The number of microvascular complications is associated with an increased risk for severity of periodontitis in type 2 diabetes patients: Results of a multicenter hospital-based cross-sectional study.

AIMS/INTRODUCTION: To explore the relationships between periodontitis and microvascular complications as well as glycemic control in type 2 diabetes patients. MATERIALS AND METHODS: This multicenter, hospital-based, cross-sectional study included 620 patients with type 2 diabetes. We compared the prevalence and severity of periodontitis between patients with ≥1 microvascular complication and those without microvascular complications. We also compared the prevalence and severity of periodontitis among patients with different degrees of glycemic control. RESULTS: After adjusting for confounding factors, multiple logistic regression analysis showed that the severity of periodontitis was significantly associated with the number of microvascular complications (odds ratio 1.3, 95% confidence interval 1.1-1.6), glycated hemoglobin ≥8.0% (64 mmol/mol; odds ratio 1.6; 95% confidence interval 1.1-2.3), and older age (≥50 years; odds ratio 1.7; 95% confidence interval 1.1-2.6). However, the prevalence of periodontitis was not significantly associated with the number of microvascular complications, but was associated with male sex, high glycated hemoglobin (≥8.0% [64 mmol/mol]), older age (≥40 years), longer duration of diabetes (≥15 years) and fewer teeth (≤25). Furthermore, propensity score matching for age, sex, diabetes duration and glycated hemoglobin showed that the incidence of severe periodontitis was significantly higher among patients with microvascular complications than among those without microvascular complications (P < 0.05). CONCLUSIONS: The number of microvascular complications is a risk factor for more severe periodontitis in patients with type 2 diabetes, whereas poor glycemic control is a risk factor for increased prevalence and severity of periodontitis.

Evaluation of postextraction bleeding incidence to compare patients receiving and not receiving warfarin therapy: a cross-sectional, multicentre, observational study.

OBJECTIVES: We investigated incidence and risk factors for postextraction bleeding in patients receiving warfarin and those not receiving anticoagulation therapy. DESIGN: Cross-sectional, multicentre, observational study. SETTING: 26 hospitals where an oral surgeon is available. PARTICIPANTS: Data on 2817 teeth (from 496 patients receiving warfarin, 2321 patients not receiving warfarin; mean age (SD): 62.2 (17.6)) extracted between 1 November 2008 and 31 March 2010, were collected. Warfarin-receiving patients were eligible when prothrombin time-international normalised ratio (PT-INR) measured within 7 days prior to the extraction was less than 3.0. INTERVENTIONS: Simple dental extraction was performed, and incidence of postextraction bleeding and comorbidities were recorded. PRIMARY AND SECONDARY OUTCOME MEASURES: Postextraction bleeding not controlled by basic haemostasis procedure was clinically significant. RESULTS: Bleeding events were reported for 35 (7.1%) and 49 (2.1%) teeth, of which 18 (3.6%) and 9 (0.4%) teeth were considered clinically significant, in warfarin and non-warfarin groups, respectively, the difference between which was 3.24% (CI 1.58% to 4.90%). The incidence rates by patients were 2.77% and 0.39%, in warfarin and non-warfarin groups, respectively (incidence difference 2.38%, CI 0.65% to 4/10%). Univariate analyses showed that age (OR 0.197, p=0.001), PT-INR (OR 3.635, p=0.003), mandibular foramen conduction anaesthesia (OR 4.854, p=0.050) and formation of abnormal granulation tissue in extraction socket (OR 2.900, p=0.031) significantly correlate with bleeding incidence. Multivariate analysis revealed that age (OR 0.126, p=0.001), antiplatelet drugs (OR 0.100, p=0.049), PT-INR (OR 7.797, p=0.001) and history of acute inflammation at extraction site (OR 3.722, p=0.037) were significant risk factors for postextraction bleeding. CONCLUSIONS: Our results suggest that there is slight but significant increase in the incidences of postextraction bleeding in patients receiving warfarin. Although absolute incidence was low in both groups, the bleeding risk is not negligible.

A toothbrush impalement injury of the floor of mouth in autism child.

Penetrating injuries in the oral cavity are common in children. However, penetrating injuries with retained foreign bodies are rare. We report a case of a toothbrush impalement injury of the floor of the mouth in a child with autism. A 5-year-old boy with autism presented with an accidentally impaled toothbrush in the oral cavity. He was taken to the operation room and examined under general anesthesia. The handle of the toothbrush was cut off using rib scissors for mask ventilation, and intra-oral intubation was performed. The toothbrush was located approximately 2.5 cm into the floor of the mouth. The toothbrush was removed uneventfully. Intravenous antibiotic therapy was instituted during hospitalization, and discharge from the hospital occurred 4 days after the operation.

