bluuebunny/crossref_metadata_2025 huggingface dataset:
A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes P1 A1 from Pl A2
Authors: Weiss E. J. , Goldschmidt‐Clermont P. J. , Grigoryev D. , Jin Y. , Kickler T. S. , Bray P. F.
Published: December 2008

Abstract: Neonatal alloimmune thrombocytopenia (NATP) and post‐transfusion purpura (PTP) are acquired bleeding disorders caused by alloimmune thrombocytopenia. In most cases, the thrombocytopenia is due to an alloantibody directed against the platelet glycoprotein IIb‐IIIa (GPIIb‐IIIa) complex. During the course of routine studies on the role of GPIIb‐IIIa in inherited and acquired bleeding and thrombotic disorders, we unexpectedly identified an individual whose platelets reacted by non‐reduced Western blot analysis with anti‐GPIIIa polyclonal antisera, but did not react with a commercially available monoclonal antibody (SZ21) specific for GPIIIa. We screened all 14 GPIIIa exons for possible nucleotide changes which might alter amino acids and found variations in only exons 3 and 10. Nucleotide sequencing revealed that only the exon 3 alteration changed the predicted amino acid sequence. This variation was caused by homozygosity for the uncommon PlA2 allele of the GPIIIa gene. Platelets from two additional unrelated normal individuals known to be homozygous for PlA2 also lacked reactivity with SZ21 by Western blot. Using flow cytometry with intact platelets, we observed a markedly reduced binding of SZ21 to platelets with the PlA2 genotype. Scatchard analyses indicated that SZ21 bound to P1A1/A1 platelets with a Kd of 8.26times 10‐10 M, and to P1A2/A2 platelets with a Kd of 5.58times10‐9 M. Thus, we have characterized a readily available monoclonal antibody able to distinguish between the two P1A alleles of the GPIIIa gene. Because incompatibility for this platelet polymorphism is the most common cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura, and because platelet immunophenotyping reagents lack specificity and are not easily available, this monoclonal antibody could facilitate the management of patients with these disorders.
Evaluation of the mixed lymphocyte culture (MLC) assay as a method for selecting unrelated donors for marrow transplantation
Authors: Mickelson E. M. , Longton G. , Anasetti C. , Petersdorf E. , Martin P. , Guthrie L. A. , Hansen J. A.
Published: December 2008

Abstract: The utility of the MLC assay as a test of HLA‐D region matching and predictor of graft‐versus‐host disease (GvHD) was evaluated in 435 patients receiving marrow grafts from unrelated donors. Donors and recipients were phenotyped for HLA‐A, B and DR antigens by serology, tested in MLC, and retrospectively genotyped for DRB1, B3, B4, B5, DQB1 and DPB1 alleles by PCR/SSOP. Of the 244 HLA‐A, B, DR‐identical donor‐recipient pairs with evaluable MLC and DRB1 typing results available, 208 were matched for HLA‐A, B and DRB1, while 36 were matched for HLA‐A and B and mismatched for a DRB1 allele. Donor anti‐recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from 7.2 to 100%, with a median of 4.0%. A comparison of reactivity in MLC between pairs matched versus mismatched for DRB1 alleles showed a significant overlap in the distribution of RRs. Using optimally‐defined RR cutoffs of 4 and 16%, no correlation between MLC results and risk of developing clinically significant grades III‐IV GvHD (p=0.6 and 0.5, respectively) was found when the contribution of DRB1 mismatch was accounted for. Matching for DRB1 alleles, in contrast, was a better predictor of clinically significant GvHD, with DRB1‐matched transplant recipients less likely to develop grades III‐IV GvHD than DRB1‐mismatched recipients (p=0.14). Among the 208 patients and donors matched for DRB1 alleles, the MLC, although reactive (RR > 4.0%) in 45% of cases, did not predict GvHD. Overall, these results underscore the limitations in using the MLC to predict DRB1 matching or risk of clinically significant GvHD among patients receiving unrelated marrow grafts. The availability of DRB1 allele matching by sequence‐specific oligonucleotide probes (SSOP) or by direct sequencing provides a method for donor matching that is rapid, precise and superior to the MLC for predicting clinically relevant outcome.
Different contribution of HLA‐DR and ‐DQ genes in susceptibility and resistance to Insulin‐dependent diabetes mellitus (IDDM)
Authors: Yasunaga S. , Kimura A. , Hamaguchi K. , Rønningen K. S. , Sasazuki T.
Published: December 2008

