A Potential New Risk Factor for Stroke: Streptococcus Mutans With Collagen-Binding Protein.

BACKGROUND: Among human oral bacteria, particular kinds of Streptococcus mutans (SM) known as dental caries pathogens contain a collagen-binding protein, Cnm, and show platelet aggregation inhibition and matrix metalloproteinase-9 activation. We have previously reported that these strains may be a risk factor for intracerebral hemorrhage. As a major sample-providing hospital, we report the clinical details, including intracranial aneurysms and ischemic stroke. METHODS: After the study received approval from the Ethical Committee, 429 samples of whole saliva were obtained from patients who were admitted to or visited our hospital between February 16, 2010, and February 28, 2011. The study cohort comprised 48 patients with cardioembolic stroke (CES), 151 with non-CES infarct, 54 with intracerebral hemorrhage (ICH), 43 with ruptured intracranial aneurysm (RIA), and 97 with unruptured intracranial aneurysm (UIA). Cultured SM was identified as Cnm-positive when the corresponding gene was positive. The results were compared with those from 79 healthy volunteers. Relationships between Cnm-positive SM and known risk factors, including hypertension, diabetes, hyperlipidemia, smoking, and alcohol consumption, were analyzed. RESULTS: A statistically significant high Cnm-positive rate was observed in patients with CES, non-CES infarct, ICH, and RIA (P = 0.002, 0.039, 0.013, and 0.009, respectively). There were no relationships between Cnm-positive SM and known risk factors. CONCLUSIONS: Specific types of oral SM can be a risk factor for cardioembolic infarct, intracerebral hemorrhage, and intracranial aneurysm rupture. Further study is needed.

Current knowledge among Japanese experienced general dentists regarding prevention of infective endocarditis.

Infective endocarditis (IE), a life-threatening condition predominantly occurring in patients with underlying heart disease, is mainly caused by bacteremia induced by invasive dental treatment. However, the amount of related information shared between cardiologists and dentists appears to be inadequate. In the present study, a survey regarding prevention of IE, composed of 13 major questions, 2 of which also allowed free comments, was sent to approximately 3000 dentists belonging to a prefectural dental association in Japan. Of the 13.6% who returned the forms, more than 80% were general dentists with more than 20 years of experience. Approximately, 55% of the responders reported that they had opportunities to prescribe antibiotics prior to performing treatments with risk of IE, though noted difficulties with designation of which patients with heart disease were at risk. Most of the dentists considered that oral surgery procedures have a high risk for IE, whereas less invasive procedures were considered to be not associated with the disease. Approximately, 35% selected oral amoxicillin, with a dose of 2.0 g (20%) or 500 mg (27%) prescribed for adults, and 50 mg (10%) or 30 mg (12%) per kg of body weight for children. However, the timing of the antibiotics administration varied. The present results reveal current knowledge regarding prevention of IE among general dentists in Japan, and should be valuable for construction of a protocol to establish consensus between dentists and cardiologists.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.

Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis.

Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm(+)/PA(-) group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm(+)/PA(-) strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm(+)/PA(-) strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens.

Identification and functional analysis of an ammonium transporter in Streptococcus mutans.

Streptococcus mutans, a Gram-positive bacterium, is considered to be a major etiologic agent of human dental caries and reported to form biofilms known as dental plaque on tooth surfaces. This organism is also known to possess a large number of transport proteins in the cell membrane for export and import of molecules. Nitrogen is an essential nutrient for Gram-positive bacteria, though alternative sources such as ammonium can also be utilized. In order to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell. However, the ammonium transporter in S. mutans remains to be characterized. The present study focused on characterizing the ammonium transporter gene of S. mutans and its operon, while related regulatory genes were also analyzed. The SMU.1658 gene corresponding to nrgA in S. mutans is homologous to the ammonium transporter gene in Bacillus subtilis and SMU.1657, located upstream of the nrgA gene and predicted to be glnB, is a member of the PII protein family. Using a nrgA-deficient mutant strain (NRGD), we examined bacterial growth in the presence of ammonium, calcium chloride, and manganese sulfate. Fluorescent efflux assays were also performed to reveal export molecules associated with the ammonium transporter. The growth rate of NRGD was lower, while its fluorescent intensity was much higher as compared to the parental strain. In addition, confocal laser scanning microscopy revealed that the structure of biofilms formed by NRGD was drastically different than that of the parental strain. Furthermore, transcriptional analysis showed that the nrgA gene was co-transcribed with the glnB gene. These results suggest that the nrgA gene in S. mutans is essential for export of molecules and biofilm formation.

