Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Effects of green tea extract combined with brisk walking on lipid profiles and the liver function in overweight and obese men: A randomized, double-blinded, placebo-control trial.

This study was aimed to investigate the effect of green tea extract (GTE) combined with brisk walking on lipid profiles and the liver function in overweight and obese men. Twenty-four participants were randomized to either the GTE group or the placebo group for 12 weeks with a 4-week follow-up. The walking program consisted of four 60-min-sessions/week and all participants were asked to consume two GTE (150mg) or placebo tablets daily. After 12-week intervention, GTE group resulted in a significant difference in the low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels when compared to placebo group (P < 0.01). There was also a significant reduction in the aspartate aminotransferase levels (P < 0.01) in the GTE group, but no change in the placebo group (P >0.05). There was no change in the triglyceride or high-density lipoprotein cholesterol (HDL-C) levels in the placebo group, but a significant reduction was noted in the HDL-C levels in the GTE group (P < 0.05). GTE combined with brisk walking resulted in a significant change in the LDL-C and TC levels, however, a significant reduce in HDL-C in the GTE group. The study has a more positive effect on the liver function than brisk walking alone.

Pulmonary Alveolar Microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a genetic lung disorder that is characterized by the accumulation of calcium phosphate deposits in the alveolar spaces of the lung. Mutations in the type II sodium phosphate cotransporter, NPT2b, have been reported in patients with PAM. PAM progresses gradually, often producing incremental dyspnea on exertion, desaturation in young adulthood, and respiratory insufficiency by late middle age. Treatment remains supportive, including supplemental oxygen therapy. For patients with end-stage disease, lung transplantation is available as a last resort. The recent development of a laboratory animal model has revealed several promising treatment approaches for future trials.

Adjunct Antimicrobial Therapy and Periodontal Surgery to Treat Generalized Aggressive Periodontitis: A Case Report.

Here we report a case of generalized aggressive periodontitis treated with periodontal therapy including adjunct antimicrobial therapy and periodontal surgery. The patient was a 22-year-old woman who presented with the chief complaint of gingival recession. Baseline examination revealed generalized plaque deposition and gingival inflammation. Thirty-nine percent of the sites had a probing depth (PD) of 4-6 mm and 2% a PD of ≥7 mm; 63% exhibited bleeding on probing (BOP). Radiographic examination revealed vertical bone loss in the molars and horizontal bone loss in other teeth. Microbiological examination of subgingival plaque revealed the presence of Aggregatibacter actinomycetemcomitans and Tannerella forsythia. Oral health-related quality of life was assessed as a measure of patient-reported outcome. Based on a clinical diagnosis of generalized aggressive periodontitis, initial periodontal therapy and adjunct antimicrobial therapy were implemented. After reducing inflammation and subgingival bacteria, open flap debridement was performed for teeth with a PD of ≥4 mm. Reevaluation showed no sites with a PD of ≥5 mm, a minimal level of BOP, and a marked reduction in the level of the targeted periodontal pathogens. The patient's oral health-related quality of life was slightly worsened during supportive periodontal therapy (SPT). Implementation of adjunct antimicrobial therapy targeting periodontal pathogens and subsequent periodontal surgery resulted in improvement in periodontal and microbiological parameters. This improvement has been adequately maintained over a 2-year period. However, additional care is necessary to further improve the patient's oral health-related quality of life during SPT.

In vitro Effect of Mouthrinse Containing Essential Oils on Proliferation and Migration of Gingival Epithelial Cells.

We aimed to investigate in vitro the effects of mouthrinses containing essential oils (EOs) on proliferation and migration of gingival epithelial cells. Human gingival epithelial cells were treated with predetermined dilutions of commercially available EO mouthrinses with or without ethanol and a mouthrinse containing cetyl pyridinium chloride (CPC) for 60 s. Cell proliferation was evaluated using WST-1 assay. Cell migration was assessed using a wound closure model. Within 10 s of exposure to EO mouthrinse without ethanol, the epithelial cells became aberrant and shrank. No statistically significant difference in cell migration or proliferation was observed among cells pretreated by the EO mouthrinse with ethanol, CPC mouthrinse and control (phosphate buffered saline). In contrast, the EO mouthrinse without ethanol significantly reduced cell proliferation (p < 0.001) to approximately 20% relative to control. As for the EO mouthrinse without ethanol, it was not possible to assess its effect on cell migration using this model, because treated cells could be easily detached from the culture plate upon scratch, possibly because of the surfactant ingredient in the formulation. Within the limitations of the study, the EO mouthrinse with ethanol exerted no inhibitory effect on proliferation and migration of the gingival epithelial cells. Copyright © 2016 John Wiley & Sons, Ltd.

