bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of cell surface antigens and regulation of phagocytic activity mediated by CD11b in the monocyte‐like cell line U937 in response to lipopolysaccharide
Authors: Ikewaki Nabunae , Tamauchi Hidekazu , Inoko Hideteshi
Published: December 2008

Abstract: Modulation of the cellular antigens and regulation of the phagocytic activity of the monocyte‐like cell line U937 after culture with lipopolysaccharide (LPS) were investigated. CD14 expression was induced on the surface of the U937 cells after 48 h of culture with LPS and then they became adhesive with numerous filamentous filopodia extruded on the cell surface, exhibiting the enhanced expression of CD16 and CD23, the activation cell surface markers for differentiation into macrophage. However, no induction or enhancement of the cell surface expression was observed with respect to CD11b, CD18, HLA‐A, B, C, HLA‐DR, DQ, DP or CD57. These U937 cells also acquired the ability to produce superoxide anions and to phagocytose the Salmonella enteritidis strain, 116–54. This phagocytosis was inhibited by the anti‐CD11b monoclonal antibodies, but not by the anti‐CD14, anti‐CD16, anti‐CD18, anti‐CD23, anti‐HLA‐A, B, C or anti‐HLA‐DR monoclonal antibodies. These findings indicate that the phagocytic activity against Salmonella enteritidis 116–54 induced by LPS is mediated mainly via the CD11b molecule, but is not associated with the increased expression of CD11b. Puromycin and cycloheximide, inhibitors of protein synthesis, or a divalent cation‐chelating agent, EDTA completely inhibited this phagocytic activity. Interestingly, EDTA was found to suppress specifically the CD11b expression on the U937 cells cultured with LPS. No phagocytic activity was induced when the U937 cells cultured with LPS were incubated at 4°C, but restored to the control level when shifted up to 37°C. This experimental system provides an excellent model for the investigation of the molecular mechanism underlying modulation of the cell surface antigens and regulation of the phagocytic activity mediated by CD11b in the U937 cells in response to LPS.
Widespread tissue distribution of aminopeptidase A, an evolutionarily conserved ectoenzyme recognized by the BP‐1 antibody
Authors: Li Li , Wu Qi , Wang Jiyang , Bucy R. Pat , Cooper Max D.
Published: December 2008

Abstract: Early B‐lineage cells in mice express a cell surface glycoprotein, recognized by the BP‐I and 6C3 monoclonal antibodies, that has been identified as aminopeptidase A (APA E.C.3.4.11.7). In the present studies we obtained evidence by DNA ”zoo‐blot” analysis that the APA gene is highly conserved. This ectoenzyme catalyzes the removal of N‐terminal Glu‐ and Asp‐residues to convert angiotensin II to angiotensin III. a degradation step important in local regulation of blood pressure in mammals. To gain further insight into the physiology of this molecule, which is shared between immune and vascular systems, we examined the tissue distribution of BP‐l mRNA using a cDNA probe and of the protein antigen using the BP‐I antibody for immunohistology. APA transcripts were present in all tissues examined. Abundant BP‐I IAPA was found in the intestinal brush border of the small intestine, renal glomeruli, proximal renal tubules, pulmonary alveolar walls and vascular endothelium in many organs. Other tissues containing the BP‐I antigen included stromal cells in the thymus cortex, bile canaliculi in liver, gall bladder epithelium, interlobular ducts in pancreas, the ovarian theca interna, basement membrane of the epididymis and the splanchnopleure in placenta. APA enzyme activities have been identified in most of these locations in keeping with identification of the BP‐1/6C3 antigen as APA. The data suggest this ectopeptidase may serve diverse physiologic roles in a broad spectrum of tissues.
Multi‐locus analysis of HLA class II genes in DR2‐positive IDDM haplotypes in Finland
Authors: Reijonen Helena , Ilonen Jorma , Åkerblom Hans K. , Knip Mikael , Dosch Hans‐Michael ,   “Childhood Diabetes in Finland” (DiMe) Study Group
Published: December 2008

Abstract: In this study we characterized the haplotypes found in IDDM patients that normally confer resistance to the disease in order to localize the polymorphisms relevant for the protection. We studied 15 DR2‐positive subjects with IDDM for their DRB1, DRB5 and DQB1 genes using RFLP, polymerase chain reaction (PCR), oligonucleotide typing, and in some specific cases direct sequencing after allele‐specific PCR. In addition we analyzed 39 DR2‐positive, IDDM non‐associated haplotypes representing those haplotypes that are not inherited to probands and hence are present only in healthy family members. The frequency of the DRB1*1501‐DRB5*0101‐DQB1*0602 haplotype was slightly decreased among diabetic patients (80% vs. 92%). In addition, two unconventional haplotypes DRB1*1501‐DRB5*0101‐DQB1*05031 and DRB1*1501‐DRB5*0101‐DQB1*0502 were found in patients with IDDM while all the control ones were conventional. The sequencing of the DQB1*0602 allele present in IDDM haplotypes showed no differences when compared to the controls. These results support the primary but not absolute role of DQ in the protection against IDDM. An additional role of factors centromeric to DQB1 gene was suggested by findings based on the biallelic TaqI RFLP polymorphism of the DQA2 gene. All DR2‐DQB1*0602 IDDM haplotypes were associated with the 2.1‐kb fragment while in the control group the 2.1‐kb and 1.9‐kb fragments were evenly distributed.
Molecular characterization of the HLA‐DR2LUM haplotype
Authors: Young N. T. , Darke C.
Published: December 2008

