Hangeshashinto for prevention of oral mucositis in patients undergoing hematopoietic stem cell transplantation: a randomized phase II study.

PURPOSE: Oral mucositis (OM) is a side effect associated with cancer treatment. Hangeshashinto (HST), a Kampo medicine, was originally prescribed to treat diarrhea, gastritis, and stomatitis. Several reports have described the effects of HST for OM induced by chemotherapy in patients with gastric or colorectal cancer. In this study, the effects of HST for prevention of OM were investigated in patients undergoing hematopoietic stem cell transplantation (HSCT). METHODS: Thirty patients scheduled to receive allogeneic grafts were enrolled from July 2020 to December 2021. They were randomly assigned to two groups and instructed to wash their mouth using HST dissolved in saline solution or using only saline solution three times a day. The observation period was from the initiation date of the conditioning regimen to the date of engraftment, and the end point was the incidence of OM. RESULTS: Eighteen patients developed OM, the most severe of which was Grade (G)3. There was no significant difference in the incidence of OM between the HST group and the control group. However, a negative correlation tended to be observed between the duration using HST use and the duration of OM (G2-3: P = 0.027, G3: P = 0.047). CONCLUSIONS: The present study demonstrated that HST use did not clearly inhibit onset of OM but showed a tendency to inhibit OM exacerbation. However, further studies are necessary to fully understand the effects of HST on OM in patients undergoing HSCT. TRIAL REGISTRATION: This study was registered in the Japan Registry of Clinical Trials on 7 May 2020 (jRCTs071200012).

Effects of anti-mouse RANKL antibody on orthodontic tooth movement in mice.

Background/purpose: Orthodontic tooth movement is achieved by alveolar bone remodeling, and therefore the balance of bone resorption and formation is important. Receptor activator of nuclear factor-κB ligand (RANKL) plays a crucial role in bone resorption. We previously reported that tumor necrosis factor-α (TNF-α) is also important in bone resorption during tooth movement. In this study, we focused on bone and root resorption during orthodontic tooth movement in mice using anti-mouse RANKL antibody (anti-mRANKL ab). Materials and methods: Anti-mRANKL ab was administered intraperitoneally to mice that subsequently underwent orthodontic tooth movement. After 10 days, tissues around the moved teeth were histologically evaluated. To confirm the effects of anti-mRANKL ab on TNF-α induced bone resorption, TNF-α was administered with and without anti-mRANKL ab into the supracalvaria and the sutures of the calvaria were histologically evaluated. Results: Orthodontic tooth movement was suppressed in mice treated with anti-mRANKL ab. Root resorption was observed after orthodontic tooth movement, but not in mice treated with anti-mRANKL ab. In the calvarial experiment, the number of TRAP-positive cells in the calvarial sutures was lower in mice administered TNF-α with anti-mRANKL ab than in mice administered TNF-α alone. Conclusion: Our findings suggest that anti-mRANKL ab suppressed orthodontic tooth movement. This needs to be considered when orthodontic tooth movement is required in patients using anti-RANKL antibody.

A preliminary study of suppression of candida infection by miconazole mucoadhesive tablets in oral or oropharyngeal cancer patients undergoing radiotherapy.

Elevated numbers of candida in the oral cavity often lead to oral candidiasis development in patients undergoing radiotherapy for oral or oropharyngeal cancer. This study aimed to verify the effect of miconazole mucoadhesive tablets on suppression of oral candida infection during radiotherapy. For this preliminary interventional study, miconazole mucoadhesive tablets were attached to the oral mucosa for 14 days from when grade 2 oral mucositis appeared in patients with oral or oropharyngeal cancer receiving radiotherapy, and the incidence of oral candidiasis was investigated. Various clinical factors were examined; univariate and multivariate Cox regression analyses were performed to investigate and compare the efficacy of this drug in preventing oral candidiasis with results of our previous study as historical control. Miconazole mucoadhesive tablets were administered to 18 patients, and oral candidiasis was observed in one patient (5.6%) after treatment completion. Among 144 historical control patients, 43 (29.9%) developed oral candidiasis. Multivariate Cox regression showed that miconazole mucoadhesive tablets significantly reduced oral candidiasis development during radiotherapy (p = 0.049, Hazard ratio 0.136, 95% confidence interval 0.019-0.994). This preliminary study suggests the efficacy of miconazole mucoadhesive tablets in preventing oral candidiasis in oral or oropharyngeal cancer patients treated with radiotherapy.Trial registration: Japan Registry of Clinical Trials (jRCT), jRCTs071190023. Registered 3 September, 2019.

