Synergistic effect of Toll-like receptor 2 ligands and alendronate on proinflammatory cytokine production in mouse macrophage-like RAW-ASC cells is accompanied by upregulation of MyD88 expression.

OBJECTIVES: Toll-like receptors (TLRs) recognize whole cells or components of microorganisms. Alendronate (ALN) is an anti-bone-resorptive drug that has inflammatory side effects. The aim in this study was to examine whether ALN augments TLR2 ligand-induced proinflammatory cytokine production using mouse macrophage-like RAW264.7 cells transfected with murine apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) gene (hereafter, referred to as "RAW-ASC cells"). METHODS: RAW-ASC cells were pretreated with or without ALN and then incubated with or without TLR2 ligands. The levels of secreted mouse IL-1α, IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in culture supernatants and the activation of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were measured using enzyme-linked immunosorbent assay (ELISA). The expressions of myeloid differentiation factor 88 (MyD88), caspase-11, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), ASC, NF-κB p65, and actin were analyzed via Western blotting. TLR2 expression was analyzed using flow cytometry. RESULTS: ALN substantially upregulated the Pam3CSK4-induced release of IL-1α, IL-1ß, IL-6, and TNF-α and MyD88 expression in RAW-ASC cells. ST-2825, a MyD88 inhibitor, inhibited the ALN-augmented release of these cytokines. Pretreatment with ALN augmented Pam3CSK4-induced NF-κB activation in RAW-ASC cells and upregulated AP-1 activation. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein and ALN synergically upregulated the release of IL-1α, IL-1ß, IL-6 and TNF-α in RAW-ASC cells. CONCLUSIONS: Our findings suggest that ALN augments TLR2 ligand-induced proinflammatory cytokine production via the upregulation of MyD88 expression, and this augmentation is accompanied by the activation of NF-κB and AP-1 in RAW-ASC cells.

Alendronate augments lipid A­induced IL­1ß release by ASC­deficient RAW264 cells via AP­1 activation.

Alendronate (ALN) is an anti-bone-resorptive drug with inflammatory side effects. ALN upregulates lipid A-induced interleukin (IL)-1α and IL-1ß release by J774.1 cells via apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) activation. The present study examined whether ALN augmented lipid A-induced proinflammatory cytokine production using ASC-deficient mouse macrophage-like RAW264 cells. Pretreatment of RAW264 cells with ALN significantly augmented lipid A-induced IL-1ß release, although ALN did not upregulate the expression of Toll-like receptor 4, myeloid differentiation factor 88 (MyD88) and caspase-11. Moreover, pretreatment of caspase-11-deficient RAW264.7 cells with ALN significantly augmented lipid A-induced IL-1ß release. Notably, ALN upregulated the activation of FosB, c-Jun or JunD, but not c-Fos or NF-κB in RAW264 cells. Furthermore, pretreatment with the activator protein 1 (AP-1) inhibitor SR11302, but not the c-Fos inhibitor T-5224, before addition of ALN inhibited ALN-augmented IL-1ß release by lipid A-treated RAW264 cells. SR11302 also reduced ALN-augmented lactate dehydrogenase release by the cells. These findings collectively suggested that ALN augmented lipid A-induced IL-1ß release and cell membrane damage in ASC-deficient RAW264 cells via activation of AP-1, but not NF-κB.

Comparative Pan-Genome Analysis of Oral Veillonella Species.

The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies.

Alendronate Augments Lipid A-Induced IL-1α Release via Activation of ASC but Not Caspase-11.

Nitrogen-containing bisphosphonates (NBPs), such as alendronate (ALN), are anti-bone-resorptive drugs that have inflammatory side effects. We previously reported that ALN augmented lipid A-induced interleukin (IL)-1ß production and NOD-like receptor pyrin domain-containing-3 (NLRP3)/apoptosis-associated speck-like protein containing a CARD (ASC)-dependent cell death. The present study aimed to examine whether ALN augments lipid A-induced IL-1α release and necroptosis, which is induced by the activation of receptor-interacting protein kinase (RIPK) 3. Treatment of J774.1 cells with ALN augmented lipid A-induced IL-1α release, which was not inhibited by Ac-IETD-CHO, a caspase-8 inhibitor. ALN also activated mixed lineage kinase domain-like (MLKL), a key mediator of the necroptosis pathway, and upregulated the expression of caspase-11, a lipid A receptor. GSK'872, a RIPK3 inhibitor, suppressed the ALN-upregulated expression of caspase-11 and augmented lipid A-induced caspase-8 activation. Moreover, ALN induced the release of NLRP3 and ASC into culture supernatants. GSK'872, but not Ac-IETD-CHO, reduced the ALN-induced release of NLRP3, but not ASC, into culture supernatants, and reduced ALN-induced cell death, but not ALN-induced LDH release. Antibodies against NLRP3 and ASC upregulated caspase-11 expression in the cytosol by inhibiting ALN-induced cell death. However, pretreating cells with an antibody against ASC, but not NLRP3, before ALN addition also inhibited lipid A-induced IL-1α release. Pretreating cells with an antibody against caspase-11 before the addition of ALN or lipid A did not downregulate lipid A-induced production of IL-1α. Taken together, our findings suggest that ALN augments lipid A-induced IL-1α release via activation of ASC, but not caspase-11.

