Graduate Program in Experimental Pathology · by reducing pain and inflammation in mice..... 27 abstracts selected for ... adherence and efficacy of olfactory training as a treatment

Graduate Program in Experimental Pathology

PROCEEDINGS

III International Symposium os Experimental Pathology

Editorial board

Eduardo José de Almeida Araújo, PhD

Alessandra Lourenco Cecchini, PhD

Ana Paula Franco Punhagui, MSc

Rafaela Pires Erthal, MSc

Taylon Felipe Silva, MSc

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 / BRASIL

Contact: Eduardo José de Almeida Araújo

Telephone: +55 43 3371-5498

Graduate Program in Experimental Pathology | Londrina-Brazil | 3rd Edition | p.1-187 | 2019

The authors of the abstracts are totally responsible for the veracity of the information.

The Scientific Committee is not responsible for 'spell-checking' and 'grammar-checking' of the

abstracts.

All authors agreed with the publication of the accepted abstracts in the Annals of

III International Symposium of Experimental Pathology without any consideration regarding

copyrights.

Catalogação na publicação elaborada pela Divisão de Processos Técnicos da

Biblioteca Central da Universidade Estadual de Londrina.

Dados Internacionais de Catalogação-na-Publicação (CIP)

Bibliotecária responsável: Marlova Santurio David CRB 9/1107

I61a International Symposium of Experimental Pathology (3. : 2019 :

Londrina, PR)

Annals [of the] III International Symposium of Experimental

Pathology [livro eletrônico] / Editorial board: Eduardo José de

Almeida Araújo ...[et al.]. – Londrina : UEL, 2019.

1 Livro digital.

Vários autores.

Inclui bibliografia.

Disponível em: http://www.uel.br/eventos/simposiopato/

ISBN 978-85-302-0047-3

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. Título.

CDU 616-092

PROCEEDINGS

III International Symposium os Experimental Pathology

Organizing Committees

Sc

ien

tific

Qasim Aziz, PhD, Queen Mary of London - UK

Madusha Peiris, PhD, Queen Mary of London - UK


Ana Paula Franco Punhagi, MSc

Stephanie Badaró Garcia, MSc


So

cia

l Mariana Marques Bertozzi, MSc



Virgínia Marcia Concato, BSc

Ch

air

s


Alessandra Lourenco Cecchini, PhD

Sp

on

sor

Tiago Henrique Zaninelli, MSc

Mariana Marques Bertozzi, MSc

Ana Paula Franco Punhagi, MSc

Natália Almeida de Barros, MSc

Virgínia Marcia Concato, MSc


Matheus Decoro, BSc

Thiago Nascimento, BSc

Dis

clo

sure


Tiago Henrique Zaninelli, MSc

Natália Almeida de Barros, MSc

Virgínia Marcia Concato, MSc

Andréia Carla Eugenio Pupim, MSc

Camila Cristina Alves Machado, MSc

Matheus Decoro, BSc

Thiago Nascimento, BSc

Fin

an

cia

l

Camila Cristina Alves Machado, MSc

Andréia Carla Eugenio Pupim, MSc

CONTENTS

ABOUT US .................................................................................................................................... 14

PROGRAM .................................................................................................................................. 15

SHORT-COURSES ........................................................................................................................ 19

ABSTRACTS SELECTED FOR ORAL PRESENTATION .................................................................... 21

Undergraduate Selected Abstracts.............................................................................................

CROSS-SECTIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS INFECTION AND

CYTOLOGICAL ABNORMALITIES IN GYNECOLOGICAL OUTPATIENTS IN NORTHERN

PARANÁ, BRAZIL ........................................................................................................................ 22

RESOLVIN D2 REDUCES SKIN INFLAMMATION INDUCED BY UVB IRRADIATION IN MICE .... 23

SILDENAFIL (VIAGRA®, PFIZER) ALTERS INFLAMMATORY RESPONSE AND AGGRAVATES THE

CLINICAL CONDITION IN A MURINE MODEL OF PARKINSON’S DISEASE .............................. 24

Graduate Selected Abstracts.......................................................................................................

COMBINED EFFECTS OF LOW DOSE OF PLASMA AND IONIZING RADIATION IN MURINE

MELANOMA CELL LINE B16F10. ................................................................................................ 25

SARCOPLASMIC RETICULUM VOLUME LOSS PARTICIPATION ON FIBER-SPECIFIC SKELETAL

MUSCLE WASTE IN PRECACHEXIA INDUCED BY WALKER-256 CARCINOSARCOMA .......... 26

THE LIPID MEDIATOR LIPOXIN A4 AMELIORATES TITANIUM DIOXIDE (TiO2)-INDUCED ARTHRITIS

BY REDUCING PAIN AND INFLAMMATION IN MICE ................................................................ 27

ABSTRACTS SELECTED FOR POSTER PRESENTATION ................................................................. 28

Clinical Pathology..........................................................................................................................

ADHERENCE AND EFFICACY OF OLFACTORY TRAINING AS A TREATMENT FOR PERSISTENT

OLFACTORY LOSS ...................................................................................................................... 29

BIOLOGICAL BEHAVIOR IN DIFERENTE PARAMETERS OF OXIDATIVE STRESS AND

INFLAMMATORY CYTOKINES, AND DETERMINATION OF THE CRITICAL DIFFERENCE IN

HEALTHY MALE SUBJECTS AGED 18 TO 28 YEARS ................................................................... 30

CORRELATION ANALYSIS OF CXCR4 AND CXCR7 mRNA LEVELS WITH CLINICAL OUTCOMES

IN AGGRESSIVE BREAST CANCER SUBTYPES ............................................................................ 31

GSSH/GSH RATIO: A BIOMARKER THAT CAN DIFFERENTIATE THE TYPES OF NON-MELANOMA

SKIN CANCER ............................................................................................................................. 32

MONITORING THE BIOCHEMICAL PARAMETERS OF STOCK INJURIES, DURING THE STORAGE

OF CONCENTRATES OF DOGS 'HEMACIES, FOR 42 DAYS IN PLASTIC BAGS CPD/SAG-M 33

OLFACTORY FUNCTION, NASAL OBSTRUCTION AND QUALITY OF LIFE IN PATIENTS WITH

CHRONIC RHINOSINUSITIS WITH AND WITHOUT NASAL POLYPOSIS ...................................... 34

OXIDATIVE STRESS AND INFLAMMATION PLAY A ROLE IN THE DEVELOPMENT OF SQUAMOUS

CELL CARCINOMA ASSOCIATED WITH OTHER SKIN NEOPLASIAS ......................................... 35

OXIDATIVE STRESS BIOMARKERS INCREASE DURING 42 DAYS OF STORAGE IN PACKED RED

BLOOD CELLS OF DOGS ........................................................................................................... 36

SAFETY AND EFFICACY OF SUPERIOR TURBINATE BIOPSIES AS A SOURCE OF OLFACTORY

EPITHELIUM APPROPRIATE FOR MORPHOLOGICAL ANALYSIS .............................................. 37

SUPPLEMENTATION OF OMEGA-3 FATTY ACID IN DIABETIC NEPHROPATHY - SYSTEMATIC

REVIEW ........................................................................................................................................ 38

SYSTEMIC CORTISOL AND POOR PROGNOSIS IN OVERWEIGHT/OBESE WOMEN WITH

BREAST CANCER ........................................................................................................................ 39

Epidemiology.................................................................................................................................

CHALLENGES TO HUMAN LEISHMANIASIS: DISCUSSION OF THE DISEASE BASED ON ITS

BEHAVIOR ................................................................................................................................... 40

DEATHS FROM RECTO-SIGMOID SEGMENT CANCER IN SOUTHERN BRAZIL, A DATA SERIES

ANALYSIS, BY AGE AND SEX, FROM 2000 TO 2015. ................................................................ 41

DENGUE: THE SCIENTIFIC PRODUCTION IN BRAZIL RELATED TO THE NURSING ASSISTANCE IN

PUBLIC HEALTH ........................................................................................................................... 42

EVALUATION OF CARDIOVASCULAR RISK FACTORS IN RENAL TRANSPLANTATION PATIENTS

..................................................................................................................................................... 43

OCCURRENCE OF SCHISTOSOMIASIS MANSONI AND INTESTINAL PARASITOSES IN PATIENTS

ATTENDED AT THE AMBULATORY OF SPECIALTIES OF LONDRINA UNIVERSITY HOSPITAL,

BRAZIL, FROM TO 2017 .............................................................................................................. 44

PROFILE OF ATTEMPTED SUICIDE BY CHEMICAL SUBSTANCES ASSISTED BY POISON CONTROL

CENTER OF LONDRINA .............................................................................................................. 45

REGULATION OF LIPID METABOLISM BY AEROBIC PHYSICAL TRAINING AND OMEGA-3

INTAKE IN PROSTATE OF WISTAR RATS SUBMITTED TO HIGH FAT DIET .................................... 46

RELATION BETWEEN SOCIAL INDICATORS AND THE INCIDENCE OF MULTIDRUG RESISTANT

TUBERCULOSIS ............................................................................................................................ 47

THE PREVALENCE OF SYPHILIS IN PRENATAL CARE WOMEN IN PARANA STATE, BRAZIL ..... 48

General Pathology........................................................................................................................

A CARACTERIZATION OF CARBOXYMETHYLQUITOSAN HIDROGEL INCORPORATED WITH

HYALURONIC ACID WITH POTENTIAL HEALING ACTIVITY ON SECOND DEGREE BURNS

EXPERIMENTALLY INDUCED ON MICE ...................................................................................... 49

A CARACTERIZATION OF CARBOXYMETHYLQUITOSAN HIDROGEL INCORPORATED WITH

SILVER WITH POTENTIAL HEALING ACTIVITY ON SECOND DEGREE BURNS EXPERIMENTALLY

INDUCED ON MICE .................................................................................................................... 50

ACQUIRED DOXORUBICIN RESISTANCE IN MCF-7 AND MDA-MB-231 INVOLVES

PROTECTION AGAINST OXIDATIVE STRESS AND APOPTOSIS AND MODULATION OF GENES

RELATED WITH TRANSFORMING GROWTH BETA 1 PATHWAY ................................................ 51

ANALYSIS OF HISTOPATHOLOGY INTRAPERITONEAL INFECTION RELATED BY Escherichia coli

IN LARGE INTESTINE AND KIDNEYS IN FEMALE SWISS MICE .................................................... 52

ANALYSIS OF OXIDATIVE STRESS PARAMETERS IN PATIENTS WITH PAPILLARY THYROID

CARCINOMA AND HASHIMOTO'S THYROIDITIS ...................................................................... 53

ANTI-PROLIFERATIVE ACTIVITY OF TRANS-CHALCONE IN VITRO IN HUMAN

HEPATOCELLULAR CARCINOMA HUH7.5 LINE ........................................................................ 54

ASSOCIATION BETWEEN LACTOFERRIN GENE POLYMORPHISM AND DENTAL CARIES IN

PRIMARY DENTITION .................................................................................................................. 55

ASSOCIATION OF THE IL-10 POLYMORPHISM c. -592 C>A (rs1800872)IN WOMEN WITH

CERVICAL CANCER .................................................................................................................. 56

Brucella abortus INTERFERES IN THE INFLAMMATORY PROFILE IN A SYNTHETIC MATRIX

IMPLANT IN MICE IN A TYPE IV SECRETION SYSTEM-DEPENDENT FASHION ........................... 57

CEREBRAL ISCHEMIA AND REPERFUSION PROMOTES ALTERATION IN CALICIFORM CELLS IN

THE JEJUNO OF WISTAR RATS ................................................................................................... 58

CHARACTERIZATION OF METFORMIN ACTIVITY IN VIVO MURINE METASTATIC MELANOMA

MODEL ........................................................................................................................................ 59

CHARACTERIZATION OF THE HISTORY OF RISK FACTORS IN PATIENTS WITH COLORECTAL

CANCER TREATED AT HOSPITAL UNIVERSITÁRIO REGIONAL DO NORTE DO PARANÁ ....... 60

CHIKUNGUNYA VIRUS INDUCED MECHANICAL HYPERALGESIA IS MEDIATED BY

RECOMBINANT E2 PROTEIN AND RELATED TO TRPV1 CHANNELS ACTIVITY ........................ 61

CREATINE SUPPLEMENTATION EXACERBATES ETHANOL-INDUCED HEPATIC DAMAGE IN

MICE ............................................................................................................................................ 62

DENGUE VIRUS INDUCES MECHANICAL HYPERALGESIA BY INCREASING CYTOKINE

PRODUCTION ............................................................................................................................. 63

EFFECTS OF PHYSICAL TRAINING ASSOCIATED OR NOT TO L-ARGININE SUPPLEMENTATION

ON CARDIOVASCULAR AND AUTONOMIC PARAMETERS IN OBESE RATS ........................... 64

EFFECTS OF TYPE 1 DIABETES MELLITUS AND AGARICUS BLAZEI MURRILL EXTRACT ON THE

MYENTERIC NEURONS OF THE PROXIMAL AND DISTAL COLON OF RATS ............................ 65

EVALUATION OF METFORMIN EFFECTS IN OXIDATIVE STRESS, TGF-Β1 AND GENES RELATED

TO CELL PROLIFERATION, INVASION AND METASTASIS IN HUMAN BREAST CANCER CELLS.

..................................................................................................................................................... 66

EVALUATION OF MYENTERIC AND SUBMUCOUS NEURONS OF THE DISTAL COLON OF RATS

SUBMITTED TO SUCROSE-RICH DIET AND TREATED WITH PTEROSTILBENE .............................. 67

EVALUATION OF SYSTEMIC OXIDATIVE STRESS PROFILE IN PATIENTS WITH COLORECTAL

CANCER ..................................................................................................................................... 68

EXPRESSION OF RECEPTOR-TYPE PROTEIN-TYROSINE PHOSPHATASE ZETA/PHOSPHACAN IN

COLONIC ENTERIC NEURONS IS CHANGED BY DEXTRAN SULFATE SODIUM INDUCED-

COLITIS ........................................................................................................................................ 69

FOXP3 INTRON -1 POLYMORPHISMS ARE INDEPENDENT PREDICTORS OF HUMAN

PAPILLOMAVIRUS INFECTION AND HIGHGRADE SQUAMOUS INTRAEPITHELIAL LESIONS: A

CLINIC-BASED CASE-CONTROL ............................................................................................... 70

FRAGILE X SYNDROME IN MALE SIBLINGS: A CASE REPORT IN THE GENETIC COUNSELING

SERVICE OF LONDRINA ............................................................................................................. 71

FREE RADICAL SCAVENGER POTENTIAL OF Hylocereus undatus, Hylocereus costaricensis

AND Selenicereus megalanthus .............................................................................................. 72

GENERATION OF OXIDATIVE STRESS AND DEATH PATHWAY INDUCED BY METFORMIN IN

MCF-7 HUMAN BREAST CANCER CELLS. ................................................................................. 73

HESPERIDIN-METHYL-CHALCONE ATTENUATES PAIN AND INFLAMMATION IN A ZYMOSAN-

INDUCED ARTHRITIS MODEL IN MICE ....................................................................................... 74

HIGH-THROUGHPUT COMBINATORIAL SCREENING IDENTIFIES MITOCHONDRIAL TARGETING

DRUGS THAT SHOW SYNERGISTIC EFFECT WITH COLD PHYSICAL PLASMA AGAINST

MELANOMA CELLS .................................................................................................................... 75

HISTOPATHOLOGICAL EVALUATION OF THE INTRADERMAL INJECTION OF CRUDE EXTRACT

AND THE FRACTION F1 OF Fusarium oxysporum .................................................................... 76

HOW SKELETAL MUSCLE LOSS DIFFERENTIALLY PROGRESS DURING CACHEXIA

DEVELOPMENT AFTER EHRLICH CARCINOSARCOMA INOCULATION: A COMPARISON

BETWEEN PRECACHEXIA AND CACHEXIA .............................................................................. 77

MYELOPATHY ASSOCIATED WITH HUMAN TYPE I CELL LYMPHOTROPIC VIRUS ................... 78

Hylocereus undatus SEEDS PROTECTS CELL MEMBRANE FROM LIPOPEROXIDATION

INDEPENDENTLY OF IRON CHELATION .................................................................................... 79

IMPACT OF NEONATAL METFORMIN AND HYPERLIPID INTAKE IN ADULTHOOD ON RAT

VENTRAL PROSTATE MORPHOLOGY AND SPERM MOTILITY .................................................. 80

JEJUNUM MORPHOLOGY OF RATS SUBMITTED TO SUCROSE-RICH DIET AND TREATED WITH

PTEROSTILBENE ........................................................................................................................... 81

MAST CELL QUANTIFICATION IN THE DUODENAL MUCOSA OF WALKER-256 TUMOR-

BEARING RATS ADMINISTRATED WITH 1% L-GLUTATHIONE .................................................... 82

METFORMIN ACTIVITY IN THE INDUCTION OF DACARBAZINE RESISTANCE IN B16F10 MURINE

MELANOMA CELLS ON ANTIOXIDANT PARAMETERS ............................................................. 83

METFORMIN PREVENTS SKIN AND SYSTEMIC OXIDATIVE DAMAGE AND IMMUNE

ALTERATIONS INDUCED BY UVB RADIATION. .......................................................................... 84

MORPHOLOGICAL EVALUATION OF HEPATOCYTES NUCLEUS OF WALKER-256 TUMOR-

BEARING RATS SUPPLEMENTED WITH LGLUTATHIONE 1% ....................................................... 85

MORPHOMETRIC ANALYSIS OF MYENTERIC NEURONS IMMUNOREACTIVE TO HuC/D

PROTEIN AND ENTERIC GLIAL CELLS IMMUNOREACTIVE TO s100 PROTEIN IN THE JEJUNUM

OF WALKER256 TUMOR-BEARING RATS TREATED WITH 1% L-GLUTATHIONE ........................ 86

NEURONAL AND COLONIC MOTILITY CHANGES CAUSED BY DEXTRAN SODIUM SULFATE-

INDUCED ACUTE COLITIS: AN EXPERIMENTAL MODEL ........................................................... 87

NFKB1/NFKBIA POLYMORPHISMS PLAY A PROTECTIVE ROLE AGAINST HPV INFECTION .... 88

PRECACHEXIA STAGE OCCURS 5 DAYS AFTER WALKER-256 TUMOR SOLID INOCULATION.

..................................................................................................................................................... 89

PREVENTIVE CREATINE SUPPLEMENTATION DOES NOT INFLUENCE TUMOR AGGRESSIVENESS

IN WALKER-256 TUMOR-BEARING RATS ................................................................................... 90

PREVENTIVE PUNCTUAL APPLICATION OF LED IN SKELETAL MUSCLE REDUCES THE LEVEL OF

OXIDATIVE STRESS CAUSED BY ACUTE PHYSICAL EXERCISE. .................................................. 91

QUANTITATIVE ANALYSIS OF SUBMUCOSAL NEURONS IMMUNOREACTIVE TO THE HUC/D

PROTEIN AND SUBMUCOSAL ENTERIC GLIAL CELLS IMMUNOREACTIVE TO THE s100 PROTEIN

IN THE JEJUNUM OF WALKER-256 TUMOR-BEARING RATS TREATED WITH 1% L-GLUTATHIONE

..................................................................................................................................................... 92

TENASCIN X DEFICIENCY ALTERS THE EXPRESSION OF RECEPTOR- TYPE PROTEIN-TYROSINE

PHOSPHATASE ZETA/PHOSPHACAN IN ENTERIC COLONIC NEURONS OF MICE ................ 93

THE EFFECT OF TROLOX AND CURCUMIN ON THE ACTION OF DACARBAZINE ON MURINE

MELANOMA CELLS (B16F10). .................................................................................................... 94

TREATMENT WITH SYSTEMIC OR CENTRAL ANTIOXIDANT ATTENUATE CARDIOVASCULAR,

AUTONOMIC AND OXIDATIVE PARAMETERS IN METABOLIC SYNDROME ........................... 95

Immunology...................................................................................................................................

APOBEC3A/B DELETION POLYMORPHISM IS CORRELATED WITCLINICOPATHOLOGICAL

AND PROGNOSTIC PARAMETERS IN BREAST CANCER SUBTYPES .......................................... 96

Arthrographis kalrae FORMS BIOFILM AND INHIBITS THE PRODUCTION OF NITRIC OXIDE BY

MACROPHAGES IN VITRO ........................................................................................................ 97

CELL ADHESION MOLECULES AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 IN

PATIENTS WITH PROSTATE CANCER: ASSOCIATION WITH THE PRESENCE OF METASTASIS AND

RISK STRATIFICATION .................................................................................................................. 98

CRANBERRY JUICE DECREASES DISEASE ACTIVITY IN WOMEN WITH RHEUMATOID ARTHRITIS

..................................................................................................................................................... 99

DIFFERENTIAL PROFILE OF ADHESION MOLECULES IN SYSTEMIC LUPUS ERYTHEMATOSUS

VERSUS RHEUMATOID ARTHRITIS: A MACHINE LEARNING STUDY ........................................ 100

DISABILITY IN MULTIPLE SCLEROSIS IS PREDICTED BY A TH17 CYTOKINE PROFILE, CARBONYL

PROTEINS, METABOLIC SYNDROME AND METABOLIC BIOMARKERS: RESULTS OF

MULTIVARIATE AND MACHINE LEARNING PROCEDURES .................................................... 101

FOXP3 POLYMORPHISMS rs3761548 AND rs2232365 DO NOT INFLUENCE INTERLEUKIN-10

LEVELS IN HPV-INFECTION ....................................................................................................... 102

INCREASED ADIPONECTIN, TH2, AND TREG RESPONSES IN PATIENTS WITH SYSTEMIC

SCLEROSIS: USE AS A PREDICTOR OF DIAGNOSIS ................................................................ 103

INFLAMMATORY MONOCYTES CONTRIBUTE TO AN EXACERBATE INFLAMMATION AND

ORGAN DAMAGE DURING SEPSIS ......................................................................................... 104

INHIBITION OF Trypanosoma cruzi INVASION INTO THP-1, A HUMAN MONOCYTIC CELL LINE,

BY NIMESULIDE DRUG .............................................................................................................. 105

METABOLIC SYNDROME AGRAVATES CARDIOVASCULAR, OXIDATIVE AND

INFLAMMATORY DYSFUNCTION DURING THE ACUTE PHASE OF TRYPANOSOMA CRUZI

INFECTION IN MICE .................................................................................................................. 106

POLYMORPHISMS IN GENES RELATED TO IMMUNOSUPPRESSION ARE ASSOCIATED WITH

CERVICAL CANCER ................................................................................................................. 107

PREVENTIVE HEALTH WITH A FOCUS ON IMMUNIZATION: A LITERATURE REVIEW .............. 108

PROINFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINE PROFILES IN PSORIASIS: USE AS

LABORATORY BIOMARKERS AND DISEASE PREDICTORS ...................................................... 109

SUPPLEMENTATION WITH FISH OIL IMPROVES LIFE QUALITY, AND DECREASES

INFLAMMATORY STATUS AND OXIDATIVE STRESS IN PSORIASIS ........................................... 110

THE PATTERN OF CELL MIGRATION AND TNF-ALPHA PRO-INFLAMMATORY CYTOKINE

SECRETION, PROVIDED BY THE INITIAL INTRAPERITONEAL INFECTION OF Escherichia coli IN

FEMALE SWISS MICE ................................................................................................................. 111

THIOL GROUP AS A BIOMARKER FOR THE DIAGNOSIS OF PROSTATE CANCER AND

METASTASIS PREDICTOR .......................................................................................................... 112

TNF-β +252 A>G (rs909253) POLYMORPHISM IS INDEPENDENTLY ASSOCIATED WITH

PRESENCE OF AUTOANTIBODIES IN RHEUMATOID ARTHRITIS PATIENTS .............................. 113

TRACE ELEMENTS ASSOCIATED WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND INSULIN

RESISTANCE .............................................................................................................................. 114

Trypanosoma cruzi: THE BLOCKING EFFECT OF COX-2 WITH CELECOXIB ON THE

ACTIVATION AND INVASION OF MACROPHAGES DERIVING FROM THP-1, A LINEAGE OF

HUMAN MONOCYTES. ............................................................................................................ 115

TUMOR NECROSIS FACTOR α AND ITS SOLUBLE RECEPTORS ARE ASSOCIATED WITH DISABILITY, DISEASE PROGRESSION AND CLINICAL FORMS OF MULTIPLE SCLEROSIS ............................................................................................................................... 116

Microbiology and Parasitology....................................................................................................

ACTIVITY OF A Piper solmsianum COMPOUND AGAINST Mycobacterium tuberculosis 117

ANALYSIS OF THE NUMBER OF VIPERGIC SUBMUCOSAL NEURONS IN THE DUODENUM OF

WISTAR RATS SUBMITTED TO ACUTE Toxoplasma gondii INFECTION .................................. 118

ANTIVIRAL ACTIVITY OF BOTRYOSPHAERAN AND ITS SULFONATED MOLECULES IN THE

REPLICATION OF DENGUE VIRUS ............................................................................................ 119

ASSOCIATION BETWEEN INFECTION BY Staphylococcus aureus AND DEATH OF CHILDREN:

AN EIGHTEEN YEARS RETROSPECTIVE STUDY ......................................................................... 120

CHRONIC INFECTION WITH Toxoplasma gondii OOCYSTS CAUSE HYPOPLASIA AND

HYPERTROPHY OF MOTOR ENTERIC NEURONS IN THE PROXIMAL COLON OF RATS ......... 121

CORRELATION ANALYSIS OF MOUSE MAMMARY TUMOR VIRUS-LIKE DNA SEQUENCE WITH

CLINICOPATHOLOGICAL PARAMETERS IN BREAST CANCER SUBTYPES ............................. 122

DETECTION OF GENES ASSOCIATED WITH VIRULENCE OF Proteus mirabilis ISOLATED FROM

CHICKEN CARCASS ................................................................................................................. 123

EFFECS OF CHRONIC Toxoplasma gondii INFECTION ON JEJUNUM ................................. 124

EFFECTS OF ACUTE AND CHRONIC Toxoplasma gondii INFECTION ON DUODENUM GOBLET

CELLS ......................................................................................................................................... 125

PREVALENCE OF MICROORGANISMS RESISTANT TO MULTIPLE DRUGS IN NEWBORNS. ... 126

Escherichia coli BLOODSTREAM INFECTION IN PATIENTS OF A UNIVERSITY HOSPITAL:

VIRULENCE FACTORS AND CLINICAL CHARACTERISTICS. ................................................... 127

EVALUATION OF ADHESION PATTERN, BIOFILM FORMATION AND CYTOTOXICITY OF Proteus

mirabilis ISOLATED FROM CHICKEN CARCASS ..................................................................... 128

EXCRETION / SECRETION OF LARVAE OF FLY DIPTERANS OF THE SPECIES Lucilia cuprina

PRESENT DIRECT ACTION ON TRYPOMASTIGOTE FORMS OF Trypanosoma cruzi ............. 129

GENOTYPIC AND EPIDEMIOLOGICAL STUDY OF CARBAPENEM RESISTANT

ENTEROBACTERIACEAE ISOLATES OF PATIENTS FROM THE UNIVERSITY HOSPITAL OF

LONDRINA STATE UNIVERSITY, FROM 2009 TO 2016.............................................................. 130

HERPES SIMPLEX VIRUS IS INHIBITED BY Myrciaria dubia POLYSACCHARIDES ................... 131

HPV INFECTION AND APOBEC3A/B DELETION POLYMORPHISM IN OROPHARYNGEAL

SQUAMOUS CELL CARCINOMA PATIENTS ............................................................................ 132

IN VITRO ANTIVIRAL ACTIVITY OF LASIODIPLODAN AND ITS CARBOXIMETILATED AND

SULFATED MOLECULES IN THE REPLICATION OF HERPES SIMPLEX 1 .................................... 133

INFECTION BY Leishmania (Viannia) braziliensis DO NOT PROMOTE MORPHOLOGICAL

CHANGES IN THE ENTEROCYTES OF HAMSTERS COLON ..................................................... 134

Klebsiella pneumoniae: EPIDEMIOLOGY, RESISTANCE TO CARBAPENENS AND

POLYMYXINS AND GENETIC DIVERSITY .................................................................................. 135

MANGIFERIN CREAM EXHIBITS ACTIVITY AGAINST HERPES SIMPLEX VIRUS RESISTANT TO

ACYCLOVIR .............................................................................................................................. 136

POSSIBLE ASSOCIATION BETWEEN Paracoccidioides brasiliensis and Trichosporon asahii IN

SOIL ........................................................................................................................................... 137

PRESENCE OF VIRULENCE FACTORS AND PATHOGENICITY ISLANDS IN EXTRAINTESTINAL

PATHOGENIC ESCHERICHIA COLI ISOLATES FROM DIFFERENT CLINICAL MATERIALS ...... 138

REDUCTION OF GOBLET CELLS IN HAMSTERS INFECTED BY Leishmania (Viannia) braziliensis

................................................................................................................................................... 139

REDUCTION OF INTRAEPITHELIAL LYMPHOCYTES IN HAMSTERS INFECTED BY Leishmania

(Viannia) braziliensis ................................................................................................................ 140

Staphylococcus aureus CA-MRSA EMERGING AS PATHOGEN IN HOSPITAL ENVIRONMENT.

................................................................................................................................................... 141

THE INFECTION BY DIFFERENT STRAINS OF THE Leishmania (Viannia) braziliensis CAUSE

ALTERATIONS IN THE QUANTITY OF INTESTINAL MAST CELLS ................................................ 142

THE INFECTION BY Toxoplasma gondii PROMOTE QUANTITATIVE AND MORPHOMETRIC

ALTERATIONS IN PANETH CELLS IN THE INTESTINE OF C57BL/6 MICE .................................. 143

THE OCCURRENCE OF ANTIGENS OF CANINE DISTEMPER VIRUS IN THE OVARY AND UTERUS

OF DOGS: IMPLICATIONS OF A POSSIBLE REPRODUCTIVE DISEASE PATHOGEN ............... 144

VENOM OF THE SCORPION Tityus bahiensis, IN LOW CONCENTRATIONS, REDUCES IL-8 AND

THE PROLIFERATION OF TACHIZOITES OF Toxoplasma gondii (CEPA RH) IN HeLa CELLS 145

Pharmacology...............................................................................................................................

AMY WINEHOUSE’S DEATH: AN ALCOHOL INTOXICATION CASE STUDY ........................... 146

ANTIPROLIFERATIVE ACTIVITY of 3,3',5,5'-tetramethoxybiphenyl-4,4'-diol ON THE HUMAN

CELL LINE OF NON-SMALL CELL LUNG CARCINOMA A549 ................................................ 147

BRACHYDIN FLAVONOIDS DECREASE MIGRATION AND INVASION OF PROSTATE TUMOR

CELLS DU-145 ........................................................................................................................... 148

CAN THE SALMON TROUT CONSUMPTION BE CONSIDERED A RISK FACTOR FOR HEPATIC

STEATOSIS? ............................................................................................................................... 149

CHANGES IN THE SPERMATOZOIDS OF MICE EXPOSED TO SIBUTRAMINE .......................... 150

CHIKUNGUNYA VIRUS INDUCES ARTICULAR HYPERALGESIA IN MICE ................................ 151

CHIKUNGUNYA VIRUS RECOMBINANT E2 PROTEIN-INDUCED ARTICULAR HYPERALGESIA IS

INHIBITED BY MONOCLONAL ANTIBODIES IN MICE. ............................................................. 152

CONTRACEPTIVE POTENTIAL OF CYCLOSPORIN A IN SHORT-TERM TREATMENT ............... 153

CYTOTOXICITY OF THE STEAM PHASE FROM Jasminum officinale (JASMINE) AND

Pogostemon cablin (PATCHOULI) ESSENTIAL OILS ON LUNG CANCER CELLS ................. 154

EFFECT AND ROLE OF 15-EPI-LIPOXIN A4 IN ASPIRIN MECHANISM OF ACTION ON UVB-

IRRADIATION INDUCED SKIN INFLAMMATION AND OXIDATIVE STRESS IN HAIRLESS MICE 155

EFFECT OF ACUTE RESTRAINT STRESS IN THE RECRUITMENT OF NEUTROPHILS TO THE

PERITONEAL CAVITY INDUCED BY CARRAGEENAN ............................................................. 156

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED COLLAGEN

DEGRADATION, AND PRODUCTION OF CYTOKINES IN THE SKIN OF MICE, AND EFFECT OF

THE BOC-2 IN THE EFFICACY OF BML-111 IN THIS MODEL .................................................... 157

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED MAST CELL, IN THE

EFFICACY OF BML-111 IN THIS MODEL SUNBURN CELL, AND TNF-α LEVELS IN THE SKIN OF

MICE, AND EFFECT OF THE BOC2 ........................................................................................... 158

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED OXIDATIVE STRESS IN THE

SKIN OF MICE, AND EFFECT OF THE BOC-2 IN THE EFFICACY OF BML-111 IN THIS MODEL

................................................................................................................................................... 159

EFFECTS OF PTEROSTILBENE ON THE HEPATIC OXIDATIVE STRESS OF RATS SUBMITTED TO

SUCROSERICH DIET. ................................................................................................................. 160

EFFICACY OF Rosmarinus officinalis EXTRACT-LOADED FORMULATION AGAINST UV-

INDUCED OXIDATIVE STRESS ................................................................................................... 161

EFFICACY OF THE ASPIRIN AND 15-EPI-LIPOXIN A4 IN SKIN DAMAGE INDUCED BY UVB

IRRADIATION, AND EFFECT OF THE BOC-2 IN THE EFFICACY OF ASPIRIN AND 15-EPILIPOXIN

A4 IN THIS MODEL .................................................................................................................... 162

EFFICACY OF THE BML-111 ON UV IRRADIATION-INDUCED INFLAMMATION IN THE SKIN OF

MICE, AND EFFECT OF THE BOC-2 IN THE EFFICACY OF BML-111 IN THIS MODEL ............. 163

ESCITALOPRAM INDUCES CHANGES IN MICE ODONTOGENESIS ....................................... 164

EVALUATION OF AORTA REACTIVITY IN MALE RATS TREATED WITH TOPIRAMATE DURING

ADOLESCENCE ........................................................................................................................ 165

EVALUATION OF THE HYPOGLYCEMIC ACTIVITY OF THE BARK ETHANOLIC EXTRACT FROM

Spondias dulcis AND Spondias purpurea IN ALOXAN-INDUCED DIABETIC RATS ............. 166

EXPOSURE OF ZOLPIDEM DURING SPERMATOGENESIS PROMOTES CONGENITAL

MALFORMATION IN PROLE ..................................................................................................... 167

EXPOSURE TO BUPROPION HYDROCHLORIDE IN PREGNANCY MICE COMPROMISES

FERTILITY AND PROMOTES CONGENITAL MALFORMATIONS ............................................... 168

HISTORY OF MULTIPLE ALLERGIES: A CASE STUDY ................................................................. 169

LOW DOSES OF MALATHION CAUSE ATROPHY OF JEJUNAL WALL IN RATS ...................... 170

MARESIN-1 ATTENUATES INFLAMMATION IN TITANIUM DIOXIDE (TIO2)-INDUCED CHRONIC

ARTHRITIS IN MICE. ................................................................................................................... 171

ACUTE AND LATE METABOLIC EFFECTS OF TOPIRAMATE TREATMENT DURING CHILDHOOD

................................................................................................................................................... 172

MORPHOMETRIC ANALYSIS OF MYENTERIC NEURONS IMMUNOREACTIVE TO HUC/D

PROTEIN OF JEJUNUM FROM ARTHRITIC RATS TREATED WITH QUERCETINLOADED

MICROCAPSULES ..................................................................................................................... 173

MORPHOPHYSIOLOGICAL ALTERATIONS OF RAT COLON EXPOSED TO LOW DOSES OF

MALATHION .............................................................................................................................. 174

IN UTERO AND LACTATIONAL EXPOSURE TO TRICLOCARBAN DID NOT ALTER SPERM

PARAMETERS IN MALE RATS .................................................................................................... 175

PERIPUBERAL EXPOSURE OF MALE RATS TO LOW DOSES OF MALATHION IMPAIRS

EPIDIDYMAL MORPHOLOGY AND PHYSIOLOGY ................................................................. 176

PHOTOCHEMOPROTECTIVE EFFECTS OF TOPICAL FORMULATION WITH 15-DEOXY-Δ12,14-

PROSTAGLANDIN J2 IN UVB-INDUCED INFLAMMATION AND OXIDATIVE STRESS IN HAIRLESS

MICE .......................................................................................................................................... 177

PLATELET ACTIVATING FACTOR INVOLVEMENT IN ACUTE LUNG INJURY CAUSED BY

SCORPION VENOM ................................................................................................................. 178

PROTECTIVE EFFECT OF TOPICAL FORMULATION CONTAINING VANILLIC ACID AGAINST

UVB-INDUCED OXIDATIVE STRESS IN HAIRLESS MICE ............................................................ 179

RESOLVIN D2 ATTENUATES UVB RADIATION-INDUCED SKIN OXIDATIVE DAMAGE IN MICE

................................................................................................................................................... 180

RESOLVIN D5 REDUCES FORMALIN OVERT PAIN-LIKE BEHAVIOR IN MICE. ........................ 181

SUCROSE AND PTEROSTILBENE ALTER THE HEPATIC MORPHOLOGY OF RATS ................... 182

THE PRO-RESOLUTION 17-R-RVD1 LIPID MEDIATOR REDUCES PAIN, JOINT EDEMA,

LEUKOCYTE RECRUITMENT, PROINFLAMMATORY CYTOKINES PRODUCTION, AND

OXIDATIVE STRESS IN A MURINE MODEL OF GOUTY ARTHRITIS............................................ 183

THE ROLE OF ESTROGEN IN VASCULAR REACTIVITY OF FEMALE PROGENY EXPOSED TO

FLUOXETINE DURING EARLY DEVELOPMENT ......................................................................... 184

THE ROLE OF SPINAL ASTROCYTES, MICROGLIAL CELLS AND NF-κB ON THE TRYPANOSOMA

CRUZI EXPERIMENTAL INFECTION-INDUCED PAIN ................................................................ 185

TREK-1 IS INVOLVED IN BRAIN CHANGES ON A DEPRESSION ANIMAL MODEL ................. 186

REALIZATION, SPONSORSHIP AND SUPPORT .......................................................................... 187

ABOUT US The Postgraduate Program in Experimental Pathology of the Londrina

State University announces the III International Symposium of Experimental

Pathology (ISEP 2019), to be held in Londrina, PR, Brazil, on May 22nd to

24th, 2019.

The first five editions of this event were promoted in order to reach

junior and senior researchers from Londrina. After becoming a local well-

established event we decided to expand to a larger conference to join

more researchers who studies in the experimental pathology field.

The ISEP 2019 will provide a program with international and

Brazilian speakers including lectures, round table and short-courses.

Besides, we do encourage you to present your work during the Symposium

as poster or oral presentation. We are sure that will be an excellent

opportunity to discuss your basic and clinical research with numerous

colleagues from Brazil and other countries.

14

PROGRAM

15

16

WEDNESDAY (MAY 22nd, 2019)

2 – 4 PM Registration and putting

up to posters 7:30 - 8:30 PM Opening Lecture

4 – 6:30 PM Poster session

8:30 PM Reception

7 PM Opening ceremony

THURSDAY (MAY 23rd, 2019)

8 – 9:30 AM Short-courses I to X 2:30 – 4 PM Round Table 1

9:30 – 10

AM Coffee Break 4 – 4:30 PM Coffee Break

10 – 12 AM Short-courses I to X 4:30 – 5 PM Lecture II

12 – 1:30

PM Lunch Time 5 – 6:30 PM Round Table II

1:30 – 2:30

PM Lecture I 6:30 – 7 PM Lecture III

FRIDAY (MAY 24th, 2019)

8 – 9:30 AM Short-courses XI to XX 3 – 4 PM Lecture IV

9:30 – 10

AM Coffee Break 4 – 4:30 PM Coffee Break

10 – 12 AM Short-courses XI to XX 4:30 – 6 PM Round Table III

12 – 1:30

PM Lunch Time

6 PM Awards and Closing

Ceremony

1:30 – 3 PM Oral presentation: Best

Works

17

LECTURESOpening lecture: TRANSLATIONAL MEDICINE, FACTS AND CHALLENGESMarcelo José Villar, PhD – Universidad Austral - Pilar, Buenos Aires, Argentina

Lecture I: CHARACTERIZATION OF A NOVEL G-PROTEIN COUPLED RECEPTOR IN MOUSE AND HUMAN ENTERIC NERVOUS SYSTEM Madusha Peiris, PhD – Queen Mary University of London – UK

Lecture II: APPLICATIONS OF COLD PHYSICAL PLASMA IN ANTI CANCER THERAPYRajesh Kumar Gandhirajan, PhD – Leibniz-Institute for Plasma Science and Technology – (INP Greifswald) Greifswald – Germany

Lecture III: METHODOLOGY OF THE POPULARIZATION OF SCIENTIFIC KNOWLEDGE IN HEALTHDébora de Mello Gonçales Sant’ana, PhD – Maringá State University – BR

Lecture VI: VOLTAGE-GATED CALCIUM CHANNELS AS TARGETS FOR PAIN THERAPEUTICSGerald W. Zamponi, PhD – Department of Physiology & Phamacology Calgary University – Calgary, Canada

18

ROUND TABLES

Round Table 1: ARBOVIRUSES: WHAT DO WE KNOW UNTIL THENSession Chair / Speakers: Milena M. Miranda-Sapla, PhD / Tiago Campos Pereira, PhD / Anderson de Sá Nunes, PhD

Round Table 2: OBESITY: A DISEASE IN ALERT SIGNALSession Chairs / Speakers: Hugo Tourinho Filho, PhD / Marli Cardoso Martins Pinge, PhD / Kesia G. Palma Rigo Wutzow, PhD

Round Table 3: LONGEVITY REVOLUTION: HOW DO WE WORK WITH AGING?Session Chairs / Speakers: Lígia Carreira, PhD / Rodrigo T. C. S. Rodrigues, PhD / Marcos A. Sarria Cabrera, PhD

SHORT-COURSES Th

urs

da

y –

Ma

y 2

3rd

I “Macrophage: a divergent cell”

Phileno Pinge Filho, PhD

Londrina State University

CCB

Room 271b

II

“Oxidative Stress and Diseases”

Flávia Alessandra Guarnier, PhD

State University of Londrina

CCB

Experimental

Pathology

Classroom

III

“Editing and production of photographic images for

scientific publishing”

Thiago Fernandes, PhD


LMEM

Electron

Microscopy

IV

“Primers: who are they and how to outline them?”

Eliandro Reis Tavares, PhD


LABESC

Room 4

V

“Methodology of the popularization of scientific

knowledge in health”

Débora de Mello Gonçales Sant’ana, PhD

Maringá State University

CCB

Classroom 250

VI

“Cell culture: a master move in the study of cancer”

Poliana Camila Marinello, PhD


CCB

Classroom 209

VII

“Western blotting: principles and applications”

Emerson José Venâncio, PhD


CCB

Classroom 241

VIII

“Congenital toxoplasmosis: challenges of a neglected

diseases”

Regina Mitsuka-Breganó, PhD


CCA

Veterinary

Hospital

IX

“Principles of immunohistochemistry”



CCB

Histology

Room 369

X

“Tissue explants as alternative model for

experimentation”

Ana Paula Frederico Rodrigues Loureiro Bracarense, PhD


LABESC

Room 8

19

Fri

da

y –

Ma

y 2

4th

XI

“Molecular identification of microbial pathogens by

bioinformatic tools”

Laurival Vilas Boas, PhD

Rogerio Fernandes de Souza, PhD


LABESC

Room 3

XII

“Markers of tumor susceptibility”

Maria Angélica Ehara Watanabe, PhD


CCB

Classroom 209

XIII

“Detection of Toxoplasma gondii oocysts in

environmental samples”

Regina Mitsuka-Breganó, PhD

Felippe Danyel Cardoso Martins, MD

Fernanda P. Ferreira, MD


CCA

Veterinary

Hospital

XIV

“Leishmania strategies and application”

Ivete Conchon Costa, PhD


CCB

Pathology

Costa’s Lab

XV

“Macrophage: a divergent cell”

Phileno Pingge Filho, PhD


CCB

Pathology

Pinge-Filho's

Lab

XVI

“Toxic agents in male infertility”

Glaura Scantamburlo Alves Fernandes, PhD


CCB

Fernandes’

Lab

XVII “Notions of molecular biology”

Eliandro Reis Tavares, PhD


CCB

Classroom 241

XVIII

“Epigenetics and its basic principles”

Gislaine Garcia Pelosi Gomes, PhD

State University of Londrina

Leandro Vaz Toffoli, PhD

University of North Parana (UNOPAR)

CCB

Classroom 201

XIX

“Strategies for identification of immunomodulatory

salivary molecules from arthropod vectors with

biotechnological potential”

Anderson Sá-Nunes, PhD

University of Sao Paulo

CCB

Classroom 250

XX

“Electronic microscopy”

Admilton Gonçalves de Oliveira Junior, PhD


LMEM

Electron

Microscopy

20

ABSTRACTS Selected for Oral Presentation

21

CROSS-SECTIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS INFECTION AND CYTOLOGICAL

ABNORMALITIES IN GYNECOLOGICAL OUTPATIENTS IN NORTHERN PARANÁ, BRAZIL

Mangieri, L. F. L2; Esposito, A.1; Trugilo, K. P.1; Cezar-dos-Santos, F.1; Okuyama, N. C. M.1;

Singi P.1; Sena, M. M.1; Pereira, A. P. L.1; Curti, R. R. J,1; Pereira, E. R.1; Ferreira, R. S.1;

Bonaldo, A. L. L.1; Jesus Carlos, N.1; Oliveira, K. B.1

¹ Laboratory of Molecular Genetics and Immunology, Department of Pathological Sciences, State

University of Londrina, Londrina – PR, Brazil.

² Department of Ginecology and Obstetrics, State University of Londrina, Londrina – PR, Brazil.

[email protected]

Introduction and objectives: Londrina metropolitan area is the second largest urban agglomerate in the

Paraná, Brazil, and the greatest health care demand in the State Northern region. Epidemiological data

about HPV occurrence in Paraná are scarce. Therefore, this cross-sectional study aimed to determine

the occurrence of HPV infection and cytological abnormalities and to investigate potential

predisposing factors such as sociodemographic characteristics, sexual behavioral habits and

gynecological and obstetric background.

Material and methods: This study was approved by the Institutional Ethics Committee Involving Humans

Beings of the State University of Londrina, Londrina-PR, Brazil (CEP/UEL 133/2012; CAAE

05505912.0.0000.5231). Cervical samples were examined for HPV DNA presence by PCR from 429

consenting women. Two groups were formed: non-infected (n = 219, controls) and infected women (n

= 210). The HPV infection susceptibility was assessed regarding the potential susceptibility factors data.

Results: A greater proportion of women infected by HPV were younger than 25 years of age (p < 0.001),

single (p < 0.001), with monthly income up to one minimum wage (p = 0.018), smokers (p = 0.014), who

had their first sexual intercourse before the age of 18 years (p = 0.012), had at least four sexual partners

in their lifetime (p < 0.001), and had not been pregnant (p = 0.008). The multivariate binary logistic

regression analysis showed that age lower than 25 years increased around 5 times the chances of

infection (OR = 4.92; CI95% = 1.67 – 14.52; p = 0.004), and both married or civil partner (OR = 0.45; CI95%

= 0.23 - 0.88; p = 0.020) and monthly income of 1 to 3 minimum wage (OR = 0.59; CI95% = 0.36 - 0.95; p

= 0.030) offered protection against HPV infection. HPV positive group was also analyzed for squamous

intraepithelial lesion (SIL) presence. SIL was more frequent in women who smoked (p = 0.017), earned

up to one minimum wage monthly (p = 0.019), and declared spontaneous abortion (p = 0.042). Binary

logistic regression analysis showed that monthly income ranging of 1 to 3 minimum wage protected

against SIL development in HPV patients (OR = 0.49; CI 95% = 0.26 - 0.93; p = 0.028).

Conclusion: In conclusion, the results suggest that age, marital status and monthly income are important

cofactors for the HPV infection and SIL development in Northern region of Paraná State.

Keywords: HPV, cervical cancer, risk factors, squamous intraepithelial lesions

Grants: CAPES, CNPq and PPSUS

22

RESOLVIN D2 REDUCES SKIN INFLAMMATION INDUCED BY UVB IRRADIATION IN MICE

Pinto, I. C.1; Bezerra, J.R.1; Saito, P.1; Martinez, R. M.1; Rodrigues, C. C. A.1; Kumagai, C.

M.1; Melo, C. P. B.1; Bussmann, A. J. C.2; Baracat, M. M.1; Georgetti, S. R.1; Verri, W. A.2;

Casagrande, R.1* 1 State University of Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil

2 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil

*Email: [email protected]

Introduction and objectives: The skin is a highly metabolic and complex organ and represents a

protective physical barrier against external agents. Ultraviolet B (UVB) radiation is the main factor for the

development of photocarcinogenesis. Excessive exposure to UVB irradiation generates reactive oxygen

species (ROS) and activates signaling pathways such as NF-κB, triggering the inflammatory response of

the skin. In this context, therapies for the control of the UVB induced inflammatory would be an

alternative. Resolvin D2 (RvD2) is endogenous lipid mediators and studies have demonstrated potential

activity in some disease models, but there is no knowledge of the role of RvD2 in the UVB irradiation-

induced cutaneous lesion model. Therefore, we investigated the photoprotective effect of RvD2 in this

model.

Material and methods: This study was approved by the Animal Ethics Committee (CEUA) of the State

University of Londrina, following its standards (process number 1447.2015.10). Inflammation was induced

in hairless mice by UVB irradiation (4.14 J/cm2). The animals were randomly assigned to five experimental

groups (n=6): non-irradiated, irradiated and treated with RvD2 (0.3; 1.0 or 3.0 ng/mouse,

intraperitoneally) 1 hour before the irradiation. The skin edema was measured as an increase in dorsal

skin weight. The leukocyte migration was evaluated by myeloperoxidase (MPO) activity. The activity of

metalloproteinase-9 (MMP-9) is determined by the sodium dodecyl sulfate polyacrylamide gel (SDS-

PAGE) zymography assay. Cytokines levels were measured by enzyme-linked immunosorbent assay

(ELISA). For the histopathological analysis of the skin, the samples embedded in paraffin were stained

with hematoxylin-eosin, toluidine blue and masson’s trichrome and subsequently examined by light

microscopy for determination of epidermal thickness, number of apoptotic keratinocytes and mast cells

and damage to collagen fibers. Data were analyzed by one-way ANOVA followed by the Tukey’s test,

p<0.05.

Results: Systemic treatment with RvD2 protected the skin against inflammatory damage induced by UVB

radiation, as it significantly reduced the parameters of edema, myeloperoxidase and

metalloproteinase-9 activity, production of different cytokines (TNF-α, IL-33, IL-1β, TGF-β and IL-10),

collagen fibers degradation, epidermal thickness, keratinocyte apoptosis and number of mast cells.

Conclusion: These results suggest that the lipid mediator RvD2 has great potential of use for the control

of UVB irradiation-induced inflammation.

Keywords: photocarcinogenesis, UVB irradiation, resolvin, skin damage.

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional

de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária.

