bluuebunny/crossref_metadata_2025 huggingface dataset:
DR4 subtypes and their molecular properties in a population‐based study of Swedish childhood diabetes
Authors: Sanjeevi C. B. , Höök P. , Landin‐Olsson M. , Kockum I. , Dahlquist G. , Lybrand T. P. , Lernmark Å.
Published: December 2008

The aim of this study was to determine the association between childhood insulin‐dependent diabetes mellitus (IDDM) and HLA‐DR4 subtypes and to test in a population‐based investigation whether the DR4 association has an effect independent to that of DQ. First, HLA genotyping identified DR4 in 337/425 (79%) patients and 148/367 (40%) controls (Odds Ratio 5.67; p<0.01). Second, a total of 14 DR4 subtypes were detected by PCR and sequence specific oligo probes. Only two DR4 subtypes, DRB1*0401 (62% patients and 25% controls; OR 4.95, p<0.01) and *0404 (16% patients and 10% controls; OR 1.67, p<0.05) were however positively associated with the disease. These two subtypes were positively associated only when linked to DQB1*0302‐DQA1*0301 (DQ8) (56% patients and 14% controls; OR 7.69, p<0.01; 15% patients and 10% controls; OR 1.55, p<0.05, respectively). When DRB1*0401 was linked to DQB1*0301‐DQA1*0301 (DQ7) (6% patients and 11% controls; OR 0.52, p<0.05), this DR4 subtypes was negatively associated with IDDM. Third, tests of strongest association allowed the following ranking of alleles or haplotypes: DQB1*0302‐DQA1*0301 (DQ8) >DQB1*0302 > DRB1*0401 >DRB1*0404 and the association of DRB1*0401 has a significant effect in DQ8 positive IDDM patients. We conclude that the DR4 association with IDDM is secondary to DQ by linkage disequilibrium, which support the role of HLA‐DQ as a primary genetic risk factor for IDDM.
HLA‐A9 antibodies and epitopes
Authors: Fussell H. , Thomas M. , Street J. , Darke C.
Published: December 2008

Fifty pregnancy alloantisera directed towards HLA‐A23 and/or A24 antigens were investigated serologically in titration studies against the three sequenced HLA‐A9 specificities, A23 (A*2301), A24 (A*2402) and A2403 (A*2403). The reaction patterns of the antisera fell into five categories which allowed the three HLA‐A9 specificities to be easily differentiated. Based on the various titre cytotoxicity scores of the antisera five possible antibody specificities were defined: anti‐A23; ‐A24; ‐A23/24; ‐A24/2403 and anti‐A23/24/2403. One antiserum crossreacted with HLA‐A1 and A24. Inspection of the amino acid sequences of 136 HLA‐A, B and C molecules allowed the prediction of five unique epitopes corresponding to these antibody specificities, a possible epitope unique to A2403 and confirmation of a likely epitope shared by A1 and A24. These, together with the previously suggested epitopes HLA‐A9/ A2/A28 and A1/A23/A24 together with the presence of Bw4 on the three HLA‐A9 antigens suggests that the HLA‐A9 family of antigens is characterized by a minimum of nine serologically definable epitopes.
Haplotypic association of two new HLA class I alleles: Cw*15052 and B*0706: evolutionary relationships of HLA‐Cw*15 alleles
Authors: Sanz L. , Vilches C. , de Pablo R. , Bunce M. , Moreno M. E. , Kreisler M.
Published: December 2008

A novel HLA‐Cw*15, B7 haplotype has been found in Caucasians. Molecular cloning studies demonstrate that this haplotype is constituted by the new alleles Cw*15052 and B*0706, which seem to be intermediate steps in the diversification of their respective allelic families. A pathway for the evolution of Cw*15 alleles is proposed.
Molecular and functional phenotypes of melanoma cells with abnormalities in HLA Class I antigen expression
Authors: Wang Z. , Margulies L. , Hicklin D. J. , Ferrone S.
Published: December 2008

Analysis of melanoma cell lines with abnormalities in HLA Class I antigen expression has identified two serological phenotypes caused by distinct molecular defects. One is characterized by lack of HLA Class I antigen expression which is not induced by IFN‐γ or by incubation at 25°C for 24 hrs. This phenotype reflects structural changes in the β2m gene which interfere with its transcription and/or translation or result in the synthesis of a defective β2‐μ polypeptide unable to associate with HLA Class I heavy chains. The other phenotype manifests very low HLA Class I antigen expression which is enhanced by IFN‐γ or by incubation at 25°C for 24 hrs. This phenotype reflects abnormalities in TAP heterodimer expression, which cause defects in stable assembly and intracellular transport of the HLA Class I antigen trimolecular complex. Loss of HLA Class I antigens renders melanoma cells resistant to lysis by HLA Class I antigen‐restricted cytotoxic T cells which specifically recognize melanoma associated antigens. Therefore, abnormalities in HLA Class I antigen expression may have a negative impact on the outcome of T cell based immunotherapy. Characterization of the molecular defects underlying loss of HLA Class I antigens may suggest approaches to restore their expression. Inclusion of these approaches in the protocols of T cell based immunotherapy may improve its efficacy.
Immunodominant deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro
Authors: Van Waes C. , Monach P. A. , Urban J. L. , Wortzel R. D. , Schreiber H.
Published: December 2008