Complete Genome Sequence of Veillonella nakazawae JCM 33966T (=CCUG 74597T), Isolated from the Oral Cavity of Japanese Children.

We report the complete genome sequence of Veillonella nakazawae JCM 33966T (=CCUG 74597T). This bacterium is a member of the oral Veillonella and has the potential to be anticariogenic as an oral probiotic seed.

Etidronate down-regulates Toll-like receptor 2 ligand-induced chemokine production by inhibiting MyD88 expression and NF-κB activation.

OBJECTIVE: Pretreatment of J774.1 cells with etidronate, a non-nitrogen-containing bisphosphonate (non-NBP) used as an antibone resorptive drug, was previously reported to inhibit Toll-like receptor (TLR) 2 agonist-induced proinflammatory cytokine production. The present study aimed to examine the effects of etidronate on chemokine production by human monocytic U937 cells incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a TLR2 ligand) and lipid A (a TLR4 ligand). METHODS: U937 cells were pretreated with or without etidronate, and then incubated with or without Pam3CSK4 or lipid A. Levels of secreted human interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants and activation of nuclear factor-κB (NF-κB) p65 were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) activity in supernatants. Expression of intracellular adhesion molecule (ICAM)-1 and MyD88 was analyzed by flow cytometry and Western blot analysis, respectively. RESULTS: Etidronate down-regulated IL-8 and MCP-1 production and NF-κB p65 activation induced by Pam3CSK4, but not lipid A, in U937 cells. Etidronate also inhibited MyD88 expression in U937 cells incubated with Pam3CSK4. CONCLUSION: Etidronate down-regulates IL-8 and MCP-1 production in U937 cells by inhibiting both the expression of MyD88 and activation of NF-κB p65 in the TLR2, but not TLR4, pathway.

Veillonella nakazawae sp. nov., an anaerobic Gram-negative coccus isolated from the oral cavity of Japanese children.

Two strains of previously unknown Gram-negative cocci, T1-7T and S6-16, were isolated from the oral cavity of healthy Japanese children. The two strains showed atypical phenotypic characteristics of members of the genus Veillonella, including catalase production. Sequencing of their 16S rRNA genes confirmed that they belong to genus Veillonella. Under anaerobic conditions, the two strains produced acetic acid and propionic acid as metabolic end-products in a trypticase-yeast extract-haemin medium containing 1 % (w/v) glucose, 1 % (w/v) fructose and 1 % (v/v) sodium lactate. Comparative analysis of the 16S rRNA, dnaK, rpoB and gltA gene sequences revealed that the two strains are phylogenetically homogeneous and comprise a distinct, novel lineage within the genus Veillonella. The sequences from the two strains shared the highest similarity, at 99.9, 95.8, 96.9 and 96.7 %, using the partial 16S rRNA, dnaK, rpoB and gltA gene sequences, respectively, with the type strains of the two most closely related species, Veillonella dispar ATCC 17748T and Veillonella infantium JCM 31738T. Furthermore, strain T1-7T shared the highest average nucleotide identity (ANI) value (94.06 %) with type strain of the most closely related species, V. infantium. At the same time, strain T1-7T showed the highest digital DNA-DNA hybridization (dDDH) value (55.5 %) with the type strain of V. infantium. The two strains reported in this study were distinguished from the previously reported species from the genus Veillonella based on catalase production, partial dnaK, rpoB and gltA sequences, average ANI and dDDH values. Based on these observations, the two strains represent a novel species, for which the name Veillonella nakazawae sp. nov. is proposed. The type strain is T1-7T (JCM 33966T=CCUG 74597T).

