bluuebunny/crossref_metadata_2025 huggingface dataset:
Latex‐induced occupational asthma: time trend in incidence and relationship with hospital glove policies
Authors: Vandenplas O. , Larbanois A. , Vanassche F. , François S. , Jamart J. , Vandeweerdt M. , Thimpont J.
Published: December 2008

Background:  Natural rubber latex (NRL) has become as a major cause of occupational asthma (OA) in workers using NRL gloves. Few population‐based studies have assessed the impact of changes in the patterns of glove usage on the incidence of NRL‐induced OA.

Objective:  To characterize the time trends in incident cases of NRL‐induced OA in Belgium and examine whether incidence rates were related to the types of gloves used in hospitals.

Methods:  Incident cases of NRL‐induced OA were identified through a retrospective review of all claims submitted to the Workers’ Compensation Board up to December 2004. Based on the results of diagnostic procedures, the diagnosis of NRL‐induced OA was categorized as definite, probable, unlikely, or indeterminate. The patterns of glove usage were characterized through a questionnaire survey of Belgian hospitals.

Results:  A total of 298 claims for NRL‐induced OA were identified, including 127 subjects with definite OA and 68 with probable OA. Categorized by the year of asthma onset, the incident cases of definite and probable NRL‐induced OA markedly decreased from 1999 onwards. The use of powdered NRL gloves fell from 80.9% in 1989 to 17.9% in 2004. Powdered NRL gloves were predominantly substituted with NRL‐free gloves, especially in the case of non‐sterile procedures.

Conclusion:  These national compensation‐based data confirm that a persistent decline in the incidence of NRL‐induced OA has occurred since late 1990s. This downward trend has temporally been associated with a decreasing usage of powdered NRL, further supporting a beneficial role of changes in glove policies.
Activation of cytotoxic T lymphocytes in HLA‐A, ‐B and ‐C‐identical responder‐stimulator pairs I
Authors: Breuning Martijn H. , Breur Birgitta S. , Engelsma Marian Y. , Ivänyi Pavol
Published: December 2008

Cytotoxic T lymphocytes were activated in primary one‐way mixed lymphocyte cultures of cells matched for serologically defined HLA‐A, ‐B and ‐C antigens. In 16 out of the 29 combinations mismatched for the HLA‐D/DR antigens, cell‐mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA‐Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T‐target cells enriched by resetting with sheep red blood cells, whereas target cells from the ‘non‐T’fraction were strongly lysed, indicating that antigenic determinants associated with Class‐II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA‐typed third‐party target cells. One cytotoxic T‐lymphocyte population preferentially lysed HLA‐DR2‐positive target cells. Family studies, including a family with a recombination between HLA‐B and ‐D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T‐lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA‐A‐, ‐B‐ and ‐C‐identical, D/DR‐different combinations, cell‐mediated lympholysis of stimulator target cells could not be detected, not even on enriched ‘non‐T’target cells. Thus, after primary mixed lymphocyte culture of HLA‐A‐, ‐B‐and C‐identical, HLA‐D/DR‐non‐identical cells, cytotoxic T lymphocytes directed against sensitizing Class‐II molecules can be detected in some combinations, but not in others.
A cytotoxic anti HLA‐AB monoclonal antibody which in dilution becomes specific to HLA‐A3 crossreacting group
Authors: Mazzilli M. C. , Cascino I. , Porrini S. Costanzi , Lavaggi M. V. , Lulli P. , Morellini M. , Testa L. , Gandini E.
Published: December 2008

XI 20.4 monoclonal antibody belongs to the IgM class. It precipitates two polypeptide chains characteristic of HLA Class I antigens. At the highest dilutions it is cytotoxic against lymphocytes carrying antigens of the HLA‐A3 crossreacting group. Lysostrip experiments show that, at the lowest dilutions, the antibody reacts either with HLA‐A, or B antigens.
An association between cutaneous sensitivity to histamine and HLA phenotype
Authors: Stuckey Martin S. , Witt Campbell , Lattimore Margaret , Warlow Robet , Schmitt Lincoln H. , Dawkins Roger L.
Published: December 2008

Four hundred and twenty randomly chosen subjects from a normal population were HLA typed and tested for cutaneous sensitivity to histamine by prick testing with 5 concentrations of histamine (10−3, 10−2, 10−1, 1, 10 mg ± ml−1). Positive responses to 10−1 mg ± ml−1 histamine occurred in 41% of the subjects, and particularly those with HLA‐B7 (55%) (p < 0.005). It is concluded that genes within the major histocomptability complex influence cutaneous responses to histamine.
HLA‐A, ‐B and ‐C specificities in a sample of the Nile Delta population, Egypt
Authors: Sawy M. El , Helmy‐Khalil S. , Zaky A. , Marcelli‐Barge A.
Published: December 2008

HLA‐A, ‐B and ‐C specificities were determined in 100 Egyptians living and originating from the region of the Nile Delta. The pattern of antigen frequencies were similar to those of Berber populations living in North Africa with some exceptions compatible with the Semitic contribution to the Egyptian population.
DR4 related antisera pattern differences in insulin dependent diabetes mellitus and rheumatoid arthritis
Authors: Tait B. D. , Boyle A. , Solty S. , Cunningham T. , Mandel T. , Martin F. I. R. , Dorans T.
Published: December 2008

