Topical application of glycyrrhetinic acid in the gingival sulcus inhibits attachment loss in lipopolysaccharide-induced experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Attachment loss of the junctional epithelium and alveolar bone destruction are signs of periodontitis, which is mainly caused by an inflammatory response to dental plaque. Glycyrrhetinic acid (GA), a component of the licorice herb, has been shown to have important anti-inflammatory activities; however, there are no previous reports on the ability of its inhibitory effects to prevent periodontal diseases. Hence, in this study, using our experimental periodontitis model, we attempted to evaluate whether GA had an effect on the prevention of attachment loss and alveolar bone loss. MATERIAL AND METHODS: Rats were intraperitoneally immunized with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 5) received 3 topical applications of 50 µg/µL of LPS followed by one application of the vehicle (propylene glycol:ethyl alcohol:phosphate-buffered saline [PBS] = 8:1:1) into the gingival sulcus. This protocol was repeated twice per day for 10 days. The low (n = 5) and high (n = 5) groups received topical application of LPS and 0.03% or 0.3% GA, respectively. The control group received topical application of PBS and vehicle. The rats were killed on the 10th day. Attachment loss, alveolar bone level and inflammatory cell infiltration were investigated histometrically. The formation of immune complexes and infiltration of LPS were evaluated immunohistologically. RESULTS: Attachment loss, formation of immune complexes and infiltration of inflammatory cells were increased in the LPS group compared with the control group, and were completely inhibited in the low and high groups compared with the LPS group. The LPS group showed greater alveolar bone destruction compared with the control group and GA-treated groups. In addition, invasion of LPS was detected in the LPS group, was absent in the control group and was weaker in the GA-treated groups than in the LPS group. CONCLUSION: In the present study, we showed that GA inhibits periodontal destruction in the rat experimental periodontitis model.

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1ß precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. MATERIAL AND METHODS: HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. RESULTS: Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers. CONCLUSION: Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis.

The histopathological comparison on the destruction of the periodontal tissue between normal junctional epithelium and long junctional epithelium.

BACKGROUND AND OBJECTIVE: The barrier function of long junctional epithelium is thought to be important after periodontal initial therapy and periodontal surgery. Although the difference between long junctional epithelium and normal junctional epithelium regarding their resistance to destruction of periodontal tissue has been investigated, the mechanism still remains unclear. Using our rat experimental periodontitis model in which loss of attachment and resorption of alveolar bone is induced by the formation of immune complexes, we investigated the resistance of periodontal tissue containing long junctional epithelium and normal junctional epithelium to destruction. MATERIAL AND METHODS: Rats were divided into four groups. In the immunized long junctional epithelium (I-LJE) group, rats were immunized with lipopolysaccharide (LPS), and curettage and root planing procedures were performed on the palatal gingiva of the maxillary first molars to obtain reattachment by long junctional epithelium. In the immunized normal junctional epithelium (I-JE) group, rats were immunized without curettage and root planing procedures. In the nonimmunized long junctional epithelium (nI-LJE) group, rats were not immunized but curettage and root-planing procedures were performed. In the control group, neither immunization nor curettage and root-planing was performed. In all rats, periodontal inflammation was induced by topical application of LPS into the palatal gingival sulcus of maxillary first molars. The rats were killed at baseline and after the third and fifth applications of LPS. Attachment loss and the number of inflammatory cells and osteoclasts in the four groups were compared histopathologically and histometrically. RESULTS: After the third application of LPS in the I-LJE group, attachment loss showed a greater increase than in control and nI-LJE groups, and inflammatory cell infiltration and osteoclasts were increased more than in the other groups. After the fifth application of LPS, attachment loss was greater and there was a higher degree of inflammatory cell infiltration in nI-LJE and I-LJE groups than in control and I-JE groups. CONCLUSION: Our findings suggest that the destruction of periodontal tissue is increased in tissue containing long junctional epithelium compared with normal junctional epithelium and that the immunized condition accelerates the destruction by forming immune complexes.

