Hisashi Suguro

Poly I:C-induced expression of intercellular adhesion molecule-1 in intestinal epithelial cells

Intercellular adhesion molecul‐1 (ICAM‐1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM‐1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen‐associated molecular patterns. One of these stimuli, double‐stranded RNA (dsRNA), is a by‐product of viral replication and can be recognized by its cognate receptor Toll‐like receptor 3 (TLR‐3). In spite of expression of both TLR‐3 and ICAM‐1 in IECs, correlation between TLR‐3‐signalling and ICAM‐1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM‐1 in IEC line, HT‐29. Poly I:C‐stimulation up‐regulated the expression of ICAM‐1 mRNA by real‐time polymerase chain reaction. Enhanced expression of ICAM‐1 was confirmed in protein level by immunofluoresense cell staining and enzyme‐linked immunosorbent assay by measuring the released soluble ICAM‐1 in culture supernatant. As the stimulation effect was reduced by pre‐treatment of the cells with anti‐TLR‐3 antibody, poly I:C‐binding signal was thought to be sensed by TLR‐3 on the surface of HT‐29. The results of luciferase assay and nuclear factor kappa‐b (NF‐kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF‐kB. All these results demonstrated the connection between TLR‐3 signalling and ICAM‐1 expression in HT‐29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

Multiple cleavage sites for polymeric immunoglobulin receptor

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild‐type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild‐type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.

Characterization of human dental pulp-derived cell lines.

AIM To establish and characterize different types of fibroblastic cell lines derived from dental pulp tissue. METHODOLOGY Human dental pulp tissue-derived cells were transfected with SV40 large T antigen by Lipofectamine transfection method. Geneticin (G418)-resistant cells were selected and different cell lines were established by a limiting dilution method. To characterize the lineages of cells, each clone was immunofluorescently stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. Total RNA was extracted from each clone and subjected to a differential display experiment. RESULTS By transfecting SV40 large T antigen, nine different cell clones were obtained. All these cell clones were positively stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. With differential display experiment, eight different genes, the expression levels of these genes were varied amongst each cell clone, were detected. After sequencing and database search, one gene was revealed to be identical to T-cell marker, Thy-1. Thy-1 expression in dental pulp tissue was confirmed by immunohistochemical staining. CONCLUSION Fibroblastic cell lines derived from human dental pulp tissue possessed different gene expression profiles suggesting the existence of subpopulations.

Active synthesis of mouse polymeric immunoglobulin receptor in the epithelial cells of the distal urinary tubule in kidney.

The tissue distribution of mouse polymeric immunoglobulin receptor (pIgR) has been demonstrated. By Northern blot hybridization, pIgR mRNA expression was detected in liver, intestine, stomach, lung and kidney. A weak expression was also detected in thymus by reverse transcriptase‐polymerase chain reaction. The pIgR expression in kidney was further studied and confirmed that pIgR protein was actively synthesized in the epithelial cells of distal urinary tubule and of Henles loop. Immunoelectron microscopical analysis showed the accumulation of pIgR‐containing vesicles in the apical portion of distal urinary tubule epithelial cells.

Differential Distribution of Mouse Polymeric Immunoglobulin Receptor (mpIgR): Establishment of Enzyme‐Linked Immunosorbent Assay System for mpIgR

In spite of the relevance of the mouse as an experimental animal for immunological research, there is no specific monoclonal antibody (MoAb) against mouse polymeric immunoglobulin receptor (mpIgR) molecule. The aim of this study was to generate MoAb against mpIgR and to develop an enzyme‐linked immunosorbent assay (ELISA) system. For this purpose, a mammalian expression vector encoding the extracellular part of mpIgR cDNA (also known as mouse free secretory component: mfSC) was injected into rats and mice. Specific responses were induced in both animals. The lymphocytes were collected from immunized mice and fused with myeloma cells to establish hybridomas secreting a MoAb against mpIgR. The purified MoAb reacted with mpIgR specifically and could be used in several biochemical and morphological experiments, such as Western blotting, immunoprecipitation and cell staining. By combining the two different MoAb, clone No.7 and No.19, which recognize different epitopes on the mpIgR molecule, a sandwich ELISA system was established. With this system, the pIgR concentrations in homogenates of several different mice organs were estimated. As a result, the homogenates of the small intestine were shown to contain higher amounts of pIgR compared with those from the large intestine, the liver or the kidney. These results indicated a differential distribution of the mpIgR molecule along the intestinal tract. This ELISA system should expand our knowledge about the mucosal immune system in mice.

Nickel chloride administration prevents the growth of oral squamous cell carcinoma

The effect of NiCl2 on oral squamous cell carcinoma-derived cell line HSC3 was examined. Incubation with 1 mM NiCl2 significantly reduced the expression of MMPs at mRNA and protein levels. The in vivo orthotopic implantation model was established by injecting highly metastatic subcell line HSC3-M3 to nude mouse tongue. After 1 week of injection, mice were fed with or without 1 mM NiCl2-containing water for two to three weeks. Immunohistochamical examination revealed that MMP9 expression was drastically reduced in NiCl2-fed mice. By CT images, cancer mass was observed as a translucent area in control mice. In NiCl2-fed mice, much highly translucent area was observed within the translucent area. Histologically, this area corresponded to the necrotic area in the tumor mass. Real-time PCR analysis revealed the reduced expression of angiogenic factors such as IL-8 and VEGF mRNA in NiCl2-fed mice. To further examine the effect of NiCl2 on metastasis, human β-globin gene expression in regional lymphnodes was compared. The β-globin gene was totaly absent in NiCl2-fed mice. Moreover, various cancer metastasis-related genes were inhibited in NiCl2-fed mice by PCR array analysis. The results indicated that NiCl2 might be a promising new anti-cancer therapeutics for the oral cancer treatment.

Microcomputed tomographic evaluation of techniques for warm gutta-percha obturation

Transparent epoxy resin root canal models were used to evaluate vertical condensation techniques for obturating lateral canals. The root canal model was configured with a straight main root canal and four right-angled lateral canals at 1.0 and 3.0 mm from the apex. Root canal obturation was performed with Thermafil, Obtura II, or NT condenser. Obturation volume in lateral canals was measured by three-dimensional microcomputed tomography, and one-way analysis of variance was used to analyze differences between groups. Lateral canals at 1.0 and 3.0 mm were uniformly filled by all obturation methods. Among the three obturation methods, Thermafil resulted in the highest obturation volumes for all lateral canals.

Comparison of gene expression profiles of gingival carcinoma Ca9-22 cells and colorectal adenocarcinoma HT-29 cells to identify potentially important mediators of SLPI-induced cell migration

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor whose expression level is positively correlated with tumor aggressiveness and metastatic potential. However, the mechanism underlying SLPI-induced enhancement of malignant phenotype is not completely understood. The malignancy of cancer cells is highly dependent on cell migration activity. Our previous study revealed that gingival carcinoma Ca9-22 cells, but not colorectal adenocarcinoma HT-29 cells, expressed SLPI. Therefore, we investigated the migration activity of these two cell types to understand the nature of SLPI-mediated tumor aggressiveness and metastatic potential. In vitro wound healing assay indicated that HT-29 cells and SLPI-deleted Ca9-22 cells showed lower migration activity than wild-type Ca9-22 cells, suggesting that SLPI-induced cell migration plays an important role in tumor aggressiveness and metastatic potential. In addition, our gene expression profiling study based on microarray data for the three cell types identified a number of candidates, including LCP1 and GLI, that could be key molecules in the mechanism of SLPI-induced cell migration.

Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.