bluuebunny/crossref_metadata_2025 huggingface dataset:
Polylactosamine sugar chains expressed by epithelia of Henle's loop and collecting duct in rat and human kidney are selectively recognized by human cold agglutinins anti‐I/i
Authors: Raghunath Michael , Grupp Clemens , Neumann Ilka , Heidtmann Antje , Roelcke Dieter
Published: December 2008

Abstract: Normal rat and human kidney was mapped immunohistochemically with human monoclonal cold agglutinins against the biochemically related erythrocyte glycoconjugate antigens I and i, which represent branched and linear polylactosamines, respectively. The antibodies worked well with both cryostat and paraffin‐embedded material. Anti‐i gave specific staining of collecting duct cells (mostly principal cells) as evidenced by double labelling with antibodies to band 3 and a 23 kD protein of intercalated cells. In contrast, anti‐I turned out to be the first exclusive marker for the ascending and descending thin limb of Henle's loop. In addition, in rat kidney, the binding of anti‐I/i was preserved in primary cultures both of principal cells of the papillary collecting duct and cells of the thin limb of Henle's loop. Our results suggest that both antibodies might be interesting tools for in vitro studies of renal cell physiology and the investigation of kidney development.
SSOP typing of the Tenth International Histocompatibility Workshop reference cell lines for HLA‐C alleles
Authors: Levine John E. , Yang Soo Young
Published: December 2008

Abstract: HLA‐C gene products are the most poorly understood of the HLA class I molecules because they express at low level on the cell surface compared to HLA‐A and ‐B. However, recent evidence shows that HLA‐C molecules are functionally competent in eliciting T‐cell responses and in controlling NK‐cell recognition. Approximately 20 to 50% of HLA‐C alleles type “blank” in most populations. To provide a better definition of the HLA‐C alleles, we analyzed 98 extensively characterized B‐cell lines from the 10th International Histocompatibility Workshop. Selective HLA‐C‐specific DNA amplification of exons 2 and 3 from DNA prepared from the cell panel was achieved with the use of two sets of locus‐specific primers. We used 64 sequence‐specific oligonucleotide probes (SSOPs) complementary to variable sites in exons 2 and 3 to generate hybridization patterns. Twenty‐five alleles were found among these patterns, including seven new alleles in the homozygous cell lines and seven potential new alleles in heterozygous cell lines. Differences between the new alleles and known alleles were generally small. Five major groups were identified in the Cw “blank” cells by the SSOP patterns. In addition, linkage between HLA‐B specificities and HLA‐C alleles was similar to previous observations. The present study demonstrated that SSOP typing was effective in identifying new alleles in homozygous typing cells but not in the heterozygous cells. Also, DNA typing can facilitate the identification of all HLA‐C alleles, including those that serologically type as blanks. The HLA‐C locus may be more polymorphic than was previously recognized.
Susceptibility to insulin‐dependent diabetes mellitus and short cytoplasmic ATP‐binding domain TAP2*01 alleles
Authors: Martinez‐Laso Jorge , Martin‐Villa Jose M. , Alvarez Miguel , Martinez‐Quiles Narcisa , Lledo Gregorio , Arnaiz‐Villena Antonio
Published: December 2008

Abstract: TAP2 genes are placed within the HLA complex, have limited genetic variability and encode two main groups of peptide transporter proteins, the so‐called TAP2*01 alleles, with a short ATP‐binding domain, and the TAP2*0201 allele with a long domain. These transporters carry antigenic peptides from cytoplasm across the endoplasmic reticulum membrane to release them into nascent HLA class I molecules, which will then travel towards the plasma membrane. The shorter TAP2*01 alleles are present in 99% of diabetics and 90% of controls; these alleles may add slight, although significant and independent, susceptibility to diabetes, particularly in subjects carrying non‐Asp 57 at βDQ. Moreover, this increased suscpetibilty is not due to linkage disequilibrium with other HLA markers (i.e.: DR4), which does not exist in our Spanish population.
Analysis of HLA‐B*44 alleles encoded on extended HLA haplotypes by direct automated sequencing
Authors: Petersdorf Effie W. , Setoda Tracey , Smith Anajane G. , Hansen John A.
Published: December 2008