Variant cancer cells which arise from the parent tumor during tumor progression can escape immunity but retain antigens. We have mixed highly immunogenic (A+B+) murine parental cancer cells with less immunogenic (A‐B+) variant cancer cells to construct a model of a cancer containing escape variants. When such mixtures of cancer cells were injected into normal mice, the variant cells grew out because immune responsiveness to the B antigen on the variant was hindered by dominance of the A antigen on the surrounding parental tumor cells. However, A‐B+ variant cells inoculated alone at a separate site induced B specific cytolytic T cells and were rejected. Moreover, mice immunized with A‐B+ cells rejected a challenge which contained a mixture of variant and parental cancer cells, while immunization with A+B+ cells was ineffective. Thus, variant tumor cells selected from parental tumor cells by cytolytic T cells in vitro can be used to induce protective immunity against variants expected to escape tumor immunity in vivo. The immunodominance of the A antigen may be related to its ability to induce a much more rapid CTL response than the B antigen, since we show in another model that the preexistence of a CTL response to one antigen prevented the subsequent induction of CTL to another antigen injected at the same site, even if both antigens were equally efficient at inducing CTL. These results indicate that immunodominance can affect strong as well as weak antigens. Vaccination with individual antigens at separate sites rather than with multiple antigens at one site may, therefore, be needed to prevent tumor escape and tumor recurrence or to counteract infectious diseases.
Expression of MHC Class II and B7–1 and B7–2 costimulatory molecules accompanies tumor rejection and reduces the metastatic potential of tumor cells
Authors: Ostrand‐Rosenberg S. , Baskar S. , Patterson N. , Sciences V. K. ClementsDepartment of Biological
Published: December 2008

Mouse tumor cells transfected with syngeneic MHC class II genes are highly immunogenic in the autologous host, and induce a potent tumor‐specific immunity against wild type tumor. Previous studies with sarcoma tumor cells expressing transfected class II gene products with truncated cytoplasmic domains suggested that during the process of tumor rejection costimulatory molecules are induced on the tumor cells, contributing to the cells' ability to stimulate immunity. In the present study we directly demonstrate that tumor cells containing full‐length class II heterodimers are induced to express B7–1 and B7–2 costimulatory molecules during the rejection process. In contrast, tumor cells expressing class II heterodimers truncated for their cytoplasmic tails are not induced to express B7–1 and/or B7–2. Blocking the interaction of the induced costimulatory molecules with their corresponding receptors on T cells prevents tumor rejection. These results support the hypothesis that the cytoplasmic domain of the MHC class II molecule is involved in induction of costimulatory molecule expression, perhaps via intracellular signalling pathways. Because class II, B7 transfected tumor cells are such effective immunogens against ascites and solid tumors, they have also been tested in metastatic disease. K1735 and B16BL6 mouse melanomas, when transfected with syngeneic MHC class II and B7–1 genes, are significantly less metastatic than parental cells, and immunization with the transfectants protects against subsequent challenge with wild type tumor.
Japanese cedar pollinosis and HLA‐DP5
Authors: Hori T. , Kamikawaji N. , Kimura A. , Sone T. , Komiyama N. , Komiyama S. , Sasazuki T.
Published: December 2008

Japanese cedar pollinosis is a type I allergic disease caused by Japanese cedar (Cryptomeria japonica) pollen. We investigated the association between the disease and HLA class II alleles by HLA‐DNA typing using a PCR‐SSOP method and found that the frequency of HLA‐DP5 (DPA1*02022 and DPB1*0501) was significantly increased in the patients. To investigate whether the HLA‐DP5 molecule is directly involved in the pathogenesis of the disease, Japanese cedar pollen antigen (CPAg)‐specific T cell lines were established from 3 patients who possessed HLA‐DP5 (DPA1*02022/DPB1*0501). By using these CPAg‐specific T cell lines and HLA class II‐expressing L‐cell transfectants, we found that disease‐associated HLA‐DP5 restricted T cells specific for CPAg existed in the patients. Furthermore, among 38 synthesized overlapping peptides spanning the entire length of one of the major Japanese cedar pollen allergens, Cry j 1, an immunodominant peptide which induced HLA‐DP5 restricted Th2 was identified. These observations suggest that the HLA‐DP5 may be involved, at least in part, in the pathogenesis, by helping the IgE antibody production against CPAg.
Typing the HLA‐B locus by a nested primer approach and oligonucleotide hybridization
Authors: Inamdar A. , Sintasath D. M. , Husted L. , Henson V. , Ng J. , Hartzman R. J. , Hurley C. K.
Published: December 2008

A system for intermediate level identification of the HLA‐B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA‐B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group‐specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR‐SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large‐scale DNA‐based typing of the HLA‐B locus.
Expression of LA45 reactive β2‐microglobulin free HLA Class I α‐chains on activated T‐cells is regulated by internalization, constitutive and protein kinase C inducible release
Authors: Pickl W. F. , Holter W. , Stockl J. , Majdic O. , Knapp W.
Published: December 2008

HLA Class I molecules on activated T cells are expressed as mAb W6/32 reactive heterodimers associated with β2‐microglobulin (β2‐m) and also as mAb LA45 reactive β2‐m free HLA Class I α‐chains. However, the regulation of free α‐chain expression remained enigmatic. Here we show, that the amount of cell surface expressed free heavy chains is influenced by two distinct mechanisms. Firstly, a proportion of expressed molecules are cleaved and give rise to a soluble pool of HLA Class I molecules. We provide evidence that, besides the previously described constitutive release of free alpha chains, a second phorbol ester inducible release mechanism involving activation of protein kinase C (PKC) does exist. We demonstrate that both the constitutive and the enhanced release of LA45 reactive HLA Class I α‐chains are the consequence of a cell membrane bound proteolytic activity with the characteristics of a 1, 10 phenanthroline sensitive metalloprotease. Secondly, we report that a distinct fraction of mAb tagged free a‐chains is internalized via an n‐ethylmaleimide sensitive pathway. Together, this data suggests that the expression of free α‐chains is regulated by pathways governing release and internalization.
HLA DQ‐DR haplotype and susceptibility to cervical carcinoma: indications of increased risk for development of cervical carcinoma in individuals infected with HPV 18
Authors: Allen1 M. , Kalantari M. , Ylitalo N. , Pettersson B. , Hagmar B. , Scheibenpflug L. , Johansson B. , Petterson U. , Gyllensten U.
Published: December 2008