Abstract: Molecular studies of HLA‐DRB, ‐DRA and ‐DQB1 genes in the variant DR2 haplotype, DR2LUM, were performed using the homozygous lymphoblastoid cell line, CTS. The results of HLA Class II gene RFLP and PCR analyses suggest that DR2LUM was created by a homologous recombination event between HLA‐DR1 and HLA‐DR15 haplotypes. Evidence for the presence of a recombinational “hotspot” in haplotypes possessing a DRB6 pseudogene is presented. The results of this study have important implications for detection of HLA‐DR2 alleles in DRB gene oligotyping strategies, and suggest that the CTS cell line will be a useful addition to cell panels for characterizing HLA antisera.
DNA polymorphism of HLA class II genes in systemic lupus erythematosus
Authors: Cowland Jack B. , Andersen Vagn , Halberg Poul , Morling Niels
Published: December 2008

Abstract: We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA‐DRB, ‐DQA, ‐DQB, ‐DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3‐ and DRw6‐associated 7.00 kb DRB TaqI DNA fragment was found in SLE patients compared to normal controls (83.3% vs 35.5%; RR = 9.1, p < 10‐4). The frequencies of the DQA1*0501‐associated 4.56 kb DQA TaqI fragment and the DRB3*01/03‐associated 9.79 kb TaqI fragment were also found to be significantly increased in SLE patients (70.8% vs 29.7%; RR = 5.8, p < 10‐2 for the DQA fragment and 70.8% vs 36.1%; RR = 4.3, p < 0.05 for the DRB3 fragment). Less extensive and insignificant increases of the frequencies of the DR3‐associated DQB and DPB fragments were observed. The frequencies of the DR2‐associated DRB, DQA, and DQB fragments were comparable to those found in normal controls.
Molecular characterization of HLA class II genes in celiac disease patients of Latin American caucasian origin
Authors: Herrera Mariana , Theiler Graciela , Augustovski Federico , Chertkoff Lilien , Fainboim Leonardo , DeRosa Susana , Cowan Elliot P. , Satz M. Leonardo
Published: December 2008

Abstract: In the present study, the polymorphic domain of HLA class II genes present in a pediatric population of Argentinian celiac disease patients was analyzed by hybridization to sequence‐specific oligonucleotides and DNA sequencing. Sixteen out of 16 DR5/7 heterozygous patients bore the DQA1*0501 and DQB1*0201 alleles implicated in the DQ2 risk specificity. The second exon of DQA1, DQB1 and DRB1 genes from 2 DR5/7 patients was characterized by DNA sequencing. The following alleles were found in both patients: DRB1*1101 and DRB1*0701; DQB1*0301 and DQB1*0201; DQA1*0501 and DQA1*0201. Previous serological analysis in this population had shown the presence of DQ2 in 95% of the patients (40% in controls) and a negative association with DQ1 haplotypes, suggesting the presence of other “permissive” or neutral alleles. The following HLA‐DQB1 alleles, besides DQB1*0201, were identified in 31 CD patients: DQB1*0301, 0302, 0401 and 0402. All these alleles share a common pattern of residues between positions 84 and 90, and distinct from that present in DQ1‐related alleles.
RFLP characterization of the upstream regulatory region of the HLA‐DQA1 gene
Authors: Maffei Antonella , Reed Elaine F. , McManus Peter , Guardiola John , Foca Nicole Suciu
Published: December 2008

Abstract: We have performed population and family studies of the distribution of DNA restriction length polymorphisms (RFLPs) in the 5′ region of the HLA‐DQA1 gene using a probe which corresponds to a sequence of the 5′ flanking region of HLA‐DQA1. Southern analysis detected four polymorphic fragments (X1, X2, X3 and X4) with XbaI and three fragments (E1, E2 and E3) with EcoRI. Family segregation studies showed that these RFLPs segregated in cis with the parental HLA haplotypes. Analysis of haplotypic associations of the X and E polymorphisms with each other and with HLA‐DQA1 alleles demonstrates that DQA1 alleles can be further subtyped according to the particular XE combination which they carry. Hence, definition of these alleles provides new markers for HLA haplotyping and allows further splitting of otherwise identical DQA1 alleles. This information may be helpful for studies of association of disease susceptibility and autoimmunity with HLA haplotypes.
HLA‐B15: A widespread and diverse family of HLA‐B alleles
Authors: Hildebrand William H. , Domena John D. , Shen Susan Y. , Lau Marie , Terasaki Paul I. , Bunce Mike , Marsh Steven G. E. , Guttridge Martin G. , Bias Wilma B. , Parham Peter
Published: December 2008

