The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

The Immunomodulatory Action of Sialostatin L on Dendritic Cells Reveals Its Potential to Interfere with Autoimmunity

Sialostatin L (SialoL) is a secreted cysteine protease inhibitor identified in the salivary glands of the Lyme disease vector Ixodes scapularis. In this study, we reveal the mechanisms of SialoL immunomodulatory actions on the vertebrate host. LPS-induced maturation of dendritic cells from C57BL/6 mice was significantly reduced in the presence of SialoL. Although OVA degradation was not affected by the presence of SialoL in dendritic cell cultures, cathepsin S activity was partially inhibited, leading to an accumulation of a 10-kDa invariant chain intermediate in these cells. As a consequence, in vitro Ag-specific CD4(+) T cell proliferation was inhibited in a time-dependent manner by SialoL, and further studies engaging cathepsin S(-/-) or cathepsin L(-/-) dendritic cells confirmed that the immunomodulatory actions of SialoL are mediated by inhibition of cathepsin S. Moreover, mice treated with SialoL displayed decreased early T cell expansion and recall response upon antigenic stimulation. Finally, SialoL administration during the immunization phase of experimental autoimmune encephalomyelitis in mice significantly prevented disease symptoms, which was associated with impaired IFN-gamma and IL-17 production and specific T cell proliferation. These results illuminate the dual mechanism by which a human disease vector protein modulates vertebrate host immunity and reveals its potential in prevention of an autoimmune disease. The Journal of Immunology, 2009, 182: 7422-7429.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappa B ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers` expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright (C) 2007 John Wiley & Sons, Ltd.

In vitro effect of low-level laser on odontoblast-like cells

The aim of this study was to evaluate the metabolism of odontoblast-like MDPC-23 cells subjected to direct LLL irradiation. The cells were seeded (20,000 cells/well) in 24-well plates and incubated for 24 hours at 37 degrees C. After this period, the culture medium (DMEM) was replaced by fresh DMEM supplemented with 2 or 5% (stress induction by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to laser doses of 2, 4, 10, 15 and 25 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 +/- 3 nm, 40 mW). One control group (sham irradiation) was established for each experimental condition (laser dose x FBS supplementation). Three and 72 hours after the last irradiation, cells were analyzed with respect to metabolism, morphology, total protein expression and alkaline phosphatase (ALP) activity. Higher metabolism and total protein expression were observed 72 hours after the last irradiation at the doses of 15 and 25 J/cm(2) (Mann-Whitney; p<0.05). Higher ALP activity was obtained with 5% FBS when the cells were irradiated with doses of 2 and 10 J/cm(2). For the dose of 25 J/cm(2), the highest ALP activity was observed with 10% FBS. It was concluded that the LLLT parameters used in this study stimulated the metabolic activity of the MDPC-23 cells, especially at the doses of 15 and 25 J/cm(2).

Concentrações séricas de progesterona, 17 b-estradiol e cortisol durante o final do próestro, estro e diestro gestacional em cadelas

Neste estudo, foram utilizadas 7 cadelas sem raça definida, adultas e hígidas, copuladas com macho hígido, após exame andrológico, para acompanhamento das variações hormonais de progesterona, 17b-estradiol e cortisol a partir do final do próestro, durante o estro e diestro gestacional em fêmeas da espécie canina. As avaliações séricas do cortisol foram iniciadas no período de estro. A citologia vaginal esfoliativa foi utilizada como parâmetro auxiliar para a determinação das fases do ciclo estral, mais especificamente para análise do melhor momento para cópula, através da presença das células superficiais queratinizadas visibilizadas nas lâminas. Os resultados obtidos mostraram concentrações médias de progesterona elevando-se discretamente no final do próestro (de 1,56 para 2,85ng ml-1), concomitante com o início do declínio dos valores de estradiol no mesmo período (de 20,93 para 18,81pg ml-1). Durante a gestação pôde-se observar concentrações elevadas de progesterona (36,90ng ml-1), havendo declínio apenas no terço final (4,10ng ml-1), quando também pôde ser notada, por um momento, ligeira elevação das concentrações médias de 17b-estradiol (2,46pg ml-1). O aumento do cortisol sérico foi notado na última semana da gestação havendo, antes disso, alterações significativas (P<0,05) nas concentrações séricas baseadas nos padrões descritos em literatura. No pós-parto imediato a redução do cortisol sérico (6,52ng ml-1) foi considerada relevante de acordo com as concentrações detectadas na última semana da gestação (22,27ng ml-1). A progesterona esteve mantida em níveis basais no pós-parto imediato (< 1ng ml-1).

