Makoto Seki

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3′,5-triiodo-l-thyronine (Km, 0.9μ m), thyronine, and rT3 in a Na+-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The ...

Requirement of B7 Costimulation for Th1-Mediated Inflammatory Bone Resorption in Experimental Periodontal Disease

The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.

Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1

BACKGROUND & AIMS Recently, we isolated a new complementary DNA (cDNA) encoding human liver-specific organic anion transporter (LST-1), representing the multispecificity of human liver. The aim of this study was to isolate a rat counterpart of human LST-1 and examine the expression regulation of its messenger RNA (mRNA) to clarify the molecular basis of cholestasis. METHODS A rat liver cDNA library was screened with human LST-1 cDNA as a probe. Xenopus oocyte expression system was used for functional analysis. Northern blot analyses were performed using the isolated cDNA (termed rlst-1). The bile duct ligation model and the cecum ligation and puncture model were used for expression analyses. RESULTS rlst-1 encodes 652 amino acids, predicting at least 11 transmembrane regions. The overall homology with human LST-1 was 60.2%, which is the highest among all known organic anion transporters. rlst-1 also belongs to the same new gene family as human LST-1, located between the organic anion transporter family and the prostaglandin transporter. rlst-1 preferably transports taurocholate (K(m), 9.45 micromol/L) in an Na(+)-independent manner. The rlst-1 mRNA is exclusively expressed in the liver. In both the bile duct ligation model and the cecum ligation and puncture model, mRNA expression levels of rlst-1 were down-regulated. CONCLUSIONS rlst-1 is a counterpart of human LST-1 and is one of the important transporters in rat liver for the clearance of bile acid. The expression of rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue

Anti‐microbial peptides produced from mucosal epithelium appear to play pivotal roles in the host innate immune defence system in the oral cavity. In particular, human beta‐defensins (hBDs) and the cathelicidin‐type anti‐microbial peptide, LL‐37, were reported to kill periodontal disease‐associated bacteria. In contrast to well‐studied hBDs, little is known about the expression profiles of LL‐37 in gingival tissue. In this study, the anti‐microbial peptides expressed in gingival tissue were analysed using immunohistochemistry and enxyme‐linked immunosorbent assay (ELISA). Immunohistochemistry revealed that neutrophils expressed only LL‐37, but not hBD‐2 or hBD‐3, and that such expression was prominent in the inflammatory lesions when compared to healthy gingivae which showed very few or no LL‐37 expressing neutrophils. Gingival epithelial cells (GEC), however, expressed all three examined anti‐microbial peptides, irrespective of the presence or absence of inflammation. Moreover, as determined by ELISA, the concentration of LL‐37 in the gingival tissue homogenates determined was correlated positively with the depth of the gingival crevice. Stimulation with periodontal bacteria in vitro induced both hBD‐2 and LL‐37 expressions by GEC, whereas peripheral blood neutrophils produced only LL‐37 production, but not hBD‐2, in response to the bacterial stimulation. These findings suggest that LL‐37 displays distinct expression patterns from those of hBDs in gingival tissue.

Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease

Periodontal disease involves multi‐bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor‐kB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (Treg)‐associated cytokine, interleukin (IL)‐10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL‐1β, showed positive correlation. Also, according to the fluorescent‐immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double‐positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL‐10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen‐specific manner. Taken together, these results suggested that FoxP3/CD25 double‐positive Treg cells may play a role in the down‐regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ Treg cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.

Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29+/OmpA− E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29−/OmpA− E. coli. While the entry of Aa and Omp29+/OmpA− E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

Ultraviolet Light Exposure Suppresses Contact Hypersensitivity by Abrogating Endothelial Intercellular Adhesion Molecule-1 Up-Regulation at the Elicitation Site

Hapten sensitization through UV-exposed skin induces systemic immune suppression, which is experimentally demonstrated by inhibition of contact hypersensitivity (CHS). Although this UV-induced effect has been shown to be mediated by inhibition of the afferent phase of the CHS, the UV effects on the efferent (elicitation) phase remain unknown. In this study, UV effects on endothelial ICAM-1 expression at elicitation sites were first examined. Mice were sensitized by hapten application onto UV-exposed back skin, and ears were challenged 5 days later. ICAM-1 up-regulation at nonirradiated elicitation sites following hapten challenge was eliminated by UV exposure on sensitization sites distant from elicitation sites. To assess whether loss of the ICAM-1 up-regulation at elicitation sites contributed to UV-induced immunosuppression, we examined CHS responses in UV-exposed ICAM-1-deficient (ICAM-1−/−) mice that genetically lacked the ICAM-1 up-regulation. ICAM-1−/− mice exhibited reduced CHS responses without UV exposure, but UV exposure did not further reduce CHS responses in ICAM-1−/− mice. Furthermore, ICAM-1 deficiency did not affect the afferent limb, because ICAM-1−/− mice had normal generation of hapten-specific suppressor and effector T cells. This UV-induced immunosuppression was associated with a lack of TNF-α production after Ag challenge at elicitation sites. Local TNF-α injection before elicitation abrogated the UV-induced CHS inhibition with increased endothelial ICAM-1 expression. TNF-α production at elicitation sites was down-regulated by IL-10, a possible mediator produced by hapten-specific suppressor T cells that are generated by UV exposure. These results indicate that UV exposure inhibits CHS by abrogating up-regulation of endothelial ICAM-1 expression after Ag challenge at elicitation sites.

Effective production of the hepatitis C virus core antigen having high purity in Escherichia coli

The amino-terminal half of putative nucleocapsid (core) protein (amino acids 1-115) of hepatitis C virus (HCV) was directly overproduced in Escherichia coli under the control of the tac promoter. Overproduction of core antigen was achieved by inserting several target genes and by optimizing the culture conditions, whereas a large amount of directly expressed and purified core antigen has not yet been reported. Although the level of expression was comparable to that of the conventional E. coli fused expression system, our recombinant proteins contain only HCV amino acid sequence. Using recombinant E. coli, overproduced large-scale culture system was achieved in jar-fermenter. A highly purified sample of the expressed protein was obtained by ion-exchange and gel permeation column chromatography in the presence of 8 M urea. From a 3.5 l culture, approximately 440 mg of recombinant core protein was obtained after a two-step purification procedure. An enzyme-linked immunosorbent assay developed using the highly purified antigen satisfactorily diagnosed hepatitis C.