Abstract: HLA‐B15 embraces a multiplicity of antigenic specificities which vary in their distribution amongst human populations. To correlate B15 molecular structure with the serological picture we have sequenced alleles encoding the various subspecificities of the B15 antigen: B62, B63, B75, B76 and B77, and a number of “variants” of these antigens including the 8w66 split of B63. HLA‐B63 (B*1517) and 8w66 (B*1516) heavy chains have sequence identity to B17 in the α1 helix correlating with the antigenic crossreactivity of these molecules. HLA‐B77(B*1513) and B75 (B*1502) heavy chains differ solely in segments determining the Bw4 and Bw6 public epitopes, consistent with the serological description of the B77 and B75 antigens. One allele encoding the B76 antigen (B*1512) appears to be the product of gene conversion between the HLA‐A and ‐B loci and differs from B*1501 in codons 166 and 167. In contrast, a second allele encoding the B76 antigen (B*1514) differs from B*1501 by an unrelated substitution in codon 167 which confers similarily with B45, an antigen crossreactive with B76. A third allele encoding B76. B*1519, differs from B*1512 by a unique point substitution in exon 4. Three alleles encoding variant B15 and B62 antigens (B*1508, B*1511 and B*1515) differ from B*1501 by localized clusters of substitutions that probably result from interallelic conversion. The B15 sequences described in this paper, in combination with those previously determined, define a family of 22 alleles, including those encoding the B46 and B70 antigens. Within this family the patterns of allelic substitution are analogous to those of other HLA‐A and ‐B families, in that pairwise differences almost always involve functional positions of the antigen recognition site and recombination is the major agent of diversification.
Comprehensive typing of DQB1 alleles by PCR‐RFLP
Authors: Sengar Dharmendra P. S. , Goldstein Rose
Published: December 2008

Abstract: The protocols represented in this report can resolve all 22 DQB1 alleles. The second exon of DQB1 was subjected to PCR using two group‐specific primers to obtain DQB1 group 1 (DQ5 and DQ6) and group 2 (DQ2, DQ3, DQ4) specific amplified products, respectively. Three endo‐nucleases, Apal, BssHII and Ncil, can provide typing of DQ5 and DQ6 based on easy‐to‐read uncleaved, cleaved and a combination of uncleaved/cleaved patterns. Similarly, two endonucleases, FokI and BgII can define the specificities DQ2, DQ3 and DQ4. Moreover, all 13 group 1 DQB1 alleles and all but one of their 78 possible heterozygotes can be unambiguously resolved using an extended panel of 10 endonucleases. The remaining pair of heterozygotes, DQB1*05031/0603 and 05032/0608, can however be resolved by double digestion with BsmFI and SfaNI. RsaI splits the previously unresolved alleles DQB1*0602 and 0603 in the amplified products of the modified primer SDQ‐01. Fnu4HI can resolve DQB1*0606 from 0605. DQB1*0603, 0607 and 0608 can be resolved by SfaNI and the new endonuclease BsmFI. The comprehensive typing of group 2 DQB1 alleles can be achieved using five endonucleases. All 9 group 2 DQB1 alleles and all but one pair (DQB1*0301/0302 from DQB1*03032/0304) of 36 possible heterozygotes can be resolved. Thus, PCR‐RFLP remains a simple, inexpensive and reliable method for DQB1 typing. The PCR‐RFLP can be used for comprehensive DQB1 typing either independently or to complement the PCR‐SSP and PCR‐SSOP methods.
Both class I and class II HLA antigens are thyroid cancer susceptibility factors
Authors: Rigopoulou Demetra , Martinez‐Laso Jorge , Martinez‐Tello Francisco , Alcaide Juan F. , Benmamar Djamal , Hawkins Federico , Arnaiz‐Villena Antonio
Published: December 2008

Abstract: It has been established that HLA antigens are susceptibility factors for different cancers, including thyroid tumors. However, the diversity and sometimes weak and contradictory associations found have frequently led to the view that the HLA and tumorigenesis links might be the result of statistical errors. However, it has recently been established that it is indeed a currently complex and unexplained but real phenomenon, which may be crucial in preventing several types of cancer. In the present work we have found in a relatively large series of thyroid cancer patients (n = 161) that both HLA class I (B35) and class II (DR11) antigens are susceptibility factors only in the papillary tumor group of patients, B35 association p value is found at the limit of significance (pc(120) = 0.05); the follicular group did not show any HLA association, suggesting that the etiopathogenesis of each type of cancer is different. HLA‐B35 and DR11 are not working together to induce tumorigenesis and each of them seems to confer susceptibility by using different pathways or by being markers of distinct neighboring susceptibility genes. DR4 has also been found in 86% (n = 6) of Hürthle cell carcinoma. No association has been found between HLA and disease activity. HLA mechanisms of association to cancer are discussed and a world‐wide HLA/tumorigenic study is proposed to obtain a clear picture of the puzzling and controversial susceptibility markers found in different tumors and in different ethnic groups.
The DQA1*0104 allele is carried by DRB1*1001‐ and DRB1*1401‐positive haplotypes in Caucasians, Africans and Orientals
Authors: Fogdell Anna , Olerup Olle
Published: December 2008

