The CD14 monocyte differentiation antigen maps to a region encoding growth factors and receptors.

CD14 is a myelomonocytic differentiation antigen expressed by monocytes, macrophages, and activated granulocytes and is detectable with the monoclonal antibodies MO2, MY4, and LeuM3. Analyses of complementary DNA and genomic clones of CD14 show that it has a novel structure and that it maps to chromosome 5 within a region containing other genes encoding growth factors and receptors; it may therefore represent a new receptor important for myeloid differentiation. In addition, the CD14 gene is included in the "critical" region that is frequently deleted in certain myeloid leukemias.

Dual recognition of a human cytotoxic T-cell clone for melanoma antigens.

It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tumor antigens in MHC-restricted and MHC-unrestricted manners. We have established two alpha beta CTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2-restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CD8, and alpha beta T-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single alpha beta TCR. Both CTL clones contained the same Valpha and Vbeta gene segments although they carried different Jalpha and Jbeta gene segments. Taken together, these results confirm that CTL clones that carry a single alpha beta TCR recognize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo.

Contribution of CD4+ and CD8+ T cells and interferon-gamma to the progress of chronic rejection of kidney allografts: the Th1 response mediates both acute and chronic rejection.

T cells mediating chronic rejection (CR) of human kidney allografts were characterized by comparing them with those mediating acute rejection (AR). Two lines of analysis were performed using biopsy specimens (23 CR and 8 AR). First, the extent of infiltration of CD4+ and CD8+ T cells into allografts was assessed from mRNA expression of CD4 and CD8. The group of CR specimens was not significantly different from the group of AR specimens in terms of the extent of CD4+ and CD8+ T cell infiltration, underlining the importance of the immunological contribution to the progress of CR. Second, Th1/Th2 polarization in infiltrating T cells was investigated by measuring mRNA expression of interferon gamma (IFN-gamma; a Th1 cytokine) and interleukin 4 (IL-4; a Th2 cytokine). IFN-gamma expression was detected in most CR specimens, and was not significantly different between the group of CR specimens and the group of AR specimens. On the other hand, IL-4 expression was detected in only two CR specimens and one AR specimen; from its pathological features, the AR in this last case was concomitant with CR. These results suggest that most cases of CR and of AR are mediated by Th1 mechanisms, although some cases of CR show features of both Th1 and Th2.

The mouse/human cross-species heterodimer of leucine-rich repeat kinase 2: possible significance in the transgenic model mouse of Parkinson's disease.

Leucine-rich repeat kinase (LRRK2) is the causal molecule of autosomal dominant Parkinson's disease (PD). We previously reported that intracellular degradation of wild-type (WT) LRRK2 is promoted by formation of heterodimers with the I2020T mutant LRRK2. In the present study, we investigated whether this is also the case for mouse/human cross-species heterodimers, which could be formed in transgenic mice. First, by co-transfection and immunoprecipitation, we identified the cross-species heterodimer of mouse LRRK2 and human LRRK2. Next, we found that the protein level of mouse LRRK2 decreased when co-transfected with human I2020T LRRK2, but not with human WT LRRK2. These results suggested that degradation of mouse LRRK2 was promoted by formation of a cross-species heterodimer with the mutant LRRK2. In I2020T LRRK2-transgenic mice, the lower protein level of brain LRRK2 in comparison with control mice, together with higher expression of the mRNA, suggested that endogenous LRRK2 was degraded by formation of cross-species heterodimers. Our results suggest a new concept of cross-species dimer/oligomer formation in transgenic disease-model mice.

Rapid DNA typing utilizing immobilized oligonucleotide probe and a nonradioactive detection system. Application to HLA-DR typing of the Japanese population.

We established a rapid and simple method of HLA-DR genotyping, and applied it for analysis of the Japanese population. Our method includes rapid preparation of DNA samples from buccal mucosa, incorporation of biotin-dATP into DRB genes during amplification by the polymerase chain reaction, hybridization with sequence-specific oligonucleotide (SSO) probes immobilized on nylon membranes via poly (dT) tails, and detection of the hybridization signal as chemiluminescence. We carried out DR typing of 30 Japanese donors using 20 different immobilized SSO probes, and obtained unambiguous typing signals showing perfect correlation with their serologic DR types. The genotyping also enabled us to identify several DR types unique to the Japanese population, such as DRw12b (DRB1*1202), DRw14c (DRB1*1405), and serology blank type, DR'JX6' (DRB1*1403). The method presented here would be suitable for routine DR typing in tissue-typing laboratories.