23

SILDENAFIL (VIAGRA®, PFIZER) ALTERS INFLAMMATORY RESPONSE AND AGGRAVATES THE CLINICAL

CONDITION IN A MURINE MODEL OF PARKINSON’S DISEASE

D. F. Almeida1; A. P. Bonfanti1,2; G. Peron2; J. Oliveira2; N. B. Santos1,2; L. Melo-Thomas3; L. Verinaud2; C.

Raposo1,2

¹Universidade Estadual de Campinas (UNICAMP), Faculdade de Ciências Farmacêuticas (FCF),

Campinas, SP, Brazil;

²UNICAMP, Departamento de Biologia Estrutural e Funcional, Instituto de Biologia (IB); ³Department

of Psychology, Philipps University of Marburg, Marburg, Germany. Email:

[email protected]

Introduction and objectives: Studies about the effects of Phosphodiesterase Inhibitors (PDE-Is), such as

Sildenafil (Viagra®, Pfizer), on Parkinson's Disease (PD) are scarce in the literature. On the other hand,

there is a consolidated role of these drugs in the control of other neurodegenerative diseases, such as

Multiple Sclerosis, through a mechanism that involves immunomodulation. Therefore, an evaluation of

the clinical condition and a correlation with immunological parameters were made in the present study,

in a PD-murine model treated with sildenafil.

Material and methods: The parkinsonian model was induced in C57BL/6 male mice (CEUA Protocol:

4708-1/2017 and 4708-1(A)/2018) in two ways: Rotenone (Rot) 1 mg/kg (s.c.) for 14 days associated to

0.2 mg/kg (i.p.) Lipopolysaccharide (LPS) in the first 5 days, to achieve a mild clinical condition; or to

induce a more severe clinical condition, Rot 2 mg/kg and LPS 0.1 mg/Kg were given. Animals were

randomly divided into the following groups: Control - no treatment; Preventive - received sildenafil (10

mg/kg, s.c.) for 7 days prior Rot/LPS administration and continued for another 14 days; Therapeutic - 7

days after the initiation of Rot/LPS, mice received sildenafil for 7 days; a Naïve group (without Rot/LPS

and no treatment) and a Sildenafil Control group (without ROT/LPS but receiving sildenafil at the same

posology as the Preventive group) were also performed. All animals were clinically analyzed by means

of weight register, motor test (RotaRod) and behavior (Open Field test). Only for animals receiving Rot

1 mg/kg/LPS 0.2 mg/Kg, cells isolated from the spleen were submitted to Flow Cytometry, with markers

for Th1 and Treg Lymphocytes; Blood serum was analyzed by ELISA to detect cytokines TNF-α, IL-6 and

IL-10.

Results: Rotarod showed that ROT/LPS led to significant motor impairment, compared to Naïve in both

induced clinical conditions; however, for Rot 2 mg/kg/LPS 0.1 mg/Kg, motor impairment was more

severe. Sildenafil did not improve the clinical condition, but it worsened in the 2 mg/kg/LPS 0.1 mg/Kg

group. ROT/LPS induced an increase in IL-1β levels, whereas it did not alter IL-10, compared to Naïve.

Preventive (but not Therapeutic) sildenafil significantly decreased IL-1β, TNF-alpha and IL-6 and

increased IL-10, compared to Naïve and Disease groups. T cell analysis showed that ROT/LPS did not

change the percentage of Th1, but reduced Treg cells, compared to Naïve. Both Preventive and

Therapeutic sildenafil significantly reduced Th1; and Preventive sildenafil increased Treg, compared to

the Disease group. Immunofluorescence of the brain sections showed that sildenafil aggravated the

neuroinflammation, increasing GFAP and Iba-1, compared to the Disease group. No differences were

detected between Sildenafil and Naïve control groups.

Conclusion: The study contributed to a better understanding of the sildenafil effects in a murine PD

model. Interestingly, although PDE5-Is have been shown to contribute to the treatment of other

neuroinflammatory diseases, we have demonstrated here that sildenafil did not clinically protect PD

mice. Experiments are underway to respond whether the worsening of neuroinflammation induced by

this PDE5-I is responsible for the aggravation of the clinical condition.

Keywords: T lymphocytes; Neuroinflammation; Microglia; Astrocytes; Rotenone.

Grants: FAPESP (#2015/04194-0; #2017/19799-0); CNPq (#431465/2016-9).

24

COMBINED EFFECTS OF LOW DOSE OF PLASMA AND IONIZING RADIATION IN MURINE MELANOMA CELL

LINE B16F10.

Pasqual Melo, G.1; Gandhirajan, R.1; Rückert, M.2; Frey, B.2; Gailp, U.S.2; Bekeschus, S.1

¹ Leibniz-Institute for Plasma Science and Technology (INP), ZIK plasmatis, Greifswald, Germany.

² Universität Klinikum Erlangen, Departament of Radiation Oncology, Erlangen, Germany.

[email protected]

Introduction and objectives: Metastatic melanoma presents severe challenges in clinical therapies,

and, palliation is often problematic due to large numbers of fast growing metastasis. Radiotherapy (RT)

is one of the most frequently used standard treatments for cancers. Although melanoma is a radio

resistant tumor entity, cancer cells can be rendered visible to the immune system by RT especially in

combination with hyperthermia. Cold physical plasma is a partially ionized gas that generates oxygen

and nitrogen species and selectively kills cancer cells in vitro. In plasma onco-therapy, several groups

reported an immuno-stimulatory effect of plasma treatment besides the plasma-mediated killing of

tumor cells. Hence, we sought out to combine RT and plasma aiming at additive effects towards

melanoma toxicity with hallmarks of immunogenic cell death.

Material and methods: Murine B16F10 melanoma cells were plated in a 24 wells-plate. Still in suspension,

cells were treated with plasma (kINPen) for 10s followed by ionizing radiation (2Gy or 8Gy), or vice versa.

In the second and third day the cells that were treated with ionizing radiation received another dose of

2Gy or 8Gy. 24hours after the last treatment, cells were harvested with accutase and analyzed by FACS

for cell death (AnnexinV/PI), cell cycle (DAPI) and gamma H2AX. The cell supernatant was collected

and storage at -20°C. A bead-based sandwich ELISA (LEGENDplex) was used to investigate different

cytokines. Immature DCs (jaws II) were incubated with the collected supernatants, and 96 hours later

checked for DCs activation surface markers via FACS. DCFH-DA was used to analyze the levels of ROS

in different time points after treatment with plasma or ionizing radiation.

Results: Ionizing radiation as well as plasma were able to increase the ROS levels 5 minutes after the

treatment, however, plasma can still keep the levels high after 1 or 24 hours. The combination of

radiotherapy and plasma induced DNA damage, evidenced by increase in gamma-H2AX staining.

AnnexinV/PI staining showed that both plasma alone and radiotherapy alone (8Gy) as well as the

combined treatment were able to decrease the cell viability, nevertheless in the single treatment it was

found more apoptotic cells while in the combination necrosis seems to be the more prominent. It is well

known that is important that anticancer therapies also induces an antitumor immune response.

Dendritic cells play an important role in antigen presentation, triggering an immune response. The

combined treatment seems to be able to improve the DCs maturation increasing MHCII.

Conclusion: These results reveal an innovative field to be explored in melanoma treatment as combined

treatment with low dose radiotherapy and physical plasma may promote cytotoxicity with

immunogenic cell death.

Keywords: Reactive oxygen and nitrogen species, radiotherapy, immune response.

Grants: This work was supported by grants funded by the German Federal Ministry of Education and

Research (BMBF), grant number 03Z22DN11.

25

SARCOPLASMIC RETICULUM VOLUME LOSS PARTICIPATION ON FIBER-SPECIFIC SKELETAL MUSCLE WASTE

IN PRECACHEXIA INDUCED BY WALKER-256 CARCINOSARCOMA

Blegniski, F. P.1; Vieira, N. A.2; Yamaguchi, E. Y1.; Signori. L.1.; Bordini, H.P.1; Pecorai, C.3; Protasi, F.3;

Gomes, M. D.2; Boncompagni, S.3.; Guarnier, F. A1.

¹Department of General Pathology, LAFAM, Universidade Estadual de Londrina, Londrina, Brazil.

²Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, Universidade

de São Paulo, Ribeirão Preto, São Paulo, Brazil.

³Center for Research on Ageing and Department of Neuroscience and Imaging, Università G.

d’Annunzio, Chieti, Italy [email protected]

Introduction and objectives: Cachexia is a typical cancer-related disorder clinically classified in 3 stages

by severity: precachexia, cachexia and refractory cachexia. Cancer progression promotes

mitochondrial functional and morphological abnormalities in the skeletal muscle, what directly

influences patient´s survival. During cachexia syndrome, oxidative muscle fibers are more resistant to

atrophy than glycolytic. Oxidative stress in moderate level, especially protein oxidation, has been

related to muscle loss. So far, no data have related other structure dynamics, as sarcoplasmic reticulum

(SR), and oxidative stress in specific fiber type in the early stages of cachexia syndrome. The purpose of

this study was to investigate SR ultrastructure changes correlated with its protein alteration expression

and oxidative stress in muscles with diverse fiber types, in a very early stage of cachexia induced by

Walker-256 tumor.

Material and methods: Male Wistar rats (200-230g) were divided into three groups: Control (C), Walker-

256 tumor-bearing rats (T) and Walker-256 tumor-bearing rats treated with Nacetylcysteine 1% in drinking

water for 5 days (TNAC). After 5 days of tumor inoculation (precachexia), animals were euthanized

(Ethics Committee on Animal Experimentation/Universidade Estadual de Londrina, ref.9775). Body,

tumor, gastrocnemius, soleus and EDL weights were registered. Atrogin-1, IGF1 and PI15 atrophy-related

gene expression were determined by qRT-PCR in gastrocnemius. Lipid peroxidation was determined on

EDL and soleus homogenates. Sarcoplasmic reticulum (SR) volume was quantified using a stereology

point-counting technique through electronic microscopy micrographs. SR Ryr and CASQ-1 protein levels

were evaluated through immunohistochemistry and western blotting assays, respectively. Non-

parametric values were compared using Kruskall–Wallis test followed by Dunn’s multiple comparison.

Multiple time point analysis were compared by two-way ANOVA. Differences were considered

statistically significant when p<0.05.

Results: EDL showed significant reduction of 15.33% on wet weight compared to C. Gastrocnemius (-

10.30%) and soleus (-5.14%), but no statistical significance. Muscle weight loss was partially protected by

NAC treatment. Atrogin-1 expression showed threefold increasing on day 5 after tumor inoculation

(p=0.0112). T expressed significantly lower levels of PI15 when compared to C (p<0.05). IGF1 expression

showed no statistical difference among groups. As revealed by electron microscopy quantitative

analysis, the SR volume area decreased in T, both in EDL muscle (18.89%, p<0.005) and soleus muscles

(22.09%, p<0.0001) when compared to C. In none of the evaluated muscles, the antioxidant treatment

revealed significant protection. EDL showed significant increase in T chemiluminescence curve when

compared to C, which was partially diminished in TNAC (p<0.0001, for all comparisons). On the other

hand, soleus analysis showed no statistical difference when T curve was compared to C. However, TNAC

produced the highest emission compared to C and T, showing a time-course highly significant curve

elevation during the entire analysis (p<0.0001). Interestingly, RyR and CASQ-1 showed specific

differences in the comparison with C.

Conclusion: The present study demonstrated that in precachexia stage, where body mass waste is not

well established, EDL are already sensitive to SR dynamic alterations, which corresponds straightly to

oxidative stress parameters, participating on skeletal muscle mass loss modulation before clinical

cachexia comes to light. Keywords: EDL, soleus, sarcoplasmic reticulum, precachexia, N-acetylcysteine

Grants: Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do

Paraná.

26

THE LIPID MEDIATOR LIPOXIN A4 AMELIORATES TITANIUM DIOXIDE (TiO2)-INDUCED ARTHRITIS BY

REDUCING PAIN AND INFLAMMATION IN MICE

Telma S. dos Santos1, Tiago H. Zaninelli1, Ketlem C. Andrade1, Marília F. Manchope1, Nayara A. Antero1,

Stephanie Badaro-Garcia1, Larissa Staurengo-Ferrari1, Rúbia Casagrande2 and Waldiceu A. Verri Jr1*. 1Universidade Estadual de Londrina-UEL, Departamento de Ciências Patológicas, Londrina, Paraná,

Brasil. 2 Universidade Estadual de Londrina-UEL, Departamento de Ciências Farmacêuticas, Londrina,

Paraná, Brasil. E-mail: [email protected]

Introduction and objectives: Total knee joint arthroplasty is a common orthopedic procedure effective

for the treatment of patients with arthritis, relieving discommodity and improving functional state.

Despite this, about 15% of these proceedings tend to fail due to the release of metallic nanoparticles,

such titanium dioxide (TiO2) that creates a pro inflammatory microenvironment, which results in the

prothesis rejection and the need of surgical revision. Currently therapies available to patients with

arthritis include non-steroidal anti-inflammatory drugs, corticosteroids and opioids, that have limited

efficacy and offer intense adverse effects. Hence, a therapeutic alternative is the lipoxin A4 (LXA4), a

pro-resolving lipid mediator derived from arachidonic acid (AA) which acts in the range of nanograms

and have analgesic, antiinflammatory and resolutive properties. Therefore, the present study aims to

evaluate the effect and mechanisms of Lipoxin A4 in TiO2-induced chronic arthritis in mice, a model

resembling prosthesis and implant inflammation.

Material and methods: Animal care and handling procedures were in accordance with the

International Association for Study of Pain (IASP) guidelines and were approved by the Universidade

Estadual de Londrina Ethics Committee on Animal Research and Welfare (process number

11147.2016.40). Male Swiss mice were stimulated with 3 mg of TiO2 on the knee joint. After 24h, mice

were treated with LXA4 (0.1, 1 or 10 ng/animal) or vehicle (ethanol in saline) over 30 days. The disease

phenotype, treatment toxicity, leukocytes recruitment, oxidative stress, cytokines production and mRNA

expression of receptor activator of nuclear factor kappa B (RANK) were evaluated at the 2nd and 30th

days of treatment.

Results: The results demonstrate that LXA4 reduced TiO2-induced mechanical hiperalgesia in a dose-

dependent manner, in addition acting to reduce TiO2-induced edema and leukocytes recruitment

without inducing kidney, liver or stomach toxicity. LXA4 modules cytokines such as TNF-α, IL-1β, IL-6 and

IL-10, as well as the expression of IL-1β and IL-10. Furthermore, LXA4 reestablished the antioxidant

capacity (GSH and ABTS assays) by increasing the expression of Nrf2 mRNA and reducing bone

resorption through RANK mRNA expression. Conclusion: Therefore, the present result suggests that LXA4

possess antioxidant, antiinflammatory and analgesic proprieties in this model of joint prosthesis-induced

arthritis.

Keywords: Lipid mediator; Arthritis; Arthroplasty; Resolution of inflammation; Pain.

Grants: This work was supported by Programa para o Sistema Único de Saúde (PPSUS) grant

intermediated by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and

supported by Fundação Araucária and Secretaria Estadual de Saúde, Paraná (SESA-PR, Brazil) and,

Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

27

ABSTRACTS Selected for Poster Presentation

28

ADHERENCE AND EFFICACY OF OLFACTORY TRAINING AS A TREATMENT FOR PERSISTENT OLFACTORY

LOSS

Monteiro, R. S.1; Miyazawa, I. M. I. 1; Silva, G. S. 1; Garcia, E. C. D.2; Lopes, N. M. D.3; Fornazieri, M. A.2

1 Londrina State University, Departament of Medical Clinic, Londrina, PR, Brazil.

2 Londrina State University, Departament of Clinical Surgery, Londrina, PR, Brazil.

3 Londrina State University, Departament of General Pathology, Londrina, PR, Brazil.

Introduction and objectives: Patients deprived of normal olfaction have difficulty performing daily

activities such as cooking and evaluating their hygiene. They also become more susceptible to

accidents due to kitchen gas leaks, food poisoning and depression. Upper respiratory tract infections,

cranial traumas, inflammatory and obstructive nasal diseases constitute 60% of the etiologies in

patients with this disorder. Other common causes are presbyosmia, olfactory loss due to aging,

idiopathic Parkinson's and Alzheimer’s diseases. Among emerging therapies, olfactory training (OT)

has been proposed as a potential treatment for persistent olfactory loss. Through this treatment, the

olfactory function would be improved by sensorineural modulation as a result of repeated odor

exposure. However, due to the extended treatment period demanded, the patient’s lack of

adherence seems to be high. In this study, we verified both the adherence and the efficacy of OT in

patients with persistent post-infectious, post-traumatic and idiopathic olfactory loss.

Material and methods: Prospective observational study. Twenty-five patients with persistent olfactory

loss underwent two types of OT. Twelve patients received a box containing four bottles containing 1

ml of essences of the classical training. The second group with thirteen patients used odors of

commercial products from previously determined brands found in supermarkets (modified training).

Adherence and olfactory function with the University of Pennsylvania Smell Identification Test (UPSIT)

were measured three and six months after initiation of treatment. A minimum improvement of five

points in the test was considered significant. All patients signed a consent statement and study

approved by the Research Ethics Committee Involving Human Beings (approval number: 1.073.331).

Results: Adherence of the patients after three months was 88% and, at after 6, 56%. The percentage

of clinical improvement was 23.5% in the third month and 25% six months after the beginning of the

training. There was no relation of age, sex, time of olfactory loss, race, the degree of olfactory loss,

etiology, education, and type of training with adherence and treatment efficacy percentages.

Conclusion: Adherence to training remains high in the first three months of OT, but declines

moderately at the sixth month. Regarding the efficacy of this therapy during these periods, olfactory

function improvement in our sample was similar to spontaneous recovery without treatment.

Keywords: Smell, Olfaction Disorders, Treatment Adherence, Compliance.

Grants: Fundação Araucária

29

BIOLOGICAL BEHAVIOR IN DIFERENTE PARAMETERS OF OXIDATIVE STRESS AND INFLAMMATORY

CYTOKINES, AND DETERMINATION OF THE CRITICAL DIFFERENCE IN HEALTHY MALE SUBJECTS AGED 18

TO 28 YEARS

Natália Almeida de Barros¹, Fernanda Paschoal Blegniski ¹, Flávia Alessandra Guarnier¹1

¹ Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil E-

mail:[email protected]

Introduction: The critical difference is the minimal change that must occur before true biological

change can be established. When examining the concentration of a metabolite after a stimulus, a

mathematical change can occur (p <0.05) that is biologically insignificant, or means with no clinical

relevance. In order to study the critical difference one must understand the biological variation of

the individual mainly during the day. There are many techniques used to quantify oxidative stress

and cytokines systemically. However the results of these analysis are inconsistent in the literature, and

may be caused by biological variability and methods applied, and it is necessary to establish the

critical difference. The aim of the present study was to determine the critical difference of

parameters of oxidative stress and levels of circulating cytokines in physically active individuals.

Methods: Young adults, aged between 18 and 28 years old, were recruited, all of whom were

recruited for blood sampling from one hour to one day, from 9:00 to 17:00. Blood samples were

centrifuged and stored separately from plasma erythrocyte for analysis of oxidative stress

parametertotalthiols, advanced protein oxidation products and analysis of the systemic markers of

TNFα and ILβinflammation.

Results: The critical difference of the techniques for catalase, total thiol and AOPP, the values

obtained were 14.8%, 124.9% and 84.3%, respectively. The critical difference values obtained for ILb

and TNFalpha were 23.9% and 34.5%. The values of CVA found were 0.2%, 2.7%, 3.4%, 6.4% and 4.1%

for total thiol, catalase, AOPP TNF-alpha and IL1-b. In the analysis of the biological variation the values

found were 41.1%, 4.6%, 30.3%, 10.7% and 7.6% in the analysis of the respective techniques mentioned

above.

Conclusions: There is a great difference in the values found on biological variability, however, the

parameters of analytical variation are within the desirable standard in oxidative stress techniques.

The catalase values showed a lower variation of the values because it was an enzymatic technique,

with greater sensitivity. These findings refer to a population between 18 and 28 years of age.

Keywords: Critical difference; oxidative stress; cytokines; biological variation; analytical variation.

30

CORRELATION ANALYSIS OF CXCR4 AND CXCR7 mRNA LEVELS WITH CLINICAL OUTCOMES IN

AGGRESSIVE BREAST CANCER SUBTYPES

Bruna Karina Banin-Hirata1,2; Mayara Bocchi1; Sarah Lott Moretto1; Julie Massayo Maeda

Oda3; Alberto Yoichi Sakaguchi1; Carolina Batista Ariza 2; Roberta Losi Guembarovski1;

Carlos Eduardo Coral de Oliveira4; Maria Angelica Ehara Watanabe 1

1 State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

2Philadelphia University Center, Collegiate of Biomedicine, Londrina, PR, Brazil

3Federal University of Mato Grosso do Sul, Três Lagoas, MS, Brazil 4Pontifical Catholic

University of Paraná, Londrina, PR, Brazil [email protected]

Introduction and objectives: The breast cancer (BC) is the most frequently diagnosed tumor in

women. This carcinoma is considered as a heterogenous disease, characterized by different

biological features and prognosis. The CXCL12 chemokine and its receptors CXCR4 and CXCR7

has been widely studied in this tumor type, however several studies have demonstrated that these

different axis develops distinct roles in tumor microenvinroment. In this context, the present study

aimed to investigate the correlation of CXCR4 and CXCR7 mRNA expression with clinical

outcomes in aggressive BC subtypes, including luminal B HER2-positive (LB), HER2-enriched

(HER2+) and triple-negative (TN).

Material and methods: The present study was approved by Human Ethics Committee of Londrina

State University, Paraná, Brazil (CAAE-171231134000005231) and a consent term was obtained

from all BC patients. In total, were obtained 67 BC samples, of which 22 were diagnosed as LB, 16

as HER2+ and 29 as TN subtype. The CXCR4 and CXCR7 relative expression was investigated by

Quantitative real time PCR (qRT-PCR). A commercial pool of human normal mammary gland RNA

was used as a non-neoplastic sample. The mRNA expression was correlated with clinical outcomes

collected from medical records, which included TNM staging, tumor size, histopathological grade,

lymph node commitment, presence of metastasis, Ki-67 status, hormonal receptors and HER2

status. The statistical analysis was performed by Kendal tau-b test, using statistic software SPSS

version 22.0.

Results: The expression level of CXCR4 mRNA was correlated with aggressiveness of BC subtype

(p=0.038; τ=0,205). The LB, HER2+ and TN presented 1.2, 2.5 and 2.8 fold higher CXCR4 mRNA

expression relative to normal mammary gland. The CXCR7 expression was not correlated with BC

subtype, however the LB, BC subtype of better prognosis, presented 1,7 lesser mRNA, while HER2+

and TN presented 1.4 higher expression of CXCR7 mRNA expression relative to normal mammary

gland. Although no significant correlations between CXCR4 mRNA with clinical outcomes were

found, tendencies with metastasis (p=0,057; τ=0,213) and HER2 expression (p=0,054; τ=-0,198) were

observed, which is in accordance with literature. Curiously, the CXCR7 mRNA relative expression

were correlated with progesterone receptor status (p=0,022, τ=-0,227).

Conclusion: The results of present study shows the higher mRNA levels of CXCR4 and CXCR7 in

aggressive BC subtypes, such as HER2-enriched and triple-negative, which frequently are

associated with metastasis. Furthermore, the correlation analysis suggests different roles of CXCR4

and CXCR7 in tumor microenvironment, emphasizing its potential as prognostic markers in breast

cancer and the need for further studies in this area.

Keywords: breast cancer, aggressive subtypes, chemokine receptors, CXCR4, CXCR7.

31

GSSH/GSH RATIO: A BIOMARKER THAT CAN DIFFERENTIATE THE TYPES OF NON-MELANOMA SKIN

CANCER

Brito, W. A. S1; Marinello, P. C.1; Sanches, L. J. 1; Lopes, N. M. D.1; Fumegali, W. C.1; Moreira, C. R.2;

Reis, S. G.2, Melhado, I. P.2, Armani, A.3; Luiz, R. C.1; Gon, A. S.2; Cecchini, R.4; Cecchini, A. L.1.

1 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil. 2 State University of Londrina, Department of Internal Medicine, Londrina, PR, Brazil.

3 State University of Londrina, Department of Surgical Clinic, Londrina, PR, Brazil. 4 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil.

Email: [email protected]

Introduction and objectives: Non-melanoma skin cancer (NMSC) is the neoplasia with the highest

incidence in the world. The cure of NMSC is the surgical removal of the lesions, which can be often

mutilating, causing social and psychological impact on patients. Basal cell carcinoma (BCC) and

squamous cell carcinoma (SCC) are the most common type of NMSC. It is known that oxidative

stress (OS) has an important role in skin carcinogenesis, which occurs mainly due to ultraviolet

radiation, which is also related to inflammatory process. However, systemic OS and inflammatory

profile in patients with different types of NMSC have never been investigated. Thus, the objective

of this work was to analyze the systemic oxidative and inflammatory profiles of patients with NMSC.

Material and methods: Participants were categorized in 3 groups: Control (without skin cancer

history; n=64); BCC (n=82) and SCC (n=19) (CEP-UEL process n. 3.146.725). Blood samples were

collected, erythrocytic catalase and superoxide dismutase (SOD) activity, oxidized and reduced

glutathione levels (GSSG and GSH, respectively) and lipid peroxidation was assessed by

chemiluminescence stimulated by tert-butyl-hydroperoxide (CL). Malondialdehyde (MDA), total

thiols, TNF-α, IL-10, TGF-β1 and advanced oxidative protein products (AOPP) concentrations in

plasma were determined. C-reactive protein (CRP), ferritin and free iron levels were measured in

serum, and activity of gamma-glutamyl transpeptidase (GGT). Qualitative data were analyzed

by Chi-square test or Fisher’s exact test. Quantitative data were analyzed by ANOVA (one-way

or two-way, when applicable) or Kruskal-Wallis after the verification of normality and

homogeneity. All results with p<0.10 in the univariate analyses were included in the multivariate

logistic regression model. The statistical analysis was performed with SPSS (version 20.0) and

GraphPad Prism (version 6) and p<0.05 was considered significant.

Results: The characterization of population studied showed that BCC and SCC groups presented

significant differences in some of the parameters when compared to control group: age

(p=0.001), Fitzpatrick’s phototype skin classification (p=0.0086), sun exposure (p=0.0024) and

location of tumor (p=0.0055), which the latter three are risk factors for the development of NMSC.

Characteristics such as gender, smoking and chronic diseases (diabetes mellitus and systemic

arterial hypertension) did not demonstrate significant differences between groups. In univariate

analysis, BCC and SCC patients demonstrated a reduction in GSH (p=0.007) and GSSG (p<0.0001)

levels when compared to control. A reduction in GSSG/GSH ratio was observed in BCC group

(p<0.0001) and activity of SOD was reduced in SCC group (p=0.017) when compared to control.

BCC group showed an increase in lipid peroxidation (p<0.0001) (CL) when compared with control.

The other parameters did not shown significant differences between groups. The multivariate

analysis were used as a way to knowing if any of these parameters are actually associated with

the presence of NMSC and if there are any that can differentiate the types of NMSC. Thus, SCC

group demonstrated reduced GSSG levels (p=0.045) and reduced GSSG/GSH ratio (p=0.007)

when compared with control, however this group showed an increase in GSSG/GSH ratio

(p=0.046) when compared to BCC group.

Conclusion: The GSSG/GSH ratio may be a biomarker that differentiates types of NMSC.

Keywords: Basal cell carcinoma, Squamous cell carcinoma, Oxidative stress, Inflammation.

Grants: Coordination of Improvement of Higher Level Personnel (CAPES)

32

MONITORING THE BIOCHEMICAL PARAMETERS OF STOCK INJURIES, DURING THE STORAGE OF

CONCENTRATES OF DOGS 'HEMACIES, FOR 42 DAYS IN PLASTIC BAGS CPD/SAG-M

Danielle Venturini1; Ana Paula Michelin1; Andressa Keiko Matsumoto1; Raquel Reis Martins2;Júlia

Frare1; Barbara Fiori Silva1; Gabriela de Oliveira Pacheli1; Patrícia Mendes Pereira2 1 Universidade

Estadual de Londrina, DepartmentofPathology,

ClinicalAnalysisandToxicological, Londrina, PR, Brazil 2 Universidade

Estadual de Londrina, Small Animal Medical Clinic, Londrina, PR, Brazil

Email [email protected]

Introduction and objectives: Preservative solutions maintain energy metabolism of erythrocytes

through glycolysis, ensuring cell viability during the storage period and even choosing the most

appropriate type of packaging and preservative solution, the blood undergoes biochemical

changes depending on the time. The objective of this study was to evaluate weekly lesion

measurements of packed red blood cells stored for 42 days at CPD / SAG-M bags.

Material and methods: 40 donor dogs were selected and donated 450 mL of whole blood, after

approval of the ethics committee (CEUA 17632.2015.23). The glucose, lactate and hematocrit

levels were analyzed at T0 moments (immediately after centrifugation), weekly until T6. Data and

statistical management were performed with SPSS Statistic® 20 (IBM, Armonk, NY, USA). The normal

distribution (Shapiro-Wilk test) and homogeneity of variance (Levene’s test) were verified. If these

criteria were reached (p ≥ 0.05), the variables were evaluated by analysis of variance, followed

by the Friedman test with Bonferroni. The results were expressed in median (minimum - maximum).

Results: In the hematocrit (HCT) T0 was higher than T1 and T2, and T2 and T3 presented lower values

when compared to T5 and T6. In addition, T1 had lower values of hematocrit when compared to

T3, T4, T5 and T6, with T4 and T5 presenting a significant difference between T6, which is increased

in relation to them. Hematocrit values at each time point were, respectively, 52.44 (42.5-52.2);

50.24 (41.2-59.6); 50.69 (40.4-61.8); 51.93 (41.8-62.8); 53.36 (44.5-65.8); 55.39 (46.2-68.9); 57.15 (47-

69.2). All the moments presented reduced glycemic levels when compared to T0, with T1

presenting higher glucose values when compared to T2, T3, T4, T5 and T6. T2 also had higher

glucose levels when compared to T3, T4, T5 and T6. In addition, T5 and T6 demonstrated glucose

levels lower than T3 and

T4. The glucose levels were, respectively, 630-06 (548-751); 501.24 (325-684); 385.89 (217595306,48

(136-493), 236,37 (71-444), 162,82 (0-373), 115-37 (5-337). it was possible to verify a significant

increase when all times were compared to T0, the same occurs when T1 was compared with T2,

T3, T4, T5 and T6, in addition, T2 also presented lower levels of lactate when compared to T3, T4,

T5 and T6, and T3 and T4 values were significantly lower when compared to T5 and T6. Lactate

levels at the times verified were, respectively, 2.02 (0.9-4), 14.65 (7 -28), 22.79 (11.2-32) and 28.82

(16.9-40.9).) 32.86 (20.2-43-5), 35.32 (2246) and 35.67 (21-43.8 ) ).

Conclusion: With the passage of weeks HCT increases from T3. Soon after the collection, the

glucose that is very high due to its presence in the preservative solution, falls considerably, being

a substrate used for energy production to produce ATP through glycolysis, during storage,

generating a series of metabolites, among them lactate. It was concluded with this study that the

storage produces gradual changes in the quality of the packed red blood cells.

Keywords: blood bag; dogs; biochemical parameters

33

OLFACTORY FUNCTION, NASAL OBSTRUCTION AND QUALITY OF LIFE IN PATIENTS WITH CHRONIC

RHINOSINUSITIS WITH AND WITHOUT NASAL POLYPOSIS

Ellen Cristine Duarte Garcia1; Jéssica Taynara Moreira Oliveira 2; Marco Aurélio

Fornazieri.1 1 Universidade Estadual de Londrina, Department of Clinical Surgery, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Olfactory dysfunction is one of the possible consequences of chronic

rhinosinusitis, which is often neglected. This dysfunction may result in difficulties in perceiving

kitchen gas leaks and personal hygiene; cause disturbances of appetite and changes in

emotional and sexual behavior. In addition, there are other symptoms related to chronic

rhinosinusitis that could affect the quality of life of patients, but it is not known the impact of the

presence of polyposis on these symptoms. Therefore the objective of this work was to evaluate

and compare the quality of life, olfactory function and nasal obstruction of patients with chronic

rhinosinusitis with and without polyps.

Material and methods: Twenty patients with chronic rhinosinusitis, 15 with polyps and 5 without

polyps were analyzed. In all patients, the SNOT-22 (Sino-Nasal Outcome Test-22) test was used to

evaluate the symptoms that impact the quality of life, the UPSIT (University of Pennsylvania Smell

Identification Test) for measuring smell capacity and the VAS (visual analog scale) for subjective

perception of nasal obstruction, olfactory function and discomfort with loss of smell. This clinical

study was conducted at the State University of Londrina and approved by the Research Ethics

Committee involving human beings (approval number: 1.024.603). All patients were oriented

about the study and signed an informed consent form, after reading the same and agreeing to

participate in the research. Statistical analysis was done using Student's t-test, after confirming the

normal distribution of the data. Was considered significant p<0.05.

Results: Patients with chronic rhinosinusitis with polyps presented a greater subjective olfactory loss

(p <0.01), discomfort with this deficit (p = 0.01) and a trend of a lower score in the olfactory test.

The symptoms of nasal obstruction and quality of life were similar between patients with and

without nasal polyposis.

Conclusion: Patients with nasal polyposis had a higher complaint of olfactory loss and annoyance

with the reduction of smell than patients without polyposis. On the other hand, other symptoms

such as nasal obstruction and quality of life were similar between the two groups.

Keywords: smell, polyposis, anosmia

Grants: The authors thank Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) and Fundação Araucária for for financial support.

34

OXIDATIVE STRESS AND INFLAMMATION PLAY A ROLE IN THE DEVELOPMENT OF SQUAMOUS CELL

CARCINOMA ASSOCIATED WITH OTHER SKIN NEOPLASIAS

Moreira, C. R.1, Marinello, P. C.2; Brito, W. A.S. 2; Lopes, N. M. D.2, Sanches, L. J. 2, Veronez, A. C. P. 1,

Reis, S. G. 1, Melhado, I. P. 1, Armani, A. 3, Luiz, R. C. 2, Gon, A. S. 1, Cecchini, R. 2, Cecchini, A. L2.

1 State University of Londrina, Department of Internal Medicine, Londrina, PR, Brazil.

2State University of Londrina, Department of General Pathology, Londrina, PR, Brazil.

3 State University of Londrina, Department of Surgical Clinic, Londrina, PR, Brazil.

Introduction and objectives: The squamous cell carcinoma (SCC) is the second more common and

the most aggressive type of non-melanoma skin cancer (NMSC). Oxidative stress (OS) and

inflammation are closely related to skin carcinogenesis, mainly due to ultraviolet radiation. Despite

the well-known participation of oxidative stress (OS) and inflammation in the development of NMSC,

little is known about their relationship with different clinical outcomes. Thus, the objective of this study

was to compare OS and systemic inflammation in patients who present only SCC with patients who

present SCC associated with others skin neoplasias.

Material and methods: Participants were categorized in 3 groups: Control (without skin cancer history;

SCC (n= 13) and SCC associated with others skin neoplasias (SCCML; n=10) (CEP-UEL process n.

3.146.725). Blood samples were collected and erythrocytic catalase and superoxide dismutase

activity, oxidized and reduced glutathione levels (GSSG and GSH, respectively) was determined.

Lipoperoxidation was assessed by chemiluminescence stimulated by tert-butyl-hydroperoxide. The

levels of tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10) and transforming growth factor beta

1 (TGF-β1) were determined on plasma by ELISA. The statistical analysis was performed using one-

way ANOVA (one-way or two-way, when applicable) or Kruskal-Wallis after the verification of the

normality of the data, p<0.05 was considered significant.

Results: The levels of reduced glutathione did not alter, however, the relation between oxidized and

reduced glutathione decreased in SCC-ML when compared to control (no history of skin cancer,

n=53). Superoxide Dismutase activity significantly decreased in SCC-ML when compared to control.

The SCC-ML group presented higher levels of lipoperoxidation in comparison with control and SCC

group. In relation to the analyses of the systemic inflammatory profile, the levels of TNF-α, IL-10, C

reactive protein, ferritin, iron and gamma glutamyl transpeptidase did not alter. However, TGF-β1

significantly reduced in SCC-ML group when compared to control.

Conclusion: The analysis of the results indicate that although patients of SCC-ML presented preserved

systemic antioxidant proteins, they showed increased levels of lipoperoxidation and reduced levels

of the anti-inflammatory cytokine TGF-β1, which suggest the importance of systemic OS and

inflammation in the development of multiple NMSC.

Key words: Non melanoma skin cancer, oxidative stress, inflammation

35

OXIDATIVE STRESS BIOMARKERS INCREASE DURING 42 DAYS OF STORAGE IN PACKED RED BLOOD

CELLS OF DOGS

Danielle Venturini1; Ana Paula Michelin1; Andressa Keiko Matsumoto1; Raquel Reis Martins2; Júlia

Frare1; Barbara Fiori Silva1; Gabriela de Oliveira Pacheli1; Patrícia Mendes Pereira2

1 Universidade Estadual de Londrina, DepartmentofPathology, ClinicalAnalysisandToxicological,

Londrina, PR, Brazil

2 Universidade Estadual de Londrina, Small Animal Medical Clinic, Londrina, PR, Brazil


Introduction and objectives: Preservation of erythrocytes for transfusion purposes has begun with

hypothermic storage for inhibition of temperature-dependent metabolic processes, which

deplete critical cellular metabolites and accumulate cell damage. Among these lesions are the

extravasation of the content (procoagulant and proinflammatory substances) of microvesicles.

Therefore, the objective of this study was to monitor the levels of oxidative stress (OS) during the

storage of red blood cell concentrates, weekly, for 42 days in plastic bags CPD/SAG-M.

Material and methods: A total of 40 red blood cells were collected from blood bags of donor dogs

sorted according to age, behavior, weight, physical examination and laboratory tests after

approval by the ethics committee (CEUA 17632.2015.23). Inclusion criteria were dogs between

two and eight years of age, physically healthy, with no skin lesions and / or complaints of diseases

by the guardians. The levels of advanced protein oxidation products (AOPP), Superoxide

dismutase (SOD) and lipid hydroperoxides (LOOH) were analyzed at the following moments:T0

(immediately after centrifugation), T1 (seven days storage), T2 (14 days), T3 (21 days), T4 (28 days),

T5 (35 days) and T6. Data management and statistics were performed with SPSS Statistic® 20

(IBM,Armonk, NY, USA). Initially, normal distribution (Shapiro-Wilk test) and homogeneity ofvariance

(Levene’stest) were checked. If these criteria were reached (p ≥ 0.05), variables wereevaluated

by analyses of variance, followed by Friedman Test with Bonferroni. The results were expressed in

median (minimum - maximum).

Results:In the AOPP assay the T0 was decreased in relation to all the times, and T3 was decreased

in relation to T5 and T6, presenting, respectively, the values of 97.22 (7.04-297.05); 727.71 (21.30-

5214.17) ;837.85 (25.49-3816.89);431.44 (40.92-1604.73);641.84 (51.14-3448.41); 1073.31 (64.39-

11987.68) and 2272.57(63.38-14014.9). SOD was reduced at T4 and T5 times when compared to

T0, as well as T4 presented decreased values in relation to T1, presenting, respectively, the values

of 90.77(14.94-164.1); 83.67(18.21-167.6); 58.05(10.26-127.58); 58.79(2.01-133); 37.64(2.76-129.38);

40.29(2.03-110) and 62.21(1.5-163.38). In LOOH assay, T0 was significantly decreased in relation to

all times, and T1 presented lower values when compared to T3, T4 and T5. In addition, T2 also

decreased compared to T4 and T5, respectively, 1.99(1.01-4.12); 2.96(1.01-4.87); 3.2(1.27-4.28);

3.6(2.32-5.56); 3.73(1.894.91); 3.72(0.66-5.33) and 3.00(0.66-4.7).

Conclusion: This study demonstrated an imbalance between pro-oxidant biomarkers in blood

bags from donor dogs during 42 days of storage.

Keywords: dogs; oxidative stress; blood bags

36

SAFETY AND EFFICACY OF SUPERIOR TURBINATE BIOPSIES AS A SOURCE OF OLFACTORY EPITHELIUM

APPROPRIATE FOR MORPHOLOGICAL ANALYSIS

Ellen Cristine Duarte Garcia1; Ana Carolina Rossaneis 2; Alexandre Salvatore Pipino1; Gustavo

Vasconcelos Gomes1 ; Waldiceu Aparecido Verri Jr 2, Marco Aurélio Fornazieri 1.

1 Universidade Estadual de Londrina, Department of Clinical Surgery, Londrina, PR,

Brazil 2 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Olfactory epithelium biopsy is useful for several types of studies,

including the research of the pathophysiology of various neurodegenerative diseases and the

obtaining of stem cells. However, there are descriptions of olfactory deficit after this procedure. A

gold standard technique for obtaining human olfactory epithelium in vivo is still lacking. We aimed

to determine the efficacy of obtaining good-quality olfactory epithelium specimens suitable for

pathological analysis from the lower half of the superior turbinate and verify the safety of this

procedure in maintaining bilateral (total and specific) and unilateral olfactory capacity.

Material and methods: This clinical study was conducted at the State University of Londrina and

approved by the Research Ethics Committee involving human beings (approval number:

1.024.603). All patients were informed about the study and signed the informed consent form, after

reading the same and agreeing to participate in the research. Twenty-one individuals without

olfactory complaints underwent olfactory epithelium biopsy in superior turbinate during

septoplasty. Unilateral and bilateral olfactory function was assessed with the University of

Pennsylvania Smell Identification Test (UPSIT), adapted and validated version, before and one

month after the procedure. Specimens were marked with the olfactory marker protein for

confirmation of olfactory epithelium presence using immunohistochemistry technique. Continuous

variables, such as age and UPSIT scores, were expressed as means and standard deviations.

Comparisons of bilateral and unilateral UPSIT mean scores and percentages of correct answers

for each odorant pre- and post-procedure were performed and the respective 95% confidence

intervals were determined.

Results: Olfactory epithelium appropriate for epithelial characterization was obtained in 62% of

the patients, although 90% of the samples contained olfactory epithelium. There was no

deterioration of olfactory function bilaterally or unilaterally in the nasal cavity where the biopsy

was performed. Patients also maintained the ability to identify individual odorants, precluding the

possibility of selective anosmia.

Conclusion: Biopsies of the lower half of the superior turbinate do not affect olfactory function and

show moderate efficacy in yielding olfactory epithelium appropriate for morphological analysis.

Future studies assessing the safety of this procedure in other olfactory epithelium regions are

necessary.

Keywords: immunohistochemistry, smell, smell tests.

Grants: The authors thank Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and

Fundação Araucária for MSc, PhD and Post-Doc for financial support.

37

SUPPLEMENTATION OF OMEGA-3 FATTY ACID IN DIABETIC NEPHROPATHY - SYSTEMATIC REVIEW

Kátia Calvi Lenzi Almeida1, Artur Ferrer Sandres Melo2, Adail Orrith Liborio Neto2, Daniel José de

Carvalho Pereira3 , Vitor Ferrer Sandres Melo4,

1. Universidade Federal do Rio de Janeiro, Professor at School of Medicine, Macaé, Rio de

Janeiro, Brazil

2. Universidade Federal do Rio de Janeiro, Undergraduate student at School of Medicine,

Macaé, Rio de Janeiro, Brazil.

3. Universidade Federal do Rio de Janeiro, Undergraduate student at School of Medicine, Ilha do

Fundão, Rio de Janeiro, Brazil.

4. Doctor residing in General Surgery of Stella Maris Hospital, Guarulhos, São Paulo, Brazil.


Introduction and objectives: Diabetes mellitus is characterized mainly by hyperglycemia as a result

of defects in insulin production and/or action. Chronic hyperglycemia is associated with long-term

damage, dysfunction and insufficiency of various organs such as kidneys, eyes, heart and peripheral

nerves. Diabetic nephropathy is one of the most serious complications and the renin-angiotensin-

aldosterone system has assumed an important role in this pathological condition. Metabolic and

hemodynamic factors, together with the interaction of the renin-angiotensin-aldosterone system

and metabolic changes present in diabetes mellitus, are responsible for a significant part of the

progression and development of nephropathy. Multiple studies have shown to the likely benefits of

supplementation of omega-3 polyunsaturated fatty acids in patients with diabetic nephropathy,

but the results are still controversial. In this sense, the purpose of this study is understand the

relationship between diabetic nephropathy and omega-3 fatty acid supplementation, contributing

to the growing demand for complementary strategies in the treatment of this disease.

Material and methods: A systematic review was carried out through an search in the databases

Pubmed, Biblioteca Virtual em Saúde (BVS) and Scientific Electronic Library Online (SciELO). For this,

the descriptors diabetic nephropathy and omega-3 fatty acids were applied, with the following

filters: temporal window from 2008 to 2017 (10 years); Portuguese, English and Spanish; Journal article

and Review.

Results: A total of 109587 studies were found related to the descriptors used. First, 56993 articles were

removed because they were not within the period from Jan/2008 to Dec/2017. 52594 articles were

analysed and 50890 articles were removed because they were duplicates, case reports, letters,

guidelines, editorials or because they did not meet the inclusion criteria (Journal articles, Reviews,

pathophysiology of diabetic nephropathy, omega3 effects and omega-3 use in nephropathy). 1691

studies were selected to read the abstracts and, of these, 1579 abstracts were excluded because

they were not in accordance with the objectives of the present review. This left 112 articles for

reading, of which 64 were excluded. Therefore, 48 articles were adequate to the inclusion criteria

and analyzed in a complete way in this study.

Conclusion: Results suggest a potential renoprotective effect of n-3 PUFA supplementation in

patients with diabetic nephropathy, reaffirming this as a complementary therapeutic in the

management of this pathological condition.

Keywords: Omega-3, nephropathy diabetic, systematic review.

38

SYSTEMIC CORTISOL AND POOR PROGNOSIS IN OVERWEIGHT/OBESE WOMEN WITH BREAST CANCER

Aedra Carla Bufalo Kawassaki1,2; Elaine Minatti Dias 2; Daniel Rech 3; Carolina Panis2; Wander

Rogério Pavanelli1

¹Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

²Universidade Estadual do Oeste do Paraná, Health Sciences Center, Francisco Beltrão,

PR, Brazil

³Cancer Hospital of Francisco Beltrão, CEONC, Francisco Beltrão, PR, Brazil.

[email protected]

Introduction and objectives: Excessive body fat promotes several negative effects on the

pathogenesis of breast cancer. Studies have shown that both the factors of excessive fat and cancer

modify the physiological effects of the hormone cortisol, which could possibly affect cancer

progression and disease prognosis. Our study analyzed the correlation between systemic levels of

cortisol and prognostic parameters in patients with breast cancer categorized according to the

body mass index (BMI).

Material and methods: This is a cross-sectional observational study whose participant women aged

15–86 years with indicative of cancer breast were recruited from Cancer Hospital of Francisco Beltrão

(CEONC), Paraná, Brazil, over the period from 2015 to 2017. Our analysis was approved by the

National Commission of Ethics in Research (CONEP), protocol number 35524814.4.0000.0107 and all

participants signed informed consent terms. We collected peripheral blood samples from 128 women

diagnosed with breast cancer out of the peak of morning cortisol, between 2 p.m. and 5 p.m., aiming

at investigating whether cortisol levels were deregulated during the day. Cortisol levels were

measured using an enzyme immunoassay kit and correlated to clinicopathological data (molecular

subtypes of tumors, histological grade of the tumor, lymphnodal metastasis, presence or absence of

clots, age at diagnosis and presence or absence of menopause at diagnosis). Our investigation

selected the participants diagnosed with breast cancer (with diagnosis confirmed after biopsy) to

be categorized according to their BMI – values defined for adults by the World Health Organization

– as eutrophic (BMI ≤ 24.9Kg/m2), overweight (BMI ≥ 25.0 Kg/m2 or ≤ 29.9 Kg/m2); 3) or obese (BMI ≥

30.0 Kg/m2). Results were analyzed and significant results were considered when p≤0.05.

Results: Circulating cortisol levels were significantly higher in obese women patients in relation to the

eutrofic ones (10.02±0.88 g/dL to eutrophic, 12.46±0.9 g/dL for obese, p=0.051). We found

differences among the molecular subtypes of breast cancer regarding their cortisol levels by

comparing eutrophic and overweight patients who carried Luminal A tumors (5.9±0.82 g/dL for

eutrophic and 14.05±1.63 g/dL for overweight, p = 0.0108). For Luminal B subtype, obese women

presented higher levels than either eutrophic (p=0.0211) or overweight (p=0.0191) women (16.78±1.13

g/dL for obese, 11.2±1.5 g/dL for overweight, and 11.58±1.49 g/dL for eutrophic). Overweight

patients bearing triple negative tumors had augmented cortisol levels in relation to both eutrophic

(p=0.0310) and obese (0.0296) groups (9.89±2.25 g/dL for eutrophic, 17.13±1.35 g/dL for

overweight, and 10.62±1.69 g/dL to obese). Overweight patients (16.9±1.48 g/dL) also exhibited

augmented cortisol when diagnosed with high grade tumors in relation to both eutrophic (9.17±2.52

g/dL, p=0.0401) and obese women (10.21±1.19 g/dL, p=0.0024). In addition, the presence of

lymphnodes metastasis correlated to high cortisol and overweight/obesity (7.6±1.74 g/dL for

eutrophic; 12.54±1.45 g/dL for overweight, p=0.0449; and 13.82±1.15 g/dL for obese, p=0.0052).

Conclusion: These data indicate that excessive body fat has a negative effect on the disease

prognosisas well as that cortisol levels can be associated with poor prognosis in breast cancer.

Keywords: breast cancer, cortisol, prognosis, overweight, obesity.

Grants: This work was supported by the National Council for Scientific and Technological

Development (CNPq); Programa Pesquisa para o SUS (PPSUS) – Fundação Araucária/Ministério da

Saúde; Universidade Estadual do Oeste do Paraná, PR, Brazil.

39

CHALLENGES TO HUMAN LEISHMANIASIS: DISCUSSION OF THE DISEASE BASED ON ITS BEHAVIOR

Cínthia Akemi Tanoshi1 ; Isabella Harumi Yonehara Noma1 ; Regivaldo Florentino Rodrigues1; Lincoln

Luís Silva¹; Diogo Francisco Rossoni²; Raíssa Bocchi Pedroso¹; Sandra Mara Alessi Aristides¹.

1State University of Maringá, Program in Health Science, Maringá, PR, Brazil.

²State University of Maringá, Statistical Department, Maringá, PR, Brazil.

E-mail: [email protected]

Introduction: Leishmaniasis is a disease caused by different Leishmania species. Brazil is one of seven

countries with the majority of new cases of leishmaniasis in the world and Paraná state has

concentrated about 90% of the illness of Southern region. Among the manifestations there are fever,

weight loss and hepatosplenomegaly by visceral leishmaniasis (VL); deforming lesions by cutaneous

leishmaniasis (CL); and when disseminate it can develop a severe form of mucocutaneous

leishmaniasis (ML), with destruction of cartilage. Even death can occur in consequence of secondary

infections, functional impairment and the toxicity of the treatment.

Objective: Analyse and describe the behavior of the Leishmania’s epidemiology and epidemic

outbreaks in recent years.