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Expressão imuno-histoquímica de IL-17, TGF-β1 e FOXP3 em ganulomas periapicais, cistos radiculares e cistos radiculares residuais

Periapical lesions are chronic inflammatory conditions of periradicular tissues considered direct consequences of infectious diseases resulting from pulp necrosis and subsequent progression to periapical region. The participation of the immune response and bone resorption in the formation of these lesions has been investigated, so that different cell types and cytokines have been identified as contributors to this process. In this perspective, this study aimed to evaluate the immunohistochemical expression of IL-17, TGF-β1 and FoxP3 in periapical granulomas (PGs), radicular cysts (RCs) and residual radicular cysts (RRCs), seeking a better understanding of the etiopathogenesis these periapicopatias. To this end, we selected 20 cases of GPs, 20 CRs and 10 RRCs to undergo morphological analysis and immunohistochemistry for biomarkers above, the latter being performed quantitatively using scores and average percentages of immunostaining for the analysis of IL-17 and TGF- β1, while for the FoxP3 were counted only the positive lymphocytes. The results showed statistically significant differences between TGF-β1 and FoxP3 imunoexpressions, in relation to the periapical lesions studied (p = 0.002, p <0.001, respectively) but not between IL-17 and these (p = 0.355). Furthermore, the analysis of lymphocytes FoxP3-positive revealed significant statistical differences in that refers to the intensity of inflammatory infiltrate (p = 0.003) and also regarding thickness of the epithelial lining (p = 0.009). Finally, it was observed in the case of PGs, strong positive correlation between the amount of FoxP3- positive lymphocytes and the immunohistochemical expression of TGF-β1 (r = 0.755, p<0.001), as well as moderate positive correlation between IL-17 and TGF-β1 imunoexpressions (r = 0.503, p = 0.024). Thus, we can conclude that interactions between Th17 and Treg cells seem to be established at the site of injury, suggesting the involvement of both pro-inflammatory and immunoregulatory cytokines in the pathogenesis of periapical lesions

Imunoexpressão da IL-17 e RORγt em carcinomas de células escamosas de lábio e língua

Th17 cells have been strongly associated to the pathogenesis of inflammatory and autoimmune diseases, although their influence on the carcinogenesis is still little known, there are reports of anti-tumor and protumoral actions. The objective of this study is to research the presence of Th17 lineage in lip and tongue SCC, using the analysis of the immunoexpression of IL-17 and RORγt, relating this immunoexpression with clinical and morphological findings in the attempt to better comprehend the role of these cells on the tumoral immunity of OSCCs. The results were submitted to non-parametric statistical tests with significance level of 5%. On the histomorphological analysis, it was observed the predominance of low level lesions on lip and high level lesions on tongue (p=0,024). It was not observed statistical significance between clinical stage and histological gradation of malignancy (p=0,644). For the immunohistochemical study, 5 random fields with greater immunoreactivity of the peritumoral inflammatory infiltrate were photomicrographed on the 400x magnification. It was done the count of lymphocytes which showed cytoplasmic and pericytoplasmic staining for the IL-17 cytokine as well as nuclear and cytoplasmic staining for RORγt. It was observed statistical significance difference on the quantity of immunopositive lymphocytes to IL-17 between the groups of SCC of lip and tongue (p=0,028). For the RORγt it was not observed statistical significance difference between the groups of SCC of lip and tongue (p=0,915). It was not observed statistical difference between the immunostaining of IL-17 and RORγt with histological gradation of malignancy and clinical staging. The findings of this research suggest a possible anti-tumor role of IL-17 for cases of lip. The results of the analysis of the RORγt are possibly due to the wide duality of the anti-tumor and protumoral role of the Th17 cells and their plasticity which, in the presence of different cytokines expressed on the tumor microenvironment, can alter its phenotype.

Killing of Paracoccidioides brasiliensis yeast cells by IFN-gamma and TNF-alpha activated murine peritoneal macrophages: evidence of H(2)O(2) and NO effector mechanisms

Paracoccidioidomycosis is a deep mycosis, endemic in Latin America, caused by Paracoccidioides brasiliensis. Macrophage activation by cytokines is the major effector mechanism against this fungus. This work aimed at a better understanding of the interaction between yeast cells-murine peritoneal macrophages and the cytokine signals required for the effective killing of high virulence yeast-form of P. brasiliensis. In addition, the killing effector mechanisms dependent on the generation of reactive oxygen or nitrogen intermediates were investigated. Cell preincubation with IFN-gamma or TNF-alpha, at adequate doses, resulted in effective yeast killing as demonstrated in short-term (4-h) assays. Both, IFN-gamma and TNF-alpha activation were associated with higher levels of H(2)O(2) and NO when compared to nonactivation. Treatment with catalase (CAT), a H(2)O(2) scavenger, and N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor, reverted the killing effect of activated cells. Taken together, these results suggest that both oxygen and L-arginine-nitric oxide pathways play a role in the killing of highly virulent P. brasiliensis.

Genotoxicity and cytotoxicity of glass ionomer cements on Chinese hamster ovary (CHO) cells

Glass ionomer cements are widely used in dentistry as restorative materials and adhesives for composite restorations. However, the results of genotoxicity studies using these materials are inconclusive in literature. The goal of this study was to examine the genotoxic and cytotoxic potential of three different glass ionomer cements available commercially (Ketac Cem, Ketac Molar and Vitrebond) by the single cell gel (comet) assay and trypan blue exclusion test, respectively. For this, such materials were exposed to Chinese hamster ovary (CHO) cells in vitro for 1 h at 37 degrees C. Data were assessed by Kruskall-Wallis nonparametric test. The results showed that the powder from Ketac Molar displayed genotoxicity only in the maximum concentration evaluated (100 mu g/mL). In the same way, the liquid from Vitrebond at 0.1% dilution caused an increase of DNA injury. Significant differences (P < 0.05) in cytotoxicity provoked by all powders tested of glass ionomer cements were observed for exposure at 1000 mu g/mL concentration. With respect to liquids of glass ionomer cements evaluated, the major toxic effect on cell viability was produced at 10%, beginning at the dilution of 0.5% for Vitrebond. Taken together, we conclude that some components of glass ionomer cements show both genotoxic and cytotoxic effects.

Cytogenetic damage in circulating lymphocytes and buccal mucosa cells of head-and-neck cancer patients undergoing radiotherapy

This study evaluated cytogenetic damage by measuring the frequency of micronucleated cells (MNC) in peripheral blood and buccal mucosa of head-and-neck cancer patients undergoing radiotherapy.MNC frequencies were assessed in 31 patients before, during, and after radiotherapy, and in 17 C, healthy controls matched for gender, age, and smoking habits. Results showed no statistically significant difference between patients and controls prior to radiotherapy in cytokinesis-blocked lymphocytes or buccal mucosa cells. During treatment, increased MNC frequencies were observed in both cell types. Micronucleated lymphocyte levels remained high in samples collected 30 to 140 days after the end of treatment, while MNC frequency in buccal mucosa decreased to values statistically similar to baseline values. There is controversy over the effects of age, smoking habit, tumor stage, and/or metastasis on MNC frequency. However, increased frequency of micronucleated buccal mucosa cells was seen in patients under 60 years old and in those with tumors >4cm.In conclusion, the data show that radiotherapy has a potent clastogenic effect in Circulating lymphocytes and buccal mucosa cells of head-and-neck cancer patients, and that the baseline MNC frequency in these two tissues is not a sensitive marker for head-and neck neoplasm.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)