Abstract: Previous studies have indicated that certain alleles of HLA‐DR and ‐DQ genes were strongly associated with susceptibility and resistance to insulin‐dependent diabetes mellitus (IDDM), and the role of DQ molecule in IDDM has been suggested. To further clarify the association of DQ alleles with IDDM, we determined the nucleotide sequences of full‐length cDNA from 13 DQA1 alleles and 14 DQB1 alleles. The sequencing analysis revealed sequence polymorphisms outside the hypervariable region of DQ genes. We then analyzed the DQA1 and DQB1 polymorphisms along with that of DRB genes in 86 B‐lymphoblastoid cell lines (B‐LCLs) from various ethnic groups and in healthy unrelated Japanese and Norwegian individuals. The allelic and haplotypic distributions in each population revealed the characteristic haplotypic formation in the HLA class II region. HLA genes in 139 Japanese and 100 Norwegian IDDM patients were analyzed. DQB1*0301 was negatively associated with IDDM in both ethnic groups, irrespective of associated DRB1 and DQA1 alleles. In DQB1*0302 positive populations, which represented a positive association with IDDM in both ethnic groups, DRB1*0401, *0404, *0802 haplotypes increased in the patients, whereas DRB1*0406 haplotype decreased. Considering about the hierarchy in DRB1 alleles with IDDM susceptibility (DRB1*0401>*0404>*0403 in Norwegian and DRB1*0802>*0403>*0406 in Japanese), the genetic predisposition to IDDM is suggested to be defined by the combination of DR‐associated susceptibility and DQ‐associated susceptibility and by the DQ‐associated resistance which is a dominant genetic trait.
Novel alleles HLA‐B*7802 and B*51022: evidence for convergency in the HLA‐B5 family
Authors: Prilliman K. , Steiner N. , Ellexson M. , Stewart D. , Lau M. , Terasaki P. , Hurley C. , Hildebrand W.
Published: December 2008

Abstract: We have characterized two novel HLA‐B alleles, B*7802 and B*51022. The Caucasian‐derived variant B*7802 most resembles the African‐derived B*7801, from which B*7802 differs by two nucleotides. Only one of these modifications, however, is translated: a tyrosine for aspartate substitution occurs at residue 74 in B*7802, while the second nucleotide difference reflects a proximal synonymous substitution in codon 23. A second variant, B*51022, differs synonymously only at codon 23 from B*51021. Comparative analysis of the B5 CREG demonstrates that other pairs of B5 alleles differ synonymously only at codon 23 or synonymously at codon 23 and non‐synonymously at a second more distal location. Contrary to the genesis of like pairs of B5 alleles via introduction of coordinate yet distant mutagenic events onto a single B5 progenitor, we postulate that synonymously different B5 progenitor molecules, B5ATT and B5ATC, are evolving in convergence to generate homologous B5 allele pairs differing silently at codon 23. Our finding that B*7802 is a single amino acid away from complete convergence with B* 7801 and that B*51022 and B*51021 are in complete convergence is exemplary of such evolution.
Molecular and serological characterization of HLA‐B71 in association with different class I haplotypes or in different ethnic groups
Authors: Rodriguez S. G. , Bei M. , Inamdar A. , Stewart D. , Johnson A. H. , Hurley C. K.
Published: December 2008

Abstract: The HLA‐B70 antigen is among the most common antigens present in African Americans; however, monospecific serologic reagents defining B70 and its subtypes, B71 and B72, are rare. We have recently reported the molecular characterization of a B71 allele (B*1510) from an African American individual carrying the haplotype HLA‐A30, Cw3, B71(w6). In order to better define the degree of polymorphism of molecules carrying the B71 serological specificity in the human population, we have used serology, cDNA sequencing, and PCR/SSOP typing to characterize B71 alleles from additional individuals from different ethnic populations and carrying different class I haplotypes. All carried either B*1510 or B*1518 alleles. Other HLA‐B alleles isolated from these individuals (B*5001, B*4901, B*3501, B*3701) were identical to previously reported sequences except for a novel B41 allele (B*4102) identified in one Hispanic individual. This allele has concurrently been identified by Rufer and colleagues in Caucasian individuals. The B*4102 allele differs from B*4101 at codons 95 (Leu/Trp) and 97 (Ser/Arg). In addition, the B*4102 allele differs from B*4101 by two silent substitutions at codons 94 (ACC/ACT) and 99 (TAC/TAT). Since the polymorphic sequence present in B*4102 is also present in other HLA‐B alleles (e.g., B*2707, B*4002, B*0702), it may represent a gene conversion cassette. The allelic diversity at the class I loci and the scarcity of monospecific alloantisera support the importance of the application of molecular based methods to identify HLA class I alleles in matching unrelated donor/recipient pairs for bone marrow transplantation.
HLA‐DRB1, DQA1, DQB1 DNA polymorphism in the Bulgarian population
Authors: Ivanova R. , Naoumova E. , Lepage V. , Djoulah S. , Yordanov Y. , Loste M. N. , Charron D.
Published: December 2008