Abstract: The DQA1*0104 allele is known to differ from DQA1*0101 by a single nucleotide in the sequenced part of the first exon. DQA1*0104 has a guanine in the second position of the second expressed codon, whereas DQA1*0101 and all other sequenced DQA1 alleles have an adenine in that position, changing aspartic acid to glycine. The DQA1*0104 allele was originally described in African Americans with the DRB1*12, DRB3*0101, DQA1*0104, DQB1*0501, DRB1*12, DRB3*0202, DQA1*0104, DQB1*0605 or DRB1*14, DQA1*0104, DQB1*0503 haplotypes. When developing DQA1 typing by PCR amplification with sequence‐specific primers (PCR‐SSP), we observed that all DR10‐ and DR14‐positive samples carried the DQA1*0104 allele, wheres all DRB1*01‐positive DNAs carried the closely related DQA1*0101 allele. In the present study, samples representing the major ethnic groups with DR‐DQ haplotypes known to carry the DQA1*0104 allele or the very similar DQA1*0101 allele were investigated by TaqI RFLP analysis, PCR‐SSP typing and nucleotide sequencing. The DQA1*0104 allele was found to differ from DQA1*0101 not only in the second expressed codon, but also by a productive mutation in the signal peptide. All investigated DRB1*1001‐(n = 24) and DRB1*1401‐positive (n = 25) haplotypes, defined by homozygosity or association, of Caucasian, African or Oriental origin carried the DQA1*0104 allele, whereas the DQA1*0101 allele was found on all DRB1*01‐positive (n = 32) haplotypes. These findings demonstrate that in the assignment of HLA class II alleles, polymorphism outside the second exon sometimes must be considered. The maintenance of the DQA1*0104 allele on a few distinct haplotypes indicates that the allele is old and might also be compatible with a functional difference between the DQA1*0101 and DQA1*0104 alleles.
HLA class I self peptides isolated from a T‐cell leukemia reveal the allele‐specific motif of HLA‐B38
Authors: Colovai Adriana I. , Suciu‐Foca Nicole , Baiulescu George Emil , Harris Paul E.
Published: December 2008

Abstract: Naturally‐processed self peptides bound to HLA class I molecules of a T‐cell leukemia (HLA‐A1, A31, B38, B58) were isolated for sequence analysis. Acid‐eluted peptides were subjected to reversed‐phase HPLC separation and single‐fraction sequencing was performed by Edman degradation. The peptides were found to be mostly nonamers and could be grouped into three distinct structural motifs. One of the peptide groups consistently displayed histidine at position 2 and a bulky hydrophobic residue at the C‐terminus (XHXPXXXXY/F). The only HLA class I structure expressed by this T‐cell leukemia which is consistent with the binding of peptides carrying this sequence motif is HLA‐B38. A peptide binding assay confirmed this assignment. HLA‐B38 is present in 10–12% of the Jewish population and is associated with several autoimmune disorders. The HLA‐B38 motif may be an important tool for identifying potential T‐cell epitopes involved in these diseases and for designing peptide vaccines.
A method for typing polymorphism at the HLA‐A locus using PCR amplification and immobilized oligonucleotide probes
Authors: Bugawan Teodorica L. , Apple Raymond , Erlich Henry A.
Published: December 2008

Abstract:We have developed a simple and rapid method for DNA typing of the HLA‐A locus using PCR amplification and hybridization of the PCR product, labeled with biotinylated primers, to an array of immobilized oligonucleotide probes in a single hybridization reaction (reverse dot or line blot). A single primer set (RAP1007 and DB337) is used to specifically amplify a 990‐bp fragment containing the HLA‐A locus exons 1, 2, and 3 from genomic DNA. This primer set is locus‐specific and amplifies all HLA‐A alleles. A set of 51 sequence‐specific oligonucleotide (SSO) probes, 25 for exon 2 and 26 for exon 3, was immobilized to a nylon membrane by UV‐crosslinking oligonucleotide probes containing a poly‐thymidine “tail” added with terminal transferase. In the line blot format, all 50 SSO probes plus a control probe are immobilized on a single nylon membrane strip. The probe array was used for typing in a hybridization reaction with DNA amplified from a variety of samples. These probes can identify 37 homozygous HLA‐A alleles. In the analysis of heterozygous samples, 604 heterozygous types out of 633 (95.4%) possible heterozygous probe patterns can be detected as a unique probe reactivity pattern. A simple computer program has been developed to assign the alleles and genotypes based on the probe hybridization pattern.
HLA‐DQA1, ‐DQB1 and ‐DRB1 genotyping in Japanese pemphigus vulgaris patients by the PCR‐RFLP method
Authors: Niizeki Hironori , Inoko Hidetoshi , Mizuki Nobuhisa , Inamoto Nobuko , Watababe Kyoko , Hashimoto Takashi , Nishikawa Takeji
Published: December 2008

