Universidade Estadual De Londrina Graduate Program in Experimental Pathology

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Universidade Estadual de Londrina Graduate Program in Experimental Pathology

Editorial Board Alessandra Lourenço Cecchini Armani – Chair Eduardo José de Almeida Araújo – Chair Gláucia Eloisa Munhoz de Lion Siervo Glauco Akelinghton Freire Vitiello Kleber Paiva Trugilo Paulo da Silva Watanabe Victor Fattori (Universidade Estadual de Londrina)

Responsible for publishing: UNIVERSIDADE ESTADUAL DE LONDRINA PROGRAMA DE PÓS-GRADUAÇÃO EM PATOLOGIA EXPERIMENTAL

Rodovia Celso Garcia Cid. PR445 Km 380, Campus Universitário / CEP: 86057-970 LONDRINA / PR / BRAZIL Contact: Eduardo José de Almeida Araújo / Alessandra Lourenço Cecchini Armani Telephone: +55 43 3371-5498 / +55 43 3371-4387

Graduate Program in Experimental Pathology Londrina-PR 1st Edition p.1-186 2016

Catalogação na publicação elaborada pela Divisão de Processos Técnicos da Biblioteca Central da Universidade Estadual de Londrina.

Dados Internacionais de Catalogação-na-Publicação (CIP)

I61a International Symposium of Experimental Pathology (1. : 2016 : Londrina, PR) Annals [of the] I International Symposium of Experimental Pathology [e do] VI Simpósio de Patologia Experimental da UEL [livro eletrônico] / Editorial board: Eduardo José de Almeida Araújo... [ et al.]. – Londrina : UEL, 2016. 1 Livro digital.

Vários autores. Textos em português e inglês. Inclui bibliografia. Disponível em: http://www.uel.br/eventos/simposiopato/ ISBN 978-85-7846-411-0

1. Patologia experimental – Congressos. I. Araújo, Eduardo José de Almeida. II. Universidade Estadual de Londrina. Centro de Ciências Biológicas. Programa de Pós-graduação em Patologia Experimental. III. Queen Mary University of London. IV. Simpósio de Patologia Experimental da UEL (6. : 2016 : Londrina, PR). V. Título. VI. Annals [do] VI Simpósio de Patologia Experimental da UEL.

CDU 616-092

SUMMARY

ABSTRACTS ...... 13 NEGLECTED DISEASES CHALLENGES IN PARACOCCIDIOIDOMYCOSIS: NON-REACTIVE IMMUNODIFFUSION ...... 14 PHYSICAL TRAINING MODULATES NITRIC OXIDE PRODUCTION AND CYTOKINE IMPROVING CARDIOVASCULAR RESPONSE AND RESISTANCE TO INFECTION TO Trypanosoma cruzi ...... 15 Caryocar coriaceum EXERTS EFFECTS AGAINST PROMASTIGOTE FORMS OF Leishmania amazonensis ...... 16 ANTI-AMASTIGOTE EFFECT OF DIFFERENT EXTRACTS OF Caryocar coriaceum, A BRAZILIAN CERRADO PLANT ...... 17 INHIBITION OF Paracoccidioides brasiliensis GROWTH BY SOIL YEAST CRUDE EXTRACT ...... 18 NANOTECHNOLOGY IN THE TREATMENT OF TOXOPLASMOSIS: A REVIEW ...... 19 ROSUVASTATIN INHIBIT INFECTION AND PROLIFERATION OF TACHYZOITES OF Toxoplasma gondii (RH STRAIN) IN HeLa CELLS ...... 20 LYMPHOTROPIC VIRUS INFLUENCE OF HUMAN T CELL TYPE 1 IN PATIENTS COINFECTED WITH HELMINTH Strongyloides stercoralis - A SYSTEMATIC REVIEW ...... 21 MACROPHAGES ACTIVATED WITH CONCANAVALIN-A INCREASE LEISHMANICIDE ACTIVITY BY REACTIVE OXYGEN SPECIES PRODUCTION ...... 22 CONCANAVALIN-A INDUCES TNF-α AND NITRIC OXIDE PRODUCTION BY PERITONEAL CELLS PRETREATED IN EXPERIMENTAL LEISHMANIASIS ...... 23 ACUTE Toxoplasma gondii INFECTION ALTERS THE MYELOPEROXIDASE ACTIVITY IN RAT DUODENUM ...... 24 COMBINED BENZNIDAZOLE AND ASPIRIN CHEMOTHERAPY IN MICE INFECTED WITH Trypanosoma cruzi ...... 25 CROSSTALK BETWEEN CYCLOOXYGENASE PATHWAY AND ADENYLATE-CYCLASE IN THE REGULATION OF ANTI-Trypanosoma cruzi ACTIVITY BY HUMAN MONOCYTES ...... 26 NON-COMMUNICABLE DISEASES HORMONE THERAPY ATTENUATES THE SYSTEMIC PROOXIDANT STATUS IN POSTMENOPAUSAL WOMEN ...... 27 PROTECTIVE ASSOCIATION OF TGF-βR2 GENE POLYMORPHISM AND CYTOKINE PLASMA LEVELS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA...... 28 PROBUCOL ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION BY REDUCING LEUKOCYTE RECRUITMENT, CYTOKINE PRODUCTION AND NF-КB ACTIVITY ...... 29 HASHIMOTO’S THYROIDITIS: A REVIEW ARTICLE ...... 30 MUSCLE RECONDITIONING IN CHRONIC OBSTRUTIVE PULMONARY DISEASE ...... 31

EXPERIMENTAL MODEL PADRONIZATION OF MURINE ORAL’S MELANOMA: PILOT STUDY ...... 32 iNOS PARTICIPATION IN CARDIOVASCULAR AND OXIDATIVE PARAMETERS OF FEMALE RATS WITH ENDOTOXEMIA INDUCED BY LPS ...... 33 INVOLVEMENT OF NITRIC OXIDE SYNTHASE ISOFORMS IN CARDIOVASCULAR, AUTONOMIC AND OXIDATIVE ASPECTS IN FEMALE RATS ...... 34 GENETIC POLYMORPHISMS AND EXPRESSION OF FOXP3 mRNA IN AGGRESSIVE BREAST CANCER SUBTYPES ...... 35 CYTOKINE PROFILE IN PATIENTS WITH PROGRESSIVE MULTIPLE SCLEROSIS AND ITS ASSOCIATION WITH DISEASE PROGRESSION AND DISABILITY ...... 36 HISTOPATHOLOGICAL EVALUATION FOR ABERRANT CRYPT FOCI (ACF) STUDY IN EXPERIMENTAL MODEL OF COLORECTAL CANCER IN INDUCED BY 1,2-DIMETILHIDRAZINA (DMH) IN RATS WISTAR...... 37 INTRAUTERINE AND LACTATIONAL EXPOSURE TO FLUOXETINE INHIBITED THE AORTIC ADAPTATION INDUCED BY ACUTE RESTRAIN STRESS IN RATS ...... 38 DAMAGE CAUSED BY YELLOW FEVER VACCINE ON THE GESTATIONAL DEVELOPMENT ...... 39 MATERNAL EXPOSURE TO METFORMIN DID NOT INTERFERE WITH VASCULAR ENDOTHELIAL FUNCTION OF ADULT MALE OFFSPRING ...... 40 GENE EXPRESSION ANALYSIS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA: CHEMOTHERAPY INFLUENCE IN KEY REGULATING GENES ...... 41 BARK ETHANOLIC EXTRACT FROM Spondias dulcis FORST F. PROTECTS AGAINST ALTERATIONS OF THE REDOX STATUS INDUCED BY CYCLOPHOSPHAMIDE AND BENZO[a]PYRENE IN MICE ... 42 THE VANILLIC ACID INHIBITS THE EHLICH-TUMOR CELLS INDUCED PAIN LIKE-BEHAVIOR ...... 43 POSSIBLE EFFECTS OF TGFB1 SIGNAL PEPTIDE AND PROMOTER REGION GENETIC POLYMORPHISMS AND HAPLOTYPE STRUCTURES: POSSIBLE ROLE AS A SUSCEPTIBILITY MARKER ...... 44 VITAMIN D DEFICIENCY IS ASSOCIATED WITH ACUTE ISCHEMIC STROKE, C-REACTIVE PROTEIN, AND SHORT-TIME OUTCOME ...... 45 IMMUNE RESPONSE AND VIRULENCE FACTORS ASSOCIATED CANDIDIASIS IN DIABETES: A REVIEW ...... 46 EVALUATION OF METABOLIC AND CARDIOVASCULAR PARAMETERS OF MALE AND FEMALE RATS EXPOSED TO METFORMIN DURING GESTATIONAL AND LACTATION PERIOD ...... 47 MULTIPLE ENDOCRINE NEOPLASIA TYPE 2A: CASE REPORT ...... 48 PROTECTIVE EFFECTS OF NICOTINAMIDE ON MICE OFFSPRING EXPOSED TO CYCLOPHOSPHAMIDE DURING PREGNANCY ...... 49 ALTERATIONS IN REPRODUCTIVE PARAMETERS OF MICE EXPOSED TO WINE DURING SPERMATOGENESIS ...... 50 ANTIBIOTIC SUSCEPTIBILITY OF Staphylococcus aureus ISOLATED FROM BLOODSTREAM INFECTIONS IN A UNIVERSITY HOSPITAL SOUTH BRAZIL FOR A PERIODO OF FIFTEEN YEARS ... 51

EVALUATION OF PHYSICAL AND REFLEXOLOGICAL DEVELOPMENT OF MICE OFFSPRING EXPOSED TO PROPOFOL IN DIFFERENT GESTACIONAL PERÍODS ...... 52 NITRIC OXIDE-GLUTAMATE INTERACTIONS IN ROSTRAL VENTROLATERAL MEDULLA OF OBESE RATS: INVOLVEMENT OF iNOS ISOFORM ...... 53 SKELETAL MUSCLE WASTING PROMOTES LIPID PEROXIDATION AND SARCOPLASMIC RETICULUM ALTERATIONS IN A FIBER TYPE DEPENDENT MANNER ...... 54 KAURENOIC ACID REDUCES EOSINOPHIL RECRUITMENT IN ASTHMA BY REDUCING TH2 CYTOKINE PRODUCTION IN MICE...... 55 MACROPHAGES-EXPRESSING FOXP3 TRANSCRIPTION FACTOR: DO THEY REALLY EXIST? ...... 56 EFFECT OF METFORMIN IN ACUTE MODEL OF MURINE UVB IRRADIATION AND THE INVOLVEMENT OF SYSTEMIC ROS ...... 57 ROLE OF ROS AND IMMUNE RESPONSE OF LANGERHANS CELL IN AN ACUTE UVB IRRADIATION MURINE MODEL TREATED WITH METFORMIN ...... 58 ALUMINIUM CHLORIDE, DURING THE PERIBUBERTAL PERIOD, AFFECT THE CYTOKINES LEVEL AND MORFOLOGY IN TESTES OF RATS ...... 59 SYSTEMIC OXIDATIVE STRESS PROFILE DURING THE PROGRESSION OF EXPERIMENTAL METASTASIS OF MURINE MELANOMA ...... 60 SYSTEMIC OXIDATIVE STRESS PROFILE DURING THE PROGRESSION OF EXPERIMENTAL METASTASIS OF MURINE MELANOMA ...... 61 CYTOTOXICITY EFFECT IN TM3 CELL LINE (LEYDIG CELL) CAUSED BY BPA (BISPHENOL A) ...... 62 TANSFORMING GROWTH FACTOR TYPE 2 RECPTOR (TGFβR2) PROMOTER REGION POLYMORPHISM EXERT SUBTYPE SPECIFIC ROLES IN BREAST CANCER ...... 63 THE ROLE OF TRANSFORMING GROWTH FACTOR BETA 1 (TGFB1) FUNCTIONAL POLYMORPHISMS IN GENE EXPRESSION AND CYTOKINE PRODUCTION ...... 64 METFORMIN REGULATES SKIN REDOX PROFILE IN UVB-INDUCED SQUAMOUS SKIN CELL CARCINOMA ...... 65 Rhopalurus rochai VENOM-INDUCED INFLAMMATORY PAIN, OVERT-PAIN LIKE BEHAVIOR DEPENDS ON CELL RECRUITMENT AND CYTOKINES PRODUCTION ...... 66 Tityus serrulatus VENOM INDUCES INFLAMMATORY PAIN IN MICE ...... 67 MALIGNANT MELANOMA SKIN ...... 68 OBSTRUCTIVE PULMONARY DISEASE: PATHOPHYSIOLOGY AND REHABILITATION PULMONARY ...... 69 DOES MATERNAL EXPOSURE TO FLUOXETINE INDUCE METABOLIC ALTERATIONS IN ADULT MALE OFFSPRING? ...... 70 KAURENOIC ACID REDUCES DICLOFENAC-INDUCED ACUTE KIDNEY INJURY THROUGH INHIBITION OF OXIDATIVE STRESS IN MICE ...... 71 EVALUATION OF BIOCHEMICAL PARAMETERS OF DIABETICS RATS SUBMITTED TO FOOD RESTRICTION DIET ...... 72

CITRAL HAS CYTOTOXIC ACTION IN MURINE MELANOMA CELLS (B16F10) BY GENERATING REACTIVE OXYGEN SPECIES ...... 73 EFFECTS OF NATURAL RUBBER LATEX MEMBRANES STABILIZED WITH DIFFERENT CONCENTRATIONS OF AMMONIA IN CHO-K1 CELLS ...... 74 LATEX C-SERUM FROM Hevea brasiliensis INDUCES NECROTIC CELL DEATH IN MURINE MELANOMA CELL LINE (B16F10) ...... 75 CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION AND EVALUATION OF ERYTHROCYTIC ANTIOXIDANT DEFENSES OF PATIENTS AFFECTED BY BASAL CELL CARCINOMA IN THE NORTHERN REGION OF PARANA ...... 76 CELL ADHESION MOLECULES EVALUATION AS PREDICTORS OF DIAGNOSIS AND DISEASE ACTIVITY IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS...... 77 BARK ETHANOLIC EXTRACT FROM Spondias dulcis FORST F. PROTECTS AGAINST ALTERATIONS OF THE REDOX STATUS INDUCED BY CYCLOPHOSPHAMIDE AND BENZO[a]PYRENE IN MICE ... 78 THE PREDICTIVE VALUE OF TRANSFORMING GROWTH FACTOR-β IN WILMS TUMOR IMMUNOPATHOGENESIS ...... 79 TREATMENT WITH PIOGLITAZONE AND INSULIN IN WALKER-256 TUMOR BEARING RATS: EFFECTS ON PANCREATIC ISLETS ...... 80 CINNAMON, TURMERIC AND OKRA: EFFECTS OF DIET ON BODY WEIGHT AND BIOCHEMICAL PARAMETERS IN WISTAR RATS ...... 81 Hypericum perforatum REDUCES INFLAMMATORY PAIN IN MICE ...... 82 CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION OF PATIENTS AFFECTED BY SQUAMOUS CELL CARCINOMA ALONE AND ASSOCIATED WITH OTHERS SKIN NEOPLASIAS IN THE NORTHERN REGION OF PARANA ...... 83 SIM2 AS A RNA CIRCULANTING MARKER FOR PROSTATE CANCER DIAGNOSIS AND PROGNOSIS ...... 84 Tityus bahiensis VENOM INDUCES INFLAMMATORY PAIN IN MICE ...... 85 ANALGESIC AND ANTI-INFLAMMATORY EFFECT OF NARINGENIN IN TITANIUM DIOXIDE- INDUCED ARTHRITS ...... 86 CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION OF PATIENTS AFFECTED BY MULTIPLE SKIN NEOPLASIAS IN THE NORTHERN REGION OF PARANA...... 88 EVALUATION OF rs11781889 POLIMORPHYSM OF NKX3.1 GENE AND PROSTATE CANCER RISK ...... 89 POLYMORPHISM ANALYSIS OF GENE AR IN PATIENTS WITH PROSTATE CANCER ...... 90 INVERTED PAPILLOMA. CASE REPORT ...... 91 CLINICO-PATHOLOGICAL PARAMETERS INVOLVED IN THE ASSOCIATION BETWEEN DIFFERENTIATED THYROID CANCER AND HASHIMOTO THYROIDITIS: A REVISION OF THE LITERATURE ...... 92

INDUCTION OF LIPID PEROXIDATION AND OXIDATIVE INJURY OF DNA BY CURCUMIN IN HUMAN BREAST CANCER CELLS MCF7 AND MDA-MB-231 ...... 93 SEARCH OF HUMAN MAMMARY TUMOUR VIRUS (MMTV-LIKE) DNA IN BREAST CANCER MICROENVIROMENT ...... 94 OXIDATIVE AND NITROSATIVE STRESS AND AUTOANTIBODIES ARE ASSOCIATED WITH LUPUS NEPHRITIS ...... 95 CREATINE SUPPLEMENTATION INCREASES LIVER OXIDATIVE STRESS AND LIPIDS ACCUMULATION IN A MODEL OF MURINE ALCOHOLIC STEATOSIS ...... 96 METFORMIN PRETREATMENT PREVENTS DOXORUBICIN RESISTANCE INDUCTION IN MCF-7 AND MDA-MB-231 HUMAN BREAST CANCER CELLS THROUGH OXIDATIVE STRESS GENERATION, INDUCTION OF P53 AND TGF-Β1 AND DECREASE OF NRF2 ...... 97 COLON HISTOPATOLOGICAL EVALUATION OF RATS WISTAR FOR ABERRANT CRYPT FOCI (ACF) STUDY AND ADENOMATOUS INTESTINAL TUMOR ...... 98 VINPOCETINE AS ANTI-INFLAMMATORY AGENT – A REVIEW ...... 99 ALBUMIN AND PROTEIN OXIDATION ARE PREDICTORS THAT DIFFERENTIATE RELAPSING- REMITTING FROM PROGRESSIVE CLINICAL FORMS OF MULTIPLE SCLEROSIS ...... 100 EVALUATION OF ANTINFLAMMATORY AND ANTIOXIDANT EFFECTS OF HESPERIDIN-METHYL- CHALCONE IN LPS-INDUCED ACUTE LUNG INJURY IN MICE...... 101 EVALUATION OF OCCUPATIONAL EXPOSURE AT PESTICIDES USING BIOCHEMICAL, GENOTOXIC AND OXIDATIVE MARKERS ...... 102 VINPOCETINE AMELIORATES ACETIC ACID-INDUCED COLITIS BY INHIBITING OXIDATIVE STRESS AND PRO-INFLAMMATORY CYTOKINES PRODUCTION IN MICE ...... 103 SYSTEMIC CHANGES IN THE REDOX STATUS IN A MURINE MODEL OF UVB-INDUCED NONMELANOMA SKIN CANCER ...... 104 CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION AND EVALUATION OF ERYTHROCYTIC ANTIOXIDANT DEFENSES OF PATIENTS AFFECTED BY ACTINIC KERATOSIS AND NONMELANOMA SKIN CANCER IN THE NORTHERN REGION OF PARANA...... 105 EFFECTS OF SLEEP RESTRICITON DURING PERIPUBERTY ON TESTICULAR DEVELOPMENT OF RATS ...... 106 INFLAMMATION AND OXIDATIVE STRESS IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS WITH METABOLIC SYNDROME – A REVIEW ...... 107 COMMUNICABLE DISEASES LEPROSY IN PRUDENTÓPOLIS: THE PORTRAIT OF ONE OF PARANÁ'S INLAND CITIES ...... 108 P2 ALLELE OF PVUII GENETIC POLYMORPHISM OF LOW-DENSITY LIPOPROTEIN RECEPTOR IS ASSOCIATED WITH INCREASED LIPID PEROXIDATION AND LIVER INJURY IN CHRONIC HEPATITIS C PATIENTS ...... 109 THE PROFILE OF OXIDATIVE STRESS MARKERS IS DEPENDENT ON VITAMIN D LEVELS IN PATIENTS WITH CHRONIC HEPATITIS C ...... 110

POLYMORPHISM rs3087465 IN THE TGFBR2 GENE IS ASSOCIATED WITH HUMAN PAPILLOMAVIRUS INFECTION ...... 111 SYPHILIS: MOTHER TO SON ...... 112 THE INFLUENCE OF TGFB1 POLYMORPHISM RS1800469 (C.-1347C>T) IN THE HPV INFECTION AND CERVICAL LESIONS IMMUNOPATHOGENESIS ...... 113 TGFB1 SIGNAL PEPTIDE POLYMORPHISMS INFLUENCE HPV INFECTION AND CERVICAL LESIONS DEVELOPMENT ...... 114 INTESTINAL INFLAMMATION GENERATED BY TOXOPLASMOSIS PROMOTE LOSS NEURONAL AND GLIAL POPULATION OF ILEUM SUBMUCOSAL PLEXUS OF RATS Wistar ...... 115 COINFECTION BETWEEN HPV AND Chlamydia trachomatis AND ITS ROLE IN CERVICAL LESIONS DEVELOPMENT ...... 116 ANTIBODIES IgY ANTI-NP OF THE VIRUS H1N1 FOR DEVELOPMENT OF DIAGNOSTIC TESTS FOR THE DETECTION OF THE INFLUENZA VIRUS ...... 117 INFLUENCE OF CXCL12 RS1801157 POLYMORPHISM IN HPV INFECTION AND LESION DEVELOPMENT ...... 118 GENETIC AND NON GENETIC FACTORS IN THE ETIOLOGY OF CARIES IN CHILD PRESCHOOL ... 119 PHYSIOLOGICAL AND MOLECULAR CHARACTERISTICS OF CARBAPENEM RESISTANCE IN Klebsiella pneumoniae AND Enterobacter aerogenes ...... 120 PLACENTAL HISTOPATHOLOGIC ALTERATIONS AS A TOOL IN CONGENITAL ZIKA VIRUS INFECTION DIAGNOSIS: A CASE REPORT ...... 121 CYTOKINE PROFILE FOLLOWING INTRAPERITONEAL INFECTION BY Escherichia coli ENTEROHEMORRHAGIC IN SWISS MICE ...... 122 EXPANDED ABSTRACTS ...... 123 CARACTERIZAÇÃO DE POLIMORFISMOS DO TGFB1 E EXPRESSÃODO TGFB1, TGFBR1 e TGFBR2 EM PACIENTES COM CÂNCER CERVICAL ...... 124 AVALIAÇÃO DOS EFEITOS DA DIETA HIPERLIPÍDICA ISOLADA OU ASSOCIADA AO HIPOTIREOIDISMO E HIPERTIREOIDISMO SOBRE O TESTÍCULO E EPIDÍDIMO DE CAMUNONGOS C57BL/6: ANÁLISES MORFOFISIOLÓGICAS E MOLECULARES ...... 128 AVALIAÇÃO DA PERFORMANCE REPRODUTIVA DE CAMUNDONGOS EXPOSTOS AO CLORIDRATO DE BUPROPIONA DURANTE A ESPERMATOGÊNESE E DOS POSSIVEIS EFEITOS TERATOGÊNICOS, DESENVOLVIMENTO PÓS NATAL E COMPORTAMENTAIS NA PROLE ...... 132 AVALIAÇÃO DOS EFEITOS DO INSETICIDA MALATION SOBRE O DESENVOLVIMENTO DO SISTEMA GENITAL MASCULINO DE RATOS DESDE O PERÍODO JUVENIL ATÉ A PUBERDADE ... 135 O PAPEL DE AGRECANAS EM DISTÚRBIOS GASTROINTESTINAIS DE HUMANOS E CAMUNDONGOS ...... 139 O PAPEL DE FOSFACANAS EM DISTÚRBIOS GASTROINTESTINAIS DE HUMANOS E CAMUNDONGOS ...... 143

ANÁLISE DO CÓLON DE RATOS SUBMETIDOS A DIFERENTE DURAÇÕES DE INFECÇÃO CAUSADA POR Toxoplasma gondii ...... 147 ESTUDO COMPARATIVO DA FISIOPATOLOGIA NO ESCORPIONISMO INDUZIDO POR PEÇONHAS DOS Tityus serrulatus E Rhopalurus rochai ...... 150 ESTABELECIMENTO DA DIFERENÇA CRITICA EM PARÂMETROS DE ESTRESSE OXIDATIVO UTILIZANDO PROTOCOLO DE EXERCÍCIO EXCÊNTRICO ...... 153 ESTRESSE OXIDATIVO E PERFIL INFLAMATÓRIO SISTÊMICO NA DOENÇA ARTERIAL OCLUSIVA PERIFÉRICA (DAOP) EM HUMANOS ...... 157 ESTUDO DOS NÍVEIS DE VITAMINA D E POLIMORFISMO VRD-FOKI EM PACIENTES COM CÂNCER COLORRETAL: CORRELAÇÃO COM O ESTADIAMENTO DA DOENÇA...... 161 AVALIAÇÃO DA REATIVAÇÃO DO CITOMEGALOVÍRUS EM PACIENTES COM SEPSE BACTERIANA EM UNIDADES DE TERAPIA INTENSIVA ...... 165 AVALIAÇÃO DOS EFEITOS ESPINAIS DO MEDIADOR LIPÍDICO PRÓ-RESOLUÇÃO RESOLVINA D1 EM MODELO DE GOTA INDUZIDA POR CRISTAIS DE MONOURATO DE SÓDIO (MSU) ...... 169 AVALIAÇÃO DOS EFEITOS ESPINAIS DO MEDIADOR LIPÍDICO PRÓ RESOLUÇÃO RESOLVINA D2 EM MODELO DE ARTRITE GOTOSA INDUZIDA POR CRISTAIS DE MONOURATO DE SÓDIO (MSU) ...... 173 AVALIAÇÃO DO EFEITO ANTINOCICEPTIVO E ATIVIDADE ANTIOXIDANTE DO DIOSMIN EM MODELO DE DOR NEUROPÁTICA INDUZIDA PELA CONSTRIÇÃO DO NERVO CIÁTICO EM CAMUNDONGOS ...... 177 INDEX ...... 181

PROGRAM

ABSTRACTS

CHALLENGES IN PARACOCCIDIOIDOMYCOSIS: NON-REACTIVE IMMUNODIFFUSION

Adriane Lenhard-Vidal1, João Paulo Assolini1, Nilson de Jesus Carlos1, Eiko Nakagawa Itano1 1 Laboratório de Imunologia Aplicada, Depto de Ciências Patológicas/CCB, Universidade Estadual de Londrina, Rodovia Celso Garcia Cida (PR-445), km 380, 86057-970 Londrina, PR, Brasil.

Introduction: Paracoccidioidomycosis (PCM) is caused by the thermodimorphic fungi Paracoccidioides spp. In the Brazilian public health system, its diagnosis relies on the use of immunodiffusion (ID), that despite having low costs and being easy to carry out, has lower sensibility than other tests. Objective: to evaluate the rate of misdiagnosis of PCM by the use of ID. Material and methods: Current research was approved by the Internal Scientific Commission and the Research Bioethics Committee of the State University of Londrina (n. 33455 2011.06, CAEE 0314.0.268.000-11). The 32 sera were from patients already clinically diagnosed, living in the northern and west regions of Paraná State, Brazil (HC-HURNP-COU UEL; Municipal Laboratory Foz do Iguaçu; HC UNIOESTE). Cell free antigens (CFA) and exoantigens (ExoAg) were produced from P. brasiliensis strain B339. The ID tests were performed in duplicates, with the antigens in the central position and titration of the sera (pure and up to 1/32) in the surrounding wells (25 μL/well). In addition, IgG was detected by indirect ELISA (CFA 25 µg/mL, ExoAg 1/4000). Results: Ten samples (31%) were negative in ID, out of which seven were negative in both CFA and ExoAg ID. If ELISA was co-employed as part of the diagnosis, these patients would be considered positive, but the majority (70%) had low or intermediate titers/optical density readings. In addition to the difficulty to compare results among institutions due to the lack of antigen standardization (strain, production, concentration, etc.), the employment of solely ID is another obstacle. The use of a second immunological method when ID turns out negative seems advisable. Conclusion: There would be a high rate of false negative results if only ID was employed. Although ID may be cost-effective and easy to perform in any laboratory, it is necessary to investigate other more sensitive methods.

Keywords: ELISA Immunodiagnosis; Immunoenzymatic assay. Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária/PR and Pró-reitorias de Pós-graduação e Extensão from Londrina State University (PROPPG/PROEX/UEL). Thematic area: Neglected diseases.

PHYSICAL TRAINING MODULATES NITRIC OXIDE PRODUCTION AND CYTOKINE IMPROVING CARDIOVASCULAR RESPONSE AND RESISTANCE TO INFECTION TO Trypanosoma cruzi

Bruno Fernando Cruz Lucchetti1, Nágela Ghabdan Zanluqui1, Hiviny de Ataides Raquel1, Maria Isabel Lovo-Martins1, Vera Lúcia Hideko Tatakihara1, Mônica de Oliveira Belém1, Lisete Compagno Michelin2, Eduardo José de Almeida Araújo1, Phileno Pinge-Filho1, Marli Cardoso Martins Pinge1. 1Universidade Estadual de Londrina, Londrina-PR, 2Universidade de São Paulo, São Paulo-SP

Introduction: Trypanosoma cruzi infection causes innate immune response initiated in the acute phase responsible for host resistance to the parasite. It is observed progressive heart inflammation, fibrosis with changes in the architecture and functionality of the heart. Recent studies have shown that chronic physical activity (PA) of moderate intensity can act as a resistance factor against T. cruzi infection in animals. However, the cardiovascular parameters of mean arterial pressure and heart rate (HR), and nitric oxide levels (NO) accompanying the acute phase of infection in animals previously trained, are not clear. Objective: Evaluate the effects of previous physical conditioning on inflammatory, cardiovascular and biochemical aspects of resistance to infection by T. cruzi. Material and methods: Study approved by CEUA:28105.2014.72. Swiss mice were randomized into four groups: control sedentary, control trained, sedentary infected (SI), and trained infected (TI). Physical activity was performed on a treadmill for nine weeks. After PA mice were infected with 5x10³ trypomastigotes of T. cruzi (y strain). Results: We observe resting bradycardia and better performance in trained animals compared to sedentary. On the 20th day post-infection (dpi), we found a decrease in HR in SI compared with TI animals (699.73±42.37vs742.11±25.35bpm). There was also an increase in NO production in heart tissue in 20 dpi in SI, and normalized in the TI group (20.73±2.74vs6.51±1.19). An increase in the plasma concentration of pro-inflammatory cytokines (IL- 12, TNF-α, IFN-γ,) and MCP-1 was found in the SI group, compared to TI. An increase in parasitaemia in 15 and 17 dpi in the SI group, since the TI group this increase was attenuated. In addition, TI animals showed increased survival compared to the SI. Conclusion: Our results suggest that previous PA plays a preventive role in resistance to infection by T. cruzi, modulating the inflammatory response and NO levels in infected mice.

Keywords: Chagas disease; Inflammation; Arterial pressure; Heart rate Grants: CAPES and CNPq. Thematic area: Neglected diseases.

Caryocar coriaceum EXERTS EFFECTS AGAINST PROMASTIGOTE FORMS OF Leishmania amazonensis

Fernanda Tomiotto-Pellissier1; Daniela Ribeiro Alves2; Selene Maia de Morais 2, Milena Menegazzo Miranda-Sapla1, Bruna Taciane da Silva Bortoleti1, João Paulo Assolini1, Manoela Daiele Gonçalves1, Virginia Marcia Concato1, Cláudia Stoeglehner Sahd1, Alan Ferreira Chagas1, Idesania Nazareth Costa1, Ivete Conchon-Costa1, Wander Rogério Pavanelli1. 1 State University of Londrina, Londrina 86057-970, Paraná, Brazil 2 State University of Ceará, Fortaleza 60.714.903, Ceará, Brazil

Introduction: Leishmaniasis is caused by species of the protozoa Leishmania. The lack of effective drugs and the side effects, coupled with the inability of host to eliminate the parasite, have been the driving force behind the search for new chemotherapeutic agents. Plants of Caryocar genus are widely distributed Brazilian cerrado trees, that have been used as food and popular medicine. Studies have demonstrated several biological effects of Caryocar sp., as activity against bacteria, fungi, protozoa and tumor cells. Objective: In this sense, the aim of the present study was to verify the leishmanicide effect of extracts from different parts of the plant C. coriaceum on promastigotes forms of L. amazonensis. Material and methods: It was used different plant extracts, according to the origin and solvent, respectively: (1) fruit pulp, ethanol; (2) fruit peel, ethanol; (3) leaf, ethyl acetate. The extracts were tested at 0.05 mg/mL. Promastigotes forms of L. amazonensis were maintained in 199 medium. The antipromastigote assay was performed through neubauer chamber counting and samples were analyzed by Scanning Electron Microscopy (SEM). Results: Our data showed that all the tested extracts were able to reduce the amount of viable pomastigotes after 24 (p<0.001), 48 (p<0.0001) and 72 (p<0.0001) hours of treatment. In addition, by SEM we observed changes in cellular structure, as signs of shrinkage and flagella with alterated lengths, of treated parasites when compared to the control (medium alone). Conclusion: The results demonstrate that extracts from fruit and leaf of C. coriaceum can act on free forms of L. amazonensis, prompting future studies aiming the understanding the mechanism of action of these extracts on the parasite.

Keywords: Scanning Electron Microscopy; Leishmanicidal; Leishmaniasis. Thematic area: Neglected diseases.

ANTI-AMASTIGOTE EFFECT OF DIFFERENT EXTRACTS OF Caryocar coriaceum, A BRAZILIAN CERRADO PLANT

Fernanda Tomiotto-Pellissier1; Daniela Ribeiro Alves2; Selene Maia de Morais 2, Milena Menegazzo Miranda-Sapla1, Bruna Taciane da Silva Bortoleti1, João Paulo Assolini1, Manoela Daiele Gonçalves1, Virginia Marcia Concato1, Cláudia Stoeglehner Sahd1, Idesania Nazareth Costa1, Ivete Conchon-Costa1, Wander Rogério Pavanelli1. 1 State University of Londrina, Londrina 86057-970, Paraná, Brazil 2 State University of Ceará, Fortaleza 60.714.903, Ceará, Brazil

Introduction: The currently available chemotherapy for the treatment of American Tegumentar Leishmaniasis (ATL) has toxicity, low efficiency and difficulty of administration, prompting a search for alternative treatments. Plants of the Caryocar genus are found in brazilian cerrado, where are used as food and in folk medicine. Studies have demonstrated several biological effects, however, there are no studies that verify the leishmanicidal potential of Caryocar coriaceum extracts. Objective: The objective of this study was to verify the activity of different extracts of C. coriaceum on intracellular forms of L. amazonensis. Material and methods: In the present study it was used different extracts, according to the origin and solvent, respectively: (1) pulp of the fruit, ethanol; (2) peel of the fruit, ethanol; (3) leaf, ethyl acetate; (4) leaf, methanol. The extracts were tested at 0.025; 0.05 and 0.1 mg/mL. To evaluate the anti-amastigote activity, peritoneal macrophages from BALB/c mice (ethics committee: 13134.2016.62) were infected with the parasite and treated with the extracts for 24h, after that were analyzed the percentage of infected macrophages and the number of amastigotes per macrophage by optical microscopy. In order to confirm this experiment, after the described time, the macrophages were transferred under optimal conditions for differentiation of viable amastigotes into extracellular promastigotes. The number of viable parasites was counted in Neubauer chamber. Results: When evaluating the anti-amastigote effect, all extracts were able to reduce the percent of infected macrophages after 24h of treatment (p<0.001), however, the number of amastigotes per macrophages was not affected by the treatment. When analyzed recovery of viable forms of the parasite, all extracts showed significant reduction (p≤0.01) after 48 and 72h of experiment, confirming the leishmanicide effect of the extracts. Conclusion: Together, our data showed that the tested extracts have an effect on the intracellular amastigote forms of L. amazonensis.

Keywords: Leishmaniasis; Leishmanicide; Macrophages. Thematic area: Neglected diseases.

INHIBITION OF Paracoccidioides brasiliensis GROWTH BY SOIL YEAST CRUDE EXTRACT

Giovana Gomes de Carvalho1; Raquel Pires Nakama1; Mario Augusto Ono1 1 Londrina State University, Biological Sciences Center, Pathological Sciences Department, Animal Immunology Laboratory, Londrina, PR, Brazil.

Introduction: Fungi of the Paracoccidioides genus are the etiological agents of paracoccidioidomycosis, a systemic granulomatous mycosis most prevalent in Latin America and Brazil has the highest number of cases. The habitat of Paracoccidioides spp. is not yet defined, although studies suggest that it inhabits the soil as other human pathogenic fungi. The study of interaction between Paracoccidioides spp. and other organisms from the soil will help to clarify eco- epidemiological aspects of paracoccidioidomycosys. Objective: This study aimed to evaluate the effect of extracts of yeasts isolated from soil on the growth of P. brasiliensis. Material and methods: Six yeasts isolated from soil were cultured in brain heart infusion broth at 25 or 36ºC under constant agitation for 5 days and then the media were either filtrated in 0,22 micrometer filter or autoclavated at 121ºC for 20 minutes and filtrated in filter paper. Three isolates of P. brasiliensis (B339, Pb18 and LBR1) were inoculated into plates containing Sabouraud agar medium with extracts and incubated at 36ºC. The diameter of isolates was measured after 14 days. T Student’s test was applied to verify differences between groups (results significant when p<0,05). Results: Filtered extracts inhibited more P. brasiliensis growth than autoclavated ones (p<0,05). Regarding the effect of temperature, there was no significant difference between groups. Pb18 was the most inhibited isolate, being negatively affected by 5 autoclavated supernatants produced at 25ºC, 3 produced at 36ºC and all filtrated extracts. B-339 and LBR1 isolates were less inhibited by the autoclavated extracts in comparison to the filtrated ones. Interestingly, some extracts (mainly autoclavated ones) stimulated P. brasiliensis growth, especially LBR1. This may indicate the presence of some thermosensitive component in the extracts. Conclusion: These results suggest that the inhibition by yeasts may one of the factors that hinders the isolation of Paracoccidioides spp. from soil.

Keywords: Paracoccidiodomycosis; Inhibition; soil yeasts. Thematic area: Neglected diseases.

NANOTECHNOLOGY IN THE TREATMENT OF TOXOPLASMOSIS: A REVIEW

João Paulo Assolini1; Virginia Márcia Concato1; Manoela Daiele Gonçalves1; Amanda Cristina Machado Carloto1; Ivete Conchon-Costa1; Wander Rogério Pavanelli1; Francine Nesello Melanda1; Idessania Nazareth Costa1 1 State University of Londrina, Londrina 86057-970, Paraná, Brazil

Introduction: Toxoplasmosis is an infectious disease caused by the intracellular parasite Toxoplasma gondii, which affects about a third of the world population, and the cats are definitive hosts and humans, other mammals and birds, intermediate hosts. Currently, the most effective treatment against infections caused by T. gondii is the association of pyrimethamine and sulfadiazine, however this treatment has a high toxicity. The ability to cross biological barriers, achieve a specific target and minimize toxic effects are important characteristics for an efficient and desirable drug. Thus the use of nanomaterials, characterized by having sizes smaller than 1000 nm, may have these characteristics, acting as drugs carriers. Objective: Thus, the aim of this review is to present recent advances in nanotechnology as an alternative therapy in toxoplasmosis. Methods: The studies about the subject were searched in databases: PubMed, LILACS, MEDLINE, Science Direct and Web of Science. Results: Polymeric nanoparticles of alginate-chitosan have ability to decrease the parasitic load, by increasing ROS and NO and were effective in inhibiting the growth of parasites, furthermore the combination of chitosan nanoparticles and AgNP were also able to cause deformities in tachyzoites. Nanorods and nanospheres conjugated with antibodies binding to surface of tachyzotes, decreasing the CHO-K1 cells infection. Other nanomaterial used are the liposomes, acting as carrier of IFN-γ, triclosan, usnic acid, improving the activity of these compounds, changing the structure of parasites, reducing the number of parasites and mice mortality. Furthermore, it was also shown that atovaquone nanosuspensions were able to increase the concentration of drug and presented protective effect in model of toxoplasmic encephalitis. In addition, resveratrol nanocomplexos showed anti-Toxoplasma and anti-inflammatory effects. Conclusion: This way nanomedicine presents to be a tool potential as a therapeutic alternative against toxoplasmosis.

Keywords: Toxoplasma gondii; Nanoparticles; Treatment. Thematic area: Neglected diseases.

ROSUVASTATIN INHIBIT INFECTION AND PROLIFERATION OF TACHYZOITES OF Toxoplasma gondii (RH STRAIN) IN HeLa CELLS

Raquel Arruda Sanfelice1, Gabriela de Alcântara Dalevedo1, Laís Fernanda Machado1, Larissa Rodrigues Bosqui1, Milena Menegazzo Miranda-Sapla1, Fernanda Tomiotto-Pellissier1, Dielle Ioris1, Guilherme Fonseca Reis2, Luciano Aparecido Panagio2, Italmar Teodorico Navarro3, Juliano Bordignon4, Ivete Conchon-Costa1, Wander Rogério Pavanelli1, Ricardo Sergio Almeida2, Idessania Nazareth Costa1. 1 Departamento de Patologia Experimental, Laboratório de Parasitologia, Universidade Estadual de Londrina, PR, Brasil. 2 Departamento de Microbiologia, Universidade Estadual de Londrina, PR, Brasil. 3 Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, PR, Brasil. 4Laboratório de Virologia Molecular, Instituto Carlos Chagas, ICC/ Fiocruz, Curitiba, Paraná, Brasil.

Introduction: Toxoplasmosis is an infection caused by the protozoan Toxoplasma gondii, obligate intracellular parasite capable of infecting nucleated cells of warm-blooded animals. Due to the toxicity of conventional drugs (pyrimethamine and sulfadiazine), other drugs are being studied for treating this infection, and statins; especially rosuvastatin - compound capable of inhibiting the initial processes of isoprenoid biosynthesis in human and parasite. Objective: Evaluate the activity of rosuvastatin in HeLa cells front to infection by T. gondii RH strain. Material and methods: For the experiment, HeLa cells (1x105) were infected with tachyzoites of T. gondii (5x105); then treated with rosuvastatin (50 g/mL and 25 g/mL) and with the combination of pyrimethamine and sulfadiazine (50 g/mL and 25 g/mL). After the phagocytic assays, we assessed the number of infected cells and intracellular tachyzoites. In addition, culture supernatants were collected for determination of IL- 6 and IL-17 by citometric bead array (CBA). Results: Rosuvastatin significantly reduced the number of infected cells by 37% and 27% for concentrations of 50 g/mL and 25 g/mL respectively. The combination of sulfadiazine and pyrimethamine (50 g/mL and 25 g/mL) decreased 27% when compared to the control. The intracellular parasite proliferation significantly reduced in 71% when treated with rosuvastatin (50 g/mL) and 54% at a concentration of 25 g/mL, whereas the conventional treatment, sulfadiazine, and pyrimethamine (50 g/mL and 25 g/mL), promoted a 48% reduction in cell proliferation. There was also a reduction in levels of IL-6 and IL-17 cytokines in the supernatant of infected cells and treated with rosuvastatin as compared to untreated cells. Conclusion: Isolated rosuvastatin has anti-proliferative activity on tachyzoites forms of T. gondii. The data presented show promise for further studies, enabling investigations of rosuvastatin as an alternative therapy in toxoplasmosis.

Keywords: Rosuvastatin; Toxoplasmosis; Statins. Thematic area: Neglected diseases.

LYMPHOTROPIC VIRUS INFLUENCE OF HUMAN T CELL TYPE 1 IN PATIENTS COINFECTED WITH HELMINTH Strongyloides stercoralis - A SYSTEMATIC REVIEW

Larissa Rodrigues Bosqui1, Taylon Felipe Silva1, Raquel Arruda Sanfelice1, Laís Fernanda Machado1, Érika Caroline Steinle1, Bianca A. Bertasso1, Marina L. R. Mantovani1, Larissa Dolfini Alexandrino1, Bruno Bevenutto Lucas1, Naara C. C. dos Santos1, Ivete Conchon-Costa1, Wander Rogério Pavanelli1, Francine Nesello Melanda1, Idessania Nazareth Costa1. 1 Department of Pathological Sciences - Experimental Parasitology Laboratory, Universidade Estadual de Londrina, Londrina, Paraná, Brazil.

Introduction: Strongyloides stercoralis is the helminth that causes strongyloidiasis, a neglected infection, with overall estimated prevalence of 60 million people mainly in tropical regions. The proliferation of this parasite occurs mainly in immunocompromised hosts, especially in infected patients with Human T cell lymphotropic virus type 1 (HTLV-1), which is responsible for infections of immune cells. Objective: Discuss correlation aspects reported in the main studies that investigated concomitant infection among patients with HTLV-1 and infected with S. stercoralis. Material and methods: This is an exploratory research, a systematic review of the literature description, with data collection in electronic databases PubMed, Scielo, Web of Science, Science Direct and Virtual Health Library - VHL. Data research took place between 4 and 25 August 2016. Articles should be in English or Portuguese, having the descriptors in the title and / or summary, published in the last 20 years and are inherent in the guiding question. Results: The available studies allowed us to infer that factors such as coincidence in vulnerable groups to diseases, immunomodulation promoted by strongyloidiasis and difficulties related to the diagnosis of strongyloidiasis are important factors in severity of cases of these infections and should be taken into consideration especially in the regions where cases of coinfection are more frequent. Conclusion: The search for HTLV-1 infections by S. stercoralis is highly recommended, especially when other risk factors are apparent in order to start fast and efficient treatment against this coinfection.

Keywords: Strongyloidiasis; HTLV-1; Coinfection. Thematic area: Neglected diseases.

MACROPHAGES ACTIVATED WITH CONCANAVALIN-A INCREASE LEISHMANICIDE ACTIVITY BY REACTIVE OXYGEN SPECIES PRODUCTION

Daniele Sapede Alvarenga1; Luciana Mendes1; Milena Miranda Sapla1; Fernanda Tomiotto Pellissier1; Renan Cajuca1; Francine Neselo Melanda1; Idessania Nazareth Costa1; Wander Rogério Pavanelli1; Ivete Conchon Costa1. 1 Department of Experimental Pathology – Laboratory of Experimental Protozoology, State University of Londrina, Londrina, Brazil.

Introduction: Cutaneous leishmaniasis is caused by protozoans of genus Leishmania spp, transmitted by sandflies. This zoonosis is considered a disease of great importance by deformities occurrences and the impact generated in society. Macrophages represents essential element in leishmaniasis and immune response for being the population of host cells and by production of effectors molecules capable to destroy Leishmania spp as reactive oxygen species (ROS). Leishmania (L.) amazonensis has been described as numerous escape mechanisms. In CBA mice, L. amazonensis can triggers less ROS production by macrophages in vitro at 30 min post infection. Several studies have been shown the immunomodulatory potential of Concanavalin-A (Con-A) activating macrophages and increasing their leishmanicide activity with ROS production. Objective: The aim of this study is to verify if the pretreatment intraperitoneal with Con-A in BALB/c mice is efficient to eliminate promastigotes forms by activated macrophages and inducing ROS production. Material and methods: BALB/c mice were pretreated with Con-A or PBS for 72h and infected with 107 promastigotes of L. amazonensis for 30min. Peritoneal cells were collected for phagocytosis test, recovery promastigotes assay and ROS analysis by fluorescence. This study was approved by Londrina State University Ethics Committee for Animal Experimentation No. 056/2013. Results: The percentage of macrophages phagocytosing pretreated with Con-A was significantly higher than the PBS group. Macrophages pretreated with Con-A were able to destroy more amastigotes than PBS group. We observed ROS production in 30min post infection in Con-A group but not in PBS. Conclusion: BALB/c mice are susceptible to L. amazonensis infection, however, the pretreated with Con-A induced a ROS production and therefore, we find a lower number of free promastigotes in a recovery assay indicating an increase of leishmanicide activity of these macrophages.

Keywords: Leishmania (L.) amazonensis; Concanavalin-A; ROS. Thematic area: Neglected diseases.

CONCANAVALIN-A INDUCES TNF-α AND NITRIC OXIDE PRODUCTION BY PERITONEAL CELLS PRETREATED IN EXPERIMENTAL LEISHMANIASIS

Daniele Sapede Alvarenga1; Luciana Mendes1; Milena Miranda Sapla1; Fernanda Tomiotto Pellissier1; Renan Cajuca1; Francine Nesello Melanda1; Idessania Nazareth Costa1; Wander Rogério Pavanelli1; Ivete Conchon Costa1. 1 Department of Experimental Pathology – Laboratory of Experimental Protozoology, State University of Londrina, Londrina, Brazil.

Introduction: Leishmaniasis is a zoonosis caused by protozoans of genus Leishmania spp., considered the sixth disease of great importance by World Health Organization (WHO). Leishmania (L.) amazonensis can modulate the inflammation, reducing oxide nitric (NO) production in macrophages by increasing the expression of arginase, and inducing anti-inflammatory response. Previous studies have shown the immunomodulatory potential of Concanavalin-A (Con-A) activating lymphocytes and macrophages. Objective: The aim of this study is to verify TNF-α and NO production by peritoneal cells after treatment with Con-A and infection with L. amazonensis. Material and methods: BALB/c mice were pretreated intraperitoneally with Con-A (250μg mL-1 PBS) or PBS (250μL) for 72h and infected with 107 promastigotes of L. amazonensis for 2h, 6h, 18 and 24h. Exudates were collected and supernatant separated for cytokine analysis by ELISA and NO by Griess method. This study was approved by Londrina State University Ethics Committee for Animal Experimentation No. 056/2013. Results: We found a significant increase of TNF-α level at 2h post infection in Con-A group but not in PBS mice; and observed increase of NO levels in Con-A group infected at 18h post infection. Conclusion: Con-A can activate cells in the peritoneum, leading to TNF-α production. TNF-α may activate macrophages and inducing NO production, important element to pathogeny. In our model, we found NO production in vivo after infection, but not in PBS group, showing that Con-A is able to modulate the infection by L. amazonensis.

Keywords: Leishmaniasis; Concanavalin-A; TNF-α Thematic area: Neglected diseases.

ACUTE Toxoplasma gondii INFECTION ALTERS THE MYELOPEROXIDASE ACTIVITY IN RAT DUODENUM

Paulo da Silva Watanabe1, Saulo Euclides Silva Filho2, João L. Garcia1, Roberto Kenji Nakamura Cuman2, Débora de Mello Gonçales Sant’Ana2, Gessilda Alcântara Nogueira de Melo2 1 Universidade Estadual de Londrina - Brazil 2 Universidade Estadual de Maringá - Brazil

Introduction: Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis, zoonosis common among vertebrate. There is a high incidence of disease worldwide. For the parasite to spread in the host body it is necessary to transpose the intestinal barrier and reach the bloodstream causing inflammatory reactions. Polymorphonuclear leukocytes first line of defense against infectious agents possess the enzyme myeloperoxidase (MPO), that is responsible for generating hypochlorous acid, important in driving the inflammatory response. Objective: It was to evaluate the activity MPO enzyme at different times of acute T. gondii infection. Material and methods: The entire experimental protocol was approved by the Ethics Committee on Animal Experiments of the Universidade Estadual de Maringá, on the advice of approval n ° 079/2013. We used 24 male Wistar rats with 60 days reallocated ages randomly into control group (CG) received saline orally and in infected groups, with a suspension of 5000 oocysts in saline for 6 hours (G6), 12 hours (G12), 24 hours (G24) 48 hours (G48) 72 hours (G72). The animals were euthanized and 0.5 cm of the duodenum of each animal was collected. Samples were suspended in a volume of 20 times in hexadecyltrimethylammonium buffer and homogenised for 1 min. Thereafter were centrifuged at 5000 rpm for 15 min at a temperature of 4 °C, the supernatants were pipetted in duplicate into 96 potions plate, was added to each well a solution containing o-dianisidine, distilled water, phosphate buffer sodium and hydrogen peroxide. After 5 minutes the reaction was quenched with the addition of sodium acetate. The reading was done in ELISA reader at 460nm. Results: There was an increase in MPO activity 12 hours after infection. Conclusion Acute infection with T. gondii causes significant changes in MPO enzyme activity within 12 hours of infection. Keywords: Toxoplasma gondii; Acute infection; Myeloperoxidase. Grants: CAPES. Thematic area: Neglected diseases.

COMBINED BENZNIDAZOLE AND ASPIRIN CHEMOTHERAPY IN MICE INFECTED WITH Trypanosoma cruzi

Rito Santo Pereira1; Aparecida Donizette Malvezi1; Bruno Fernando Cruz Lucchetti1; Maria Isabel Lovo-Martins3, Marli Cardoso Martins Pinge2, Phileno Pinge Filho1 1Experimental Immunopathology Laboratory, Department of Pathological Sciences, Londrina State University, Londrina, Parana, Brazil. 2Cardiovascular Physiology Laboratory, Department of Physiological Sciences, Londrina State University, Londrina, Parana, Brazil. 3Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil.

Introduction: Chagas disease (CD) affects millions of people living in Latin America. Nifurtimox and benznidazole (BZ), drugs used since 1970, are specific to the Trypanosoma cruzi (Tc), can induce collateral effects and are ineffective in chronic phase. Different studies have shown positive and negative effects of cyclooxygenase inhibitors such as aspirin (ASA) on the course of Tc infection, but none them evaluated the combination BZ+ASA. Objective: Evaluate the efficacy of combined treatment of BZ+ASA on the acute and chronic experimental CD. Material and methods: The study was approved by CEUA (Process number 4628.2016.40). Until the moment, experiments were conducted using 60 Balb/C mice of 8 to 12 weeks of age. Animals were randomized into different groups: infected untreated, infected treated with ASA 25 mg/kg/day, infected treated with BZ 25 and 15 mg/kg/day, infected and treated with BZ 25 and 15 mg/kg/day + ASA 25 mg/kg/day. Mice were infected intraperitoneally with Tc (Y strain, 5x103 blood trypomastigotes).The therapy by gavage was initiated two days after infection. On days 8, 14, 20, 30, 40 and 50 after infection cardiovascular parameters were evaluated using CODA system. The parasitemia was performed using the Brener’s method. Results: As expected BZ treatment was effective in the control of parasitemia and ameliorates some cardiovascular parameters (as mean arterial pressure and heart rate). ASA treatment increased parasitemia and mortality, corroborating previous data from literature. Combined therapy (BZ+ASA) controlled better the parasitemia when compared to the group only treated with BZ) and improving cardiovascular parameters. Conclusion: Our data suggest that BZ+ASA therapy was able to improve some parameters related to the disease, such as blood pressure, parasitic load and animal survival. Taken together, the results indicate that BZ+ASA therapy had a healthful effect on the host than BZ and increased the efficacy of the reference drug against Tc infection.

Keywords: Benznidazole; Aspirin; Acute and Chronic Chagas Disease. Grants: CAPES, CNPq, UniZambeze and MCTESTP Mozambique. Thematic area: Neglected diseases.

CROSSTALK BETWEEN CYCLOOXYGENASE PATHWAY AND ADENYLATE-CYCLASE IN THE REGULATION OF ANTI-Trypanosoma cruzi ACTIVITY BY HUMAN MONOCYTES

Rafael Carvalho de Freitas1, Sandra Cristina Heim Lonien1, Aparecida Donizette Malvezi1, Guilherme Ferreira Silveira2, Pryscilla Fanini Wowk2, Rosiane Valeriano da Silva1, Lucy Megumi Yamauchi3, Sueli Fumie Yamada-Ogatta3, Luiz Vicente Rizzo4, Juliano Bordignon2, Phileno Pinge- Filho1

1Laboratório de Imunopatologia Experimental, Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, 86051-970, Londrina, Paraná, Brasil 2Laboratório de Virologia Molecular, Instituto Carlos Chagas – ICC/Fiocruz, Curitiba, 81350-010, Paraná, Brasil 3Laboratório de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, 86051-970, Londrina, Paraná, Brasil 4Hospital Israelita Albert Einstein, Avenida Albert Einstein 627-701, Subsolo Bloco A.,05651-901 São Paulo, São Paulo, Brasil

Introduction: Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Acute Chagas disease elicits a strong inflammatory response that modulates the immune response of host. Objective: In this study, we evaluated the effect of aspirin, a non-selective cyclooxygenase (COX) inhibitor, and SQ 22536, an adenylate-cyclase inhibitor, on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. Material and methods: Study was approved by the Ethical Committee in Research Involving Human Subjects, Process number: 5491/2012, CONEP number: 5231. Human blood taken from healthy volunteers and peripheral blood mononuclear cells were isolated using lymphocyte separation medium (density 1.077 g/ml, Lonza, Walkersville, MD USA). Human monocytes were selected from mononuclear cells via adherence. Human monocytes were pretreated with different concentrations of aspirin for 60 min at 37°C before inoculation. Cells were allowed to interact with trypomastigote forms added in a ratio of 5 parasites per cell, for 24 h, at 37°C in a 5% CO2 atmosphere. Other treatments included combined incubation with SQ 22,536 (20 µM) for 30 minutes at 37°C. Levels of prostaglandin E2 (PGE2), interleukin-10 (IL-10), interleukin- 12 (IL-12) and nitric oxide (NO) were determined in the cultures supernatants. Results: The pretreatment of monocytes with aspirin or SQ 22536 before T. cruzi infection induced a significant inhibition of infection. On the other hand, the treatment of monocytes with SQ 22536 after aspirin restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in NO and PGE2 production, and an increase of IL-10 and IL-12. Conclusion: These results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of T. cruzi invasion.

Keywords: Trypanosoma cruzi; Aspirin; SQ 22536. Grants: CAPES, CNPq and Araucária Foundation. Thematic area: Neglected diseases.

HORMONE THERAPY ATTENUATES THE SYSTEMIC PROOXIDANT STATUS IN POSTMENOPAUSAL WOMEN

Adriano Martin Felis Aranome¹; Vanessa Jacob Victorino¹; Rubens Cecchini¹; Flávia Alessandra Guarnier¹; Carolina Panis²; Alessandra Lourenço Cecchini¹. ¹Laboratório de Fisiopatologia e Radicais Livres, Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, Paraná, Brasil ²Laboratório de Mediadores Inflamatórios, Universidade Estadual do Oeste do Paraná (UNIOESTE), Campus Francisco Beltrão, Paraná, Brasil.

Introduction: Postmenopausal is a naturally phenomenon characterized by physiological changes as reducing the activity of ovarian hormones. Due to these changes, menopause is undoubtedly one of the major current themes at the adverse effects caused by this period. On the other hand, hormone therapy (HT) performed to replenish estrogen levels, progesterone or both, has been extensively studied to attenuate the adverse symptoms of postmenopause. However; until the moment it is not completely known its actual effect. Objective: Our study aimed to characterize the oxidative profile of women in postmenopause and after the completion of hormone therapy (HT). Material and methods: We recruited 10 healthy postmenopausal women consuming HT, and 25 healthy postmenopausal women without the use of HT as a control group and fresh blood was collected (CAAE 35524814.4.0000.0107). To evaluate the prooxidant parameters, we quantified lipid peroxidation levels by a highly sensitive chemiluminescense (CL) method, malondialdehyde (MDA) levels by high performance liquid chromatography (HPLC), advanced products protein oxidation (AOPP) employing a semi-automated method and nitrite estimated as nitric oxide (NO). To antioxidants parameters we determined the total antioxidant capacity (TRAP) by CL, assessment of overall levels of thiols and uric acid. Results: Our results showed that HT significantly decreased susceptibility to oxidative stress, as measured by CL and decreased levels of MDA. There was a positive correlation between the levels of NO and AOPP in HT group. The total thiol levels significantly decreased in HT group. We detected no differences between body mass index, NO, TRAP and uric acid between groups. Conclusion: Our study was the first to demonstrate a characterization between pro-oxidant and antioxidant mediators in women using HT, with a strict criterion for exclusion of participants. We hope that this study may help to understand the important role of redox metabolism of some possible complications climacteric period.

Keywords: Climacteric; Oxidative stress; Antioxidant. Thematic area: Non-transmitted diseases.

PROTECTIVE ASSOCIATION OF TGF-βR2 GENE POLYMORPHISM AND CYTOKINE PLASMA LEVELS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

Alberto Yoichi Sakaguchi1, Marla Karine Amarante1, Carlos Eduardo Coral de Oliveira1, Diego Lima Petenuci1, Thiago Cezar Fujita1, Fausto Trigo2, Maria Angelica Ehara Watanabe1 1Laboratory of Study and Application of DNA Polymorphisms, Department of Pathological Sciences, State University of Londrina, Londrina, PR, Brazil 2Londrina Cancer Hospital, Londrina, PR, Brazil

Introduction: Acute Lymphoblastic Leukemia (ALL) is a neoplasic disease that affects hematopoiesis, leading to deregulated proliferation of lymphoid lineage progenitors cells in bone marrow. Many studies have been demonstrated association of TGF-β1-activated signaling pathway in malignant hematologic processes resulting from activation of TGF-β receptor II (TGF-βR2). Moreover, alterations of this receptor may act on in regulation of differentiation and immune response. The identification of single nucleotide polymorphisms (SNPs) in TGF-β1 and TGF-βR2 genes and plasma concentration may help predicting disease risk. Objective: The aim of this study was to evaluate the association between two polymorphisms of TGF-β1 and TGF-βR2 genes; cytokine plasma levels in childhood ALL susceptibility, relapse and death. Material and Methods: This study was approval by human ethics committee of UEL (CAAE N° 0164.0.268.000-09). We performed a case-control study involving 81 ALL children patients and 106 controls, genotyping for rs1800470, rs1800471 and rs3087465 polymorphisms using RFLP-PCR assay and the cytokine was quantified by ELISA. Genotype frequencies were submitted to chi-square test to detect a deviation from Hardy-Weinberg equilibrium. For comparison of polymorphism frequency between groups, Odds Ratio (OR) analysis, with 95% confidence interval (CI), was performed. Cytokine levels were analyzed by t test (Mann-Whitney U test). Results: All samples were within the Hardy-Weinberg equilibrium. The heterozygous GA genotype and G allele of TGF-βR2 polymorphism conferred protection from ALL pathogenesis (OR 0.50/ 95%CI 0.27-0.93, and OR 0.63/ 95%CI 0.41-0.98, respectively). Moreover, protective role was observed for G allele carriers compared to homozygous AA genotype (OR 0.50/ 95%CI 0.27-0.90). The TGF-β1 plasma concentration was significantly reduced in ALL patients. Furthermore, chemotherapy demonstrated re-establishment of this cytokine. Conclusion: Our results demonstrate that rs3087465 polymorphism may confer significant protection from ALL in south Brazilian population and plasma TGF-β1 could be a possible marker of prognostic this disease.

Keywords: ALL; TGF-β1; Genetic polymorphism. Grants: Secretaria da Família e Desenvolvimento Social. Thematic area: Non-transmitted diseases.

PROBUCOL ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION BY REDUCING LEUKOCYTE RECRUITMENT, CYTOKINE PRODUCTION AND NF-КB ACTIVITY

Amanda Z. Zucoloto1; Marília F. Manchope1; Felipe A. Pinho-Ribeiro1; Ana C. Zarpelon1; José C. Alves-Filho2, Thiago M. Cunha2, Gustavo B. Menezes3, Fernando Q. Cunha2, Rubia Casagrande4, Waldiceu A. Verri, Jr.1

1Departament of Pathology, State University of Londrina, Rod. Celso Garcia Cid PR445 km 480, CEP 86057-970, Londrina, Paraná Caixa Postal 10.011, Brasil 2Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão Preto, São Paulo, Brasil 3Departament of Morphology, Federal University of Minas Gerais, Avenida Presidente Antonio Carlos 6627, CEP 31270-901 Belo Horizonte, Minas Gerais, Brasil 4Departament of Pharmaceutical Sciences, State University of Londrina, Londrina, Av. Robert Koch, 60, CEP 86038-350, Londrina, Paraná, Brasil

Introduction: Inflammation and pain underlie nearly every pathophysiological process. Probucol is a synthetic molecule clinically used for prevention and treatment of hypercholesterolemia and atherosclerosis. However, due to its antioxidant and anti-inflammatory activities, the beneficial use of probucol has been evaluated under varied disease models where inflammation plays a pivotal role such as diabetes and brain ischemia. Objective: The study aimed to evaluate the effect of probucol in inflammatory hyperalgesia and leukocyte recruitment in mice. Materials and methods: Probucol at 0.3-30mg/kg was administrated to male Swiss mice (n=6) per oral 1h before intraplantar or intraperitoneal lipopolysaccharide administration. Mechanical and thermal hyperalgesia induced by lipopolysaccharide were determined using an electronic anesthesiometer and hot plate apparatus, respectively. Leukocyte recruitment was evaluated by direct count or by determination of myeloperoxidase activity. Antioxidant ability was determined by measurement of GSH levels, ABTS, TBARS and NBT assays. Cytokine production was evaluated by ELISA. NF-кB activity was measured in vitro using RAW 264.7 macrophages expressing luciferase on NF-кB responsive promoter. This study was approved by The Animal Welfare and Ethics Committee (process number 1012.2015.74). Data were analyzed by ANOVA followed by Tukey’s post-hoc. p<0.05 was considered significant. Results: Probucol at 3mg/kg reduced lipopolysaccharide-induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx and cytokine production in both paw skin and peritoneum exudate. Probucol did not alter lipopolysaccharide-induced tissue oxidative stress at anti-inflammatory/analgesic dose. On the other hand, probucol inhibited lipopolysaccharide-induced NF-кB activity in vitro. Conclusion: Probucol presents analgesic and anti-inflammatory activities by employing mechanisms other than its antioxidant properties. These mechanisms involve targeting of pro-inflammatory cytokines and NF- κB. The inhibition of NF-кB activity in macrophages could explain the reduced levels of cytokines. In addition, the decrease in leukocyte recruitment could be due to downregulation of adhesion molecules, which are also targets of NF-кB.

Keywords: Acute Inflammation; Hyperalgesia; LPS. Grants: CAPES, CNPq, Fundação Araucária. Thematic area: Non-transmitted diseases.

HASHIMOTO’S THYROIDITIS: A REVIEW ARTICLE

Janaine Aparecida Fortunato1; Ana Lúcia Sanches1; Márcia Regina Terra1. 1Community College of Londrina - INESUL, Londrina, Paraná, Brazil. Avenue Duque de Caxias, 1290, New London Garden, Londrina, Paraná, Brazil. CEP: 86015-000. [email protected]

Introduction: Hashimoto's thyroiditis (HT) is a chronic inflammation of the thyroid gland first described more than a century ago, but still incomplete etiopathogeny set. Objective: Because the exposed problematic this study aimed to carry out a literature review about the theme Hashimoto’s thyroiditis. Material and methods: This study is of the literature review which was carried out the databases such as periodic CAPES and SciELO. The descriptors used were “hypothyroidism” and “Hashimoto's disease”. Results: Hashimoto's thyroiditis (HT) is a chronic inflammation of the thyroid gland resulting in low expression of hormones triiodothyronine (T3) and thyroxine (T4) and increased production of thyroid stimulating hormone (TSH). Currently, TH is a autoimmune disease the most common, and the most common endocrine disorder, as well as the most common cause of hypothyroidism. Hypothyroidism affects 0.7% to 5.7% of the general population and the main cause for such a condition in the population of iodine-sufficient areas is chronic lymphocytic thyroiditis – HT. The pathological lesions of HT involve both the interstitium around the thyroid follicles and the thyroid cells themselves, and have distinct features in the various forms. The diagnosis of HT is currently established by a combination of clinical features, presence of serum antibodies against thyroid antigens (mainly to T3 and T4). The signs and symptoms affecting the gastrointestinal system (constipation), cardiovascular (bradycardia), skeletal muscle (muscles appear falsely hypertrophic due to myxedema infiltration of tissue), pulmonary (bradypnea and hypoxia), hematopoietic (anemia), reproductive (oligomenorrhea and/or menometrorrhagia) and skin and appendages (dry skin, cold and yellow). Conclusion: Treatment of TH is important given the various local and systemic changes will originate in loss of function of the thyroid gland and subsequent primary hypothyroidism, where the signs and symptoms of hypothyroidism are numerous and variable.

Key words: Hashimoto’s thyroiditis; Hypothyroidism; Thyroid disease. Thematic area: Non-transmitted diseases.

MUSCLE RECONDITIONING IN CHRONIC OBSTRUTIVE PULMONARY DISEASE

Janaina Aparecida Fortunato1; Ana Lúcia Sanches1; Vanessa Almeida Nascimento2; Cristhiane Da Silva Ferreira Gonçalves3; Uili Andrey De Souza4; Luana Aparecida Cossentini5 1Graduate student Physiotherapy of INESUL - Instituto de Ensino Superior de Londrina. 2Graduate student Physiotherapy of UNOPAR – Universidade do Norte do Paraná. E-mail: [email protected] 3Graduated in Nursing; Specialization in Emergency; Coordinator and teacher in CIE – Centro Integrado de Educação and INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 4Graduated in Nursing; Specialization Audit in Health; Degree in Biological Sciences; Coordinator and teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 5Graduated in Biomedicine; Master in Experimental Pathology; PhD student in Experimental Pathology at the Regional University Hospital of Londrina; Teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected].

Introduction: The Chronic Obstructive Pulmonary Disease (COPD) is an irreversible disease. Caused by smoking, environmental pollution and toxic gases. Among the main symptoms characterized by COPD are chronic airflow limitation, presence of cough, sputum production and breathlessness. Systemically is decreased muscle strength, especially of the lower limbs. Muscle weakness contributes to exercise intolerance in patients with pulmonary disease and the strength exercise is rational option in the pulmonary rehabilitation process. Objective: The aim of this study is to describe strategies for muscular reconditioning and the importance of pulmonary rehabilitation in the treatment of COPD. The study also seeks to report the change skeletal muscle in individuals with COPD, analyze whether these variations are related to the severity of the disease and comprises the use of pulmonary rehabilitation and muscle reconditioning. Material and methods: It was adopted as a technical procedure for the study, a literature with systematic review covering scientific articles with time frame of 2005 and 2015 with the qualitative approach problem. Conclusion: Studies have shown that individuals with COPD have an impaired quality of life, due to the decreased exercise tolerance and loss of respiratory muscle strength. Still, several studies on the muscle reconditioning suggest that physical training of patients is based on the improvement in aerobic capacity and are few exercises that assess a specific approach to the changes of the rib cage and chest muscles.

Keywords: Chronic obstructive pulmonary disease; Muscular reconditioning; Pathophysiology. Thematic area: Non-transmitted diseases.

EXPERIMENTAL MODEL PADRONIZATION OF MURINE ORAL’S MELANOMA: PILOT STUDY

Barbara Prevital1; Gabriella Pasqual Melo1; Rodrigo Luiz Cabral1; Alessandra Lourenço Cecchini1 1Laboratório de Patologia Molecular, Universidade Estadual de Londrina.

Introduction: The oral’s melanoma (OM) affects only 0.5% of all oral’s neoplasias, showing high aggressiveness, poor prognosis and high mortality rate. Objective: To set a standard experimental model of murine OM. Materials and Methods: (6738.2015.40). B16F10 cells were grown in culture medium DMEM 10% FBS. The cells were kept in DMEM without serum, counted and prepared in the inoculation’s solution. It was chosen two ways for development of OM, the left superior edge of tongue (1x105 cells in 50 µL of DMEM without serum, group MT) and the left edge of gum (1x105 cells in 20 µL of DMEM without serum, group MG). Animals were sacrificed after seven or fourteen days of melanoma cells injection and were named according to the site of inoculation and day of sacrifice: MT7, MT14, MG7 and MG14. The tumor mass was withdrawn and a portion of the tissue with the tumor was dissected and included in paraffin for histological analysis (HE). An exploratory investigation of all organs was performed after tumor removal. Results: The MG group showed that the tumor mass was considerably developed in MG7 and MG14. The satellites linfonodes were normal in MG7 and increased contralateral linfonode in MG14. The MT group had similar MG tumor mass. There was no apparent increase in linfonodes size. The HE microscopic analysis showed moderate inflammatory cells in the muscular layer in MG7 and presence of tumor cells with melanin and intense inflammatory figures in MG14. Also, MT14 had an intense inflammatory cells and tumor cells with melanin. Conclusion: This pilot study allowed determining that the B16F10 cells could be used for the development of an experimental model of OM when inoculated in tongue or gum. However it is necessary to continue the study to determine the best method of inoculation and experimental time to study.

Keywords: Mucosal melanoma; B16F10 cells; C57BL/6 mice. Thematic area: Non-transmitted diseases.

iNOS PARTICIPATION IN CARDIOVASCULAR AND OXIDATIVE PARAMETERS OF FEMALE RATS WITH ENDOTOXEMIA INDUCED BY LPS

Blenda Hyedra de Campos1; Jaqueline Costa Castardo de Paula1; Lorena de Jager1; Nagela Ghabdan Zanluqui1; Carine Coneglian de Farias1; Luciana Higashi1; Décio Sabbatini Barbosa1; Phileno Pinge-Filho1; Marli Cardoso Martins-Pinge1. 1 State University of Londrina, Londrina – Paraná

Introduction: Literature demonstrates that female are less susceptible to sepsis and that estrogen increases the bioavailability of nitric oxide. Nevertheless, any study had evaluated the interaction of both in the cardiovascular and oxidative function during sepsis. Objective: Evaluate cardiovascular responses to LPS endotoxemia in female rats during the estrus (SHAM), compared to ovariectomized rats (OVX) and ovariectomized rats treated with estradiol; measurement the nitric oxide (NO), radical-trapping antioxidant parameter (TRAP), lipid peroxidation (LOOH) and paraoxonase 1 activity (PON1), in the plasma of animals, pretreated with S-methylisothiourea (SMT), that is an inhibitor of iNOS, or saline. Materials and methods: CEUA (8541/276.2013.81). We used Wistar rats, after 8 weeks of ovariectomy, treated or not with estradiol 1mg/Kg/day, or false-OVX (SHAM), and evaluated the cardiovascular parameters, before and after the LPS injection (5mg/Kg, IV), preceded by saline (0,9%) or SMT (3mg/Kg) intravenous injection. 2 hours after LPS injection, it was collected plasma sample to measure nitrite levels, LOOH, TRAP and PON1 activity. Results: Pretreatment with SMT caused reduction of mean arterial pressure (MAP) in OVX+E rats, after 2 hours of LPS, when compared to SHAM and to the moment of injection (ΔMAP mmHg: SHAM= 0,8±3, OVX= -8,3±3, OVX+E= -13,3±4, n=8-9/group); every groups had tachycardia. LPS decreased the TRAP of OVX and the treatment with estradiol prevented this fall (TRAP uM trolox: SHAM+LPS= 379±24, OVX+LPS= 153±8, OVX+E+LPS= 312±34, N=5-11/group). When pretreated with SMT, after 2 hours of LPS, TRAP didn’t reduce (OVX SMT-LPS= 521±29), and the estradiol reduced the lipid peroxidation (x103 URL: SHAM SMT+LPS= 4714±563, OVX+E SMT+LPS= 1991±310). Conclusion: The results suggest that, in the cardiovascular system, the treatment with estradiol in this concentration used in this study has protector effects in inflammatory challenge when iNOS is inhibited.

Keywords: Endotoxemia; Nitric oxide; Estrogen. Grants: CNPq. Thematic area: Non-transmitted diseases.

INVOLVEMENT OF NITRIC OXIDE SYNTHASE ISOFORMS IN CARDIOVASCULAR, AUTONOMIC AND OXIDATIVE ASPECTS IN FEMALE RATS

Introduction: Cardiac autonomic modulation responds to estrogen, what increases the availability of nitric oxide (NO). Nevertheless, it is unknown how estrogen can affect cardiovascular, autonomic and oxidative parameters, after an acute inhibition of NO isoforms (NOS). Objective: Investigate the role of estrogen in these aspects in female rats, before and after an injection of NOS inhibitor. Materials and methods: CEUA (8541/276.2013.81). It was used ovariectomized Wistar rats (OVX), ovariectomized rats treated with estradiol 1mg/Kg/day (OVX+E) or false-OVX (SHAM), subordinated to a L-nitroarginine-methyl-ester (L-NAME, 10mg/Kg), or S-methylisothiourea (SMT, 0,3mg/Kg), or saline (0,9%) injection. It was registered the medium arterial pressure (MAP) and heart rate (HR) before and after the injection of the inhibitors. It was collected the plasma to measure nitric oxide level (NO), radical-trapping antioxidant parameter (TRAP), lipid peroxidation (LOOH) and paraoxonase 1 activity (PON1) and evaluated the systolic blood pressure variability (SPV), heart rate variability (HRV), and baroreflex sensitivity (BRS). Results: In the basal period, OVX reduced BRS, didn’t change SPV and increased HR. After 2 hours, all saline groups had reduction of BRS. L-NAME increased MAP and reduced LF of SPV, and the raise of MAP was lower in OVX. OVX group had a reduction on PON1 activity. This reduction on PON 1 activity caused by L-NAME was prevented in OVX+E. In all groups, SMT promoted a raise in MAP and LF of SPV. OVX+E+SMT presented lesser NO than OVX+SMT. Conclusion: Estradiol didn’t prevent the increase on sympathetic modulation caused by ovariectomy. The cNOS and iNOS seem to carry out opposite functions on autonomic nervous system. Besides that, cNOS contribute to PON1 activity in OVX rats, while iNOS keep the NO levels in OVX+E rats.

Keywords: Estrogen; Nitric oxide; Autonomic. Grants: CNPq. Thematic area: Non-transmitted diseases.

GENETIC POLYMORPHISMS AND EXPRESSION OF FOXP3 mRNA IN AGGRESSIVE BREAST CANCER SUBTYPES

Bruna Karina Banin Hirata1; Roberta Losi Guembarovski1; Karen Brajão de Oliveira1, Carolina Batista Ariza1, Carlos Eduardo Coral de Oliviera1, Marla Karine Amarante1, Alda Fiorina Maria Losi Guembarovski2, Marina Okuyama Kishima2, Glauco Akelinghton Freire Vitiello1, Maria Angelica Ehara Watanabe1 1 Laboratory of Study and Application of DNA Polymorphisms , Department of Pathological Sciences, Biological Sciences Center, State University of Londrina, Celso Garcia Cid highway Pr 445 Km 380, Londrina, Paraná, Brazil. 2 Laboratory of Human Pathology, Department of Pathology, Clinical Analysis and Toxicology, State University of Londrina.

Introduction: Studies have demonstrated breast tumor cells expressing a transcription factor termed forkhead box protein 3 (FOXP3), which is believed to be restrictively expressed in regulatory T cells (Treg). FOXP3 genetic polymorphisms, as well as their expression level, have been associated with development and prognosis of breast cancer (BC). Objective: The present study aimed to investigate the influence of two FOXP3 polymorphisms, A-924G (rs2232365) and C-3279A (rs3761548) on its mRNA expression in aggressive BC subtypes, including Luminal B HER2-positive (LB), HER2-enriched (HER2+) and triple-negative (TN). Material and methods: This study was approved by Human Ethics Committee of the State University of Londrina (CAAE- 171231134000005231). 58 BC samples were obtained, including peripheral blood and tumor tissues. FOXP3 polymorphisms genotyping were performed by polymerase chain reaction (PCR) followed by enzymatic restriction, and mRNA expression quantification was performed by quantitative real time PCR (RT-qPCR). Results: LB subtype presented 1.7 fold lesser FOXP3 mRNA expression when compared to normal mammary gland, while the TN presented a slightly higher expression (1.6 folds). However, the HER2+ subtype presented an increase of 4.2 fold of FOXP3 mRNA. The C-3279A polymorphism was correlated with mRNA expression (p=0.045). The FOXP3 mRNA expression was correlated with higher histological grade in general sample (p=0.03), lower proliferation index Ki-67 in LB (p=0.045, rho=-0.26) and negative lymph nodes commitment, in HER2+ subtype (p=0.02). Conclusion: Our results demonstrated that the C-3279 polymorphism may be involved in the gene transcription regulation. Furthermore, the prognostic value of FOXP3 mRNA expression may vary depending on the breast cancer subtype, appearing to be favorable in LB and HER2+. More studies are necessary to clarify if the FOXP3 mRNA expression is related to breast tumor cell or to Treg infiltration, providing new insights into this transcription factor impact in progression and prognosis of different breast cancer subtypes.

Keywords: FOXP3; Genetic polymorphism; Breast cancer. Grants: Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação Araucária. Thematic area: Non-transmitted diseases.

CYTOKINE PROFILE IN PATIENTS WITH PROGRESSIVE MULTIPLE SCLEROSIS AND ITS ASSOCIATION WITH DISEASE PROGRESSION AND DISABILITY

Daniela Frizon Alfieri1, Brunna Emanuella França Robles1, Ana Paula Kallaur1, Sayonara Rangel Oliveira1; Andréa Name Colado Simão3, Tamires Flauzino1, Wildéa Jennings Pereira De Carvalho2, Caio de Meleck Proença1, Lorena Flor da Rosa Franchi Santos1, Bruna Miglioranza Scavuzzi1, Damácio Ramón Kaimen-Maciel2, Michael Maes4,5, Edna Maria Vissoci Reiche3.

1Research Laboratory in Applied Immunology – University Hospital from State University of Londrina, Londrina, Paraná, Brazil. 2Department of Neurology – State University of Londrina, Londrina, Paraná, Brazil. 3Department of Pathology, Clinical Analysis and Toxicology – State University of Londrina, Londrina, Paraná, Brazil. 4Impact Strategic Research Centre, School of Medicine, Deakin University, Geelong, VIC, Australia. 5Department of Psychiatry, King Chulalongkorn Memorial Hospital, Chulalongkorn, Bangkok, Thailand.

Introduction: Inflammation is the driving force for brain injury in patients with multiple sclerosis (MS). Objective: The present study is to delineate the serum cytokine profile in patients with progressive MS in a Southern Brazilian population compared with healthy controls and patients with relapsing- remitting MS (RRMS) and its associations with disease progression and disability. Material and methods: The protocol was approved by the Institutional Research Ethic Committee of the State University of Londrina, and a written consent form was obtained from all of the individuals. We included 32 patients with progressive MS, 126 with RRMS, and 40 healthy controls. The patients were evaluated using the Expanded Disability Status Scale (EDSS) and magnetic resonance imaging (MRI) with gadolinium. Serum interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-10, IL-4, and IL-17 levels were assessed using an enzyme-linked immunosorbent assay. Results: IL-1β, IL-6, TNF-α, IFN-γ, IL-17, IL-4, and IL-10 levels were higher in progressive MS than in controls. Increased IL-1β and IFN-γ and decreased IL-12 and IL-4 levels were found in progressive MS compared with RRMS. Patients with progressive MS with disease progression presented higher TNF-α, IFN-γ, and IL-10 levels than those without disease progression. Patients with progressive MS with disease progression showed a higher frequency of positive gadolinium-enhanced lesions in MRI; higher TNF-α, IFN-γ, and IL-17 levels; and decreased IL-12 levels compared with RRMS patients with progression. There was a significant inverse correlation between IL-10 levels and EDSS score in patients with progressive MS. Conclusion: The results underscore the complex cytokine network imbalance exhibited by progressive MS patients and show the important involvement of TNF-α, IFN-γ, and IL-17 in the pathophysiology and progression of the disease. Moreover, serum IL-10 levels were inversely associated with disability in patients with progressive MS.

Keywords: Multiple Sclerosis; Progressive Multiple Sclerosis; Cytokines. Thematic area: Non-transmitted diseases

HISTOPATHOLOGICAL EVALUATION FOR ABERRANT CRYPT FOCI (ACF) STUDY IN EXPERIMENTAL MODEL OF COLORECTAL CANCER IN INDUCED BY 1,2- DIMETILHIDRAZINA (DMH) IN RATS WISTAR.

Bruno Bueno Pimenta1, Raíssa Coracini Varago1, Tiago Peres da Silva Suguiura1, Sara Santos Bernardes2, Isolde Previdelli1, Edilson Noboioshi Kaneshima1, Alice Maria de Souza-Kaneshima1, Tânia Cristina Alexandrino Becker1. 1Universidade Estadual de Maringá, Av. Colombo 5790, Maringá - PR, Brasil, 87020-900 2Universidade Federal da Grande Dourados, R. João Rosa Góes 176, Dourados - MS, Brasil 79825-070.

Introduction: Colorectal cancer is on the top list of cancer diagnoses and is a major cause of death. The developing of colonic cancer is a multistage process with pathological alterations that represents a series of genetic mutations. Aberrant cript foci (ACF) are formed by aberrant cript (AC) and identified as pre-neoplasic lesions due to the presence of tecidual dysplasia. Objectives: Characterize the ACF development by colorectal carcinogenesis induction in an animal model. Material and methods: Under the opinion Nº104/2014 of the Ethics Committee on Animal Use (CEUA-UEM), it was inoculated 1,2-dimethylhydrazine (DMH) in 12 Wistar rats divided into short (P2-pré) and medium (P2) term trials with 6 animals in each group. The colon mucosa was examined and ACF were classified according to their number and distribution pattern throughout the colon, as well as the number of aberrant crypt/focus, by optical microscopy, at 10x magnification, with methylene blue staining 0.1%. Through the technique “Swiss roll", paraffin blocks were produced, which were cut in semi-automatic microtome and adhered to the histological slides. The slides were stained with hematoxylin-eosin (HE). Tumor analysis enabled the classification for the presence and degree of tissue dysplasia. The statistical model used for the analysis was the “Quasipoisson” with support of the software R. Results: There was dominance of ACF with 01 and 02-03 AC in the distal colon compared with the proximal part and no adenomatous tumor was observed. These results showed no statistical difference between the experimental times. Conclusion: The proposed carcinogenesis induction protocol showed AC induction efficacy and therefore we proved this model as a valid study tool for colonic carcinogenesis because the positive predictive value of the presence of ACF as precursor lesion of neoplasia is extremely important to the study of preventive substances or the ones used in the colorectal cancer treatment.

Keywords: Aberrant Crypt Foci (ACF); 1,2-Dimethylhydrazine (DMH); Colorectal Carcinogenesis (CRC) Thematic area: Non-transmitted diseases

INTRAUTERINE AND LACTATIONAL EXPOSURE TO FLUOXETINE INHIBITED THE AORTIC ADAPTATION INDUCED BY ACUTE RESTRAIN STRESS IN RATS

Bruno Vinicius Duarte Marques¹, Daniella Regina Barrionuevo da Silva Novi1, Nagela Ghabdan Zanluqui², Carolina Matias Higashi¹, Rafaela Picinin1, Phileno Pinge-Filho2, Gislaine Garcia Pelosi Gomes1, Graziela Scalianti Ceravolo1 1 State University of Londrina - UEL - Physiological Sciences 2 State University of Londrina - UEL - Pathological Sciences

Introduction: It has been demonstrated that intrauterine and lactational exposure to fluoxetine (FLX) can interfere with the offspring behavioral response to stress. Objective: Evaluate whether exposure to FLX during development alter vascular adaptations promoted by acute restraint stress. Material and methods: (CEUA: 16166-2012.12) Female Wistar rats were treated by gavage with: FLX (5mg/kg/day) or water (CTL) during pregnancy and lactation. The experiments were carried out in male offspring, 75 days old, treated or not with reserpine. FLX and CTL rats were submitted to a single stress session (ST) for 1 hour, in a metal tube. Cumulative concentration-effect curves to phenylephrine (PHE), in the absence or presence of L-NAME, were performed in thoracic aorta with (E+) and without (E-) endothelium. Aortic nitric oxide (NO) was evaluated by Griess method. Results expressed as mean±SEM of maximal response (MaxR), (n) represents number of rats/group. Statistical analysis: three-way ANOVA followed by Bonferroni's test, differences with p<0.05. Results: The MaxR for PHE in E+ rings were similar between CTL (20) and FLX (14) rats. The acute stress reduced MaxR to PHE in E+ rings of CTL-ST rats compared to CTL not stressed [CTL: 2.67±0.11 (20) vs CTL-ST: 1.93±0.11 (16)], and L-NAME equaled those MaxR. In FLX, stress (8) didn’t change the MaxR to PHE in E+ aortic rings. Reserpine treatment restored the vasoconstriction in E+ ring from CTL-ST, but didn’t interfere with MaxR in rings from FLX. The contraction induced by PHE in E- rings was similar between the groups. The NO concentration was higher in aorta from CTL-ST (6) compared to CTL (5) rats, and was similar between FLX (6) and FLX-ST (6) groups. Conclusion: The endothelial and NO modulation induced by acute stress were abolished in FLX- maternally exposed rats, probably by a mechanism involving reduced activity of sympathetic activation during acute restrain stress.

Keywords: Vascular Reactivity; Antidepressant exposure; Acute Stress Grants: CNPq Thematic area: Non-transmitted diseases

DAMAGE CAUSED BY YELLOW FEVER VACCINE ON THE GESTATIONAL DEVELOPMENT

Caio Cezar Nantes Martins1; Priscila Marianno1; Maria José Sparça Salles1 1Department of General Biology, Londrina State University, Londrina - PR - Brazil

Introduction: The yellow fever is a viral acute disease with variable severity, transmitted by a mosquito bite. The etiologic agent is an arbovirus of the genus Flavivirus. The most efficient prevention method is the vaccination with the live-attenuated yellow fever 17D virus. Vaccine's risk to embryofetal health is unknown. Objective: Thus, we investigated the live-attenuated yellow fever 17D vaccination effects on mice's intrauterine development. Material and methods: Approval of the ethics committee: 124.2011.21. Pregnant female mice were distributed in 6 groups, being 3 control groups and 3 experimental groups, with 15 animals each. The experimental group was divided in: experimental group treated at 5th pregnancy day (GD5), experimental group treated at 10th pregnancy day (GD10) and experimental group treated at 15th pregnancy day (GD15). Those groups received a 2,0 log 10 PFU subcutaneous dose of the vaccine. In control groups (C5, C10 and C15), injection water was administered on the same experimental way. On the 18th day of pregnancy, the females were euthanized and the analyzed parameters were: fetal weight and size, reabsorption rate and placental weight. On statistical analyzes the absolute data with normal distribution was utilized ANOVA and non-normal distributions was analyzed by Kruskal-Wallis. Results: The data showed that GD5 females presented more reabsorptions (C5=0.6±0.27; GD5=3.13±0.76). In addition, GD5 fetuses presented lower weight than the control group (C5=1.359±0.02; GD5=1.278±0.02) and placental weight lower than the control group (C5=0.66±0.003; GD5=0.07±0.002). Based in statistical results, we could infer that the yellow fever vaccine applied on 5th pregnancy day lowers the fetal viability and altered the placental index. Conclusion: We concluded that the vaccine should not be administered in the first pregnancy weeks, because it increases the embryolethality and compromises the nutrients and gas exchanges due to decreased size of the placenta.

Keywords: Yellow Fever; Vaccine; Embryotoxicity. Thematic area: Non-transmitted diseases

MATERNAL EXPOSURE TO METFORMIN DID NOT INTERFERE WITH VASCULAR ENDOTHELIAL FUNCTION OF ADULT MALE OFFSPRING

Camila Borecki Vidigal1, Daniella Regina Barrionuevo da Silva Novi1, Simone Forcato2, Daniela Cristina Ceccatto Gerardin2, Graziela Scalianti Ceravolo1 1Laboratory of Vascular Pharmacology, Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR. 2Laboratory of Reproduction Pharmacology, Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR.

Introduction: Metformin (MET) is used for the treatment of pregnant with polycystic ovary syndrome or gestational diabetes. Although the MET crosses the placenta and has been detected in the umbilical cord, at similar concentrations of maternal blood, it is considered a safe drug throughout gestation. It has been demonstrated that maternal exposure to MET may increases body weight, visceral fat deposit, the fasting glycemia and glucose intolerance, when the offspring is exposed to high-fat diet, indicating that MET may cause in the adult offspring metabolic syndrome such as obesity and diabetes. Metabolic syndrome is strongly related to the development of endothelial dysfunction. Objective: investigate if maternal exposure to MET could interfere with aortic endothelial function. Material and methods: Wistar female rats were treated with metformin 293mg/kg/day, by gavage from gestational day (GD) 0 to the GD 21 (METG) or water by gavage at the same period (CTRG). It was evaluated in male offspring (75 days) the aortic reactivity to phenylephrine (Phe), acethylcoline (Ach) and sodium nitroprussiate (SNP) in the presence (E+) or absence (E-) of endothelium. Data were expressed as mean±SEM and compared by one-way ANOVA (*p<0.05). The study was approved by the CEUA/UEL (6996.2015.02). Results: The contraction induced by Phe in rings E+ or E- was similar between METG and CTRG [E+: 2.23±0.11 (10) vs 2.18±0.23 (8); E-: 3.64±0.46 (7) vs 3.45±0.27 (8)]. The % of relaxation to Ach was similar between METG and CTRG groups [91.40±2.18 (8) vs 90.36±2.19 (6)] and as well as the response to SNP [METG: 94.29±0.94 (10) vs CTRG: 94.56±0.81 (7)]. Conclusion: These results suggest that maternal exposure to metformin did not interfere with the vascular endothelial function in adult male offspring.

Keywords: Vascular Reactivity; Endothelial Function; Maternal Exposure to Metformin. Thematic area: Non-transmitted diseases.

GENE EXPRESSION ANALYSIS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA: CHEMOTHERAPY INFLUENCE IN KEY REGULATING GENES

Carlos Eduardo Coral de Oliveira1; Marla Karine Amarante1; Alberto Yoichi Sakaguchi1; Fausto Celso Trigo 2; Glauco Akelinghton Freire Vitiello1; Maria Angelica Ehara Watanabe 1 1 State University of Londrina, Laboratory of Study and Application of DNA Polymorphisms, Dept. Pathological Sciences, Biological Sciences Center, Londrina-PR, Brazil. 2 Londrina Cancer Hospital, Division of Hematology and Oncology, Londrina-PR, Brasil.

Introduction: Acute lymphoblastic leukemia (ALL) is a hematological malignancy, and the main childhood cancer. To date, approximately 70% of pediatric patients with ALL have excellent long- term response to combined chemotherapy, however, the absence of molecular predictors of less favorable outcome and disease recurrence have concerned oncologists and have been addressed by researchers. Recent reports support a differential gene expression profiling for ALL, which may lead to distinct outcomes and determine prognosis factors. Objective: Here, we present gene expression data of 64 children acute lymphoblastic leukemia (ALL) patients compared to 30 neoplasia-free children. Material and methods: This retrospective study was approved by Human Ethics Committee of the State University of Londrina (No. 214/09-CAAE N°. 0164.0.268.000-09). qPCR was used to determine mRNA expression of human CXCR4, CXCR7, CXCL12, TGFB1 and FOXP3 target genes, ACTB and GAPDH reference genes, from leukocytes of peripheral blood. Graphpad Prism 6 was used to calculate differential expression patterns. Results: While CXCL12 have presented regular expression, FOXP3, CXCR4, CXCR7 and TGFB1 genes were down regulated in ALL patients (almost 4 fold lower). Chemotherapy treatment modified gene expression, modulating CXCR4 and TGFB1 mRNA. At diagnosis and during treatment, CXCR4 remains down regulated almost 4 fold lower than controls. Notably, leukocytes from patients under remission expressed regular CXCR4 mRNA (compared to controls), which was significantly different from patients during treatment (p=0.01) and at diagnosis (p=0.03). Furthermore, TGFB1 gene have shown significant down expression, which was maintained during treatment (almost 5 fold lower), but decreased during remission phase (p=0.02). Conclusion: Gene expression analysis revealed that key regulating genes are down regulated in ALL patients. Therapeutic approach in ALL is able to regulate important key genes, such as CXCR4, restoring its normal expression, and TGFB1, a negative regulator of hematopoiesis, but a possible marker of therapeutic success.

Keywords: ALL; Chemotherapy; Regulatory Genes. Grants: Scholarship from CAPES/FAADCT-PR (Nº 41.080), Research grant from CNPq (Nº 442319/2014-2). Thematic area: Non-transmitted diseases.

BARK ETHANOLIC EXTRACT FROM Spondias dulcis FORST F. PROTECTS AGAINST ALTERATIONS OF THE REDOX STATUS INDUCED BY CYCLOPHOSPHAMIDE AND BENZO[a]PYRENE IN MICE

Caroline de Souza Araujo1; Lorrane Davi Brito1; Marcos Alberto Zocoler2; Leandra Ernst Kerche- Silva1. 1 Faculdade de Artes, Ciências e Letras, Universidade do Oeste Paulista, 19050-920, Presidente Prudente-SP, Brazil. 2 Faculdade de Farmácia, Universidade do Oeste Paulista, 19050-920, Presidente Prudente-SP, Brazil.

Introduction: Spondias dulcis is widely used for medicinal purposes. However, no in vivo study concerning its effects on the redox status has been conducted. Objective: The aim of this study was to assess the effects of bark ethanolic extract from S. dulcis on cyclophosphamide (CP)- and benzo[a]pyrene (B[a]P)-induced oxidative stress in liver, kidney and total blood of mice. Material and methods: 10 animals per group were used for the tests and three concentrations of the extract were used, 500, 1000 and 1500 mg/kg bw. Benzo[a]pyrene (B[a]P) and Cyclophosphamide (CP) were used to evaluate the protective effects. B[a]P (9 mg/kg bw) and CP (40 mg/kg bw) were used as oxidative stress inducers. 24 h after the treatment the animals were euthanized and liver, kidney and total blood were collected. Liver and kidney were investigated for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH), and total blood was investigated for GSH. Local Ethics Committee for Animal Use (CEUA) of Unoeste approved this study, register No. 2950/2016. Results: The extract did not raise TBARS levels in liver and kidney and it was able to deplete TBARS levels associated to B[a]P and CP. The extract also did not deplete the levels of GSH in liver, kidney and total blood, and were able to increase the levels of GSH associated to B[a]P and CP. The most protective concentration was 500 mg/kg bw. Conclusion: These results show that the treatment with this extract attenuates B[a]P- and CP-induced oxidative stress. More studies are being accomplished.

Keywords: Spondias dulcis; Oxidative Stress; GSH. Grants: UNOESTE, CAPES. Thematic area: Non-transmitted diseases.

THE VANILLIC ACID INHIBITS THE EHLICH-TUMOR CELLS INDUCED PAIN LIKE-BEHAVIOR

Cássia Calixto de Campos1; Thacyana Teixeira de Carvalho1; Camila Rodrigues Ferraz1; Marília Fernandes Manchope1; João Eurípedes Calixto de Campos1; Rubia Csagrande2; Waldiceu Aparecido Verri Junior1 1Universidade Estadual de Londrina, Centro de Ciências Biológicas, Londrina - Pr, 2Universidade Estadual de Londrina, Centro de Ciências da Saúde, Londrina - Pr

Introduction: Cancer pain is a severe clinical health problem for these patients and currently the treatment for this pain is inadequate enhancing this problem. Vanillic acid is a flavoring agent found in edible plants and fruits. These compounds have received considerable attention. However, the effect of vanillic acid in models of cancer has not been investigated. Objective: Investigate the analgesic effect of vanillic acid, and their mechanism of action in Ehrlich cells-induced cancer pain in mice. Material and methods: Male Swiss (20-25g; n6) were used for experiments (CEUA n° 16888.2015.41). Firstly, mice received 1x106 Ehrlich tumor cells, intraplantar i.pl., and after 8 days was accessed the dose (10, 30 and 100 mg/kg intraperitoneal i.p) and time response (1-7 hours) for acute treatment with vanillic acid (diluted in 5% Tween and saline). Mechanical (digital analgesymeter, von Frey) and thermal hyperalgesia (hot plate 52°C) and paw thickness (caliper rule) were accessed for investigate the effect of treatment. After, mice were daily treated during during 12 days after injection of 1x106 cells. Mechanical and thermal hyperalgesia and paw thickness were accessed with intervals of 24h. On the 12th day, mice were terminally euthanized and the cutaneous plantar tissue was collected for the determination of myeloperoxidase (MPO) and n- acetylglucosaminidade (NAG) activity. Results: The acute treatment with vanillic acid reduced time and dose dependent manner the mechanical and thermal hyperalgesia, but not alter the paw thickness. The dose of 100 mg/kg i.p was choice for the next experiments. The chronic treatment reduced the mechanical and thermal hyperalgesia, paw thickness, MPO and NAG activity. Conclusion: The vanillic acid inhibits the cancer pain induced by Ehrlich tumor cells by anti- inflammatory mechanism.

Keywords: Cancer Pain; Hyperalgesia; Vanillic Acid Grants: We acknowledgements from support of Capes, CNPq and Fundação Araucária Thematic area: Non-transmitted diseases

POSSIBLE EFFECTS OF TGFB1 SIGNAL PEPTIDE AND PROMOTER REGION GENETIC POLYMORPHISMS AND HAPLOTYPE STRUCTURES: POSSIBLE ROLE AS A SUSCEPTIBILITY MARKER

Cintya Mayumi Ishibashi1, Carolina Batista Ariza1, Roberta Losi Guembravoski2, Carlos Eduardo Coral de Oliveira1, Glauco A. Freire Vitiello1, Marina Okuyama Kishima3, Daniela Rudgueri Derossi4; Daniela Sartori5; Alda Losi Guembarovski3, Fernanda Pelisson Massi2, Maria Helena P. Fungaro2, Maria Angelica Ehara Watanabe1

1Laboratory of Study and Application of DNA Polymorphisms, State University of Londrina, Londrina, Parana, Brazil 2Department of General Biology, State University of Londrina, Londrina, Parana, Brazil 3Department of Pathology, Clinical Analysis and Toxicology, Heath Sciences Center, State University of Londrina, Londrina, Parana, Brazil 4Laboratory of Pathology, Cancer Hospital of Londrina, Londrina, Parana 5Department of Biochemistry, State University of Londrina, Londrina, Parana, Brazil

Introduction: Wilms' tumor (WT) is an embrionary neoplasia that accounts for approximately 6% of all childhood tumors. The microenvironment is critical in the pathogenesis of cancer, and the transforming growth factor beta 1 (TGFB1) is a pleiotropic cytokine associated with tumor development. In this context, several TGFB1 polymorphisms were studied, including polymorphisms in the promoter region and signal peptide. Objective: To investigate possible effects of rs1800468 (c.-1638G>A) and rs1800469 (c.-1347C>T) polymorphisms, both the promoter region, and rs1800470 (c.29C>T) and rs1800471 (c.74G>C), signal peptide, and their haplotype structures in susceptibility to TW. Material and methods: The project was approved by the Ethics Committee of the State University of Londrina (CEP/UEL 189/2013 – CAAE 17123113400005231) and included 22 tumor tissue samples and 99 samples of children free of neoplasia. DNA was obtained and the genetic polymorphisms were analyzed by PCR-RFLP analysis followed by polyacrylamide gel electrophoresis stained with silver. The control-case study for TW susceptibility was assessed through odds ratio analysis and Fisher's exact test, performed using Prism 6 for Windows and the TGFB1 haplotypes were determined using PHASE version 2.1.1 software. Results: The case-control study indicated association of the T allele of the c.-1347C>T polymorphism in a recessive manner with increased susceptibility to TW (OR: 10.68; CI: 3.685 to 30.95; p<0.0001). Similarly, the C allele of c.29C>T polymorphism in a recessive manner, showed a tendency to risk (OR: 2.800; CI: 0.9692 to 8.089, p:0.0644). Was observed a significant difference in the distribution of haplotype structures between control children and WT (p:0.001), whose haplotype GCTG was associated with protection (OR: 0.2361 CI: 0.1045 to 0.0534; p:0.0002) and GTTG haplotype associated with susceptibility to WT (OR: 12.00, Cl: 4.202 to 34.27; p<0.0001). Conclusion: These results suggest that this gene could be used, after further studies, as prognostic marks to WT susceptibility.

Keywords: Wilms Tumor; Polymorphism; TGFB1 Grants: Hospital do Câncer de Londrina, Secretaria da família e desenvolvimento social, Fundação Araucária, CNPq Thematic area: Non-transmitted diseases

VITAMIN D DEFICIENCY IS ASSOCIATED WITH ACUTE ISCHEMIC STROKE, C-REACTIVE PROTEIN, AND SHORT-TIME OUTCOME

Daniela Frizon Alfieri1; Márcio Francisco Lehmann1; Lígia Grecco Costa Dall’Aqua2; Sayonara Rangel Oliveira1; Tamires Flauzino1; Francieli Delongui1; Maria Caroline Martins de Araújo1; Rafaele Maria Tirolla1; Isaias Dichi1; Vinícius Daher Delfino1; Andréa Name Colado Simão1; Edna Maria Vissoci Reiche1. 1Universidade Estadual de Londrina/Centro de Ciências da Saúde 2Universidade Estadual de Londrina/Centro de Ciências Biológicas

Introduction: Vitamin D deficiency (VDD) and elevated levels of high sensitivity C-reactive protein (hsCRP) can be risk factors for strokes. However, the presence of vitamin D indicates neuroprotective effect, reducing sequels in poststroke patients. Objective: The aim of this study was to investigate whether VDD is associated with acute ischemic stroke, inflammatory markers, and short-time outcome. Material and methods: 168 acute ischemic stroke patients and 118 subjects were included. The modified Rankin Scale (mRS) was applied up to eight hours of stroke and after three-month follow-up, and blood samples were obtained up to 24 hours of stroke to evaluate serum levels of 25-hydroxivitamin D [25(OH)D] determined using chemiluminescent microparticle immunoassay, and inflammatory markers (Leukocyte counts; hsCRP; Ferritin; INF-α; IL-6). Vitamin D status classified the individuals in sufficient (VDS ≥30 ng/mL), insufficient (VDI 20-29.9 ng/mL), and deficient (VDD <20.0 ng/mL). Results: Patients presented with lower levels of 25(OH)D and higher frequency of VDD (43.45% vs. 5.08%, p<0.0001), and higher inflammatory markers than controls (p<0.05). After adjustment for the classical variables associated with the occurrence of this ischemic event, such as age, sex, ethnicity, BMI, smoking status, presence of diabetes, hypertension, dyslipidemia and drugs, patients with VDD were 18.40 more likely to present acute ischemic stroke when compared to those with VDS status (OR: 18.40, 95% CI: 6.14-55.175, p<0.0001). Patients with VDD presented higher hsCRP levels than those with VDS (p=0.043); those with poor outcome presented lower 25(OH)D levels than those with good outcome (p<0.008); moreover, 25(OH)D levels were negatively correlated with mRS after three-month follow-up (r=- 0.239, p=0.005). Conclusion: The associations between VDD and higher hsCRP levels in acute ischemic stroke, and between 25(OH)D levels and poor outcome at short-term of acute ischemic stroke patients suggest the important role of vitamin D in the inflammatory response and pathophysiology of this ischemic event.

Keywords: Vitamin D; Acute Ischemic Stroke; High Sensitivity C-Reactive Protein. Thematic area: Non-transmitted diseases.

IMMUNE RESPONSE AND VIRULENCE FACTORS ASSOCIATED CANDIDIASIS IN DIABETES: A REVIEW

Luciana Mendes1; Thais Peron da Silva1; Maria Eduarda Tesser1; Mariana Barbosa Detoni1; Renan Cajuca1; Daniele Sapede Alvarenga1; Wagner Loyola1; Wander Rogério Pavanelli1; Idessania Nazareth Costa1; Francine Neselo Melanda1; Ivete Conchon Costa1. 1 Department of Experimental Pathology – Laboratory of Experimental Protozoology, State University of Londrina, Londrina, Brazil.

Introduction: Systemic fungal infections have increased dramatically in prevalence and severity in the last decades, in accordance with increasing number of patients with immune dysfunctions, principally in patients with Diabetes Mellitus (DM). The uncontrolled glycemic rate resulting in a predisposition to opportunistic infections, especially Candida species. The immune response of host and virulence factors of Candida spp. determines the nature and magnitude of the infection. Objective: Thus, this review aims to describe the immune response of diabetic patients and the virulence factors associated with candidiasis. Material and methods: It was realized a systematic literature search through periodical available electronically in the period 1985 – 2016, using the bases National Center for Biotechnology Information (NCBI), Science Direct e Web of Science. Results: Thirty articles were selected for this study. In patients with DM, occurs an increase in expression of the enzyme myeloperoxidase and NET formation in neutrophils. Studies show an increase in the enzymatic activity of hemolysin, proteinase and esterase in Candida spp. The frequency of switching and adherence to host cells was bigger in diabetic patients. Conclusion: The mechanisms involved in fungal infections in diabetic patients are still not well understood. Hyperglycemia appears to be an important modulator agent of the immune response, and promote the proliferation of Candida spp. and exacerbates virulence factors involved in the infection.

Keywords: Candida spp.; Diabetes Mellitus; Infection. Thematic area: Non-transmitted diseases

EVALUATION OF METABOLIC AND CARDIOVASCULAR PARAMETERS OF MALE AND FEMALE RATS EXPOSED TO METFORMIN DURING GESTATIONAL AND LACTATION PERIOD

Daniella Regina Barrionuevo da Silva Novi1; Simone Forcato1; Camila Borecki Vidigal1; Guilherme Henrique Loiola1; Daniela Ceccatto Gerardin1; Graziela Scalianti Ceravolo1. 1Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina/PR.

Introduction: Maternal exposure to drugs during the intrauterine development may influence the health of offspring in adulthood. Metformin is used for the treatment of gestational diabetes. Treatment of obese and diabetics subjects with metformin causes weight loss and reduction of adipose mass. On the other hand, exposure to this drug during gestation can increase offspring’s body weight and mesenteric fat after high fat diet. Objective: To evaluate the metabolic and cardiovascular parameters in adult rats female and male exposed to metformin during pregnancy and lactation. Materials and methods: Wistar female rats were treated with metformin 293mg/kg/day, by gavage from gestational day (GD) 0 to the GD 21 (METG) or GD 0 until lactation day 21 (METGL). Controls dams received water by gavage at the same periods (CTRG and CTRGL). It was evaluated in male and female offspring with 75 days of age: Lee index, the weight of the perigonadal and retroperitoneal fat pads (g/100 of body weight), body weight (g), mean arterial blood pressure (MAP, mmHg) and heart rate (HR, bpm). Data expressed as meanSEM, (n) number of rats/group. Results were considered statistically significant if p<0.05 and compared by Student t-test (CEUA/UEL: 6996.2015.02).Results: Lee index and body weight of males or females metformin rats of different treatments were similar to their respective controls of the same sex. In female offspring, the weight of the perigonadal fat pad were reduced in METG [0.44±0.02 (10) vs CTRG [0.68±0.11 (8)]. In male offspring, metformin exposure reduced retroperitoneal fat pad in [CTRGL 1.51±0.12 (7) vs METGL 1.06 ±0.13 (7)]. The MAP and HR were similar between metformin rats and their respective control of the same sex. Conclusion: It was possible to conclude that exposure to metformin during gestational and lactation did not interfere with metabolic and cardiovascular parameters of adult progeny.

Keywords: Maternal Treatment; Obesity; Blood Pressure. Grants: CAPES Thematic area: Non-transmitted diseases

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2A: CASE REPORT

Poliana Camurça1, Denis Massatsugu Ueda1, Murilo Carlos Gimenes1, Alvaro Ferdinando Scremin1, Rafael Yoshio Kanashiro1, Mauricio Pacheco Reis1, Andre Armani1 1Londrina State University, Rodovia Celso Garcia Cid, Km 380, Campus Universitário, Londrina - PR

Introduction: Multiple endocrine neoplasia type 2A (MEN2A) is characterized by patients with medullary thyroid cancer (MTC), pheochromocytoma and primary hyperparathyroidism. Objective: To report a case of MEN2A and highlight the importance of the correct management. Case report: R.P, 17-year-old, was admitted at our head and neck surgery service presenting hypertensive crisis, palpitations, diaphoresis and tremors. The possibility of secondary hypertension was considered, therefore, laboratory tests and computed tomography (CT) were requested. They revealed high levels of urinary metanephrines and tumors in right and left adrenal, so pheochromocytoma was diagnosed. Metaiodobenzylguanidine (MIBG) scan excluded extra-adrenal involvement. After preoperative preparation with alpha and beta blocker the patient underwent bilateral adrenalectomy. After surgery, the patient remained normotensive and was discharged with prednisone 5mg/day. The patient underwent further endocrinological research, in which a firm thyroid nodule was found and laboratory tests showed parathyroid hormone (PTH) of 148.9 and calcitonin of 397. Fine-needle aspiration (FNA) of thyroid nodules and parathyroid scintigraphy were performed. Findings of FNA were compatible with MTC and scintigraphy evidenced suggestive parathyroid hyperactivity in the left lower pole. Thus, the patient underwent total thyroidectomy, neck dissection and resection of parathyroid hyperactivity. Pathology was able to confirm MTC and parathyroid adenoma. The patient is in endocrinological monitoring with levothyroxine 175mcg and prednisone. In paralel, we made a screening of patient's relatives to exclude MEN2A and its variants. Discussion: Early diagnosis in first-degree relatives of MEN2A patients is the main focus nowadays. The family screening is done with pentagastrin test, catecholamine dosages, urinary metanephrines and genetic evaluation of the RET gene, since the absence of mutation establishes that the person has the same chances of developing a MEN2A than general population. Conclusion: Knowledge of MEN2A in their clinical, laboratory and screening aspects is extremely important, considering early diagnosis is directly related to the reduction of mortality.

Keywords: MEN2A; RET gene. Grants: There is no financial support or relationships that may pose conflict of interest to declare. Thematic area: Non-transmitted diseases

PROTECTIVE EFFECTS OF NICOTINAMIDE ON MICE OFFSPRING EXPOSED TO CYCLOPHOSPHAMIDE DURING PREGNANCY

Eliane Swely Amador Monteiro Sanches1, Vinicius Popolin1, Felipe Tsuzuki1, Maria José Sparça Salles1 1Department of General Biology, Londrina State University, Londrina - PR - Brazil.

Introduction: Nicotinamide participates in activities related to the maintenance of the original structure of the DNA. Cyclophosphamide is an alkylating agent prescribed to treat different types of carcinomas. Objective: Thus, we investigated the effects in animals undergoing treatment with nicotinamide and its possible protective effects on toxic and teratogenic model induced by cyclophosphamide. Material and methods: Approved by CEUA-UEL n. 5265.2015.28. Pregnant females were divided into six experimental groups, each one with 15 animals. The groups 1 and 2 (nicotinamide 100mg/kg and 200mg/kg respectively); Group 3 (saline); Group 4 (cyclophosphamide 50 mg/kg); Associated Treatment 5 and 6 (nicotinamide 100mg/kg and 200mg/kg associated with cyclophosphamide 50mg/kg respectively). On the 18th day of pregnancy females were euthanized and conducted to evaluate the intrauterine development of the offspring and the presence of congenital malformations. The absolute data with normal distribution were analyzed by ANOVA followed by Tukey’s test and with non-normal distribution and the analyses of maternal parameters were analyzed by student’s t test. Data for external, visceral and skeletal malformations were analyzed by Fisher's exact test. Results: The groups G1 and G2 did not show any parameter of toxicity. Associated Treatment 5 and 6 have not been able to minimize the adverse effects induced by cyclophosphamide on maternal toxicity and congenital malformations. However, the group associated with nicotinamide at a dose of 200mg/kg showed a protective effect on the increase in reabsorption rate (G4: 42.58 ± 10.32; G6: 19.20 ± 9.54) and nicotinamide in the two tested doses showed protective effects in intra-uterine growth retardation (G4: 0,83±0,11; G5: 1,05±0,16; G6: 1,16±0,13) and placental weight (G4: 0,05±0,07; G5: 0,06±0,010; G6 :0,07±0,01) induced by cyclophosphamide. Conclusion: The results of this study are promising, nicotinamide in a dose- dependent manner exerted protective effect, reducing the number of abortions, the morbidity and mortality induced by cyclophosphamide.

Keywords: Antineoplastic Agents; Reproductive Performance; Teratogenesis. Thematic area: Non-transmitted diseases

ALTERATIONS IN REPRODUCTIVE PARAMETERS OF MICE EXPOSED TO WINE DURING SPERMATOGENESIS

Fábio Augusto Joinhas1; Fernanda Dias Figueira1; Maria José Sparça Salles1. 1Department of General Biology, Londrina State University, Londrina - PR - Brazil.

Introduction: Chronic alcohol consumption by men is associated with impotence, decreased testicular steroids and atrophy of the gonads. It is the most consumed drug abuse and prevalence among youth. Objective: To evaluate possible alterations induced by wine in the fertility of male mice when administered during spermatogenesis. Materials and methods: Ethics Committee for Animal Experimentation of the Londrina State University of number 8198.2013.22. Male Swiss mice were divided into two groups, the group treated with wine (G1) at a concentration of 1.9 mL / kg of ethanol and the control group (G0) which was administered PBS under the same experimental design. The course of treatment was intraperitoneally for a period of 35 days, equivalent to a sperm cycle in mice that day, the animals were put to mate with females without any treatment. The animals continued to be treated for more 10 days, corresponding to two estrous cycles in female, allowing fertilization to occur. On the 46th day, blood was collected by cardiac puncture for testosterone dosage and euthanasia by cervical dislocation. After laparotomy and removal of organs, the reproductive parameters were evaluated. Results: The evaluation of reproductive performance showed the following statistically significant alterations compared to the control group: reduction of testosterone level (G0: 259,5 ± 55,68 e G1: 183,0 ± 49,08) and decreasing in mating frequency (G0: 100% e G1: 70%). Morphological analysis of sperm showed abnormalities in the head (G0: 1,7% e G1: 16,25%) and tail (G0: 16,8% e G1: 27,35%), significant in the group treated with wine. The organs: lung, kidney, liver, testis and epididymis showed no significant alteration. Conclusion: Wine consumption in the tested concentration alters spermatogenesis and compromise reproductive performance.

Keywords: Spermatogenesis; Reproductive Performance; Alcohol Thematic area: Non-transmitted diseases

ANTIBIOTIC SUSCEPTIBILITY OF Staphylococcus aureus ISOLATED FROM BLOODSTREAM INFECTIONS IN A UNIVERSITY HOSPITAL SOUTH BRAZIL FOR A PERIODO OF FIFTEEN YEARS

Felipe Crepaldi Duarte1; Marcia Regina Eches Perugini2. 1Master´s student of the State University of Londrina 2Department of Pathology, Clinical Analysis and Toxicology, State University of Londrina

Introduction:Staphylococcus aureus are among the most common etiologic agents, both in health care associated infections (HAIs) such as those acquired in the community. Objective: The purpose of this study was to evaluate the resistance profile of Staphylococcus aureus over 15 years bloodstream infections and evaluate the epidemiological change in the resistance profile of S. aureus and enhance the clinical management. Material and Methods: The results of susceptibility testing of 1.432 positive blood cultures collected at the Laboratory of Clinical Microbiological Analysis (LACM) of a university hospital in southern Brazil were analyzed. This study was approved by the committee of ethics (CAAE número 0015.0.268.000-11). Results and Conclusion: The results show that there was continued resistance to some antimicrobials like ciprofloxacin (42%), clindamicin (50.29%), eritromicin (54.2%), oxacilin (42.28%), penicillin (92.91%) and significant drop in resistance to the antibiotic gentamicin (6.96%), rinfampicina (3.46%), trimethoprim sulfamethoxazole (0,56%) and tetracilclin (1.96%). With this study we showed that there was a decrease in the resistance rate of some antimicrobials and lifting others in the past 15 years. This may change the empirical treatment of bacteremia whose etiologic agent is Staphylococcus aureus.

Keywords: Staphylococcus aureus; Antimicrobials; Susceptibility Thematic area: Non-transmitted diseases

EVALUATION OF PHYSICAL AND REFLEXOLOGICAL DEVELOPMENT OF MICE OFFSPRING EXPOSED TO PROPOFOL IN DIFFERENT GESTACIONAL PERÍODS

Felipe Tsuzuki1; Lucimara Aparecida Sensiate1; Maria José Sparça Salles1 1Department of General Biology, Londrina State University, Londrina - PR - Brazil.

Introduction: Propofol (2,6-diisopropylphenol), is a phenol derivative used as anesthetic. Patients undergoing anesthesia and surgery during pregnancy, have incidences of miscarriage and perinatal mortality. However, little is known about the postnatal development. Objective: To investigate the physical and reflexological development of mice offspring who were exposed to propofol in different periods of pregnancy. Material and Methods: Approved by CEUA-UEL, No. 13035/2008. Pregnant mice were divided into six groups, three groups treated with propofol concentration of 15 mg / kg-1 intravenously at 5th; 10th or 15th gestational day (GD) and the control group received saline under the same experimental design. The birth was natural to allow the analysis of parameters related to the physical and reflexological development. The results were analyzed by Student's t test and by Mann-Whitney using the GraphPad Prism program. Results: The offspring whose mothers received propofol on the 15th gestational day showed significant delay in detachment of the pinna (Control: DG5 = 3.96 ± 0.14, 3.59 ± DG10 = 024, DG15 = 3.96 ± 0.13; Treaties: DG5 = 4.09 ± 0.21, 4.14 ± 0.24 = DG10, DG15 = 4.42 ± 0.13) and increase in anogenital distance of male offspring (Control: DG5 = 7.92 ± 0.29, 7.95 ± 0.46 = DG10, DG15 = 7.12 ± 0.41; Treaties: DG5 = 7.28 ± 0.43, 7.48 ± 0.36 = DG10 and DG15 = 8 56 ± 0.53) in millimeters. Though not statistically significant in physical parameters such as: eruption of the incisor teeth, opening of the ear canal, eye opening, testicular descent / vaginal opening, appearance of nipples and anogenital distance female puppies. Regarding Reflexive parameters, the negative geotaxia and adult have not changed. Conclusion: Propofol affected postnatal development of the parameters of anesthetized mothers of offspring at the end of pregnancy, indicating a possible teratogenic effect of the drug.

Keywords: Propofol; Teratogenesis; Postnatal. Thematic area: Non-transmitted diseases.

NITRIC OXIDE-GLUTAMATE INTERACTIONS IN ROSTRAL VENTROLATERAL MEDULLA OF OBESE RATS: INVOLVEMENT OF iNOS ISOFORM

Fernanda Novi Cortegoso Lopes1; Hiviny de Ataides Raquel1; Nagela Ghabdan Zanluqui2; Sueli Fumie Yamada-Ogatta3; Phileno Pinge-Filho2; Marli Cardoso Martins-Pinge1 1Department of Physiological Sciences, State University of Londrina, Londrina, Parana Region, Brazil. 2Department of Pathological Sciences, State University of Londrina, Londrina, Parana Region, Brazil. 3Department of Microbiology, State University of Londrina, Londrina, Parana Region, Brazil.

Introduction: Nitric oxide (NO) plays an important role in cardiovascular function, and the production via inducible (iNOS) is linked to inflammatory processes, as obesity. In rostral ventrolateral medulla (RVLM), glutamate is an important neurotransmitter and NO modulates its effects. Objective: Our aim was to evaluate the effects of L-glutamate (GLU) microinjection in RVLM on mean arterial pressure (MAP) and heart rate (HR) before and after microinjection of iNOS inhibitors; gene expression of iNOS and dosage of NO (nitrite) in punchs of RVLM in control (CTR) and obese (MSG) rats. Material and methods: Approved by ethics committee UEL nº 33645/2010-29. Wistar rats received 4 mg/g body weight of monosodium glutamate or equimolar saline within the first 5 days of life. At 90 days were implanted guide cannulas to RVLM and artery catheterization after 3 days. After 24 hours was performed the record of MAP and HR recordings. Data analized by two-way Anova/ Bonferroni test. The effects of Glu microinjection (5nm/100nL) were evaluated before and after aminoguanidine (AG) or S-methylisothiourea (SMT)(250pmol/100nL) or physiological saline (sal)(100nL) at RVLM. Results: Obesity was evidenced by increased Lee index MSG groups in relation to CTR (n=6) and increased weight of the perigonadal and retroperitoneal fat. The increased MAP by Glu was higher in MSG group after AG (before: 40 ± 7.93 mm Hg; after: 61 ± 4.39 mmHg, p<0.05) and SMT (before: 45 ± 7.58 mm Hg, after: 65 ± 2.42 mmHg,p<0.05). HR was increased after AG in CTR group (before 78 ± 12.12 bpm, after: 132 ± 12.12 bpm, p<0.05) and SMT (before 43±46 bpm, after 119±29 bpm, p<0.05). Nitrite measurement showed no differences as gene expression for iNOS. Conclusion: Our data suggest a functional modulation of nitric oxide in the RVLM of obese rats, but it may not be only derived from iNOS source.

Keywords: Sympathetic; Nitric Oxide; Arterial Pressure. Grants: CAPES. Thematic area: Non-transmitted diseases.

SKELETAL MUSCLE WASTING PROMOTES LIPID PEROXIDATION AND SARCOPLASMIC RETICULUM ALTERATIONS IN A FIBER TYPE DEPENDENT MANNER

Fernanda Paschoal Blegniski1; Feliciano Protasi2; Claudia Pecorai2; Rubens Cecchini 3; Simona Boncompagni 2; Flávia Alessandra Guarnier1 1 Pathophysiology Laboratory of Muscular Adaptations, Department of Pathological Sciences, Universidade Estadual de Londrina, 86051-990 Londrina, Brazil 2 Center for Research on Ageing and Department of Neuroscience and Imaging, Università G. d’Annunzio, 66100 Chieti, Italy 3 Pathophysiology Laboratory of Free Radicals, Department of Pathological Sciences, Universidade Estadual de Londrina, 86051-990 Londrina, Brazil

Introduction: Skeletal muscle wasting is a very common complication in cancer progression. There is no literature data that relates muscle ultrastructure changes and sarcoplasmic reticulum volume alteration during muscle mass loss in cancer development. Objective: The objective of the present study was to determine differences in ultrastructure of muscle fibers from extensor digitorum longus (EDL) and soleus, and its relation to lipidperoxidation in an experimental model of cancer cachexia. Material and Methods: Male Wistar rats (200-230g) were divided into three groups: Control (C5), Walker-256 tumor bearing rats (T5) and Walker-256 tumor bearing rats treated with N-acetylcysteine 1% (T5NAC). Animals were euthanized (approved by Ethics Committee on Animal Experimentation from the Universidade Estadual de Londrina, Brazil (ref.9775). Body, tumor, soleus and EDL weights were registered. Results: On skeletal muscle homogenates, lipid peroxidation was determined by chemiluminescence (CL) and carbonyl protein content was quantified. Histological parameter of muscle adaptation was determined by soleus and EDL cross-sectional area (CSA). Sarcoplasmic reticulum (SR) volume was measured using a stereology point-counting technique through electronic microscopy (ME) transversal sections micrographs. Data were compared by Kruskall-Wallis test followed by Dunn´s multiple comparison test. Protein carbonyl content showed no significant difference in all groups. In contrast, T5 CL curves demonstrated to be different when compared to C5 and T5NAC in soleus and EDL. The CSA frequency distribution of fibers showed qualitative differences between groups. The 50th, 75th and 90th percentile of fiber area in T5 groups were reduced in soleus and EDL when compared to control groups. The treatment was efficient to revert fiber area to control levels in all analyzed percentiles. The relative fibers volume occupied by SR was significant altered in soleus T5, but not in EDL. Conclusion: The present study demonstrated ultrastructural alterations correspondingly to oxidative stress parameters forward to challenges that modulate muscle mass.

Keywords: Sarcoplasmic reticulum, extensor digitorum longus and soleus. Thematic area: Non-transmitted diseases

KAURENOIC ACID REDUCES EOSINOPHIL RECRUITMENT IN ASTHMA BY REDUCING TH2 CYTOKINE PRODUCTION IN MICE

Talita P. Domiciano1; Fernanda S. Rasquel de Oliveira1, Nilton S. Arakawa2, Rubia Casagrande2, Waldiceu A. Verri Jr.1

1 Departamento de Ciências Patológicas - Centro de Ciências Biológicas, Universidade Estadual de Londrina, 86051990 Londrina, Brazil. 2 Departamento de Ciências Farmacêuticas - Centro de Ciências de Saúde, Universidade Estadual de Londrina, Londrina, Paraná, Brazil.

Introduction: Bronchial asthma is a chronic inflammatory disease of the airways in which some studies have been attempting to find strategies for treatment, including the use of medicinal plants. Kaurenoic acid (KA) is a natural compound which is known by an anti-inflammatory and analgesic actions. Objective: To investigate the effect of kaurenoic acid in the cellular recruitment and cytokine production in a murine model of asthma. Material and methods: Isolation and identification of KA was done by collecting roots of Sphagneticola trilobata. Male Swiss mice (n=6/group) were used. Care and handling procedures were approved by the Ethics Committee of the Universidade Estadual de Londrina (process number 1440.2011.09). For asthma model, mice were sensitized by ovalbumin (OVA) plus aluminum hydroxide by intraperitoneal injection. The challenge was made by nebulization with OVA using an ultrasonic nebulizer. For treatment, mice received per oral KA (0.1, 1 and 10 mg/kg) or vehicle 1h before nebulization. To obtain the bronchoalveolar lavage fluid (BALF) samples, mice were sacrificed 24h after the last challenge with OVA and it was obtained by instillation followed by aspiration with saline into lungs. Total and differential cell count was performed. The cytokine production was determined by ELISA. Results: KA reduced in a dose-dependent manner the recruitment of total leukocytes (up to 90%) in the BALF of mice immunized against OVA. There was also inhibition of eosinophil recruitment (up to 89%). In agreement, kaurenoic acid reduced the production of Th2 cytokines IL-4 (up to 100%), IL-5 (up to 100%) and IL-33 (up to 71%). There was no significant modulation of IL-10 levels. Conclusion: These results support further investigation on the use of kaurenoic acid to reduce the airway inflammation in asthma, and that its anti-inflammatory mechanism is related to inhibition of Th2 cytokine production.

Keywords: Kaurenoic Acid; Asthma; Cytokines

Thematic area: Non-transmitted diseases

MACROPHAGES-EXPRESSING FOXP3 TRANSCRIPTION FACTOR: DO THEY REALLY EXIST?

Fernando Cezar dos Santos1; Rodolfo Sanches Ferreira1; Kleber Paiva Trugilo1, Nádia Calvo Martins Okuyama1; Fernanda Costa Brandão Berti1; Adriano Martin Felis Aranome1; Érica Romão Pereira1; Guilherme Cesar Martelossi Cebinelli1; Michelle Mota Sena1; Gabriela Cristine Queiroz Maria1; Luis Fernando Lasaro Mangieri1; Karen Brajão de Oliveira1 1Departament of Pathological Sciences, Londrina State University, Londrina, Parana, Brazil

Introduction: In an imposing work published in The Journal of Experimental Medicine (JEM), Zorro- Manrique et al. showed that mouse macrophages express FOXP3, are immunosuppressants and promote tumor growth. Once there are no studies showing FOXP3 expression in macrophages, this assertion caused polemic in scientific community and three notes were published, disproving this finding. Objective: We aimed to discuss these data critically. Methods: Our discussion will be based on these four papers. Results: Put et al. not detected FOXP3 mRNA or protein expression in bone marrow (BM)/spleen macrophages in vivo (i.e., collagen-induced arthritis) and in vitro (i.e., under GM-CSF/IL-4 stimulation), postulating that the results shown by Zorro-Manrique et al. are artefacts. Meanwhile, the investigation performed by Put et al. was restricted to two tissues and not used a cancer animal model. Possibly, FOXP3-expressing macrophages may occur in a tissue-specific manner in different pathologies. Mayer et al. not found FOXP3 expression in macrophages from WT and melanoma-bearing mice, although a CD11b+FOXP3low subset was found. Li et al., using flow citometry, demonstrated that autofluorescence contributes to false-positive FOXP3 staining in macrophages, once that the same cells did not show FOXP3 expression by qPCR. At the request of Dean for Research of Mayo Clinic Arizona and laboratories worldwide that attempted, unsuccessfully, reproduce the findings of Zorro-Manrique et al., the JEM editors sent the data of these laboratories to Zorro-Manrique team, demanding a retraction. The senior investigator, Dr. Joseph Lustgarten, passed away, and we transcribe an excerpt of this study retracted by his team: ‘’The surviving authors maintain a strong conviction that the paper's substance and the underlying science of regulatory macrophages, are valid’’. Conclusion: Considering that the works presented above are controversial, the question about the existence of regulatory macrophages remains unsolved. Extensive designs are required to minutely elucidate the immunosuppressive function of macrophages.

Keywords: Macrophages; FOXP3; Gene Expression. Thematic area: Non-transmitted diseases

EFFECT OF METFORMIN IN ACUTE MODEL OF MURINE UVB IRRADIATION AND THE INVOLVEMENT OF SYSTEMIC ROS

Fernando Souza Neto1; Poliana Camila Marinello1; Gabriella Pasqual Melo1; Rubens Cecchini1; Leandra Naira Zambelli Ramalho2; Alessandra Cecchini1. 1 Laboratory of Molecular Pathology, State University of Londrina, Brazil. 2 Laboratory of Molecular Pathology, University of São Paulo, Ribeirão Preto,Brazil.

Introduction: Ultraviolet radiation (UVr) induces an inflammatory and oxidative response in epidermal exposed cells, due to mitochondrial and cytosolic alterations. Skin suffers the effects of irradiation, and radiation causing immunosuppression, reactive oxygen species (ROS) increase, which can lead to skin cancer. UVB effects are not only observed on the skin, but also reach systemic levels. Metformin is an oral anti-hyperglycemic agent derived from Galega officinalis, with anticancer effects. Therefore, we studied the use of metformin in murine model of acute radiation and systemic involvement of ROS. Objective: Investigate in murine model of acute irradiation the effect of metformin in systemic levels of ROS and analyzing immunohistochemically and the meaningful participation of ROS on the skin. Material and methods: The ethics committee were approved (CEA14636.2011), were used protocol to get skin of C57BL/6 for evaluate immunohistochemically marked for p53, hydroxynonenal (4-HNE) and nitrotyrosine (NT). Metformin has been used in clinical dose i.p 2%(w/v) for 11 days before irradiation (400mj/cm2). There are 4 groups of 5 mice per group: Control with or without metformin the same for irradiated. Activity of superoxide dismutase (SOD) and catalase (CAT), as well as reduced glutathione levels (GSH) were measured in erythrocytes. The statistics used was parametric test (ANOVA) with Tukey pos test, significance was set at p<0.05. Results: The histochemical analysis of the irradiated group showed loss of epidermis, with partial recover by metformin. The immunohistochemistry for 4-HNE, p53 and NT for the irradiated group presented increased marking on the epidermis when compared to metformin. SOD, GSH, CAT were decreased in the irradiated group and metformin reverses this of condition. Conclusion: Metformin reestablishes the epidermis and partially protects the skin from oxidative injuries. Systemically, metformin decrease the oxidative stress induced by irradiation, by keeping antioxidants at control levels.

Keywords: UVB; Metformin; Immunohistochemically. Thematic area: Non-transmitted diseases

ROLE OF ROS AND IMMUNE RESPONSE OF LANGERHANS CELL IN AN ACUTE UVB IRRADIATION MURINE MODEL TREATED WITH METFORMIN

Fernando Pinheiro de Souza Neto1,2*; Adrián Friedrich2; Valeria Campos2; Mariela Paz2; Juliana Leoni2; Daniel Gonzalez Maglio2; Alessandra L. Cecchini1. 1 Laboratory of Molecular Pathology, State University of Londrina, Brazil. 2 Instituto de Estudios de la Inmunidad Humoral (IDEHU - CONICET). Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.

Introduction: UVB irradiation is very harmful for the human skin. UVB is a source of reactive oxygen species (ROS), and has a strong correlation with skin inflammation and skin cancer. Langerhans cell (LC) are present in the skin epidermis. Knowing that irradiation is able to suppress the systemic immune response, it’s important to know if acute irradiation changes the role of LC. New studies with Metformin suggest that it prevents inflammation, contributing to cancer treatment. Objective: We evaluated the use of metformin in murine model of acute radiation and its action on the immune system of the skin and the involvement of ROS. Material and methods: The ethics committee according to the UBA. Were used protocol to get LC from epidermis of C57/BL6. It was used flow cytometry to evaluate metabolically active cells with the probe DiOC6 and mitochondrial superoxide - anions (O2 ) with MitoSox. Metformin was used in intraperitoneally injections 2% (w/v) for 11 days before irradiation (400mj/cm2). There were 4 groups of 5 mice per group: Control with or without metformin, and the same for irradiated mice. The cytokines were evaluated (IL-6, TNF-α, IL-10) and the statistics used was parametric test (ANOVA) with Newman-Keuls post test (p<0.05). Results: Acute irradiation was sufficient to change the cytokine profile in the epidermis and treatment with metformin showed greater inflammatory profile, raising IL-6 levels (Irrad+PBS mean 71.59pg/mg vs. Irrad+Met 143.99pg/mg) and reducing the immunosuppression caused by UVB-induced IL-10 (Irrad+PBS mean 750pg/mg vs. Irrad+Met 630pg/mg). Irradiation significantly reduces the population LC and metformin does not reverse it, but the metformin improves the metabolism activity increasing the fluorescence. Conclusion: Metformin changes the profile of cytokines in the skin, and increased metabolism, we suggest that this is due to the increased of superoxide anions.

Keywords: UVB; Metformin; Superoxide anions

Thematic area: (2- Non-transmitted diseases)

ALUMINIUM CHLORIDE, DURING THE PERIBUBERTAL PERIOD, AFFECT THE CYTOKINES LEVEL AND MORFOLOGY IN TESTES OF RATS

Franciely A. V. Dantas1,2; Gessica D. Gonçalves1,2; Larissa Staurengo-Ferrari2; Victor Fattori2; Fernando Q. Cunha3; Waldiceu Ap. Verri Jr2; Glaura S. A. Fernandes1

1General Biology Department, University State University of Londrina (UEL), Londrina, Paraná, Brazil. 2Pathological Science Department, University State University of Londrina (UEL), Londrina, Paraná, Brazil. 3Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil.

Introduction: Aluminum (Al) is the most widely distributed metal in the environment and the third most abundant element in the earth's crust. Puberty comprises a complex sexual development to acquisition of reproductive capacity. Objective: This study aimed to assess whether exposure to aluminum chloride during peripubertal period could lead to damage to testicular development in rats. Material and methods: Male Wistar rats were divided into three groups. Two groups were treated with aluminum chloride (AlCl3–in sterile 0.9% saline buffer) at doses of 7 mg/kg or 34 mg/kg body weight intraperitoneally in the peripubertal period (post natal day PND 36-66). The control group received only the vehicle. In the 67 PND rats were anesthetized and euthanized. The sperm from the vas deferens were used for sperm morphology and motility assay, the testes were used for histopathological analysis, evaluation of myeloperoxidase (MPO) and N-acetyl glicosidase (NAG) activity and cytokine levels (IL1-β, IL-6, IL-10 and TNF-α). Experimental procedures were approved by the CEUA/UEL - 059.2015. Results: Analysis of sperm morphology demonstrated a significant reduction in the number of normal sperm in the Al7 group. The Al34 group of animals presented a reduction in the number of mobile sperm compared to other groups. In the Al34 group, histopathological analysis showed a significant increase in the number of abnormal seminiferous tubules and a reduction in the number of Sertoli cells in relation to the control and Al7 groups. There was an increase in MPO activity and TNF-α dosage in the Al34 group and a decrease in IL-10 level, while the Al7 group presented an increase in the level of IL-10. The other cytokines were similar between groups. Conclusion: Aluminium chloride, as both doses, affects sperm parameters and testicular development in peribubertal rats, and cytokines could be the mechanism action for this toxic substance.

Keywords: Aluminium; Puberty; Testes. Thematic area: Non-transmitted diseases

SYSTEMIC OXIDATIVE STRESS PROFILE DURING THE PROGRESSION OF EXPERIMENTAL METASTASIS OF MURINE MELANOMA

Gabriella Pasqual Melo1; Iriana Morato Carrara1; Rodrigo Luiz Cabral; Alessandra Lourenço Cecchini1 1Laboratório de Patologia Molecular, Universidade Estadual de Londrina

Introduction: Melanoma is the most serious of malignant skin cancers and has high rates of mortality due its highly invasive feature. Objective: To draw a profile of systemic oxidative stress (OS) in a murine metastatic melanoma model using cells B16F10. Materials and methods: (6738.2015.40). B16F10 cells were grown in culture medium DMEM 10% FBS. About 1x105 viable cells were resuspended in 50 µL DMEM without serum and injected intravenously in C57BL/6 mice (8-12 weeks) through ophthalmic plexus. Control group received 50 µL DMEM without serum. Mice were sacrificed 3, 7, 14 or 21 days after inoculation (Groups: M3, M7, M14 and M21, n = 8 each group). Lung was removed for metastatic nodules counting and heparinized blood was collected by cardiac puncture. Plasma was used for oxidative stress (OS) and Total Radical Trapping Capacity (TRAP) analysis. Erythrocyte was used for OS analysis, catalase (CAT) activity and reduced glutathione (GSH) levels measurement. Statistical analysis was performed using One-way ANOVA with Tukey’s post-hoc testing. Results: Metastatic nodules counting showed a significant increase in M14 (p<0.0001) and M21 (p<0.0001) groups when compared to control. Plasma OS was increased in M3 (p<0.01) group compared to control and decreased in M14 (p<0.01) and M21 (p<0.001) groups compared to M3 group. Erythrocyte OS was decreased in M21 (p<0.05) compared to M3. Regarding antioxidant status, M14 and M21 groups showed an increase in TRAP (p<0.05), CAT activity showed to be increased in M7 (p<0.001), M14 (p<0.0001) and M21 (p<0.01) groups and GSH was increased in M7 (p<0.01) and M14 (p<0.05), compared to control. Conclusion: Systemic OS decreases during progression of metastasis and the initial increase may reflect an attempt to contain metastatic cells present in bloodstream. However, lung metastasis leads to antioxidants increase in later stages of disease, which may represent a response to tumor local growth.

Keywords: Melanoma; Metastasis; Oxidative Stress. Thematic area: Non-transmitted diseases

SYSTEMIC OXIDATIVE STRESS PROFILE DURING THE PROGRESSION OF EXPERIMENTAL METASTASIS OF MURINE MELANOMA

Gabriella Pasqual Melo1; Iriana Morato Carrara1; Rodrigo Luiz Cabral; Alessandra Lourenço Cecchini1 1Laboratório de Patologia Molecular, Universidade Estadual de Londrina

Keywords: Melanoma; Metastasis; Oxidative Stress. Thematic area: Non-transmitted diseases

CYTOTOXICITY EFFECT IN TM3 CELL LINE (LEYDIG CELL) CAUSED BY BPA (BISPHENOL A)

Gessica Dutra Gonçalves1,2; Simone Cristine Semprebon1; Mario Sergio Mantovani 1; Glaura Scantamburlo Alves Fernandes1.

1 Department of General Biology, University State of Londrina (UEL), Londrina, Paraná, Brazil. 2 Department of Pathology, University State of Londrina (UEL), Londrina, Paraná, Brazil.

Introduction: Bisphenol A is a xenoestrogen used in numerous products, studies have reported anti- androgenic activity and decreased sperm production after exposure to BPA. Objective: This present study reports on the evaluation of cytotoxic effects of BPA on Leydig cell line Mus musculus (TM3). Methods: Was used different concentrations (0.5 uM to 500 uM) of BPA for 24 and 48 hours treatments. To perform the MTT assay and RTCA, cells were seeded at a density of 3.125x103/well into in 96 well plates and E-plate 16 respectively. After 24 hours of stabilization treatments were added. For MTT, the solution of 0.5 mg / ml was added and cells were incubated for 4 h at 37 ° C. The absorbance of the formazan was measured at 540 nm. For the RTCA cells were incubated with the compounds and monitored for 24 and 48 h at 37 °C in a 5% CO2 atmosphere. The RTCA 1.2.1 software was used to calculate the maximal inhibitory concentration half (IC50) and cell index values. All experiments were performed in duplicate, in three independent repeats. The data were submitted to analysis of variance followed by Dunnett's test (p <0.05). Results: The BPA concentrations from 50 uM to 500 uM showed decreased cell viability both in concentrations of 24 to 48 hours, dosages of 5 uM and 10 uM showed a decrease in cell viability just in time 24 hours, furthermore using RTCA assay was also observed changes in cell proliferation at doses from 5 uM to 500 uM. The IC50 were 6.21 uM confirming the decrease in the cell viability. Conclusion: Our results show that BPA strong cytotoxic effect of BPA on TM3 cells. Therefore, the BPA can be harmful to male fertility since the Leydig cells are of great importance during spermatogenesis.

Keywords: Bisphenol A; TM3; Cytotoxicity. Thematic area: Non-transmitted diseases

TANSFORMING GROWTH FACTOR TYPE 2 RECPTOR (TGFβR2) PROMOTER REGION POLYMORPHISM EXERT SUBTYPE SPECIFIC ROLES IN BREAST CANCER

Glauco Akelinghton Freire Vitiello1; Bruna Karina Banin Hirata1; Alberto Yoichi Sakaguchi1; Marcos Henrique Rosa1; Cintya Mayumi Ishibashi1; Nathalia de Souza Pereira1; Alda Losi Guembarovski1; Mayara Tiemi Enokida1; Marla Karine Amarante1; Carlos Eduardo Coral de Oliveira1; Roberta Losi Guembarovski1; Maria Angelica Ehara Watanabe1 1 Laboratory of Studies and Applications of DNA Polymorphisms, Department of Pathological Sciences, State University of Londrina, Londrina, Parana, Brazil.

Introduction: Transforming growth factors β (TGFβs) are pleiotropic cytokines that act through two transmembrane receptors (TGFβR1 and TGFβR2) generating cell- and context-dependent responses. In Breast Cancer (BC) TGFβ inhibits growth of poorly aggressive subtypes such as luminal A (LA), but favors tumor progression in highly aggressive tumors, such as triple negative (TN) and HER2+ cancers. Objective: This work aimed to investigate whether a polymorphism of the promoter region of TGFβR2 (rs3087465, G-875A) affects on the susceptibility and clinical parameters of different BC subtypes. Material and methods: 321 BC patients and 405 control women were enrolled in the study. Genotypes were determined by RFLP-PCR. Associations were assessed through binary logistic regressions controlled by age and clinical correlations were measured through Kendall’s Tau-b rank correlation coefficient. This study was approved by the Ethic Committee for Research Involving Human Subjects of State University of Londrina (CEP/UEL: 189/2013 – CAAE: 17123113400005231). Results: G-875A polymorphism was a protective factor in general BC in the dominant model (OR=0.52; CI95%=0.38-0.71; p<0.001) and this effect was more evident in LA cancers (OR=0.50; CI95%=0.35-0.70; p<0.001), while in TN BCs this association was marginal (OR=0.52; CI95%=0.27-0.99; p=0.046). In general BC this polymorphism correlated positively with histopathological grade in dominant model (Tau-b=0.116; p=0.03). In LA tumors the presence of A allele negatively correlated with tumor size (Tau-b=-0.135; p=0.02), while in TN cancers this correlation was positive (Tau-b=0.26; p=0.04). Conclusion: These results are consistent with TGFβ signaling effects in different BC subtypes, and indicate this polymorphism as a potential susceptibility and prognosis marker in BC subtypes.

Keywords: TGFβ; Breast Cancer; Prognosis Grants: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação Araucária – Governo do Paraná Thematic area: non-transmitted diseases

THE ROLE OF TRANSFORMING GROWTH FACTOR BETA 1 (TGFB1) FUNCTIONAL POLYMORPHISMS IN GENE EXPRESSION AND CYTOKINE PRODUCTION

Guilherme Cesar Martelossi Cebinelli¹; Kleber Paiva Trugilo¹; Stephanie Badaró Garcia¹; Nádia Calvo Martins Okuyama¹; Michelle Mota Sena¹; É rica Romão Pereira¹; Ana Paula Lombardi¹; Rodolfo Sanches Ferreira¹; Gabriela Cristine Queiroz Maria¹; Adriano Martin Felis Aranome¹; Fernando Cezar dos Santos¹; Fernanda Costa Brandão Berti¹; Luis Fernando Lasaro Mangieri¹; and Karen Brajão de Oliveira¹. ¹Immunology and Molecular Genetics Laboratory, Pathological Sciences Department, Biologic Science Center, Londrina State University, Paraná, Brazil.

Introduction: Transforming Growth Factor β (TGFB) is a multifunctional cytokine that plays a role in several biological processes. The TGFBI is the most abundant expressed isoform and it is associated with susceptibility to various diseases. Several polymorphisms were described in the TGFB1 gene structure, and some of them have been associated with functional implications. Objectives: Investigate the TGFB1 functional polymorphisms and their molecular implications. Material and methods: We conduct a systematic literature review using PubMed and Google Academics searching the terms: TGFB1 SNP, TGFB1 functional polymorphisms, EMSA and TGFB1, and Luciferase and TGFB1. Results: Until now, 7 single nucleotide polymorphisms (SNPs) and one deletion/insertion polymorphism were associated with a functional impact on TGFB1 levels. The rs2317130 SNP is located in an undetermined region upstream to second enhancer region; rs11466313 deletion/insertion polymorphism in the second enhancer region; rs1800468 SNP in the enhancer region 1; rs1800469 SNP in the first negative regulatory region; and rs11466316 SNP is located in the TGFB1 first promoter region; and these polymorphisms interferes at transcription regulation by affecting transcription factors binding; and the rs1800471 SNP and rs1800470 SNP are located in signal peptide region, and interfere at protein production. These polymorphisms have been associated to different types of diseases (i. e. cancers, cardiac diseases, inflammatory diseases, and others) and could be used as susceptibility biomarkers. Conclusion: Various polymorphisms were described to affect the TGFB1 levels and have been associated with clinical implications. In fact, a cluster of polymorphisms is more reliable to contribute to pathological effects than a single one. Therefore, in order to control this bias, haplotype analysis of TGFB1 polymorphism could be more efficient to certificate a real genetic influence on disease susceptibility.

Keywords: TGFB1 polymorphisms; Luciferase; Haplotype. Thematic area: Non-transmitted diseases

METFORMIN REGULATES SKIN REDOX PROFILE IN UVB-INDUCED SQUAMOUS SKIN CELL CARCINOMA

Iriana Moratto Carrara1; Gabriella Pasqual Melo1; Sara Santos Bernardes1; Fernando Souza-Neto1; Leandra Naira Zambelli Ramalho3; Rodrigo Cabral Luiz1; Rubens Cecchini2; Alessandra Lourenço Cecchini1.

1Laboratory of Molecular Pathology, State University of Londrina, Brazil, Rodovia Celso Garcia Cid, PR-445, km 380, Campus Universitário, 86051-990 Londrina, PR, Brazil. 2Laboratory of Pathophysiology and Free Radicals, State University of Londrina, Rodovia Celso Garcia Cid, PR-445, km 380, Campus Universitário, 86051-990 Londrina, PR, Brazil. 3Department of Pathology, Ribeirão Preto Medical School, University of São Paulo

Introduction: Exposure to solar UV radiation leads to oxidative stress on the skin. Long-term effect, accumulation of oxidative damage may trigger photocarcinogenesis and squamous skin cell carcinoma (SCC) development. There is scientific evidence metformin is able to exert antineoplastic effect on tumour cells and this may be related to cell redox status changes. Objective: To verify the effect of metformin in a UVB-induced SCC experimental model by analyzing the skin redox profile. Material and methods: Hairless mice HRS/J (CEA 6738.2015.40) were divided into: one non irradiated group treated with metformin (MET, n=8) and two experimental irradiated groups treated with metformin (MET+UVB, n=15) and vehicle (PBS+UVB, n=15). The experimental period consisted of 15 weeks and groups were treated (i.p.) daily from first to the last day of the experiment. MET+UVB and PBS+UVB groups were exposed to 0.228 mJ/cm2 of UVB, five times a week for 15 weeks (17.1 mJ/cm 2). Animals were sacrificed and dorsal skin containing lesions were removed from irradiated groups for H&E staining and 3-NT and 4-HNE labbeling by immunohistochemistry. Lesion-free irradiated dorsal skin samples were removed and stored at -80°C for oxidative stress analysis. Differences in statistical significance were analyzed using Student’s T test with Tukey’s post-hoc testing, considering p<0.05 as significant. Results: Incidence of SCC was lower in MET+UVB group (p<0.05), compared to PBS+UVB group. Furthermore, MET+UVB group showed a significant increase of catalase activity, GSH and Total Radical-Trapping Capacity levels (p<0.05) accompanied by a significant diminished expression of 3-NT and 4-HNE (p<0.0001) in the surrouding tissue of SCC, compared to PBS+UVB group. Conclusion: Metformin regulates skin redox profile observed by maintenance of skin antioxidant system and diminished presence of oxidative products within the surrounding tissue of the tumour and this event was associated with a lower incidence of SCC in animals treated with the drug.

Keywords: Metformin; Oxidative Stress; Squamous Skin Cell Carcinoma.

Thematic Area: Non-transmitted diseases

Rhopalurus rochai VENOM-INDUCED INFLAMMATORY PAIN, OVERT-PAIN LIKE BEHAVIOR DEPENDS ON CELL RECRUITMENT AND CYTOKINES PRODUCTION

Camila Rodrigues Ferraz1; Marília Fernandes Manchope2; Jackson Gabriel Miyamoto2; Ketlem Cristine Andrade2; Fábio Henrique Kwasniewski2; Waldiceu Aparecido Verri Jr2. 1 Health Science Center, Department of Health Science, Londrina State University 2 Biological Science Center, Department of Pathology, Londrina State University

Introduction: Rhopalurus rochai (Rr) is a very common scorpion species found in the semiarid areas in northeastern of Brazil. There is no information about the mechanisms of Rhopalurus rochai venom- induced pain. Objective: To evaluate the peripheral mechanisms involved in nociception induced by Rr venom. Material and methods: Male Swiss mice were used (20-25g; n=6) and the experiments were approved by the Institutional Ethics Committee under the protocol 21344.2015.72. Mechanical hyperalgesia was assessed by an electronic pressure meter, an electronic version of the von Frey filaments. Thermal hyperalgesia was assessed by hot plate (50o C). Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity by colorimetric assay. Cytokine (TNF-α and IL-1β) production by ELISA assay. Mice received intraplantar (i.pl.) injection of Rr venom (0.2, 0.6, 2.4 µg/paw, diluted in saline) and mechanical and thermal hyperalgesia were evaluated betweem 0.5-6h. MPO and NAG activity was determined 6h after Rr venom injection. Cytokine levels was determined 0.5h, 1h and 3h after Rr venom injection. The induced overt pain-like behaviors was assessed by paw flinches and time spent licking paw for 30 min after Rr venom injection. Results: The Rr venom induced mechanical and thermal hyperalgesia in a dose-depend manner. The doses 0.6 and 2.4 µg/paw induced mechanical hyperalgesia until 6h. All the doses induced thermal hyperalgesia until 6h. The Rr venom were induced increase of MPO and NAG activity in a dose- depend manner. The dose 2.4 µg/paw induced increase of TNF-α levels 3h and IL-1β levels 0.5h, 1h and 3h after Rr venom injection and that dose induced increase of paw flinches and time spent licking paw. Conclusion: The Rr venom induced overt-pain like behavior, mechanical and thermal hyperalgesia, neutrophil and macrophage recruitment and hyperalgesic cytokines.

Keywords: Inflammation; Hyperalgesia; Rhopalurus rochai venom. Grants: CAPES, CNPq and Paraná State Government. Thematic area: Non-transmitted diseases

Tityus serrulatus VENOM INDUCES INFLAMMATORY PAIN IN MICE

Introduction: Tityus serrulatus (Ts) is considered the most dangerous scorpion in South America and Ts is main scorpion species of medical importance in Brazil. The pain is a common symptom in victims of these accidents, but there is few information about the mechanisms of pain these accidents. Objective: To evaluate the peripheral mechanisms involved in nociception induced by Ts venom. Material and methods: Male Swiss mice were used (20-25g; n=6) and the experiments were approved by the Institutional Ethics Committee under the protocol 21344.2015.72. Mechanical hyperalgesia was assessed by an electronic pressure meter, an electronic version of the von Frey filaments. Thermal hyperalgesia was assessed by hot plate (50o C). Myeloperoxidase (MPO) and N- acetyl-β-D-glucosaminidase (NAG) activity by colorimetric assay. Cytokine (TNF-α and IL-1β) production by ELISA assay. Mice received intraplantar (i.pl.) injection of Ts venom (0.2, 0.6 or 2.4 µg/paw, diluted in saline) and mechanical and thermal hyperalgesia were evaluated betweem 0.5- 6h. MPO and NAG activity was determined 6h after Ts venom injection. Cytokine levels was determined 0.5h, 1h and 3h after Ts venom injection. The overt pain-like behaviors was assessed by paw flinches and time spent licking paw for 30 min after Ts venom injection. Results: The injection of Ts venom induced mechanical hyperalgesia in a dose-depend manner. All doses of Ts venom induced thermal hyperalgesia until 6h. The doses 0.6 and 2.4 µg/paw induced significant mechanical hyperalgesia until 6h. All doses of Ts venom were also able to induce significant increase of MPO and NAG activity. The dose 2.4 µg/paw induced increase of TNF-α, IL-1β levels 1h and 3h after Ts venom injection and that dose induced increase of paw flinches, and time spent licking paw. Conclusion: The Ts venom induced overt-pain like behavior, mechanical and thermal hyperalgesia, neutrophil and macrophage recruitment and hyperalgesic cytokines.

Keywords: Inflammation; Hyperalgesia; Tityus serrulatus venom. Grants: CAPES, CNPq and Paraná State Government. Thematic area: Non-transmitted diseases

MALIGNANT MELANOMA SKIN

Janaine Aparecida Fortunato1, Ana Lúcia Sanches1, Márcia Regina Terra2.

1Student of the Bachelor course in Physiotherapy of the Community College of Londrina – INESUL. 2Professor of the Community College of Londrina - INESUL, Master in Microbiology from the University of Londrina UEL.

Introduction: The Cutaneous Malignant Melanoma (MMC) has shown many decades an aggressive malignancy influencing the growing cause of mortality reaching approximately 4% of malignant tumors, which the skin, the main because of its production of metastases. Its beginning comes from melanocytes, your primary site is the skin with 90% of the extension, but can spread to other organs, but in metastasis, its primary site is unknown. It is presented in four ways and they Melanoma Lentigo malign; Superficial malignant melanoma or pagetoid; Acral litigious melanoma and nodular melanoma. The prognosis is obtained through analysis of the Clark level and Breslow thickness and clinical examinations. Exposure to UVB light is the increasing ratio of melanoma cases in Brazil, Rio Grande do Sul (RS), this MMC predisposed to be due to the large population of fair skin, on account of its geographical position and UVB exposure. The treatments are varied but none with higher rates of survival the education activities promoting early, should be directed at all to reduce the incidence and mortality of patients with the disease. Objective: to report histopathology identifying the causes, treatments, prognosis and incidence paying attention to early diagnosis of Cutaneous Malignant Melanoma. Methods: bibliographic analyzes based on sciello, lilacs, med line using MMC writer’s skin and skin cancer in addition to extending dermatological books and pathology. Conclusion : We conclude that the MMC is an aggressive malignant neoplasm of dermatologic factor of high incidence, a skin disease that exhibits high metastatic ability and sun exposure the biggest factors of pre disposition, your primary site is unknown, but affects the general people Clear Skin. The treatment did not show higher survival rates but recognition prognostic factors and early diagnosis is reconverted healing.

Keywords: Cutaneous Malignant Melanoma; Skin-to-skin neoplasms; Prognosis. Thematic area: Non-transmitted diseases.

OBSTRUCTIVE PULMONARY DISEASE: PATHOPHYSIOLOGY AND REHABILITATION PULMONARY

Janaina Aparecida Fortunato1; Ana Lúcia Sanches1; Vanessa Almeida Nascimento2; Cristhiane da Silva Ferreira Gonçalves3; Uili Andrey de Souza4; Luana Aparecida Cossentini5

1Graduate student Physiotherapy of INESUL - Instituto de Ensino Superior de Londrina. 2Graduate student Physiotherapy of UNOPAR – Universidade do Norte do Paraná. E-mail: [email protected] 3Graduated in Nursing; Specialization in Emergency; Coordinator and teacher in CIE – Centro Integrado de Educação and INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 4Graduated in Nursing; Specialization Audit in Health; Degree in Biological Sciences; Coordinator and teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 5Graduated in Biomedicine; Master in Experimental Pathology; PhD student in Experimental Pathology at the Regional University Hospital of Londrina; Teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected].

Introduction: Chronic Obstructive Pulmonary Disease (COPD) is a disease that is initially related to the variation in pulmonary function. May lead to the disorder of the skeletal muscles favoring the restriction of tolerance to physical activities. Basically occurs limitation of airflow characteristic of the destruction of the alveolar. Patients with COPD have an impaired quality of life due to symptoms that affect, which is extremely important attention to patients. Pulmonary rehabilitation is comprehensive and integrated physical training, patient discipline, oxygen therapy, psycho social support and nutritional intervention. Objective: Describe attributes of COPD pathogenesis and as the factors determining the exercise tolerance for patients with this disease and the use of pulmonary rehabilitation. Material and methods: This study systematic literature review on COPD approached literature reviews, scientific articles with time frame 2005 and 2016 published in English and Portuguese, through national and international electronic databases such as MEDLINE, SCIELO. Conclusion: Pulmonary rehabilitation has been shown to be effective against symptoms such as dyspnea, ability to develop exercises and closely the quality of life of patients. The study of the pathophysiology of this relationship becomes important for the advancement of treatments.

Keywords: Chronic obstructive pulmonary disease; Rehabilitation; pathophysiology. Thematic area: Non-transmitted diseases

DOES MATERNAL EXPOSURE TO FLUOXETINE INDUCE METABOLIC ALTERATIONS IN ADULT MALE OFFSPRING?

Kawane Fabricio Moura¹; Bruno Vinicius Duarte Marques¹; Carolina Matias Higashi¹; Gabriela Matias Costa¹; Daniella Regina Barrionuevo¹; Graziela Scalianti Ceravolo¹.

¹Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina/PR.

Introduction: Depression is a multifactorial disease that affects different age groups. Fluoxetine (FLX) is an antidepressant worldwide prescribed throughout life stages, including pregnancy and breastfeeding. The FDA classifies FLX as category C. This drug crosses placental barrier and is secreted in breast milk. It is has been reported that early developmental exposure to fluoxetine affects central nervous system areas related to neuroendrocrine regulation. Objective: Therefore, the objective of the present study was evaluate whether intrauterine and lactation exposure to fluoxetine causes metabolic changes related with diabetes and obesity in adult male offspring. Methods: Male Wistar rats 75 days of age, whose mother was treated with fluoxetine (FLX) (5mg/kg) or water (C) during pregnancy and lactation were used. Lee’s obesity index was calculated as follows: body weight1/3(g)/nasal-anal length (cm) ×100. Visceral adipose tissue (periepididymal and retroperitoneal) were excised and weighted and the values expressed as fat pad weight/100g of body. After 4 hour of food deprivation, the basal glycemia and insulin tolerance test (kITT) were evaluated. For statistical analysis, T-student test was used and differences considered with P<0.05. Values expressed as mean ±S.E.M, (n) is the number of rats/group (CEUA nº16166-2012.12). Results: The body weight (C: 288.00±18.61 (7) vs FLX: 310.10±14.59 (8)), Lee’s index (C: 29.85±0.31 (7) vs FLX: 30.81±0.35 (8)) and the periepididymal fat (C: 0.95±0.04 (6) vs FLX: 0.91±0.04 (7)) and retroperitoneal fat (C: 0.79±0.08 (6) vs FLX: 0.80±0.05 (7)) were similar between FLX and C rats. Also, the baseline glycemia (C: 123.10±4.98 (7) vs FLX: 124.30±6.25 (6)) and kITT (C: 2.57±0.11 (7) vs FLX: 2.42±0.27 (6)) did not differ between the groups. Conclusion: The results reveal that maternal exposure to FLX (5mg/Kg) during pregnancy and lactation did not induced obesity or insulin resistance in adult male offspring.

Keywords: Maternal Treatment; Obesity; Insulin Resistance. Grants: CNPq Thematic area: Non-transmitted diseases

KAURENOIC ACID REDUCES DICLOFENAC-INDUCED ACUTE KIDNEY INJURY THROUGH INHIBITION OF OXIDATIVE STRESS IN MICE

Ketlem C. Andrade1; Yuri L. Gonzalez1; Mariana M. Bertozzi1; Allan J.C. Bussmann2; Nilton S. Arakawa3; Victor Fattori1; Rúbia Casagrande3; Waldiceu A. Verri Jr.1* 1Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rod. Celso Garcia Cid PR445 KM380, 86057-970 Londrina, Paraná, Brasil. 2Laboratório de Anatomia Patológica, Centro de Ciências de Saúde, Universidade Estadual de Londrina, 86038-350, Londrina, Paraná, Brasil. 3Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Hospital Universitário, Universidade Estadual de Londrina, Av. Robert Koch, 60, 86038-350 Londrina, Paraná, Brasil. *author for correspondence: [email protected]

Introduction: Diclofenac is one of the most used non-steroidal anti-inflammatory drugs in the clinics. Although considered safe, growing body of evidence points to the potential risk of renal damage due to diclofenac ingestion. Diclofenac-induced renal lesion occurs through induction of oxidative stress by affecting kidney mitochondrial complex I, leading to reduction of ATP formation, and consequently kidney apoptosis. Kaurenoic acid (KA, ent-kaur-16-en-19-oic acid) is a diterpene presented in various plants and the major constituent of Sphagneticola trilobata (L.) Pruski. Studies have shown that KA possesses anti-inflammatory, antioxidant, and antinociceptive activity, which can be attributed to the inhibition of NF-kB activation. Objective: We aim to investigate the efficacy of Kaurenoic acid in diclofenac-induced acute kidney injury (AKI). Material and methods: The experiments were conducted in male Swiss mice with Londrina State University Ethics Committee on Animal Research and Welfare approval under process number 18650.2016.00. Mice were treated with diclofenac (200mg/kg, p.o.) 30 min prior to the KA administration (0.3, 1, or 3mg/kg, i.p.). All samples were collected 24h after the stimulus. Urine was collected for the determination of proteinuria by Lowry’s method. Blood was collected for the determination urea and creatinine plasma levels. Kidney was collected for the determination of superoxide anion levels (NBT assay), levels of thiobarbituric reactive substances (TBARS assay), and total antioxidant capacity (ABTS and FRAP assays). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc. Results: KA treatment dose- dependently reduced urea and creatinine plasma levels. Given that there were no differences between the dose of 1 and 3mg/kg, the lower dose was chosen for the following experiments. KA 1mg/kg decreased superoxide anion and lipid peroxidation formation, and proteinuria. In addition, KA 1mg/kg restored total antioxidant capacity. Conclusion: This study demonstrates that KA ameliorates diclofenac-induced AKI through inhibition of kidney oxidative stress.

Keywords: Diclofenac; Nephrotoxicity; Kaurenoic acid Financial support: Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secretaria da Ciência, Tecnologia e Inovação (SETI), Fundação Araucária, and Paraná State Government. Thematic area: Non-transmitted diseases

EVALUATION OF BIOCHEMICAL PARAMETERS OF DIABETICS RATS SUBMITTED TO FOOD RESTRICTION DIET

Jéssica Men de Campos1; Carlos Vinicius Dalto da Rosa1; Larissa Carla Lauer Schneider1; Maria Raquel Marçal Natali2; Vilma Ferreira de Godoi3 1Post-graduating on Program of Cellular Biology, State University of Maringa, Maringa, Brazil 2Departament of Morphological Sciences, State University of Maringa, Maringa, Brazil 3Departament of Physiological Sciences, State University of Maringa, Maringa, Brazil

Introduction: Type 2 diabetes mellitus (T2DM) is a metabolic disease in which is noted significant changes in biochemical parameters which are measured to confirm the diagnosis and to monitor treatment. Food restriction (FR) can be an alternative treatment fo T2DM. Objectives: To evaluate biochemical parameters in diabetic rats submitted to FR. Materials and methods: 20 (n=5/group) Wistar rats (CEUA/UEM Nº.7590050415) were divided into 4 groups: control(C), control with food restriction (CR), diabetics (D) and diabetics with food restriction (DR). For 2 initial months, controls (C and CR) received standard chow and water ad libitum; diabetics (D and DR) received streptozotocin intravenously (35 mg/Kg body weight), and then cafeteria diet style (33% standard+33% condensed milk Nestlé®+7% sugar+water+sugar water (32%). At the end of the initial 2 months until the end of treatment: control (C) received standard diet ad libitum; and control+restriction (CR), subjected to FR of 50% from the standard diet for 2 months. Diabetic (D) received ad libitum only standard chow and water for the last 2 months of treatment; and the diabetic+restriction (DR) group was subjected to FR of 50% from the standard diet for the last 2 months. At the end of 4 months the animals blood samples were collected for biochemical analysis: High-density lipoprotein (HDL), cholesterol, triglycerides and fructosamine and determinate how these parameters were influenced in animals subjected to FR compared to those who were not submitted. Data (mean±SE) were subjected to ANOVA and Tukey's test (95%) Results: T2DM was confirmed by hyperglycemia, hyperphagia, polydipsia and diarrhea. Fructosamine (mg/dL) (C=0,28±0,03, CR=0,21±0,01, D=0,32±0,02, DR=0,31±0,04); Triglycerides (mg/dL) (C=137,3±35, CR=41±6,5, D=126,4±40,2, DR=26±7,3); Total cholesterol (mg/dL) (C=63,1±9,9, CR=57,1±9,2, D=73,2±10,7, DR=44,1±9,1); HDL (mg/dL) (C=46,5±12,7, CR=40,5±6,2, D=58,2±9,9, DR=35,5±10,4). Conclusion: T2DM biochemical parameters measured showed significant improvement when the animals were subjected to FR, except fructosamine that remained constant in all groups.

Keywords: Diabetes Mellitus; Food Restriction; Serum parameters Thematic area: Non-transmitted diseases.

CITRAL HAS CYTOTOXIC ACTION IN MURINE MELANOMA CELLS (B16F10) BY GENERATING REACTIVE OXYGEN SPECIES

Larissa Juliani Sanches1; Poliana Camila Marinello1; Carolina Panis2; Rubens Cecchini.3 Alessandra Lorenço Cecchini Armani1; Rodrigo Cabral Luiz1 1Laboratório de Patologia Molecular, Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Brasil. 2Universidade Estadual do Oeste do Paraná, Brasil. 3Laboratório de Fisiopatologia e Radicais Livres, Depto. Ciências Patológicas – CCB, Universidade Estadual de Londrina (UEL)

Introduction: Currently, 132,000 melanoma skin cancers cases occur worldwide each year. In Brazil, skin cancer accounts for 25% of all malignant tumors registered, 4% of these tumors represent the metastatic form of cutaneous melanoma (CM) is the deadliest form, and generally refractory to conventional chemotherapy treatments. For this reason, it is very important to search for new antitumor agents for this type of tumor. The plants are known sources of natural compounds among these compounds we found citral, a mixture of neral (cis) and geranial (trans), which has been used in the food industry and in the production of fragrance due to its lemon flavor. There are reports in the scientific literature cytotoxic effects and apoptosis induction in human leukemia cells (NB4) and breast cancer cells (MCF7). Objective: The present work evaluated the participation of oxidative stress as a possible mechanism of citral cytotoxicity in in vitro murine metastatic melanoma cell (B16F10). Material and methods: Cells were treated (24h) with citral (0.1, 0.5, 1.0 and 2.5 µM) and results were compared to control (p<0.05), were made test Griess-Cadmium, GSH, MDA and testing with scavagers. Results: Nitrite levels (Cadmium-Copper-Griess method) decreased with citral 0.5, 1.0 and 2.5 µM, as it has been reports as an iNOS inhibitor. Citral at 0.5, 1.0 and 2.5 µM concentrations decreased GSH levels. Citral 2.5 µM showed increased MDA levels, suggesting oxygen reactive species generation. Citral 0.5 µM reduced 20% of cell viability (MTT assay). Citotoxic effect was abolished when reactive species scavangers were used (histidine 10 mM - singlet oxygen; trolox 50 µM - peroxyl and tempol 50 µM – superoxide). Conclusion: Our results suggesting that an oxidative stress burst is involved in citral cytotoxicity.

Keywords: Citral; Melanoma; Cytotoxic. Thematic area: Non-transmitted diseases

EFFECTS OF NATURAL RUBBER LATEX MEMBRANES STABILIZED WITH DIFFERENT CONCENTRATIONS OF AMMONIA IN CHO-K1 CELLS

Dalita Gomes Silva Moraes Cavalcante1; Nathália Oliveira Braga1; Leandra Ernst Kerche-Silva1,2; Andressa Silva Gomes1; Caroline de Souza Araujo2; Eidi Yoshihara3; Aldo Eloizo Job1.

1 Departamento de Química, Física e Biologia, Unesp, Presidente Prudente-SP, Brazil. 2 Faculdade de Artes, Ciências e Letras, Universidade do Oeste Paulista, Presidente Prudente-SP, Brazil. 3 Agência Paulista de Tecnologia dos Agronegócios, APTA, Presidente Prudente-SP, Brazil.

Introduction: Since fresh latex can spontaneously coagulate and putrefy shortly after it leaves the tree, ammonia is used to preserve natural rubber (NR) and so it can be used as membranes that can be used as delivery system of proteins, drugs and nanoparticles. Objective: The purpose of this study was to assess in vitro cytotoxicity of NR latex membranes stabilized with different concentrations of ammonia. Material and methods: CHO-k1 cells were exposed to NR membranes stabilized with 0%, 0.25%, 0.50%, 1.00% and 2.00% for 24 h. Cytotoxicity was assessed by the MTT assay and the oxidative stress parameters were established by reduced glutathione (GSH) and malondialdehyde (MDA) levels. Results: The results show that ammonia present in NR latex membranes can deplete cell viability and enhance GSH consumption in the cells. NR latex membranes stabilized with 2.00% of ammonia were cytotoxic to the cell lineage used in this study. Conclusion: From these results it is possible to infer that the use of ammonia to stabilize NR latex membranes with biomedical purposes has to be done carefully, to avoid possible harmful effects to human health.

Keywords: Natural rubber latex; Ammonia; Cytotoxicity Grants: FAPESP, CNPq and Unesp. Thematic area: Non-transmitted diseases.

LATEX C-SERUM FROM Hevea brasiliensis INDUCES NECROTIC CELL DEATH IN MURINE MELANOMA CELL LINE (B16F10)

Leandra Ernst Kerche-Silva1; Poliana Camila Marinello2; Iriana Morato Carrara2; Dalita Moraes Gomes Silva Cavalcante1; Aldo Eloizo Job1; Alessandra Lourenço Cecchini2. 1 Departamento de Física, Química e Biologia, Unesp, Presidente Prudente-SP, Brazil. 2 Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina-PR, Brazil.

Introduction: Latex from Hevea brasiliensis has no known primary metabolic function although its biological role is a plant defense system. Latex can be separated in three parts after centrifugation: rubber cream, C- and B-sera. C-serum is a rich source of proteins and contains a multitude of organic compounds. Objective: The aim of this study was to evaluate the antiproliferative effects of latex C- serum on murine melanoma cell line (B16F10). Material and methods: MTT cell viability (0.02, 0.2, 1, 2, 5, 10, 20, 50, 100 and 200 µg/ml), comet assay and morphologic evaluation of the cells using the fluorescent dyes Hoechst 33342 and Propidium Iodide (PI) (5, 50 and 100 µg/ml) were used to evaluate the cytotoxic effects of latex C-serum on B16F10 cell lines. Results: MTT test showed that latex C-serum exerted antiproliferative effects on B16F10 cells in the concentrations ranging from 10 to 200 µg/ml. Comet assay showed that Latex C-serum induced genotoxicity of class 2 for the concentrations of 50 and 100 µg/m in the cells. Morphologic evaluation of the cells using Hoechst 33342 and PI showed statistical significative raise in the levels of necrotic cell death. Conclusion: These results show that Latex C-serum were able to exert antiproliferative effects on murine melanoma cells inducing necrotic death in these cells. More studies are being accomplished.

Keywords: Hevea brasiliensis; Necrosis; Murine melanoma cells. Grants: FAPESP, CNPq, UEL and Unesp. Thematic area: Non-transmitted diseases.

CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION AND EVALUATION OF ERYTHROCYTIC ANTIOXIDANT DEFENSES OF PATIENTS AFFECTED BY BASAL CELL CARCINOMA IN THE NORTHERN REGION OF PARANA

Leonardo Bodner de Freitas1; Poliana Camila Marinello1; Mateus H. Cunha da Silva1; Mariani Lima Garcia1; Walison A. da Silva Brito1; Natália M. Dias Lopes1; Vanessa Gheno2; Théo Nicolacópulos2; Priscila D. Pavezzi2; Fernanda T. Ortega2; Mariana O. Okamura2; André Armani3; Airton dos Santos Gon2; Rubens Cecchini4; Alessandra L. Cecchini1.

1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, Brazil. 2 Department of Internal Medicine, State University of Londrina, Londrina, PR, Brazil. 3 Department of Surgical Clinic, State University of Londrina, Londrina, PR, Brazil. 4 Laboratory of Physiopathology and Free Radicals, State University of Londrina, Londrina, PR, Brazil.

Introduction: Taking into account the elevated incidence of basal cell carcinoma (BCC) worldwide, the identification of factors related with its development is essential to enable effective prevention. Objective: To identify the profile of patients with BCC and with BCC associated with other skin neoplasias (squamous cell carcinoma and actinic keratosis), using epidemiological, clinical and biochemical variables. Material and methods: Participants were categorized in 3 groups: Control (without skin cancer history; n=38); BCC (n= 50) and BCC associated with others skin neoplasias (BCC-ML; n=12) (CEP-UEL process n. 1.077.557). Blood samples were collected and erythrocytic catalase activity, oxidized and reduced glutathione levels (GSSG and GSH, respectively) were determined. The clinical-epidemiological characterization was performed by applying a questionnaire to participants. The statistical analysis was performed using one-way ANOVA or Kruskal-Wallis (quantitative results) and Chi-square test (qualitative results); p<0.05 was considered significant. Results: Patients from BCC presented reduced levels of GSH, which was not observed in BCC-ML group. GSSG levels did not change. Catalase activity did not alter in BCC, but significantly increased in BCC-ML. The patients presented the same age and gender distribution and no differences about the presence of comorbidities and tabagism were observed. The most of patients fit in the category of light skin (type I/II in Fitzpatrick’s Classification) and lesions were main found in the region of head and neck. Obesity was more common in BCC-ML group and occupational UV exposure shows to be more related to BCC than to BCC-ML. Conclusion: The results indicates that systemic oxidative stress could be related to the development of BCC associated with other skin neoplasias, since BCC patients presents lower levels of erythrocytic antioxidant defenses than BCC-ML. It is possible to conclude also that some nonmelanoma skin cancer risk factors could be differently involved in the pathophysiology of these two pathological conditions.

Keywords: Basal cell carcinoma; Skin cancer; Epidemiology Thematic area: Non-transmitted diseases

CELL ADHESION MOLECULES EVALUATION AS PREDICTORS OF DIAGNOSIS AND DISEASE ACTIVITY IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS.

Lorena Flor da Rosa Franchi Santos1; Nicole Perugini Stadtlober1; Ligia Grecco Costa Dall'Aqua1; Bruna Miglioranza Scavuzzi2; Poliana Macedo Guimarães2; Tamires Flauzino2; Marcell Alysson Batisti Lozovoy3; Tathiana Veiga Mayumi Iriyoda4; Edna Maria Vissoci Reiche3; Isaias Dichi5; Michael Maes6; Andréa Name Colado Simão3. 1 Graduate Program in Pathology, Clinical Analysis and Toxicology – University of Londrina, Brazil. 2 Graduate Program in Health Sciences – University of Londrina, Brazil. 3 Department of Pathology, Clinical Analysis and Toxicology – University of Londrina, Brazil. 4 Department of Rheumatology – University of Londrina, Brazil. 5 Department of Internal Medicine – University of Londrina, Brazil. 6 IMPACT Strategic Research Centre, School of Medicine – Deakin University, Geelong, VIC, Australia.

Introduction: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease and some studies have shown that the increased adhesion molecules may correlate with disease activity and clinical manifestations in patients with SLE. Objective: To delineate disorders in adhesion molecules in SLE and to assess whether cortisol, nuclear autoantibody (ANA) titers and the metabolic syndrome (MetS) are associated with adhesion molecules in SLE. Methods: 48 healthy individuals and 171 SLE patients were enrolled. Disease activity was determined by SLEDAI (SLE Disease Activity Index) score. Adhesion molecules and cortisol levels were evaluated. The study protocol was fully approved by the Ethical Committee of the University of Londrina (Paraná, Brazil) (CAAE 01865212.0.0000.5231, CEP/UEL 205.328). Results: Platelet endothelial cell adhesion molecule 1 (PECAM-1), Vascular cell adhesion molecule 1 (VCAM-1), E-selectin, P-selectin and Plasminogen activator inhibitor type-1 (PAI-1) were significantly higher in SLE patients (p<0.001). Mycophenolate treatment significantly decreased intercellular adhesion molecule 1 (ICAM-1) (p=0.045) and increased E-selectin (p=0.012) levels. Binary logistic regression analysis showed that PECAM-1 and PAI-1 predicted SLE with a sensitivity of 86.5% and a specificity of 81.3%. ANA was significantly and positively associated with PECAM-1 (p=0.001), VCAM-1 (p=0.001), E-selectina (p=0.003) and PAI- 1 (p=0.024), whilst cortisol was negatively associated with PCAM-1 (p=0.017) and ICAM-1 (p=0.045). There were significant associations between MetS and E-selectin (p<0.001) and PAI-1 (p=0.027). 18.2% of the variance in SLEDAI score was explained by increased PECAM-1 values and DNA titers and the MetS. Conclusion: Our data confirm that adhesion molecules play a role in the pathophysiology of SLE and show that increased adhesion molecule levels, especially PECAM-1, can be used as an external validating criterion for the diagnosis SLE. MetS, ANA, and cortisol modulate the adhesion molecule concentrations but do not explain the increased levels in SLE Increased levels of adhesion molecules are a new drug target in SLE.

Keywords: Systemic Lupus Erythematosus; Adhesion Molecules; Biomarkers. Thematic area: Non-transmitted diseases

BARK ETHANOLIC EXTRACT FROM Spondias dulcis FORST F. PROTECTS AGAINST ALTERATIONS OF THE REDOX STATUS INDUCED BY CYCLOPHOSPHAMIDE AND BENZO[a]PYRENE IN MICE

Introduction: Spondias dulcis is widely used for medicinal purposes. However, no in vivo study concerning its effects on the redox status has been conducted. Objective: The aim of this study was to assess the effects of bark ethanolic extract from S. dulcis on cyclophosphamide (CP)- and benzo[a]pyrene (B[a]P)-induced oxidative stress in liver, kidney and total blood of mice. Material and methods: 10 animals per group were used for the tests and three concentrations of the extract were used, 500, 1000 and 1500 mg/kg bw. Benzo[a]pyrene (B[a]P) and Cyclophosphamide (CP) were used to evaluate the protective effects. B[a]P (9 mg/kg bw) and CP (40 mg/kg bw) were used as oxidative stress inducers. 24 h after the treatment the animals were euthanized and liver, kidney and total blood were collected. Liver and kidney were investigated for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH), and total blood were investigated for GSH. Local Ethics Committee for Animal Use (CEUA) of Unoeste approved this study, register No. 2950/2016. Results: The extract did not raise TBARS levels in liver and kidney and it was able to deplete TBARS levels associated to B[a]P and CP. The extract also did not deplete the levels of GSH in liver, kidney and total blood, and were able to increase the levels of GSH associated to B[a]P and CP. The most protective concentration was 500 mg/kg bw. Conclusion: These results show that the treatment with this extract attenuates B[a]P- and CP-induced oxidative stress. More studies are being accomplished.

Keywords: Spondias dulcis; Oxidative Stress; GSH. Grants: UNOESTE, CAPES. Thematic area: Non-transmitted diseases.

THE PREDICTIVE VALUE OF TRANSFORMING GROWTH FACTOR-β IN WILMS TUMOR IMMUNOPATHOGENESIS

Diego Lima Petenuci1, Marla Karine Amarante1; Carlos Eduardo Coral de Oliveira1; Carolina Batista Ariza1; Alberto Yoichi Sakaguchi1; Cintya Mayumi Ishibashi1; Maria Angelica Ehara Watanabe1. 1State University of Londrina, Londrina-Paraná, Brazil.

Introduction: Wilms tumor (WT) is the most common kidney malignancy in children, especially under the age of 6 years. Although therapeutic approach has reached successful rates, there is still room for improvement. Objective: This review attempt to summarize the involvement of TGF-β in the environment surrounding Wilms tumor. Material and methods: Papers were searched on Pubmed using the keywords: wilms tumor and TGF-β. Results: Studies demonstrate the TGF-β induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Furthermore TGF-β has the ability to repress the WT1 that is essential for podocyte function. Analyzing the gene expression profile of pediatric WT with anaplastic histology and adjacent normal tissues it was revealed that 15 genes are up-regulated and 16 genes are down-regulated in WT tissue. Moreover, genes involved in TP53 and TGF-β signaling pathways were the most affected within anaplastic histology WT. In WT, positive expression of TGF-β1 was correlated with tumor invasion and disease progression. Conclusion: Understanding the TGF-β role in human cancer is of paramount importance for the development of new therapeutic strategies to specifically block the metastatic signaling pathway of TGF-β without affecting its tumor suppressive effect.

Keywords: Wilms tumor; TGF-β; Tumor microenvironment. Thematic area: Non-transmitted diseases

TREATMENT WITH PIOGLITAZONE AND INSULIN IN WALKER-256 TUMOR BEARING RATS: EFFECTS ON PANCREATIC ISLETS

Ramalho, M.C1; Galia, W.B.S.1; Congo, N.A.J.1; De Fatima Silva, F1,2; Carpinelli, A. R.2; De Souza, H. M1; Graciano, M. F.1. 1Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina/PR; 2Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas I, Universidade de São Paulo, São Paulo/ SP.

Introduction: Cachexia is characterized by progressive weight loss caused by depletion of muscle and adipose tissues associated with insulin resistance. The model of cachexia induced by Walker- 256 tumor showed hypoinsulinemia and increased insulin resistance. Insulin sensitizers may improve peripheral insulin sensitivity in patients with cancer, which could prevent the dysfunction of insulin secretion. Objective: Our aim was to evaluate the effect of pioglitazone therapy with or without combination with insulin on plasma insulin and insulin secretory capacity of pancreatic islets from Walker-256 tumor-bearing rats. Material and Methods: Male Wistar rats inoculated with tumor cells (8x107) were treated with oral administration of pioglitazone (5 mg/Kg) once daily during 12 days, or oral administration of pioglitazone associated with subcutaneous insulin (1U/Kg) twice daily. After treatment, pancreatic islets were isolated from pancreas and blood samples were collected to measure plasma insulin. Insulin secretion and intracellular insulin content of pancreatic islets were analyzed in response to glucose. Results: Plasma insulin of tumor-bearing animals was reduced by 80% compared with healthy animals. Treatment of Walker tumor-bearing rats with pioglitazone partially reversed the hypoinsulinemia: plasma insulin was reduced by 60% compared with healthy rats. Insulin secretion from islets of tumor-bearing animals incubated at 16.7 mM glucose was reduced by 60% compared with healthy rats. This effect was not improved by the treatments (n≥4). Intracellular insulin content of the islets from tumor bearing-rats was reduced by 40% compared with healthy rats, with no improvement by the treatments (n≥4). Conclusion: Cachexia induced by Walker-256 tumor affects both insulin secretion stimulated by glucose and insulin synthesis. This is correlated with reduced plasma insulin of cachectic animals. Treatments with pioglitazone and insulin did not improve secretory function stimulated by glucose and the synthesis of insulin by islets.

Keywords: Cachexy; Insulin resistance; Insulin secretion Grants: PROIC-UEL Thematic area: Non-transmitted diseases

CINNAMON, TURMERIC AND OKRA: EFFECTS OF DIET ON BODY WEIGHT AND BIOCHEMICAL PARAMETERS IN WISTAR RATS

Marcio Scarone1, Flávia Imanishi Ruzon1, Kamila Falchetti Damasco2, Eduardo Vignoto Fernandes1, Miriele Caroline da Silva1, Emerson José Venâncio1 and Raúl Jorge Hernan Castro- Gómez1

1State University of Londrina, Km 380, Celso Garcia Cid Road, Londrina, Brazil 2University of Northern Paraná, Av Paris, 675, Jardim Piza, Londrina, Brazil

Introduction: Plants in almost their whole have medicinal properties. Although they present actions with similar aspects to modern drugs, they are less aggressive and safer. Cinnamon, turmeric and okra are used in the treatment of various diseases. Objective: This study is aimed to investigate the effects of cinnamon, turmeric and okra on body mass and biochemical parameters of Wistar rats, fed with these plants. Commercial samples of cinnamon, turmeric and okra were used in powder form. Material and methods: The experiment with adult male Wistar rats was conducted as follows: 4 animals per box, light/dark cycle of 12 hours, food and water ad libitum and controlled temperature (25±1 °C). The animals were randomly distributed in 4 groups of n=12: Control (CO), Cinnamon (CI) Turmeric (T) and Okra (O). The plant samples were administered by gavage, in phosphate-buffered saline (cinnamon and turmeric, 50 mg/day and okra, 12.5 mg/day) or vehicle only (CO) for 14 days. The animals were weighed and blood samples were collected before and after the trial period. The results were analyzed statistically (P<0.05). The project was approved by CEUA/UEL (n.8185.2014.18). Results: When comparing the pre and post-experiment, in CO, O and CI, there was a significant increase in body mass, while in T, that didn’t happen. The same occurred regarding triglyceride levels. The analysis of total cholesterol showed no significant increase in CI, contrary to what happened with the other groups. Relative to blood glucose levels, in all groups no significant differences were observed between the two moments. Conclusion: The results show that there was an increase in body weight and in the total cholesterol and triglyceride levels of CO animals throughout the experiment, while in the groups of animals fed with cinnamon or turmeric it was observed the maintenance of levels of one or more biomarkers.

Keywords: Medicinal plants; Foods; Prevention of diseases. Grants: Londrina State University, Coordination for the Improvement of Higher Education Personnel (CAPES) Thematic area: Non-transmitted diseases

Hypericum perforatum REDUCES INFLAMMATORY PAIN IN MICE

Larissa Ferrari1; Ana Carolina Rossaneis 1; Mariana Marques Bertozzi 1; Wadiceu Ap. Verri Junior 1 1 Londrina’s State University, Biological Sciences Center, Department of Pathological Sciences, Highway Celso Garcia Cid, Pr 445, KM 380,Cx Postal 10,011. | CEP 86057-970 | Londrina - PR – Brazil

Introduction: Hypericum perforatum is the scientific name of the popular St. John's Wort used especially in the treatment of moderate depression. Recently, the analgesic effect of H. perforatum extract (HPE) has been demonstrating in inflammatory conditions. Objective: Herein, we tested the analgesic effect of HPE in several models of inflammatory pain. Material and methods: Mice received treatment with commercially obtained HPE (Iperisan®, Marjan Farm, Santo Amaro-SP, Brazil) (30-300 mg/kg, po) or vehicle (20% tween 80 plus saline) prior the inflammatory stimuli injection. For acute pain, we used acetic acid- and PBQ-induced visceral pain (abdominal writhings), and formalin, and CFA-induced paw flinching and licking. By using an electronic version of von Frey filaments, we also investigated the effects of HPE on mechanical hyperalgesia after carrageenan or CFA intraplantar injection. To evaluate the role of NO/cGMP/PKG/KATP channel signaling pathway in the analgesic effect of HPE, mice received L-NAME (NOS inhibitor, 90mg/kg,i.p.,1h), ODQ (a soluble guanylate cyclase inhibitor, 0.3mg/kg,i.p.,30min), KT5823 (an inhibitor of PKG, 0.5µg/mouse,i.p.,5min) or GLY (an ATP-sensitive K+ channels blocker, 0.3mg/kg,i.p.,45min). Mechanical hyperalgesia were evaluated 1, 3, 5 and 7h after treatment with HPE. Results: HPE (300 mg/kg, per oral) inhibited acute pain behaviors induced by all testes stimuli as PBQ and Acetic acid, including both phases of formalin test, suggesting a direct nociceptor modulatory effect of HO besides its anti-inflammatory properties. In agreement, HPE (30mg/kg) also reduced the increased sensivity to mechanical stimulus induced by carrageenan. The daily post treatment with HPE (30mg/kg) during 7 days also reduced CFA-induced mechanical hyperalgesia without gastric or hepatic toxicity. Mechanistically, the effects of HPE (30mg/kg) involve the reduction of IL-1β production. HPE (30mg/kg) also activated the analgesic NO pathway to inhibit carrageenan-induced mechanical hyperalgesia. Conclusion: These results demonstrate the efficacy of HPE as a new analgesic drug.

Keywords: Hypericum perforatum; Inflammatory pain; Cytokine. Thematic area: Non transmitted diseases.

CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION OF PATIENTS AFFECTED BY SQUAMOUS CELL CARCINOMA ALONE AND ASSOCIATED WITH OTHERS SKIN NEOPLASIAS IN THE NORTHERN REGION OF PARANA

Mariani de Lima Garcia1; Poliana Camila Marinello1; Mateus Henrique Cunha da Silva1; Leonardo Bodner Freitas1; Walison Augusto da Silva Brito1; Natália Medeiros Dias Lopes1; Vanessa Gheno2; Théo Nicolacópulos 2; Priscila Daiane Pavezzi,2; Fernanda Teixeira Ortega2; Mariana Onuki Okamura2; André Armani 3; Airton dos Santos Gon2; Rubens Cecchini4; Alessandra Lourenço Cecchini 1.

Introduction: The squamous cell carcinoma (SCC) is the second more common and the most aggressive type of non-melanoma skin cancer (NMSC). It is not restrict to sun unprotected areas, also affecting the mucous. The identification of factors related with its development is essential to enable effective prevention. Objective: To characterize clinical and epidemiologically the patients with SCC and SCC associated with others skin neoplasias in hospitals from the northern region of Parana. Material and methods: Participants were categorized in 3 groups: Control (without skin cancer history; n=38); SCC (n= 9) and SCC associated with others skin neoplasias (SCC-ML; n=10) (CEP-UEL process n. 1.077.557). The clinical-epidemiological characterization was performed by applying a questionnaire to participants, with questions about tabagism, presence of chronic diseases, other types of cancer, lesion localization, occupational UV exposure, skin phototype (Fitzpatrick’s Classification) and obesity. The statistical analysis was performed using Chi-square test; p<0.05 was considered significant. Results: The patients presented the same age and gender distribution, and no differences about skin phototype, occupational UV exposure and cancer history were observe. The patients from both SCC and SCC-ML groups have head and neck as the principal localization for lesions. It was noted that obesity was more common in SCC-ML than SCC patients and tabagism was more frequent in SCC patients. Conclusion: The analysis of the results suggests that some NMSC risk factors could be differently involved in the pathophysiology of SCC and SCC- ML, with obesity being more related to the development of SCC-ML and tabagism more related with patients only affected by SCC.

Keywords: Squamous cell carcinoma; Skin cancer; Non-melanoma skin cancer Thematic area: Non-transmitted diseases

SIM2 AS A RNA CIRCULANTING MARKER FOR PROSTATE CANCER DIAGNOSIS AND PROGNOSIS

Marilesia Ferreira de Souza1, Hellen Kuasne2, Mateus de Camargo Barros2, Heloísa Lizotti Cilião1, Milene Roldão de Souza1, Fabio Albuquerque Marchi2, Paulo Emilio Fuganti3, Silvia Regina Rogatto2, Ilce Mara de Syllos Cólus1

1 Department of General Biology, State University of Londrina, Londrina, Paraná, Brazil. 2AC Camargo cancer center, São Paulo, São Paulo, Brazil 3Hospital of Londrina, Londrina, Paraná, Brazil

Introduction: Prostate cancer is the second most commonly diagnosed neoplasia in men. Currently screening methods for this disease presents low sensitivity and specificity. Thus, the developments of new screening methods are important to reduce the clinical impact of this disease. Cell-free nucleic acid is a new molecular tool that can be useful for diagnosis and prognosis for several diseases, as prostate cancer. These molecules in plasma have been indicated as a promising non-invasive tool for disease screening. Objective: The aim of this study was identify new markers for prostate cancer diagnosis and prognosis. Material and methods: This study was approved by Human Research Ethics Committee of the State University of Londrina (CAAE19769913.0.0000.5231). In Silico analysis using TCGA data (The Cancer Genome Atlas) was performed for identify candidate genes. These genes were validated in plasma of 102 prostate cancer patients and 50 healthy controls (PSA<4ng/ml). The peripheral blood was collected and the plasma cell-free was obtained by centrifugations. RNA from plasma was extracted using miRNeasy Mini kit. RT-qPCRs were performed using Sybr green. The Statistical analysis was performed using 7900TH software and the relative expression was done by 2^∆∆ct method. Results: In silico analysis showed that the SIM gene was upregulated in prostate cancer tissue when compared with surrounding normal tissue (FC=7.85; p<0.001). This gene was validated in plasma samples, the upregulation was kept (FC=1.88; p=0.02). When the expression levels of SIM2 was compared to histopathological characteristics, we observed that the SIM2 levels was associated with aggressiveness disease, as seminal vesicle (FC=3.50; p=0.04), perineural (FC=7.28; p=0.01) and lymph node (FC=7.61; p=0.03) invasion. Conclusion: This study showed that the levels of SIM2 circulating RNA are related with prostate cancer and its levels as directly proportional with disease aggressiveness. Thus, this molecular marker may be useful for prostate cancer diagnosis and prognosis.

Keywords: Prostate cancer; Cell-free RNA; SIM2 Grants: Fundação Araucária, CAPES, CNPq and FAPESP. Thematic area: Non-transmitted diseases

Tityus bahiensis VENOM INDUCES INFLAMMATORY PAIN IN MICE

Camila Rodrigues Ferraz1; Marília Fernandes Manchope2; Jackson Gabriel Miyamoto2; Ketlem Cristine Andrade2; Fábio Henrique Kwasniewski2; Waldiceu Aparecido Verri Jr2.

1 Health Science Center, Department of Health Science, Londrina State University 2 Biological Science Center, Department of Phatology, Londrina State University

Introduction: The Tityus bahiensis (Tb) is the specie that cause accidents to humans in Brazil. Scorpion envenoming is a public health problem in Brazil, and is an important cause of morbidity and mortality, especially among children. There are quite a few studies in literature about the mechanisms of pain these accidents. Objective: To evaluate the peripheral mechanisms involved in nociception induced by Tb venom. Material and methods: Male Swiss mice were used (20-25g; n=6) and the experiments were approved by the Institutional Ethics Committee under the protocol 21344.2015.72. Mechanical hyperalgesia was assessed by an electronic pressure meter, an electronic version of the von Frey filaments. Thermal hyperalgesia was assessed by hot plate (50o C). Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity by colorimetric assay. Cytokine (TNF-α and IL-1β) production by ELISA assay. Mice received intraplantar (i.pl.) injection of Tb venom (0.2, 0.6, 1.2 or 2.4 µg/paw, diluted in saline) and mechanical and thermal hyperalgesia were evaluated betweem 0.5-6h. MPO and NAG activity was determined 6h after Tb venom injection. The overt pain- like behaviors was assessed by paw flinches and time spent licking paw for 30 min. Results: The injection of Tb venom induced mechanical hyperalgesia in a dose-depend manner. The doses 0.6, 1.2 and 2.4 µg/paw induced significant mechanical hyperalgesia until 6h. All doses of Tb venom induced thermal hyperalgesia until 6h. All doses of Tb venom were induced increase of MPO and NAG activity. The dose 2.4 µg/paw induced increase of TNF-α levels 1h and 3h and IL-1β levels 0.5h, 1h and 3h after Tb venom injection and that dose induced increase of paw flinches and time spent licking paw. Conclusion: The Tb venom induced overt-pain like behavior, mechanical and thermal hyperalgesia, neutrophil and macrophage recruitment and hyperalgesic cytokine.

Keywords: Inflammation; Hyperalgesia; Tityus bahiensis venom. Grants: CAPES, CnPQ and Paraná State Government. Thematic area: Non-transmitted diseases

ANALGESIC AND ANTI-INFLAMMATORY EFFECT OF NARINGENIN IN TITANIUM DIOXIDE- INDUCED ARTHRITS

1 2 1 2 Marília F. Manchope ; Nayara Anitelli Artero ; Sandra Satie Mizokami ; Rubia Casagrande ; Waldiceu A. Verri Jr.1

1 Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina 2 Departamento de Ciências Farmacêuticas, Centro de Ciências de Saúde, Universidade Estadual de Londrina

Introduction: Titanium dioxide (TiO2), a white and odorless powder, is used as white pigment in paint, food colorant, sunscreens and cosmetic creams. Moreover, it is used in the production of orthopedic prosthesis. However, prosthesis wear releases TiO2, which induces inflammation and osteolysis in peri-prosthetic tissues. Naringenin is a flavonoid found in citrus fruit. Naringenin presents analgesic and anti-inflammatory effects in several models of inflammatory pain. Objective: The study aimed to evaluate the effects of naringenin in pain and inflammation in TiO2-induced arthritis. Material and methods: This work was approved the ethics committee in animal experimentation (nº 11849.2015.19). Male mice (20-25g) were grouped in 6 per group. Arthritis was induced by intra-articular injection of 3 mg of TiO2 in the right knee joint. After 24h of TiO2-induced arthritis mice were treated daily with naringenin (16.7, 50 and 150 mg/kg, p.o.) for 30 days. Mechanical hyperalgesia (electronic version of von Frey’s filament) and edema were evaluated 0, 1, 3, 5, 7, and 24 h after naringenin treatment on the 1st day, and every other day from the 2nd to the 30th day. On the 30th day, joint cavity washes and knee joint were harvested to assess leukocytes recruitment and oxidative stress (superoxide anion production [NBT reduction] and lipid peroxidation), respectively, in TiO2-induced arthritis. Results: All doses of naringenin inhibited th mechanical hyperalgesia and naringenin at 50 mg/kg inhibited edema. On the 30 day, naringenin at 50 mg/kg inhibited mechanical hyperalgesia (96%), edema (77%), total leukocyte recruitment (74%), polymorphnuclear (50%) and mononuclear (96%). Naringenin at 50 mg/kg was chosen for the next experiment. Naringenin inhibited NBT reduction (76%) and lipid peroxidation (56%) in knee joint. Conclusion Naringenin is analgesic and anti-inflammatory in TiO2-induced arthritis. Naringenin’s mechanisms of action in TiO2-induced arthritis seem to depend on inhibition of leukocyte recruitment and oxidative stress.

Keywords: Arthritis; Titanium; Naringenin. Thematic area: Non-transmitted diseases

INVESTIGATING THE INFLUENCE OF CXCR4 (C/T RS2228014) GENETIC POLYMORPHISM AND GENE EXPRESSION IN BREAST CANCER SAMPLES

Marina Okuyama Kishima1; Karen Brajão de Oliveira2; Carolina Batista Ariza2; Carlos Eduardo Coral de Oliveira2; Roberta Losi Guembarovski2; Walter Jorge Sobrinho3; Clodoaldo Zago Campos4; Alda Losi Guembarovski1; Felipe Campos de Almeida2; Maria Angelica Ehara Watanabe2

1 Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, State University of Londrina, Londrina, Parana, Brazil. 2 Department of Pathological Sciences, Biological Science Center, State University of Londrina, Londrina, Parana, Brazil. 3 Gineco-Med Clinical of Mastology, Londrina, Parana, Brazil. 4 Department of Oncology, Cancer Hospital of Londrina, Londrina, Parana, Brazil.

Introduction: Genetic polymorphisms of CXCR4 have been associated with development of cancer, as well as its expression level has been associated with poor prognosis Objective: The aim of this study was to investigate the influence of CXCR4 (C/T rs2228014) genetic polymorphism in its gene expression in breast cancer samples. Material and methods: total DNA and RNA was extracted from breast cancer tissue from 74 patients. CXCR4 genotyping was performed by RFLP-PCR, CXCR4 genetic expression was measured by qRT-PCR and tissue protein expression was assessed through immunohistochemical analyses. Results: The frequencies in breast cancer patients demonstrated that 88.24% patients presented CC genotype, 11.76% the CT genotype. Overall, breast cancer tissue samples presented higher gene expression of CXCR4 (7.21 folds) in relation to normal mammary gland RNA, but no significant differences were observed when comparing this relative expression among polymorphic allelic variants and immunohistochemical analysis. All T allele carriers patients were negative for p53 however, only one was HER2 positive whom presented increased CXCR4 mRNA (18 folds) with lymph node involvement. An inverse correlation was verified according to breast cancer nodal status with CXCR4 cytoplasmic expression. Conclusion: Although our results suggests that the polymorphism could not be influencing for the higher gene expression of CXCR4 observed in breast tumor samples, this increasing chemokine receptor in breast tumor cells could indicate the involvement of this gene in the breast cancer pathogenesis.

Keywords: CXCR4; Genetic polymorphism; Immunohistochemistry Grants: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação Araucária – Governo do Paraná Thematic area: Non-transmitted disease

CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION OF PATIENTS AFFECTED BY MULTIPLE SKIN NEOPLASIAS IN THE NORTHERN REGION OF PARANA.

Mateus Henrique Cunha da Silva1; Poliana Camila Marinello1; Mariani de Lima Garcia1; Leonardo Bodner de Freitas1; Walison Augusto da Silva Brito1; Natália Medeiros Dias Lopes1; Vanessa Gheno2; Théo Nicolacópulos2; Priscila Daiane Pavezzi2; Fernanda Teixeira Ortega2; Mariana Onuki Okamura2; Andre Armani3; Airton dos Santos Gon2; Rubens Cecchini4; Alessandra Lourenço Cecchini1.

Introduction: Non-melanoma skin cancer (NMSC) is the most common type of cancer in white populations and basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most common types of NMSC. The Actinic Keratosis (AK) is a premalignant lesion with high ability of progression to the SCC. Considering the elevated incidence of skin neoplasias, the identification of factors related with its development is essential to enable effective prevention. Objective: To characterize clinical and epidemiologically the patients with multiple skin neoplasias in hospitals from the northern region of Parana. Material and methods: Participants were categorized in 2 groups: Control (without skin cancer history; n=45) and multiple skin neoplasias (MN; n=16)(CEP-UEL process n. 1.077.557). In the MN group was included patients with at least two types of skin neoplasias (BCC, SCC and AK). The clinical-epidemiological characterization was performed by applying a questionnaire to participants, with questions about tabagism, presence of chronic diseases, other types of cancer, lesion localization, occupational UV exposure, skin phototype (Fitzpatrick’s Classification) and obesity. The statistical analysis was performed using Chi-square and Fisher’s-exact test; p<0.05 was considered significant. Results: The patients presented the same age and gender distribution that control, and no differences about occupational UV exposure, cancer history, smoking and presence of comorbidities were observed. Patients with multiple skin lesions presented more frequently white skin and higher sensibility to solar burns (type I/II in Fitzpatrick’s classification) when compared with control. Moreover, the prevalence of obesity is higher in multiple skin lesions group (45,45%) than in control group (2,27%). Conclusion: The results confirms the importance of skin UV sensibility in the development of multiple skin lesions and suggests that obesity is also an important risk factor. Keywords: Photocarcinogenesis, skin cancer, non-melanoma skin cancer Thematic area: Non-transmitted diseases

EVALUATION OF rs11781889 POLIMORPHYSM OF NKX3.1 GENE AND PROSTATE CANCER RISK

Milene Roldão de Souza¹; Marilesia Ferreira de Souza¹; Heloísa Lizotti Cilião¹; Paulo Emilio Fuganti², Ilce Mara de Syllos Cólus¹

1Department of General Biology, Center of Biological Sciences, State University of Londrina. Londrina, Paraná, Brazil 2Cancer Hospital of Londrina, Londrina, Paraná, Brazil

Introduction: The prostate cancer (PCa) is the second most frequent cancer and the sixth cause of death in men worldwide. In Brazil the PCa is the most common cancer among men. The lack of screening methods for this disease further several studies in order to provide better diagnosis and treatment for PCa patients. Among the interest genes associated with this disease, the NKX3.1 gene a prostate specific tumor suppressor has been highlight. This gene is known as a marker of prostate growth, is expressed from the initial prostate development to adulthood, at all stages of differentiation; and plays an essential role in the normal development of the gland. Objective: The aim of this study was to evaluate the allelic variant rs11781889 of NKX3.1 gene and correlate with susceptibility and progression of prostate cancer. Material and methods: This study was approved by Human Research Ethics Committee of the State University of Londrina (CAAE19769913.0.0000.5231). A case-control study was conducted with 284 patients with histopathological confirmation of PCa and 284 health controls (PSA<2 ng/mL) matched by race, age, tobacco and alcohol consumption. The genotyping assay was performed by real time PCR using TaqMan methodology. The association between the occurrence of PCa and polymorphisms were calculated by odds ratio (OR) with 95% confidence interval, obtained by univariate logistic regression using SPSS 20.0. Results: The polymorphism rs11781889 is associated with low levels of mRNA of NKX3.1 gene. Studies showed that this polymorphism is associated with prostate cancer risk. In this study we found no association with rare genotypes (CT and/or CC) and PCa (OR=0.93; IC95%0.93-1.79; p=0.13). The differences between our results and the others in the literature may be because the miscegenation that occurs in Brazilian population. Conclusion:. In this study we found no significant association between the polymorphism rs11781886 and prostate cancer.

Keywords: Prostate cancer; NKX3.1; Tumor suppressor. Grants: Fundação Araucária, CAPES, CNPq and FAPESP. Thematic area: Non-transmitted diseases

POLYMORPHISM ANALYSIS OF GENE AR IN PATIENTS WITH PROSTATE CANCER

Monyse de Nóbrega¹; Marilesia Ferreira Souza¹; Milene Roldão Souza¹; Heloísa Lizotti Cilião¹; Paulo Emílio Fuganti²; Ilce Mara Syllos Cólus¹.

1 Department of General Biology, State University of Londrina, Londrina, Paraná, Brazil. 2 Hospital of Londrina, Londrina, Paraná, Brazil

Introduction: Prostate cancer (PCa) is the second most frequent cancer in men.The development of this cancer can be influency by multiple factors, such as hereditary, race, age and androgens. The androgen pathway is critical in the normal development and progression of prostate cancer, since these processes are hormone dependent. Despite these hormones alone does not induce malignant changes in the prostate cells, the functional status of androgen receptor (AR) is important mediator of prostate carcinogenesis. Objective: Thus, the aim of this study was to investigate the association between the polymorphism rs17302090 of AR gene and susceptibility for PCa. Material and methods: This study was approved by Human Research Ethics Committee of the State University of Londrina, Brazil (CAAE19769913.0.0000.5231). A case-control study was done with 281 patients with histopathological confirmation of PCa and 281 individuals healthy controls, matched by race, smoking and alcohol consumption habits. DNA was extracted from peripheral blood of individuals using the PCR kit High Pure Template Preparation Kit (Roche). Polymorphism analysis was performed by real time PCR using TaqMan probes. Odds Ratio analysis (OR) with a confidence interval (IC) of 95% was obtained by univariate logistic regression using the statistical program BioEstat 5.0. . Results: The evaluation of the rs17302090 polymorphism showed no association with prostate cancer risk [OR=0.62; IC95% 0.34-1.11; p=0.14]. This polymorphism is located in the promoter region of the AR gene.The studies in literature about this polymorphism and PCa are controversial.But, most of then showed no association between this polymorphism and PCa, our results corroborate with this findings. In conclusion, our results showed that the rs17302090 polymorphism no have association with prostate cancer.

Keywords: Androgen receptor; Prostate cancer; rs17302090. Financial Support: Fundação Araucária; CAPES; CNPq-PQ. Thematic area: Non-transmitted diseases

INVERTED PAPILLOMA. CASE REPORT

Murilo Carlos Gimenes1, Denis Massatsugu Ueda1, Alvaro Ferdinando Scremin1, Rafael Yoshio Kanashiro1, Mauricio Pacheco Reis1, Poliana Camurça1, Andre Armani1 1Londrina State University, Rodovia Celso Garcia Cid, Km 380, Campus Universitário, Londrina - PR,

Introduction: Inverted papilloma (IP) is a rare benign nose/sinus neoplasia with incidence of 0.75 ± 1.5 cases / 100 thousand inhabitants. Responsible for 0,5% to 4% of all nasal tumors, although benign, it has aggressive growth and great invasion potential, reaching 10% of cases with association with squamous cell carcinoma (SCC). Objective: To report a case of inverted papilloma and the importance of the correct management. Case report: M.L.B.S, female, 64-year-old, smoker, looked for the head and neck surgery department with complaints of purulent rhinorrhea, decreased visual acuity on the left eye and nasal obstruction - this last symptom started 1 year ago. On physical examination, left front and periorbital hyperemia and edema were shown. The nasal tumor was viewed by nasopharyngoscopy. The magnetic resonance showed large expansive lesion of 8,1x 5.0x 5.2 cm with contrast uptake, necrotic areas in the left orbital roof, paranasal sinuses, intra- and extracranial. It was opted for surgical intervention by neurosurgery and head and neck surgery being performed excision of craniofacial injury and left eyepiece exenteration including eyebrow. After the biopsy sample was submitted, it determined moderately differentiated SCC, frontal sinus with neoplastic infiltration and IP, as well as compromised surgical margins. The patient underwent chemotherapy and radiotherapy sessions. The surgery was performed a month ago, the patient maintain outpatient treatment with good evolution. Discussion: Because of the risk of malignant evolution to SCC, ranging from 5-15%, the IP should be approached as invasive and aggressive lesion, with surgical treatment and follow-up, since there is possibility of recurrence, especially in the first 2 years after surgery. Conclusion: The knowledge of IP is of great importance, since its malignant potential provides high morbidity and mortality if diagnosis, proper approach and early treatment are not performed.

Keywords: Inverted papilloma; Malignancy Grants: There is no financial support or relationships that may pose conflict of interest to declare. Thematic area: Non-transmitted diseases

CLINICO-PATHOLOGICAL PARAMETERS INVOLVED IN THE ASSOCIATION BETWEEN DIFFERENTIATED THYROID CANCER AND HASHIMOTO THYROIDITIS: A REVISION OF THE LITERATURE

Natália Medeiros Dias Lopes1 ; Poliana Camila Marinello 1 ; Walison Augusto da Silva Brito1 ; André Armani1 ; Rubens Cecchini 2; Alessandra Lourenço Cecchini Armani1 .

1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, BR. 2 Laboratory of Pathophysiology and Free Radicals, State University of Londrina, Londrina, PR, BR

Introduction: The thyroid cancer is considered the most common type of cancer of the head and neck worldwide. Its progression and incidence is increasing every year, being the Differentiated Carcinoma of the Thyroid (DCT) the most frequent. The papillary thyroid carcinoma (PTC) is the most common type of DCT and is considered the cancer with the higher increase in the world. The Hashimoto's thyroiditis (HT) is the leading cause of hypothyroidism, and the combination of PTC and HT has been studied. It has been suggested that there are a relationship between thyroiditis and the development of the thyroid carcinoma. Although the related pathogenesis are not clear, oxidative stress seems to play a role in the their differentiation. The clinical relationship and clinico-pathological parameters of both diseases shows that the coexistence of PTC and HT presents a better prognosis, related with recurrence and mortality than PTC without HT. Objective: This review aims to investigate the clinico-pathological parameters involved in the association between PTC and HT. Materials and methods: The revision was conducted using the database available on PubMed in the last 15 years, using the following key words: Thyroid cancer, Hashimoto’s thyroiditis, oxidative stress. Results: The studies indicates that patients with PTC and HT have, in general, tumors at earlier stages and a better prognosis compared to patients with DCT without the presence of HT, showing a possible association between the two diseases. The lymphocytic inflammatory infiltrate of HT seems to be a predisposing factor for the DCT, with the increase in oxidative stress, but also appears to be an immune surveillance factor for defense against this type of cancer. Conclusion: Although the literature reports the association between DCT and HT, it is not possible to establish the precise mechanisms of biological events, or if oxidative stress connects both pathologies.

Keywords: Thyroid cancer; Hashimoto’s thyroiditis; Oxidative stress. Thematic area: Non-transmitted diseases

INDUCTION OF LIPID PEROXIDATION AND OXIDATIVE INJURY OF DNA BY CURCUMIN IN HUMAN BREAST CANCER CELLS MCF7 AND MDA-MB-231

Cassio Fernando Nunes da Silva1; Natália Medeiros Dias Lopes 1 ; Poliana Camila Marinello1; Kaliana Larissa Machado 1; Walison Augusto Silva Brito 1; Rodrigo Cabral Luiz 1 Rubens Cecchini 2; Alessandra Lourenço Cecchini Armani 1.

1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, BR. 2 Laboratory of Pathophysiology and Free Radicals, State University of Londrina, Londrina, PR, BR.

Introduction Breast cancer is the most common female malignancy in the world population. Curcumin has been investigated as a possible adjuvant in the treatment of this type of cancer. Objective: To investigate the role of oxidative stress in curcumin toxicity mechanisms in breast cancer cells MCF-7 and MDA-MB-231 evaluating the metabolic activity, proliferation and death, oxidative stress and DNA damage. Material and methods: Cells were exposed to curcumin (10 and 40μM) for 24 hours. Metabolic activity was analyzed by MTT assay and the proliferation by counting in a Neubauer chamber. Staining with ethidium bromide/acridine orange was performed to quantify apoptosis and necrosis. Oxidative stress was evaluated by lipid peroxidation (chemiluminescence), total thiols and catalase activity (spectrophotometry). The DNA damage was analyzed by comet assay and the quantification of 8-hydroxy-2-deoxy-guanosine (8-OHdG). The results were analyzed by one-way ANOVA and variations between groups were investigated using the Bonferroni post-test. Results: In MCF-7 cells curcumin reduced metabolism and cell proliferation, predominantly induced apoptosis, increased lipid peroxidation and total thiols and decreased catalase activity in the concentration of 40μM. DNA oxidation was observed (8-OHdG), but without significant damage (comet assay). In MDA-MB-231 cells, decreased cell proliferation and oxidative DNA injury were observed with 10μM drug, while reducing metabolic activity and increased lipid peroxidation, reduction of catalase activity and cell death (only necrosis) were found at 40μM. The levels of total thiols did not change. Conclusion: Although the cells have presented different behaviors, curcumin was able to induce oxidative stress and death in both strains. However, MCF-7 cells were able to activate antioxidant protective mechanisms with increasing thiol levels and protection from damage to the genetic material, which was not observed in MDA-MB-231 cells. These results suggest the clinical potential of curcumin as an adjuvant in the treatment of these two breast cancer subtypes.

Keywords: Breast cancer; Curcumin; Oxidative stress. Grants: Araucária Foundation (grant number 973/2013) Thematic area: Non-transmitted diseases

SEARCH OF HUMAN MAMMARY TUMOUR VIRUS (MMTV-LIKE) DNA IN BREAST CANCER MICROENVIROMENT

Nathália de Sousa Pereira1; Marla Karine Amarante1; Carlos Eduardo Coral de Oliveira1, Glauco Akelinghton Freire Vitiello1, Bruna Karina Banin Hirata1, Maria Angelica Ehara Watanabe1 1 Laboratory of Study and Application of DNA Polymorphisms, Department of Pathological Sciences, State University of Londrina, Londrina, PR, Brazil

Introduction: Breast cancer (BC) is a complex disease whose evolution depends on the tumor-host interaction. Human mammary tumor virus (MMTV-like) has been suggested as a candidate for the viral etiology of some cases of BC. Virus associated tumor cells can show viral antigens, and, as a result, representing a potential antigenic target, indicating a promising treatment for BC. Increasing knowledge has emerging related to MMTV-like, However, several key issues remain unclear, such as the clinical significance of its infection. Objective: The aim of this study was to detect the DNA virus in tumot tissue samples from patients with BC, and correlate with clinicopathological parameters. Material and methods: This project was approved by Ethic Committee from UEL (CAEE 47709015.2.0000.5231). Genomic DNA was extracted from 218 mammary tumor tissue samples, MMTV-like env gene sequence was amplified through nested PCR using specific primers for MMTV-like, and all products were analyzed on polyacrylamide gel (10 %), stained with silver nitrate. Statistical analyses were performed using relative risk test (GraphPad Prism 6, USA). Results: Viral sequence was detected in 18.8% (n=41) of BC samples. There was no significant association between the presence of the virus with clinical outcome including tumor size, stage, metastasis, lymphonodes involvement, estrogen receptor, HER-2 overexpression, p53 expression and Ki67 expression. However, we found tendency between the MMTV-like presence and progesterone receptor (p=0.06). Conclusion: Other studies should be conducted to demonstrate the involvement MMTV-like virus and clinical effects in BC patients and thus, characterize a possible association of this virus and BC pathogenesis.

Keywords: MMTV-like; Breast cancer; Clinicopathologic parameters. Grants: CNPq, Fundação Araucária, Hospital do Câncer de Londrina. Thematic area: Non-transmitted diseases.

OXIDATIVE AND NITROSATIVE STRESS AND AUTOANTIBODIES ARE ASSOCIATED WITH LUPUS NEPHRITIS

Nicole Perugini Stadtlober1, Tatiana Mayumi Veiga Iriyoda2, Lorena Flor Da Rosa Franhci Santos1, Brunna Emanuella França Robles1, Daniela Frizon Alfieri1, Tamires Flauzino1, Ligia Grecco Costa Dall’aqua1, Bruno Alexandre Sekiguchi1, Beatriz Sardinha Sabino1, Marcell Alysson Batisti Lozovoy3, Neide Tomimura Costa2, Edna Maria Vissoci Reiche3, Isaias Dichi4, Andréa Name Colado Simão3

1 Research Laboratory in Applied Immunology - University Hospital from University of Londrina, Londrina, Paraná, Brazil. 2 Department of Rheumatology - University of Londrina, Londrina, Paraná, Brazil. 3 Department of Pathology, Clinical Analysis and Toxicology - University of Londrina, Londrina, Paraná, Brazil. 4 Department of Internal Medicine - University of Londrina, Londrina, Paraná, Brazil.

Introduction: The pathophysiology of systemic lupus erythematosus (SLE) is multifactorial, and evidences has shown that immune-inflammatory response and oxidative and nitrosative stress (O&NS) are involved in the development and complication of SLE. Lupus Nephritis (LN) is one of the most serious complication of SLE. Although several studies have demonstrated the involvement of autoantibodies in the LN, the data is still controversial about their exact role in the pathophysiology of the disease. Objective: evaluate if the association of anti-dsDNA and anti-NCS and biomarkers of O&NS could be used as indicators of the presence and activity of LN. Methods: We selected 152 patients with SLE without nephritis and 48 with nephritis (36 in remission and 12 in activity). CAAE:01865212.0.0000.5231 Results: Patients with LN had higher serum levels of anti-dsDNA (p=0.033) and anti-nucleosome antibodies (anti-NCS) (p=0.002). Patients with nephritis showed decreased advanced oxidation protein products (AOPP, p=0.010) and increased nitric oxide metabolites (NOx, p=0.030) compared to the group without nephritis. AOPP, anti-NCS antibodies, and BMI were associated to LN independently of age. However, anti-NCS antibodies lacked the association with LN when the results were controlled for mycophenolate use (OR: 1.004, CI 95%:0.993-1.015, p=0.459), whereas AOPP (OR: 0.973, CI 95%: 0.953-0.992, p=0.007) and BMI (OR: 1.186, CI 95%: 1.018-1.382, p=0.029) maintained their significance. When patients with nephritis were divided into remission and in active disease, the later patients had increased AOPP (p=0.022) and decreased NOx (p<0.0001) compared to those in remission. AOPP was directly and independently (OR: 1.027, CI 95%: 1.000-1.055, p=0.046) and NOx was inversely and independently (OR: 0.920, CI 95%: 0.847-0.999, p=0.049) associated with LN activity. Conclusion: Anti-NCS antibodies showed to be good predictors of the presence of LN but not of its activity. Concomitant evaluation of O&NS and anti-NCS antibodies are useful tools in identifying and monitoring disease activity in LN.

Keywords: Systemic Lupus Erythematosus; Lupus Nephritis; Autoantibodies. Grants: The study was supported by Institutional Program for Scientific Initiation Scholarship (PIBIC) of the National Council for Scientific and Technological Development (CNPq) and Fundação Araucária. Thematic area: Non-transmitted diseases.

CREATINE SUPPLEMENTATION INCREASES LIVER OXIDATIVE STRESS AND LIPIDS ACCUMULATION IN A MODEL OF MURINE ALCOHOLIC STEATOSIS

Poliana Camila Marinello1,2; Mayra Tardeli de Jesus Testa1; Fabrício Azevedo Voltarelli 1,3; Paola Sanches Cella1; Fernando Pinheiro de Souza Neto2; Phillippe Bovo Guirro1; Fernando Henrique Borges1; Camila de Souza Padilha2; Alessandra Lourenço Cecchini2; Rubens Cecchini2; José Alberto Duarte4; Rafael Deminice1.

1Department of Pathological Sciences, State University of Londrina, Londrina, PR, BR. 2Department of Physical Education, State University of Londrina, Londrina, PR, BR 3Faculty of Physical Education, Federal University of Mato Grosso, Cuiabá, MT, BR 4Faculty of Sport, University of Porto, Porto, POR.

Introduction: Creatine has demonstrated protective effect on the liver lipids accumulation in different experimental models of steatosis by regulating β-oxidation involved genes. However, its action in models of alcoholic hepatic steatosis (AHS) is poorly described. Objective: To investigate the effects of creatine supplementation (CrS) in the pathophysiology of AHS by evaluating liver oxidative stress and inflammatory profile, fat accumulation and tissue morphological changes. Material and methods: Male Swiss mice (35 - 40g) were divided into 4 groups (n = 8/group): control (C), control creatine (CC), ethanol (E) and ethanol creatine (EC) (CEUA-UEL: Process n. 222/12). Animals were fed for 15 days with Lieber-DeCarli diet (5% ethanol; groups E and EC); C and CC received an isocaloric diet without ethanol. Creatine (1%) was added to the diet of CC and EC. After euthanasia, the liver was removed and weighed, and a tissue fragment was fixed in 4% paraformaldehyde for histological processing; the remaining tissue was frozen for biochemical analysis. Liver architecture was blindly evaluated after hematoxilin/eosine (H&E) staining by crosses method. Oxidative stress (malondialdehyde-MDA; carbonilated proteins; reduced and oxidized glutathione -GSH and GSSG, respectively) and inflammatory (IL-10, IL-6 and TNF-α) markers were assessed on liver. Results: Although liver steatosis was observed in H&E staining, E group did not present significantly alterations in liver weight, hepatic lipids, IL-10, IL-6, TNF-α and oxidative stress parameters. However, CrS significantly intensified steatosis as well as increased liver weight, hepatic lipids, carbonilated proteins, MDA and GSH when compared to E group. The hepatic levels of all cytokines investigated and the tissue collagen deposition did not alter in EC when compared with E group. Conclusion: The analysis of the results indicates that CrS intensified hepatic ethanol toxicity through the increased oxidative damage to lipids and proteins, which favored the lipids accumulation in the liver.

Keywords: Creatine; Alcoholic steatosis; Oxidative stress Thematic area: Non-transmitted diseases

METFORMIN PRETREATMENT PREVENTS DOXORUBICIN RESISTANCE INDUCTION IN MCF-7 AND MDA-MB-231 HUMAN BREAST CANCER CELLS THROUGH OXIDATIVE STRESS GENERATION, INDUCTION OF P53 AND TGF-Β1 AND DECREASE OF NRF2

Poliana Camila Marinello 1; Carolina Panis 2, 3; Thamara Nishida Xavier da Silva 2; Fernando Henrique Borges 2; Adriano Martin Felix Aranome 2; Natália Medeiros Dias Lopes1; Rodrigo Cabral Luiz 1; Rubens Cecchini 2; Alessandra Lourenço Cecchini 1.

1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, BR. 2 Laboratory of Pathophysiology and Free Radicals, State University of Londrina, Londrina, PR, BR. 3 Laboratory of Inflammatory Mediators, State University of West Parana, Unioeste, Francisco Beltrão, PR, BR.

Introduction: The metformin effects in the sensitization of chemoresistant breast cancer cells has been investigated. However, the mechanism regarding its interference during the chemoresistance induction is unknown. Objective: To investigate metformin action during doxorubicin resistance induction in MCF-7 and MDA-MB-231 cells, evaluating the cellular oxidative status, nitric oxide levels (NO), and p53, nuclear factor kB (NF-kB), nuclear factor (erithroid-related-2)-like 2 (Nrf2), and cytoplasmic transforming growth factor beta 1 (TGF-β1). Material and methods: Chemoresistance was induced exposing cells to increasing concentrations of doxorubicin (10nM to 100 nM) along seven sequential passages. The chemoresistance induction was confirmed by the MTT assay. Metformin (6 µM) treatment was performed before (seven passages) and simultaneously to the doxorubicin administration. Samples of each cell passage were collected for the analysis of oxidative stress parameters, such as total thiols levels (spectrophotometry) and lipoperoxidation (chemiluminescence), for the detection of NO levels (chemiluminescence), p53, NF-kB, Nrf2 and TGF-β1 (immunocytochemistry). Data were analyzed using one-way ANOVA with Bonferroni post- test. p<0.05 was considered statistically significant. It was compared the difference between cells at the several passages with the cells at the first passage. Results: The induction of chemoresistance progressively reduced lipoperoxidation and increased antioxidant defenses and NO, in addition to the reduction in nuclear p53 and cytoplasmic TGF-β1, and increase in NF-kB and Nrf2, in both cell lines. The metformin pretreatment prevented the resistance induction in both cell lines and sensitized MCF-7 cells to doxorubicin. The prevention of chemorresistence occurred through the generation of oxidative stress, reduction in NO levels, increase in nuclear p53 and cytoplasmic TGF-β1, and reduction in nuclear Nrf2, in both cell lines. Conclusion: The analysis of the results suggests that metformin pretreatment could exert a beneficial role in the prevention of the doxorubicin chemoresistance, highlighting its potential therapeutic investigation in patients bearing chemoresistant breast tumors.

Keywords: Breast cancer; Metformin; Chemoresistance Grants: Araucaria Foundation (grant number: 973/2013) Thematic area: Non-transmitted diseases

COLON HISTOPATOLOGICAL EVALUATION OF RATS WISTAR FOR ABERRANT CRYPT FOCI (ACF) STUDY AND ADENOMATOUS INTESTINAL TUMOR

Raíssa Coracini Varago¹, Bruno Bueno Pimenta¹, Tiago Peres da Silva Suguiura¹, Sara Santos Bernardes², Isolde Previdelli1, Edilson Noboioshi Kaneshima¹, Alice Maria de Souza-Kaneshima¹, Tânia Cristina Alexandrino Becker¹, 1Universidade Estadual de Maringá, Av. Colombo 5790, Maringá - PR, Brasil, 87020-900 2Universidade Federal da Grande Dourados, R. João Rosa Góes 176, Dourados - MS, Brasil 79825- 070.

Introduction: Colorectal cancer is the third most frequent in men and the second most frequent in women. Colorectal carcinogenesis is a complex process, where the benign lesions considered precursors may develop into highly metastatic malignant tumors. Animal models of study consist of a precious tool in the field of biochemical research, including toxicology, carcinogenesis and cancer treatment and prevention. Aberrant crypt foci (ACF) formed by aberrant crypts (AC) are identified as precancerous lesions due to the presence of dysplasia tissue. Thus, in animal models for carcinogenesis study, the presence of ACF has been analyzed. Objective: Describe the development of precancerous lesions (ACF) by means of histopathological methods. Material and methods: Under the opinion Nº104/2014 of the Ethics Committee on Animal Use (CEUA-UEM), it was inoculated 1,2-dimethylhydrazine (DMH) in Wistar rats divided into short (P1-pré) and medium (P1) term trials. The colon mucosa was examined and ACF were classified according to their number and distribution pattern throughout the colon, as well as the number of aberrant crypt/focus, by optical microscopy, at 10x magnification, with methylene blue staining 0.1%. Through the technique “Swiss roll", paraffin blocks were produced, which were cut in semi-automatic microtome and adhered to the histological slides. The slides were stained with hematoxylin-eosin (HE). Tumor analysis enabled the classification for the presence and degree of tissue dysplasia. The statistical model used for the analysis was the “Quasipoisson” with support of the software R. Results: There was a predominance of ACF with 02-03 AC/focus on P1-pré group and 04-09 AC/focus on the P1 group, besides 12 hyperplastic / dysplastic adenomas presenting varying degrees and intramucosa adenocarcinoma in the P1 group. Conclusion: ACF are precursor lesions to colonic tumors and the medium term protocol can be used for chemical induction models of colonic carcinogenesis studies.

Keywords: Aberrant crypt foci (ACF); 1,2-dimethylhydrazine (DMH); Colorectal carcinogenesis (CRC) Grants: Thanks for the Araucaria Foundation for funding for the project. Thematic area: Non-transmitted diseases

VINPOCETINE AS ANTI-INFLAMMATORY AGENT – A REVIEW

Renata Streck Fernandes1; Eduardo José de Almeida Araújo1. 1State University of Londrina, Londrina, Paraná - Brazil.

Introduction: Vinpocetine is a nootropic drug widely used on the treatment of cognitive and cerebrovascular diseases. Its anti-inflammatory effect has been demonstrated on in vitro and in vivo studies performed during the last years using different tissues, such as liver, gastrointestinal tract and retina. Objective: To review the applicability of vinpocetine as anti-inflammatory agent and its possible mechanisms. Material and methods: Original papers were searched on Pubmed using the key words: “vinpocetine and inflammation”. There was included on the review only papers which described mechanisms. Results: Although vinpocetine is selective inhibitor of phosphodiesterase, studies showed that its anti-inflammatory activity is dependent of NF-kB inhibition, with consequent reduction of TNF-α, IL-1β, IL-6 and IL-33. Mice treated with vinpocetine presented reduction of neutrophil recruitment and of myeloperoxidase activity, providing protection of the oxidative stress- induced damage. The literature also reveals that vinpocetine is able to inhibit that process via depletion of GSH, reduction of superoxide anions, nitric oxide and lipid peroxidation. Vinpocetine was also able to inhibit inflammasome formation by preventing NF-kB activation, ratifying its protective role. Vinpocetine also appears to be effective in the treatment of inflammatory pain caused by the sensitization of nociceptors neurons, because of its ability to inhibit the production of pro- inflammatory cytokines and reactive oxygen species. It was observed in vitro that macrophages are involved on the effects of vinpocetine, since RAW 264.7 cells challenged with lipopolysaccharide were inhibited. Conclusion: The literature shows that vinpocetine is a safe and promising anti- inflammatory agent and its most well-known mechanism of action in the inhibition of NF-kB activation.

Keywords: Vinpocetine; Inflammation; Anti-inflammatory therapy. Thematic area: Non-transmitted diseases

ALBUMIN AND PROTEIN OXIDATION ARE PREDICTORS THAT DIFFERENTIATE RELAPSING-REMITTING FROM PROGRESSIVE CLINICAL FORMS OF MULTIPLE SCLEROSIS

Sayonara Rangel de Oliveira1, Ana Paula Kallaur1, Tamires Flauzino1, Daniela Frizon Alfieri1, Wildea Lice de Carvalho Jennings Pereira1, Luana Aparecida Cossentini1, Beatriz Sardinha Sabino1, Bruna Miglioranza Scavuzzi1, Lorena Flor da Rosa Franchi Santos1, Brunna Emanuella França Robles1, Edna Maria Vissoci Reiche2, Damácio Ramon Maciel-Kaimen3, Marcell Alysson Lozovoy2, Helena Kaminame Morimoto2, Michael Maes4, Isaias Dichi3, Andrea Name Colado Simão2 1 Research Laboratory in Applied Immunology, University Hospital, State University of Londrina, Londrina, Paraná, Brazil 2 Department of Pathology, Clinical Analysis, and Toxicology, Health Sciences Center, University Hospital, State University of Londrina, Londrina, Paraná, Brazil 3 Department of Clinical Medicine, Health Sciences Center, University of Londrina, Londrina, Paraná, Brazil 4 IMPACT Strategic Research Centre, School of Medicine, Deakin University, Geelong, VIC, Australia

Introduction: Multiple sclerosis (MS) is an inflammatory and degenerative neurological disease in which damage to the central nervous system causes widespread dysfunction. A growing body of evidences has shown that activated immune-inflammatory response, oxidative, and nitrosative stress pathways play an important role in MS pathophysiology. Objective: The aim of the present study was to evaluate inflammatory, oxidative, and nitrosative stress (IO&NS) blood markers as possible predictors of MS and its clinical forms. Material and methods: This study included 258 MS patients (175 with relapsing-remitting MS (RRMS) and 83 with progressive MS clinical forms) and 249 healthy individuals. Peripheral blood samples were obtained to determine serum levels of albumin, ferritin, C-reactive protein (CRP), total protein, lipid hydroperoxide by tert-butyl hydroperoxide-initiated chemiluminescence (CL-LOOH), carbonyl protein content, advanced oxidation protein products (AOPP), nitric oxide metabolites (NOx), and total radical-trapping antioxidant parameter (TRAP). MS patients showed higher ferritin (p < 0.001) and CL-LOOH (p < 0.001) and lower albumin (p = 0.001), TRAP (p < 0.001), AOPP (p = 0.013), and NOx values (p < 0.001) than controls. Results: Difference was not observed in CRP, total protein, and carbonyl proteins between patients and controls. In the logistic regression age-adjusted, ferritin and CL-LOOH showed positive association with MS and were predictors of MS development (OR: 1.006, 95 % CI: 1.003-1.009, p < 0.001 and OR: 1.029, 95 % CI: 1.007-1.052, p = 0.009, respectively). Albumin, TRAP, AOPP, and NOx were negatively associated with MS (p = 0.019, p = 0.003, p = 0.001, and p = 0.003, respectively). Moreover, other logistic regression age-adjusted showed that MS patients with progressive clinical forms had lower albumin and higher AOPP than those with RRMS (p = 0.037). Conclusion: Ferritin, albumin, and biomarkers of IO&NS, such as CL-LOOH, AOPP, TRAP, and NOx were predictors of MS diagnosis, whereas albumin and AOPP were predictors that differentiated RRMS from the progressive clinical forms of MS.

Keywords: Multiple Sclerosis; Predictors; Protein Oxidation. Thematic area: Non-transmitted diseases

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EVALUATION OF ANTINFLAMMATORY AND ANTIOXIDANT EFFECTS OF HESPERIDIN- METHYL-CHALCONE IN LPS-INDUCED ACUTE LUNG INJURY IN MICE.

Talita Perdigão Domiciano1, Stephanie Badaró Garcia1, Larissa Staurengo-Ferrari1, Felipe A. Pinho-Ribeiro, Rubia Casagrande2, Waldiceu Ap. Verri Junior 1 1Departamento de Ciências Patológicas - Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, Brazil 2Departamento de Ciências Farmacêuticas - Centro de Ciências de Saúde, Universidade Estadual de Londrina, Londrina, Paraná, Brazil,

Introduction: Acute lung injury (ALI) is characterized by acute alveolar damage resulting from excessive accumulation of neutrophils in the lung and production of inflammatory mediators. In this context, the flavonoids act as effective antioxidants, protect the actions of oxygen radicals and are also known to inhibit the production of pro-inflammatory cytokines. The flavonoid hesperidin methyl chalcone (HMC) is used in the treatment of venous diseases, but its bioactivity as anti-inflammatory and antioxidant remains unclear. Objective: To demonstrate the efficacy of HMC on ALI induced by LPS in mice. Material and methods: Male Swiss mice care and handling procedures were approved by the Ethics Committee of the Universidade Estadual de Londrina (process number 1440.2011.09). To accomplish ALI model, mice received intratracheal instillation of LPS (50µg/50µl) from Escherichia coli diluted in PBS, while control mice received PBS. HMC treatment (10-300 mg/kg, i.p) was performed 1h before stimulus. The bronchoalveolar lavage (BALF) was achieved by instillation followed by aspiration of PBS into the lungs 6h after stimulus with LPS and leukocyte profile and cytokine production was determined. Further, the effect of HMC on LPS-induced oxidative stress and-MPO activity in lung samples was assessed. The differences between groups were analyzed by one-way ANOVA followed by the Tukey’s test. Results: HMC treatment (10-300 mg/kg) reduced in a dose-dependent manner leukocyte recruitment in BALF, and the 300 mg/kg dose of HMC was significantly different of lower doses. We also observed that HMC treatment reduced levels of NO, IL-1β, IL-6, TNF-, IL-10 and IL-33 in BALF. Regarding the antioxidant mechanisms, HMC treatment prevented the decrease of ferric-reducing ability potential and free-radical scavenger ability in lung - tissue. In addition, the MPO activity, O2 (NBT reduction) were also reduced. Conclusion: Our results suggest that HMC represents a therapeutic approach to treat ALI by targeting oxidative stress and cytokine production.

Keywords: Hesperidin-methyl-chalcone; Acute lung injury; cytokines. Thematic area: Non-transmitted diseases

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EVALUATION OF OCCUPATIONAL EXPOSURE AT PESTICIDES USING BIOCHEMICAL, GENOTOXIC AND OXIDATIVE MARKERS

Elaine Eloiza Cortellini Landivar1, Bárbara Lidiane Kummer Mallmann1, Jeferson Noslen Casarin1, Eduardo Ottobelli Chielle2, Tiago Mateus Andrade Vidigal2 1Laboratory of Biochemistry and Molecular Biology of the University of the West of Santa Catarina - Unoesc, Oiapoque Street, 211, Agostini São Miguel do Oeste - SC – Zip code:89900-000. 2Department of Health Sciences, Laboratory of Biochemistry and Molecular Biology of the University of the West of Santa Catarina -, Unoesc, Oiapoque Street, 211, Agostini São Miguel do Oeste - SC – Zip code:89900-000.

Introduction: The dosages of acetyl and butirilcolinesterase are widely used to assessment of pesticide exposure, however these markers are not sufficient to assess comprehensively and long- term an occupational exposure. Objective: This study aimed to employ hepatic, toxicological, oxidative and genotoxic markers to evaluate the harmful effects of pesticide exposure in farming workers. Material and methods: The experiment was approved by Ethics Committee on Human Research by the protocol: 55809116.0.0000.5367. Were evaluated 200 subjects divided between test and control groups. using a longitudinal design. The test group was composed by 100 farmers exposed to pesticides for at least five years and the control group was composed by 100 individuals not exposed occupationally. Were performed the dosage of acetylcholinesterase and butyrylcholinesterase, AST/SGOT, ALT/SGPT, GGT, ALP, and albumin; in addition to Markers of oxidative stress: SOD and TBARs and the genotoxic tests comet assay and micronulei test. The comparison of means between the groups was made with the Mann-Whitney test for nonparametric data. Results: Both acetyl and butyrylcholinesterase levels were significantly reduced in the test group, which also found significantly increased levels of AST / SGOT, ALT / SGPT, GGT and ALP, TBARs and SOD. Test group had higher scores of the comet assay and a statistically increased frequency of micronuclei. Conclusion: Dosages of acetyl and butyryl cholinesterases indicate that there is overexposure to pesticides in the subjects of the test group. In addition, the test group showed higher rates of injury to liver cells and genetic material. These lesions may be associated with increased oxidative stress from exposure to pesticides. Probably the highest levels of SOD in the test group, correspond to a daptative response.

Keywords: Oxidative stress; Genotoxicity; Pesticides Thematic area: Non-transmitted diseases

102

VINPOCETINE AMELIORATES ACETIC ACID-INDUCED COLITIS BY INHIBITING OXIDATIVE STRESS AND PRO-INFLAMMATORY CYTOKINES PRODUCTION IN MICE

Victor Fattori1; Bárbara B. Colombo1; Carla F. S. Guazelli1; Kenji W. Ruiz-Miyazawa1; Allan C. Bussmann2; Rúbia Casagrande3; Waldiceu A. Verri, Jr.1 1 Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rod. Celso Garcia Cid PR445 KM380, 86057-970 Londrina, Parana, Brasil. 2 Laboratório de Anatomia Patológica, Centro de Ciências de Saúde, Universidade Estadual de Londrina, 86038-350, Londrina, Paraná, Brasil. 3 Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Hospital Universitário, Universidade Estadual de Londrina, Av. Robert Koch, 60, 86038-350 Londrina, Parana, Brasil.

Introduction: The idiopathic inflammatory bowel diseases (IBD) comprise two types of chronic intestinal disorders: Crohn's disease and ulcerative colitis. Neutrophils and macrophages recruitment towards intestinal tissues is an important event given that in the inflammatory foci, these cells produce ROS and NF-κB-dependent pro-inflammatory cytokines, which contribute to tissue damage. A seminal advance was the introduction an anti-TNF-α monoclonal antibody as a treatment for IBD patients. This therapy is often limited by a loss of efficacy due to the development of adaptive response, underscoring the need for novel therapies. Vinpocetine is a nootropic drug known to have anti-inflammatory properties, partly by inhibition of NF-κB and downstream cytokines, in addition to its antioxidant effect. Objective: In the present study, we investigated the efficacy of vinpocetine in a model of acid acetic-induced colitis. Material and methods: Experiments were conducted in male Swiss mice with Londrina State University Ethics Committee on Animal Research and Welfare approval under process number 3307.2015.37. Colitis was induced by intracolonic injection of acetic acid 7.5% (n = 8 mice per group). Vinpocetine treatment was divided in two different protocols: (I) for oxidative stress analysis, mice were treated with vinpocetine 10 and 4h before acetic acid injection, and 2h after. In this protocol, distal colon was collected 4h after colitis induction for evaluation of total antioxidant capacity (ABTS assay) and GSH levels. (II) For cytokine production and neutrophils recruitment, mice were treated with vinpocetine 2h before acetic acid injection, and 4, 10, and 16h after. In this protocol, distal colon was collected 18h after colitis induction for MPO analysis (neutrophils recruitment) and cytokines production, by ELISA. Results: Vinpocetine at 30 mg/kg reduced acetic acid-induced MPO activity, and thereby IL-33, TNF-α, and IL-β production. In addition, vinpocetine restored total antioxidant capacity and GSH levels. Conclusion: Vinpocetine is a promising molecule for the treatment of IBD.

Keywords: Colitis; Neutrophils; Vinpocetine Financial support: Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secretaria da Ciência, Tecnologia e Inovação (SETI), Fundação Araucária, and Paraná State Government. Thematic area: Non-transmitted diseases

103

SYSTEMIC CHANGES IN THE REDOX STATUS IN A MURINE MODEL OF UVB-INDUCED NONMELANOMA SKIN CANCER

Iriana Moratto Carrara1; Gabriella Pasqual Melo1; Walison Augusto da Silva Brito1; Fernando Pinheiro Souza-Neto1; Sara Santos Bernardes1; Poliana Camila Marinello1; Rodrigo Cabral Luiz1; Leandra Náira Zambelli Ramalho3, Rubens Cecchini2, Alessandra Lourenço Cecchini1 1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, BR. 2 Laboratory of Pathophysiology and Free Radicals, State University of Londrina, Londrina, PR, BR. 3 Department of Pathology and Forensic Medicine, University of São Paulo, Ribeirão Preto, SP, BR.

Introduction: Nonmelanoma skin cancer (NMSC) is the most common type of cancer in white populations. Although it is known that oxidative stress (OS) is related to UVB-induced skin photocarcinogenesis, the systemic redox status in NMSC is poorly described. Objective: To characterize the systemic and local redox status in a UVB-induced NMSC model, using female hairless mice. Materials and Methods: Mice (20-25g) were divided in two groups: control (C; non- irradiated; n=8) and NMSC (irradiated animals; n=15) (CEUA/UEL: Process n. 6738.2015.40). Animals were exposed to 0.228 mJ/cm2 of UVB during 16 minutes, five days a week, for 15 weeks. After this period, blood was collected by cardiac puncture to assess systemic OS and animals were euthanized. A piece of dorsal skin was removed for subsequent histopathological and imumunohistochemical analysis (p53, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT)). The results were analyzed by Student’s t test with Tukey post-hoc test or Mann-Whitney U test. p<0.05 was considered significant. Results: The histopathological analysis demonstrated that the animals developed actinic keratosis, in situ carcinoma and squamous cell carcinoma (SCC). The NMSC group showed increased 3-NT, 4-HNE and p53 in skin lesions when compared to control. It was also observed a significant increase in OS index (due to consumption of reduced glutathione and increased levels of oxidized glutathione), in the advanced oxidation protein products and a reduction in the total antioxidant capacity and malondialdehyde levels in the NMSC group. No significant differences were observed in total thiol levels and catalase activity. Conclusion: The analysis of the results indicates that the process of UVB-induced photocarcinogenesis is accompanied with both local and systemic OS. With this knowledge, a better therapeutics and a chemoprevention could be investigated.

Keywords: Nonmelanoma skin cancer; Oxidative stress; Systemic biomarkers. Thematic area: Non-transmitted diseases.

104

CLINICAL-EPIDEMIOLOGICAL CHARACTERIZATION AND EVALUATION OF ERYTHROCYTIC ANTIOXIDANT DEFENSES OF PATIENTS AFFECTED BY ACTINIC KERATOSIS AND NONMELANOMA SKIN CANCER IN THE NORTHERN REGION OF PARANA.

Walison Augusto da Silva Brito1; Poliana Camila Marinello1; Mariani Lima Garcia1; Mateus Henrique Cunha da Silva1; Leonardo Bodner Freitas1; Natália Medeiros Dias Lopes1; Vanessa Gheno2; Théo Nicolacópulos2; Priscila Daiane Pavezzi2; Fernanda Teixeira Ortega2; Mariana Onuki Okamura2; André Armani3; Airton dos Santos Gon2; Rubens Cecchini4; Alessandra Lourenço Cecchini1. 1 Laboratory of Molecular Pathology, State University of Londrina, Londrina, PR, BR. 2 Department of Internal Medicine, State University of Londrina, Londrina, PR, BR. 3 Department of Surgical Clinic, State University of Londrina, Londrina, PR, BR. 4 Laboratory of Physiopathology and Free Radicals, State University of Londrina, Londrina, PR, BR.

Introduction: Taking into account the elevated incidence of nonmelanoma skin cancer (NMSC), the identification of factors related with its development is essential to enable effective prevention. Objectives: To evaluate erythrocytic antioxidant defenses and to characterize clinical and epidemiologically patients with NMSC and actinic keratosis (AK) in hospitals from the northern region of Parana. Material and Methods: Participants were categorized in 4 groups: Control (without skin cancer history; n=38); Actinic keratosis (AK; n= 8); Basal cell carcinoma (BCC; n=50) and squamous cell carcinoma (SCC; n= 9) (CEP-UEL process n. 1.077.557). Blood samples were collected and erythrocytic catalase activity, oxidized and reduced glutathione levels (GSSG and GSH, respectively) were determined. The clinical-epidemiological characterization was performed by applying a questionnaire to participants. The statistical analysis was performed using one-way ANOVA or Kruskal-Wallis (quantitative results) and Chi-square test or Fisher’s exact test (qualitative results); p<0.05 was considered significant. Results: Patients from BCC presented reduced levels of GSH when compared to control, which were not observed in AK and SCC. Catalase activity and GSSG did not change. The patients presented the same age and gender distribution and no differences about the presence of relapses and comorbidities were observed. It was noted that most patients from BCC and AK fit in the category of light skin (type I/II in Fitzpatrick’s Classification), most of the lesions were found in head and neck regions in BCC patients and in upper limb in SCC and AK patients. BCC patients presented the higher occupational UV exposure. Obesity was more common in AK. Conclusion: The results indicates that BCC patients presents lower levels of erythrocytic antioxidant defenses than the other groups, observed by reduced GSH levels. The clinical- epidemiological characterization of the patients suggests that some NMSC risk factors could be differently involved in the pathophysiology of BCC, SCC and AK.

Keywords: Glutathione system; Catalase activity; Risk factors. Thematic area: Non-transmitted diseases.

105

EFFECTS OF SLEEP RESTRICITON DURING PERIPUBERTY ON TESTICULAR DEVELOPMENT OF RATS

Gláucia Eloisa Munhoz de Lion Siervo1,2, Fernanda Mithie Ogo1,2, Larissa Staurengo-Ferrari2, Tathiana Aparecida Alvarenga3, Rubens Cecchini2, Waldiceu Aparecido Verri Jr.2, Flávia Alessandra Guarnier2, Monica Levy Andersen3, Glaura Scantamburlo Alves Fernandes1 1Department of General Biology, Biological Sciences Center, State University of Londrina – UEL, Londrina, Paraná, Brazil 2Department of Pathological Sciences, Biological Sciences Center, State University of Londrina – UEL, Londrina, Paraná, Brazil 3Departamento de Psicobiologia, Universidade Federal de São Paulo – UNIFESP, São Paulo, SP, Brazil

Introduction: Puberty represents a period of complex sexual development, which leads to sexual maturation and reaching reproductive capability. Sleep alterations affect the neuroendocrine reproductive control and redox balance. Objective: The aim of this study is to evaluate whether sleep restriction during the peripubertal period could impair testicular postnatal development in rats. Material and methods: (CEUA/UEL protocol number 3467.2014.86). Thirty male Wistar rats (postnatal day - PND 40) were used. The Sleep Restriction (SR) group was exposed to 21 days of sleep restriction by the modified multiple-platform method (18h of SR and 6h of sleep, per day). The Control group (C) was maintained in their home-cages during all the experiment. At PND 62, the rats were weighed, anesthetized and euthanized. Testis was removed, weighted and used to sperm count, oxidative stress markers, histological and morphometric parameters as well as inflammatory profile evaluation. Spermatozoa from vas deferens were used to sperm morphology and motility. Results: The final body weight of SR animals was decreased as well as absolute weight of vas deferens. The testicular absolute weight was similar among the groups, while the relative testicular weight was increased in SR group. There is an increase in lipid peroxidation in SR animals, while the total antioxidant capacity remains unchanged. The seminiferous epithelium height was decreased after SR period, and the tubular seminiferous diameter did not change. The Sertoli cells number were decreased in SR group. The neurophil and macrophages number in testis were similar between the groups. The percentage of mobile spermatozoa decreased in the SR group. The sperm number in testis and the daily sperm production were not altered. The histopathological analysis, spermatogenesis kinetics and sperm morphology were not affected by SR period. Conclusion: SR during peripuberty affects testicular postnatal development and sperm motility, with a crucial role of oxidative stress.

Keywords: Sleep loss; Puberty; Postnatal development. Thematic area: Non-transmitted diseases

106

INFLAMMATION AND OXIDATIVE STRESS IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS WITH METABOLIC SYNDROME – A REVIEW

Tatiana Mayumi Veiga Iriyoda1; Marcell Alysson Batisti Lozovoy1, Isaías Dichi1, Andréa Name Colado Simão1.

1State University of Londrina, Londrina, Parana - Brazil.

Introduction: Metabolic syndrome (MetS) comprises pathological conditions that include insulin resistance (IR), visceral obesity, arterial hypertension and dyslipidemia, which favors the development of cardiovascular (CV) diseases. Patients with systemic lupus erythematosus (SLE) have a high prevalence of MetS and this syndrome may constitute a common link between the increased CV risk and higher levels of inflammation. Inflammation and oxidative stress (OS) have been associated with IR and MetS. Objective: To review the role of inflammation and OS in SLE patients with MetS. Methods: Original and review articles were searched on Pubmed using the keywords beneath. Results: SLE is associated with higher circulating levels of high sensitivity C- reactive protein (CRP), interleukin-18 and tumour necrosis factor alfa (TNF-α) leading to clinical and biochemical manifestations of the MetS. Inflammatory cytokine TNF-α can induce IR and suppression of Glut4 expression. IL-6 also inhibits insulin signal transduction in hepatocytes. Lower concentrations of adiponectin, with pro-inflammatory and atherogenic effects and higher concentrations of leptin, with anti-inflammatory and anti-atherogenic effects, were associated with IR and CRP in patients with SLE, with controversy related to disease activity. Circulating levels of markers of endothelial activation and injury such as CRP, homocysteine, and thrombomodulin have been significantly higher in SLE patients with MetS and have been implicated in the development of CV diseases. Inflammatory processes may favor OS increases in patients with SLE and MetS. SLE patients with IR show higher advanced oxidation protein products, lower sulfhydryl groups of proteins, lower serum 8-isoprostanes and carbonyl protein levels and lower total radical-trapping antioxidant parameter when compared to SLE patients without IR. Conclusion: The literature shows that systemic chronic inflammation and OS have a prominent role in the pathogenesis of MetS and this syndrome directly contributes to further increase the inflammatory status and OS of SLE patients.

Keywords: Inflammation; Metabolic syndrome; Systemic lupus erythematosus Thematic area: Non-transmitted diseases

107

LEPROSY IN PRUDENTÓPOLIS: THE PORTRAIT OF ONE OF PARANÁ'S INLAND CITIES

Jéssica Loraine Drugik1; Marcio Lupepsiw2; Adriane Lenhard-Vidal1

1 Faculdade Campo Real, Rua Comendador Norberto, 1299, Bairro Santa Cruz, 85015-240, Guarapuava, Paraná, Brasil. 2 Setor de Hanseníase e Tuberculose, Secretaria de Saúde, Rui Barbosa 1780, Centro, 84400- 000, Prudentópolis, Paraná, Brasil 3 Laboratório de Imunologia Aplicada, Depto de Ciências Patológicas/CCB, Universidade Estadual de Londrina, Rodovia Celso Garcia Cida (PR-445), km 380, 86057-970 Londrina, PR, Brasil.

Introduction: Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae, that primarily affects the skin and peripheral nerves. Currently, Brazil is the second country in the world in absolute numbers of leprosy cases. Objective: to establish the epidemiological profile of patients with leprosy in a small city of Paraná, Brazil. Material and methods: Current research was approved by the Internal Scientific Commission and the Research Bioethics Committee of State University of the Central West (Guarapuava – PR), under n. 740.635 from August 5th 2014 (CAEE 34246514.5.0000.010). This is a retrospective epidemiological study performed in Prudentópolis, PR. Data were collected from the investigation forms and the notifications of the Notifiable Diseases Information System (SINAN), from patients registered from January 2006 to December 2014. Data gathered were age, sex, residence in rural or urban area, entry mode, bacilloscopy, number of skin lesions, clinical presentation, operational classification, therapeutic regimen and degree of disability. The prevalence coefficient was calculated by dividing the number of registered cases in each year by the mean of the city population (49.000 inhabitants). Analysis was performed in Microsoft Excel. Results: In 103 registries (11.4 cases/year), there was a predominance of cases in the rural area (54%), of male patients (68%), with age ranging from 46 to 60 years (34%), of multibacillary patients (88%), with leprosy, lepromatous (86%), that were treated with 12 doses-polychemotherapy. The mean prevalence rate was 2.63 cases/10,000 inhabitants, which is still above the recommended by the World Health Organization. Conclusion: Prudentópolis remains an endemic area for leprosy, requiring even further attention for its control.

Keywords: Epidemiological monitoring, Hansen’s disease, Mycobacterium leprae Grants: no funding was received during this research. Thematic area: Transmitted diseases

108

P2 ALLELE OF PVUII GENETIC POLYMORPHISM OF LOW-DENSITY LIPOPROTEIN RECEPTOR IS ASSOCIATED WITH INCREASED LIPID PEROXIDATION AND LIVER INJURY IN CHRONIC HEPATITIS C PATIENTS

Lucas S Liberati1, Camila Cataldi de Alcantara2, Jorge PS de Almeida1, Elaine RD de Almeida3, Ana P Kallaur1, Isaias Dichi4, Andrea NC Simão3, Edna M V Reiche3

1Postgraduate Program of Health Sciences, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 2Postgraduate Program of Clinical Analysis, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 3Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 4Department of Internal Medicine, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil.

Introduction: The HCV infects cells through interactions between its proteins with host cellular receptors. When the virus is associated with apolipoproteins is called lipoviroparticles (LVPs) which interacts with the low-density lipoprotein receptor (LDLR) expressed of hepatocytes. Objective: The aim of this study was to evaluate the influence of PvuII intron 15 low-density lipoprotein receptor (LDLR) polymorphism on liver injury, oxidative stress, and hepatitis C virus (HCV) core antigen in individuals chronically infected with HCV. Methods: Forty-six consecutive untreated chronic HCV patients and 87 healthy controls were evaluated with biochemical markers, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (ɣGT), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), triglyceride, uric acid, ferritin, iron, AST-to-platelet index (APRI) and with the viral maker HCV core antigen. Oxidative stress markers were also evaluated including tert-butyl hydroperoxide-initiated chemiluminescence (CL-LOOH), advanced oxidation protein products (AOPP), oxidative stress index (OSI), and total radical-trapping antioxidant parameter (TRAP). The PvuII LDLR polymorphism was genotyped using PCR-RFLP. The patients were divided in two groups, carriers and non-carriers of the P2 allele. Results: The patients showed higher AST, ALT, ɣGT, ferritin, iron, hydroperoxide, and OSI, and lower TC, LDL-C (p <0.0001), triglycerides (p = 0.0395), and TRAP (p = 0.0302) than controls. Patients carrying the P2 allele exhibited higher AST (p=0.0438), ALT (p=0.0271), hydroperoxide (p=0.0398), and APRI (p=0.0265), and lower HCV core antigen (p=0.0235) that those with the P1P1 genotype. Conclusions: The P2 allele of PvuII intron 15 LDLR polymorphism was associated with increased lipid peroxidation and liver injury, as well as with decreased HCV core antigen, suggesting that this genetic variant may influence viral replication and oxidative stress, and therefore, could contribute to liver injury in chronically HCV-infected patients.

Keywords: genetic polymorphism; hepatitis C virus; low-density lipoprotein receptor (LDLR); Grants: The study was supported by grants from Coordination for the Improvement of Higher Level of Education Personnel (CAPES) of Brazilian Ministry of Education; Institutional Program for Scientific Initiation Scholarship (PIBIC) of the National Council for Scientific and Technological Development (CNPq); and State University of Londrina (PROPPG). We thank the University Hospital of State University of Londrina for technical supports. Thematic area: Transmitted diseases

109

THE PROFILE OF OXIDATIVE STRESS MARKERS IS DEPENDENT ON VITAMIN D LEVELS IN PATIENTS WITH CHRONIC HEPATITIS C

Jorge P Sales de Almeida1, M.Sc.; Camila Cataldi de Alcantara2; Lucas Silva Liberatti1, Marcell A Batisti Lozovoy, Ph.D3; Bruna Miglioranza Scavuzzi1, Edna Maria V Reiche3; Isaias Dichi, MD4, PhD; Andréa N Simão3 1Postgraduate Program of Health Sciences, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 2Postgraduate Program in Clinical Analysis, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 3Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil. 4Department of Internal Medicine, Health Sciences Center, University of Londrina, Av. Robert Koch, 60, CEP 86.038-440, Londrina, Paraná, Brazil.

Introduction: Although vitamin D deficiency can change liver injury progression in Hepatitis C Virus (HCV) patients, the main molecular mechanisms involved are largely unknown. Objective: The first aim of this study was to evaluate the association between oxidative stress and hypovitaminosis D in patients with HCV. A second objective was to verify if oxidative stress is involved in the molecular mechanisms related to liver injury. Matherial and Methods: The study included 147 subjects: 89 controls and 58 HCV patients (Vit D <30, n=32 and vit D >30, n=26). Results: Patients with HCV and hypovitaminosis D presented significantly higher aminotransferase-to-platelet ratio index (APRI) (p=0.0464) and viral load (p=0.0426) compared to the patients with HCV without hypovitaminosis D. Regarding oxidative stress, HCV patients with hypovitaminosis D had higher advanced oxidation protein products (AOPP) (p=0.0409), nitric oxide metabolites (p=0.0206) levels and oxidative stress index (OSI) (p=0.0196), whereas total radical-trapping antioxidant parameter (TRAP) (p=0.0446) levels were significantly lower than HCV patients without hypovitaminosis D. Vitamin D in HCV patients showed inverse correlations with levels of iron (r= -0.407, p= 0.0285), ferritin (r= -0.383, p=0.0444), APRI (r= -0.762, p= 0.0280) and glutamyl transpeptidase (r= -0.453, p=0.0154) and also with plasma lipid hydroperoxides levels (r= -0.426, p= 0.0189). The register number of the ethical approval is CEP/UEL 035/2012, CAAE: 01116612.0.0000.5231, on June 12, 2012. Conclusion: Vitamin D insufficiency contributes to the inflammatory process and oxidative stress imbalance in patients with HCV. The profile of oxidative stress markers in HCV patients depends on vitamin D levels, which probably change intracellular signaling pathways and increase inflammation and liver injury.

Keywords: hepatitis C virus; vitamin D insufficiency; inflammation; liver injury markers; iron metabolism Grants: The study was supported by grants from Coordination for the Improvement of Higher Level of Education Personnel (CAPES) of Brazilian Ministry of Education; Institutional Program for Scientific Initiation Scholarship (PIBIC) of the National Council for Scientific and Technological Development (CNPq); and State University of Londrina (PROPPG). We thank the University Hospital of State University of Londrina for technical supports. Thematic area: Transmitted Diseases.

110

POLYMORPHISM rs3087465 IN THE TGFBR2 GENE IS ASSOCIATED WITH HUMAN PAPILLOMAVIRUS INFECTION

É rica Romão Pereira¹; Guilherme Cesar Martelossi Cebinelli¹; Kleber Paiva Trugilo¹; Stephanie Badaró Garcia¹; Nathália Tatakihara¹; Nádia Calvo Martins Okuyama¹; Michelle Mota Sena¹; Ana Paula Lombardi Pereira¹; Rodolfo Sanches Ferreira¹; Gabriela Cristine Queiroz Maria¹; Fernando Cezar dos Santos¹; Adriano Martin Felis Aranome¹; Fernanda Costa Brandão Berti¹; Luis Fernando Lasaro Mangieri¹; and Karen Brajão de Oliveira¹. ¹Immunology and Molecular Genetics Laboratory, Pathological Sciences Department, Biologic Science Center, Londrina State University, Paraná, Brazil.

Introduction: Cervical cancer is the third most common type of cancer among Brazilian women, caused mainly by persistent HPV infection. Many cytokines are produced in this microenvironment playing several roles in lesions development and persistent virus infection. As a multifunctional cytokine, the transforming growth factor-β1 (TGFB1) regulates a number of important cellular responses, participating in the cell differentiation, apoptosis, cell migration, immune responses, angiogenesis and extracellular matrix formation. TGFB1 action is dependent on binding with the transforming growth factor-β receptor 2 (TGFBR2). Genetic variations such as single nucleotide polymorphisms (SNP) has been associated with expression and production of this receptor. Objectives: Investigate the influence of TGFBR2 SNP (rs3087465) and epidemiological data in HPV infection. Material and methods: The evaluation of polymorphism and HPV detection were performed by polymerase chain reaction (PCR), and epidemiological data obtained through a structured questionnaire. Results: This case-control study comprised 347 patients, 189 (54.5%) were included in non-detected HPV group (control), and 158 (45.5%) were infected (case group). Epidemiological analysis showed that age (p=0.017) and tobacco usage (p=0.019) were independently associated with HPV infection. Patients younger than 25 years old had more chance to HPV infection compared to older groups, and tobacco usage increased 1.93-fold the chances of HPV infection (CI95% 1.07–3.48). Binominal logistic regression model using age and tobacco status as confounding factors demonstrated that the c.-875GG genotype was associated with HPV infection and GG women were more likely to have HPV than the ones presenting c.-875AA genotype (OR=2.24, CI95% 1.09–4.96; p=0.043). Additionally, c.-875GG genotype has been suggested to promote lower gene expression than c.-874AA genotype. Thus, a reduction in TGFB1 signaling favors the HPV persistence by contributing to viral genome maintenance. Conclusion: Tobacco usage, age younger than 25 years and the presence of -875GG genotype are risk factors to HPV infection.

Keywords: Cervical cancer, TGFBR2, HPV, epidemiological data. Thematic area: Transmitted diseases.

111

SYPHILIS: MOTHER TO SON

Gabriel Zanotto dos Santos1; Luana Aparecida Cossentini2; Marcia Regina Terra3

1Graduate student Nurse of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 2Graduated in Biomedicine; Master in Experimental Pathology; PhD student in Experimental Pathology at the Regional University Hospital of Londrina; Teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected]. 3Master in Microbiology from the University of Londrina – UEL. Teacher of INESUL - Instituto de Ensino Superior de Londrina. E-mail: [email protected].

Introduction: Congenital syphilis, it has occupied a prominent place in the world, despite being a preventable disease; particularly in developing countries, its incidence is increasing alarmingly. This pathological process is the result of infection by T. pallidum in the blood of pregnant women coming in contact with the fetus through the placenta, so-called vertical transmission. Congenital syphilis is a major cause of perinatal morbidity and mortality. The vertical transmission of syphilis remains a major public health problem in Brazil as well. From 2001 to 2010, were confirmed 132 cases of congenital syphilis in metropolitan Londrina, number considered small in relation to the Curitiba region with 507 confirmed cases of infection with congenital pox. Objective: To understand how does the congenital syphilis, as well as observing the high rate of global incidence and location of this disease that can be devastating. Designating preventive methods for congenital cases. Methods: This study is a systematic review of the scientific literature carried out from the databases PubMed, LILACS and Scielo. Studies selected Were Conducted in Brazil, published in Portuguese, Spanish or English and indexed in the databases mentioned from 2009 to 2015. Conclusion: The alarming data underscore the negligence by the treatment. Once treated, no spread of the parasite and therefore transplacental infection from mother to fetus. The biggest cause of many cases is related mainly to the lack of information and irresponsibility on the part of individuals sexually active.

Keywords: Pathogenesis; incidence; transmission. Thematic area: Transmitted diseases

112

THE INFLUENCE OF TGFB1 POLYMORPHISM RS1800469 (C.-1347C>T) IN THE HPV INFECTION AND CERVICAL LESIONS IMMUNOPATHOGENESIS

Guilherme Cesar Martelossi Cebinelli¹; Kleber Paiva Trugilo¹; Stephanie Badaró Garcia¹; Nádia Calvo Martins Okuyama¹; Michelle Mota Sena¹; É rica Romão Pereira¹; Ana Paula Lombardi¹; Rodolfo Sanches Ferreira¹; Gabriela Cristine Queiroz Maria¹; Adriano Martin Felis Aranome¹; Fernando Cezar dos Santos¹; Fernanda Costa Brandão Berti¹; Luis Fernando Lasaro Mangieri¹; Glauco Akelinghton Freire Vitiello²; Maria Angelica Ehara Watanabe²; and Karen Brajão de Oliveira¹.

¹Immunology and Molecular Genetics Laboratory, Pathological Sciences Department, Biologic Science Center, Londrina State University, Paraná, Brazil. ²Laboratório de Estudos Aplicados a Polimorfismos de DNA, Pathological Sciences Department, Biologic Science Center, Londrina State University, Paraná, Brazil.

Introduction: Cervical cancer is the third most common type of cancer among Brazilian women, it is associated with an infection by the Human Papillomavirus (HPV). The pathogenesis comprises low-grade intraepithelial lesions (LIEBG), which are usually eliminated by the immune system or can progress to high grade intraepithelial lesions (HSIL) and cervical cancer. The immune response plays a role in the resolution of the infection and their suppression is correlated with viral persistence. In this context, immunosuppressive cytokines such as TGFBI are important to the viral infection susceptibility and cervical lesions progression. Genetic variations, such as single nucleotide polymorphisms (SNP), can lead differences in TGFB1 expression and production, and could be a risk factors for the immunopathogenesis of HPV infection. Objectives: Thus, this study aimed to evaluate the influence of TGFB1 polymorphism rs1800469 (c.-1347C>T) in viral infection and cervical lesions, and to identify risk factors related to epidemiological data. Material and methods: This case-control study included 424 patients that was evaluated TGBFB1 polymorphism and HPV detection by polymerase chain reaction (PCR), epidemiological data through a questionnaire, and clinical data through cytopathology result. Results: Patients younger than 25 had independently more chance to HPV infection (p=0.001), and monthly income less than 1 minimum wage (p=0.047), early first sexual intercourse (p=0.012), smoking (p=0.011), and more partners (p=0.001), contributing to increase the risk of positivity to this group. Already, the large number of pregnancies (p=0.019), monthly income less than 1 minimum wage (p=0.032), and hormonal contraceptives use (p=0.008) are risk factors independently associated with cervical lesions development in HPV positive patients. Regarding the influence of the polymorphism c.-1347C>T of TGFB1, the c.-1347TT genotype is associated independently to the infection (p=0.030) and could be associated with viral maintenance, for contributing to the HPV virulence factors. However, no association was found for the lesions development (p=0.464). Conclusion: Thus, in order to ensure the effect of this polymorphism in infection and maintenance of HPV is necessary haplotype analysis to certify the independently function of this SNP in relation to other polymorphisms and perform quantitative studies to observe the effect of the influence of alleles levels TGFB1 of the studied sample groups.

Keywords: TGFB1 polymorphism; Cervical lesion; HPV infection. Thematic area: Transmitted diseases.

113

TGFB1 SIGNAL PEPTIDE POLYMORPHISMS INFLUENCE HPV INFECTION AND CERVICAL LESIONS DEVELOPMENT

Kleber Paiva Trugilo1; Guilherme Cesar Martelossi Cebinelli1, Fernanda Costa Brandão Berti1, Nadia Calvo Martins Okuyama1, Fernando Cezar dos Santos1, Nathália Tatakihara1, Ana Paula Pereira Lombardi1, Érica Romão Pereira1, Michelle Mota Sena1, Adriano Martin Felis Aranome1, Rodolfo Sanches Ferreira1, Maria Angelica Ehara Watanabe1, Karen Brajão de Oliveira1 1Londrina State University, Rodovia Celso Garcia Cid, Pr 445 Km 380, Campus Universitário, CEP 86057-970, Londrina – PR. Introduction: Persistent Human Papillomavirus (HPV) infection is closely associated with the development of high-grade squamous intraepithelial lesion (HSIL). However, HPV alone is not sufficient for malignant progression. Exogenous and endogenous factors may influence the risk of cervical cancer. Transforming Growth Factor β (TGFB), a cytokine that regulates cell growth, maturation, and differentiation, is important in the progression of HPV infection to HSIL. Additionally, single nucleotide polymorphisms (SNPs) in the TGFB1 gene may alter the protein production and influence disease. Objective: To assess the effect of c.29C>T and c.74G>C polymorphisms in the TGFB1 signal peptide on HPV infection and cervical lesions development. Material and methods: This case-control study was approved by Ethics Committee Involving Humans (CEP/UEL 133/2012; CAAE 05505912.0.0000.5231). Cervical swabs and blood samples were obtained from 349 outpatient women, along with socio-demographic and sexual behavioral data. The study population was stratified by lesion grade, as well as by absence or presence of HPV DNA, as tested by PCR. TGFB1 signal peptide polymorphisms were genotyped by PCR-restriction fragment length polymorphism. Results: HPV DNA was detected in 172 (49.3%) patients. c.74G>C and the combined c.29CC+CT/c.74GC genotype were more frequent in infected patients (35.1% and 15.7%) than in uninfected women (6.2% and 14.7%). Accordingly, using adjusted analysis, these genotypes were associated with higher risk of HPV infection, with odds ratio and 95% confidence interval 2.81 and 1.35-5.86 (p=0.004) for c.74G>C and 3.14 and 1.42-6.94 (p=0.004) for the combined genotype. High-grade lesions were also 2.48 times more likely to occur in c.29CC patients than in c.29TT patients, with 95% confidence interval 1.01-6.08 (p=0.047). Conclusion: Although further studies are necessary, the data demonstrate that c.74G>C and c.29C>T polymorphisms are significantly associated with risk of HPV infection and high-grade squamous intraepithelial lesions, respectively. Thus, the results suggest the TGFB1 signal peptide polymorphisms as potential susceptibility markers.

Keywords: High-grade squamous intraepithelial lesion; Combined genotype; Susceptibility marker. Grants: Capes, PPSUS, CNPq, Fundação Araucária Thematic area: Transmitted diseases.

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INTESTINAL INFLAMMATION GENERATED BY TOXOPLASMOSIS PROMOTE LOSS NEURONAL AND GLIAL POPULATION OF ILEUM SUBMUCOSAL PLEXUS OF RATS Wistar

Larissa Carla Lauer Schneider1; Stephanie Carvalho Borges1; Evandro José Beraldi1; Paulo da Silva Watanabe2; Débora de Mello Sant’Ana1; Nilza Cristina Buttow1 1 State University of Maringa /UEM 2 State University of Londrina/UEL

Introduction: The affinity of the Toxoplasma gondii for nervous tissues and the prevalence of diarrhea in infected patients demonstrate that the ENS is a target of parasite. Objective: The aim of this study was to evaluate the effects of acute and chronic T. gondii infection on neurons and glial cells of the submucosal plexus. Material and methods: 20 male Wistar rats (protocol n. 5529220915/2015) were randomly distributed into 4 groups (n = 5): uninfected (CG) and infected for 24 hours (IG24h), 72 hours (IG72h) and 30 days (IG30d) after inoculation with 5000 sporulated oocysts of T. gondii (Me-49 strain, genotype II). Ileum segments were used to quantify the levels of lipid hydroperoxidation (LOOH), reduced glutathione (GSH) and nitrite (NO2-), and the enzymatic activity of superoxide dismutase (SOD), glutathione S-transferase (GST) and myeloperoxidase (MPO). Immunofluorescence was also performed for marking HuC/D, nNOS, CALR and VIP submucosal neurons, and S100 glial cells. Results: Neuronal loss of the HuC/D was gradual up to 30 days of infection. Glial cells proliferated in the acute phase, but reduced in the chronic phase. nNOS-IR neuronal density increased at acute phase, returning to control values in the chronic phase. CALR-IR neuronal density increased after 72 hours, remaining high up to 30 days of infection. VIP- IR neuronal density in the acute phase increased in IG24h and decreased in IG72h group, while in the chronic phase values were similar to controls. LOOHs and GSH levels did not change, but NO2- levels reduced after 24 hours and remained low up to 30 days of infection. The enzymatic activity of SOD and GST were similar to control, but MPO showed higher levels in GI30d group. Conclusion: The changes of submucosal neurons were not due to generation of oxidative stress but can be explained by the inflammation caused by T. gondii.

Keywords: Toxoplasma gondii; Enteric Nervous System; Oxidative stress Grants: CAPES Thematic area: Transmitted diseases

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COINFECTION BETWEEN HPV AND Chlamydia trachomatis AND ITS ROLE IN CERVICAL LESIONS DEVELOPMENT

Michelle Mota Sena1; Érica Romão Pereira1; Guilherme Cesar Martelossi Cebinelli1; Ana Paula Lombardi Pereira1; Nathalia Tatakihara1; Rodolfo Sanches Ferreira1; Gabriela Cristine Queiroz Maria1; Nadia Calvo Martins Okuyama1; Adriano Martin Felis Aranome1; Fernanda Costa Brandão Berti1; Kleber Paiva Trugilo1; Fernando Cezar dos Santos1; Luis Fernando Lasaro Mangieri1; Karen Brajão de Oliveira1. 1 State University of Londrina, Celso Garcia Cid Highway, PR 445 km 380, Campus, Londrina-PR

Introduction: Human Papillomavirus (HPV), the main etiologic agent of cervical cancer is transmitted by sexual contact and is responsible for about 270,000 deaths around the world, while Chlamydia trachomatis is the most common bacterial sexually transmitted disease attaining more than 100 million new cases annually. It has been suggested that the infection by C. trachomatis increased risk of acquiring HPV, promotes the persistency of carcinogenic types, as well as difficults preexisting virus removal. Objective: Analyze the presence of C. trachomatis in female patients with positive molecular diagnosis for HPV attended by cervical cancer prevention programs of public health sector of northern of Paraná. Material and Methods: This study was approved by the Ethics Committee for Research Involving Human Beings of State University of Londrina, CEP/UEL 133/2012. Cervical secretion samples from 204 women who agreed to participate voluntarily by signing the Instrument of Consent were obtained. Genomic DNA was extracted with subsequent molecular detection of HPV and C. trachomatis. Statistical analysis included the Odds Ratio calculation for sociodemographic variables and sexual behavior, using the SPSS software 22.0, considering p < 0.05 as statistically significant. It was also performed binary logistic regression to determine the influence of confounding factors in HPV+ patients lesions development. Results: It was observed association for coinfection between HPV and C. trachomatis (OR: 3.16 [95% CI: 1.081 – 9.214]) and that C. trachomatis infection was dependently associated with the contraceptive use and viral types in the lesions development. Conclusion: Our data demonstrated a positive association for the coinfection and that bacteria itself does not interfere in the lesions development. However, further analysis with a higher number of samples are necessary to elucidate the possible way in which these microorganisms act together in the cervical microenvironment.

Keywords: Sexual transmitted disease; Molecular diagnosis; Cervical cancer. Grants: CNPq; Fundação Araucária; PPSUS Thematic area: Transmitted diseases

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ANTIBODIES IgY ANTI-NP OF THE VIRUS H1N1 FOR DEVELOPMENT OF DIAGNOSTIC TESTS FOR THE DETECTION OF THE INFLUENZA VIRUS

Miriele Caroline da Silva1; Kamila Falchetti Damasco2; Márcio Scarone1; Paola Singi1; Aline Miquelin Nascimento1; Rejane Schaefer3; Emerson José Venancio1 1Departament of Pathological Science, Biological Science Center, State University of Londrina, Km 380, Celso Garcia Cid Road, Londrina, PR, Brazil 2University of Northern Paraná, Av Paris, 675, Jardim Piza, Londrina, PR, Brazil 3Animal Health/Virology Swine, Brazilian Company of Agricultural Research, Swine and Poultry Research National Center, BR153, Km 110, Vila Tamanduá, Concórdia, SC, Brazil

Introduction: Influenza is a zoonotic viral disease that represent a threat to health. The H1N1 subtype is swine specific, but can be found in humans and other animals. Swine influenza is a infection with low mortality, but has a high morbidity rate. The nucleoprotein (NP) of influenza virus has been used in rapid diagnostic detection kits. These tests are based on the production of polyclonal antibodies in mammals. However, due to concerns related to animal welfare, alternative production of antibodies in mammals has been investigated. The IgY is an antibody present in birds, are easily obtained from egg yolk of chickens. It has been studied for immunotherapy and immunodiagnosis research. Objective: To obtain anti-NP IgY antibodies from laying hens. Material and methods: The project was approved by CEUA/UEL (11976/2012.14). The NP protein was provided by EMBRAPA Swine and Poultry. Three chickens, of White Leghorn line, were immunized with 20 ug of NP protein. Booster doses were administered on days 14, 28, 42, 84, 126 and 168 of the experiment. Eggs were collected after 7 days of the seventh immunization. The IgY antibodies in the yolks were extracted by precipitation with ammonium sulfate. The levels of antibodies were determined by ELISA. The specificity of anti-NP IgY was verified by Western blotting. Results: Animals immunized with NP protein produced high levels of specific antibodies after the seventh immunization, whereas animals were not immunized showed low levels of anti-NP antibodies. Furthermore, it was observed by Western blotting that the antibodies obtained from the immunized chickens recognize specifically the NP protein. Conclusion: The results show the obtainment of specific antibodies IgY to the NP protein of H1N1 influenza virus. These antibodies will be used for the development of diagnostic tests for the detection of influenza virus.

Keywords: Influenza; Laying hens; IgY. Thematic area: Transmitted diseases

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INFLUENCE OF CXCL12 RS1801157 POLYMORPHISM IN HPV INFECTION AND LESION DEVELOPMENT

Nádia Calvo Martins Okuyama¹; Nathália Tatakihara1; Guilherme Cesar Martelossi Cebinelli1; Kleber Paiva Trugilo1; Michelle Mota Sena¹; É rica Romão Pereira¹; Ana Paula Lombardi Pereira¹; Stephanie Badaró Garcia1; Fernando Cezar dos Santos¹; Rodolfo Sanches Ferreira¹; Gabriela Cristine Queiroz Maria¹; Adriano Martin Felis Aranome¹; Fernanda Costa Brandão Berti¹; Luis Fernando Lasaro Mangieri¹ and Karen Brajão de Oliveira¹. ¹Immunology and Molecular Genetics Laboratory, Pathological Sciences Department, Biologic Science Center, Londrina State University, Paraná, Brazil.

Introduction: Cervical carcinoma is considered an important public health issue. It is the third most common type of cancer in Brazilian women and the fourth worldwide. The disease is strongly associated to Human Papillomavirus (HPV) which is present in 99.7% of the cervical cancer cases and it is classified according to its carcinogenic potential in high-risk, undetermined risk and low-risk. Evidences suggest that chemokines are important regulators in development of viral infections and immune responses and genetic variations such as single nucleotide polymorphisms (SNP) may promote alterations in this molecules expression and production. Objectives: The aim of the present study was to evaluate the role of CXCL12 polymorphism rs1801157 in HPV infection and lesions development. Material and methods: This case-control study was approved by the Institutional Ethics Committee Involving Humans at State University of Londrina, Londrina – PR, Brazil (CEP/UEL 133/2012; CAAE 05505912.0.0000.5231). Genomic DNA was obtained from cervical cytobrushes and peripheral blood, from 360 women, for detection of HPV and polymorphism analysis respectively, by polymerase chain reaction (PCR). Epidemiological data was obtained through a structured questionnaire and clinical data through cytopathology result. Results: Significant results were found for knowledge about HPV (p=0.024), number of sexual partners (p=0.007), smoking (p=0.001), factors that may contribute for the infection. A significant difference in polymorphism genotype distribution was observed between control group (non-infected women) and HPV infected patients. A higher frequency of allele A carriers was observed in HPV patients (p<0.001) which was confirmed by codominant, dominant and recessive models. No association was found between this polymorphism and lesion development. Conclusion: It is the first time that the polymorphism rs1801157 of CXCL12 was shown to be associated to HPV infection, which is the major factor for cervical cancer development.

Keywords: CXCL12 polymorphism; HPV infection; cervical cancer. Thematic area: Transmitted diseases.

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GENETIC AND NON GENETIC FACTORS IN THE ETIOLOGY OF CARIES IN CHILD PRESCHOOL

Paola Singi1; Carla Cristiane da Silva2; Emerson José Venancio1

1Department of Pathological Sciences/ Center of Biological Sciences/ State University of Londrina, Londrina, Paraná, Brazil 2Department of Physical Education/ Center of Health Sciences/ University of North Paraná/ Jacarezinho, Paraná, Brazil

Introduction: Dental caries is a chronic infectious disease with multifactorial etiology and with worldwide spread. It is a complex disease resulting from the interaction between genetic and not genetic factors. Objective: To perform a narrative review describing the genetic and not genetic factors contributing to the establishment and progression of caries in children age 2 to 5. Material and methods: For the definition of genetic factors and not genetic ones related to the establishment and the progression of child preschool caries; firstly, it was performed a flow chart showing the relationship of these factors with dental caries. Then, it was performed a bibliographic survey in Pubmed (February 2011 to June 2016) using the following keywords "dental caries", "child preschool", "diet”, “cariogenic", "genetic polymorphism". Results: Initially 94 items were found and 9 were selected. Several hosting and environmental factors demonstrated great association with the development of dental caries in child preschool. Previous studies showed the importance of saliva, in particular of antimicrobial molecules present in saliva, on protection against dental caries. In addition, studies about genetic factors associated with the occurrence of dental caries have allowed the identification of genes and their polymorphisms related to the establishment and progression of dental caries. Conclusion: The results obtained show that a better understanding of the factors associated with dental caries may provide new approaches that lead to the development of new appropriate treatments, particularly before the onset of irreversible damage.

Keywords: Dental caries; Child preschool; Genetics. Thematic area: Transmissible diseases

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PHYSIOLOGICAL AND MOLECULAR CHARACTERISTICS OF CARBAPENEM RESISTANCE IN Klebsiella pneumoniae AND Enterobacter aerogenes

Rito Santo Pereira1, Vanessa Cordeiro Dias1, Alessandra Barbosa Ferreira-Machado1, Juliana Alves Resende1, Vânia Lúcia da Silva1, Cláudio Galuppo Diniz1 1Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil

Introduction: Bacterial resistance is a growing concern in the nosocomial environment in which Klebsiella pneumoniae and Enterobacter aerogenes play an important role due to their opportunism and carbapenemase-production. Objective: This work aimed to evaluate physiological and molecular characteristics of carbapenem-resistant K. pneumoniae and E. aerogenes isolated in a Brazilian tertiary hospital. Material and method: In total, 42 carbapenem-resistant bacteria isolated from clinical specimens: urine, catheter, blood, respiratory tract and wound secretion were included (21 K. pneumoniae and 21 E. aerogenes). Drug-sensitive K. pneumoniae (n = 27) were also included. Antimicrobial susceptibility and biocide tolerance patterns, hemolytic activity, tolerance to oxidative stress, and aggregative ability were assessed. Genetic markers related to carbapenem resistance, or ESBL-production were screened by PCR. This study is in accordance with current legislation on scientific research involving human beings (resolution 196/96 CNS) and was approved by the Research Ethics Committee of University Federal of Juiz de Fora (protocol nr 2587.327.2011). Results: Compared to drug-sensitive strains, carbapenem-resistant K. pneumoniae were more tolerant to biocides and to oxidative stress, and they displayed an increase in biofilm formation. The genetic markers blaKPC (95.2%) and blaTEM (90.5%) were the most frequent. Among the carbapenem- resistant E. aerogenes strains, blaKPC and blaTEM were detected in all bacteria. Drug-sensitive E. aerogenes were not isolated in the same period. blaSHV, blaVIM and blaCTX markers were also observed among carbapenem-resistant bacteria. Conclusion: Results suggest that carbapenemase-producing enterobacteria might show peculiar characteristics regarding their physiology associated with their environmental persistency, virulence, and multidrug resistance. The observed phenomenon may have implications not only for antimicrobial chemotherapy, but also for the prognosis of infectious diseases and infection control.

Keywords: KPC enterobacteria; Antimicrobial resistance; Biocide tolerance. Grants: CNPq, FAPEMIG, Cortes Villela Clinical Laboratory, FCS/UniZambeze and MCTESTP Mozambique Thematic area: Transmitted diseases.

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PLACENTAL HISTOPATHOLOGIC ALTERATIONS AS A TOOL IN CONGENITAL ZIKA VIRUS INFECTION DIAGNOSIS: A CASE REPORT

Sara Santos Bernardes1; Silvana Beutinger Marchioro1; Agruslávia Rezende de Souza2; Flora Martinez Figueira Moreira1; Júlio Henrique Rosa Croda1.

1 Laboratório de Pesquisa em Ciências da Saúde, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum km 12, Dourados, Mato Grosso do Sul, Brazil. 2 Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum km 12, Dourados, Mato Grosso do Sul, Brazil.

Introduction: Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but the low viremia and the lack of proper serological tests to date makes the late infection diagnosis a challange. Here we report a case of a 24-year-old woman with cutaneous rash in the first trimester of pregnancy, without laboratorial tests to confirm the infection. In the third trimester, microcephaly was detected in the fetus during a routine ultrasound, accompanied by dilatation of the lateral ventricles, mega cisterna magna and Dandy-Walker like malformations. In this point, RT-PCR test for ZIKV in the mother serum and urine were negative. The infant was born full term, with 29 cm of head circumference, and realized STORCH laboratorial tests and ZIKV rapid test for IgM and IgG. Objective: Detect pathological alterations in the placenta in a case of suspected microcephaly associated with congenital infection with ZIKV. Material and methods: After birth, cranium computed tomography and immunological tests for STORCH and ZIKV in the infant serum were performed. Fragments of the placental disc were stored in ethanol 70% after fixation in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin for histopathological analysis. This study was approved by the Institutional Research Ethics Committee, under protocol no. 1.627.790. Results: Cranium CT scan showed multiple calcifications in brain tissue, overriding of sutures and dilation of the supratentorial ventricular system. Laboratorial tests for STORCH were all negative, and rapid test for ZIKV negative for IgM and positive for IgG. In placenta, calcified areas were observed, with absence of inflammatory infiltrate, compatible with ZIKV infection. Conclusion: While immunohistochemical tests for the detection of ZIKV are unavailable, histopathological analysis of the placenta can reinforces the Zika diagnosis in newborn with microcephaly.

Keywords: Zika virus; Placenta; Microcephaly. Thematic area: Transmitted diseases.

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CYTOKINE PROFILE FOLLOWING INTRAPERITONEAL INFECTION BY Escherichia coli ENTEROHEMORRHAGIC IN SWISS MICE

Telma Saraiva dos Santos1, Bruna Santos Marnieri1, Anelise Franciosi2, Juan Josue Puño Sarmiento3, Ionice Felipe2, 3, Nathalia Maria Fioreto Campois1, Karina de Almeida Gualtieri1, Tacito Graminha Campois1.

1 Filadélfia University Center. Av. Juscelino Kubitschek, 1.870 - Londrina - PR 2 Pathology Experimental Graduate Program at the State University of Londrina, highway Celso Garcia Cid, Pr 445 Km 380 – Londrina - PR. 3 Microbiology Graduate Program State University of Londrina, Paraná.

Introduction: Escherichia coli it's a part of our microbiota and can be found in the gut of mammals, being a commensal strain, however, can cause disease in hosts with broken gastrointestinal barrier. The virulence mechanisms differ according the pathotypes, one of them is Escherichia coli Enterohemorrhagic (EHEC). Objective: Aimed to establish the relationship between the cellular response in the early stage of infection by Escherichia coli through the profile of cytokines Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in mice infected with diarrheagenic and not diarrheagenic strains. Material and methods: This experimental study was approved by the Ethics Committee Animal of the Filadélfia University Center (Opinion No. 011/2016), 52 Swiss female mice were used, divided into 3 groups (control, ATCC and EHEC) at different times of infection. Inoculation was performed intraperitoneally of the 105 suspension and the peritoneal exudate was obtained by euthanasia to perform the dosage of cytokines by ELISA. The results were analyzed by the Prism 5.0 program and the differences between the experimental groups using the ANOVA test, where p <0.05 was considered statistically significant. Results: Evaluating the profile of IL-1 was a significant difference between the groups in the times studied in the EHEC and ATCC strain, where the ATCC strain presented higher dosages, changing profile in 24 hours and 48 hours of infection. The peak of IL-1 of EHEC strain occurred in 12 hours, declining afterwards, however, without returning the control level. Regarding the dosage of TNF, EHEC strains showed large variation in 6 hours and 24 hours. Conclusion: The pattern of IL-1β cytokine observed in animals infected by EHEC, compared to ATCC reveals a highly pathogenic feature and the importance of infectious and inflammatory processes generated by EHEC as well as the cytokine profile to decrease occurrence of SHU and mortality.

Keywords: Diarrheagenic; Escherichia coli Enterohemorrhagic; Pro-inflammatory. Grants: Fundação Araucária. Thematic area: Transmitted diseases

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EXPANDED ABSTRACTS

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CARACTERIZAÇÃO DE POLIMORFISMOS DO TGFB1 E EXPRESSÃODO TGFB1, TGFBR1 e TGFBR2 EM PACIENTES COM CÂNCER CERVICAL

Adriano Martin Felis Aranome1, Kleber Paiva Trugilo1, Guilherme Cesar Martelossi Cebinelli1, Nádia Calvo Martins Okuyama1, Fernando Cezar dos Santos1, Érica Romão Pereira Maria1, Fernanda Costa Brandão Berti1, Michelle Mota Sena1, Nathália Tatakihara1, Rodolfo Sanches Ferreira1, Angelica Ehara Watanabe2, Carolina Panis3, Karen Brajão de Oliveira1. e-mail: [email protected]

1Laboratório de Genética Molecular e Imunologia, Universidade Estadual de Londrina /Departamento de Ciências Patológicas 2 Laboratório de Estudos e Aplicações de Polimorfismos de DNA, Universidade Estadual de Londrina /Departamento de Ciências Patológicas 3Laboratório de Mediadores Inflamatórios, Universidade Estadual do Oeste do Paraná, Campus Francisco Beltrão, Paraná, Brasil

Palavras-chave: HPV, SNP, Câncer do colo do útero.

TGFBR1, TGFBR2, estadiamento e desenvolvimento do câncer cervical, Resumo infecção pelo HPV, perfil sócio-demográfico O câncer cervical está entre as e de comportamento sexual. Para isso neoplasias mais frequentes na população serão coletadas amostras do tumor do colo feminina mundial, acarretando uterino de 100 pacientes atendidas pelo aproximadamente 265.000 mortes por ano, Hospital do Câncer de Londrina, e 150 sendo associado à presença do controles HPV negativas, de Unidades Papilomavírus Humano (HPV). O sistema Básicas de Saúde de Londrina. A detecção imunológico possui importância tanto na do HPV será feita por PCR, a tipagem viral resolução, como no desenvolvimento das e a genotipagem dos polimorfismos por lesões pré-malignas e do câncer cervical. PCR-RFLP, a quantificação da citocina e Uma citocina importante nesta modulação é seus receptores serão feitas por Imuno- o Fator de Tranformação do Crescimento histoquímica e características sócio- Beta 1 (TGFB1), cuja sinalização no demográficas e dados clínicos serão microambiente inflamatório está associada obtidos por meio dos prontuários médicos e a várias neoplasias. Uma vez que entrevista semi-estruturada. Diferenças na polimorfismos genéticos podem interferir distribuição genotípica entre grupos nos níveis desta citocina, este estudo controle e câncer serão avaliadas pelo teste objetivou avaliar a associação dos 2. Modelos de regressão logística polimorfismos rs1800468, rs1800469, ajustados para possíveis confundidores rs1800470 e rs1800471, e distribuição serão utilizados para análise de associação haplotípica, com a expressão do TGFB1, e susceptibilidade, apresentando valores

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ajustados de Odds Ratio com intervalo de imunológica. Portanto, faz-se necessária confiança de 95%. O nível de significância uma maior compreensão do microambiente adotado será de p<0,05. tumoral, no qual destaca-se a molécula do TGFB1 e seus receptores, a fim de que Hipótese novos marcadores de susceptibilidade Polimorfismos no gene TGFB1 possam ser caracterizados e utilizados na influenciam na sinalização imunológica do prática clínica, objetivando um melhor mesmo, contribuindo para a persistência acompanhamento e tratamento destas viral e para o desenvolvimento do câncer pacientes. cervical.

Métodos Introdução

Aspectos éticos O câncer cervical acarreta Este estudo foi aprovado pelo aproximadamente 265.000 mortes por ano. Comitê de Ética em Pesquisa com Seres Sabe-se que a regressão ou progressão Humanos da Universidade Estadual de das lesões pré-malignas, depende da ação Londrina de acordo com a resolução do sistema imunológico (SCHIFFMAN et al., 466/12, CAAE 56738316.3.0000.5231, 2007). Neste contexto, o fator de parecer número 1.590.141. Antes da coleta, transformação do crescimento β (TGFB) o projeto será explicado para as pacientes e possui um papel paradoxal na patogênese caso aceitem participar assinarão o termo das neoplasias malignas, atuando como de consentimento livre e esclarecido. supressor tumoral, em fases iniciais, ou estimulador da progressão, invasão e metástase tumoral, em fases avançadas (ODA; GUEMBAROVSKI; WATANABE, Pacientes e amostras 2012). Ao longo das últimas décadas, Amostras de tumor do colo uterino, estudos vêm mostrando a associação de previamente fixadas e incluídas em parafina polimorfismos no gene do TGFB1 a de 100 pacientes, atendidas pelo Hospital diversas neoplasias (CHANG et al., 2014; do Câncer de Londrina, serão coletadas. FAN et al., 2014; POOJA et al., 2013). Entretanto, a associação ao câncer cervical O grupo controle será composto por ainda não está bem estabelecida. amostras de sangue periférico e de secreção cervico-vaginal de 150 pacientes HPV negativas, livre de lesão cervical, Justificativa obtidas em Unidades Básica de Saúde de Londrina. As características clínicas e de O câncer cervical é a terceira comportamento sexual serão obtidas pelos neoplasia feminina mais comum no Brasil, prontuários médicos, e características e está associado à presença do HPV. A sócio-demográficas e comportamentos maioria das mulheres é infectada pelo HPV sexuais por meio de um questionário ao longo da vida sexual e apenas uma estruturado. pequena porcentagem desenvolve a doença invasiva, uma vez que a maioria destas infecções é resolvida pela resposta Extração do DNA

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O DNA será extraído a partir do submetido à clivagem pelas enzimas tecido tumoral dos blocos de parafina MspA1-I, para análise do polimorfismo conforme descrito por Isola et al. (1994). O rs1800470 (Alelo C: 149, 67, 40, 26 e 12pb; DNA do grupo controle será obtido a partir Alelo T: 161, 67,40 e 26 pb), Bgl-I, para do sangue periférico, utilizando-se o Kit análise do polimorfismo rs1800471 (Alelo Biopur Mini Spin Plus Kit (Biometrix, G: 131, 103 e 60pb; Alelo C: 163 e 131pb). Curitiba-PR, Brasil), enquanto que o da Todas as reações de restrição enzimática secreção será extraído utilizando o seguirão as instruções fornecidas pelo reagente DNAzol (Invitrogen Inc., Carlsbad, fabricante. CA, USA), seguindo as instruções do fabricante. O material genético será quantificado por espectrofotometria em Imunohistoquímica 260nm e a pureza calculada pela razão A260/A280. Os cortes histológicos serão tratados durante 40 min à temperatura ambiente com 2% de soro albumina bovina, e incubados durante a noite a 4°C com Detecção Molecular do HPV anticorpo primário contra TGB1, TGFBR1 A presença do DNA viral será ou TGFBR2 . Será adicionado o anticorpo verificada por PCR nas amostras de DNA secundário e incubado durante 2 horas. A de secreção vaginal, utilizando os primers atividade da peroxidase será visualizada consenso MY09 e MY11 os quais pelo tratamento com H2O2 e 3,3'- amplificam uma região conservada de cerca diaminobenzidina (DAB) durante 5 min. No de 450pb do gene que codifica a proteína último passo, as secções serão L1 do HPV (BAUER et al., 1991). contracoradas com hematoxilina. A intensidade e localização da

imunorreatividade serão avaliadas com um Genotipagem dos polimorfismos fotomicroscópio. Para identificação dos polimorfismos rs1800468 e rs1800469, em região Análises Estatísticas promotora, e rs1800470 e rs1800471, no peptídeo sinal do TGFB1, serão utilizados aproximadamente 100ng de DNA genômico Diferenças na distribuição para amplificação com primers específicos, genotípica entre os grupos controle e com sintetizados de acordo com a sequência do 2 GenBank J04431.1 e NG_013364.1, câncer serão avaliadas pelo teste  . A respectivamente. Após amplificação, os distribuição haplotípica será avaliada pelo fragmentos serão submetidos à restrição software Phase 2.1.1 (STEPHENS; enzimática para determinação dos SCHEET, 2005). Modelos de regressão diferentes genótipos de cada polimorfismo. logística ajustados para possíveis O produto contendo o fragmento de 598pb confundidores serão utilizados para análise será submetido à clivagem pela enzima Tai- de associação e suscetibilidade. Análises l para análise do polimorfismo rs1800468 estatísticas serão feitas pelo software SPSS (Alelo G: 195 e 402; Alelo A: 598), enzima Statistics 22.0 (SPSS Inc., Chicago, Illinois, Eco81-I para o polimorfismo rs1800469 USA), utilizando valor de p<0,05. (Alelo C: 109 e 488; Alelo T: 598), enquanto o produto com fragmento de 294pb será

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Resultados Esperados ISOLA J, DEVRIES S, CHU L, GHAZVINI AS,WALDMAN F. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded Espera-se que com este estudo tumor samples. American Journal oh possamos compreender melhor o papel da Pathology, v. 145, n. 6, p. 1301-1308, 1994. molécula TGFB1 no microambiente tumoral, bem como identificar possíveis STEPHENS M, SCHEET P. Accounting for Decay of Linkage Disequilibrium in Haplotype marcadores moleculares de suscetibilidade Inference and Missing-Data Imputation. Am. J. ao desenvolvimento do carcinoma cervical. Hum. Genet. v. 76, p. 449–462, 2005. Agradecimentos ODA, J. M. M.; GUEMBAROVSKI, R. L.; WATANABE, M. A. E. Transforming Growth À Capes, CNPq e Fundação Factor b ( TGF- b ) and Regulatory T Cells ( Treg Araucária, pelo apoio financeiro para o ): The Interface of Tumor and Host Immunity. deste estudo. European Journal of Clincal and Medical Oncology, v. 4, n. 1, p. 27–32, 2012.

POOJA, S. et al. Strong impact of TGF-β1 gene Referências polymorphisms on breast cancer risk in Indian women: a case-control and population-based

study. PloS one, v. 8, n. 10, p. e75979, Oct. BAUER, H. M. et al. Genital human 2013. papillomavirus infection in female university RIVA E, et al. Markers of human students as determined by a PCR-based papilomavirus infection and their method. Journal of the American Medical correlation with cervical dysplasia in Association, v. 265, n. 4, p. 472–477, 1991. human immunodeficiency vírus-positive CHANG, W. et al. An updated meta-analysis of women. Clin Microbiol Infect. v. 13, n. transforming growth factor-β1 gene: three 1, p. 86-108, 2007. polymorphisms with gastric cancer. Tumour Biology, v. 35, n. 4, p. 2837–2844, Apr. 2014. SCHIFFMAN, M. et al. Human papillomavirus and cervical cancer. Lancet, v. 370, n. 9590, p. FAN, H. et al. Transforming growth factor-β1 890–907, Sep. 2007. rs1800470 polymorphism is associated with lung cancer risk: a meta-analysis. Medical Science Monitor, v. 20, p. 2358–2362, 2014.

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AVALIAÇÃO DOS EFEITOS DA DIETA HIPERLIPÍDICA ISOLADA OU ASSOCIADA AO HIPOTIREOIDISMO E HIPERTIREOIDISMO SOBRE O TESTÍCULO E EPIDÍDIMO DE CAMUNONGOS C57BL/6: ANÁLISES MORFOFISIOLÓGICAS E MOLECULARES

Ana Paula Franco Punhagui1,2, Francemilson Goulart da Silva3, Glaura Scantamburlo Alves Fernandes1 E-mail: [email protected]

1Universidade Estadual de Londrina/Departamento de Biologia Geral. 2Universidade Estadual de Londrina/Departamento de Ciências Patológicas. 3Universidade de São Paulo/Departamento de Fisiologia e Biofísica.

Palavras-chave: Obesidade, Hipertireoidismo, Hipotireoidismo.

análises morfométricas, histopatológicas e reações de imunohistoquímica (IL6 e TNF- Resumo ), além de análise semi-quantitativa de proteínas por Western blotting e contagem espermática. O testículo e o fêmur serão A obesidade pode ser associada a submetidos a análises citogenéticas. O alterações fisiológicas, incluindo sangue será coletado para dosagem de modificações nas concentrações hormônios, como TSH, T3, T4, LH, FSH, circulantes de hormônios. Alterações no testosterona e leptina. eixo hipotálamo-hipófise promovem desequilíbrio na liberação de hormônios tireoidianos e esteroides sexuais. Com isso, Hipótese o objetivo é avaliar o efeito da ingestão de dieta hiperlipídica, associada ou isolada às disfunções tireoidianas (hipotireoidismo e A ingestão de dieta hiperlipídica, hipertireoidismo) sobre o sistema genital associada ou isolada às disfunções masculino de camundongo. Para isso, tireoidianas causam danos sobre a serão utilizados camundongos C57BL/6 morfofisiologia testicular e epididimária (n=30/grupo) distribuídos em grupos: prejudicando a via espermática de controle (CON, dieta padrão), hipercalórico camundongos C57BL/6 adultos. (HPL, dieta hiperlipídica), hipotireoidismo (CON/HPO), hipercalórico com hipotireoidismo (HPL/HPO), hipertireoidismo (CON/HPE) e hipercalórico Introdução com hipertireoidismo (HPL/HPE), tratados por 12 semanas. Ao final deste período, testículos e epidídimos serão coletados e O papel dos hormônios tireoidianos, submetidos à rotina histológica para triiodotironina (T3) e tiroxina (T4), na

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morfologia e função testicular tem sido na espermatogênese e na esteroidogênese discutido desde quando observaram que os (BIALAS et al., 2009). testículos de animais adultos eram Isto posto, objetiva-se com o sensíveis aos hormônios da tireoide presente trabalho verificar o efeito da dieta (OPPENHEIMER; SCHWARTZ; SURKS, hiperlipídica, associada ou isolada às 1974). Constataram que alterações na disfunções tireoidianas (hipotireoidismo e produção desses hormônios estão hipertireoidismo), sobre a função e associadas não só com anormalidades da morfofisiologia testicular e epididimária de morfologia e função testicular, mas também camundongos C57BL/6. com a diminuição da fertilidade e alterações na atividade sexual de homens (KRASSAS; PERROS, 2003). Justificativa O hipotireoidismo neonatal transitório induzido pelo composto propiltiouracil (PTU) altera mecanismos O acúmulo excessivo de gorduras diversos no sistema genital masculino de resultantes da obesidade causa prejuízos à camundongos (JOYCE; PORCELLI; saúde dos indivíduos e figuram um grave COOKE, 1993). problema de saúde pública mundial. Além O hipertireoidismo neonatal disso, alterações nos níveis de hormônios transitório tem como resultado um tireoidianos são cada vez mais frequentes encerramento precoce da proliferação das em homens e mulheres, com uma série de células de Sertoli. Visto que esta célula mudanças na fisiologia do organismo, suporta um número limitado de células incluindo o sistema genital. germinativas, esta redução pode prejudicar a capacidade reprodutiva e a produção de espermatozoides (ZAMONER, 2006). Em Métodos conjunto com espécies reativas de oxigênio

(ROS) e radicais livres, esta disfunção causa mudanças nas defesas antioxidantes Serão utilizados camundongos do testículo (CHOUDHURY; CHAINY; adultos C57BL/6 machos, mantidos no MISHRO, 2003). biotério do Departamento de Fisiologia e Biofísica do ICB-USP, em condições A obesidade prejudica a adequadas e padronizadas. Os animais homeostase metabólica sistêmica e, assim, serão distribuídos em 6 grupos induz uma resposta de estresse crônico (n=20/grupo) e tratados por 12 semanas. induzido pelo acúmulo de gordura (HOTAMISLIGIL, 2006). Sabe-se que a 1) Controle (CON): ração padrão (10% expressão de receptores para TNF-α em de gordura); indivíduos obesos é duas vezes maior que 2) Hipercalórico (HPL): ração em não obesos (KERN et al., 1995). A IL-6 hiperlipídica (60% de gordura); é considerada uma citocina de ação 3) Hipotireoidismo (CON/HPO): ração parácrina produzida, em testículo humano, padrão + PTU; por células de Sertoli, como resultado da 4) Hipercalórico + hipotireoidismo liberação de FSH, testosterona e (HPL/HPO): ração hiperlipídica + PTU; 5) Hipertireoidismo (CON/HPE): ração neuropeptídios. A expressão de citocinas padrão + T3; pró-inflamatórias em gônadas masculinas 6) Hipercalórico + hipertireoidismo pode ser um dos principais riscos de falha (HPL/HPE): ração hiperlipídica + T3.

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O hipotireoidismo será induzida por processo espermatogênico será avaliado PTU 0,1% adicionado à água de beber classificando os túbulos seminíferos nos (BARCELÓ et al., 1997; RODRIGUEZ- estágios I-VI, VII-VIII, IX-XI e XIV. SOSA et al., 2012; TONG et al., 2007). O Citogenética hipertireoidismo será induzido por via intraperitoneal de T3 2mg/g de massa Serão analisados cromossomos corpórea/dia (MINAMISAWA et al., 2006). mitóticos retirados da medula óssea do Os estados de hipotireoidismo e fêmur e etapas da meiose em testículo, hipertireoidismo serão confirmados por além da montagem de cariótipo para dosagem sérica de T3, T4 e TSH, bem verificar o número diploide e alterações como testosterona, LH, FSH e leptina. cromossômicas. Após 84 dias experimentais, os Imunohistoquímica e quantificação de animais serão anestesiados com xilazina e citocinas quetamina e mortos por punção cardíaca. Cortes de 5µm de testículo e Contagem, morfologia e motilidade epidídimo serão submetidos à detecção de espermática receptores de andrógeno (AR), mastócito e Os testículos direitos descapsulados das citocinas Il-6 e TNF-. A quantificação serão congelados e homogeneizados para dessas citocinas será realizada por contagem de espermátides resistentes e Western-Blot determinação da produção diária de Estatística espermatozoides. Os epidídimos direitos serão separados em porções Os dados serão analisados por teste (cabeça+corpo e cauda), congelados e paramétrico de análise de variância homogeneizados para determinação do (ANOVA) com a posteriori de Tukey- trânsito espermático. Ambos utilizando a Kramer, ou teste não paramétrico de câmara de Neubauer. Kruskall-Wallis com a posteriori de Dunn, de acordo com a variável, através do programa Espermatozoides do ducto GraphPad Prism 6.01. deferente direito e esquerdo serão utilizados, respectivamente, para avaliação de morfologia e motilidade do gameta. Resultados Esperados Análises: histopatológicas, estereológica, morfométricas e espermatogênica Espera-se com os resultados do Os testículos e epidídimos serão presente estudo contribuir no fixados em Methacarn para avaliação esclarecimento da extensão dos danos histopatológica e morfométrica. A análise relacionados ao sistema genital masculino e estereológica do epidídimo será realizada sua correlação com a infertilidade através de acordo com teste de pontos de Weibel da dieta hiperlipídica e dos hormônios (1963). tireoidianos, associados ou isolados, em Nas análises morfométricas no testículos e epidídimos. testículo serão feitas em túbulos seminíferos no estágio IX-XI da espermatogênese com quatro medições do Agradecimentos diâmetro do túbulo e outras quatro da espessura do epitélio germinativo. O

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Agradeço à CAPES pela bolsa de necrosis factor in human adipose tissue. estudos, ao programa de pós-graduação Regulation by obesity, weight loss, and Stricto Sensu em Patologia Exprtimental, à relationship to lipoprotein lipase. The Journal comissão organizadora do evento, à minha of clinical investigation, v. 95, n. 5, p. 2111– 2119, 1995. orientadora Profa Dra Glaura Scantamburlo Alves Fernandes e ao meu co-orientador KRASSAS, G. E.; PERROS, P. Thyroid disease Prof Dr Francemilson Goulart da Silva pelo and male reproductive function. Journal of apoio e conhecimento. endocrinological investigation, v. 26, n. 4, p. 372–80, abr. 2003.

MINAMISAWA, S. et al. Post-transcriptional Referências downregulation of sarcolipin mRNA by triiodothyronine in the atrial myocardium. FEBS letters, v. 580, n. 9, p. 2247–52, 17 abr. 2006.

BARCELÓ, A. C. et al. Unmodified OPPENHEIMER, J. H.; SCHWARTZ, H. L.; erythropoietic response to a beta adrenergic SURKS, M. I. Tissue differences in the agonist in hypothyroid mice. Acta concentration of triiodothyronine nuclear physiologica, v.47, n.4, p.251–3, 1997. binding sites in the rat: liver, kidney, pituitary, BIALAS, M. et al. The role of IL-6, IL-10, TNF-α heart, brain, spleen, and testis. Endocrinology, and its receptors TNFR1 and TNFR2 in the v. 95, n. 3, p. 897–903, set. 1974. local regulatory system of normal and impaired RODRIGUEZ-SOSA, J. R. et al. Endocrine human spermatogenesis. American Journal of modulation of the recipient environment affects Reproductive Immunology, v. 62, n. 1, p. 51– development of bovine testis tissue ectopically 59, 2009. grafted in mice. Reproduction (Cambridge, CHOUDHURY, S.; CHAINY, G. B. N.; England), v. 144, n. 1, p. 37–51, jul. 2012. MISHRO, M. M. Experimentally induced hypo- TONG, H. et al. Age-related learning and and hyper-thyroidism influence on the memory impairments in adult-onset antioxidant defence system in adult rat testis. hypothyroidism in Kunming mice. Physiology Andrologia, v. 35, n. 3, p. 131–140, 2003. & behavior, v. 91, n. 2–3, p. 290–8, 8 jun. HOTAMISLIGIL, G. S. Inflammation and 2007. metabolic disorders. Nature, v. 444, n. 1476– ZAMONER, A. Ações dos Hormônios 4687 (Electronic), p. 860–867, 2006. Tireoidianos sobre o Sistema Reprodutor e JOYCE, K. L.; PORCELLI, J.; COOKE, P. S. o Sistema Nervoso Central: Vias de Neonatal goitrogen treatment increases adult Sinalização, Mecanismos de Ação e Modulação do Citoesqueleto. [s.l.] testis size and sperm production in the mouse. Journal of andrology, v. 14, n. 6, p. 448–55, Universidade Federal do Rio Grande do Sul, 1993. 2006.

KERN, P. A et al. The expression of tumor

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AVALIAÇÃO DA PERFORMANCE REPRODUTIVA DE CAMUNDONGOS EXPOSTOS AO CLORIDRATO DE BUPROPIONA DURANTE A ESPERMATOGÊNESE E DOS POSSIVEIS EFEITOS TERATOGÊNICOS, DESENVOLVIMENTO PÓS NATAL E COMPORTAMENTAIS NA PROLE

Eliane Swely Amador Monteiro Sanches1,2, Felipe Tsuzuki1, Fábio Joinhas1, Estefânia Gastaldello Moreira3, Glaura Scantamburlo Alves Fernandes1, Maria José Sparça Salles1

e-mail: [email protected]

Universidade Estadual de Londrina/ 1Departamento Biologia Geral, 2Departamento de Ciências Patológicas, 3 Departamento de Ciências Fisiológicas Palavras chave: Bup, toxicidade paterna, toxicidade fetal Resumo será realizado a avaliação da toxicidade paterna e performace reprodutiva.

O cloridrato de bupropiona é considerado um antidepressivo atípico, Hipótese usado nas terapias de alívio dos sintomas de privação tabágica e compulsão Considerando-se que o cloridrato de alimentar, além de ser usado no tratamento bupropiona apresenta capacidade de alternativo para défice de atenção e ultrapassar a barreira hematotesticular a transtorno do desejo sexual hipoativo. hipótese testada será a verificação das Estima-se que há um elevado número de possíveis alterações que este fármaco pode homens em idade reprodutiva que causar na performace reprodutiva e na prole necessitam de tratamento com este de camundongos machos adultos. fármaco. Desta forma, o presente estudo terá como objetivo avaliar a toxicidade e as possíveis alterações na performance Introdução reprodutiva em camundongos machos O cloridrato de bupropiona (BUP) expostos ao cloridrato de bupropiona (CH13H18CINO) é considerado um durante a espermatogênese, analisar a presença de malformações congênitas, o antidepressivo atípico não-tricíclico, desenvolvimento pós-natal e as alterações pertencente à classe das aminocetonas. comportamentais da prole. Camundongos Por meio de inibição da recaptação da machos serão distribuídos em 2 grupos dopamina e noradrenalina, possui pouco experimentais: o grupo tratado receberá efeito na recaptura de serotonina e seu diariamente cloridrato de bupropiona mecanismo de ação inclui a liberação pré- (40mg/Kg), por um período de 45 dias e sináptica dessas catecolaminas grupo controle que receberá água destilada (BALDESSARINI, 2010). É o fármaco mais sob o mesmo delineamento experimental. utilizado para o tratamento da dependência No 35º de tratamento esses animais serão de nicotina, também utilizado no tratamento acasalados com fêmeas não tratadas para alternativo de défice de atenção e a análise de malformações congênitas, dependência de metanfetaminas, no desenvolvimento pós-natal e as alterações tratamento da compulsão alimentar, no comportamentais da prole. No 46º dia os transtorno do desejo sexual hipoativo e no machos serão submetidos a eutanásia e tratamento de disfunção sexual associada à diabetes. A BUP é um fármaco de primeira escolha por não induzir efeitos nocivos

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sobre a pressão arterial e frequência acasalamento. No 46° dia os animais serão cardíaca (SILVA, et al. 2016). submetidos a eutanasia por decaptação. Hueletl-Soto (2014) mostrou que a Avaliação da toxicidade paterna e administração de BUP em ratos interferiu no performance reprodutiva gerador espinal para a ejaculação desses animais. Segundo Sukoff Rizzo et al. (2008) Acompanhamento da massa corpórea e verificaram que o farmaco induziu à observação de sinais clínicos (presença de diminuição da ereção peniana em ratos piloereção, olhos avermelhados, diarréia, adultos. coordenação motora e morte). Após a O acompanhamento pós natal da eutanásia os órgãos como coração, prole de ratos expostos à BUP durante a pulmão, fígado, rins, testículos e epidídimos gestação aumentou a suscetibilidade ao serão retirados, analisados externamente e stress e apresentou mecanismo de pesados. O sangue coletado será recompensa semelhante à induzida por submetido à dosagem de testosterona e do cocaína (HSIAO et al., 2005). LH pelo método de radioimunoensaio. Do material coletado serão realizados: Justificativa avaliação da morfologia dos espermatozoides (WYROBEK et al., 1983) A segurança do uso do BUP durante e analise histológica da espermatogênese a reprodução masculina ainda não foi nos testículos (JOHNSEN, 1970). estabelecida. A escassa literatura que mostra dados de exposição do Avaliação do desenvolvimento intrauterino medicamento sobre o processo da e embriofetotoxicidade espermatogênese, síntese hormonal, comportamento sexual e teratogenese na As fêmeas serão submetidas a eutanasia no prole, não permitem uma avaliação de risco 18º dia de prenhez para avaliação do específica. Considerando-se o elevado desenvolvimento intrauterino, verificando- número de homens em idade reprodutiva se a presença de reabsorções, número de que necessitam de tratamento com este fetos vivos e mortos, peso fetal e fármaco, o presente trabalho justifica-se em placentário, comprimento fetal, e avaliação virtude da falta de conhecimentos dos das malformações estruturais externas. efeitos deste fármaco sobre a performance Com estes dados serão calculados: taxa de reprodutiva e possíveis efeitos na prole. reabsorção, taxa de perdas pós- implantacionais, taxa de viabilidade fetal e a Métodos frequência de dominantes letais. Para a avaliação das malformações congênitas, a Aprovação CEUA-UEL, processo n. análise visceral será realizada pela 8722.2016.33. Camundongos machos metodologia de Barrow & Taylor (1969), e a serão distribuídos em 2 grupos análise esquelética pelo método de Taylor experimentais: grupo tratado receberá (1986). diariamente BUP na dose de 40mg/Kg, diluídos em água destilada, por um período Avaliação do desenvolvimento pós-Natal de 45 dias e grupo controle que receberá (desenvolvimento físico e reflexológico) água destilada sob o mesmo delineamento experimental. O período de administração foi baseado no período do ciclo O nascimento de parte da prole ocorrerá espermatogênico em camundongos, que é de maneira natural. O dia do parto será de 35 dias (ADLER, 2000), nesse dia, os considerado dia zero de vida pós-natal machos serão colocados para acasalar com (DPN0). Os parâmetros envolvidos na fêmeas e continuarão a ser tratados por avaliação do desenvolvimento físico e mais 10 dias, período para permitir o neurocomportamental da prole, propostos por Alder & Zbinden (1977), serão observados diariamente após o parto. No

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DPN60 será colhido o sangue de uma KWAIGROCH, T. E. Evaluation of embriotoxic fêmea e um macho de cada ninhada para effects in animals. Stuttgard; Geoge Thieme. p. dosagens de estrogênio, progesterona e 175-185. 1977. testosterona, pelo método de BALDESSARINI, R. J. Drug Therapy of radioimunoensaio. Depression and Anxiety Disorders. In: Goodman & Gilman's. The Pharmacological Basis of Therapeutics. 12ª edição. Edição on- Avaliação comportamenal (atividade line, 2010. motora e ansiedade) BARROW, M.V; TAYLOR, W.J. A rapid method for detecting malformations in rat fetuses. Os testes serão aplicados nos filhotes em Journal of Morphology. n. 127, p. 291-306. duas idades distintas: DPN 25 e 60. Para 1969. cada idade será utilizado um macho e uma HSIAO, S. Y., et al. Prenatal bupropion fêmea de cada ninhada, segundo exposure enhances the cocaine reward and metodologia descrita por Weiss & stress susceptibility in adult mice. The Chinese Greenberg (1998). journal of physiology. v. 48(4), p. 223-9. 2005. Análise Estatística HUELETL-SOTO, M. E., CARRO-JUÁREZ, M., RODRIGUEZ-MANZO, G. Effects of bupropion on the ejaculatory response of male rats. Os dados absolutos com distribuição International journal of impotence research. normal serão analisados por Teste T ou v. 26(6), n. 205-12. 2014. ANOVA (one way) seguido de Teste de JOHNSEN, S.G.Testicular biopsy score count - Tukey, os com distribuição não-normal por a method for registration of spermatogenesis in Kruskal-Wallis/Dunn e os dados de human testes: normal values and results in 335 frequência pelo teste qui-quadrado. O nível hypogonadal mals. Hormones. v. 1(1), p.2- de significância considerado será de 5%. 25.1970 SILVA, P. S., et al. Influence of smoking cessation drugs on blood pressure and heart rate in patients with cardiovascular disease or Resultados esperados high risk score: real life setting. BMC Cardiovascular Disorders. v. 16(2). 2016. Os resultados sinalizarão para a SUKOFF RIZZO, S. J., SCHECHTER, L. E., segurança do uso do BUP em homens que ROSENZWEIG-LIPSON, S. A novel approach se encontram em idade reprodutiva, for predicting antidepressant-induced sexual trazendo informações sobre os efeitos dysfunction in rats. Psychopharmacology. v. deste fármaco em relação a aspectos 195(4), n. 459-67. 2008 reprodutivos, desenvolvimento intrauterino, TAYLOR, P. Skeletal examination. Practical pós-natal e comportamental da prole, Teratology. p.77-100. 1986 WEISS, E., GREENBERG, G. Open field possibilitando que estratégias de prevenção procedures. In: GREENBERG, G.; contra possíveis danos sejam adoptadas. HARAWAY, M. M. Comparative psychology: Referências a handbook, New York: Garland Publishers; p. 257–263, 1998. WYROBEK, A.J et al. An evaluation of the ADLER, I.D. Spermatogenesis and mouse sperm morphology test and other sperm mutagenicity of environmental hazards: tests in non-human mammals: a report of the extrapolation of genetic risk from mouse to US. Environmental Protection Agency Gene- man. Andrologia. n. 32, p. 233-237. 2000 Tox Program. n. 115, p.1-72. 1983. ALDER, S., ZBINDEN, G. Methods for the evaluation pf physical, neuromuscular and behavior development of rats in early postnatal life. In: NEUBERT, D., MERKER, H. J.,

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AVALIAÇÃO DOS EFEITOS DO INSETICIDA MALATION SOBRE O DESENVOLVIMENTO DO SISTEMA GENITAL MASCULINO DE RATOS DESDE O PERÍODO JUVENIL ATÉ A PUBERDADE

Rafaela Pires Erthal1, Glaura Scantamburlo Alves Fernandes1 e-mail: [email protected]

1Universidade Estadual de Londrina / Departamento de Biologia Geral

Palavras-chave: malation, desenvolvimento, machos

para a análise hormonal de testosterona, LH e FSH. Os testículos e epidídimos serão Resumo pesados e utilizados para avaliação da Doenças atualmente consideradas contagem espermática ou fixados para de grande problema de saúde pública como posterior avaliação histopatológica, dengue, chikungunya e zika são causadas morfométrica (testículo) e estereológica por meio do mosquito vetor do gênero (epidídimo). Espermatozoides do ducto Aedes. A prevenção dessas doenças deferente serão utilizados para análises de depende do controle dos vetores através de morfologia e motilidade. Espera-se com os inseticidas químicos, sendo o malation resultados deste estudo obter informações utilizado em grande escala. A absorção por mais precisas sobre o efeito do malation no via oral possui grande importância em desenvolvimento testicular e epididimário intoxicações ambientais através do hábito durante o período peripuberal, e deste de comer ou preparar alimentos durante a modo auxiliar nas normas de segurança aplicação desses compostos. Além disso, a para seu uso. literatura sugeriu danos em parâmetros reprodutivos após exposição ao malation. Sabendo-se da importância da Hipótese peripuberdade como período crítico para o A exposição de ratos machos a desenvolvimento pós-natal do sistema baixas doses de malation durante o genital masculino, o objetivo do presente período juvenil e peripuberal interfere em trabalho é avaliar se a exposição ao parâmetros reprodutivos. malation durante o período peripuberal poderá causar danos para o desenvolvimento testicular e epididimário. Para tanto, ratos machos Wistar serão Introdução tratados com malation nas doses de 10 Doenças ocasionadas pelos vírus da mg/Kg ou 50 mg/Kg de peso corpóreo via dengue, zika e chikungunya representam gavage do DPN 25 ao 65. Ao final do um dos maiores problemas de saúde período experimental, os animais serão pública nas regiões tropicais e subtropicais, anestesiados e submetidos à eutanásia por sendo declarada situação de emergência a punção cardíaca e o sangue será coletado nível nacional (BRASIL, 2015a). O mosquito

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do gênero Aedes aegypt é o vetor de tais expostos ao malation (200 mg/kg de peso doenças, sendo a fêmea mais importante no corpóreo DPN 21) durante 30 dias. processo de transmissão. Por conta disso, Adolescentes e jovens que estão tem-se tomado medidas nacionais crescendo durante esse período de controle buscando combater esse vetor transmissor do mosquito Aedes aegypt são expostos ao desses vírus em cumprimento ao Plano malation pelos meios já descrito. Esse Nacional de Enfrentamento à Microcefalia período de puberdade envolve mudanças (BRASIL, 2015b). O combate ao mosquito físicas, comportamentais e hormonais, adulto é feito por meio do uso de inseticidas sendo um período crítico para a maturação químicos (WHO, 2009). O malation é um sexual e a obtenção da capacidade inseticida do grupo dos organofosforados reprodutiva (GOLUB et al., 2008). utilizado para erradicação do Aedes aegypt. Durante a aplicação, tais compostos são A maioria dos estudos relacionando redepositados e dispersos no ambiente, exposição ao malation e efeitos sobre o atingindo o solo e corpos d’água (CHAIM, trato genital masculino ocorrem em animais 1989). Por isso, a população está exposta a no período adulto. Dado a relevância social tais compostos em regiões de combate ao do inseticida em questão e o período crítico mosquito vetor. O malation também tem de desenvolvimento, a puberdade, o sido utilizado como praguicida em culturas presente estudo busca avaliar os efeitos agrícolas. De acordo com a Agência para devido à exposição do malation durante o Substâncias Tóxicas e Registro de Doencas período peripuberal, em duas doses, sobre (ATSDR, 2003), órgão governamental dos o sistema genital masculino. EUA, mais de 100 alimentos podem ser tratados com o praguicida, e aproximadamente metade das aplicações Justificativa nos EUA são em alfafa, algodão, arroz e trigo. Dessa maneira, a população está Crianças e adolescentes tem-se exposta diretamente a tais alimentos desenvolvido durante períodos de combate contaminados. ao mosquito Aedes utilizando inseticidas químicos. Uma classe de inseticidas O malation pode ser absorvido pelo utilizada em grande escala é a dos organismo por via dérmica, respiratória ou organofosforados que são inibidores da digestiva, sendo essa última, de acetilcolinesterase. A absorção por via oral fundamental importância nas intoxicações possui grande importância em intoxicações acidentais através do manuseio do ambientais principalmente em crianças, e composto durante o hábito de comer ou em adultos, no hábito de comer ou preparar preparar alimentos (LARINI, 1996). A DL50 alimentos durante o trabalho de aplicação aguda do malation administrado por via oral de tais compostos. Dessa maneira, essas em ratos varia de 1500 a 2000 mg/kg (U.S. exposições são recorrentes e acontecem de EPA, 2000). maneira não intencional. Há alguns anos, o Os danos em decorrência da inseticida indicado para combate do exposição ao malation abrange os mosquito A. aegypt no Brasil era a diferentes sistemas do organismo. Em deltametrina, mas com o passar dos anos, relação ao sistema genital masculino, o mosquito adquiriu resistência contra o Slimen et al. (2015) mostraram alterações mesmo. Como alternativa, tem-se utilizado histopatológicas, bioquímicas e o organofosforado malation. Estudos moleculares na função reprodutiva de ratos mostram que o malation pode causar danos em parâmetros reprodutivos de ratos

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machos expostos durante a vida adulta. Por a cada três dias e receberão água e ração outro lado, não existem estudos ad libidum. O consumo de água e ração e o apresentando exposição a baixas doses de peso corporal serão aferidos durante todo malation durante a peripuberdade. Essa período experimental. Esse projeto foi fase trata-se de um período crítico para o aprovado pelo Comitê de Ética no Uso de desenvolvimento do sistema genital Animais (CEUA/UEL) sob número masculino. 137/2016. Portanto, este estudo tem grande aplicabilidade ao avaliar os efeitos da Coleta dos materiais exposição ao malation em diferentes doses no sistema genital masculino durante a No 65º dia experimental os ratos juventude e peripuberdade. As serão anestesiados com a associação de metodologias propostas para este projeto xilazina e quetamina e mortos por punção são plenamente relevantes, aplicáveis e cardíaca para a coleta do sangue em tubo pertinentes ao tema proposto. As mesmas heparinizado (heparina sódica) para têm sido publicadas com êxito em dosagens hormonais. Dez animais de cada periódicos internacionais por autores da grupo experimental terão os testículos e presente área do conhecimento. epidídimos direitos retirados, pesados em balança analítica de precisão e congelados

a –20ºC para posterior determinação do Métodos número de espermátides maduras. Os testículos esquerdos desses animais serão congelados a -80ºC e destinados à Delineamento experimental detecção de receptores de andrógenos por Western Blotting. O ducto deferente direito Serão utilizados 45 ratos macho da será pesado e dele serão obtidos os linhagem Wistar, com idade inicial de 22 espermatozoides para avaliação da dias (40-60 g), provenientes do Biotério morfologia espermática. Os Central da Universidade Estadual de espermatozoides do ducto deferente Londrina – UEL e serão aclimatados ao esquerdo serão utilizados para avaliação da novo ambiente (Biotério do Laboratório de motilidade espermática. As vesículas Biologia da Reprodução) durante cinco dias seminais (cheias e vazias, sem a glândula que antecedem o início do período coaguladora) e as próstatas ventrais serão experimental. Os animais serão distribuídos pesadas e descartadas. De outros 5 casualmente em três grupos experimentais animais de cada grupo os testículos e (n=15 animais/grupo). Dois grupos de epidídimos direitos serão utilizados para animais serão tratados com malation nas análises histopatológicas e morfométricas, doses de 10 mg/Kg ou 50 mg/Kg de peso e testículos e epidídimos esquerdos serão corpóreo via gavage. Essas doses utilizados para determinação do perfil correspondem a 0,5% e 2,5%, inflamatório. Parte dos testículos e medula respectivamente, da DL50 oral para ratos óssea serão utilizados para análise (DL50 oral =2000 mg/kg) (U S EPA, 2000). citogenética de meiose e mitose, O outro grupo (grupo controle) receberá respectivamente. apenas o veículo (óleo de soja) em igual volume. O período de tratamento será entre o DPN 25 e 65 (CLEGG, 1960; OJEDA et Resultados Esperados al., 1980). Todos os animais serão pesados

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A partir do presente estudo, espera- importância Nacional (ESPIN) por alteração se contribuir com o desenvolvimento do padrão de ocorrência de microcefalias científico e social, uma vez que o tema do no Brasil. presente estudo é um grave problema social BRASIL. MINISTÉRIO DA SAÚDE, 2015b. na atualidade que é o combate ao vetor das Diretriz geral SNCC: sistema de doenças dengue, chikungunya e zika; coordenação e controle para intensificar as conscientizar e reforçar dados científicos ações de mobilização e combate ao relacionados ao crescimento do número de mosquito. casos e doenças no homem e em animais CHAIM, A. Processos de aplicação de em consequência da utilização do malation; produtos fitossanitários e contaminação esclarecer se o declínio na qualidade ambiental. EMBRAPA, CNPDA, 24p., 1989. espermática evidenciado nas últimas décadas pode estar relacionado à GOLUB, M. S.; COLLMAN, G.W.; FOSTER, exposição ao malation durante o período P.M.D.; KIMMEL, C.A.; MEYETS, E.R.D.; REITER, E.O.; SHARPE, R. M.; SKAKKEBAEK, peripuberal; determinar seu efeito em N. E.; TOPPARI, J. Public health implications baixas doses sobre o desenvolvimento pós of altered puberty timing. Pediatrics, 2008; natal do sistema genital masculino. 121: S218 -S230

LARINI, L. Praguicidas. In: OGA, S. Fundamentos de toxicologia. São Paulo: Agradecimentos Atheneu, 1996. 515p.

À Universidade Estadual de SLIMEN, S.; SALOUA, E. F.; NAJOUA, G. H. Londrina, ao Programa de Pós Graduação Oxidative stress and cytotoxic potential of em Patologia Experimental pela anticholinesterase insecticide, malathion in oportunidade; à CAPES pelo financiamento, reproductive toxicology of male adolescent à Professora Dra Glaura Scantamburlo mice after acute exposure. Iran J Basic Med Alves Fernandes pela orientação. Sci 2014; 17:522-530. UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES. Agency for Toxic Referências Substances and Disease Registry (ATSDR). 2003. Malathion Technical Summary.

BRASIL. MINISTÉRIO DA SAÚDE, 2015a. Declara Emergência em Saúde Pública de

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O PAPEL DE AGRECANAS EM DISTÚRBIOS GASTROINTESTINAIS DE HUMANOS E CAMUNDONGOS

Anelise Franciosi1, Jorge Mali Júnior1, Laurence Ashley Blackshaw2, Eduardo José de Almeida Araújo1. e-mail: [email protected]

1 Universidade Estadual de Londrina/Departamento de Histologia, Brasil 2 Queen Mary University of London/Wingate Institute for Neurogastroenterology, Reino Unido

Palavras-chave: Agrecana, Sistema Nervoso Entérico.

a realização de ensaios de avaliação funcional do plexo submucoso (secreção de Resumo coleto em câmara de Ussing) e do plexo mioentérico (esvaziamento gástrico e manometria intestinal). Cortes histológicos A agrecana é uma proteoglicana revelaram a presença de agrecana no TGI, presente na matriz extracelular (MEC) tanto de humanos como de camundongos. pertencente à família das lecticanas. A marcação apresentava-se na base do Estudos têm demonstrado que a ausência epitélio de revestimento da mucosa, tecido de alguns tipos de proteínas da MEC em conjuntivo da submucosa e camadas da células do sistema nervoso entérico (SNE) musculatura externa. Percebeu-se co- tem provocado distúrbios gastrointestinais. localização com corpos celulares de No entanto, não se sabe se agrecana está neurônios entéricos. Conclui-se, até o presente no SNE. Assim, o objetivo deste momento, que agrecana está associada a projeto é verificar se agrecana está neurônios do sistema nervoso entérico. presente no trato gastrointestinal (TGI) de humanos e camundongos C57BL/6, sobretudo de forma associada a neurônios Hipótese entéricos. Caso esteja presente, pretende- se ainda avaliar o papel fisiológico dessa proteína em neurônios entéricos. Para localização de agrecanas no TGI, amostras A agrecana é uma proteína da rede de intestino delgado e grosso de humanos perineuronal (RNN) que pode estar e camundongos foram submetidas a presente nos gânglios entéricos e técnicas de imunofluorescência tanto em apresentar relação com distúrbios cortes como em preparados totais. gastrointestinais do trato gastrointestinal. Preparados totais serão utilizados para uma análise quantitativa. Será avaliado também Introdução se agrecanas possuem associação específica com subpopulações neuronais. Em seguida, serão utilizados camundongos A MEC no SNC é um complexo molecular com gene silenciado para agrecana visando amorfo e difuso, que ocupa o espaço entre

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as células do tecido nervoso. Ao redor de Justificativa alguns neurônios, a MEC torna-se uma estrutura estável e densa conhecida como rede perineuronal (RPN) (KWOK et al., Os distúrbios gastrointestinais 2011). RPN é constituída por ácido funcionais são alterações recorrentes do hialurônico, lecticanas, proteínas de ligação TGI que afeta milhões de pessoas no e tenascinas. As Lecticanas são mundo inteiro, sem uma causa aparente de proteoglicanos de sulfato de condroitina, sua fisiopatologia. Caracterizar e que se subdividem em agrecana, versicana, correlacionar proteínas da MEC que neurocana e bevicana. A agrecana possam estar envolvidas com estes apresenta um núcleo proteico com três distúrbios pode contribuir para a elucidação domínios de ligação. O domínio G1, desse problema que causa grandes localizado na porção N-terminal da transtornos ao paciente. molécula, o qual possui sítio de ligação para o ácido hialurônico, o G2 é característico da agrecana e ainda não tem sua função Métodos conhecida e o G3, encontrado na porção C- terminal da estrutura, permite a ligação da agrecana com outras lecticanas como a versicana, e proteínas de adesão com a Todos os procedimentos já estão tenascina C e X. A interação destas aprovados pelo Comitê de Ética em proteínas leva a formação de agregados na Pesquisa Envolvendo Seres Humanos da cartilagem, onde a agrecana é capaz de UEL (1.073.326) e pela Comissão de Ética sequestrar íons e moléculas de água no Uso de Animais da UEL (032/2015). promovendo o inchaço hidrodinâmico, Amostras do trato gastrointestinal essencial para a sustentação (ASPBERG, (estômago, jejuno e cólon) de cinco 2012). No SNC, a agrecana está presente camundongos C57BL/6 e de três seres em toda a extensão da RPN (GALTREY et humanos já foram coletadas, fixadas em al., 2008). paraformaldeído 4% e congeladas em gel próprio para congelamento (optimum Embora não haja descrição de RPN no cutting temperturate compound – OCT). sistema nervoso entérico, estudos recentes Essas amostras estão armazenadas num demonstram que proteína da MEC, ultrafreezer a -80oC. Essas amostras estão Tenascina X, (AKTAR, 2016) está passando por microtomia em criostato. fortemente associada com distúrbios Parte das amostras de TGI de camundongo gastrointestinais. Porém, não se conhece está sendo microdissecada para obtenção até o momento se outras proteínas da MEC de preparados totais de submucosa também estão associadas a esse tipo de (contendo o plexo submucoso) e de doença. muscular externa (contendo o plexo mioentérico). Cortes histológicos obtidos Por isso, o objetivo deste projeto é verificar por microtomia em criostato foram se agrecana está presente no trato submetidos à técnica de gastrointestinal de humanos e imunofluorescência para evidenciação de camundongos, sobretudo de forma neurônios entéricos gerais (anti-PGP9.5 associada a neurônios entéricos. Caso 1:1000; ABCAM, AB8189) ou colinérgicos esteja, pretende-se ainda avaliar o papel (anti-ChAT 1:50; MERK MILLIPORE, funcional dessa proteína no TGI e verificar AB144P), em dupla marcação específica se a deficiência de agrecana pode estar para agrecana (anti-ACAN 1:250, NOVUS associada a distúrbios gastrointestinais.

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BIO, NB100-74350). As análises foram Resultados e Discussão feitas em fotomicroscópio trinocular de fluorescência (ZEISS AXIO IMAGER.A1) visando identificar marcações positivas Até o momento, observou-se para agrecana em toda a parede intestinal, marcação positiva para agrecana na base observando também se estavam do epitélio de revestimento da mucosa, no associadas com neurônios dos plexos tecido conjuntivo da submucosa e nas submucoso e mioentérico. Além disso, camadas da musculatura externa. Quanto à amostras de intestino de camundongo associação com neurônios entéricos, serão microdissecadas para obtenção de observou-se intensa co-localização com preparados totais de submucosa (contendo corpos celulares de neurônios tanto do o plexo submucoso) e de muscular externa plexo submucoso como do plexo (contendo o plexo mioentérico) que serão mioentérico evidenciados com PGP9.5. submetidos à imunofluorescência e Quando analisamos a subpopulação neural analisados pelo mesmo fotomicroscópio colinérgica, observamos que também para quantificação dos neurônios entéricos existia co-localização com agrecana (Fig. que expressam agrecana. A partir desse 1). resultado, serão utilizados camundongos silenciados para agrecana visando realizar A literatura mostra que na testes fisiológicos de secreção de cloreto cartilagem, este proteoglicano é essencial em câmara de Ussing, esvaziamento para o seu funcionamento adequado gástrico e manometria intestinal. Assim será (GIBSON; BRIGGS, 2016), assim podemos possível dimensionar o papel funcional de inferir que a agrecana possa estar envolvida agrecana no TGI. no funcionamento normal da mucosa, submucosa e camadas musculares, uma vez que se observa sua presença nesses estratos.

Figura 1 – Imunomarcação de Agrecana na parede intestinal. 1.A – Imunomarcação de PGP9.5 em glânglio do plexo mioentérico do intestino de camundongo. 2.A - Imunomarcação de Agrecana em glânglio do plexo mioentérico do intestino de camundongo. 3.4 – Imunocolocalização de PGP9.5 com Agrecana em glânglio do plexo mioentérico do intestino de camundongo. 4.A - Imunomarcação de ChAt em glânglio do plexo mioentérico do reto de humano. 5.A - Imunomarcação de Agrecana em glânglio do plexo mioentérico do reto de humano. 6.A - Imunocolocalização de ChAT com Agrecana em glânglio do plexo mioentérico do reto de humano. 141

Conclusão Gatroenterology Journal 150: p. 346, 2016.

Podemos concluir, preliminarmente, que a agrecana está presente no TGI de humanos e camundongos, sobretudo de forma associada com neurônios entéricos.

Agradecimentos

Os autores agradecem à CAPES pelo financiamento do projeto (processo: 88881.068190/2014-01).

Referências

ASPBERG A. The different roles of aggrecan interaction domains. J Histochem Cytochem. 60(12): p. 987–96, 2012.

GALTREY CM, KWOK JC, CARULLI D, et al. Distribution and synthesis of extracellular matrix proteoglycans, hyaluronan, link proteins and tenascin-R in the rat spinal cord. Eur J Neurosci 27: p. 1373-1390, 2008.

GIBSON BG, BRIGGS MD. The aggrecanopathies; an evolving phenotypic spectrum of human genetic skeletal diseases. Orphanet Journal of Rare Diseases 11: p. 86, 2016.

KWOK JCF, DICK G, WANG D, et al. Extracellular matrix and perineuronal nets in CNS repair. Dev Neurobiol 71: p. 1073-1089, 2011.

MORAWSKI M, BRUCKNER G, JAGER C, et al. Neurons associated with aggrecan-based perineuronal nets are protected against tau pathology in subcortical regions in Alzheimer’s Disease. Neuroscience 169: 1347- 1363, 2010.

AKTAR R, PEIRIS M, TOUGH I.R, et al. Sa1688 Multiple Functional Roles for the Extracellular Matrix Glycoprotein Tenascin X.

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O PAPEL DE FOSFACANAS EM DISTÚRBIOS GASTROINTESTINAIS DE HUMANOS E CAMUNDONGOS

Joana D’Arc de Lima Mendes; Jorge Mali-Júnior1 Laurence Ashley; Blackshaw2; Eduardo José de Almeida Araújo1 [email protected]

1UEL-Universidade Estadual de Londrina/Departamento de Histologia, Brasil. 2Queen Mary University of London/Wingate Institute for Neurogastroenterology, Reino Unido.

Palavras-chave: Fosfacana, trato gastrointestinal, neurônios entéricos.

dessas proteínas podem estar envolvidos em distúrbios gastrointestinais. No entanto, Resumo até o presente momento, não há descrição da presença e ação do RPTP ζ/fosfacana nos plexos nervosos do trato A matriz extracelular (MEC) gastrointestinal. Portanto, este projeto tem desempenha um papel fundamental no por objetivo avaliar a presença e função do desenvolvimento neuronal atuando na RPTP ζ/fosfacana no sistema nervoso sinaptogenese, estabilidade sináptica e entérico de humanos e camundongos. Para celular. No sistema nervoso central (SNC), avaliação da presença de RPTP a MEC apresenta-se distinta ao redor de ζ/fosfacana, amostras de tecido alguns neurônios, o que constitui as redes gastrointestinais humano e de perineuronais (PNN). Estas são camundongos serão submetidas técnicas constituídas por moléculas de CSPGs, de imunofluorescência. A partir da ácido hialurônico e tenascinas. As principais localização e associação de RPTP moléculas de CSPGs são agrecana, ζ/fosfacana em neurônios entéricos, serão versicana, neurocana, brevicana e o definidos ensaios de avaliação funcionais receptor tirosina fosfatase ζ/fosfacana envolvendo o plexo submucoso (secreção (RPTP ζ/fosfacana). Essas moléculas de cloreto) e o plexo mioentérico promovem interação célula-célula e célula- (esvaziamento gástrico e manometria MEC em diversas ações fisiológicas. O intestinal). RPTP ζ/fosfacana é um receptor tipo tirosina fosfatase com alta atividade durante o desenvolvimento neuronal, promovendo Hipótese crescimento, fasciculação e mielinização de axônios. Estudos recentes têm demonstrado que alterações nas moléculas A RPTP ζ/fosfacana é uma proteína da PNN estão relacionadas com a da PNN que pode estar presente nos morfologia e funcionamento de neurônios gânglios entéricos e apresentar relação entéricos. Portanto, defeitos ou deficiências

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com distúrbios gastrointestinais do trato keratam. O RPTP ζ/fosfacana pode se ligar gastrointestinal. a mais de um ligante promovendo diferentes atividades nas células (DWYER et al., Introdução 2015). Segundo Mohebiany et al. (2013), quando o RPTP ζ/fosfacana se liga a Contractina -1 (CNTN1) ocorre Segundo Zimmermann (2008), a diferenciação dos precursores de MEC é uma rede composta em sua grande oligodendrócitos, além de sua migração e a maioria por moléculas e proteínas mielinização dos axônios neuronais. No sintetizadas pelas células locais. Tem como entanto, quando o receptor se liga à função dar sustentação aos tecidos, adesão pleitrofina (PTN) ocorre dimerização do celular e comunicação célula-célula. A MEC receptor e consequentemente inativação de é composta por proteínas fibrosas como o sua atividade de fosfatase. colágeno, elastina, fibronectina, laminina, glicosaminoglicanas (GAGs) e Devido a associações com proteoglicanas. No SNC, a MEC tem uma neurônios, estudos recentes têm região especializada ao redor de alguns demonstrado que proteínas da MEC podem neurônios, chamada de PNN. Dentre as estar envolvidas em distúrbios moléculas que compõe a PNN, destacam- gastrointestinais de origem neural(AKTAR se as moléculas de CSPG: agrecanas, et. al. 2016). Por isso, neste projeto versicanas, brevicanas, neurocanas e pretende-se caracterizar a presença de RPTP ζ/fosfacanas, as quais estão RPTP ζ/fosfacana nos neurônios entéricos dispostas em corpos celulares, axônios e e sua possível correlação com distúrbios dendritos. Tais moléculas promovem a funcionais do TGI. sinalização entre as células e MEC, proporcional regulação de várias atividades fisiológicas como crescimento, migração e Justificativa diferenciação celular (HAYASHI et al., 2005). Maurel et al. (1993) foram os Os distúrbios gastrointestinais primeiros a descrever o RPTP ζ/fosfacana funcionais são alterações recorrentes do da família de proteínas tirosina fosfatase TGI que afeta milhões de pessoas no (PTP). As PTP são proteínas com atividade mundo inteiro, sem uma causa aparente de antagonista às proteínas kinases (PK), sua fisiopatologia. Caracterizar e removendo grupamentos fosfatos correlacionar proteínas da MEC que adicionados pelas PK (DEN HERTOG et al., possam estar envolvidas com estes 1999). PTPs estão subdivididas em 2 distúrbios pode contribuir para a elucidação grandes grupos: clássicas e desse problema que causa grandes citoplasmáticas. RPTP ζ/fosfacana é um transtornos ao paciente. receptor tipo tirosina fosfatase pertencente às PTP clássicas, a qual é composta por uma porção intracelular com dois domínios Métodos fosfatase (D1 e D2), e uma porção extracelular com um domínio N-terminal de anidrase carbônica, uma ligação de Todos os procedimentos já foram fibronectina III, uma ligação de sulfato de aprovados pelos comitês de ética (Seres condroitina e uma ligação de sulfato de humanos: 1.073.326 e Animais: 032/2015)

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da Universidade Estadual de Londrina C57BL/6 nocauteados para o gene desta (UEL). Amostras do trato gastrointestinal proteína. Nestes camundongos serão (estômago, intestino delgado e grosso) de realizados testes fisiológicos de secreção camundongos C57BL/6 (n=5) e de seres de cloreto em câmara de Ussing, humanos (n=3) já foram coletadas, fixadas esvaziamento gástrico e manometria em paraformaldeído 4% e congeladas em intestinal. OCT. Amostras do TGI de seres humanos vieram da margem de segurança de pacientes oncológicos submetidos à Resultados e Discussão ressecção cirúrgica no Hospital do Câncer de Londrina. Para se considerar que os resultados em camundongos possam ser Até o momento foram obtidos extrapolados para seres humanos é resultados de imunofluorescência para essencial inicialmente se confirmar a localização da RPTP ζ/fosfacana no TGI de presença da RPTP ζ/fosfacana em tecidos humanos e camundongos. Observou-se a de camundongos e humanos. Para isso, presença da RPTP ζ/fosfacana em todos os essas amostras estão sendo submetidas à segmentos do TGI, especificamente nos técnica de imunofluorescência para avaliar gânglios nervosos, tanto de camundongos a presença de RPTP ζ/fosfacana em como nas amostras humanas (Fig. 1). Em neurônios entéricos de camundongos humanos, a marcação foi mais evidente do C57BL/6 e de humanos. A análise dessas que a encontrada nos camundongos, mas o amostras está sendo realizada em padrão de imunomarcação no corpo celular fotomicroscópio de fluorescência (ZEISS de neurônios foi semelhante nas duas AXIO IMAGER. A1) visando identificar espécies. Em preparados totais, observou- marcações para RPTP ζ/fosfacana nos se imunorreatividade para RPTP gânglios do SNE, observando também se ζ/fosfacana em neurônios dos plexos do estão associadas com neurônios (corpo mioentérico e submucoso, tanto no corpo celular e prolongamentos) dos plexos celular como também nos prolongamentos submucoso e mioentérico. Será quantificado o número de neurônios entéricos que expressam RPTP ζ/fosfacana. Para avaliar o papel da RPTP ζ/fosfacana em neurônios entéricos, serão utilizados

Figura 1 – A) marcação com anti-PGP9.5 e anti-RPTP/Fosfacana em corte de intestino humano (40X). B) marcação com anti-β-tubulina e anti-RPTP/Fosfacana em intestino de camundongos camundongo (40X).

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dos neurônios evidenciados pelo anti- neurons and neural cell-adhesion molecules, is PGP9.5 e anti-β-tubulina. an extracellular variant of a receptor-type protein tyrosine phosphatase. Biochemistry, v. 91, n.7, p. 2512-2516, 1994. Conclusão MOHEBIANY A. N. et al. Receptor-type tyrosine phosphatases ligands: looking for the needle in the haystack. The FEBS Journal Author manuscript; available in PMC, v. 280, Neurônios mioentéricos e issue 2, p. 388–400, 2014. submucosos de humanos e camundongos expressam RPTP ζ/fosfacana. Futuros ZIMMERMANN, D. R.; DOURS- estudos deverão revelar se a presença da ZIMMERMANN, M. T. Extracellular matrix of the RPTP ζ/fosfacana está associada com a central nervous system: from neglect to função destes neurônios no TGI. challenge. Histochemistry and Cell Biology, v.130, issue 4,p 635–653, 2008.

Agradecimentos

A CAPES pelo financiamento do projeto (processo: 88881.068190/2014-01).

Referências

AKTAR, R. et al Multiple funcional roles for the extracellular matrix glycoprotein tenascin X. Gastroenterology., v. 150, n. 4, p. s346, 2016.

DEN HERTOG J. et al Receptor protein- tyrosine phosphatase signaling in development. The International journal of Developmental Biology, v. 43, n 7 p. 723-733, 1999.

DWYER, C. A. et al Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase (RPTP)/Phosphacan with Cell-specific O- Mannosyl lycans in the Developing Brain. The Journal Of Biological Chemistry, v. 290, n. 16, p. 10256-10273, 2015.

HAYASHI, N. et al. Chondroitin sulfate proteoglycan phosphacan associates with parallel fibers and modulates axonal extension and fasciculation of cerebellar granule cells. Molecular and Cellular Neuroscience, v. 30, issue 3, p. 364-377, 2005.

MAUREL P. et al Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with

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ANÁLISE DO CÓLON DE RATOS SUBMETIDOS A DIFERENTE DURAÇÕES DE INFECÇÃO CAUSADA POR Toxoplasma gondii

Camila Cristina Alves Machado1, Marcelo Biodaro Góis2, Débora de Mello Gonçales Sant´Ana 2, Eduardo José de Almeida Araújo1. email: [email protected]

1 Universidade Estadual de Londrina/Departamento de Histologia, Brasil. 2 Universidade Estadual de Maringá/Departamento de Ciências Morfológicas, Brasil.

Palavras-chave: Toxoplasmose, sistema nervoso entérico, plasticidade neural.

Resumo: As alterações provocadas pelo T. O Toxoplasma gondii é um coccídio gondii sobre o SNE ocorrem principalmente que se encontra presente em quase todo o durante a fase aguda da infecção. mundo. Este parasito tem tropismo por alguns tecidos, sobretudo o tecido nervoso. Estudos prévios demostram que o T. gondii Introdução: é capaz de causar alterações no sistema nervoso entérico (SNE). No entanto, pouco A toxoplasmose é uma doença de se conhece sobre essas lesões durante o ampla distribuição mundial causada pelo curso da infecção toxoplásmica. Sendo protozoário Toxoplasma gondii. Os felídeos assim, o objetivo deste projeto é verificar as são o hospedeiro definitivo deste parasito, alterações provocadas pelo T. gondii sobre enquanto os demais animais de sangue neurônios entéricos do cólon durante a quente são hospedeiros intermediários. A infecção aguda e crônica. Para isso, ratos transmissão do T. gondii pode acontecer Wistar machos serão distribuídos em por várias formas, sendo mais comum no grupos de acordo com o tempo de infecção: homem, a transmissão via ingestão de controle (n=5), 6h (n=5), 12h (n=5), 24h oocistos presentes em alimentos e água (n=5), 72h (n=5) e 30 dias (n=5). Os ratos já contaminados (JONES; DUBEY, 2012). foram inoculados com 5000 oocistos Normalmente, este protozoário parasita esporulados da cepa ME-49 e cólon foi seus hospedeiros sem trazer prejuízos coletado. Os cólons serão dissecados para maiores, porém, dependendo de sua cepa e obtenção de dois tipos de preparados totais: da susceptibilidade imunológica do um contendo o plexo submucoso (PS) e hospedeiro, a infecção pode desencadear outro contendo o plexo mioentérico (PM). alterações clínicas relevantes (HILL; Todos os preparados totais serão CHIRUKANDOTH; DUBEY, 2005). submetidos à técnica de Durante seu ciclo biológico, imunofluorescência para marcação pan- buscando atingir a circulação sistêmica, neuronal (HuC/HuD). este parasito atravessa o epitélio intestinal por intermédio de várias estratégias Neurônios VIPérgicos e colinérgicos serão (HARKER; UENO; LODOEN, 2015). A marcados em preparados totais contendo o intensa proliferação e invasão celular do PS. Neurônios nitrérgicos e colinérgicos parasita no intestino desencadeia um serão marcados em preparados totais processo inflamatório local. contendo o PM. Em todos os preparados Inflamações na parede intestinal são totais serão realizadas análises de uma das principais causas de perda de densidade populacional e morfométrica. neurônios do sistema nervoso entérico (SNE). Halliez e Buret (2015) consideram Hipótese: que o T. gondii é um exemplo de parasito que induz alterações no SNE, incluindo

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modificação da distribuição neuronal, Amostras do colón proximal destes mudanças nos níveis neuroquímicos e animais foram coletadas e fixadas em deficiência funcional. Estudos anteriores paraformaldeído 4% durante três horas. revelaram que a infecção por T. gondii é Cada cólon será microdissecado para capaz de causar alterações no SNE remoção da mucosa e submucosa, (Sugauara et al.,2009; Odororizzi et resultando na obtenção de dois tipos de al.,2010; Silva et al.,2011; Araújo et al., preparados totais: um contendo o plexo 2015; Trevizan et al.,2016), porém pouco se submucoso (PS) e outro contendo o plexo sabe como essas alterações ocorrem no mioentérico (PM). decorrer do tempo de infecção. Por isso, o objetivo deste projeto é verificar as Análise quantitativa e morfométrica alterações provocadas pelo T. gondii sobre Os preparados totais obtidos serão neurônios entéricos do cólon de ratos submetidos à técnica de durante a infecção aguda e crônica. imunofluorescência para marcação pan- neuronal (HuC/HuD). Neurônios VIPérgicos Justificativa e colinérgicos serão marcados em Como o T. gondii apresenta preparados totais contendo o PS. tropismo pelo sistema nervoso, nos últimos Neurônios nitrérgicos e colinérgicos serão anos a literatura descreve alterações marcados em preparados totais contendo o causadas por este parasito sobre neurônios PM. do SNE. A maioria dos estudos Para análise da densidade experimentais foi realizada na fase crônica populacional e morfométrica, trinta e duas e, pouco se sabe quando as lesões imagens de cada preparado total serão começam e se intensificam. Por isso, capturadas num fotomicroscópio de estudos avaliando lesões neuronais durante fluorescência (ZEISS AXIO IMAGER.A1) o curso da infecção se fazem necessários. utilizando-se a objetiva de 20X. Cada imagem obtida corresponde a 0,0014 cm2, Métodos totalizando uma área de análise de 0,0448 cm2 por rato. Os resultados de densidade Aprovação ética: populacional encontrados para cada rato Todos os procedimentos detalhados serão projetados para 1 cm2. a seguir foram aprovados pelo Comitê de Além disso, para cada animal, serão Ética no Uso de Animais da Universidade mensurados 200 corpos celulares de Estadual de Maringá (UEM), protocolo neurônios da população total e a mesma número 079/2013. quantidade de neurônios das subpopulações. Desenho experimental: Ratos Wistar machos foram Análise estatística distribuídos em grupos controle (n=5) e Os resultados serão analisados infectados: 6h (n=5), 12h (n=5), 24h (n=5), utilizando-se o Bioestat 5.3 software. Será 72h (n=5) e 30 dias (n=5). Os animais verificado o tipo de distribuição dos dados, infectados receberam oralmente um inóculo a variância e a comparação entre os grupos. de 5000 oocistos esporulados da cepa ME- Para todos os testes o nível de significância 49 de T. gondii suspendidos em 1 mL de será de 5%. salina estéril. Os ratos do grupo controle receberam apenas 1 mL de salina estéril por Resultados e Discussão gavagem. Todos os animais passaram por teste de aglutinação direta para verificar a Até o momento, observou-se que o presença de anticorpos anti-T. gondii. número de neurônios mioentéricos totais e nitrérgicos tenderam a decrescer no Obtenção das amostras: decorrer da infecção aguda: 6h, 12h e 24h, porém sem diferença estatisticamente

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significativa (p > 0,05), como demonstrado na Tabela 1.

Tabela 1 – Densidade populacional (em 1 cm2) de neurônios mioentéricos colônicos totais e nitrérgicos de ratos infectados com T. gondii. GRUPOS HuC/HuD nNOS

Controle 30223.2 (29040.2; 30758.9) 10223.2 (9933.0; 10625.0)

6 h 31205.4 (30982.1; 32388.4) 9732.1 (9174.1; 10111.6)

12 h 29330.4 (27075.9; 29821.4) 8169.6 (7700.9; 10000.0)

24 h 24531.3 (24263.4; 25312.5) 7589.3 (6919.6; 10580.4)

Conclusão host. Parasite Immunology, 2015, 37, 141- 149. Resultados preliminares do projeto HILL, D. E.; CHIRUKANDOTH, S.; DUBEY, demonstram que durante a infecção aguda J. P. Biology and epidemiology of (6h, 12h e 24h) causada por oocistos da Toxoplasma gondii in man and animals. cepa ME-49 de T. gondii não se observa Anim Health Res Rer. 2005 Jun; 6(1): 41- alterações de densidade populacional no 61. cólon de ratos. JONES, Jeffrey L.; DUBEY, J. P. Foodborne Agradecimentos Toxoplasmosis. Clin Infect Dis 2012; 55: 845–851 À Coordenadoria de ODORIZZI, Leandro et al. Quantitative and Aperfeiçoamento de Pessoal de Nível morphometric changes of subpopulations of Superior (CAPES) pela bolsa de mestrado myenteric neurons in swines with da primeira autora e pelo financiamento do toplasmosis. Autonomic Neuroscience: projeto. Basic and Clinical. 155 (2010) 68-72. SILVA, Lilia S. et al. Toxoplasma Gondii: Referências Myenteric neurons of intraperitoneally ARAÚJO, E. J. et al. Toxoplasma gondii inoculated rats show quantitative and causes death and plastic alteration in the morphometric alterations. Experimental jejunal myenteric plexus. World Journal of Parasitology. 129 (2011) 5-10. Gastroenterology. April 28, 2015. v. SUGAUARA, Elaine Y. Y. et al. Hypertrophy 21(16): 4829-4829. of the neurons in the ileum of rats infected HALLIEZ, Marie C. M.; BURET, André G. with cysts of Toxoplasma gondii (genotype Gastrointestinal Parasites and the Neural II). Acta Scientiarum. Biological Controlo f Gut Functions. Frontiers in Sciences. Maringá, v. 31, n. 2, p. 195-201, Cellular Neuroscience. November 2015. 2009 v.9, Article 452. TREVIZAN, Aline R. et al. Kinetics of acute HARKER, K. S.; UENO, N.; LODOEN, M. B. innfection with Toxoplasma gondii and Toxoplasma gondii dissemination: a histopathological changes in the duodenum parasite´s journey through the infected of rats. Experimental Parasitology. 165 (2016) 22-29.

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ESTUDO COMPARATIVO DA FISIOPATOLOGIA NO ESCORPIONISMO INDUZIDO POR PEÇONHAS DOS Tityus serrulatus E Rhopalurus rochai

Jackson Gabriel Miyamoto1, Fabio Henrique Kwasniewski1 e-mail: [email protected] 1Universidade Estadual de Londrina/Departamento de Ciências Patológicas Palavras-chave: Tityus serrulatus, Rhopalurus rochai, fisiopatologia pulmonar. Resumo Verificar a capacidade da peçonha do R. rochai de induzir alterações O escorpionismo é importante pulmonares, comparando-as com as ações assunto de saúde pública no Brasil, e em da peçonha do T. serrulatus. vários outros países, sendo os escorpiões da família Buthidae envolvidos nos Introdução acidentes de importância médica. No Brasil, os principais escorpiões dessa família são O escorpionismo é um problema de do gênero Tityus, cujos representantes saúde pública de países na América causam a maioria dos acidentes. Nesses Central, África e Ásia e na América do Sul acidentes, quando graves, são encontradas (onde o Brasil é o maior acometido), com o manifestações sistêmicas e possivelmente número de acidentes anuais no mundo edema pulmonar, relacionado ao óbito dos excedendo os 1,2 milhões (CHIPPAUX; pacientes especialmente quando as vítimas GOYFFON, 2008). Entre as 16 famílias de são crianças. Sendo o Tityus serrulatus o escorpiões descritas, os acidentes de principal desencadeador de acidentes importância médica (escorpionismo) são graves no Brasil, a maioria dos estudos é originados pelos representantes da família desenvolvida com sua peçonha. Buthidae devido às suas potentes toxinas Experimentalmente foi demonstrado que a (LORET; HAMMCOCK, 2001). Na fauna peçonha desse escorpião, e de outros da brasileira os representantes Buthidae família Buthidae, desencadeiam inflamação envolvidos em acidentes são geralmente do sistêmica e pulmonar, tendo sido descrito gênero Tityus, figurando os T. serrulatus edema pulmonar, leucocitose e migração (Lutz & Mello 1922) entre os que possuem de leucócitos para os pulmões. Os as peçonhas mais potentes da América Rhopalurus rochai pertencem à Latina. Os T. serrulatus são os mais escorpiofauna brasileira e são da família amplamente distribuídos, encontrados em Buthidae, ocupando região de cerrado todas as regiões do país (SOUZA et al., pouco habitada; não há estudos 2009). Devido ao fato de que o T. serrulatus epidemiológicos ou experimentais acerca ser o mais comumente associado aos casos da atividade biológica sua peçonha no graves de envenenamento, deu-se maior aparelho respiratório. Nesse projeto será importância ao estudo das ações de sua estudada a fisiopatologia experimental com peçonha. Sendo assim, dados sobre a ação a peçonha dos T. serrulatus e do R. rochai das peçonhas de outros animais da avaliando parâmetros de lesão pulmonar, escorpiofauna brasileira são escassos ou como o aumento da permeabilidade inexistentes; não podemos descartar que vascular e a hemorragia, bem como o outros escorpiões de nossa fauna possam influxo de células inflamatórias (neutrófilos desencadear acidentes graves. Ainda que e monócitos), e resposta sistêmica os casos devam ser comunicados, existe (mobilização de leucócitos para o sangue). subnotificação e a identificação do animal causador do acidente nem sempre é Hipótese possível.

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As alterações inflamatórias comitê de ética, e parte de um projeto desencadeadas pelas peçonhas podem ser (número 08690; CEUA/UEL número locais ou sistêmicas, culminando em óbito 16583.2013.29) em execução, ambos sob devido o edema pulmonar e choque cardio- coordenação do orientador desse trabalho respiratório (BUCARETCHI et al., 1995), (Fábio H. Kwasniewski, Departamento de também presentes no escorpionismo Ciências Patológicas do CCB/UEL). experimental com a peçonha do T. serrulatus (FREIRE-MAIA; MATOS, 1993). Métodos Devido às similaridades com a síndrome da As peçonhas dos T. serrulatus angústia respiratória aguda (SARA), (pTs) e R. rochai (pRr) foram cedidas patologia pulmonar inflamatória aguda liofilizadas pelo Laboratório de Venenos desencadeada por uma série de fatores e com letalidade elevada, foi proposto que o do Instituto Butantan, armazenadas em o termo edema pulmonar no escorpionismo -20 C, no momento do uso serão fosse abandonado em favor de “síndrome diluídas em salina apirogênica e da angústia respiratória desencadeada pela administradas por via intravenosa (iv); a peçonha de escorpião” (D´SUZE et al., pTs na dose de 200 µg/kg e a pRr nas 1999). Os eventos iniciais da SARA são o doses de 200 e 400 µg/kg. Como grupos aumento da permeabilidade alvéolo-capilar controles serão utilizados ratos nos e o infiltrado de neutrófilos; o primeiro já foi quais apenas salina isotônica identificado em uma vítima fatal do T. apirogênica. Os ratos machos (200 a serrulatus (AMARAL et al., 1994) e 250 g) serão cedidos pelo biotério da experimentalmente (FREIRE-MAIA; UEL e permanecerão com água e ração MATOS 1993), e o segundo tem sido descrito experimentalmente (COELHO et ad libitum e ciclo claro/escuro de 12 al., 2007). horas. O aumento da permeabilidade O projeto busca investigar em vascular será avaliado através do modelo de escorpionismo experimental no extravasamento do corante azul de Evans, rato, efeitos da peçonha do R. rochai administrado iv (20 mg/kg) em conjunto com comparando-os aos da peçonha do T. a peçonha (pTs ou pRr). Passados 30 serrulatus. minutos os animais serão sacrificados por inalação de CO e exsangüinados. A seguir Justificativa 2 a circulação pulmonar será perfundida com Importante em nosso país, o salina (25 mL/min) através de uma cânula escorpionismo é relativamente pouco inserida no tronco pulmonar. O aparelho estudado. Logo, os resultados contribuirão respiratório será retirado e a traquéia, para desvendar o potencial toxicológico da brônquios externos e internos e o peçonha dos R. rochai, que têm potencial parênquima pulmonar serão isolados e de causar acidentes de importância médica, divididos em dois fragmentos: um colocado visto que segundo Chippaux e Goyffon em formamida para a extração do azul de (2008), escorpiões Buthidae que excedam 5 Evans e o outro acondicionado na estufa cm (caso do R. rochai) devem ser (60oC por 48 horas) para determinação da considerados potenciais causadores desse razão peso seco/peso úmido. Após 24 tipo de acidente. Além disso, os resultados horas a formamida será coletada e aumentarão o conhecimento acerca do distribuída em poços de placas de 96 poços escorpionismo por T. serrulatus. com água destilada e a absorbância determinada por espectrofotometria 630 O estudo proposto engloba o projeto nm. (número 10426; CEUA/UEL 19958.2016.54) aprovado no departamento A hemorragia será avaliada pela e pelo CCB e aguardando parecer do concentração de hemoglobina tecidual,

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dosada pelo método colorimétrico da Referências cianometahemoglobina. Após a pesagem dos órgãos, serão colocados em líquido de AMARAL, C.F.S. et al. Scorpion sting-induced pulmonary oedema: evidence of increased Drabkin; após 24 horas o sobrenadante alveolocapillary membrane permeability. será recolhido e a absorbância determinada Toxicon: Official Journal of the International (em 540 nm). Society on Toxinology, v. 32, n. 8, p.999- 1003, 1994. O número e a identificação dos leucócitos serão avaliados 4 e 24 horas BUCARETCHI, F. et al. A comparative study of após o envenenamento. Uma alíquota de severe scorpion envenomation in children sangue será colocada em líquido de Turk caused by Tityus bahiensis and Tityus para a contagem de células totais, e outra serrulatus. Revista do Instituto de Medicina será utilizada para contagem diferencial Tropical de São Paulo, v. 37, n. 4, p.331-336, realizada por esfregaço corado com 1995. Giemsa. CHIPPAUX, J.P.; GOYFFON, M. Epidemiology of scorpionism: a global appraisal. Acta Nos mesmos tempos acima tropica, v.107, n. 2, p. 71-9, 2008. avaliaremos a migração de leucócitos aos pulmões, coletados por lavado bronco- COELHO, F.M. et al. Platelet activating factor alveolar (LBA). O LBA será centrifugado e o receptors drive CXC chemokine production, botão celular ressuspenso; uma alíquota neutrophil influx and edema formation in the será diluída em líquido de Turk e outra lungs of mice injected with Tityus serrulatus venom. Toxicon: Official Journal of the alíquota centrifugada em citocentrífuga e a International Society on Toxinology, v. 50, n. lâmina submetida à coloração com Giemsa. 3, p.420-427, 2007. A presença de leucócitos no interstício pulmonar, será avaliada pela dosagem de DINIZ, C.R. et al. Effect of scorpion venom from mieloperoxidase – MPO (evidenciando Tityus serrulatus (Tityustoxin) on the neutrófilos) e N-acetil-β-D-glicosaminidase acetylcholine release and fine structure of the nerve terminals. Experientia, v. 30, n. 11, (macrófagos). p.1304-1305, 1974. Resultados Esperados D’SUZE, G. et al. Tityus discrepans venom Alterações das vias respiratórias de produces a respiratory distress syndrome in ratos no envenenamento com ambas rabbits through an indirect mechanism. Toxicon: Official Journal of the International peçonhas: permeabilidade vascular Society on Toxinology, v. 37, n. 1, p.173-180, (extravasamento de azul de Evans), 1999. hemorragia (hemoglobina tecidual), mobilização de leucócitos para o espaço FREIRE-MAIA, L.; MATOS, I.M. Heparin or a aéreo, interstício pulmonar e sangue dos PAF antagonist (BN 52021) prevents the acute animais envenenados. pulmonary edema induced by Tityus serrulatus scorpion venom in the rat. Toxicon: Official Além disso, os resultados poderão Journal of the International Society on ser interpretados de modo comparativo, Toxinology, v. 31, n. 9, p.1207-1210, 1993. permitindo melhor entendimento do LORET, E.; HAMMOCK, B. Structure and escorpionismo. Isso pode ser importante na neurotoxicity of venoms. In: BROWNELL, P., possível formulação de ações aplicáveis POLIS, G. (Eds.). Scorpion biology and aos pacientes vítimas do escorpionismo research. New York: Oxford University Press, (especialmente nos casos moderados a 2001. p. 204-233 graves) sejam essas ações adjuvantes ou SOUZA, C.A.R. et al. On the Tityus stigmurus não ao tratamento atual, que consiste na complex (Scorpiones, Buthidae). Zootaxa, utilização de soro hiperimune, que pode 1987, p. 1-38, 2009. nem sempre estar disponível para uso.

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ESTABELECIMENTO DA DIFERENÇA CRITICA EM PARÂMETROS DE ESTRESSE OXIDATIVO UTILIZANDO PROTOCOLO DE EXERCÍCIO EXCÊNTRICO

Natália Almeida de Barros1, Thâmara Alves1, Rubens Cecchini1, Alessandra Lourenço Cecchini Armani1, Gareth Davison2, Flávia Alessandra Guarnier1 e-mail: [email protected]

1Universidade Estadual de Londrina/Departamento de Patologia 2University of Ulster/North Ireland, UK

Palavras-chave: estresse oxidativo, diferença crítica, exercício excêntrico determinação colorimétrica da atividade da superóxido dismutase e da catalase, Resumo quimiluminescência estimulada por t-butil

hidroperóxido, glutationa total e glutationa Apesar de o exercício intenso estar oxidada. Espera-se, com este estudo, tradicionalmente associado com acúmulo elucidar a importância de se distinguir a estatisticamente significante de marcadores significância biológica da significância sistêmicos de estresse oxidativo, ainda é estatística quando o estresse oxidativo necessário que se determine se a resposta induzido pelo exercício é avaliado. ao estresse oxidativo possui significância biológica. Então, para avaliar a significância Hipóteses: biológica de alguns marcadores de estresse oxidativo frequentemente utilizados para resposta sistêmica ao exercício em sangue 1 – O exercício aumenta significativamente de humanos, será calculada a diferença o estresse oxidativo; crítica destes parâmetros em 20 voluntários 2 – A magnitude da mudança após o entre 18 e 28 anos. Para que o conceito seja exercício excêntrico excede o valor da provado, os mesmos parâmetros serão diferença crítica para cada parâmetro de avaliados nos mesmos indivíduos após um estresse oxidativo avaliado. protocolo de exercício conhecido por promover estresse metabólico e lesão muscular. A diferença crítica será Introdução determinada no plasma através da quantificação colorimétrica das substâncias reativas ao ácido tiobarbitúrico, Muitos estudos realizados na malondialdeído por cromatografia líquida de literatura têm sugerido o aumento da alta performance, proteína carbonílica pela suscetibilidade a lesões por radicais livres técnica de imunoensaio enzimático em várias doenças (DAVISON, et al. 2012). (ELISA), quimiluminescência estimulada Estes estudos comumente possuem o por t-butil hidroperóxido, glutationa total, e objetivo de descrever o estado metabólico e produtos avançados de oxidação de bioquímico em grupos saudáveis ou protéinas. Para determinação no eritrócito portadores de alguma doença, além de serão utilizadas as técnicas de examinar os efeitos de intervenções como,

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muito frequentemente, o exercício sobre a ocorrência de mudanças em (DAVISON, et al., 2012). qualquer fenômeno fisiológico, bioquímico ou patológico (DAVISON, et al., 2012). O exercício resulta na produção de espécies reativas de oxigênio(ERO) e O conceito e importância da nitrogênio(ERN), e consequentemente em diferença crítica é bem reconhecido na estresse oxidativo. Este efeito é observado bioquímica clínica, mas frequentemente em todos os tipos de exercício e consistente negligenciado no campo da bioquímica do em modelos experimentais, em exercício. Há poucos dados de referência delineamentos com humanos, em para metabólitos quantificados nas análises diferentes tipos celulares, fluidos corporais de estresse oxidativo, já que os resultados e tecidos (LEWIS, et al., 2016). As ERO e podem variar bastante intra-indivíduo, intra- ERN servem como importantes moléculas ensaio e entre indivíduos. sinalizadoras da biogênese mitocondrial, Tendo em vista o exposto, este transporte de glicose, e da hipertrofia trabalho se propõe a (a) determinar a muscular (LEWIS, et al., 2016), tendo um diferença crítica de parâmetros de estresse papel fundamental no processo de oxidativo frequentemente utilizados na adaptação ao exercício. No entanto, rotina de laboratórios experimentais; e (b) conclusões sólidas em humanos são comparar os valores de diferença crítica difíceis pela variação nos resultados obtidos nos parâmetros de avaliação do encontrados. No exercício, na maior parte estresse oxidativo antes e depois de um das vezes, o sangue é o material de escolha protocolo de exercício conhecido por para a análise por possibilitar uma avaliação promover estresse metabólico e lesão sistêmica do estresse metabólico. muscular. O estado metabólico de um indivíduo é representado por medidas Justificativa obtidas em um período específico do dia; no entanto, possui forte influência do ritmo circadiano, possuindo variações importantes durante o ciclo (DAVISON, et Como exposto, existem muitas al., 2012). Com isso em mente, alguns técnicas utilizadas para quantificar a estudos têm chamado atenção para o fato resposta ao estresse oxidativo, de que algumas quantificações variam de sistemicamente. No entanto, a resposta indivíduo para indivíduo, com a maioria dos sistêmica ao estresse oxidativo frente ao indivíduos apresentando flutuações em mesmo protocolo de exercício ainda sofre medidas repetidas (DAVISON, et al., 2012). de inconsistência quando diversos De acordo com Fraser & Frogarty (1989), trabalhos são comparados, na literatura. Diferença Crítica é a mudança que deve Esta inconsistência pode ser provocada ocorrer em um parâmetro antes de uma pela falta de conhecimento sobre a verdadeira mudança biológica ser atingida. variabilidade biológica dos métodos Enquanto uma resposta ao exercício pode aplicados. Desta forma, torna-se promover, matematicamente, a mudança necessário, antes de qualquer avaliação de um metabólito (p<0,05), a magnitude sistêmica em modelos de exercício, o desta diferença pode ser biologicamente estabelecimento da diferença crítica para insignificante. Desta forma, uma clara que a significância matemática seja distinção entre significância biológica e diferenciada da significância biológica. significância estatística deve ser determinada antes de qualquer conclusão

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Métodos colorimétrica de glutationa total e glutationa oxidada.

Após todas as coletas e cálculo de Fase 1: todos os resultados, a diferença analítica Após a aprovação pelo Comitê de (CVA), a variação biológica (CVB), e a Ética em Pesquisa envolvendo Seres diferença crítica (DC) serão calculadas Humanos, da Universidade Estadual de pelas fórmulas descritas em (Davison et al., Londrina, serão recrutados 20 adultos 2012). jovens, do sexo masculino, com idade entre

18 e 28 anos. Fase 2: Os participantes serão informados sobre os procedimentos e objetivo da Os mesmos indivíduos serão pesquisa e após concordarem, assinarão novamente recrutados para o teste de um termo de consentimento livre e estresse metabólico pelo exercício. Os esclarecido, em que ficará assegurada a participantes realizarão um protocolo de privacidade dos mesmos. Os indivíduos exercício envolvendo um total de 100 saltos serão orientados a se abster do uso de de uma altura de 60 cm. Após aterrissarem, qualquer antioxidante por 6 semanas antes eles serão encorajados a saltar da fase experimental, e também instruídos verticalmente com a máxima força que a refrear a prática de exercícios, além do puderem. Serão realizadas 5 séries de 20 consumo de cafeína e álcool por 24h, saltos, com 10 segundos de intervalo entre mantendo suas dietas diárias padrão. cada salto e 2 minutos de descanso entre cada série. Já foi assegurado que este

protocolo produz dano muscular expressivo Protocolo Experimental: em indivíduos não treinados. Os exercícios serão realizados no Laboratório de Após orientação para realização de Fisiopatologia das Adaptações Musculares. 12 horas de jejum, e consumo, pela manhã O sangue será colhido antes, de um kit contendo uma refeição padrão imediatamente depois e após 1h do término (recebido na véspera), os sujeitos deverão da atividade. comparecer ao local de realização do estudo, onde terão peso e estatura Os mesmos parâmetros de estresse registrados. Amostras de sangue serão oxidativo usados para o cálculo da diferença colhidas por profissional habilitado, a cada crítica serão analisados. hora (das 9:00 às 17:00 do mesmo dia), a partir da primeira coleta, considerada basal (volumes de 5mL). Análise estatística (ambas as fases): Eritrócitos e plasma serão utilizados Os dados serão analisados usando para a análise de estresse oxidativo, na estatística para dados paramétricos após avaliação do plasma serão utilizadas as confirmação matemática de distribuição tecnicas: TBARS, MDA, ELISA, QL, GT, normal pelo teste de Shapiro-Wilk. Os AOPP. No eritrócito: avaliação colorimétrica vários momentos de coleta, no tempo, serão da atividade da superóxido dismutasee da analisados por One-way ANOVA com teste catalase, quimiluminescência estimulada a posteriori de Tukey. As coletas pré e pós por t-butil hidroperóxido, avaliação exercício serão avaliadas usando teste t de student para dados pareados. Alfa será

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estabelecido em p<0,05 (95% do intervalo de confiança), e todos os valores serão Coordenação de Pessoal de Nível descritos por meio da média ± desvio Superior; Universidade Estadual de padrão. Londrina.

Referências Resultados Esperados

DAVISON G. W, ASHTON T, MCENENY J, Encontrar quais dentre as técnicas YOUNG I, DAVIES B, BAILEY D M. Critical para verificar o estresse oxidativo são mais difference applied to exercise-induced oxidative sensíveis para detectar mínimos valores stress: the dilemma of distinguishing biological from statistical change. Journal Physiol que sejam não apenas estatisticamente Biochem, 68, 377–384, 2012. significantes, mas clinicamente relevantes. LEWIS N A, NEWELL J, BURDEN R, Na segunda fase desse estudo, será HOWATSON G, PEDLAR C. Critical Difference aplicado um protocolo de exercício and Biological Variation in Biomarkers of excêntrico com objetivo de induzir níveis de Oxidative Stress and Nutritional Status in estresse oxidativo no sangue e espera-se Athletes. Journal Plos One, march, 2016. com isso, encontrar valores que FRASER C. G, FOGARTY Y. Interpreting ultrapassem a diferença crítica nas técnicas laboratory results. Journal Bm ,298, p.1659, que estão sendo testadas e dessa forma 1989. estabelecer qual a mais sensível.

Agradecimentos

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ESTRESSE OXIDATIVO E PERFIL INFLAMATÓRIO SISTÊMICO NA DOENÇA ARTERIAL OCLUSIVA PERIFÉRICA (DAOP) EM HUMANOS

Paola Gomes Benício Souza1; Rubens Cecchini1

[email protected]

1Universidade Estadual de Londrina/Departamento de Ciências Patológicas, Londrina, Paraná, Brasil.

Palavras-chave: estresse oxidativo, inflamação, doenças vasculares periféricas. isquemia e reperfusão induzido pela DAOP os níveis de estresse oxidativo, perfil de Resumo citocinas inflamatórias sistêmicas e biomarcadores de lesão celular sistêmica. Alterações nesses níveis poderão estar A Doença Arterial Oclusiva Periférica associadas à redução da capacidade física (DAOP) é uma doença crônica responsável e funcional em pacientes com DAOP. por alto grau de morbidade e mortalidade principalmente em homens com idade mais avançada. Estudos mostram que pacientes Hipótese portadores desta doença, tem sua capacidade física e funcional reduzidas, cuja causa é parcialmente representada pela redução do Pacientes com DAOP aporte de sangue em membros inferiores e apresentam aumento no estresse oxidativo disfunção mitocondrial. Todavia, evidências e perfil pró-inflamatório, alterações estas obtidas em modelos experimentais de que levam a uma síndrome de resposta isquemia e reperfusão demonstram a inflamatória sistêmica. ocorrência de lesões sistêmicas. O estresse oxidativo desempenha um papel central no desenvolvimento da síndrome da resposta Introdução inflamatória sistêmica. A DAOP mimetiza um modelo contínuo de isquemia e reperfusão quando se estabelece a condição atividade física/repouso intitulada A Doença Arterial Oclusiva Claudicação Intermitente. O estudo será Periférica (DAOP) é uma condição em que feito com indivíduos divididos em grupo ocorre o estreitamento e endurecimento das controle e grupos com DAOP de acordo com artérias que transportam o sangue para os a classificação de Rutherford. membros inferiores do corpo, como as A partir de amostras biológicas serão pernas e os pés conforme a Sociedade dosados padrões inflamatórios, perfis Brasileira de Angiologia e de Cirurgia metabólicos e oxidativos. Assim, nosso Vascular Regional São Paulo (2016) sendo propósito é investigar a partir do modelo predominantemente decorrente de

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fenômenos ateroscleróticos sistêmicos e O estresse oxidativo causa lesões atingindo com mais frequência homens na progressivas, e possivelmente, provoca faixa de 50 a 70 anos, fumantes, alterações nos mecanismos de sinalização hipertensos, sedentários e com celular que hipoteticamente podem estar hiperlipidemia (DIRETRIZES SBACV, ligadas aos defeitos mitocondriais já 2015). demonstrados e expressão de citocinas pró- inflamatórias (PIPINOS, 2008b) A claudicação intermitente (CI) é o principal sintoma da DAOP, caracterizada A geração de lesão oxidativa na pela dor que ocorre nos membros acome- isquemia-reperfusão corrobora com a nossa tidos pela doença durante a caminhada, hipótese de que em pacientes com DAOP onde a demanda sanguínea é maior e cessa ocorre estresse oxidativo e resposta pró- rapidamente com o repouso (demanda inflamatória sistêmica, e que esta condição menor de sangue) sendo que esse sintoma leva a redução do desempenho físico e gera uma importante limitação de capacidade funcional geral do paciente com locomoção, ocasionando redução nos esta doença. níveis de atividade física dos pacientes

(BARBOSA, et al., 2012). Esta condição, está relacionada a um pior prognóstico da Justificativa doença, o que pode aumentar a morbidade e mortalidade dos pacientes em decorrência de complicações como infarto do miocárdio A DAOP, é uma doença que afeta 10 ou acidente vascular cerebral isquêmico milhões de pessoas, principalmente idosos. (DURAZZO et al., 2005). Por ano, 260.000 procedimentos de A classificação de Rutherford é revascularização são realizados e mais de utilizada pela Sociedade Internacional de 100.000 amputações são feitas somente Cirurgia Vascular e é realizada com base na nos Estados Unidos de acordo com Pipinos categoria funcional da DAOP a partir da (2008b). Este quadro não deve ser muito clínica médica e consiste em categoria I diferente no Brasil. Com o aumento da como claudicação leve, a categoria II como população em idade mais avançada no claudicação moderada, a categoria III como mundo, DAOP representa significante e claudicação severa e a categoria IV dor crescente causa de morbidade e isquêmica em repouso (VIDAL, 2009; mortalidade. Assim, um estudo desta DIRETRIZES SBAC, 2015). doença em humanos, procurando compreender os mecanismos que levam à O duplo comprometimento muscular uma redução da capacidade física e na DAOP, ou seja, reduzido aporte de funcional, com perspectivas de evidenciar oxigênio provocado pela demanda e marcadores sanguíneos e indicação de reduzida capacidade mitocondrial de terapêuticas coadjuvantes, que reduzam a metabolizar o oxigênio segundo Garbaisz e mortalidade e morbidade é de fundamental colaboradores (2014), produz uma importância. condição muito particular de geração de espécies reativas de oxigênio (ERO) e espécies reativas de nitrogênio (ERN) Métodos promovendo um desequilíbrio entre espécies pró-oxidantes e antioxidantes (PIPINOS, 2008b) Delineamento

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O presente estudo está em quantificação de Peroxinitrito por submissão ao Comitê de Ética (CEP-UEL). Luminescência e avaliação do consumo de A amostra será composta de: GRUPO oxigênio eritrocitário e plasmático. CONTROLE com 50 indivíduos do sexo masculino com mais de 65 anos sem doença inflamatória sistêmica comprovada. Resultados Esperados GRUPO CLAUDICAÇÃO INTERMITENTE com 25 indivíduos apresentando níveis I e II na classificação de Rutherford, sem outra A avaliação do estresse oxidativo e doença inflamatória sistêmica comprovada. fatores inflamatórios sistêmicos, pode GRUPO CLAUDICAÇÃO INTERMITENTE contribuir para estabelecimento de com 25 indivíduos apresentando níveis III e marcadores de lesão sistêmica no sangue IV na escala de Rutherford sem outra de pacientes com DAOP com a finalidade doença inflamatória sistêmica comprovada. de um melhor conhecimento dos mecanismos da doença. Obtenção das amostras Agradecimentos Serão coletadas amostras de urina e do sangue periférico serão obtidas Ao meu orientador Rubens Cecchini, amostras de plasma, a partir de punção pelo suporte, e companheirismo. venosa. As coletas serão realizadas no Hospital Universitário de Londrina–HU A Fundação Araucária, pelo apoio e concomitantemente com os exames pré- incentivo. definidos em consulta médica.

Referências Análise das amostras

BARBOSA, J. P. et al. Nível de atividade física em Analisaremos o perfil bioquímico indivíduos com doença arterial periférica: uma (HDL, LDL, Colesterol Total e Triglicerídeo), revisão sistemática. Jornal Vascular as enzimas Creatina-quinase (CK) e Brasileiro. Escola Superior de Educação Física da Universidade de Pernambuco (UPE) – Recife (Pe), Desidrogenase Lática (LDH) e dosaremos Brasil., p. 22-28. jan. 2012. os níveis séricos de potássio. O perfil inflamatório será traçado através da COWLED, P. A. et al. Simvastatim Plus Nitric dosagem de IL-6 e TNF-α por Elisa. A injúria Oxide Synthase Inhibition Modulates Remote renal será avaliada por quantificação Organ Damage Following Skleletal Muscle bioquímica de sódio e creatinina Ischemia - Reperfusion Injury. Journal Of plasmáticos juntamente com a Investigative Surgery. University Of determinação de nitrogênio ureico. Adelaide, p. 119-126. fev. 2008 Serão analisados diferentes parâmetros pró-oxidantes como quantificação de Diretrizes SBACV. DOENÇA ARTERIAL Malondialdeído (MDA) no plasma por PERIFÉRICA OBSTRUTIVA DE MEMBROS HPLC, quantificação dos metábolitos de INFERIORES DIAGNÓSTICO E óxido nítrico (NO) pela técnica de Griess; TRATAMENTO. Sociedade Brasileira de

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Angiologia e Cirurgia Vascular, São Paulo, Surgery, Nebrasca, v. 42, n. 2, p.101-112, p.1-33, gestão 2012-2015. 2008b.

DURAZZO, A. E. S., et al. Doença arterial SOCIEDADE BRASILEIRA DE ANGIOLOGIA E obstrutiva periférica: que atenção temos DE CIRURGIA VASCULAR REGIONAL SÃO dispensado à abordagem clínica dos pacientes? PAULO. Doença Arterial Obstrutiva Jornal Vascular Brasileiro. São Paulo, p. Periférica (DAOP). Disponível em: 255-264. ago. 2005.. . Acesso em: 12 out. 2016.

GARBAISZ, D. et al. Attenuation of Skeletal Muscle and Renal Injury to the Lower Limb VIDAL, L. Avaliação do sistema de following Ischemia - Reperfusion Using mPTP classificação de risco do pé, proposto Inhibition NIM-811. Plos One, Georgia, v. 9, n. pelo grupo de trabalho internacional 6, p.1-12, jun. 2014. sobre o pé diabético, Hospital da Polícia Militar de Minas Gerais, 2002-2007. 2009. 170 f. Dissertação (Mestrado) - Curso de PIPINOS, I. I. et al. The Myopathy of Peripheral Medicina, Universidade Federal de Minas Artherial Occlusive disease: Part I. Functional Gerais, Belo Horizonte - Minas Gerais, 2009. and histomorphological Changes and Evidence for Mitochondrial Dysfunction. Vascular And Endovascular Surgery, Nebrasca, v. 41, p.481-489, 2008a.

PIPINOS, I. I. et al. The Myopathy of Peripheral Artherial Occlusive Disease: Part 2. Oxidative Stress, Neuropathy, and Shift in Muscle Fiber Type. Vascular And Endovascular

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ESTUDO DOS NÍVEIS DE VITAMINA D E POLIMORFISMO VRD-FOKI EM PACIENTES COM CÂNCER COLORRETAL: CORRELAÇÃO COM O ESTADIAMENTO DA DOENÇA.

Priscilla Ferreira Crespo Gutierrez1, Rodrigo Cabral Luiz1 e-mail: [email protected]

1Universidade Estadual de Londrina/Departamento de Ciências Patológicas

Palavras-chave: Vitamina D, VDR-Fokl, câncer colorretal

pareados por idade, sexo e tipo de pele de acordo com a classificação de Fitzpatrick. Resumo Serão analisadas amostras de sangue periférico para a dosagem dos níveis de vitamina D plasmática e a presença ou O câncer colorretal (CCR) ausência do polimorfismo VDR-FokI. representa uma das principais causas de morte por neoplasia em todo mundo sendo o quinto câncer mais diagnosticado no Hipótese Brasil. Recentemente os baixos níveis séricos da vitamina D, têm sido relacionados com o aumento no risco do Existe diferença no padrão alimentar desenvolvimento do câncer colorretal. Os especifico de fonte de vitamina D bem como níveis séricos de vitamina D podem variar da exposição a luz solar em uma população de acordo com a cor da pele, dieta, estilo de brasileira de pacientes com câncer vida e exposição solar. Seus efeitos colorretal. Estas diferenças podem resultar biológicos são decorrentes da interação de no melhor ou pior da doença prognostico da sua forma ativa, o calcitriol, com o receptor doença em comparação com artigos de vitamina D, o VDR. Logo polimorfismos publicados em outros países, presentes no gene do VDR, também têm principalmente se levarmos em sido considerados fatores de interferência consideração o polimorfismo VDR-Fokl, que sobre a atividade fisiológica da vitamina D. deve estar em uma taxa particular na Dentre eles, o polimorfismo de maior população brasileira, devido à sua destaque é o VDR-FokI. Esta pesquisa tem miscigenação genética. como objetivo principal, determinar o nível de calcidiol em pacientes com diagnóstico clínico de câncer colorretal no Instituto de Câncer de Londrina e em controles sem o Introdução diagnóstico clínico da doença residentes na O câncer colorretal (CCR) mesma região geográfica. A amostra será representa uma das principais causas de composta de 80 a 120 pacientes morte por neoplasia em todo mundo sendo selecionados com diagnóstico de CCR e o o quinto câncer mais diagnosticado no mesmo número de controles saudáveis Brasil ocupando o terceiro lugar na região

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Sul. Mais de 95% dos cânceres colorretal apenas de 4 à 6 horas e além disso é são esporádicos, com forte influência encontrada em níveis extremamente baixos hereditária e de estilo de vida o que dificulta seu diagnóstico (HIBLER et (sedentarismo, dieta rica em gorduras e al, 2010). carnes vermelhas, pobre em fibras, etc.). Os efeitos biológicos do calcitriol são Recentemente os baixos níveis séricos da mediados pela interação com o receptor de vitamina D, têm sido relacionados com o vitamina D (VDR), que é um receptor de aumento no risco do desenvolvimento localização nuclear e atua como um fator de câncer de diversos tipos de câncer transcrição ao se ligar ao calcitriol. Ele incluindo o colorretal (LEE, 2011). regula 3% do genoma humano, incluindo Para os seres humanos a principal genes relacionados a regulação da fonte de vitamina D é a síntese endógena densidade mineral óssea, a secreção e (70 a 80%) que ocorre na pele a partir da sensibilidade da insulina. A ligação do conversão do 7-desidrocolesterol calcitriol ao VDR induz mudanças dependente da exposição à radiação conformacionais que permitem ao VDR ultravioleta. De forma que o tempo e tipo de formar um heterodímero com o receptor X exposição solar, bem como a localização de ácido retinóico (RXR) e também ligar-se geográfica e a cor de pele do indivíduo são a proteínas co-ativadoras transcripcionais, fatores que afetam os níveis séricos desta que atuam diretamente na proliferação e vitamina (HOLICK, 2008). Outra fonte de diferenciação celular (HIBLER et al, 2010). vitamina D é a alimentação (20 a 30%) onde Pesquisas demonstram que se observa a vitamina D3 em peixes polimorfismos no VDR afetam a resposta gordurosos de água fria (ex. atum e salmão) frente à vitamina D. Dentre os polimorfismos e a vitamina D2 em cogumelos comestíveis já identificados, o de maior destaque é o (HIBLER et al, 2010). No Brasil há VDR-FokI (rs2228570; C>T), que resulta na problemas na distribuição de renda da formação de uma proteína levemente população, de forma que a fonte principal de truncada com três aminoácidos a menos vitamina D é a exposição solar. (ALIMIRAH, 2011). Classicamente a função da vitamina D é restrita ao metabolismo ósseo e a Resultados de estudos regulação da concentração de cálcio epidemiológicos correlacionando a plasmático. Recentemente novas funções vitamina D com o câncer são têm sido atribuídas à vitamina D, dentre elas controversos, pois muitas vezes o podemos citar a redução de proliferação de polimorfismo do VDR e o estadiamento células epiteliais, a promoção da do câncer dos pacientes não é levado diferenciação de diversos tipos celulares, em consideração (ALIMIRAH, 2011; incluindo as células da mucosa colorretal MCCULLOUGH, 2009; VANDEWALLE, (WANG, 2005). Seu principal metabolito 2004; LEE, 2011; GARLAND, 2006). circulante é o calcidiol (25-hidroxi-vitamina D) e é a forma mais utilizada para avaliação Desta forma, a presente pesquisa irá laboratorial, uma vez que sua meia vida tem avaliar os níveis de vitamina D em pacientes uma duração de até três semanas com câncer colorretal no Instituto de Câncer diferentemente do calcitriol que é a sua de Londrina (ICL) e correlacionar com forma ativa (1,25-di-hidroxivitamina D), esta fatores alimentares, exposição solar e cor é produzida no fígado a partir do calcidiol da pele e a frequência do polimorfismo pela ação da CYP27B1 uma enzima da VDR-FokI com o estadiamento do tumor. família P450, porém, tem uma meia vida de

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Justificativa Fitzpatrick. Esses dados serão obtidos no período pré-cirúrgico, quando o paciente já estiver internado. O prontuário clínico será Evidencias cientificas associam um acessado para obter informações sobre o maior risco do câncer colorretal em estadiamento do tumor (classificação TNM). indivíduos com hipovitaminose D. Não Serão coletadas amostras de sangue em 1 existem pesquisas desta natureza tubo à vácuo com EDTA para coleta do realizadas no Brasil, um país com plasma. O plasma será utilizado para abrangente miscigenação, alimentação dosagem dos níveis plasmáticos da típica e um padrão de exposição solar que vitamina D (dosagem de calcidiol), que diferem das pesquisas até então serão determinados pelo método de publicadas. De forma que os resultados quimioluminescência, por meio da Elecsys obtidos na população brasileira contribuirão 2010, Roche® (MONTICIELO et al, com as divergências apontadas em 2012). Definiu-se como níveis séricos literatura científica. normais de calcidiol valores > 30 ng/mL, e deficiente quando < 30 ng/mL (HOLICK, 2008). A fração leucocitária será utilizada para extração do DNA, que será feita a Métodos partir do método convencional de salting- out. A genotipagem do polimorfismo VDR- FokI será realizada pela reação em cadeia Amostra da polimerase (PCR), a partir do DNA genómico, tampão de polimerase, primers e Para este estudo serão enzima de restrição especificos. Os primers selecionados de 80 a 120 pacientes com utilizados serão: 5’- diagnóstico de CCR, que aceitem participar AGCTGGCCCTGGCACTGACTC da pesquisa, do Instituto do Câncer de TGCTCT-3’ e 5’-ATGGAAACACCTTGCTT Londrina, Paraná. Serão excluídos deste CTTCTCCCTC-3’ (MONTICIELO et al, grupo pacientes etilistas e tabagistas. E 2012). para o grupo controle serão selecionados o mesmo número de indivíduos, com Os produtos da PCR serão equiparação de etnia, faixa etária, sexo e digeridos com a Fok I (Behringer exposição solar, sendo excluídos do grupo Mannheim) de acordo com as controle pessoas com histórico de câncer, indicações do fabricante. Os fragmentos tabagistas e etilistas. resultantes da digestão serão revelados, por meio de electroforese em Coleta e análise dos dados gel de agarose. O produto de amplificação não reconhecido pela Fok I apresenta 265 bp e será designado por Após a concordância de alelo F, enquanto que a presença do participação na pesquisa e assinatura do sítio de restrição da enzima (com a TCLE será aplicado um questionário para obtenção de duas bandas, uma de 196 coletar dados demográficos, etnia, sexo, bp e outra de 69 bp) será designado por idade, padrão de exposição solar, uso de alelo f, desta forma, após as análises as protetor solar, hábitos alimentares, estilo de amostras serão divididas em 3 grupos: o vida, consumo de alimentos fonte de de sujeitos homozigotos para o vitamina D, tabagismo, alcoolismo e cor da polimorfismo (f / f), de sujeitos sem o pele de acordo com a classificação de

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polimorfismo (F / F) e de sujeitos Lee J, Li H, Chan A, Hollis B, Lee I, Stampfer M. heterozigotos para o polimorfismo (F / f). Circulating levels of vitamin D and colon and rectal cancer: the Physicians Health Study and a meta-analysis of prospective studies. Cancer Resultados e Discussão Prev Res 2011; 4: 735-743

Esperamos encontrar correlações Monticielo O, Brenol J, Chies J, Longo M, Rucatti entre o nível plasmático de vitamina D e o G. The role of BsmI and FokI vitamin D receptor maior acometimento de câncer colorretal e gene polymorphisms and vitamin D in Brazilian patients com LES. 2012 Jan;21(1):43-52. Epub entre o polimorfismo de VDR e o nível 2011 Oct 12. plasmático de vitamina D. O que contribui para a prevenção e prognóstico do câncer colorretal. McCullough M, Bostick R, Mayo T. Vitamin D gene pathway polymorphisms and risk of colorectal, breast, and prostate cancer. Annu Referências Rev Nutr 29: 2009.

Alimirah F, Peng X, Murillo G, Mehta RG. Wang T, Tavera-Mendoza L, Laperriere D, Libby Functional significance of vitamin D receptor E, et al. Large-scale in silico and microarray- FokI polymorphism in human breast cancer based identification of direct 1,25- cells. PLoS One. 2011. dihydroxyvitamin D3 target genes. Mol Endocrinol. 2005.

Bouillon R, Carmeliet G, Verlinden L, et al. Vitamin D and human health: lessons from vitamin D receptor null mice. Endocr Rev.

2008;29(6):72-76.

Garland C, Garland F. Do sunlight and vitamin D reduce the likelihood of colon cancer? Int J Epidemiol. 2006; 35: 217–20.

Holick MF. Vitamin D: a D-Lightful health perspective. Nutr Rev. 2008; 66.

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AVALIAÇÃO DA REATIVAÇÃO DO CITOMEGALOVÍRUS EM PACIENTES COM SEPSE BACTERIANA EM UNIDADES DE TERAPIA INTENSIVA

Taylon Felipe Silva1, Ivete Conchon Costa1, Wander R. Pavanelli1, Idessânia Nazareth Costa1, Francine Nesello Melanda1, Lucy Megumi2, Zuleica Naomi Tanno3, Cintia Magalhães Carvalho Grion3 e-mail: [email protected]

1Universidade Estadual de Londrina/Departamento de Ciências Patológicas. 2Universidade Estadual de Londrina/Departamento de Microbiologia 3Universidade Estadual de Londrina/Centro de Ciências da Saúde/Clínica Médica

Palavras-chave: citomegalovírus, sepse, unidades de terapia intensiva.

intensiva. Portanto, para realização deste estudo serão avaliados pacientes nestas Resumo condições de saúde e sorologicamente positivos para o vírus. Serão coletadas O Citomegalovírus Humano (CMV) é amostras de sangue periférico para membro da família Herperviridae, com realização de diagnóstico sorológico taxas altas de infecção e a soro prevalência através de ensaio imunoenzimático, tem aumento na faixa etária de 40 a 49 anos antigenemia da proteína pp65 para avaliar a em cerca de 65%, podendo chegar a 91% reativação viral através de acima dos 80 anos. O contágio pelo CMV imunocitoquímica, dosagem de citocinas e ocorre pelo contato e a infecção é avaliação da carga viral por Reação em geralmente assintomática, mas pode Cadeia da Polimerase em tempo real, além manifestar-se semelhantemente a um dos aspectos clínicos pela análise de quadro de mononucleose infecciosa. A prontuários, visando analisar as variáveis e reativação do vírus tem sido amplamente possíveis correlações com a cinética associada à sepse bacteriana e imunológica dos pacientes. provavelmente resulta do processo inflamatório e do comprometimento temporário do sistema imune, induzindo respostas exageradas a infecções graves Hipótese que podem ser prejudiciais ao hospedeiro. Assim, a sepse bacteriana tem sido Devido à escassez de estudos que identificada como um fator de risco para a avaliem estes eventos em conjunto, a reativação de CMV em populações questão que surge é, quais características heterogêneas que se encontram em estado estão envolvidas na reativação do crítico de saúde. Diante do proposto este Citomegalovírus em pacientes com sepse estudo tem por objetivo analisar os internados em Unidades de Terapia principais aspectos fisiopatológicos Intensiva, qual a relação entre os perfis de associados à reativação do CMV em reposta imunológica Th1, Th2 e Th17 frente pacientes com quadro de sepse bacteriana a esse evento e quais os fatores que levam internados em unidades de terapia a uma alteração nas taxas de

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morbimortalidade desta população? Nossa afetados pela reativação de CMV. Contudo, hipótese é que os fatores imunológicos nos últimos 10 anos tem sido observado um presentes durantes os quadros de sepse aumento nas taxas de reativação de CMV estejam intimamente ligados a reativação em pacientes transplantados renais, do citomegalovírus e, consequentemente, à mesmo com a ausência dos mecanismos evolução clínica destes pacientes. típicos de imunossupressão comumente usados (PANNUTI, 1985 e PEREYRA,

2004). Além disso, tem crescido o número Introdução de evidências que infecções por CMV têm sido consideradas subestimadas em O Citomegalovírus Humano (CMV) é pacientes em estado crítico (AYRE, 2009). um vírus membro da família Herperviridae, Diante do proposto, este trabalho que possui uma dupla fita linear quando terá como objetivo analisar os principais ativo e que se torna circular em períodos de aspectos fisiopatológicos associados da latência (MOCARSKI, 1996, PANNUTI, reativação do CMV em pacientes com 2009 e CRHOUGH, 2009). A infecção com quadro de sepse bacteriana internados em CMV é comum e a soro prevalência unidades de terapia intensiva, analisar a aumenta a partir dos 40 em 65% chegando sorologia e a reativação do CMV nestes a 91% em pessoas com 80 anos ou mais pacientes, correlacionar às taxas de (STARAS et al., 2006). expressão de algumas das principais O material genético do vírus é citocinas presentes durantes os eventos de dividido em duas cadeias centrais, que sepse bacteriana e reativação de CMV e codificarão proteínas do capsídeo que estabelecer um perfil de morbimortalidade envolvem o genoma, proteínas do envelope que articule o quadro de sepse bacteriana viral e proteínas de matriz ou tegumento, com a reativação de CMV e cinética da principalmente fosfolipídeos pp150 e pp65, resposta imunológica, considerando-se os esta última sendo relacionada com o ciclo principais agravos no âmbito das unidades replicativo do vírus e, um alvo importante no de terapia intensiva. diagnóstico de infecção ativa do CMV em neutrófilos do sangue periférico (MOCARSKI, 1996). Assim como muitos Justificativa outros vírus, possuí a capacidade de manter seu material genético junto ao DNA do Aproximadamente um terço de hospedeiro em células sanguíneas, pacientes em estado crítico desenvolvem principalmente monócitos, mas sem reativação de citomegalovírus durante sua replicação ativa, estabelecendo um quadro estadia em Unidades de Terapia Intensiva e de infecção latente, que pode ou não ser a duração dessa permanência, bem como o reativada ao longo da vida. Portanto, tempo de ventilação mecânica, tornam-se distúrbios no equilíbrio entre o sistema mais prolongados em relação a pacientes imune e os vírus inativados é o gatilho para que não apresentam essa morbidade, reativação do CMV que pode resultar na caracterizando um maior agravo a saúde do associação com alta morbimortalidade em paciente e piores chances de sobrevivência paciente imunodeprimidos (TAYLOR, 2003 (KALIL e FLORESCU, 2009). Neste e HERFARTH, 2010). sentido, estudos que avaliem a correção entre sepse e reativação de citomegalovírus Geralmente, pacientes em estado são fundamentais para garantir a estes crítico em Unidades de Terapia Intensiva pacientes uma melhor chance de sobrevida, sem imunossupressão exógena não são

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principalmente quando se verifica a escassez de trabalhos na área, Resultados esperados especialmente os que avaliam cinética imunológica. Torna-se importante estabelecer quais características estão envolvidas na reativação do Citomegalovírus, qual a Métodos relação entre os perfis de reposta imunológica Th1, Th2 e Th2 frente a esse Para realização deste estudo serão evento e quais os fatores que levam a uma analisados paciente internados em UTIs alteração nas taxas de morbimortalidade que possuam quadro de sepse por 7 ou desta população. Assim sendo, este estudo mais dias e que tenham 18 anos ou mais. visa contribuir com uma análise dos Para aquisição das amostras, será coletado principais aspectos fisiopatológicos sangue venoso periférico dos participantes associados à reativação do CMV em através de punção venosa em dois tubos de paciente com quadro de sepse bacteriana 5 mL contendo EDTA em três momentos internados em unidades de terapia distintos após o diagnóstico da sepse, nos intensiva. dias 7, 14 e 21.

Para avaliar a soropositividade utilizaremos o método imunoenzimático Referências (ELISA) para detecção tanto de anticorpos IgG como de IgM. O teste direto de AYRE, K.; WARREN, B. F.; JEFFERY, K.; imunocitoquímica, para detecção do TRAVIS, S. P. The role of CMV in steroidresistant ulcerative colitis: a systematic antígeno pp65 em leucócitos do sangue review. J Crohns Colitis, v. 3, n. 3, p. 141-8, periférico aderentes em lamínulas usando o 2009. método streptavidina-biotina (Universal Dako LSAB®+kit; DAKO Japan, Kyoto, CROUGH, T; KHANNA, R. Immunobiology of Japan). O soro dos pacientes com sepse e human cytomegalovirus: from bench to bedside. reativação com CVM ou apenas com sepse Clin. Microbiol Ver, v. 22, n. 1, p. 76-98, 2009. serão utilizados para mensurar os níveis de citocinas do perfil Th1, Th2 e Th17, por meio HERFARTH, H. H.; LONG, M. D.; RUBINAS, T. do ensaio imunoenzimático C.; SANDRIDGE, M; MILLER, M. B. Evaluation (eBiosciences®, EUA). A análise da carga of a non-invasive method to detect cytomegalovirus (CMV)-DNA in stool samples of viral de CMV será realizada por análise da patients with inflammatory bowel disease (IBD): quantificação absoluta, através de PCR-real a pilot study. Dig Dis Sci, v. 55, n. 4, p. 1053-8, time. Para traçar o perfil de 2010. morbimortalidade serão escrutinados os dados clínicos e fisiopatológicos do MANSFIELD, S.; GRIEßL, M.; GUTKNECHT, paciente, como: tempo de internação, M.; COOK, C. H. Sepsis and cytomegalovirus: patologia ou intervenção cirúrgica inicial, foes or conspirators? Medical microbiology dias livres de ventilação mecânica, infecção and immunology, v. 204, n. 3, p. 431-437, que causou a sepse, presença de choque 2015. séptico, transfusão de concentrado de MOCARSKI, E. S.; KEMBLE, G. W.; LYLE, J. M.; hemácias, intervenções cirúrgicas, GREAVES, R. F. A deletion mutant in the human APACHE III (Acute Physiology and Chronic cytomegalovirus gene encoding IE1 (491aa) is Health Evaluation III) e SOFA (Sepsis- replication defective due to a failure in Related Organ Failure Assessment). autoregulation. Proceedings of the National

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PANNUTI, C. S.; VILAS-BOAS, L. S.; ANGELO, SUASSUNA, J. H.; LEITE, L. L.; VILLELA, L. H. M. J.; CARVALHO, R. P.; SEGRE, C. M. Prevalence of cytomegalovirus infection in Congenital cytomegalovirus infection. different patient groups of an urban university in Occurrence in two socioeconomically distinct Brazil. Rev Soc Bras Med Trop, v. 28, n. 2, p. populations of a developing country. Rev Inst 105-8, 1995. Med Trop, Sao Paulo, v. 27, n. 2, p. 105-7, 1985. TAYLOR, G. H. Cytomegalovirus. Am Fam PANNUTI, C. S. Citomegalovirose. In: Physician, v. 67, n. 3, p. 519-24, 2003. VERONESI, R.; FOCACCIA, R. Tratado de Infectologia. 4º ed: Ateneu; 2009. p. 363-71. KALIL, A. C., et al: Metaanalysis: The efficacy of strategies to prevent organ disease by PEREYRA, F.; RUBIN, R. H. Prevention and cytomegalovirus in solid organ transplant treatment of cytomegalovirus infection in solid recipients. Ann Intern Med, v. 143, p. 870–8, organ transplant recipients. Curr Opin Infect 2005. Dis, v. 17, n. 4, p. 357-61, 2004.

STARAS, S. A. S., et al. Seroprevalence of cytomegalovirus infection in the United States,

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AVALIAÇÃO DOS EFEITOS ESPINAIS DO MEDIADOR LIPÍDICO PRÓ-RESOLUÇÃO RESOLVINA D1 EM MODELO DE GOTA INDUZIDA POR CRISTAIS DE MONOURATO DE SÓDIO (MSU)

Tiago Henrique Zaninelli1, Stephanie Badaró Garcia1, Victor Fattori1, Kenji William Ruiz Myazawa1, Sergio Marques Borghi1, Rubia Casagrande2, Waldiceu Aparecido Verri Junior1 e-mail: [email protected]

1,2Universidade Estadual de Londrina/Departamento de Ciências Patológicas. 2Departamento de Ciências Farmacêuticas

Palavras-chave: artrite gotosa, mediadores lipídicos, resolvina D1.

tempos 1, 3, 5, 7 e 15h após o estímulo. As amostras de tecido serão coletadas para Resumo análise dos parâmetros inflamatórios através da atividade das enzimas mieloperoxidase e N-acetilglucosamina, e A artrite gotosa (AG) ou gota é dosagem de citocinas. A ativação glial será caracterizada por sinais típicos da analisada por imunofluorescência e RT- inflamação que afeta frequentemente qPCR. Além disso, será avaliada a adultos, com prevalência aumentada na expressão dos canais iônicos TRPV1 e população idosa. O quadro inflamatório é Nav1.8 no gânglio da raiz dorsal por RT- desenvolvido pela deposição de cristais de qPCR. Desta forma, será investigada a urato monossódico (MSU) nas articulações efeito farmacológico espinal do mediador decorrendo da hiperuricemia. A dor intensa lipídicos RvD1 em modelo de gota induzida e o edema são as principais queixas dos por MSU. pacientes. Atualmente, o tratamento para “ataques” agudos da gota se restringe à colchicina, anti-inflamatórios não Hipótese esteroidais, corticoides e antagonista de receptor de IL-1, que embora eficazes, apresentam alto custo e vários efeitos O presente estudo buscará avaliar colaterais, tornando o tratamento clínico se a RvD1 apresenta efeito anti-inflamatório limitado. Considerando o papel fundamental e atividade em modelo de gota induzida por da ativação espinal de células da glia cristais de MSU. Até o momento não (astrócitos e micróglia) na dor, o presente existem trabalhos descritos sobre o efeito projeto visa avaliar os mecanismos espinais da RvD1 neste modelo de AG. do mediador lipídico pro-resolução Resolvina D1 (RvD1) em modelo de gota. Para isso, camundongos swiss machos serão submetidos à AG por meio de injeção Introdução intra-articular com MSU. A hiperalgesia mecânica e edema serão avaliados nos

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A AG ou gota é caracterizada por Além disso, ensaios clínicos demonstram sinais típicos da inflamação, como calor, que a suplementação com ω-3 reduz a dor rubor, edema e dor intensa que afeta reportada em pacientes com artrite frequentemente adultos, com prevalência reumatoide (FATTORI et al., 2016). Deste na população idosa (ZHU et al., 2010). O modo, o presente estudo visa avaliar a desenvolvimento da AG está relacionado à participação das células da glia na hiperuricemia, causada por alterações na nocicepção provocada pela artrite gotosa produção e excreção de ácido úrico através em modelo animal, assim como os do catabolismo das purinas (WORTMANN, mecanismos de ação e efeitos do mediador 2005). O nível aumentado de ácido úrico lipídico pró-resolução RvD1 no contexto da leva à deposição de cristais de urato inflamação. monossódico (MSU) nas articulações (EGGEBEEN, 2007). Apesar de auto-limitante, os Justificativa “ataques” agudos da gota causam dor intensa nos pacientes o que os leva a buscar tratamento (EGGEBEEN, 2007). As terapias contemporâneas ainda Dentre os diferentes mecanismos que são restritas a medicamentos que podem contribuir para a dor crônica, a apresentam diversos efeitos colaterais, ativação espinal de células da glia eficácia limitada, alto custo e, em alguns (astrócitos e micróglia) vem ganhando casos, toxicidade. Assim, se faz necessário destaque em diversos estudos recentes o desenvolvimento de novas terapias (RU RONG JI et al. 2014; ZARPELON et al. capazes de reduzir a inflamação e dor 2016). Entretanto, não há ainda estudos articular durante os ataques agudos. Até o que evidenciem a participação e a momento, há apenas trabalhos modulação das células da glia na dor demonstrando o efeito farmacológico provocada pela gota. Atualmente, o periféricos de moléculas em modelos de tratamento dos “ataques” agudos da dor na gota. No presente projeto, iremos avaliar o artrite gotosa é restrito à colchicina, anti- efeito espinal do mediador lipídico pró- inflamatórios não esteroidais, corticoides resolução RvD1 e sua modulação sobre as (WORTMANN, 2005) e antagonista de células da glia. receptor de IL-1 (SO et al., 2007), que embora eficazes, apresentam altos custo e vários efeitos colaterais, tornando o Métodos tratamento clínico limitado (KEITH & GILLILAND, 2007). A resolvina D1 (RvD1) é um Animais e tratamento com RvD1 mediador lipídico pró-resolução derivado do Os experimentos serão realizados ácido graxo ω-3, e possui um papel em camundongos Swiss machos (20-25 g), endógeno regulador essencial, juntamente provenientes do Biotério Central-UEL e com outros mediadores da mesma origem. serão mantidos em ciclo de claro/escuro De fato, sua presença está aumentada em (12/12 h) com livre acesso à água e ração. pacientes com artrite (ARNARDOTTIR et Os procedimentos de cuidado e manuseio al., 2016). O tratamento intratecal com de animais estarão de acordo com as RvD1 reduz a dor em modelo de dor diretrizes da Associação Internacional de neuropática depedente da inibição do NF- Estudo da Dor (IASP). O tratamento com κB e downstream citocinas e quimiocinas.

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RvD1 será realizado através da As amostras de medula espinal administração intratecal, nas doses de 3, 10 serão coletadas 15h após o estímulo para e 30 ng, 30 min antes do estímulo com MSU dosagem das citocinas TNF-α, IL-β, IL-33, (100g/10L/animal i.a). O projeto foi IL-6 e IL-10 por ELISA, utilizando kits aprovado pelo Comitê de Ética no uso de comerciais, de acordo com as instruções do Animais (CEUA-UEL), sob número fabricante (eBioscience kits). 22186.2016.37.

Imunofluorescência Avaliação da hiperalgesia mecânica e Os segmentos de medula espinal edema L4-L6 serão dissecados e pós-fixados em A hiperalgesia mecânica será PFA, e em seguida ficarão em tampão de avaliada pelo método de von Frey com um fosfato (PB) com sacarose. Os blocos analgesímetro eletrônico (Insight®), no embebidos serão seccionados em um qual, a pressão necessária para evocar o criostato. As secções serão bloqueadas e movimento de retirada da pata pelo animal incubadas com os seguintes anticorpos é registrada automaticamente em gramas. policlonais primários: rabbit anti-Iba-1 O edema será avaliado através das (marcador de micróglia) e rabbit anti-GFAP alterações no volume da articulação, e (marcador de astrócito). As secções serão serão determinadas utilizando auxílio de um lavadas e incubadas com os anticorpos paquímetro. Os testes serão realizados secundários específicos. Após antes e nos intervalos 1, 3, 5, 7 e 15 h após procedimentos de imunocoloração, as o estímulo com MSU (100g/10L/animal secções serão examinadas usando um i.a). microscópio confocal de varrimento a laser (FV1000, Olympus, Tóquio, Japão).

Ensaio de atividade da mieloperoxidase (MPO) e N-acetilglucosamina (NAG) Reação da transcripitase reversa e PCR quantitativo (RT-qPCR) As amostras de tecido articular serão coletadas após 15 horas do estímulo As amostras de medula espinal e com MSU de camundongos previamente gânglio da raiz dorsal serão submetidos a eutanásia e colocadas em homogeinezadas em reagente Trizol e o solução tampão fosfato com HTAB. As RNA será extraído utilizando o kit SV Total amostras serão homogeneizadas e RNA Isolation System (Promega). Serão centrifugadas e o sobrenadante coletado. A avaliadas a expressão de Gfap e Iba-1 atividade da MPO e NAG serão analisadas (medula espinal), e TRPV1 e Nav1.8 a partir da adição do substrato cromógeno (gânglio da raiz dorsal). O qPCR será ao sobrenadante e as absorbâncias realizado no LightCycler Nano Instrument registradas em 450nm e 400nM, (Roche, Mississauga, ON, USA) utilizando o respectivamente em espectrofotômetro de sistema de detecção Platinum SYBR Green microplacas (Multiskan GO Microplate qPCR SuperMix UDG (Invitrogen, USA). Spectrophotometer, ThermoScientific,

Vantaa, Finland). Análise estatística

Será utilizado ANOVA de duas vias Dosagem de citocinas seguida do pós-teste de Tukey para

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comparar os grupos e doses em todos os Pharmacological Research, v. 112, p. 84-98, tempos (curvas). Será utilizada ANOVA de 2016. uma via seguido pelo pós-teste de Tukey JI R.R., XU Z.Z., GAO Y.J., Emerging targets in para experimentos com tempos específicos. neuroinflammation-driven chronic pain. Nature Serão consideradas significativas Reviews Drug Discovery. 2014, (13) 533-548. diferenças para P < 0,05 para ambas análises. KEITH MP, GILLILAND WR. Updates in the management of gout. Am J Med. 2007

Mar;120(3):221-4. Resultados Esperados SERHAN, C. N, CHIANG, N. DALLI. J. The resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution. Demonstrar a participação dos Seminars in Immunology. 2015, Mar; 1044- mecanismos espinais envolvidos na artrite 5323 gotosa, bem como o efeito do mediador SERHAN, C. N. Systems approach with lipídico pró-resolução RvD1. inflammatory exudates uncovers novel anti- inflammatory and pro-resolving mediators. Prostaglandins, Leukotrienes and Essential Agradecimentos Fatty Acids. 2008, 157–163. WORTMANN RL. Recent advances in the management of gout and hyperuricemia. Curr Agradecemos aos membros Opin Rheumatol. 2005 May;17(3):319-24. envolvidos no projeto e as agencias de ZARPELON AC, RODRIGUES FC, LOPES AH, fomento pelo apoio financeiro. SOUZA GR, CARVALHO TT, PINTO LG, XU D, FERREIRA JCA, MCINNES IB, RYFFEL B, QUESNIAUX VFJ, REVERCHON F, VERRI WA Referências Jr. Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain. The FASEB

Journal. 2016, 30:54-65. ARNARDOTTIR HH, DALLI J, NORLING LV, ZHU Y, PANDYA B, CHOI H. Increasing gout COLAS RA, PERRETTI M, SERHAN CN. prevalence in the US over the last two decades: Resolvin D3 is dysregulated in arthritis and the national health and nutrition examination reduces arthritic Inflammation. The Jor. of Immu. survey (NHANES). 74th Annual Scientific 2016. Meeting of the American College of EGGEBEEN AT. Gout: an update. Am Fam Rheumatology, 2010 Nov 7-11; Atlanta (GA). Physician. 2007 Sep 15;76(6):801-8

FATTORI, V.; AMARAL, F. A. ; VERRI, W. A. Neutrophils and arthritis: Role in disease and pharmacological perspectives.

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AVALIAÇÃO DOS EFEITOS ESPINAIS DO MEDIADOR LIPÍDICO PRÓ RESOLUÇÃO RESOLVINA D2 EM MODELO DE ARTRITE GOTOSA INDUZIDA POR CRISTAIS DE MONOURATO DE SÓDIO (MSU)

Stephanie Badaró Garcia1, Tiago Henrique Zaninelli1, Victor Fattori1, Kenji William Ruiz Myiazawa1, Sergio Marques Borghi1, Rúbia Casagrande2, Waldiceu Aparecido Verri Junior1 e-mail: [email protected] 1,2Universidade Estadual de Londrina / 1Departamento de Ciências Patológicas / 2Departamento de Ciências Farmacêuticas

Palavras-chave: artrite gotosa, mediadores lipídicos, resolvina D2 5, 7 e 15h após o tratamento. As amostras de tecido articular serão coletadas para o Resumo ensaio de atividade da mieloperoxidase, N- acetilglucosamina e dosagem de citocinas. A ativação das células da glia será A artrite gotosa (AG) é consequente analisada por imunofluorescência e RT- do depósito de cristais de urato qPCR. Além disso, será avaliada a monossódico (MSU) nas articulações expressão dos canais iônicos TRPV1 e decorrente do aumento da produção e baixa Nav1.8 no gânglio da raiz dorsal por RT- excreção de ácido úrico. A dor intensa e o qPCR. Desta forma espera-se demonstrar a edema são as principais queixas dos participação dos mecanismos espinais pacientes com AG. Dentre os diferentes envolvidos na AG, assim como o efeito do mecanismos que podem contribuir para a mediador lipídico pró-resolução RvD2. manifestação e persistência da dor, a ativação espinal de células da glia, astrócitos e micróglia, vem ganhando Hipótese destaque. As terapias convencionais disponíveis são muitas vezes inadequadas e restritas à medicamentos que apresentam O atual estudo buscará avaliar se a efeitos adversos. Assim, torna-se de grande resolvina D2 apresenta efeito anti- importância a investigação de novas inflamatório e atividade em modelos de gota terapias para o tratamento da AG. Deste induzida por cristais de MSU. Até o modo, considerando o papel fundamental momento não há nenhum trabalho da ativação espinal na dor, o presente relatando sobre o efeito da RvD2 em trabalho visa avaliar a participação espinal modelos de artrite gotosa. e o efeito farmacológico do mediador lipídico pró-resolução resolvina D2 (RvD2) em modelo de AG. Para isso, camundongos Swiss machos serão induzido à AG por Introdução injeção de cristais de MSU, e os animais serão tratados com RvD2. A hiperalgesia mecânica e o edema serão avaliados 1, 3,

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A artrite gotosa é consequente do inflamatórias e pró-resolução (SERHAN et depósito de cristais de urato monossódico al., 2008). Estudos recentes apontam a nas articulações (DIEPPE, 1982) RvD2 como um potente inibidor endógeno decorrente ao aumento da produção e baixa para TRPV1/A1 e dor inflamatória, porém, excreção de ácido úrico. O quadro clínico da seus mecanismos analgésicos ainda não artrite gotosa é caracterizado por sinais são bem estabelecidos. Deste modo, o típicos da inflamação, como calor, rubor e objetivo deste estudo será avaliar a edema, além de dor intensa (WORTMANN, participação de células da glia espinal na 2005). hiperalgesia produzida pela artrite gotosa em modelo animal bem como a avaliação Dentre os mecanismos que podem dos efeitos e mecanismos de ação do contribuir para a manifestação e mediador lipídico pró-resolução RvD2. persistência da dor, a ativação espinal de células da glia promove a liberação de mediadores inflamatórios (MILLIGAN et al., Justificativa 2008) e já foi demonstrada em modelos de dor inflamatória, neuropática e no câncer ósseo e de pele (PAVÃO-DE-SOUZA et al., 2012). Entretanto, não há ainda estudos As terapias atuais são restritas a que evidenciem a participação desse medicamentos que apresentam diversos mecanismo na dor provocada pela gota. efeitos colaterais, eficácia limitada, alto custo e toxicidade. Desta forma, é Atualmente, o tratamento é restrito à necessário o desenvolvimento de novas colchicina, anti-inflamatórios não terapias capazes de reduzir a inflamação e esteroidais, corticoides (WORTMANN, dor articular. Até o momento, há apenas 2005) e antagonista de receptor de IL-1 que trabalhos demonstrando os mecanismos embora eficazes, apresentam alto custo e periféricos de doença bem como o efeito diversos efeitos colaterais, (KEITH; farmacológico de moléculas. No presente GILLILAND, 2007). Sendo assim, a projeto, iremos avaliar os mecanismos importância da investigação de novos alvos espinais envolvidos na gota, e o efeito farmacológicos e desenvolvimento de espinal do mediador lipídico pró-resolução novas terapias para o tratamento da gota é RvD2 e sua modulação sobre as células da fundamental. Neste contexto, os glia. mediadores lipídicos pró-resolução possuem um papel endógeno regulador e ensaios clínicos demonstram que a Métodos suplementação com ômega-3 (precursores dos mediadores lipídicos pró-resolução) reduz a dor em pacientes com artrite Animais e tratamento com RvD2 reumatoide (FATTORI et al., 2016). Além disso, os mediadores lipídicos pró- Os experimentos serão realizados resolução agem em receptores presentes em camundongos Swiss machos, em neurônios sensoriais diminuindo seu provenientes do Biotério Central – UEL. Os limiar de ativação (SERHAN et al., 2015). animais serão divididos de acordo com os grupos experimentais com livre acesso à A resolvina D2 (RvD2) é um dos água e ração. Os procedimentos de cuidado mediadores lipídicos proveniente do e manuseio de animais estarão de acordo ômega-3, que desempenha papel com as diretrizes da Associação endógeno importante em ações anti- Internacional de Estudo da Dor (IASP). O

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tratamento com RvD2 será realizado Dosagem de citocinas através da administração intratecal, nas As amostras de medula espinal doses de 3, 10, e 30ng, 30 min antes do serão coletadas 15h após o estímulo para estímulo com MSU (100µg/10µL/animal ia). dosagem de citocinas TNF-α, IL-β, IL-33, IL- O projeto foi aprovado pelo do Comitê de 6 e IL-10 por ELISA, utilizando kits Ética no Uso de Animais (CEUA-UEL), no comerciais, de acordo com as instruções do 22186.2016.37 fabricante (eBioscience kits).

Avaliação da hiperalgesia mecânica e Imunofluorescência avaliação do edema Os segmentos de medula espinal A hiperalgesia mecânica será L4-L6 serão dissecados e pós-fixados em avaliada pelo método de von Frey, que PFA e em seguida ficarão em solução com consiste na aplicação de pressão pontual sacarose a 30% num tampão de fosfato. Os crescente na pata posterior do animal com blocos embebidos serão seccionados em uma ponteira de polipropileno associada a um criostato e armazenado em PBS por um analgesímetro eletrônico. O edema será imunofluorescência. As secções serão avaliado através das alterações volume da bloqueadas e incubadas com os seguintes articulação e serão determinadas com o anticorpos policlonais primários: rabbit anti- auxílio de um paquímetro. As avaliações Iba-1 (marcador de micróglia) e rabbit anti- serão realizadas antes e em medidas GFAP (marcador de astrócito). As secções únicas 1, 3, 5, 7 e 15h após o estímulo com serão lavadas e incubadas com os MSU (100μg/10 µL/animal, i.a.). anticorpos secundários específicos. Após imunocoloração, as secções serão examinadas usando um microscópio Ensaio de atividade da mieloperoxidase confocal de varrimento a laser (FV1000, (MPO) e N-acetilglucosamina (NAG) Olympus, Tóquio, Japão). As amostras de tecido articular serão coletadas após 15h do estímulo com MSU de camundongos e colocadas em Reação da transcriptase reversa e PCR solução tampão fosfato com HTAB. As quantitativo (RT-qPCR) amostras serão homogeneizadas, Os RNAs das amostras de medula centrifugadas e o sobrenadante coletado. espinal e gânglio da raiz dorsal serão As absorbâncias das amostras serão extraídos utilizando o kit SV Total RNA registradas em espectrofotômetro de Isolation System (Promega). Serão microplacas e a atividade da MPO será avaliadas a expressão de Gfap e Iba-1, e comparada a uma curva padrão de TRPV1 e Nav1.8. O qPCR será realizado no neutrófilos e o resultado expresso como LightCycler Nano Instrument (Roche, atividade da mieloperoxidase (nº de Mississauga, ON, USA) utilizando o sistema neutrófilos x 105/mg tecido). A atividade de de detecção Platinum SYBR Green qPCR NAG será determinada da mesma forma e SuperMix UDG (Invitrogen, USA). os resultados serão expressos como atividade de NAG mieloperoxidase (nº de macrófagos x 105/mg tecido). Análise estatística

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Será utilizado ANOVA de duas vias pharmacological perspectives. seguida do pós-teste de Tukey para Pharmacological Research, v. 112, p. 84-98, experimentos com tempos diferentes e 2016. ANOVA de uma via seguido pelo pós-teste Keith MP, Gilliland WR. Updates in the de Tukey para experimentos com tempos management of gout. Am J Med. 2007 específicos. Serão consideradas Mar;120(3):221-4. significativas diferenças para P < 0,05 para Milligan ED, Sloane EM, Watkins LR. Glia in ambas análises. pathological pain: a role for fractalkine. J Neuroimmunol. 2008 Jul 31;198(1-2):113-20. Resultados Esperados PAVAO-DE-SOUZA GF, ZARPELON AC, TEDESCHI GC, MIZOKAMI SS, SANSON JS, CUNHA TM, FERREIRA SH, CUNHA FQ, CASAGRANDE R, VERRI WA JR. Acetic Demonstrar a participação dos acid- and phenyl-p-benzoquinone-induced overt mecanismos espinais envolvidos na artrite pain-like behavior depends on spinal activation gotosa, assim como o efeito do mediador of MAP kinases, PI(3)K and microglia in mice. lipídico pró-resolução RvD2. Pharmacol Biochem Behav. 2012 May;101(3):320-8.

SERHAN, C. N, Chiang, N. Dalli. J. The Agradecimentos resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution. Seminars in Immunology. 2015, Mar; 1044- Agradecemos aos membros 5323 envolvidos no projeto e as agências de SERHAN, C. N. Systems approach with fomento CAPES e Fundação Araucária. inflammatory exudates uncovers novel anti- inflammatory and pro-resolving mediators. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2008, 157–163. Referências WORTMANN RL. Recent advances in the management of gout and hyperuricemia. Curr DIEPPE P.A , DOHERTY M. The role of particles Opin Rheumatol. 2005 May;17(3):319-24. in the pathogenesis of joint disease. Curr Top Pathol. 1982;71:199-233. Fattori, V.; Amaral, F. A. ; Verri, W. A. Neutrophils and arthritis: Role in disease and

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AVALIAÇÃO DO EFEITO ANTINOCICEPTIVO E ATIVIDADE ANTIOXIDANTE DO DIOSMIN EM MODELO DE DOR NEUROPÁTICA INDUZIDA PELA CONSTRIÇÃO DO NERVO CIÁTICO EM CAMUNDONGOS

Mariana Marques Bertozzi1, Waldiceu Aparecido Verri Junior1 e-mail: [email protected]

1Universidade Estadual de Londrina/Departamento de Ciências Patológicas

Palavras-chave: Neuropatia, Hiperalgesia, Flavonoide.

Resumo Hipótese O estudo tem como objetivo avaliar o efeito antinociceptivo e antioxidante do De acordo com a literatura, diosmin no modelo experimental de dor flavonoides são compostos que apresentam neuropática induzida por constrição crônica potencial terapêutico. Desse modo, o atual do nervo ciático (CCI). Camundongos Swiss estudo buscará avaliar se o flavonoide machos passarão pela cirurgia e após 7 diosmin apresenta efeito antinociceptivo e dias serão tratados com diosmin (i.p) em atividade antioxidante em modelos de dor diferentes doses e a hiperalgesia mecânica crônica em modelos de constrição de nervo e térmica serão avaliadas após 1, 3, 5, 7 e ciático. Além disso, até o momento não há 24h. Para avaliar a participação das nenhum trabalho relatando sobre o efeito do possíveis vias analgésicas de ação do diosmin em modelos de dor crônica. diosmin serão avaliadas as vias do NO/cGMP/PKG/K+ATP e da Heme- oxigenase. Os animais CCI serão pré- tratados com diferentes inibidores das vias Introdução e posteriormente tratados com diosmin e A dor é um dos sintomas clássicos avaliadas a hiperalgesia mecânica e do processo inflamatório. Esta é decorrente térmica após 1, 3, 5 e 7h. A atividade da sensibilização dos nociceptores, antioxidante na medula espinal (L4-L6) será produzindo a hiperalgesia e a produção e analisada por meio de ensaios liberação de mediadores inflamatórios. colorimétricos. A toxicidade do tratamento Entretanto, quando esta sensibilização dos prolongado (7 dias) com diosmin será neurônios não cessa, desencadeia o avaliada utilizando como parâmetros níveis processo de dor crônica (ROBBINS, 2008). plasmáticos de AST/ALT, ureia e creatinina A dor neuropática se encaixa nesse quadro e atividade de mieloperoxidase no crônico de dor e é uma síndrome complexa estômago. Além disso será feita avaliação resultante de lesão ou disfunção de fibras da ativação de células da glia micróglias e nervosas. (WOOLF e MANNION, 1999). As astrócitos por imunofluorescência e RT- terapias farmacológicas disponíveis qPCR, além da expressão de moléculas atualmente utilizadas na prática clínica para envolvidas na dor neuropática por RT- o tratamento da dor neuropática têm se qPCR. mostrado insuficientes, além de

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apresentarem efeitos adversos e baixa pequena incisão na altura superior do fêmur eficácia nestes casos (DWORKIN et al., onde a musculatura será cuidadosamente 2003). O crescente interesse no estudo de divulsionada para expor o nervo ciático. Em compostos naturais se deve principalmente seguida, com auxílio de agulha e fio ao fato de muitos destes apresentarem cirúrgico, será realizada uma ligadura (“nó”) propriedades benéficas e baixa toxicidade. no nervo ciático sem que ocorra secção do O diosmin é um flavonoide, que além de nervo, apenas constrição. Nos animais do apresentar várias atividades biológicas, é grupo controle (falso-operados), o nervo considerado um composto seguro, com boa será exposto utilizando o mesmo tolerabilidade e não exerce efeito tóxico. procedimento cirúrgico, porém sem sofrer Desse modo, o objetivo desse estudo foi constrição. O projeto foi aprovado pela avaliar o efeito antinociceptivo e CEUA/UEL n°28995.2014.39 antioxidante do flavonoide diosmin em modelos de dor crônica neuropática induzida pela constrição do nervo ciático. Avaliação da hiperalgesia mecânica e térmica

A hiperalgesia mecânica será avaliada pelo Justificativa método eletrônico de von Frey (CUNHA et Devido à falta de terapias eficazes al., 2004). Já para hiperalgesia térmica no tratamento da dor neuropática a camundongos serão colocados no aparelho utilização de derivados naturais vem sendo de placa quente, mantida a 52°C aplicada como uma nova abordagem (CATERINA et al, 2000). A latência máxima terapêutica. Neste sentido, pretendemos (cut-off) foi estabelecida em 20 segundos avaliar a ação do efeito antinociceptivo e a para evitar danos nos tecidos (HSIEH et al., possível atividade antioxidante para o 2008). Os animais serão testados antes e controle de dor crônica. após a cirurgia de constrição do nervo ciático. O tratamento com diosmin será realizado sempre uma semana após a Métodos cirurgia de constrição do nervo ciático e serão avaliados nos intervalos de 1, 3, 5, 7 Animais e 24h. Serão utilizados camundongos Swiss machos (20-25g) provenientes do Biotério Central/UEL e serão mantidos sob ciclo Tratamento dos animais claro/escuro (12/12h) em ambiente Após 7 dias da cirurgia, receberão um único climatizado, com exaustão do ar e livre tratamento, via intraperitoneal (i.p.) com o acesso à água e ração. flavonoide diosmin 1-30mg/kg (diluído em DMSO 2%). Experimentos posteriores, será avaliado o efeito do tratamento com diosmin Modelo de dor neuropática induzida pela na dose mais eficaz (Xmg/kg) porém em um constrição de nervo ciático (CCI) período prolongado de tratamento 7 dias). O procedimento cirúrgico para indução da dor neuropática será realizado conforme previamente descrito por Bennett e Xie Efeito de inibidores da via óxido nítrico (1988) com algumas modificações. Os Será feita a avaliação do papel da via de animais serão anestesiados e será feita sinalização do NO/ cGMP/PKG/K+ATP na

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analgesia promovida pelo diosmin. Os Avaliação dos níveis plasmáticos de camundongos CCI serão pré tratados com AST/ALT, ureia e creatinina L-NAME (inibidor da NOS, 90mg/kg, Os níveis plasmáticos das enzimas i.p.,1h), ODQ (inibidor da guanilato ciclase aspartato aminotransferase (AST), alanina solúvel, 0,3mg/kg, i.p., 5min) ou GLY aminotransferase (ALT), ureia e creatinina (bloqueador dos canais de K+ATP serão avaliados através de método cinético- sensíveis, i.p. 45min). Será avaliada a colorimétrico. hiperalgesia mecânica e térmica 1,3,5 e 7h após o tratamento com diosmin (Xmg/kg, i.p.). Avaliação da ativação das micróglias e astrócitos espinais e periférico, e moléculas envolvidas na dor neuropática por PCR Avaliação de inibidores da via da heme- tempo real e por imunofluorescência oxigenase Os camundongos serão anestesiados com Para verificar a participação da via de isoflurano e eutanasiados, depois o sinalização CO/BVD/Fe possível efeito seguimento (L4-L6) da medula espinhal e o antinociceptivo do diosmin na dor gânglio da raiz dorsal será rapidamente neuropática induzida pela CCI, no 7º dia removido e devidamente armazenado. O após CCI os animais serão tratados com ensaio de RT-qPCR quantitativo em tempo ZnPPIX (inibidor da HO, s.c.), Hemin real será utilizado para analisar as IL-1β, (substrato do grupo Heme, s.c.) e BVD TNF-α, IL-33, Nav1.8, TRPV-1, NRF2, HO- (produto da degradação do grupo Heme, 1 e ATF3. Para imunofluorescência os s.c.). Será avaliada a hiperalgesia mecânica animais serão anestesiados terminalmente e térmica 1,3,5 e 7h após o tratamento com com isoflurano e perfundidos através do diosmin (Xmg/kg, i.p.). ventrículo esquerdo com 20ml de solução salina tamponada (PBS 0,01M) e subsequentemente com 20mL de solução Avaliação do estresse oxidativo de paraformaldeído 4% (PFA). As medulas Os animais serão terminalmente serão cuidadosamente retiradas e anestesiados por inalação de isoflurano criopreservados após imersão em solução 1,5%. Após a eutanásia, amostras do tecido de sacarose 30%, durante 48 horas. Após o medular (L4-L6) serão coletadas e processo de crioproteção, as medulas rapidamente congeladas a -80 °C para os serão congelada e estocadas a -80º C até ensaios colorimétricos (GSH, ABTS, FRAP, serem utilizadas. As amostras serão TBARS e NBT). marcadas para avaliação de IBA (micróglia) e GFAP (astrócitos), conforme metodologia descrita por Chiu et al 2013, as lâminas Avaliação da atividade de mieloperoxidase serão montadas em Vectashield com DAPI (MPO) (Vector Labs) e analisadas usando microscópio confocal Leica TCS SP8. Para investigação de lesão da mucosa gástrica será determinada a atividade de MPO no tecido gástrico após o tratamento Análise estatística prolongado (7dias) com diosmin (Xmg/kg). Análise de variância two-way, seguida de pós-teste de comparações múltiplas de Tukey realizada a cada tempo. Serão

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consideradas significativas diferenças para DWORKIN, R.H.; BACKONJA, M.; P<0.05. ROWBOTHAM, M.C.; ALLEN, R.R.; ARGOFF, C.R.; BENNETT, G.J.; BUSHNELL, M.C.; FARRAR, J.T.; GALER, B.S.; HAYTHORNTHWAITE, J.A.; HEWITT, D.J.; Resultados Esperados LOESER, J.D.; MAX, M.B.; EL-SHAFAE, A.M.; EL-DOMIATY, M.M. Improved LC methods for the determination of diosmin and/or hesperidin in Demonstrar a possível ação plant extracts and pharmaceutical formulations. farmacológica do flavonoide diosmin em J Pharm Biomed Anal. v.24, n.4, p.539-45. Nov modelo de dor neuropática induzida pela 2001. constrição do nervo ciático. HSIEH, Y.L.; CHIANG, H.; TSENG, T.J.; HSIEH, S.T. Enhancement of cutaneous nerve

regeneration by 4-methylcatechol in Agradecimentos resiniferatoxin-induced neuropathy. J Neuropathol Exp Neurol. v.67, n.2, p.93-104. Agradeço a todos os envolvidos no Feb 2008. projeto e às agências de fomento pelo apoio ROBBINS, S. L.; KUMAR, V. (ed.); ABBAS, A.K. financeiro. (ed.); FAUSTO, N. (ed.). Patologia: Bases Patológicas das doenças. 7ª ed. Rio de Janeiro: Elsevier, 2005. Referências WOOLF, C.J.; MANNION, R.J. Neuropathic BENNETT, G.J.; XIE, Y.K. A peripheral pain: aetiology, symptoms, mechanism and mononeuropathy in rat that produces disorders management. The Lancet. v.353, p. 1959-64, of pain sensation like those seen in man. Pain. 1999. v.33, n.1, p.87-107, Apr, 1988.

CATERINA, M.J.; LEFFLER, A.; MALMBERG, A.B.; MARTIN, W.J.; TRAFTON, J.; PETERSEN-ZEITZ, K.R.; KOLTZENBURG, M.; BASBAUM, A.I.; JULIUS, D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. v.288, n.5464, p.306-313, 2000.

CHIU, I.M.; HEESTERS, B.A.; GHASEMLOU, N.; VON HEHN, C.A.; ZHAO, F.; TRAN, J.; WAINGER, B.; STROMINGER, A.; MURALIDHARAN, S.; HORSWILL, A.R.; BUBECK WARDENBURG, J.; HWANG, S.W.; CARROLL, M.C.; WOOLF, C.J. Bacteria activate sensory neurons that modulate pain and inflammation. Nature. 501, n.7465,p.52-7. Sep, 2013.

CUNHA, T.M.; VERRI, W.A. JR.; VIVANCOS, G.G.; MOREIRA, I.F.; REIS, S.; PARADA, C.A., CUNHA, F.Q.; FERREIRA, S.H.; An electronic pressure-meter nociception paw test for mice. Braz J Med Biol Res. v.37, n.3, p.401-407, Mar 2004.

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INDEX

Adrián Friedrich, 58 Andréa Name Colado Simão, 36, 45, 77, Adriane Lenhard-Vidal, 14, 109 95, 108 Adriano Martin Felis Aranome, 27, 56, 64, Andrea NC Simão, 110 112, 114, 115, 117, 119, 125 Andressa Silva Gomes, 74 Adriano Martin Felix Aranome, 97 Anelise Franciosi, 123, 140 Agruslávia Rezende de Souza, 122 Angelica Ehara Watanabe, 125 Airton dos Santos Gon, 76, 83, 88, 106 Aparecida Donizette Malvezi, 25, 26 Alan Ferreira Chagas, 16 Bárbara B. Colombo, 104 Alberto Yoichi Sakaguchi, 28, 41, 63, 79 Bárbara Lidiane Kummer Mallmann, 103 Alda Fiorina Maria Losi Guembarovski, Barbara Prevital, 32 35 Beatriz Sardinha Sabino, 95, 100 Alda Losi Guembarovski, 44, 63, 87 Bianca A. Bertasso, 21 Aldo Eloizo Job, 74, 75 Blenda Hyedra de Campos, 33, 34 Alessandra Barbosa Ferreira-Machado, Bruna Karina Banin Hirata, 35, 63, 94 121 Bruna Miglioranza Scavuzzi, 36, 77, 100, Alessandra Cecchini, 57 111 Alessandra L. Cecchini, 58, 76 Bruna Santos Marnieri, 123 Alessandra Lorenço Cecchini Armani, 73 Bruna Taciane da Silva Bortoleti, 16, 17 Alessandra Lourenço Cecchini, 27, 32, 60, Brunna Emanuella França Robles, 36, 95, 61, 65, 75, 83, 96, 97, 105, 106 100 Alessandra Lourenço Cecchini Armani, Bruno Alexandre Sekiguchi, 95 92, 93, 155 Bruno Bevenutto Lucas, 21 Alessandra Lourenço Cecchini1, 88 Bruno Bueno Pimenta, 37, 98 Alice Maria de Souza-Kaneshima, 37, 98 Bruno Fernando Cruz Lucchetti, 15, 25 Aline Miquelin Nascimento, 118 Bruno Vinicius Duarte Marques, 38, 70 Allan C. Bussmann, 104 Caio Cezar Nantes Martins, 39 Allan J.C. Bussmann, 71 Caio de Meleck Proença, 36 Alvaro Ferdinando Scremin, 48, 91 Camila Borecki Vidigal, 40, 47 Amanda Cristina Machado Carloto, 19 Camila Cataldi de Alcantara, 110, 111 Amanda Z. Zucoloto, 29 Camila Cristina Alves Machado, 148 Ana C. Zarpelon, 29 Camila de Souza Padilha, 96 Ana Carolina Rossaneis, 82 Camila Rodrigues Ferraz, 43, 66, 67, 85 Ana Lúcia Sanches, 30, 31, 68, 69 Carine Coneglian de Farias, 33, 34 Ana P Kallaur, 110 Carla Cristiane da Silva, 120 Ana Paula Franco Punhagui, 129 Carla F. S. Guazelli, 104 Ana Paula Kallaur, 36, 100 Carlos Eduardo Coral de Oliveira, 28, 41, Ana Paula Lombardi, 64, 114 44, 63, 79, 87, 94 Ana Paula Lombardi Pereira, 112, 117, Carlos Eduardo Coral de Oliviera, 35 119 Carlos Vinicius Dalto da Rosa, 72 Ana Paula Pereira Lombardi, 115 Carolina Batista Ariza, 35, 44, 79, 87 Andre Armani, 48, 88, 91 Carolina Matias Higashi, 38, 70 André Armani, 76, 83, 92, 106 Carolina Panis, 27, 73, 97, 125 Andréa N Simão, 111 Caroline de Souza Araujo, 42, 74, 78 Andrea Name Colado Simão, 100 Carpinelli, A. R, 80 Cássia Calixto de Campos, 43

181

Cassio Fernando Nunes da Silva, 93 Elaine Eloiza Cortellini Landivar, 103 Cintia Magalhães Carvalho Grion, 167 Elaine RD de Almeida, 110 Cintya Mayumi Ishibashi, 44, 63, 79 Eliane Swely Amador Monteiro Sanches, Claudia Pecorai, 54 49, 133 Cláudia Stoeglehner Sahd, 16, 17 Emerson José Venancio, 118, 120 Cláudio Galuppo Diniz, 121 Emerson José Venâncio, 81 Clodoaldo Zago Campos, 87 Érica Romão Pereira, 56, 115, 117 Congo, N.A.J., 80 É rica Romão Pereira, 64, 112, 114, 119 Cristhiane da Silva Ferreira Gonçalves, Érica Romão Pereira Maria, 125 69 Érika Caroline Steinle, 21 Cristhiane Da Silva Ferreira Gonçalves, Estefânia Gastaldello Moreira, 133 31 Evandro José Beraldi, 116 Dalita Gomes Silva Moraes Cavalcante, Fabio Albuquerque Marchi, 84 74 Fábio Augusto Joinhas, 50 Dalita Moraes Gomes Silva Cavalcante, Fabio Henrique Kwasniewski, 152 75 Fábio Henrique Kwasniewski, 66, 67, 85 Damácio Ramón Kaimen-Maciel, 36 Fábio Joinhas, 133 Damácio Ramon Maciel-Kaimen, 100 Fabrício Azevedo Voltarelli, 96 Daniel Gonzalez Maglio, 58 Fausto Celso Trigo, 41 Daniela Ceccatto Gerardin, 47 Fausto Trigo, 28 Daniela Cristina Ceccatto Gerardin, 40 Feliciano Protasi, 54 Daniela Frizon Alfieri, 36, 45, 95, 100 Felipe A. Pinho-Ribeiro, 29, 102 Daniela Ribeiro Alves, 16 Felipe Campos de Almeida, 87 Daniela Rudgueri Derossi, 44 Felipe Crepaldi Duarte, 51 Daniela Sartori, 44 Felipe Tsuzuki, 49, 52, 133 Daniele Sapede Alvarenga, 22, 23, 46 Fernanda Costa Brandão Berti, 64, 112, Daniella Regina Barrionuevo, 70 114, 119 Daniella Regina Barrionuevo da Silva Fernanda Costa Brandão Berti, 56, 115, Novi, 38, 40, 47 117, 125 De Fatima Silva, F, 80 Fernanda Dias Figueira, 50 De Souza, H. M, 80 Fernanda Mithie Ogo, 107 Débora de Mello Gonçales Sant’Ana, 24 Fernanda Novi Cortegoso Lopes, 53 Débora de Mello Gonçales Sant´Ana, 148 Fernanda Paschoal Blegniski, 54 Débora de Mello Sant’Ana, 116 Fernanda Pelisson Massi, 44 Décio Sabbatini Barbosa, 33, 34 Fernanda S. Rasquel de Oliveira, 55 Denis Massatsugu Ueda, 48, 91 Fernanda T. Ortega, 76 Diego Lima Petenuci, 28, 79 Fernanda Teixeira Ortega, 83, 88, 106 Dielle Ioris, 20 Fernanda Tomiotto Pellissier, 22, 23 Edilson Noboioshi Kaneshima, 37, 98 Fernanda Tomiotto-Pellissier, 16, 17, 20 Edna M V Reiche, 110 Fernando Cezar dos Santos, 56, 64, 112, Edna Maria V Reiche, 111 114, 115, 117, 119, 125 Edna Maria Vissoci Reiche, 36, 45, 77, 95, Fernando Henrique Borges, 96, 97 100 Fernando Pinheiro de Souza Neto, 58, 96 Eduardo José de Almeida Araújo, 15, 99, Fernando Pinheiro Souza-Neto, 105 140, 144, 148 Fernando Q. Cunha, 29, 59 Eduardo Ottobelli Chielle, 103 Fernando Souza Neto, 57 Eduardo Vignoto Fernandes, 81 Fernando Souza-Neto, 65 Eidi Yoshihara, 74 Flávia Alessandra Guarnier, 27, 54, 107, Eiko Nakagawa Itano, 14 155

182

Flávia Imanishi Ruzon, 81 Iriana Moratto Carrara, 65, 105 Flora Martinez Figueira Moreira, 122 Isaias Dichi, 45, 77, 95, 100, 110, 111 Francemilson Goulart da Silva, 129 Isaías Dichi, 108 Francieli Delongui, 45 Isolde Previdelli, 37, 98 Franciely A. V. Dantas, 59 Italmar Teodorico Navarro, 20 Francine Nesello Melanda, 19, 21, 23, 167 Ivete Conchon Costa, 22, 23, 46, 167 Francine Neselo Melanda, 22, 46 Ivete Conchon-Costa, 16, 17, 19, 20, 21 Gabriel Zanotto dos Santos, 113 Jackson Gabriel Miyamoto, 66, 67, 85, 152 Gabriela Cristine Queiroz Maria, 56, 64, Janaina Aparecida Fortunato, 31, 69 112, 114, 117, 119 Janaine Aparecida Fortunato, 30, 68 Gabriela de Alcântara Dalevedo, 20 Jaqueline Costa Castardo de Paula, 33, Gabriela Matias Costa, 70 34 Gabriella Pasqual Melo, 32, 57, 60, 65, 105 Jeferson Noslen Casarin, 103 Gabriella Pasqual Melo1, 61 Jéssica Loraine Drugik, 109 Galia, W.B.S, 80 Jéssica Men de Campos, 72 Gareth Davison, 155 Joana D’Arc de Lima Mendes, 144 Gessica D. Gonçalves, 59 João Eurípedes Calixto de Campos, 43 Gessica Dutra Gonçalves, 62 João L. Garcia, 24 Gessilda Alcântara Nogueira de Melo, 24 João Paulo Assolini, 14, 16, 17, 19 Giovana Gomes de Carvalho, 18 Jorge Mali Júnior, 140 Gislaine Garcia Pelosi Gomes, 38 Jorge Mali-Júnior, 144 Gláucia Eloisa Munhoz de Lion Siervo, Jorge P Sales de Almeida, 111 107 Jorge PS de Almeida, 110 Glauco A. Freire Vitiello, 44 José Alberto Duarte, 96 Glauco Ackelinghton Freire Vitiello, 41 José C. Alves-Filho, 29 Glauco Akelinghton Freire Vitiello, 35, 63, Juan Josue Puño Sarmiento, 123 94, 114 Juliana Alves Resende, 121 Glaura S. A. Fernandes, 59 Juliana Leoni, 58 Glaura Scantamburlo Alves Fernandes, Juliano Bordignon, 20, 26 62, 107, 129, 133, 136 Júlio Henrique Rosa Croda, 122 Graciano, M. F, 80 Kaliana Larissa Machado, 93 Graziela Scalianti Ceravolo, 38, 40, 47, 70 Kamila Falchetti Damasco, 81, 118 Guilherme Cesar Martelossi Cebinelli, 56, Karen Brajão de Oliveira, 64, 112, 114, 64, 112, 114, 115, 117, 119, 125 119 Guilherme Ferreira Silveira, 26 Karen Brajão de Oliveira, 35, 56, 87, 115, Guilherme Fonseca Reis, 20 117, 125 Guilherme Henrique Loiola, 47 Karina de Almeida Gualtieri, 123 Gustavo B. Menezes, 29 Kawane Fabricio Moura, 70 Helena Kaminame Morimoto, 100 Kenji W. Ruiz-Miyazawa, 104 Hellen Kuasne, 84 Kenji William Ruiz Myazawa, 171 Heloísa Lizotti Cilião, 84, 89, 90 Kenji William Ruiz Myiazawa, 175 Hiviny de Ataides Raquel, 15, 53 Ketlem C. Andrade, 71 Idessania Nazareth Costa, 16, 17, 19, 20, Ketlem Cristine Andrade, 66, 67, 85 21, 22, 23, 46 Kleber Paiva Trugilo, 56, 64, 112, 114, 115, Idessânia Nazareth Costa, 167 117, 119, 125 Ilce Mara de Syllos Cólus, 84, 89 Laís Fernanda Machado, 20, 21 Ilce Mara Syllos Cólus, 90 Larissa Carla Lauer Schneider, 72, 116 Ionice Felipe, 123 Larissa Dolfini Alexandrino, 21 Iriana Morato Carrara, 60, 61, 75 Larissa Ferrari, 82

183

Larissa Juliani Sanches, 73 Maria Caroline Martins de Araújo, 45 Larissa Rodrigues Bosqui, 20, 21 Maria Eduarda Tesser, 46 Larissa Staurengo-Ferrari, 59, 102, 107 Maria Helena P. Fungaro, 44 Laurence Ashley Blackshaw, 140 Maria Isabel Lovo-Martins, 15, 25 Laurence Ashley; Blackshaw, 144 Maria José Sparça Salles, 39, 49, 50, 52, Leandra Ernst Kerche-Silva, 42, 74, 75, 78 133 Leandra Naira Zambelli Ramalho, 57, 65 Maria Raquel Marçal Natali, 72 Leandra Náira Zambelli Ramalho, 105 Mariana Barbosa Detoni, 46 Leonardo Bodner de Freitas, 76, 88 Mariana M. Bertozzi, 71 Leonardo Bodner Freitas, 83, 106 Mariana Marques Bertozzi, 82, 179 Ligia Grecco Costa Dall’aqua, 95 Mariana O. Okamura, 76 Lígia Grecco Costa Dall’Aqua, 45 Mariana Onuki Okamura, 83, 88, 106 Ligia Grecco Costa Dall'Aqua, 77 Mariani Lima Garcia, 76 Lisete Compagno Michelin, 15 Mariani de Lima Garcia, 83, 88 Lorena de Jager, 33, 34 Mariani Lima Garcia, 106 Lorena Flor da Rosa Franchi Santos, 36, Mariela Paz, 58 77, 100 Marilesia Ferreira de Souza, 84, 89 Lorena Flor Da Rosa Franhci Santos, 95 Marilesia Ferreira Souza, 90 Lorrane Davi Brito, 42, 78 Marília F. Manchope, 29, 86 Luana Aparecida Cossentini, 31, 69, 100, Marília Fernandes Manchope, 43, 66, 67, 113 85 Lucas S Liberati, 110 Marina L. R. Mantovani, 21 Lucas Silva Liberatti, 111 Marina Okuyama Kishima, 35, 44, 87 Luciana Higashi, 33, 34 Mario Augusto Ono, 18 Luciana Mendes, 22, 23, 46 Mario Sergio Mantovani, 62 Luciano Aparecido Panagio, 20 Marla Karine Amarante, 28, 35, 41, 63, 79, Lucimara Aparecida Sensiate, 52 94 Lucy Megumi, 167 Marli Cardoso Martins Pinge, 15, 25 Lucy Megumi Yamauchi, 26 Marli Cardoso Martins-Pinge, 33, 34, 53 Luis Fernando Lasaro Mangieri, 56, 64, Mateus H. Cunha da Silva, 76 112, 114, 117, 119 Mateus de Camargo Barros, 84 Luiz Vicente Rizzo, 26 Mateus Henrique Cunha da Silva, 83, 88, Manoela Daiele Gonçalves, 16, 17, 19 106 Marcell A Batisti Lozovoy, 111 Mauricio Pacheco Reis, 48, 91 Marcell Alysson Batisti Lozovoy, 77, 95, Mayara Tiemi Enokida, 63 108 Mayra Tardeli de Jesus Testa, 96 Marcell Alysson Lozovoy, 100 Michael Maes, 36, 77, 100 Marcelo Biodaro Góis, 148 Michelle Mota Sena, 56, 64, 112, 114, 115, Marcia Regina Eches Perugini, 51 117, 119, 125 Marcia Regina Terra, 113 Milena Menegazzo Miranda-Sapla, 16, Márcia Regina Terra, 30, 68 17, 20 Márcio Francisco Lehmann, 45 Milena Miranda Sapla, 22, 23 Marcio Lupepsiw, 109 Milene Roldão de Souza, 84, 89 Marcio Scarone, 81 Milene Roldão Souza, 90 Márcio Scarone, 118 Miriele Caroline da Silva, 81, 118 Marcos Alberto Zocoler, 42, 78 Mônica de Oliveira Belém, 15 Marcos Henrique Rosa, 63 Monica Levy Andersen, 107 Maria Angelica Ehara Watanabe, 28, 35, Monyse de Nóbrega, 90 41, 44, 63, 79, 87, 94, 114, 115 Murilo Carlos Gimenes, 48, 91

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Naara C. C. dos Santos, 21 Raquel Arruda Sanfelice, 20, 21 Nadia Calvo Martins Okuyama, 115, 117 Raquel Pires Nakama, 18 Nádia Calvo Martins Okuyama, 56, 64, Raúl Jorge Hernan Castro-Gómez, 81 112, 114, 119, 125 Rejane Schaefer, 118 Nágela Ghabdan Zanluqui, 15 Renan Cajuca, 22, 23, 46 Nagela Ghabdan Zanluqui, 33, 34, 38, 53 Renata Streck Fernandes, 99 Natália Almeida de Barros, 155 Ricardo Sergio Almeida, 20 Natália M. Dias Lopes, 76 Rito Santo Pereira, 25 Natália Medeiros Dias Lopes, 83, 88, 92, Rito Santo Pereira1, 121 93, 97, 106 Roberta Losi Guembarovski, 35, 63, 87 Nathália de Sousa Pereira, 94 Roberta Losi Guembravoski, 44 Nathalia de Souza Pereira, 63 Roberto Kenji Nakamura Cuman, 24 Nathalia Maria Fioreto Campois, 123 Rodolfo Sanches Ferreira, 56, 64, 112, Nathália Oliveira Braga, 74 114, 115, 117, 119, 125 Nathalia Tatakihara, 117 Rodrigo Cabral Luiz, 65, 73, 93, 97, 105, Nathália Tatakihara, 112, 115, 119, 125 163 Nayara Anitelli Artero, 86 Rodrigo Luiz Cabral, 32, 60, 61 Neide Tomimura Costa, 95 Rosiane Valeriano da Silva, 26 Nicole Perugini Stadtlober, 77, 95 Rubens Cecchini, 76 Nilson de Jesus Carlos, 14 Rubens Cecchini, 27, 54, 57, 65, 73, 83, 88, Nilton S. Arakawa, 55, 71 92, 93, 96, 97, 105, 106, 107, 155, 159 Nilza Cristina Buttow, 116 Rubia Casagrande, 29, 55, 86, 102, 171 Paola Gomes Benício Souza, 159 Rúbia Casagrande, 71, 104, 175 Paola Sanches Cella, 96 Rubia Csagrande, 43 Paola Singi, 118, 120 Sandra Cristina Heim Lonien, 26 Paulo da Silva Watanabe, 24, 116 Sandra Satie Mizokami, 86 Paulo Emilio Fuganti, 84, 89 Sara Santos Bernardes, 37, 65, 98, 105, Paulo Emílio Fuganti, 90 122 Phileno Pinge Filho, 25 Saulo Euclides Silva Filho, 24 Phileno Pinge-Filho, 15, 26, 33, 34, 38, 53 Sayonara Rangel de Oliveira, 100 Phillippe Bovo Guirro, 96 Sayonara Rangel Oliveira, 36, 45 Poliana Camila Marinello, 57, 75, 76, 83, Selene Maia de Morais, 16, 17 88, 92, 93, 96, 97, 105, 106 Sergio Marques Borghi, 171, 175 Poliana Camila Martinello, 73 Silvana Beutinger Marchioro, 122 Poliana Camurça, 48, 91 Silvia Regina Rogatto, 84 Poliana Macedo Guimarães, 77 Simona Boncompagni, 54 Priscila D. Pavezzi, 76 Simone Cristine Semprebon, 62 Priscila Daiane Pavezzi, 83, 88, 106 Simone Forcato, 40, 47 Priscila Marianno, 39 Stephanie Badaró Garcia, 64, 102, 112, Priscilla Ferreira Crespo Gutierrez, 163 114, 119, 171, 175 Pryscilla Fanini Wowk, 26 Stephanie Carvalho Borges, 116 Rafael Carvalho de Freitas, 26 Sueli Fumie Yamada-Ogatta, 26, 53 Rafael Deminice, 96 Tacito Graminha Campois, 123 Rafael Yoshio Kanashiro, 48, 91 Talita P. Domiciano, 55 Rafaela Picinin, 38 Talita Perdigão Domiciano, 102 Rafaela Pires Erthal, 136 Tamires Flauzino, 36, 45, 77, 95, 100 Rafaele Maria Tirolla, 45 Tânia Cristina Alexandrino Becker, 37, 98 Raíssa Coracini Varago, 37, 98 Tathiana Aparecida Alvarenga, 107 Ramalho, M.C, 80 Tathiana Veiga Mayumi Iriyoda, 77

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Tatiana Mayumi Veiga Iriyoda, 95, 108 Virginia Marcia Concato, 16, 17 Taylon Felipe Silva, 21, 167 Virginia Márcia Concato, 19 Telma Saraiva dos Santos, 123 Wadiceu Ap. Verri Junior, 82 Thacyana Teixeira de Carvalho, 43 Wagner Loyola, 46 Thais Peron da Silva, 46 Waldiceu A. Verri Jr, 71, 86 Thâmara Alves, 155 Waldiceu A. Verri Jr., 55 Thamara Nishida Xavier da Silva, 97 Waldiceu A. Verri, Jr, 29, 104 Théo Nicolacópulos, 76, 83, 88, 106 Waldiceu Ap. Verri Jr, 59 Thiago Cezar Fujita, 28 Waldiceu Ap. Verri Junior, 102 Thiago M. Cunha, 29 Waldiceu Aparecido Verri Jr, 66, 67, 85, Tiago Henrique Zaninelli, 171, 175 107 Tiago Mateus Andrade Vidigal, 103 Waldiceu Aparecido Verri Junior, 43, 171, Tiago Peres da Silva Suguiura, 37, 98 175, 179 Uili Andrey de Souza, 69 Walison A. da Silva Brito, 76 Uili Andrey De Souza, 31 Walison Augusto da Silva Brito, 83, 88, Valeria Campos, 58 92, 105, 106 Vanessa Almeida Nascimento, 31, 69 Walison Augusto Silva Brito, 93 Vanessa Cordeiro Dias, 121 Walter Jorge Sobrinho, 87 Vanessa Gheno, 76, 83, 88, 106 Wander R. Pavanelli, 167 Vanessa Jacob Victorino, 27 Wander Rogério Pavanelli, 16, 17, 19, 20, Vânia Lúcia da Silva, 121 21, 22, 23, 46 Vera Lúcia Hideko Tatakihara, 15 Wildéa Jennings Pereira De Carvalho, 36 Victor Fattori, 59, 71, 104, 171, 175 Wildea Lice de Carvalho Jennings Vilma Ferreira de Godoi, 72 Pereira, 100 Vinícius Daher Delfino, 45 Yuri L. Gonzalez, 71 Vinicius Popolin, 49 Zuleica Naomi Tanno, 167

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