The association of HLA class II DQB1 and DRB1 alleles with the development of cervical carcinoma was studied in 150 Swedish patients using PCR‐based HPV and HLA typing. The association of cervical carcinoma with alleles encoding the DQ3 antigen, previously found among German and Norwegian patients, was not observed in the Swedish patients. Five DQ‐DR haplotypes were indicated to be positively associated with development of cervical carcinoma in the Swedish patients. Two of these HLA associations were specific for HPV 18 infected patients, suggesting that the ability of the oncogenic HPV 18 to cause more rapid‐transit tumors than other high risk HPV types may be due to a deficiency in antigen presentation by the HLA molecules encoded by carried on these haplotypes.
Nomenclature for factors of the HLA system, update April 1996
Authors: Marsh Steven G. E.
Published: December 2008

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

Below are listed the newly assigned sequences, confirmations of previously reported sequences and some sequences which are corrections of those originally reported. The accession number of each sequence is given and these can be used to retrieve the sequence files from either the EMBL, GenBank or DDBJ data libraries. Although accession numbers have been assigned by the data libraries and most sequences are already available, there is still the possibility that an author may not yet have allowed the sequence to be released, in such a case you will have to contact the submitting author directly

Sequences correcting previously reported sequences
Six newly identified HLA‐DRB alleles: DRB1*1121, *1419, *1420, *1421, DRB3*0203 and DRB5*0103
Authors: Verduyn W. , Anholts J. D. H. , Versluis L. F. , Parlevliet J. , Drabbels J. , de Meester J. , Tilanus M. G. J. , Doxiadis I. I. N. , Giphart M. J. , Schreuder G. M. T.
Published: December 2008

Seven samples with irregular PCR‐SSO hybridization patterns, observed during routine HLA‐DRB typing, were studied in more detail. Group‐specific amplification, followed by hybridization with relevant SSOs strengthened the suggestion that these samples contained new DRB alleles. DRB exon 2 segments were amplified, cloned and sequenced and revealed: DRB1*1121 [MUL] is similar to DRB 1*1102 in which codon 85 changed from GTT(V) into GTC(A); DRB1*1419 [AKKAL] is similar to DRB1*1402 with codon 71 changed from AGG(R) into AAG(K); DRB1*1420 [OND‐52971] is a DRB1*1406 with codon 37 changed from AAC(N) into TTC(F); DRB1*1421 [TGI] is similar to DRB1*1417 with codon 71 changed from AGG(R) into AAG(K); DRB3*0203 [POS] is similar to DRB3*0202 in which codons 37–38 are changed from TAC GCG(YA) into TCC GTC(SV); DRB5*0103 was found in two unrelated individuals of Oriental origin [IND‐24 and IND‐59] and is similar to DRB5*0102 in which codon 71 AGG(R) changed into ACG(T). This particular sequence variation at position 71 has not yet been described. The new DRB sequences were confirmed using the sequencing based typing technique. Low resolution PCR‐SSP typing failed to amplify two of the DRB1*14 variants, whereas high resolution PCR‐SSP resulted in aberrant patterns. Class II alloantisera identify the codon 71 changes in DRB1*1419 and *1421 with respect to the MC1(‘DR1+DR4’) epitope.
Apoptosis following interleukin‐2 withdrawal from T cells: evidence for a regulatory role of CD 18 (β2‐integrin) molecules
Authors: Röpke C. , Gladstone P. , Nielsen M. , Borregaard N. , Ledbetter J. A. , Svejgaard A. , Ødum N.
Published: December 2008

Following a successful immune response against invading microorganisms, the majority of activated T cells is eliminated, while a minor fraction survives as memory T cells. A decline in T lymphocyte growth factors such as interleukin‐2 (IL‐2) appears to play a role in the elimination of previously activated T cells. Thus, removal of IL‐2 from proliferating T cells not only induces growth arrest, but triggers a massive cell death due to apoptosis. While the apoptotic response involves a series of well‐described events, it remains less clear how apoptosis is regulated following IL‐2 withdrawal. Here, we provide evidence that CD 18 molecules (β2‐integrins) play a regulatory role in the apoptotic response following removal of IL‐2 from previously activated, antigen specific CD4+ T cell lines. Thus, CD 18 mAb inhibited the apoptotic response to IL‐2 deprivation, whereas mAb against other adhesion molecules (CD28, CD29, CD49d, CD80, CD86) did not. Secondly, IL‐2 withdrawal resulted in a retarded apoptotic response in LFA‐1 (CD11a/CD 18) negative T cells obtained from a leukocyte adhesion deficiency (LAD) patient, as compared to LFA‐1 positive T cell lines. Thirdly, co‐culture of LFA‐1 positive‐ and negative‐ T cells at different ratios induced apoptotic responses that were higher than expected, had the two lymphocyte populations not been interacting and significantly higher than that seen in pure LFA‐1 negative T cells. Supema‐tants from LFA‐1 positive T cell cultures undergoing apoptosis did not induce an enhanced apoptotic responses in LFA‐1 negative T cells, and, reversely, culture supernatants from LFA‐1 negative T cells did not rescue LFA‐1 positive cells from undergoing apoptosis. The apoptotic response was partly blocked by IL‐15, a newly identified T cell growth factor. Taken together, these findings suggest that CD 18 molecules (β2‐integrins) play a regulatory role in the apoptotic response following cytokine withdrawal, and that the regulation is mediated, at least partly, through T‐T cell interactions. Thus, apoptotic death following IL‐2 deprivation appears to be under “social” control by surrounding T cells.
Nucleotide sequence of the corrected DQB1*06011 allele
Authors: Schranz P. , Nessler G. , Schindera F. , Seelig R.
Published: December 2008