Abstract: We developed a PCR‐based approach to sequence exons 2 and 3 of HLA‐B44 alleles from genomic DNA. We applied this method to determine the B44 alleles encoded on extended HLA‐A, B, DRB1, DQB1 haplotypes and the degree of mismatching for B44 alleles among marrow transplant patients and their unrelated donors (URD). A total of 81 samples was studied and included 38 patients, 42 donors and the cell “FMB”; the 80 clinical samples were comprised of 8 unpaired patients, 12 unpaired donors, and 30 URD‐recipient pairs. Three alleles encoding B44 were identified, B*4402 (N=51), 4403 (N=32) and a new allele designated B*44KB and named B*4405 (N=4). Of the 27 patients for whom family study was available, there were 13 different B*4402, 7 different B* 4403 and 2 new B*4405 haplotypes. HLA‐A2, Cw*0501, B*4402, DRB1* 0401, DQB1*0301 (n=2); A2, Cw*0501, B*4402, DRB1*1501, DRB5* 0101, DQB1*0602 (n=2); and HLA‐A29, Cw*1601, B*4403, DRB1* 0701, DQB1*0201 (n=5) comprised the most common patient haplotypes. Of 30 URD‐recipient transplant pairs studied, 27 were HLA‐A, B serologically matched and DRB1, DRB3, DRB5, DQB1 allele matched, and 3 pairs were DRB1‐mismatched. All B44 allele mismatching (N=3) occurred among the 27 matched pairs. The novel B*4402‐variant sequence, HLA‐B*4405, was identified in 4 individuals, and in each case was associated with an HLA‐B44, Cw*02022, DRB1*0101, DQB1*0501 haplotype. HLA‐B*4405 and B*4402 are identical in exon 2; in exon 3 however, B*4405 encodes T instead of G at nucleotide position 75 which translates to a substitution of tyrosine for aspartic acid at codon 116. Finally, the published B*4402 sequence derived from cell “FMB” was found to contain an error; the corrected B*4402 sequence encodes G rather than C at position 146 of exon 3.
Insulin‐dependent diabetes mellitus and the major histocompatibility complex peptide transporters TAP1 and TAP2: no association in a population with a high disease incidence
Authors: Cucca Francesco , Congia Mauro , Trowsdale John , Powis Stephen H.
Published: December 2008

Abstract: Although many studies have established an association between insulin‐dependent diabetes mellitus (IDDM) and the class II region of the human major histocompatibility complex (MHC), it has been difficult to assign susceptibility to a single locus. Recently, two antigen‐processing genes, TAP1 and TAP2, have been identified within the region. Previous studies have reached conflicting conclusions as to the role of these genes in IDDM; it is uncertain whether an increased frequency of the allele TAP2A and a concomitant decrease in TAP2B are independent disease associations or secondary to linkage disequilibrium (LD) between TAP2A and HLA‐DR3. To further investigate this question, we have characterized TAP1 and TAP2 alleles in 129 IDDM patients from Sardinia, a population with limited genetic heterogeneity and a high disease incidence. When compared to 90 random controls, the only significant difference was a decrease in the minor allele TAP2C in patients. However, when HLA‐DR and ‐DQ matched controls were compared, this difference disappeared. Further analysis suggested that TAP2C was in LD with HLA‐DRB1*1401 and subtypes of HLA‐DRB1*11, alleles which were not observed in the IDDM population. LD was also observed between other TAP and HLA‐DR alleles, in particular between TAP2A and HLA‐DR3 in both patients and controls. Our data supports the conclusion that there is no primary association between TAP2 alleles and IDDM, and that previously reported associations may be due to LD with other class II loci.
Selection of unrelated bone marrow donors by PCR‐SSP typing and subsequent nonradioactive sequence‐based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles
Authors: Knipper A. J. , Hinney A. , Schuch B. , Enczmann J. , Uhrberg M. , Wernet P.
Published: December 2008

Abstract: HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR‐SSP (PCR‐sequence‐specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA‐DRB identity by PCR‐SSP analysis. After solid‐phase single‐strand separation, direct sequencing of the allele‐ or group‐specific DRB amplified products was performed by applying fluorophor‐labelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group‐specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor‐recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.
Characterization of natural peptide ligands for HLA‐B*4402 and ‐B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA‐B44 heavy chain
Authors: Fleischhauer K. , Avila D. , Vilbois F. , Traversari C. , Bordignon C. , Wallny H.‐J.
Published: December 2008

Abstract: This study describes the characterization of endogenous peptides associated with the two major subtypes of HLA‐B44. The two subtypes differ for a single amino acid substitution from Asp (HLA‐B*4402) to Leu (HLA‐B*4403) in position 156 of the α2 domain, causing strong alloreactivity in vivo. In order to study the involvement of peptides in this phenomenon, the peptide motifs of the two subtypes were determined from natural peptide pools using Edman degradation. The motif was found to be essentially identical for HLA‐B*4402 and ‐B*4403, with a strong predominance for Glu at position 2, Tyr or Phe at positions 9 and 10 and hydrophobic residues, especially Met, at position 3. Two individual naturally processed ligands of HLA‐B*4403 were sequenced and shown to be derived from intracellularly expressed proteins found in protein sequence databases. The sequence of these natural peptide ligands conform well to the determined motif. These data will allow the prediction of HLA‐B44 restricted peptide epitopes from viral and tumor antigens of known amino acid sequences. Moreover, they indicate that the peptide repertoire presented by HLA‐B*4402 and ‐B*4403 is very similar, suggesting that the strong alloresponse between these two subtypes is not due to presentation of a different set of self peptides.
High resolution HLA‐DRB1 SSP typing for cadaveric donor transplantation
Authors: Savelkoul Paul H. M. , de Bruyn‐Geraets Daisy P. , van den Berg‐Loonen Ella M.
Published: December 2008