PLGA artificial nerve conduits with dental pulp cells promote facial nerve regeneration.

A number of recent studies have shown the effectiveness of tubulation, using neural progenitor cells or Schwann cells, for promoting nerve regeneration. However, the use of neural cells from other neural donor tissues has potentially serious clinical complications. Therefore, we focused on dental pulp as a new cell source for use in such artificial conditions. Previously, we showed that silicone tubes filled with dental pulp cells (DPCs) promoted facial nerve regeneration in rats. However, the use of silicone tubes requires a secondary removal operation because they may give rise to chronic inflammation and pain. Therefore, to avoid this procedure, a new artificial device was prepared from a degradable poly-DL-lactide-co-glycolide (PLGA) tube containing DPCs, and its effectiveness for repairing gaps in the facial nerves of rats was investigated. A PLGA tube containing rat DPCs embedded in a collagen gel was transplanted into a gap in a rat facial nerve. Five days after transplantation, the facial nerves connected by the PLGA tubes containing DPCs were repaired more quickly than the control nerves. The PLGA tubes were resorbed in vivo and nerve regeneration was observed 2 months after the transplantation. Immunostaining showed that Tuj1-positive axons were present in the regenerated nerves 2 months after transplantation, and osmium-toluidine blue staining showed no mineralization of the regenerated nerves in those tubes containing myelinated fibres after 9 weeks. PLGA tubes filled with DPCs promoted nerve regeneration and were readily resorbed in vivo.

In vivo 3D analysis with micro-computed tomography of rat calvaria bone regeneration using periosteal cell sheets fabricated on temperature-responsive culture dishes.

Transplantable cell sheets containing osteoblasts were fabricated from periostea on temperature-responsive culture dishes. This study demonstrated the time-course of bone regeneration in living small animals. This continuous observation of bone regeneration was achieved by micro-computed tomography (µCT), which assessed the osteogenic capability of periosteal cells without biodegradable scaffolds. Real-time bone regeneration was non-invasively monitored in a rat calvarial bone defect model, using µCT. Three-dimensional (3D) images obtained over time by µCT clearly showed that two different bone regeneration modes, specific to the control and experimental groups, were observed. In the control group, bone was regenerated only from the periphery of the defect edges. In the experimental group, bone regeneration was observed in several small regions within the central portions of the defects that were covered by the transplanted cell sheets. However, bone regeneration observed after periosteal cell sheet transplantation was limited. The results of ALP staining and the time-course observations concluded that periosteal cell sheets contained a small fraction of cells that could differentiate osteoblasts. Fibroblasts in transplanted cell sheets or from around subcutaneous tissues suppressed bone regeneration. The periosteal cell sheets had a capability to produce ectopic regenerated bones. Therefore, to increase the content of osteogenic cells in harvested cell sheets, the enrichment of cells that could produce osteoblasts was expected by the modification of the initial cell preparation and the culture conditions. With further possible improvements, scaffold-free periosteal cell sheet fabricated on temperature-responsive culture dishes will be a valuable method for inducing and accelerating bone regeneration.

A protocol for immunofluorescence staining of floating neurospheres.

This protocol describes the immunofluorescence staining of floating neurospheres in culture plates. Although this protocol is similar to conventional immunofluorescence staining, the staining procedure of floating neurospheres in multiwell culture plates and the washing procedure are different. Neurospheres in culture plates are transferred to a 12-well plate using a 200-1000microL pipette. The spheres are precipitated by gravity for 3min. Then, the 12-well plate is tilted slightly, and the culture medium is aspirated by the pipette. After aspiration, the spheres are visually verified to be at the bottom of the wells. PBS (400microL) is added to the well for washing the spheres. This procedure is repeated three times. This protocol is easier than a conventional procedure using cryostat sections and can give clear sphere structures.

Irsogladine is effective for recurrent oral ulcers in patients with Behçet's disease : an open-label, single-centre study.