The DQB1*06011 allele first classified and registered with the codon ACC at position 51(1) was recently corrected to ACG by Dr. Akinori Kimura (2) and in independently confirmed in our laboratory (3). The correct nucleotide sequence for this allele is shown below. The DQB1*06011 allele was found in two sisters of Turkish nationality who had been serologically typed for class I as HLA‐A11, A33, B44, B52, Cw4. Nucleotide sequencing based typing of HLA class II alleles disclosed DRB1*0701, *15021, DRB4*01011/*0103, DRB5*0102, DQA1*0103, *0201, DQB1*02, *06011, DPB1*0401,*11011.
Ligation based HLA‐B*27 typing
Authors: Fischer G. F. , Faé I. , Moser S. , Pelrasek M. , BäBler H. M. S. , Menzel G. R. , Mayr W. R.
Published: December 2008

We have established a ligation based typing method to detect HLA‐B*27 alleles at the DNA level. The method requires amplification of exon 2 of the HLA‐B locus from genomic DNA by the polymerase catalyzed chain reaction (PCR) using group specific primers. An aliquot of the PCR amplification product, heat stable ligase and a pair of oligonucleotide probes, designed to hybridize adjacently to HLA‐B*27 specific sequences of the amplified DNA are subsequently thermocycled. If the probes are perfectly complementary they become ligated otherwise they stay separated. The ligation of probes can be detected through their different labels by an enzyme linked immunosorbent assay (ELISA). Ligation based detection of beta‐actin sequences which have been co‐amplified serves as positive control for each PCR reaction. We observed complete concordance when typing 76 HLA‐B*27 positive and 107 HLA‐B*27 negative individuals either by serology or by the ligation based approach. We conclude that ligation based typing is a reliable tool for the DNA based detection of HLA‐B*27 alleles. The procedure allows automation to a large extent and should be easily applicable to the typing of other HLA‐class I alleles.
Results of Expedition Humana
Authors: Trachtenberg E. A. , Keyeux G. , Bernal J. E. , Rhodas M. C. , Erlich H. A.
Published: December 2008

HLA class II variation was analyzed in nine Native American populations of Colombia using PCR/SSOP typing methods. Under the auspices of the Expedition Humana, approximately 30 unrelated native Colombian Indian samples each from the Tule (NW Pacific Coast), Kogui (Sierra Nevada), Ijka (Sierra Nevada), Ingano (Amazonas), Coreguaje (Amazonas), Nukak (Amazonas), Waunana (Pacific), Embera (Pacific) and Sikuani (Northeastern Plains) were collected and analyzed at the DRB1, DQA1, DQB1 and DPB1 loci. The number of different DRB1, DQA1, DQB1 and DPB1 alleles in the Colombian Indians is markedly reduced in comparison with neighboring African Colombian populations, which exhibit a very high degree of class II variability, as discussed in an accompanying paper. In the Colombian Amerindian groups, DR2 (DRB1*1602), DR4 (DRB1*0407, *0404, *0403 and *0411), DR6 (DRB1*1402) and DR8 (DRB1*0802) comprise >95% of all DRB1 alleles. We also found an absence of DR3 in all populations, and DR1, DR7 and DR9 allelic groups were either very rare or absent. Each Colombian Amerindian population has a predominant DRB1 allele (f=˜0.22–0.65) and DRB1‐DQA1‐DQB1 haplotype. Several novel DR‐DQ haplotypes were also found. At the DPB1 locus, DPB1*0402 (f=0.28‐0.82), *1401 (f=0.03–0.45), and *3501 (f=0.03–0.27), were the three most prevalent alleles, each population maintaining one of these three alleles as the predominant (f>0.26) DPB1 allele. The reduction of diversity for the HLA class II alleles in the Colombian Indians is suggestive of a population bottleneck during the colonization of the Americans, with little to no subsequent admixture with neighboring African Colombian populations in the last˜300 years.
Inhibition of the human allogeneic mixed lymphocyte response by cyclosporin A: relationship with the IL‐12 pathway
Authors: Bonnotte B. , Pardoux C. , Bourhis J. H. , Caignard A. , Burdiles A.‐M. , Chehimi J. , Mami‐Chouaib F. , Chouaib S.
Published: December 2008

