Tacrolimus, FK506, promotes bone formation in bone defect mouse model.

OBJECTIVES: Some studies have reported that tacrolimus (FK506), an immunosuppressant, may have positive effects on bone formation. However, the precise effects of FK506 on bone repair or osteoblasts remain inadequately elucidated, and limited research has explored the outcomes of its use in an in vivo mouse model. This study aims to examine the effects of FK506 on bone repair and osteoblast functions using bone defect and BMP-2-induced ectopic ossification mouse models, as well as cultured primary mouse osteoblasts treated with FK506. METHODS: We established mouse models of femur bone defect and BMP-2-induced ectopic ossification to evaluate the effect of FK506 on new bone formation, respectively. Additionally, primary mouse osteoblasts were cultured with FK506 and examined for gene expressions related to osteoblast differentiation. RESULTS: While FK506 promoted the repair of bone defect areas in the femur of the bone defect mouse model, it also led to widespread abnormal bone formation outside the intended area. Additionally, following the implantation of a collagen sponge containing BMP-2 into mouse muscle tissue, FK506 was found to promote ectopic ossification and enhance BMP-2-induced osteoblast differentiation in vitro. Our findings also revealed that FK506 increased the number of immature osteoblasts in the absence of BMP-2 without affecting osteoblast differentiation. Furthermore, direct effects were observed, reducing the ability of osteoblasts to support osteoclastogenesis. CONCLUSIONS: These results indicate that FK506 increases new bone formation during bone repair and influences the proliferation of immature osteoblasts, as well as osteoblast-supported osteoclastogenesis.

Metagenomic Analysis of Bacterial Microflora in Dental and Atherosclerotic Plaques of Patients With Internal Carotid Artery Stenosis.

Background: Internal carotid artery stenosis is primarily attributed to atherosclerosis in the carotid artery bifurcation. Previous studies have detected oral bacteria in atherosclerotic lesions, suggesting an association between oral bacteria and atherosclerosis. In this study, we compared the bacterial flora of the atherosclerotic plaque in the carotid artery and dental plaque of patients with internal carotid artery stenosis using 16S ribosomal RNA (16S rRNA) metagenomic sequencing. Methods: Fifty-four patients who underwent internal carotid endarterectomy for internal carotid artery stenosis at the Showa University Hospital between April 2016 and February 2018 were included. Polymerase chain reaction targeting the 16S rRNA gene detected bacterial DNA in the carotid plaques of 11 cases, of which only 5 could be further analyzed. Thereafter, DNA extracted from the carotid and oral plaques of these 5 cases were analyzed using metagenomic sequencing targeting 16S rRNA. In addition, their general condition and oral conditions were evaluated. The patients were classified into symptomatic and asymptomatic groups based on the presence or absence of symptoms of transient ischemic attack, and their bacterial flora was evaluated. Results: The results demonstrated that the microflora of carotid plaques (n = 5) contained bacterial species from 55 families and 78 genera. In addition, 86.5% of the bacteria detected in the carotid plaques were also detected in oral plaques. Cariogenic and periodontopathic bacteria accounted for 27.7% and 4.7% of the bacteria in the carotid plaques, respectively. Conclusions: These results suggest that oral bacteria are directly or indirectly involved in the pathogenesis of atherosclerosis. More extensive studies of oral commensal bacteria detected in extra-oral lesions are warranted to comprehensively investigate the role of oral bacteria in the pathogenesis of systemic diseases.

Planar catechin increases bone mass by regulating differentiation of osteoclasts in mice.

OBJECTIVES: While catechins have been reported to exhibit potential to benefit osteoporosis patients, the effects of planar catechin (PCat), synthesized during the development of drugs for Alzheimer's disease, have not been clearly elucidated. Here, we examined the effects of PCat on mouse bone metabolism both in vivo and in vitro. METHODS: Six week old female mice were orally administered PCat (30 mg/kg) every other day for four weeks, and their femurs were analyzed using micro-computed tomography imaging. Osteoclasts and osteoblasts were collected from mice and cultured with PCat. Subsequently, osteoclast formation and differentiation and osteoblast differentiation were observed. RESULTS: Mice orally administered PCat displayed significantly increased femur bone mass compared to the control group. Quantitative polymerase chain reaction findings indicated that PCat addition to osteoclast progenitor cultures suppressed osteoclast formation and decreased osteoclast marker expression without affecting the proliferative potential of the osteoclast progenitor cells. Addition of PCat to osteoblast cultures increased osteoblast marker expression. CONCLUSIONS: PCat inhibits osteoclast differentiation and promotes osteoblast differentiation, resulting in increased bone mass in mice. These results suggest that PCat administration is a promising treatment option for conditions associated with bone loss, including osteoporosis.

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state.

OBJECTIVES: Commensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions. METHODS: We used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively. RESULTS: We found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils. CONCLUSIONS: The presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

Particle Size Analysis in Aerosol-Generating Dental Procedures Using Laser Diffraction Technique.