Material and methods: The cross-sectional survey was performed using a secondary data of a public

health database, DATASUS, with notifications sent to the Information System for Notifiable Diseases -

SINAN [Sistema de Informação de Agravos de Notificação]. Through option Diseases and Notifiable

diseases - from 2007, the variables related on Leishmaniasis in Paraná between 2007 to 2017 were

selected and clustered in tables. The database was organized since January 2019 until March 2019

and after, the statistical analysis were conducted using the RStudio ™ program, while spatial

modelling require QGIS ™ (3.4) and GeoDa™ softwares. The result was compared to numbers of

leishmaniasis in Brazil. Ethical committee submission was not required by reason of DATASUS being a

public domain without nominal identification.

Results: Over the years leishmaniasis kept constant despite of some abrupt increase numbers. A

number of 4371 cases were diagnosticated only in Paraná between the period analysed

representing 89.53% of southern region, almost 400 cases by year. According to the health region of

residence - CIR, Cianorte (n=726), Maringá (n=672), Metropolitan region (n=439), Campo Mourão

(n=374) and Londrina (n=345) are the focus of the disease in the state. The most reported cases

(99.09%) were registered as American Tegumentary Leishmaniasis (ATL), that include mucocutaneous

leishmaniasis (ML), cutaneous leishmaniasis (CL) and clinical aspects that are similar to them. These,

72.02% (n=3119) culminated on cure. On visceral leishmaniasis, 55.00% of 40 cases (n= 22) tend to

heal and 15.00% (n=6) decease.

Conclusion: According the datas it was possible to comprehend the distribution of the parasite

disease. Monitoring actions are fundamental for the purpose of taking intervention strategies in

surveillance improving health care applications, as reducing the prevalence of the disease, focusing

on specific and spatial areas.

Keywords: leishmania; leishmaniasis; epidemiology; health care; public health; spatial modeling;

spatial analysis.

Grants: CAPES (Coordination for the Improvement of Higher Education Personnel) [Coordenação

de Aperfeiçoamento de Pessoal de Nível Superior]

40

DEATHS FROM RECTO-SIGMOID SEGMENT CANCER IN SOUTHERN BRAZIL, A DATA SERIES ANALYSIS, BY

AGE AND SEX, FROM 2000 TO 2015.

Daniel Augusto Nunes de Lima1, Aline Amenencia Souza1, William Augusto de Melo2, Miguel

Machinski Junior4, Raissa Bocchi Pedroso3, Sandra Marisa Pelloso4, Maria Dalva de Barros Carvalho4

Thaís Gomes Verzignassi Silveira4, Tânia Cristina Alexandrino Becker4.

1. Post Graduate Program in Health Sciences (PCS) State University of Maringá, Maringá/PR.

2. Professor - Health Sciences Center, State University of Paraná (UNESPAR) - Paranavaí/PR.

3. Graduate Program of Medicine, Federal University of Paraná, Curitiba/PR

4. Post-Doctorate - Post Graduate Program in Health Sciences (PCS), State University of

Maringá, Maringá/PR.

5. Professor – Post Graduate Program in Health Sciences (PCS) State University of Maringá,

Maringá/PR.


Introduction and Objectives: According to World Health Organization (2018), colorectal cancer

(CRC) is the fourth leading cause of cancer and is the third leading cause of cancer related mortality.

In Brazil, at 2018 there were 36,360 new cases, with 17,380 men and 18,980 women (INCA, 2018). The

analysis of the trend of mortality and morbidity of diseases throughout the years, allows the

evaluation of the reflex of quality of life improvement for the health of population and public health

actions. The aim of this work was to analyze the trend of mortality rate of the neoplasia in the lower

segment of the gastrointestinal tract though 2000-2015 at Paraná state.

Material and Methods: An epidemiological study of ecological type of time series by CRC mortality

in the macro regions of the state of Paraná / Brazil between the years 2000 to 2015. The mortality

data from the Mortality Information System of the Ministry of Health and the demographic data of

the IBGE were obtained at Department of Informatics of the Ministry of Health (DATASUS) website

with exemption from submission to the research ethics committee according to Resolution CNS nº

506/2016 available on the COPEP/UEM. The temporal trend was realized by joinpoint regression by

which mortality rates from colorectal neoplasms were analyzed as dependent variables (y) and the

years 2000 to 2015 as independent variables (x), considering only the best model in the value with

significance of p value <0.01.

Results: Mortality rates for recto-sigmoid neoplasia throughout the state of Paraná showed a positive

tendency (p < 0.01), both for males and females. In the period of 2000-2015 referring to males, a

percentage of increase of (APC*: 8.5; CI**: 6.3-10.7) higher compared to female gender (APC: 6.7;

CI: 3.3 – 10.3). When compared by age groups, the 25-49-year-old group (APC: 7.6; CI: 1.2-14.4) had

the increase in its higher percentage compared to the group of < 50 years (APC: 7; CI: 4.5-9.6). In the

period analyzed, a joinpoint with a negative tendency was observed for the female group (APC: 3.3;

CI:-5.3-12.8) in the West macro region, but without statistical significance. * APC: Annual percent

changes calculated by Joinpoint Regression Analysis; ** CI: confidence interval.

Conclusion: The observed results demonstrated a considerable increase in the mortality rate by this

neoplasm, both for women and for men during the study period. Despite advances in public policies

of oncology care in Paraná, it is necessary to strengthen health surveillance in order to subsidize

preventive policies for the formulation of screening and early diagnosis programs, assisting in this way,

in decrease of the incidence of death by this neoplasm.

Keywords: Neoplasms; Colorectal Neoplasms; Sigmoid Neoplasms; Temporal Trend;

41

DENGUE: THE SCIENTIFIC PRODUCTION IN BRAZIL RELATED TO THE NURSING ASSISTANCE IN PUBLIC

HEALTH

SOUZA, C.W ¹; CHAVES, B.C.1; PEREIRA, J. 1; FERREIRA, A.R.O. 2; MACHADO, M.F.³

¹ Undergraduate student in nursing, Faculty Adventista Paranaense, Ivatuba, PR, Brasil.

²Postgraduate Program in Biosciences and Pathophysiology, State University of Maringá, Paraná,

Brazil.

³ Professor of the Nursing Department at the Faculty Adventista Paranaense, Paraná, Brasil.

²[email protected]

Introduction and objectives: Dengue is an infectious disease, occurring in tropical and subtropical

countries and its transmission is through the infected Aedes Aegypti mosquito bite. Worldwide, the

Health Department estimates that 2.5 billion people live in areas where risk of virus transmission exist,

resulting in 20 thousand annual deaths. In this context, performing adequate nursing assistance

becomes of crucial importance because the triage allows the professional to correctly identify and

intervene in all clinical manifestations of the disease. In this context, the objective of this research is

to evaluate the scientific production in Brazil about the nurse contribution related to dengue in the

public health, more specifically, to analyze the strategies pertaining to the promotion of health

education to the population and verify the quality of the information displayed in websites related

to the subject.

Material and methods: Qualitative study of revised literature, in which articles available in the

database SCIELO and CAPES were selected. These articles are related to the researched subject

and were published in Portuguese between 2015 and 2017. Incomplete articles, monographies,

theses, and dissertations or articles that did not have relation to the researched subjected were

excluded.

Results: 8 articles were selected. As evidenced by the articles it is necessary that nurses promote the

execution of health education of the population in order to provide greater empowerment to the

community, by raising the awareness of the responsibility for the natural environment, in order to

promote increased society life quality, decreased costs related to public service and management

strengthening. For the execution of health education, it is necessary to perform an expansion of

actions of prevention and of health promotion, for example instruction in schools, cleaning taskforces

and pamphlet distribution. Regarding the available websites discussing dengue, though they help in

the education about health matters, professionals need to pay attention to the quality of such

websites, checking the accuracy of the information provided because unfortunately only 5 Brazilian

websites, amongst all that were evaluated, received the seal of ethical and scientific quality issued

by HON (Health on the Net) foundation.

Conclusion: In this research we realized the importance of the nurse contribution in the

implementation of strategies of health education, so that the nurse is able to correctly instruct the

population about the disease, raising their awareness about the importance of actions to prevent

dengue.

Keywords: Nursing assistance, dengue and public health.

42

EVALUATION OF CARDIOVASCULAR RISK FACTORS IN RENAL TRANSPLANTATION PATIENTS

RIBEIRO, M. V.G.1; ZANESCO, C.2; SILVA, D. T. de R.3

1Department of Clinical Analysis and Biomedicine, State University of Maringá, Paraná, Brazil. 2Master of Science in Health, Nurse the Health Strategy in the city Abelardo Luz, Santa Catarina,

Brazil. 3Professor department of Nursing and Medicine, Federal University of Southern Frontier, Chapecó,

Santa Catarina, Brazil.


Introduction and objectives: Chronic kidney disease belongs to chronic noncommunicable diseases.

It is understood as a clinical syndrome characterized by the slow, gradual and progressive reduction

of the renal excretory, endocrine and metabolic functions. When the glomerular filtration rate

becomes lower than 15 mL min. -1 1,73 m -2, the endstage kidney disease occurs, and the renal

transplant became a form of treatment. Cardiovascular disease accounts for a large amount of the

mortality and comorbidity present in renal transplant patients. The aim of this study was to evaluate

cardiovascular risk factors in patients with different ages of renal transplantation.

Material and methods: The study was conducted in a clinic for the renal treatment located at the

West of Santa Catarina state, evaluating patients with kidney transplantation, of both sexes, that

carried out clinical follow-up in the referred clinic. Patients over 75 years of age, who died, who lost

the kidney graft, or discontinued follow-up were not evaluated. The normality of the data was verified

by the Kolmogorov-Smirnov test. The data were submitted to analysis of variance (one-way ANOVA)

and when significant differences were observed, the means were compared by the Tukey test at the

5% level of significance. All analyzes were performed using the GraphPad Prism 6.0 statistical

software.

Results: Patients with more than five years after renal transplantation presented a high risk of develop

cardiovascular disease. The parameters of low-density lipoprotein (LDL-C), high density lipoprotein

(HDL-C) and triglycerides (TG) in the group with more than five years of renal transplantation

presented high and worrisome chance of the develop dyslipidemia. Patients with more than five

years of renal transplantation have elevated risk for developing cardiovascular disease in the next

decade of life.

Conclusion: The Framingham risk score generates important and necessary information to elaborate

manners of decrease the risk of developing a cardiovascular disease, and consequently, increase

the quality of life and survival of patients after renal transplantation.

Keywords: Chronic Kidney Disease. Renal transplant. Cardiovascular diseases. Risk factors.

43

OCCURRENCE OF SCHISTOSOMIASIS MANSONI AND INTESTINAL PARASITOSES IN PATIENTS ATTENDED

AT THE AMBULATORY OF SPECIALTIES OF LONDRINA UNIVERSITY HOSPITAL, BRAZIL, FROM TO 2017

Mariana Barbosa Detoni1; Fernanda Tomiotto-Pollissier1; Thaïs Peron da Silva1; Luryan

Silvério Fidelis Ortiz1; Valter Abou Murad2, Luiz Antonio Custódio2; Francisco José de Abreu

Oliveira1; Ivete Conchon-Costa1; Idessania Nazareth Costa1; Wander Rogério Pavanelli1;

Francine Nesello Melanda3; Milena Menegazzo Miranda Sapla1

1State University of Londrina, Department of Pathology Sciences, Londrina, PR, Brazil 2State University of Londrina, Department of Clinical and Toxicological Analysis, Londrina,

PR, Brazil 3Federal University of Mato Grosso, Institute of Collective Health, Cuiabá, MT, Brazil


Introduction and objectives: Schistosomiasis mansoni and intestinal parasites are a serious health problem in

developing countries. In these regions, the parasitism occurs in high intensity, resulting from precarious sanitation

conditions, low economic level, inadequate hygiene and hot and humid environment that favors the life cycle

of such parasites. According to the World Health Organization, schistosomiasis is responsible for 11.4% of the

global burden of disease, being estimated about 779 million people at risk of infection, while intestinal parasites,

in general, 1.4 billion people are infected by Ascaris lumbricoides, 1.3 billion by hookworms and about 200 million

by Giardia lamblia. However, many cases are not reported, resulting in problems like diarrhea, malnutrition and

impairment of the development for affected individuals. For this reason, the present study aimed to describe

the occurrence of schistosomiasis mansoni and intestinal parasitoses in the city of Londrina from 2006 to 2017,

seeking to strengthen the currently scarce data in the region.

Material and methods: It was performed a descriptive epidemiological study of the analysis of medical records

of coprological exams of patients attended at Ambulatory of Specialties of Londrina University Hospital from

2006 to 2017. The coprological exams were performed using the Kato-Katz, Hoffman, Pons and Janer methods,

and method of Faust by Londrina University Hospital laboratory staff. Data were analyzed using Microsoft Excel

and SPSS, version 19.0. software. The QGIS 2.18.25 software was used for map creation. The data were expressed

as percentage or absolute number. Significant differences between the categorical data were determined

through chi-square test. Differences were considered statistically significant upon p ≤ 0.05.

Results: The coprological exams performed in the studied period included a total of 26,974 individuals. Of these,

0.19% were positivity for schistosomiasis mansoni and 24.33% positive for some intestinal parasitoses. Regarding

to individuals infected by Schistosoma mansoni, there was no significance statistical difference between the

sexes (p=0.131), but 63.16% (n=32) were male. From individuals with intestinal parasitoses, 51.45% corresponded

to female subjects and 48.29% for male individuals (p≤0.05). In relation to the prevalence of parasite species,

86.15% were protozoa and 13.85% were helminths (p≤0.05). In relation to protozoa, there was a prevalence of

commensal species: Endolimax nana (43.21%) and Entamoeba coli (25.58%), and the pathogenic Giardia

lamblia (12.37%). Regarding the helminths species, 4.49% were hookworms followed by 2.49% of Enterobius

vermicularis. In relation to the most affected age group by S. mansoni, 26.92% were 31 to 40 years old (p≤0.05),

while in individuals infected by intestinal parasites the highest age group was 0 to 15 years (37.23%) (p≤0.05).

Regarding the spatial distribution of S. mansoni infected individuals, the highest density of positive cases was

located in the east region of Londrina.

Conclusion: The work showed that the prevalence of individuals infected by schistosomiasis mansoni and

intestinal parasitoses in the study sample of the Londrina city remains considerable, being affected both

economically active population and individual children, besides residents of the risk area, especially the ones

who live in the east regions, where the sanitation conditions are precarious.

Keywords: Schistosomiasis, epidemiology, parasitic diseases

Grants: Ambulatory of Specialties of Londrina University Hospital

44

PROFILE OF ATTEMPTED SUICIDE BY CHEMICAL SUBSTANCES ASSISTED BY POISON CONTROL CENTER

OF LONDRINA

Mariana Biscaia Falanga¹; Nathália Cristine Florêncio²; Edmarlon Girotto³; Camilo Molino Guidoni³

¹ Universidade Estadual de Londrina, Medical Student, Londrina, PR, Brazil

² Universidade Estadual de Londrina, Graduation in Pharmaceutical Sciences, Londrina, PR, Brazil

³ Universidade Estadual de Londrina, Department of Pharmaceutical Sciences, Londrina, PR Brazil

[email protected]

Introduction and objectives: Suicidal behavior is a complex phenomenon influenced by synergism of

psychological, social, cultural, environmental and biological aspects, such as psychiatric and mood

disorders, drugs of abuse, financial crisis, loss of hope in the future, family history of suicide and

genetic/biological factors. Suicide is the fourth cause of death among 15 and 29 years old in Brazil.

In 2015, the mortality rate for this cause increased to 5.7/100,000 inhabitants. Brazil is a signatory to

the World Health Organization's (WHO) Plan of Action on Mental Health, whose goal is to reduce the

suicide rate to 10% by 2020. Thus, it is necessary to undertake studies that contribute to the

identification of causal factors of this event and enable assertive preventive policies. This study aimed

to describe the profile of suicide attempts by chemical substances assisted by the Poison Control

Center (CIATox) of Londrina-PR in 2017.

Material and methods: Observational and cross-sectional study of suicide attempts by chemical

substances assisted by CIATox-Londrina in 2017. The data stored in the electronic base (DATATOX®)

were analyzed descriptively. The variables were age, gender, schooling, occupation, place of

exposure and attendance, severity classification, route of administration, seasonality, agents and

outcome. The study was approved by the Research Ethics Committee of the State University of

Londrina (UEL) (CAAE 45986415.1.0000.5231)

Results: 832 cases of attempted suicide were identified, with female prevalence (70.3%), age range

of 20-39 years (51.6%), students (41.0%) with complete secondary education (44.2%). The residence

was the most frequent place of exposure (96%). Patients were referred to hospitals or Emergency

Care Units (UPAs), with initial classification as mild, and the minority required hospitalization. The

predominant route of administration was oral (98.9%) and intoxicating agents were medications

(68%) (mostly Clonazepam, Amitriptyline and Acetaminophen), pesticides, drugs of abuse, raticides,

household chemicals and inseticides, residential or industrial chemicals, veterinary products, food

and others. The most frequent association was medication and drug abuse (25.6%). There was peak

in October, December and February, especially on Sundays and Saturdays. Death occurred in seven

cases and the predominant outcome was mild clinical manifestations.

Conclusion: The predominant profile of users studied was women aged 20 to 39 years, with complete

high school, students, who attempted to commit suicide in their own residence on Sundays, mainly

in October, by benzodiazepine medications, orally, without associations, and did not require

hospitalization due to mild clinical manifestations. This risk group should be targeted of selective

psychosocial intervention, according to WHO.

Keywords: Health Information Systems. Poison Control Centers. Suicide, Attempted.

Grants: CIATox – Londrina.

45

REGULATION OF LIPID METABOLISM BY AEROBIC PHYSICAL TRAINING AND OMEGA-3 INTAKE IN

PROSTATE OF WISTAR RATS SUBMITTED TO HIGH FAT DIET

Allice Santos Cruz Veras1; Rayana Loch Gomes2; Maria Eduarda de Almeida Tavares3; Giulia

Fonseca dos Santos3; Rafael Ribeiro Correia3; Sabrina Alves Lenquiste2; Giovana Rampazzo

Teixeira1,3.

1Postgraduate Program in Movement Sciences, Department of Physical Education, School of

Technology and Sciences, UNESP campus of Presidente Prudente, Brazil.

2University of West Paulista - Unoeste, Presidente Prudente, São Paulo-SP, Brazil.

3Department of Physical Education, School of Technology and Sciences, UNESP campus of

Presidente Prudente, São Paulo-SP, Brazil.


Introduction and objectives: The prostate issues occur due to principally poor lifestyle as the

excessive high fat diet intake and insufficient physical exercise practice. There are a specific family

that coordinately activate the expression of genes cascades associated with lipids and cholesterol

called Sterol regulatory element-binding protein-1 (SREBP-1). On the other hand, the fatty acid

synthase realized by the FAS protein, is responsible to produce these lipids, known as fatty acids.

However, recent studies has demonstrated that some animal lipids can benefit health and the

prostate functioning as omega-3 polyunsaturated fatty acid. The exercise use these lipids as fuel

during session, protecting against prostate disorders and helping in metabolism. Based on that, we

investigated the effects of SREBP-1 and FAS mechanism of action associated with omega-3 and

aerobic physical training in prostate of Wistar rats supplemented with high fat diet.

Material and methods: Thirty-five male Wistar rats were used, divided in 7 groups (n=5): CT - received

standard diet and water ad libitum; HF - the animals were supplemented with high fat diet; HF+n-3 -

received the high fat diet and fish oil intake; HF+Ex - the animals received the high fat diet and

performed aerobic physical training; HF+n-3+Ex - supplemented with high fat diet, omega-3 intake

and performed aerobic physical training; HF+Chia - the animals received the high fat diet and chia

oil; HF+Chia+Ex - were supplemented with high fat diet, chia oil and performed the aerobic physical

training. The high fat diet was composed by roasted peanuts, milk chocolate and crackers (3:2:2),

and the omega-3 was applied by gavage as well as water in other groups to induce the same kind

of stress. The swimming protocol training was 3 times per week, for 30 minutes, and approved by

Ethics Committee on the Use of Animals (protocol number 3962).

Results:Our findings demonstrated that the HF animals increased the SREBP-1 expression (11.58±0.69)

compared to HF+Chia+Ex (10.19±1.00), HF+Ex (11.39±0.27) and HF+Chia+Ex (10.19±1.00). Similar data

were found in HF group for FAS expression (10,99±1,12) while HF+n-3+Ex (9,41±1,12), HF+Chia+Ex

(8,15±0,66), HF+Ex (10,38±0,71) and HF+Chia+Ex (8,15±0,66) reduced this expression. The HF+Chia

group had presented most expression (9,92±0,39) compared to HF+Chia+Ex (8,15±0,66).

Conclusion: Nevertheless, these results help to conclude lipids overexpression caused by FAS

expression even regulated by SREBP-1. The lipids overexpression were reduced in omega-3 group

and by the physical training that which is responsible for increasing the energy expenditure, which

favors the loss of weight and the reduction of adiposity, favoring the prostatic functioning.

Grants: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior - Brasil (CAPES) - Finance Code 001.

46

RELATION BETWEEN SOCIAL INDICATORS AND THE INCIDENCE OF MULTIDRUG RESISTANT

TUBERCULOSIS

Lincoln Luís Silva 1,2; Tamires Leite Valverde 1,2; Raíssa Bocchi Pedroso 2;

Amanda de Carvalho Dutra 2, Luciano de Andrade 2, Maria Dalva de Barros Carvalho 2,

Sandra Marisa Pelloso 2, Regiane Bertin de Lima Scodro1,2

1 State University of Maringá, Department of Clinical Analysis and Biomedicine, Brazil;

2 State University of Maringá, Program of Post-Graduation in Health Sciences, Brazil;

[email protected]

Introduction and objectives: Multidrug-resistant tuberculosis (MDR-TB) represents a major obstacle in

the fight against tuberculosis (TB) around the world. There were an estimated 10.4 million cases of TB

and more than 460,000 cases of MDR-TB globally in 2017. Beyond that, 1.3 million people died as

result of the disease in the same year. Brazil is among the twenty countries with the highest number

of TB cases in the world and with several cases of MDRTB, where Rio de Janeiro is one of the state

with the highest number of MDR-TB cases. Improving the outcome for MDR-TB is one of the priority

actions recommended by the World Health Organization. The success of actions will depend on the

ability to identify where it occurs and who is at risk. Therefore, the aim of this study was to identify the

social indicators associated with the incidence of MDR-TB in Rio de Janeiro from 2013 to 2017.

Material and methods: An ecological study was performed to verify what social indicators are related

to the incidence of MDR-TB. Data of MDR-TB cases were collected from Sistema de Informação de

Tratamentos Especiais da Tuberculose. All patients with MDR-TB who started treatment during the

period from January 2013 to December 2017 were included in the study. Patients that started

treatment before or after this period, has the diagnosis changed, extra pulmonary forms and other

forms of resistance were excluded from the database. In order to overcome non reported data, we

applied the Multivariate imputation by chained equations, a data imputation strategy which assigns

values to missing data using R (version 3.5.1) with MICE package. Data for social indicators (life

expectancy at birth, expectation of years of study, illiteracy rate, per capita income, proportion of

people vulnerable to poverty, and Human Development Index) was obtained from the Brazilian

Census of 2010. TB rates were obtained from DATASUS. To verify associations between MDR-TB rates

and social indicators, Ordinary Least Square regression (OLS) and Spatial Lag Model (SLM) based on

Lagrange Multiplier (LM) was applied in QGIS (version 2.16) and GeoDa (version 1.12). To ensure the

robustness of the analysis, a sensitivity test was conduct and chose the model with the lowest

multicollinearity value of Akaike information criterion (AIC) and considered significant with P <0.05.

This study was approved by, Research Ethics Committee Involving Human Being, of the State

University of Maringá (COPEP/UEM) according to the process number 22/2018.

Results: Three independent variables were statistically significant associated to MDR-TB incidence in

OLS regression model. The illiteracy rate (18 to 24 year olds who can not read or write), income

(maximum per capita income of the 40% poorest) and percentage of people vulnerable to poverty

(people with per capita incomes of $160.00 and who spend more than one hour traveling to the

workplace) had a positive tendency.

Conclusion: Three social indicators were associated to the incidence rate of MDR-TB in Rio de Janeiro.

This demonstrates that the spread of the disease is not only related to microorganism characteristics.

Keywords: Mycobacterium tuberculosis, multidrug-resistance, epidemiology, spatial analysis.

Grants: CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

47

THE PREVALENCE OF SYPHILIS IN PRENATAL CARE WOMEN IN PARANA STATE, BRAZIL

Isabella Harumi Yonehara Noma1; Cínthia Akemi Tanoshi 1; Dário Sodré dos Santos2; Regivaldo

Florentino Rodrigues1; Lincoln Luís Silva1; Marcia Edilaine Lopes Consolaro1; Eniuce Menezes de

Souza2; Sandra Marisa Pelloso1; Raíssa Bocchi Pedroso1.

1Universidade Estadual de Maringá, Post-graduate Program in Health Sciences, Maringa, PR, Brazil

2 Universidade Estadual de Maringá, Department of Statistics, Maringa, PR, Brazil

Email ([email protected])

Introduction and objectives: Syphilis is a chronic infectious disease, potentially fatal, caused by the

bacterium Treponema pallidum, which is sexually and vertically (mother-to-child) transmitted. Two

million pregnant women are infected with syphilis globally, considering it as a global public health

problem. In 2016, there were approximately 661,000 cases of congenital syphilis in the world, resulting

in more than 200,000 stillbirths and neonatal deaths. During pregnancy, syphilis is of high-risk for fetuses

if untreated or inadequately treated. It is associated with adverse pregnancy outcomes such as

miscarriage, stillbirth, preterm birth, hydrops or, when transmitted to the fetus, it can present low

birthweight, sequelae including neurological impairment and bone deformities. The seroprevalence

of women with syphilis attending prenatal care is estimated to be highest in Latin America (3.90%)

and Africa (1.98%). Therefore prenatal care is necessary for screening and prevent congenital

infections. The aim of this study is to evaluate and analyze syphilis detected during prenatal care in

the last 10 years.

Material and methods: A cross-sectional study of syphilis cases in pregnant women in Parana state

form 2008 to 2018, were obtained at Department of Informatics of the Unified Health System

(DATASUS), a public domain information database. The data were collected at January and March

2019. The data obtained was organized and analysed with Microsoft Excel® software.

Results: From 2008 to 2018, there were 14,298 cases of gestational syphilis, and of these, only 5,014

women underwent prenatal care corresponding to 35.08% of the cases. The cases of syphilis in

pregnant women had a significant increase of 14 times in the last 10 years, in 2008 from 181 cases, to

2,596 cases in 2018 with 2013 heading with a increased of more than a thousand cases. Among the

14,298 cases of syphilis in pregnant women, 67.25% (n=9,619) were white, the majority in the age

group of 20-39 years with 70.65% (n=10,102) followed by the age of 1519 years with 25.80% (n=3,689).

Regarding maternal educational level was more prevalent in women with less than 8 years of

schooling.

Conclusion: The age range of women and schooling indicates the importance of information as

preventive measures against sexually transmitted diseases (STDs), especially for young women with

lower education. Cases of syphilis that were not diagnosed at prenatal will be at puerperium or with

the newborn. The late diagnosis can lead inadequately treated or untreated syphilis causing to fetus

spontaneous abortion, prematurity or other sequelaes. However through services antenatal syphilis

screening and reproductive health programs, can reduce syphilis perinatal deaths and stillbirth

incidence. Therefore, it is important to encourage pregnant woman to follow-up prenatal and to

increase antenatal screening policies.

Keywords: syphilis, prenatal, pregnant women

48

A CARACTERIZATION OF CARBOXYMETHYLQUITOSAN HIDROGEL INCORPORATED WITH HYALURONIC

ACID WITH POTENTIAL HEALING ACTIVITY ON SECOND DEGREE BURNS EXPERIMENTALLY INDUCED ON

MICE

Dias, Yuri Santana Pereira1; Rosa, Luciana Martins1; Gonçalves, Randys Caldeira 1 Junior, Ruy de

Souza Lino1 1Universidade Federal de Goías, Institute of Tropical Pathology and Public Health, Goiânia,

GO, Brazil [email protected]

Introduction and objectives: On the market, there are multiple products destined to treating burns,

however, there is not a gold-standard treatment. Considering this, studies that seek the production

of hydrogels capable of improving the cicatricial process are ones of great relevance and are geting

considerable acknowledgement from the scientific milieu. The objectif of this paper was to avaliate

the carboxymethylchitosan hydrogel incorporated with hyaluronic acid efect over the burn

cicatrization of partial thickness in rats.

Material and methods: Carboxymethylchitosan hydrogels were synthetized and later incorporated

with hyaluronic acid. The experiments were done using Wistar rats, in wich were inflicted partial

thickness burns using boiling water. Then, it was stablished 4 groups, each one containing 15 rats, in

wich it were applied the following compounds in daily bandages: G1 (silver sulfadiazine); G2

(hyaluronic acid); G3 (carboxymethylchitosan hydrogel); G4 (carboxymethylchitosan hydrogel

incorporated with hyaluronic acid). For each compound, the 15 animals were divided in three

experimental days: 7, 14 and 30 days. For each experimantal day, it were euthanized five rats.

Macroscopic avaliation by wound mesuring was morphometrically done at day 0 and at euthanasia

day. For the calculation of the wound área, the obtained images were transfered to the ImageJ

software (data were presented as contraction area percentage ± standard deviation). The statistical

test applied was ANOVA and post test Tukey (p<0,05). Data of the macroscopic parameters

avaliation are presented as medians (maximum and minimum ratings) utilizing Kruskal-Wallis and post

test Dunn’s for the statistic analisis.

Results: When comparing the wound contraction 30 days after the lesion induction, it was perceived

that G2 (99,6 ± 0,89), G3 (94,3 ± 2,62) and G4 (99,9% ± 0,04) had a significatively bigger contraction

rate (p<0,05) than G1 (84,28% ± 3,37). At day 7, inflamatory phase, the presence of necrosis was

significatively (p<0,05) smaller in G1 (0, from 0 to 1) than in G2 (3, from 3 to 3), G3 (3, from 2 to 3) and

G4 (3, from 2 to 3). When it comes to the presence of granulation tissue and reepithelization there

was no significative variation. At day 14, proliferative phase, the presence of granulation tissue was

significatively (p<0,05) smaller in G1 (1, from 0 to 2) than in G2 (3, from 3 to 3), G3 (3, from 3 to 3) and

G4 (3, from 3 to 3). When it comes to the presence of necrosis and reepithelization there was no

significative variation. At day 30, remodeling phase, the presence of granulation tissue was

significatively (p<0,05) bigger in G1 (1, from 0 to 1) than in G2 (0, from 0 to 0), G3 (0, from 0 to 0) and


significative variation.

Conclusion: It is possible to conclude that G4 has a greater wound contraction capability, necrosis

inhibition and production of granulating tissue when compared to G1. The results sugest that

carboxymethylchitosan hydrogels incorporated with hyaluronic acid may accelerate the burn

healing process.

Keywords: carboxymethylchitosan; hyaluronic acid; burn; cicatrization;

49

A CARACTERIZATION OF CARBOXYMETHYLQUITOSAN HIDROGEL INCORPORATED WITH SILVER WITH

POTENTIAL HEALING ACTIVITY ON SECOND DEGREE BURNS EXPERIMENTALLY INDUCED ON MICE

Rosa, Luciana Martins1; Dias, Yuri Santana Pereira1; Gonçalves, Randys Caldeira 1 Junior,

Ruy de Souza Lino1

1Universidade Federal de Goías, Institute of Tropical Pathology and Public Health, Goiânia, GO,

Brazil

Email:[email protected]

Introduction and objectives: In developing countries deaths by burns are very frequent, due to higher

incidence and deficient treatment. Despite its relevancethere, there is no goldstandard treatment

for this kind of lesion. Considering this, the development of curatives minimally manipulated, atoxic,

hypoallergenic, antimicrobial and wich favor the cicatrization process is one of great relevance.

There are papers that support the effectiveness of silver and chitosan as antimicrobials over wound

healing. However there is no paper evaluating the action of carboxymethylchitosan hydrogels

incorporated with silver until now. With that being said, the objective of the research was to evaluate

the action of this compound over the cicatrization process of partial thickness burns in rats.

Material and methods: Carboxymethylchitosan hydrogels were synthetized, purified and later

incorporated with silver. The experiments were done using female Wistar rats, in wich were inflicted

partial thickness burns using boiling water. Then, it was stablished 3 groups, each one containing 15

rats, in wich it were applied the following compounds in daily bandages: G1(silver sulfadiazine);

G2(carboxymethylchitosan hydrogel); G3(carboxymethylchitosan hydrogel incorporated with

silver). For each compound, the 15 animals were divided in three experimental days: 7, 14 and 30

days. For each experimantal day, it were euthanized five rats. Macroscopic avaliation by wound

mesuring was morphometrically done at day 0 and at euthanasia day. For the calculation of the

wound área, the obtained images were transfered to the ImageJ software (data were presented as

contraction area percentage ± standard deviation) and the statistical test applied was ANOVA and

post test Tukey (p<0,05). Data of the macroscopic parameters avaliation are presented as medians

(maximum and minimum ratings), utilizing Kruskal-Wallis and post test Dunn’s for the statistic analisis.

Results: When comparing the wound contraction 30 days after the lesion induction, it was perceived

that G2(94,2 ± 2,62) and G3(96,7 ± 3,97) had a significatively bigger contraction rate (p<0,05) than

G1(84,28% ± 3,37). At day 7, inflamatory phase, the presence of necrosis was significatively (p<0,05)

smaller in G1(0, from 0 to 1) than in G2(3, from 2 to 3) and G3(2,5, from 2 to 3). When it comes to the

presence of granulation tissue and reepithelization there was no significative variation. At day 14,

proliferative phase, the presence of granulation tissue was significatively (p<0,05) smaller in G1(1,

from 0 to 2) than in G2(3, from 3 to 3) and G3(2, from 1 to 3). When it comes to the presence of

necrosis and reepithelization there was no significative variation. At day 30, remodeling phase, the

presence of granulation tissue was significatively (p<0,05) bigger in G1(1, from 0 to 1) than in G2 and


significative variation.

Conclusion: It is possible to conclude that G3 has a greater wound contraction capability, and

necrosis inhibition when compared to G1 and G2. The results sugest that carboxymethylchitosan

hydrogels incorporated with silver may accelerate the burn healing process.

Keywords: burn; carboxymethylchitosan; silver; cicatrization.

50

ACQUIRED DOXORUBICIN RESISTANCE IN MCF-7 AND MDA-MB-231 INVOLVES PROTECTION AGAINST

OXIDATIVE STRESS AND APOPTOSIS AND MODULATION OF GENES RELATED WITH TRANSFORMING

GROWTH BETA 1 PATHWAY

Marinello, P. C.1; Cavalcanti, L. F.1; Panis, C.2; Silva, T. N. X1, Binato, R.3; Abdelhay, E.3;Rodrigues, J.

A.4; Mencalha, A. L4; Lopes, N. M. D.1; Luiz, R. C.1; Cecchini, R.1; Cecchini, A. L.1*

¹State University of Londrina, Department of General Pathology, Londrina, PR, Brazil

²Laboratory of Tumor Biology, State University of West Parana, Unioeste, Francisco Beltrão, PR, Brazil

³Bone Marrow Transplantation Unit, National Cancer Institute (INCA), Rio de Janeiro, RJ, Brazil. 4Laboratory of Cancer Biology, Department of Biophysics and Biometrics, State University of Rio de

Janeiro, Rio de Janeiro, RJ, Brazil.


Introduction and objectives: The establishment of resistance to chemotherapy is an important factor

that limits successful treatment of breast cancer. The generation of oxidative stress is implied in the

mechanism of action of several chemotherapeutic drugs. In this scenario, it is known that some

cancer cells develop a chemoresistant phenotype during this pro-oxidant treatment, and may

develop additional mechanisms to protect themselves against this chemotherapy-driven oxidative

injury. Based on this, the objective of this work was to evaluate certain oxidative stress-related

pathways during doxorubicin chemoresistance induction in human estrogen and progesterone

receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells.

Material and Methods: The induction of doxorubicin-chemoresistant phenotype (DOX-res) was

achieved exposing cells to increasing concentrations of doxorubicin (10 nM to 100 nM) during

sequential passages. Samples were collected to the evaluation of oxidative stress parameters,

immunocytochemistry (P53, NF-kB and Nrf2) and gene expression by real-time quantitative array PCR

(qRT-PCR). Oxidative stress was assessed by the evaluation of lipoperoxidation, determined by

chemiluminescence stimulated by tert-butyl-hydroperoxide (CL), total thiols levels and nitric oxid

(NO) determination. The normality of the data and the presence of outliers were investigated by

Shapiro-Wilk and Rout test, respectively. Data were expressed as mean ± standard deviation of the

mean and analyzed using the paired Student t test. P < 0.05 was considered statistically significant

Results: The DOX-res group presented increased thiols and reduced lipoperoxidation, increased

levels of NO, nuclear NF-kB and Nrf2, and reduced nuclear p53 labelling. Analysis of the expression

of genes enrolled with TGF-β1 signaling pathway by RT-PCR array showed that DOX-res cells

presented altered expression of different genes related to the development of an adaptive

response, such as resistance against apoptosis and OS. Some genes involved in this process were

upregulated in both MCF-7 and MDA-MB-231 DOX-res cells, such as HERPUD1 and RHOA. Both cell

lines also presented upregulation of PTK2B and VEGFA. MDA-MB-231 DOX-res cells presented

upregulation in genes related to highly aggressive characteristics, such as HEY1, EMP1 and CREB1.

Conclusion: In conclusion, the results of this study indicate that the prooxidant environment induced

by doxorubicin exposure modulated the profile of gene expression in both breast cancer cells

analyzed, which was responsible to the increased intracellular antioxidant defenses and reduced

activation of pathways related to apoptosis, important mechanisms related with doxorubicin

cytotoxicity.

Keywords: Breast cancer, chemoresistance, oxidative stress, TGF-β1

Grants: Fundação Araucária, Capes.

51

ANALYSIS OF HISTOPATHOLOGY INTRAPERITONEAL INFECTION RELATED BY Escherichia coli IN LARGE

INTESTINE AND KIDNEYS IN FEMALE SWISS MICE

Luana Carvalho Silva1; Bruna Santos Marnieri1, Telma Saraiva dos Santos2, Stéfane Frazão de

Morais Cabral1 , Eliza Pizarro Castilha1, Gerson Nakazato3, Anelise Franciosi1,2, Eduardo

José de Almeida Araújo2, Karina de Almeida Gualtieri1, Náthalia Maria Fioreto Campois1 e Tacito

Graminha Campois1.

1 Centro Universitário Filadélfia, Departament of Immunology and Patology, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

3Universidade Estadual de Londrina, Department of Microbiology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Escherichia coli (E.coli) is a microorganism typically colonizing of the

intestinal microbiota. However, in immunocompromised individuals, this infection may become

pathogenic, leading to several problems in the patient. The diarrheogenic strain, enterohemorrhagic

Escherichia coli (EHEC), is known to cause widespread disease in humans, including asymptomatic

cases, mild diarrhea, hemorrhagic colitis, and the most severe form of uremic hemolytic syndrome

(HUS). Studies suggest that the presence of the StX2 toxin produced by the EHEC strain is strongly

associated with the development of HUS. This condition may be characterized by hemolytic anemia,

thrombocytopenia and acute renal failure, which may be fatal to the patient. The American Type

Collection Culture (ATCC) strain is used as the control strain, because of low virulence and non-

toxigenic.

Material and methods: This work aimed to establish a relationship between the histopathological

results of EHEC and ATCC infection in mice, by an alternative route of infection, the intraperitoneal

route. In this experiment, 60 female Swiss mice were divided into three groups (control, ATCC and

EHEC). 24 hours after infection containing 1x105 E.coli, the animals were submitted to euthanasia,

following the rules established by the UniFil Ethics Committee. 1 cm of the cecum of the 3 groups of

animals were removed, prepared and placed to histology, with subsequent microtomy and staining

in Hematoxylin and Eosin (HE). During the evaluation of the histological results by means of

photographs using an optical microscope, we may observe the tissue changes of the gastrointestinal

tract in the portion of the cecum.

Results: The changes observed in this intestinal segment show the presence of superficial epithelium

destruction, focal areas of evident inflammation, neutrophil infiltration in the lamina propria,

formation of crypt abscesses, edema and hemorrhages, when compared to the control group and

the ATCC strain. Also, in the kidneys, it was possible to observe changes such as the presence of

hemorrhage, provided by the action of the toxin produced by the diarrheogenic strain,

corroborating previous studies.

Conclusion: By these analyzes, we can conclude that the studied strains have different forms of tissue

invasion and histological alterations, and also demonstrates the tropism of the bacteria by

enterocytes, independent of the infection pathway.

Keywords: histopathological Changes; Diarrheogenic strain; Escherichia coli


52

ANALYSIS OF OXIDATIVE STRESS PARAMETERS IN PATIENTS WITH PAPILLARY THYROID CARCINOMA

AND HASHIMOTO'S THYROIDITIS

Natália Medeiros Dias Lopes1; Hannah Hamada Mendonça Lens1; Poliana Camila Marinello1;

Walison Augusto da Silva Brito1; Rubens Cecchini2; André Armani3; Alessandra Lourenço Cecchini1.

1Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil. 2Universidade Estadual de Londrina, Laboratory of Physiopathology and Free Radicals, Londrina,

PR, Brazil. 3Universidade Estadual de Londrina, Department of Surgical Clinic, Londrina, PR, Brazil.

[email protected]

Introduction and objectives: Thyroid cancer is considered the most frequent type of endocrine

neoplasms, with the papillary subtype being the most common. An important risk factor for the

development of this type of cancer is the presence of preexisting thyroid diseases, such as

Hashimoto's Thyroiditis, which is the most prevalent autoimmune thyroid disease. The clinical

association between these two diseases is being widely reported, however the biological events that

would be occurring between them have not yet been fully clarified, so a better analysis of this

association is a necessity. Thus, the objective of this work was to analyze the preliminary results,

obtained with a reduced sample, of some parameters of oxidative stress presented by patients with

thyroid cancer, Hashimoto's thyroiditis as well as the association between them.

Material and methods: The patients were divided into 4 groups: Control (n=15), Papillary thyroid

carcinoma (PTC; n=4), Hashimoto's thyroiditis (HT; n=4) and a group with Papillary thyroid carcinoma

and Hashimoto's thyroiditis (PTC-HT; n=4), according to histopathological diagnosis obtained after

analysis of thyroid tissue removed during surgery. Blood samples were collected before

thyroidectomy and some parameters of oxidative stress were analyzed. Oxidized and reduced

glutathione levels (GSSG and GSH, respectively), superoxide dismutase (SOD) and catalase activity

was determined in erythrocytes. Membrane hydroperoxides was assessed by chemiluminescence

stimulated by tert-butyl-hydroperoxide (CL) in erythrocytes. Levels of Malondialdehyde (MDA) was

determined in plasma. The statistical analysis was performed using one-way or two-way ANOVA, or

Kruskal-Wallis after the verification of the normality of the data, p<0.05 was considered significant.

This project was approved by the ethics committee (CEP-UEL process n. 2.793.785).

Results: No statistically significant difference was found between the groups when antioxidant

parameters were analysed (GSH, GSSG, Catalase and SOD. Patients from PTC group presented

increased hydro-peroxides levels when compared with patients of the HT group, but no alterations

was found in MDA levels.

Conclusion: Our results are preliminary and the sample size is small which could compromise the

statistical analysis. However, although preliminary, the results point to the existence of differences in

erythrocyte lipoperoxidation in patients with PTC and HT, indicating the necessity of increase sample

size and to continue the study of the differences between these groups considering the relevance

of thyroid cancer.

Keywords: Papillary thyroid carcinoma, Hashimoto thyroiditis, Oxidative stress.

53

ANTI-PROLIFERATIVE ACTIVITY OF TRANS-CHALCONE IN VITRO IN HUMAN HEPATOCELLULAR

CARCINOMA HUH7.5 LINE

Elaine da Silva Siqueira¹; Vírgínia Márcia Concato¹; Fernanda Tomiotto-Pellissier¹,5; Bruna Taciane da

Silva Bortoletti¹,5;Tatiane Renata Fagundes¹; João Paulo Assolini¹; Taylon Felipe Silva¹; Aedra Carla

Bufalo Kawassaki¹,²; Waldiceu Aparecido Verri Junior3, Idessania Nazareth Costa¹; Juliano

Bordignon5; Milena Menegazzo Miranda-Sapla¹; Mário Sérgio Mantovani4; Carolina Panis², Wander

Rogério Pavanelli¹, Ivete Conchon-Costa¹.

¹State University of Londrina, Laboratory of Immunoparasitology of Neglected Diseases and

Cancer, Londrina, PR, Brazil.

²State University of Western Paraná, Laboratory of Inflammatory Mediators, Health Sciences Center,

Francisco Beltrão, PR, Brazil.

³State University of Londrina, Laboratory of Research on Pain, Neuropathy and Inflammation,

Londrina, PR, Brazil. 4State University of Londrina, Laboratory of Genetic Toxicology, Department of General Biology,

Londrina, PR, Brazil. 5Carlos Chagas Institute, Laboratory of Molecular Virology, Curitiba, PR, Brazil.


Introduction and objectives: The search for new compounds with antitumor potential and that

respond selectively to cancer cells with low cytotoxicity has been the focus of the development of

many studies. However, the aim of this study was to investigate the antitumor action of trans-

chalcone (TC) in vitro on human Hepatocellular Carcinoma (HCC) HuH7.5 cells.

Material and methods: Tumor cells of the HuH7.5 lineage was treated with TC at increasing

concentrations and the 50% inhibitory concentration of the cells (IC50) at the 48 hour time by MTT

was determined. From this, assays were performed to determine the morphological changes by light

microscopy, cell viability by tripan blue, as well as the mechanisms of death involved by the Muse®

cell analyzer.

Results: We observed that TC treatment was able to promote reduction of cell viability of the HuH7.5

CHC line and to induce changes in the morphology of these cells. Later, we verified that TC

treatment was able to promote mitochondrial depolarization but did not alter the production of NO

and EROs. It was also found that TC induced cell cycle arrest in G2/M and, consequently, led to cell

death. In addition, it was found that the compound, at the concentrations tested, did not present a

hemolytic potential, suggesting the selectivity of the compound to tumor cells.

Conclusion: TC treatment was able to reduce the viability of HuH7.5 cells, causing mitochondrial

alterations and concomitant stoppage in the cell cycle in the G2/M phases.

Keywords: Hepatocellular Carcinoma; HuH7.5 cells; trans-chalcone.

54

ASSOCIATION BETWEEN LACTOFERRIN GENE POLYMORPHISM AND DENTAL CARIES IN PRIMARY

DENTITION

Paola Singi1; Ana Claudia Poletto2; Rosário Mamani Barri2; Kleber Paiva Trugilo1; Nádia Calvo

Martins Okuyama1; Érica Romão Pereira1; Thaílla Cristina Faria Pacheco1; Nilson de Jesus Carlos1;

Maria Angelica Ehara Watanabe1, Emerson José Venancio4; Cássia Cilene Dezan Garbelini3; Karen

Brajão de Oliveira1

¹Laboratory of Molecular Genetics and Immunology, Department of Pathological Sciences, State

University of Londrina, Londrina, PR, Brazil.

²Master in Dentistry, State University of Londrina, Londrina, PR, Brazil.

³Associate Professor, Department of Oral Medicine and Dentistry for Children, State University of

Londrina, Londrina, PR, Brazil. 4Associate Professor, Department of Pathological Sciences, State University of Londrina, Londrina,

PR, Brazil.


Introduction and objectives: Dental caries is a dynamic and continuous process that can present

different stages of evolution, from undetected visible changes to complete destruction of tooth

structure. Saliva plays an important role in maintaining the oral cavity in conditions that prevent the

development of caries. One of the major salivary components is lactoferrin. This multifunctional

salivary glycoprotein presents modulating role in the development and aggregation of cariogenic

bacteria, such as Streptococcus mutans. The aim of this study was to investigate the association

between the polymorphism rs1126478 in the Lactoferrin (LTF) gene and dental caries in preschool

children attended at the Department of Pediatric Dentistry of State University of Londrina.

Material and methods: Ethical approval was obtained from the University Ethics on Research

Committee (CAAE: 56077916.2.0000.5231) in compliance with Brazilian National Health Council

Resolution 4666/12. All parents/guardians received information about study risks and benefits, and

signed a statement of informed consent. The present retrospective cohort study included 119 healthy

preschool patients aged 34-79 months. Patients were divided into three groups according to ICDAS

(International Caries Detection and Assessment System) criteria: 11 with no caries or white-spot lesions

(caries-free group = ICDAS 0); 64 with visual change in enamel (enamel caries group = ICDAS 1 and

2); 44 with clinical visual signs of dentine involvement (dentin caries group = ICDAS 3,4,5,6). Samples

of unstimulated saliva were collected in the morning. The LTF rs1126478 polymorphism alleles were

genotyped by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism).

Continuous data (age) were expressed by median and interquartile range (IQR). Between-groups

differences in age were evaluated by Kruskal–Wallis test followed by post-hoc Dunn's test. The

association between polymorphism and caries status was assessed using multivariate logistic

regression. The significance level was set at 0.05 for all tests.

Results: The groups differed in age (H= 10.013; P=0.007), which the caries-free group with median age

43.0 months and IQR=16 were younger than the dentin caries group, whose median age was 60.5

months and IQR=18 (P=0,005). No between-group differences were verified between enamel caries

group (median age 55.0 months and IQR=20) and other groups. The frequency of the LTF rs1126478

AA genotype was 45.5%, 35.9% and 31.8%, while AG genotype was 36.4%, 40.6% and 34.1%, and

finally GG genotype was 18.2%, 23.4% and 34.1% in caries-free, enamel caries and dentin caries

group, respectively. No significant difference was found in the LTF rs1126478 genotypes distribution

among the groups.

Conclusion: The LTF rs1126478 polymorphisms showed no association with the different status of caries

in preschool patients.

Keywords: Dental caries, Genetic polymorphism, Lactoferrin

Grants: CAPES, CNPq

55

ASSOCIATION OF THE IL-10 POLYMORPHISM c. -592 C>A (rs1800872)IN WOMEN WITH

CERVICAL CANCER

Pereira, A. P. L. 1;; Trugilo, K. P.1;; Okuyama, N. C. M.1;; Ferreira, R. S.1;; Sena, M. M.1; Esposito,

A.1;; Bonaldo, A. L. L.1;; Singi, P.1;; Carlos, N. J.1;; Couto Filho, J. d’O.2;; Oliveira, K. B.1

¹Universidade Estadual de Londrina, Departamento de Patologia Geral, Londrina, Brazil

²Hospital do Câncer de Londrina, Londrina, Brazil

[email protected]

Introduction and objectives: Cervical cancer (CC) is the fourth most incident cancer among women

worldwide. This neoplasia affects the cervix epithelium and is a result of pre-malignant squamous

intraepithelial lesions (SIL) initiated by persistent infection caused by Human papillomavirus (HPV)

carcinogenic types. The majority of SIL cases will be cleared by the immune response and regress

spontaneously in 36 months, only a minor percentage will progress to cancer. Thereby, HPV infection is

necessary but not sufficient for cancer outcome. In this context, the immune response plays a crucial

role determining progression or inhibition of tumor development. Cytokines are small proteins responsible

for immune signaling and coordination, which cooperate to establish the immune response profile.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti inflammatory and immunosuppressive

role. Higher cervical and serum concentration of IL-10 have been associated with a poor prognostic

for CC. The c. -592 C>A polymorphism of IL-10 (rs1800872), seems to modify IL-10 transcription,

leading to a different inflammatory pattern. Thereby, the aim of this study was to better understand

the role of c. -592 C>A IL-10 polymorphism in CC development.

