Ihachi Kato

Structural requirements of muramylpeptides for induction of Toll-like receptor 2-mediated NF-κB activation in CHO cells

We previously demonstrated that Gram-positive bacteria activated immune cells via CD14 and Toll-like receptor 2 (TLR2). Although peptidoglycan, a major constituent of the bacterial cell wall, substituted for whole organisms, the essential structure of muramylpeptides required to stimulate the cells is not clear. We further investigated the critical determinant for recognition by CD14 and TLR2. Chinese hamster ovary (CHO) fibroblasts, which do not express a functional TLR2 transcript, were transfected with TLR2 or TLR4. These cells were exposed to freeze-dried Staphylococcus epidermidis and were subsequently subjected to the pro-inflammatory transcription factor nuclear factor-κB (NF-κB)-dependent CD25 expression assay. Heterologous expression of human TLR2, but not TLR4, in CHO cells conferred immune responsiveness to freeze-dried S. epidermidis. A preparation of peptidoglycan from S. epidermidis substituted for whole organisms. Staphylococcus aureus lytic enzyme-digested product (SEPS) from peptidoglycan retained the activity, but hydrolysis of the glycan backbone in SEPS by M-1 endo-N-acetylmuramidase resulted in loss of the activity. These findings showed that cellular activation by Gram-positive cell wall components was mediated by TLR2, but not TLR4, and indicated that the glycan backbone of peptidoglycan is critical for TLR2-mediated NF-κB activation.

HISTOPATHOLOGICAL STUDY OF THE ROLE OF CD4- AND CD8-POSITIVE T CELLS ON BONE RESORPTION INDUCED BY ESCHERICHIA COLI ENDOTOXIN

Abstract. The purpose of this study was to clarify the involvement of CD4+ and CD8+ T cells on bone resorption induced by Escherichia coli endotoxin. Two kinds of monoclonal antibodies, anti-CD4 and/or anti-CD8, were employed for the depletion of each or both T cell subsets. E. coli endotoxin was injected into mouse mesial gingiva of the first molar of the left mandible every 48 hours for up to 14 days (7 injections). The mice were divided into four groups: CD4-depleted, CD8-depleted, T cell-depleted, and normal. The mice were sacrificed on the day after the 1st, 3rd, 4th, 5th, and 7th injection and alveolar bone was examined histopathologically and histomorphometrically. Bone surface in contact with osteoclasts was defined as the site of active resorption and the ratios of active resorption were compared among the four groups. In addition, sections obtained after the 1st, 4th, and 7th injection were immunohistologically stained in order to confirm the presence or absence of CD4+ or CD8+ T cells. Alveolar bone resorption gradually increased in normal mice as the number of injections increased. In contrast, alveolar bone resorption was significantly weaker in each or both subset-depleted mice. For the duration of the experimental period, the number of CD4+ T cells in CD8-depleted and normal mice significantly increased with increasing bone resorption. Considering the function of CD4+ and CD8+ T cells, these results suggest that each subset preferentially acts as a macrophage activator in the early period of bone resorption induced by E. coli endotoxin.

A histopathological study of the role of periodontal ligament tissue in root resorption in the rat.

Whether periodontal ligament (PDL) tissue is capable of inducing root resorption was examined. The distal root of the rat molar was sectioned at the furcation and the PDL tissue removed from the root (non-PDL group, n=40). The distal root with the PDL intact was also prepared (PDL-intact group, n=40). The roots were transplanted into the dorsal skin of the rat. On the 1st, 3rd, 5th, 7th, 10th, 14th, 21st or 28th day after transplantation, the roots were removed together with surrounding dorsal subcutaneous tissue and were fixed, demineralized and embedded in paraffin. Serial sections from each block were stained with haematoxylin and eosin or by the tartrate-resistant acid phosphatase (TRAP) method to observe root-resorbing cell formation. Cyclo-oxygenase-2 (COX2) was also detected immunohistologically to examine prostaglandin E(2) production. On the 7th day after transplantation, multinucleated root-resorbing cells with TRAP were observed in the PDL-intact group. The number of TRAP-positive cells peaked on the 10th day after transplantation. COX2-positive cells were observed in PDL during the early experimental stages. No root resorption was seen in the non-PDL group. These results suggest that PDL tissue is involved in the formation of root-resorbing cells and root resorption.

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: Identification by immunological and enzymological methods

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous, cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in, GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.

Extracellular 37-kDa Antigenic Protein from Actinobacillus actinomycetemcomitans Induces TNF-α, IL-1β, and IL-6 in Murine Macrophages

The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced IL-1β, IL-6, and TNF-a release from murine macrophages. The IL-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans- associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.

Possible roles of medullasin in nifedipine-induced human gingival overgrowth

In order to clarify a possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, the distributions of medullasin-positive cells immunostained in specimens from patients with NF-induced gingival overgrowth and chronic marginal gingivitis were compared in three different biopsy areas. Twenty gingival biopsies were obtained from five patients with gingival overgrowth and 20 biopsies from another five patients with chronic marginal gingivitis. In the marginal gingivitis group, the mean percentage of positive cells in the vicinity of pocket epithelium (zone I) was significantly higher than in the areas of connective tissue of the mid-portion (zone II) and adjacent to oral epithelium ( zone III) (p < 0.05). In the gingival overgrowth group, on the contrary, the positive cells significantly increased in zone II as compared with zones I and III (p < 0.05). Further, medullasin-positive cells of zones II and III in the overgrowth group had infiltrated more extensively than those in the gingivitis group (p < 0.001), indicating the participation of this enzyme in the mechanism of NF-induced gingival overgrowth. These observations suggest that medullasin may play a part in NF-induced overgrowth both in host defence and in immunoregulation, possibly cytotoxically.

