bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural definition of the A*74 group: implications for matching in bone marrow transplantation with alternative donors
Authors: Blasczyk R. , Wehling J. , Önaldi‐Mohr D. , Rebmann V. , Chandanayingyong D. , Grosse‐Wilde H.
Published: December 2008

We have identified two new A*74 alleles (A*7402 and 7403) in two unrelated individuals. A*7402 differs from A*7401 by a single amino acid substitution in the signal peptide and may be the result of a gene conversion event at the 3′ end of exon 1. A*7403 differs from A*7401 by a single amino acid exchange in the α1 domain and is most likely due to a point mutation in exon 2, since no HLA class I donor allele has been found. Since A*7402 appears to be the ancestor of the other two A*74 alleles, it is possible that A*7401 and 7403 have been created by successive point mutations. The sequences of the expressed proteins of A*7401 and 7402 are identical. The heavy chain sequence of A*7403 differs from these alleles at the crucial residue 79 which is located in the sequence stretch of the al α‐Helix where the Bw4/Bw6 determinants have been identified and which probably affects TCR interaction. This variation can therefore be expected to stimulate alloreactive T cells, graft rejection and graft versus host disease emphasizing the relevance for matching in bone marrow transplantation with alternative donors.
Nomenclature for factors of the HLA system, update June 1996
Authors: Marsh Steven G. E.
Published: December 2008

The following sequences have been submitted to the Nomenclature Committee since the May 1996 Nomenclature update and, following agreed policy, have been assigned official allele designations. Full details of all sequences will be published in the forthcoming report ‘Nomenclature for factors of the HLA System, 1996’. Below are listed the newly assigned sequences, confirmations of previously reported sequences and some sequences which are corrections of those originally reported. The accession number of each sequence is given and these can be used to retrieve the sequence files from either the EMBL, GenBank or DDBJ data libraries. Although accession numbers have been assigned by the data libraries and most sequences are already available, there is still the possibility that an author may not yet have allowed the sequence to be released, in such a case you will have to contact the submitting author directly.
NK cell receptor gene of the KIR family with two IG domains but highest homology to KIR receptors with three IG domains
Authors: Selvakumar A. , Steffens U. , Dupont B.
Published: December 2008

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) cells and some T cells. They recognize polymorphic human HLA class I molecules. Two families of KIRs have been identified and named p58 and p70. The p58 family of genes encode type I membrane proteins with two extracellular immunoglobulin (Ig) domains, while the p70 genes have three Ig domains. We here report the cloning and characterization of a novel KIR cDNA obtained from tumor cell lines with NK reactivity (YT and NK‐92). This gene is also expressed in the normal cell line NK 3.3 and in NK cells obtained from some but not all normal donors. The clone, KIR103AS, has an open reading frame consistent with a KIR with two extracellular Ig domains, a transmembrane region and a 114 amino acid long cytoplasmic domain containing a single src homology 2 (SH2) binding motif. The membrane distal Ig domain of KIR 103AS has highest homology with the first Ig domain of p70 KIRs and differs significantly from the first Ig domain of p58 KIRs. The second, membrane proximal Ig domain of KIR103AS has similar and high homology with the membrane proximal Ig domains of both p70 and p58 KIRs. The extracellular domains of KIR 103AS therefore share characteristic features with both p70 and p58 genes: the domain structure is identical to p58 KIRs but the sequence homology matches closely with p70 KIRs. The putative transmembrane and cytoplasmic domains are distinctly different from all previously reported KIR cDNAs.
Genetic heterogeneity between type 1 a and type lb insulin‐dependent diabetes mellitus: HLA Class II and TAP gene analysis
Authors: Maugendre D. , Alizadeh M. , Gauthier A. , Guilhem I. , Pouillaud C. , Genetet B. , Allannic H. , Semana G.
Published: December 2008

The aim of this study was to compare the genetic susceptibility linked to the HLA Class II region genes of the Major Histocompatibility Complex in isolated insulin‐dependent diabetes mellitus (la‐IDDM) and insulin‐dependent diabetes mellitus associated with another autoimmune endocrinopathy (lb‐IDDM). HLA genes DRB1, DQA1 and DQB1 were studied at the genomic level, as well as genes TAP1 and TAP2. One hundred and seventy‐nine la‐IDDM diabetic patients were compared with 83 lb‐IDDM patients. While it appeared that common genetic traits characterize diabetes regardless of the subtype (la or lb), certain features differentiate the two forms of IDDM. Extending the analysis of risk haplotypes DRB1*03 and DRB1*04 to TAP genes elicited a difference between la‐IDDM and lb‐IDDM patients. Haplo‐type DRB1*03 was thus characterized in la‐IDDM patients by a lower frequency of alleles TAP1‐B (13.5%) and TAP2‐B (16.2%), not found in lb‐IDDM patients (33.3% for each allele). Likewise, haplotype DRB1*04 is characterized in lb‐IDDM patients by a lower frequency of alleles TAP1‐C (4.0%) and TAP2‐B (8.0%) than in la‐IDDM patients (22.2% and 25.9%, respectively). In total, this study showed that extending the characterization of HLA Class II haplotypes to TAP genes discriminates between the forms of diabetes restricted to a specific pancreatic affection and those reflecting a wider autoimmune disorder affecting several organs.
Molecular analysis of DLA‐DRBB1 polymorphism
Authors: Wagner J. L. , Burnett R. C. , Works J. D. , Storb R.
Published: December 2008