Material and methods: This study was approved by the Institutional Ethics Committee Involving Humans

Beings of the State University of Londrina (Londrina, PR, Brazil) (CEP/UEL 133/2012;; CAAE

05505912.0.0000.5231). All subjects agreed to participate and sample collection was performed after

properly authorization. Genomic DNA was extracted from peripheral blood and tumor biopsy in patients

of the Hospital do Cancer de Londrina, Hospital Erasto Gaertner and two Basic Health-Care Units.

Socio-demographic, sexual behavior and reproductive characteristics data were also collected. The

c. -592 C>A polymorphism genotyping was conducted by PCR (Polymerase Chain Reaction) followed

by restriction fragment length polymorphism (RFLP).

Results: High frequency of CC was found in women at age ³ 55 (p=0.001), married (p=0.018), smoker

(p=0.001), had educational level up to elementary schooling (p=0.001), use oral contraceptive

hormonal method (p=0.011), and had more than one parturition (p=0.009). Considering the genotypes,

C/A was the most frequent among cancer group in an independent manner. The co-dominant model

in logistic binary regression adjusted for confounders, showed that patients presenting C/A genotype

had 2.15 times more chances for developing cervical cancer (OR= 2.15;; IC95%= 1.02 – 4.56) meanwhile

A/A genotype was not associated. The dominant model, C/A + A/A, was also associated with 2.71

times more chances for cervical cancer development than CC patients (OR= 2.71;; IC95%= 1.05 – 4.47)

and considering the alleles frequency we observed that the variant A carriers have 1.55 times more

chances in developing CC (OR= 1.55;; IC95%= 1.05 – 2.29).

Conclusion: Our study analyses show for the first time the association between cervical cancer

and c. -592C>A polymorphism of IL-10 in Brazilian population (mixed race). The C/A genotype and

the allele A are independently associated with CC, suggesting the correlation with the variant allele

A. Older age, smoking status, use of oral contraceptive method and educational level are also

independently associated. Thus, a profile of susceptibility for CC can be stated using the IL-10

(rs1800872) genotype herewith socio-demographic data, supporting accurate screening for women.

Keywords: Cervical cancer. Interleukin-10. Polymorphism. rs1800872.

Grants: This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária,

Programa Pesquisa para o SUS (PPSUS) and by Londrina State University Graduate Coordination

(PROPPG-UEL).

56

Brucella abortus INTERFERES IN THE INFLAMMATORY PROFILE IN A SYNTHETIC MATRIX IMPLANT IN

MICE IN A TYPE IV SECRETION SYSTEM-DEPENDENT FASHION

Silva, M. F.¹*.;Batista, D. F. A.¹; Eckstein, C. ²; Costa, F. B.²;

Ferreira, M. A. N. D.¹; Campos, P. P¹.; Santos, R. L.².; Paixão, T. A¹.

¹Departamento de patologia Geral, Instituto de Ciências biológicas, Universidade Federal de

Minas Gerais, Belo Horizonte, MG - Brazil

²Departamento de Clinica e Cirurgia Veterinarias, Escola de Veterinária, Universidade Federal

de Minas GeraisBelo Horizonte, MG - Brazil


Introduction and objectives: Human brucellosis caused by bacteria of the genus Brucella spp. is considered a

debilitating chronic zoonotic disease. B. abortus is a Gram-negative, intracellular, facultative bacterium, agent

of bovine and human infections considered endemic in some regions of world. Brucella spp. virulence factors

contribute to its evasion of the immune system, induction of insidious inflammatory response favoring the and its

persistence in the host and establishment of chronic infection. Polyester-polyurethane-based synthetic sponges

is a widely used model in the study of acute and chronic inflammatory response, angiogenesis, fibroplasia, but

was not yet explored to evaluate the influence of pathogen infection in the inflammatory response. Thus, the

objective of this study was to evaluate the inflammatory process induced by Brucella abortus infection and the

influence of the Type IV Secretory System (T4SS) in the process at 7 and 14 days after implantation of synthetic

matrix in mice.

Material and methods: A total of 30 female 6-8-week-old BALB/c mice provided by the central vivarium of the

Universidade Federal de Minas Gerais (UFMG) were used in this study. All procedures were approved by the

animal research ethics committee (CEUAUFMG, Brazil, protocol 91/2018). The inflammatory response was

evaluated in four experimental groups, at 7 and 14 days after implantation of the synthetic sponge (dpie)

corresponding to six and 13 days post infection (dpi) with B. abortus, with the following experimental treatments:

group B1: infected with B. abortus 2308 (n = 5); group B2: B. abortus ΔvirB2 (n = 5), control group: inoculated with

sterile PBS (n = 5). On day 0, two sponges were implanted in the subcutaneous of the cervical region of

anesthetized mice. One dpi, mice were manually contained and 50 μL of PBS (control) or 50 μL of suspension

containing 1x104 CFU of B. abortus 2308 wild type (wt), or B. abortus ΔvirB2 (ΔvirB2) were inoculated directly into

each sponge. At 7 and 14 dpi, mice were euthanized and samples from the sponge and spleen were collected

for bacteriological analysis, and the sponge for histological and biochemical analysis of the inflammatory

response.

Results: This is the first study of the inflammatory profile induced by B. abortus infection in this in vivo model of

synthetic biomaterial. B. abortus wt was able to survive and replicate in this model while ΔvirB2 was attenuated,

confirming its inability to cause persistent infection. B. abortus wt is able to modulate the inflammatory response

in sponge differently from ΔvirB2. We observed that B. abortus wt induced lower production of inflammatory

mediators, lower influx of inflammatory cells as neutrophils and macrophages and inhibited formation of giant

multinucleate cells, and fibrovascular tissue at 14 dpi compared to the control or ΔvirB2-infected groups.

Conclusion: B. abortus 2308 has the ability to inhibit inflammation in vivo in a TSS4dependent manner in the

synthetic matrix model.

Keywords: Brucella abortus, Type 4 Secretion System, inflammation, innate immunity

Grants: We gratefully acknowledge to financial support came from Coordination of Improvement of Higher

Level Personnel (CAPES), National Council for Scientific and Technological Development (CNPq) and

Foundation for Research Support of the State of Minas Gerais (FAPEMIG).

57

CEREBRAL ISCHEMIA AND REPERFUSION PROMOTES ALTERATION IN CALICIFORM CELLS IN THE JEJUNO

OF WISTAR RATS

Vanessa de Brito Pereira 1; Anne Caroline Santa Rosa 2; Julia Estuani ³; Humberto Milani ⁴;

Nilza Cristina Buttow 5

1State Universit of Maringa, Department of Morphological Science, undergraduate in

Biology,Maringa,PR, Brazil 2 State Universit of Maringa, Department of Morphological Science, undergraduate in Biology,

Maringa, PR, Brazil

³ State Universit of Maringa, Department of Morphological Science, Postgraduate Department of

Cell Biology- DBC, Maringa, PR, Brazil

⁴ State Universit os Maringa, Department of Pharmacology and Therapeutic, Health Sciences

Center, Postgraduate, Maringa, PR, Brazil 5 State Universit of Maringa, Department of Morphological Science, Postgraduate Department of

Cell Biology- DBC, Maringa, PR, Brazil

Email: 1 [email protected]; 2 [email protected]; 3

[email protected]; [email protected] 4 ; [email protected] 5

Introduction and objectives: The brain and intestine maintains communication through the vagus

nerve called the cerebellar-gut axis. This communication directly influences gastrointestinal activities

through the plexuses present in the intestinal wall, with the function of preserve local integrity. Injuries

that occur in the brain can conduce the gastrointestinal tract to dysfunction, such as lack of motility.

Ischemia-reperfusion (I / R) is a type of injury, characterized by interruption of blood flow to an organ

or tissue and reestablishment of that flow, respectively. In our study we evaluated the effect of fifteen

minute cerebral ischemia followed by fifty-two day reperfusion on the jejunal goblet cells of Wistar

rats.

Material and methods: The experiment was conducted with Rattusnovergicus of the Wistar lineage.

The animals were randomly separated into two groups, Group GC (n-5): control rats, without surgical

procedures and Group G (n-5): rats submitted to Fifteen-minute Global and Transient Cerebral

Ischemia Induction (ICGT, model 4 -GRANDFATHER). The jejunal follow-up was collected fifty-two

days after surgery prepared for routine hitological techniques. The laminae were stained with Alcian

Blue pH 1.0 (sulphomucins) and pH 2.5 (sialomucins) and Schiff's Periodic Acid (Neutral PAS). We

quantified the goblet cells in the villus axis, in a 10x objective lens. We used the program

GrangPadPrism® V.5. The data were expressed with 95% confidence interval of error submitted to

the Student t test. (Ethical Approval by CEUA n ° 44232208171 UEM).

Results: We could observed that neutral mucins profile did not show changes, but the whereas those

producing acid mucins, showed a significant difference between the groups. A 25% increase in

sialomucins was observed in the ischemic animals while the sulfomucines reduced about 12%.

Inflammatory processes caused by injuries, changes the integrity of the epithelial barrier by providing

bacterial entry. The 25% increase in sialomucnas is related to the protective role against pathogen

entry in the intestinal epithelium.

Conclusion: Fifteen-minute cerebral ischemia followed by reperfusion of fifty-two days promotes

changes in goblet cells in the jejunum of rats.

Keywords: Gastrointestinal, sulfomucines, neutral mucins, sialomucins.

Grants: CNPq- National Council for Scientific and Technological Development

58

mailto:[email protected]

CHARACTERIZATION OF METFORMIN ACTIVITY IN VIVO MURINE METASTATIC MELANOMA MODEL

Sanches, L. J. 1; Marinello, P. C. 1; Brito, W.A.S.1; Lopes, N. M. D. 1; Blegniski, F.P2; Luiz, R. C. 1; Cecchini,

R.2 ; Cecchini, A. L. 1.

1Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil.

2 Universidade Estadual de Londrina, Laboratory of Pathophysiology and Free Radicals, Londrina,

PR, Brazil.

[email protected]

Introduction and objetives: Melanoma represents the most aggressive type of skin cancer, and is the

cause of the largest number of skin cancer-related deaths worldwide with increasing incidence in

recent decades. When it becomes metastatic, the prognosis is poor, being refractory to available

treatments. Dacarbazine is considered one of the most widely used chemotherapeutic agents in

Brazil. However, due to the aggressiveness and nature of melanoma, the effects of

chemotherapeutic alone are unsatisfactory, the most commonly discussed treatment strategy is the

combination of therapies. Metformin is the drug most prescribed for the treatment of type 2 diabetes.

Studies have shown beneficial effects of metformin in the treatment of various types of cancers,

especially melanoma. This work aims to evaluate the effect of metformin in the treatment of

metastatic murine melanoma.

Material and Methods: After approval of the animal ethics committee of the State University of

Londrina (CEUA N⁰ 13281.2017.07), C57BL / 6 mice were inoculated with 4X106 B16F10 cells in the

ophthalmic plexus and divided into clusters. MET: Metformin 2mg metformin intraperitoneally for 14

days, DTIC: treated with dacarbazine 50mg / kg from the 5th to the 10th day via intraperitoneal, and

MET-DTIC: treated with metformin 2mg from 1 to 14 days and dacarbazine 50mg / Kg from 5 to 10

days, both drugs intraperitoneally. Control - was also performed, where the animals were not

inoculated with B16F10 cells and received no medications. On the 14th day the animals were

euthanized. The number of pulmonary nodules were counted, oxidative stress tests such as GSH,

MDA, QL and catalase activity were performed in the tumor microenvironment, pulmonary tissue,

and systemic in the blood. Weight and food consumption analyzes were performed

Results: Treatment with metformin reduced the number of nodules, but significantly only in the MET-

DTIC group. There was an increase in feed intake for the MET-DTIC group, however, there were no

significant changes for weight. In the tumor microenvironment there was elevation in GSSG levels for

the MET-DTIC group and reduction for MDA. At the systemic level, there was a reduction in the GSSG

levels in the MET-DTIC group and an increase in catalase activity, the MDA was high for the DTIC, and

the curve for the QL was higher for the MET-DTIC and lower for the MET.

Conclusion: Our results indicate that metformin is able to reduce nodule numbers, however, its action

is more significant when given in conjunction with dacarbazine. Metformin was able to decrease the

chemotherapeutic side effects. In addition to being involved in oxidative stress in both the tumor and

systemic microenvironment. Aiming a beneficial effect of metformin on the pulmonary metastasis

model.

Keywords: Metastatic melanoma, metformin, dacarbazine, C57BL / 6.

Grants: CAPES.

59

CHARACTERIZATION OF THE HISTORY OF RISK FACTORS IN PATIENTS WITH COLORECTAL CANCER

TREATED AT HOSPITAL UNIVERSITÁRIO REGIONAL DO NORTE DO PARANÁ

Silva, I. C.1; Nascimento, T. H. D.1; Blegniski, F. P.1; Lens, H. H. M.1; Liberatti, M2; Luiz, R. 1 C.1;

1Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Ciências

Patológicas, Laboratório de Patologia Molecular, Londrina, PR, Brazil. 2Universidade Estadual de Londrina, Hospital Universitário Regional do Norte do Paraná,

Departamento de Clínica Médica, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Colorectal cancer (CRC) is the third most common cancer worldwide,

and it is characterized by abnormal tissue growth in colon or rectum. Estimates of new CRC cases in

Brazil are 36,600 per year (17,380 for men and 19,980 for women). Only 5 to 10% of the cases are

familial. For sporadic cases the main risk factors are age (over 50 years old), intestinal constipation,

high consumption of red meat and sausages, sedentarism, among others. The objective of this study

was to characterize the history of risk factors in patients with CRC.

Material and methods: A exploratory descriptive study was carried out with the application of a

questionnaire for 21 patients with CRC treated at Hospital Universitário Regional do Norte do Paraná.

The research was previously approved by the research ethics committee CAAE no.

56874216.0.00005231. Data were obtained on age, gender, ethnicity, family history of cancer,

physical activity, history of intestinal transit, exposure to toxic substances, immune system response,

presence or absence of other chronical diseases, alcohol intake, tobacco consumption, and eating

habits. The data were tabulated in Excel® software version 2016. Statistical comparisons were not

performed.

Results: The patients presented a predominance of the age range between 51 to 60 years old

(47.6%). We observed a slight predominance of men (52.3%) and a predominance of Caucasians

(66.7%). Most of the patients (90.5%) reported cases of cancer among relatives (31.6% in siblings and

26.3% among parents), the most reported types of cancer were prostate cancer (23.8%), intestine

(14.3%) and lung (14.3%). The highest percentage of patients (42.9%) reported a history of mild

physical exercise (dancing and/or walking, 1-3 times/week), while 38.1% reported a sedentary

lifestyle. The history of regular intestinal transit (7 times/week) was reported by 71.4% of the patients.

Only two patients reported exposure to pesticides. Most patients did not report diagnosis of

hypertension or diabetes (62%), nor abnormalities in response to infections (85.8%). Many patients did

not report alcohol intake (47.6%), but 33.3% of the patients reported a moderate consumption. For

tobacco, we observed a predominance of patients with no history of smoking (52.3%), while 42.9%

reported a history of intense tobacco use. For eating habits, 81% of the patients reported

consumption of alimentary fibers (4-7 times/week), and a consumption of red meat (71.4%, 4-7

times/week), and sausages (47.7%, 1-3 times/week).

Conclusion: The present study emphasizes the importance of some risk factors for CRC development,

such as the age range (over 50 years) and the history of tobacco, red meat, and sausage

consumption. Our work also demonstrates that CRC prevention campaigns addressing risky dietary

habits are necessary. In contrast, the report of regularity in intestinal transit differs from that expected

in this population, probably associated with difficulties related to recall of the history of intestinal

transit. We also observed a high report of relatives with CCR, which exceeds 10% of family cases

proposed by the scientific literature, that needs to be elucidated by increasing the sample size.

Keywords: Colorectal cancer, risk factors, epidemiology.

Grants: CAPES, PPSUS/Fundação Araucária.

60

CHIKUNGUNYA VIRUS INDUCED MECHANICAL HYPERALGESIA IS MEDIATED BY RECOMBINANT E2

PROTEIN AND RELATED TO TRPV1 CHANNELS ACTIVITY

Carina Z. Segato-Vendrameto1, Victor Fattori1, Larissa Staurengo-Ferrari1, Stephanie Badaro-

Garcia1, Tiago H. Zaninelli1, Mariana M. Bertozzi1, Telma Saraiva dos Santos1,

Felipe Pinho-Ribeiro1, Sérgio M. Borghi1, Camila Zanluca2, Rubia Casagrande1, Cláudia N.

Duarte dos Santos2, Waldiceu A. Verri, Jr1.


²Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil


Introduction and objectives: Chikungunya fever is a disease caused by Chikungunya virus (CHIKV),

an arthritogenic alphaviruses transmitted by mosquitoes of Aedes genus. The initial stage of the

disease considered acute is characterized as an abrupt clinical onset of crippling joint pains, high

fever and rash, being easily confused with dengue. Polyarthralgia is one of the most common

complain among infected patients when chronic stage is stablished and it may persist for months

even after clearance of the virus from the blood. The virus genome consists in a single-strand RNA

molecule encoding four nonstructural (nsP1-4) and three structural proteins (C, E1, E2), which have

received attention over the last few years as targets to immunization and therapy. Knowing that

TRPV1 channels are involved in nociceptor sensory neuron sensitization and activation resulting in

pain, and that CHIKV envelop proteins are capable of induce joint swelling and inflammation, we

aimed to investigate the relation between E2 recombinant protein and TRPV1 channels in

hyperalgesia induction after CHIKV infection.

Material and methods: Male C57-bl/6 and TRPV1 knockout mice received intra-articular (i.a.)

injection of inactivated Chikungunya virus (iCHIKV: 100 FFU, 10ul), recombinant E2 protein (rE2: 100

ng, 10ul) or Mock (control, 10ul) and 5 minutes later, C57-bl/6 received intrathecal treatment with

TRPV-1 antagonist AMG-9810 (100 ng). Mechanical hyperalgesia was evaluated 1 – 7 hours after

stimulus injection. In another set of experiments, the dorsal root ganglia (DRG from L4-L6 spinal cord

segments) were dissected seven hours after stimulus injection to determine cellular activation and

TRPV1 expression by immunofluorescence. DRG were also collected and processed and calcium flux

was observed using confocal microscopy. All the procedures using animals were properly approved

by The Ethics Committee for Animal Research of the Universidade Estadual de Londrina (process

number 13216.2017.97).

Results: We have observed that iCHIKV and rE2-induced hyperalgesia was inhibited in the absence

or blockage of TRPV1 channels. We also have demonstrated by immunofluorescence that iCHIKV

and rE2-activated DRG cells are expressing TRPV-1 channels, leading us to relate this channels activity

to induced pain after virus infection. DRG neurons from iCHIKV and rE2-stimulated mice also

presented a higher baseline level of calcium influx than DRG neurons from Mock mice, an indicative

of neuron activation after stimulation.

Conclusion: All together, these data suggest that DRG neurons activation by Chikungunya virus is

mediated by E2 protein and related to TRPV1 channel activity, but the specific mechanisms of this

interaction are still being investigated.

Keywords: Chikungunya virus, hyperalgesia, TRPV1 channels, Dorsal root ganglia.

61

CREATINE SUPPLEMENTATION EXACERBATES ETHANOL-INDUCED HEPATIC DAMAGE IN MICE

Marinello, P. C.1,2; Cella, P. S.2; Testa, M.T.J.1; Guirro, P. B. 1; Brito, W. A. S.2; Borges F. H.2; Cecchini R2;

Cecchini A. L.2; Duarte, J. A.3; Deminice, R1.

¹Universidade Estadual de Londrina, Department of Physical Education, Londrina, PR, Brazil

²Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

³University of Porto, CIAFEL, Faculty of Sport, Porto, Portugal.

[email protected]

Introduction and objectives: The ethanol metabolism in liver is related with its toxicity through the

generation of oxidative stress-related lesions, which induces fat accumulation and inflammation.

Creatine supplementation has demonstrated a protective effect in models of nonalcoholic fatty liver

disease (NAFLD); however, its effects on ALD remain poorly known. The objective of this study was to

investigate the effects of creatine supplementation on early stages of ethanol-induced hepatic

damage

Material and methods: Male swiss mice (SWR/J; ~25g body weight) obtained from the animal house

of the Biological Sciences Centre of the State University of Londrina (Brazil) were randomly divided

into three groups (n=12/group): Control (C), ethanol (E) and ethanol supplemented with creatine

(EC). Animals from E and EC groups were fed with Lieber-De Carli diet (Rhoster, São Paulo, Brazil),

diluted in mineral water, containing 5% (vol/vol) of 95% ethanol. Control mice (C) received an

isocaloric diet without ethanol. Animals from different groups were pair-fed in relation to E group.

After 14 and 28 days, six animals from each group were euthanized and the liver was removed,

weighed and prepared for hematoxilin/eosine staining, biochemical (oxidative stress parameters)

and molecular analysis (real-time PCR). The experimental protocol was approved by the Institutional

Animal Care and Use Committee of the State University of Londrina (CEUA/UEL: 21179.2016.78). The

normality of the data and the presence of outliers were investigated by Shapiro-Wilk and Rout test,

respectively. Data shown as mean ± standard deviation or as median and interquartile range when

nonparametric. One-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test were used

for parametric data analysis. The nonparametric data were tested using Kruskal-Wallis, followed by

Dunn’s post-hoc

Results: Ethanol intake induced mild cell degeneration, liver damage, oxidative lesions and

inflammation. Surprisingly, ethanol intake combined with creatine exacerbated cell degeneration

and fat accumulation, hepatic expression of genes related with ethanol metabolism (cytochrome

P450, family 2, subfamily e, polypeptide 1- Cyp2e1; aldehyde dehydrogenase 2- ALDH2), oxidative

stress (superoxide dismutase 1-Sod1; NADPH oxidase 4 - Nox4), and inflammation (tumor necrosis

factor alpha- Tnfα; Interleukin 6 - IL-6; interleukin 1 beta - IL-1β). The association between ethanol and

creatine supplementation also promoted oxidative stress (malondialdehyde, advanced oxidized

protein products and reduced glutathione levels) and elevated plasma alanine aminotransferase

levels (p<0.05), which indicates hepatic damage.

Conclusion: The analysis of the results indicates that creatine supplementation associated with

ethanol is able to interfere in the alcohol metabolism and oxidative stress and to exacerbate ethanol-

induced hepatic damage. These new findings are opposite to the observed in several studies where

protective effects of creatine in a wide variety of injury models, including NAFLD, were described.

Keywords: Alcoholic liver disease, hepatic injury, creatine

Grants: Capes-PVE (Process nº 88881.068035/2014-01)

62

DENGUE VIRUS INDUCES MECHANICAL HYPERALGESIA BY INCREASING CYTOKINE PRODUCTION

Carina Z. Segato-Vendrameto1, Victor Fattori1, Amanda Z. Zucoloto1, Tiago H. Zaninelli1, Stephanie

Badaro-Garcia1, Larissa Staurengo-Ferrari1, Ana C. Rossaneis1, Allan H. D. Cataneo2, Rubia

Casagrande1, Juliano Bordignon2, Waldiceu A. Verri, Jr1 1 University, Department, City, Country

1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 2

Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil

[email protected]

Introduction and objectives: Dengue is the most rapidly spreading mosquito-borne viral disease in

the world affecting more than 50 million people annually. The virus transmission among humans is

mediated by the bites of Aedes (Stegomyia) mosquitoes, specially Ae. aegypti. Dengue fever (DF),

one of the clinical manifestations of Dengue infection, involves an abrupt febrile illness which can

last around 7 days, and is characterized by acute fever accompanied by two or more of the

following manifestations: nausea, vomiting, rash, headache, petechiae, positive tourniquet test,

leukopenia, retroorbital pain, myalgia, and arthralgia of such great intensity. Since there is no

effective antiviral agents specific to treat dengue fever or the severe forms of dengue infection, the

current treatment remains largely supportive. It is well known that intense pain, like myalgia and

polyarthralgia, is one of the main complains among Dengue infected patients and that there is still

few information about the mechanisms involved in hipernociception process. So, the aim of the

current study was to investigate the participation of inflammatory cytokines in the mechanisms of

pain induction in Dengue infection.

Material and methods: Male swiss mice were injected intraperitoneally with Dengue virus (DENV 20,

200 and 2000 FFU/200µl saline) or Mock (non-infected control, 200 µL, i.p.) and the mechanical and

thermal thresholds were evaluated 1, 3, 5 and 7 h after the stimulus injection and daily during seven

days. Samples were collected in different time intervals to determine cytokine (TNFa, IL-1b, and IL-10)

levels. In an attempt to confirm the importance of the cytokines in the nociception induced by DENV,

72 hours after the stimulus mice received intrathecal treatment with one of the following inhibitors at

indicated doses: Etanercept (10 ng, i.t.), IL-1ra (10ng, i.t.) or recombinant IL-10 (3 ng, i.t.) and the

mechanical threshold was measured during 1, 3, 5 and 7 hours after. All the procedures using animals

were properly approved by The Ethics Committee for Animal Research of the Universidade Estadual

de Londrina (process number 13325.2017.97).

Results: Infection with Dengue virus increased the intensity of mechanical hyperalgesia starting at 3rd

hour after stimulus injection. The highest dose (2000FFU) maintained the increased hyperalgesia until

day 7 post infection but there was no significant increase of thermal hyperalgesia by anyone of the

tested doses. The pro-inflammatory cytokines TNF-α, IL-1β levels were significantly increased after 1h

of DENV infection and we also observed increased levels of the anti-inflammatory cytokine IL-10 from

3 to 48 hours after infection. In another set of experiments, we demonstrated that Dengue virus

induced mechanical hyperalgesia was significantly reduced after treatment with the cytokines

inhibitors Etanercept, IL-1ra and recombinant IL-10.

Conclusion: Our results demonstrated that the pro-inflammatory cytokines TNF-α and IL-1β and the

ant-inflammatory cytokine IL-10 have an important role in the DENV-induced hyperalgesia. However,

the involvement of different cell types and the specific mechanisms of this interaction are still under

investigation.

Keywords: Dengue virus, Hyperalgesia, Pro-inflammatory cytokine

63

EFFECTS OF PHYSICAL TRAINING ASSOCIATED OR NOT TO L-ARGININE SUPPLEMENTATION ON

CARDIOVASCULAR AND AUTONOMIC PARAMETERS IN OBESE RATS

Gabriela de Souza Reginator1; Beatriz Geovana Leite Vacario 2; Lorena de Jager1; Fernanda Novi

Cortegoso Lopes 1; Marli Cardoso Martins-Pinge1

1 Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina,

Londrina - PR, Brazil

2 Biomedicine Graduation course, Philadelphia University Center (UniFil), Londrina – PR, Brazil


Introduction and objectives: Obesity is the major risk factor for the development of cardiovascular

diseases, but not all the mechanisms that act in this process have been clarified. Nitric oxide (NO)

plays an important role in cardiovascular and autonomic function, in processes that regulate energy

balance such as hunger and satiety and also in the inflammatory process as both antioxidant and

pro-oxidant. L-arginine is considered a fundamental substrate for the synthesis of nitric oxide (NO)

and its uptake and availability are limiting factors for the production of NO. In addition, the regular

practice of aerobic exercise is capable of promoting various beneficial effects to the body,

especially the cardiovascular system, modulating NO bioavailability among other effects. The aim of

the present study was to evaluate the interference of physical training by treadmill, associated or not

with L-arginine supplementation on autonomic and cardiovascular modulation in obese rats.

Material and methods: All the procedures were approved by the Committee of Ethics and Research

of Animals of the State University of Londrina with the protocol number: 21195.2017.56. Obesity was

induced by subcutaneous administration of 4 mg / g monosodium glutamate (MSG) from the 1st to

5th day of life and control rats (CTR) received equimolar saline solution. The animals were divided into

8 experimental groups: sedentary control treated with water; sedentary control treated with L-

arginine; trained control treated with water; trained control treated with L-arginine; obese sedentary

treated with water; obese sedentary treated with L-arginine; obese trained treated with water; and

obese trained treated with L-arginine. Treadmill physical training and L-arginine supplementation

occurred concomitantly for eight weeks. After this period, the animals were submitted to femoral

artery catheterization surgery to record cardiovascular parameters and to calculate blood pressure

variability, heart rate variability and baroreflex sensitivity.

Results: The results of this study show that Lee's index and the perigonadal and retroperitoneal fats

of obese animals were increased when compared to their controls and the physical training

associated with L-arginine supplementation was able to reduce perigonadal fat. L-arginine

supplementation and physical training was also able to reduce the increased heart rate of obese

animals. Baroreflex sensitivity was decreased in sedentary obese animals and physical training was

able to recover this sensitivity.

Conclusion: Therefore, we concluded that supplementation with L-arginine associated or not with

physical training brings benefits to the health of obese individuals.

Keywords: MSG obesity, physical training, l-arginine supplementation, cardiovascular parameters

64

EFFECTS OF TYPE 1 DIABETES MELLITUS AND AGARICUS BLAZEI MURRILL EXTRACT ON THE MYENTERIC

NEURONS OF THE PROXIMAL AND DISTAL COLON OF RATS

Indyanara Inacio Barreto1; Luan Vitor Alves de Lima1; Ana Paula da Silva Barbosa1; Carlos Vinícius

Dalto da Rosa1; Ana Paula de Santi Rampazzo2; Maria Raquel Marçal Natali2; João Paulo Ferreira

Schoffen1

1Universidade Estadual do Norte do Paraná (UENP), Center of Biological Sciences, Bandeirantes,

PR, Brazil 2Universidade Estadual de Maringá (UEM), Department of Morphological Sciences, Maringá, PR,

Brazil


Introduction and aims: Absolute insulin deficiency resulting from type 1 diabetes mellitus is associated

with abnormalities in metabolism. Disorders in the gastrointestinal tract, such as dysphagia and

diarrhea are common in diabetes, and are related to changes in intestinal morphology and in the

enteric nervous system. Antioxidant rich compounds have been used as prevention or treatment of

diabetes. Agaricus blazei Murrill (ABM) has great pharmacological interest due to anti-inflammatory,

hypoglycemic and antioxidant properties. The objective of this study was to evaluate and compare

the effects of ABM on the intrinsic innervation of the proximal and distal colon in an experimental

diabetes model.

Materials and methods: After approval by the Committee of Ethics in Animal Experimentation of the

Universidade Estadual de Maringá (Protocol 042/2014), 20 Wistar rats were distributed in 4 groups

(n=5): normoglycemic (N), diabetic (D) and with supplementation (NB and DB) by gavage of the

hydroalcoholic extract of Agaricus blazei (200 mg/kg) during 120 days. Type 1 diabetes was induced

by streptozotocin (45mg/Kg) intravenously. Samples of the proximal and distal colon were fixed in

Giemsa fixative and subsequently dissected to obtain the wholemount of the muscular tunica. The

whole-mounts were stained with the Giemsa technique and mounted on a slide. Under 40x objective,

images of 50 myenteric ganglia per animal were captured, and had the neuronal cell bodies

quantified. Within the same ganglia, the morphometry of the cell body, nucleus and cytoplasm area

of 100 neurons per animal was performed. The data were statistically analyzed by one-way ANOVA

and Tukey test.

Results: There was maintenance of the myenteric neuronal population of the proximal colon, in the

number, cell body and cytoplasm sizes, in the diabetic and supplemented groups. The size of the

nucleus increased in the groups supplemented with ABM in relation to the control, without influence

of diabetes. In the distal colon, on the other hand, it was observed that diabetes promoted a

significant reduction in the neuron number, besides the increase of the size of the cell body and

cytoplasm. Supplementation with ABM did not prevent neuronal loss or morphometric alteration of

the myenteric neurons of the distal colon.

Conclusions: The myenteric innervation of the proximal and distal colon responds differently to type

1 diabetes mellitus, with the distal colon being more vulnerable to diabetic neuropathy. The

hydroalcoholic extract of ABM (200 mg/kg) had no effect on the morphoquantitative parameters of

myenteric neurons.

Keywords: Hyperglycemia. Cogumelo do sol. Enteric neurons. Large intestine.

Grants: Fundação Araucária - Governo do Estado do Paraná/SETI.

65

EVALUATION OF METFORMIN EFFECTS IN OXIDATIVE STRESS, TGF-Β1 AND GENES RELATED TO CELL

PROLIFERATION, INVASION AND METASTASIS IN HUMAN BREAST CANCER CELLS.

Poliana Camila Marinello1, Lucas Rennan de Oliveira Andrade 1, Carolina Panis2, Thamara Nishida

Xavier da Silva1, Renata Binato3, Eliana Abdelhay3, Juliana Alves Rodrigues4, André Luiz

Mencalha4, Natália Medeiros Dias Lopes1, Rodrigo Cabral Luiz1, Rubens Cecchini1, Alessandra

Lourenço Cecchini1 1 State University of Londrina (UEL), Department of Pathological Sciences, Londrina, PR, Brazil.

[email protected]

Introduction and objectives: Metformin is an anti-diabetic medication and a putative antineoplastic

drug. Recent studies have shown that metformin can induce oxidative stress (OS) and transforming

growth factor β1 (TGF-β1) expression in human breast cancer cells. OS and TGF- β1 induction are two

important events in breast cancer and they can both have tumor suppressor or promotor effects.

The purpose of this study was to evaluate OS and TGF- β1related pathways in MCF-7 and MDA-MB-

231 human breast cancer cells treated with a noncytotoxic dosage of metformin.

Material and methods: Two cell lines were used: a human breast cancer cell line positive for estrogen

and progesterone receptors and negative for HER-2 receptor, MCF-7, and a triple negative cell line,

MDA-MB-231. Cells were exposed to 6 µM of metformin for seven consecutive passages. Samples

were collected to evaluation of oxidative stress parameters, immunocytochemistry and gene

expression by real-time quantitative array PCR (qRT-PCR). Oxidative stress was assessed by the

evaluation of lipoperoxidation, determined by chemiluminescence stimulated by tert-butyl-

hydroperoxide (CL), total thiols levels and nitric oxid (NO) determination. P53, NF-kB and Nrf2 nuclear

labeling were measured by immunocytochemistry. The normality of the data and the presence of

outliers were investigated by Shapiro-Wilk and Rout test, respectively. Data were expressed as mean

± standard deviation of the mean and analyzed using the paired Student t test (comparisons

between P7 and P0 cells). P < 0.05 was considered statistically significant

Results: Biochemical parameters indicated that metformin induced OS in both breast cancer cells

(increased lipoperoxidation and reduction in total thiols). Furthermore, metformin also decreased NO

levels. Metformin altered signaling pathways related to oxidative stress and cell survival, since

immunocytochemistry showed increased levels of p53-labeling and decreased p65 Nf-kB-labeling in

both cell lines. Nrf2 nuclear labeling did not change. The RT-qPCR array for the genes related with

TGF-β1 pathway demonstrated that metformin upregulated the expression of genes involved in OS

generation and apoptosis, and downregulated genes associated with metastasis and epithelial

mesenchymal transition (EMT) in MCF-7 cells. In MDA-MB-231 cells, which represents more aggressive

cases of breast cancer, metformin downregulated several genes involved with cell invasion, viability

and proliferation.

Conclusion: The analysis of our results indicates that, whereas cell line specific, consecutive

treatments with a non-cytotoxic dosage of metformin can change the expression of numerous

genes involved in the aggressiveness of breast cancer, promoting a less aggressive profile in these

cells.

66

EVALUATION OF MYENTERIC AND SUBMUCOUS NEURONS OF THE DISTAL COLON OF RATS SUBMITTED

TO SUCROSE-RICH DIET AND TREATED WITH PTEROSTILBENE

Wesley Ladeira Caputo1; Joice Moraes Menezes1; Carlos Vinícius Dalto da Rosa1; Cristiano Massao

Tashima1; Fábio Rodrigues Ferreira Seiva1; João Paulo Ferreira Schoffen1


PR, Brazil


Introduction and objectives: Currently the consumption of beverages containing high rates of sugar

has been used in large scale by the population. Excessive consumption of sucrose drinks, coupled

with sedentary lifestyle, may lead to obesity and gastrointestinal tract disorders, compromising

intestinal intrinsic innervation, which is responsible for controlling the motility, secretion and blood flow

of the digestive tract. In order to alleviate the deleterious effects of sugar, natural compounds with

medical properties arouse interest in the field of research. Pterostilbene, a polyphenol found in

grapes, blackberries and plums, has been highlighted by promoting lower glycemia, antioxidant

effects, inhibit carcinogenesis and increase the survival of brain neurons, among other effects. The

objective of this work was to evaluate the effects of the sucrose-rich diet and the treatment with

pterostilbene on the enteric neurons of the distal colon of rats.

Material and methods: After approval by Committee of Ethics in Animal Experimentation of the

Universidade Estadual do Norte do Paraná (Declaration nº 05/2017), Wistar rats were distributed into

groups: Control (C), Control + Pterostilbene (CP), Sucrose (S), Sucrose + Pterostilbene (SP). During 195

days, groups C and CP received filtered water and commercial feed ad libitum, whereas groups S

and SP received commercial ration and water plus sucrose 50% ad libitum. Pterostilbene, at doses of

40mg/kg was administered daily via gavage for 45 consecutive days, from the 151st day of the

experiment. After euthanasia of the animals, samples of the distal colon were dissected to obtain

whole-mounts preparations of the muscularis externa and the submucosa tunics, and stained by the

Giemsa method for quantitative and neuronal morphometric study. The neurons were counted in 60

random microscopic fields and measured in the area of the cell bodies, nuclei and cytoplasm of 100

neurons per animal. The data were statistically analyzed by one-way ANOVA and Tukey test.

Results: No statistically significant changes were observed in the quantitative and morphometric

parameters of the myenteric plexus neurons of the distal colon of the rats, when submitted to a

sucrose-rich diet and/or treated with pterostilbene. However, a tendency to increase neuronal

numbers was observed in response to pterostilbene. For submucosal plexus neurons, no significant

morphoquantitative change was observed between the groups.

Conclusion: Treatment with sucrose over a period of 195 days is not able to induce

morphoquantitative changes in the enteric neurons of the distal colon. Neurogenic properties of

pterostilbene may be associated with a tendency to increase neuronal number.

Keywords: Diet. Antioxidant. Enteric neurons. Large intestine.

Grants: Fundação Araucária - Governo do Estado do Paraná/SETI.

67

EVALUATION OF SYSTEMIC OXIDATIVE STRESS PROFILE IN PATIENTS WITH COLORECTAL CANCER

Nascimento. T. H. D.¹; Silva. I. C; Sanches. L. J¹; Blegniski. F. P.¹; Lopes. N. M¹; Brito. W. A. S¹; Marinello,

P. C. 1; Liberatti, M²; Cecchini, A. L1; Luiz, R. C.¹.

1Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Ciencias

Patológicas, Laboratório de Patologia Molecular, Londrina, PR, Brasil.

2Universidade Estadual de Londrina, Hospital Universitário Regional do Norte do Paraná,

Departamento de Medicina Clínica, Londrina, PR, Brasil.

Introduction and objetives: Colorectal cancer (CRC) is one of the most common type of cancer,

and the second leading cause of cancer death worldwide. In Brazil, the increase of CRC mortality

rate is associated to difficulties in accessing health services for early diagnosis and adequate

treatment. CRC is characterized by an intense inflammatory process in the tumor tissue. The

inflammation induces the tumor progression, but little is known about its effects at a systemic level.

Oxidative stress (OE) occurs along the inflammatory process, it is characterized by an imbalance

between the generation of reactive oxygen and nitrogen species and the antioxidant defenses.

There are few scientific studies that address the relationship between OE and CRC, so the present

work aimed to characterize the profile of systemic OE in CRC patients.

Material and Methods: for this study, up to now, 20 patients with CRC were selected at the North

Parana Regional University Hospital (HURNP). This research was previously approved by the Ethics

Committee CAAE no. 56874216.0.0000.5231. Blood samples where obtained from CRC patients and

control group (5ml serum, 5ml heparin). For CRC patients, blood collection occurred before

chemotherapy. We evaluated the erythrocyte levels of reduced (GSH) and oxidized glutathione

(GSSG), oxidative stress index (GSSG/GSH) and membrane lipid peroxidation, and plasma levels of

malondialdehyde (MDA). 2 way ANOVA was used for the statistic analysis of QL, and Mann-Whitney

test was used for the other oxidative stress parameters, p values ≤0.05 were considered significant.

Results: CRC patients presented significantly lower levels of erythrocyte GSSG (531.1 ± 72.32, p =

0.0136) and membrane lipoperoxidation (1,879 ± 37.87, p = 0.0515) in comparison to the control

group (1,231.1 ± 310.4 and 4,050 ± 73.08, respectively). For the other markers, no significant difference

was observed.

Conclusion: For the biomarkers analyzed to date, we observed that CRC patients tend to have

lower levels systemic EO. With the increase of the sample group and the use of more EO

parameters, we will elucidate this trend and assess the correlation of EO levels with the prognosis of

the disease.

Keywords: COLORECTAL CANCER, OXIDATIVE STRESS, BIOMARKERS.

Grants: CAPES.

68

EXPRESSION OF RECEPTOR-TYPE PROTEIN-TYROSINE PHOSPHATASE ZETA/PHOSPHACAN IN COLONIC

ENTERIC NEURONS IS CHANGED BY DEXTRAN SULFATE SODIUM INDUCED- COLITIS

MENDES1, Joana D’arc de Lima; MACHADO1, Camila Cristina Alves ; WATANABE1, Paulo da Silva;

MIQUELOTO2,Carlos Alberto; AKTAR3, Rubina; PEIRIS3, Madusha; Blackshaw3, L Ashley; AZIZ3, Qasim;

ARAÚJO1, Eduardo José de Almeida.

1State University of Londrina, Department of Histology, Londrina, PR, Brazil; 2State University of Londrina, Department of General Biology, Londrina, Paraná, Brazil;

3Queen Mary University of London, London, United Kingdom


Introduction and objectives: The Enteric Nervous System (ENS) is the intrinsic nervous system of the gastrointestinal

tract, which control motility, secretion, digestion and absorption. Recently, evidences are showing that a

specialized extracellular matrix (ECM) exists in the ENS likewise the perineuronal net found around some neurons

in the central nervous system. RPTPζ/phosphacan is among the ECM molecules that plays important role on the

nervous tissue. Importantly, it is well known that the inflammatory process might alter the distribution of ECM

molecules and so change the morphophysiology of neurons and glial cells. In this sense, this study aimed to

assess the association of PTPRζ/phosphacan with enteric neurons in DSS-induced ulcerative colitis.

Material and methods: All procedures were approved by the Ethical Committee for Use of Animals at UEL

(032/2015), Brazil. Male C57bl/6 mice had free access to 3% of dextran sulfate sodium DSS (molecular weight 36-

45 kDa) diluted in tap water for 7 days (colitis group, n=5). Control mice (n=5) had access to drinking tap water

for 7 days. Scores for weight loss, stool consistency and blood presence in stool were used the calculate the

Disease Activity Index (DAI). After euthanasia, the colon was dissected in order to obtain two types of whole-

mount preparations: one consisting the submucosa layer (including the submucosal plexus) and the other

consisting the longitudinal muscular layer (including the myenteric plexus). The submucosal whole-mount

preparations were incubated with anti- RPTPζ/phosphacan and anti-PGP9.5 (general marker for

neurons) or anti- RPTPζ/phosphacan and anti-calretinin (marker for non-cholinergic secretomotor neurons) or

anti- RPTPζ/phosphacan and anti-ChAT (marker for cholinergic secretomotor neurons). The longitudinal

muscular whole-mount preparations were incubated with anti- RPTPζ/phosphacan and anti-PGP9.5, anti-

RPTPζ/phosphacan and anti-nNOS (marker for inhibitory motor neurons), anti- RPTPζ/phosphacan and anti-ChAT

(marker for excitatory motor neurons and interneurons). The number of neurons was counted in 50 (submucosal)

or 100 ganglia (myenteric) per animal and a total of 50 (submucosal) or 100 (myenteric) cell body area were

measured. GraphPad Prism6 software (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical

analysis and the groups were compared considering the significance level was set to 5%.

Results: The DAI of all mice exposed to DSS increased progressively during the experiment indicating

development of colitis (p<0.05). The consumption of water and feed was unchanged, but the colitis mice lost

weight during the experiment (p<0.05). The number and the weight of fecal pellets reduced in colitis mice

(p<0.05). Colitis caused reduction in the number and cell body area of submucosal neurons which produce

RPTPζ/phosphacan (p<0.05).The non-cholinergic secretomotor/valodilator neurons (Calretinin+) had no

change in number, but they were significantly hypertrophied (p<0.05). The cholinergic submucosal neurons were

atrophied (p<0.05). In the myenteric plexus, general (PGP9.5+) and cholinergic (ChAT+) neurons were strongly

atrophied in DSS-induced colitis mice (p<0.05). The number of inhibitor (nNOS+) and excitatory colonic motor

neurons and interneurons (ChAT+) increased in colitis mice in relation to the control (p<0.05).

Conclusion: The DSS-induced colitis is able to change the number and morphology of colonic enteric neurons

that express of RPTPζ/phosphacan.

Keywords: Receptor-Like Protein Tyrosine Phosphatases - Class 5, RPTPζ/phosphacan, Gastrointestinal Tract,

Extracellular Matrix

Grants: CAPES/PVE (process number 88881.068190/2014-01)

69

FOXP3 INTRON -1 POLYMORPHISMS ARE INDEPENDENT PREDICTORS OF HUMAN PAPILLOMAVIRUS

INFECTION AND HIGHGRADE SQUAMOUS INTRAEPITHELIAL LESIONS: A CLINIC-BASED CASE-CONTROL

Cesar-dos-Santos, F.1, Ferreira, R. S.1; Okuyama, N. C. M. 1; Trugilo, K. P.1; Sena, M. M.1; Pereira, E. R.1;

Pereira, A. P. L.1; Watanabe. M. A. E.1; Oliveira, K. B.1 1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: FOXP3 is a bona fide marker of the T regulatory (Treg) cell subset and

drives its function and homeostasis. Its expression maintains the host immunosuppressive state that

favors persistence of human papillomavirus (HPV) infection and squamous intraepithelial lesions (SIL)

appearance. We evaluated the effects of the rs3761548 and rs2232365 intronic polymorphisms on

HPV infection and SIL diagnosis in HPV-infected and uninfected women.

Material and methods: HPV DNA-based detection in cervical specimens was performed by

polymerase chain reaction (PCR). FOXP3 polymorphisms were genotyped by PCRrestriction fragment

length polymorphism and haplotype structures were inferred for 208 HPV-infected and 218 HPV-

uninfected women diagnosed or not with low- or high-grade intraepithelial lesions. Case-control

analyses were carried out by logistic regression adjusted for several socio-demographic, sexual

lifestyle and clinical data. This study was approved by the Institutional Ethics Committee Involving

Humans Beings of the State University of Londrina, Londrina-PR, Brazil (CEP/UEL 133/2012; CAAE

05505912.0.0000.5231).

Results: The homozygous genotype of the rs3761548 polymorphism (A/A) (related to decreased

FOXP3 expression) protected against HPV infection in women (ORAj: 0.60; 95% CI = 0.36 – 0.99; p =

0.049) this genotype is also an independent predictor of protection against HSIL development (ORAj:

0.28; 95% CI = 0.11–0.68; p = 0.006). Additionally, the homozygous genotype (G/G) of the rs2232365

polymorphism (related to increased FOXP3 expression) was an independent risk factor for HPV

infection (ORAj: 2.10; 95% CI = 1.06– 4.15; p = 0.033). Haplotype analysis revealed no significant

associations in the present study.

Conclusion: Our results reveal the significant and independent associations between FOXP3 genetic

variants and susceptibility to HPV infection and SIL diagnosis and their role as biomarkers of HPV

infection and cervical lesions management.

Keywords: Cervical lesion, Polymerase chain reaction, HPV

Grants: This study was supported by Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

Fundação Araucária, Programa Pesquisa para o SUS (PPSUS) and by Londrina State University

Graduate Coordination (PROPPG-UEL).

70

FRAGILE X SYNDROME IN MALE SIBLINGS: A CASE REPORT IN THE GENETIC COUNSELING SERVICE OF

LONDRINA

Mariana Biscaia Falanga¹; Viviane Mestre²; Maria Eliane Longhi Barroso³; Wagner José Martins

Paiva³

¹ Universidade Estadual de Londrina, Medical Student, Londrina, PR, Brazil

² Universidade Estadual de Londrina, Department of Health Sciences, Londrina, PR, Brazil ³

Universidade Estadual de Londrina, Department of General Biology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Fragile X Syndrome is a X-linked inheritance caused by quantitative

expansion of CGG trinucleotides above 200 repetitions in promoter region of Fragile mental

retardation I (FMR-1) gene. The mutation generates hypermethylation of CpG island of the gene and

loss or heavy reduction of Fragile mental retardation protein (FMRP), which regulates synaptic

plasticity and maturation of learning and memory regulatory neurons. The prevalence is 1:5000-7000

men and 1:4000-6000 women. It is characterized by intellectual disability, psychomotor retardation,

behavioral alterations, hyperactivity, facial dysmorphisms - as proeminent ears -, language disorders,

propensity to obesity, orthopedic affections and macroorchidia. The phenotypic variability and

absence of pathognomonic signs makes it advisable to investigate the syndrome in all boys with

idiopathic intelectual disability. Genetic counseling results in decrease in the incidence of the

syndrome. The objective of this study was to present clinical and genetic aspects of Fragile X

Syndrome in male siblings assisted by the Genetic Counseling Service of State University of Londrina

(SAG-UEL) and highlight the importance of genetic counseling in this case.

Material and methods: Observational and cross-sectional study of two users’ files who were referred

to SAG-UEL in 2017 due to suspicion of Fragile X Syndrome. Authorizations were obtained through the

Informed Consent Form.

Results: Male patients, 15 and 21 years old, siblings, were referred by neuropediatrician to SAG-UEL

due to learning difficulties and behavioral alterations. The younger individual has difficulty of learning

and social interaction and started to speak and walk only at age three. The older individual presents

anxiety disorder, hyperactivity, learning disabilities and obesity. Both have proeminent ears. Four

maternal and paternal relatives have intellectual disability and/or unspecified psychiatric disorders.

The kariotyping in culture of lymphocytes by GTG banding was performed in the Laboratory of

Human Cytogenetics of UEL and did not show alterations. Molecular screening was performed by

amplification of FMR-1 gene by PCR Multiplex (Asuragen FMR1 PCR Kit, Inc.) of purified genomic DNA

and CGG repetitions was counted by electrophoresis platform (Applied Biosystems Genetic

Analyzer). The patients presented more than 200 trinucleotide repetitions. The devolution to the

parents was performed by the geneticist, with psychological referral and follow-up of the case.

