC cocultured with autologous PBL. The results suggest that CMV modulates the immunogenic potential of BEC, which is important for the HLA and CMV‐mediated pathomechanisms in vivo.
Complete coding regions of two novel HLA‐B alleles detected by prototyping (PCR‐SSP) in the British Caucasoid population: B*5108 and B*5002
Authors: Vilches C. , Bunce M. , de Pablo R. , Murray A. K. , McIntyre C. A. , Kreisler M.
Published: December 2008

Two previously reported PCR‐SSP variants of the HLA‐B locus, B51GAC and B45v, were investigated by RT‐PCR cloning and nucleotide sequence analysis of their complete coding regions. They have been shown to correspond to the new alleles B*5108 and B*5002, both of which differ from the common B*5101 and B*5001 subtypes, respectively, by amino acid replacements at their α‐2 domain α‐helices. The primary structure of B*5002, intermediate between those of B*4501 and B*5001, raises further concern about the current classification of B*45 as a B12 rather than as a B*50 subtype.
Identification of four new DR52‐associated DRJB1 alleles: DRB1*1424, *1425, *1323 and *1324
Authors: Ellis J. M. , Robbins F. , Wang J. , Tang T. , Heine U. , Mason J. M. , Sese D. , Milford E. , Hurley C. K.
Published: December 2008

Four previously unreported DR52‐associated DRB1 alleles have been characterized through DNA sequencing, contributing to the diversity of the HLA system. DRB1*1424 is nearly identical to DRB1*1402 in the second exon, except that it contains the “I—A” motif found at codons 67–71 common to the DRB1*15 alleles. DRB1*1425 contains the “A—H” motif, found in DRB1*1401 at codons 57–60, in a sequence otherwise identical with the second exon of DRB1*1307. Compared with DRB1*11012, DRB1*1323 contains three predicted amino acid changes at codons 58 (ala→glu), 67 (phe→ile), and 71 (arg→glu). The sequence of DRB1*1324 is identical to exon 2 of DRB1*1103, except that DRB1*1324 does not contain the GAG at codon 58 characteristic of the DRB1*11 alleles. These new alleles may have arisen through gene conversion, and they contribute to the complexity of the DR6 family.