The polymorphism of the canine major histocompatibility complex class II DRB gene, DRBB1, was analyzed. Exon 2 that encodes the polymorphic 81 domain was amplified by the polymerase chain reaction (PCR). The PCR product from 250 dogs was cloned and sequenced. Eighteen alleles were identified. Most of the variation in amino acid composition occurred at positions in the peptide binding site. Inheritance of these sequences showed Mendelian segregation with one or two alleles per dog. Cluster analysis of the nucleotide and predicted amino acid sequences subdivided the canine DRBB1 alleles into three major allelic groups. The number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites indicative of positive selection. The data generated can serve as a basis for developing a typing assay for the canine DRBB1 gene.
Extensive conservation of upstream C4 promoter sequences: a comparison between C4A and C4B
Authors: Ulgiati D. , Abraham L. J.
Published: December 2008

Basal levels of expression of complement C4 vary in individuals but it is unknown whether this is due to gene copy number, protein clearance rates or differences in expression. To investigate whether differences in the promoter region may influence transcription of the C4 genes, we sequenced the promoter region of the C4B1 gene from the HLA‐A1, B8, C4AQ0, C4B1, DR3 haplotype and compared this with an equivalent C4A3 region. The promoter regions of the C4 genes were highly conserved, indicating that transcriptional differences are unlikely. An unusual feature of the alignment was the higher level of polymorphism in the RP genes past the breakpoint of deletion/duplication in a region where the highest conservation would be expected.
Significance of antibodies to streptococcal M protein in psoriatic arthritis and their association with HLA‐A*0207
Authors: Muto M. , Date Y. , Ichimiya M. , Moriwaki Y. , Mori K. , Kamikawaji N. , Kimura A. , Sasazuki T. , Asagami C.
Published: December 2008

Psoriatic arthritis (PA) is an immune disease associated with HLA‐A2 in the Japanese population. To investigate mechanisms the association between HLA‐A2 and PA, we examined in vivo immune responsiveness to Streptococcus pyogenes. Recombinant M proteins for the subtype specific N‐terminal half (AB region) and conserved C‐terminal half (C region) were produced separately. IgG antibody level against each region was measured by ELISA in 31 PA patients, 88 patients with psoriasis vulgaris, 6 patients with rheumatoid arthritis and 77 healthy controls. We found that IgG antibody levels against the C region were markedly higher in the PA patient group than in the other disease groups or controls. Further, IgG antibody levels were higher in PA patients with spondylitis and polyarticular arthritis than in PA patients with rheumatoid‐like arthritis and arthritis mutilans. In contrast, no significant difference in the IgG antibody levels against the AB region was observed among the tested groups. HLA‐A2 DNA typing showed that HLA‐A*0207 was associated with PA (RR= 17.6; Pcorr<0.01) and the IgG antibody responses to the C region correlated well with the presence of HLA‐A*0207. These results suggest that streptococcal infection may be involved in the pathogenesis of PA by participating in the HLA‐linked immune responsiveness.
Inefficient assembly limits transport and cell surface expression of HLA‐Cw4 molecules in C1R
Authors: Zemmour J.
Published: December 2008

HLA‐C antigens are expressed to the cell surface at roughly 10% the level of HLA‐B or ‐A, and their serological definition remains persistently difficult. To characterize the factors limiting surface expression, the processes of assembly and intracellular transport of HLA‐Cw4 molecules were investigated in the C1R cell line. When appropriate peptides were added to cultured cells or in cell lysates significant amounts of conformed HLA‐C molecules that associate with β2‐microglobulin (β2m) are detected, but are indeed not sufficient to restore expression to the level observed for HLA‐A or ‐B molecules. Furthermore, a precursor/product relationship exists between the free class I heavy chain and the mature conformation of HLA‐Cw4 molecules. Thus, HLA‐C assembly promotes the conversion of HC‐10‐reactive molecules (weakly‐β2m‐associated non‐ligand associated free HC form) into the β2m‐associated class I molecules recognized by W6/32. To further investigate the factors that regulate cell surface expression, intracellular transport of HLA‐Cw4 was studied in pulse chase analysis. In contrast to some HLA‐A and B, maturation of HLA‐Cw4 heavy chains and their export to the medial and trans‐Golgi compartments are quite inefficient. After 4 h of chase period, roughly half of the pulse‐labeled HLA‐Cw4 molecules have transited to the medial‐Golgi and acquired complex oligosaccharides characteristic of mature form. In addition, treatment with γ‐interferon does not appear to improve maturation of HLA‐Cw4 heavy chains, suggesting that increased supply of peptides does not influence intracellular transport. Moreover, only a small fraction in the pool of HLA‐Cw4 molecules was subsequently transported through the trans‐Golgi network, as indicated by their acquisition of sialic acids. Taken together these studies show that HLA‐Cw4 molecules are inefficiently transported through the Golgi apparatus and presumably retained in the endoplasmic reticulum or cis‐Golgi compartment.
HLA haplotypes and class II molecular alleles in Sardinian and Italian patients with pemphigus vulgaris
Authors: Carcassi C. , Cottoni F. , Floris L. , Vacca A. , Mulargia M. , Arras M. , Boero R. , La Nasa G. , Ledda A. , Pizzati A. , Cerimele D. , Contu L.
Published: December 2008