Abstract: We performed HLA‐DQA1, ‐DQB1 and ‐DRB1 genotyping using the PCR‐RFLP (polymerase chain reaction‐restriction fragment length polymorphism) method for 32 Japanese pemphigus vulgaris (PV) patients. There was a significant association of either DQB1*0503 or DRB1*1405 with PV, and a negative association of either DQA1*0103 or DQB1*0601 with PV was found. Since the DQB1*0503+ patients had various DR14‐related alleles, we concluded that the association with DQB1 is primary and that the association with DRB1 is simply due to linkage disequilibrium between the DQ and DR genes. These results may indicate that specific HLA class II antigens confer the susceptibility to PV among Japanese.
Characterization of a novel bovine leukocyte protein involved in cell‐cell adhesion
Authors: Taylor Bernadette C. , Mattapallil Joseph , Moore Peter F. , Scibienski Robert J. , Stott Jeffrey L.
Published: December 2008

Abstract:Preliminary characterization of an apparently novel bovine leukocyte adhesion protein is described. Two IgG1monoclonal antibodies, UC‐C1 and UC‐H5, raised against established cultures of IL‐2‐dependent bovine peripheral blood lymphocytes (PBL) were found to react with an antigen expressed by the majority of bovine peripheral blood leukocytes. Immunoprecipitation and polyacrylamide gel electrophoresis of the antigen produced a distinct protein band of molecular weight 160 000, and additional diffuse protein bands of approximate molecular weight 180 000, 175 000 and 150 000. Two‐color flow cytometric analyses showed that the antigen was expressed at low density on a small proportion of circulating B lymphocytes, but was highly expressed on all circulating T lymphocytes. The majority of monocytes and all granulocytes expressed the antigen at a density lower than that of T lymphocytes. Peripheral blood lymphocytes stimulated with concanavalin A had an approximately 3‐fold increased expression of the antigen, which was apparent within 18 h and remained stable in long‐term cultures. Expression of the antigen in thymus, analyzed by the immunoperoxidase technique, was predominantly restricted to thymocytes in the immediate subcapsular cortex and medulla; expression in lymph nodes and spleen was predominantly confined to lymphocytes in T‐cell areas. Flow‐cytometric analysis demonstrated that thymocytes and the majority of peripheral and mesenteric lymph node‐derived T cells had relatively low surface density of antigen compared to circulating T cells. Binding of UC‐C1 or UC‐H5 to the antigen on lymphocytes induced homotypic aggregation. UC‐C1 completely blocked binding of FITC‐conjugated UC‐H5 to blood mononuclear cells, suggesting that the antibodies recognize the same epitope or proximal epitopes.
A new simplified method of gene typing
Authors: Chia David , Terasaki Paul , Chan Henry , Acalinovich Andrew , Maruya Etsuko , Saji Hiroh , Ware Krista
Published: December 2008

Abstract: SSP‐PCR (sequence‐specific primer) DNA typing was performed in Terasaki trays using 1.5 μ1 of DNA, and the ethidium‐stained PCR product was measured by direct fluorometric reading. Elimination of the gel electrophoresis step greatly simplified the SSP method. 17 serological DR specificities were discriminated for 239 DNA samples utilizing the new method, standard SSP, sequence‐specific oligonucleotide probe (SSOP), and restriction fragment length polymorphism (PCR‐RFLP). Results showed 98% concordance between the SSP‐PCR assay and conventional methods. DRB1 alleles were determined by PCR‐RFLP in 59 samples, by SSP in 110 samples, and by consensus (all methods) in the remaining samples.
Locus‐specific amplification of HLA class I genes from genomic DNA: locus‐specific sequences in the first and third introns of HLA‐A, ‐B, and ‐C alleles
Authors: Cereb N. , Maye P. , Lee S. , Kong Y. , Yang S. Y.
Published: December 2008

Abstract: We have identified locus‐specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA‐A, ‐B, and ‐C loci in the amplification of genomic DNA. PCR products generated using each of the locus‐specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non‐classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin‐ddUTP‐labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus‐specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR‐based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.
HLA class I variation in Australian Aborigines: characterization of allele B*1521
Authors: Lienert Kristin , McCluskey James , Bennett Greg , Fowler Craig , Russ Graeme
Published: December 2008