Interleukin‐12 (IL‐12) is an important cytokine in the control of cell‐mediated immunity. We have previously shown that endogenous IL‐12 plays a role in the development of human allogeneic response. In the present study, we investigated the relationship between Cyclosporin A (CsA)‐inhibitory effect and IL‐12 pathway during human alloreaction in vitro. CsA addition at the sensitizing phase of primary mixed lymphocyte reaction (MLR) resulted in the inhibition of both p40 and p70 IL‐12 production in a dose‐dependent manner. In contrast, CsA had no effect on IL‐12‐receptor β1 chain (IL‐12 Rβ) expression in T cells induced upon allogeneic activation. Addition of exogenous IL‐12 significantly restored CsA‐inhibited alloreactive cytotoxic T lymphocyte (CTL) generation and had a marginal effect on T cell proliferative response. The IL‐12‐induced restoration of CTL generation was IFNγ‐mediated, as it was significantly altered when anti‐IFNywas added. The restoration of CTL activity by exogenous IL‐12 correlated with the capacity of this cytokine to partially restore granzyme B mRNA expression in alloreactive CTL. This study indicates that inhibition of IL‐12 production is a novel additional mechanism for the inhibitory effect of CsA on the development of human allogeneic cytotoxic response.
Specificity of two anti‐class IHLA monoclonal antibodies that block class I recognition by the NKB1 killer cell inhibitory receptor
Authors: Gumperz J. E. , Paterson J. C. M. , Litwin V. , Valiante N. , Lanier L. L. , Parham P. , Little A.‐WI.
Published: December 2008

Cytolysis by NK cells that possess the NKB 1 killer cell inhibitory receptor is inhibited by target cell expression of Bw4+ HLA‐B molecules. The inhibitory effect can be prevented by addition of mAbs which block recognition of class I molecules by NKB 1. The epitopes recognized by two anti‐class I mAbs, DX15 and DX16, which inhibit the interaction of NKB 1 with class I have been characterized. Binding of DX15 and DX16 to class I allotypes was investigated by flow cytometric analysis of transfected cell lines which express just one HLA‐A, B, or C allele, and by immunoprecipitation of class I molecules from HLA typed B‐lymphoblastoid cell lines, followed by isoelectric focusing. The DX16 mAb recognizes class I allotypes which possess alanine at position 71 of the α1 helix, and therefore has a specificity resembling that of the ME 1 mAb but with broader specificity. Class I recognition by DX15 is affected by polymorphisms of the C‐terminal part of the α1 helix, and the N‐terminal part of the α2 helix. DX15 thus appears to recognize a complex epitope near the end of the peptide binding groove which may be conforma‐tionally determined. Both antibodies are as effective as the anti‐NKB 1 mAb (DX9) in preventing class I recognition by the NKB 1 receptor. DX16 also blocked recognition by a B*0702 allospecific CTL clone, whereas DX15 did not.
Expression of MHC class I and class II antigens in pancreatic adenocarcinomas
Authors: Scupoli M. T. , Sartoris S. , Tosi G. , Ennas M. G. , Nicolis M. , Cestari T. , Zamboni G. , Martignoni G. , Lemoine N. R. , Scarpa A. , Accolla R. S.
Published: December 2008

The antigens encoded by the major histocompatibility complex (MHC) are cell surface glycoproteins that play a fundamental role in the regulation of the immune response. Anomalous MHC expression in tumor cells has been viewed as an important feature to escape tumor recognition by immune cells. Low or absent MHC class I expression as well as ectopic MHC class II expression have been often observed to correlate with high grade malignancy and metastatic potential in a variety of human cancers. To date, very little investigation of MHC (HLA in man) class I and class II expression inhuman pancreatic cancer has been reported. We investigated this aspect on frozen sections of 8 pancreatic adenocarcinomas and 18 established in vitro cell lines. HLA class I was expressed in all but two cancers whereas de novo HLA class II expression was detected in 3 of 8 cancers. Interestingly, a hierarchy in the expression of the various subsets of HLA class II was found with HLA‐DR>‐DP>‐DQ. Results on cell lines strongly resembled the ones obtained in cancer tissues. However, a peculiar feature was observed in certain cell lines. HLA class II antigens were expressed in only a few cell lines and in some of them a mixed population of positive and negative cells was found. Sorting and cloning of the two populations confirmed the existence of tumor cell clones with stable and distinct HLA class II phenotype. Taken together, these results indicate the cellular heterogeneity of pancreatic cancer cells with regard to the qualitative and quantitative expression of major histocompatibility complex genes, and may provide new insights for a better understanding of the tumor‐host relationships in this extremely severe form of neoplasia.
Binding of peptides naturally presented by HLA–B27 to the differentially disease–associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism
Authors: Galocha B. , Lamas J. R. , Villadangos J. A. , Albar J. P. , de Castro J. A.López
Published: December 2008

B*2704 and B*2706 are closely related HLA–B27 subtypes of which the former but not the latter is associated to ankylosing spondylitis. Their peptide specificity relative to other disease–associated subtypes was analyzed by testing binding of self–peptides naturally presented by B*2705 or B*2702, and synthetic analogs, to B*2704, B*2706, and site–specific mutants mimicking their changes. Peptides with basic, aliphatic or aromatic C–terminal residues bound to B*2705 with similar affinity. In B*2704 C–terminal aliphatic/aromatic residues were preferred. B*2706 discriminated drastically between polar and nonpolar C–terminal residues, showing strong preference for Leu and Phe, and less than B*2704 for basic and Tyr residues. Loss of single acidic charges (D>S77, D>Y116) increased preference for C–terminal Leu and Phe, but allowed efficient binding of peptides with basic residues or Tyr. Their gain (V>E152, H>D114) maintained wide C–terminal specificity, but severely impaired binding, presumably by disrupting interactions with internal peptide residues. This was compensated by Y116 in the double Dl 14Y116 mutant. The specificity of B*2704 and B*2706 was explained only partially by the separate effects of single mutations, indicating that novel properties arise from concomitant changes at various positions. For instance, specificity of B*2706 for nonpolar C‐terminal residues required simultaneous removal of Asp77 and Asp116. B*2706 differed from B*2705, B*2702, and B*2704 in its lower suitability for C‐terminal Tyr, suggesting that this feature might be relevant for HLA–B27 association to spondyloarthropathy.
S‐Endo 1, a pan‐endothelial monoclonal antibody recognizing a novel human endothelial antigen
Authors: Bardin N. , George F. , Mutin M. , Brisson C. , Horschowski N. , Francés V. , Lesauie G. , Sampol J.
Published: December 2008