Modeling pulmonary alveolar microlithiasis by epithelial deletion of the Npt2b sodium phosphate cotransporter reveals putative biomarkers and strategies for treatment.

Pulmonary alveolar microlithiasis (PAM) is a rare, autosomal recessive lung disorder associated with progressive accumulation of calcium phosphate microliths. Inactivating mutations in SLC34A2, which encodes the NPT2b sodium-dependent phosphate cotransporter, has been proposed as a cause of PAM. We show that epithelial deletion of Npt2b in mice results in a progressive pulmonary process characterized by diffuse alveolar microlith accumulation, radiographic opacification, restrictive physiology, inflammation, fibrosis, and an unexpected alveolar phospholipidosis. Cytokine and surfactant protein elevations in the alveolar lavage and serum of PAM mice and confirmed in serum from PAM patients identify serum MCP-1 (monocyte chemotactic protein 1) and SP-D (surfactant protein D) as potential biomarkers. Microliths introduced by adoptive transfer into the lungs of wild-type mice produce marked macrophage-rich inflammation and elevation of serum MCP-1 that peaks at 1 week and resolves at 1 month, concomitant with clearance of stones. Microliths isolated by bronchoalveolar lavage readily dissolve in EDTA, and therapeutic whole-lung EDTA lavage reduces the burden of stones in the lungs. A low-phosphate diet prevents microlith formation in young animals and reduces lung injury on the basis of reduction in serum SP-D. The burden of pulmonary calcium deposits in established PAM is also diminished within 4 weeks by a low-phosphate diet challenge. These data support a causative role for Npt2b in the pathogenesis of PAM and the use of the PAM mouse model as a preclinical platform for the development of biomarkers and therapeutic strategies.

Effect of collaborative intervention by medical and dental professionals on adherence to smoking abstinence.

The need for smoking cessation care is widely recognized. It is, however, difficult to achieve continued smoking abstinence, even when cessation has initially been achieved. The aim of this study was to determine the effectiveness of a collaborative smoking cessation program involving both medical and dental professionals on smoking abstinence. A total of 10 patients visiting our Smoking Cessation Outpatient Clinic were followed up and monitored for smoking abstinence. All received smoking cessation care consisting mainly of counseling by dental and medical professionals and pharmacotherapy. They also concurrently received an oral examination, instruction on oral hygiene, and professional tooth cleaning. The 4-week smoking abstinence rate was 90.0% on completion of the program. One patient failed to complete the program. At one month after the program, 8 out of 9 patients remained abstinent (4-month abstinence; 88.9%). At 3 months after the program, 7 patients remained abstinent (6-month abstinence; 77.8%). Follow-up was impossible in one patient. Within the limitations of the present study, it is suggested that such collaborative intervention including subsequent dental care has the potential to promote short-term adherence to smoking abstinence.

Changes in antimicrobial susceptibility profile and prevalence of quinolone low-sensitive strains in subgingival plaque from acute periodontal lesions after systemic administration of sitafloxacin.

This study aimed to assess changes in antimicrobial susceptibilities of subgingival bacteria in acute periodontal lesions following systemic administration of a new-generation fluoroquinolone, sitafloxacin and to monitor the occurrence and fate of quinolone low-sensitive strains. Patients with acute phase of chronic periodontitis were subjected to microbiological assessment of their subgingival plaque samples at baseline (A1). Sitafloxacin was then administered systemically (100 mg/day for 5 days). The microbiological examinations were repeated one week after administration (A2). Susceptibilities of clinical isolates from acute sites to various antimicrobials were determined using broth and agar dilution methods. At A2, subgingival bacteria with low sensitivity to levofloxacin were identified in four patients, and they were subjected to a follow-up microbiological examination at on the average 12 months after sitafloxacin administration (A3). The patients received initial and supportive periodontal therapy during the period A2 to A3. From the examined subgingival sites, 8 and 19 clinical isolates were obtained at A2 and A3, respectively. Some Streptococcus strains isolated at A2 were found to be resistant to levofloxacin (MIC 16-64 µg/ml), azithromycin (MIC 2->128 µg/ml) or clarithromycin (MIC 1->32 µg/ml). At A3, isolated streptococci were highly susceptible to levofloxacin (MIC 0.5-2 µg/ml), while those resistant to azithromycin or clarithromycin were still isolated. It is suggested that the presence of the quinolone low-sensitive strains in initially acute lesions after sitafloxacin administration was transient, and they do not persist in the subgingival milieu during the periodontal therapy.