Three allosera patterns of reactivity are described which appear to define subtypes of DR4 and segregate in families with different DR4 bearing haplotypes. There appears to be a preponderance of one particular subtype (termed DR4.3) occurring in type 1 insulin dependent diabetes mellitus, while in rheumatoid arthritis the distribution of subtypes within DR4 individuals is similar to that seen in controls. Although we refer to DR4 subtypes the proof that these sera are detecting a determinant on the DR molecule and not a product of another locus, an allele of which is linkage disequilibrium with DR4, awaits immunochemical confirmation.
HLA‐DR phenotypes and blood levels of T cell subsets
Authors: Caruso Calogero , Lio Domenico , Palmeri Patrizia , Cillari Enrico
Published: December 2008

Blood mononuclear cell and T cell subsets values were analyzed in 53 Sicilian individuals according to HLA‐DR phenotypes. The results demonstrate that DR1‐positive subjects show a significant increase of blood T cell subsets whereas DR3‐positive subjects show a non‐significant decrease of these values. These results suggest that gene(s) associated with HLA‐DR could be one of the factors which affect blood levels of T cell subsets.
Study of HLA segregation in 479 thalassemic families
Authors: Contu L. , Arras M. , Mulargia M. , Nasa G. La , Carcassi C. , Leone A. L. , Ledda A. , Goddi F.
Published: December 2008

Abstract: 479 families, each with a proband affected by homozygous β‐thalassemia, were typed for HLA. 224 families with a total of 1020 members were typed for the HLA‐A, B, C, DR and DQ loci and 255 families with 1046 family members, were typed for the HLA‐A, B, and C loci. Altogether, 896 A, B, C, DR and DQ haplotypes and 1020 A, B and C haplotypes were defined. At the same time, 120 healthy unrelated individuals from the same population were typed and used as controls. The analysis of the results was carried out at antigen, allele, haplotype, genotype and sex ratio level with the aim of looking on the one hand, for the existence of heterogeneity between the probands, the unrelated individuals and the healthy siblings and, on the other, for the existence of any distortion whatsoever of the HLA segregation in either the probands or in the healthy siblings in respect of the expected values according to the Mendelian equilibrium. No significant differences were evident between the probands and the controls by the tests carried out at different levels of the HLA system. This leads us to exclude the existence of an association between β‐thalassemia and HLA in the population studied. Moreover, the analysis of the transmission of the alleles, the haplotypes, the genotypes and the sex‐ratio by parents to both affected and to healthy children did not show any clear evidence of segregation distortion in respect of the theoretical values.
HLA‐DPB1 allele mismatches between unrelated HLA‐A,B,C,DR (generic) DQA1‐identical unrelated individuals with unreactive MLC
Authors: Salazar M. , Yunis I. , Alosco S. M. , Chopek M. , Yunis E. J.
Published: December 2008

Abstract: We have used a PCR‐RFLP method with one generic amplification of HLA‐DPB1 second exon and 6 endonucleases to differentiate the 19 HLA‐DPB1 alleles and 171 heterozygous combinations. The set of primers used in our studies produced fragment sizes different from those published before (1). The HLA‐DPB1 alleles in Caucasians showed a higher frequency of DPB 1*0401 and DPB 1*0402, when compared to a small group of Colombians who showed a higher frequency of DPB 1*0402 and DPB 1*0201. We found three HLA‐DPB1 alleles associated with two HLA haplotypes that result from non‐random association of alleles: DPB1*0401 with HLA‐A26, B38, DR4, DQA1*0301 and DPB1*0101 and DPB1*0401 with HLA‐A1, B8, DR3, DQA1*0501. We also report that 70% of combinations between HLA (generic A,B,C,DR) and DQA1‐identical MLC‐unreactive cell mixtures showed HLA‐DPB1 mismatches, suggesting that HLA‐DPB1 differences are not important in MLC reactivity.
Progression of HIV‐related disease is associated with HLA DQ and DR alleles defined by restriction fragment length polymorphisms
Authors: Donald J. A. , Rudman K. , Cooper D. W. , Baumgart K. W. , Garsla R. J. , Gatenby P. A. , Rickard K. A.
Published: December 2008

Abstract:A cohort of 139 hemophiliacs was typed for HLA D region genes by means of restriction fragment length polymorphisms (RFLPs) detected by HLA DQ and DR gene probes. Disease progression was studied in the 65 HIV antibody‐positive patients, who were infected by contaminated clotting factor before 1985. Strong associations were found between disease progression in HIV‐infected patients and allelic DNA fragments revealed by a DQα cDNA probe. A 5.5 kb fragment was reduced in frequency and a 4.6 kb fragment increased in frequency (p < 0.005) in the faster progressing group, as measured both by development of CDC Category IV clinical symptoms and CD4 number <200 × 106/1. These results correlate with DR types deduced from the RFLP patterns revealed by DRβ and DQα gene probes. A decrease in DR4 and an increase in both DR5 and the DR3 subtype found in the A1 B8 DR3 haplotype were associated with disease progression (p < 0.05).
Association of HLA‐B51 and lack of association of class II alleles with Behcet's disease
Authors: Mizuki Nobuhisa , Ohno Shigeaki , Tanaka Hiroshi , Sugimura Kazuhito , Seki Takeshi , Mizuki Nobuhiko , Kera Jiro , Inaba Goro , Tsuji Kimiyoshi , Inoko Hidetoshi
Published: December 2008