Green tea extract inhibits the onset of periodontal destruction in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Green tea extract exerts a variety of biological effects, including anti-inflammatory activities. However, there has been no report on the effect of green tea extract on loss of attachment, which is an important characteristic of periodontitis. Here, we examined the inhibitory effects of green tea extract on the onset of periodontitis in a rat model. MATERIAL AND METHODS: Rats were immunized intraperitoneally with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 12) received a topical application of LPS onto the palatal gingival sulcus every 24 h. The green tea extract group (n = 12) received a topical application of LPS mixed with green tea extract, sunphenon BG, every 24 h. The phosphate-buffered saline (PBS) group (n = 6) received a topical application of PBS every 24 h. The levels of anti-LPS immunoglobulin G (IgG) in serum were determined using ELISA. Rats in the LPS and green tea extract groups were killed after the 10th and 20th applications. Rats in the PBS group were killed after the 20th application. Loss of attachment, level of alveolar bone and inflammatory cell infiltration were investigated histopathologically and histometrically. RANKL-positive cells and the formation of immune complexes were evaluated immunohistologically. RESULTS: There was no significant difference in the serum levels of anti-LPS IgG between the LPS group and the green tea extract group. In contrast, loss of attachment, level of alveolar bone, inflammatory cell infiltration and RANKL expression in the green tea extract group were significantly decreased compared with those in the LPS group. CONCLUSION: These findings demonstrate that green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat model of experimental periodontitis.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

BACKGROUND AND OBJECTIVE: Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. MATERIALS AND METHODS: Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. RESULTS: Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. CONCLUSIONS: Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive bacteria are able to induce periodontal destruction.

Topical application of lipopolysaccharide into gingival sulcus promotes periodontal destruction in rats immunized with lipopolysaccharide.

BACKGROUND AND OBJECTIVE: The causes of periodontitis are bacteria and the host immune system, but the role of the immune system in the onset and progression of periodontal disease is still unclear. Our previous report showed that the formation of an immune complex in the gingival sulcus induces periodontal destruction. This study was carried out to investigate how the immune system, particularly immunization, is involved in periodontal destruction. MATERIAL AND METHODS: Animals immunized intraperitoneally with lipopolysaccharide (LPS) were used as the immunized group. The nonimmunized group received only phosphate-buffered saline. LPS was applied daily onto the palatal gingival sulcus in both groups 1 d after the booster injection. Serum levels of anti-LPS IgG were determined. Loss of attachment and the level of alveolar bone were histopathologically and histometrically investigated. RANKL-bearing cells and the expression of C1qB were immunohistologically evaluated. RESULTS: The serum levels of anti-LPS IgG were elevated in the early experimental period in the immunized group. There were significant increases in loss of attachment, level of alveolar bone and the number of RANKL-bearing cells in the immunized group. C1qB was observed in the junctional epithelium and adjacent connective tissue. The nonimmunized group showed similar findings at and after the time when the serum level of anti-LPS IgG was elevated. CONCLUSION: Topical application of LPS as an antigen induced periodontal destruction when the serum level of anti-LPS IgG was elevated in rats immunized with LPS. The presence of C1qB suggests that the formation of immune complexes is involved in this destruction.

Peptidoglycan and lipopolysaccharide synergistically enhance bone resorption and osteoclastogenesis.

BACKGROUND AND OBJECTIVE: Peptidoglycan (PGN) and lipopolysaccharide (LPS) are bacterial cell wall constituents that are able to induce bone resorption by stimulating Toll-like receptor (TLR) 2 and TLR4, respectively. The fragments of PGN also stimulate inflammatory responses via nucleotide-binding oligomerization domain (NOD) 1 and NOD2, although there are differences in the NOD-stimulatory activities between gram-positive and gram-negative PGNs. The TLR and NOD signaling pathways are known to engage in cross-talk to enhance the production of inflammatory cytokines. In the present study, we investigated the effects of gram-negative and gram-positive PGNs on bone resorption and osteoclastogenesis in the presence or absence of LPS. MATERIAL AND METHODS: We injected Escherichia coli PGN or Staphylococcus aureus PGN with or without LPS into mouse gingiva, and histopathologically assessed alveolar bone resorption by tartrate-resistant acid phosphatase staining. We also stimulated osteoclast precursors from mouse bone marrow macrophages with these PGNs in vitro and assessed osteoclastogenesis. The cells were also stimulated with synthetic ligands for NOD1; γ-D-glutamyl-meso-DAP NOD2; muramyl dipeptide or TLR2; Pam(3) CSK(4) with or without LPS to analyse the signaling cross-talk. RESULTS: S. aureus PGN, but not E. coli PGN, induced alveolar bone resorption, as did LPS. However, PGN from both sources significantly enhanced the bone resorption in the mice co-injected with LPS. Both types of PGNs induced osteoclastogenesis and accelerated osteoclastogenesis when the cells were co-stimulated with LPS in vitro. All synthetic ligands synergistically induced osteoclastogenesis by co-stimulation with LPS. CONCLUSION: Gram-positive or gram-negative PGN worked synergistically with LPS to induce bone resorption and osteoclastogenesis, possibly by co-ordinating the effects of TLR2, NOD1, NOD2 and TLR4 signaling.