RANK-RANKL signaling upregulates Il-10 mRNA expression in mucosal Candida infection in vivo.

Osteoprotegerin (OPG) prevents binding of receptor activator of nuclear factor-kappa B ligand (RANKL) to RANK. Recent studies have reported that immune cell RANK-RANKL interactions are critical to the infection process. Candida albicans is an opportunistic pathogenic fungus and a common cause of candidiasis. This study utilized an orally inoculated mouse model of C. albicans infection to determine whether superficial or systemic candidiasis was associated with alterations in RANK/RANKL/OPG expression. Invasive systemic C. albicans infection increased serum OPG levels in mice. In addition, tongue Opg, Rankl, and Rank mRNA expression were upregulated in mice with superficial oral cavity C. albicans infection. Moreover, administration of exogenous soluble RANKL upregulated Rank and interleukin-10 (Il-10) mRNA in superficially infected tissue, suggesting suppression of localized inflammation. Taken together, these findings suggested that RANK/RANKL/OPG signaling contributes to the pathogenesis of candidiasis. This is the first in vivo study to identify a relationship between this opportunistic infection and the RANK/RANKL/OPG axis.

MPMBP down-regulates Toll-like receptor (TLR) 2 ligand-induced proinflammatory cytokine production by inhibiting NF-κB but not AP-1 activation.

MPMBP is a novel non-nitrogen-containing bisphosphonate (non-NBP) which possesses anti-bone resorptive activity and an antioxidant side chain. This study aimed to assess the effects of MPMBP on the production of proinflammatory cytokines and chemokines by the macrophage-like cell line, J774.1, in the presence of Toll-like receptor (TLR) agonists. J774.1 cells were pretreated with or without MPMBP for 5 min, and then incubated with or without Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a TLR2 agonist) or lipid A (a TLR4 agonist) for 24 h. MPMBP down-regulated TLR2 ligand-induced production of IL-6, MCP-1, MIP-1α, and TNF-α, but not TLR4 ligand-induced proinflammatory cytokine production, and was not cytotoxic in J774.1 cells. Cu-CPT22, a TLR2 antagonist, down-regulated Pam3CSK4-induced production of IL-6, MCP-1, and MIP-1α, but not TNF-α. MPMBP inhibited the translocation of NF-κB p65, but not p50, RelB, or p52, and inhibited the activation of JNK, but not p38 MAPK or ERK, in J774.1 cells stimulated with Pam3CSK4. Moreover, MPMBP did not down-regulate AP-1 activation in J774.1 cells stimulated with Pam3CSK4 or lipid A. Our findings suggest that MPMBP inhibits proinflammatory cytokine production in J774.1 cells by suppressing NF-κB p65 activation in the TLR2, but not TLR4, pathway.

Heat-killed Candida albicans augments synthetic bacterial component-induced proinflammatory cytokine production.

Candida albicans can enhance the invasion of oral epithelial cells by Porphyromonas gingivalis, although the fungus is not a periodontal pathogen. In this study, we investigated whether C. albicans augments proinflammatory cytokine production by mouse macrophage-like J774.1 cells incubated with synthetic bacterial components. Mouse macrophage-like J774.1 cells, mouse primary splenocytes, human THP-1 cells, and A549 cells were pretreated with or without heat-killed C. albicans (HKCA) or substitutes for C. albicans cell wall components in 96-well flat-bottomed plates. Cells were then washed and incubated with Pam3CSK4, a Toll-like receptor (TLR) 2 ligand, or lipid A, a TLR4 ligand. Culture supernatants were analyzed by ELISA for secreted IL-6, MCP-1, TNF-α, and IL-8. HKCA augmented TLR ligand-induced proinflammatory cytokine production by J774.1 cells, mouse splenocytes, and THP-1 cells, but not A549 cells. However, IL-6, MCP-1, and TNF-α production induced by Pam3CSK4 or lipid A was not augmented when cells were pretreated with curdlan, a dectin-1 ligand, or mannan, a dectin-2 ligand. In contrast, pretreatment of cells with TLR ligands upregulated the production of IL-6 and TNF-α, but not MCP-1, induced by Pam3CSK4 or lipid A. The results suggest that C. albicans augments synthetic bacterial component-induced cytokine production by J774.1 cells via the TLR pathway, but not the dectin-1 or dectin-2 pathway.

Extracellular galectin-1 enhances adhesion to and invasion of oral epithelial cells by Porphyromonas gingivalis.