A Study on the Effects of Continuous Mechanical Irritation to the Periodontal Tissues of Germfree and Conventional Rats

The changes of the periodontal tissues irritated mechanically by small active elastic rings on the survival portion of the right first molar were observed microscopically in germfree and conventional JCL: SD strain rats.The results obtained were as follows:1. Pocket formation, slight infiltration of inflammatory cells into the periodontal tissues and hyaline degeneration or disappearance of the gingival fibers in some degree were noticed in germfree rats.2. More sever inflammatory changes of the periodontal tissues such as epithelial acanthosis, ulceration, disappearance of the gingival fibers, wide spread inflammatory cell infiltration, alveolar bone destruction and abscess formation in the bifurcation of the teeth in conventional rats were distinctly recognized than in the germfree rats.3. The depth of the pockets in conventional rats was 0.8mm to 0.9mm and about twice of the depth in germfree rats.

Role of medullasin in nifedipine-induced gingival overgrowth in rats

To clarify the possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, a rat model of gingival overgrowth was first established using a diet containing NF. The relation between histopathological changes and the distribution of the proteinase was then investigated. Thirty-two, specific pathogen-free 20 day-old, male, Fisher 344 rats were fed a diet containing NF and killed at 2, 8, 16 and 32 weeks. Control rats (n = 32) were fed the same diet but without the drug. The mandible of each rat was resected and sectioned at 4-microm thickness buccolingually between the first and second molars. Computer image analysis was used to evaluate the extent of overgrowth in the approximal gingiva of each sample. To examine medullasin activity, the mean percentage of medullasin-positive cells per total cells counted in the pocket epithelium and the connective tissue adjacent to the epithelium of approximal gingiva was determined immunohistochemically. The height of the mid-portion and the area in NF-treated group increased significantly with time (with the exception of area at 2 weeks) compared with the corresponding regions in the control group. A marked inflammatory-cell infiltration and elongated rete pegs, especially in the mid-portion of approximal gingiva, were seen in the NF-treated group. The mean percentages of medullasin-positive cells in the NF-treated group at 8, 16 and 32 weeks were significantly higher than those of the control. Although medullasin-positive cells were mainly neutrophils, in several samples of the NF-treated group they were recognized as macrophage-like. These findings suggest that medullasin may be involved in host defence and immunoregulation in a NF-induced rat model of gingival overgrowth.

Extracellular Antigens of Actinobacillus actinomycetemcomitans Y4 in Severe Alveolar Bone Loss Patients Studied by Two-Dimensional Electrophoresis and Western Blots

The human immune response to extracellular substances (ES) from Actinobacillus actinomycetemcomitans Y4 were analyzed. Twenty‐nine periodontal patients with generalized severe alveolar bone loss and 13 healthy volunteers were examined for serum IgG antibody titers against ES. Among the patients, 17 had higher antibody titers than healthy individuals (high‐titer patients) but the remainder (low‐titer patients) did not. Two‐dimensional electrophoresis (2DE) and Western blots demonstrated that two proteins (40 and 37 kDa) and three smeared substances reacted with IgGs from high‐titer patients, but not with IgGs from healthy volunteers. The low‐molecular‐mass smear at the acid side reacted with over 90% of all patients. This smear reacted with anti‐A. actinomycetemcomitans lipopolysaccharide (LPS) monoclonal antibody which recognized LPS from each A. actinomycetemcomitans serotype. The high molecular mass smear at the acid side might be serotype‐specific O‐antigens of LPS. Another high molecular mass smear which located from the alkaline to the neutral side reacted with anti‐serotype‐b‐specific capsular polysaccharide monoclonal antibody. Moreover, the 40‐ and 37‐kDa proteins reacted with this anti‐capsular polysaccharide antibody. This results suggested that 40‐and 37‐kDa proteins which reacted with 100% or 88% of high‐titer patients might be glycoproteins linked with capsular polysaccharide.

In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe: Digoxigenin (1)

In this article, protocols are described for preparation of digoxigenin-labeled oligo-DNA probe and their use in in situ hybridization. As has been shown previously (Koji et al. 1988, 1989; Koji and Brenner 1993; Koji and Nakane 1990, 1996), nonradioactive synthetic oligo-DNA probes can be very useful tools. The 3’ -end of oligo-DNA can be labeled enzymaticallywith several possible haptens, such as digoxigenin and biotin. Digoxigenin, a plant steroidal aglycone, is especially suitable for immunocytochemical detection because its epitope is not present in animal cells. Also, it has recently been confirmed that under appropriate conditions, there is no substantial difference in the sensitivity of detection between [35S] cRNA (Denjin et al. 1990) or [35S] cDNA (Unger et al. 1991) probes and nonradioactive probes.