Abstract: An HLA‐DRB1 typing procedure by means of sequence‐specific primer (SSP) amplification was developed for 65 different DRB1 subtypes. Subtyping is achieved by the performance of two subsequent PCR assays (PCR‐1 assay and PCR‐2 assay) using a limited number of reactions. The PCR‐1 assay determined low‐resolution HLA‐DRB1 typing, i.e. the serologically defined specificities DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9 and 10. The second exon of the DRB1 gene is amplified also in this PCR‐1 assay. High‐resolution subtyping for positively identified alleles was performed in the PCR‐2 assay with the exon‐2 product from PCR‐1 assay as DNA template. PCR reactions were carried out using unpurified primers in reaction volumes of 20 μ1 and 100 ng of chromosomal DNA. After 3 hours, the results of the PCR‐1 assay were analyzed and subsequently subtyping results in the PCR‐2 assay were obtained in another 1.5 hours. A total of 249 DNA samples was typed by this method. No false positive nor false negative results were obtained in DRB1 typing of 32 homozygous cell lines, 56 serologically well‐defined panel cells and 125 unrelated individuals. Segregation of the amplification patterns was investigated in 36 members of 7 two‐generation families. DRB1 subtyping revealed codominant Mendelian segregation for all subtypes investigated. In conclusion, LR‐HR‐PCR‐SSP typing is a fast and reliable typing technique for routine DNA typing purposes which gives complete DRB1 subtyping within 4.5 h. Besides low‐resolution DRB typing, also high‐resolution DRB subtyping for prospective HLA‐DR matching in cadaveric renal transplantation is possible by this method.
Significance of the HLA‐DQB matching in one‐haplotype identical kidney transplant pairs and the matching analysis by the polymerase chain reaction (PCR) — heteroduplex — polymorphism method
Authors: Fukuda Y. , Kimura A. , Hoshino S. , Tashiro H. , Furukawa M. , Shintaku S. , Hori H. , Sasazuki T. , Dohi K.
Published: December 2008

Abstract: Sixty‐five living related kidney transplant pairs were analyzed for matching at HLA class II loci by the polymerase chain reaction (PCR)‐ sequence specific oligonucleotide probe (SSOP) method. The retrospective HLA matching study revealed that there were many early graft loss cases despite the DQB compatibility, contrary to our expectation. There were 54 DRB1 one‐mismatched cases, in which 7 of the 11 (64%) DQB zero‐mismatched cases had lost their grafts, while the graft loss cases were only 10 of the 43 (23%) DQB one‐mismatched pairs (P value=0.0006). The DQB matching of these cases was studied in detail, because the PCR‐SSOP methods are based on the detection of sequence polymorphisms in a relatively narrow range, i.e., recognized sequences by SSOPs. The PCR ‐ heteroduplex ‐ polymorphism analysis method was developed to analyze the polymorphism in exon 2 of the DQB1 gene. However, all the pairs proved to be compatible for the DQB, demonstrating that the DQB compatibility was associated with a harmful influence on the graft outcome. These observations suggested that the DQB1 incompatibility might exert the low responsiveness to HLA haplo‐identical allogeneic transplants.
Major histocompatibility complex class II and C4 alleles in Mexican Americans with systemic lupus erythematosus
Authors: Reveille John D. , Moulds Joann M. , Arnett Frank C.
Published: December 2008

Abstract: Few data exist on associations of class II and class III alleles of the major histocompatibility complex (MHC) and susceptibility to systemic lupus erythematosus (SLE) in Mexican Americans, a group of predominantly mixed Spanish and Native American ancestry. Therefore, MHC class II alleles (HLA‐DRB1, DQA1, DQB1, DPA1 and DPB1 alleles) and C4 allotypes were determined in 52 Mexican American SLE patients and 105 ethnic‐matched controls. HLA‐DRB1*0301 and C4A*Q0 were each increased in the SLE patients, especially HLA‐DRB1*0301 in those with anti‐Ro/SSA autoantibodies. C4A*Q0 was associated with HLA‐DRB1*0301 only in a minority of patients and controls. Anti‐U1‐RNP antibodies were significantly associated with the presence of HLA‐DQB1*0302, and the risk for the production of anti‐Ro antibodies was heightened by the presence of at least three (out of four possible) DQA1 chains possessing a glutamine at position 34 and/or DQB1 chains a leucine at position 26 of their outermost domains. Thus the HLA class II and C4 null allele associations that have been noted in other ethnic groups are also found in Mexican Americans, suggesting shared susceptibility factors across ethnic lines in predisposition to SLE.
Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302
Authors: Viken H. D. , Thorsby E. , Gaudernack G.
Published: December 2008