We describe for the first time the use of PCR based techniques to analyze the MHC class II polymorphism of the Bulgarian population. The present study provides the HLA‐DRB, DQB1 allele frequencies in 116 Bulgarian individuals and DQA1 alleles frequencies in 100 subjects. DNA from these individuals was typed for DRB and DQB1 typed by the PCR‐ Allele Specific Amplification (PCR‐ASA) method and DQA1 by PCR followed by hybridization using Sequence Specific Oligonucleotides (PCR‐SSO). Allele and haplo‐type frequencies and linkage disequilibria are computed by the standard methods used for the XIth International Histocompatibility Workshop. The highest frequencies are 0.159, 0.109 and 0.085 for DRB1*1101, DRB1*1601 and DRB1*1301 respectively. Among the eight DQA1 alleles detected, DQA1*0501 (0.344) is found to be much more frequent than the two most frequent alleles DQA1*0102 (0.225) and DQA1*0101 (0.151). Twelve DQB1 alleles are found and three of them, DQB1*0301 (0.280), DQB1*0502 (0.153) and DQB1*0201 (0.133) showed the highest frequencies. The haplo‐type DRB1*1101‐DQA1*0501‐DQB1*0301 (0.079) predominate clearly, followed by DRB1*1601‐DQA1*0102‐DDQB1*0502 (0.055) and DRB1*0101‐DQA1*0101‐DQB1*0501. These results indicate that the Bulgarian population is characterized by features representative of the European anthropological type with a substantial contribution from the Southern Belt of Europe.
Novel HLA‐B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types
Authors: Hurley C. K. , Steiner N. , Hoyer R. J. , Menchaca E. , Mitton W. , Simonis T. , Hartzman R. J. , Johnson A. H. , Ng J.
Published: December 2008

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA‐B alleles by 2–3 nucleotide substitutions resulting in 1–2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA‐B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA‐B molecule. The newly defined HLA‐B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B “blank” or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA‐B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.
Rapid allelic diversification and intensified selection at antigen recognition sites of the Mhc class II DPB1 locus during hominoid evolution
Authors: Gyllensten U. , Bergström T. , Josefsson A. , Sundvall M. , Erlich H. A.
Published: December 2008

The evolution of polymorphism at the Mhc class II DPB1 locus was studied by comparison of chimpanzee (Pan troglodytes), pygmy chimpanzee (Pan paniscus), gorilla (Gorilla gorilla) and human DPB1 alleles. Extensive polymorphism was found in all hominoids. The clustering of sequences in the phylogenetic tree is consistent with rapid generation of the DPB1 polymorphism. Analysis of the substitution pattern for human alleles shows an excess of nonsynonymous changes to synonymous changes at antigen recognition sites, indicating that the amino acid polymorphism at these sites is being maintained by selection. By contrast, no excess of nonsynonymous changes was found at the antigen recognition sites of nonhuman hominoid species. Thus, it appears that diversifying selection on the DPB1 polymorphism has intensified in the lineage leading to humans. No evidence was found for the existence of ancient allelic lineages predating the divergence of the hominoid species. The number of synonymous differences among DPB1 alleles is lower than among DQB1 and DRB1 alleles, indicative of a more recent origin for the DPB1 polymorphism and consistent with the more rapid evolution suggested by the phylogenetic tree.
MHC class II alleles associated with clinical and immunological manifestations of HIV‐1 infection among children in Catalonia, Spain
Authors: Just J. J. , Casabona J. , Bertrán J. , Montané C. , Fortuny C. , Rodrigo C. , Mur A. , Bosque M. , Jovane L. , King M. C.
Published: December 2008

Children with perinatally‐acquired HIV‐1 infection were studied to determine if major histocompatibility complex (MHC) genes are involved in progression to pediatric AIDS. Molecular genetic techniques were used to genotype loci in the class II region (DRB1, DQA1, DQB1, DPA1, DPB1, LMP2 and LMP7). HIV‐infected children were classified by clinical manifestations and degree of immunosuppression using age‐specific CD4 T‐lymphocyte counts at enrollment. Alleles at the DPB1 and DQB1 loci showed independent and opposite associations; DPB1*0301 showed a trend toward protection while DQB1*0201 appeared to be a risk factor for developing severe immunosuppression and severe clinical outcomes. Presence of DQB1*0201 conferred a greater than 10‐fold increased odds of having severe clinical manifestations and a 2.8‐fold increased odds of severe immunosuppression. Presence of DPB1*0301 was associated with a greater than 8‐fold decreased odds of severe immunosuppression and severe clinical manifestations. These results support host genetic influences on HIV‐1 outcomes in children.
Distribution of HLA‐DQA1 and ‐DQB1 alleles and DQA1‐DQB1 genotypes among Senegalese patients with insulin‐dependent diabetes mellitus
Authors: Cissé A. , Chauffert M. , Chevenne D. , Parfait B. , Julier C. , Assouline Z. , Michel S. , Trivin F.
Published: December 2008

Transracial analysis is one method for distinguishing primary associations between insulin‐dependent diabetes mellitus (IDDM) and HLA II alleles from those related to linkage disequilibrium. Black people have different DR‐DQ relationships from other races and are a useful group to investigate HLA‐D regions associated with IDDM. In this study, we compared the frequencies of HLA‐DQA1 and DQB1 alleles in Senegalese IDDM and control subjects. DQA1*0301 was positively associated with insulin‐dependent diabetes mellitus (p<10‐9, OR 5.21), as were DQB1*0201 and *0302 (p<10‐7 OR=3.55, p<10‐3 OR=3.20, respectively). The positive associations with DQA1*0301, DQB1*0201 and DQB1*0302 are consistent with all racial groups investigated. However, taken together, the data in Senegalese population show that susceptibility and resistance to IDDM are associated both with particular haplotypes and DQA1‐DQB1 heterodimers.
T‐cell receptor usage in alloreactivity against HLA‐B*2703 reveals significant conservation of the antigenic structure of B*2705
Authors: Barber D. F. , Obeso D. , García‐Hoyo R. , Villadangos J. A. , de Castro J. A. López
Published: December 2008