HLA class II antigens and DRB1, DQA1, DQB1 alleles were studied in 16 Italian and in 16 Sardinian patients with pemphigus vulgaris (PV). In the last group the complete HLA A‐DQ haplotypes, including the complotypes, were defined by family studies. As in other populations, two PV susceptibility haplotypes were found: HLA‐DRB 1*0402, DQA1*0301, DQB1*0302 and HLA‐DRB 1*1401, DQA1*0104, DQB 1*0503. The first haplotype was largely prevalent in the Sardinian patients and was a part of the extended haplotype HLA‐A2, Cw4, B35, S31, DR4, DQ8. The strength of the allele associations to PV is in agreement with the view that the main PV susceptibility genes are the DRB 1*0402 and DQB 1*0503 alleles. A genetic resistance to PV seems to be conferred by the HLA‐DR3, DQ2 haplotype in the Sardinian population.
Molecular analysis of HLA‐B in the Malaysian aborigines
Authors: Hirayama K. , Zaidi A. S. M. , Hakim S. Lokman , Kimura A. , Ong K. J. , Kikuchi M. , Nasuruddin H. A. , Kojima S. , Mak J. W.
Published: December 2008

We have examined 56 unrelated individuals from Malaysian aborigines for their DNA polymorphism of the HLA‐B gene by sequence specific oligonucleotide probe (SSO) method. Using the SSO hybridization, we found that one specific DNA allele with a B*1513 like pattern of epitope combination (ECB1513) was dominant among the Melayu Asli (Af=41.9%) and the Senoi (Af=24%). To determine the nucleotide sequences of ECB1513, a DNA fragment spanning from the beginning of exon 1 to the middle of exon 4 of the HLA‐B gene was amplified by polymerase chain reaction (PCR) from two ECB1513 positive individuals, and the PCR products were cloned and sequenced. This sequencing analysis confirmed that ECB1513 was identical to HLA‐B*1513 in exon 1,2, 3, and 4. Amino acid sequence of this major allele, HLA‐B*1513, in the aborigines especially around the peptide binding groove (B and F pockets), was compared with that of African B*5301 that had been suggested to confer resistance to malaria infection in Africa. The amino acid residues composing of the F pocket were completely identical in B*1513 and B*5301. These observations suggest that acommon environmental factor, the malaria infection, might have independently enhanced the selection of functional change in the polymorphic portion of HLA‐B gene in Africa and in South‐East Asia.
HLA‐B27 presents a peptide from a polymorphic region of its own molecule with homology to proteins from arthritogenic bacteria
Authors: Garcia F. , Marina A. , Albar J. P. , de Castro J. A. López
Published: December 2008

A possible mechanism for the pathogenesis of HLA‐B27‐associated spondyloarthropathies is that peptides from arthritogenic bacteria with homology to endogenous self‐peptides presented by HLA‐B27, including those derived from HLA‐B27 itself, could elicit an autoimmune T‐cell response upon infection. We report here that an undecamer corresponding to the polymorphic region of HLA‐B27 spanning residues 169–179 is presented in vivo by the B*2701, B*2704 and B*2706 subtypes, but was not detected in the B*2703‐bound peptide pool. This peptide binds to B*2705 in vitro with sufficient affinity to allow its natural presentation by this subtype, but it binds with low affinity to B*2703. In spite of homology of this peptide to proteins from arthritogenic bacteria, its binding specificity does not correlate with current evidence concerning association of HLA‐B27 subtypes to ankylosing spondylitis, suggesting that presentation of this peptide is not the critical feature that determines linkage of HLA‐B27 to this disease.
HLA‐DRB4 genotyping by PCR‐RFLP: diversity in the associations between HLA–DRB4 and DRB1 alleles
Authors: Naruse T. K. , Ando R. , Nose Y. , Kagiya M. , Ando H. , Kawata H. , Nabeya N. , Isshiki G. , Inoko H.
Published: December 2008

The serologically defined HLA‐DR53 antigen is associated with HLA‐DR4, ‐DR7, and ‐DR9 antigens, and these haplotypes contain two functional genes, DRB1 and DRB4, and two pseudogenes, DRB7 and DRB8. The DRB4 gene encodes the DR53 antigen, and has been officially recognized to contain three allelic variants (DRB4*0101, 0102, and 0103). In this study, we have established the polymerase chain reaction ‐ restriction fragment length polymorphism (PCR‐RFLP) method for DRB4 genotyping and analyzed genetic polymorphism of the DRB4 gene in Japanese population. DRB4*0101, DRB4*0102, and DRB4*0103 could be observed at the frequencies of 0.5%, 1.1% and 32.7%, respectively. The same DRB1 allele does not necessarily share an identical DRB4 allele. Further, a tight linkage disequilibrium was found between DRB4*0I02 and DRB1*0401 in Japanese population, whereas DRB1*0401 was associated with DRB4*0101 or *0103 in Caucasian population. These findings reveal extensive diversity of the HLA‐DRB1 and ‐DRB4 haplotypes and may have important implications for HLA‐disease associations and donor selection in unrelated transplantation.
Lack of carboxyl–terminal tyrosine distinguishes the B2706–bound peptide repertoire from those of B2704 and other HLA–B27 subtypes associated with ankylosing spondylitis
Authors: García F. , Marina A. , de Castro J. A. López
Published: December 2008