Galectin-1 and galectin-3 are C-type lectin receptors that bind to lipopolysaccharide in the cell wall of gram-negative bacteria. In this study, we investigated the effects of galectin-1 and galectin-3 on adhesion to and invasion of the human gingival epithelial cell line Ca9-22 by Porphyromonas gingivalis, a periodontal pathogenic gram-negative bacterium. Recombinant galectin-1, but not galectin-3, enhanced P. gingivalis adhesion and invasion, although both galectins bound similarly to P. gingivalis. Flow cytometry also revealed that Ca9-22 cells express low levels of galectin-1 and moderate levels of galectin-3. Ca9-22 cells in which galectin-3 was knocked-down did not exhibit enhanced P. gingivalis adhesion and invasion. Similarly, specific antibodies to galectin-1 and galectin-3 did not inhibit P. gingivalis adhesion and invasion. These results suggest that soluble galectin-1, but not galectin-3, may exacerbate periodontal disease by enhancing the adhesion to and invasion of host cells by periodontal pathogenic bacteria.

Gastrointestinal colonisation and systemic spread of Candida albicans in mice treated with antibiotics and prednisolone.

Normally, Candida albicans is a commensal microbe that resides in the human oral cavity, gut and vagina. However, the fungus can cause mucosal and systemic infections in immunocompromised individuals. The mechanism by which local mucosal infections progress to systemic candidiasis is poorly understood. Here, a murine model of gastrointestinal (GI) candidiasis was developed by inoculation of the oral cavity, followed by treatment with tetracycline (TC) and prednisolone (PSL). Temporal progression from a local infection of the oral cavity to a systemic infection was then monitored. Histological analysis of tissues from mice treated with both TC and PSL revealed massive infiltration of the tongue and stomach by hyphae. PSL increased the fungal burden in the tongue, stomach and small intestine, and facilitated dissemination to the spleen, kidney and liver within 3 days post-infection. Treatment with both TC and PSL supressed interferon (IFN)-γ and interleukin (IL)-17 (cytokines that play key roles in host defence against fungal infection) levels in the tongue, which were induced by C. albicans infection. In addition, the mucosal layer of the small intestine of mice treated with both TC and PSL was almost destroyed by the fungal infection; this may be a critical event that allows passage of the fungus across the mucosa and into the systemic circulation. Thus, this mouse model is useful for studying mechanisms underlying progression of C. albicans from a local infection of the oral cavity to a systemic infection in immunocompromised individuals.

Alendronate augments lipid A-induced IL-1ß release and Smad3/NLRP3/ASC-dependent cell death.

Alendronate (ALN) is a nitrogen-containing bisphosphonate (NBP) that inhibits bone resorption. NBPs have inflammatory side effects, and ALN augments bacteria-induced interleukin (IL)-1ß production. The present study aimed to examine whether ALN induces pyroptosis, a form of cell death associated with IL-1ß release, in macrophage-like J774.1 cells incubated with lipid A, a component of gram-negative bacteria. Pretreatment of J774.1 cells with ALN increased lipid A-induced IL-1ß production and cell death, but not IL-6 and TNF-α production. Ac-YVAD-CHO, a caspase-1 inhibitor, inhibited ALN-augmented IL-1ß production induced by lipid A, although it did not affect ALN-induced cell death. Moreover, Ac-IETD-CHO, a caspase-8 inhibitor, and Z-VAD-FMK, a pan-caspase inhibitor, did not inhibit ALN-induced cell death, suggesting that the effects of ALN are exerted independently of caspase activation. We also demonstrate that a Smad3 inhibitor (SIS3) suppressed ALN-augmented IL-1ß production. Moreover, SIS3 attenuated ALN-augmented release of LDH and caspase-1. These results suggest that ALN augments IL-1ß production, cell death, and caspase-1 release in a manner dependent on Smad3. We then investigated whether ALN-augmented IL-1ß production and cell death are dependent on apoptosis-associated speck-like protein containing a CARD (ASC) and NOD-like receptor pyrin domain containing-3 (NLRP3), which are associated with Smad3 activation. Both anti-ASC and anti-NLRP3 antibodies suppressed ALN-induced cell death and caspase-1 release, but only anti-ASC antibody inhibited ALN-augmented IL-1ß production. Our findings suggest that ALN-augmented IL-1ß production and cell death require Smad3 and ASC activation, and that SIS3 and anti-ASC antibodies may serve as palliative agents for necrotizing inflammatory diseases caused by ALN.

