Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material.

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with L-N(6)-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A(2) (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. L-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA(2) but not PI-3K-dependent fashion.

Effect of exogenous nitric oxide on murine splenic immune response induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide.

The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-gamma and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-gamma suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.

Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide.

BACKGROUND/AIM: Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells). METHODS: Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l-NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically. RESULTS: The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l-NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin. CONCLUSION: These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14-TLR4 molecule complex, a cAMP-PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.

Effect of colchicine on the murine immune response induced by Aggregatibacter actinomycetemcomitans.

The aim of the present study was to test the hypothesis that colchicine may alter Aggregatibacter actinomycetemcomitans-induced immune response and abscess formation in mice. BALB/c mice were either sham-immunized or immunized with heat-killed A. actinomycetemcomitans. Spleen cells were stimulated with heat-killed A. actinomycetemcomitans in the presence or absence of colchicine. Specific IgG subclass antibodies, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and cell proliferation were determined. The animals were sham-immunized (group I) or immunized with heat-killed A. actinomycetemcomitans (groups II-VII). Colchicine was administered intraperitoneally before (group III), on the same day of (group IV), or after (group V) the primary immunization and on the same day of (group VI) or after (group VII) the secondary immunization. All groups were challenged with viable A. actinomycetemcomitans. The levels of serum-specific IgG subclasses and both IFN-gamma and IL-4 before and after bacterial challenge were assessed. The diameter of skin lesions was assessed. The results showed that colchicine augmented splenic-specific IgG1 and IL-4 as well as cell proliferation but suppressed specific IgG2a and IFN-gamma levels. Enhancement of serum-specific IgG1 and IL-4 levels, suppression of specific IgG2a and IFN-gamma levels as well as DTH response, and delayed healing of the lesions were observed in groups IV and VI, but not in the remaining groups of animals. Therefore, these results suggest that colchicine may induce a T helper 2 (Th2)-like immunity specific to A. actinomycetemcomitans in vitro and that colchicine administered on the same day as the immunization may stimulate a non-protective Th2-like immunity in A. actinomycetemcomitans-induced infections in mice.

Effect of exogenous nitric oxide on murine immune response induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Elevated nitric oxide (NO) has been associated with destructive periodontal disease. The aim of the present study was to test the hypothesis that exogenous NO may inhibit a protective immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in a murine model. MATERIAL AND METHODS: Mice of the BALB/c strain were sham immunized, immunized with A. actinomycetemcomitans LPS, treated with S-nitroso-N-acetyl penicillamine (SNAP; a NO donor) and immunized with A. actinomycetemcomitans LPS or treated with SNAP plus 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) and immunized with A. actinomycetemcomitans LPS. All animals were then challenged subcutaneously with viable A. actinomycetemcomitans. The serum-specific immunoglobulin G (IgG) subclasses and both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) as well as splenic inducible nitric oxide synthase (iNOS) activity before and after bacterial challenge were assessed. The diameter of skin lesions was determined. Groups of mice were treated with l-N(6)-(1-iminoethyl)-lysine (l-NIL), an iNOS inhibitor, or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, prior to injections with SNAP and/or A. actinomycetemcomitans LPS, and the skin lesions were assessed. RESULTS: Treatment with SNAP increased the iNOS activity, suppressed both serum-specific IgG2a and IFN-gamma levels, and delayed the healing of the lesions. These SNAP-induced immune alterations were restored by treatment with carboxy-PTIO. Pretreatment with l-NIL resulted in partial healing, whereas pretreatment with ODQ induced a delayed healing of the lesions. CONCLUSION: The present study suggests that exogenous NO may suppress a protective T helper 1-like murine immune response to A. actinomycetemcomitans LPS by an endogenous NO-independent but a cyclic GMP-dependent mechanism.

Effect of inhibition of inducible nitric oxide synthase (iNOS) on the murine splenic immune response induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans lipopolysaccharide.

