Establishment of optimized ELISA system specific for HLA-G in body fluids.

Recently, human leukocyte antigen-G (HLA-G) has been a focus in the field of reproductive immunology, tumor progression and transplantation, because of its inhibitory function as ligand to the inhibitory receptors leukocyte immunoglobulin-like receptors (LILR) B1 and LILRB2. The HLA-G is expressed in distinct mRNA isoforms, one of which encodes a soluble HLA-G (sHLA-G) protein, detectable by sandwich ELISA. Therefore, sHLA-G ELISAs have been used as a noninvasive diagnosis system. While a number of sHLA-G-specific ELISAs have been described, our prior studies showed that data obtained by the conventional ELISA system detecting sHLA-G in body fluids was not consistent with the data obtained from immunoprecipitation (IP)/immunoblotting (IB). Therefore, we established an optimized ELISA system described in this report, which yields results consistent with IP/IB analysis. Using this system, we determined sHLA-G protein in amniotic fluids, and found that sHLA-G levels at preterm (∼36 weeks) were clearly higher than those at term (37-41 weeks). These data and supporting experiments showed that the ELISA system we established can be an useful tools for the detection of sHLA-G protein in body fluids than the conventional ELISA system.

Histochemical reactivity of soybean agglutinin with blood group antigens and their precursor substances in acinar cells of human pancreas.

In human pancreas, soybean agglutinin (SBA) conjugated to horseradish peroxidase reacted with the acinar cells secreting blood group A and/or H antigen, but not with those secreting only B antigen. For detailed histochemical characterization of SBA staining, the effects of treatment with unlabeled lectins and of digestion of certain enzymes on SBA staining were investigated in formalin-fixed, paraffin-embedded pancreatic tissue from individuals of different blood groups. Pre-incubation of sections with unlabeled Dolichos biflorus agglutinin to block A antigen eliminated subsequent SBA staining in the cells secreting A antigen, although failing to induce any effects in those secreting H antigen. In contrast, pre-incubation with unlabeled Ulex europaeus agglutinin-I (UEA-I) to block H antigen abolished SBA staining in cells secreting H antigen but not in those secreting A antigen. Treatment with galactose oxidase yielded the same results as those with unlabeled UEA-I, i.e., SBA reactivity was significantly diminished in cells secreting H antigen but not in those secreting A antigen. Digestion with beta-galactosidase resulted in a slight decrease of SBA staining in the cells secreting H antigen. Accompanying the decrease of SBA staining, reactivity with Griffonia simplicifolia agglutinin-II (GSA-II) appeared for the first time in the enzyme-susceptible, SBA-reactive cells secreting H antigen. Pre-treatment with galactose oxidase abolished this effect of beta-galactosidase. The GSA-II reactivity disclosed by treatment with galactosidase was completely eliminated by digestion with beta-N-acetylhexosaminidase, indicating that GSA-II staining after digestion with galactosidase is due to exposed penultimate beta-N-acetyl-D-glucosamine residues. These results demonstrate that at least two substances react with SBA in acinar cells of human pancreas, one being terminal beta-N-acetyl-D-galactosamine residues of A antigen, and the other being terminal beta-D-galactose-(1----3 or 1----4)-beta-N-acetyl-D-glucosamine dimers in the precursor of blood group H antigen. Such dimers may exist in close proximity to L-fucose residues of H antigen, since unlabeled UEA-I blocked SBA staining.

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism.

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3' untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.

[Mercury in hair of patients with ALS].

In middle of Kii peninsula, one of the biggest mercury mine in Japan had been present until about 10 years ago. The mercury contents in water and fish are reported to be higher in this district. So we investigated the mercury in hair of patients and normal controls. In this study the subjects are 23 cases of ALS including 15 cases in Nara and Mie and 8 cases in other prefectures except in Kii peninsula, 14 cases with ataxia, 11 cases with other degenerative diseases like Parkinson's disease and Alzheimer's disease, 25 cases of cerebrovascular disease as compared to 26 normal controls. The hair are taken from 3 areas on head of patients and normal controls. They are washed in 2% sodium lauryl sulfate and stirred in distilled water several times, and they are soaked in acetone and dried in filter paper. They are inserted in fire and vaporized mercury are measured (Zeeman Effect Mercury Analyzer) in ppm. The hair mercury concentration is 2.81 ppm in ALS in total, 3.62 ppm in ALS in Nara and Mie and 1.39 ppm in outside of Kii Peninsula, 2.34 ppm in ataxia, 1.83 ppm in other degenerative diseases, 1.66 ppm in cerebrovascular disease and 1.44 ppm in normal controls. Statistically it is significant (p less than 0.05) between that in ALS in Nara and Mie and that in normal controls. 6 cases (40%) with ALS in Nara and Mie have the value above the mean +2 standard deviation of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

[Paternity probability in the cases of incest].