Conclusion: The clinical features of the younger individual are intellectual disability, social behavioral

alterations and proeminent ears. The older individual has learning difficulties, hyperactivity, obesity,

anxiety disorder and proeminent ears. Molecular screening in both siblings showed more than 200

repetitions of the CGG trinucleotide of the FMR-1 gene, confirming the suspicion of Fragile X

Syndrome. The genetic counseling performed by SAGUEL enabled therapeutic intervention,

psychological referral, clarification to parents about the syndrome and possible future family

planning.

Keywords: Genetic Counseling Service. Intellectual disability. Fragile X Syndrome.

Grants: SAG-UEL.

71

FREE RADICAL SCAVENGER POTENTIAL OF Hylocereus undatus, Hylocereus costaricensis AND

Selenicereus megalanthus

Lens, H.H.M.1; Lone, A.B. 2; Blegniski, F.P. 3; Borges, F. H.4, Banin-Hirata, B.K.5 Panis, C. 6; Takahashi,

L.S.A. 7; Faria, R.T. 7; Cecchini, A.L. 1; Victorino, V.J.8; Cecchini, R. 4

1 Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil

2 Empresa de Pesquisa e Extensão Rural de Santa Catarina (EPAGRI) – Estação Experimental de

Itajaí, Itajaí, SC, Brazil

3 Universidade Estadual de Londrina, Laboratory of pathophysiology of muscular adaptations,


4 Universidade Estadual de Londrina, Laboratory of General Pathology, Londrina, PR, Brazil

5 Universidade Estadual de Londrina, Laboratory of studies and applications of DNA polymorphisms,


6 Universidade do Oeste do Paraná, Laboratory of Inflammatory Mediators, Francisco Beltrão, PR,

Brazil

7 Universidade Estadual de Londrina, Departament of Agronomy, Londrina, PR, Brazil

8 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro- Campus Engenheiro

Paulo de Frontin, Engenheiro Paulo de Frontin, RJ, Brazil

[email protected]

Introduction and objectives: Compounds ingested in the diet may exert antioxidant protection against oxidative

stress. This defense is responsible for controlling reactive species and their effects. Antioxidants can be produced

by the body itself or acquired through diet. Considering the high potential of pitaya fruits for industrial use and

its nutritional value, the goal of this study was to evaluate the antioxidant capacity of the bark, pulp and seed

of the three pitaya species: Hylocereus undatus, Hylocereus costaricensis and Selenicereus megalanthus

through the verification of their influence in a free radical analyzing their behavior and effect in one reactive

specie.

Material and methods: The fruits of H. undatus, H. costaricensis and S. megalanthus (n = 7 of each species), were

processed to obtain a sample of the peels, pulp and seed, separately. We determined antioxidant capacity for

seeds, pulp and peels, assessed whether they could scavenger DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical.

The DPPH is based on its capture by antioxidants, producing a decrease in absorbance at 515 nm. For our

positive control, we used butylated hydroxytoluene (BHT), a known antioxidant, diluted in DPPH solution and

incubated at room temperature, and compared the antioxidant capacity in scavenging DPPH radical for the

three species, using pitaya’s samples with DPPH solution at room temperature. Results were expressed as

percentage of DPPH inhibition. Differences among groups were assessed by One Way analysis of variance

(ANOVA) with Turkey as a post-hoc. A p< 0,005 was considered statistically significant.

Results: Our results show that, seeds from H. undatus, H. costaricensis and S. megalanthus significantly decreased

DDPH radical formation (p<0.001). Moreover, H. undatus and H. costaricensis seeds were more effective in

decreasing DPPH radical formation as compared to S. megalanthus seeds (p<0.05). Pulps from pitaya were also

able to decreased DPPH radical generation (p<0.05) but in small capacity than seeds. H. costaricensis pulp

decreased DPPH radical greater than H. undatus and S. megalanthus pulps (p<0.01); on the other side, peels

from H. undatus and H. costaricensis did not decrease DPPH radical formation, while, peel from S. megalanthus

was able in reducing DPPH radical (p<0.01). We next compared the antioxidant capacity in scavenging DPPH

radical for the sample of three species. Again, the greater antioxidant capacity in scavenging DPPH radical was

ascribed for seeds (p<0.001).

Conclusion: This work showed that seeds have greater antioxidant activity as compared to pulp and peel. And

between species, seeds from H. undatus, and H. costaricensis, showed greater capacity antioxidant than S.

megalanthus, which showed better results for the antioxidant capacity of the peel.

Keywords: Antioxidant, pitaya, free radical.

Grants: CAPES, CNPq, Fundação Araucária.

72

GENERATION OF OXIDATIVE STRESS AND DEATH PATHWAY INDUCED BY METFORMIN IN MCF-7 HUMAN

BREAST CANCER CELLS.

Bianchi, J. K.1; Lopes, N. M. D.1*; Marinello, P. C.1; Sanches, L. J. 1; Brito, W. A. S. 1; Martins, M. I. L.2;

Pinge-Filho, P. 2; Luiz, R. C. 1; Cecchini, R. 1; Cecchini, A. L. 1. [email protected]

¹Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil.

²Universidade Estadual de Londrina, Laboratory of Experimental Immunopathology, Londrina, PR,

Brazil.

³Universidade Estadual de Londrina, Laboratory of Physiopathology and Free Radicals, Londrina,

PR, Brazil.

Introduction and objective: Metformin (MET), a drug used to treat type 2 diabetes, has been studied

as an adjuvant in the treatment of breast, from the generation of oxidative stress (OE). However, no

studies have been conducted to characterize through which cell death pathways MET acts. Thus,

the present study analyzed the action of metformin after the inhibition of apoptosis, necroptosis and

ferroptosis in MCF-7 human breast cancer cells. For this evaluation, the following inhibitors were used:

a) Z-vad: a pan-caspase inhibitor, capable of blocking apoptosis; b) Necrostatin-1 (Nec-1): an

inhibitor of RIPK1 activity, capable of blocking necroptosis; c) Deferoxamine (DFO): an iron ion

chelator, capable of blocking ferroptosis.

Materials and methods: MCF-7 was treated with two concentrations of MET (1mM and 5mM)

concomitantly with the inhibitors of death, Z-vad (10 μM), Necrostatin1 (50 μM) and Deferoxamine

(100 μM). After 24 hours of treatment, cell viability, proliferation and cell death were analyzed. In

relation to the EO parameters, the total levels of thiol, reduced and oxidized glutathione (GSH and

GSSG, respectively) and malonaldehyde (MDA) were evaluated.

Results: We confirmed that MET is able to generate EO, with reduction of GSH, total thiol and increase

of MDA. When cells were treated with MET and inhibitors, antioxidants were restored, but MDA levels

increased except when cells were treated with Nec-1 and MET 5mM. Cell viability was reduced when

MCF-7 cells were treated with the two concentrations of MET and, when added separately, each

inhibitor was able to restore cell viability.

Conclusion: Metformin does not induce only a single type of cell death in MCF-7 cells, because all

the inhibitors were able to restore cell viability. It is possible that generation of EO is the mechanism

of cytotoxicity of the drug, interfering strongly in cellular metabolism and inducing at least three

different mechanisms of death (apoptosis, necroptosis and ferroptosis).

Key words: Metformin, cell death, breast cancer, oxidative stress.

73

HESPERIDIN-METHYL-CHALCONE ATTENUATES PAIN AND INFLAMMATION IN A ZYMOSAN-INDUCED

ARTHRITIS MODEL IN MICE

Fernanda Soares Rasquel de Oliveira1; Marilia Fernandes Manchope 1; Larissa Staurengo-Ferrari1;

Tiago Henrique Zaninelli1; Waldiceu Aparecido Verri Jr. 1

1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Zymosan is a polysaccharide derived from the fungus Saccharomyces

cerevisiae. The administration of this polysaccharide in joints is capable of inducing arthritis as an

inflammatory response in mice. Hesperidin-methyl-chalcone (HMC) is a flavonoid with therapeutic

efficacy demonstrated by several studies. It presents analgesic effects against some inflammatory

stimuli, offers protection against UVB radiation, inhibits the inflammatory process, and oxidative stress.

However, the HMC effects in the zymosaninduced joint inflammation are still unknown. Therefore, the

present study aims to evaluate the effects and mechanisms of action of the flavonoid HMC in a

zymosan-induced arthritis model.

Material and methods: A dose-response curve was initially developed to determine the most

effective dose to reduce pain in the arthritis model by testing the mechanical hyperalgesia by a

digital analgesimether. The effects of treatment with the best HMC dose were evaluated on knee

joint edema and cell recruitment – direct assessment by total and differential leukocyte count.

Further, knee joint samples were collected to GSH, FRAP, ABTS and NBT assays as the parameter of

oxidative stress, production of cytokines (IL-33, TNF-α and IL-6), and expression of gp91phox, peproET-

1 and HO-1 at 3-7h post-zymosan stimulus. In addition, histopathological analysis of the femoral-tibial

joint were performed to examine the joint injury. The present study was submitted to evaluation by

the Animal Research Ethical Committee (CEUA) – protocol number 5943.2017.85, in Circular Letter nº

78/2017.

Results: The results on the dose-response curve demonstrated that the 100 mg/kg HMC dose reduced

more effectively the inflammatory pain induced by zymosan. Also, the best predetermined dose

treatment was effective in reducing knee joint edema, as well as the total and differential leukocyte

recruitment. In addition, the pretreatment with HMC, significantly reduced proinflammatory cytokine

levels IL-33, TNF-α, IL-6, and peproET-1 expression at 3h post-inflammatory stimulus. Corroborating,

morphological analysis was also effective to prevent synovitis as observed by a reduction in the cell

infiltrate as determined by HE analysis.

An increase in antioxidant capacity was also observed by the GSH, FRAP, ABTS and NBT colorimetric

assays, as well as a decrease in gp91phox and, and an increase in HO-1 expression at 7h post zymosan

stimulus. Statistical differences were considered significant when p < 0.05.

Conclusion: Our study demonstrated, pre-clinically, the analgesic and anti-inflammatory potential of

hesperidin-methyl-chalcone in a zymosan-induced arthritis model.

Keywords: Pain. Flavonoid. Inflammation. Hyperalgesia. Joint.

74

HIGH-THROUGHPUT COMBINATORIAL SCREENING IDENTIFIES MITOCHONDRIAL TARGETING DRUGS

THAT SHOW SYNERGISTIC EFFECT WITH COLD PHYSICAL PLASMA AGAINST MELANOMA CELLS

Pasqual Melo, G.1; Gandhirajan, R.1; von Woedtke, T. 1; Bekeschus, S.1

1 Leibniz-Institute for Plasma Science and Technology (INP), ZIK plasmatis, Greifswald, Germany.

[email protected]

Introduction and objectives: Cutaneous melanoma is a highly aggressive malignancy and has

rapidly increased over the past several decade. The use of combination therapies is an emerging

field and may be a strategy to overcome resistance and decrease toxicity of melanoma treatment.

Cold physical plasma is a partially ionized gas that generates oxygen and nitrogen species and

selectively kills cancer cells in vitro. Previously, we demonstrated that inhibiting mitochondrial

cytochrome c oxidase (CcO) sensitizes melanoma cells towards cold plasma induced cell death.

Hence, we sought out to identify potential therapeutic targets in the mitochondria, which can show

synergistic or additive effects towards melanoma toxicity.

Material and methods: 5000 cells were seeded in 100µl of DMEM 10%FBS in a 96 well plate and treated

with different drugs in RPMI 2%FBS. It was generated dose response curves using a library of 46

targeting mitochondria drugs in combination with cold plasma (16h after drug treatment) in murine

B16F10 and human SKMel-28 melanoma cell lines. A metabolic activity assay (alamar blue) was used

to determine the IC50 of the drug alone or in the combination. The 11 drugs that showed better

outcome ([IC50 drug alone/IC50 combination] ≥5) were tested against a normal keratinocyte cell line

HaCaT. The drugs that were also toxic for the nonmalignant cell line ([IC50 HaCat/IC50 SKMel-28] ≤2)

were excluded for the next analysis. The resulting five therapeutic candidates were analyzed after 6

and 24 hours of plasma treatment for cytotoxicity and mitochondrial membrane potential with high

content imaging, using Sytox green and TMRE staining, respectively. Cell proliferation was also

analyzed with high content imaging after 24 hours. The cytotoxicity was confirmed using a 3D

spheroids in vitro model stained with Sytox green and Hoechst. To understand mode of cell death

induced, the cells were pretreated with necrostatin-1s and Z-VAD-FMK, 30 min before plasma

treatment. All parameters were analyzed comparing to the vehicle control group (DMSO).

Results: The metabolic activity assay showed that several mitochondrial targeting drugs induced

melanoma cell death in monotherapy or in combination with cold physical plasma. We selected 11

drugs that induced cytotoxicity against the murine and human melanoma cell lines with the IC50 at

least five times higher for the drug alone, when compared to the combination. Five drugs that did

not show cytotoxicity towards HaCaT cells were included in the further analysis. The temporal analysis

showed that the combination induced progressive cell death for all drugs with decrease in

mitochondrial membrane potential after 6 hours for B16F10 cells and after 24 hours for SKMel-28. For

all drugs the combination also induced decrease in cell proliferation and increase in cell death in

spheroids model, compared to drug alone. Z-VADFMK prevented the cytotoxicity for the most of

drugs in both cell lines, indicating that this is a caspase dependent cell death.

Conclusion: In this study, we demonstrated that targeting mitochondrial pathway in conjunction with

cold plasma derived oxidants is a potential therapeutic strategy in treatment of malignant

melanoma inducing apoptotic cell death.

Keywords: Reactive oxygen and nitrogen species, oncotherapy, mitochondrial inhibitors.

Grants: This work was supported by grants funded by the German Federal Ministry of Education and

Research (BMBF), grant number 03Z22DN11.

75

https://www.sciencedirect.com/topics/medicine-and-dentistry/degree-of-ionization

https://www.sciencedirect.com/topics/medicine-and-dentistry/degree-of-ionization

HISTOPATHOLOGICAL EVALUATION OF THE INTRADERMAL INJECTION OF CRUDE EXTRACT AND THE

FRACTION F1 OF Fusarium oxysporum

Gustavo Henrique Doná Rodrigues Almeida1; Mariana Cristina Vicente Umada Zapater1; Kátia

Cristina Sibin Melo1; José Rosa Gomes3; Terezinha Inez Estivalet Svidzinski2; Luzmarina Hernandes1

¹Universidade Estadual de Maringá, Department of Morphological Sciences, Maringá, PR, Brazil

²Universidade Estadual de Maringá, Department of Clinical Analysis and Biomedicine, Maringá,

PR, Brazil

³Universidade Estadual de Ponta Grossa, Department of General Biology, Ponta Grossa, PR, Brazil

[email protected]

Introduction and objectives: Fusarium oxysporum is a filamentous fungus that produces mycotoxins

which cause fusarioses in plants, animals and humans. It is an emerging pathogen with a high rate

of morbidity and mortality and reports of Fusarium infections in immunocompetent patients are

increasingly more frequent. The objective of this study was to evaluate the response of cutaneous

tissues to the metabolites produced by F. oxysporum in immunocompetent Wistar rats, comparing

tissue morphology after contact with the crude extract and with a F1 fraction obtained from crude

extract.

Material and methods: Initially, from a culture of F. oxysporum, which was isolated from a patient with

onychomycosis in Laboratory of Teaching and Research in Clinical Analyzes (LEPAC) of the State

University of Maringá (UEM), the crude extract was obtained. From this, the F1 fraction was obtained

using the techniques of protein precipitation and electrophoresis of the same. Subsequently, eighty

male animals (CEUA protocol number 005/2010, and register 080/2010), divided into four groups were

used according to the intradermal administration received: crude extract (0,5 mg/ml); 0,9% saline

solution control; F1 fraction (0,05 mg/ml) and control gel (solution obtained after dialysis of the 12%

polyacrylamide gel). The animals were sacrificed by sodium thiopental anesthetic overdose at 3, 6,

12 and 24 hours after application of the solutions. Five animals were used for each group / time. Skin

samples were collected, which were submitted to histological processing for the accomplishment of

different histological stains and immunohistochemistry techniques (detection of MMP-9

metalloproteinase). The images were captured through the Olympus BX41 optical microscope with

a camera attached. Quantitative analyzes were performed in the Image Pro Plus ® 4.5 program and

the statistical analyzes of the data by the GraphPad Prism ® 3.0 program.

Results: It was observed a more intense inflammatory response in the skin of the animals injected

with the F1 fraction in comparison to the crude extract, mainly from the 6 hours and increased with

time, presenting with polymorphonuclear and mononuclear cells, besides vacuolization of

keratinocytes and detachment of the epidermis. In all the analyzed periods, there was the

extravasation of red blood cells, however, evidently, in a smaller quantity in the group that received

the fraction than those with the crude extract. There was a significant increase in the number of mast

cells in both situations, as well as morphological and structural changes of the collagen in the dermis.

The expression of MMP-9 was more intense in the animals injected with F1 fraction, which was intense

mainly in keratinocytes, fibroblasts and polymorphonuclear cells, in relation to the crude extract.

Conclusion: We conclude that the histopathological changes resulting from the injection of F1

fraction caused drastic alterations in the tissue morphology compared to the injection of the crude

extract.

Keywords: Fungus, skin, morphology, MMP-9.

Grants: My thanks to CNPq for the financial support for the project.

76

HOW SKELETAL MUSCLE LOSS DIFFERENTIALLY PROGRESS DURING CACHEXIA DEVELOPMENT AFTER

EHRLICH CARCINOSARCOMA INOCULATION: A COMPARISON BETWEEN PRECACHEXIA AND

CACHEXIA

Bordini, H.P1; Blegniski, F.P1; Guarnier, F.A1.

1 Universidade Estadual de Londrina, LAFAM, Department of General Pathology, Londrina, PR,

Brazil [email protected]

Introduction and objectives: Cachexia is directly related to 20% of all cancer deaths, that are

frequently related to the amount of skeletal muscle loss. However, clinically, this syndrome is highly

neglected, once it is rarely diagnosed (2.4% of cancer patients) or even treated (6.4% of cancer

patients). Recently, an international consensus stratified the syndrome in 3 phases, according to its

progression: precachexia (weigh loss <5%, anorexia, metabolic alterations), cachexia (weight loss

>5%, anorexia, systemic inflammation) and refractory cachexia (high catabolism, non-

responsiveness to cancer treatment, low life expectancy). Experimental models rarely classify

cachexia when purposing new treatment approaches, and different tumor behaviors are scarcely

considered. In addition, the evolution and mechanisms of muscle loss and atrophy during cachexia

evolution are not completely elucidated. The aim of this study is to propose Ehrlich tumor (a

mamarian carcinosarcoma) as a new and reliable experimental model to comprehend skeletal

muscle loss during cachexia progression considering prechachexia and cachexia phases.

Material and methods: A pilot study determined times of 14 and 28 days after tumor implantation as

cachexia and pre cachexia, respectively. Swiss male mice (n=80) weighing 25-35g (CEUA-UEL,

n°12549.2017.35) were randomly divided as: (i) control 14 days (C14d); (ii) control 28 days (C28d); (iii)

precachexia group (PreCaq) – s.c. inoculated with 1.0 x 106 Ehrlich tumor cells in the back region, in

phosphate buffer saline (PBS), and monitored for 14 days; (iv) cachexia group (Caq) – inoculated

with 1.0 x 106 Ehrlich tumor cells and monitored for 28 days. C14d and C28d received s.c. PBS

inoculation. Tumor size, weight loss, food consumption, hand grip strength test, rectal temperature,

leptin, inflammatory cytokines and muscle fibers cross sectional area (CSA) were analyzed to

establish the differences between cachexia and precachexia. One-way ANOVA with p<0.05 were

used to determine statistical differences among groups.

Results: Tumor size differed between PreCaq and Caq (0.38 ± 0.31; 1.11 ± 0.49 mg). PreCaq showed

significant reduced final body weight (37.82 ± 4.18 g) and weight gain (4.58 ± 2.54 g) when compared

to C14d (44.10 ± 6.56; 7.85 ± 3.51g, respectively). Muscle loss was considered significant in PreCaq

when compared to C14d (0.22 ± 0.03; 0.31 ± 0.07 mg). Food consumption reduced significantly in

PreCaq and Caq groups (156.1 ± 3.46; 197.5 ± 3.41 g) compared to C14d (189.1 ± 3.41 g). Hand grip

strength test demonstrated to be reduced in PreCaq (70.81 ± 8.5 g) and Caq (78.56 ± 2.43 g) when

compared to C14d (93.99 ± 1.19 g). Interleukins, leptin, and CSA followed a classical pattern

observed in cancer cachexia.

Conclusion: It was possible to establish Ehrlich carcinosarcoma as a reliable model to study

cachexia, and define precisely which its phases are. Skeletal muscle loss is the main cachexia

characteristic and influences directly patient´s survival. In this model, we also showed how skeletal

muscle loss progress during cachexia development. These data can contribute significantly on

establishing more consistent treatments on cachexia syndrome and when is the ideal period to start

treatments.

Keywords: Ehrlich tumor, cachexia, cancer.

Grants: Capes

77

MYELOPATHY ASSOCIATED WITH HUMAN TYPE I CELL LYMPHOTROPIC VIRUS

Jose Melo1; Francis Fregonesi Brinholi 2

1,2 University of Pythagoras Unopar, Londrina, PR, Brazil


Introduction and objectives: Human T cell lymphotropic virus (HTLV) type 1 (I) is a retrovirus associated

with several different diseases and clinical conditions (DELAZELI, 2012; ZIHLMANN et al., 2017). In Brazil,

it is present in all states with varying prevalence rates and it had been estimated that around 2.5

million Brazilians are infected (ROMANELLI et al., 2010; SODRÉ, 2010). The HTLV has no cure, is

asymptomatic, and its diagnosis is rarely made before the onset of diseases to which the virus is

associated, among them the myelopathy (AMORIM; MIRANDA, 2015). It is estimated that between 2

to 5% of individuals with HTLV-1 may develop myelopathy (CARVALHO, 2014). HTLV-1 Associated

myelopathy (HAM) is a neurodegenerative disease, with the involvement of the gray and white

matter in the spinal cord, characterized by demyelination and axonal degeneration. This condition

affects the spinal cord which is characterized by greater involvement of the proximal muscles of the

lower limbs back-lumbar (PARANHOS et al., 2016). In this context, the objective of this study was to

associate the clinical profile of patients HAM.

Material and methods: To the search strategy, PubMed, SciELO and Google Scholar

databases were examined using the following keywords: Clinical profile; Human T Cell Lymphotropic

Virus; Myelopathy. This study just used bibliographic references published between 2009 and 2019.

Results: HAM presents a clinical condition characterized by individuals affected predominantly in the

fourth and fifth decades of life, rarely before 20 years or after 70 years, with insidious onset and slowly

progressive evolution (ORGE et al., 2009; COUTINHO, 2011). There is a predominance of females,

mainly before menopause, and this gender difference could be attributed to female sex hormones

(GLORIA et al., 2015). Infection stands out among individuals associated with low schooling and lower

socioeconomic power (CARVALHO, 2014). This can be explained by the fact that these patients do

not have access to the necessary information regarding basic health, especially regarding

mechanisms for prevention of sexually transmitted diseases (GLORIA et al., 2015). Motor symptoms

characterized by gait disturbances, weakness, and stiffening of the lower limbs are a consequence

of the gradual decrease of muscle strength and spasticity in the myotomes affected, being the first

symptom to be perceived (SANTOS et al., 2010). The patient gradually decreases ambulation,

requiring overtime assistance with locomotion until evolve to use a wheelchair (CHAMPS et al., 2010;

SANTOS et al., 2017). During evolution, bladder dysfunction is common, with incontinence or urinary

retention and voiding urgency. Other signs such as intestinal constipation, low back pain, erectile

dysfunction, and prostatitis are also observed in HAM (SHUBLAQ et al., 2010).

Conclusion: HAM is a serious and disabling disease that presents high morbidity, leading to a

compromise in the activities and quality of life. Negative effects of the disease are directly

determined by the level of restriction and inability to perform essential human being activities.

Knowing the clinical profile in population can help the health professionals with suspect about the

diagnosis more frequently while promoting the investigation of these disease by associated

symptoms.

Keywords: Clinical profile; Human T Cell Lymphotropic Virus; Myelopathy.

78

Hylocereus undatus SEEDS PROTECTS CELL MEMBRANE FROM LIPOPEROXIDATION INDEPENDENTLY OF

IRON CHELATION

Lens, H.H.M.1; Lone, A.B. 2; Blegniski, F.P. 3; Borges, F. H.4, Banin-Hirata, B.K.5

Panis, C. 6; Takahashi, L.S.A. 7; Faria, R.T. 7; Cecchini, A.L. 1; Victorino, V.J.8; Cecchini, R. 4

1 Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil

2 Empresa de Pesquisa e Extensão Rural de Santa Catarina (EPAGRI) – Estação Experimental de

Itajaí, Itajaí, SC, Brazil

3 Universidade Estadual de Londrina, Laboratory of pathophysiology of muscular adaptations,


4 Universidade Estadual de Londrina, Laboratory of General Pathology, Londrina, PR, Brazil

5 Universidade Estadual de Londrina, Laboratory of studies and applications of DNA polymorphisms,


6 Universidade do Oeste do Paraná, Laboratory of Inflammatory Mediators, Francisco Beltrão, PR,

Brazil

7 Universidade Estadual de Londrina, Departament of Agronomy, Londrina, PR, Brazil

8 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro- Campus Engenheiro

Paulo de Frontin, Engenheiro Paulo de Frontin, RJ, Brazil

[email protected]

Introduction and objectives: One of the consequences of oxidative stress is the process of lipoperoxidation,

which can damage the cell membrane. This state of oxidative stress is not restricted to a single cell, which may

interfere with tissue and even organ homeostasis. Therefore, it is necessary for the body to avoid this damage

through an antioxidant defense system. This defense is responsible for controlling and combating reactive

species and their effects and can be produced by the body itself or acquired through diet. The pitaya, known

in other regions as dragon fruit, has been promising for studies about its antioxidant properties. The objective of

this work was to evaluate the antioxidant profile of the bark, pulp and seed of the white pitaya specie,

Hylocereus undatus, through the verification of the ability to protect biological membranes.

Material and methods: The fruits of H. undatus (n = 7) were processed to obtain a sample of the bark, pulp and

seed, separately. Hepatic microsomes were also obtained from healthy rats for lipoperoxidation assay. The test

groups were divided into control (microsome + phosphate buffer solution (PBS) + tert-butil), oxidized microsome

(microsome + FeCl3 + ascorbic acid + PBS + tert-butil), pitaya with control microsome (pitaya + microsome + PBS

+ tert-butil) and pitaya with oxidized microsomes (microsome + FeCl3 + ascorbic acid + tert-butil). The

pretreatment of the microsomes was performed with a sample of bark, pulp and pitaya seed. After 12 hours of

incubation at 37 ° C, oxidative stress was evaluated by chemiluminescence. To confirm the antioxidant activity

of pitaya, a spectrophotometric evaluation of Fe3+ complex formation was performed. Statistical analyzes were

performed by Student t-tests, One Way ANOVA for area comparison and Two-Way ANOVA followed by

Bonferroni as post-test for analysis of the chemiluminescence curve, adopting p <0.05 as significant.

Results: Our results show that, based on the significant increase in lipoperoxidation of the oxidized microsome

group when compared to the control group (p <0.001), only the Hylocereus undatus seed sample was able to

significantly prevent the microsome lipoperoxidation (p <0.001). In evaluating whether the antioxidant

protection of pitaya would be related to the iron chelation of the lipoperoxidation system, we showed that there

is no formation of pitaya complex with Fe3+.

Conclusion: This work showed that the white pitaya seed, Hylocereus undatus, protects membranes from the

lipoperoxidation process and also testified that the mechanism of protection presented does not occur through

iron chelation.

Keywords: Antioxidant, pitaya, oxidative stress

Grants: CAPES, CNPq, Fundação Araucária.

79

IMPACT OF NEONATAL METFORMIN AND HYPERLIPID INTAKE IN ADULTHOOD ON RAT VENTRAL

PROSTATE MORPHOLOGY AND SPERM MOTILITY

Celso Vitor Calomeno1; Henrique Rodrigues Vieira2; Josana Klagenberg2; Paulo Cezar de Freitas

Mathias2; Jaqueline de Carvalho Rinaldi1

1State University of Maringa, Department of Morphological Science, undergraduate in

Biotechnology, Maringa, PR, Brazil. 2State University of Maringa, Department of Biotechnology, Genetics and Cell Biology, Maringa,

PR, Brazil 3State University of Maringa, Department of Clinical Science, Postgraduate Program in

Biosciences and Pathophysiology – PBF, Maringa, PR, Brazil.


Introduction and objectives: Metformin has been used to control the glucose blood levels, but it has

also been shown that metformin acts on a variety of organs including the reproductive system.

Literature suggested that metformin could ameliorate the reproductive harms caused by obesity

and also type-2-diabetes. In this sense, our study aimed to evaluate the impacts of metformin

exposure during early lactation on ventral prostate (VP) and vas deferens (VD) of rats that were feed

hyperlipid diet in adulthood.

Material and methods: Twelve dams (9 pups each) were randomized distributed in two experimental

groups: saline (S; 0.9% of NaCl/day) and metformin (M; 100mg/ Kg/day) were the male offspring

received S or M daily via intraperitoneal injection until postnatal day 12 (PDN12). On PDN21, male

and female offspring from each group were separated. On

PDN60 males from S and M group were subdivided in normal fat diet (S-NFD and M-NFD; 4.5% fat) or

high fat diet (S-HFD and M-HFD; 35% fat). After 30 days (PDN90) the animals were euthanized, VP and

vas deferens were dissected and weighted. VP was used to hystophatological and

immunohystochemical analysis. The sperm from VD was used to motility analysis. The data was

analyzed by Two-way ANOVA with post-hoc Turkey’s test (Ethical Approval by CEUA/UEM

5869020418).

Results: It was observed an increase in body weight gain in both groups feed with HFD in adulthood

in relation to their controls (S-NFD vs. S-HFD groups: p<0.0001, M-NFD vs. MHFD: p<0.001). There is no

statistical difference in VP and VD relative weight among groups. Analyzing sperm motility, there was

a reduction in number of mobile and immobile sperms in the S-HFD group compared to S-NFD

(p<0.05). Histophatological analysis revealed prostatitis in VP from S-HFD and M-HFD; hyperplasia and

epithelial mucinous degeneration in VP from S-HFD but not in M-HFD. The proliferative marker Ki-67

was detected by immunohistochemistry in all groups. The hyperlipid intake increased the number of

epithelial ki-67 positive cells, suggesting higher proliferative rates on S-HFD and M-HFD groups.

Conclusion: In summary, the preliminary findings showed the neonatal exposure to metformin

ameliorate the injuries by adulthood hyperlipic intake on prostate histoarchitecture and sperm

motility.

Keywords: proliferation; histopathology; immunohistochemistry; reproductive system.

Grants: CNPq

80

JEJUNUM MORPHOLOGY OF RATS SUBMITTED TO SUCROSE-RICH DIET AND TREATED WITH

PTEROSTILBENE

Joice Moraes Menezes1; Wesley Ladeira Caputo1; Ana Paula da Silva Barbosa1; Carlos Vinícius

Dalto da Rosa1; Fábio Rodrigues Ferreira Seiva1; João Paulo Ferreira Schoffen1


PR, Brazil


Introduction and objectives: Foods and drinks rich in sucrose are being increasingly consumed by the

world's population. Products with a high caloric content, when ingested in excess and associated

with sedentary lifestyle, are responsible for the obesity epidemic observed in the 21st century.

Excessive intake of food leads the cells to a state of oxidative stress and may cause damage to

important components of the gastrointestinal tract. Pterostilbene, a synthetic analogue of resveratrol,

reduces glycemia, improves lipid profile, inhibits the production of reactive oxygen species,

enhances brain neuronal survival, inhibits carcinogenesis, and other effects. The objective of this work

was to evaluate the effects of the sucrose-rich diet and pterostilbene on the wall and population of

goblet cells of the jejunum of rats.

Material and methods: After approval by the Committee of Ethics in Animal Experimentation of the

Universidade Estadual do Norte do Paraná (Declaration nº 05/2017), Wistar rats were divided into

groups: Control (C), Control + Pterostilbene (CP), Sucrose (S), Sucrose + Pterostilbene (SP). During 195

days, groups C and CP received filtered water and commercial ration ad libitum, while groups S and

SP received commercial ration and water plus sucrose 50% ad libitum. Pterostilbene, at a dose of

40mg/kg, was administered daily via gavage for 45 consecutive days, starting on the 151st day of

the experiment. After euthanasia of the animals, jejunum samples were processed and stained with

hematoxylin-eosin for morphometric analysis of the total wall, mucosal and muscular tunics, villi

height and crypt depth (100 measurements/animal) and with Periodic Acid Schiff to determine the

number of goblet cells (2500 epithelial cells per animal were recorded to obtain the percentage of

labeled to unlabeled cells). The data were statistically analyzed by one-way ANOVA and Tukey test.

Results: There were maintenance of intestinal morphometric parameters, such as total wall, mucosa

tunica, villus height, crypt depth and muscle tunic thickness in all the studied groups. There were no

changes in the total number of goblet cells.

Conclusion: The sucrose-rich diet and the treatment with pterostilbene (40mg/kg) do not interfere in

the morphoquantitative aspects of the intestinal wall, a fact that indicates the preservation of the

functions of the jejunum in this experimental model.

Keywords: Diet. Pterostilbene. Intestinal wall. Goblet cells.

Grants: Universidade Estadual do Norte do Paraná.

81

MAST CELL QUANTIFICATION IN THE DUODENAL MUCOSA OF WALKER-256 TUMOR-BEARING RATS

ADMINISTRATED WITH 1% L-GLUTATHIONE

Sestak, S. S.1; Bagon, N. P.2; Lima, F. G. da M.1; Oliveira, A. P. de1; Casagrande, L.3; Silva, B. T.4;

Bossolani, G. D. P.3; Almeida, G. H. D. R.2; Cícero L. R.3; Perles, J. V. C. M.5; Zanoni¹, J. N.5

1 State University of Maringá, Department of Physiology Sciences, Maringá, PR, Brazil.

2 State University of Maringá, Department of Clinical Analysis and Biomedicine, Maringá, PR, Brazil.

3 State University of Maringá, Department of Pharmaceutical Sciences, Maringá, PR, Brazil.

4 State University of Maringá, Department of Biological Sciences, Maringá, PR, Brazil

5 State University of Maringá, Department of Morphological Sciences, Maringá, PR, Brazil. Email:

[email protected]

Introduction and objectives: Walker-256 tumor cells are an experimental model frequently used in

research to induce cachexia. Cancer cachexia is characterized by systemic inflammation and may

exhibit gastrointestinal dysmotility and damage to enteric innervation induced by oxidative stress.

The aim of this study was to analyze the quantification of mast cells in the intestinal mucosa of Walker-

256 tumor-bearing rats given 1% L-glutathione.

Material and methods: This study was approved by the Animal Research Ethical Committee of the

State University of Maringá (UEM) under the approval number 7434160316. 24 male Wistar rats with

55 days of age were used. Rats were randomly assigned to four groups of six animals each: control

group (C), control administered with 1% L-glutathione (GT), Walker-256 (TW) tumor and Walker-256

tumor administered with 1% of L-glutathione. (TWG) Cancer induction for the TW and TWG groups

was performed by inoculating a suspension containing 8.0 x 10 7 viable tumor cells in 0.5 ml of

phosphate buffered saline (PBS) on the right flank of each animal. Groups C and GT received only

PBS at the same site. Food and water were available ad libitum. The GT and TWG rats were

administered with L-glutathione, which was incorporated into the standard diet at the concentration

of 1% (1g / 100g of feed). After 14 days of treatment, the animals were euthanized and two

centimeters of the duodenum removed, opened along the mesenteric border, adhered in

styrofoam, rinsed in PBS (0.1 M, pH 7.4) and fixed in Bouin for 6 h. After fixation, the samples were

dehydrated and embedded in paraffin to obtain semisilary histology of 4 μm thickness. The sections

were then stained with Toluidine Blue. Quantification of mast cells was performed at 30 villi per animal.

Results: Mast cell count increased approximately 29.9% in the GT group in relation to the control

group (p = 0.04556). The TW group presented a 102.1% increase in the number of mast cells in relation

to the control group (p = 0.00001). While in the TWG group a reduction of 14% (p = 0.055) was

observed in relation to the TW group and it was not statistically significant.

Conclusions: Changes in the quantification of mast cells in diseased groups were already expected,

since these cells are responsible for the storage of potent chemical mediators of inflammation.

However, as observed in the results, treatment with L-glutathione did not promote significant

reduction in the increase of mast cells due to cancer, as well as attenuated the amount of mast cells

in the mucosa of supplemented healthy animals.

Keywords: Cancer, Intestinal mucosa, Duodenum.

82

METFORMIN ACTIVITY IN THE INDUCTION OF DACARBAZINE RESISTANCE IN B16F10 MURINE

MELANOMA CELLS ON ANTIOXIDANT PARAMETERS

Sanches, L. J. 1; Marinello, P. C. 1; Brito, W.A.S.1; Lopes, N. M. D. 1; Luiz, R. C. 1; Cecchini, R.2 ; Cecchini,

A. L. 1.

1Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil.

2 Universidade Estadual de Londrina, Laboratory of Pathophysiology and Free Radicals, Londrina,

PR, Brazil.

[email protected]

Introduction and objectives: Melanoma is a malignant tumor with a strong capacity for invasion and

metastasis, high rates of recurrence and mortality, and limited response to currently available

treatments. Dacarbazine (DTIC) is the oldest chemotherapeutic used for the treatment of metastatic

melanoma, but the results are not satisfactory, due to the resistance mechanisms of melanoma.

Metformin is the drug most prescribed for type 2 diabetes and has shown antitumor effects against

various types of cancer, including melanoma. The objective of this work was to develop the

phenotype of DTIC resistance in B16F10 cells and to evaluate the participation of metformin in the

induction of resistance to DTIC.

Material and Methods: We induced resistance by exposing cells to increasing doses of DTIC, and to

study the action of metformin in that process, the cells were exposed to metformin for 7 passages

and in the sequences were induced to chemoresistance along with metformin treatment. At the end

of the experimental time, the MTT, thiol, MDA and immunocytochemistry tests were performed for

p53, 8-OH-DG, Nrf2 and NF-κB.

Results: Our results showed that the cells of the DTIC group were more resistant to chemotherapy

than those of the MET-DTIC group. We observed that the levels of MDA were higher than those of the

control, and the thiol levels of the MET-DTIC group were higher in relation to the DTIC group. 8-OH-

DG is a marker for oxidative DNA damage, the DTIC group showed higher marking for 8-OH-DG, in

the MET-DTIC group the marking was slightly lower. P53 is involved in several cellular processes, such

as apoptosis and cell cycle arrest, the labeling for p53 was lower in the DTIC group than in the MET-

DTIC. Some chemotherapeutics induce resistance by NF-kB activation, in the DTIC group the labeling

for NF-kB was higher than in the MET-DTIC. Nrf2 is also related to chemoresistance, and its marking

was higher in the DTIC group and lower in the MET-DTIC group

Conclusion: Our results indicate that we were able to develop the resistance phenotype to DTIC,

and that metformin can block the induction of resistance to DTIC. We show this by increasing the

labeling for 8-OH-DG in the DTIC group that is related to DNA damage caused by oxidative stress.

The reduction in the labeling for p53 in the DTIC group and the increase in the MET-DTIC group

indicates that we were able to generate the resistant phenotype, and it indicates that metformin is

able to prevent this induction. The increase in labeling for NF-kb in the DTIC group and reduction in

the MET-DTIC is another fact that proves the induction of resistance in DTIC and blockade by

metformin in MET-DTIC, since NF-kB activation is associated with resistance to chemotherapeutics.

Finally, the high DTIC and reduced DTIC-DTIC marking shows that we have achieved our goals of

inducing resistance to chemotherapy in B16F10 cells, and of analyzing the performance of

metformin, it is in fact acting to prevent the induction of resistance.

Keywords: Melanoma, chemoresistance, dacarbazine, oxidative stress, metformin

Grants: CAPES.

83

METFORMIN PREVENTS SKIN AND SYSTEMIC OXIDATIVE DAMAGE AND IMMUNE ALTERATIONS

INDUCED BY UVB RADIATION.

Souza-Neto, F.P 1; Souza, P.G.B 1; Melo, G.P 1; Cela, E.M 2; Campo, V.E 2; Ramalho, L.Z.N 3; Maglio,

D.H.G 2; Cecchini, R 1; Cecchini, A, L1.

1 Universidade Estadual de Londrina, Department of Molecular Pathology, Londrina, PR, Brazil 2 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Inmunología,

Buenos Aires, Argentina 3 Departament of Patology and Legal Medicine, University of São Paulo, Ribeirão Preto, Brazil.

[email protected]

Introduction and objectives: Solar radiation is composed by ultraviolet (UV), visible and infrared

radiations. Great attention is given to UV radiation because of its high energy that allows it to react

with DNA, contributing to skin carcinogenesis. The UV radiation that reaches the Earth's surface

consists of approximately 95% of UVA radiation (321-400nm) and a small percentage (<5%) of UVB

(281-320nm). Skin exposure to Ultraviolet B (UVB) radiation leads to local oxidative stress and

inflammation, systemic Immunosuppression and, ultimately, skin cancer development. Metformin is

the most prescribed drug for type 2 diabetes, and it has been shown that chronic treatments with

this drug reduces skin cancer incidence. Our aim was to analyze metformin effects on UVB-induced

local and systemic acute damage.

Material and Methods. The experimental protocol was approved by the Institutional Animal Care and

Use Committee of the Universidade Estadual de Londrina (CEUA/UEL: 14636.2011). C57/BL6 mice

were pretreated with 90 mg/kg of metformin for 11 days and exposed to 400 mJ/cm2 of UVB

radiation. Twenty-four hours later we obtained skin and blood samples to analyzed oxidative stress

and inflammatory response. Alternatively, a group of animals were used to determine metformin

effect on UVB-induced immunosuppression, using a CHS reaction to oxazolone.

Results. We observed that metformin did not prevent UVB-induced epidermal damage, Langerhans

cells loss or epidermal cells’ mitochondrial alterations. However, it improved the skin reducing state,

increasing antioxidant molecules that led to a reduced 4-hidroxynonenal and nitrotyrosine labeling.

Local inflammatory mediators increased by UVB radiation, such as IL-6 and IL-1β, were also reduced

by metformin. On the other hand, systemic alterations induced by UVB radiation were also reverted

by metformin, increasing thiol in erythrocytes and decreasing serum malondialdehyde and IL-6.

Finally, metformin partially prevented the development of UVB-induced immunosuppression.

Conclusion. We demonstrate that metformin reduces UVB-induced local and systemic damage,

changes that may contribute to explain its antitumoral effect. In this way, the use of metformin as a

potential agent to maintain skin homeostasis and prevent UVB-induced lesions could be evaluated.

Financial support : CAPES- Coordenação de Aperfeiçoamento de Pessoal de nível superior

Keywords: Skin. Oxidative stress. UVB. Immunosuppression. Acute irradiation. Metformin.

84

MORPHOLOGICAL EVALUATION OF HEPATOCYTES NUCLEUS OF WALKER-256 TUMOR-BEARING RATS

SUPPLEMENTED WITH LGLUTATHIONE 1%

Bernardo, C.C.O. 1, Lima, F.G.M. 2, Sestak, S.S. 2, Oliveira, A.P. 2, Sanches, M.R. 1, Cícero, L. R. 1, Silva,

M.P.A. 3, Martins, J.V.C.P. 4, Zanoni, J. N.4 1 State University of Maringa, Department of Pharmaceutical Sciences, Maringá, Brazil 2 State University of Maringa, Department of Physiology, Maringá, Brazil 3 State University of Maringa, Department of Biology, Maringá, Brazil 4 State University of Maringa, Department of Morphological Sciences, Maringá, Brazil


Introduction: World Health Organization (WHO) has announced that cancer cases may increase in

70% by 2018 and estimates 600,000 new cases per year for Brazil, in the 2018/2019 biennium.

Cachexia syndrome is one of the most aggravating consequences of cancer, and leads to an

exacerbated weight loss, which impairs the body and causes the oxidative stress process. It is found

in literature that L-glutathione is one of the main endogenous antioxidants and participates in cellular

protection, by removing reactive oxidative species (ROS), including peroxides formed in oxygen

metabolism. Because the liver is a central gland with numerous basic metabolism vital functions, and

the principal involved in oxidant and antioxidant process as well as free radicals’ reactions, the aim

of this study was to verify the antioxidant effects of L-glutathione in hepatic morphology of sick rats,

using the Tumor of Walker-256 experimental model.

Methods and materials: all the procedures described in this study were approved by the Ethics

Committee in Animal Experimentation (UEM) by the 099/2012 protocol. The liver of Wistar male adult

rats were used, with the animals randomly distributed in four groups (n=6): control (C), control

supplemented with 1% L-glutathione (CG), tumor of Walker-256 (TW) and Tumor of Walker-256

supplemented with 1% L-glutathione (TWGT). By the end of 14 days, the animals were euthanized

with thiopental overdose (150 mg/kg) and lidocaine (10 mg/ml), the liver was collected and process

in automatized histotechnical machine. The slides were made e submitted to hematoxylin and eosin

routine coloring to morphological analysis of 100 hepatocytes by measuring the nucleus and

cytoplasm areas and calculating the nucleus/cytoplasm ratio, in five cuts per animal. The results were

submitted to One-Way variance analysis (ANOVA), followed by Tukey’s post-test with significance

level of 95%.

Results: there was a 15.32% nuclear area increase and a 18.46% nucleus/cytoplasm ratio increase

when comparing TW and C groups (p<0.05). TWGT presented a reduction of 8.40% in nuclear area

and a raise of 22.94% in nucleus/cytoplasm ratio when compared to TW group (p<0.05). It was also

possible to notice a 9.82% (p<0.05) reduction in nuclear area and a 4.61% (p<0.05) reduction in

nucleus/cytoplasm ratio between CGT and C.

Conclusions: In this study, it was clear that 1% L-glutathione supplementation tends to prevent or

reverse the hepatocytes morphological changes observed in TW group, getting closer to control

animals values.

Keywords: Liver, L-Glutathione, Hepatocytes

85

MORPHOMETRIC ANALYSIS OF MYENTERIC NEURONS IMMUNOREACTIVE TO HuC/D PROTEIN AND

ENTERIC GLIAL CELLS IMMUNOREACTIVE TO s100 PROTEIN IN THE JEJUNUM OF WALKER256 TUMOR-

BEARING RATS TREATED WITH 1% L-GLUTATHIONE

Silva, M. P. A.1; Lima, F. G. M.2; Sestak, S. S.2, Oliveira, A. P.2, Ramalho, F. V.3, Silva, B. T.1, Frez, F. C. V.4,

Bernardo, C. C. O.4, Bagon, N. P.5, Perles, J. V. C. M.6, Zanoni, J. N.6

1 Universidade Estadual de Maringá, Department of Biological Sciences, Maringá, PR, Brasil 2 Universidade Estadual de Maringá, Department of Physiological Sciences, Maringá, PR,

Brasil 3 Universidade Estadual de Maringá, Department of Biotechnology, Genetics and Cell

Biology, Maringá, PR, Brasil 4 Universidade Estadual de Maringá, Department of Pharmaceutical Sciences, Maringá, PR,

Brasil 5 Universidade Estadual de Maringá, Department of Clinical Analyses and Biomedicine,

Maringá, PR, Brasil 6 Universidade Estadual de Maringá, Department of Morphological Sciences, Maringá, PR,

Brasil [email protected]

Introduction and objectives: Cancer cachexia is defined as a continuous loss of fat and skeletal

muscle mass that can't be treated exclusively with nutritional support. It is also possible to observe

systemic inflammation and oxidative stress associated to this condition. In the enteric neural system,

there are neuronal and glial cells that affect the gastrointestinal homeostasis regulation. Diseases

that causes inflammation can affect the expression of the markers HuC/D, used for immunoreactive

neurons, and s100, used for immunoreactive enteric glial cells. The treatment with L-glutathione, an

antioxidant, aims to influence on the said symptoms. The objective was the morphometric analysis of

myenteric neurons immunoreactive to HuC/D and enteric glial cells immunoreactive to s100 in the

jejunum of Walker-256 tumor-bearing rats treated with 1% L-glutathione.


State University of Maringá (UEM) under de approval number #7434160316. 20 male Wistar rats were

used and divided in four groups: control (C), control treated with 1% Lglutathione (CGT), tumor of

Walker-256 (TW) and tumor of Walker-256 treated with 1% Lglutathione (TWGT). The control rats were

fed with Nuvilab® balanced standard rodents ration, while the treated groups were given ration

pellets with 1% L-glutathione. The animals were euthanized at the age of 69 days and the jejunum

were collected for HuC/D and s100 immunohistochemistry on the myenteric plexus. The

morphometric analysis of neuronal cell bodies was made using the software Image Pro® Plus 4.5. The

data comparison was made by One-Way ANOVA block design followed by Fischer post-test using

Software Statistics® 7.1 and GraphPad Prism® 6.1.

Results: In the HuC/D analysis, between groups C and TW, there was an increase of 16.5% in the area

of the neurons (p<0.05). There was also an increase of 8.12% between C and CGT (p<0.05). There

was decrease of 14.96% in the area of the neurons comparing TW and TWGT (p<0.05). In the s100

analysis, comparing groups C and TW there were no significative differences (p>0.05), while the

comparison between groups C and CGT showed an increase of 3.32% in the area of the enteric glial

cells (p<0.05). Groups TW and TWGT showed an increase of 5.3% in the area (p<0.05).

Conclusion: The treatment of healthy animals with 1% L-glutathione can increase the area of the

neurons and enteric glial cells. The treatment was also capable of reducing the neuronal area of

Walker-256 tumor-bearing animals to values close to the control, but the same didn't happen to the

enteric glial cells.

Keywords: neurons; enteric glial cells; cancer cachexia; L-glutathione.

86

NEURONAL AND COLONIC MOTILITY CHANGES CAUSED BY DEXTRAN SODIUM SULFATE-INDUCED

ACUTE COLITIS: AN EXPERIMENTAL MODEL

Paulo da Silva Watanabe1; Joana D’Arc de Lima Mendes1; Andreza Manzato1; Eduardo José de

Almeida Araújo1

1 Universidade Estadual de Londrina, Histologia - CCB, Londrina, PR, Brazil Email

[email protected]

Introduction and objectives: Inflammatory bowel diseases currently represent a major concern in

gastroenterology. Patients with ulcerative colitis (UC) have mucus diarrhea, rectal bleeding,

abdominal pain, severe loss of appetite and weight, and also risk of colorectal carcinoma.

Experimental UC can be induced by different methods, but the exposure to dextran sodium sulfate

(DSS) is the most common used in the world. Despite many evidences support intestinal motility

changes due UC, there is no an experimental model defined to investigate the morphophysiology

of the colonic myenteric plexus during the inflammation process caused by DSS and, therefore, this

was the objective of this study.

Material and methods: Experimental protocols were previously approved by the ethics committee of

the State University of Londrina (4023.2017.31). Young male C57BL/6 mice were distributed into two

groups: control (CG) and ulcerative colitis (UCG). Acute ulcerative colitis was induced by exposing

the mice to 3% DSS in the drinking water for seven days. CG had pure water. During DSS exposure,

body weight, consistency and presence of blood in stool were evaluated in order to obtain the

disease development index (DAI). After 7 days of DSS exposure, the gastrointestinal transit time was

measured by administering the carmine red dye by gavage and then mice were euthanized. Spleen

was removed and weighed. Colon was removed and its total length and width were measured.