Abstract: Eighty‐one Behcet's disease patients have been studied for HLA association by HLA‐DRB1, ‐DQA1, ‐DQB1 and ‐DPB1 genotyping with the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) technique and for NcoI RFLP in the tumor‐necrosis factor (TNF β) gene by Southern hybridization in addition to serological typing. In serological typing, the frequency of HLA‐B51 was significantly increased in the patients. In PCR genotyping, there was a significant increase in the HLA‐DRB1*0802, DQA1*0301 and DQB1*0303 alleles, whereas the frequencies of DRB1*1502, DQA1*0103, DQAl*0101, DQB1*0601 and DQB1*0501 showed a significant decrease in the patients. No significant difference was observed in any HLA‐DPB1 alleles. Among them, B51 was found to be a genetic marker most strongly associated with Behcet's disease (p<0.00005, δ2=46.47, pc[corrected p] < 0.005). The positive and negative associations of class II alleles with the disease can be explained by linkage disequilibrium with B51, and do not reach statistical significance by the corrected p‐value test. In NcoI RFLP typing in the TNFβ gene, 250 kb centromeric of the HLA‐B gene, the frequency of a 5.5 kb fragment was considerably decreased in the patients when compared to the controls, although the decrease was not statistically significant. These results indicate that the HLA class II genes or other non‐HLA genes mapping within the class II region are not involved in the development of Behcet's disease but that HLA‐B51 itself or a non‐HLA gene located around the HLA‐B gene region, more closely linked to HLA‐B than the TNFβ gene, does determine the genetic susceptibility to Behcet's disease.
HLA antigens in Tlingit Indians with rheumatoid arthritis
Authors: Nelson J. Lee , Boyer Georgiana , Templin David , Lanier Anne , Barrington Robert , Nisperos Brenda , Smith Anajane , Mickelson Eric , Hansen John A.
Published: December 2008

Abstract: HLA‐DR4 has been described in association with rheumatoid arthritis (RA) in multiple populations. We have studied HLA antigens in Alaskan Tlingit Indians. HLA‐DR4 was decreased in the RA group (n = 32) compared with controls (n = 62) (6% vs 21% p=0.07). The predominant DR4 allele observed was DRB1*0403 (Dw13.1). The most striking observation in these studies was a marked predominance of the DRB1*1402 allele encoding Dw16 (DRw14). This allele was present in 91% of RA cases, but was also highly prevalent in controls (80%, OR = 2.4 p = 0.20). DRB1*1402 only was observed in 47% of cases and 31% of controls. The DRB3*0101 (DRw52), and the DQA*0501 and DQB*0301 alleles encoding a subset of DQw3 were associated with DRB1*1402 in cases and in controls. HLA‐Bw62 was increased in RA cases (28%) compared with controls (8%) (OR = 4.5, p = 0.01, corrected p = ns).
HLA class II alleles associated with celiac disease susceptibility in a Southern European population
Authors: Tighe M. Richard , Hall Margaret A. , Barbado Maria , Cardi Ectore , Welsh Kenneth I. , Ciclitira Paul J.
Published: December 2008

Abstract: Susceptibility to celiac disease in Northern Europe is associated with the human leukocyte antigens (HLA) B8, DR3 and DQ2, which exist together on an extended haplotype. The strong predominance of this haplotype within the Northern European celiac populations, together with the linkage disequilibrium which occurs between these loci, does not allow identification of the gene(s) primarily associated with disease susceptibility. Studies from Southern Europe using both serology and examination of restriction fragment length polymorphisms (RFLP) have demonstrated associations with DR3, DR7 and DQ2, suggesting that the DQ locus is primarily involved. We investigated 43 celiac patients and 41 healthy controls from Rome, Italy, using sequence‐specific oligonucleotide (SSO) probes, in conjunction with gene amplification by the polymerase chain reaction (PCR), to determine alleles at the DRB, DQA1, DQB1 and DPB1 loci: 19% of celiac patients possessed the alleles DRB1*0301 DRB3*0101, 33% DRB1*0301 DRB3*0201 and 33% of celiac patients were heterozygous for DRB1*1101–1201/DRB1*0701. The strongest association with celiac disease susceptibility was the combination of alleles DQA1*0501 DQB1*0201 (91% celiac patients vs. 12% controls; p = 0.000002). There was no additional susceptibility associated with alleles at the DPB locus. This study confirms the hypothesis that susceptibility is associated with a particular combination of DQ alleles and the ethnic variation in DR frequencies is secondary to linkage disequilibrium with these DQ alleles.
Effect of an anti‐HLA class I monoclonal antibody on the antigenic and transcriptional expression of HLA class I genes in U937 cells
Authors: Guillaudeux Thierry , Gaudin Annette , Collet Brigitte , Fauchet Renée , Toujas Louis
Published: December 2008

The phenomenon of antigenic modulation was studied in the histiocytic lymphoma line U937. A redistribution of cell surface HLA antigen after incubation of U937 cells with the monomorphic anti‐HLA class I monoclonal antibody W6/32 was demonstrated by immunofluorescence analysis. As assessed by hybridization of RNA obtained from W6/32‐treated U937 cells with a probe corresponding to the α3 domain of HLA Cw3, prolonged W6/32 incubation (24 to 72 hours) induced a decrease in HLA class I transcript abundance. This decrease was about 25% as compared with untreated control cells. These data indicate that W6/32 incubation can induce changes in HLA class I gene expression not only at the antigenic but also at the transcriptional level. Possible implications for the molecular basis of antigenic modulation are discussed.
New polymorphic HLA‐DR epitopes recognized by three monoclonal antibodies produced against DR103 transfected L cells
Authors: Cayrol Corinne , Moro Franĉoise , Sommer Evelyne , Tkaczuk Jean , Ohayon Elie , Cambon‐Thomsen Anne
Published: December 2008