A case of difficult management of fluid-electrolyte imbalance in choroid plexus papilloma.

A 22-month-old boy presented with nausea and gradual deterioration of gait disturbance. Computed tomography (CT) demonstrated an intraventricular mass lesion in the right lateral ventricle. He was referred to our department 3 weeks after onset. Acute hydrocephalus gradually proceeded 4 days after admission, and external ventricular drainage (EVD) was performed. EVD revealed cerebrospinal fluid (CSF) overproduction (800-1,500 mL/day) under constant pressure of 10 cm H2O above external auditory meatus. Magnetic resonance imaging showed a multi-lobular mass in the inferior horn of the right lateral ventricle. A choroid plexus tumor was suspected. The ratio of blood urea nitrogen:creatinine (BUN:Cre) remained between 30 and 40, and hemoglobin was between 14.0-17.0 mg/dL, suggesting marked dehydration. Serum sodium varied between 117 and 140 mmol/L, and serum potassium between 2.2 mmol/L and 6.9 mmol/L. The amount of EVD was unstable and fluid balance management was difficult. Hypotonic fluid with sodium chloride supplement was used to adjust the fluid and electrolyte imbalance. Surgical removal of the tumor was performed 6 days after EVD and tumor was grossly and totally removed. The high BUN:Cre ratio decreased to about 15 and hemoglobin recovered to 7.5-9.0 mg/dL after removal. Electrolytes returned to the normal range. Overproduction of CSF also markedly improved to < 300 mL/day. Histopathological examination diagnosed choroid plexus papilloma.We experienced a case of choroid plexus papilloma associated with fluid-electrolyte imbalance due to over-drainage after EVD, which could not be effectively controlled before tumor removal. Cautious fluid management and emergent surgical resection might be required to manage the overproduction of CSF and fluid-electrolyte imbalance.

Endocannabinoid system: potential novel targets for treatment of schizophrenia.

Accumulating epidemiological evidences suggest that cannabis use during adolescence is a potential environmental risk for the development of psychosis, including schizophrenia. Consistently, clinical and preclinical studies, using pharmacological approaches and genetically engineered animals to target endocannabinoid signaling, reveal the multiple varieties of endocannabinoid system-mediated human and animal behaviors, including cognition and emotion. Recently, there has been substantial progress in understanding the molecular mechanisms of the endocannabinoid system for synaptic communications in the central nervous system. Furthermore, the impact of endocannabinoid signaling on diverse cellular processes during brain development has emerged. Thus, although schizophrenia has etiological complexities, including genetic heterogeneities and multiple environmental factors, it now becomes crucial to explore molecular pathways of convergence of genetic risk factors and endocannabinoid signaling, which may provide us with clues to find novel targets for therapeutic intervention. In this review, epidemiological, clinical, and pathological evidences on the role of the endocannabinoid system in the pathophysiologies of schizophrenia will be presented. We will also make a brief overview of the recent progress in understanding molecular mechanisms of the endocannabinoid system for brain development and function, with particular focus on cannabinoid receptor type 1 (CB1R)-mediated cascade, the most well-characterized cannabinoid receptor. Lastly, we will discuss the potential of the endocannabinoid system in finding novel therapeutic targets for prevention and treatment of schizophrenia.

Phase II trial of radiotherapy after hyperbaric oxygenation with multiagent chemotherapy (procarbazine, nimustine, and vincristine) for high-grade gliomas: long-term results.

PURPOSE: To analyze the long-term results of a Phase II trial of radiotherapy given immediately after hyperbaric oxygenation (HBO) with multiagent chemotherapy in adults with high-grade gliomas. METHODS AND MATERIALS: Patients with histologically confirmed high-grade gliomas were administered radiotherapy in daily 2 Gy fractions for 5 consecutive days per week up to a total dose of 60 Gy. Each fraction was administered immediately after HBO, with the time interval from completion of decompression to start of irradiation being less than 15 minutes. Chemotherapy consisting of procarbazine, nimustine, and vincristine and was administered during and after radiotherapy. RESULTS: A total of 57 patients (39 patients with glioblastoma and 18 patients with Grade 3 gliomas) were enrolled from 2000 to 2006, and the median follow-up of 12 surviving patients was 62.0 months (range, 43.2-119.1 months). All 57 patients were able to complete a total radiotherapy dose of 60 Gy immediately after HBO with one course of concurrent chemotherapy. The median overall survival times in all 57 patients, 39 patients with glioblastoma and 18 patients with Grade 3 gliomas, were 20.2 months, 17.2 months, and 113.4 months, respectively. On multivariate analysis, histologic grade alone was a significant prognostic factor for overall survival (p < 0.001). During treatments, no patients had neutropenic fever or intracranial hemorrhage, and no serious nonhematologic or late toxicities were seen in any of the 57 patients. CONCLUSIONS: Radiotherapy delivered immediately after HBO with multiagent chemotherapy was safe, with virtually no late toxicities, and seemed to be effective in patients with high-grade gliomas.

