Report of a new DRB1*13 allele: DRB1*1336.

This brief communication describes the characterization of a new allele, DRB1*1336.

Ergonomics oriented to processes becomes a tool for continuous improvement.

A holistic view is essential for quality initiatives such as Total Quality Management (TQM), Standard No. ISO 9001:1994 (International Organization for Standardization [ISO], 1994), Concurrent Engineering, Business Reengineering, and Business Process Improvement. The challenge is knowing how to transition from this theoretical concept to implementation. The relationship between quality interest and an ergonomics program will be the focus of this discussion. An ergonomics oriented improvement program includes (a) ergonomics or fitting the job to the person; (b) integration of operations management, safety engineering, medical management, and employees as co-owners of the process; (c) the emphasis of ergonomic precepts in the engineering of new processes and improvement of current processes; and (d) the emphasis of employees taking responsibility for their own well being and the improvement of their work environment. The parallel between the continuous improvement process delineated by the quality-system requirements in Standard No. ISO 9001:1994 (ISO, 1994) and the improvement contributions of ergonomics are very revealing (Getty, Abbott, & Getty, 1995). It is the contention of this approach that if the precepts of ergonomics were applied to the work environment, it would support the objective of world class quality and productivity, resulting in improved global competitiveness of businesses.

DQB1*0202 and the new DQB1*0203 allele: a fourth pair of DQB1 alleles differing only at codon 57.

Amino acid 57 of DQ beta 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 phenotype 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.

Functional dissociation of CD8 alpha's Ig homologue and connecting peptide domains.

The contribution of the CD8 alpha.IgV homologue domain to class I MHC binding was evaluated using a series of chimeric human CD8 alpha:Fc polypeptides incorporating alternative CD8 alpha extracellular domain components. Using a nonisotopic cellfree physical binding assay, those Fc chimeras encompassing the CD8 alpha.IgV homologue domain only (dissociated from the 48-amino acid CD8 alpha connecting peptide) were shown to retain the capacity of the complete CD8 alpha extracellular domain to bind to a recombinant soluble class I MHC alpha 3 domain unit or to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha 3 domain interaction was verified by mAb and soluble polypeptide blocking experiments. Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha.IgV homologue domain and a recombinant soluble class I MHC alpha 3 domain unit was accomplished. In addition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD8 alpha.IgV homologue domain was generated via chimerization with the GPI signal sequence from decay-accelerating factor. GPI anchorage for this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was shown in a cell:cell binding assay. The findings indicate that the CD8 alpha.IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing retains class I MHC binding capacity. This further establishes the principle that Ig superfamily domains from receptor:counter-receptor pairs can interact with each other as isolated units, providing an experimental path for tailoring therapeutically useful IgSF protein derivatives.

Class I MHC alpha 3 domain can function as an independent structural unit to bind CD8 alpha.

Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the alpha 3 domain of the class I MHC molecule. We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids. This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, alpha 3/MalE, in which the alpha 3 domain has been expressed independently of its neighboring polypeptide domains on the native class I MHC molecule and beta 2-microglobulin (beta 2-m). These proteins were produced using eukaryotic and prokaryotic expression systems, respectively. Our data demonstrated specific, saturable binding between soluble CD8 alpha (sCD8 alpha) and alpha 3/MalE, and the Kd of this interaction was determined to be 4.5 x 10(-7) M. Monoclonal antibodies (mAb) directed against either CD8 or the alpha 3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and beta 2-m did not. Our data suggest that the interaction between CD8 alpha and the alpha 3 domain of class I MHC does not require the participation of neighboring class I sequences or beta 2-m.

Hematopoietic placental protein 14. An immunosuppressive factor in cells of the megakaryocytic lineage.

Placental protein 14 (PP14), an immunosuppressive molecule previously known to be expressed in the female and male reproductive tracts only, was shown to be expressed by hematopoietic cells of the megakaryocytic lineage. Northern blot analysis confirmed the induction specificity of PP14 mRNA in phorbol ester-treated K562 cells. Potent immunosuppressive activity in conditioned medium from phorbol ester-treated K562 cells was attributed to hematopoietic PP14 by anti-PP14 antibody blocking. Immunoprecipitation with anti-PP14 antibodies from conditioned medium revealed two distinct PP14 protein isoforms, designated PP14.1 and PP14.2. Polymerase chain reaction cloning and analysis demonstrated the presence of distinct mRNA counterparts to PP14.1 and PP14.2 that had not been resolved by Northern blot analyses. Hematopoietic PP14.1 mRNA corresponds in size to endometrial PP14 mRNA, whereas the smaller hematopoietic PP14.2 mRNA displays an internal in-frame 66-nucleotide deletion that can be explained by alternative splicing and predicts a 22-amino-acid deletion in the encoded gene product. Both PP14 mRNA isoforms were additionally detected by reverse transcriptase polymerase chain reaction analyses in two human megakaryocytic cell lines and in normal human megakaryocytes and platelets. PP14 mRNA was not detected by reverse transcriptase polymerase chain reaction in a panel of nonhematopoietic, nonendometrial tissues examined. The finding of hematopoietic PP14 within the megakaryocytic lineage provides an additional regulatory link between the coagulation and immune systems in normal and pathological settings.

Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis.

Recombinant GPI-anchored HLA-A2.1 (HLA-A2.1-GPI/beta 2m) was used as a protein transfer vehicle to deliver a hepatitis B virus antigenic peptide to the surfaces of cytotoxic T cell targets. Empty HLA-A2.1-GPI/beta 2m was first produced in D. melanogaster cotransfectants and immunoaffinity purified. Cell coating with HLA-A2.1-GPI/beta 2m was shown to occur rapidly, and to be protein concentration dependent. Protein-transferred HLA-A2.1-GPI/beta 2m effectively presented a hepatitis B virus peptide to peptide-specific HLA-A2.1-restricted T cell clones in cytotoxicity assays. Protein transfer of functional GPI-modified class I MHC-antigenic peptide complexes represents a novel strategy for delivering functional antigenic complexes to cell surfaces that bypasses limitations of gene transfer and permits control of antigenic peptide densities at cell surfaces.

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.

Glycolipid reanchoring of T-lymphocyte surface antigen CD8 using the 3' end sequence of decay-accelerating factor's mRNA.

Decay-accelerating factor (DAF) is one of a family of cell-associated proteins that undergo posttranslational modifications in which glycolipid anchoring structures are substituted for membrane-spanning sequences. The signals that direct the covalent substitution reaction in these proteins are unknown. Human DAF was expressed in Chinese hamster ovary (CHO) cells and murine BW lymphocytes. In both cases, the xenogeneic DAF in transfectants incorporated a glycolipid anchor. A chimeric CD8-DAF cDNA, encompassing the extra-cellular region of the T-lymphocyte surface antigen CD8 and the 3' end of DAF mRNA (encoding the C-terminal region of mature DAF as well as the hydrophobic extension peptide), was expressed in human leukemia lines after transfection with an Epstein-Barr virus-based episomal vector. The chimeric protein in transfectants demonstrated glycolipid anchoring, whereas unaltered CD8 in control experiments did not. The signals directing glycolipid anchoring in eukaryotic cells are thus evolutionarily conserved and contained in the 3' end of the DAF sequence.

Cloning and characterization of cDNAs encoding the complete sequence of decay-accelerating factor of human complement.

cDNAs encoding the complement decay-accelerating factor (DAF) were isolated from HeLa and differentiated HL-60 lambda gt cDNA libraries by screening with a codon preference oligonucleotide corresponding to DAF NH2-terminal amino acids 3-14. The composite cDNA sequence showed a 347-amino acid protein preceded by an NH2-terminal leader peptide sequence. The translated sequence beginning at the DAF NH2 terminus encodes four contiguous approximately equal to 61-amino acid long repetitive units of internal homology. The repetitive regions contain four conserved cysteines, one proline, one glycine, one glycine/alanine, four leucines/isoleucines/valines, one serine, three tyrosines/phenylalanines, and one tryptophan and show striking homology to similar regions previously identified in factor B, C2, C4 binding protein, factor H, C1r, factor XIII, interleukin 2 receptor, and serum beta 2-glycoprotein I. The consensus repeats are attached to a 70-amino acid long segment rich in serine and threonine (potential O-glycosylation sites), which is in turn followed by a stretch of hydrophobic amino acids. RNA blot analysis of HeLa and HL-60 RNA revealed three DAF mRNA species of 3.1, 2.7, and 2.0 kilobases. The results indicate that portions of the DAF gene may have evolved from a DNA element common to the above proteins, that DAF cDNA predicts a COOH-terminal anchoring polypeptide, and that distinct species of DAF message are elaborated in cells.

Arteria intercarotica caudalis and its homologue in the domestic animals.

The arteria intercarotica caudalis was observed to be present in the dog, horse and cat but was reticulated in the case of cattle, sheep, goat, and pig. The latter structure was a homologue of the former and represented an important intercarotid communication present in most of vertebrate.

Presence of the arteria caroticobasilaris in the horse.

The consistent presence of the caroticobasilar artery was observed and discussed in view of the anatomical normalities in the horse. The persistence of the above vessel was correlated with the developmental changes in the cranial and cerebral arteries.