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non‐MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103‐transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA‐DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B‐lymphoblastoid cell lines (B‐LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA‐DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B‐LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA‐DR products and react in western blots with DR α/β‐dimer but not with free α‐ or β‐chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.
Flow microfluorometric analysis of murine spermatozoa fails to detect H‐2 antigens
Authors: Harding George B. , Wellhausen Samuel R.
Published: December 2008

The presence of class 1 and class 2 histocompatibility antigens on murine sperm was investigated by flow microfluorometry. Monoclonal anti‐H‐2Kk (class 1), anti‐Iak (specificity 2, class 2) and allo‐anti‐Iak (class 2) antisera were used in direct or indirect fluorescence labelling experiments to probe the expression of class 1 and class 2 antigens on epididymal mouse spermatozoa. Sperm‐specific antibodies were generated by intraperitoneal immunization of both male and female C3H/HeN mice with syngeneic spermatozoa. Sperm‐specific antigens were detected in 68–85% of syngeneic mouse sperm and 65–90% of allogeneic mouse sperm examined. Conversely, these antibodies did not stain syngeneic or allogeneic lymphocytes above the background of the negative control. Mouse sperm samples failed to exhibit specific fluorescence above the background of negative control values with antibodies against either class 1 or class 2 MHC antigens. We have established the sensitive, objective and economical assay of sperm membrane antigens with fluorochrome‐labelled antibodies by flow microfluorometry. By use of this sensitive and objective technique we have not detected MHC antigens on murine sperm. We conclude that these MHC antigens are not expressed on sperm at a level to be practically detectable.
HLA‐DRB1 first domain sequence for a novel DR4 allele designated DRB1*0413
Authors: Petersdorf Effie W. , Smith Anajane G. , Martin Paul J. , Hansen John A.
Published: December 2008

A novel DR4 allele, DRB1*0413, was identified in a Caucasian individual “LEV” having the HLA phenotype A2; B51,14; DR4,7; DQw3,w2. DRB1*0413 is a DRB1*0401‐variant differing from DRB1*0401 only at codon 86 where valine is present instead of glycine.
DPB1 polymorphism in rheumatoid arthritis: Evidence of an association with allele DPB1 0401
Authors: Perdriger A. , Semana G. , Quillivic F. , Chalès G. , Chardevel F. , Legrand L , Meadeb J. , Fauchet R. , Pawlotsky Y.
Published: December 2008

Abstract: HLA‐DP polymorphism was examined in 71 rheumatoid arthritis patients and 148 controls, using dot‐blot analysis with 14 synthetic oligonucleotide probes specific for the variable region of the DPB1 second exon. The DPB1 0401 allele was found to be significantly more frequent in RA patients than in controls (77.46% vs 55.40%, p< 0.002, pc<0.03, relative risk value:2.74). An association between DPB1 0401 and seropositivity for rhumatoid factors was also observed: 44 of the 55 seropositive RA patients were DPB1 0401 (p<0.001). Analyzing the HLA DPB1 alleles frequencies in 57 HLA‐DR‐typed RA patients did not show any linkage between the DPB1 0401 and the DR4 specificities. Furthermore, the DPB1 0401 homozygous frequency was increased in DR4‐negative RA patients. Our findings suggest an independent role of the DPB1 0401 allele in the genetic susceptibility to RA.
Genes for insulin‐dependent diabetes mellitus (IDDM) in the major histocompatibility complex (MHC) of African‐Americans
Authors: Fernandez‐Vlña Marcelo , Ramirez Luis C. , Raskin Philip , Stastny Peter
Published: December 2008

Mapping the MHC‐associated susceptibility and resistance factors for insulin‐dependent diabetes mellitus (IDDM) has been difficult due to the strong linkage disequilibrium within the HLA‐DR‐DQ region. Previous analyses have suggested that the study of IDDM‐associated haplotypes in different races might be useful for identifying the responsible genes. We have performed complete HLA class II genotyping to study susceptibility and resistance to IDDM in 34 randomly selected African‐American IDDM patients and 69 ethnically‐matched controls. IDDM patients showed highly significant increases of DRB1*0301, DRB1*0401, DRB1*0405, DQA1*0301, DQA1*0302, DQB1*0201 and DQB1*0302. Analysis of DQA1‐DQB1 associations showed that DQA1*03 combined with both DQB1*0201 and DQB1*0302 gave the highest odds ratio, suggesting a synergistic effect due to formation of heterodimers encoded both in cis and in trans. Among the subsets of DR4, only DRB1*0401 and DRB1*0405 were increased in diabetic patients. Interestingly, DQB1*0602 and DQB1*0301, which have previously been thought to encode resistance factors in Caucasians, were not significantly decreased and, after removal of known susceptibility haplotypes, were found to have essentially identical frequencies in patients and controls.
A human monoclonal hybridoma antibody (TrJ1) specific for HLA‐DQ2
Authors: Ge Jun , Bartnes Kristian , Hannestad Kristian
Published: December 2008