T-cell receptor variable gene analysis of renal allograft-infiltrating cells in biopsy specimens using a nonradioisotopic micromethod.

BACKGROUND: A sensitive micromethod for T-cell receptor (TCR) analysis is needed for clonality analysis of renal allograft-infiltrating T cells (RAITs) obtained by needle biopsy. METHODS: TCR cDNA was amplified by the anchored polymerase chain reaction and was hybridized with 28 different TCR beta variable (TCRBV) genes fixed on nylon membranes, and the percentage of each TCRBV gene was measured spectrophotometrically. RESULTS: The specificity and linearity of the hybridization technique and the constancy of the TCRBV percentages over a wide range of sample amounts were demonstrated by control experiments. Analysis of RAITs of biopsy specimens from four patients showed broad or skewed TCRBV usage, indicating the presence of polyclonal and oligoclonal RAIT populations, respectively. In one patient who received OKT3 immunosuppressive treatment, the TCRBV skewness was dramatically reduced after the treatment. CONCLUSION: We have established a powerful method for analyzing RAIT clonality, which is especially useful for monitoring RAIT dynamics after immunosuppression therapy.

Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.

Linkage between HLA-DRB1 and -DRB3 types in the Japanese population analyzed by oligonucleotide genotyping.

We analyzed linkage between HLA-DRB1 and -DRB3 types in 219 Japanese donors by oligonucleotide genotyping. In the Japanese population, DRB1*1201 was linked with DRB3*0101 in all donors analyzed; in contrast, most Caucasian DRB1*1201 is known to be linked with DRB3*02(01/02) (*0201 or *0202). However, most DRB1*1202 was linked with DRB3*0301. Thus, the two DRw12-related DRB1 types are linked with DRB3 types distinct from each other. All the three DRw14-related DRB1 types, DRB1*1401, DRB1*1402, and DRB1*1405, were linked with DRB3*02(01/02) in the Japanese population, contrasting with the known linkage between DRB1*1402 and DRB3*0101 in other ethnic populations. The serologically "blank" DR type, DRB1*1403, was linked with DRB3*0101. Other DRB1 types, DRB1*0301, DRB1*11(01/04) (*1101 or *1104), and DRB1*13(01/02) (*1301 or *1302) in the Japanese population were linked mostly with the same DRB3 types, like those known in other ethnic populations.

A novel human class II specificity, DQ "Wa," resides on DQ molecules of DR4,Dw15 and DRw8,Dw8 B-cell lines identified as DQ "blank".

The molecular localization of a novel human class II specificity, DQ "Wa," was investigated. A monoclonal antibody, HU46, which has previously been shown to react with DR4, Dw15 and DRw8, Dw8 B cells that type as DQ "blank," was used for the isolation and structural characterization of class II molecules bearing the DQ "Wa" determinant. The partial N-terminal sequence analysis of class II molecules bearing the DQ "Wa" determinant, purified from two B-cell lines, EBV-Wa (DR4, Dw15, DQ "blank") and GI (DRw8, Dw8, DQ "blank"), shows that the alpha and beta chain sequences are homologous to HLA-DQ. Within the limits of our analysis, the alpha and beta chains from both cell lines are identical. Both beta chains possess a phenylalanine residue at position 9 that differs from the tyrosine residue present at this position in beta chains of DQ alleles. These studies indicate that a novel human class II specificity, DQ "Wa," resides on a new allelic form of DQ molecules found in DR4, Dw15 and DRw8, Dw8 cells that are DQ "blank."

A single universal primer for the T-cell receptor (TCR) variable genes enables enzymatic amplification and direct sequencing of TCR beta cDNA of various T-cell clones.

We designed a primer for the PCR directed against a highly conserved sequence of the TCR V beta gene. The V beta-universal primer, in combination with a constant region-specific primer, enabled us to amplify TCR beta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without prior knowledge of their V beta sequences. The amplified TCR cDNA was purified by agarose gel electrophoresis and subjected to direct sequencing. In nine of ten T-cell clones analyzed, direct TCR sequencing gave readable sequence ladders, including two-thirds of V beta, junctional, and J beta regions. One T-cell clone gave an unreadable mixed-profile sequence ladder, indicating that this clone expressed more than one major TCR beta transcript. Even in this case, however, it was possible to determine two different TCR beta sequences separately using sequence primers specific to one of the 13 J beta segments deduced from the mixed ladder. Thus, direct sequencing utilizing the single V beta-universal primer enabled a simple, rapid, and reliable sequence determination of TCR beta cDNA of all T-cell clones analyzed.