The formation of immune complexes is involved in the acute phase of periodontal destruction in rats.

BACKGROUND AND OBJECTIVE: Loss of clinical attachment and alveolar bone destruction are major symptoms of periodontitis, caused by not only the destructive effect of periodontopathic bacteria but also the overactive response of the host immune system against periodontal pathogens. The details of the participation of the immune system in the onset and progression of periodontitis are unclear. In this study, we attempted to determine whether the host immune system, and in particular the formation of immune complexes, is involved in the periodontal destruction. MATERIAL AND METHODS: We applied ovalbumin or lipopolysaccharide (LPS) as antigens and their specific immunoglobulin G (IgG) antibodies purified from rat serum to rat gingival sulcus alternately. Loss of attachment, alveolar bone destruction and the numbers of inflammatory cells infiltrating the periodontal tissue and osteoclasts on the alveolar bone surface were investigated histometrically. The formation of immune complex was confirmed by immunohistological staining of complement C1qB. RESULTS: Loss of attachment and the presence of C1qB were observed histopathologically in both experimental groups. The group that had been treated with LPS and anti-LPS IgG showed greater loss of attachment. The number of inflammatory cells in the periodontal tissue was increased in both experimental groups, while osteoclasts at the alveolar bone crest were observed only in the group that had been treated with LPS and anti-LPS IgG. CONCLUSION: In the present study, we showed that the formation of immune complex appears to be involved in the acute phase of periodontal destruction and that the biological activity of antigens is also important.

Membrane-bound CD40 ligand on T cells from mice injected with lipopolysaccharide accelerates lipopolysaccharide-induced osteoclastogenesis.

BACKGROUND AND OBJECTIVE: T cells infiltrate the inflammatory site of periodontitis and consequently stimulate the loss of periodontal bone. We previously reported that T cells from lipopolysaccharide (LPS)-injected mice (LPS-T cells) accelerated osteoclastogenesis in the presence of LPS. Ηowever, the detailed mechanism of this acceleration is still unclear. In this study, we analyzed the mechanism of osteoclastogenesis accelerated by LPS-T cells. MATERIAL AND METHODS: We examined the mechanism of osteoclastogenesis acceleration. First, to determine the effect of cell-to-cell contact, we co-cultured T cells and bone marrow macrophages, prestimulated with RANKL for 48 h (R-BMMs), in the presence of LPS for 24 h, in a Transwell. Second, to determine the effect of CD40 ligand (CD40L), we co-cultured T cells and R-BMMs in the presence of LPS and anti-CD40L immunoglobulin. Third, we examined the effect of recombinant mouse CD40L (rCD40L) in the presence of LPS in vitro and in vivo. Lastly, we examined the expression of membrane-bound CD40L (mCD40L) by fluorescence-activated cell sorting (FACS). RESULTS: Blocking cell-to-cell contact between LPS-T cells and R-BMMs completely inhibited the acceleration of osteoclastogenesis. Anti-CD40L immunoglobulin also completely inhibited the acceleration of osteoclastogenesis. Moreover, rCD40L accelerated osteoclastogenesis in the presence of LPS in vitro and in vivo. Finally, the expression of mCD40L on LPS-T cells was higher than that on T cells isolated from mice not injected with LPS. CONCLUSION: The results demonstrate that CD40L accelerates osteoclastogenesis in the presence of RANKL and LPS. The results also suggest that mCD40L on LPS-T cells accelerates osteoclastogenesis.