Abstract: Traditional methods of serological typing have largely used antisera of Caucasoid origin, which can overlook HLA heterogeneity in non‐Caucasoid populations. Therefore, we have used molecular techniques to evaluate potential polymorphism in HLA class I molecules of Aborigines from the central desert and northern coast of Australia. The DNA sequence of common Aboriginal HLA‐A and B antigens were compared with serological reaction patterns which suggested new polymorphisms. Although serological data indicated that long and short variants of A34 may exist, regardless of the serological pattern, all individuals carried the A*3401 allele. Therefore, the variation in A34 reaction pattern observed serologically was not attributable to primary sequence variation in the HLA A*3401 allele. Similarly, there was no detectable polymorphism in the sequences of selected HLA‐B alleles, even though some of these alleles showed unusual serological reaction patterns. However, a new allele of B15 (B*1521) was detected in two individuals carrying this serotype. The cells from both of these individuals showed ambiguous reaction patterns with monospecific B62 and B75 sera. cDNA sequencing of the HLA B15 gene from these cells revealed a B15 allele that differed from B*1502 by a single nucleotide change. This change occurred at position 272, resulting in a C to G substitution at residue 67 in the consensus B15 cDNA sequence. Hence, the Australian Aborigines as an ethnic group show very little primary sequence polymorphism within the class I loci, consistent with the results obtained from previous serological studies.
Low resolution DNA typing of the HLA‐B5 cross‐reactive group by nested PCR‐SSP
Authors: Hein Joachim , Böttcher Kerstin , Grundmann Regina , Kirchner Holger , Bein Gregor
Published: December 2008

Abstract: We have established a DNA typing system for the HLA‐B5 serologically cross‐reactive group (CREG) by means of a two‐step PCR amplification with nested sequence‐specific primers (nPCR‐SSP). The present study provides a low resolution definition of the HLA‐B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group‐specific amplification of all HLA‐B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA‐B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n=50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA‐B specificities by nPCR‐SSP.
Sequencing‐based typing reveals new insight in HLA‐DPA1 polymorphism
Authors: Rozemuller Erik H. , Bouwens Anne G. M. , van Oort Erica , Versluis Leone F. , Marsh Steven G. E. , Bodmer Julia G. , Tilanus Marcel G. J.
Published: December 2008

Abstract: An HLA‐DPA1 sequencing‐based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3′ end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the — corrected — DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201. As is found in other class II genes, all exons of DPA1 show some polymorphism, but the polymorphism in exon 2 is sufficient to identify the different alleles.
Comprehensive, serologically equivalent DNA typing for HLA‐B by PCR using sequence‐specific primers (PCR‐SSP)
Authors: Bunce M. , Fanning G. C. , Welsh K. I.
Published: December 2008

Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one‐dimensional isoelectric focusing (1D‐IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence‐specific primers (PCR‐SSP). We describe here a comprehensive HLA‐B PCR‐SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one‐step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA‐B PCR‐SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele‐specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR‐SSP systems for HLA‐Class II and HLA‐Class I in that the same PCR conditions and controls are used which allows us to do one step PCR‐SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.
HLA in familial and nonfamilial Mediterranean Kaposi's sarcoma in Greece
Authors: Kaloterakis A. , Papasteriades C. , Filiotou A. , Economidou J. , Hadjiyannis S. , Stratigos J.
Published: December 2008

Abstract: Fifty‐four (54) unrelated patients with Mediterranean Kaposi's sarcoma (MKS) and 8 patients members of 4 unrelated families with familial MKS were serotyped for HLA‐A,B and DR antigens. The diagnosis was histologically confirmed and all patients were negative for anti‐HIV antibodies. An increased frequency of HLA‐B18 (44.4% vs 14.2% in the controls, p<0.001, RR=4.8) and HLA‐DR5 (57.6% vs 37.2% in the controls, p<0.025, RR=2.29) was observed in the group of patients with MKS. Seven (7) of the 8 family members with FMKS possessed HLA‐DR5, and the affected members in the 3 families shared a common haplotype which included HLA‐DR5. These findings support the hypothesis that genetic factors linked to HLA‐DR5 antigen may contribute to the pathogenesis of MKS.
Comparison of two HLA‐DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon‐86 valine or glycine PCR segregation
Authors: Peponnet C. , Schaeffer V. , Lepage V. , Chatelain F. , Rodde I. , Alsayed J. , Boucher P. , Hermans P. , de Linval N. Monplaisir/Cassius , Charron D.
Published: December 2008

Abstract: Two rapid, nonisotopic, high‐resolution HLA‐DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic amplification (1GA) method amplifies all HLA‐DRB alleles in the same reaction mix. The 2 Allelic Subset amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal‐size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.
Modification of an HLA‐B PCR‐SSOP typing system leading to improved allele determination
Authors: Middleton D. , Williams F. , Cullen C. , Mallon E.
Published: December 2008

Abstract: Modifications have been introduced to a previously reported HLA‐B PCR‐SSOP typing system. This has enabled further definition of alleles, determination of the probe pattern of some alleles not previously examined and identification of patterns of possible new alleles. However there are still some alleles that cannot be differentiated and there are several alleles which when present as a homozygote have the same pattern as in combination with another allele. When the method was applied to the typing of 66 consecutive cadaveric donors there were, three donors whose type differed from the serological type.
The contribution of the HLA‐A, ‐B, ‐C and ‐DR, ‐DQ DNA typing to the study of the origins of Spaniards and Basques
Authors: Martinez‐Laso J. , De Juan D. , Martinez‐Quiles N. , Gomez‐Casado E. , Cuadrado E. , Arnaiz‐Villena A.
Published: December 2008