Distal colon were fixed in paraformaldehyde 4% for 3h. Samples were processed for histological

analysis in section stained with haematoxylin and eosin or dissected to obtain whole-mount

preparations containing the myenteric plexus. Immunofluorescence for general neuronal (anti-

PGP9.5) and nitrergic neurons (anti-nNOS) was performed in whole-mount preparations aiming

analysis of the neuronal counting and morphometry. Colonic motility was determined using a

multilumen perfusion manometry apparatus.

Results: Progressive increase of DAI was observed during the DSS exposure reaching scores

compatible with UC (p<0.05). The gastrointestinal transit time and the spleen weight were not

significantly changed in the UCG. The colonic length was intensely shortened in the UCG vs

CG (p<0.05). The microscopic analysis has shown massive presence of inflammatory infiltrates in the

mucosa and submucosa. In addition, submucosa layer was swollen. The inflammation did not cause

loss of general myenteric neurons. However, the number of nitrergic neurons was reduced and the

remained cells were atrophied. Regarding the manometric assessment, the frequency of colonic

contractions in UC mice were reduced and the time of the migratory motor complex was increased

when compared to control mice. In the meantime, no change was observed in the amplitude and

area under the curve of the contractions.

Conclusion: We conclude that DSS-induced ulcerative colitis causes reduced frequency of colonic

contractions in mice due impairment of nitrergic myenteric neurons.

Keywords: colonic motility; dextran sodium sulfate; enteric nervous system; ulcerative colitis.

Grants: CAPES

87

NFKB1/NFKBIA POLYMORPHISMS PLAY A PROTECTIVE ROLE AGAINST HPV INFECTION

Sena, M. M.1; Okuyama, N. C. M.1; Trugilo, K. P.1; Pereira, E. R.1; Pereira, A. P. L.1; Cesardos-Santos,

F.1; Ferreira, R. S.1; Esposito, A.1; Singi, P.1; Couto-Filho, J. D’O2; Oliveira, K. B.1.

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 2Hospital do Câncer de Londrina, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Human Papillomavirus (HPV) is the etiological agent of the

anogenital tract most frequently transmitted infection. In general, the infection is asymptomatic,

however, the persistence of oncogenic types added to the characteristics of the host, such as

genetic background, immunological status and behavioral profile, may lead to the formation of

squamous intraepithelial lesions, which, once untreated, can progress to cervical cancer (CC).

The NF-kB pathway plays an important role in controlling the expression of several genes essential

to cellular activity, such as those related to immune response, proliferation, differentiation,

apoptosis, among others. Thus, the study purpose was to evaluate the influence of NFKB1

rs28362491 and NFKBIA rs696 polymorphisms on HPV infection and on pre-neoplastic and

neoplastic lesions development.

Material and methods: Samples were submitted to Polymerase Chain Reaction (PCR) for viral

detection and subsequent enzymatic restriction for genotyping. Results were observed in 10%

polyacrylamide gel stained with silver nitrate. Through the application of questionnaire, patients’

information about socio-demographic and sexual behavior features were obtained. This study was

approved by the Institutional Ethics Committee Involving Humans Beings of the State University of

Londrina, Londrina-PR, Brazil (CEP/UEL 133/2012; CAAE 05505912.0.0000.5231). The purpose of the

study as well as the procedures were explained to all participants, and the written consent was

obtained prior to the interview and the material collection.

Results: A total of 334 patients were recruited, of who 163 (48.8%) were HPV infected, and of these,

58 (37.4%) presented precursor lesions and 26 (16.8%) were diagnosed with CC. It was observed that

among the infected women, the majority reported not knowing the virus (P=0.038), aged ≤24 years

(P=0.001), smoking habit (P=0.043), single (P=0.007) and reported ≥4 sexual partners during their

lifetime (P=0.024). When considering the lesions presence, patients with precursor lesions reported

having had more sexual partners than the other groups (P=0.006), whereas CC patients reported

having had low education level (P=0.004), monthly income <1 minimum wage (P=0.001) and ≥5 full-

term pregnancies (P<0.001). Regarding to the polymorphisms analyses, the combination of

homozygous insertion genotype for NFKB1 (II) and AA genotype for NFKBIA showed to be a protective

factor against HPV infection [P=0.014; OR=0.259 CI95% (0.088-0.765)], whereas it was not associated

with lesion progression and CC development.

Conclusion: The present study demonstrated for the first time that rs28362491 and rs696

polymorphisms may present a protective role against HPV infection in the analyzed population. From

this promising finding, it will be possible to begin the search for a better understanding of the

mechanisms by which these genetic alterations are involved in protection against HPV.

Keywords: Cervical cancer, SNP, restriction fragment length polymorphism.

Grants: This study was supported by Conselho Nacional de Desenvolvimento Científico e


Fundação Araucária, Programa Pesquisa para o SUS (PPSUS) and by Londrina State University

Graduate Coordination (PROPPGUEL).

88


PRECACHEXIA STAGE OCCURS 5 DAYS AFTER WALKER-256 TUMOR SOLID INOCULATION.

Yamaguchi, E. Y.1; Signori, L.1; Kamakura, L.S.1; Blegniski, F. P.1; Guarnier, F. A.1

1 Department of General Pathology, LAFAM, Universidade Estadual de Londrina, Londrina,


Introduction and objectives: Cachexia is a typically cancer-related disorder clinically classified in 3

stages of evolution and severity: Precachexia, the initial and often barely perceptible beginning of

cancer cachexia trajectory, classified as an involuntary weight loss (i.e., ≤ 5%); Cachexia is

characterized as weight loss ≥ 5% or weight loss ≥ 2% associated with BMI ≤ 20 Kg/m2 or sarcopenia;

in refractory cachexia, the cachexia can be clinically refractory as a result of very or the presence

of rapidly progressive cancer unresponsive to anticancer therapy. The clear classification of stages

in cachexia provides context for early multimodal intervention (precachexia) or symptom-control

intervention (refractory cachexia). The objective of the preset study was to determine cachexia

stage after 5 days of Walker-256 solid tumor implantation.

Material and methods: Male Wistar rats (200-230g) were divided into two groups: Control and Walker-

256 tumor-bearing rats (Tumor). Control group received 0.5 mL vehicle injection and Tumor group

received a Walker-265 cell suspension (8x107 cells in 0.5 mL of PBS, s.c. into the hindlimb flank). In order

to determine cachexia stage, food intake, body weight and core temperature were controlled daily

in all groups. Handgrip strength test was performed with a commercial digital grip force meter.

Animals were familiarized with the device for 4 days previously cachexia induction. After tumor

inoculation, all animals were exposed to the grip force test daily. The mean of four consecutive trials

was recorded (g) and further normalized by body weight. Body mass index (BMI), defined as body

weight in grams divided by the square of nose-anus length in centimeters (g/cm2), was registered on

the same way. After 5 days, animals were euthanized (approved by Ethics Committee on Animal

Experimentation/Universidade Estadual de Londrina, ref.9775). Parametric values were compared by

One-way ANOVA followed by Tukey post-test. Comparison between groups in multiple time point

analysis, were compared by two-way ANOVA. Differences were considered statistically significant

for p values <0.05.

Results: While the Control group presented, in the time interval of 5 days, weight gain of 10.51±2.78%

when compared to its initial weight, Tumor showed loss of 2.77±9.11% of general body wasting

(p<0.05). Whilst Control group kept a regular amount of food during the five days, Tumor group

achieved 16.49% less consumption on the 5th day (from 22.37±0.75 on day 1 to 18.68 ±1.43

g/day/animal on day 5; p<0.05). Core temperature showed to be decreased on Tumor group during

almost the entire experimental design (from 34.91±0.16 °C on day 1 to 35.40±0.22 °C on day 5) when

compared to Control (from 35.70±0.18 °C on day 1 to 35.85±0.26 °C on day 5). Hand grip strength

test and BMI did not show statistical difference between groups.

Conclusion: In the present study, as weight loss demonstrated to be around 2% (mean), no consistent

sarcopenia, strength and BMI loss was found, and parameters of anorexia and decreased

metabolism could be observed, we could establish that precachexia occurs 5 days after Walker-256

tumor inoculation, a previous stage to massive muscle catabolism.

Keywords: Precachexia, cancer cachexia stage, muscle mass loss.

Grants: Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do

Paraná.

89

PREVENTIVE CREATINE SUPPLEMENTATION DOES NOT INFLUENCE TUMOR AGGRESSIVENESS IN WALKER-

256 TUMOR-BEARING RATS

Cella, P. S.1; Marinello, P.C. ¹,2; Padilha, C. 1; Testa, M.T.J ¹; Ribeiro D. F.¹; Guirro, P. B.¹;

Cecchini R. ²; Duarte, J.A ³.; Guarnier F. A. ²; Deminice R.1

¹State University of Londrina, Department of Physical Education, Londrina, PR, Brazil.

²Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR,Brazil

³University of Porto, CIAFEL, Faculty of Sport, Porto, Portugal. [email protected]

Introduction and objectives: Recent studies have shown that creatine supplementation has anti-

cancer effects such as prevention of cancer cachexia, chronic inflammation and tumor growth.

However, the effects of creatine supplementation on tumor development and microenvironment is

not known. Considering that creatine is known as a fast source of energy for tissues with high

energetic demand, is important to determine whether creatine modulates tumor cells

microenvironment, which frequently presents higher anaerobic energy rates than non-neoplastic

cells. Thus, the aim of this study was to analyze the effect of creatine supplementation on tumor

microenvironment, evaluating parameters of tumor aggressiveness.

Material and methods: twenty-four male Wistar rats (initially weight ~220g) were randomly assigned

in three groups (n=8/group): sham inoculated with PBS (S), tumor-bearing (T) and tumor-bearing

supplemented with creatine (TCr). After 11 days of supplementation, Walker256 tumor cells (7.0x107

cells in 0.5 ml of PBS) were inoculated in animals from the T and TCr groups. In TCr, creatine was

added in drinking-water (8g/L) for 21 days. Control rats were inoculated s.c. in the right flank with 0.5

ml of PBS. Ten days after tumor implantation, animals were weighed and euthanized. Tumors were

then excised, weighed and prepared for tumor microenvironment evaluation such as cell

proliferation and apoptosis determination by Ki67 immunohistochemistry and TUNEL assay,

respectively. Collagen deposit and tumor capsule thickness were assessed by picrosirius red staining

and the evaluation of malignant tumor morphology was performed after hematoxylin/eosin staining,

blindly. Water and food intake were monitored daily. For statistical analysis, student t test was used.

All experimental procedures were approved by the Animal Experimentation Ethics Committee of

State University of Londrina (CEUA nº 17628.2012.6).

Results: Tumor weight corresponded to approximately 3.5% of animal body weight in T and TCr.

Creatine supplementation did not accelerate tumor growth or increased tumor size. The

histopathological analysis demonstrated the presence of nuclear pleomorphisms and atypical

nuclei, with the presence of low-differentiated tumor cells, in both groups. The Scarff-Bloom-

Richardson histological grade analysis demonstrated no changes in tubule formation, nuclear

pleomorphism or in mitotic rates between T and TCr groups. Apoptosis and cell proliferation were not

altered on the same way. Thickness of tumor capsule and collagen deposition remained the same

between groups.

Conclusion: The analysis of the results indicates that preventive creatine supplementation in Walker-

256 tumor-bearing rats did not induce significant changes on tumor development, as well as no

interference in parameters of tumor aggressiveness, such as the level of cell differentiation and

proliferation.

Keywords: Tumor aggressiveness, creatine supplementation, Walker-256, carcinoma

Grants: CAPES (processo nº 88881.068035/2014-01).

90

PREVENTIVE PUNCTUAL APPLICATION OF LED IN SKELETAL MUSCLE REDUCES THE LEVEL OF OXIDATIVE

STRESS CAUSED BY ACUTE PHYSICAL EXERCISE.

Signori, L.1; Yamaguchi, Y. E.1; Kamakura, S. L.1; Correa, J. C. M.2; Ramos, S.de P.2;

Freitas, V. H.1; Blegniski, F. P.1; Guarnier, F. A.1

1Department of General Pathology, LAFAM, Universidade Estadual de Londrina, Brazil

2 Department of Histology, Universidade Estadual de Londrina, Brazil.

[email protected]

Introduction and objectives: The performance of acute physical exercise promotes increased

oxidative stress on skeletal muscle, that can be defined as an unbalance between free radicals and

antioxidants in the tissue. Oxidative stress can cause modifications in proteins, lipids, or DNA, leading

to impaired muscle function. The use of Light-Emiting-Diode (LED) therapy has been assigned in the

treatment or prevention of several muscle injuries, aiming increased ATP synthesis, modulation of

reactive oxygen species, or apoptosis reduction. The objective of the present study was to evaluate

the effects of LED therapy on skeletal muscle of adult rats submitted to high intensity physical training

and its relationship with oxidative stress.

Material and methods: Adult male Wistar rats (6 m.o.) were divided into three groups: Control (C),

trained (TRE) and trained+LED therapy (TLED). The TRE and TLED groups were submitted to a

familiarization period with the aquatic environment for five days. On the sixth day, animals received

LED irradiation (940nm) on both gastrocnemius muscles (4 J/cm2) and were immediately submitted

to 100 min swimming exhaustive exercise in a plastic container (37cm depth, 35cm diameter) under

continuous supervision and constant water temperature of 30-32ºC. After physical training, animals

were euthanized and gastrocnemius muscle collected (Ethics Committee on Animal

Experimentation/Universidade Estadual de Londrina, ref.08356). On skeletal muscle homogenates,

lipid peroxidation was determined by tert-butyl hydroperoxide-initiated chemiluminescense (CL),

and carbonyl protein content was determined. Non-parametric values were compared using

Kruskall– Wallis test followed by Dunn’s multiple comparison. Comparison between groups in multiple

time point analysis were compared by two-way ANOVA. Differences were considered statistically

significant when p<0.05.

Results: Gastrocnemius muscle showed a significance increased in TRE CL curve when compared to

C group, which was partially diminished in TLED (p<0.001 for all comparisons). The kinetic analysis in

the ascending part of TRE curve showed increase of 23.62% at initial speed, when compared to C.

LED therapy showed CL decreased levels (-15.19%) in the comparison with TRE. Similarly, TRE maximal

rate emission (565.9 ± 179,5 RLU/g tissue) increased 21.77% when compared to C (464.7 ± 148.6 RLU/g

tissue), which was partially diminished on LED therapy group (-15.19%) on the same way. Area under

the curve (AUC) and carbonyl protein content showed no significant difference in all comparisons.

Conclusion: Preventive punctual application of LED on the skeletal muscle showed decreased

effects caused by oxidative stress during exhaustive exercise, revealing one of the pathways

recruited to the mechanism involved on LED treatment.

Keywords: LED therapy, oxidative stress, skeletal muscle.

91

QUANTITATIVE ANALYSIS OF SUBMUCOSAL NEURONS IMMUNOREACTIVE TO THE HUC/D PROTEIN AND

SUBMUCOSAL ENTERIC GLIAL CELLS IMMUNOREACTIVE TO THE s100 PROTEIN IN THE JEJUNUM OF

WALKER-256 TUMOR-BEARING RATS TREATED WITH 1% L-GLUTATHIONE

Lima, F. G. M.1 ; Sestak, S. S.1, Oliveira, A. P.1, Ramalho, F. V.2, Silva, B. T.3, Frez, F. C. V.4, Sanches, M.

R.4, Cícero, L. R.4, Silva, M. P. A.3, Perles, J. V. C. M., Zanoni, J. N.5

¹ Universidade Estadual de Maringá, Department of Physiological Sciences, Maringá, PR, Brazil

² Universidade Estadual de Maringá, Department of Biotechnology, Genetics and Cellular Biology,

Maringá, PR, Brazil

³ Universidade Estadual de Maringá, Department of Biological Sciences, Maringá, PR, Brazil 4Universidade Estadual de Maringá, Department of Pharmaceutical Sciences, Maringá, PR, Brazil 5Universidade Estadual de Maringá, Department of Morphological Sciences, Maringá, PR, Brazil


Introduction and objectives: Cancer is a disease characterized by disordered cell growth that leads

to damage to tissues. Cachexia syndrome is the most common comorbidity present in oncological

patients and it is defined as an exacerbated weight loss, affecting the muscular and adipose tissues.

It causes oxidative stress and inflammation, that impairs the function of the enteric nervous system

components and causes losses on the gastrointestinal homeostasis regulation. In the attempt to

minimize such effects, antioxidants like L-glutathione come as an important tool. The aim was to

evaluate the neural and glial densities in submucosal plexus in the jejunum of Walker-256 tumor-

bearing rats supplemented with 1% Lglutathione.


State University of Maringá (UEM) under the approval number #7434160316. 20 male Wistar rats were

used and divided in four groups, with 5 animals per group: control (C), control treated with 1% L-

glutathione (CGT), tumor of Walker-256 (TW) and tumor of Walker-256 treated with 1% L-glutathione

(TWGT). The control rats were fed with Nuvilab® balanced standard rodents ration, while the treated

groups were given ration pellets with 1% L-glutathione. The animals were euthanized at the age of 69

days and the jejunum were collected for HuC/D and s100 immunohistochemistry of the submucosal

plexus. The quantitative analysis of neuronal cell bodies and enteric glial cells was made using the

software Image Pro® Plus 4.5. The data comparison was made by One-Way ANOVA block design

followed by Fischer post-test using Software Statistics® 7.1 and GraphPad Prism® 6.1.

Results: In the HuC/D analysis, between groups C and TW, there was a decrease of 28% in the

neuronal density (p<0.05). Also, there was a decrease of 11.5% between C and CGT groups (p<0.05)

and 18% between groups TW and TWGT (p<0.05). In the s100 analysis, it was also possible to observe

a 39.36% reduction between groups C and TW (p<0.05), a 14.76% decrease comparing C and CGT

groups (p<0.05) and no significative changes comparing TW and TWGT groups (p>0.05)

Conclusion: With these analyses, it was observable that the cachexia establishment itself can diminish

both neuronal and enteric glial populations, and that treatment with 1% L-glutathione was not

effective in the attempt to reestablish these populations. Also, by analyzing the data from healthy

groups that received 1% L-glutathione, it was clear that the supplementation itself is capable of

diminish both populations as well.

Keywords: Cancer Cachexia; Enteric Nervous System; L-glutathione

Grants: Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES)

92

TENASCIN X DEFICIENCY ALTERS THE EXPRESSION OF RECEPTOR- TYPE PROTEIN-TYROSINE

PHOSPHATASE ZETA/PHOSPHACAN IN ENTERIC COLONIC NEURONS OF MICE

Mendes1, Joana D’arc de Lima; Machado1, Camila Cristina Alves ; Watanabe1, Paulo da Silva;

Miqueloto2,Carlos Alberto; Aktar3, Rubina; Peiris3, Madusha; Blackshaw3, L Ashley; Aziz3, Qasim;

Araújo1, Eduardo José de Almeida.

1 State University of Londrina, Department of Histology, Londrina, PR, Brazil;

2 State University of Londrina, Department of General Biology, Londrina, Paraná, Brazil;

3 Queen Mary University of London, London, United Kingdom


Introduction and objectives: Perineuronal net (PNN) is a specialized extracellular matrix (ECM) around some

neurons in the central nervous system. It is predicted that PNN also exist in the enteric nervous system (ENS). The

RPTPζ/phosphacan is among the ECM molecules and it plays important role on cells of the nervous tissue. Little

is known about the presence and function of RPTPζ/phosphacan in structures that innervate the gastrointestinal

tract (GIT). Deficiency of specific ECM molecules alters the distribution of other molecules in the neuronal

microenvironment. Therefore, it is necessary studies that evaluate the involvement of ECM in gastrointestinal

disorders. In this sense, the aim of the present study was to assess the association of PTPRzeta/phosphacan with

the ENS in tenascin X knockout mice.

Material and methods: All procedures were approved by the Ethical Committee for Use of Animals at UEL

(032/2015), Brazil. Tenascin X-knockout mice (TNX-KO) and wildtype C57bl/6 mice (n= 5 each) were used. After

euthanasia, colon was removed and dissected in order to obtain two types of whole-mount preparations: one

consisting the submucosa layer (including the submucosal plexus) and the other consisting the longitudinal

muscular layer (including the myenteric plexus). The submucosal whole-mount preparations were incubated

with anti-PTPRzeta/phosphacan and anti-PGP9.5 (general marker for neurons) or antiPTPRzeta/phosphacan and

anti-calretinin (marker for non-cholinergic secretomotor neurons) or anti-PTPRzeta/phosphacan and anti-ChAT

(marker for cholinergic secretomotor neurons). The longitudinal muscular whole-mount preparations were

incubated with anti-PTPRzeta/phosphacan and anti-PGP9.5 (general marker for neurons) or anti-

PTPRzeta/phosphacan and anti-nNOS (marker for inhibitory motor neurons) or antiPTPRzeta/phosphacan and

anti-ChAT (marker for excitatory motor neurons and interneurons). The number of neurons was counted in 30

(submucosal) or 50 ganglia (myenteric) per animal and a total of 50 (submucosal) or 100 (myenteric) cell body

area were measured. GraphPad Prism6 software (GraphPad Software Inc., San Diego, CA, USA) was used for

all statistical analysis and the groups were compared considering the significance level was set to 5%.

Results: In the submucosal plexus, TNX-KO mice had less PGP9.5+ neurons producing PTPRzeta/phosphacan than

the wildtype mice (p<0.05). The number of non-cholinergic (Calretinin+) and cholinergic

secretomotor/vasodilator neurons producing PTPRzeta/phosphacan was greater in TNX-KO mice compared to

control (p<0.05). In addition, general (PGP9.5+) and cholinergic submucosal neurons were atrophied and

noncholinergic (Calretinin+) neurons were hypertrophied in TNX-KO mice (p<0.05). In the myenteric plexus, the

number of general neurons (PGP9.5+) was similar between both group of mice and the number of inhibitory

(nNOS+) and excitatory motor neurons and interneurons (ChAT+) increased in the TNX-KO mice in relation to the

wildtype mice (p<0.05). The cell body area of general (PGP9.5+) and cholinergic myenteric neurons were

atrophied and nitrergic neurons were hypertrophied in TNX-KO mice when compared to control (p<0.05).

Conclusion: The expression of PTPRzeta/phosphacan in the colonic neurons of mice can be changed due

Tenascin X deficiency.

Keywords: Receptor-Like Protein Tyrosine Phosphatases - Class 5, RPTPζ/phosphacan, Gastrointestinal Tract,

Extracellular Matrix

Grants: CAPES/PVE (process number 88881.068190/2014-01)

93

THE EFFECT OF TROLOX AND CURCUMIN ON THE ACTION OF DACARBAZINE ON MURINE MELANOMA

CELLS (B16F10).

Souza, P.G.B 1; Cecchini, R 1; Souza-Neto, F.P 1; Cecchini, A, L1.

1 Universidade Estadual de Londrina, Department of Molecular Pathology, Londrina, PR, Brazil

[email protected]

Introduction and objectives The annual increase in the number of new cases of melanoma in the

world is of greater concern due to its high mortality rate and lower efficacy of the treatment with

Dacarbazine (DTIC) of around 20%. Therefore several search seek for improvements in DTIC treatment

and / or new treatment alternatives. DTIC is the standard treatment in the most severe stages of

melanoma. Curcumin is widely used as a food dye and has been extensively investigated as a

natural antioxidant that acts to inhibit free radicals and lipid peroxidation by acting on the cellular

protection of cell macromolecules including DNA. Acts by inhibiting the enzymes of the p-450

complex responsible for the degradation of DTIC potentiating the effect of chemotherapy. For the

achievement the research, we made use of the murine melanoma cell line B16F10. The objective of

this study was to elicit the role of DTIC-induced oxidative stress in B16F10 murine melanoma cells and

to observe the effect of a pre-treatment with antioxidants on the action of Dacarbazine using

curcumin (Curcuma longa extract) and antioxidant standard (Trolox is the vitamin E analogue).

Material and methods: Three groups pre treatment (24h before treatment with DTIC) were make with

Trolox Group (500μM), Curcumin group (10 μM) and Trolox (500μM) + Curcumin

(10 μM) group. B16F10 cells were exposed to DTIC treatment (500 μg/mL) for 24h. Assays were

performed with cytotoxicity/proliferation and lipoperoxidation.

Results We found a higher cytotoxicity and decrease proliferation at the DTIC group in the cell line

B16F10 demonstrating chemotherapeutic effect on this cells (p<0.05). The pretreatment of Curcumin

group showed a potentiation the effect cytotoxic and antiproliferative of DTIC (p<0.05). Whereas

environment oxidative contributes to melanoma cells spread, it was observed also the formation of

membrane lipoperoxides at the groups. We found that the pre- treatment curcumin group presented

higher level of lipoperoxidation (p<0.05),

Conclusion It concludes that curcumin showed cytotoxic activity and antiproliferative effect on

murine melanoma B16F10 cells, improving the action of the chemotherapeutic DTIC by inhibiting the

process of degradation of the chemotherapeutic also increasing lipoperoxidation in the cells B16F10.

Financial support Araucária Foundation

Keywords: Dacarbazine, B16F10, Curcumin, Oxidative stress, Melanoma.

94

TREATMENT WITH SYSTEMIC OR CENTRAL ANTIOXIDANT ATTENUATE CARDIOVASCULAR, AUTONOMIC

AND OXIDATIVE PARAMETERS IN METABOLIC SYNDROME

Fernanda Novi Cortegoso Lopes1; Natalia Veronez da Cunha Bellinati 1; Marli Cardoso Martins-

Pinge 1

1Universidade Estadual de Londrina, Department of Phisiological Sciences, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Studies have shown that treatment with vitamin C (vit C), an antioxidant

substance, can improve metabolic, cardiovascular and central parameters present in hypertensive

animals. In this condition the autonomic nervous system seems to contribute to the altered

mechanisms. The metabolic syndrome (MS) that is characterized by hypertension, obesity and insulin

resistance, in addition to a low degree of inflammation and oxidative stress, has been investigated

in the autonomic aspects, however little is known about the participation of antioxidant substances

on cardiovascular dysfunction in this condition.

Thus, the objective of our study was to evaluate the effects of vit C treatment on peripheral and

central parameters of cardiovascular, autonomic, as well as the oxidative stress profile in obese non-

anesthetized adult rats.

Material and methods: Approved by ethics committee UEL nº 33645.2010.29, and 19678.2016.07.

Wistar rats received 4 mg/g body weight of monosodium glutamate or equimolar saline within the

first 5 days of life. The study was divided into 4 groups: control treated with water (CTR) and metabolic

syndrome (MSG) group treated with water, and CTR and MSG treated with vit C (50 mg / kg) orally

for 21 consecutive days from the 69th day of life. Other CTR and MSG animals were evaluated with

microinjection of vit C (10nm / 100nl) or sterile saline (100nl) in the rostroventrolateral medulla (RVLM)

in unanesthetized rats. At 90 days a catheterization of artery and femoral vein were performed, with

central guidecannula implantation directed to RVLM. After 24 hours, mean arterial pressure (MAP)

and heart rate (HR) were evaluated with rats with free movement and unanesthetized.

Results: MSG rats presented higher mean arterial pressure in relation to CTR (MAP CTR=103±6mmHg,

MAP MSG=121±4mmHg, p<0.05). After treatment with Vit C during 21 days, MSG rats presented

reduction on MAP without changes in heart rate (HR). Vit C treatment reduced LF normalized band

(MSG=49±7; MSGvit=26±4, p<0.05) increase HF normalized band (MSG=51±6; MSGvit=74±4, p<0.05)

and decrease LF/HF ratio (MSG=1.1±0.3; MSG vit=0.4±0.1, p<0.05) of pulse interval. MSG rats

presented increased plasma lipid peroxidation reverted by Vit C (p<0.001). MSG rats with implanted

guide cannulas to the RVLM also presented higher MAP in relation to CTR (p<0.05). Microinjection of

Vit C in the RVLM promoted increase in MAP in both groups although a lower increase in MSG group

(CTR: 9,42± 10,71 mmHg,; MSG: 6,72±1,92 mmHg, p<0.05).

Conclusion: Our results suggest that oxidative stress is involved in the pathophysiology of

hypertension derived from MSG obesity and the use of vit C attenuates these effects.

Keywords: Metabolic Syndrome; Arterial Pressure; Oxidative Stress.

Grants: CAPES

95

APOBEC3A/B DELETION POLYMORPHISM IS CORRELATED WITCLINICOPATHOLOGICAL AND

PROGNOSTIC PARAMETERS IN BREAST CANCER SUBTYPES

Vitiello, G. A. F.1; Sousa-Pereira, N.1; Motoori-Fernandes, C. Y.1; Bocchi, M.1;

Moretto, S. L.1; Pinsetta, M. O.1; Munuera, M.1; Spolador, L. H.1; Banin-Hirata, B.

K.1; Amarante, M. K.1; Losi-Guembarovski, R.2; Watanabe, M. A. E.1 1Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of General Biology, Londrina, PR, Brazil


Introduction and objectives: APOBEC3 is a family of cytidine deaminases whose genes are clustered

on chromosome 22 and participate in immune response to viruses by promoting site-specific

mutagenesis in virus-derived nucleic acids. Recently a signature related to APOBEC3-mediated

mutagenesis was identified in the genome of several cancers, including breast cancer (BC) and

APOBEC3A and APOBEC3B were implicated in carcinogenic process. A 29.5 kilobase deletion

polymorphism joining the 3’UTR from APOBEC3B with APOBEC3A exons was associated with BC by

increasing APOBEC3A activity and mutational burden. In the present work we aimed to investigate

the possible association between this polymorphism and susceptibility and clinical presentation for

BC subtypes in a Brazilian sample.

Material and methods: This study was approved by the Londrina State University Ethics Committee for

Research Involving Human Subjects (CEP/UEL 189/2013 – CAAE 17123113400005231) and all volunteer

donors signed a free-informed consent term before sample collection. Genomic DNA was extracted

from peripheral blood or from fresh tumor tissues from women diagnosed with BC and from neoplasia-

free women with no familial history of BC for control group, both from Londrina city. In total,

APOBEC3A/B (A3A/B) deletion polymorphism was analyzed through allele-specific PCR in 397 control

women and 341 BC patients. Ageadjusted logistic regression analyses were performed for case-

control study and correlation between polymorphism and clinicopathological features was

evaluated through Kendall’s Tau-b rank correlation test.

Results: The frequency of wild-homozygotes, heterozygotes and deleted homozygotes were,

respectively, 314 (79.1%), 74 (18.6%) and 9 (2.3%) in control group and 286 (83.8%), 51 (15.0%) and 4

(1.2%) in BC group. There was no association between A3A/B and BC susceptibility in any subtype

analyzed. However, in luminal A subtype A3A/B was positively correlated with tumor size (Taub = 0.12,

p = 0.03) and proliferation index (Ki67; Tau-b = 0.144, p = 0.04) and inversely correlated with lymph

node metastasis (Tau-b = -0.159, p = 0.02), while in triple negative BCs, it was positively correlated

with histopathological grade (Tau-b = 0.169, p = 0.04).

Conclusion: These results indicate that, differently from those observed in other populations

worldwide, A3A/B deletion polymorphism may not be a risk factor for BC in Brazilian population. The

correlation with clinicopathological parameters indicates that this polymorphism may have

prognostic implications in different BC subtypes: in luminal A this role seems paradoxical, since A3A/B

was correlated with enhanced proliferation of malignant cells but with diminished probability of

lymph node metastasis; in triple-negative tumors, A3A/B indicated poorly cellular differentiation.

These results might be explained by the enhanced mutational potential of A3A/B, which increases

genomic instability and anaplasia, while give rise to neoepitopes favoring immunologic recognition

and elimination of neoplastic cells. In conclusion, A3A/B deletion polymorphism may participate in

breast carcinogenesis, being a promisor candidate for prognosis biomarker in BC subtypes.

Keywords: APOBEC, mutagenesis, susceptibility, breast cancer, polymorphism.

Grants: CNPq, CAPES, Fundação Araucária e FAUEL.

96

Arthrographis kalrae FORMS BIOFILM AND INHIBITS THE PRODUCTION OF NITRIC OXIDE BY

MACROPHAGES IN VITRO

Bianca Dorana Oliveira Souza da Fonseca1; Janneth Josefina Escobar Arcos2; Eiko Nakagawa

Itano1 1State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

2State University of Londrina, Department of Microbiology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Arthrographis kalrae is a thermodymorphic fungus that causes a

complex neurological syndrome and lesions in different organs in mice. It is a fungus considered rare

in humans, but in different countries it has been described in different human clinical manifestations,

such as keratitis, sinusitis, meningitis, eumicetoma and onychomycosis. Considering the scarcity of

studies involving virulence factors and the pathogenicity of A. kalrae, the present study aimed to

demonstrate in vitro biofilm formation by this fungus and to analyze the immunopathological

response of macrophages infected with A. kalrae.

Material and methods: The A. kalrae IFM55165 isolate maintained at 37 °C in 4% Sabouraud dextrose

agar culture medium was used, with five days of growth. Biofilm assay was performed on 96-well

polystyrene plates with different incubation times (4, 12, 24, 36 and 48 hours), with the concentration

of fungal cells being adjusted to 1x108 cells/ml in supplemented RPMI 1640 medium with 2% glucose.

200 μl of the fungal suspension were deposited in the wells of the plates, which were then incubated

at 37 °C and 5% CO2. After the pre-adhesion time for 4 hours, the supernatant was removed from

each well and the total biofilm biomass of the 4 hours plate was determined. In the other plates, 200

μl of RPMI 1640 medium supplemented with 2% glucose and 10% fetal bovine serum (FBS) were

added, these plates were again incubated until their respective evaluation times. The biofilm total

biomass quantification was done through a 0.1% crystal violet solution and the reading was made

at 550 nm. For the evaluation of the immunopathological response, RAW 264.7 macrophages were

treated and/or infected according to the following groups: treated with LPS (positive control),

treated with LPS and infected with planktonic cells (LPS + P), treated with LPS and infected with

biofilm cells (LPS + B), infected with planktonic cells (P) and infected with biofilm cells (B) for 15, 24

and 48 hours. Subsequently, the production of nitric oxide by macrophages was quantified by the

accumulation of nitrite present in the culture supernatants through the Griess reagent. Statistical

analysis was performed on Graphpad Prism 6.01 software (Graphpad Software, San Diego,

California, USA) and assumed to be significant p-value <0.05.

Results: The results showed that A. kalrae is able to form biofilm in vitro and in 24 hours of growth, there

is formation of mature biofilm. A. kalrae was also able to inhibit the production of nitric oxide from

infected macrophages (p <0.0001) and A. kalrae biofilm cells had an even greater capacity for

inhibition (p <0.0001).

Conclusion: This study represents the first demonstration of biofilm formation by this fungus and its

effect on the production of nitric oxide by macrophages. Further studies are needed to verify if the

capacity of biofilm formation is an important virulence factor of A. kalrae and whether the inhibition

of nitric oxide production in macrophages represents one of its mechanisms of pathogenicity.

97

CELL ADHESION MOLECULES AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 IN PATIENTS WITH

PROSTATE CANCER: ASSOCIATION WITH THE PRESENCE OF METASTASIS AND RISK STRATIFICATION

Ana Gabriela da Silva Bonacini1; Brunna França Robles1, Alexsandro Koike2 , Edna Maria Vissoci

Reiche3, Rubens Cecchini4, Andréa Name Colado Simão3

1Laboratory of Research in Applied Immunology, University of Londrina, Londrina, Paraná, Brazil

2 Cancer Institute of Londrina, Laboratory of Research in Applied Immunology, University of

Londrina, Londrina, Paraná, Brazil

3 Department of Pathology, Clinical Analysis and Toxicology, Laboratory of Research in Applied

Immunology, University of Londrina, Londrina, Paraná, Brazil

4 Department of Pathology Sciences, University of Londrina, Londrina, Paraná, Brazil

[email protected]

Introduction and objectives: Prostate cancer (PCa) is the most frequent prostatic diseases in aging

men. Cancer metastasis is the culmination of a series of steps: cell chemotaxis, cell adhesion, and

selective tumor growth. Cell adhesion molecules (CAMs) are complex network molecules that

mediate cell-cell and cell-matrix interactions and play a crucial role in metastasis processes.

Abnormal expression of plasminogen activator inhibitor type-1 (PAI-1) is closely related to cancer. To

determine the profile of CAMs and PAI-1 in PCa patients and to associate them with the presence

of metastasis and the different degrees of risk for metastasis in this disease.

Material and methods: This is a prospective case-control study that consisted of 213 men with age

from 30 to 90 years; of them 149 patients with PCa diagnosed at the Uro-oncology clinic of the

Cancer Hospital of Londrina and 64 controls. First, PCa group was subdivided in two groups: without

metastasis (Met-, n=108) and with metastasis (Met+, n=41). Subsequently, the PCa group was

subdivided according to the stratification of risk for metastasis following the guidelines of the National

Comprehensive Cancer Network (NCCN): Group A: very low and low risk (n=28); group B: favorable

and unfavorable intermediate risk (n=41); Group C: high and very high risk (n=77). Anthropometric

and clinical data were obtained according to a standardized data collection questionnaire for this

study. Plasma levels of platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell

adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin

and PAI-1 were measurement. Metabolic Syndrome (MetS) was defined following the Adult

Treatment Panel III criteria. Informed consent was given by all the subjects included in this work. The

Human Ethics Committee from the State University of Londrina approved the protocol number: CAAE

56182916.7.0000.5231.

Results: Patients with PCa Met+ were significantly distinguished from controls by decreased PECAM-

1 (p=0.029; OR: 1.000; 95% CI: 0.999-1.000) and PAI-1 (p=0.020; OR:1.000; 95% CI:1.0001.000); and

differed from PCa Met- by decreased VCAM-1 (p=0.043; OR: 1.000; 95% CI: 1.000-1.000). Levels of P-

selectin (p= 0.031; OR: 1.000; 95% CI: 1.000-1.000) and PAI-1 (p=0.017; OR: 1.000; 95% CI: 1.000-1.000)

decreased between patients with PCa Met- when compared to controls. The results were adjusted

by age, inflammation, prostate size and presence of MetS. Levels of PECAM-1 among the high risk

group were lower than intermediated risk (p=0.039; OR: 1.000; 95% CI: 1.000-1.000). The results were

adjusted by age and inflammation.

Conclusion: Our data demonstrated that PECAM-1, P-selectin, and PAI-1 have an important role in

pathophysiology of PCa, but only VCAM-1 may be differ metastatic PCa, independently of age,

prostate size, presence of MetS, and inflammation. PECAM-1 may be differentiate patients with high

risk for metastasis from those with intermediate risk, independently of age and inflammation.

Keywords: Prostate Cancer, Cell Adhesion Molecules, Plasminogen Activator Inhibitor Type-1,

Metastasis, VCAM-1, PECAM-1.

98

CRANBERRY JUICE DECREASES DISEASE ACTIVITY IN WOMEN WITH RHEUMATOID ARTHRITIS

Thimóteo, N.S.B1; Mari, N.L 1; Iryioda, T.M.V 1; Alfieri, D.F1; Rego, B.E.F1; Scavuzzi, B.M1; Fatel,E.2,

Lozovoy, M.A.B 1,3; Simão,A.N.C.1,3; and Dichi,I.4.

1 Research Laboratory in Applied Immunology – University of Londrina, Londrina, Paraná,

Brazil. 2 Department of Nutrition – Universidade Federal Fronteira Sul, 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.

[email protected]

Introduction and objectives: RA is characterized by the progressive destruction of the joints as well as

by extra-articular involvement and is thus classified as a systemic inflammatory disease. The disorder

likely develops from individuals who are genetically susceptible to abnormal imune responses and

have been exposed to specific environmental factors. Studies have shown that cranberry

(Vaccinium macrocarpon) has antiinflammatory and antioxidant effects, however, the effects of

cranberry juice consumption has not been studied in patiens with rheumatoid arthritis (RA). Thus, the

aim of this study was to verify the effect of cranberry juice consumption on several inflammatory

biomarkers and on the disease activity of patients with RA.

Material and methods: A prospective study was conducted with 41 female patients diagnosed with

RA. The disease activity measured by DAS28 (Disease Activity Score 28) and anti-cyclic citrullinated

peptide (anti-CCP) antibodies, and several inflammatory and biochemical biomarkers were

analyzed. The first group (control group C, n = 18) maintained the usual diet, the second group

(cranberry group, n = 23) consumed 500mL / day of low calorie cranberry juice. This study was

conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures

involving human subjects/patients were approved by the Ethical Committee of the University of

Londrina, Paraná, Brazil (CAAE: 13426014.6.0000.5231). Written informed consent was obtained from

all patients.

Results: Regarding the baseline values, the group who received the cranberry juice intervention

presented a decrease in the values of DAS 28 (p = 0.048) and anti-CCP (p = 0.034) after 90 days of

treatment, whereas changes in inflammatory biomarkers were not found.

Conclusion: The presente study indicates that cranberry juice decreased disease activity and

therefore have beneficial effects in RA patients, although larger and long term studies are needed

to definitely probe this effect and to clarify the mechanisms involved

Keywords: cranberry, inflammation, bioactive compounds, polyphenols, rheumatoid arthritis.

Grants: This research was supported by the National Research Council of Brazil - CNPq. Juxx

Company supplied the cranberry juice.

99

DIFFERENTIAL PROFILE OF ADHESION MOLECULES IN SYSTEMIC LUPUS ERYTHEMATOSUS VERSUS

RHEUMATOID ARTHRITIS: A MACHINE LEARNING STUDY

Lorena Flor da Rosa Franchi Santos1, Nicole Perugini Stadlober1, Tatiana Mayumi Veiga Iriyoda2,

Neide Tomimura Costa3, Tamires Flauzino4, Marcell Alysson Batisti Lozovoy5, Edna Maria Vissoci

Reiche5, Sira Sriswasdi6, Michael Maes7, Isaias Dichi, Andréa Name Colado Simão5

1 University of Londrina, Department of General Pathology, Laboratory of Research in Applied

Immunology, Londrina, PR, Brazil 2 Pontifical Catholic University of Paraná (PUC/PR), Department of Rheumatology, Londrina,

PR, Brazil 3 University of Londrina, Department of Rheumatology, Londrina, PR, Brazil

4 University of Londrina, Laboratory of Research in Applied Immunology, Londrina, PR, Brazil 5 University of Londrina, Department of Pathology, Clinical Analysis and Toxicology,

Laboratory of Research in Applied Immunology, Londrina, PR, Brazil 6 The Wistar Institute, Center for Systems and Computational Biology and Molecular and

Cellular Oncogenesis Program, Philadelphia, United States 7 Deakin University, IMPACT Strategic Research Centre, School of Medicine, Geelong, VIC,

Australia 8 University of Londrina, Department of Internal Medicine, Laboratory of Research in Applied

Immunology, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are chronic

autoimmune diseases and the development of both diseases requires the combination of genetic susceptibility

factors and environmental influences. Despite many factors involved in the pathophysiology of SLE and RA, it is

known that cell adhesion molecules (CAM) play an important role. CAM are strongly related to inflammation,

endothelial dysfunction, onset of cardiovascular diseases, and insulin resistance with a high prevalence of

metabolic syndrome (MetS). The main aims of the present study were to compare the CAM profile in SLE and

RA and to examine the impact of the MetS on CAM.

Material and methods: 104 SLE patients and 124 RA patients were enrolled. CAM were determined by Luminex®

platform. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the

Ethical committee of the University of Londrina, Paraná, Brazil approved all procedures involving SLE (CAAE

01865212.0.0000.5231) and RA patients (CAAE 06405812.1.0000.5231). Written informed consent was obtained

from all subjects.

Results: Platelet endothelial cell adhesion molecule 1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-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 when compared to RA patients. Machine learning showed that

CAM highly significantly discriminated groups and that increased PECAM-1, VCAM-1 and P-selectin was the

most important features of SLE. Neural Network analysis showed that SLE was significantly discriminated from RA

with a sensitivity of 96.8% and a specificity of 85.4% and an area under the ROC curve of 0.956. SIMCA analysis

showed that both SLE and RA are qualitatively different classes with respect to CAM levels. Patients with SLE

showed a higher incidence of MetS and atherogenic index of plasma (AIP) than those with RA, with VCAM-1,

E-selectin and PAI-1 being increased in MetS. CAM levels have significant effects on blood pressure, AIP and B

cell function.

Conclusion: Our data demonstrate that CAM are significantly higher in SLE versus RA and that a combination of

VCAM-1, PECAM-1 and E-selectin can be used to discriminate SLE from RA. MetS increases VCAM-1, E-selectin

and PAI-1, while different CAM may modulate metabolic variables including blood pressure and AIP.

Keywords: Systemic Lupus Erythematosus; Rheumatoid Arthritis; adhesion molecules.

100

DISABILITY IN MULTIPLE SCLEROSIS IS PREDICTED BY A TH17 CYTOKINE PROFILE, CARBONYL PROTEINS,

METABOLIC SYNDROME AND METABOLIC BIOMARKERS: RESULTS OF MULTIVARIATE AND MACHINE

LEARNING PROCEDURES

Tamires Flauzino1, Claudia Mara Ribeiro1, Andrea Name Colado Simão2, Wildea Lice de Carvalho Jennings

Pereira3, Daniela Frizon Alfieri1, Sayonara Rangel Oliveira2, Ana Paula Kallaur1, Marcell Alysson Batisti Lozovoy2,

Damacio Ramón Kaimen-Maciel4, Michael Maes5,6 Edna Maria Vissoci Reiche2

1Laboratory of Applied Immunology, Health Sciences Center, University of Londrina, Paraná, Brazil; 2Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, University of Londrina,

Paraná, Brazil; 3Department of Clinical Medicine- University of Londrina, Londrina, Paraná, Brazil;

4Santa Casa de Misericórdia, Londrina, Paraná, Brazil; 5 Impact Strategic Research Centre, School of Medicine, Deakin University, Geelong, Victoria, Australia;

6Department of Psychiatry, King Chulalongkorn Memorial Hospital, Chulalongkorn, Bangkok, Thailand;

[email protected]

Introduction and objectives: Multiple sclerosis (MS) is a chronic immune-mediated disease that affects the

central nervous system (CNS) and characterized by demyelination and neurodegeneration. The etiology of MS

is multifactorial and it has been suggested that an autoimmune inflammation in early MS is primarily mediated

by adaptive immune responses driven by a Th1, Th17, B cell, and autoantibodies, while the later, chronic stages

of MS are characterized by a compartmentalized immune response in the CNS with activated microglia and

macrophages. Mounting evidence suggests that autoreactive T cells generated in the periphery that have the

ability to enter to the CNS by crossing the brain blood barrier. There, they persist and generate an inflammatory

cascade and increase reactive oxygen and nitrogen species leading to formation of lesions and neurologic

deficits. MS is a heterogeneous disease and the search for more adequate biomarkers predicting disease course

is currently an active area of research. The concomitant evaluation of immuneinflammatory (Th1, Th17 and Treg

response), metabolic pathways (MetS, lipid metabolism, homocysteine) and oxidative stress biomarkers may

provide more accurate predictive models for the evaluation of disease disability in MS patients. Hence, the aim

of the present study was to evaluate in the immune-inflammatory, metabolic and oxidative and nitrosative stress

(IMO&NS) biomarkers as predictors of disability in MS patients.

Material and methods: The protocol was approved by the Institutional Research Ethics Committees of University

of Londrina, Paraná, Brazil (CAAE: 22290913.9.0000.5231) and all of the individuals invited were informed in detail

about the research and gave written informed consent. The study included 122 patients with MS, at admission

(baseline), classified according to the clinical form as relapsing-remitting MS (RRMS) (n =103) or progressive MS

(n=19), such as primary progressive MS (PPMS, n=3) and secondary progressive MS (SPMS, n=16). The disability

was evaluated using the Expanded Disability Status Scale (EDSS) (baseline) and categorized as mild (EDSS<3) or

moderate/high (EDSS≥). IMO&NS biomarkers were evaluated in peripheral blood samples.

Results: Patients with moderate/high disability were older and showed higher homocysteine, uric acid,

advanced oxidized protein products (AOPP) and low-density lipoprotein (LDL)cholesterol and higher rate of

metabolic syndrome (MetS), while high-density lipoprotein (HDL)-cholesterol was significantly lower than in

patients with mild disability; 84.6% of all patients were correctly classified in these EDSS subgroups with an area

under the receiving operating curves (AUC/ROC) of 0.842. Moreover, 36.3% of the variance in EDSS score was

explained by age, a Th17/T regulatory (zTh17/Treg) profile, LDL/HDL ratio and homocysteine (all positively

related) and body mass index (BMI) (negatively related). In addition, neural network analysis showed that

carbonyl proteins were associated with an increased EDSS score.

Conclusion: Taken together, the results showed that a pro-inflammatory Th17 profile coupled with age and

increased oxidative stress (carbonyl proteins) were the most important biomarkers that could predict high EDSS

followed at a distance by homocysteine, MetS and LDL/HDL ratio. These data underscore that IMO&NS

pathways play a key role in increased disability in MS patient and may be possible new targets for the treatment

of MS. Moreover, a panel of these laboratory biomarkers may be used to predict severity of disability in MS.

Keywords: Multiple sclerosis; disability; EDSS; inflammation; oxidative stress; biomarkers

101

FOXP3 POLYMORPHISMS rs3761548 AND rs2232365 DO NOT INFLUENCE INTERLEUKIN-10 LEVELS IN

HPV-INFECTION

Fernando Cezar-dos-Santos1; Kleber Paiva Trugilo1; Fernanda Costa Brandão Berti1; Ana Paula

Lombardi Pereira1; Nádia Calvo Martins Okuyama1; Michelle Mota Sena1; Érica Romão Pereira1;

Rodolfo Sanches Ferreira1; Paola Singi1; Aline Esposito1; Ana Luiza Labbate Bonaldo1; Maria

Angelica Ehara Watanabe1; Karen Brajão de Oliveira1.

1State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Introduction and objectives: Forkhead box P3 (FOXP3) transcription factor is the main molecule that

regulates CD4+ CD25+ T cell regulatory (Treg) development and function. Treg has

immunosuppressive activity, with high production of TGF-ß and IL-10. These immunoregulatory

cytokines induce an immune tolerant microenvironment in viral infections, inhibiting viral clearance;

in HPV infection, IL-10 favors HPV immune escape. Single nucleotide polymorphisms (SNPs) in FOXP3

regulatory regions may alter Treg activity by regulating FOXP3 gene transcription. Thus, we aim to

assess the influence of FOXP3 SNPs rs3761548 and rs2232365 in intron-1 region on IL-10 plasmatic and

cervical levels in an HPV infection-context.

Material and methods: The project was approved by the Institutional Ethics Committee Involving

Humans of the State University of Londrina, Londrina- PR, Brazil (CEP/UEL 133/2012; CAAE

05505912.0.0000.5231). Study population was composed by 308 women recruited from several health

services in Londrina (Paraná, Southern Brazil), between March 2015 and December 2016. All study

subjects received clear instructions about the project and signed a formal consent, prior to sample

collection (cervical secretion and peripheral blood). FOXP3 SNPs genotyping was carried out by

polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and

HPV detection was made by PCR. IL10 plasmatic and cervical levels were measured by enzyme-

linked immunosorbent assay (ELISA). Interactions between the IL-10 levels and FOXP3 inheritance

models, considering HPV infection, were analyzed by Two-Way Analysis of Variance (ANOVA) and

Tukey’s posthoc test. All tests were two-tailed with a significance level set at 0.05.

