Biomedical subjects

Kouichi Watanabe

Publications and source records attributed to Kouichi Watanabe.

At least 19 recordsLinked to original sources

PubMed2006

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice.

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP(Sc)). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP(c)) to PrP(Sc). Therefore, PrP(c) expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP(c) in the gut is still a matter of controversy. We therefore investigated the localization of PrP(c) in the bovine and murine small intestine. In cattle, most PrP(c) positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP(c) was expressed in serotonin producing cells. In bovine Peyer's patches, PrP(c) was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP(c) was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP(c) was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer's patches. In this study, we demonstrate that there are a number of differences in the localization of PrP(c) between the murine and bovine small intestines.

Animals↗

PubMed2006

Toll-like receptor 2 and 9 are expressed and functional in gut-associated lymphoid tissues of presuckling newborn swine.

To clarify the crucial role of Toll-like receptor (TLR) 2 and TLR9 in immature gut-associated lymphoid tissues (GALT), we focused on the expression of TLR2 and TLR9 and the immune responses induced by their ligands in the GALT of presuckling newborn swine. Quantitative real-time PCR revealed that TLR2 and TLR9 mRNA were expressed at detectable levels in all tested tissues (heart, thymus, lung, spleen, liver, kidney, skeletal muscle, duodenum, jejunum, ileum, ileal Peyer patches (Pps), and mesenteric lymph nodes (MLN)). In particular, in immature intestinal tissues and GALT, TLR2 and TLR9 mRNA were expressed at higher levels in ileal Pps and MLN than in the duodenum, jejunum, and ileum. We confirmed that the TLR2 and TLR9 proteins were also highly expressed and that their ligands were preferentially recognized by TLR2- or TLR9-expressing cells in the MLN and ileal Pps. Zymosan, CpG2006, and lactic acid bacteria could promote mitogenesis and production of multiple cytokines by the MLN and ileal Pps. In addition, double immunostaining for cytokeratin 18 and either TLR2 or TLR9 revealed that both TLR2 and TLR9 are strongly expressed in the columnar membranous (M) cells. Interestingly, while the apical membrane of the columnar M cells strongly expressed TLR2 protein and preferentially recognized zymosan, both "TLR2 expression on the apical membrane" and "TLR2-mediated zymosan binding" were negligible in neighboring enterocytes. These results indicate that TLR2 and TLR9 allow MLN and ileal Pps to respond to a variety of bacterial components immediately after birth, thereby providing newborns with a host defense system.

Animals↗

PubMed2006

Staining patterns for actin and villin distinguish M cells in bovine follicle-associated epithelium.

M cells play a central role in the initiation of mucosal immune responses. However, a primary source of difficulty for investigations of this is the lack of an available specific marker for bovine M cells. As M cells possess irregular and short microvilli, we investigated the distribution and localization of the microvillar proteins actin and villin by immunohistochemistry of the gut of calves. In ileum of the calf, actin and villin were clearly and continuously immunostained in the brush border of the villous epithelia, however, discontinuous immunostaining with patches of no staining were observed in follicle-associated epithelium (FAE). Electron microscopy revealed that M cells had irregular microvilli and lacked the typical brush border, and it was inferred that these patches of no staining might be the intercellular crevices of M cells. As the microvilli of M cells were very sparse, there were several areas of weak immunostaining in calf jejunal FAE. These results suggest that M cells in calf FAE are detectable by the absence of staining for actin and villin.

Actins↗

PubMed2006

Two-stage reconstructive surgery of a patient with head trauma resulting in extensive cranial bone and dura mater loss caused by postoperative infection: usefulness of a pericranial flap for dura mater reconstruction.

After external decompression for a case of head trauma, epidural abscess formation resulted in extensive cranial bone and dura mater loss, for which two-stage reconstructive surgery was conducted. In the first operation, after thorough debridement of the infected wound, the dura mater was reconstructed using a bipedicle pericranial flap with posterior and anterior pedicles. After the infection had completely subsided, the second operation was performed, reconstructing the cranium with grafted outer-table calvarial bone and cutting bone to reposition an old zygomatic fracture. At this point, the pericranial flap used for dura mater reconstruction in the first operation became a satisfactory graft bed for the grafted bone. The postoperative course was satisfactory, and there was no cranial bone absorption after roughly 2 year, and a favorable shape has been maintained for an extremely satisfying result.

Adult↗

PubMed2006

Comparison of T cell subsets between somatic cloned and normal cow.