Locally administered interferon-γ accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation.

BACKGROUND AND OBJECTIVE: Interferon-γ (IFN-γ) potently inhibits RANKL-induced osteoclastogenesis in vitro. In contrast, previous studies have shown that an increase in IFN-γ expression is correlated with an increase in lipopolysaccharide (LPS)-induced bone loss in vivo. However, it is not clear whether local IFN-γ accelerates osteoclastogenesis or not in vivo. Therefore, the aim of this study was to clarify the role of local IFN-γ in LPS-induced osteoclastogenesis. MATERIALS AND METHODS: We induced bone loss in calvaria by injecting LPS. One group of mice received an IFN-γ injection together with LPS injection, while another group received IFN-γ 2 d after LPS injection. Bone resorption was observed histologically. Next, we stimulated murine bone marrow macrophages with macrophage-colony stimulating factor and RANKL in vitro. We added different doses of IFN-γ and/or LPS at 0 or 48 h time points. Cells were stained with tartrate-resistant acid phosphatase at 72 h. RESULTS: Local administration of IFN-γ together with LPS injection did not affect osteoclast formation. However, IFN-γ injected after LPS injection accelerated osteoclast formation. Also, we confirmed that IFN-γ added at 0 h inhibited RANKL-induced osteoclastogenesis in vitro. However, inhibition by IFN-γ added at 48 h was reduced compared with that by IFN-γ added at 0 h. Interestingly, IFN-γ together with a low concentration of LPS accelerated osteoclast formation when both were added at 48 h compared with no addition of IFN-γ. CONCLUSION: The results suggest that local IFN-γ accelerates osteoclastogenesis in certain conditions of LPS-induced inflammatory bone loss.

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

T cells are able to promote lipopolysaccharide-induced bone resorption in mice in the absence of B cells.

BACKGROUND AND OBJECTIVE: T cells and their cytokines are believed to be key factors in periodontal disease and bone resorption. We previously showed that T cells transferred to nude mice were related to inflammatory bone resorption in vivo. However, it has not been clarified whether T cells can induce bone resorption in the absence of B cells. In this study, we therefore investigated the ability of T cells to induce bone resorption without B cells, using both T cell- and B cell-deficient mice with severe combined immune deficiency (SCID). MATERIAL AND METHODS: Escherichia coli lipopolysaccharide (LPS) was injected into the gingivae of SCID mice reconstituted by T cells (SCID + T mice). Wild-type C.B-17 mice and SCID mice were used as control animals. Alveolar bone resorption and production of cytokines in the gingivae were then compared histopathologically and immunohistologically. RESULTS: The degree of bone resorption in SCID + T mice was significantly greater than that in SCID mice but less than that in wild-type mice. The same tendency was found for expression of receptor activator of nuclear factor kappaB ligand. The number of interferon-gamma-positive cells in SCID + T mice was the highest of the three groups. In contrast, interleukin-4-positive cells were detected in wild-type mice but not in SCID + T and SCID mice. CONCLUSION: The results suggest that T cells are able to promote LPS-induced bone resorption in the absence of B cells. The expressions of cytokines in the presence of B cells are quite different.

Expression of receptor activator of nuclear factor kappa B ligand relates to inflammatory bone resorption, with or without occlusal trauma, in rats.