Role of ABC transporter proteins in stress responses of Streptococcus mutans.

Streptococcus mutans possesses a number of ATP-binding cassette (ABC) transporters, which function as multiple sugar metabolism transporters and are promising targets for antimicrobial strategies. In the present study, we performed functional analyses of SMu0836 and SMu0837 products, which are possible ABC transporters. Isogenic mutant strains Δ0836 and Δ0837 were generated by insertional inactivation of SMu0836, and SMu0837, respectively, of strain MT8148 and found to be more sensitive to antibiotics as compared to MT8148. In addition, assays of membrane transport functions using a fluorescent probe showed that export pumps did not function properly in strain Δ0836. Expression of those genes was elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline, as well as when exposed to heat shock, osmotic stress, and oxidative stress conditions. Furthermore, the expressions of other genes including SMu0374, SMu0215, SMu0475, SMu0986, and SMu1051, which encode possible ABC transporters, were also elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline. Together, these results suggest that the products of SMu0836 and SMu0837 are ABC transporters, which may function in stress response.

[Accuracy estimation of non-invasive X-ray output analyzer].

This study estimated the accuracy of an X-ray analyzer by comparing it with an ionization chamber and a tube voltage current meter, and investigated whether it was usable as a substitute for a reference meter for output measurements for quality control purposes. The X-ray output analyzer used was a Piranha (RTI Electronics), a non-invasive instrument. The two subjects of measurements were as follows: the tube voltage, exposure, and half-value layer used in ordinary X-ray radiographic system equipment, and the exposure and half-value layer in X-ray equipment for mammographic systems. The results for a conventional radiographic system showed the error rates for tube voltage, exposure, and half value layer to be within ±1.0%, ±1.8%, and ±4.3%, respectively. The Piranha is not influenced by the dependence of the beam quality in a range of the tube voltage in clinical use. In X-ray equipment for mammographic system results, error rates for exposure and half value layer were ±2.2% and within ±4.0%, respectively. We conclude that it is possible to use the Piranha as an alternative reference meter for quality control of X-ray equipment for typical radiographic and mammographic systems.

Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis.

BACKGROUND: Infective endocarditis (IE) is known to be a life-threatening disease and prevention of its onset is important. Oral amoxicillin (AMPC) is generally prescribed to patients at risk for IE prior to undergoing risky procedures, such as invasive dental treatments. We previously found that approximately 5% of systemically healthy Japanese subjects harbor strains highly resistant to AMPC. In the present study, the prevalence of strains in patients at risk for IE was investigated. METHODS AND RESULTS: Thirty-four Japanese children and adolescents designated at risk for IE by their cardiovascular surgeons participated. Dental plaque specimens were obtained at recall examinations for dental checkups and placed in sterile phosphate-buffered saline, then diluted and streaked onto selective media for oral streptococci and also media containing AMPC. Nine strains with a minimum inhibitory concentration of AMPC of 16µg/mL or more were isolated from 7 of the subjects (20.6%), each of which was also resistant to other antibiotics analyzed except for new quinolone drugs. The 16S rRNA sequence of each strain demonstrated that all were oral streptococcal species. In addition, dental plaque specimens collected from 5 subjects after an additional interval of 3-4 months showed that 2 harbored the same clones at different time points. CONCLUSIONS: These findings suggest a higher prevalence of AMPC-resistant strains in children and adolescents at risk for IE as compared to systemically healthy subjects. Thus, alternative antibiotics should be considered for such subjects when performing prophylaxis procedures.

Multilocus sequence typing of Streptococcus mutans strains with the cbm gene encoding a novel collagen-binding protein.