Association of neutrophil-to-lymphocyte ratio with severe radiation-induced mucositis in pharyngeal or laryngeal cancer patients: a retrospective study.

BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation that informs clinical decisions regarding recurrence and overall survival in most epithelial cancers. Radiotherapy for head and neck cancer leads to mucositis in almost all patients and severe radiation-mucositis affects their quality of life (QOL). However, little is known about the NLR for severe mucositis. Therefore, this study aimed to show the association between the NLR and severe radiation-induced mucositis in hypopharyngeal or laryngeal cancer patients. METHODS: In this retrospective study, we determined the incidence of grade 3 mucositis in 99 patients who were receiving definitive radiotherapy or chemoradiotherapy (CRT) for hypopharyngeal or laryngeal cancer. We performed univariate and multivariate logistic regression analyses to investigate the characteristics of grade 3 mucositis. Kaplan-Meier curves and log-rank tests were used to evaluate the occurrence of grade 3 mucositis between two groups with high (NLR > 5) or low (NLR < 5) systemic inflammation. RESULTS: The incidence of grade 3 mucositis was 39%. Univariate logistic regression analysis showed that the NLR (Odd ratio [OR] = 1.09; 95% confidence interval [CI] = 1.02-1.16; p = 0.016) and smoking (OR = 1.02; 95% CI = 1.00-1.03; p = 0.048) were significantly associated with grade 3 mucositis. Multivariate logistic regression analysis showed that the NLR was independently associated with grade 3 mucositis (OR = 1.09; 95% CI = 1.01-1.17; p = 0.021). Kaplan-Meier curves also showed that patients with higher NLR (NLR > 5) prior to radiotherapy developed grade 3 mucositis more frequently than those with lower NLR during radiotherapy (p = 0.045). CONCLUSION: This study suggests that a higher NLR is a risk factor and predictor of severe radiation-induced mucositis in hypopharyngeal or laryngeal cancer patients.

Effect of Cancer Treatment on the Worsening of Periodontal Disease and Dental Caries: A Preliminary, Retrospective Study.

PURPOSE: To investigate the effect of cancer treatment on the worsening of dental caries and periodontal disease. MATERIALS AND METHODS: Fifty-three adult cancer patients who underwent panoramic radiography before cancer treatment and 1-2 years later were enrolled in this study. They received professional oral care, including oral hygiene instruction, scaling and root planing, professional mechanical tooth cleaning, and dental treatment or extraction of any tooth with the source of infection. Age, sex, smoking habit, probing pocket depth, alveolar bone loss, oral hygiene, number of teeth, leukocytes, haemoglobin, and albumin counts, cancer treatment, the administration of immunosuppressants, worsening of dental caries, and alveolar bone loss after 1-2 years were examined. Factors related to the worsening of dental caries and alveolar bone loss were analysed using logistic regression analysis. RESULTS: Dental caries and periodontal disease worsened in 20.8% of the patients. Smoking habit and chemotherapy were independent risk factors for the worsening of dental caries, while alveolar bone loss greater than 1/3 and chemotherapy were independent risk factors related to worsening periodontal disease. CONCLUSION: Anticancer drug treatment is an exacerbating factor for dental caries and periodontal disease.

High-mobility group box 1 released by traumatic occlusion accelerates bone resorption in the root furcation area in mice.

BACKGROUND AND OBJECTIVE: Traumatic occlusion can cause bone resorption without bacterial infection. Although bone resorption in periodontitis has been relatively well studied, little is known about bone resorption by traumatic occlusion. High-mobility group box 1 (HMGB1) is released from damaged tissue and has been recently shown to promote bone resorption in a murine periodontitis model and may also promote bone resorption by traumatic occlusion. The present study aimed to examine whether HMGB1 accelerates bone resorption by traumatic occlusion in mice. MATERIALS AND METHODS: Occlusal trauma was induced in the lower left first molar of mice by bonding a wire to the upper left first molar, and bone resorption and osteoclast formation were evaluated histochemically. The expression of HMGB1, Toll-like receptor 4 (TLR4; the receptor for HMGB1), and receptor activator of NFκB ligand (RANKL; an essential osteoclast differentiation factor) was evaluated immunohistologically. In addition, mice were administrated with an anti-HMGB1-neutralizing antibody to analyze the role of HMGB1. RESULTS: Bone resorption and osteoclast formation gradually increased until day 5 at the furcation area after the application of traumatic occlusion. Expression of HMGB1 was observed at the furcation area on day 1, but was attenuated by day 3. Expression of RANKL gradually increased until day 3, but was attenuated by day 5. Administration of anti-HMGB1 antibody significantly reduced the number of osteoclasts and the expression of RANKL and TLR4 at the furcation area. CONCLUSION: Release of HMGB1 in the root furcation area accelerated bone resorption by up-regulating RANKL and TLR4 expression in mice with traumatic occlusion.