Abstract: To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA‐DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B‐lymphoblastoid cell lines (B‐LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302‐3, 0401‐2, 0501‐3, 0601‐4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45–47 of the DQ β chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQP1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B–LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1 *0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39–46 of DR‐and DQ‐ and 37–44 of DP p chains.
Analysis of HLA‐A, C, B, DR and DQ loci in an Italian sample of possible Celtic origin
Authors: Matullo G. , Griffo R.M. , Mangione A.M. , Cappello N. , Rendine S. , Piazza A.
Published: December 2008

Abstract: Trino Vercellese, a village of Piedmont (Italy), was selected with the aim at reconstructing the genetic history of a putative Celtic sample known to be settled in Italy with the name of Rigomagus since pre‐roman times. The HLA‐A, Cw, B, DR and DQ antigens of 101 unrelated individuals have been typed. The antigens characterizing this sample for their higher frequency are shown to be A3, All, A32, B35, B39, Bw52, Cw4, DRw11, DRw13, DQw7. Gene frequencies are estimated by maximum likelihood and Hardy‐Weinberg equilibrium was tested with no deviant genetic locus. Two‐locus haplotype frequencies were also estimated and those with significant associations tabulated. “Extended” haplotypes were reconstructed: the three most frequent haplotypes (covering a total frequency of 11.5%) share the same Cw, B, DR and DQ alleles. Comparisons with other Italian and European samples are indicated to challenge archeological evidence of a pre‐roman genetic stratification of the people living in our old Rigomagus.
Direct sequencing of SSP‐PCR‐amplified cDNA to identify new alleles in the DR52‐associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319
Authors: Robbins F. , Tang T. , Yao H. , Ng J. , Hartzman R. J. , Hurley C. K.
Published: December 2008

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.
Comparative analysis of HLA polymorphism at the serologic and molecular level in Moroccan and Ashkenazi Jews
Authors: Roitberg‐Tambur A. , Witt C. S. , Friedmann A. , Safirman C. , Sherman L. , Battat S. , Nelken D. , Brautbar C.
Published: December 2008

Abstract: The Jewish people comprise two major groups, one encompassing the Jews of Ashkenazi (Central and Eastern European) origin and the other including those of Sephardic (Middle Eastern and North African) descent. To the latter belong the Jews of Moroccan stock, who form the largest Jewish subgroup among the non‐Ashkenazi population living in Israel. As the members of each of these groups differ in physiognomy and life style, it was of interest to investigate whether these differences are also reflected in their respective HLA compositions. To this end, 132 subjects of Ashkenazi and 113 individuals of Moroccan origin residing in Israel were tested and the results compared with data for other populations made available by the 11th International Histocompatibility Workshop. Comparison between their HLA profiles and those of non‐Jews revealed that the Jewish groups in some aspects resembled one another but in others showed disparities. The dissimilarities between the various groups are expressed in terms of gene and haplotype frequencies, as well as in HLA‐disease associations (as for example rheumatoid arthritis, erosive lichen planus, primary Sjögren's syndrome, pemphigus vulgaris). However, both Jewish groups shared some unique features with respect to HLA class II allelic frequencies, pointing to a common ancestry.
Determination of HLA class II alleles by genotyping in a Manchu population in the northern part of China and its relationship with Han and Japanese populations
Authors: Geng L. , Imanishi T. , Tokunaga K. , Zhu D. , Mizuki N. , Xu S. , Geng Z. , Gojobori T. , Tsuji K. , Inoko H.
Published: December 2008

Abstract: The genetic polymorphism of the HLA class II loci was investigated in a Manchu population resident in the northern part of China and compared with those of other Asian populations including Japanese and Han. In 8 DQA1 alleles, the most frequent allele was DQA1*03 with the gene frequency of 25.5%. Of 15 DQB1 alleles tested, 11 were observed and the most common allele was DQB1*0301 with the gene frequency of 24.5%. Among 19 DPB1 alleles, 11 were detected and DPB1*0501 (43.8%) was the most frequent allele as observed in other Asian populations such as Japanese, Chinese and Korean. Of 43 DRB1 alleles tested, 21 were detected and DRB1*0901 (14.0%), *1501 (11.0%), *1201 (11.0%), *07 (9.0%) and *1401 (9.0%) were highly predominant and account for the high frequencies of DR9, DR2, DR5, DR7 and DR6. In the DRB3 gene (DR52), DRB3*0202 (18.0%) was the most frequent. With respect to the DRB4 gene (DR53), the gene frequency of DRB4*0101 was 35.0%. Of 3 DRB5 alleles detected, DRB5*0101 (11.0%) was highly predominant. Comparison of HLA class II allele frequencies in Manchu with those in Japanese and Han Chinese populations (South & North) detected some significant differences and genetic divergence between these Oriental populations. The dendrogram constructed by the neighbor‐joining (NJ) method based on the allele frequencies of DQA1, DQB1, DPB1 and DRB1 of 10 representative populations over the world suggested that Manchu is the closest, but at the same genetic distance to both Northern and Southern Han Chinese.
Increased serum concentration of soluble HLA‐DR antigens in HIV infection and following transplantation
Authors: Filaci G. , Contini P. , Brenci S. , Lanza L. , Scudeletti M. , Indiveri F. , Puppo F.
Published: December 2008