OBJECTIVE: Streptococcus mutans, an oral pathogen associated with infective endocarditis (IE), possesses two genes encoding collagen-binding proteins, namely cnm and cbm. In this study, we used multilocus sequence typing (MLST) of S. mutans with the cbm gene. DESIGN: Forty-five S. mutans strains including 15 strains with the cnm gene, 15 strains with the cbm gene, and 15 strains without these two genes were analysed by MLST. In addition, the collagen-binding properties as well as the abilities to adhere to and invade human umbilical vein endothelial cells (HUVEC) were also evaluated for all strains. RESULTS: In the groups of cnm-positive and cbm-positive strains, all properties, including collagen binding, adhesion, and invasion were significantly greater than those of the cnm-cbm-negative group. Moreover, MLST revealed three clonal complexes of S. mutans possessing the cbm gene. These three clones showed no close relatedness with clones of strains containing the cnm gene. Among three clones harbouring the cbm gene, two clones belong to serotype k, and appeared to be associated with the pathogenesis of IE due to their strong collagen binding and relatively enhanced abilities to adhere to and invade endothelial cells. However, such properties were relatively weak in the other non-serotype k clone possessing the cbm gene. CONCLUSIONS: MLST indicated a difference in evolution between S. mutans strains with the cbm gene and those with the cnm gene. In addition, this technique also suggested the importance of cbm-positive S. mutans clones relative to the pathogenesis of IE.

Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype.

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be a pathogen for periodontitis in dogs. Approximately 41kDa filamentous appendages on the cell surface (FimA) encoded by the fimA gene are regarded as important factors associated with periodontitis. The fimA genotype was classified into two major types and strains in type B were shown to be more virulent than those in type A. In the present study, we characterized a strain with a novel fimA genotype and designated it as type C. The putative amino acid sequence was shown to be similar to the genotype IV fimA of Porphyromonas gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell carcinoma cell line derived from tongue primary lesions revealed that the type C strain inhibited proliferation and scratch closure more than genotype A and B strains. In addition, experiments using a mouse abscess model demonstrated that the type C strain could induce much higher systemic inflammation when compared with strains of the other genotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs demonstrated that the detection frequencies of P. gulae and the genotype C in the periodontitis group were significantly higher than those in the periodontally healthy group. These results suggest that FimA of P. gulae is diverse with the virulence of genotype C strains the highest and that molecular identification of genotype C P. gulae could be a possible useful marker for identifying dogs at high risk of developing periodontitis.

Complete genome sequence of the serotype k Streptococcus mutans strain LJ23.

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.

Trps1 is necessary for normal temporomandibular joint development.

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Infection of specific strains of Streptococcus mutans, oral bacteria, confers a risk of ulcerative colitis.

Although oral bacteria-associated systemic diseases have been reported, association between Streptococcus mutans, pathogen of dental caries, and ulcerative colitis (UC) has not been reported. We investigated the effect of various S. mutans strains on dextran sodium sulfate (DSS)-induced mouse colitis. Administration of TW295, the specific strain of S. mutans, caused aggravation of colitis; the standard strain, MT8148 did not. Localization of TW295 in hepatocytes in liver was observed. Increased expression of interferon-γ in liver was also noted, indicating that the liver is target organ for the specific strain of S. mutans-mediated aggravation of colitis. The detection frequency of the specific strains in UC patients was significantly higher than in healthy subjects. Administration of the specific strains of S. mutans isolated from patients caused aggravation of colitis. Infection with highly-virulent specific types of S. mutans might be a potential risk factor in the aggravation of UC.

Diversity of fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs.

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis. METHODS: The detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis. RESULTS: The detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT. CONCLUSIONS: Infection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Amoxicillin-resistant oral streptococci identified in dental plaque specimens from healthy Japanese adults.