B*2704 and B*2706 are two closely related HLA‐B27 subtypes, which differ from the common B*2705 by the Asp>Ser77, Val>Glu152, and Ala>Gly211 amino acid changes. In addition, B*2706 differs from B*2704 by the His>Asp114 and Asp>Tyrl 16 changes. In spite of their similarity B*2704, but not B*2706, was associated to ankylosing spondylitis in a same population. We have carried out pool sequence analyses of the peptides naturally bound to each of these subtypes, and of several individual peptide ligands. B*2704 and B*2706 shared with B*2705, among other features, their selectivity for Arg2 and their allowance for some aliphatic and aromatic C‐terminal residues in their bound peptides. The main features that distinguished both subtypes from B*2705 were: 1) their failure to present peptides with C‐terminal basic residues, and 2) their allowance for both polar and nonpolar residues at peptide position 3. A major difference between B*2704 and B*2706 was that C‐terminal Tyr was prominent among the peptides bound to B*2704, but was not detected among those from B*2706. The use of Tyr as a C‐terminal anchor motif is the only functional feature shared by the disease‐associated B*2705, B*2702, and B*2704 subtypes that is absent in B*2706. This suggests that the ability of HLA‐B27 to present peptides with C‐terminal Tyr might be critical for its association to spondyloarthropathy,
TNF microsatellite a2, b3 and d2 alleles are associated with systemic lupus erythematosus
Authors: Hajeer A. H. , Worthingtan J. , Davies E. J. , Hillarhy M. C. , Poulton K. , Oilier W. E. R.
Published: December 2008

We have investigated TNF microsatellite polymorphisms in SLE and their association with both HLA class II alleles and disease expression. A total of 91 Caucasoid SLE and 109 matched Caucasoid controls were recruited for this study. TNF microsatellites a, b and d were typed using fluorescent based semi‐automated gene scanning. TNF a2, b3 and d2 allele frequencies were significantly increased in the SLE group compared to controls. These alleles were found to be part of an extended HLA‐DRB 1*0301 haplotype and have previously been associated with high TNF‐α production. When the SLE group was analyzed according to presentation of certain clinical features, photosensitivity and Raynaud̂s phenomenon, the frequency of these alleles (TNF a2, b3 and d2) were also significantly increased. No significant increase in the allele frequencies of TNF a2, b3 and d2 was demonstrated in the group of patients with renal involvement. These data suggest that TNF microsatellite alleles are not independent of HLA associations in SLE and may be important in the expression of certain clinical features in SLE.
Canine major histocompatibility complex genes DQA and DQB in Irish setter dogs
Authors: Polvi A. , Garden O. A. , Elwood C. M. , Sørenssn S. H. , Batt R. M. , Mäki M. , Partanen J.
Published: December 2008

Information about genetic variation within the canine major histocompatibility complex (MHC) class II genes is limited. In common with most other vertebrate species the canine MHC, or DLA, includes genes which are homologous to human DR, DQ, and DP. Recently, at least one functional DLA DQ gene‐pair has been characterized, but so far systematic screening efforts have been lacking. In the present study, we sequenced both cDNA and genomic clones derived from DLA DQ genes of Irish setter dogs. This breed was of interest, since it shows a high prevalence of gluten sensitive enteropathy (GSE), which may be a useful animal model for celiac disease (CD) of man. Interestingly, few of the alleles found in Irish setters were identical to those previously detected in other breeds. Three novel DLA DQA and four novel DLA DQB alleles were discovered in 19 unrelated dogs. Strong association between certain HLA DQ alleles and CD of man prompted us to screen the DQ alleles of members of a family of gluten‐sensitive Irish setter dogs. No haplotypes or alleles were shared by all affected dogs, but one frequent haplo‐type in this family was also detected in an unrelated gluten‐sensitive Irish setter; this haplotype was absent in the healthy dogs. This observation warrants further investigation by screening the DQ alleles of a large population of unrelated gluten‐sensitive Irish setters.
Identification of a new HLA‐B*08 variant, HLA‐B*0804
Authors: Eberle M. , Lorentzen D. F. , Iwanaga K. K. , Hennes L. F. , Watkins D. I.
Published: December 2008

The HLA‐B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically‐defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA‐B*08 variant, HLA‐B*0804. found in Caucasian siblings JH and PF serologically typed as HLA‐B51/B59 and HLA‐B59/B60, respectively. Additionally, DNA‐based typing by the polymerase chain reaction using sequence‐specific primers (PCR‐SSP) identified HLA‐B*51 in JH and HLA‐B*4001 in PF. However, PCR‐SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA‐B*08 variant, HLA‐B*0804. The presence of this allele was confirmed by cloning and sequencing. HLA‐B*0804 differed from HLA‐B*0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR‐SSP. This striking difference between both the serologically typed antigen and the PCR‐SSP‐identified allele compared to the sequenced allele supports the use of sequence‐based typing for the analysis of HLA class I locus alleles.
The complete primary structure of Cw*1701 reveals a highly divergent HLA class I molecule
Authors: Herrero M. J. , Vilches C. , de Pablo R. , Puente S. , Kreisler M.
Published: December 2008

Genotyping of the HLA‐C locus by PCR‐SSP has previously shown 100% association of B41 and B42 with a new allelic variant. Partial sequencing studies (exons 2–4) demonstrated that this PCR‐SSP variant corresponded to the new allele Cw*1701. In this study we have characterized the whole coding region of Cw*1701 from a Bubi individual of Equatorial Guinea. Our results partially confirm the previously reported sequence and reveal that Cw*1701 has many new polymorphisms at several exons, including a 18‐bp insertion in exon 5. Cw*1701 is thus a most unusual HLA‐C molecule denning a third allelic lineage of this locus.
HLA‐DRB1 in eight Finnish monozygotic twin pairs concordant for rheumatoid arthritis
Authors: de Vries N. , Tijssen H. , Jarvinen P. , Aho K. , van de Putte L. B. A.
Published: December 2008