B*2703 is an exceptional HLA‐B27 molecule in that it differs from the most common B*2705 subtype by a unique amino acid change (His59) altering N‐terminal peptide anchorage. To assess how this unusual feature affects the antigenic structure of HLA‐B27, TCR usage by alloreactive CTL raised against B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 but not or at a lower level B*2705. Limited heterogeneity of these CTL was revealed by: 1) identity of TCR in two pairs of such CTL clones, 2) identity of β chains, paired to distinct α chains, in two clonotypes, and 3) almost identical fine specificity of these two clonotypes with site‐specific HLA‐B27 mutants. These results indicate that B*2703 “private” epitopes are rare. TCR usage among anti‐B*2703 CTL was analogous as in anti‐B*2705 responses in the predominant and donor‐independent usage of Vβ segments from homology subgroup 4, more moderate and donor‐dependent Vα skewing, N+Dβ diversity limited by motifs shared among clonotypes, and restricted Jα heterogeneity. Homology of N+Dβ motifs and Jα segments of anti‐B*2703 with anti‐B*2705 TCR suggested significant sharing of peptide‐associated epitopes between both subtypes. The results indicate that allospecific TCR are recruited by B*2703 following similar rules as in the anti‐B*2705 response, and suggest that the B*2703 change keeps unaltered much of the antigenic structure of the molecule relative to B*2705. Therefore, most of the peptides bound to B*2703 should be the same and keep a similar conformation as in B*2705.
Localization of the recombination points in a family with two DR/DP recombinations
Authors: Thomsen M. , Cullen M. , Carrington M. , Foissac A. , Abbal M. , de Préval C. , Crouau‐Roy B. , Cambon‐Thomsen A.
Published: December 2008

In a family with a maternal DR/GLO recombination, cellular DP typing showed it to be located between DR and DP. RFLP studies done during the 9th international histocompatibility workshop gave anomalous segregation patterns of DPA and DPB bands that could be interpreted as being due to a second, paternal DR/DP recombination. This assumption was confirmed later by PCR‐SSO typing. A more precise mapping has been done by new markers showing the maternal recombination to be within the TAP2 locus and the paternal recombination to be between DQB1 and DQB3. This supports earlier suggestions of a hot spot of recombination in the TAP region. The recombinations involve parental haplotypes that presently show DR/DP linkage disequilibrium in the French population and it is proposed that DR/DP recombinations occur randomly while B/DR recombinations preferentially occur on haplotypes without strong linkage disequilibrium. Existing DR/DP linkage disequilibria in a given population will thus be broken down with time. The mixed lymphocyte culture response towards an isolated DP difference was tested in this and another DR/DP recombinant family. It showed that an alloresponse towards DP may be highly variable and this suggests that it might be important to define the rules for the strength of this reaction and the possible implications for allotransplantation.
Analysis on allelic variation of the HLA‐DMB gene in Japanese by PCR‐RFLP as well as direct DNA sequencing and identification of a new DMB allele, DMB*0105
Authors: Naruse T. K. , Kawata H. , Ishihara M. , Ando A. , Kagiya M. , Nose Y. , Isshiki G. , Inoko H.
Published: December 2008

In the HLA‐D region, one of the class II genes, DMA and DMB have been identified between the DQ and DP genes, and four allelic polymorphisms in each of the DMA (DMA*0101–0104) and DMB (DMB*0101–0104) genes have been so far recognized. Several recent studies suggested that the DM molecule is required for class II antigen presentation pathway especially by promoting the binding of antigenic peptides to the classical HLA class II molecule. In this study, we have analyzed genetic polymorphism and allelic variation of the DMB gene in a Japanese population by the direct DNA sequencing technique and also by the polymerase chain reaction — restriction fragment length polymorphism (PCR‐RFLP) method, and could recognize DMB*0101 (49.3%), DMB*0102 (23.2%), DMB*0103 (23.2%), and DMB*0104 (0.4%). Further, a new DMB allele, DMB*0105 characterized by the presence of Val and Iie at two polymorphic sites, codons 144 and 179, respectively was identified. Strong linkage disequilibria were found between DMB*0101 and DRB1*0101, DPB1*0402 and DRB1*1502, and also between DMB*0103 and DRB1*1501 and DQB1*0602. HLA‐DMB genotyping using the PCR‐RFLP method established here will provide accurate evaluation of the effects of sequence allelism in the DMB gene on the HLA class II disease associations.
Allele assignment for HLA‐A, ‐B, and ‐C genes to the Tenth International Histocompatibility Workshop cell lines
Authors: Prasad V. K. , Yang S. Y.
Published: December 2008

Development of DNA typing for Class I HLA alleles has lagged behind that of class II for a variety of technical reasons. Following the recognition of locus specific sequences in the first and the third intron, and acquiring the ability to amplify genomic DNA by intron‐based PCR primer, we have devised DNA typing of class I alleles by SSOP and direct sequencing. In this study using these techniques we provide the allelic typing of HLA‐A, ‐B, and ‐C genes for the B‐lymphoblastoid reference cell lines from the Tenth International Histocompatibility Workshop. We also describe some common associations of the C alleles with HLA‐A and HLA‐B alleles.
Development and characterization of rabbit antisera to human MHC‐linked transporters associated with antigen processing
Authors: Hicklin D. J. , Kageshita T. , Ferrone S.
Published: December 2008

The limited availability of sera to human MHC‐linked transporters associated with antigen processing (TAP) has hampered the analysis of the role of these molecules in the reduced HLA Class I antigen expression by normal and transformed cells. To overcome these limitations, anti‐human TAP1 and anti‐human TAP2 xenoantisera have been generated and characterized. To this end rabbits have been immunized with TAP 1‐specific or TAP2‐specific peptides which correspond to nonhomologous, hydrophilic regions of each transporter subunit. The immunized rabbits developed high titer IgG antibodies which displayed specific reactivity with the immunizing peptides in ELISA. Both anti‐TAPl and anti‐TAP2 antisera immunoprecipitated the 70–76 kDa TAP complex from TAP1+‐TAP2+ cell lines WALK and Colo 38, but precipitated no component from TAP1+‐TAP2+ cell lines T2 and SK‐MEL‐19. Furthermore, in immunode‐pletion experiments anti‐TAPl and anti‐TAP2 antisera removed the molecules recognized by each of them in a lymphoid cell extract. Lastly, in Western blotting assays anti‐TAPl and anti‐TAP2 antisera reacted specifically with isolated TAP1 and TAP2, respectively. The latter results in conjunction with those of the immunodepletion experiments indicate thatTAPl and TAP2 are not detectable as isolated subunits in a cell extract and that TAP heterocomplex is the major, if not the only detectable molecular species in cells. Anti‐TAPl and anti‐TAP2 antisera were evaluated in immunohistochemical staining of both frozen and formalin fixed sections of skin and primary malignant melanoma lesions. Both antisera stained the cytoplasm of keratinocytes in normal skin and of melanoma cells in malignant lesions. The antisera we have elicited with TAP1‐ and TAP2‐specific peptides appear to be useful reagents to characterize the role of TAP in abnormalities of HLA Class I antigen expression.
Nomenclature for factors of the HLA system, update May 1996
Authors: Marsh Steven G. E.
Published: December 2008

The following sequences have been submitted to the Nomenclature Committee since the April 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.
A novel monoclonal antibody mNI‐58A against the α‐chain of leukocyte function‐associated antigen‐1 (LFA‐1) blocks the homotypic cell aggregation and actively regulates morphological changes in the ohorbol myristate acetate (PMA)‐activated human monocyte‐like cell line, U937
Authors: Ikewaki N. , Yamada A. , Sonoda A. , Inoko H.
Published: December 2008

A monoclonal antibody (mAb), designated mNI‐58A, was produced by immunizing mice with the lipopolysaccharide (LPS)‐stimulated monocyte‐like cell line, U937. The antigen defined by mNI‐58A was widely expressed on various lymphoid cells and all cell lines examined except the erythroid cell line, K562. When the reactive patterns between mNI‐58A and the mAbs to various human differentiation antigens (CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD 102, CD 106, HLA‐class I and ‐class II antigen) were compared, that of mNI‐58A was found to be similar to those of the leukocyte function‐associated antigen‐1 (LFA‐1) mAbs. Using a competitive immunofluorescence binding assay it was found that the preincubation with one of the CD11a mAbs, 2F12 completely blocked the subsequent binding of mNI‐58A. mNI‐58A prevented the homotypic cell aggregation of the phorbol myristate acetate (PMA)‐activated U937 cells (referred to as PMA‐U937) and PMA‐activated Epstein‐Barr virus (EBV)‐transformed B cell lines, B‐85 and Mann. mNI‐58A markedly induced the spread formation of the PMA‐U937 cells following this blocking of the homotypic cell aggregation, whereas 2F12 did not under the same condition. The spread formation induced by mNI‐58A was completely blocked by cytochalasin B (CyB), cytochalasin D (CyD), cycloheximide (CHX) or protein kinase C inhibitors, sphingosine and H‐7. The U937 cells markedly adhered to the tumor necrosis factor‐α (TNF‐α)‐stimulated human umbilical vein endothelial cells (HUVECs) and also to the extracellular matrix protein, fibronectin, but mNI‐58 A did not enhance or block these adhesion processes. mNI‐58A precipitated two glycoproteins with molecular weight 180 kDa and 95 kDa as determined by SDS‐PAGE analysis, which were identical to the LFA‐α (CD 11 a) and β (CD 18) chains of leukocyte integrin precipitated by the CD 11 a mAbs, respectively. Sequential immunoprecipitation studies using the CD1 la mAb (2F12) also indicate that mNI‐58 A recognizes an epitope on the α‐chain of the LFA‐1 motecule. The ability of mNI‐58 A to block the PMA‐U937 cells and to induce the spread formation of these cells suggests that mNI‐58A is a novel mAb reacting with an epitope on the α‐chain of LFA‐1 different from those recognized with the existing CD1 la mAbs.
Results of Expedicion Humana:II. Analysis of HLA class II alleles in three African American populations from Colombia using the PCR/SSOP: identification of a novel DQB1*02 (*0203) allele
Authors: Trachtenberg E. A. , Keyeux G. , Bernal J. , Noble J. A. , Erlich H. A.
Published: December 2008