Effectiveness of weekly percutaneous maxacalcitol injection therapy in patients with secondary hyperparathyroidism.

Percutaneous injection therapy with vitamin D has been applied in the treatment of hyperparathyroidism (HPT); however, the application of percutaneous injection therapy with vitamin D lacks established guidelines regarding the volume of injected solution and the frequency of injection. We have developed an outpatient treatment regimen using percutaneous maxacalcitol injection therapy (PMIT) on a weekly basis for 4-6 weeks following dialysis without major complications. Intact parathyroid hormone decreased from 797 +/- 178 pg/mL to 253 +/- 25 pg/mL, and the parathyroid gland volume initially increased during the first week, but thereafter, it gradually decreased with weekly PMIT (wPMIT). Finally, the parathyroid gland volume decreased from 1.27 +/- 1.06 cm(3) to 0.24 +/- 0.15 cm(3) after wPMIT. The benefits of our method were confirmed on weekly ultrasonographic examinations, which detailed the gradual reduction in gland size following an initial increase after the first injection. Therefore, we conclude that our carefully implemented PMIT method would be an effective treatment against refractory secondary HPT.

Increased expression of a proline-rich Akt substrate (PRAS40) in human copper/zinc-superoxide dismutase transgenic rats protects motor neurons from death after spinal cord injury.

The serine-threonine kinase, Akt, plays an important role in the cell survival signaling pathway. A proline-rich Akt substrate, PRAS40, has been characterized, and an increase in phospho-PRAS40 (pPRAS40) is neuroprotective after transient focal cerebral ischemia. However, the involvement of PRAS40 in the cell death/survival pathway after spinal cord injury (SCI) is unclear. Liposome-mediated PRAS40 transfection was performed to study whether overexpression of pPRAS40 is neuroprotective. We further examined the expression of pPRAS40 after SCI by immunohistochemistry and Western blot using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats and wild-type (Wt) littermates. We then examined the relationship between PRAS40 and Akt by injection of LY294002, a phosphatidylinositol 3-kinase (PI3K) pathway inhibitor, or Akt inhibitor IV, a compound that inhibits Akt activation after SCI. Our data demonstrated that increased pPRAS40 resulted in survival of more motor neurons compared with control complementary DNA transfection. Phosphorylated PRAS40 increased in the Wt rats after SCI, whereas there was a greater and prolonged increase in the SOD1 Tg rats. Coimmunoprecipitation showed that binding of pPRAS40 with 14-3-3 increased 1 day after SCI in the Wt rats, whereas there was a significant increase in the Tg rats. The inhibitor studies showed that phospho-Akt and pPRAS40 were decreased after injection of LY294002 or Akt inhibitor IV. We conclude that an increase in pPRAS40 by transfection after SCI results in survival of motor neurons, and overexpression of SOD1 in the Tg rats results in an increase in endogenous pPRAS40 and a decrease in motor neuron death through the PI3K/Akt pathway.

Superoxide dismutase 1 overexpression reduces MCP-1 and MIP-1 alpha expression after transient focal cerebral ischemia.

Proinflammatory cytokines and chemokines are quickly upregulated in response to ischemia/reperfusion (I/R) injury; however, the relationship between I/R-induced oxidative stress and cytokine/chemokine expression has not been elucidated. We investigated the temporal profile of cytokine and chemokine gene expression in transient focal cerebral ischemia using complementary DNA array technology. Among 96 genes studied, 10, 4, 11, and 5 genes were increased at 6, 12, 24, and 72 h of reperfusion, respectively, whereas, 4, 11, 8, and 21 genes, respectively, were decreased. To clarify the relationship between chemokines and oxidative stress, we compared the gene and protein expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in wild-type (WT) mice and copper/zinc-superoxide dismutase (SOD 1) transgenic (Tg) mice. Monocyte chemoattractant protein-1 and MIP-1 alpha mRNA were significantly upregulated at 6 to 12 h of reperfusion. In the SOD 1 Tg mice, however, MCP-1 and MIP-1 alpha mRNA expression was significantly decreased 12 h postinsult. In the WT mice, MCP-1 and MIP-1 alpha protein expression peaked 24 h after onset of reperfusion determined by immunohistochemistry. In the SOD 1 Tg mice, MCP-1 and MIP-1 alpha immunopositive cells were reduced, as were concentrations of these proteins (measured by enzyme-linked immunosorbent assay) at 24 h of reperfusion. Our results suggest that MCP-1 and MIP-1 alpha expression is influenced by I/R-induced oxidative stress after transient focal stroke.