This study presents the results of HLA‐DRB1 typing of the eight monozygotic twin pairs with both members affected by rheumatoid arthritis (RA), sampled in the nationwide Finnish twin cohort. The shared epitope, associated with RA in case‐control studies, was present in all eight twin pairs, being significantly more frequent than among RA patients in a recent Dutch case‐control study. Furthermore, 4 out of 8 twin pairs were homozygous for the shared epitope, while in 73 Dutch healthy controls encoding the shared epitope only 13 (18%) were homozygous; this suggests a gene dose effect in RA susceptibility. Combining these results with data from other sources may help to clarify the contribution of HLA alleles in the genetic predisposition to RA.
Nomenclature for factors of the HLA system, update November/December 1996
Authors: Marsh Steven G. E.
Published: December 2008

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

Below are listed the newly assigned sequences, confirmations of previously reported sequences and some sequences which are corrections of those originally reported. The accession number of each sequence is given and these can be used to retrieve the sequence files from either the EMBL, GenBank or DDBJ data libraries. Although accession numbers have been assigned by the data libraries and most sequences are already available, there is still the possibility that an author may not yet have allowed the sequence to be released, in such a case you will have to contact the submitting author directly.
Serologic ambiguity and allelic frequency of the HLA‐B40 family in the Korean population
Authors: Lee K. W. , Kim Y. S.
Published: December 2008

The most frequently identified HLA‐B type in Koreans is HLA‐B40 (13.4%). Due to the lack of mono‐specific alloantisera and cross reactivity of sera used as typing reagents, discrimination between the serologic splits of B40, B60 and B61, has been a problem in tissue typing laboratories. In this study, an efficient PCR‐SSP typing system was established to distinguish B60 and B61 and to assess the difficulty in serologic assignment for these types. The SSP system was also used to elucidate the frequency of B40 alleles (B*4001‐B*4008) encoding B40 molecules in the Korean population. Eighty eight unrelated individuals identified serologically as B40 positive were selected from 358 consecutive volunteers from the unrelated bone marrow registry. Seven sets of PCR that amplify exons 2 and 3 of the HLA‐B gene using 10 sequence specific primers (SSP) were used for discrimination between B60 and B61, and for B40 allelic typing. A clear discrimination of B60 and B61 was possible in all samples including 48 serologically ambiguous samples (B60 – 14/48; B61 – 34/48) and 5 potentially B40 homozygous samples (B60/B61 heterozygotes – 4/5; B60 homozygote ‐ 1/5). Therefore, the use of a focused SSP approach enhances serologic definition of HLA types in routine clinical testing. In allelic typing, all B60 samples (26) appeared to be B*4001, but B61 samples revealed more heterogeneity (B*4002 – 36/58, B*4003 – 4/58, B*4006 – 18/58). In addition, B*4003 seemed to be closely associated with the A24‐Cw3‐DRB1*02 haplotype (3/4). The characterization of allele frequency as well as haplotypic association will be helpful in determination of the optimal size of the volunteer marrow donor pool in the Korean population.
Molecular analysis of HLA‐B35 alleles and their relationship to HLA‐B15 alleles
Authors: Cereb N. , Kim C. , Hughes A. L. , Yang S. Y.
Published: December 2008

The HLA‐B35 serotype is one of the largest allelic groups of HLA class I molecules and includes four isotypes. Of the four, the B35 variant isoform is relatively rare and is the most acidic form. DNA sequencing of die rare isoforms revealed three alleles, B*1522, B*3511, and B*3517. A phylogenetic tree of HLA‐B15‐ and HLA‐B35‐related alleles for the exon 2 and 3 nucleotide sequences showed that exon 2 of B* 1522 clusters with B35 alleles whereas exon 3 clusters with B15 alleles. Branches of the tree suggest that the serodeterminants of B35, B62, B63, and B70 may reside in the αl domain, encoded by exon 2. The B*1520 and B*1522 genes, which type as B62 and B35, respectively, are hybrid molecules alternatively using exon 2 and exon 3 sequences of B*3501 and B*1501. A comparison of intron 2 sequences for B*3501, B*1501 and B*1522 suggests that the recombination site may have been in the region at the 3′ end of intron 2. Despite being flanked by two highly polymorphic exons (exons 2 and 3), intron 2 is relatively well conserved in the B‐locus, and it is characterized by seven to eight tandem repeats of the CGGGG pentanucleotide. A high degree of sequence homology and repetitive sequences are essential for a significant frequency of recombination. In this report, we reveal more about the complex evolutionary history of the HLA‐B alleles
Monoclonal antibodies specific for porcine monocytes/macrophages: macrophage heterogeneity in the pig evidenced by the expression of surface antigens
Authors: Bullido R. , Moral M. Gómezdel , Alonso F. , Ezquerra A. , Zapata A. , Sánchez C. , Ortuño E. , Alvarez B. , Dominguez J.
Published: December 2008