PCR/SSOP typing methods were used to analyze the HLA Class II DRB1, DQA1, DQB1 and DPB1 loci of samples from three African American populations of Colombia. Forty samples from the Cauca (Pacific), and twenty samples each from the Choco (North Pacific Coast) and the Providencia (Caribbean island) populations, were collected and the Class II loci analyzed under the auspices of the Expedicion Humana. Despite the limited number of samples analyzed, the African Colombian populations exhibit a very high degree of class II polymorphism. A great diversity of DRB1 alleles was found, with representatives from all serological classes, including 19 DRB1 alleles in the Providencia, 16 in the Cauca and 14 in the Choco groups. In addition, a novel DQB1*02 allele (*0203) was found in two individuals from the Cauca population of the Pacific Coast. The sequence of the DQB1*0203 allele, associated with DR3, differs from DQB 1*0201 by only one nucleotide substitution (C←A) in the second position of codon 57, resulting in an Ala to Asp change. The addition of DQB1*0203 brings the total number of DQB1 alleles identified to date to 26. HLA class II diversity is much greater in these African Colombian populations than that seen in nearby Amerindian populations. Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.
Molecular analysis and polymorphism of the DLA‐DQA gene.
Authors: Wagner J. L. , Burnett R. C. , DeRose S. A. , Storb R.
Published: December 2008

A full‐length cDNA clone and two overlapping genomic clones corresponding to the canine DQA class II gene were isolated and sequenced. Restriction mapping and sequence data allow identification and orientation of the five exons corresponding to the alpha (α) chain. Sequence analysis of exon 2 amplified from 17 unrelated dogs of various breeds identified seven alleles. The structure of the canine DQA gene is similar to HLA‐DQA1 and other mammalian DQA genes. This study will serve as a reference for developing a typing system for the DLA‐DQA gene for donor and recipient matching in the canine model for organ and bone marrow transplantation.
Phosphorylation of each of the distal three tyrosines of the CD28 cytoplasmic tail is required for CD28‐induced T cell IL‐2 secretion
Authors: Teng J. M. C. , King P. D. , Sadra A. , Liu X. , Han A. , Selvakumar A. , August A. , Dupont B.
Published: December 2008

Signaling by the CD28 T cell costimulatory receptor is known to involve recruitment and activation of phosphatidylinositol 3‐kinase (PI3‐kinase) which is dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail, present in a YMNM motif. However, whether this phosphorylation is required for CD28 costimulation and whether or not phosphorylation of any of the other three tyrosines of the CD28 cytoplasmic tail (tyrosines 188, 191 and 200) is also important for CD28 induced responses is unclear. To address this we examined the ability of chimeric receptors, consisting of the extracellular plus transmembrane membrane domain of human CD8α linked to different mutated human CD28 cytoplasmic tails, to induce IL‐2 secretion in Jurkat T leukemia cells in the presence of PMA and ionomycin. A receptor in which tyrosine 173 of the CD28 tail was mutated to phenylalanine was able to induce IL‐2. By contrast, receptors which contained single tyrosine 188, 191 or 200 to phenylalanine substitutions were unable to induce IL‐2. These results imply that in this system phosphorylation of tyrosine 173 and hence activation of PI3‐kinase is not required for CD28 induced IL‐2 secretion. Further, they imply that phosphorylation of each of tyrosines 188, 191 and 200 is necessary for this response. Despite an apparent requirement for phosphorylation of all three of these tyrosines, however, receptors which contain tyrosine only at positions 191 or 200 and a truncated receptor which does not contain tyrosine 200 induce normal IL‐2. These last findings, therefore, illustrate the complexity of CD28 mediated activation signals.
Signal transduction properties of the T cell activation monoclonal antibody panel: ubiquitous increase in cellular substrate tyrosine phosphorylation in the absence of detectable calcium mobilization
Authors: Levine B. L. , Ueda Y. , Faith A. , June C. H.
Published: December 2008

A panel of coded monoclonal antibodies (mAbs) submitted to the T cell section of the Vth International Workshop and Conference on Human Leukocyte Differentiation Antigens was examined for the ability to induce cellular activation. Intracellular calcium levels were examined by loading resting T cells with the Ca++‐specific dye Indo‐1, incubating the cells with Ab, cross‐linking with goat anti‐mouse Ab and analyzing by flow cytometry. Only 11 out of 68 Abs induced a detectable rise in Ca++; two of these Abs induced a substantial rise in Ca++. In resting T cells, 63 of 68 Abs induced increased tyrosine phosphorylation of substrates compared to negative control. Approximately half of the Abs that induced tyrosine phosphorylation induced substrates different from those seen following treatment of cells with anti‐CD3 Ab. All mAbs that induced a rise in Ca++ also induced increased tyrosine phosphorylation. These experiments show a distinct difference in the ability of cross‐linked Ab to induce changes in tyrosine phosphorylation and intracellular free Ca+. Furthermore, these results indicate that transient increases in cellular substrate phosphorylation may have questionable biologic significance in T cells.
The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies
Authors: Zola H. , Fusco M. , Ridings J. , Flego L. R. , Weedon H. M. , Nicholson I. , Organ N. , Roberton D. M. , Macardie P. J.
Published: December 2008