To calculate the paternity probability in the cases of incest where the alleged father was either the father or the brother of the plaintiff's mother, some algebraic expressions applicable to a simple codominant diallelic genetic marker system were derived by modifying the formulas of Essen-Möller and Komatsu (the both formulas gave the same result). The paternity probability in the incest case is generally lower than that in usual case, because in the former case an allele present in the mother is sometimes found in both the alleged father and the child (plaintiff), even if the alleged father is not true father. The paternity probability in the incest case, however, becomes higher than that in usual case when an allele is common to both the alleged father and the child but not to the mother. The mean value of paternity probability becomes lower, as the relationship becomes closer between the alleged father and the mother.

[A method to calculate the probability of paternity between relatives--a paternity case where the putative father was a deceased granduncle].

To test paternity in a case where the putative father was a deceased uncle of mother (plaintiff's granduncle), we designed a new method to calculate the probability of paternity likelihood. The putative father's genotypes of red cell antigens, HLA and short tandem repeat (STR) polymorphism were estimated from those of mother and sister of the plaintiff. When the probability was calculated from the frequencies in the unrelated individuals (the standard method), a significant bias might be introduced since the putative father and the plaintiff were likely to have the same alleles come from their common ancestry. Therefore, we designed a new method to calculate the likelihood ratio from the frequencies in the group of mother's uncles estimated from mother's genotypes. The probability (0.9299) calculated with our method was found to be lower than that (0.9992) done with the standard method indicating that the new method could remove the bias introduced from the incest.

Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens.

We have investigated HLA-G mRNA expression in cells and tissues expressing the gene. This analysis has demonstrated that the HLA-G primary transcript is alternatively spliced to yield at least three distinct mature mRNAs. Sequencing of the transcripts has shown that the largest mRNA is essentially that previously characterized, encoding a leader sequence, three external domains, a transmembrane region, and a cytoplasmic sequence. Of the two smaller messages, a 900-base mRNA does not include exon 3, resulting in a predicted protein sequence with the alpha 1 and alpha 3 external domains joined. The smallest mRNA results from splicing out exons 3 and 4, connecting the alpha domain directly to the transmembrane sequence. Alternative splicing of HLA-G mRNA was found in placental tissues and in eye tissue as well as in HLA-G-transfected cell lines. In term placental tissue the smallest mRNA appeared to be more abundant than the full-length form, while in a cell line derived from an earlier developmental stage the larger form predominated. Immunoprecipitation of [35S]methionine-labeled cell lysates showed that three different HLA-G proteins were present in transfected cells, with sizes corresponding to those predicted from the three alternative mRNA sequences. These findings are discussed in terms of potential functions of the alternative HLA-G proteins.

A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4.

The HLA-G primary transcript is alternatively spliced to yield mRNAs encoding three alternative membrane bound proteins. In addition to these forms, a soluble HLA-G protein has been described which is not encoded directly by any of the three alternative mRNAs. To explain the process which might lead to the expression of a soluble HLA-G Ag, we investigated the potential roles proteolytic processing and additional alternative splicing of HLA-G RNA might play. By generating transfected cells with HLA-G cDNA expression driven by a retroviral promoter, it was possible to rule out proteolytic processing of the membrane-bound HLA-G as a mechanism of generating soluble HLA-G, resulting in our focus on alternative splicing as an explanation. Analysis of PCR-amplified cDNA revealed a relatively abundant transcript present in all samples examined which consisted of the full length HLA-G mRNA sequence interrupted by intron 4 sequence. The open reading frame in this mRNA continues into intron 4 terminating 21 amino acids after the alpha 3 domain, thus excluding the transmembrane encoding region and yielding a protein with a highly charged carboxyl terminus. Transfection of the intron 4 containing cDNA, inserted into a retroviral expression vector, into LCL .221 followed by comparison of the class I protein to native soluble G by two dimensional isoelectric focusing/SDS-PAGE analysis, demonstrated this message encoded the soluble HLA-G protein. In addition, a similar intron containing message derived from the HLA-G2 mRNA was found, suggesting the existence of a soluble form of this alternative HLA-G protein. These findings are discussed in relation to other soluble class I molecules and with regard to potential functions of the soluble HLA-G Ag.

HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences.

Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.

Aberrant expression of HLA-G antigen in interferon gamma-stimulated acute myelogenous leukaemia.

We have analysed the expression of HLA-G in 40 leukaemia samples of various subtypes [seven cases of acute lymphoblastic leukaemia (ALL), 28 cases of acute myelogenous leukaemia (AML), three cases of chronic myelogenous leukaemia (CML) and two cases of chronic lymphocytic leukaemia (CLL)] by flow cytometry using HLA-G-specific monoclonal antibody. No leukaemia samples expressed HLA-G without incubation with interferon (IFN)-gamma. However, six out of 28 (21%) AML samples expressed HLA-G upon incubation with IFN-gamma. These six samples derived from one out of seven M2, two out of eight M4 and three out of five M5. The results indicated that AML cells, especially myelomonocytic leukaemia samples, are capable of expressing the HLA-G molecule.

Personal identification by HLA typing of cultured fibroblasts derived from cadaveric tissues.

A method for HLA typing of cultured fibroblasts derived from abdominal skin was developed and applied for personal identification of a cadaver. Successful results were obtained using the conventional NIH HLA typing method for lymphocytes after a slight modification.

Definitive high resolution typing of HLA-E allelic polymorphisms: Identifying potential errors in existing allele data.

A set of robust PCR-SSP reactions were developed for each of the five polymorphic sites that define the five alleles of the HLA class Ib gene, HLA-E. This method was developed using 28 homozygous cell lines and further tested in a sample of African-Americans, a sample of Japanese, and a core panel of cell lines compiled for the 13th International Histocompatibility Workshop. Three alleles were found in each of these four sample groups, HLA-E*0101 (64.29, 50.00, 32.00 and 56.58%, respectively), *01031 (5.36, 20.65, 39.00 and 18.42%) and *01032 (30.35, 29.35, 29.00, and 25.00%). HLA-E*0102 was not detected in any of these samples nor in the cell line, LCL 722.221, in which this allele was originally described. HLA-E*0104 was not found either. This latter allele was originally reported in Japanese at a frequency of 1/22 (4.5%), which should have been high enough to have resulted in multiple occurrences of the *0104 allele in the samples tested in this study. We propose that the existence of the HLA-E*0102 and E*0104 alleles should be questioned.

Mapping HLA for single nucleotide polymorphisms.

Knowledge of DNA sequence variation may help us understand how genetic variability gives rise to functional variability and, in so doing, revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1,000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human leukocyte antigen (HLA) complex. Interestingly, the precise molecular defects in the HLA genes are unknown and the notion that non-HLA genes, within the same chromosomal region, are involved remains a formal possibility. We have determined the nucleotide sequence of a contiguous 2.2 Mbp segment of chromosome six that includes all of the HLA class I region, and have identified over 10,000 SNPs therein. Because of the derivative knowledge of gene and SNP content and position, the scientific community is now uniquely poised to identify disease-contributory SNPs that lie within the MHC.

The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association.

The class Ib antigen HLA-G is expressed as a membrane-bound protein like classical class Ia molecules (M.HLA-G) but, unlike typical class I, is also expressed as a soluble protein (S.HLA-G) with a unique C terminus. Our results show that, similar to classical class I proteins, the membrane-bound form of HLA-G associated with TAP, as evidenced by the ability to immunoprecipitate HLA-G class I heavy chain with TAP antisera. In contrast, the soluble G protein did not appear to associate with TAP in the same manner, since similar immunoprecipitation experiments failed to detect soluble G complex. A detailed analysis of peptides bound to the soluble and membrane HLA-G proteins expressed in the B lymphoblastoid cell line 721.221 showed that, like class Ia complexes, both HLA-G proteins consist of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. The two proteins bind essentially the same set of peptides, which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptides contain Leu at the C terminus and Pro or small hydrophobic amino acids in position 3 followed by Pro or Gly in position 4. The complexity of the bound peptides is lower than that found for some class Ia complexes, but is more similar to class Ia than to the limited repertoire of some murine class Ib molecules.