Macrophages are widely distributed in most tissues of the body, where they play important roles in host defense and repair of tissue damage. In this report we describe the production and characterization of a panel of six monoclonal: antibodies (mAb) against porcine macrophages and their use for phenotyping tissue macrophages. All mAbs were produced by immunizing mice with porcine alveolar macrophages. Three of them (2A10/11, 3B11/11 and3F7/ll) react mainly with macrophages and, at a lower extent, blood monocytes, whereas the others (1E12/11, 2C12/10 and 4E9/11) also recognize granulocytes. Antigens recognized by these antibodies could be characterized by Western blot and/or immunoprecipitation, with the exception of that one recognized by 2C12/10. By their behavior in SDS‐PAGE under reducing and nonreducing conditions, all seem to be single polypeptides, whose apparent molecular weight under reducing conditions are: 1E12/11 and 3B11/11 larger than 204 kDa; 2A10/11, 150 kDa; 4E9/I1, 125–170 kDa; and 3F7/11, 135 kDa. Immunohistochemical analyses of both lymphoid and non‐lymphoid organs using these mAbs reveal important antigenic heterogeneity among tissue macrophages. These mAbs are, therefore, useful tools for the study of porcine macrophage maturation and differentiation and for determining their heterogeneity both in normal and pathological conditions.
HLA‐DQA1 genotyping by bi‐directional sequencing of PCR‐amplified DNA spanning exon 2
Authors: Craig M. A. , Elliott J. F.
Published: December 2008

We describe a simple, reliable technique for HLA‐DQA1 genotyping based on direct DNA sequencing of PCR amplified fragments from genomic DNA. The alleles of DQA1 can be divided into two subsets, with one subset demonstrating a 3 base deletion in exon 2 relative to the other. Typing heterozygous individuals who possess one allele from each sub‐group can be difficult using a direct sequencing approach, since the two overlapping DNA sequences move out of phase by 3 nucleotides once the point of deletion is reached. The complete sequence is obtained by performing two separate sequencing reactions with fluorescent dye primers, coming from either end of the template. This allows all heterozygous positions in exon 2 to be unambiguously assigned.
Biochemical analysis of plasma‐soluble invariant chains and their complex formation with soluble HLA‐DR
Authors: Rebmann V. , Dornmair K. , Grosse‐Wilde H.
Published: December 2008

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA‐DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units±1.04 SD (range 0.17‐4.31). Approximately 10% of the samples had increased amounts of sCD74 (3.0 relative units). Complexes of sCD74 and sHLA‐DR were detected in all samples and their quantities were positively correlated (r=0.83, p0.001) with the sCD74 concentrations. SDS‐PAGE analysis of plasma samples with high sCD74 concentrations (3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA‐DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA‐DR molecules.
The HLA system in the population of Mongolia
Authors: Chimge N.‐O. , Kashiwase K. , Ayush D. , Tokunaga K. , Saji H. , Akaza T. , Batsuuri J. , Juji T.
Published: December 2008

The frequencies of HLA‐A, B, C and DRB1 alleles and haplotype frequencies for HLA‐A, B, C and A, B, DR loci were studied in 201 healthy unrelated Khalkha‐Mongolians. The most common class I antigens were A24 (25.8%), A2 (23.4%), B61 (12.6%), B51 (8.5%), Cw10 (14.2%), Cw9 (14.1%), Cw7 (13.3%), Cwl (11.8%) and Cw6 (10.2%). A total of 35 DRB1 alleles were identified in this group of samples. The most frequent DRB1 allele was DRB1*0301 (11.1%), followed by DRB 1*0701 (9.7%) and DRBl*H01 (8.5%). One novel DRB1 allele (14MV) and three rare types, DRB1*1111, DRB 1*1504 and DRB 1*1412, recently described in Jews, the Dai minority of China and Japanese, respectively, were identified in Mongolians. The phylo‐genetic tree constructed by UPG method put Mongolians in the Northeast Asian cluster. A comparison of three locus haplotype distributions with world populations, revealed that Mongolians share several characteristics in common with other Mongoloids as well as with Caucasoids and Amerindians. The most common A, B and DR haplotype in Mongolians, A33‐B58‐DR3, was shared with Thai, Thai Chinese and Singapore Chinese. These data support that unique genetic background of Mongolians played a major role in ethnic formation and differentiation of Mongoloid populations.
MICA gene and ankylosing spondylitis: linkage analysis via a transmembrane‐encoded triplet repeat polymorphism
Authors: Goto K. , Ota M. , Ohno S. , Mizuki N. , Ando H. , Katsuyama Y. , Maksymowych W. P. , Kimura M. , Bahram S. , Inoko H.
Published: December 2008

In order to address the possibility that the MICA gene located 47 kb upstream from HLA‐B is involved in the pathogenesis of ankylosing spondylitis (AS), we have investigated microsatellite polymorphism in the transmembrane region of MICA in Caucasian patients with AS. The microsatellite allele consisting of 4 repetitions of GCT/AGC was present at significantly higher frequency in the patient group (Pc<0.0000001) than in the ethnically matched control group. However, the frequency of the (GCT/AGC)4 allele was significantly low in the B27‐positive patients than in the B27‐positive healthy controls (Pc=0.0145). These observations suggest that B27 itself remains the primary genetic marker for AS, although the significantly dissimilar pheno‐type frequency of the (GCT/AGC)4 allele in B27‐positive patients and healthy individuals may reflect the existence of other genetic factor(s) in the HLA‐B27 haplotype involved in the development of AS.
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 DRB1*1501) and some haplotypes of DRB1‐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.
Genomic organization and allelic polymorphism of the human killer cell inhibitory receptor gene KIR 103
Authors: Selvakumar A. , Staffens U. , Palanisamy N. , Chaganti R. S. K. , Dupont B.
Published: December 2008