Abstract: The high polymorphism of the HLA system has been used as a powerful genetic tool to single out individuals and populations. By studying characteristic allele frequencies and extended HLA haplotypes in different populations, it is possible to identify ethnic groups and establish the genetic relationships among them. In the present study, HLA‐A, ‐B, ‐C, ‐DR and ‐DQ typing at the serological/antigenic and the DNA level has been used for the first time to assign specific HLA frequencies and haplotypes to Spaniards and Basques and compare them with frequencies in other populations, particularly with North Africans. Allelic frequencies do not significantly differ between Spaniards and Basques. HLA genetic distances and their respective dendrogram together with the results on complete HLA haplotypes place Basques and Spaniards closer to paleo‐North African populations than to other Europeans. This goes in favour of the Basques being a relative genetic isolate coming from the primitive Iberian/paleo‐North African people. In addition, a tentative assignment of the most common Spanish HLA haplotypes to the different people who populated Iberia according to historical records has been done.
Dissection of positive regulatory elements in the upstream region of the HLA–DBP1 gene
Authors: Koide Y. , Yoshida T. O.
Published: December 2008

Abstract: We investigated the cis‐acting sequences that function in the B‐cell‐specific expression of the HLA‐DPB1 gene. Class II B major histocom‐patibility genes contain a conserved upstream sequence that is important in the expression of these genes. This region has been divided into three major elements, the W, X, and Y boxes. In this paper, we identified an additional positive regulatory element upstream from the DPB1 W box. Using 5′ deletion mutants and a substitution mutant, we mapped a positive element, called the W box, between‐184˜‐169 bp. Sequence comparison revealed that the W box shares homology with the W box. Electrophoretic mobility shift assay confirmed that the W box binds proteins that also recognize the W box. Furthermore, deletion and substitution mutants indicate that the W and W boxes effectively enhance CAT activities only when the X and Y boxes exist.
Definition of a new HLA‐B7 subtype (B*0704) by isoelectric focusing, family studies and DNA sequence analysis
Authors: Kubens B. S. , Arnett K. L. , Adams E. J. , Parham P. , Grosse‐Wilde H.
Published: December 2008

Abstract: During screening of potential bone marrow donors, a previously undescribed banding position for the serologically defined HLA‐B7 antigen was identified in three unrelated families using one dimensional isoelectric focusing and class I specific Western blot analysis. The isoelectric point of the new variant is more acidic than the two HLA‐B7 variants that had been defined before. In each family the new B7 variant was found linked to HLA‐A2 and ‐Cw7. Cloning and sequencing of full‐length clones of complementary DNA showed that the new allele (B*0704) differs from B*0702, the common allele encoding HLA‐B7, by three nucleotide substitutions within the codon for residue 156 of the mature heavy chain. As a result of these differences amino acid 156 is changed from arginine to aspartic acid, a difference consistent with the isoelectric points. The group of three nucleotide substitutions that distinguish B*0704 from B*0702 is present in other HLA‐B alleles.
Allelic variation at the TAP 1 locus influences disease phenotype in HLA‐B27 positive individuals with ankylosing spondylitis
Authors: Maksymowych W. P. , Tao S. , Li Y. , Wing M. , Russell A. S.
Published: December 2008

Abstract: The objective was to determine the role of the TAP 1 gene in influencing the phenotype of disease in adult patients with ankylosing spondylitis (AS). The distribution of TAP 1 gene alleles was determined using the PCR RFLP method and restriction enzymes Bcl I and Ace I. The study population included 115 HLA‐B27 positive patients with well‐documented AS and 41 HLA‐B27 positive normal controls. No significant difference in distribution of TAP 1 alleles was noted in comparisons of all AS patients with normal controls. However, AS patients with extraspinal disease were noted to have a significantly increased prevalence of the TAP 1B allele (17.0%) as compared to AS patients without extraspinal disease (2.9%) (P=0.005) or normal controls (1.9%) (P=0.005). Polymorphism at the TAP 1 locus may influence disease outcome in patients with AS.
Increased surface expression of class I MHC molecules on immunogenic cells derived from the xenogenization of P815 mastocytoma cells with 8‐methoxypsoralen and long‐wavelength ultraviolet radiation
Authors: Schmitt I. M. , Moor A. C. E. , Patrignelli R. , Chimenti S. , van Henegouwen G. M. J. Beijersbergen , Edelson R. L. , Gasparro F. P.
Published: December 2008