Animal studies suggest that inducible nitric oxide synthase (iNOS) may be associated with destructive periodontal disease. l-N(6)-(1-Iminoethyl)-lysine (L-NIL) has been shown to inhibit iNOS in a selective manner, and hence the aim of the present study was to test the hypothesis that treatment with l-NIL may induce a T-cell helper 1 (Th1)-like immune response by Aggregatibacter (Actinobacillus) actinomycetemcomitans lipopolysaccharide (LPS)-stimulated murine spleen cells in vitro. BALB/c mice were either sham-immunized or immunized with A. actinomycetemcomitans LPS. Spleen cells were stimulated with A. actinomycetemcomitans LPS in the presence or absence of L-NIL. Nitric oxide (NO), iNOS activity, specific IgG subclass antibodies, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) levels and cell proliferation were determined. The results showed that treatment with L-NIL suppressed both NO production and iNOS activity but enhanced specific IgG2a, IFN-gamma levels, and increased cell proliferation following stimulation with A. actinomycetemcomitans LPS-stimulated cells. The results of the present study suggest that inhibition of iNOS activity by L-NIL may skew the A. actinomycetemcomitans LPS-stimulated murine splenic immune response towards the Th1-like immune profile in vitro.

Effect of L-N6-(1-iminoethyl)-lysine, an inducible nitric oxide synthase inhibitor, on murine immune response induced by Actinobacillus actinomycetemcomitans lipopolysaccharide.

BACKGROUND AND OBJECTIVES: Inducible nitric oxide synthase (iNOS) activity is known to regulate the immune response. The present study was carried out to determine the effect of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, on the induction of immune response to Actinobacillus actinomycetemcomitans lipopolysaccharide in mice. MATERIAL AND METHODS: BALB/c mice were sham-immunized (group I), immunized with A. actinomycetemcomitans lipopolysaccharide (group II) or treated with L-NIL and immunized with A. actinomycetemcomitans lipopolysaccharide (group III). All animals were then challenged with viable A. actinomycetemcomitans. The levels of serum nitric oxide (NO), specific immunoglobulin G (IgG) isotypes and both interferon-gamma and interleukin-4, as well as spleen cell-derived iNOS activity, before and after bacterial challenge, were assessed. The diameter of skin lesions was also determined. Serum and spleen cells from the above groups were adoptively transferred to the recipients that were then subsequently challenged with live bacteria. RESULTS: Treatment with L-NIL suppressed serum NO and splenic iNOS activity, but enhanced serum-specific IgG2a antibody and interferon-gamma levels. The lesions in L-NIL-treated mice healed much more rapidly. Transfer with serum and cells from L-NIL-treated and A. actinomycetemcomitans lipopolysaccharide-immunized donors resulted in rapid healing of the lesions in the recipients. CONCLUSION: It is suggested that treatment with L-NIL in mice immunized with A. actinomycetemcomitans lipopolysaccharide may shift the immune response towards a protective T helper 1-like immunity against A. actinomycetemcomitans-induced infection.

The role of cyclic-AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

AIMS: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. MATERIALS AND METHODS: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. RESULTS: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). CONCLUSION: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.

The role of CD4+ and CD8+ T cells on antibody production by murine Peyer's patch cells following mucosal presentation of Actinomyces viscosus.

The aim of this study was to determine the role of CD4 and CD8 cells on specific antibody production by murine Peyer's patch (PP) cells after oral immunization with Actinomyces viscosus in mice. Female DBA/2 mice were orally immunized with three low doses of heat-killed A. viscosus. Sham-immunized mice served as a control group. Mice were depleted of CD4 or CD8 cells by intraperitoneal injection of anti-CD4 or anti-CD8 antibodies daily for 3 days before oral immunization. One week after the last oral immunization, PPs were removed and cell suspensions were cultured with A. viscosus. Specific antibody production in the culture supernatants was assessed by enzyme-linked immunosorbent assay. The results showed that oral immunization with A. viscosus induced a predominant specific immunoglobulin A (IgA) response by PP cells and, to a lesser extent, IgM antibodies. Depletion of CD4 but not CD8 cells suppressed the production of specific antibodies. These results suggest that oral immunization with low doses of A. viscosus may induce the production of specific antibodies by murine PP cells in a CD4-cell-dependent fashion.