Sequence analysis and oligonucleotide genotyping of HLA-DR"JX6", a DR"blank" haplotype found in the Japanese population.

We analyzed one of the HLA-DR"blank" haplotypes found in the Japanese population using serologic studies, sequence determination, and genotyping with sequence-specific oligonucleotide (SSO) probes. The DR"blank" haplotype, designated DR"JX6", segregated in a family in association with the DRw52 and the DQw7 specificities. The cDNA and genomic DNA of the DRB1 gene originating from the DR"JX6" haplotype were amplified enzymatically and sequenced after cloning into a plasmid vector. The amino acid sequence of the first domain in the DR beta 1 chain of the DR"JX6" haplotype was different from those of other DR haplotypes sequenced so far, but in the first hypervariable region, the sequence was identical to those of the DRw11, DRw13, DRw14, and DRw17 haplotypes. SSO probes were synthesized on the basis of the DR"JX6" haplotype sequence as well as known sequences of the DRB1, DRB3, and DRB4 genes of other DR haplotypes. These SSO probes were used for the genotyping of Japanese donors whose DRB genes were amplified enzymatically and found to show a hybridization profile that was consistent with the results of serologic studies on the DR"JX6" haplotype.

Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions.

Genetic analysis of hydatidiform moles utilizing the oligonucleotide-DNA typing of the HLA-DRB gene.

The genetic origin of hydatidiform moles was analysed utilizing HLA-DNA typing. Using HLA-DR type-specific oligonucleotide probes, the DRB types of seven moles were determined and compared with the parental DRB types to determine the paternal and/or maternal origin of the moles. In four cases, the molar tissues showed single DRB types of paternal origin, although in one, the molar DRB type was also possessed by the mother. These four moles were, therefore, considered to be androgenetic in origin. Chromosomal karyotyping was carried out for three of these cases and confirmed the DR-DNA typing results. Two moles demonstrated a DRB-type triplet, which strongly suggested triploidy. Although one mole showed a heterozygous DRB type, karyotyping indicated triploidy (69, XXX) and suggested that this mole was caused by dispermy-fertilization, in which both of the sperms had the same DRB type. Although the majority (about 80%) of partial hydatidiform moles have been reported to be triploid as a result of dispermy, four of the moles analysed in this study (cases 1, 2, 3 and 4), diagnosed as partial macroscopically and/or histopathologically, were found to be androgenetic in origin using karyotyping and DR-DNA typing. Therefore, HLA-DR DNA typing, combined in some cases with karyotyping, provides an accurate method for diagnosing androgenesis and triploidy in complete and partial hydatidiform moles.

Allogeneic recognition of HLA-DRB1*0406 by T cells with HLA-DRB1*0403: role of amino acid residue 37 on the beta sheet in T cell recognition.

Five T cell clones reactive with allogeneic HLA-DR molecules were obtained by stimulating CD4+ T cells (DRB1*0403) with DRB1*0406-homozygous KT13 cells whose DR beta chain differed by a single amino acid residue (37) on the beta sheet from the DRB1*0403 product. Except for one T cell clone which had both auto- and alloreactivities, these clones proliferated by stimulation with KT13 but not with autologous cells, indicating that the single substitution at position 37 on the HLA-DR molecule was sufficient to alter the alloantigenicity of the DR molecule and to elicit an allogeneic T cell response. Two clones reacted with some but not all B cell lines with DRB1*0406, suggesting the possible involvement of a certain peptide whose distribution is restricted to some cells which form the alloantigenic structure recognized by these clones. The two remaining clones showed broad but distinct anti-DR specificity in addition to anti-DRB1*0406 reactivity, suggesting that they recognize the DRB1*0406-peptide complex whose antigenic structures also occur in some combinations of other DRB1 alleles with certain peptides bound to these alleles. The T cell clone with both auto- and alloreactivity was found to react with autologous monocytes but not with autologous B or T cells and to express lower TCR alpha beta than other T cell clones which showed no autoreactivity. The possible recognition molecule for this autoreactive T cell clone is discussed.

