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A/B-Transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen, which is responsible for the synthesis of glycoprotein- and glycolipid-conjugates termed A/B-antigens. One polymorphism that causes the Pro234Ser substitution in B-transferase was recently found in a genotyping study, and might be cis-AB. In the present study, we analyzed the phenotypes arising from the enzymatic specificity of B-transferase with the Pro234Ser mutation. To evaluate the effect of the P234S mutation on enzymatic specificity, we generated an expression plasmid for B-transferase with Pro234Ser as well as A-transferase with Leu266Met, which is frequently found in cis-ABs. Transfection of B-transferase/P234S or A-transferase/L266M cDNA into HeLa cells, an O-blood group cell line, resulted in an AB-phenotype by absorption-elution testing and immunostaining, whereas A- and B-transferase-expressing HeLa cells exhibited only their own activity. Molecular simulation indicated that the P234S mutation causes a conformational change in the substrate pocket making it suitable for N-acetylgalactosamine.

The high-affinity IgE receptor, FcεRI, which is composed of α-, β-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of β mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIβ). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIβ, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIβ transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.

To examine the immune-modulatory effects of probiotics during early infancy, Bifidobacterium breve M-16V (B. breve) was administered to rat pups during the newborn or weaning period, and the expression of inflammatory genes was investigated using a cDNA microarray and real-time PCR.After B. breve administration, significant increases in the numbers of Bifidobacterium in both the cecum and colon were confirmed during the newborn period. The numbers of upregulated and downregulated genes were greater during the weaning period than in the newborn period and were greatest in the colon, with fewer genes altered in the small intestine and the fewest in the spleen. The expression of inflammation-related genes, including lipoprotein lipase (Lpl), glutathione peroxidase 2 (Gpx2), and lipopolysaccharide-binding protein (Lbp), was significantly reduced in the colon during the newborn period. In weaning rat pups, the expression of CD3d, a cell surface receptor-linked signaling molecule, was significantly enhanced in the colon; however, the expression of co-stimulatory molecules was not enhanced.Our findings support a possible role for B. breve in mediating anti-inflammatory and antiallergic reactions by modulating the expression of inflammatory molecules during the newborn period and by regulating the expression of co-stimulatory molecules during the weaning period.Gene expression in the intestine was investigated after feeding 5 × 10(8) cfu of B. breve every day to the F344/Du rat from days 1 to 14 (newborn group) and from days 21 to 34 (weaning group). mRNA was extracted from intestine, and the expression of inflammatory gene was analyzed by microarray and real-time PCR.

Background The human IL1RL1/ST2 gene encodes IL33 receptor. Recently, IL33 has been recognized as a key molecule for the development of Th2 response. Although mast cells and basophils are major targets of IL33 and play important roles in IL33-mediated Th2-type immune responses, the expression mechanism of ST2 in mast cells and basophils is largely unknown. In the present study, we analyzed regulation mechanism of the human ST2 promoter in the human mast cell line LAD2 and basophilic cell line KU812. Methods Promoter activity was determined by reporter assay with plasmids carrying the wild-type ST2 promoter obtained from human genomic DNA and its mutant. The transcription factor binding to the identified cis- element was identified by an electrophoretic mobility shift assay (EMSA). The effect of candidate transcription factor on ST2 expression was confirmed by analyzing ST2 mRNA level in siRNA-introduced cells. Results: Reporter assay demonstrated that a cis-element of typical Ets-family binding sequence was critical for promoter activity in LAD2 and KU812. An Ets-family transcription factor PU.1 bound to this element in an EMSA. When PU.1 expression was suppressed by siRNA, sT2 mRNA level was significantly reduced in KU812. Conclusions These observations indicated that PU.1 positively regulates the ST2 promoter as a transcription factor that directly transactivates the ST2 promoter via Ets-family-related cis -element in mast cells and basophils.

Fibrodysplasia ossificans progressiva (FOP) is a rare, congenital disorder caused by heterozygous mutation of the bone morphogenetic protein type I receptor ACVR1. Various forms of atypical FOP have recently been identified, and a recurrent mutation, ACVR1 (p.Arg258Ser) was reported. We encountered a 17-year-old Japanese female patient with sporadic occurrence of FOP. At the age of 7 years, radiological examination revealed progressive heterotopic ossification and cervical spine malformations. Although great toe malformation was not observed, we diagnosed her as having FOP. Then, ACVR1 was analyzed and a recurrent mutation of p.Arg258Ser was identified. We noticed that there may be phenotypic differences between c.774G>T and c.774G>C, which lead to the same amino-acid change, p.Arg258Ser. Genotype-phenotype correlation was discussed with the review of the previous reports.

In this study, we investigated the role of a transcription factor, PU.1, in the regulation of CD80 and CD86 expression in dendritic cells (DCs). A chromatin immunoprecipitation assay revealed that PU.1 is constitutively bound to the CD80 and CD86 promoters in bone marrow–derived DCs. In addition, co-expression of PU.1 resulted in the transactivation of the CD80 and CD86 promoters in a reporter assay. The binding of PU.1 to cis-enhancing regions was confirmed by electromobility gel-shift assay. As expected, inhibition of PU.1 expression by short interfering RNA (siRNA) in bone marrow–derived DCs resulted in marked down-regulation of CD80 and CD86 expression. Moreover, overexpression of PU.1 in murine bone marrow–derived lineage-negative cells induced the expression of CD80 and CD86 in the absence of monocyte/DC-related growth factors and/or cytokines. Based on these results, we conclude that PU.1 is a critical factor for the expression of CD80 and CD86. We also found that subcutaneous injection of PU.1 siRNA or topical application of a cream-emulsified PU.1 siRNA efficiently inhibited murine contact hypersensitivity. Our results suggest that PU.1 is a potential target for the treatment of immune-related diseases.

Induction of alphaEbeta7 expression on T cells by transforming growth factor (TGF)-beta is thought to be important for intestinal intraepithelial T lymphocyte (IEL) entry into the epithelial compartment. However, there has been no in vivo evidence that up-regulation of alphaEbeta7 expression on T cells by TGF-beta is critical for the selective localization of intestinal IEL in the epithelial area. We have recently established transgenic mice expressing Smad7 under the control of a distal lck promoter where TGF-beta/Smad signaling is specifically blocked in mature T cells. Here we showed that TGF-beta-mediated up-regulation of alphaEbeta7 was impaired on T cells isolated from the Smad7 transgenic mice associated with reduced numbers of intestinal IEL when compared with that in wild-type littermates. These results indicated that failure to induce alphaEbeta7 on T cells by TGF-beta resulted in reduced numbers of intestinal IEL, suggesting the importance of alphaEbeta7 expression by TGF-beta in selective localization of intestinal IEL.