Role of protein kinase C in hydroxyapatite- induced phagocytosis by a murine macrophage cell line (RAW264.7).

The role of protein kinase C (PKC) in hydroxyapatite (HA)-induced phagocytosis by RAW 264.7 cells was investigated. The cells were incubated with HA particles at various incubation time and the levels of PKC activity were determined from the cell lysate. To determine the role of PKC, particles were incubated with the cells pretreated with the various concentrations of bisindolylmaleimide, a PKC inhibitor, and phagocytosis was then assessed at 60 min. Latex beads were used as a control. Our results showed that following incubation with HA particles, the levels of PKC activity in RAW264.7 cells was highest at 7 min and then decreased to reach the baseline levels of the controls at 30 min. Pretreatment of the cells with bisindolylmaleimide significantly reduced phagocytosis of HA particles in a dose-dependent pattern. The results of our present study suggest therefore that ingestion of HA by RAW264.7 cells may depend on PKC activity that may act in the early stages of phagocytosis.

The induction of oral tolerance to Actinomyces viscosus in mice.

OBJECTIVES: To determine whether oral tolerance with the oral bacterium Actinomyces viscosus was inducible in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) and then intraperitoneally (i.p.) immunized with heat-killed A. viscosus. A control group of mice received only saline. A delayed type hypersensitivity (DTH) response and the levels of isotype specific antibodies were assessed. Spleen cells from mice that were i.g. immunized with A. viscosus were transferred to A. viscosus-primed mice in vivo and in vitro. Furthermore, mice were i.g. immunized with saline or A. viscosus and then challenged i.p. with saline, A. viscosus, or Porphyromonas gingivalis. RESULTS: Intragastric immunization with A. viscosus suppressed both DTH and serum specific antibodies to A. viscosus. DTH suppression lasted until week 4, while serum immunoglobulin (Ig)A and both IgG and IgM specific antibody levels remained suppressed up to week 8 and 12 respectively. IgG specific antibody suppression was transferable. The DTH response and serum antibodies specific to A. viscosus were suppressed in mice after i.g. challenged with A. viscosus but not P. gingivalis. CONCLUSION: Mucosal presentation of A. viscosus in mice led to the suppression of immune response to this bacterium in an antigen-specific fashion. Tolerance of DTH response was short lived, while suppression of antigen-specific IgG antibodies in mucosally tolerized mice was long-lasting.

Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

AIMS: The aim of the present study was to determine whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells). METHODS: RAW264.7 cells were treated with A. actinomycetemcomitans-lipopolysaccharide or lipopolysaccharide from Escherichia coli for 24 h. The effect of polymyxin B, l-norvaline, dl-norvaline, dexamethasone and cytokines (interferon-gamma and interleukin-4) on arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells was also determined. The cells were pretreated with anti-CD14, anti -toll-like receptor 2, or anti-toll-like receptor 4 antibody prior to stimulation with A. actinomycetemcomitans-lipopolysaccharide. Arginase activity was determined by a colorimetric assay. RESULTS: A. actinomycetemcomitans-lipopolysaccharide stimulated arginase activity in RAW264.7 cells in a dose-dependent manner, but was less potent than E. coli-lipopolysaccharide. Polymyxin B and l-norvaline, but not dl-norvaline, blocked the arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. Dexamethasone and interleukin-4 but not interferon-gamma augmented arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. Treatment of the cells with anti-CD14 and anti-toll-like receptor 4 but not anti-toll-like receptor 2 antibody decreased arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. CONCLUSION: The results of the present study suggest that lipopolysaccharide from A. actinomycetemcomitans via CD14/toll-like receptor 4 complex molecules and the regulatory control of glucocorticoid and cytokines may stimulate arginase activity in RAW264.7 cells.

Role of CD4 and CD8 T-cells in the induction of oral tolerance to Actinomyces viscosus in mice.