TrJ1 is a cytotoxic human hybridoma mAb (IgMλ). Its reaction pattern with a panel of 42 HLA‐defined lymphoblastoid B‐cell lines correlated precisely with expression of DQ2. By flow cytometry it was shown that the binding of TrJ1 to DQ2 was efficiently blocked by the murine anti‐DQ2 mAb 358.4, indicating that the TrJ1 and 358.4 epitopes overlap. TrJ1 reacted much better with EBV‐transformed B cells than with B cells freshly isolated from blood. TrJ1 seemed suitable for typing freshly isolated B cells provided the incubation with complement lasted for 115 min in Terasaki plates. One or more of the DQ2‐specific polymorphic amino acids E46, F52, L52, L55, K74 or A74, situated on the α‐helix of the DQ2 β chain, are probably critical for the TrJ1 epitope.
HLA‐DQB1 and ‐DQA1 typing by PCR amplification with sequence‐specific primers (PCR‐SSP) in 2 hours
Authors: Olerup Olle , Aldener Anna , Fogdell Anna
Published: December 2008

In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence‐specific primers (PCR‐SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterozygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA‐DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR‐SSP technique, as well as with the HLA‐DQβ57 primers. The concordance between PCR‐SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB‐DQA‐DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR‐SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post‐amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well‐suited for routine clinical use.
Isoelectric focusing subtypes of HLA‐A can be defined by oligonucleotide typing
Authors: Oh Seung‐Hoon , Fleischhauer Katharina , Yang Soo Young
Published: December 2008

This study describes a simple and direct method for sequence‐specific oligonucleotide probe (SSOP) typing of the A locus of HLA class I genes. Genomic DNA from a panel of over 200 cells which have been characterized by the methods of serology and isoelectric focusing (IEF) for the HLA class I antigens was used for locus‐specific PCR amplification of HLA‐A sequences. Dot blot hybridization of the amplified products was performed with 28 SSOPs derived from hypervariable regions in exon 2 and 3. Co‐amplification of three alleles of HLA‐H pseudogene in apparent linkage disequilibrium with HLA‐A2 and A10 was observed but did not interfere with the typing of HLA‐A alleles. Using short SSOPs (15 nucleotides each) in single temperature tetramethylammonium chloride (TMAC) hybridization and washing steps, 30 IEF‐definable isotypes of HLA‐A antigens could be unambiguously defined by their hybridization patterns. Moreover, comparison of the typing results with available nucleotide sequences of HLA‐A alleles showed that the conditions used allowed faithful detection of single codon mismatches between probe and template. Thus, these alleles can be identified by their unique hybridization patterns generated by the SSOPs. Nucleotide sequence analysis of any new HLA‐A allele will further permit its rapid and unambiguous characterization by SSOP typing.
Biochemical and functional characteristics of soluble MHC molecules derived from H‐2Ld/Q10d chimeric gene
Authors: Urbero B. , Gillet D. , Frangoulis B. , Pla M. , Kress M.
Published: December 2008

Abstract: We have constructed a chimeric class I gene in which the 5’half of the H‐2Ld gene is linked to the 3’half of Q10d. The resulting H‐2Ld/Q10d protein is homologous to the native H‐2Ld heavy chain for the three external domains except for an Arg to His substitution at position 260. The transmembrane and intracytoplasmic domains of the H‐2Ld chain are replaced by the short low hydrophobic transmembrane‐like domain of the Q10d chain. Following DNA‐mediated gene transfer into mouse L cells, transformants were selected for the presence of specific mRNA. Radiolabelling and immunoprecipitation analysis revealed secretion of a 48‐46 kd chain weakly associated with β2‐microglobulin. This molecule reacts with H‐2Ld‐specific mAb that identify determinants on the first and second domains as well as with an anti‐Q10 carboxyl‐terminal peptide antiserum, but is not recognized by a mAb specific for a determinant of H‐2Ld third domain. The integrity of antibody reactivity of the first and second domains together with β2‐microglobulin association suggest that our molecule may be considered a good soluble counterpart of the native membrane H‐2Ld molecule with which to perform functional studies. In order to analyze the immunogenic capacities and T‐cell recognition of the soluble H‐2Ld molecules, T‐cell lines were produced from mice of various inbred strains immunized with supernatant from H‐2Ld/Q10d‐transfected fibroblasts. Characterization of these T cells revealed that they expressed a CD4−CD8− phenotype, and recognized H‐2Ld/Q10d products in a class II‐restricted manner.
Soluble MHC class I antigens (sHLA) and anti‐HLA antibodies in heart and kidney allograft recipients
Authors: Zavazava N. , Böttcher H. , Ruchholtz W. Müller
Published: December 2008

Abstract: Soluble HLA (sHLA) antigens were measured in a panel of 50 renal and 50 cardiac graft recipients by a quantitative ELISA over periods ranging between 2 months and 3 years. 72% of the renal patients and 68% of the cardiac graft patients experienced episodes of acute rejection during the observation period. sHLA were elevated to over 2–5 times the normal levels up to 10 days before histological evidence of rejection. This duration was for each individual patient very variable. Cytomegalovirus (CMV) infections did not appear to elevate sHLA levels. Donor‐specific HLA‐A2 was measured in only 1 cardiac recipient. 15% of the sera obtained from cardiac patients had cytotoxic anti‐HLA antibodies, when tested against a panel of 50 test cells. After thermal inactivation at 56 ° C, 42.5% of the sera became positive against at least 10% of the test cells. This finding suggests thermal dissociation of immune complexed anti‐HLA antibodies. sHLA measurement and careful monitoring of serum for the presence of cytotoxic anti‐HLA antibodies in graft patients are useful techniques which could help predict the long‐term fate of an allograft.
A human monoclonal antibody, produced following in vitro immunization, recognizing an epitope shared by HLA‐A2 subtypes and HLA‐A28
Authors: Mulder Arand , Kardol Marrie , Blom Joke , Jolley Weldon B. , Melief Cornells J. M. , Bruning Hans
Published: December 2008