Carbohydrate-binding activity of concanavalin A containing various numbers of calcium and manganese ions.

After treatment with EDTA, fragment-free concanavalin A (Con A), F3, was fractionated into three major fractions, f1, f2, and f3, by CM-cellulose chromatography. Fully metallized fragment-free Con A, F3M, was prepared by incubation of F3 with excess metal ions and also fractionated into f1M, f2M, and f3M by the same method as for F3. The Con A preparations obtained were analyzed for metal content by atomic absorption spectrophotometry and for number of carbohydrate-binding sites by ultraviolet absorbance change on binding of p-nitrophenyl alpha-D-mannopyranoside (PNP . Man) to Con A. Both f1 (Ca: 0.2, Mn: 0.1) and f1M (Ca: 0.4, Mn: 0.2) had less than 0.6 carbohydrate-binding sites per tetramer of Con A. f2 (Ca: 2.1, Mn: 0.9) and f2M (Ca: 2.1, Mn: 1.9) had 1.9 and 1.8 carbohydrate-binding sites, respectively. f3 (Ca: 4.1, Mn: 2.1) and f3M (Ca: 3.8, Mn: 4.0) had 4.1 and 4.2 carbohydrate-binding sites, respectively. Carbohydrate-binding sites of Con A were in a stoichiometric relation to bound Ca2+. The molar absorptivity change at 317 nm was 2 X 10(3) M-1 . cm-1. Precipitation activity of the above Con A preparations towards glycogen was also estimated using glycogen labeled covalently with Remazolbrilliant Blue R. The precipitation activity of Con A as well as PNP . Man-binding was strongly affected by the number of bound Ca2+. A slight contribution by Mn2+ was also detected in the precipitation activity.

Clonality analysis of T cells mediating acute and chronic rejection in kidney allografts.

The clonality of T-cell populations mediating acute and chronic rejection (AR and CR, respectively) of kidney allografts was ascertained by investigating the diversity of TCRBV genes expressed by allograft-infiltrating T cells. Both oligoclonality and polyclonality cases were found in biopsy specimens of AR as well as CR. These results indicated that the T-cell clonality in each specimen did not correlate directly with the mode of rejection. When AR and CR specimens were compared, however, the CR specimen group was significantly more polyclonal (or less oligoclonal) than the AR group. This result may reflect the higher chance of epitope spreading in the more slowly progressing CR than in AR.

An improved system for selection of forward mutations in an Escherichia coli supF gene carried by plasmids.

An improved system to examine forward mutations that occurred in the supF gene of Escherichia coli carried on a multicopy plasmid is described. The system was validated by measuring spontaneous mutations of supF plasmids propagated in wild-type, recA- and mutM- mutY- E. coli strains, the mutation frequencies of which were 1.3 x 10(-7), 6.3 x 10(-7) and 1.5 x 10(-6), respectively. Sequence analysis of the supF mutant plasmids revealed that G:C-->T:A and G:C-->C:G transversions dominated. This improved system allows rapid scoring and sequencing forward mutations in the supF gene, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria and mammalian cells.

Polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis of the human HLA-DQB regions.

Genetic diagnosis of 13 alleles of HLA-DQB1 (0501, 0502, 5031, 5032, 0601, 0602, 0604, 0201, 0301, 0302, 3032, 0401 and 0402) from 65 human DNA samples was achieved by applying single-strand conformation polymorphism (SSCP) analysis to DNA fragments amplified by the polymerase chain reaction (PCR) using a convenient primer set for DQB1 (recommendation of the International Histocompatibility Workshop, 1991). Differences between strand images (narrow/distinct or broad/diffuse) from the individual alleles and their electrophoretic mobilities are regarded as criteria for confirming the genetic diagnosis of DQB1 alleles. This primer set amplifies not only DNA fragments belonging to DQB1, but also to DQB2, and classification of 3 phenotypes (1.1, 1.2 and 1.1/1.2) in the presence of two alleles at the latter locus was suggested. Consequently, PCR/SSCP of DNA amplified by this primer enables classification of the phenotypes, at least under our experimental conditions, into 3 x 91 groups. Two advantages of SSCP analysis over VNTR with regard to the use of amplified DNA in forensic practice are described.