A murine monoclonal antibody (mAb) S‐Endo 1 has been produced to detect circulating endothelial cells detached from blood vessels in pathological conditions. We have demonstrated that the associated‐antigen (S‐Endo 1 Ag) was highly expressed on human vascular structure irrespective of tissue origin or vessel caliber. Its expression was not restricted to endothelium, since it was also detected at low level on smooth muscle cells, stroma cells and follicular dendritic cells. But its absence on hematopoietic cells made S‐Endo 1 a helpful reagent to specifically discriminate endothelium from hematopoietic tissues. Biochemical characterization showed that S‐Endo 1 recognizes a monomelic structure of ˜ 118 kDa on cultured endothelial cells. S‐Endo 1 was submitted to the 5th International Workshop (Boston, 1993) and did not cluster in any of the old or new endothelial clusters discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggested that S‐Endo 1 defines a previously undescribed endothelial molecule.
A new HLA–DQB 1*0306 allele sharing motifs from DQB 1*03032 and DQB1*04 sequences
Authors: Saito S. , Ota S. , Hashizume K. , Yamada E. , Kaneshige T. , Kinoshita T. , Hashimoto M. , Oguchi H. , Ishii E. , Fukushima H.
Published: December 2008

We have discovered a new HLA‐DQB 1 allele in a Japanese family, MAT. In the family the new allele segregates in three generations and demonstrates the positive association with DRB 1*0901. We observed a novel RFLP pattern in the course of examining the modified PCR‐RFLP method for HLA‐DQB 1 genotyping. The PCR‐SSOP analysis also showed a new hybridized pattern. Sequence analysis of the allele indicates that it was generated by a gene conversion‐like event between the HLADQB 1*03032 and one of DQB 1*04 contemporary alleles. This new allelic product did not react with all of allosera and monoclonal antibodies against DQ1, DQ2, DQ3, DQ4 and DQ7. The HLA molecule encoded by the allele is not defined by serology. This new allele was officially recognized and named DQB 1*0306 by the WHO Nomenclature Committee in November 1995.
Role of the human CD38 molecule in B cell activation and proliferation
Authors: Funaro A. , Morra M. , Calosso L. , Zini M. G. , Ausiello C. M. , Malavasi F.
Published: December 2008

Human CD38 is a surface molecule which has been attributed the function of a signaling channel leading to cellular activation and proliferation, an ectoenzyme with multiple function as well as an inducer of Ca2+ mobilization from cytoplasmic stores. The effect mediated by CD38 have been studied in different cell populations: the results obtained in human B cells are apparently contradictory, with CD38 simultaneously leading to apoptosis in early B cells while increasing survival in cells derived from lymph node germinal center. Other effects recently reported concern a different potential in terms of signaling in early B cells and derived cell lines or in more detailed disease models of human leukemia, namely B chronic lymphocytic leukemia cells.

To complete the picture of the effects mediated by CD38 in the B cell compartment, we have studied the signals elicited by ligation of the human molecule in mature B cells from circulating pool and also from spleen of normal individuals. The information obtained completes the picture of CD38 and mature B cells, where we also studied the contribution of relevant cytokines involved in maintenance and differentiation of these normal cells, namely IL‐1 α, IL‐2, IL‐4 and IL‐6. Our results indicate that human CD38 plays a key role as a co‐receptor in mature B cells from normal individuals.
Diversity and evolution of the DRB1*03 family: description of DRB1*03022, *0307, **0308
Authors: Ellis J. M. , Steiner N. , Wang J. , Tang T. T. , Hurley C. K.
Published: December 2008

Three previously unreported DRB1*03 alleles are described, adding to the diversity of the DRB 1 family of alleles. DRB1*03022 contains a silent substitution at codon 77. DRB1*0307 differs from DRB1*03011 by a substitution at codon 26 resulting in a predicted change from tyrosine to phenylalanine. DRB1*0308 is almost identical to DRB1*03011 differing at codon 58 which specifies the glutamic acid residue commonly found in DRB1*11 alleles. The new alleles (DRB1*03022, *0307, *0308) may have arisen by gene conversion‐like events and add to the increasing complexity of the HLA system.
Polymorphism at the TNF loci in rheumatoid arthritis
Authors: Vinasco J. , Beraun Y. , Nieto A. , Fraile A. , Mataran L. , Pareja E. , Martin J.
Published: December 2008

The purpose of this work was to analyze the possible influence of TNF loci polymorphism on the susceptibility and/or the disease profile of rheumatoid arthritis (RA). Tumor necrosis factor (α and β) genotypes were determined in 60 patients with RA and 102 healthy subjects by a method based on PCR‐RFLP with amplification‐created restriction sites. The results obtained in the present study showed that there is not a significant association of either TNFα promoter variation (at positions ‐308 and ‐238) or TNFp polymorphism with susceptibility to RA. However, a significant difference in the mean age at disease onset was found between‐238 TNFβgenotypes. In addition, a difference in the presence of nodular disease was observed between‐308 TNFα genotypes. The results of this study suggests that the TNFα gene may play a role in the disease profile of rheumatoid arthritis.
Polymorphic MHC class II promoters exhibit distinct expression pattern in various antigen‐presenting cell lines
Authors: Janitz M. , Mitchison A. , Reiners‐Schramm L. , Lauster R.
Published: December 2008