The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue, but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N‐glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N‐glycosylation.
HLA gene and haplotype frequencies in Dutch blood donors
Authors: Schipper R. F. , Schreuder G. M. T. , D'Amaro J. , Oudshoorn M.
Published: December 2008

We analyzed the HLA‐A, ‐B, ‐C, ‐DR and ‐DQ phenotypes of 2,440 healthy, unrelated, Dutch Caucasoid blood donors and of 20, 814 Dutch blood donors who were registered as volunteer bone marrow or platelet donors. Phenotype and gene frequencies, Hardy‐Weinberg equilibrium fit and homozygosity were calculated as well as 2‐ and 3‐locus haplotype frequencies, deltas, relative deltas and significance levels of the deltas. The population appears to be in Hardy‐Weinberg equilibrium. Many haplotypes are in strong positive linkage disequilibrium. A phylogenetic tree, based on the HLA‐A, ‐B and ‐DR gene frequencies of blood donors in different Dutch regions, reflects the limited but manifest heterogeneity of the Dutch population. Additionally we introduce a stepwise test for Hardy‐Weinberg equilibrium and discuss the applicability of this test and of the single test for Hardy‐Weinberg equilibrium for tissue typing quality control and for selection of split antigens prior to gene and haplotype frequency analyses.
Complete coding region of the new HLA‐DQB1*0612 allele, obtained by RT‐PCR
Authors: Vilches C. , Garcia‐Pacheco J.‐M. , de Pablo R. , Puente S. , Kreisler M.
Published: December 2008

A novel HLA‐DQB1 allele was detected by oligotyping in the Bubi population of Equatorial Guinea. In order to characterize the new allelic variant, a RT‐PCR method which permitted the cloning of its complete coding region was designed. With this method, we have determined the nucleotide sequence of the new DQB1*0612 allele, related to *0604 and *0609 but differing from them at polymorphic codon 70. A proposal for the improvement of the sequencing strategies of HLA class II alleles is made.
HLA–G transcription studies during the different stages of normal and malignant hematopoiesis
Authors: Amiot L. , Onno M. , Renard I. , Drénou B. , Guillaudeux T. , Bouteiller P. Le , Fauchet R.
Published: December 2008

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

The functional significance of polymorphism among MHC class II promoters in man and mouse is here reviewed, mainly in terms of the hypothesis of differential expression. The hypothesis proposes that differences between antigen‐presenting cells in MHC class II expression exert a co‐dominant effect on the Th1 ‐ Th2 cytokine balance, such that class II molecules of one type come to control to a greater extent the production of one group of cytokines, and those of another type the production of the alternative group. The survey deals with the influence of signal strength and antigen‐presenting cell type on T‐cell subset differentiation; functional differences between MHC class n molecules not obviously related to determinant selection; disease protection mediated by HLA alleles; mechanisms possibly responsible for allotypic and isotypic bias; overdominance (heterozygous advantage) in selection for expression of class II alleles; MHC class II promoter structure and function; inter‐locus and inter‐allele variability within human MHC class II gene upstream regulatory regions; a comparison of these polymorphisms in mouse and man; read‐out of class II promoter function; and a comparison with expression of MHC class I. We conclude that the evidence that this variation is functionally active (i.e. controls expression) is increasing, but is not yet compelling. The crucial test still to come, we suggest, is whether or not the biological effects attributable to this polymorphism will line up with molecular studies on expression.
High resolution HLA‐C typing by PCR‐SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology
Authors: Bunce M. , Barnardo M. C. N. M. , Procter J. , Marsh S. G. E. , Vilches C. , Welsh K. I.
Published: December 2008

Recent evidence indicates that HLA‐C molecules are biologically relevant by eliciting T‐cell responses and exerting control over NK cell function. In addition, HLA‐C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA‐C has however proved difficult because detection of HLA‐C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA‐C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA‐C blank alleles. Recently, amplification of DNA using sequence‐specific primers (PCR‐SSP) has proved a reliable, accurate and rapid method for medium resolution HLA‐C typing. We have now developed high resolution HLA‐C typing by PCR‐SSP utilizing allele and group‐specific PCR‐SSP reactions which can identify all HLA‐C alleles (except non‐coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA‐C, PCR‐SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA‐C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High‐resolution HLA‐C typing of 112 International Histocompatibility Workshop cell lines has also been performed.
The Ly‐1. 1 and Ly‐1. 2 epitopes of murine CD5 map to the membrane distal scavenger receptor cysteine‐rich domain
Authors: Starling G. C. , Llewellyn M. ‐B. C. , Whitney G. S. , Aruffo A.
Published: December 2008

