Further diversity of the 5' promoter region of the MHC class I-related chain B gene.

We have now found a total of 15 individual MICB promoter sequences, varying by combination of 18 polymorphic positions within the MICB minimal promoter sequence. Sequence-based typing and cloning characterized the three new 5' promoter sequences as MICB-P13, MICB-P14 and MICB-P15.

Two novel 5' promoter sequences of the MHC class I-related chain A gene.

In this study, we have characterized two novel polymorphism of the 5' promoter sequence of MICA gene, MICA-P13 and MICA-P14, by sequence-based typing and cloning.

Diversity and characterization of polymorphic 5' promoter haplotypes of MICA and MICB genes.

The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are ligands for the natural killer group 2, member D (NKG2D) activating receptor expressed on natural killer (NK) cells, natural killer T (NKT) cells, CD8+ T cells and γδ T cells. Natural killer group 2, member D (NKG2D) ligand expression is stress-related and upregulated by infected or oncogenic cells leading to cytolysis. MICA and MICB genes display considerable polymorphism among individuals and studies have investigated allelic association with disease and relevance of MICA in transplantation, with variable success. It is now known that promoters of MICA and MICB are polymorphic with some polymorphisms associating with reduced expression. We sequenced International Histocompatibility Workshop (IHW) cell line DNA to determine promoter types and alleles encoded by exons 2-6. We found 8 of 12 known MICA promoter polymorphisms and although promoter P7 dominated, other promoters associated with the same allele. For example, MICA*002:01 had promoters P3, P4 or P7 and the common MICA*008:01/04 type had P1, P6 or P7. Similarly, we sequenced 8 of 12 known MICB promoter haplotypes. Some coding region defined MICB alleles had a single promoter, for example, MICB*002:01 and promoter P9, whereas the promiscuous MICB*005 allele had promoters P1, P2, P5, P6, P10 or P12. The results indicate potential for variation in expression of MICA and MICB ligands between individuals with the same allelic types. If differential expression by polymorphic MICA and MICB promoters is confirmed by functional studies, involvement of these genes in disease susceptibility or adverse transplantation outcomes may require knowledge of both promoter and allelic types to make meaningful conclusions.

Further polymorphism of the RAET1E/ULBP4 gene in humans.

Description of a novel RAET1E/ULBP4 allele characterized by sequence-based typing and cloning: RAET1E*011.

Three novel allelic variants of the RAET1E/ULBP4 gene in humans.

Discovery of three novel alleles of RAET1E/ULBP4 by sequence-based typing: RAET1E*008, RAET1E*009 and RAET1E*010.

Two novel MICA alleles, MICA*054 and MICA*056.

We report the identification of two novel major histocompatibility complex (MHC) class I-related chain A (MICA) alleles. MICA*054 has a nucleotide substitution of A to G at position 871 (codon 268), encoding an amino acid change of serine to glycine in the alpha-3 domain. MICA*056 has a nucleotide substitution at position 758 of G to C resulting in the substitution of tryptophan for serine at codon 230, also in the alpha-3 domain.

Interaction of choriocarcinoma cells and human peripheral blood lymphocytes. Resistance of cultured choriocarcinoma cells to cell-mediated cytotoxicity by mitogen-activated lymphocytes.

Cultured choriocarcinoma (Be Wo) cells exist that share many of the morphologic and bio-synthetic properties of normal human trophoblasts. In an attempt to develop a model for the immunologic relationship between a sensitized mother and fetus, we mixed Be Wo cells with mitogen-activated cytotoxic lymphocytes in vitro. Be Wo cells were resistant to the cytolytic effects of the activated lymphocytes despite 24-h exposure and intimate cell-to-cell contact as determined by microscopy. Control target cells, a line of human hepatoma cells, were readily destroyed. Cytotoxicity was measured by determining residual radioactivity of [(3)H]thymidine-labeled target cells after exposure to activated lymphocytes. Employing the quantitative assay, we confirmed the morphologic results and showed that Be Wo and a number of other choriocarcinoma cell lines were resistant to the cytotoxic effects of lymphocytes activated by phytohemagglutinin, pokeweed mitogen, and allogeneic cells in mixed lymphocyte cultures. Moreover, Be Wo cells were resistant to injury over a wide range of killer to target cell ratios. Significant killing of the Be Wo cells occurred only after prolonged exposure (48 and 72 h) to the activated lymphocytes. We suggest that one mechanism that may assist the fetus (or a choriocarcinoma) in its immunologic survival is the intrinsic resistance of trophoblast cells to lymphocyte-mediated cytotoxicity.

