Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.     

Angiotensin-1-converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides

Circulating angiotensin-1-converting enzyme (ACE) is a highly heritable trait, and a major component of the genetic variance maps to the region of the ACE gene. The strong effect of the locus, and the interest in ACE as a candidate gene for cardiovascular disorders, has led to extensive investigation of its relationship to the ACE phenotype, providing one of the most complete examples of quantitative trait locus (QTL) analysis in humans. Resequencing of ACE followed by haplotype analysis in families of British and French origin has shown that the genetic variants that are primarily associated with the ACE trait map to an 18 kb interval flanked by two intragenic, ancestral recombination breakpoints. This critical interval contains dozens of ACE-associated variants in Caucasians, but identification of which of these directly influence ACE concentration is ambiguous because of the almost complete linkage disequilibrium in European populations. In a complementary sequencing and genotyping study of individuals from West African families, we show that this population has much greater haplotype diversity across the gene. Through analysis of the contrasting relationships of the trait phenotype with haplotypes that carry different allelic combinations from those observed in Caucasians, we demonstrate that (at least) two major intragenic sites within the critical interval and (at least) one minor promoter site are associated with the ACE quantitative trait through additive effects. These results point to the importance of analysing diverse populations with different gene genealogies in gene-association studies.     

Polymorphism in RANTES chemokine promoter affects extent of sarcoidosis in a Japanese population

RANTES, a member of C-C chemokine, is known to be produced at sites of granulomatous reactions in the lung of sarcoidosis. RANTES is a potent eosinophil and lymphocyte attractant with particular preference for CD45RO+ T cells and eosinophils. Polymorphism of the RANTES promoter has recently been shown to be related to allergic and infectious diseases; atopic dermatitis, asthma, and polymyalgia rheumatica. Considering that this might affect sarcoidosis, we studied polymorphism of the RANTES gene in 114 patients with sarcoidosis and 136 healthy control subjects. Their genotypes were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Although no difference in the genotype distribution between healthy control subjects and sarcoidosis patients was identified, the difference in the frequencies of the patients with three or more organ involvement was significant (P<0.01) with the frequency of those in AA genotype being elevated (P<0.05). BAL findings in 48 out of 114 patients who underwent bronchoscopy were reviewed. The CD4/8 ratio of lymphocytes in bronchoalveolar lavage fluid in the patients with AA genotype was significantly increased (P<0.05). From the results, we suggest that in RANTES gene polymorphism the homozygous A allele might be a genetic risk factor for extent disease of sarcoidosis.     

Evidence for endocytosis-independent infection by human rotavirus

Summary The effects of five lysosomotropic drugs (NH₄Cl, chloroquine, methylamine, amantadine and dansylcadaverine) and cytochalasin B on human rotavirus (HRV KUN strain) and vesicular stomatitis virus (VSV Indiana strain) infection in monkey MA 104 cells were examined. These drugs had little effect on HRV yield but greatly reduced VSV yield. The results strongly suggest that HRV does not require endocytotic activity and intracellular acidic vesicles for the initial stage of infection and support our postulate that HRV enters the target cell by direct penetration of its nucleoid through the cell membrane.     

Significance of aberrant (cytoplasmic/nuclear) expression of beta-catenin in pancreatoblastoma

This study concerns the significance of aberrant (nuclear/cytoplasmic) expression of beta-catenin in pancreatoblastoma (PBL). On immunohistochemistry, all seven PBLs examined showed nuclear/cytoplasmic expression of beta-catenin, predominantly in the squamoid corpuscles (SCs). In areas with acinar/ductular differentiation, few tumour cells displayed nuclear/cytoplasmic expression of beta-catenin and more than half of the tumour cells showed membranous expression. Two out of five (40%) tumours examined showed missense mutations in codons 33 and 37 of exon 3 of the beta-catenin gene. No mutation of the adenomatous polyposis coli (APC) gene was detected in two of the remaining three tumours. Amplifiable DNA for APC analysis was not obtained from the one other tumour. Immunoreactivity for cyclin D1, one of the nuclear targets of beta-catenin, was found predominantly in the SCs of the seven tumours. In contrast, the Ki-67 labelling index was 2-4% (median 3%) in the SCs and 8-18% (median 12%) in the other areas, indicating a negative correlation with nuclear cyclin D1 reactivity. These results imply that in PBLs, nuclear/cytoplasmic accumulation of beta-catenin and overexpression of its target gene cyclin D1 are not associated with the induction of tumour cell proliferation. Nuclear/cytoplasmic accumulation of beta-catenin may be related to the morphogenesis of the SCs that are considered most characteristic for PBL.     