Abstract: In vitro immunization and subsequent immortalization of peripheral blood cells of a multiparous woman has resulted in the production of a stable human mouse heterohybridoma, 5C2A2, secreting an HLA‐A2/A28‐specific human monoclonal antibody. Although possibly exposed to HLA‐A2 by transfusions, the cell donor showed no HLA‐A2‐specific serum antibodies. The present protocol for in vitro immunization includes the elimination of suppressor cells from the responder cell population, the presence of irradiated allogeneic lymphocytes as a source of antigen, as well as stimuli – recombinant interleukin‐2 and a B‐cell specific nucleoside analogue – causing the proliferation of B lymphocytes, prior to immortalization. The ability of the antibody 5C2A2 to detect all known HLA‐A2 subtypes, except A2.3, and A28, allows identification of the serological epitope on the HLA‐A2 molecule. Application of this in vitro immunization method allows the production of a set of HLA monoclonal antibody‐secreting human hybridomas, independent of the existence of serum HLA antibodies in the lymphocyte donors.
DNA‐binding proteins for transcription enhancing region of HLA class I gene
Authors: Park Jean Han , Lee Hang Woog , Fleischhauer Katharina L. , Kim† Chul Gun , Sheffery† Michael , Yang Soo Young
Published: December 2008

Abstract: Class I regulatory complex (CRC) located in the 5′‐upstream region of MHC class I gene contains transcriptional enhancing sequences, called Enh A. This Enh A region contains tandem‐arranged κB‐like sites, one of which has a well‐conserved perfect palindromic sequence. The second κB‐like site, juxtaposed to the perfect palindrome, contains an imperfect palindromic sequence. In B‐cell nuclear extracts, we have identified at least four sequence‐specific protein complexes; three shared the repeated κB enhancer as their binding motifs. The perfect palindromic sequence facilitates the binding of a complex termed BI, while the imperfect palindrome provides the binding sites for two other complexes, BII and BIII. The BII and BIII complexes exhibited binding crossreactivity with other κB‐related motifs and recognized both the perfect and imperfect palindromic sequences, whereas the BI complex was specific for the perfect palindromic sequence which is unique to the class I promoters. A DNA segment outside the repeated κB enhancers probably binds the fourth complex, BIV. These complexes, except for the perfect palindrome‐binding complex, differ from those described for the murine class I promoter. The binding characteristics of these factors suggest that the mechanism controlling the class I transcription may be quite complex.
Functional dissection of the serological DR LYGUE and genotypic DRB1*1303 specificities using a tetanus toxin‐specific T‐cell clone
Authors: Martínez‐Seria Eduardo , Tiercy Jean‐Marie , Beffy Pascale , Taban‐Marín Pascale , Jaques Dominique , Bétuel Hervé , Jeannet Michel , Mach Bernard , Irlé Claudius
Published: December 2008

Abstract: The DRB1 sequence of the homozygous cell line HAG (DR13‐DwHAG‐DQ7) represents a new DRB allele assigned DRB1*1103, whereas its DRB3 sequence corresponds to the previously described DRB3*0101 (DR52a) allele. The DRB1*1303 gene product is undetectable by current sera used in routine serology typing. We report here direct evidence that the MHC molecule encoded by the DRB1*1303 gene is ‐functional in antigen presentation and in T‐cell restriction. We describe a T‐cell clone specific for tetanus toxin whose restriction pattern strictly follows the DRB1*1303 allele, as defined by oligonucleotide typing. It also follows the serologic reactivity with the serum LYGUE and also the DwHAG MLC‐defined specificity pattern, with one exception. The potential functional sites for the DRB1*1303 gene product involved in T‐cell restriction were deduced from sequence comparisons between DRB1*1303 and closely related DRB1 alleles. The relevant aa substitutions were located within close proximity to each other on the HLA class II structural model. Our results demonstrate that 1) DRB1*1303 is functional in antigen presentation and T‐cell restriction 2) the functional region involved in antigen presentation and T‐cell restriction by DRB1*1303 can be defined structurally.
Large‐scale oligonucleotide typing for HLA‐DRB1/3/4 and HLA‐DQB1 is highly accurate, specific, and reliable
Authors: Ng Jennifer , Hurley Carolyn Katovich , Baxter‐Lowe Lee Ann , Chopek Michael , Coppo Patricia A. , Hegland Janet , KuKuruga Debra , Monos Dimitri , Rosner Gayle , Schmeckpeper Barbara , Yang Soo Young , Dupont Bo , Hartzman Robert J.
Published: December 2008

Abstract: DNA typing of HLA class I1 alleles of the DRB1/3/4 and DQBI loci using sequence‐specific oligonucleotide probes and polymerase chain reaction‐amplified DNA was used for the large‐scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the first 7 months of the project show the typing to be highly accurate, specific, and reliable. The percent of correctly classified HLA oligotypes based on 1652 DRB1 and 1652 DQBI assignments was greater than 99% for DRBI/DRB3/DRM and greater than 98% for DQBI. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 9011 donor samples tested at the same time by the laboratories.
Rapid DNA typing for HLA‐C using sequence‐specific primers (PCR‐SSP): Identification of serological and non‐serologically defined HLA‐C alleles including several new alleles
Authors: Bunce Mike , Welsh Kenneth I.
Published: December 2008