The promoter regions of MHC class II genes are characterized by the presence of conserved sequence motifs called S, X and Y boxes, which are crucial for regulation of transcription of these genes. In humans, promoter polymorphism is known to result in differential transcriptional activity at both inter‐locus and inter‐allelic levels, but it is not yet known how this relates to tissue‐specific expression of MHC class II molecules. We sequenced the 5′ regulatory regions of α and β genes of I‐A and I‐E molecules from four mouse haplotypes and found allelic polymorphisms which were mainly confined to the X box. The promoter sequences of I‐Ea genes were non‐polymorphic. Transfection of four antigen‐presenting cell types with promoter‐reporter gene constructs revealed that the promoter sequence polymorphisms result in distinct allele‐ and tissue‐specific activity patterns. Mutagenesis experiments in which the X2 box was reshuffled between I‐Aβ alleles demonstrated that this box contributes to regulation of differential MHC class II expression in the four cell types. The possibility is discussed that tissue‐specific MHC class II expression may control differentiation of T‐cell subsets.
Analysis of allelic variation of the TAP2 gene in sarcoidosis
Authors: Ishihara M. , Ohno S. , Ishida T. , Naruse T. , Kagiya M. , Mizuki N. , Maruya E. , Saji H. , Inoko H.
Published: December 2008

Sarcoidosis is a systemic granulomatous disease and the DRB1 gene of the DR subregion has been implicated for determining the genetic susceptibility to the disease. We evaluated the allelic variation of the TAP2 gene using the PCR‐RFLP method as well as the mismatched PCR‐RFLP method in 82 Japanese patients with sarcoidosis and 92 healthy controls. A new allele, TAP2*0103 and a new polymorphic variation at codon 577 in addition to TAP2*0101, TAP2*0102 and TAP2*0201 have been recognized in the Japanese subjects. No significant differences were observed in the frequencies of any TAP2 alleles or dimorphism at codon 577 between the patients and healthy controls. Polymorphic variation of the TAP2 gene does not confer the susceptibility to sarcoidosis.
HLA‐B44 subtyping in a Spanish population: further evidence of Caucasian population diversity
Authors: Santos S. , Vicario J. L. , Merino J. L. , Balas A.
Published: December 2008

HLA‐B44 is the most frequent HLA‐B allele in Caucasian populations. Several B44 subtypes, B*4402‐B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR‐SSO method, based on a B12‐ specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA‐A.‐C.‐DRB1,‐DRB3/DRB4/DRB5.‐DQA1 and ‐DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44‐bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.
Development of PCR‐SSOP for the identification of HLA‐A*02 subtypes and determination of HLA‐A*02 frequencies within different ethnic populations
Authors: Williams F. , Middleton D. , Savage D. , Gorodezky C. , Wilson D. W. L. , Fitzgerald J. M. , Urbaniak S. J.
Published: December 2008

A PCR‐SSOP typing method, involving a single PCR amplification in conjunction with 19 digoxigenin labelled oligonucleotide probes, has been developed for the identification of 17 known HLA‐A*02 alleles. The method has been applied to four populations (Northern Ireland, Singapore Chinese, Shetland Island and Mexican) and percentages of HLA‐A*02 alleles determined within each population.
Identification of HLA‐C alleles using PCR—single‐strand‐conformation polymorphism and direct sequencing
Authors: Wang H. , Tokunaga K. , Akaza T. , Tadokoro K. , Shibata Y. , Juji T.
Published: December 2008

Alleles encoding HLA‐C antigens in Japanese were identified by polymerase chain reaction followed by single strand conformation polymorphism (PCR‐SSCP) and nucleotide sequencing analyses. The results showed that at least sixteen different alleles code for eight serologically detectable antigen groups and undetectable blanks. Cwl was mainly encoded by Cw*0102, whereas two split antigens of Cw3, Cw9 and Cw10, were encoded by Cw*0303 and Cw*0304, respectively. Cw4 and Cw6 were encoded by Cw*0401 and Cw*0602, respectively. Seven alleles, Cw*0801, Cw*0803, Cw*1202, Cw*1203, Cw*1402, Cw*1403 and Cw*1502, were found to encode serological HLA‐C “blanks” in Japanese. Moreover, errors in the published nucleotide sequences of Cw*0501 and Cw*1201 were corrected. Twenty‐one HLA‐C alleles were distinguished from each other by means of group‐specific PCR amplification followed by the SSCP method developed in the present study. The system using genomic DNAs can be used effectively for identification of new HLA‐C alleles.
A new DRB1*15 allele (DRB1*1506) identified by sequence‐based typing
Authors: van den Berg‐Laonen E. , Rani R. , Singal D. , Tilanus M.
Published: December 2008

A new DRB1*15 allele (DRB1*1506) was detected during the studies of the 12th International Histocompatibility Workshop within the Allele and Haplo‐type Society #11 which studied the DR2 and DR51 antigens. The new allele was found in four unrelated Asian Indian individuals by sequence‐based typing. It has a base substitution from T to C in codon 50 at a previously considered conserved position.
DQB1*0202 and the new DQB1*0203 allele: a fourth pair of DQB1 alleles differing only at codon 57
Authors: Olerup O. , Aldener‐Cannavá A. , Fogdell‐Hahn A. , Getty R. R. , Wagenknecht D. R. , McIntyre J. A.
Published: December 2008