Anionic copolymerization of β-lactones in correlation with the mode of fission. studies on the syntheses of aliphatic polyesters. XI

The copolymerization behavior of β-propiolactone (PL) and α.α-bischloromethyl β-propiolactone (α-BCPL) is correlated with the mode of fission. With sodium acetate as catalyst, the formation of β-acetoxy propionic acid at the initiation reaction was observed and α-BCPL was more than five times as reactive as PL in copolymerization. Thus propagation by carboxylate anion through alkyl-oxygen fission was presumed in the case of pyridine and the acetate of Na, Li, and Mg as catalyst. On the other hand, the formation of ethyl β-hydroxy propionate and the decreased reactivity of α-BCPL in copolymerization was observed with magnesium and aluminum ethoxide as catalyst. In the case of the ethoxide of Na, Li, and Al, and Et₂Mg or Et₃Al as catalyst, propagation by alkoxide anions through acyl-oxygen fission of the coordinated lactones was presumed.     

Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes

A mAb J43 has been produced against the product of the mousePD-1 gene, a member of the Ig gene superfamily, which was previouslyisolated from an apoptosis-induced T cell hybridoma (2B4.11)by using subtractive hybridization. Analyses by flow cytometryand immunoprecipitation using the J43 mAb revealed that thePD-1 gene product is a 50–55 kDa membrane protein expressedon the cell surface of several PD-1 cDNA transfectants and 2B4.11cells. Since the molecular weight calculated from the aminoacid sequence is 29,310, the PD-1 protein appears to be heavilyglycosylated. Normal murine lymphoid tissues such as thymus,spleen, lymph node and bone marrow contained very small numbersof PD-1⁺ cells. However, a significant PD-1⁺ population appearedin the thymocytes as well as T cells in spleen and lymph nodesby the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1antigen expression was strongly induced in distinct subsetsof thymocytes and spleen T cells by in vitro stimulation witheither anti-CD3 mAb or concanavalin A (Con A) which could leadT cells to both activation and cell death. Similarly, PD-1 expressionwas induced on spleen B cells by in vitro stimulation with anti-IgMantibody. By contrast, PD-1 was not significantly expressedon lymphocytes by treatment with growth factor deprivation,dexamethasone or lipopolysaccharide. These results suggest thatthe expression of the PD-1 antigen is tightly regulated andinduced by signal transduction through the antigen receptorand do not exclude the possibility that the PD-1 antigen mayplay a role in clonal selection of lymphocytes although PD-1expression is not required for the common pathway of apoptosis.     

Detection of serum thermolabile beta-2 macroglycoprotein (Hakata antigen) by enzyme-linked immunosorbent assay using polysaccharide produced by Aerococcus viridans

Although a serum thermolabile beta-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of D-glucose, N-acetyl-D-glucosamine, D-mannose, and D-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56 degrees C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.     

Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model

Tissue-engineered vascular autografts (TEVAs) were made by seeding 4-6 x 10(6) of mixed cells obtained from femoral veins of mongrel dogs onto tube-shaped biodegradable polymer scaffolds composed of a polyglycolid acid (PGA) nonwoven fabric sheet and a copolymer of L-lactide and caprolactone (n = 4). After 7 days, the inferior vena cavas (IVCs) of the same dogs were replaced with TEVAs. After 3, 4, 5, and 6 months, angiographies were performed, and the dogs were sacrificed. The implanted TEVAs were examined both grossly and immunohistologically. The implanted TEVAs showed no evidence of stenosis or dilatation. No thrombus was found inside the TEVAs, even without any anticoagulation therapy. Remnants of the polymer scaffolds were not observed in all specimens, and the overall gross appearance similar to that of native IVCs. Immunohistological staining revealed the presence of factor VIII positive nucleated cells at the luminal surface of the TEVAs. In addition, lesions were observed where alpha-smooth muscle actin and desmin positive cells existed. Implanted TEVAs contained a sufficient amount of extracellular matrix, and showed neither occlusion nor aneurysmal formation. In addition, endothelial cells were found to line the luminal surface of each TEVA. These results strongly suggest that "ideal" venous grafts with antithrombogenicity can be produced.