In vitro and in vivo activity of olamufloxacin (HSR-903) against Legionella spp.

The activity of the fluoroquinolone olamufloxacin (HSR-903) against Legionella spp. was studied in vitro and in vivo. The olamufloxacin MIC at which 50% of isolates are inhibited (MIC50) for 81 different Legionella spp. strains (59 type strains and 22 clinical isolates) was 0.008 mg/L, which was identical to sparfloxacin, whereas the MIC50s for erythromycin, levofloxacin and ciprofloxacin were 0.25, 0.032 and 0.032 mg/L, respectively. Olamufloxacin and sparfloxacin (at 0.008 mg/L) inhibited intracellular growth and subsequent cytotoxicity of L. pneumophila 80-045 in J774.1 macrophages, whereas levofloxacin and ciprofloxacin did not, at the same concentration. When olamufloxacin was given to the infected guinea pigs orally (5 mg/kg of body weight), peak levels in the lung were 3.02 mg/kg at 2 h post-administration, with a half-life of 3.41 h and an AUC0-12 of 12.31 mg.h/kg. The 2 day post-infection bacterial burden of the lung in the animals treated with olamufloxacin (5 and 1.25 mg/kg given orally twice a day) was much lower than in those treated with levofloxacin (same dose as olamufloxacin) or erythromycin (10 mg/kg given orally twice a day). When treated with olamufloxacin (5 mg/kg given orally twice a day) for 7 days, 11 of 12 L. pneumophila-infected guinea pigs survived for 14 days post-infection, as did all 12 guinea pigs treated with levofloxacin (5 mg/kg given orally twice a day) for 7 days. In contrast, only two of 12 animals treated with erythromycin survived and 10 of 11 died in the physiological saline group. Olamufloxacin was as effective as levofloxacin in a guinea pig model of Legionnaires' disease. These data warrant further study of whether olamufloxacin is an option for the treatment of Legionella infections.

Prospective trial of radiotherapy after hyperbaric oxygenation with chemotherapy for high-grade gliomas.

Twenty-one patients with high-grade gliomas were enrolled in a prospective trial of radiotherapy after hyperbaric oxygenation (HBO). Radiotherapy was administered in daily 2-Gy fractions up to a total dose of 60 Gy, and each fraction was delivered immediately after HBO. The current study indicated that radiotherapy immediately after HBO with chemotherapy was feasible for high-grade gliomas.

[Postoperative analgesia with morphine with or without diclofenac after shoulder surgery].

Balanced analgesia using a narcotic and a nonsteroidal anti-inflammatory drug has been successfully tested for postoperative analgesia. This study was designed to examine the efficacy of such combination therapy after shoulder surgeries. Twenty ASA physical status I or II patients, scheduled for shoulder surgeries under general anesthesia, were randomly assigned to either morphine (M) group (n = 10), who received IV morphine patient-controlled analgesia (PCA) alone (2 mg as a bolus, lock-out interval of 10-minutes, and 10 mg as 1-hour limit for 48 hours), or morphine + diclofenac (M + D) group (n = 10), who received, in addition to morphine PCA, diclofenac suppositories 50 mg.8 h-1 starting immediately before surgical incision for 48 hours. Postoperative analgesic profiles, such as visual analog scale (VAS) at rest and on movement, and cumulative morphine consumption, the incidence and extent of side effects (nausea, vomiting, and time till the first bowel movement), and other complications were recorded. The two groups were similar demographically. Patients in the M + D group required 15.1 +/- 9.0 mg of morphine within 48 hours after surgery, while those in the M group required 30.5 +/- 21.0 mg of morphine (P < 0.05). No significant differences in VAS at rest and on movement were observed between the two groups. The time till the first bowel movement was significantly shorter in the M + D group. Our data suggest that diclofenac suppositories 50 mg.8 h-1 starting immediately before surgery for 48 h are effective adjuvant in reducing post-shoulder surgery morphine requirement and retardation of bowel movement.