Amino acid 57 of DQ β chains is of functional importance as it influences peptide binding, is part of B and T cell epitopes, and is associated with susceptibility and resistance to insulin‐dependent diabetes mellitus and humoral immunodeficiencies. Polymorphism of codon 57 is conserved in primates and in HLA class II B genes implying that balancing selection operates on this residue. Previously, three DQB1 allele pairs have been described, that only differ at residue 57. In an African‐American Black individual with the HLA pheno‐type A23, 30; B58, 63; Cw6; DR18, 12; DR52; DQ5, 2, we found a fourth example of this dimorphism; the new DQB1*0203 allele, that was identical to DQB1*0202 except for codon 57, which encodes aspartic acid and alanine respectively in the two alleles. The class II haplotype carrying the new allele was deduced to be DRB1*0302, DRB3*0101, DQA1*05011, DQB1*0203
CpG methylation patterns in the 5 part of the nonclassical HLA‐G gene in peripheral blood CD34+ cells and CD2+ lymphocytes
Authors: Onno M. , Amiot L. , Bertho N. , Drenou B. , Fauchet R.
Published: December 2008

A dominant goal of research focused on the nonclassical human leukocyte antigen G (HLA‐G) gene is to understand the molecular mechanism involved in its limited expression. In the present report, we examined DNA methylation as a potential regulatory mechanism of HLA‐G transcription in two cell types of the adult lymphomyeloid lineage: CD2+ lymphocytes express several mRNA isoforms while transcripts are undetectable in CD34+ hematopoietic cells. The methylation status of 63 CpG sites in the promoter and in the 5 CpG island was established using bisulfite‐treated genomic DNA sequencing. Methylation was first analyzed by the direct sequencing of bisulfite‐treated and amplified products. The general patterns of CpG methylation in the 5 part of the gene were found to be similar for CD34+ cells and CD2+ lymphocytes: the distribution of methylation was not uniform across the 63 CpG sites. In the promoter region, both CpG dinucleotides were partially or fully methylated whereas in the CpG island, several CpG sites were totally demethylated. Unexpectedly, in HLA‐G positive CD2+ lymphocytes, a great number of CpG dinucleotides displayed a higher frequency of methylation relative to that found in CD34+ cells. However, the sequence analysis of cloned products revealed that the molecules have different methylation patterns which suggests that the HLA‐G gene is differentially expressed in CD2+ cells. Our results suggest that methylation is not the sole mechanism that achieves the repression of HLA‐G transcription in immature CD34+ cells.
Human TAP1 polymorphisms detected by denaturing gradient gel electrophoresis
Authors: Shi L. , Yan G. , Fu Y. , Ma L , Penfornis A. , Faustman D.
Published: December 2008

Presentation of endogenous peptides by major histocompatibility complex class I (MHC) molecules is controlled, in part, by the Tap1 and Tap2 genes in the MHC class II region that encode a heterodimeric peptide transporter. Polymorphisms of human Tap1 in normal individuals have now been investigated systematically by denaturing gradient gel electrophoresis (DGGE) analysis of fragments of genomic DNA generated by the polymerase chain reaction. Polymorphisms identified by distinctive DGGE band patterns were confirmed by DNA sequencing. In addition to four previously described polymorphisms in the open reading frame, DGGE detected three new polymorphisms: a GT substitution in the promoter region, a 10‐bp insert in intron 9, and a GT substitution 80‐bp downstream of the translation termination codon.
Comparison of DRB sequence‐based typing using different strategies
Authors: Voorter C. E. M. , Rozemuller E. H. , de Bruyn‐Geraets D. , van der Zwan A.‐W. , Tilanus M. G.J. , van den Berg‐Loonen E. M.
Published: December 2008

Sequence‐based typing (SBT) has become an important tool in the identification of HLA alleles. In this study a comparison was made between SBT of DRB 1/3/4/5 alleles performed in two laboratories each using a different strategy for SBT. The laboratories in Utrecht and in Maastricht performed direct sequencing of PCR amplified genomic DNA from 30 selected samples. Primers and conditions for PCR amplification were different. Sequencing was either performed with T7 polymerase, using internal sequencing primers, or with cycle sequencing using an M13 tailed system. Two different automated DNA sequencers were used; the ALFexpress from Pharmacia and Applied Biosystems 373 A. We concluded that nor the method of sequencing nor the sequencing machine influences typing results. However the PCR reaction used for generating template DNA is the most critical step. Different primers and different conditions can lead to false negative reactions. The fact that these false negative reactions can occur with different alleles in different combinations but not in all, implicates that extensive quality control is needed to assure correct typing results.
Frequency of HLA‐B27 subtypes in a Danish population and in Danish patients with ankylosing spondylitis
Authors: Baech J. , Schmidt‐Qlsen S. , Steffensen R. , Varming K. , Grunne N. , Jersild C.
Published: December 2008

Polymerase chain reaction in combination with sequence‐specific oligonucleotide probes were used to analyze nine HLA‐B27 subtypes among 51 healthy I HLA‐B27 positive Danish blood donors and 30 Danish HLA‐B27 positive patients with ankylosing spondylitis (AS). In the group of healthy Danes we found two subtypes, B*2705 (90.2%) and B*2702 (9.8%), however, among the AS patients only the B*2705 subtype was detected. We did not find a significant evidence for associations between AS and a particular HLA‐B27 subtype in a Danish population.