Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Photoprotective and antioxidant effects of Rhubarb: Inhibitory action on tyrosinase and tyrosine kinase activities and TNF-α, IL-1α and α-MSH production in human melanocytes

Background: Exposure to ultraviolet (UV) radiation causes various forms of acute and chronic skin damage, including immunosuppression, inflammation, premature aging and photodamage. Furthermore, it induces the generation of reactive oxygen species, produces proinflammatory cytokines and melanocyte-stimulating hormone (MSH) and increases tyrosinase activity. The aim of this study was to evaluate the potential photoprotective effects of Rheum rhaponticum L. rhizome extract on human UV-stimulated melanocytes.Methods: The effects of Rheum rhaponticum rhizome extract on tyrosine kinase activity, and on interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α), and α-MSH production in human epidermal melanocytes were evaluated under UV-stimulated and non-stimulated conditions. Antioxidant activity was evaluated by lipid peroxidation and 1,1-dyphenyl-2-picryl-hydrazyl (DPPH) assays, while anti-tyrosinase activity was evaluated by the mushroom tyrosinase method.Results: Rheum rhaponticum L. rhizome extract showed in vitro antioxidant properties against lipid peroxidation, free radical scavenging and anti-tyrosinase activities, and inhibited the production of IL-1α, TNF-α, α-MSH, and tyrosine kinase activity in melanocytes subjected to UV radiation.Conclusions: These results support the inclusion of Rheum rhaponticum L. rhizome extract into cosmetic, sunscreen and skin care products for the prevention or reduction of photodamage. © 2013 Silveira et al; licensee BioMed Central Ltd.

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing >= 50% inhibition property against CHIKV at 10 mu M were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 mu M and 7.1 mu M. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity -inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Perfil global de expressão de micro-RNAS circulantes como marcadores de resposta terapêutica na leucemia mielóide crônica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

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

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O

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

Relação da expressão de fatores de crescimento celular (IGF-1) e (SCF) com fatores prognósticos e o alvo da rapamicina em mamíferos (m-TOR) em mastocitomas cutâneos caninos

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

Actinic cheilitis and squamous cell carcinoma of the lip: clinical, histopathological and immunogenetic aspects

Queilite actínica é a principal lesão pré-neoplásica do lábio. O carcinoma espinocelular do lábio é incluído nas estatísticas brasileiras junto com os cânceres de boca e, em conjunto, somam 40% dos cânceres de cabeça e pescoço. Há certo desconhecimento médico e odontológico em geral quanto aos fatores relacionados à carcinogênese e à progressão de tumores de boca. Genes de supressão tumoral e proteínas regulatórias de proliferação celular exercem papel na evolução da queilite actínica para carcinoma espinocelular e no comportamento biológico deste. O conhecimento de marcadores de diagnóstico e prognóstico e sua investigação têm utilidade no acompanhamento de tais pacientes.

Serum inhibin A and angiogenic factor levels in pregnancies with previous preeclampsia and/or chronic hypertension: are they useful markers for prediction of subsequent preeclampsia?

OBJECTIVE: Our objective was to determine whether measurement of placenta growth factor (PLGF), inhibin A, or soluble fms-like tyrosine kinase-1 (sFlt-1) at 2 times during pregnancy would usefully predict subsequent preeclampsia ( PE) in women at high risk. STUDY DESIGN: We analyzed serum obtained at enrollment (12(0/7) to 19(6/7) weeks) and follow-up (24-28 weeks) from 704 patients with previous PE and/or chronic hypertension (CHTN) enrolled in a randomized trial for the prevention of PE. Logistic regression analysis assessed the association of log-transformed markers with subsequent PE; receiver operating characteristic analysis assessed predictive value. RESULTS: One hundred four developed preeclampsia: 27 at 37 weeks or longer and 77 at less than 37 weeks (9 at less than 27 weeks). None of the markers was associated with PE at 37 weeks or longer. Significant associations were observed between PE at less than 37 weeks and reduced PLGF levels at baseline (P =.022) and follow-up (P <.0001) and elevated inhibin A (P <.0001) and sFlt-1 (P =.0002) levels at follow-up; at 75% specificity, sensitivities ranged from 38% to 52%. Using changes in markers from baseline to follow-up, sensitivities were 52-55%. Associations were observed between baseline markers and PE less than 27 weeks (P <=.0004 for all); sensitivities were 67-89%, but positive predictive values (PPVs) were only 3.4-4.5%. CONCLUSION: Inhibin A and circulating angiogenic factors levels obtained at 12(0/7) to 19(6/7) weeks have significant associations with onset of PE at less than 27 weeks, as do levels obtained at 24-28 weeks with onset of PE at less than 37 weeks. However, because the corresponding sensitivities and/or PPVs were low, these markers might not be clinically useful to predict PE in women with previous PE and/or CHTN.

Effects of Coccoloba uvifera L. on UV-stimulated melanocytes

Exposure to ultraviolet (UV) radiation induces generation of reactive oxygen species, production of proinflammatory cytokines and melanocyte-stimulating hormone (MSH) as well as increase in tyrosinase activity. The potential photoprotective effects of Coccoloba uvifera extract (CUE) were evaluated in UV-stimulated melanocytes.Human epidermal melanocytes were used as an in vitro model to evaluate the effects of CUE on the production interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and alpha-MSH under basal and UV-stimulated conditions. Antioxidant and anti-tyrosinase activities were also evaluated in membrane lipid peroxidation and mushroom tyrosinase assay, respectively.Coccoloba uvifera L. showed antioxidant and anti-tyrosinase activities and also inhibited the production of IL-1 alpha, TNF-alpha and alpha-MSH in melanocytes subjected to UV radiation (P < 0.01). Moreover, CUE inhibited the activity of tyrosine kinase in cell culture under basal and UV radiation conditions (P < 0.001), corroborating the findings of the mushroom tyrosinase assay.This study supports the photoprotective potential of CUE.

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals.Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLC gamma/Ca(2+) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. on the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action.Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cellsthrough direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma

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

p16INK4A immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16 INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.