Effects oxidative on splenic inflammatory response of rats exposed to different concentrations of formaldehyde

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1 Effects oxidative on splenic inflammatory response of rats exposed to different concentrations of formaldehyde Marçal, M. C. C. S., Campos, K. K. D., Soares, N.P., Lima, W. G., Bezerra, F. S., Introducao: Departamento de Ciências Biológicas - UFOP The Formaldehyde is a flammable gas, reactive, colorless, and soluble in water, ethanol and diethyl ether. Studies have shown that continuous and prolonged exposure to this substance and its easy absorption through the respiratory and gastrointestinal tract can cause extensive damage to the human body. Objetivos: This study aimed to evaluate the oxidative damage caused to spleen in animals exposed to different concentrations of formaldehyde. Metodos: The study was approved by the Commission of Ethics in the Use of Animals of the Federal University of Ouro Preto (CEUA 2011/01). Male Fischer rats (n = 28, adults, grams) were divided in four groups: control (GC), exposed to ambient air and three groups with the following formaldehyde solutions: 1% (FA1), 5% (FA5) and 10% (FA10). An ultrasonic nebulizer (Ultra-Relief Inhaler Multipower - Unique Group) coupled to a chamber was used to produce air suspension, containing 1%, 5% and 10% formaldehyde solution during twenty minutes, three times per day during five days. Twenty four hours after the last exposure the animals were euthanatized and spleens were collected for analysis of the absolute mass, oxidative damage (reactive substances to thiobarbituric acid - TBARS) and activity of antioxidant enzyme catalase. The normal distribution of each variable was evaluated using the Kolmogorov Smirnov test and was expressed as means ± SEM. For comparison among groups, one-way ANOVA followed by the Bonferroni post-test was performed (p < 0.05). Resultados: The exposure to formaldehyde decreased the absolute mass (g) of FA10% (164.8 ± 5.36) compared to CTR (184.5 ± 2.60). The analysis of lipid peroxidation (nmol/mg protein) in spleen showed an increase of TBARS content in FA10% (2.08 ± 0.15) and FA5% (1.95 ± 0.06) compared to

2 FA1%(0.73 ± 0.05) and CTR (0.78 ± 0.02). There was a increase of enzyme activity (U / mg protein) in FA10% (2.88 ± 0.21) compared to FA5% (1.87 ± 0.22), FA1% (1.83 ± 0.25) and CTR (0.94 ± 0.19). Conclusão: The results showed that exposure to formaldehyde caused oxidative damage in the spleen, which was evidenced by increase of activity catalase and increased of TBARS content. Apoio Financeiro: The results showed that exposure to formaldehyde caused oxidative damage in the spleen, which was evidenced by increase of activity catalase and increased of TBARS content. Comitê de Ética: 2011/01

3 High levels of HbA1c from T2DM patients positively correlated with increased of oxidative stress biomarker and IL-6 levels Volpe, C. M. O., Abreu, L. F. M., Soares, A. N., Anjos, P. M. F., Fagundes-netto, F. S., Introducao: Nogueira-machado, J. A., Núcleo de Pós-Graduação e Pesquisa - IEP-BH Type 2 diabetes mellitus (T2DM) is an inflammatory disease showing the pathological involvement of the immune system. Although many studies have demonstrated the involvement of elevated levels of inflammatory biomarkers in the pathogenesis of diabetes complications, a systematic definition of the inflammatory profile in T2DM patients has not been determined. The use of only one biomarker could be problematic and inconclusive, however, the combined association among biomarkers of glycemic control (HbA1c), oxidative stress (malonaldehyde) and innate immunity (IL-6) could be an effective and reliable alternative to determine the inflammatory profile in these patients. Objetivos: To determine the association between T2DM and inflammation based on the plasma levels of pro-inflammatory cytokines (IL-1b, IL-6, TNF-a and IFN-g), lipid peroxidation (malonaldehyde), vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) associated with glycated hemoglobin (HbA1c). Metodos: This study was approved through the Ethical Committee of Santa Casa Hospital, Belo Horizonte - MG, Brazil (022/2010). The expression of oxidative stress markers and the plasma levels of angiogenic factor and inflammatory cytokines were determined in 51 T2DM patients compared with 38 non-diabetic controls (ND), ranging years of age. The levels of cytokines (IL-1b, IL-6, TNF-a and IFN-g), angiogenic factor (VEGF) and CRP were measured using ELISA. Lipid peroxidation (oxidative stress biomarker) was determined through malonaldehyde (MDA) levels using the TBARS assay, and HPLC was used to measure the HbA1c levels. The values were presented as the means ± standard deviation (SD). Comparisons between groups were performed using unpaired Student t-tests. Within-group correlations were performed using Pearson s r calculation. All analyses were considered signi?cant at p-values<0.05. Resultados: As expected, individuals with T2DM had signi?cantly higher fasting glucose values (145±49.1 vs. 89.2±8.1) and levels of HbA1c (8.3±1.3 vs. 5.8±0.3) compared with ND controls (p<0.05). The

4 plasma concentrations of IL-6 (123.1±30.4 vs ±21.6), TNF-a (78.2±34.2 vs. 59.9±34.5), IL-1b (9.8±2.4 vs. 8.6±1.2) VEGF (42.9±8.4 vs. 36.0±6.7) and MDA (15.0±4.0 vs. 8.1±3.7) were signi?cantly higher (p<0.05) in T2DM patients compared with ND controls. The plasma levels of IFN-g (12.1±1.8 vs. 12.8±1.84) and CRP (0.89±0.65 vs. 0.87±0.66) were similar between these groups (p>0.05). Correlation studies of the glycemic control (HbA1c levels) versus IL-6 levels and the HbA1c concentration versus MDA levels indicated a high positive correlation for T2DM patients (r = 0.68 and r = 0.67, respectively), but not for ND (r = 0.32 and r = 0.16, respectively). Conclusão: The results of this study show that the combined association of biomarkers for glycemic control (HbA1c) with pro-inflammatory cytokine (IL-6) and oxidative stress biomarker (MDA) might be a promising tool for the follow-up and evaluation of diabetes complications, although further studies are needed. Apoio Financeiro: The results of this study show that the combined association of biomarkers for glycemic control (HbA1c) with pro-inflammatory cytokine (IL-6) and oxidative stress biomarker (MDA) might be a promising tool for the follow-up and evaluation of diabetes complications, although further studies are needed. Comitê de Ética: 022/2010

5 EFEITO DO EXTRATO ETANÓLICO DA CASCA DO CAULE DE COPAIFERA MULTIFUGA HAYNE SOBRE AS CÉLULAS TUMORAIS DE EHRLICH Albiero, L. R., Nery, E. F., Dalazen, J. C., Kelly, T. O., Pereira, D. L., Castoldi, L., Introducao: Enfermagem - UFMT Ciências Ambientais - UFMT As copaibeiras são árvores comuns na América Latina, sendo encontradas nas regiões Sudeste, Centro-oeste e Amazônica do Brasil. Da árvore pode-se extrair um óleo-resina utilizado popularmente por suas propriedades antiinflamatória, cicatrizante, antiséptica, antitumoral, antibacteriana, expectorante, diurética e analgésica. Objetivos: Avaliar o efeito do extrato etanólico obtido da casca do caule da Copaifera multijuga Hayne sobre as células do tumor de Ehrlich. Metodos: A casca da planta foi retirada a 5 cm de profundidade do caule e retalhada em pedaços menores com serra. Ao material seco (40oC durante sete dias) e triturado (granulometria média de 1,0 mm) foi adicionado, na proporção de 1:20 (g/v) e em duas etapas de sete dias, hexano e, posteriormente, acetato de etila. Ao resíduo obtido foi adicionado etanol absoluto em cinco períodos de sete dias. Em todas as etapas de extração, o material vegetal permaneceu em repouso e ao abrigo da luz. O extrato foi filtrado e concentrado à vácuo em evaporador rotativo e dessecador. A atividade citotóxica do extrato foi avaliado pelo ensaio de viabilidade celular através do teste de exclusão do Azul de Tripan. O extrato (1mg/ml, 0,5mg/ml e 0,25mg/ml) foi incubados durante 24 horas a 37ºC 5% CO? com uma suspensão de células esplênicas ou células ascíticas do Tumor de Ehrlich. O grupo controle basal foi composto pelas células incubadas com meio RPMI 20% SBF (solução veículo diluente). O experimento seguiu as recomendações do Colégio Brasileiro de Experimentação Animal (COBEA) e do Comitê de Ética no Uso de Animais (CEUA) da Universidade Federal de Mato Grosso UFMT. ( /13-0). O desenvolvimento in vivo do Tumor de Ehrlich também foi avaliado. Neste caso, camundongos Swiss (n=10; 45g; 90 dias) foram inoculados com 1x10? células tumorais na região do flanco direito e tratados diariamente (gavagem) com diferentes concentrações do extrato (100, 150 e 200mg/kg) ou solução veículo (PBS estéril) durante 30 dias. Após este período, os animais foram sacrificados e a massa tumoral extraída e pesada. Os dados obtidos foram avaliados pelo teste de Análise de Variância (ANOVA), seguido pelo teste de médias Tukey, sendo consideradas as diferenças com p<0,05 (Software Graphpad Instat, San Diego, Califórnia - USA).

6 Resultados: A análise estatística dos resultados da viabilidade celular demonstrou diferença significativa na concentração de 1mg/ml, na qual o extrato etanólico da copaíba apresentou efeito citotóxico sobre as células esplênicas e tumorais (Ehrlich, Basal: 95,73±3,76; Extrato 1mg/ml: 49,38±19,33; Extrato 0,5mg/ml: 88,32±4,13; Extrato 0,25mg/ml: 97,88±1,30; Esplenócitos, Basal: 56,57±6,42; Extrato 1mg/ml: 44,54±6,98; Extrato 0,5mg/ml: 39,33±4,08; Extrato 0,25mg/ml: 49,17±9,02). A viabilidade das células esplênicas também foi significativamente reduzida na presença do extrato na concentração de 0,5mg/ml. A avaliação do desenvolvimento tumoral in vivo demonstrou que o tratamento com extrato na concentração de 200mg/kg foi capaz de reduzir em 33% o crescimento tumoral (EHR/extrato 100mg/kg: 1,49±0,52; EHR/extrato 150mg/kg: 1,29±0,55; EHR/extrato 200mg/kg: 0,87±0,53 e ERH/PBS: 1,31±0,67), apesar de não apresentar diferença significativa em relação ao controle. Conclusão: A análise dos resultados demonstrou que o extrato etanólico da casca de copaíba apresenta efeitos sobre as células tumorais de Ehrlich, com redução da viabilidade celular in vitro bem como na redução do desenvolvimento tumoral in vivo. Apoio Financeiro: A análise dos resultados demonstrou que o extrato etanólico da casca de copaíba apresenta efeitos sobre as células tumorais de Ehrlich, com redução da viabilidade celular in vitro bem como na redução do desenvolvimento tumoral in vivo. Comitê de Ética: /13-0

7 mtorc1 inhibition in DCs diminishes Sepsis Symptoms Latancia, M. T., Castoldi, A., Miyagi, M. T., Miyagi, M. Y. S., Correa-costa, M., Burgos, M., Silva, R. C., Hiyane, M., Aguiar, C. F., Oliveira, V. A., Ferreira, R. R., Camara, N. O. S., Amano, M. T., Immunology - ICB Central Animal Laboratory - ICB - USP Introducao: Dendritic cells (DCs) have a major role in the immune system and can be activated by Toll like receptors (TLR), which recognize patterns of pathogens such as lipopolysaccharide (LPS). The mammalian target of Rapamycin (mtor) pathway is associated to metabolism and cell survival and it is composed by mtorc1 (Raptor) and mtorc2 (Rictor). The first one is directly inhibited by Rapamycin (RAPA), used as an imunossupressor agent. Although this pathway is activated in DCs, little is known about the mtorc1/2 in these cells. Since sepsis is an overwhelming immune response related to TLR activation, possible modulation of inflammation by mtor pathway could diminishes sepsis symptoms. Objetivos: Our aim was to unravel how mtorc1 complex is modulated by TLR4 in DCs and observe the influence of this activation during sepsis. Metodos: Animal procedures were approved by CEUA-ICB/USP. We used male B6, RaptorCD11cCRE and TSCCD11cCRE mice (6 weeks old, 20g). We generated bone marrow-derived DCs from B6 mice (4/group) and used GM-CSF (20ng/ml) during 6 days and on day 6 we added LPS (50ng/ml). To inhibit mtorc1 we used RAPA (10ng/mL). We analyzed Raptor and Rictor expression by qpcr. To detect mtorc1 and mtorc2 activation we analyzed phosphorylation of AKT and S6k by Western blot. To verify DCs phenotype used FACS. Dosage of cytokines was evaluated by CBA. We induced sepsis in mice by cecal ligature and puncture and to estimate acute kidney injury (AKI) we used colorimetric methods for creatinine and urea levels. The statiscal analysis was performed by One way ANOVA with Bonferroni post test. Resultados: We analyzed the expression of molecules of each mtor complex and observed that LPS increased the expression of Raptor (n=3, 0.9±0.3) and Rictor (n=3, 0.9±0.1) when compared to control (n=3, 0.8±0.3). We sought to define whether LPS stimulation could modulate mtorc1 and mtorc2 activation, and we observed that LPS increased activation of both mtorc1 and 2. We next

8 investigated if mtorc1 inhibition could change DCs phenotype, we treated DCs with RAPA and we observed no alteration in co-stimulatory molecules expression (n=3, CD80: LPS=47±7, LPS+RAPA=42±11; CD86: LPS=39±21, LPS+RAPA=36±22; CD40: LPS=11±7, LPS+RAPA=13±5; MHC: LPS=44±8, LPS+RAPA=46±6). However, TNF levels were higher in RAPA treatment (1349±14) in comparison to LPS presence (1183±23). These results suggests that mtorc1 inhibition increases DCs activation. Therefore, we sought to determine the physiological consequences of mtorc1 modulation in DCs. We observed that absence of mtorc1 protected against AKI induced by sepsis using animals treated with RAPA (n=6, vehicle sepsis=295±32, RAPA sepsis=241±38 urea leves) and RaptorCD11cCRE [n=6, wild type (wt) sepsis=361±119, mutant sepsis=264±44 urea levels]. We also observed less DCs activation in RaptorCD11cCRE with CD40 (n=6, wt sepsis=9.9±6.4, mutant sepsis=4.4±2.5) and CD80 (n=6, wt sepsis=62±88, mutant sepsis=5.8±1.6) and diminished TNF expression (n=6, wt sepsis=22±26, mutant sepsis=1.1±0.8). Moreover, we found these mice (n=10) survived 2-fold longer than wt (n=10) accompanied by an increase of bacteria in peritoneal cavity (468±123x109) compared to control (non detected [ND]), while TSCDC11cCRE mice (n=10) had a decreased survival 1.5-fold less than wt (n=10) with less bacteria in peritoneal cavity (308±295x107) compared to control (ND). Conclusão: Our data suggest that mtor complexes are modulated by LPS and mtorc1 inhibition increases DCs activation. However, in a sepsis context mtorc1 inhibition in DCs seems to affect the whole environment in tolerogenic way, decreasing AKI and prolonging survival. Apoio Financeiro: Our data suggest that mtor complexes are modulated by LPS and mtorc1 inhibition increases DCs activation. However, in a sepsis context mtorc1 inhibition in DCs seems to affect the whole environment in tolerogenic way, decreasing AKI and prolonging survival. Comitê de Ética: 60 fls 06 livro 03

9 ANALYSIS OF OXIDANT EFFECTS OF EXPOSURE TO MEDICAL OXYGEN ON LIVER AND SPLEEN OF BALB/C MICE ADULTS Soares, N. P., Campos, K. K. D., Pena, K. B., Lima, W. G., Bezerra, F. S., Introducao: Departamento de Ciências Biológicas - UFOP Supplementary oxygen is routinely used in cardiac and pulmonary diseases, on the attempt to reverse hypoxemia and it s commonly prescribed without careful evaluation of their potential side effects on clinical practice. The oxidative damage induced by hyperoxia, have been mentioned as a factor for installation of systemic lesions. Objetivos: This study aimed to analyze the redox imbalance and cellular injury in liver and spleen of BALB/c mice exposed to hyperoxia. Metodos: Thirty male BALB/c were divided in three groups: Control (CTR), which was exposed to ambient air, exposed to oxygen on a concentration of 100% for 24 hours (H24) and 48 hours (H48). The exposure was made in a chamber, with O2 100% in a 10L/min flow. One day after the exposure the animals were euthanized, liver and spleen were collected for analysis of oxidative damage (reactive substances to thiobarbituric acid - TBARS) and activity of enzyme catalase. Data were expressed as mean ± SEM. For comparison among groups, one-way ANOVA followed by Bonferroni`s multiple comparison test were performed (p<0.05). Resultados: The group H48 (0.42±0.03, n=5) revealed higher lipidic peroxidation (p=0,0011) in liver compared to H24 (0.29 ±0.04, n=5) and CTR (0.20± 0.02, n=6). The CAT activity was higher (p=0,001) in H24 (87.69± , n=7) compared to CTR (22.13±5.18, n=6) and H48 (79.62±10.36, n=6). In spleen, the groups H48 (0.81±0.03, n=7) and H24 (0.43 ± 0.03, n =6) presented an elevated TBARS content (p<0.0001) compared to CTR (0.25 ± 0.01, n=8). The group H48 (0.81±0.03, n=7) revealed higher lipidic peroxidation compared to H24 (0.43±0.03, n=6). There were no statisticals differences between the groups in the CAT activity. Conclusão: Our initial results showed that hyperoxia promoted an oxidative damage in liver and spleen evidenced by the enhancement of catalase activity. Apoio Financeiro: Our initial results showed that hyperoxia promoted an oxidative damage in liver and spleen

10 evidenced by the enhancement of catalase activity. Comitê de Ética: CEUA

11 Caracterização temporal da sinoviopatia diabética em modelo experimental de ratos Andrade, P. C., Velosa, A. P. P., Catanozi, S., Atayde, S. A., Cruz, I. C. B., Frediane, D., Parra, Introducao: E. R., Fuller, R., Capelozzi, V. L., Teodoro, W. R., Patologia - FMUSP Endocrinologia - FMUSP Reumatologia - FMUSP Emergências Clínicas - FMUSP O diabetes mellitus apresenta disfunções nos componentes articulares, relacionadas ao dano microvascular. Estas alterações estão relacionadas ao remodelamento das fibras colágenas que sofrem modificações ocasionadas por ligações cruzadas com produtos de glicação avançada (AGEs) resultantes da hiperglicemia crônica. A sinóvia se destaca por sua função e alta vascularização, parece ser o primeiro órgão afetado e provavelmente o gatilho para os distúrbios articulares observados nestes pacientes. Objetivos: Analisar a evolução do remodelamento sinovial da articulação do joelho de ratos diabéticos induzidos por estreptozotocina. Metodos: Sessenta ratos Wistar (2,5 meses de idade e g) foram divididos em três grupos de acordo tempo de indução {7 (G1=20), 30 (G2=20) e 60 (G3=20) dias}. Os ratos diabéticos (GD1=10, GD2=10 e GD3=10) foram induzidos pela infusão de estreptozotocina (35mg/kg) e os ratos controle (GC1=10, GC2=10 e GC3=10) receberam mesmo volume de soro fisiológico. Após eutanásia, peso, glicemia e a carboximetilisina (CML) plasmática foram analisadas. Foi realizada a análise histomorfométrica (Hematoxilina e Eosina (H&E), Picrosírius e Imunofluorescência do COL I, III e V, quantificação pelo programa Image pró plus 6.0), quantificação do colágeno total pela 4-hidroxiprolina e RNAm para COL I, III e V por qpcr em Tempo Real. Resultados: Aumento glicêmico significante nos grupos diabéticos (>200mg/dl), quando comparados com os grupos controle (p <0,005). Progressiva perda de peso dos animais diabéticos (p <0,001) e aumento da CML no GD em relação ao GC (p<0,05). A análise morfológica da histoarquitetura do tecido sinovial (H&E) mostrou substituição do tecido adiposo subsinovial por um tecido fibrótico com intenso depósito de fibras colágenas, principalmente ao redor dos vasos expandindo-se para a tela subsinovial (Pricrosírius), sendo significante a partir de 30 dias de indução quando comparado ao

12 GC {G1 (p>0,05),g2 (p<0,05) G3 (p<0,05)}. Essa alteração do colágeno total foi relacionado principalmente ao aumento das fibras grossas {(G1(p>0,05), G2 (p<0,05), G3 (p<0,05)} e diminuição das fibras finas nos grupos diabéticos {G1(p>0,05) G2 ( p<0,05), G3 (p<0,05)} comparado ao controle. Pela imunofluorescência observamos aumento do COLI {G1(p>0,05) G2 ( p<0,05), G3 (p<0,05)} e a diminuição do COLIII {G1(p>0,05) G2 ( p<0,05), G3 (p<0,05)} e COLI V{G1(p>0,05) G2 ( p<0,05), G3 (p<0,05)} assim como visto no mrna. Na quantificação do colágeno total (4-hidroxiprolina) observamos que estas alterações refletiram no aumento progressivo da proteína de colágeno no tecido sinovial nos GD, sendo significantes a partir do G2 {G1(p>0,05),G2(p<0,05),G3 (p<0,05)}. Conclusão: O acometimento sinovial no diabetes se inicia precocemente e é caracterizado por intenso remodelamento, principalmente ao redor dos vasos até a tela subsinovial, associando esta estrutura ao comprometimento microvascular que antecede as complicações crônicas presente nos pacientes diabéticos. Apoio Financeiro: O acometimento sinovial no diabetes se inicia precocemente e é caracterizado por intenso remodelamento, principalmente ao redor dos vasos até a tela subsinovial, associando esta estrutura ao comprometimento microvascular que antecede as complicações crônicas presente nos pacientes diabéticos. Comitê de Ética: 149/13

13 Papel da Obesidade em Modelo Experimental de Transplante de Pele Alogênico Terra, F. F., Amano, M. T., Festuccia, W., Camara, N. O. S., Silva, M. B., Introducao: Imunologia - ICB - USP Fisiologia e Biofísica - ICB - USP O excesso de tecido adiposo está associado a múltiplas comorbidades crescentes. Além de modular o metabolismo energético, o tecido adiposo também participa do sistema endócrino. Dentre os produtos bioativos que produz, a adiponectina (APN), molécula expressa de forma reduzida na obesidade, desempenha um papel importante na regulação do metabolismo e do sistema imune. Embora pesquisas indiquem que esta seja capaz de inibir a ativação pró-inflamatória de várias células do sistema imune, pouco se sabe sobre sua ação em patologias relacionadas à reatividade alogênica. Deste modo, propomos estudar o papel da obesidade e adiponectina no processo de rejeição de órgãos, utilizando um modelo experimental de transplante de pele alogênico em camundongos. Objetivos: Avaliar o papel da obesidade e em especial da adiponectina na regulação do processo de rejeição em modelo experimental murino de transplante de pele. Metodos: Para os transplantes, transferiu-se 5cm2 de pele da cauda de camundongos doadores F1 (C57BL/6j x Balb/c) machos ao dorso de camundongos C57Bl/6 selvagens (wt) ou knockout (KO) para adiponectina (8-10 semanas, 20 a 25 g). Para a determinação da sobrevida do enxerto, considerou-se rejeição o ponto quando o tecido atingiu 100% de necrose. A seguir, avaliou-se o perfil inflamatório nos linfonodos drenantes por PCR de tempo real para a expressão gênica de IFN-g e TNF-a. O projeto associado foi aprovado pela Comissão de Ética no Uso de Animais (CEUA) do ICB/USP conforme os Princípios Éticos de Experimentação Animal adotado pela Sociedade Brasileira de Ciência de Animais de Laboratório (SBCAL), sob o número 138 nas folhas 135 do livro 02 da instituição. Resultados: Animais receptores APN KO apresentaram uma menor sobrevida do enxerto em comparação aos camundongos de tipo selvagem, indicando um papel anti-inflamatório para esta adipocina na rejeição [11,17 dias ± 2,14 (wt) vs. 8,83 dias ± 0,75 (APN KO)]. Estudos envolvendo PCR em tempo real corroboram estes resultados com tendência a expressão maior dos genes pró-

14 inflamatórios IFN-g [1,01±0,19 (wt) vs. 3,64±2,24 (APN KO)] e TNF-a [1,10 ± 0,58(wt) vs. (1,47±0,18 (APN KO)] em linfonodos drenantes de receptores APN KO (p<0.05). Conclusão: Estes resultados preliminares sugerem que a APN possui um papel na modulação da rejeição de transplantes. Estudos futuros devem elucidar o papel da obesidade-apn no controle do sistema imune e os mecanismos moduladores dos mesmos no processo de rejeição. Apoio Financeiro: Estes resultados preliminares sugerem que a APN possui um papel na modulação da rejeição de transplantes. Estudos futuros devem elucidar o papel da obesidade-apn no controle do sistema imune e os mecanismos moduladores dos mesmos no processo de rejeição. Comitê de Ética: número 138 nas folhas 135

15 Expression of HLA-G molecule in patients with oral cancer Sousa, M. C. P. S., Wastowski, I. J., Magalhães, K. C. S. F., Camargo, F. A. M., Ferreira, E., Introducao: Silva, I. L. D., Soares, E. G., Simões, R. T., Biologia Molecular - IEP-Santa Casa Patologia - UFMG Biologia - UEG Patologia - USP The non-classical HLA-G molecule is responsible for several immune-modulatory activities enabling immune tolerance to the cells that express it. Thus, the expression of HLA-G could represent an additional mechanism, used by tumor cells, in order to escape an efficient anti-tumor immune response, impairing the function of host immune effectors cells against the tumor. Objetivos: The aim of this study was to investigate HLA-G tissue expression in patients with oral cavity squamous cell carcinoma (OCSCC). Metodos: This study was approved through the Ethical Committee of Santa Casa Hospital, Belo Horizonte-MG (025/2009) and Ethical Committee of HCRP-USP, Ribeirão Preto-SP (9802/2007), Brazil. Eighty-seven formalin-fixed and paraffin-embedded oral biopsies were selected from the Department of Pathology, School of Medicine of Ribeirão Preto, University of São Paulo archives between 1992 and Patients were stratified into 4 groups according to the type of lesion: oral benign lesions (n=26), oral squamous cell carcinoma in situ (n=19), oral invasive carcinoma with metastasis (n=16) and invasive carcinoma without metastasis (n=26). MEMG02 (Exbio Antibodies, CZ) and DAKO EnVision1 System (DAKO, Carpinteria, CA) were used for immunohistochemistry assays. All statistical analyses were performed with the GraphPad Instat 4.03 and MedCalc software. Resultados: HLA-G expression was detected in 78,7% (n=48) of 61 malignant oral lesions. Following histological examination, HLA-G staining was observed in tumor cells and its expression was significantly higher in invasive well differentiated cancer (56.15±22.56; p=0.04) than in invasive moderately differentiated one (37.69±21.27). The HLA-G expression was significantly higher in invasive lesions with metastasis (59.66±23.37) were compared with invasive lesions without metastasis (46.92±23.46; p=0.04). No significant correlation could be established between HLA-G expression

16 and other clinical parameters. Conclusão: Our findings suggest that the increased HLA-G expression in tumor cells is associated with high grade lesions. Apoio Financeiro: Our findings suggest that the increased HLA-G expression in tumor cells is associated with high grade lesions. Comitê de Ética: 025/ /2007

17 Identification and immunological characterization of vaccine targets against schistosomiasis isolated by bioinformatics and molecular docking associated with cell-proliferation stimulation assay Oliveira, F. M., E.v.coelho, I., Lopes, G. F., Taranto, A. G., Comar-Junior, M., Santos, L. L., Introducao: Ribeiro, R. I. M. A., Fonseca, C. T., Oliveira, F. M., Lopes, D. O., campus centro oeste - UFSJ Laboratório de esquistossomose - CPqRR Schistosomiasis remains an important parasitic disease that affects millions of individuals worldwide. Despite the availability of chemotherapy, the occurrence of constant reinfection, particularly in poor countries, demonstrates the need for additional forms of intervention. Development of a vaccine is a relevant strategy to control this parasitic disease, which continues to spread throughout the world. With the advent of genomic and bioinformatics investigations, new strategies to search for vaccine targets have been proposed, and reverse vaccinology has played a prominent role in this context. Objetivos: Identify and synthesize epitopes of Schistosoma mansoni for evaluation of immunogenicity Metodos: Initially, bioinformatics analyses of Schistosoma mansoni membrane proteins were performed in order to identify immunogenic epitopes to be used in a vaccine formulation. The epitopes selected were synthesized, and tested in mice Black C57Bl-6 (single group of ten animals) of 6-8week (approval code of the ethics committee number 12/2010) as a cocktail of immunogens to verify their ability to induce CD4+ lymphocyte proliferation. Simultaneously, peptide interaction with human and murine MHC class II molecules was evaluated through molecular docking. The epitopes that stood out in these analyses were then used in immunization trials in mice Black C57Bl-6 (ten animals per group, control and experimental and seven animals on infection control group) to evaluate the protection conferred, the profile of antibodies and cytokines of the immune response induced by this vaccine formulation and to assess their effect on hepatic granulomas. Resultados: A significant correlation was obtained between in vitro and in vivo experiments. Two epitopes yielded best results in terms of cell-proliferative stimulation* and affinity for MHC class II molecules in molecular docking analysis**. These epitopes, used as a vaccine cocktail, induced 44%-26%*** of protection against challenge infection, significant levels of IgG, IgG2c**** and IFN-γ*****, but failed to reduce liver pathology in two independent experiments******. * percentage of CD4+ cell-proliferation stimulated by two epitopes (0,504 epitope one and 0,327

18 negative control in cell culture 72 hours/3,767 epitope two and 0,761 negative control in cell culture 120 hours) ** Energy (Kcal/mol) of epitope binding to class II MHC molecules (-8,1 epitope one and -6,7 epitope two). *** mean ± standard deviation worm burden recovered (18 ± 10 negative control group and 10 ± 4 experimental group in first trial/ 27 ± 5 negative control group and 20 ± 7 experimental group in second trial). **** mean of the levels of antibodies induced on two trials in optical density 15, 30 and 90 days after first immunization (0.044, 0.048, experimental group and 0.026, 0.025, negative control group by IgG stimulated epitope one / 0.077, 0.067, experimental group and 0,025, 0,025, 0,071 negative control group by IgG stimulated epitope two / 0.046, 0.046, experimental group and 0.026, 0.020, negative control group by IgG2c stimulated epitope one / 0.066, 0.061, experimental group and 0.029, 0.031, negative control group by IgG2c estimulated epitope two). ***** mean of the levels of IFN-γ induced on two trials in culture supernatant (460,83 pg/ml experimental group and 20,75 pg/ml negative control group) ****** mean of the volume and number of granulome/area respectively on two trials (68938,3 um2 experimental group and 71786,8 um2 negative control group / 2.09 granulomes/mm2 experimental group and 2.22 granulome/mm2 negative control group) Conclusão: The results obtained by assay immunization suggest induction of immune response Th1 type and revealed a novel strategy for the characterization of vaccine targets against schistosomiasis, involving isolation by bioinformatics analysis, testing by molecular docking, and performance of cell-proliferation stimulation assay. Apoio Financeiro: The results obtained by assay immunization suggest induction of immune response Th1 type and revealed a novel strategy for the characterization of vaccine targets against schistosomiasis, involving isolation by bioinformatics analysis, testing by molecular docking, and performance of cell-proliferation stimulation assay. Comitê de Ética: 12/2010

19 DIFFERENTIAL MODULATION OF mtor COMPLEXES IN MONOCYTES ADHESION INDUCED BY LPS AND PHORBOL ESTER. Ribeiro, M. C., Silva-filho, J. L., Abreu, T. P., Peruchetti, D. B., Souza, M. C., Henriques, M. D. G. Introducao:, Caruso-neves, C., Pinheiro, A. A. S., Instituto de Biofisica Carlos Chagas Filho - UFRJ Farmacologia Aplicada - Fundação Oswaldo Cruz Monocytes are innate immune cells and the circulating precursors of tissue macrophages. Activation of both cells is mediated by its interaction with pathogen components, such as LPS (lipopolysaccharide), which induces cell adhesion and production of proinflammatory cytokines. It is known that phorbol ester (PMA) induces monocytes adhesion and their differentiation to macrophages. However, it has been shown a different behavior of monocyte adhesion induced by PMA and LPS. mtor complexes were demonstrated to regulate immune response. In human monocytes, mtorc1 complex blocks the production of pro-inflammatory cytokines and enhances the anti-inflammatory ones. mtorc2 is involved in the actin cytoskeletal rearrangement, which could infer an important role in adhesion mechanisms. Objetivos: The aim of this study is to verify whether mtor complexes are involved in LPS and PMA-activated monocyte adhesion. Metodos: Cells were incubated, in 6-well plates, with 1ug/mL LPS or 80nM PMA for different time periods with components described. Cells adhesion was quantified by trypan blue staining exclusion. PKC activity was determined by histone phosphorylation. mtorc1 and mtorc2 phosphorylation as well as the activity of these complexes were evaluated by immunoblotting. The results are expressed as percentage in relation to the control and statistical analysis was performed using absolute values. Significance, determined as p<0.05, was assessed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. Resultados: LPS induced a transient adhesion of monocytes during 24h incubation, with maximal effect (45%) after 1 h, while with PMA adhesion increased in a time-dependent manner, achieving 100% after 24 h (n=6). Both PMA and LPS induced 40% adhesion after 1h incubation M Calphostin C, a PKC inhibitor, prevented LPS or PMA-induced adhesion in 50% after 1h, suggesting a partial dependence on PKC activity (n=4). LPS induced a 4- and 7-fold increase in PKC activity in

20 non-attached and attached cells, respectively. This effect was completely reversed by 10-6 M WYE-354, inhibitor of mtorc1 and mtorc2 activities, in both cell population but, in non-attached cells, PKC activity was partially reduced (50%) by rapamicyn, an inhibitor of mtorc1 complex (n=3). Furthermore, LPS-induced monocyte adhesion was partially reversed (40%) by WYE-354 but it was not changed by rapamycin (n=4). On the other hand, PMA effect was partially reversed by both rapamycin or WYE-354. With these treatments it is still observed a 50% adhesion induced by PMA (n=3). Immunoblotting assay reveled that LPS enhanced the phosphorylation of mtorc2 at S2481, mtorc1 at S2448 and consequently, mtorc1 substrate, S6K at T389 in non-adhered and adherent cells (n=3). PKB phosphorylation at S473 was increased by LPS treatment in non-attached cells (n=3), whereas in adhered cells this phosphorylation decreased (n=4). On the other hand, PMA increased the mtorc1 and S6K phosphorylation but did not changed mtorc2 phophorylation (n=3). In PMA stimulated cells, PKB phosphorylation at S473 decreased in the supernatant and adherent cells (n=4). This effect was not changed by calphostin C (n=2). Conclusão: Our results indicate that the adhesion induced by LPS is dependent of mtorc2/pkc/pkb/mtorc1 pathway while PMA induced adhesion is dependent of PKC/mTORC1 pathway. Apoio Financeiro: Our results indicate that the adhesion induced by LPS is dependent of mtorc2/pkc/pkb/mtorc1 pathway while PMA induced adhesion is dependent of PKC/mTORC1 pathway. Comitê de Ética: THP-1 cells, a human monocyte cell line were used.