Etidronate down-regulates Toll-like receptor (TLR) 2 ligand-induced proinflammatory cytokine production by inhibiting NF-κB activation.

BACKGROUND: Etidronate is a non-nitrogen-containing bisphosphonate (non-NBP) used for anti-bone resorptive therapy as well as having inhibitory effects on atherosclerotic plaques. The present study examined the effects of etidronate on the production of proinflammatory cytokines and chemokines by the macrophage-like cell line, J774.1, incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a Toll-like receptor (TLR) 2 agonist) and lipid A (a TLR4 agonist). METHODS: J774.1 cells and human monocytic THP-1 cells were pretreated with or without etidronate for 5min, and then incubated with or without Pam3CSK4 or lipid A for 24h. Levels of secreted interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by LDH activity in the supernatants. We also examined the effects of etidronate on the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) in J774.1 cells by ELISA and Western blotting. RESULTS: Treatment of J774.1 cells with etidronate down-regulated TLR2 ligand-induced production of IL-6, TNF-α, MCP-1, and MIP-1α. Etidronate also inhibited Pam3CSK4-induced MCP-1 and TNF-α production by THP-1 cells. However, etidronate did not induce cytotoxicity and reduced lipid A-induced cytotoxicity in J774.1 cells. In addition, this agent did not down-regulate TLR4 ligand-induced proinflammatory cytokine production. Furthermore, etidronate inhibited the translocation of NF-κB but not p38 MAPK in J774.1 cells stimulated with Pam3CSK4 or lipid A. CONCLUSION: Etidronate likely inhibits proinflammatory cytokine production in J774.1 cells by suppressing NF-κB activation in the TLR2 and not the TLR4 pathway.

Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells.

The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism.

Candida albicans and Candida parapsilosis rapidly up-regulate galectin-3 secretion by human gingival epithelial cells.

Galectin-3 is a ß-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.

Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis.

Although Candida albicans has been isolated from periodontal pockets, its relationship to periodontitis is unclear. In this study, we investigated the effect of C. albicans on the adhesion and invasion of Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts by Porphyromonas gingivalis. Heat-killed C. albicans and water-soluble mannoprotein-ß-glucan complex from C. albicans (CAWS) did not enhance P. gingivalis adhesion or upregulate the expression of ß1 integrin and ICAM-1, which are required for P. gingivalis invasion; both the epithelial cells and fibroblasts expressed dectin-1, which recognizes components of the C. albicans cell wall. However, pretreatment of Ca9-22 cells and human gingival fibroblasts with heat-killed C. albicans or CAWS significantly enhanced P. gingivalis invasion. These results suggest that C. albicans may exacerbate infectious disease by enhancing the invasion of host cells by anaerobic bacteria.

Alendronate augments interleukin-1beta release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1.

Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1beta, but not TNFalpha, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1beta production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1beta production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1beta induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1beta release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs.

Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae.

It was previously reported that two oligonucleotide primer sets (spn9802 and spn9828) for discriminating Streptococcus pneumoniae from pneumococcus-like oral streptococcal isolates using PCR had been developed. In this study, PCR amplification of the lytA, ply, spn9802 and spn9828 genes was used to identify presumptive S. pneumoniae. Two genetic groups were identified by analysing sputum samples from 28 patients with community-acquired pneumonia: the lytA-positive, ply-positive, spn9802-positive and spn9828-negative group, and the lytA-positive, ply-positive, spn9802-positive and spn9828-positive group. Isolates of the former group were resistant to optochin, while those of the latter group showed susceptibility to optochin. The lytA-positive, ply-positive, spn9802-negative and spn9828-negative isolates, and lytA-positive, ply-positive, spn9802-negative and spn9828-positive isolates, were not detected in sputum from patients with pneumonia. Subsequently, a total of 92 saliva samples from healthy individuals was screened by PCR using these primer sets. The lytA-positive, ply-positive, spn9802-positive and spn9828-negative group was identified more frequently in saliva from healthy children than in saliva from older healthy individuals and patients with pneumonia. The lytA-positive, ply-positive, spn9802-positive and spn9828-positive group was found frequently in saliva from healthy children, and in saliva and sputum from patients with pneumonia. This study demonstrates a rapid, optimal screening method for the genotypic identification of presumptive S. pneumoniae by PCR using four genes highly specific for S. pneumoniae.

Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization.

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.