BACKGROUND: Infective endocarditis (IE) is known to be a life-threatening disease and invasive dental procedures are considered to be important factors. Oral amoxicillin (AMPC) is widely used for prophylaxis in patients with heart disorders who are at risk for IE. However, there is only limited information regarding the inhibition of oral bacteria by AMPC. METHODS AND RESULTS: Dental plaque specimens were obtained from 120 healthy Japanese adult subjects, then diluted and streaked onto selective medium for oral streptococci. The minimum inhibitory concentration (MIC) of AMPC was evaluated using a macro-dilution method by Clinical Laboratory Standard Institute (2006). Seven strains with an MIC of AMPC of 16µg/mL or more were isolated from 5 subjects. The bacterial species were confirmed by sequence analysis of 16S rRNA from each strain, which demonstrated that most were Streptococcus sanguinis, followed by Streptococcus oralis. Dental plaque specimens collected from these 5 subjects again after an interval of 2-3 months possessed no strains with an MIC of AMPC of 16µg/mL or more. CONCLUSIONS: These findings suggest that strains with a high MIC of AMPC are present in the oral cavities of Japanese adults, though they may be transient rather than inhabitants.

α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.

Establishment of Streptococcus mutans in infants induces decrease in the proportion of salivary α-haemolytic bacteria.

OBJECTIVE: For paediatric dentists, an indicator to assess caries risk of infants is very important. Conventionally, the number and/or proportions of Streptococcus mutans have been employed as risk indicator; however, because such figures reflect the existing situation, they are not suitable for assessing caries risk of infants that have not yet been infected with S. mutans. Thus, we searched for an indicator for the establishment of S. mutans. METHODS: To evaluate the changes caused by the establishment of S. mutans in the microbiota of the infant oral cavity, we monitored changes in the oral microbiota of two pre-dentate infants over a 3-year period and in a cross-sectional study of 40 nursery school-aged children by cultivation of saliva on nonselective blood agar, Mitis-Salivarius agar, and Mitis-Salivarius agar supplemented with bacitracin combined with identification of selected isolates. RESULTS: Two longitudinal observations suggested that the establishment of S. mutans would induce a decrease in α-haemolytic bacteria in the microbial population of the oral cavity. This suggestion was compensated with the results of cross-sectional study, and it was revealed that the establishment of 10(3) CFU/mL of mutans streptococci in saliva might be predicted by a microbiota comprising less than approximately 55% of α-haemolytic. CONCLUSION: Decrease in the proportion of α-haemolytic bacteria in saliva of infant was found to be applicable as an indicator to predict the establishment of S. mutans and to assess dental caries risk as a background for planning of dental care and treatment in the infants before infection with S. mutans.

Krüppel-like factor 4 regulates membranous and endochondral ossification.

Krüppel-like factor 4 (KLF4/GKLF/EZF) is a zinc finger type of transcription factor highly expressed in the skin, intestine, testis, lung and bone. The role played by Klf4 has been studied extensively in normal epithelial development and maintenance; however, its role in bone cells is unknown. Previous reports showed that Klf4 is expressed in the developing flat bones but its expression diminishes postnatally. We now show that in the developing long bones, Klf4 is expressed in the perichondrium, trabecular osteoblasts and prehypertrophic chondrocytes. In contrast, osteoblasts lining at the surface of the bone collar showed extremely low levels of Klf4 expression. To investigate the possible roles played by Klf4 during skeletal development, we generated transgenic mice expressing Klf4 under mouse type I collagen regulatory sequence. Transgenic mice exhibited severe skeletal deformities and died soon after birth. Transgenic mice showed delayed formation of the calvarial bones; and over-expressing Klf4 in primary mouse calvarial osteoblasts in culture resulted in strong repression of mineralization indicating that this regulation of Klf4 is through an osteoblast-autonomous effect. Surprisingly, long bones of the transgenic mice exhibited delayed marrow cavity formation. Even at E18.5, the presumptive marrow space was occupied by cartilage anlage and invasion of the vascular endothelial cells and osteoclasts were seldom observed. Instead of entering the cartilage anlage, osteoclasts accumulated at the periosteum in the transgenic mice. Significantly, osteocalcin, which is known to chemotact osteoclasts, was up-regulated at the perichindrium as early as E14.5 in the mutants. In vitro studies showed that this induction of osteocalcin by Klf4 was regulated at its transcriptional level. Our results demonstrate that Klf4 regulates normal skeletal development through coordinating the differentiation and migration of osteoblasts, chondrocytes, vascular endothelial cells and osteoclasts.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1ß and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages.