Bilirubin reversibly affects cell death and odontogenic capacity in stem cells from human exfoliated deciduous teeth.

OBJECTIVE: Hyperbilirubinemia in patients with biliary atresia causes deciduous tooth injuries such as green pigmentation and dentin hypoplasia. In patients with biliary atresia who received liver transplantation, tooth structure appears to be recovered radiographically. Nevertheless, little is known about cellular mechanisms underlying bilirubin-induced damage and suppression of deciduous tooth formation. In this study, we examined the effects of bilirubin in stem cells from human exfoliated deciduous teeth (SHED) in vitro. MATERIALS AND METHODS: SHED were cultured under exposure to excess of bilirubin and then interruption of bilirubin stimulation. RESULTS: Bilirubin induced cell death and inhibited the odontogenic capacity of SHED by suppressing AKT and extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathways and enhancing nuclear factor kappa B p65 (NF-κB p65) pathway. The interruption of bilirubin stimulation reduced cell death and recovered the inhibited odontogenic capacity of bilirubin-damaged SHED. The bilirubin interruption also normalized the impaired AKT, ERK1/2, and NF-κB p65 signaling pathways. CONCLUSION: These findings suggest that tooth hypodontia in patients with hyperbilirubinemia might be due to bilirubin-induced cell death and dentinogenic dysfunction of odontogenic stem cells via AKT, ERK1/2, and NF-κB pathways and also suggested that bilirubin-induced impairments in odontogenic stem cells were reversible when bilirubin stimulation is interrupted.

Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-κB pathways in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis. MATERIAL AND METHODS: HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting. RESULTS: AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72 hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression. CONCLUSION: AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.

Development of novel treatment with a bioabsorbable esophageal patch for benign esophageal stricture.

Using a large animal model, we examined whether circumferential stricture after esophageal endoscopic submucosal dissection (ESD) can be treated by grafting a bioabsorbable esophageal patch. Circumferential ESD was performed on the thoracic esophagus in pigs (n = 6) to create a stricture, for which one of the following interventions was performed: (1) the stricture site was longitudinally incised, and an artificial esophageal wall (AEW) was grafted after placing a bioabsorbable stent (AEW patch group, n = 3); (2) endoscopic balloon dilation (EBD) was performed every other week after stricture development (EBD group, n = 3). In both groups, esophageal fluoroscopy was performed 8 weeks after the interventions, and the esophagus was excised for histological examination of the patched site. In the AEW patch group, esophageal fluoroscopy revealed favorable passage through the patched site. Histologically, the mucosal epithelium and lamina propria had regenerated as in the normal area. In the EBD group, the circumferential stricture site showed marked thickening, and there were hypertrophic scars associated with epithelial defects on the luminal surface. Histologically, defects of the mucosal epithelium and full-thickness proliferation of connective tissue were observed. AEW patch grafting was suggested to be a potentially novel treatment strategy for post-ESD esophageal circumferential stricture.

Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats

Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

Bone structure and strength are enhanced in rats programmed by early overfeeding.

Childhood obesity is growing in prevalence. Obesity and bone dysfunctions may be related disorders, and therefore our aim was to study the impact of the early overfeeding (EO) in offspring bone health since weaning up to adulthood. To induce EO during lactation, litter size was adjusted to 3 male rats per litter (SL). Litter containing 10 pups per mother was the control (NL). Bone tissue was evaluated by dual-energy X-ray absorptiometry, computed tomography, microcomputed tomography, biomechanical tests, and serum analyses. SL offspring presented higher body weight, fat mass, lean mass from 21 up to 180 days, hyperphagia, and higher visceral fat mass. Bone analysis showed that SL offspring presented higher total bone mineral density (BMD) only at 180 days, and higher total bone mineral content and higher bone area from 21 until 180 days. At 180 days, SL offspring presented higher femur BMD and fourth lumbar vertebra (LV4) BMD, higher femoral head radiodensity and LV4 vertebral body radiodensity, lower trabecular pattern factor and trabecular separation, however with higher trabecular number, higher maximal load, resilience, stiffness and break load, and lower break deformation. SL group had, at 180 days, higher osteocalcin and lower C-terminal cross-linked telopeptide of type I collagen (CTX I). We have shown that the excess of fat mass contributed to an increased bone mass, and hypothesized that this increase could be mediated by the hypothyroidism and previous higher thyroid hormone action and hyperleptinemia at weaning. Furthermore, the increased biomechanical loading due to increased body weight probably help us to understand the protective effects obesity exerts upon bone health.

Immune therapeutic potential of stem cells from human supernumerary teeth.

Discoveries of immunomodulatory functions in mesenchymal stem cells (MSCs) have suggested that they might have therapeutic utility in treating immune diseases. Recently, a novel MSC population was identified from dental pulp of human supernumerary teeth, and its multipotency characterized. Herein, we first examined the in vitro and in vivo immunomodulatory functions of human supernumerary tooth-derived stem cells (SNTSCs). SNTSCs suppressed not only the viability of T-cells, but also the differentiation of interleukin 17 (IL-17)-secreting helper T (Th17)-cells in in vitro co-culture experiments. In addition, systemic SNTSC transplantation ameliorated the shortened lifespan and elevated serum autoantibodies and nephritis-like renal dysfunction in systemic lupus erythematosus (SLE) model MRL/lpr mice. SNTSC transplantation also suppressed in vivo increased levels of peripheral Th17 cells and IL-17, as well as ex vivo differentiation of Th17 cells in MRL/lpr mice. Adoptive transfer experiments demonstrated that SNTSC-transplanted MRL/lpr mouse-derived T-cell-adopted immunocompromised mice showed a longer lifespan in comparison with non-transplanted MRL/lpr mouse-derived T-cell-adopted immunocompromised mice, indicating that SNTSC transplantation suppresses the hyper-immune condition of MRL/lpr mice through suppressing T-cells. Analysis of these data suggests that SNTSCs are a promising MSC source for cell-based therapy for immune diseases such as SLE.

Absorption, distribution and excretion of radioactivity by male rats after a single intravenous or subcutaneous dose of 14C-azacitidine.

The absorption, distribution and excretion of radioactivity were investigated in male Sprague-Dawley rats after a single intravenous or subcutaneous dose of 14C-azacitidine. After subcutaneous administration, 14C-azacitidine was rapidly absorbed and the radioactivity was distributed to the tissues. The absorption of radioactivity after subcutaneous administration was 76.6% of that observed after intravenous administration. There were no marked differences in the tissue distribution of the radioactivity between administration routes. The concentrations of radioactivity in most tissues, including the spleen and bone marrow, which are sites of action of azacitidine, were higher than those in the plasma. Particularly high concentrations of radioactivity were detected in the spleen, kidney and liver. The accumulation of radioactivity in blood cells increased from 0.5 to 48 h. The binding of azacitidine to serum protein was low at <9%, and the cumulative urinary and fecal excretion of radioactivity for 168 h after intravenous or subcutaneous administration was >95% of the administered dose, indicating that radioactivity did not accumulate in the tissues. The radioactivity was mainly excreted in the urine.

Progesterona plasmática de ovelhas submetidas ao efeito-macho e mantidas sob diferentes condições nutricionais / Plasmatic progesterone of ewes submitted to male effect and kept at different nutritional conditions

Dosou-se a concentração plasmática de progesterona (P4) em ovelhas Santa Inês (SI), Suffolk (SU) e Romney Marsh (RM) em anestro sazonal e submetidas ao efeito-macho, as quais receberam ou não suplementação alimentar. Machos vasectomizados foram introduzidos no grupo de fêmeas após um período prévio de isolamento de 60 dias, e amostras de sangue foram colhidas antes e após a introdução dos machos. Houve efeito (P<0,01) de período, raça, interação período x raça e interação suplementação x período x raça sobre a concentração de P4. Nas ovelhas SI ocorreu aumento (P<0,01) da concentração de P4 após a introdução do macho, indicando que houve aumento na atividade cíclica reprodutiva desse grupo. A suplementação, neste caso, potencializou este aumento. Nas ovelhas SU e RM não ocorreram modificações na concentração de P4 (P>0,01) após a introdução dos machos, nem houve efeito de suplementação. O efeito-macho foi eficaz em induzir a atividade reprodutiva durante o anestro sazonal em ovelhas SI, mas não em ovelhas SU e RM, e a associação dessa prática com a suplementação alimentar é recomendada para ovelhas da raça nativa SI.

The present study aimed to verify the plasmatic progesterone (P4) concentrations in Santa Inês (SI), Suffolk (SU) and Romney Marsh (RM) ewes submitted to male effect receiving or not food supplementation during seasonal anestrous. Vasectomized rams were introduced to the group of ewes after 60 day isolation. Blood samples were collected before and after the male's introduction. Effects (P<0.01) of period, breed, period x breed interaction and period x breed x supplementation interaction on plasmatic P 4 concentrations were observed. The plasmatic P 4 concentrations of SI ewes increased (P<0.01) after the male introduction, indicating there was an increase in the reproductive activity in this group. Supplementation increased this effect. For SU and RM ewes the plasmatic P 4 concentrations did not alter (P>0.01) after the male introduction, with no supplementation. It was concluded the male effect was efficient in inducing the reproductive activity in SI ewes, but not in SU and RM ewes. The association of the effect of the male with supplementation increased this effect.

Feasibility of endoscopic submucosal dissection: a new technique for en bloc resection of a large superficial tumor in the colon and rectum.

Endoscopic submucosal dissection (ESD) is a promising procedure that enables en bloc resection of large superficial tumors in the upper gastrointestinal tract. On the other hand, ESD in the colon and rectum is technically difficult to perform because of its anatomical features. At our institution, 137 consecutive superficial colorectal tumors larger than 20 mm in diameter in 137 patients were treated by ESD between April 2007 and October 2010, and 132 lesions were successfully resected. The average procedure time was 79.2 minutes, and the rate of en bloc resection was 89.1% (122/137). The rate of complete resection, defined as en bloc resection with tumor-free lateral and vertical margins, was 85.4% (117/137). The rate of perforation was 3.6% (5/137). Colorectal ESD achieved a high rate of en bloc resection and complete resection and is applicable in the colorectum.

Expression levels of adiponectin receptors and periodontitis.

BACKGROUND AND OBJECTIVE: We recently showed that adiponectin, an adipocyte-derived cytokine, may function as a negative regulator of the Toll-like receptor signaling pathway and of osteoclast formation in periodontal disease. In this study, we investigated whether the expression levels of adiponectin receptors (AdipoR1 and AdipoR2) are related to the presence of periodontitis. MATERIAL AND METHODS: We initially examined, using RT-PCR, the expression of the AdipoR1 and AdipoR2 genes at the mRNA level in several oral tissues of C57BL mice. Next, we investigated (using real-time PCR assays) whether inflammatory cytokines, such as tumor necrosis factor-alpha, could affect the expression levels of these genes in human gingival fibroblasts. Lastly, we compared the expression levels of these receptor proteins in gingival tissues between two healthy subjects and five patients with severe periodontal disease using western blotting analysis. RESULTS: The AdipoR1 and AdipoR2 receptors were ubiquitously expressed in the oral tissues of mice. We observed that treatment with tumor necrosis factor-alpha could significantly reduce the expression levels of both AdipoR1 and AdipoR2 genes in human gingival fibroblasts. Moreover, we found that the expression of both receptors was lower in periodontal tissues from patients with severe periodontitis than in patients with healthy periodontal tissues. CONCLUSION: These observations suggest that adiponectin may not function efficiently in sites of periodontal disease because of a decrease in the number of its receptors, and this probable dysfunction may play a role in worsening periodontitis in patients.

Nocturnal plasma levels of melatonin in quails (Coturnix japonica) injected with l-5-hydroxy-tryptophan

This study aimed to demonstrate the influence of the systemic administration of l-5-hydroxy-tryptophan (L-HTP) on the plasma levels of melatonin during the dark period in quails. Throughout daylight, the plasma levels of melatonin did not differ significantly, oscillating between 110.2 ± 15.8 pg.mL-1 and 157.4 ± 34.8 pg.mL-1, from 8 to 16 hours. L-HTP (25 mg.kg-1, through the intracelomic route) administered at 18 hours lessened significantly the nocturnal increase of the plasma levels of melatonin (controls, 327.3 ± 20.1 and 315.8 ± 20.9 pg.mL-1 vs. 242.1 ± 24.8 and 217.5 ± 21 pg.mL-1, respectively, at 20 and 24 hours, P < 0.05). The results obtained showed that the administration of LHTP reduced the nocturnal melatonin release, possibly by bringing about an increase in serotonin synthesis and synaptic release in the pineal. Therefore, the serotoninergic transmission from the raphe towards the pineal would constitute a mechanism of modulation of the synthesis and melatonin release in quails.

Este trabalho objetivou demonstrar a influência da administração sistêmica de l-5-hidroxi-triptofano (L-HTP) sobre os níveis plasmáticos de melatonina durante o período noturno em codornas. Ao longo do período claro, os níveis plasmáticos de melatonina não diferiram significativamente, oscilando entre 110,2 ± 15,8 pg.mL-1 e 157,4 ± 34,8 pg.mL-1, de 8 às 16 horas. L-HTP (25 mg.kg-1, via intracelomática), administrado às 18 horas atenuou significativamente a elevação noturna dos níveis plasmáticos de melatonina (controles, 327,3 ± 20,1 e 315,8 ± 20,9 pg.mL-1 vs. 242,1 ± 24,8 e 217,5 ± 21 pg.mL-1, respectivamente, às 20 e 24 horas, P < 0,05). Os resultados obtidos mostraram que a administração de L-HTP reduziu a liberação noturna de melatonina, possivelmente por suscitar um aumento da síntese e liberação sináptica de serotonina na pineal. Portanto, a transmissão serotoninérgica da rafe para a pineal constituiria um mecanismo de modulação da síntese e/ou liberação de melatonina em codornas.

Craniofacial growth and functional change in oligodontia with ectodermal dysplasia: a case report.

A case of ectodermal dysplasia in a Japanese boy is presented in this report. After prosthetic treatment was performed with partial dentures in both jaws, mandibular movement, dental casts, and lateral and frontal cephalograms were taken at an initial visit (age 7 years 8 months), and at follow-up 1 year and 8 months later (age 9 years 3 months) to evaluate the developmental changes in masticatory movement, dental arch width, and craniofacial morphology. Stability of the jaw movements improved during the follow-up period both with and without dentures. The facial skeleton grew between the initial visit and follow-up. Mandibular length remained long compared with the average value for Japanese children. A possible genetic marker for mandibular growth (growth hormone receptor), which is considered to be an important factor in mandibular growth and development, was analysed for the gene variant and the result supported the characteristic mandibular growth in the reported case. These results indicate the characteristic craniofacial growth in this disease and emphasize the potential ability of functional development of jaw movements with oligodontia.

Annual pattern of plasma melatonin and progesterone concentrations in hair and wool ewe lambs kept under natural photoperiod at lower latitudes in the southern hemisphere.