Risk factors associated with the development of aspiration pneumonia in patients receiving radiotherapy for head and neck cancer: retrospective study.

BACKGROUND: Patients with head and neck cancer who are receiving radiotherapy can develop aspiration pneumonia. Determination of the incidence of aspiration pneumonia and the associated risk factors could facilitate the identification of high-risk patients. METHODS: In this retrospective study, we determined the incidence of aspiration pneumonia in 357 patients receiving radiotherapy along with oral care for head and neck cancer. We also performed univariate and multivariable logistic regression analyses to investigate the risk factors for this complication. RESULTS: The incidence of aspiration pneumonia was 17.6%. Hypopharyngeal cancer, grade 3 oral mucositis, and nasogastric tube feeding were independent risk factors. Moreover, the development of aspiration pneumonia was one of the major effects on the discontinuation of radiotherapy. CONCLUSION: Approximately, one-sixth of the patients developed aspiration pneumonia despite appropriate oral care during radiotherapy for head and neck cancer. Aspiration pneumonia during radiotherapy could adversely affect head and neck cancer management.

A histopathologic study of the controlling role of T cells on experimental periodontitis in rats.

BACKGROUND/PURPOSE: The onset and progression of periodontitis involve bacterial infection and the immune response. T cells function in the immune response and reportedly induce bone resorption in inflammatory bone loss. However, the exact role of T cells in periodontal destruction remains unclear. Using our experimental model of periodontitis, we aimed to investigate the influence of T cells on periodontal destruction. MATERIALS AND METHODS: Male athymic nude (Nu) and euthymic wild-type (WT) rats were divided into the immunized (I-Nu and I-WT), non-immunized (nI-Nu and nI-WT). The immunized groups were immunized intraperitoneally with lipopolysaccharide (LPS). The non-immunized groups received phosphate-buffered saline (PBS). Nothing was administered to the non-treated groups. LPS was applied to the right palatal gingival sulcus in the immunized and non-immunized groups daily for 20 days. Loss of attachment, numbers of inflammatory cells and osteoclasts, and levels of alveolar bone were investigated histopathologically and histometrically. Osteoclasts were stained with tartrate-resistant acid phosphatase. The numbers of IL-4-positive cells were evaluated immunohistologically. RESULTS: Loss of attachment, numbers of inflammatory cells, levels of alveolar bone, and the number of osteoclasts were significantly increased in the nI-WT group compared with the nI-Nu group. However, the parameters were significantly increased in the I-Nu group compared with the I-WT group. The number of IL-4-positive cells was greater in the I-WT group than in the I-Nu group. CONCLUSION: T cells promote inflammation in non-immunized animals; however, they regulate these processes in immunized animals.

T cell and periosteum cooperation in osteoclastogenesis induced by lipopolysaccharide injection in transplanted mouse tibia.

BACKGROUND/PURPOSE: We previously reported that injedctions of lipopolysaccharide (LPS) into the gingiva of mice induce inflammatory bone resorption that actively involved T cells. Receptor activator of NF-κB ligand (RANKL), which is an essential factor for osteoclastogenesis, was reportedly produced by osteoblasts, fibroblasts, and T cells in vitro; however, it has not been established which cells affect osteoclastogenesis in vivo. Here we determined the roles of T cells and the periosteum on osteoclastogenesis in LPS-induced inflammatory bone resorption. MATERIALS AND METHODS: Thirty-five BALB/c (wild-type: WT) and 10 BALB/c-nu/nu (nude: Nu) mice congenitally lacking T cells were used. Using inbred WT mice, tibias were transplanted with and without the periostea [(+) and (-), respectively, n = 15 per group] into the dorsal subcutaneous connective tissue of WT or Nu mice. Each group received four injections around the transplanted site: experimental groups were injected with LPS, and control groups were injected with phosphate-buffered saline. Isolated tissues were prepared for histopathological observation of the transplanted bone surface. RESULTS: Many infiltrating inflammatory cells were present near the surface of the tibias in the LPS-injected groups. Only the WT (+) LPS group showed osteoclasts. The number of mononuclear preosteoclasts and RANKL-positive cells was highest in the WT (+) LPS group, and there were no significant differences among the other three groups. CONCLUSION: T cells and the periosteum are closely involved in osteoclastogenesis in inflammatory bone resorption in vivo.