Abstract: HLA class I and class II antigens circulate in serum as soluble molecules. Increased concentrations of soluble HLA class I molecules have been demonstrated in viral diseases, in rejection episodes following organ transplantation and in graft versus host disease. To explore the possibility of a variation of the serum concentrations of soluble HLA class II molecules in the same pathologic conditions we developed a double determinant immune assay that detects whole soluble HLA‐DR molecules (sHLA‐DR). The mean level of sHLA‐DR antigens in sera from 23 healthy individuals was 0.64±0.72 μg/ml. Elevated serum concentrations of sHLA‐DR molecules were detected in sera from HIV infected patients in CDC2/3 and in CDC4C1 stages (2.0±1.7 m̀g/ml and 4.6±1.7 m̀g/ml, respectively), in sera from patients affected by acute rejection after liver transplantation (5.3±3.7 μg/ml) and in sera from patients affected by severe acute graft versus host disease following bone marrow transplantation (8.8±3.1 μg/ml). The increase of sHLA‐DR molecules in these sera significantly correlated with the elevation of soluble HLA class I antigens (P=0.0004). The reported data suggest that both soluble HLA class I and class II molecules serum levels increase during viral infections and strong immune reactions and could suggest the involvement of these molecules in immunoregulation.
Molecular analysis of HLA class II polymorphism in Croatians
Authors: Grubić Z. , Ẑunec R. , Naipal A. , Kaŝtelan A. , Giphart M. J.
Published: December 2008

Abstract: HLA‐class II polymorphisms have been studied in a population of 141 unrelated healthy Croatians using PCR amplification, followed by non‐radioactive oligonucleotide hybridization. Thirty one DRB1, 8 DQA1, 13 DQB1 and 16 DPB1 alleles were found in the tested population. DRB1*1601, 0701, 1501, 0101 and 1104 are the most frequent alleles at the DRB1 locus. At the DQA1 locus two alleles predominate: DQA1*0501 and 0102, while the most frequent DQB1 allele is *0301. Analysis of HLA‐DPB1 polymorphism showed that, as in other Europeans, DPB1* 0401 is the most frequent allele. Four different two locus haplotypic associations (DRB1‐DRB3, DRB1‐DRB5, DRB1‐DQB1 and DQA1‐DQB1) as well as three locus DRB1‐DQA1‐DQB1 haplotypic associations were assigned on the basis of known linkage disequilibria. Several unusual two‐locus associations have been observed: DRB1*0301‐DRB3* 0202, DRB1*1501‐DRB5*02, DRB1*1601‐DRB5*0101, DRB1*1502‐DRB5*0101, DQA1*0103‐DQB1*0503 and DQA1*0501‐DQB1*0302. Among 236 examined DRB1‐DQA1‐DQB1 haplotypic combinations, the most frequent was DRB1*1601‐DQA1*0102‐DQB1*0502 that was found with statistically significant higher frequency than in other Europeans. Twenty‐eight distinct probable haplotypes were observed just once, suggesting that the main characteristic of Croatian population is great heterogeneity of haplotypes. This study will serve as a reference for further anthropology studies, HLA and disease associations studies and for donor/recipient matching in organ and bone marrow transplantation.
Linkage disequilibrium in HLA cannot be explained by selective recombination
Authors: Termijtelen A. , D'Amaro J. , van Rood J. J. , Schreuder G. M. T.
Published: December 2008

Abstract: Some combinations of HLA‐A,‐B and ‐DR antigens occur more frequently than would be expected from their gene frequencies in the population. This phenomenon, referred to as Linkage Disequilibrium (LD) has been the origin of many speculations. One hypothesis to explain LD is that some haplotypes are protected from recombination. A second hypothesis is that these HLA antigens preferentially recombine after cross‐over, to create an LD haplotype. We tested these 2 hypotheses: from a pool of over 10,000 families typed in our department, we analyzed 126 families in which HLA‐A:B or B:DR recombinant offspring was documented. To overcome a possible bias in our material, we used the non‐recombined haplotypes from the same 126 families as a control group. Our results show that the number of cross‐overs through LD haplotypes is not significantly lower then would be expected if recombination occurred randomly. Also the number of LD haplotypes created upon recombination was not significantly increased.
Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments
Authors: Gilliland L. K. , Norris N. A. , Marquardt H. , Tsu T. T. , Hayden M. S. , Neubauer M. G. , Yelton D. E. , Mittler R. S. , Ledbetter J. A.
Published: December 2008