UNLABELLED: BACKGROU nd and objective: Behçet's disease (BD) is a polysymptomatic condition characterized by recurrent oral and genital ulceration and uveitis. Aphthous stomatitis, a common complication, is painful, recurrent and sometimes resistant to treatment. Topical or intralesional corticosteroids and local anaesthetics are used for palliative therapy. We investigated whether irsogladine, a drug for the treatment of gastritis and peptic ulcers, reduced aphthous stomatitis lesions in patients with BD. METHODS: Irsogladine 2-4 mg/day was administered orally to ten BD patients (cases 1-10), three men and seven women, with a mean age of 48.5 years, with recurrent aphthous stomatitis as the main symptom. Pre-existing treatments were not changed. All patients were followed up at our outpatient clinic once a month. The patients had no systemic neurological, gastrointestinal or vascular symptoms. Efficacy was evaluated on the basis of the macroscopic findings of aphthous lesions. We counted the number of aphthous lesions three times prior to administration of irsogladine and three times after treatment at the outpatient clinic, i.e. six times in total, and compared pre- and post-treatment mean numbers of lesions for each patient. RESULTS: Irsogladine was effective in all ten patients. The mean aphthous ulcer count decreased in all patients 3 months after administration (p < 0.0003). Cases 5 and 6 stopped taking irsogladine of their own accord when the stomatitis disappeared; however, the stomatitis reappeared, and in both patients, aphthous stomatitis healed completely soon after re-administration of irsogladine. In case 8, aphthous stomatitis reappeared 2 months after administration of irsogladine 2 mg/day. The dose of irsogladine in this patient was then increased to 4 mg/day, after which aphthous stomatitis resolved. Taking irsogladine continuously prevented recurrence of stomatitis in these three patients. CONCLUSION: Irsogladine reduces aphthous stomatitis/oral ulcers in patients with BD. The improvement in gap-junctional intercellular communication by irsogladine may contribute to the treatment of aphthous stomatitis in patients with BD. Increasing the dose of irsogladine may resolve ulcers resistant to low doses of irsogladine.

Tubulation with dental pulp cells promotes facial nerve regeneration in rats.

Dental pulp is an easily obtainable source of viable cells for potential use in peripheral nerve regeneration. We prepared artificial conditions for nerve regeneration using a silicone tube containing a collagen gel embedded with rat dental pulp cells, and we examined its effectiveness for repairing a gap in the rat facial nerve. Twelve days after transplantation, defective facial nerves connected with silicone tubes containing dental pulp cells were repaired more rapidly than control tubes containing the collagen gel alone. When a tube containing green fluorescent protein (GFP)-positive dental pulp cells was transplanted into a facial nerve gap in a GFP-negative rat, we observed regenerated nerves with GFP-positive cells at 2 weeks posttransplantation. The regenerated nerves included Tuj1-positive axons, RECA1 and GFP double-positive blood vessels, and S100 and GFP double-positive Schwann-like supportive cells. Osmium-toluidine blue staining revealed that the regenerated nerves contained myelinated fibers. Moreover, fluorescent retrograde tracing analysis by application of Fluoro-Gold into the regenerated nerves demonstrated the presence of Fluoro-Gold-positive motor neurons in the facial nucleus of the rat brain. These results suggest that the transplanted dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of normal nerve regeneration.

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.

Possible involvement of T cell co-stimulation in pustulosis palmaris et plantaris via the induction of inducible co-stimulator in chronic focal infections.

BACKGROUND: Inducible co-stimulator (ICOS) is a co-stimulatory receptor on activated T cells that provides the signals needed for Th1 and Th2 responses via its interaction with B7h. Chronic focal infections are closely related to pustulosis palmaris et plantaris (PPP), but the involvement of ICOS in PPP has not been clarified. OBJECTIVE: To investigate the effectiveness of treatments for focal infections on PPP skin lesions and the involvement of ICOS-positive T cells at focal infection sites in the tonsils and in PPP lesional skin. METHODS: In patients that had undergone a tonsillectomy or dental treatment, the clinical activities of PPP, both the skin lesions and pustulotic arthro-osteitis were followed for over 2 years. The expressions of ICOS and various other activation markers on T cells were examined in tonsil tissue from both PPP patients and non-PPP patients, and the expression levels in peripheral blood were also evaluated in PPP patients and healthy donors. ICOS-positive T cells and B7h expression in PPP and normal skin were examined immunohistochemically. RESULTS: The above treatments for focal infections led to a dramatic and persistent improvement in the PPP skin lesions and pustulotic arthro-osteitis. The expression of ICOS, but not of other activation markers, was higher in tonsil tissues from PPP patients than in tonsil tissues from non-PPP patients. B7h was upregulated without numerous ICOS-positive T cell infiltrates in the skin lesions. CONCLUSION: The activation of T cells via ICOS co-stimulation in focal infections likely triggers the skin and skeletal inflammation associated with PPP, resulting in tissue damage.

Neurosphere generation from dental pulp of adult rat incisor.

Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nervous system disorders. Here we report that adult rat dental pulp cells have the ability to form neurospheres when cultured in serum-free culture medium on super-hydrophilic plates. The cells within small spheres continued to grow, and the dental pulp-derived cells generated large spheres. Sphere formation was dependent on exogenously supplied basic-fibroblast growth factor, but not on epidermal growth factor, and the formation and growth of dental pulp-derived spheres were negatively regulated by transforming growth factor-beta. Plating cells that were dissociated from spheres on an adhesive substrate resulted in differentiation into Tuj1- and MAP2-positive neuronal cells. Analysis of the three-dimensional structure of dental pulp-derived spheres shows that they contained nestin-positive progenitors, Tuj1-positive neuronal cells and S100-positive glial cells. We found that spheres contained CD81 (TAPA1) and nestin double-positive cells, and identified a small population of CD81 and nestin double-positive cells in the odontoblast layer of the dental pulp. Flow cytometric analysis showed that CD81-positive cells were enriched in the spheres compared with the dental pulp tissue. Bromodeoxyuridine (BrdU) staining showed that nestin- and BrdU-positive cells were located only in the apical portion of the dental pulp, and the apical portion produced a large number of large-sized spheres. These data suggest that the CD81 and nestin double-positive cells localized in the odontoblast layer of the apical portion of the dental pulp may have the ability to grow and form neurospheres.

[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.

CD28 is required for induction and maintenance of immunological memory in toxin-reactive CD4+ T cells in vivo.

We previously reported that Vbeta3+ CD4+ T cells maintained a protracted expansion, with the phenotypes of memory Th2 cells, for 30 days in C57BL/6 (B6) mice implanted with SEA-containing mini-osmotic pumps. In the present study, we followed the fate of Vbeta3+ CD4+ T cells in CD28-/- mice. Vbeta3+ CD4+ T cells increased to a degree similar to that of B6 Vbeta3+ CD4+ T cells until day 10 after implantation, then declined rapidly reaching the control level by 28 days. Remaining Vbeta3+ CD4+ T cells at that time did not exhibit memory phenotypes nor Th2-deviated responses. The rapid drop in Vbeta3+ CD4+ T cells in CD28-/- mice was attributable to upregulated induction of apoptosis owing to marginal inductions of Bcl-2 and Bcl-xL. Collectively, these data indicate CD28 to play critical roles in the generation and maintenance of SEA-reactive CD4+ T cells in vivo.

Expression and function of the co-stimulator H4/ICOS on activated T cells of patients with rheumatoid arthritis.

OBJECTIVE: To investigate the expression and function of the inducible co-stimulator H4/ICOS in rheumatoid arthritis (RA) patients. H4/ICOS is the newest member of the CD28/CTLA-4 family to have been found to be expressed on activated T cells, and it participates in a variety of important immunoregulatory functions. METHODS: The levels of H4/ICOS expression on T cells among peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from 28 patients with RA were analyzed by flow cytometry. To explore the role of H4/ICOS function in the inflammation of rheumatoid joints, lymphokine production by SF CD4+ T cells co-stimulated by H4/ICOS was assayed. Expression of H4/ICOS ligand (B7RP-1) mRNA in synovial tissues from patients with RA was examined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: H4/ICOS-positive cells were increased significantly in whole, CD4+, and CD8+ T-cell fractions of SFMC compared with control PBMC. Comparison between control PB and PB from patients with active RA showed that H4/ICOS-positive whole and CD8+ T-cell fractions were increased significantly in the PB of RA patients. H4/ICOS costimulation clearly increased interferon-g, interleukin 4 (IL-4), and IL-10 production by SF CD4+ T cells. By RT-PCR, RA synovial tissue was shown to express mRNA of B7RP-1. CONCLUSION: Our results suggest that local immune responses may be modulated by H4/ICOS expressed on T cells in the joints of patients with RA, and thus H4/ICOS may be involved in the pathogenetic mechanism of RA.

[Diagnosis of the autonomic nervous function during sedation by peripheral blood flow and psychogenic perspiration].

BACKGROUND: Pain induces a variety of physiological responses, many of which are mediated by the sympathetic nervous system. Among these are a reduction in peripheral blood flow and evaporative cutaneous water loss (sweating). We therefore tested the hypothesis that adequate sedation obliterates the normal pain-induced reduction in peripheral blood flow and an increase in evaporative water loss. METHODS: We studied eight volunteers. Two different painful stimuli were randomly applied: 1) electrical pulp stimulation (200 microamperes) and 2) electrical pain stimulation on the right upper thigh (80 mA). Conscious sedating was controlled by propofol infusion titrated to a Bispectral Index near 80, or near 60. RESULTS: At each stimulation, peripheral blood flow detected by laser Doppler decreased without any relation to the level of consciousness (by Bispectral Index). On the other hand, although the psychogenic perspiration rate increased significantly at alert level, during BIS 80 or 60 level, the increase was not significant. CONCLUSION: Peripheral blood flow reacts most to pain stimulation during intravenous sedation.

[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.