Results: IL-10 plasmatic levels were obtained from 146 HPV-infected and 162 uninfected women.

From these, IL-10 cervical levels were verified in 30 HPV-infected and 70 uninfected women.

Considering the within-subjects analysis of FOXP3 genotypes and inheritance models, we were not

able to detect any significant differences both in plasmatic and cervical IL-10 levels of HPV-infected

and uninfected women. It is important to highlight that we found a significant difference in between-

subjects analysis, that is, IL-10 cervical levels between HPV-infected versus uninfected women

(P=0.026).

Conclusion: It leads us to suggest that although IL-10 is a putative binding site to FOXP3 transcription

factor, IL-10 transcription and translation may occur in a FOXP3-independent manner. This inference

would explain why the FOXP3 SNPs rs3761548 and rs2232365 (whose alleles C and G, respectively, are

known to increase FOXP3 gene expression) do not interfere with IL-10 production. Therefore, further

studies are required to better clarify whether FOXP3 transcription factor and IL-10 gene present a

physical interaction and may somehow influence on IL-10 production in different disease contexts.

Keywords: FOXP3 intronic variant, FOXP3 SNP, CD4+ CD25+ T cell, Treg

Grants: This work was supported by Conselho Nacional de Desenvolvimento científico e


State University of Londrina Coordination for Post-Graduation (PROPPG-UEL) and Fundação

Araucária do Paraná – Programa de Pesquisa para o SUS.

102

INCREASED ADIPONECTIN, TH2, AND TREG RESPONSES IN PATIENTS WITH SYSTEMIC SCLEROSIS: USE AS

A PREDICTOR OF DIAGNOSIS

Meline Angélica Cunha Rotter Ferreira1,2, Gustavo Fassina1,2, Nicole Perugini Stadtlober2, Neide

Tomimura Costa1, Cam il a Ca ta l di d e A lc a nt ar a 1, M ar c os Ri be ir o 3, Leila Droprinchinski

Martins 4, Edna Maria Vissoci Reiche 2,5, Isaias Dichi 3, Michael Maes6,7, Andréa Name Colado

Simão2,

1 Department of Rheumatology, University of Londrina, Londrina, PR, Brazil

2 Laboratory of Research in Applied Immunology, University of Londrina, Londrina, PR,

Brazil

3 Department of Internal Medicine, University of Londrina, Londrina, PR, Brazil

4 Technologic University of Paraná, Campus Londrina, Londrina, PR, Brazil


Immunology, University of Londrina, Londrina, PR, Brazil

6 IMPACT Strategic Research Centre, School of Medicine, Deakin University, Geelong, VIC,

Australia.

7 Department of Psychiatry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

[email protected]

Introduction and objectives: Immune dysregulation is a central process in the pathogenesis of

Systemic Sclerosis (SSc). Lymphocytes and macrophages produce cytokines, which induce tissue

damage, recruit additional inflammatory cells, and promote extra-cellular matrix production and

fibrosis. Hence, the main objective of this study is to evaluate cytokine profiles and the pro and anti-

inflammatory in SSc patients as well as to determine models of cytokine profiles which could help to

predict the diagnosis.

Material and methods: This study recruited 117 individuals. Forty-two patients with SSc (SSc diffuse n=7

and SSc limited n=35) were selected from the ambulatory of Rheumatology of the University Hospital

of Londrina, Paraná, Brazil and 75 were healthy controls.

Results: Pro-inflammatory profiles: M1 (IL-1β + IL-6 + TNF-α), Th1 (IL-2+ IFN-γ). The antiinflammatory

cytokines formed the profile Th2 +Treg: (IL-4 + TGF-β) and the anti-inflammatory system (IL-4 + TGF-β

+ Adiponectin). Diagnosis of SSc was strongly associated with altered cytokine levels, accounting for

67.6% of its variance, with a strong impact on IL-2 (43.7%), IL4 (26.0%), IL -6 (12.7%), TGF-β (19.3%) and

adiponectin (24.2%). The results also showed that the diagnosis is mainly involved in the Th2 and Treg

profiles, explaining 37.9% of their variation. When adiponectin was added to the anti-inflammatory

profile, the variation was increased to 45.7%. In addition, a statistical model was proposed using pro

(IL-2 and IFN-γ) and anti-inflammatory (TGF-β and adiponectin) cytokines to predict SSc with high

sensitivity (100%) and specificity (91.7%).

Conclusion: Patients with SSc have shown an increase in the pro-inflammatory and antiinflammatory

response, with anti-inflammatory response predominance. This finding may be responsible for the

fibrotic characteristic of the disease. In addition, this is the first study that demonstrated higher

adiponectin levels in SSc patients. More studies are necessary to confirm the present results as well

as to investigate the contradictory findings on adiponectin, especially verifying the levels of its

specific isoforms.

Keywords: Systemic sclerosis; Scleroderma; Cytokines; Adiponectin.

103

INFLAMMATORY MONOCYTES CONTRIBUTE TO AN EXACERBATE INFLAMMATION AND ORGAN

DAMAGE DURING SEPSIS

Guilherme C. M. Cebinelli¹; Kalil A. de Lima¹; Fernanda V. S. Castanheira¹; Carlos H. Hiroki¹; Mikhael

H. F. de Lima¹; Valter V. S. Monteiro¹; Daniele C. Nascimento¹; José C. A. Filho¹; Thiago M. Cunha¹;

Fernando Q. Cunha¹.

1 FMRP- University of Sao Paulo, Department of Pharmacology, Ribeirao Preto, Brazil. Email:

[email protected]

Introduction and objectives: Sepsis is defined as a life-threatening organ dysfunction caused by a

dysregulated host response to infection. In sepsis, neutrophils migrate to infection focus and mediate

infection control and sepsis survival. Besides neutrophils, inflammatory monocytes (IMs) are also

involved in the control of infections. However, in sepsis, the role of inflammatory monocytes still

unclear. Given that, the aim of our work was to address these open questions by using an

experimental sepsis model (CLP).

Results: Firstly, to evaluate the role of IMs during sepsis, we assessed the frequency of these cells

between moderate and grave sepsis. We observed that during both levels of sepsis, IMs emigrate

from bone marrow. However, in lethal sepsis, we observed a failure of these cells to migrate to

infection focus and an increase of the migration into organs. Moreover, we used CCR2 knockout

mice that have deficiency of IMs emigration from the bone marrow. As expected, the CCR2-/- mice

had a diminished migration of IM to the infection focus. Additionally, we observed that they were

less susceptible to sepsis, nevertheless, the bacteria load and inflammation cytokines (CCL2, CXCL1,

IL-6 and IL-10) was the same to WT. On another hand, cytokines level in serum were increased in WT

mice when compared to CCR2-/- (CXCL1, IL-6 and IL-10). The same result was observed in organs

(increase of IL6 and IL-10), as well as an increase of the IMs frequency (lung and kidney), and organ

damage markers (ALT and CK-MB). To certify the role of IMs contributing to these phenotypes, we

sorted and transferred these cells from WT to CCR2-/- mice 1h after sepsis induction, and we observed

that CCR2-/- that received cells were more susceptible to sepsis than CCR2-/- that did not received.

Conclusion: In conclusion, we suggest that inflammatory monocytes contribute to increase systemic

inflammation leading to organ damage and increase sepsis susceptibility.

Keywords: Keywords: inflammatory monocytes, sepsis, inflammation.

Grants: FAPESP, CNPq and CAPES.

104

INHIBITION OF Trypanosoma cruzi INVASION INTO THP-1, A HUMAN MONOCYTIC CELL LINE, BY

NIMESULIDE DRUG

Raquel Pirea Nakama1; Helena Tiemi Suzukawa¹, Phileno Pinge-Filho¹

State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Introduction and objectives: Chagas disease or American human trypanosomiasis is caused by

Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. Cell invasion by T. cruzi and

its intracellular replication are essential to the parasite's life cycle and for the development of disease.

The infective trypomastigote forms of the parasite can invade several human blood cell populations,

including monocytes and macrophages. The aim of this study was to investigate the involvement of

the host's cyclooxygenase-2 (COX-2) enzyme during T. cruzi invasion.

Material and methods: The experimental protocols used were approved by the Ethics Committee in

Research Involving Human Subjects (Process 24841.2016.41). The parasites used were obtained

through the blood trypomastigotes infection of Trypanosoma cruzi (Y strain) in LLCMK2 cells. The THP-

1 cells, a lineage of human monocytes, were stimulated with 5ng/mL of PMA during two days for

differentiation in macrophages. As for the internalization test, it was distributed 10⁵ cells/well plates in

plates of 24 well plates with glass coverslips and treated with nimesulide (30ug/mL, 60ug/mL,

80ug/mL) for 1 hour, followed by cell washing and infection with 5:1 trypomastigotes forms of T. cruzi

overnight. In addition, we quantified nitric oxide (NO) production by THP-1 Cells. NO was carried out

through nitrite quantification of the supernatant of macrophages culture in plates of 96 well plates,

with 2x10⁵ cells/well plates after 24 hours and 48 hours post-infection, through the Griess method. The

cell viability of the macrophages submitted to different concentrations of the drugs used was

assessed by the MTT test. The phenotypic identity of the obtained cells was determined by flow

cytometry.

Results: The THP-1 cells and the macrophages obtained after differentiation with PMA were

confirmed phenotypically through flow cytometry, evidenced by the rise of the surface marker CD14

in the cells after exposure to the PMA, which is one of the characteristics of activated cells. Our results

show that COX-2 blockade through nimesulide decreased the internalization of T. cruzi in

differentiated macrophages from THP-1 cells.

Conclusion: The use of the protocol chosen for differentiation of THP-1 cells into macrophages was

effective, and cells that presented phenotypic characteristics similar to those described in the

literature were obtained. The doses of nimesulide used were able to decrease the entry of T. cruzi

into human macrophages. The production of nitric oxide in our assays was not significant, this may

be correlated to the human origin of the cell line used and absence of IFN-γ.

Keywords: Trypanosoma cruzi, Nimesulide, Eicosanoids, nitric oxide, THP-1 cells.

105

METABOLIC SYNDROME AGRAVATES CARDIOVASCULAR, OXIDATIVE AND INFLAMMATORY

DYSFUNCTION DURING THE ACUTE PHASE OF TRYPANOSOMA CRUZI INFECTION IN MICE

Bruno Fernando Cruz Lucchetti1; Natalia Boaretto1, Fernanda Novi Cortegoso Lopes1, Aparecida

Donizette Malvezi2, Maria Isabel Lovo Martins2, Vera Lúcia Hideko Tatakihara2, Victor Fattori2, Rito

Santo Pereira2, Waldiceu Aparecido Verri Jr2, Eduardo Jose de Almeida Araujo3, Phileno Pinge-

Filho2, Marli Cardoso Martins-Pinge1

1 Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina,

Londrina - PR, Brazil 2Department of Pathological Sciences, Center of Biological Sciences, State University of Londrina,

Londrina - PR, Brazil 3Department of Histology, Center of Biological Sciences, State University of Londrina, Londrina - PR,

Brazil


Introduction and objectives: Chagas disease (CD) is one of the leading neglected tropical infectious

diseases, and people living in endemic regions are at potential risk for developing CD. The incidence

of obesity has been increasing lately in countries endemic to DC, leading to high blood pressure,

insulin resistance, increased levels of inflammation and oxidative stress. This picture promoted by

obesity may have effects on the development of CD. In the present study, we evaluated the

influence of obesity on acute infection by Trypanosoma cruzi, on cardiovascular, oxidative,

inflammatory, and metabolic parameters.

Material and methods: All the procedures as well as the maintenance of T. cruzi were approved by

the Committee of Ethics and Research of Animals of the State University of Londrina with the protocol

number: 19665.2016.03. Obese swiss mice, on the 70th day of life were submitted to intraperitoneal

infection with 5x10² trypomastigote forms of the Y strain. The cardiovascular parameters were

evaluated before and during infection. The parasitemia and the survival up to 30º dpi were analyzed.

In the 13th dpi the tissue and blood was collected. Cytokines were determined in plasma using

commercial kit CBA. Nitric oxide (NO) was determined by the cadmium and Griess technique. The

antioxidant capacity and levels of oxidative stress were determined by the ABTS, FRAP, NBT and

TBARS assays. The insulin tolerance test was performed in 13th dpi.

Results: Obese group had higher mean arterial pressure (MAP) before infection. We observed a

higher parasitemia in the infected obese group (IOG) compared to the infected control (ICG) on

days 13 and 15 post infection. All animals of IOG died until the 19th day after infection (dpi) while

87.5% of the ICG survived to 30º. Increased plasma nitric oxide in adipose tissue and aorta was

observed in IOG group. Higher concentrations of INF-ᵞ and MCP-1, and a lower concentration of IL-

10 were observed in IOG vs ICG. It was found lower insulin sensitivity in obese animals, accentuated

after infection. Higher parasitic load was found in adipose and hepatic tissue, and increased levels

of oxidative stress in cardiac, hepatic and adipose tissue of IOG group.

Conclusion: Our results showed that the association of obesity and CD during the acute phase

promoted greater damage and decreased survival to infected animals, and may be seem as a

warning sign about the maladies of metabolic syndrome influencing other pathologies such as CD.

Keywords: nitric oxide; insulin resistance; hypertension; Chagas disease; obesity

106

POLYMORPHISMS IN GENES RELATED TO IMMUNOSUPPRESSION ARE ASSOCIATED WITH CERVICAL

CANCER

Adriano Martin Felis Aranome1; Kleber Paiva Trugilo1; Érica Romão Pereira1; Michelle Mota Sena1;

Nádia Calvo Martins Okuyama1; Rodolfo Sanches Ferreira1; Ana Paula Lombardi Pereira1;

Fernando Cezar-dos-Santos1; Paola Singi1; Aline Esposito1, José D’Oliveira Couto-Filho2; Maria

Angelica Ehara Watanabe1; Karen Brajão de Oliveira1.

1State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil 2Cancer

Hospital of Londrina, Londrina, PR, Brazil.


Introduction and objectives: Cervical cancer is the fourth most frequent cancer in women worldwide. Extrinsic

and intrinsic factors are involved in the cancer initiation, promotion and progression. Among these factors, the

immune system is important to determine resolution or development of pre-malignant lesions to cervical cancer.

Immunosuppressive microenvironment is often related with a worse prognosis, favoring the progression and

metastasis. Immune molecules levels and functions may be influenced by single nucleotide polymorphisms

(SNPs). Four SNPs have been associated with malignant diseases: rs1800470 and rs1800471 in the signal peptide

region of the transforming growth factor β1 (TGFB1) gene, rs3087465 in the TGFB receptor 2 (TGFBR2) gene, and

rs3761548 in the gene of transcription factor forkhead box P3 (FOXP3). All these genes are involved in the

maintenance of an immunosuppressed microenvironment and the role of the respective SNPs in cervical cancer

is still uncertain. Therefore, this work aimed to evaluate the four immune genes SNPs rs1800470, rs1800471,

rs3087465, and rs3761548, as well as sociodemographic data, and reproductive and sexual behavior, which

may be associated with cervical cancer.

Material and methods: This study was approved by the Institutional Ethics Committee Involving Humans at the

State University of Londrina, Londrina – PR, Brazil (CEP/UEL 466/12; CAAE 56738316.3.0000.5231). The purpose of

the study and the procedures involved were explained to all participants, and written informed consent was

obtained. A total of 156 women were the study population, of whom 49 had cervical cancer diagnosis and 107

were control subjects. Clinical characteristics were collected from medical records. Socio-demographic data,

reproductive and sexual behavioral characteristics were obtained using a structured questionnaire.

Polymorphisms genotyping were performed by polymerase chain reaction (PCR) followed by restriction

fragment length polymorphism (RFLP). Logistic regression was used for association analysis.

Results: Smoking habit (P<0.01), having four or more sexual partners during life (P=0.01), absence of previous

preventive tests (P=0.03), and high number of pregnancies (P=0.02) were related to cervical cancer.

Polymorphisms showed independent association with cervical cancer. Thus, in the adjusted models FOXP3 A/A

genotype (OR= 3.46 and CI95%= 1.14 – 10.54; P=0.03), TGFBR2 A/G genotype (OR= 8.09 and CI95%= 1.27 – 51.47;

P=0.03) and G/G (OR= 10.75 and CI95%= 1.59 – 72.76; p=0.02) were associated with increased susceptibility to

cervical cancer. In contrast, TGFB1 rs1800470 C/T genotype (OR= 0.13 and CI95%= 0.03 – 0.58; P=0.01), T/T (OR=

0.24 and CI95%= 0.06 – 0.95; P=0.04), and C/T+T/T (OR= 0.19 and CI95%= 0.05 – 0.66; P=0.01) were related to

protection. Association between SNPs and staging were not significant.

Conclusion: Although a greater number of cervical cancer patients are required for a better association

assessment, the results suggest that these SNPs have the potential to be molecular markers for cervical cancer

development and may be useful for the prognosis and therapeutic management of women with pre-malignant

cervical lesions, in the future.

Keywords: FOXP3, TGFB1, TGFBR2, SNP

Grants: This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico

(470137/2013-4), Fundação Araucária—Programa Pesquisa para o SUS (48056.501.36850.17042017),

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

107

PREVENTIVE HEALTH WITH A FOCUS ON IMMUNIZATION: A LITERATURE REVIEW

FEIFER, G.P1; SOUZA, T.B.1; FERREIRA, A.R.O.²; MACHADO, M.F.³

¹ Undergraduate student in nursing, Faculty Adventista Paranaense, Ivatuba, PR, Brasil.

²Postgraduate Program in Biosciences and Pathophysiology, State University of Maringá, Paraná,

Brazil.

³ Professor of the Nursing Department at the Faculty Adventista Paranaense, Paraná, Brasil.

[email protected]

Introduction and objectives: Immunization is a set of processes that confer resistance to certain

diseases. The national immunization program, stabelished by Ministry of Health, have the aim of

eradicate diseases such as measles and tetanus, control of immune preventable diseases e a

poliomielite. The Brazil's immunization rate in 2018 has reached the lowest levels of vaccine coverage

for children in recent years, making the population more susceptible to outbreaks and reinfections

of these diseases. The aim of this study was to search, in the literature, the nursing cares for

vaccination and the reasons for its low adherence in Brazil.

Material and methods: This is a qualitative study with descriptive and exploratory character of the

literature related to immunization. Was realized a search of scientific articles in the databases Scielo,

BVS and CAPES. The articles selected are related to the topic addressed, written in the Portuguese

language and, published as of 2008. We selected 16 articles for the research.

Results: The low adherence to the vaccination process is mainly related to increased of parental

avoidance due to their values, beliefs, culturally constructed concepts, conspiracies, incident

trauma from vaccination, fear of post-vaccination events, and lack of materials in institutions. The

awareness of people is among the strategies that can be used to encourage immunization, as it

allows to understand its importance for the prevention of diseases. For children, the therapeutic toy

during the immunization process may aid in the process of acceptance of the vaccine. In the

vaccine room, the importance of continuing education of the professionals of this sector is

emphasized, aiming at a better quality assistance to the population.

Conclusion: The reduction of the immunization in Brazil has multifactorial and cultural causes. The

professional of nursing must intervene with efficient and informed actions. For efficient acting, it is

necessary trained nursing professionals, materials and the improvement of techniques, to be possible

to provide a humanized, educative and science-based assistance.

Keywords: Vaccination, Nursing and Immunization program

108

PROINFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINE PROFILES IN PSORIASIS: USE AS

LABORATORY BIOMARKERS AND DISEASE PREDICTORS

Camila Cataldi1, Naiara Lourenço Mari1, Marcell Alysson Batisti Lozovoy2, Ligia Márcia Mário Martins3,

Edna Maria Vissoci Reiche2, Michael Maes4,5, Isaias Dichi6, Andréa Name Colado Simão2.

1Laboratory of Research in Applied Immunology, University of Londrina, Londrina, PR, Brazil 2Department of Pathology, Clinical Analysis and Toxicology, Laboratory of Research in Applied

Immunology, University of Londrina, Londrina, PR, Brazil 3Department of Dermatology, University of Londrina, Londrina, PR, Brazil 4IMPACT Strategic

Research Centre, School of Medicine, Deakin University, Geelong, VIC, Australia. 5Department of Psychiatry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

6 Department of Internal Medicine, Laboratory of Research in Applied Immunology, University of

Londrina, Londrina, PR, Brazil [email protected]

Introduction and objectives: Psoriasis is an autoimmune disease characterized by chronic

inflammation of the skin related to innate and adaptive immune responses. The presence of pro-

inflammatory cells and their cytokines creates a damaging environment leading to the development

and aggravation of psoriatic lesions. The cytokine profile of psoriasis is vast and complex. The

objectives of this study were to delineate the pro and anti-inflammatory cytokine profiles of psoriasis

and cytokine profile models that externally validate the diagnosis.

Material and methods: This study recruited 70 patients with psoriasis and 76 healthy controls. Cytokine

profiles were evaluated, including pro-inflammatory M1 (IL-1+ IL-6+TNFα), Th1 (IL-2+IL-12+IFN-γ), Th17

(IL-6+IL-17), and immune-inflammatory response system (IRS=M1+Th1+Th17) profiles. Moreover, the

anti-inflammatory potential included Th2 (IL-4), Th2+T regulatory (Th2+Treg, namely IL-4+IL-10+TGF-β),

anti-inflammatory (Th2+Treg+adiponectin), and the pro-inflammatory/anti-inflammatory index.

Results: There was a highly significant association between psoriasis and cytokine levels and psoriasis

explains 82.9% of their variance, with a particularly strong impact on IL-2 (46.3%), IL-12 (45.1%), IL-10

(53.2%), and adiponectin (40.1%). TGF-β and adiponectin were significantly lower while all other

cytokines (except IFN-γ) were significantly higher in psoriasis than in controls. In addition, M1, Th1,

Th17, Th2+Treg, and IRS/Anti-inflammatory index were significantly higher in psoriasis patients than in

controls. The IRS index, Th2+Treg, and adiponectin predicted psoriasis with 97.1% sensitivity and 94%

specificity.

Conclusion: In conclusion, psoriasis is characterized by increased M1, Th1, Th2 and Th17 profiles

together with lowered TGF-β and adiponectin. In addition, we propose a model based on a higher

IRS and Th2+Treg index coupled with lower adiponectin values, which may be used to externally

validate the diagnosis of psoriasis. The most important marker in psoriasis is adiponectin levels, which

may play a role in the modulation of the chronic inflammatory response observed in psoriasis.

Therefore, adiponectin could be a new drug target to treat psoriasis.

Keywords: Psoriasis; Cytokines; Adiponectin; Inflammation.

109

SUPPLEMENTATION WITH FISH OIL IMPROVES LIFE QUALITY, AND DECREASES INFLAMMATORY STATUS

AND OXIDATIVE STRESS IN PSORIASIS

Mari, N.L.1; Santos, L.F.R.F.1; Alfieri, D.F.1; Fauzino,T.1; Batisti Lozovoy,M.A.1,2; Martin, L.M.M.3;

Kipper,J.P.3; Lena,C.P.3; Taguti,P.S.3, Simão,A.N.C.1,2 and Dichi,I4.

1Research Laboratory in Applied Immunology – University of Londrina, Londrina, Paraná, Brazil 2Department of Pathology, Clinical Analysis and Toxicology, University of Londrina, Londrina,

Paraná, Brazil 3Department of Dermatology, University of Londrina, Londrina, Paraná, Brazil

4Department of Internal Medicine, University of Londrina, Londrina, Londrina, Paraná, Brazil.

[email protected]

Introduction and objectives: Psoriasis is an immune-mediated hyperproliferative chronic

inflammatory skin disease. This study verified the effects of fish oil n-3 fatty acids on the inflammatory

profile, redox state, adhesion molecules and impact on the life quality of patients with psoriasis.

Material and methods: Forty patients with chronic plaque psoriasis were randomized to one of two

groups: fish oil group (n=20), which received 3g/d of fish oil n-3 fatty acids (10 capsules) and control

group (n=20), which maintained their usual diet. Inflammatory biomarkers, nitro-oxidative stress

including lipid hydroperoxides (LOOH) in plasma and red blood cells, advanced oxidation protein

products (AOPP), nitric oxide metabolites (NOx), sulfhydryl groups and total radical-trapping

antioxidante parameter (TRAP) were measured as well as adhesion molecules and plasminogen

activator inhibitor type-1; Psoriasis Area Severity Index (PASI) and Dermatology Life Quality Index

(DLQI) were also evaluated. All parameters were measured at baseline and after 90 days of

supplementation. This study was conducted according to the guidelines laid down in the Declaration

of Helsinki and all procedures involving human patients were approved by the Ethical Committee of

the University of Londrina Parana, Brazil. CAAE: 51826215.0.0000.5231. Written informed consent was

obtained from all patients.

Results: The group receiving fish oil showed decreased erythrocyte sedimentation rate (ESR)

(p=0,007), LOOH in red blood cells (p=0,048), AOPP (p=<0,001) and increased sulfhydryl groups

(p=0,001),TRAP (p=0,010) and DLQI. There were not significant results in PASI, adhesion molecules,

PAI-1, NOx, and plasma LOOH. Inter-group changes (p<0.05) verified an improvement in the patient’s

life quality measured by DLQI, a decrease in AOPP and an increase in SH.

Conclusion: The present study indicated that ingestion of capsules of n-3 fish oil fatty acids was able

to improve life quality, to reduce inflammatory status, to decrease lipid peroxidation and protein

oxidation, and to increase the total antioxidant capacity in patients with psoriasis.

Keywords: Psoriasis, n-3 fish oil fatty acids, inflammatory status, cell adhesion molecules, oxidative

stress.

Grants: The study was supported by grants from the Coordination for the Improvement of Higher Level

of Education (CAPES) of Brazilian Ministry of Education; Institutional Program for Scientific Initiation

Scholarship (PIBIC) of the National Council for Scientific and Technological Development (CNPq).

We thank the University Hospital of State University of Londrina for technical support.

110


THE PATTERN OF CELL MIGRATION AND TNF-ALPHA PRO-INFLAMMATORY CYTOKINE SECRETION,

PROVIDED BY THE INITIAL INTRAPERITONEAL INFECTION OF Escherichia coli IN FEMALE SWISS MICE

Luana Carvalho Silva1; Bruna Santos Marnieri1, Telma Saraiva dos Santos2, Stéfane Frazão de Morais

Cabral1 , Eliza Pizarro Castilha1, Gerson Nakazato3, Anelise Franciosi1,2, Karina de Almeida Gualtieri1,

Náthalia Maria Fioreto Campois1, Ionice Felipe3 e Tacito Graminha Campois1.

1 Centro Universitário Filadélfia, Departament of Immunology and Patology, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 3Universidade Estadual de Londrina, Department of Microbiology, Londrina, PR, Brazil (e.g)

[email protected]

Introduction and objectives: Escherichia coli (E.coli) is a microorganism typically found in the

intestinal microbiota colony, possess, usually, a beneficial relationship with this bacteria. This

microorganism support vitamins digestion and processing that are necessary for organism

functioning and the presence of this bacteria in water is an indicative of fecal contamination.

However, the infection in individuals immunocommitment or with commitment intestinal barriers can

become pathogenic, which result in several problems for this patient and may progress to Hemolytic

Uremic Syndrome (HUS). The objective of this work is an analysis of initial results obtained from

infection in Swiss mice and implement an alternative method, the intraperitoneal route, by EHEC and

ATCC strains of E.coli, dosing the secreted cytokines profile, and evaluating cell migration, both

induced by the infection of 1x105 of these bacteria, in a single dose.

Material and methods:To develop the experiment, 52 Swiss female mice were used and divided into

3 groups. After the groups selection, the animals received 4 mL of saline, for the intraperitoneal route,

at pre-set time intervals of de 0, 3, 6, 12 e 24 hours. Thus, the animals received, to obtain

intraperitoneal exudate, with would be destined for the accomplishment of the dosage of pro-

inflammatory cytokynes and also evaluation of the cell migration. After this procediment, the animals

were submitted to euthanasia, respecting the protocols established by the Animal Ethics Committee.

Results: The solution removed from the peritoneum of these mice was subjected to ELISA (enzyme-

linked immunosorbent assay). From this step, it can be observed that the action of the Shiga toxin,

produced by the enterohemorrhagic strain (EHEC), is potentiated by circulating pro-inflammatory

cytokines, such as Tumor Necrosis Factor Alpha (TNF-α), proving that the profile of the initial cytokines

of the infection by diarrheogenic strains of this bacteria behaves differently from the infection

induced by the non-diarrheogenic strain.

Conclusion: As result, we could evaluate the cell migration and observe the presence of intense

neutrophilic inflammatory infiltrates, corroborating with previous studies, since neutrophils are the

body's first line of defense against bacteria.

Keywords: Escherichia coli, proinflammatory cytokines, ELISA


111

THIOL GROUP AS A BIOMARKER FOR THE DIAGNOSIS OF PROSTATE CANCER AND METASTASIS

PREDICTOR

Ana Gabriela da Silva Bonacini1; Alexsandro Koike2, Brunna França Robles1, Edna Maria Vissoci

Reiche3, Isaias Dichi4, Michael Maes5, Rubens Cecchini6, Andréa Name Colado Simão3

1 Laboratory of Research in Applied Immunology, University of Londrina, Londrina, Paraná, Brazil

2 Cancer Institute of Londrina, Laboratory of Research in Applied Immunology, University of

Londrina, Londrina, Paraná, Brazil


Immunology, University of Londrina, Londrina, Paraná, Brazil

4 Department of Internal Medicine, University of Londrina, Londrina, Paraná, Brazil.

5 IMPACT Strategic Research Centre, School of Medicine, Deakin University, Geelong, VIC,

Australia.

6 Department of Pathology Sciences, University of Londrina, Londrina, Paraná, Brazil

[email protected]

Introduction and objectives: Oxidative stress (OS) is one of the mechanisms that trigger the development and

progression of prostatic benign hyperplasia (BPH) and prostate carcer (PCa). The search for new biomarkers to

differentiate BPH and PCa as well as to differentiate the indolent from the aggressive disease is necessary. The

new insight into the role of OS in pathogenesis of BPH and PCa could elucidate some mechanisms and

contribute to prognosis and also be a potential strategic target for PCa treatment. Therefore, the aims of this

study were: 1) To identify biomarkers of oxidative stress that could differentiate BPH and PCa; 2) To verify which

oxidative stress biomarkers could be used as predictors of PCa prognosis to improve the sensitivity and specificity

of the existing detection techniques.

Material and Methods: This prospective case-control study consisted of 204 subjects, 73 patients with PCa, 67

patients with BPH, and 64 controls (HC). Clinical examination, total and free prostate-specific antigen (PSA) test,

prostate biopsy when necessary, and determinations of Terc-Butyl Hydroperoxide-Initiated Chemiluminescence

(CL-LOOH), plasmatic carbonyl protein (PCB), advanced oxidation protein products (AOPP), total radical-

trapping antioxidant parameter (TRAP), and protein thiol groups (SH) were performed. Informed consent was

given by all the patients included in this work. The Human Ethics Committee from the State University of Londrina

approved the protocol of the study (CAAE 22 56182916.7.0000.5231)

Results: Diagnosis (HC vs BPH vs PCa) was significantly associated with PSA/Oxidative and Nitrosative Stress

(O&NS) biomarkers explaining 43.2% of the variance. Diagnosis had a significant impact on SH (37.5%), AOPP

(11.0%), PCB and CL-LOOH in Red Blood Cells (RBC) (both <10.0%). SH levels were significantly lower in PCa than

in HC and BPH, whereas there were no significant differences between BPH and HC. AOPP was significantly

increased in BPH and PCa as compared with HC, whereas PCB was higher in PCa than in HC and BPH. CL-LOOH

levels in RBC (but not plasma) were significantly higher in both BPH and PCa than in HC. TRAP levels were

decreased in PCa when compared with HC. Better prediction of PCa was obtained using PSATotal and SH

combined with PCB, whereby 91.1% of all cases were correctly classified with a sensitivity of 85.7% and a

specificity of 94.0%. The same three variables yielded also a good prediction of PCa versus BPH, whereby 87.1%

of all cases were correctly classified with a sensitivity of 87.3% and a specificity of 86.9%. SH performed well

discriminating metastasis whereby 97.1% of all cases are classified correctly with a sensitivity of 50% and a

specificity of 99.5%. SH (and not PSA) was significantly associated with metastasis with an area under the ROC

curve of 0.965.

Conclusion: SH levels were a predictor of diagnosis and could be used together with PSA measurement for the

screening of patients with BPH and PCa improving sensibility and specificity of diagnosis. This is the first study to

propose models using oxidative stress biomarkers and clinical and laboratory data to predict prognosis and

metastasis presence in prostate diseases.

Keywords: Plasma thiol groups, oxidative stress, prostate cancer, benign prostatic hyperplasia.

112

TNF-β +252 A>G (rs909253) POLYMORPHISM IS INDEPENDENTLY ASSOCIATED WITH PRESENCE OF

AUTOANTIBODIES IN RHEUMATOID ARTHRITIS PATIENTS

Fabiano Aparecido de Medeiros1, Daniela Frizon Alfieri1, Tatiana Mayumi Veiga Iriyoda2, Neide

Tomimura Costa3, Elaine Regina Delicato de Almeida4, Marcell Alysson Batisti Lozovoy4, Naiara

Lourenço Mari1, Tamires Flauzino1, Edna Maria Vissoci Reiche 4, Isaias Dichi3, Andréa Name Colado

Simão4

1 Research Laboratory in Applied Immunology– University of Londrina, Londrina, Paraná, Brazil.

2 Department of Rheumatology – PUC, Pontifícia Universidade Católica, Londrina, Paraná,

3 Department of Internal Medicine – University of Londrina, Londrina, Paraná, Brazil.

4 Department of Pathology, Clinical Analysis and Toxicology – University of Londrina, Londrina,

Paraná, Brazil.

Introduction and objectives: The TNF-β +252 A>G (rs909253) polymorphism has been associated with

a risk of development of rheumatoid arthritis (RA) and could influence plasma tumor necrosis factor

alfa (TNF-α) levels. The aim of the present study was to evaluate the association between the TNF-β

+252 A>G polymorphism with plasma TNF-α levels, the presence of autoantibodies, and the

susceptibility for RA.

Material and methods: This cross-sectional study included 261 patients with RA and 292 controls. The

polymorphism was studied using Polymerase Chain Reaction-Restriction Fragment Length

Polymorphism (PCR-RFLP). Soluble TNF-α and receptors were measured by multiplex assay.

Rheumatoid factor (RF) and anticyclic citrullinated peptide antibodies (anti-CCP) were measured

using immunoassay.

Results: No differences were observed in allele frequency and genotype distribution among patients

and controls. The presence of RF (p=0.020) and anti-CCP (p=0.001) increased 4.23-fold and 8.13-fold,

respectively, in patients with B1 allele (B1/B2+B1/B1 genotypes) independently of demographic,

clinical, and inflammatory markers. Among patients with B1/B2+B1/B1 genotypes, higher TNF-α levels

were associated with positive RF (p=0.040), anti-CCP (p=0.011), or both (p=0.038). In patients carrying

B1 allele, the increased sTNFR1 together with RF or anti-CCP or both explained about 39.0% the

variations in TNF-α level. However, in B2/B2 genotype, the presence of those autoantibodies was not

associated with TNF-α level.

Conclusion: Our findings indicate that the TNF-β +252 A>G polymorphism was not associated with

RA susceptibility and TNF-α plasma levels. However, B1 allele was associated with the presence of

autoantibodies. In addition, interaction between the presence of B1 allele and autoantibodies was

associated with the increase of plasma TNF-α level in RA patients.

Keywords: Rheumatoid Arthritis, polymorphism, tumor necrosis factor alfa, and autoantibodies

113

TRACE ELEMENTS ASSOCIATED WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND INSULIN RESISTANCE

Eliel Marcio Pedro1, Lorena Flor da Rosa Franchi Santos2, Bruna Miglioranza Scavuzzi3, Tatiana

Mayumi Veiga Iriyoda4, Tiago Severo Peixe5, Marcell Alysson Batiste Lozovoy5, Edna Maria Vissoci

Reiche5, Isaias Dichi6, Andréa Name Colado Simão5, Maria Josefa Santos1

1 University of Londrina, Department of Chemistry, Londrina, Paraná, Brazil 2 University of Londrina, Department of General Pathology, Laboratory of Research in

Applied Immunology, Londrina, PR, Brazil 3 University of Londrina, Laboratory of Research in Applied Immunology, Londrina, PR, Brazil

4 Pontifical Catholic University of Paraná (PUC/PR), Department of Rheumatology, Londrina,

PR, Brazil 5 University of Londrina, Department of Pathology, Clinical Analysis and Toxicology,

Laboratory of Research in Applied Immunology, Londrina, PR, Brazil 6 University of Londrina, Department of Internal Medicine, Laboratory of Research in Applied

Immunology, Londrina, PR, Brazil 7 [email protected]

Introduction and objectives: Systemic lupus erythematosus (SLE) is a chronic inflammatory

autoimmune disease of multifactorial origin. Studies have shown that trace elements such as zinc

and copper may help maintain optimum function of the immune system and metabolism, while toxic

metals such as lead may increase systemic autoimmunity. The current study aimed to assess the

relationship between serum concentration of lithium (Li), vanadium (V), copper (Cu), zinc (Zn),

molybdenum (Mo), cadmium (Cd) and lead (Pb) and SLE diagnosis, disease activity measured by

SLE disease activity index (SLEDAI) and insulin resistance (IR).

Material and Methods: This case-control, cross-sectional study included 225 patients, 120 healthy

controls and 105 SLE patients. SLE patients were divided into two groups using SLE Disease Activity

Index (SLEDAI) score: patients with no activity, mild or moderate activity (SLEDAI < 10); and patients

with high activity (SLEDAI > 10). Serum concentration of Li, V,

Cu, Zn, Mo, Cd and Pb were measured inductively coupled plasma mass spectrometry (ICPMS

Varian 820-MS, Palo Alto, CA, USA). This study was conducted in accordance with the ethical

standards as laid down in the 1964 Declaration of Helsinki. The Ethics Committee of State University

of Londrina approved all procedures involving human subjects (CAAE 01865212.0.0000.5231). Written

informed consent was obtained from all patients.

Results: Serum concentrations of V (p<0.001), Zn (p<0.001) and Pb (p<0.001) were lower and Mo

(p<0.001) and Li (p<0.001) were higher in patients with SLE compared to healthy controls. SLE

diagnosis was associated with higher serum Li (p<0.001) concentration and lower V (p<0.001), Zn

(p=0.003) and Pb (p=0.020). Toxic metals and trace elements were not associated with disease

activity. Levels of Cd were higher in patients with IR (p=0.042). There was no significant association

between IR and the other metals.

Conclusion: The results indicate that SLE patients have different profiles of trace elements and toxic

metals compared to healthy controls. While some toxic metals and trace elements were found to

be associated with SLE diagnosis, they had no effect on disease activity and IR.

Keywords: SLE disease activity index (SLEDAI), Heavy metals, Trace elements, Insulin resistance,

Glucose homeostasis.

114

Trypanosoma cruzi: THE BLOCKING EFFECT OF COX-2 WITH CELECOXIB ON THE ACTIVATION AND

INVASION OF MACROPHAGES DERIVING FROM THP-1, A LINEAGE OF HUMAN MONOCYTES.

Edson Lucas Pinheiro1, Helena Tiemi Suzukawa2, Phileno Pinge Filho3

State University of Londrina, Department of Biological Sciences, Londrina, PR, Brazil E-mail:

[email protected]

Introduction and objectives: The human and experimental infection by Trypanosoma cruzi, the cause

of the Chagas disease, provokes an intense immunological response initiated in the acute phase, in

which several pro-inflammatory mediators are produced, among them the prostaglandins.

Moreover, there are drugs called NSAIDs that possess anti-inflammatory activity through the inhibitory

action of cyclooxygenase enzymes (COX), among them the ones that selectively inhibit the COX-2,

being denominated coxibs. These enzymes synthetize the prostaglandins, such as the PGE2, which is

involved in the immunosuppression of the Chagas disease in the acute phase. The aim of the present

study was to standardize the obtainment of differentiated macrophages of from THP-1 cells and

investigate the effect of the cyclooxygenase 2 (COX-2) inhibition with celecoxib over the capacity

of the T. cruzi invasion.

Materials and methods: The parasites used were obtained through the blood trypomastigotes

infection of Trypanosoma cruzi (Y strain) in LLCMK2 cells. The THP-1 cells, a lineage of human

monocytes, were stimulated with 5ng/mL of PMA during two days for differentiation in macrophages.

As for the internalization test, it was distributed 10⁵ cells/well plates in plates of 24 well plates with glass

coverslips and treated with celecoxib (0.312mM, 0.625, 1.25mM) for 1 hour, followed by cell washing

and infection with 5:1 trypomastigotes forms of T. cruzi overnight. The dosage of nitric oxide (NO) was

carried out through nitrite quantification of the supernatant of macrophages culture in plates of 96

well plates, with 2x10⁵ cells/well plates after 24 hours and 48 hours post-infection, through the Griess

method. The cell viability of the macrophages submitted to different concentrations of the drugs

used was assessed by the MTT test. The phenotypic identity of the obtained cells was determined by

flow cytometry. The experimental protocols used were approved by the Ethics Committee in

Research Involving Human Subjects (Process 24841.2016.41).

Results: The THP-1 cells and the macrophages obtained after differentiation with PMA were

confirmed phenotypically through flow cytometry, evidenced by the rise of the surface marker CD14

in the cells after exposure to the PMA, which is one of the characteristics of activated cells. The

treatment with celecoxib, regardless of the dosage, did not present cytotoxic effects on the cell and,

there was a decrease in the internalization rate of trypomastigotes forms of T. cruzi in the

differentiated macrophages of from THP-1 cells. However, the blocking of the enzyme COX-2 with

celecoxib did not have any effect over the production of NO, possibly related to the human origin

of the cell used and the sensitivity of the Griess method.

Conclusion: The chosen protocol for differentiation of THP-1 cells in macrophages was effective, cells

that presented phenotypic characteristics similar to the ones described in the literature were

obtained. The celecoxib dosages used were able to decrease the entrance of T. cruzi in the

macrophages, being the concentration of 1.25mM the one that presented this effect. The oxide nitric

production in our tests was not significant, it can be correlated to the human origin of the cell lineage

used.

Keywords: Trypanosoma cruzi; Celecoxib; THP-1 cells.

Grants: To the immunopathology experimental laboratory and the funding agencies CAPES and

Fundação Araucária.

115

TUMOR NECROSIS FACTOR α AND ITS SOLUBLE RECEPTORS ARE ASSOCIATED WITH DISABILITY, DISEASE PROGRESSION AND CLINICAL FORMS OF MULTIPLE SCLEROSIS

Claudia Mara Ribeiro1; Sayonara Rangel Oliveira1,2; Daniela Frizon Alfieri1, Tamires Flauzino 1, Damacio Ramón Kaimen-Maciel3, Andréa Name Colado Simão1,2, Michael Maes4,5, Edna Maria Vissochi Reiche1,2

1Laboratory of Applied Immunology, Health Sciences Center, University of Londrina, Paraná, Brazil; 2Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, University of Londrina,

Paraná, Brazil;3Santa Casa de Misericórdia, Londrina, Paraná, Brazil;

4IMPACT Strategic Research Centre, School of Medicine, Deakin University, Geelong, Victoria, Australia, 5Department of Psychiatry, King Chulalongkorn Memorial Hospital, Chulalongkorn, Bangkok, Thailand;

[email protected]

Introduction and objectives: The association between tumor necrosis factor (TNF)-α, soluble TNF receptor (sTNFR)1 and sTNFR2 with clinical and laboratory characteristics of multiple sclerosis (MS) remains unclear. The aim of this study is to examine whether TNF-α, sTNFR1 and sTNFR2 are associated with MS diagnosis, disability, disease progression and clinical forms of MS.

Material and methods: The protocol was approved by the Institutional Research Ethics Committees of University of Londrina, Paraná, Brazil (CAAE: 22290913.9.0000.5231) and all of the individuals invited were informed in detail about the research and gave written informed consent. The study included 168 MS patients and 70 healthy controls. Disability was evaluated using Expanded Disability Status Scale (EDSS) and categorized as mild (EDSS<3.0) or moderate/high (EDSS≥3.0). Disease progression was evaluated using Multiple Sclerosis Severity Score (MSSS) and categorized as no progression (MSSS<5) and disease progression (MSSS ≥5). TNF-α, sTNFR1 and sTNFR2 levels were determined using customized chemiluminescent multiplex immunoassay

Results: The study evaluated 147 (87.5%) patients with relapsing-remitting MS (RRMS) and (21) 12.5% with progressive clinical forms (ProgMS), including 4 (2.4%) with primary progressive MS (PPMS) and 17 (10.1%) with secondary progressive MS (SPMM). The diagnosis of MS explained 44.6% and 12.3% of the TNF-α and sTNFR2 levels, respectively. Moderate/high disability was best predicted by sTNFR1 and age (both positively) and with these variables, 73.6% of all cases were correctly classified (p<0.001). Disease progression was best predicted by sTNFR1 and age (both positively) and with these variables, 66.0% of all cases were correctly classified (p<0.001). Moreover, ProgMS was best predicted by sTNFR1 (positively) and sTNFR2 (negatively), coupled with age and sex, and with this model, 86.9% of all cases were correctly classified (p<0.001) One composed score reflecting the sTNFR1/sTNFR2 ratio showed a significant and positive association with ProgMS after adjusting for age and sex, which correctly classified 86.3% of all cases of ProgMS (p<0.001)

Conclusion: Our results underscore that TNF-α and its receptors are involved in the pathogenesis of MS. While sTNFR1 is associated with disability, disease progression and ProgMS, high levels of sTNFR2 may be a compensatory response exerting a protective effect modulating the immunopathological mechanisms of MS.

Keywords: Not declared by authors

116

ACTIVITY OF A Piper solmsianum COMPOUND AGAINST Mycobacterium tuberculosis

Lincoln Luís Silva¹, Vera Lucia Dias Siqueira2, Katiany Rizzieri Caleffi-Ferracioli2, Rosilene Fressatti

Cardoso¹, ², Rosi Zanoni da Silva3, Regiane Bertin de Lima Scodro

¹State University of Maringá, Postgraduate Program in Health Sciences, Brazil.

² State University of Maringá, Postgraduate Program in Biosciences and Physiopathology, Brazil.

³ State University of Ponta Grossa, Department of Pharmaceutical Sciences, Brazil.

[email protected]

Introduction and objectives: Tuberculosis (TB) is the deadliest infectious disease in the world and most

cases occur by the bacillus Mycobacterium tuberculosis. According to the World Health

Organization, there were an estimated 10.4 million cases of TB and 1.3 million people died only in

2017 as result of the disease. The biggest challenge to control this disease is the resistance of M.

tuberculosis to the drugs used. For this reason, searching for new drugs is essential at the moment.

About one-third of present medicines are derived from natural sources and thus, was extracted a

compound from Piper solmsianum leaves, a species that belongs to Piperaceae family, which not all

its compounds were fully evaluated. The objective of this study was to determine the minimal

inhibitory concentration (MIC) of a compound extracted from Piper solmsianum, denominated

eupomatenoid-6.

Material and methods: We purified the crude extract of P. solmsianum leaves. Fractions obtained

from hexane, dichloromethane, and ethyl acetate were analyzed with thin layer chromatography

and 1H nuclear magnetic resonance, and the data were compared with the literature. We selected

the eupomatenoid-6, and evaluated its activity against the reference strain M. tuberculosis H37Rv

(ATCC 27294) using Resazurin Microtiter Assay Plate method.

The substance was serially diluted ranging concentrations from 250 to 0.97 μg/mL. Bacterial growth

was standardized according to the McFarland Scale 1 and reveled with resazurin after 7 days of

incubation at 36 ºC. Viable bacteria reduce the blue dye in the initial color to pink coloration. MIC

was defined as the lowest concentration of the substance capable of inhibiting bacterial growth.

Results: The chromatographic column purification afforded the isolation of several fractions, and the

compound eupomatenoid-6 was isolated and evaluated. This substance exhibited great activity

against M. tuberculosis H37Rv with MIC value of 3.9 μg/mL.

Conclusion: The neolignan eupomatenoid-6 extracted from the leaves of the P. solmsianum shows

important activity against Mycobacterium tuberculosis, which makes it a possible candidate for the

treatment of tuberculosis.

Keywords: Tuberculosis, minimum inhibitory concentration, Piper, resazurin.

Grants: CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

117

ANALYSIS OF THE NUMBER OF VIPERGIC SUBMUCOSAL NEURONS IN THE DUODENUM OF WISTAR RATS

SUBMITTED TO ACUTE Toxoplasma gondii INFECTION

Aline Rosa Trevizan1; Lucas Casagrande1; Lainy Leiny de Lima1; Eduardo José de Almeida Araújo2;

Débora de Mello Gonçales Sant’Ana1

¹ Universidade Estadual de Maringá, Department of Morphological Sciences, Maringá, PR, Brazil

² Universidade Estadual de Londrina, Department of Histology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Toxoplasma gondii is primarily transmitted by the ingestion of sporulated

oocysts present in water and contaminated food. During the chronic phase of infection, the parasite

tends to form cysts in the nerve tissue, which may remain viable throughout the host's life. The enteric

nervous system controls the functions of the digestive tract, and the vasoactive intestinal peptide

(VIP) is an important neurotransmitter for the functioning of the submucous plexus enteric neurons.

Therefore, our objective was to evaluate the density of immunoreactive VIP neurons in the

submucosal plexus of the duodenum of Wistar rats submitted to acute T. gondii infection.

Material and methods: The experimental protocol was approved by the Committee of Ethics in

Animal Experimentation of the State University of Maringá, under opinion No. 079/2013. We used 10

male Wistar rats at 60 days of age (n = 5), distributed in a control group (CG) that received saline

solution and a 12-hour infected group (G12) that received oral suspension containing 5000

sporulated T. gondii oocysts (strain ME-49, genotype II). We euthanized the animals and their

duodenum were collected and fixed in 4% Paraformaldehyde. Afterwards, the duodenum

submucosal plexus was dissected and processed for the immunohistochemical staining technique

of the immunoreactive VIP neurons population. Quantification was performed in the Image-Pro Plus

image analysis software, counting the immunoreactive VIP neurons present in 32 images of

microscopic fields using 20x objective lens. Images were obtained with the FSX-BSW image browser

integrated with an Olympus FSX100 light microscope with immunofluorescence filters. The number of

neurons counted was converted to mm2 in order to obtain the vipergic neuronal density. The

statistical analysis was carried out with the Bioestat 5.3 software. The data showed a normal

distribution and were expressed as mean ± standard deviation. An analysis of variance Anova-a

criterion was performed and the level of significance used was 5%.

Results: The immunoreactive VIP submucosal neurons density in the infected group G12

(40.79 ± 3.97 neurons per mm2) showed no significant difference when compared to CG values (41.89

± 10.74 neurons per mm2).

Conclusion: Acute infection by T. gondii oocysts causes no change in the number of vipergic neurons

present in the submucosal plexus of the duodenum of Wistar rats after 12 hours of infection.