Abstract: Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single‐chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2–3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly‐G tailing the first strand cDNA followed by anchor PCR with a forward poly‐C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL‐link‐VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge‐CH2‐CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti‐human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide <tag< or in a tail‐less form. Expression of CD3 (G19–4) sFv tail‐less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.
Large‐scale DRB and DQB1 oligonucleotide typing for the NMDP registry: progress report from year 2
Authors: Ng J. , Hurley C. K. , Carter C. , Baxter‐Lowe L. A. , Bing D. , Chopek M. , Hegland J. , Lee T. D. , Li T. C. , Hsu S. , KuKuruga D. , Mason J. M. , Monos D. , Noreen H. , Rosner G. , Schmeckpeper B. , Dupont B. , Hartzman R. J.
Published: December 2008

Abstract: DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence‐specific oligonucleotide probes and polymerase chain reaction amplified DNA has been 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 second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence‐specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laboratories.
HLA‐DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland
Authors: Michalski J. P. , McCombs C. C. , Arai T. , Elston R. C. , Cao T. , McCarthy C. F. , Stevens F. M.
Published: December 2008

Celiac disease (CD) has one of the strongest class II HLA associations of any human illness. We used DNA‐RFLP typing to study the class II HLA genotypes of celiac disease patients from the West of Ireland, the geographic area with the highest rate of celiac disease in the world. We confirmed the high frequency of HLA‐DR3 in this population, and we were also able to demonstrate the additional risk of developing celiac disease imparted by HLA‐DR7. This was done by clearly distinguishing DR7, DQ2 haplotypes from DR7, DQ9 haplotypes, and by “subtraction analysis” of haplotype frequencies. As reported in other populations, most of the patients without DR3 were heterozygous for DR7 and DR11 or 12 (DR5), or had DR4. We used PCR‐RFLP and direct sequencing of amplified DNA to examine HLA‐DR4 subtypes. The frequency of HLA‐DR4 was markedly decreased in patients compared with controls (p=0.000001) and there was a significant alteration of DR4 subtypes of the patients compared with controls (p=0.0227). Moreover, all of the CD patients (5 of 5) with DR4 had a haplotype associated with the DQB1*0302 allele compared with only 11 of 23 control subjects with DR4. Our results in this population with exceptionally high risk of CD strongly support the DQ heterodimer hypothesis and suggest that the recently described sequence difference between the DQB1*02 alleles of DR3 and DR7 may contribute to a synergistic increased risk when these haplotypes are inherited together. In addition, our findings suggest a role for HLA‐DQ in DR4‐associated CD.
HLA‐B alleles of the Navajo: no evidence for rapid evolution in the Nadene
Authors: Garber T. L. , McAdam S. N. , Butler L. M. , Crocker P. , Piekarczyk M. , Troup G. M. , Milford E. L. , Watkins D. I.
Published: December 2008

New HLA‐B locus alleles have been found in South American Amerindian populations but were largely absent in North American Amerindian tribes also descended from this first Paleo‐Indian migration. We have now extended these studies to the Navajo, descendants of the second Nadene migration. No new functional alleles were found at the B locus of this tribe. This limited study supports the notion that while new B locus variants are common in South American Amerindians, it is more difficult to find new B locus alleles in North American native peoples. Whether this dichotomy is due to differences in pathogen environment and/or population structures between North and South America remains a subject of speculation.
Primate DRB6 gene expression and evolution: a study in Macaca mulatta and Cercopithecus aethiops
Authors: Paz‐Artal E. , Corell A. , Varela P. , Martinez‐Laso J. , Gomez‐Casado E. , Fernandez‐Soria V. M. , Moreno M. A. , Arnaiz‐Villena A.
Published: December 2008

DRB6 has been found to be transcribed in human and apes. Promoter region and exon 1 come from a 5‘ LTR from a mammary tumour retrovirus. However, the putative protein structure would be very different to other DR molecules and it is doubtful that it may function as an antigen presenting molecule. Primate DRB6 alleles previously published together with the two new macaque sequences reported here support the existence of a strong selective pressure working on exon 2 to generate stop codons at the end of the exon (between codons 74 and 94) during at least 23 million years. The topology of dendrograms constructed with different primate DRB6 alleles supports the “trans‐species” evolution proposed for MHC class I, class II and possibly C4 genes. Finally, DRB6, which is one of the oldest DRB genes, has been lost in the HLA‐DRB3 (or DR52) group of haplotypes (DR3, DR5, DR6 and DR8) and a small DRB6 sequence is present at the exon 2 first hypervariable region of DRB4 (or DR53) gene, which is present in DR4, DR7 and DR9 haplotypes.
Human thymic epithelial cells express functional HLA‐DP molecules
Authors: Jørgensen A. , Röpke C. , Nielsen M. , Madsen H. , Svejgaard A. , Ødum N.
Published: December 2008