Mucosal presentation of Actinomyces viscosus results in the induction of antigen specific systemic suppressor cells in mice. The aim of the present study was to determine the phenotype of the suppressor cells responsible for the induction of oral tolerance to low doses of A. viscosus. When CD8 cell-depleted DBA/2 mice were intragastrically immunized and systemically immunized with A. viscosus, the delayed type hypersensitivity response was suppressed but not the levels of antigen specific serum antibodies. Adoptive transfer of orally tolerized CD4(+) cells to CD4(+)-depleted mice resulted in suppression of delayed type hypersensitivity response but not of the levels of antigen specific serum antibodies. In contrast, adoptive transfer of orally immunized CD8(+) cells to CD8(+)-depleted mice resulted in partially suppressed delayed type hypersensitivity response but significantly inhibited the levels of antigen specific serum antibodies. When orally tolerized CD8(+) cells were cocultured with systemically immunized CD8(+) cell-depleted spleen cells, splenic specific antibodies were inhibited. However, no suppression of splenic specific antibodies could be observed in the cultures containing orally tolerized CD4(+) cells and systemically immunized CD4(+) cell-depleted spleen cells. The results of the present study suggest that oral tolerance of humoral and cellular immunity induced by low doses of A. viscosus may be mediated by CD8(+) and CD4(+) cells, respectively.

Histological evaluation of rabbit gingival wound healing transplanted with human amniotic membrane.

Human amniotic membrane has been used as a material to accelerate wound healing and reconstruct damaged organs. The aim of the present study was to assess histologically human amniotic membrane transplantation on rabbit's gingival wound. Three- to 4-month-old male rabbits were divided into 2 groups, i.e., control (group I) and amniotic membrane-transplanted animals (group II). Buccal gingival wounds were created by a punch-biopsy instrument and covered by a 5-layered human amniotic membrane for group II or left uncovered for group I. Gingival biopsies were taken at days 1, 3, 5, 7 and 10, processed for paraffin sections and stained with haematoxylin-eosin or von Gieson. Thickness of epithelial layer, the number of polymorphonuclear cells (PMN), fibroblasts and new blood vessels as well as density of collagen fibres were assessed. The results showed that the number of fibroblasts and new blood vessels, but not PMN, from group II was higher than that from group I (P < 0.05). Similarly, the epithelial thickness and density of collagen fibres from group II were significantly higher than those from group I (P < 0.05). The results of the present study indicate that amniotic membrane transplantation may induce rapid epithelialization and both granulation tissue and collagen formation but suppress inflammation, suggesting that amniotic membrane transplantation may promote rapid gingival wound healing in rabbits compared to secondary healing.

Role of nitric oxide in hydroxyapatite-induced phagocytosis by murine macrophage cell line (RAW264.7).

The aim of this study was to determine the role of nitric oxide (NO) in hydroxyapatite (HA)-induced phagocytosis by a murine macrophage cell line (RAW264.7). The cells were incubated with HA particles at various incubation time and phagocytosis was assessed using phagocytic index (PI). NO production from the culture supernatants was determined by the Griess reagent. The inducible nitric oxide synthase (iNOS) expression was determined by Western blot. The particles were also incubated with cells pretreated with various concentrations of L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL) or L-arginine. Latex beads were used as a control. Our results showed that macrophage phagocytosis induced by HA was higher than that induced by the beads. However, NO production by HA-stimulated cells was lower than that by bead-stimulated cells. iNOS expression in both bead- and HA-stimulated cells was observed expressed at 7, 15, 30, and 60 min. l-Arginine enhanced but l-NIL inhibited both phagocytosis and NO production by HA-stimulated cells. The results of the present study suggest that nitric oxide may play a crucial role in HA-induced phagocytosis by RAW264.7 cells.

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide.

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW 264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Induction of suppressor cells following mucosal presentation of Actinomyces viscosus in mice.