BACKGROUND AND OBJECTIVE: Receptor activator of nuclear factor kappa B ligand (RANKL) is an important factor in osteoclast differentiation, activation and survival; however, its involvement in inflammatory bone resorption, with or without occlusal trauma, is unclear. The purpose of the present study was to investigate the distribution of RANKL-expressing cells in rat periodontium during lipopolysaccharide-induced inflammation with or without occlusal trauma. MATERIAL AND METHODS: Lipopolysaccharide was injected into rat gingiva of the lower left first molar to induce inflammation. In addition, the occlusal surface of the upper left first molar of rat was raised by placing a gold inlay to induce occlusal trauma in the lower left first molars. The distribution of RANKL-expressing cells was immunohistochemically observed. RESULTS: In the inflammatory model, many osteoclasts were observed at the apical inter-radicular septum on day 5 and they were reduced by day 10. On the other hand, in the inflammatory model with occlusal trauma, many osteoclasts were still observed on day 10. RANKL expression was similar to the changes in osteoclast number. The expression of RANKL increased in endothelial cells, inflammatory cells and periodontal ligament cells. CONCLUSION: These findings clearly demonstrated that RANKL expression on endothelial cells, inflammatory cells and periodontal ligament cells is involved in inflammatory bone resorption and the expression is enhanced by traumatic occlusion. These results suggest that RANKL expression on these cells is closely involved in the increase of osteoclasts induced by occlusal trauma.

B cells play an important role in lipopolysaccharide-induced bone resorption.

The host immune system, especially activated T cells, plays a crucial role in inflammatory bone resorption and osteoclastogenesis. Previously, we showed that T cells are involved in inflammatory bone resorption in vivo. However, little is known about whether B cells are involved in inflammatory bone resorption and how B cells take part in osteoclastogenesis. Therefore, the aim of this study was to examine whether B c ells truly influence inflammatory bone resorption in vivo. Alveolar bone resorption in normal mice, in SCID mice that lack both B and T cells, and in B cell-reconstituted SCID mice was compared histopathologically after repeated injections of lipopolysaccharide (LPS) into the gingiva. Furthermore, we examined whether the B cells that are stimulated by LPS are involved in osteoclastogenesis in vitro. As a result, the B cell-reconstituted SCID mice showed stronger inflammatory bone resorption than the SCID mice. Also, in vitro, LPS-stimulated B cells enhanced osteoclastogenesis and anti-tumor necrosis factor (TNF)-alpha antibody completely blocked osteoclastogenesis induced by LPS-stimulated B cells. These results suggest that B cells promote inflammatory bone resorption through TNF-alpha.

Immunohistochemical localization of Toll-like receptors 2 and 4 in gingival tissue from patients with periodontitis.

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.

Interferon-gamma production changes in parallel with bacterial lipopolysaccharide induced bone resorption in mice: an immunohistometrical study.

It has been reported that bacterial lipopolysaccharide (LPS) induces alveolar bone resorption and that the host immune system, especially activated T cells, plays a crucial role in osteoclastogenesis. On the other hand, interferon-gamma (IFN-g), which is produced by activated T cells, suppresses bone resorption both in vitro and in vivo. Thus, the question arises as to whether or not IFN-g production increases with increasing bone resorption. We previously demonstrated that repeated injections of Escherichia coli LPS into mouse gingiva causes osteoclast formation in alveolar bone. In the present study we observed changes in the IFN-g production of infiltrating cells in concurrence with bone resorption. Mice were repeatedly injected with 5 mg LPS 26 times every 48 hours. After the 16th injection, when the alveolar bone resorption reached a plateau, the concentration of LPS was altered (25 mg LPS or PBS alone). The level of bone resorption became significantly elevated, and the number of IFN-g- and interleukin-1 beta (IL-1b)-bearing cells also increased significantly in relation to bone resorption within the 25 mg LPS-injected group. On the other hand, few tartrate-resistant acid phosphatase positive cells, or IFN-g- and IL-1b-bearing cells, were seen in the PBS-injected group. These results suggest that alteration in IFN-g-bearing cells might play a role in counterbalancing LPS-induced bone resorption resulting from osteoclast activating cytokines such as IL-1b.

Upregulation of functional beta(3)-adrenergic receptor in the failing canine myocardium.