HLA‐DP molecules function as restriction elements in the presentation of foreign antigens to T cells by antigen presenting cells and certain HLA‐DP molecules confer susceptibility to autoimmune disease. Because HLA molecules play an essential role in thymic selection and elimination of autoreactive T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA‐DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA‐DP molecules. The expression of HLA‐DP molecules was comparable to or higher than the expression of HLA‐DQ but lower than that of HLA‐DR. Upon IFN‐γ treatment, HLA‐DP expression was strongly upregulated. Since HLA‐DQ and DR expression was upregulated in parallel, the hierarchy between MHC class II isotypes remained unchanged following interferon treatment. TEC elicited significant proliferation of HLA‐DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN‐γ treatment strongly upregulated the HLA‐DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels of functional HLA‐DP molecules.
Characterization of a gastric tumor cell line defective in MHC class I inducibility by both α‐ and γ‐interferon
Authors: Abril E. , Mendez R. E. , García A. , Serrano A. , Cabrera T. , Garrido F. , Ruiz‐Cabello F.
Published: December 2008

α/β and γ type interferons (IFN), act through distinct cell surface receptors and induce transcription of an overlapping sets of genes. MHC class I genes are inducible by both type of interferons. We have analyzed a gastric tumor cell line, AGS, which was completely defective in MHC class I response to interferon‐α and ‐γ. Northern blot analysis demonstrated that the lack of IFN response was related with the absence of up‐regulation of specific HLA class I mRNA. Electrophoretic mobility shift assays in various tumor cell lines after IFN‐α and IFN‐γ treatment showed differential binding of the transcriptional factors to MHC class I regulatory elements. Comparison of k‐B binding activity showed that IFN‐α and IFN‐γ induced opposite changes in NF‐kB binding activity in AGS cells, indicating that the absence of MHC class I response in AGS appears to be independent of k‐B activity. In contrast, there were remarkable differences in the level of transcriptional factor binding to an interferon‐responsive sequence element (IRSE), between AGS and other interferon‐responsive tumor cell lines. This result suggests that the low level of transcriptional factor binding to IRSE in AGS cells was responsible of the lack of induction of MHC class I antigens. In this context, overlapping factors in the signal transduction pathway of both type I and II interferons may be involved in the non‐responsiveness of this gastric carcinoma tumor cell line.
Characterization of the peptide‐binding specificity of HLA‐B*7301
Authors: Barber L. D. , Percival L. , Parham P.
Published: December 2008

Previous studies showed the human MHC class I heavy chain HLA‐B*7301 has a sequence very divergent from other class I alleles. Despite the unusual sequence, we predicted B*7301 would retain the peptide‐binding function typical of other HLA‐A, B and C glycoproteins, and sequence similarity to B*2705 in a region of the peptide‐binding site known as the B pocket suggested B*7301 would bind peptides with Arg at position 2. To test this hypothesis, the peptide‐binding specificity of B*7301 was investigated. Sequence analysis of peptides bound endogenously by B*7301 indeed found selectivity for nonamer peptides possessing Arg at position 2 and a preference for small nonpolar residues such as Pro or Ala at the C terminus was also revealed. B*7301 therefore possesses the potential to function as a conventional antigen presenting class I glycoprotein. Functional similarities between B*7301 and B*2705 are discussed in the context of the association of B*27 subtypes with susceptibility to ankylosing sponylitis and arthritic diseases.
Tumor associated antigens in human renal cell carcinoma: MHC restricted recognition by cytotoxic T lymphocytes
Authors: Bernhard H. , Jäger E. , Maeurer M. J. , zum Büschenfelde K. H. Meyer , Knuth A.
Published: December 2008

Based on previous studies in human melanoma leading to the molecular cloning of genes encoding peptide antigens recognized by MHC‐restricted cytotoxic T lymphocytes (CTL) we extended our efforts to renal cancer systems established in tissue culture. In two patients we obtained stable CD8+ CTL clones with high cytolytic activity for the corresponding autologous tumor cell line in vitro. Neither autologous EBV‐transformed B lymphocytes or PHA‐activated PBL nor natural killer targets K562 were lysed by these CTL clones. MZ1257‐RCC CTL5‐30 lysed autologous tumor cells as well as normal kidney cell cultures in an HLA‐A2 restricted fashion. Further specificity analysis showed cross reactivity with HLA‐A2 matched renal cancer cell lines, melanoma cell lines and tumor cell lines of other origins. HLA‐A2 negative target cells were not lysed. The restriction element for T cell recognition in another renal cancer system, MZ1973‐RCC, appeared to be HLA‐B8 as tested with CTL 5–10. Crossreactivity of CTL 5–10 was documented with one HLA‐B8 positive RCC line. Tumor cell lines of other origin were not lysed by CTL 5–10. For further study of CTL‐defined epitopes, peptides were extracted from tumor cell lines. Peptides from cultured cell lines were acid‐eluted and fractionated by reversed phase HPLC. The peptide fractions were tested in cytotoxicity assays for their ability to reconstitute RCC epitopes by addition to the HLA‐A2. 1 positive antigen processing mutant cell line 721.124xCEM.T2. One HPLC peak was identified containing an epitope for the HLA‐A2 restricted MZ1257‐RCC CTL 5–30. These peptide epitopes were present in the autologous renal cancer cell line MZ1257‐RCC as well as in an allogeneic HLA‐A2 positive RCC cell line MZ1973 and melanoma cell line SK29‐MEL, but were absent in peptides eluted from autologous EBV‐B cells. We conclude that renal cell carcinoma may induce autologous CTL. Epitopes recognized by MHC‐restricted CTL on RCC may be shared by HLA‐matched allogeneic renal cancer or even tumors of other origin, such as melanoma. The current challenge is the determination of the amino acid sequences of these CTL‐defined peptides as a step to further characterize the nature of these tumor associated antigens.
A functional monoclonal antibody recognizing the human alpha 1‐integrin I‐domain
Authors: Fabbri M. , Castellani P. , Gotwals P. J. , Kotelianski V. , Zardi L. , Zocchi M. R.
Published: December 2008