To study the annual pattern of plasma melatonin and progesterone concentrations in hair [Santa Inês (SI)] and wool [Romney Marsh (RM) and Suffolk (SU)] ewe lambs kept under natural photoperiods at 21 degrees 59'S, 12 ewe lambs (four/breed) were used. For melatonin, blood samples were collected monthly throughout the year at the onset (17:00, 19:00 and 21:00 hr) and end (04:00, 06:00 and 08:00 hr) of the night, and for progesterone the samples were collected in the morning, two to three times a week throughout the year. Plasma melatonin concentrations at different times of the day changed according to the season. In diurnal periods (17:00 and 8:00 hr) no seasonal differences were observed but they became evident in the nocturnal intervals (21:00 and 4:00 hr) and transitional night-day (6:00 hr) times. The patterns of melatonin secretion were higher in winter and autumn than in spring and summer. The patterns of plasma progesterone secretion were affected by interaction between breed and season. There was no seasonal variation in plasma progesterone concentrations for SI females. The progesterone pattern for RM and SU females varied with season. The plasma levels were higher in autumn and winter than in spring and summer. At 21 degrees 59'S hair and wool ewe lambs showed the same annual pattern of plasma melatonin concentration while the annual progesterone profiles were quite different. For SI females this pattern was constant along all seasons and for RM and SU females this pattern was higher during autumn and winter than spring and summer.

The deficiency of immunoregulatory receptor PD-1 causes mild osteopetrosis.

Recently, the involvement of immune responses in metabolic bone disease and/or local bone destruction has received much attention. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a member of the immunoglobulin (Ig) superfamily, negatively regulates T cell activation. The deficiency of CTLA-4 induces profound osteopenia with an increase in osteoclastogenesis, suggesting the important role of activated T cells in osteoclastogenesis. Programmed death-1 (PD-1) is the newly identified immunoregulatory receptor, which also belongs to the Ig superfamily. Both CTLA-4 and PD-1 are induced on activated T cells, however, there are no reports linking PD-1 with osteoclasts. In the present study, we have examined the bone phenotype in PD-1-deficient mice PD-1-/- and the role of PD-1 in osteoclastogenesis and osteoclast function. Both trabecular and cortical bone mineral densities of tibia were significantly increased, as observed in peripheral quantitative computed tomography (pQCT), at 12 weeks of age in PD-1-/- mice. Histomorphometric analysis of the PD-1-/- mice and the age-matched controls at 12 weeks of age showed a 2-fold increase in bone volume (BV/TV) with a 55% decrease in osteoclast number (N.Oc/BS). Bone formation indices were similar in both groups. The number of soluble receptor activator of nuclear factor kappaB ligand (sRANKL)-induced osteoclast-like cells (OCLs) derived from the PD-1-deficient splenocytes was significantly decreased (by 25%). On the other hand, PD-1 deficiency did not affect the bone-resorbing activity of mature osteoclasts. Our results suggest that PD-1 deficiency reduces osteoclastogenesis resulting in an osteopetrotic phenotype. Identical members of the Ig superfamily, CTLA-4 and PD-1, which negatively regulate immune responses, may differentially affect osteoclastogenesis and bone remodeling.

[Extrapleural pneumonectomy of malignant pleural mesothelioma].

We analyzed 7 patients with malignant pleural mesothelioma who underwent extrapleural pneumonectomy. Six men and a woman had a mean age of 65 years old. The postoperative mortality rate was 14% (1 death) and morbidity, 43% (3 cases). According to staging of International Mesothelioma Interest Group, 2 patients had stage I disease, 1 did stage II, 3 did stage III and 1 did stage IV. Local recurrences were found in 3 patients and metastasis in 2. In patients with local recurrences, 2 had irradiation with chemotherapy and 1, irradiation. In patients with recurrences of metastasis, 1 had chemotherapy and 1, supportive care. Seven patients with extrapleural pneumonectomy and 10 without surgery had median survivals of 16 months and 10 months, 1-year survival rates of 71% and 40% and 2-year survival rates of 57% and 0% respectively (p=0.071). Extrapleural pneumonectomy with adjuvant therapy could be effective treatment for malignant pleural mesothelioma.