Altered expression and functional responses to cardiac beta(3)-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte beta(3)-AR mRNA and protein levels and myocyte contractile, [Ca(2+)](i) transient, and Ca(2+) current (I(Ca,L)) responses to BRL-37344 (BRL, 10(-8) mol/L), a selective beta(3)-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription-polymerase chain reaction, we detected beta(3)-AR mRNA from myocyte total RNA in each animal. Using a cloned canine beta(3)-AR cDNA probe and myocyte poly A(+) RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. beta(3)-AR protein was detected by Western blot. beta(3)-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced beta(3)-AR-mediated negative modulation on myocyte contractile response and [Ca(2+)](i) regulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca(2+)](i) transient and I(Ca,L). These responses were not modified by pretreating myocytes with metoprolol (a beta(1)-AR antagonist) or nadolol (a beta(1)- and beta(2)-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (beta(3)-AR antagonists). We conclude that in dogs with pacing-induced CHF, beta(3)-AR gene expression and protein levels are upregulated, and the functional response to beta(3)-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.

Immunohistological study of interferon-gamma- and interleukin-4-bearing cells in human periodontitis gingiva.

The purpose was to investigate the balance between interferon (IFN)-gamma- and interleukin (IL)-4-bearing cells in various human inflamed gingiva by immunohistochemistry. Gingival tissues obtained from patients with gingivitis or periodontitis were divided into three groups based on the degree of histopathological inflammation, mild, moderate and severe. The tissues were also divided into four groups according to the clinical probing depth (PD). IFN-gamma- and IL-4-bearing cells in gingival tissues were stained immunohistologically and counted. The ratio of IL-4-bearing cells to IFN-gamma-bearing cells was calculated for each section. IFN-gamma-bearing cells were widespread in the connective tissue and their number increased significantly with the severity of inflammation and an increase of PD. IL-4-bearing cells were located beneath the pocket epithelium and their number showed no significant differences among the inflammation or PD groups. The ratios of IL-4-bearing cells to IFN-gamma-bearing cells in the severe inflammation or deep PD groups were significantly lower than those in the moderate inflammation or shallow PD groups. These results suggest that a low ratio of IL-4-bearing cells to IFN-gamma-bearing cells might be involved in the destruction of periodontal tissue.

Characteristics of various film cooling jets injected in a conduit.

In the present study, film cooling characteristics by the jets through various easy-to-make straight holes and slots have been investigated. In this experiment, seven kinds of injection geometries were used. They were circular, rectangular, elliptic and oval holes and slots, respectively.

Enhanced cardiac L-type calcium current response to beta2-adrenergic stimulation in heart failure.

The beta2-adrenergic receptor (beta2-AR)-mediated increase in cardiac L-type Ca2+ current (I(Ca,L)) has been documented in normal subjects. However, the role and mechanism of beta2-AR activation on I(Ca,L) in heart failure (HF) are unclear. Accordingly, we compared the effect of zinterol (ZIN), a highly selective beta2-AR agonist, on I(Ca,L) in isolated left ventricular cardiomyocytes obtained from normal control and age-matched rats with HF induced by left coronary artery ligation (4 months). I(Ca,L) was measured by using the whole-cell voltage-clamp technique. In normal myocytes, superfusion of ZIN (10(-5) M) caused a 21% increase in I(Ca,L) (9.21 +/- 0.24 versus 7.59 +/- 0.20 pA/pF) (p < 0.05). In HF myocytes, the same concentration of ZIN produced a significantly greater increase (30%) in I(Ca,L) (6.20 +/- 0.24 versus 4.75 +/- 0.17 pA/pF) (p < 0.01). This ZIN-induced increase in I(Ca,L) was further augmented in both normal and HF myocytes (normal: 59 versus 21%; HF: 71 versus 30%) after the incubation of myocytes with pertussis toxin (PTX, 2 microg/ml, 36 degrees C, 6 h). These effects were not modified by the incubation of myocytes with CGP-20712A (3 x 10(-7) M), a beta1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551 (10(-7) M), a beta2-AR antagonist. In addition, all of the effects induced by ZIN were completely prevented in the presence of an inhibitory cAMP analog, Rp-cAMPS (100 microM, in the patch-pipette solution). In conclusion, beta2-AR activation stimulates L-type Ca2+ channels and increases I(Ca,L) in both normal and HF myocytes. In HF, beta2-AR activation-induced augmentation of I(Ca,L) was increased. These effects are likely to be mediated through a cAMP-dependent mechanism and coupled with both stimulatory G protein and PTX-sensitive G protein.