Increasing polymorphism of the RAET1E/ULBP4 gene in humans.

Description of three novel RAET1E/ULBP4 allele and promoter polymorphisms identified by sequence-based typing.

HLA class I diversity in Kolla Amerindians.

Human leukocyte antigen (HLA) class I polymorphism was studied within a population of 70 unrelated Kolla Amerindians from the far northwest of Argentina close to the Bolivian border. The results indicate that the HLA-A, -B, and -C alleles typical of other Amerindian populations also predominate in the Kolla. These alleles belong to the following allele groups: HLA-A*02, *68, *31, *24, HLA-B*35, *15, *51, *39, *40, *48, and Cw*01, *03, *04, *07, *08, and *15. For the HLA-A locus, heterogeneity was seen for HLA-A*02 with A*0201, *0211, and *0222; and for A*68 with *68012 and *6817, the latter being a novel allele identified in this population. Analysis of HLA-B identified heterogeneity for all Amerindian allele groups except HLA-B*48, including the identification of the novel B*5113 allele. For HLA-C heterogeneity was identified within the Cw*07, *04, and *08 groups with Cw*0701/06, *0702, *04011, *0404, *0803, and *0809 identified. The most frequent "probable" haplotype found in this population was B*3505-Cw*04011. This study supports previous studies, which demonstrate increased diversity at HLA-B compared with HLA-A and -C. The polymorphism identified within the Kolla HLA-A, -B, and -C alleles supports the hypothesis that HLA evolution is subject to positive selection for diversity within the peptide binding site.

Application of RSCA for the typing of HLA-DPB1.

We describe the application of RSCA, for the high resolution typing of alleles encoded at the HLA-DPB1 locus. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different alleles. A total of 251 samples were typed in a blind study, of these 109 samples had been typed previously by conventional techniques. A comparison of the RSCA data with the historical typing results showed a concordance over 93%. Seven samples initially had discordant results, however, when these samples were typed by direct sequencing, the type assigned by RSCA was found to be correct in all but one case, indicating a concordance over 99%. RSCA has proved to be a simple reliable technique for the typing of the HLA-DPB1 locus, and is not limited by the ambiguous combinations of alleles determined in other conventional techniques.

An unlikely rare result of HLA-A*0236, *3601 masking the presence of a novel allele A*0114.

We identified an A*0114 allele in an Irish patient with an apparent A*0236, A*3601 type.

A new HLA-A*31 null allele, A*3114N.

A new HLA-A*31 null allele results from addition of an extra C near the beginning of exon 4 after a string of seven Cs.

Sequence of a novel HLA-A*0301 intronic variant (A*03010103).

We report here the full-length sequence of a novel HLA-A*0301 allele, A*03010103, which differs from A*03010101 by a single nucleotide substitution (G>T) at position 492 within intron 2. The variant was originally identified by Reference Strand-mediated Conformational Analysis (RSCA) and was confirmed by cloning and sequencing. The difference in RSCA mobility between A*03010101 and A*03010103 demonstrates the sensitivity of RSCA to detect single nucleotide polymorphisms.

Arylamidase of Neisseria catarrhalis.

Neisseria catarrhalis produces arylamidase intracellularly and is one of the gram-negative bacteria producing exceptionally large amounts of this enzyme. In general, gram-positive bacteria do not produce this enzyme. Arylamidase from N. catarrhalis was purified by salt fractionation, chromatography, and density gradient ultracentrifugation. Its sedimentation coefficient was 6.6; l-alanine-beta-naphthylamide (betaNA) was the most rapidly hydrolyzed amino acid-betaNA. The enzyme had pK(e) values of 6.1 and 8.7 and pK(es) values of 7.1 and 7.9; only those amino acid-betaNA compounds of the l configuration were susceptible to hydrolysis. Arylamidase catalyzed stepwise hydrolysis of dipeptide-betaNA, beginning with the N-terminal residue. Substrates having amino acid residues with larger R groups, such as leucine, interacted much more effectively with enzyme. The significance of the predominate occurrence of arylamidase activity in gram-negative bacteria and the role of this enzyme in the physiology of these organisms remain unclear. It has been established, however, that arylamidase is distinct from leucine aminopeptidase.

The bactericidal potential of various endodontic materials for primary teeth.