The global outbreak of coronavirus disease 2019 (COVID-19) has raised concerns about the risk of airborne infection during dental treatment. Aerosol-generating dental procedures (AGDP) produce droplets and aerosols, but the details of the risks of COVID-19 transmission in AGDP are not well-understood. By discriminating between droplets and aerosols, we devised a method to measure particle size using laser diffraction analysis and evaluated aerosols generated from dental devices for providing a basis for proper infection control procedures. The droplets and aerosols generated from dental devices were characterized by multimodal properties and a wide range of droplet sizes, with the majority of droplets larger than 50 µm. AGDP emitted few aerosols smaller than 5 µm, which are of concern for pulmonary infections due to airborne transmission. In addition, the use of extraoral suction was found to prevent the spread of aerosols from high-speed dental engines. This study suggests that the risk of aerosol infections is considerably limited in regular dental practice and that current standard precautions, such as mainly focusing on protection against droplet and contact infections, are sufficient. While several cases of airborne transmission of COVID-19 in general clinics and emergency hospitals have been reported, cluster outbreaks in dental clinics have not yet been reported, which may indicate that AGDP does not pose a significant threat in contributing to the spread of SARS-CoV-2.

Effects of lipid metabolism on mouse incisor dentinogenesis.

Tooth formation can be affected by various factors, such as oral disease, drug administration, and systemic illness, as well as internal conditions including dentin formation. Dyslipidemia is an important lifestyle disease, though the relationship of aberrant lipid metabolism with tooth formation has not been clarified. This study was performed to examine the effects of dyslipidemia on tooth formation and tooth development. Dyslipidemia was induced in mice by giving a high-fat diet (HFD) for 12 weeks. Additionally, LDL receptor-deficient (Ldlr-/-) strain mice were used to analyze the effects of dyslipidemia and lipid metabolism in greater detail. In the HFD-fed mice, incisor elongation was decreased and pulp was significantly narrowed, while histological findings revealed disappearance of predentin. In Ldlr-/- mice fed regular chow, incisor elongation showed a decreasing trend and pulp a narrowing trend, while predentin changes were unclear. Serum lipid levels were increased in the HFD-fed wild-type (WT) mice, while Ldlr-/- mice given the HFD showed the greatest increase. These results show important effects of lipid metabolism, especially via the LDL receptor, on tooth homeostasis maintenance. In addition, they suggest a different mechanism for WT and Ldlr-/- mice, though the LDL receptor pathway may not be the only factor involved.

Association between simple evaluation of eating and swallowing function and mortality among patients with advanced dementia in nursing homes: 1-year prospective cohort study.

BACKGROUND: A simple and predictable method of evaluating eating and swallowing has not been yet established; thus, it is difficult to implement advance care planning according to deterioration in this function. This study aimed to clarify the association between a simple evaluation of eating and swallowing function and 1-year mortality in advanced dementia patients in nursing homes in Japan. METHODS: The study included 325 residents with advanced dementia. In a baseline survey, we examined medical history, physical function, and eating and swallowing function. We recorded mortality for 1 year from baseline. Kaplan-Meier survival analysis and Cox proportional regression were performed to investigate the association between the simple evaluation of eating and swallowing function and mortality. RESULTS: Statistical analysis included data from 312 of the 325 residents who had completed the baseline survey (7 individuals with non-oral ingestion and 6 who were alive but did not reside in the nursing home 1 year later were excluded). The participants' mean age was 85.2 years, and 79.5 % of participants were female. At the 1-year follow-up, 70 patients had died. According to Cox proportional regression analysis, age, male gender, history of cerebrovascular disorder, poor results of palpation of masseter muscle tension, and modified water swallowing test were significantly associated with 1-year mortality. CONCLUSION: The results of palpation of masseter muscle tension and modified water swallowing test were associated with 1-year mortality. These routine observations can predict mortality, and may thus provide evidence of the opportunity to implement advance care planning.

Dysregulation of Intestinal Microbiota Elicited by Food Allergy Induces IgA-Mediated Oral Dysbiosis.

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

Whole Blood Platelet Aggregation Test and Prediction of Hemostatic Difficulty After Tooth Extraction in Patients Receiving Antiplatelet Therapy.

When patients on antiplatelet therapy (APT) require minor invasive surgery, APT is usually continued to limit the risk of thrombosis. However, the possibility of hemostatic difficulties necessitates the monitoring of platelet aggregation to prevent unexpected bleeding. We examined whether whole blood aggregometry as a point-of-care testing (POCT) could be useful as a tool for predicting hemostatic difficulties. Sixty-five patients receiving APT and 15 patients who were not receiving APT were enrolled in the present study; all patients were scheduled to undergo a tooth extraction. Whole blood samples were obtained and were examined using multiple electrode aggregometry. The aggregometry was performed using arachidonic acid (AA), adenosine diphosphate (ADP), and thrombin receptor activating peptide. Hemostatic difficulty was defined as a need for more than 10 minutes of compression to achieve hemostasis. The AA test results were significantly lower in patients treated with aspirin (control: 97.7 [29.0] U, aspirin: 14.5 [7.2] U, P < .001). The ADP test results were also significantly lower in patients treated with a P2Y12 inhibitor (control: 77.7 [21.7] U, P2Y12 inhibitor: 37.3 [20.4] U, P < .01). Six of the examined cases exhibited hemostatic difficulties. The cutoff values for the prediction of hemostatic difficulty were 16.5 U for the AA test (sensitivity, 0.833; specificity, 0.508) and 21 U for the ADP test (sensitivity, 0.847; specificity, 0.500). Our study showed that whole blood aggregometry was useful as a POCT for the prediction of hemostatic difficulties after tooth extraction in patients receiving APT.

Thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase levels as predictive factors of chemotherapy in oral squamous cell carcinoma.

We conducted a clinicopathologic study on protein and mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) using biopsy tissue specimens before treatment. The mRNA levels have been measured in tumor cells microdissected from paraffin-embedded specimens (Danenberg Tumor Profile method: DTP method). We studied the mRNA and protein expression as effect predictive factors in chemotherapy. The subjects consisted of 20 cases of untreated oral squamous cell carcinoma who had undergone chemotherapy with TS-1 (16 males and 4 females, tongue in 8 cases, upper gingiva in 3 cases, lower gingiva in 3 cases, buccal mucosa in 5 cases and floor of the mouth in 1 case). TS gene expressions of the responders were lower than those for the nonresponders. Furthermore, regarding males who were less than 70 years of age, stage I and II, well differentiated type and tongue, TS mRNA expression of the responders were lower than that for the nonresponders. The mRNA expression of OPRT for the male responders was lower than that for the nonresponders. No remarkable difference was observed by immunohistochemistry. In this study, the measurement of the TS levels using the DTP method may potentially act as a predictive factor of antitumor effectiveness.

[Nedaplatin (NDP)-combination therapy (NDP/5-FU,NDP/S-1) for oral cancer].

PURPOSE: The present study evaluated the efficacy and safety of nedaplatin-combination therapy (NDP/5-FU [5-FU arm] or NDP/S-1 [S-1 arm] ) for the treatment of oral squamous cell carcinoma. PATIENTS AND METHOD: Previously non-treated oral squamous cell carcinoma patients were eligible. Patients received 5-FU 600 mg/m(2)iv, as a 24-hour infusion (day 1 to 5) followed by NDP 80 to 100 mg/m(2) iv (day 1), or S-1 60 to 80 mg/m(2) orally twice a day (day 1 to 14) followed by NDP 80 mg/m(2) iv (day 8) every 28 days for one or two cycles. RESULTS: In total, 32 patients (18 in the 5-FU arm, 14 in the S-1 arm) were enrolled. Twenty patients were male and 12 were female. Median age was 57 years (range 20 years to 87 years). Thirty-one patients had a performance status (PS) oF 0, and 1 patient had a PS 1. Three patients were stage I, 12 stage III, and 12 were stage IV. The overall response rate was 69% (5-FU arm,72%;S-1 arm,64%). Two patients achieved a complete response, 20 patients a partial response, and 10 patients had no change. Grade 3 leucopenia, grade 3 and 4 thrombocytopenia and liver injury occurred in 6% (one in the 5-FU arm, and one in the S-1 arm), 9% (two in the 5-FU arm, and one in the S-1 arm), and 3% (one in the 5-FU arm), respectively. No other severe toxicities were observed. RESULTS: Response rate and toxicities were similar in both arms. However, the psychosocial stress on patients in the S-1 arm was reduced compared to that in the 5-FU arm, which required hospitalization for a longer period. The outcome in the present study needs further investigation.

[Combination chemotherapy with nedaplatin (CDGP) and 5-FU for oral cancer].

Chemotherapy using CDGP plus 5-FU was evaluated in patients with oral cancer. The subjects were patients with squamous cell carcinoma of the oral cavity who had not received any therapy, comprising 7 patients with carcinoma of the tongue, 2 with buccal carcinoma, 2 with maxillary gingival carcinoma, and 1 with carcinoma of the oral floor. There were 4 patients in Stage II, 3 patients in Stage III and 5 patients in Stage IV. Patients with a PS < or = 1, WBC > or = 4,000/mm3, Hb > or = 10 g/dl, platelet count > or = 10 x 10/mm3, and normal liver, kidney, and heart function at baseline were selected for this study. In all patients, 5-FU was administered at a dose of 600 mg/m2/day for 5 days (day 1 to day 5) by continuous infusion, for a total dose of 3,000 mg/m2. CDGP was administered on day 1 at a dose of 80 mg/m2 in 8 patients and at 100 mg/m2 in 4 patients. This treatment was one course of therapy, and patients received 1 or 2 courses. Of 12 patients who were evaluable, there were 9 partial responses and 3 no changes, for a major response rate of 75%. Toxicities experienced by patients were mild (grade 2 or lower) gastrointestinal disorders (including nausea/vomiting) and renal impairment, while grade 3 leukopenia and thrombocytopenia developed in 1 patient each and grade 4 thrombocytopenia occurred in another patient. Thus, patients receiving CDGP + 5-FU therapy should be closely monitored for hematologic toxicity. Since CDGP + 5-FU therapy achieved a good response rate (75%) in the treatment of squamous cell carcinoma of the oral cavity, we plan to use this therapy in the future and assess its benefit in a larger number of patients.