PROBLEM: Somatic cloning technology is beneficial for genetically producing excellent animals. However, many developmental problems of somatically cloned animals have been described. Some of them may cause disorders of the immune system, resulting in the fluctuation of the proportion of white blood cells (WBC), different from that of normal animals in peripheral blood. METHOD OF STUDY: In Holstein- cloned and normal cows, the fluctuation of granulocytes, monocytes, B cells and T cells, and further T cell subsets (CD4+, CD8+, gammadelta, CD8+gammadelta and WC1+gammadelta T cell) in peripheral blood were analyzed in early lactation stage (ELS) and mid to late lactation stage (MLS) by flow cytometry using specific monoclonal antibodies for cell surface markers. RESULTS: In both ELS and MLS, there were no significant differences in the proportions of granulocytes, monocytes, B cells and T cells between cloned and normal cows. In T cell subsets, gammadelta and WC1+gammadelta T cells in cloned cows were significantly less frequent than in normal cows in ELS. The decreased proportions of gammadelta and WC1+gammadelta T cells recovered to the level of normal cows in MLS. CONCLUSIONS: The population of granulocytes, monocytes, B cells and T cells, and T cell subsets except for gammadelta and WC1+gammadelta T cells in cloned cows fluctuated in a manner similar to those of normal cows during lactation. In ELS, the proportions of gammadelta and WC1+gammadelta T cells temporarily declined in cloned cows, suggesting that cloned cows may fall into an immunosuppressive state in ELS.

Animals↗

PubMed2006

Reliability of cobalt-chromium alloy aneurysm clips after long-term implantations in patients with cerebral aneurysms.

OBJECT: Aneurysm clip reliability after long-term implantation in vivo has not been examined. In this study the authors evaluated the mechanical properties and surface elemental composition of Co-Cr alloy aneurysm clips implanted for more than 10 years in patients with cerebral aneurysms. METHODS: Five aneurysm clips implanted for ruptured or unruptured intracranial aneurysms were retrieved and examined. New aneurysm clips were applied to the regrown aneurysms. The implantation period ranged from 11 to 20 years. Four new and unused aneurysm clips were also examined as controls. The mechanical properties of the clips were tested by measuring their closing force and bending strength. The surface elemental composition of the aneurysm clips was evaluated using x-ray photoelectron spectroscopy. The closing force of the retrieved clips exceeded the minimum force requirement at the time of manufacture. The bending strength was similar between the retrieved and control clips. Chromium oxide was the predominant constituent on the surface of all clips, and its concentration on the retrieved clips was higher than that on the control clips. CONCLUSIONS: Data in the present study demonstrated that Co-Cr alloy aneurysm clips retain their mechanical properties in vivo for a long time, which indicates the reliability of these clips.

Aged↗

PubMed2005

Apoptotic process of porcine intestinal M cells.

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer's patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18(+)/PCNA(+) cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18(+)/alkaline phosphatase(+) cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex.

Animals↗

PubMed2005

Identification of bovine dendritic cell phenotype from bovine peripheral blood.

Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.

Animals↗

PubMed2005

Cellular localization of IL-18 and IL-18 receptor in pig anterior pituitary gland.

Pro-inflammatory cytokine interleukin 18 (IL-18) has been proposed to have a role in modulating immuno-endocrine functions. Our previous study showed that IL-18 and IL-18 receptor (IL-18R) colocalized in somatotrophs of the bovine anterior pituitary gland, and the possibility that IL-18 acts on somatotrophs as an autocrine factor. In the present study, we investigated the localization of IL-18 and IL-18R in the pig anterior pituitary gland. RT-PCR analysis showed the expression of IL-18 and IL-18R mRNAin the pig anterior pituitary gland. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs, mammotrophs, thyrotrophs and gonadotrophs. IL-18R was localized in somatotrophs and thyrotrophs. Furthermore, the somatotrophs immunoreactive for IL-18 did not contain IL-18R. Thus, IL-18R and IL-18 were not colocalized in an identical somatotroph. These findings suggest that the localization of IL-18 in pig somatotrophs is different from that in bovine somatotrophs, although IL-18 closely associates with somatotrophs in the anterior pituitary glands in both species.

Animals↗

PubMed2005

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland.

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Ralpha) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Ralpha cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Ralpha, IL-18 and GH showed that IL-18Ralpha was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway.

Amino Acid Sequence↗

PubMed2005

Toll-like receptor 2 is expressed on the intestinal M cells in swine.

The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.

Animals↗

PubMed2005

Possible role for avPGC-1alpha in the control of expression of fiber type, along with avUCP and avANT mRNAs in the skeletal muscles of cold-exposed chickens.

Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator, plays a role in mitochondrial biogenesis, muscle fiber specialization, and adaptive thermogenesis. Because of an absence of brown adipose tissue, the skeletal muscle tissue in chickens serves as an important source of thermogenesis to counter the cold. The present experiments were conducted (i) to clone the cDNA of PGC-1alpha homologs from chicken skeletal muscle and to examine alterations to PGC-1alpha mRNA expression in the skeletal muscles of cold-exposed chickens, (ii) to study the effect of cold-acclimation on the metabolic fiber phenotype of typically fast-glycolytic (type IIB) pectoralis muscles, and (iii) to compare avANT and avUCP mRNA expression in control and cold-exposed chickens. Results show that the cloned avPGC-1alpha cDNA encodes a 796 amino-acid protein (GenBank Accession No. AB170013) showing 84% identity with rodent PGC-1alpha cDNA. Exposure of chickens to a cold environment resulted in the prompt upregulation of avPGC-1alpha expression, which preceded increments in avUCP and avANT expression in skeletal muscle mitochondria. Consistent with the morphological appearance of muscles, an increase in the number of fast-oxidative-glycolytic (type IIA) fibers in the pectoralis muscle, which contains exclusively type IIB fibers in control chickens, was observed in cold-acclimated chickens. These findings provide novel information about possible regulatory pathways in avian skeletal muscle during thermogenesis.