Dental Calculus Stimulates Interleukin-1ß Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes.

Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1ß. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1ß precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1ß, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1ß secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1ß secretion by ELISA. We found that calculus induced IL-1ß secretion in both human PMNs and PBMCs. Calculus also induced IL-1ß in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1ß induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1ß transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1ß transcription. However, it did induce IL-1ß secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1ß in mouse macrophages, and baked calculus induced IL-1ß in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1ß secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in the activation of NLRP3 inflammasome.

RANKL pretreatment plays an important role in the differentiation of pit-forming osteoclasts induced by TNF-α on murine bone marrow macrophages.

BACKGROUND: Osteoclasts differentiated from bone marrow macrophages (BMMs) induced by TNF-α alone do not have resorbing activity. When BMMs are stimulated with receptor activator of NF-κB ligand (RANKL) before TNF-α stimulation, pit-forming osteoclasts are differentiated. However, the details of the effect of RANKL pretreatment on the pit-forming osteoclast differentiation by TNF-α have not been established. The aim of this study is to examine the condition of RANKL pretreatment for differentiation of pit-forming osteoclasts induced by TNF-α. Murine BMMs were stimulated with various concentrations of RANKL for 24h in the presence of M-CSF, then the medium was changed and TNF-α was added. Osteoclasts and pits formation were examined. Osteoprotegerin (OPG), decoy receptor of RANKL, was added to the culture to examine the necessity of co-existing RANKL with TNF-α on the formation of pit-forming osteoclasts. To investigate the influence of RANKL of sufficient concentration as pretreatment for pit-forming osteoclast formation by TNF-α, dose- and time-dependent changes of osteoclast formation were checked. RESULTS: The pit formation by osteoclasts in response to TNF-α required 10ng/mL RANKL pretreatment. Stimulation with this concentration of RANKL led to the differentiation of mature osteoclasts in the 72h culture. The pit formation was not inhibited by the OPG. CONCLUSION: These results suggested that the concentration of RANKL pretreatment, which also alone can differentiate BMMs into osteoclasts, may be important in the differentiation of pit-forming osteoclasts by TNF-α. In addition, the effects of TNF-α after RANKL treatment might be independent of RANKL.

A single nucleotide polymorphism in 3'-untranslated region contributes to the regulation of Toll-like receptor 4 translation.

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.

Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses.

Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotein E-deficient (ApoE(-/-)) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-gamma and the pro-inflammatory cytokines IL-1 beta, IL-6 and tumor necrosis factor-alpha in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2(-/-)) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen.

Locally administered T cells from mice immunized with lipopolysaccharide (LPS) accelerate LPS-induced bone resorption.

T cells play important roles in bone destruction and osteoclastogenesis and are found in chronic destructive bone lesions. Lipopolysaccharide (LPS) is one of several pathological factors involved in inflammatory bone destruction. We previously described the importance of T cells in the inflammatory bone resorption that occurs after repeated LPS administration. However, whether local or systemic T cells are important for inflammatory bone resorption and whether immunization of host animals influences bone resorption remain unclear. The present study examines the effects of local extant T cells from LPS-immunized mice on LPS-induced bone resorption. T cells from LPS-immunized or non-immunized mice were injected together with LPS into the gingival tissues of mice with severe combined immunodeficiency disease that lack both T and B cells. We histomorphometrically evaluated bone resorption at sites of T cell injections and examined the influence of T cells from LPS-immunized mice on osteoclastogenesis in vitro. We found that locally administered T cells from LPS-immunized but not non-immunized mice accelerated LPS-induced bone resorption in vivo. Moreover, T cells from LPS-immunized mice increased osteoclastogenesis in vitro induced by receptor activator of NF-kappa B ligand and LPS and anti-tumor necrosis factor (TNF)-alpha antibody inhibited this increase. These results demonstrated that local extant T cells accelerate inflammatory bone resorption. Furthermore, T cells from LPS-immunized mice appear to elevate LPS-induced bone resorption using TNF-alpha.

Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs.

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.

Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis.