Killer cell inhibitory receptors (KIR) belong to the immunoglobulin super‐family of molecules and are expressed on natural killer (NK) cells. The KIRs of the p58/p50 family have two immunoglobulin domains and are ligands for HLA‐Cw antigens, whereas the p70/p70Δ family has three immunoglobulin domains and comprises ligands for HLA‐B antigens and possibly some HLA‐A antigens. Members of a third KIR family, KIR103, have two immunoglobulin domains but have highest nucleotide sequence homology to the p70 family. The ligands for KIR 103 on target cells are currently unknown. We here report the complete genomic organization of KIR103. It spans about 12 kb of DNA and consists of eight exons of which exon 1 and exon 2 encode the leader sequence. Exon 3 encodes the first immunoglobulin domain (γ1), and exon 4 encodes the main part of the second immunoglobulin domain (γ3), which also contains sequences contributed by exon 5 and exon 6. Exon 6 encodes the transmembrane domain, whereas exons 7 and 8 encode most of the cytoplasmic domain. KIR 103 is polymorphic, and two alleles, 103AS and 103LR, are defined in this study. Additional full‐length cDNA clones for KIR 103 have been isolated and are shown to be formed by alternative mRNA splicing with exon skipping. Some of these truncated KIR 103 cDNA could encode shorter transmembrane molecules, whereas others lack the transmembrane domain and are candidate genes for secreted KIR products. KIR 103 is localized to the KIR genetic region on chromosome 19q13.4.
Characterization of the porcine homologue to human platelet glycoprotein IIb‐IIIa (CD41/CD61) by a monoclonal antibody
Authors: de la Lastra J. M. Pérez , Moreno A. , Pérez J. , Llanes D.
Published: December 2008

Human gpIIb‐IIIa or CD41/CD61 is a Ca2+‐complex dependent heterodimer, abundant on platelets, that plays a key role in hemostasis. This report describes a murine monoclonal antibody, JM2E5, able to recognize and immunoprecipitate the gpIIb/IIIa surface glycoprotein from porcine platelets. Immunoprecipitation analysis showed an antigen molecular weight of 115 and 85 kDa under nonreducing conditions, and of 110, 100 and 25 kDa under reducing conditions. Immunohistochemistry analyses of frozen sections from several porcine lymphoid organs gave specific staining on platelets. EDTA treated platelets were studied by flow cytometry indicating that the epitope recognized was Ca2+‐complex independent. Western‐blotting experiments with porcine platelet extracts gave an antigen molecular weight of 85 kDa under nonreducing conditions, thus localizing the epitope recognized by JM2E5 on the complex light chain gpllla or CD61. JM2E5 was also cross‐reacting with human, bovine and horse platelets, as shown by flow cytometry. This mAb would allow further studies on this important adhesion molecule on horses, ruminants and pigs, and it should be especially useful as a general anti‐porcine platelet reagent.
HLA class IIDRB, DQA1 and DQB1 polymorphisms in the Polish population from Wielkopolska
Authors: Jungerman M. , Sanchez‐Mazas A. , Fichna P. , Lvanova R. , Charron D. , Hors J. , Djoulah S.
Published: December 2008

HLA DRB1, DQA1 and DQB1 alleles were determined by DNA PCR‐SSO typing in a sample of 99 individuals originating from Wielkopolska (midwestern Poland). A high number of alleles (38 DRB1, 8 DQA1 and 14 DQB1) was detected at each locus, many of them presenting notable frequencies in this population. The three HLA loci are thus characterized by very high heterozygosity levels (93% for DRB1, 85% for DQA1, and 88% for DQB1), which confirms the results found for other European populations. A total of 6 DRB1‐DQA1‐DQB1 haplotypes are detected with an estimated frequency higher than 5%, namely, DRB1*1501‐DQA1*0102‐DQB1*0602, DRB1*0701‐DQA1*0201‐DQB1*0201, DRB1*0101‐DQA1*0101‐DQB1*0501, DRB1*1101‐DQA1*0501‐DQB1*0301, DRB1*03011‐DQA1*0501‐DQB1*0201, and DRB1*1301‐DQA1*0103‐DQB1*0603. A genetic distance analysis between the Polish and other world populations tested for HLA class II indicates that the Wielkopolska community is close to geographically close, rather than linguistically related populations from Europe. More generally, a good agreement between genetics and geography is found for DRB1 and DQB1 polymorphisms in Europe, suggesting that these two loci are highly informative for assessing historical relationships among humans.
Brief communication Increased diversity within the HLA‐A*66 group: implications for matching in unrelated bone marrow transplantation
Authors: Binder T. , Wehling J. , Huhn D. , Blasczyk R.
Published: December 2008