Mucosal presentation of Actinomyces viscosus results in antigen-specific systemic immune suppression, known as oral tolerance. The aim of the present study was to determine the mechanism by which this oral tolerance is induced. DBA/2 mice were gastrically immunized with A. viscosus. Serum, Peyer's patch (PP) and spleen cells were transferred to syngeneic recipients which were then systemically challenged with the sameiA. viscosus strain. To determine antigen-specificity of cells from gastrically immunized mice, recipients which received immune spleen cells were also challenged with Porphyromonas gingivalis. One week after the last systemic challenge, the delayed type hypersensitivity (DTH) response was determined by footpad swelling and the level of serum IgG, IgA and IgM antibodies to A. viscosus or P. gingivalis measured by an ELISA. No suppression of DTH response or of specific serum antibodies was found in recipients which received serum from gastrically immunized mice. Systemic immune suppression to A. viscosus was observed in recipients which had been transferred with PP cells obtained 2 days but not 4 and 6 days after gastric immunization with A. viscosus. Conversely, suppressed immune response could be seen in recipients transferred with spleen cells obtained 6 days after gastric immunization. The immune response to P. gingivalis remained unaltered in mice transferred with A. viscosus-gastrically immunized cells. The results of the present study suggest that oral tolerance induced by A. viscosus may be mediated by antigen-specific suppressor cells which originate in the PP and then migrate to the spleen.

Effect of high-density lipoprotein on lipopolysaccharide-induced alveolar bone resorption in rats.

OBJECTIVE: To determine whether treatments with high-density lipoprotein (HDL) may alter the lipopolysaccharide (LPS)-induced alveolar bone resorption in rats. MATERIALS AND METHODS: Rats were injected with 500 microg of LPS from Escherichia coli at the alveolar mucosa of lower right first molar once every 2 days for 8 days. The negative and positive control were injected with phosphate buffered saline (PBS) and LPS alone, respectively. In HDL-treated animals various concentration of HDL were injected immediately before, after the third or the final LPS injection. The bone sections were stained with tartrate-resistant acid phosphatase (TRAP) and the numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption were assessed. RESULTS: The numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption in animals treated with HDL before or during LPS injections were lower than those in the positive control, but higher than those in the negative control, regardless of HDL doses. Similar results were also observed in animals treated with 250 and 500 microg of HDL after the final LPS injection. Only treatments with 1000 microg of HDL after LPS injections completely reduced the number of both osteoclasts and preosteoclasts, but only partially decreased the alveolar bone resorption. CONCLUSION: HDL treatments partially reduced the LPS-induced alveolar bone resorption in vivo in rats, suggesting that HDL may neutralize the ability of LPS to induce alveolar bone resorption.

Effect of nifedipine on the expression of p53 protein in rat gingiva.

The aim of the present study was to determine the profile of p53 protein expression in gingival tissues after treatment with nifedipine in rats. Rats were treated daily by gastric intubation with or without DMSO alone or DMSO-dissolved nifedipine at concentrations of 15, 30 or 60 mg/kg body weight for 1, 3 or 6 week(s). Gingival width and height were measured macroscopically. Monoclonal antibodies recognizing both wild-type and mutant p53 protein were applied on paraffin-embedded gingival sections using microwave pretreatment and immunohistochemical methods. The gingival width and height were increased in the animals treated with nifedipine at concentrations of 30 and 60 mg/kg body weight. Increased gingival width and height were already seen in the animals treated with 60 mg of nifedipine for 1 week, whereas treatments with 30 mg of nifedipine resulted in increased gingival width and height after treatment for at least 3 weeks. The expression of p53 protein was elevated in the animals treated with 30 or 60 mg of nifedipine. Treatments with nifedipine at the concentration of 60 mg/kg body weight for 1 week induced the expression of p53 protein in the gingival tissues. Treatment with nifedipine in rats led to the inducement of gingival hyperplasia and increase in the numbers of p53-positive gingival epithelial cells by a dose and frequency dependent mechanism, suggesting that p53 protein may play a crucial role in the regulation of nifedipine-induced gingival hyperplasia.