Abstract: Detection of HLA‐C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undectectable HLA‐C locus antigens and 9 out of the 29 sequenced HLA‐C alleles so far published encode serologically undetected antigens. In addition, HLA‐C molecules are expressed at the cell surface at about 10% of the levels of HLA‐A and HLA‐B. Recently, amplification of DNA using sequence‐specific primers (PCR‐SSP) has proved a reliable and rapid method for typing HLA‐DR, HLA‐DQA and HLA‐DQB genes. PCR‐SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA‐C alleles corresponding to the serologically defined series HLA‐Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA‐C PCR‐SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA‐C antigens can be readily identified. DNA typing for HLA‐Cw by PCR‐SSP can take as little as 130 minutes from start to finish, including DNA preparation.
Sequence analysis of serological HLA‐A11 split antigens, A11.1 and A11.2
Authors: Lin L. , Tokunaga K. , Ishikawa Y. , Bannai M. , Kashiwase K. , Kuwata S. , Akaza T. , Tadokoro K. , Shibata Y. , Juji T.
Published: December 2008

Abstract: HLA‐A11 has two serologically‐defined splits, A11.1 and A11.2, and two alleles coding for HLA‐A11, A*1101 and A*1102, have been published so far. In order to understand the relationship between the serological subtypes and the amino acid sequences, we have sequenced the alleles coding for A11.1 and A11.2. The results demonstrated that A11.1 split antigen is encoded by A*1101 and A11.2 split antigen is encoded by A*1102. Moreover, we obtained the sequences of exons 1, 5, 6, 7, and 8 of A*1102, which were not reported previously. In addition, we could distinguish A*1101 from A*1102 using a PCR‐SSCP method, confirming that A11.1 carried by different HLA haplotypes has the same sequence.
Monoclonal antibodies that define canine homologues of human CD antigens: Summary of the First International Canine Leukocyte Antigen Workshop (CLAW)
Authors: Cobbold Steve , Metcalfe Su
Published: December 2008

Abstract: A panel of 127 monoclonal antibodies against canine leukocyte antigens, including controls, was distributed to 29 laboratories that performed a variety of experiments to identify groups of antibodies against the canine equivalents of some of the human CD antigens. Cluster analysis was performed centrally, using the submitted antibody binding data from immunofluorescence, ELISA and immuno‐histology experiments. Immunoprecipitation for molecular weight determination was also performed centrally with T‐cell blasts and a B‐cell line as the sources of antigen. Clusters of three or more antibodies were found that defined the canine equivalents of the CD5, CD4, CD8 and Thy‐1 antigens, and these could be used to label T‐cell subsets from the peripheral blood. Other groups of monoclonal antibodies recognized the canine homologues of the CD11/18 group of antigens, CD44 and the CD45/CD45R antigen family: these should be useful in isolating functional subsets of CD4+ helper T cells. There was a cluster of four antibodies that bound strongly to platelets (probably CD41 antigen), three antibodies that were specific to B cells (including CD21) and two antibodies against a granulocyte antigen (possibly CD15). A number of reagents were found against canine MHC‐II and immunoglobulin, with some of the latter able to distinguish between Ig subclasses. Properties of each of the canine antigens defined by these monoclonal antibodies are discussed and compared with other species. The availability of such a panel of reagents should allow rapid improvements in the immunological diagnosis of canine disease, and there might now be a potential for testing novel therapeutic strategies in a clinical veterinary setting.
Immunosuppression of canine renal allograft recipients by CD4 and CD8 monoclonal antibodies
Authors: Watson C. J. E. , Cobbold S. P. , Davies H. ff. S. , Rebello P. R. U. B. , Thiru S. , McNair R. , Rasmussen A. , Waldmann H. , Calne R. Y. , Metcalfe S. M.
Published: December 2008

Abstract: A state of tolerance to MHC mismatched allografts can be generated in rodents by treatment with CD4 and CD8 monoclonal antibodies (mAb). In order to transpose this type of therapy to large animals and ultimately to the clinic, a suitable model is required. To this end we have generated a series of mAb to the canine CD4, CD8, and Thy‐1 antigens and have tested their ability to prevent rejection of renal allografts. Donor‐recipient pairs were selected from a colony of mongrel dogs in which untreated rejection of two haplotype‐mismatched kidneys occurred by day 7 (defined as a serum creatinine > 300 μmol/l). Therapy with either the CD4 or the CD8 mAb, using no other immunosuppression, did not prolong graft survival. Depletion of T cells by a Thy‐1 mAb prior to surgery only extended graft survival to day 9. However, treating with combinations of mAb up to day 10 (CD4 plus Thy‐1; CD4 plus CD8; or CD4 plus CD8 plus Thy‐1) prolonged renal allograft function up to 25 days. Combination of the triple mAb therapy with a sub‐therapeutic immunosuppressive drug regimen (cyclosporin A plus azathioprine that alone gave a median survival of 15 days) favored survival to a median of 38 days. This protocol also inhibited the antiglobulin response that had curtailed the effects of mAb treatment, opening the way to more extended, and potentially tolerizing, mAb plus drug regimens.
Characterization of anti‐canine cytokine monoclonal antibodies specific for IFN‐γ: Effect of anti‐IFN‐γ on renal transplant rejection
Authors: Fuller Laphalle , Carreno Manuel , Esquenazi Violet , Zucker Keith , Zheng Shiyan , Roth David , Burke George , Nery Jose , Asthana Deshratn , Olson Les , Miller Joshua
Published: December 2008