Amino Acid Sequence↗

PubMed2004

Internal porosity of cast titanium removable partial dentures: influence of sprue direction and diameter on porosity in simplified circumferential clasps.

OBJECTIVE: The behavior of molten titanium in molds of complicated shape is still insufficiently understood; consequently, definite spruing criteria are not yet available for titanium RPD frameworks. This study investigated the influence of sprue design on porosity in pressure-cast titanium circumferential clasps. METHODS: The patterns of 90 circumferential clasps were sprued with three directions (0, 30 and 60 degrees , as measured between the sprue and the symmetry plane of the clasp assembly) and three sprue diameters (1.5, 2.0 and 2.5mm). CPTi was cast in a one-chamber pressure casting machine. Pore number and size were assessed on radiographs of the castings. Statistical analysis was done by two-way analysis of variance (ANOVA), followed by Fisher's PLSD post hoc test. RESULTS: The porosity in lingual arms increased significantly with increase of sprue diameter and sprue angle, while the porosity in minor connectors had an inversely proportional distribution. Very low porosity, uninfluenced by sprue design, was found in buccal arms. In conclusion, internal porosity in titanium circumferential clasp arms can be minimized through sprue design: the 0 degrees sprue direction produced the least porosity, while for the 30 and 60 degrees directions, 1.5mm diameter sprues produced lower porosity than 2.0 and 2.5mm diameter sprues. SIGNIFICANCE: In this study, the lowest porosity in titanium circumferential clasp arms was obtained with sprues attached perpendicularly to the minor connectors, regardless of sprue diameter. Conventional sprue directions produced significantly higher porosity in clasp lingual arms, the amount of porosity increasing with sprue diameter.

Analysis of Variance↗

PubMed2004

Toll-like receptor 9 is expressed on follicle-associated epithelia containing M cells in swine Peyer's patches.

The precise distribution and expression of Toll-like receptor (TLR) 9 in gut-associated lymphoid tissues (GALTs) has not been elucidated. In this study, we investigated the expression pattern of TLR9 in adult and neonatal swine GALTs by real-time quantitative PCR, western blot, confocal laser microscopy and flow cytometric analysis. The swine TLR9 gene was preferentially expressed in adult Peyer's patches (Pps) and mesenteric lymph nodes (MLNs), which contained approximately three times higher TLR9 than the spleen. Other tissues exhibited only weak expression of TLR9. In neonatal swine, elevated expression of TLR9 was detected only in MLNs. We firstly showed that highly expressive (TLR9(+)) cells were formed in Pps and MLNs. In addition, TLR9(+) cells were present not only in immune cells such as dendritic cells and B cells but also in follicle-associated epithelia (FAE) including membranous cells (M cells) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses.

Animals↗

PubMed2004

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle.

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.

Animals↗

PubMed2004

Induction of nitric oxide production mediated by tumor necrosis factor alpha on staphylococcal enterotoxin C-stimulated bovine mammary gland cells.

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-alpha in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-alpha in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Animals↗

PubMed2004

Morphological and chemical characterizations of the interface of a hydroxyapatite-coated implant.

The present study aimed at morphological and chemical characterization of the coating-substrate interface of a commercially available dental implant coated with plasma-sprayed hydroxyapatite (HA). For this purpose, elements in the chemically and mechanically exposed substrate surfaces were analyzed by EPMA and XPS. A thin titanium oxide film containing Ca and P was found at the interface. When the implant was subjected to mechanical stress, a mixed mode of cohesive and interfacial fractures occurred. The cohesive fracture was due to separation of the oxide film from the substrate, while the interfacial fracture was due to exfoliation of the coating from the oxide film bonded to the substrate. Analysis showed diffusion of Ca into the metal substrate, hence indicating the presence of chemical bond at the interface. However, mechanical interlocking seemed to play the major role in the interfacial bond.

Calcium↗

PubMed2004

Surface properties of electrochemically buffed titanium casting.

Electrochemical buffing, a combined process of electrochemical and mechanical polishing, was applied to titanium casting. Mixture of alpha-Al2O3 suspension (average grain diameter of 5 microm) and 5% KNO3 solution was used as abrasive slurry. Specimen and experimental wheel buff were respectively connected to the positive and negative poles of a DC source, whose potential ratings ranged from 0 V (MEP) to 10 V (ECB10). Surface roughness, hardness, color, and cleanness were investigated. ECB10 surface produced a gold color and attained a mirror finish, as its roughness value was only one-quarter that of MEP. High amount of aluminum was present in MEP surface. Its bond state entirely differed from that of alpha-Al2O3, hence indicating surface alteration due to chemical reactions with the abrasive material. At higher potentials, reaction products might be dissolved anodically, so that the surface was chemically clean to some extent. The surface also became rich in OH-.

Aluminum Oxide↗