Abstract: In a previous study we demonstrated that the treatment of the highly tumorigenic cell line, P815, with 8‐methoxypsoralen and long‐wave‐length ultraviolet radiation resulted in the production of several immunogenic clones (tum—‐). Mice inoculated with the tum‐ cells survived much longer than mice inoculated with the original tumorigenic cells (tum+). It was suggested that the increased survival of mice treated with the tum‐ clones arose as a result of an increased antigenicity derived from the phototreatment. In this report we show that the tum‐ cells have a greater density of class I MHC molecules on their surface (50–157% compared to P815). Class I MHC density on the cell surface is required to elicit targeted cytotoxic responses. These results can be considered in terms of human class I MHC assays which show that many human tumor cells have a reduced expression of class I MHC. Because other DNA damaging agents have also been shown to enhance class I expression, it is suggested that in addition to the cytotoxic effects of these agents, other pleiotropic effects must be considered. Photochemotherapy may phenotypically alter cells so that the enhanced expression of class I MHC molecules on the surface of phototreated cells may be associated with the clinical responses observed in cutaneous T cell lymphoma patients.
Complete subtyping of the HLA‐A locus by sequence‐specific amplification followed by direct sequencing or single‐strand conformation polymorphism analysis
Authors: Blasczyk R. , Hahn U. , Wehling J. , Huhn D. , Salama A.
Published: December 2008

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR‐based HLA‐A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA‐A locus. The method is based on a sequence‐specific amplification identifying the serologically defined HLA‐A specificities. The PCR products generated by these group‐specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single‐strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1‐A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1‐A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid‐phase sequencing or SSCP analysis of the group‐specific PCR fragments allowed complete subtyping of the HLA‐A locus. This strategy can identify all 48 HLA‐A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA‐A locus. Only 4 out of these 1128 allele combinations remained unresolved.
Complete generic and extensive fine‐specificity typing of the HLA‐B locus by the PCR‐SSOP method
Authors: Fleischhauer K. , Zino E. , Bordignon C. , Benazzi E.
Published: December 2008

Abstract: This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA‐B locus genes. A single HLA‐B specific PCR‐product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty‐seven chemiluminescent labeled oligonucleotide probes. Thirty‐one of these probes were derived from four hypervariability zones in exon 3 of HLA‐B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty‐six distinct hybridization patterns that could be used to unequivocally define all WHO‐designated serological HLA‐B specificities except for HLA‐B54 in all homo‐ and heterozygous combinations. Furthermore, sixty‐six out of ninety‐seven molecularly defined HLA‐B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty‐four Workshop reference cell lines and of seventy‐eight randomly chosen samples (one‐hundred forty‐seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty‐five samples (one‐hundred‐thirty‐three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety‐one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA‐B locus can be used as an alternative to biochemical methods such as one‐dimensional isoelectric focusing for assignment of serologically cross‐reacting HLA‐B molecules as well as for subtype characterization of a large variety of HLA‐B alleles.
HLA phenotypes and severe Rh(D) immunization
Authors: Hildén J.‐O. , Gottvall T. , Lindblom B.
Published: December 2008

Abstract: Blood samples from 24 Rh(D) immunized women were analyzed for antibody titers and quantification of anti‐D. The HLA‐DR and ‐DQ polymorphisms were identified as RFLP. In 11 women with titers 16–256 the HLA‐ DQB1 allele *0201 was found in 18%, i e as in a reference population. In 13 women with titers ≥ 512 the HLA ‐ DQB1 allele *0201 was found in 85% indicating a correlation between severe Rh(D) immunization with high titers/quantification values and the DQB1 allele *0201. In this group the fetus was severely affected by the immunization and treatment during pregnancy was frequently needed. HLA phenotyping of women known to have anti‐D antibodies early in pregnancy seems to be an effective way to assess the probability of severe hemolytic disease of the newborn.
Definition of HLA‐C alleles using sequence‐specific oligonucleotide probes (PCR‐SSOP)
Authors: Kennedy L. J. , Poulton K. V. , Dyer P. A. , Oilier W. E. R. , Thomson W.
Published: December 2008

Abstract: Many new HLA‐C locus alleles have recently been identified by DNA sequencing, and a molecular based method for their detection using PCR with sequence specific primers has been reported. However, other methods may be more appropriate for the identification of C locus alleles in larger studies. Here we describe one such system, based on PCR sequence specific oligonucleotide probes, (SSOP) for C locus typing. Advantages of SSOP typing compared to SSP are that it is easier to detect new alleles, more cost effective and less time consuming. We have developed a DNA typing method to identify the broad C locus antigens (including those not yet defined serologically) using a minimum of probes with one amplification. We use a C locus specific sense primer in exon 2 and a consensus antisense primer in exon 3, in a two‐step PCR, giving a product of 710 bp. Probes were designed with similar melting temperatures (54–56d̀C) that would identify as many alleles as possible. The method was established using DNA from B lymphoid cell lines of known C locus type, mostly 10th workshop homozygous cell lines, plus as many other sequenced cell lines as possible. The system was able to correctly identify their C locus types using only 26 probes. DNA was tested from a panel of serologically typed individuals which included many different heterozygous combinations. We found a high concordance of results, with all discrepancies being additional antigens identified by molecular typing, filling in serological blanks. We can identify all common heterozygote combinations using this method.