Abstract: Two murine monoclonal antibodies specific for IFN‐γ, ADI‐1, and ADI‐23 (both IgG1 kappa), were generated in BALB/c mice. The ADI‐1 exhibited a higher avidity for canine rIFN‐γ than for nIFN‐γ and human rIFN‐γ. In contrast, the ADI‐23 showed equal avidity for the three IFN‐γ preparations. The anti‐canine IFN‐γ mAb did not bind to mouse and rat rIFN‐γ. The ADI‐1, and ADI‐23 mAb were also tested for binding to human rTFN‐α and, contrary to our expectations, it was found that ADI‐23 showed significant binding to human rTFN‐α and rIFN‐γ, in contrast to ADI‐1. Both anti‐canine IFN‐γ mAb stained 48‐h PHA‐induced dog lymphoblasts. A two‐site mAb ELISA was developed, which was linear in the range of 7–500 ng of canine rIFN‐γ, which indicated that the two mAb detected non‐overlapping epitopes on the canine rIFN‐γ molecule. We studied the effect of ADI‐1 on the prolongation of canine renal allografts. Recipients of kidney allografts, that were treated with ADI‐1 by continuous arterial infusion, were prolonged to 22 and 25 days, compared to 9 and 13 days for animals given the IgG1 isotype control.
The HLA‐B73 antigen has a most unusual structure that defines a second lineage of HLA‐B alleles
Authors: Parham Peter , Arnett Kelly L. , Adams Erin J. , Barber Linda D. , Domena John D. , Stewart Dod , Hildebrand William H. , Little Ann‐Margaret
Published: December 2008

Abstract: The nucleotide sequence of cDNA encoding the HLA‐B73 antigen was determined; it is unusually divergent, differing from other HLA‐B alleles by 44–77 nucleotide substitutions. Features that distinguish the B*7301 heavy chain from other HLA‐B heavy chains include multiple substitutions in the α3 domain and a duplication‐deletion within the transmembrane region that increases the length of B*7301 compared to other HLA‐B heavy chains. The duplication‐deletion is shared with subsets of B alleles from the homologous gorilla (Gogo‐B) and chimpanzee (Patr‐B) loci. Other unusual features of B*7301 are individually shared with certain alleles of the HLA‐A, HLA‐C, HLA‐F, Gogo‐B and Patr‐B loci. The B*7301 molecules has sequence elements in common with members of the B7 crossreacting group in the α1 domain and is shown to possess the ME1 epitope, which is held in common with the B7, B22, B27, B42 and B67 antigens. B*7301 has a unique cysteine at position 270 of the α3 domain which appears accessible but probably does not form disulphide‐bonded B*7301 dimers in cell membranes. B*7301 represents a newly discovered but ancient lineage of HLA‐B alleles that appears poorly represented in the modern human population.
Low‐resolution DNA typing for HLA‐B using sequence‐specific primers in allele‐ or group‐specific ARMS/PCR
Authors: Sadler A. M. , Petronzelli F. , Krausa P. , Marsh S. G. E. , Guttridge M. G. , Browning M. J. , Bodmer J. G.
Published: December 2008

Abstract: The products of the human major histocompatibility complex (HLA Class I and II) have historically been detected using serological or cellular assays. With the availability of DNA sequence information for alleles of the HLA system, and with the development of molecular biological techniques it has become possible to tissue type for allelic differences in the HLA genes themselves. We describe here a polymerase chain reaction (PCR) system, based on the principle of the amplification refractory mutation system (ARMS), for low‐resolution DNA typing of the HLA‐B gene. The technique involves a one‐step PCR from genomic DNA using sequence‐specific primers in particular combinations that determine the specificity of each reaction. A low‐resolution primer panel has been designed, based on published HLA‐B gene nucleotide sequences, consisting of 34 sequence‐specific primers (SSP) in 24 PCR reactions which cover all known HLA‐B alleles, to give allele‐specific or group‐specific amplification of DNA fragments of defined size (344–784bp). Advantages of the system are that it can be performed in under 4 hours including DNA extraction, results are easy to interpret and it does not require viable cells.
A rapid method to study the relationship between IDDM and HLA‐DQβ 57 ASP
Authors: Huang Yu‐Won , Lee Long‐Shyong , Shih Mu‐Ching , Pai Young‐Hoi , Lee Yann‐Jinn , Chang Jan‐Gowth
Published: December 2008

Abstract: We have developed a rapid and simple method to detect the relation between HLA‐DQβ 57 Asp and Chinese IDDM patients. The method involved the selective amplification of a DNA fragment from the HLA‐DQ B1 gene by using the mutagenic primers. After PCR, if the HLA‐DQβ 57 was Asp, then there was an artificially created restriction enzyme cutting site. We then can accurately obtain the results by enzyme digestion and electrophoresis. Sixty‐nine IDDM patients and 30 nondiabetic control subjects were analyzed using this method. Twenty‐two (42%) IDDM patients had non‐Asp 57 homozygous, 31 /45%) were Asp/non‐Asp 57 heterozygous, and 9 (13%) had Asp‐57 homozygous. Of the 30 control subjects, the number of cases for these three types were 6 (20%), 18 (60%), and 6 (20%), respectively. The relative risk of homozygous DQ β 57 non‐Asp in our group was 2.9 and the p value was greater than 0.05. Using this kind of approach, we were able to provide a simple, rapid, and non‐radioactive method to detect whether the HLA DQβ 57 was Asp or not.