An in vitro study of the bactericidal or bacteriostatic effects of several pulp therapy compounds and their components on selected bacterial commonly isolated from infected primary teeth was undertaken. It was determined that zinc oxide had no inhibitory effects on E. coli, Staph. aureus, and Strep. viridans; however, the addition of eugenol to this system retarded the growth of only the grampositive organisms. The inclusion of zinc acetate as a setting accelerator inhibited both gram-positive and gram-negative bacteria. Also the inhibitory effects on these three organisms could be greatly enhanced by the addition of formocresol or paraformaldehyde to the zinc oxide--eugenol--zinc acetate system. It was also noted that Sargenti's N-2 paste was apparently no more effective in retarding growth of these organisms than the mixtures. Only the lead tetroxide and hydorcortisone present in the N-2 paste was toxic to the Staph. aureus but not to E. coli or to Strep. viridans. The other heavy metals in N-2 apparently have no antibacterial activity in the manner in which they were tested. This evidence suggests, but is not conclusive, that zinc oxide--eugenol--zinc acetate, with or without formaldehyde-containing compounds, may be effective in the elimination of bacteria from pulpotomized primary teeth. Therefore, the addition of highly cytotoxic chemicals which will remain sealed in a root canal and be active for extended periods of time may not be necessary for successful treatment.

HLA-B*4413 identified in a UK Caucasoid potential bone marrow donor.

We describe a novel allele belonging to the B*44 allele group. HLA-B*4413 was identified in a Caucasoid potential bone marrow donor from the Anthony Nolan Bone Marrow Trust register. Initial HLA typing of this donor revealed unusual serological reactivity. Further investigation was carried out by reference strand conformation analysis (RSCA) and sequence-based typing (SBT) using DNA extracted from an Epstein-Barr virus (EBV)-transformed B-cell line made from this donor (AMI005AN). In this individual, two B*44 alleles were revealed upon initial investigation: B*4409 and a previously unseen allele which has been named B*4413. B*4413 is identical to B*44031 except for a unique nucleotide substitution resulting in an amino acid difference at residue 61 in the alpha1 helix.

HLA-A*6817, identified in the Kolla Amerindians of North-West Argentina possesses a novel nucleotide substitution.

Analysis of HLA polymorphism of the indigenous populations of Central and South America has identified many alleles not seen previously in other populations. We have described a novel allele, B*5113, previously in a Kolla Amerindian individual from North-West Argentina. Here we present a second novel allele from this population: A*6817, which differs from its closest neighbour A*68012 by a single substitution at nucleotide 419. This substitution of adenosine 419 A*68012 for thymidine in A*6817 results in a novel amino acid change (aspartate to valine) at residue 116.

HLA-B*8202 identified in a Caucasoid potential bone marrow donor.

Sequence analysis of HLA class I alleles has continued to reveal the true extent of polymorphism, particularly for B-locus alleles. This diversity can arise through reshuffling of polymorphic sequences generated by point mutation, resulting in interallelic recombination or intergenic recombination (1). Here we describe a new B-locus allele, B*8202, which is structurally most similar to B*8201, having only one nucleotide difference in exon 3 at nucleotide 557, resulting in an amino acid change of aspartic acid to glycine at residue 162. Glycine is the consensus amino acid for B-locus alleles, which suggests that B*8202 is older than B*8201 in evolutionary terms. B*8201 was found to be a hybrid of B*4501 and B*5602 that may have arisen through recombination events, explaining the serological patterns observed with these allotypes. The importance of high-resolution typing is emphasised here as routine typing suggested the presence of B*8201 and the new variant allele may have been missed had it not been typed further by sequence-based typing.

Complete cDNA sequences of the HLA-DRB1*14011, *1402, *1403 and *1404 alleles.

Sequencing studies of HLA class II molecules have been focused almost exclusively on the highly polymorphic exon 2. In this study the complete cDNA sequence of four alleles of the DR14 lineage (DR52 group) are reported for the first time. The HLA-DRB1*1402 and *1403 sequences were shown to be identical to the previously determined DRB1*13011 sequence, also of the DR52 group, in exons 1, 3, 4, 5 and 6. HLA-DRB1*14011 and *1404 were identical to DRB1*13011 in exons 1, 4, 5 and 6 sequences while they showed specific features within their exon 3 sequence. Both alleles showed a synonymous substitution at the third base of codon 114. However, DRB1*14011 also has a non-synonymous substitution at the first base of codon 112 which results in a histidine to tyrosine substitution. This is a novel substitution as Histidine 112 is conserved in all known HLA class II B genes.