We have identified a new A*66 allele (A*6603) in three related individuals, an Arabic patient suffering from acute myeloid leukemia and two of her relatives. The A*66 alleles differ in three amino acid residues at positions 70, 90 and 163. The closer relationship between A*6602 and A*6603, which only differ at amino acid 70, replacing GLN with HIS, suggests that the alloreactive potential in this mismatch combination is lower than in all other mismatched A*66 donor‐recipient combinations, which exhibit two (A*6601 versus A*6602) and three (A*6601 versus A*6603) differences at the pivotal positions, respectively. This emphasizes the potential role of the A*66 subtypes in bone marrow transplantation with alternative donors. For that reason, allelic subtyping should be considered in donor‐recipient matching to identify the kind of disparity.
The absence of DR51 in a DRB5‐positive individual DR2ES is caused by a null allele (DRB5*0108N)
Authors: Voorter C. E. M. , Roeffaers H. E. T. , du Toit E. D. , van den Berg‐Loonen E. M.
Published: December 2008

DR51, a protein encoded by the DRB5 gene, was shown to be present in almost all DR2‐positive haplotypes. Exceptions were reported, some DR2‐negative samples were shown to be DR51 positive and in a number of DR2‐positive samples no DR51 antigen could be demonstrated. In some of them lack of the DRB5 gene was the cause of the absence of DR51 but in others the DRB5 gene was present without resulting in a detectable gene product. Many of these variants were studied in detail in previous international workshops. One of them was DR2ES from our laboratory. She is a DR15‐positive DR51‐negative individual of oriental origin with a clearly demonstrable DRB5*01 allele when typed by molecular techniques. To unravel the molecular mechanism responsible for the defect in expression, cDNA and DNA encoding the defective DRB5 allele were analyzed. Nucleotide sequence analysis of exon 2 showed no differences from the sequence of DRB5*0102. However, when exon 3 was examined a difference in length was noticed due to a deletion of 19 nucleotides between codon 161 and 168. The deletion caused a frameshift and a premature stopcodon resulting in a null allele. The same allele could be demonstrated in 6 other unrelated individuals of oriental origin as well as in 5 individuals from South Africa. The absence of the DR51 protein was explained by the presence of an alteration in the DRB5 allele resulting in a null allele. The allele has been officially named DRB5*0108N. This is the first description of a null allele of the DRB5 gene.
Analysis of HLA‐A and ‐B serologic typing of bone marrow registry donors using polymerasee chain reaction with sequence‐specific oligonucleotide probes and DNA sequencing
Authors: Sintasath D.M. , Bel M. , Steiner N. , Ng J. , Alosco S. , Hegland J.D. , Hurley C.K.
Published: December 2008

Unrelated volunteer donors (69) recruited by the National Marrow Donor Program were HLA typed by DNA‐based methods for both the HLA‐A and ‐B loci. Each donor had been previously typed by serology by at least two independent laboratories. Of the 69 samples, all serologic laboratories were in concordance for HLA‐A in 62 typed samples and for HLA‐B in 48 typed samples. Of the serologically concordant samples, 5 samples typed for HLA‐A and 7 samples typed for HLA‐B received DNA and serology types differing in their level of resolution. One sample typed for HLA‐A and 3 samples typed for HLA‐B by DNA methods gave different results from their serologic assignments. Of the samples exhibiting disparities among the different serologic typing laboratories, the DNA‐defined types of 7 samples typed for HLA‐A and 18 samples typed for HLA‐B were consistent with at least one of the serologic assignments. The DNA types for the remaining 3 HLA‐B typed samples did not agree with the serologic assignments and their alleles were subsequently sequenced. One of these sequences was a previously undefined allele, B*1537. Sharing of polymorphic sequences among HLA allelic products creates difficulties for consistent serologic assignments of some types complicating the process of identifying potential donors from bone marrow registries. Thus, the use of DNA‐based typing techniques for characterization of donor class I types should allow a more consistent definition of types and should speed the donor selection process.
Immune phenotype of canine hematopoietic progenitor cells
Authors: Neuner E. , Schumm M. , Schneider E.‐M. , Guenther W. , EIIwart J. , Kremmer E. , Vogl C. , Buettner M. , Thierfelder S. , Kolb H. ‐J.
Published: December 2008

The immune phenotype of canine hematopoietic progenitor cells was studied by immunoseparation and culturing of separated cells. Two separation methods were used, the magnetic cell sorting system (MACS) and the fluorescence activated cell sorter (FACS). For separation rat anti dog antibodies Dog 13 and Dog 14 directed against Thy‐1, and Dog 26 as well as cross‐reactive mouse anti human antibodies IOT2a and 7.2 directed against MHC class II were used. Separated cell populations were cultured in semisolid agar before and after long‐term culture on a pre‐established irradiated stromal cell layer. After 28 days, adherent and nonadherent cells were harvested from long‐term culture. The MACS system allowed separation of cells into positive and negative fractions. Long‐term culture‐initiating cells (LTC‐IC) were found in both the Thy‐1+ and the Thy‐1‐ fraction, but the content of LTC‐IC was higher in the Thy‐1+ fraction. The MACS system did not allow separation of progenitor cells according to the expression of MHC class II antigen detected by Dog 26 and the cross‐reactive antibodies IOT2a and 7.2. In contrast to the MACS system the FACS allowed separation of negative, low‐positive and high‐positive cell populations. Low‐positive fractions were well defined for Thy‐1 and less well defined for MHC class II. CFU before and after long‐term culture were exclusively observed in the low positive fraction (Thy‐1lo+). Using MHC class II antibody Dog 26 LTC‐IC were found mainly in the negative and low positive fraction, and CFU were observed mainly in the low and high positive fraction. In conclusion pluripotent canine hematopoietic precursor cells are low positive for Thy‐1 and for MHC class II. In this respect canine hematopoietic progenitor cells are comparable to those of mouse and man.