αβ1 heterodimer is a member of the integrin receptor superfamily that has been described to be involved in cell‐matrix binding through its interaction with collagens, fibronectin and laminin. The αl integrin belongs to a subset of I‐domain containing integrins that includes αM, αL, αX and α2. In this study we describe an anti‐αl mAb (FBI2) that recognizes an epitope located in the human αl I‐domain, since the mAb can bind to human, but not to rat, recombinant I‐domain GST fusion protein. FBI2 mAb efficiently and specifically inhibits the binding of activated human lymphocytes to laminin, collagen and fibronectin. These data support the notion that the αl I‐domain itself has an important role in receptor‐ligand binding. In particular, we show that al inte‐grin‐dependent lymphocyte adhesion to fibronectin is I‐domain mediated, at variance with the RGD‐dependent adhesion which seems to be mediated by the βl rather than the αl integrin chain. Lastly, the overexpression of the αl‐integrin by stromal cells and blood vessels of solid tumors may suggest a role for this integrin in tumor biology.
CD69 expression on primitive progenitor cells and hematopoietic malignancies
Authors: Tassone P. , Turco M. C. , Tuccillo F. , Bonelli P. , Morrone G. , Cecco L. , Cerra M. , Bond H. , Nicola M. Di , Gianni A. M. , Venuta S.
Published: December 2008

CD69 is an early activation antigen of peripheral blood lymphocytes and is constitutively expressed on a wide variety of bone marrow‐derived cells. To further characterize the distribution and understand the potential biological role of the molecule in normal and malignant hematopoiesis, we used a novel high affinity anti‐CD69 mAb (UN6) and analyzed hematopoietic progenitor cells together with a panel of myeloid and lymphoid malignancies. We report that mobilized peripheral blood CD34+ cells display detectable levels of CD69 and that the density of membrane expression correlates with the immature phenotype CD34bright Thy‐1bright cells. Furthermore, during cytokine‐induced differentiation, the expression of CD69 is moderately down‐regulated. Analysis of hematopoietic malignancies revealed that CD69 expression correlates with the immature myeloid phenotype. Taken together these data suggest a role of CD69 during the early phase of hematopoiesis and in the leukemic transformation.
Sequence‐specific priming and exonuclease‐released fluorescence detection of HLA‐DQB1 alleles
Authors: Faas S. J. , Menon R. , Braun E. R. , Rudert W. A. , Trucco M.
Published: December 2008

Molecular typing of HLA DQB1 alleles, employing sequence‐specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence‐based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP‐based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single‐base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence‐specific priming and exonuclease‐released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel‐based approaches are difficult to perform. This technique may also be useful for small‐scale class I and class II molecular typing in clinically oriented laboratories.
HLA‐C typing of eleven Papua New Guineans: identification of an HLA‐Cw4/Cw2 hybrid allele
Authors: Little A. M. , Mason A. , Marsh S. G. E. , Parham P.
Published: December 2008

HLA‐C polymorphism of 11 individuals from Papua New Guinea was studied by serology and DNA typing (SSP ARMS‐PCR). To resolve certain discrepancies HLA‐C alleles were cloned and sequenced. Five alleles were identified by sequencing, four of which; Cw*0304, Cw*0401, Cw*12022 and Cw*1502 have been identified previously in other populations. The fifth allele, which was found in four individuals is a novel HLA‐C allele. The new allele, called HLA‐Cw*0403 is most similar to HLA‐Cw*0401, differing by 10 nucleotides, 9 of which are located in the region from nucleotide 98 to 218. This region of Cw*0403 is identical to both HLA‐Cw*0201 and Cw*02022. The 9 nucleotide differences between Cw*0401 and Cw*0403 result in 6 amino acid differences in the α1 domain. These amino acids in Cw*0403 may contribute to the serological typing of some, but not all Cw*0403 expressing cells. The Final difference between Cw*0401 and Cw*0403 is a coding substitution at nucleotide 979 in exon 5. The guanine found in Cw*0403 is identical to all HLA‐C alleles except HLA‐Cw*0401, which has an adenine. The Cw*0403 allele was most likely formed by a gene conversion event between Cw*02 and Cw*04, involving a minimum of 121 to a maximum of 215 nucleotides.