Toll-like receptors (TLRs) are a group of pathogen-associated molecular pattern receptors, which play an important role in innate immune signaling in response to microbial infection. It has been demonstrated that TLRs are differentially up regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. The expression of TLRs are markedly augmented in human atherosclerotic lesions and this occurs preferentially by endothelial cells and macrophages in areas infiltrated with inflammatory cells. Furthermore polymorphisms in the human gene encoding one TLR receptor (TLR4) which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with low levels of certain circulating mediators of inflammation and a decreased risk for atherosclerosis in humans. Recent advances have established a fundamental role for inflammation in mediating all stages of atherosclerosis. However, the triggers that initiate and sustain the inflammatory process have not been definitively identified. Although definitive proof of a role of infection contributing to atherogenesis is lacking, multiple investigations have demonstrated that infectious agents evoke cellular and molecular changes supportive of such a role. Evidence in humans suggesting that periodontal infection predisposes to atherosclerosis is derived from studies demonstrating that the periodontal pathogen Porphyromonas gingivalis resides in the wall of atherosclerotic vessels and seroepidemiological studies demonstrating an association between pathogen-specific IgG antibodies and atherosclerosis. Our recent work with P. gingivalis has demonstrated the effectiveness of specific intervention strategies (immunization) in the prevention of pathogen-accelerated atherosclerosis. We have also established that the inflammatory signaling pathways that P. gingivalis utilizes is dependent on the cell type and this specificity clearly influences innate immune signaling in the context of local chronic inflammation versus distant chronic inflammation. We postulate that bacterial infection mediates inflammatory responses that involve specific innate immune pathways in defined host cells. Furthermore, these inflammatory responses can be correlated with atherosclerosis and ultimately thrombotic complications.

Toll-like receptor 2 plays a critical role in the progression of atherosclerosis that is independent of dietary lipids.

OBJECTIVE: Toll-like receptors (TLRs), a group of pathogen-associated microbial pattern recognition receptors, play an important role in innate immune signaling and are differentially regulated in chronic inflammatory diseases such as atherosclerosis. However, the involvement of TLRs in the progression of atherosclerosis is still unclear. METHODS AND RESULTS: TLR2 and apolipoprotein E double knockout (Tlr2(-/-)Apoe(-/-)) mice were generated and the progressive formation of atherosclerotic plaque in the aortas was examined in mice fed a normal chow diet. We demonstrate that inactivation of TLR2 resulted in reduced progression of atherosclerosis in both male and female Apoe(-/-) mice. Likewise, TLR2 deficiency resulted in a reduction in lipid accumulation and decreased macrophage recruitment to the aortic sinus, as well as reduced monocyte chemoattractant protein-1 (MCP-1) levels. Furthermore, macrophages isolated from Tlr2(-/-)Apoe(-/-) mice demonstrated significantly reduced MCP-1 production upon stimulation with a TLR2 ligand. However, no differences in acetylated low-density lipoprotein uptake and foam cell formation were observed in macrophages isolated from Tlr2(-/-)Apoe(-/-) mice as compared to Apoe(-/-) mice. CONCLUSIONS: TLR2 plays a critical role in the progression of atherosclerosis in Apoe(-/-) mice, which is independent of dietary lipids and macrophage lipid uptake.

Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production.

The receptor activator of NF-kappaB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-alpha)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-alpha and occurred independently of RANKL, interleukin-1beta (IL-1beta), and IL-6. CS fluids from P. gingivalis-stimulated TLR2(-/-) macrophages failed to express TNF-alpha, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4(-/-) macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-alpha production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-alpha-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.

Express railway disaster in Amagasaki: a review of urban disaster response capacity in Japan.

INTRODUCTION: On the morning of 25 April 2005, a Japan Railway express train derailed in an urban area of Amagasaki, Japan. The crash was Japan's worst rail disaster in 40 years. This study chronicles the rescue efforts and highlights the capacity of Japan's urban disaster response. METHODS: Public reports were gathered from the media, Internet, government, fire department, and railway company. Four key informants, who were close to the disaster response, were interviewed to corroborate public data and highlight challenges facing the response. RESULTS: The crash left 107 passengers dead and 549 injured. First responders, most of whom were volunteers, were helpful in the rescue effort, and no lives were lost due to transport delays or faulty triage. Responders criticized an early decision to withdraw rescue efforts, a delay in heliport set-up, the inefficiency of the information and instruction center, and emphasized the need for training in confined space medicine. Communication and chain-of-command problems created confusion at the scene. CONCLUSIONS: The urban disaster response to the train crash in Amagasaki was rapid and effective. The Kobe Earthquake and other incidents sparked changes that improved disaster preparedness in Amagasaki. However, communication and cooperation among responders were hampered, as in previous disasters, by the lack of a structured command system. Application of an incident command system may improve disaster coordination in Japan.