CD5 is a member of a superfamily of proteins which contain one or more extracellular domains homologous to the type I macrophage Scavenger Receptor cysteine‐rich (SRCR) domain. The extracellular region of CD5 is composed of three SRCR domains (D1, D2, D3). Murine CD5 (mCD5) is polymorphic (Ly‐1. 1 and Ly‐1. 2 alleles), however, the only murine CD5 gene characterized to date encodes the Ly‐1. 2 allele (mCD5. 2). Likewise, the domain specificity of many of the available anti‐mCD5 mAb recognizing either Ly‐1. 1 or Ly‐1. 2 or both has not been examined. Herein we describe the isolation and characterization of cDNA encoding the Ly1. 1 allele (mCD5. 1) and map the location and molecular nature of the mCD5 allelic variation. We also determined which SRCR domain of mCD5 is recognized by a panel of anti‐mCD5 mAb. The mCD5.1 protein differs from mCD5.2 in only three amino acids, all of which map to the most amino terminal SRCR domain (D1) of mCD5. An additional seven silent substitutions were observed in the nucleotide sequence encoding mCD5 D1, D2 and transmembrane domains. Immunoglobulin (Ig) fusion proteins consisting of various combinations of mCD5. 1 or mCD5. 2 SRCR domains were produced and used to determine that allele specific mAb bound to D1, confirming sequence data. MAb against monomorphic determinants on mCD5 bound to each mCD5D1Ig.
An epitope in the third hypervariable region of the DRB1 gene is involved in the susceptibility to endemic pemphigus foliaceus (fogo selvagem) in three different Brazilian populations
Authors: Moraes M. E. , Fernandez‐Vina M. , Lazaro A. , Diaz L A. , Filho G. H. , Friedman H. , Rivitti E. , Aoki V. , Stastny P. , Moraes J. R.
Published: December 2008

Endemic pemphigus foliaceus or fogo selvagem (FS) in an organ‐specific autoimmune skin disease characterized by epidermal vesicles and mediated: by autoantibodies. Family cases are frequent and not everyone living in endemic region develops FS suggesting that host factors play a role in determining whether exposed individuals will be affected. Because our previous works with Brazilian Mestizos and with Xavante Indians have shown that particular HLA alleles confer increased risk for the disease, we decided to extend these studies to another homogeneous population, the Terena Indians. 19 out of 20 Terena patients were either positive for DRB 1*0404, 1402 or 1406 (p< .0005, RR=14). These findings were in agreement with the data obtained from the Xavante study. In Mestizos the association was with DRB 1*01. All these alleles involved in predisposition to the disease in different populations shared the same amino acid sequence at position 67–74 on the third hypervariable region of the DRB 1 gene: LLEQRRAA, suggesting that inheritance of this sequence is involved in the susceptibility to FS. When patients and controls data from different studies were pooled and analyzed disregarding the ethnic background and the HLA alleles involved, the results obtained clearly supported the hypothesis that matching for this epitope is highly significant and predictive of FS predisposition (p< .000001, RR=6.4)
HLA class II genes in Latvian patients with juvenile rheumatoid arthritis
Authors: Rumba I. , Denisova A. , Sochnev A. , Nilsson B. , Sanjeevi C. B.
Published: December 2008

PCR‐based HLA genotyping was used to analyze the association of HLA‐DR and ‐DQ genes in 127 juvenile rheumatoid arthritis patients and 111 population‐based controls from Latvia. The results show DQA1*03 to be positively associated in overall patients and DRB1*01‐DQA1*0101‐DQB1*0501 to be negatively associated with JRA in overall patients and in polyarthritis patients compared to controls. These data indicate the immunogenetic heterogeneity in the JRA patients, in the disease subgroups and in different ethnic groups. Rheumatoid factor (RF) was assayed in patients (n= 119) and controls (n=98). RF was present in patients (7/119, 6%) compared to controls (5/98, 5%). None of the DQA1, DQB1 alleles, DQ and DR‐DQ haplotypes was associated in seropositive patients compared to seropositive controls. DR1‐DQ5 (DQA1*0101‐B*0501) was decreased in seronegative patients (11/111, 10%) compared to seronegative controls (24/105, 23%), but the difference was not significant after correction of the p value.
Intrathymic maturation of CD4+ T‐lymphocytes in an MHC class II deficient transplant model
Authors: Huss R. , Deeg H. J.
Published: December 2008

Major histocompatibility complex (MHC) class II knockout (class II‐) mice fail to generate CD4+ CD8‐ T‐lymphocytes. We were interested in determining whether these class II‐ mice could be reconstituted with CD4+ CD8‐ T‐lymphocytes following marrow transplantation from normal (class II+) donors. Transplantation of class II+ marrow into lethally irradiated class II‐ recipients failed to generate peripheral CD4+ CD8‐ T‐lymphocytes. Unexpectedly, however, transplantation of class II‐ marrow into class II+ recipients also resulted in a deficiency of CD4+ CD8‐ cells. Analysis of intrathymic T cells showed normal distribution of CD4 and CD8 single and double positive or negative thymocytes in normal recipients, while class II‐ recipients always lacked CD4+ CD8‐ T cells intrathymically. These results suggest, therefore, that T‐cell maturation in mice requires the presence of MHC class II antigens not only in the thymus but also on immature, marrow‐derived pre‐thymocytes.