Keywords: Intestine, Submucosal Plexus, Toxoplasmosis.

Grants: CAPES, Fundação Araucária.

118

ANTIVIRAL ACTIVITY OF BOTRYOSPHAERAN AND ITS SULFONATED MOLECULES IN THE REPLICATION OF

DENGUE VIRUS

Jéssica Wouk1; Vitor Yuji Ito 2a; Vinicius Martinez Rino2a; Bruce Alan Lobo Sacchelli2b; José Louzinho

Lopes2b; Alexandre Orsato2b; Aneli de Melo Barbosa Dekker2b; Robert Frans Huibert Dekker3; Ligia

Carla Faccin Galhardi2a

1 Universidade Estadual do Centro-Oeste, Department of Post-graduation in Pharmaceutical

Science, Guarapuava, Brazil

2Universidade Estadual de Londrina, aDepartment of Microbiology and bDepartment of Chemistry,

Londrina, PR, Brazil 3 Universidade Tecnológica Federal do Paraná, Deparment of Post-graduation in

Environmental Engineering, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Botryosphaeran (BOT) is a fungal exocellular (1→3)(1→6)-βD-glucan

produced by the ascomycetous Botryosphaeria rhodina MAMB-05. This natural product

demonstrates several pharmacological properties, such as antigenotoxic, hypoglycemic,

hypocholesterolemic, and anticancer. When sulfonated, BOT also presented anticoagulant activity.

Sulfonated polysaccharides are known to display activity against enveloped viruses, such as dengue.

Therefore, the present work aimed to assess the antiviral activity of BOT, and its chemically-sulfonated

derivatives, against dengue virus (DENV).

Material and methods: BOT molecules were chemically modified by sulfonylation with chlorosulfonic

acid. Two sulfonated derivatives were prepared, S1 and S2, with degrees of substitution (DS) of 0.4

and 1.1, respectively. The cytotoxicity and antiviral activity of tested compounds were evaluated by

the MTT method. Anti-DENV-3 activity was also evaluated by the immunofluorescence (IF) assay

Results: DENV inhibition was weak for native BOT but remarkably stronger for S1 and S2. In both

antiviral tests performed, native BOT inhibited viral replication by 40% at 100 μg mL-1. In contrast, at

the same concentration, the sulfonated molecules showed a viral inhibition percentage (%VI) of 60%

in MTT assay. In lower concentrations (50 μg mL-1 and 25 μg mL1), sulfated molecule presented low

inhibition, approximately 30%. When analyzed by IF, S1 and S2 reduced protein synthesis by 15.4%

and 90%, respectively at 100 μg mL-1.

Conclusion: Both native and sulfonated molecules demonstrated antiviral activity against DENV. The

presence of sulfonate groups improved the antiviral activity when compared to native BOT. S2

molecule, which presented the highest DS, was able to maintain its inhibitory activity up to viral

protein synthesis. The present results corroborate with other studies which demonstrated the antiviral

activity of sulfonated polysaccharides against enveloped viruses. Further tests might be performed

to elucidate the mechanism of action of BOT and its chemically-sulfonated derivatives, which are

promising active compounds to treat patients with dengue.

Keywords: Botryosphaeria rhodina, botryosphaeran, cytotoxicity, antiviral activity

Grants: CNPq, CAPES, Fundação Araucária

119

ASSOCIATION BETWEEN INFECTION BY Staphylococcus aureus AND DEATH OF CHILDREN: AN

EIGHTEEN YEARS RETROSPECTIVE STUDY

Alexandre Casonatto1; Thilara Alessandra de Oliveira1; Fabricio Seidy Ribeiro Inoue1; Alisson

Santana da Silva1; Natalia da Paixão Figueiredo1; Tiago Danelli1; Felipe Crepaldi Duarte1; Raquel

Soares da Silva1; Gabrielle Feijó de Araújo1; Natalia Galindo Fernandes Rubituci1; Déborah Rossane

Santana Costa de Souza1; Jaqueline Dario Capobiango1; Marcia Regina Eches Perugini1.

1 State University of Londrina, Department of Pathology, Clinical and Toxicological Analysis,

Londrina, Brazil.

[email protected]

Introduction and objectives: Staphylococcus aureus are bacteria from the Gram-positive cocci

group, accountable for a wide variety of clinic syndromes, from which includes local skin and soft

tissues infections to invasive diseases as bacteremia, endocarditis, pneumonia and osteomyelitis. The

Methicillin-Resistant Staphylococcus aureus (MRSA) can cause severe diseases and even lead to

death, in addition to considerable health care costs. The aim of this study was to associate patient

discharge and death with oxacillin susceptibility profile in children infected by S.aureus admitted at

the Londrina University Hospital.

Material and methods: were analyzed the results of antimicrobial susceptibility testing of isolated S.

aureus from 1531 children by the Clinical Microbiology Analysis Laboratory (LACM) of the Londrina

University Hospital from year 2000 to 2018. This study was approved by the ethics committe (CAAE

number 0015.0.268.000-11). Patients were assorted in a dichotomic fashion according to the

microbial susceptibility profile, death/discharge. The association was analyzed by the chi-squared

testing and the association magnitude by binary logistic regression analysis.

Results: from the study population, 1117 subjects were diagnosed with Methicillin-Susceptible

Staphylococcus aureus (MSSA) and 488 with MRSA. Patients’ average age were 3,45±4,31 and

1,05±2,03 years for children infected by MSSA e MRSA, respectively. Childrens’ average age were

3,22±3,89 (discharge) and 2,00±3,33 (death). Infection by S. aureus was associated with a 26,6%

[MSSA] and 60,4% [MRSA] (p<0,001) death rate. The logistic regression pointed that MRSA diagnosed

children have 4,22 (2,93-6,07 – IC95% - p<0,001) times more chances of presenting death disclosing

cases compared to their counterparts diagnosed with MSSA.

Conclusion: thus, was observed that children infected by MRSA strains are exposed to a greater

mortality risk than children infected by MSSA strains.

Keywords: Staphylococcus aureus; MRSA; MSSA; Children; Death.

120

CHRONIC INFECTION WITH Toxoplasma gondii OOCYSTS CAUSE HYPOPLASIA AND HYPERTROPHY OF

MOTOR ENTERIC NEURONS IN THE PROXIMAL COLON OF RATS

Machado1, Camila Cristina Alves; Pupim1, Andréia Carla Eugênio; Watanabe1, Paulo da Silva;

Trevisan2, Aline Rosa; Gois2, Marcelo Biondaro; Nino3, Beatriz de Souza Lima; Garcia3, João Luis;

Blackshaw4, L Ashley; Sant’Ana, 2Débora de Mello Gonçales; Araújo1, Eduardo José de Almeida¹.

1 State University of Londrina; Department of Histology, Londrina, PR, Brazil;

2 State University of Maringa; Department of Morphological Science, Maringa, PR; Brazil;

3 Department of Preventive Veterinary Medicine, State University of Londrina, Londrina,

Paraná, Brazil;

4 Queen Mary University of London, London, United Kingdom


Introduction and Objective: Toxoplasmosis is a zoonosis with a large worldwide distribution. One-third

of the world population is probably infected with its etiological agent: Toxoplasma gondii. This

parasite proliferates quickly and invades the intestinal wall of the host causing an intense

inflammatory local response. Inflammation in the intestinal wall is an important cause of cellular loss

of the enteric nervous system (ENS). Previous studies showed that infection with T. gondii can cause

changes in colonic myenteric plexus of infected animals, but it is unknown if the enteric motor

neurons and interneurons are directly impact for this infection. The goal of this study was to evaluate

the distribution of excitatory and inhibitory myenteric motor neurons and myenteric interneurons in

the proximal colon of rats infected with T. gondii oocysts.

Methods: All performed procedures were previously approved by the Animal Ethics Committee of

the State University of Maringá, Brazil (Reference number: 079/2013) and the State University of

Londrina, Brazil (Reference number: 50/2017). Male Wistar rats were distributed into control (n=5) and

infected group (n=5 each), which were evaluated 30 days post infection (dpi). The infected rats were

orally inoculated with 5x103 T. gondii sporulated

oocysts of the strain ME-49 (genotype II). The control rats received only 1mL of sterile saline by

gavage. Proximal colonic whole-mount preparations were submitted to immunofluorescence

technique to labelling total (anti-HuC/D), nitrergic (anti-nNOS), cholinergic (anti-ChAT) and calbindin

(anti-calbindin) neurons. Neurons that showed labelling with anti-nNOS were considered inhibitory

motor neurons and those labelled with anti-ChAT were considered excitatory motor neurons or

interneurons. ChAT and Calbindin positive cell bodies were considered interneurons. The number of

neurons was counted in 50 ganglia per animal and a total of 100 cell body area were measured.

GraphPad Prism6 software (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical

analysis and the groups were compared considering the significance level was set to 5%.

Results: Infection with ME-49 T. gondii oocysts caused loss of total (HuC/D-IR), nitrergic (nNOS-IR) and

cholinergic (ChAT-IR) myenteric neurons at 30 dpi in the proximal colon. This hypoplasia was

approximately 23% (HuC/D-IR), 16% (nNOS-IR) and 40% (ChAT-IR) (p<0,05). The total, nitrergic and

cholinergic remained neurons became hypertrophied (p<0,05). Despite the intense reduction of

cholinergic neurons, the number of enteric interneurons (ChAT-IR/Calb-IR positive) kept unchanged

(p>0,05).

Conclusions: High load (5x103) of ME-49 T. gondii oocysts caused myenteric neuronal loss and

hypertrophy cell body of total, nitrergic and cholinergic neurons in the proximal colon of rats. The

infection changed mainly the motor excitatory neurons (ChAT-IR) and did not change the proportion

of interneurons.

Keywords: Toxoplasmosis, enteric nervous system, excitatory and inhibitory motor neurons.

Grants: Capes

121

CORRELATION ANALYSIS OF MOUSE MAMMARY TUMOR VIRUS-LIKE DNA SEQUENCE WITH

CLINICOPATHOLOGICAL PARAMETERS IN BREAST CANCER SUBTYPES

Sousa-Pereira, N.1; Amarante, M. K.1; Vitiello, G. A. F1; Banin-Hirata, B. K.1; Bocchi, M. 1; Moretto, S. L. 1;

Oliveira, K. B1; Watanabe, M. A. E1

1 Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Introduction and objectives: Breast cancer (BC) is a complex heterogeneous disease whose

evolution depends on the tumor-host interaction. It is generally accepted that environmental factors

play a role in the etiology of various types of cancer. The mouse mammary tumor virus (MMTV) has

been suggested as a candidate for viral etiology of BC. The identification of the similar sequence to

MMTV, called human mammary tumor virus (HMTV), has supported the theory of viral involvement in

the BC pathogenesis. However, several key issues remain unclear, such as the clinical significance of

its infection and its role in the BC pathogenesis. Therefore, this study aimed to detect the presence

of MMTV like DNA (MMTV-L) in human mammary tumor tissue and correlate with clinicopathological

parameters.

Material and methods: This project was approved by Ethic Committee from UEL (CAAE

47709015.2.0000.5231). Women with diagnosis of BC was invited to participate in this research project

during the clinical care in the specialized services and in Londrina Cancer Hospital. The form of free-

informed consent was signed by all donors before the collections. It was obtained 216 tissue samples

from human mammary tumor and in 32 peripheral blood samples. The 251bp sequence related to

the HMTV env gene was obtained by nested-PCR and the products were submitted to

electrophoresis on 10% polyacrylamide gel. Sequencing was performed to confirm the amplification

of the studied gene. Statistical analyses were performed using Kendall Tau-b correlation coefficient.

Results: MMTV-L sequence was verified in 19.0% (n=41) of BC tissue. Among these patients MMTV-L

were also present in 53.1% (n=17) of peripheral blood samples. In our study, there was no significant

correlation between the viral DNA with clinicopathological parameters in general BC nor in Luminal

A and triple negative subtypes. However, there was a significant correlation for TNM staging in

Luminal B subtype (τ = -0.382, p = 0.042). The same result was observed for HER2-enriched subtype (τ

= -0.551, p = 0.001), in which was also verified correlation with lymph node commitment (τ = -0.559, p

= 0.015). Regarding peripheral blood samples, there were a trend for age (τ = -0.355, p = 0.053) and

lymph nodes commitment (τ = -0.400, p = 0.051).

Conclusion: Based on these results, we can infer a possible correlation between the presence of

MMTV-L and breast cancer subtypes and clinical parameters in Brazilian BC patients. However, other

studies are necessary to clarify the mechanisms by which this virus can induce carcinogenesis and

progression in BC.

Keywords: HMTV; mammary tumor; DNA; clinicopathologic parameters.

Grants: CNPq, CAPES and Cancer Hospital of Londrina.

122

DETECTION OF GENES ASSOCIATED WITH VIRULENCE OF Proteus mirabilis ISOLATED FROM CHICKEN

CARCASS

Guidone, G. H M.1; Sanches, M. S. 1; Rocha, S. P. D.1

1 Universidade Estadual de Londrina, Department of microbiology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Brazil is one of the main producers and exporters of chicken meat in the

world. In this context, the state of Paraná is extremely relevant, since it accounts for more than 35%

of the exports of chicken meat produced in Brazil. In view of the relevance of this product worldwide,

it is important that the microbiological quality of chicken carcasses is guaranteed in order to avoid

the transmission of bacteria that pose a threat to consumer health. Proteus mirabilis is a Gram-

negative bacterium belonging to the Enterobacteriaceae Family and classified as an opportunistic

pathogen, as it may eventually cause infections in humans, with urinary tract infections being the

most frequent and significant due to its virulence factors. Therefore, the present work aims to

characterize phenotypically the virulence factors in P. mirabilis isolated from chicken carcass, in order

to evaluate its pathogenic potential in developing infections in humans.

Material and methods: Thirty-two P. mirabilis strains from non-eviscerated chicken carcasses were

isolated from a slaughterhouse located in the north of Paraná, Brazil. DNA extraction from all isolates

was performed by the boiling technique, followed by screening for nine virulence genes: mrpA,

pmfA, ucaA, atfA (fimbriae), ptA, zapA (proteases), hpmA, hlyA (hemolysins) and ireA (Siderophere)

by the PCR technique. PCR amplified fragments were subjected to 2% agarose gel electrophoresis

and observed in ultraviolet light translucent.

Results: All isolates presented several genes associated with virulence of P. mirabilis in Humans, in

which 32 (100%) isolates possessed the mrpA, pmfA, atfA, ptA, zapA, hpmA, and ireA genes. Of all

the genes research, ucaA was the least prevalent 18 (50%) isolates and hlyA was not found.

Conclusion: It is concluded with the present work that chicken carcasses can be considered a

reservoir of P. mirabilis that have a variety of virulence factors, such as fimbriae, proteases, and

hemolysins. Therefore, it is imperative that consumers exercise caution when preparing chicken meat

in order to avoid contact with this pathogen that has a great virulence potential.

Keywords: Zoonotic risk; Public health; Virulence

123

EFFECS OF CHRONIC Toxoplasma gondii INFECTION ON JEJUNUM

Andressa Sulamita Siqueira Menezes de Brito 1; Aline Aguiar 1; Lucas Casagrande 1; Lainy Leiny de

Lima 2; Débora de Mello Gonçalves Sant'Ana 1; Gessilda de Alcantara Nogueira de Melo 1;

Jaqueline de Carvalho Rinaldi 1;

1 State University of Maringá, Department of Clinical Analysis and Biomedicine, Post-Graduation

Program of Biosciences and Physiopathology, Maringá, Brazil 2 State University of Maringá, Department of Morphological Sciences and Post-Graduation of

Compared Biology, Maringá, Brazil

[email protected]

Introduction and objectives: Toxoplasmosis is a highly prevalent protozoan infection, affecting

approximately one-third of the world population. Toxoplasma gondii (T. gondii) is an obligate

intracellular parasite that induces a scenario of intense inflammation. In natural oral infections,

histopathological studies demonstrate parasite invasion in the gut and subsequently dissemination

throughout the body. It induces inflammatory response that dysregulate the intestinal epithelium and

can lead to changes in different cell types in the gastrointestinal tract. In this study, we analyzed the

effects of chronic T. gondii infection on blood total leukocytes and jejunum epithelial goblet cells.

Material and methods: Eight male C57BL/6 mice were distributed as a control group (CG, n=4) that

received saline; and an infected group (IG, n=4) which received a suspension with 1,000 parasite

oocysts (strain ME-49, genotype II) orally. After 60 days they were submitted to euthanasia and blood

were collected for analysis of total leukocytes. Their jejunum were removed and processed for

histopathological analysis. 4 sections of 4 μm were stained by Periodic Acid of Schiff (PAS) and used

to quantify PAS+ goblet cells in 2560 epithelium cells. Slides were imaged using light microscopy.

Results: Although T. gondii-infected mice exhibited efficient control of the bacterial burden, we

observed an increase in total leukocyte number when compared to the control group (CG=980±80

versus IG=1852±107 leukocyte/mm3). This observation reinforce that even after infection

chronification, systemic inflammation is remained. We also analyzed the action of the protozoa on

goblet cell population. Those cells are responsible for the production of mucins, a film that lubricates

and protects the intestinal epithelium against pathogens. In this study, no alteration was observed

on PAS+ epithelial goblet cells quantification (CG=7.2±0,3 and GI=7.4±0.5 goblet cells/100 epithelial

cells). PAS+ goblet cells are responsible to secrete neutral mucins. However, further analysis are

needed to evaluate the goblet cell subpopulation that produces acid and alkaline mucins.

Conclusion: Chronic infection with T. gondii increased blood total leukocytes but did not affect PAS+

goblet cells distribution on jejunum epithelium in mice.

124

EFFECTS OF ACUTE AND CHRONIC Toxoplasma gondii INFECTION ON DUODENUM GOBLET CELLS

Aline Aguiar1; Andressa Sulamita Siqueira Menezes de Brito1; Lucas Casagrande1; Lainy Leiny

deLima2; Débora de Mello GonçalvesSant'Ana1; Jaqueline de Carvalho Rinaldi1; Gessilda de

AlcantaraNogueira de Melo1.

1 State University of Maringá, Department of Clinical Analysis and Biomedicine, PostGraduation

Program of Biosciences and Physiopathology, Maringá, Brazil 2State University of Maringá, Department of Morphological Sciences and Post-Graduation of

Compared Biology, Maringá, Brazil [email protected]

Introduction and objectives: Toxoplasmosis is a highly prevalent protozoan infection. The oral route

of infection favors the disruption of the intestinal epithelium caused by inflammatory stressors and

facilitates the spread of the parasite. The infection also can lead to changes in different cell types in

the gastrointestinal system. In this study, we analyzed the effects of chronic Toxoplasma gondii(T.

gondii) infection on duodenum goblet cells.

Material and methods: Eight male C57BL/6 mice were distributed as a control group (CG, n=4) and

an infected group (IG, n=4), which received a suspension with 1,000 parasite oocysts (strain ME-49,

genotype II) orally. After 5 and 60 days they were submitted to euthanasia and had their duodenum

collected. Four sections of 4 μm were stained by periodic acid of Schiff (PAS) and used to quantify

goblet cells in 2,560 epithelium cells.

Results: Acute infection with T. gondiireduced the number of goblet cells that produce neutral mucins

(PAS+) in the duodenum of the infected animals (CG=10.82 ± 0.46 goblet cells/100 epithelial cells) in

relation to the control (IG=14.83 ± 0.79 goblet cells/100 epithelial cells). However, no alteration was

observed on the goblet cell distribution after chronic T. gondii infection (CG=5.21 ± 0.52; IG=6.42 ±

0.63). This result implies that acute T. gondiiinfection changes mucin composition. Those cells are

responsible for the production and release of mucins, which form a film that lubricates and protects

the intestinal epithelium against pathogens when in contact with the lumen.

Conclusion: These observations show how acute and chronic infection with T. gondii affects goblet

cell distribution on duodenum epithelium in mice.

Keywords: toxoplasmosis, gastrintestinal tract, experimental infection, goblet cells Grants:

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes).

Thematic area:

125

PREVALENCE OF MICROORGANISMS RESISTANT TO MULTIPLE DRUGS IN NEWBORNS.

Gobo, Elisangela de Fátima1; Oliveira, Thilara Alessandra de¹; Santana, Alisson¹.

1 State University of Londrina, Department of clinical microbiology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: The hospital setting is where high rates of health-care-associated

infections associated with multidrug-resistant microorganisms (MDR) are observed, especially in

neonatal units, since newborns do not have a developed immune system that guarantees them

defense. Hospital infections manifest themselves intensely and frequently in these environments. Pre-

mature newborns, do not have a protective microbiota, need to be colonized. The colonization

process is characterized by the presence of the microorganism in the host in the absence of clinical

manifestations and immunological response at the moment of bacterial isolation. Often, Newborns

that maintain contact with the mother skin to skin, for a long time, are colonized at birth through the

skin and mucosal surfaces, such as nasopharynx, oropharynx, conjunctiva, external genitalia and

umbilical cord. The presence of a microbiota that is not very virulent protects the child from

colonization by potentially pathogenic microorganisms, since the bacteria of the microbiota

proliferate in the various sites, competing with pathogenic organisms, with the evolution to disease

in smaller proportions. The objective of this study was to evaluate the prevalence of microorganisms

resistant to multiple drugs belonging to the newborn microbial, determining the antimicrobial

susceptibility profile of the main Enterobacteriaceae and methicillin-resistant Staphylococcus aureus

(MRSA) in surveillance swabs.

Material and methods: An observational, prospective study in Intensive Care Unit and Neonatal

Intensive Care Unit of a school hospital, to verify the prevalence and microbial resistance. Periodic

collections with surveillance swabs were performed, using a technique standardized by CLSI, 2015,

during the entire hospitalization of the newborns. Isolation and identification of bacteria were

performed according to the standard methodology for each species to be identified. For the

antimicrobial susceptibility assessment, the disc-diffusion technique was used according to the CLSI

recommendations (2015-2018). After identification, the isolated microorganisms were stored in

appropriate medium containing 20% glycerol at -80 ° C.

Results: Between August 2018 and February 2019, 1539 swab samples were taken, with a total of 216

positive cultures, with prevalence for Enterobacteriaceae MDR, some of these microorganisms are

commensals found in the normal intestinal flora, several strains of these bacteria are pathogenic and

may cause opportunistic infections in immunocompromised patients and inpatients in hospitals.

According to Giuffre et al., 2016 in an observational study, also reported a high prevalence of BGN

in its neonatal population

Conclusion: This study provides insight into the prevalence of multidrug resistant pathogens in

neonatal intensive care and therapy units. The results reinforce the fundamental importance of care

in the units and the control of implantation of antimicrobial therapy administration programs.

Keywords: Microorganism resistant to multiple drugs, neonatal colonization, Enterobacteriaceae.

126

Escherichia coli BLOODSTREAM INFECTION IN PATIENTS OF A UNIVERSITY HOSPITAL: VIRULENCE

FACTORS AND CLINICAL CHARACTERISTICS.

Ana Paula Daga1, João Gabriel Material Soncini1, Gerusa Luciana Magalhẽs1, Ariane Tiemy Tizura1,

Carolina Martins de Matos1, Vanessa Koga2, Renata Kobayashi2, Marsileni Pelisson1, Márcia Regina

Eches Perugini1, Eliana Carolina Vespero1

1 Universidade Estadual de Londrina, Laboratory of Clinical Microbiology of the University

Hospital of Londrina, Londrina, PR, Brazil. 2Universidade Estadual de Londrina, Department of Microbiology, Londrina, PR, Brazil.

[email protected]

Introduction and objectives: Escherichia coli is the Gram-negative organism most frequently isolated

in adult patients with bacteremia and in severe cases it may lead to death. The rates of bacteremia

have increased steadily in recent years. In general, E. coli constitute the normal commensal gut

microbiota of healthy human populations. Moreover, some strains can cause intestinal or

extraintestinal infections due to the specific virulence factors. The aim of this study was to

characterize E. coli isolated from the bloodstream from patients (n= 48) of University Hospital in Brazil.

Material and methods: Epidemiological data were obtained through the analysis of medical records

and laboratory tests. By Polymerase Chain Reaction (PCR) we investigated the presence of virulence

factors (VFs), pathogenicity islands (PAIs), Extended-Spectrum βLactamase (ESBL), phylogenetic

classification (A, B1, B2, C, D, E, F) and genetic variability analysis by Enterobacterial Repetitive

Intergenic Consensus (ERIC-PCR).

Results: The median age of the patients was 63 years (0 - 90). The presence of immunosuppression

was prevalent among patients (27.1%), as well as intestinal diseases (25.0%) and diabetes (18.8%).

Half of the bloodstream infections were after urinary tract infection, 12.5% secondary to abdominal

diseases. The mortality associated with E. coli bacteremia was 33.3%. Forty-one (85.4%) of E. coli had

one or more genes related to serum resistance and traT presented a high prevalence among isolates

(77.1%). In addition, 47.9% showed at least one capsule, group 2 (KpsMT II) with 45.8% and K5 specific

capsule with 20.8% of prevalence. The siderophores systems were common among isolates,

yersiniabactina (fyuA) was present in 70.8% of the isolates. The presence of PAIs was also significant,

with 81.3% of the isolates being positive for one or more PAI. PAI IV536 was present in 77.1% of isolates.

Phylogenetic analysis showed the group B2 (45.8%) was the most prevalent. However, in this study

was found a considerable prevalence isolated from group B1 (18.8%) and E (14.6%). The B2 isolates

presented the highest prevalence of VFs and PAIs. Eight (16.7%) isolates were resistant to third and

fourth generation cephalosporin. Group CTX-M-1 (CTX-M15) was the most prevalent ESBL type. The

analysis of genetic variability showed two clonal lineages and several isolates not related to each

other.

Conclusion: These results indicate that the bloodstream infection process can be mediated by

several alternative VFs, and each strain may have a unique combination of these factors. This variety

of virulence genes can be explained by several genetic factors that contribute to genome plasticity,

such as plasmids, phages, and transposable elements. This study was important because showed

clinical and molecular characteristics of E. coli bloodstream infection from Brazilian isolates.

Keywords: ExPEC; bloodstream infection; phylogenetic group.

127

EVALUATION OF ADHESION PATTERN, BIOFILM FORMATION AND CYTOTOXICITY OF Proteus mirabilis

ISOLATED FROM CHICKEN CARCASS

Guidone, G. H. M.1; Sanches, M. S. 1; Rocha, S. P. D. 1

1 Universidade Estadual de Londrina, Department of microbiology, Londrina, PR, Brazil


Introduction and objectives: Several virulence factors may be expressed by Proteus mirabilis during

the infectious process, such as adhesion capacity, biofilm formation and cytotoxicity. P. mirabilis is

well known for its ability to form biofilm, especially on abiotic surfaces such as urinary catheters, which

makes urinary tract infection more significant and persistent. In this sense, the adhesion ability is

considered crucial in the establishment of the infection, as well as in the formation of the biofilm.

Bacterial adhesion may be expressed in various patterns, such as aggregative adhesion, which

present more adhesion to inert surfaces than to cells and confers resistance against the host immune

system. Another virulence factor also very important in the infectious process, especially in

uropatogenicity, is cytotoxicity in renal cells, which is characterized by cellular damage. The

objective of the present study was to evaluate the adhesion capacity, biofilm formation and

cytotoxicity in Vero cells expressed by 32 strains of P. mirabilis isolated from chicken carcass in a

slaughterhouse in the North of Paraná, Brazil, in order to evaluate a possible zoonotic potential of

these strains.

Material and methods: The adhesion capacity of P. mirabilis strains was evaluated in Human Larynx

Carcionama cells (HEp-2), with 6 hours of interaction between cell-bacteria. Biofilm formation was

performed on 96-well polystyrene plates using violet crystal, classifying the biofilm as absent, weak,

moderate, strong and very strong. Cytotoxicity was evaluated in Vero (African Green monkey

kidney) cells and quantified by the MTT (3- [4,5dimethyl-thiazol-2-yl] -2,5-diphenyl-tetrazolium

bromide) technique to evaluate cell viability.

Results: It was observed that 32 isolates (100%) expressed the pattern of aggregative adhesion in

HEp-2 cells, similar to the pattern exhibited by Enteroaggregative Escherichia coli (EAEC). All the

isolates showed a biofilm formation capacity, 17 (53.12%) formed a strong biofilm and 15 (46.88%)

formed a very strong biofilm. Regarding cytotoxicity, 27 isolates (84.38%) exhibited cytotoxic effect.

Conclusion: It is evident from this study that P. mirabilis isolated from chicken carcasses express

virulence factors that contribute significantly to human infection, such as aggregative adhesion,

biofilm and cytotoxicity. Therefore, it is essential that consumers of chicken be cautious in preparing

the meat in order to avoid contact and possible infections that may be caused by this pathogen.

Keywords: Factors of virulence; Zoonotic risk; Broiler chickens.

128


EXCRETION / SECRETION OF LARVAE OF FLY DIPTERANS OF THE SPECIES Lucilia cuprina PRESENT

DIRECT ACTION ON TRYPOMASTIGOTE FORMS OF Trypanosoma cruzi

Ana Carolina Jacob Rodrigues1; Manoela Daiele Gonçaves1; Andra Diaz Roa1, Bruna Taciane da

Silva Bortoleti1; Virgínia Márcia Concato1; Fernanda Tomiotto-Pellissier1; Amanda Cristina Carloto1;

Pedro Isamael da Silva Junior1; Idessania Nazareth Costa1; Ivete Conchon-Costa1; Milena

Menegazzo Miranda Sapla1; Wander Rogério Pavanelli1;


[email protected]

Introduction and objectives: Chagas' disease is a zoonosis caused by the protozoan Trypanosoma

cruzi, which was considered restricted to endemic countries, but today with globalization and

migration reaches countries around the world. Approximately 7 million people are infected, 100

million live in the area at risk, 56 thousand new cases and 12 thousand deaths a year. In view of its

high prevalence and resistance of protozoan strains to conventional treatment, the search for new

drugs with trypanocidal action and low toxicity to the patient became intense. Flies of the

Calliphoridae family (popularly known as kissing bugs) play an important role in nature, participating

in the food chain and recycling biomass. Studies have shown that the excretion/secretion (ES) of

larvae in third instar of these flies, have cicatrizant, antibacterial, antifungal and leishmanicidal

activity. However, to date, there are no studies investigating the therapeutic potential of larval ES on

the protozoan T. cruzi. In this sense, this work had as objective to investigate the direct effect of larval

ES in trypomastigote forms of T. cruzi, as well as the morphological changes caused in the protozoan.

Material and methods: ES larval strain of Lucilia cuprina was obtained from the Laboratory of

Toxicology Applied to the Institute and its prosthetic quantification obtained from Nano Vue Plus

(Biochrom, Holliston, USA). The substance was tested against the parasite in different concentrations

(3.15, 6.3, 12.5, 18.5, 25, 31.5, 50, 75 μg / mL), according to data consulted in the literature. To evaluate

the trypanocidal activity, a count of the trypomastigote forms was performed after 24 hours of

treatment using the Neubauer chamber and tripan blue. Analysis of the morphology of

tripomastigote forms was performed according to Tomiotto-Pellissier et al. [18]. The parasites were

treated with 40μg / mL DHA at 24 ° C for 24h. The samples were observed under a scanning electron

microscope (FEI Quanta 200).

Results: It was observed that the ES larval (3.15-75 μg / mL) was able to reduce the numbers of

trypomastigote forms at all concentrations tested. The data obtained in the count of viable

parasites were confirmed by Scanning Electron Microscopy (SEM), where it was observed that

parasites exposed to IC 50 treatment of larval ES suffered loss of membrane integrity and exposure

of intracellular contents. Treatments with 2xIC50 of larval ES suffered in addition loss of the typical

morphology of the parasite.

Conclusion: We conclude that ES larval has potential antipromastigote activity, however further

studies will be carried out in order to elucidate the immunomodulatory mechanisms involved.

Keywords: Trypanosoma cruzi, Excretion/ Secretion Larval, trypanocidal, SEM

Grants: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

129

GENOTYPIC AND EPIDEMIOLOGICAL STUDY OF CARBAPENEM RESISTANT ENTEROBACTERIACEAE

ISOLATES OF PATIENTS FROM THE UNIVERSITY HOSPITAL OF LONDRINA STATE UNIVERSITY, FROM 2009

TO 2016.

Tizura, A. T.1; Soncini, J.G.M.1; Callado. G.T.1; Luz, T.C.1; Magalhães, G.L.G.1; Kaminami, C.M.1;

Candido, E.P.1; Mendes, E.C.R1; Pimenta, J.1; Pelisson, M.1; Perugini, M.R.E.1; Vespero, E. C.1

1Universidade Estadual de Londrina, Micriobiology Laboratory of the University Hospital of

Londrina State University, Londrina, PR, Brazil. [email protected]

Introduction and objectives: Resistance among members of the Enterobacteriaceae family,

capable of causing several types of infections, has emerged as a major public health problem. With

the emergence of carbapenem resistance, being the carbapenemases production the most

common mechanism envolved, a restriction in the pharmacotherapy of patients with these

infections could be observed. The therapeutic failure is the most significant consequence, leading

to an increase in the morbidity and mortality of these patients, which highlights the need to develop

and implement strategies for prevention and control of the dissemination of this mechanism of

resistance and infections caused by these microorganisms. The aim of this study was to evaluate the

frequency and resistance profiles of Enterobacteriaceae isolated from clinical materials of patients

from the University Hospital of Londrina from 2009 to 2016.

Material and methods: In this studied period, 23.900 Enterobacteriaceae were identified by Vitek2®

BioMérieux automated system and the susceptibility profiles were evaluated by the sensitivity test

card AST 239 and AST 238. The isolates that were resistant for one or more carbapenems were

submitted to confirmatory tests of resistance mechanisms such as: modified Hodge test, Boronic acid

test and EDTA test. PCR was performed to research blaKPC gene.

Results: The most frequently isolated microorganism was Klebsiella pneumoniae with 78.3%, followed

by Enterobacter cloacae 9.4%, Serratia spp. 3.3%, Providencia stuartii 2.8%, Escherichia coli 1.9%,

Citrobacter spp. 1.6% e Enterobacter aerogenes 1.4%. blaKPC gene was detected in 71.8% of

carbapenems resistant Enterobacteriaceae, being K. pneumoniae the most frequent, with 82.2%,

followed by E. cloacae 7.6%, E. coli 2.3%, Serratia spp. 1.9%, Citrobacter spp. 1.7%, P. stuartii 1.4%

and E. aerogenes 1.4%. Regarding the resistance profile of microorganisms to carbapenems, more

than 90% showed resistance to ertapenem, meropenem and imipenem. These isolates were also

resistant to gentamicin (72.9%) and amicacin (51.4%). In addition, 27.7% were resistant to colistin.

Through the studies, it was also verified that the majority of these microorganisms were isolated from

surveillance cultures (40.2%), respiratory tracts (23.9%) and urinary tract (23.1%). blaKPC gene was

detected in 13% (3117/23900) of the total species, and 71.8% (3117/4341) of the CRE isolates. K.

pneumoniae was the most common microorganism CR producing blaKPC gene, with 82.2%

(2564/3117) followed by E. cloacae 7.6% (238/3117), Escherichia coli 2.3% (72/3117), Serratia spp. 1.9%

(58/3117), Citrobacter spp. 1.7% (54/3117), Providencia stuartii 1.4% (44/3117) and E. aerogenes 1.4%

(43/3117).

Conclusion: These results point to high levels of carbapenem resistance among Enterobacteriaceae

in the State University of Londrina, being KPC, one of the most important resistance mechanism

among them.

Keywords: Enterobacteriaceae, blaKPC, carbapenems resistance

130

HERPES SIMPLEX VIRUS IS INHIBITED BY Myrciaria dubia POLYSACCHARIDES

Ananda Marques de Godoi1; Daniele Zendrini Rechenchoski1; Ligia Carla Faccin-Galhardi1;

Arcelina Pacheco Cunha2; Nágila Maria Pontes Silva Ricardo2; Derlange Belizário Diniz2; Rosa Elisa

Carvalho Linhares1

1Universidade Estadual de Londrina, Department of Microbiology, Londrina, PR, Brazil

2Universidade Federal do Ceará, Department of Organic and Inorganic Chemistry, Fortaleza, CE,


Introduction and objectives: Infections caused by herpes simplex virus (HSV) are highly contagious

and endemic worldwide. HSV is a linear double-stranded DNA virus, with icosahedral capsid and

envelope containing glycoproteins. The virus induces clinical manifestations with a varying severity,

from oral lesions to encephalitis, especially in immunocompromised. The sites of primary infection are

oral and genital mucosa, with the establishment of latency, difficulties for the treatment and

effectivity of vaccine. Acyclovir (ACV) and its derivatives are the drugs of choice, however, their

frequent and inadequate use has led to the emergence of resistant strains. This encourages the

search for natural products with antiviral action, because they usually present low toxicity and act in

several stages of viral replication. Myrciaria dubia (Md) is a typical Amazonian plant, popularly known

as camu-camu whose fruits are rich in vitamin C. Used popularly as antioxidant, antiinflammatory,

antimicrobial, antiatherogenic and anti-aging. This study aimed at evaluating the antiviral effect of

the in natura (AO) and sulfated (AOS) Md polysaccharide for HSV-1 KOS (ACV-sensitive) and AR-29

(ACV-resistant) strains.

Material and methods: The cytotoxicity, in Vero cells, of the polysaccharides was carried out by

colorimetric method of dimethyl-thiazolyl-diphenyltetrazolium bromide (MTT) and the antiviral activity

by plaque reduction assay (PRA) in varying treatment protocols, beyond immunofluorescence assay

(IFA) and polymerase chain reactions (PCR).

Results: The cytotoxic concentrations 50% (CC50) were > 500 μg/mL and 386 μg/mL for AO and AOS,

respectively, showing low toxicity. The AOS showed a better inhibition effect at the time 0 h of

infection for both HSV-1 strains, without significant differences between them. Moreover, the antiviral

effect was related to the initial stages of the infection and confirmed by IFA.

Conclusion: In conclusion, our results demonstrated that AOS is a compound with promising antiviral

activity, suggesting its use for the development of new anti-herpetic drugs.

Keywords: Myrciaria dubia; Herpes simplex virus; antiviral; polysaccharides

Grants: The authors gratefully acknowledge the financial support from CNPq, CAPES (Finance Code

001) and Fundação Araucária

131

HPV INFECTION AND APOBEC3A/B DELETION POLYMORPHISM IN OROPHARYNGEAL SQUAMOUS CELL

CARCINOMA PATIENTS

Vitiello, G. A. F.1; Dias-Audibert, F. L.2; Hauenstein de Mendonça, J.1; Banin-Hirata, B. K.1; Oliveira, C. E. C.3;

Piccoli de Melo, L. G.4; Nunes, S. O. V.4; Ribeiro, E. M. S. F.5; Cavalli, I. J.5; Oliveira, K. B.1; Amarante, M. K.1; Losi-

Guembarovski, R.6; Watanabe, M. A. E.1

1Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil 2Universidade Estadual de Campinas, Faculdade de Ciências Médicas, Campinas, SP, Brazil

3Pontifícia Universidade Católica do Paraná, Campus Londrina, School of Medicine, Londrina, PR, Brazil 4Universidade Estadual de Londrina, Department of Clinical Medicine, Londrina, PR, Brazil

5Universidade Federal do Paraná, Department of Genetics, Curitiba, PR, Brazil 6Universidade Estadual de Londrina, Department of General Biology, Londrina, PR, Brazil


Introduction and objectives: Viruses are implicated in the pathogenesis of many cancers. Human papilloma

virus (HPV), which is the classical etiological agent for uterine cervix cancer, have been also implicated in oral

squamous cell carcinoma (OSCC). APOBEC3-family are a group of cytidine deaminases involved in innate

immunity against viruses by promoting mutation of viral genetic material. APOBEC3A and APOBEC3B were also

shown to be mutational sources in many human cancers, and a deletion polymorphism joining the APOBEC3B

3’UTR region to APOBEC3A exons was associated with cancer susceptibility by increasing APOBEC3A mutational

potential. In the present study we aimed to investigate the prevalence of HPV in a Brazilian sample of OSCC

patients, the association of APOBEC3A/B deletion polymorphism with OSCC susceptibility and with HPV infection

in this disease and the correlation between HPV infection or APOBEC3A/B polymorphism with

clinicopathological parameters.

Material and methods: DNA was extracted from peripheral blood samples of 54 OSCC patients from Erasto

Gaertner Hospital (Curitiba, PR) and from 40 fresh tumor tissues of these patients. For control group, DNA was

extracted from 179 heavy smoker patients without any evidence of neoplasia from Erasto Gaertner and

Londrina State University Clinical Hospital (Londrina, PR). HPV was detection through PCR in DNA from fresh tumor

tissues and APOBEC3A/B genotyping was made through allele-specific PCR. Statistical analyses consisted of

age-adjusted logistic regressions for case-control study and of Kendall’s Tau-b rank test for correlation analyses.

This study was approved by institutional ethics committees on research involving human subjects from Londrina

State University (037/10 – CAAE 0027.0.268.000-10) and Erasto Gaertner (PP909-07/2003), and all volunteer donors

signed a free-informed consent prior to biological material collection.

Results: Age significantly differed (p < 0.001) between OSCC patients (median = 54, IQR = 19) and controls

(median = 47, IQR = 14). HPV was present in 21 of 40 (52.5%) tumor tissues analyzed, and its presence was

positively correlated with patients’ age (Tau-b = 0.3; p = 0.02), but not with tumor size or lymph node involvement.

Regarding APOBEC3A/B polymorphism, the genotype frequencies for wild-homozygotes, heterozygotes and

deleted homozygotes were, respectively, 153 (85.4%), 25 (14%) and 1 (0.6%) in control group and 41 (75.9%), 13

(24.1%) and 0 in OSCC group. In age-adjusted logistic-regression there was a trend towards a positive

association between APOBEC3A/B deletion and OSCC susceptibility in heterozygote (OR = 2.42, 95%CI = 0.99 –

5.94, p = 0.05) and dominant model (OR = 2.27, 95%CI = 0.94 – 5.5, p = 0.07). APOBEC3A/B was not associated

with clinicopathological parameters nor with HPV infection in OSCC patients.

Conclusion: These results indicate that the prevalence of HPV infection in Brazilian OSCC is high, and that the

infection rates are higher the higher is the age. Also, the higher frequency of APOBEC3A/B deletion

polymorphism in OSCC group, almost reaching a statistical significance, may indicate that this polymorphism

may increase OSCC risk and point it as an interesting susceptibility marker to be investigated in larger samples.

Keywords: APOBEC, human papilloma virus, oropharynx squamous cell carcinoma, polymorphism, susceptibility.

Grants: CNPq, CAPES, Fundação Araucária e FAUEL.

132

IN VITRO ANTIVIRAL ACTIVITY OF LASIODIPLODAN AND ITS CARBOXIMETILATED AND SULFATED

MOLECULES IN THE REPLICATION OF HERPES SIMPLEX 1

Jéssica Wouk1; Ligia Carla Faccin Galhardi2a; Vinicius Martinez Rino2a; Vitor Yuji Ito 2a; Aneli de Melo

Barbosa Dekker2b; Mario Antônio Alves da Cunha3; Gabrielle Cristina Calegari3; Vidiany Aparecida

Queiroz Santos3; Robert Frans Huibert Dekker4; Carlos Ricardo Maneck Malfatti1

1 Universidade Estadual do Centro-Oeste, Department of Post-graduation in Pharmaceutical

Science, Guarapuava, Brazil 2Universidade Estadual de Londrina, aDepartment of Microbiology and bDepartment of Chemistry,

Londrina, PR, Brazil 3 Universidade Tecnológica Federal do Paraná, Chemistry Department, Pato Branco, PR,

Brazil 4 Universidade Tecnológica Federal do Paraná, Deparment of Post-graduation in

Environmental Engineering, Londrina, PR, Brazil [email protected]

Introduction and objectives: β-Glucans are carbohydrate polymers produced by

wooddecomposing fungi and are increasingly becoming recognized for their rheological properties

and biological activities, with applications in foods, cosmetics, and pharmaceuticals. Lasiodiplodan

(LAS) is a (1 6)-β-D-glucan which presented antiproliferative and antiinflammatory activities.

Anticoagulant and antioxidant activities were improved when the native LAS molecule was

chemically modified by sulphation. Derivatized polysaccharides are known to display activity against

enveloped viruses, such as herpes virus. Therefore, the present work aimed to assess the antiviral

activity of LAS, and its derivatives, against herpes simplex virus type 1 (HSV-1), in Vero cells.

Material and methods: LAS was produced by the fungus Lasiodiplodia theobromae MMPI and

chemically modified by sulfation (L1 – L3) and carboxymethylation (C1- C5). The cytotoxicity and

anti-HSV-1 activity (KOS strain) of native LAS and its modified molecules were evaluated by the MTT

method.

Results: Modified substances showed the following degrees of substitution (DS) from 0.32 (C1) to 0.68

(C5) and 0.15, 1.61 and 0.18 for L1, L2, and L3, respectively. The sulfated molecules L1, L2, and L3

demonstrated CC50 of 125 μg mL-1, 171.25 μg mL-1 and 537.5 μg mL-1, respectively. The other tested

compounds presented CC50 >1000 μg mL-1. However, only L2 and L3 inhibited HSV-1 replication in

vitro. The viral inhibition percentage (%VI) found were 50% at 53.5 μg mL-1 (IC50) for L2 and 100% even

until the lowest concentration tested (25 μg mL-1) for L3.

Conclusion: From all tested compounds, L3, which present the highest DS, demonstrated the best

anti-HSV-1 activity in vitro, totally inhibiting viral replication in low concentrations. The anti-herpetic

activity of sulfated polysaccharides was also reported in other scientific studies, relating the

hydrophobic interactions between the polysaccharide and the viral envelop glycoproteins. The

present study corroborates with these results. Further tests may be performed to unveil LC mechanism

of action against HSV-1.

Keywords: Lasiodiplodia theobromae, lasiodiplodan, antiviral, HSV-1, in vitro

Grants: CNPq, CAPES, Fundação Araucária

133

INFECTION BY Leishmania (Viannia) braziliensis DO NOT PROMOTE MORPHOLOGICAL CHANGES IN

THE ENTEROCYTES OF HAMSTERS COLON

Lainy Leiny de Lima1; Amanda Gubert Alves dos Santos2; Bruna Cristina Pagliarini2; Thaís Gomes

Verzignassi Silveira2; Gessilda Alcântara de Nogueira de Melo 2; de Débora Mello Gonçales

Sant'Ana3

1 Universidade Estadual de Maringá, Department of Morphology Sciences,Postgraduation

Program in Comparative Biology - PGB, Maringá, PR, Brazil 2 Universidade Estadual de Maringá, Department of Clinical Analyzes and Biomedicine,

Postgraduate Program in Biosciences and Pathophysiology - PBF, Maringá, PR, Brazil 3 Universidade Estadual de Maringá, Department of Morphology Sciences, Postgraduate

Program in Biosciences and Pathophysiology - PBF, Maringá, PR, Brazil

[email protected]

Introduction and objectives: The tegumentary leishmaniasis is a worldwide neglected disease. The

specie Leishmania (Viannia) braziliensis is one of the most important species that cause the disease.

It can manifest in cutaneous, mucocutaneous and disseminated forms. In addition, authors describe

that the parasite can reach secondary organs, such as the intestine. Thus, the aim of the study was

to evaluated the enterocytes present in the colon of hamsters infected with L. (V.) braziliensis.

Material and methods: This study was approved by the Comissão de Ética no Uso de Animais of the

Universidade Estadual de Maringá (protocol 7587260416). Eight female hamsters were used (n=4).

The animals were distributed in two groups: Control group (CG) and Infected group (IG). The IG

received 2x107 promastigotes forms of L. (V.) braziliensis (MHOM/BR/2003/2311 strain) in the left hind

paw. After 90 days of infection, the animals were submitted to euthanasia by anesthetic deepening,

the colon was collected, washed and fixed. Semi-serialized crosssections of 4 μm thickness were

performed and subsequently stained by the hematoxylin and eosin technique. 80 enterocytes were

measured, their height and width, as well as the smaller and larger axis of their nuclei were measured

from images captured with the objective of 100x. Statistical analysis was performed using Student's

T-Test (p <0.05) and the results were presented by mean ± standard error.

Results: The height and width of enterocytes (18.38 ± 2.09, 5.48 ± 0.36) of the IG, as well as higher and

lower axis of their nuclei (8.10 ± 0.40, 3.76 ± 0.36), when compared to the CG (20.84 ± 1.03, 5.27 ±

0.095, highest and lowest axis of their nuclei 7.76 ± 0.40, 4.23 ± 0.16), did not present a significant

alterations, considering p <0.05.

Conclusion: After 90 days of the infection by L. (V.) braziliensis, was not able to promote significant

changes in the morphology of one of the most numerous cells of the intestine, mainly responsible for

the intestinal absorption.

Keywords: Leishmaniasis; Gastrointestinal tract, Intestinal epithelium

Grants: CNPq e Fundação Araucária

134

Klebsiella pneumoniae: EPIDEMIOLOGY, RESISTANCE TO CARBAPENENS AND POLYMYXINS AND

GENETIC DIVERSITY

Soncini, J. G. M.1; Magalhães, G. L. G.1; Tizura, A. T.1; Frizon, D. A.1; Daga, A. P.1; Luz, T. C.1; Candido,

E. P.1; Pimenta, Julia1; Mendes, E. C. R.1; Pelisson, M.1; Perugini, M. R. E.1; Vespero, E. C.1

1 Universidade Estadual de Londrina, Laboratory of Clinical Microbiology of the University

Hospital of Londrina, Londrina, PR, Brazil.

[email protected]

Introduction and objectives: Klebsiella pneumoniae is responsible for causing hospital infection and

has a high capacity of resistance mechanisms, including carbapenems, which are the main drugs

used to treat infections caused by this microorganism. Thus, the treatment became restricted to few

antimicrobials, returning the use of polymyxins in clinical practice. Resistance to carbapenems and

polymyxins has now become a public health problem.

Material and methods: In this context, this work aims to characterize by phenotypic, molecular and

epidemiological tests, Klebsiella pneumoniae samples resistant to carbapenems and to polymyxins

(KpRCP). We included 57 KpRCP previously identified by the VITEK® 2 system (bioMérieux).

Results: Of the clinical isolates studied, 30 (52.6%) were urine, followed by tracheal secretion 10

(17.5%), blood 9 (15.8), tissue 5 (8.8%), peritoneal fluid 2 (3, 5%) and sacral ulcer 1 (1.8%). Phenotypic

microdilution tests were performed on broth for imipenem, meropenem, colistin and polymyxin,

Polimyxin NP test (Poly NP) and agar dilution test for colistin and polymyxin. The presence of

carbapenemase-like enzymes, CTX-M ESBLs and the presence of the mcr-1 gene were characterized

by PCR. Genetic variability was performed using the ERIC-PCR technique and epidemiological data

were obtained through the analysis of medical records and laboratory tests. Based on the

phenotypic tests of VITEK® 2 and broth microdilution the resistance of the isolates was classified as

extremely resistant to drugs (PDR) (38.6%) and extensively resistant (XDR) (61.4%). Of the samples

studied, 55 (96.5%) of the strains were positive for the Poly NP test, 57 (100%), for the colistin drop test

and 54 (94.7%) for the polymyxin drop test. By the PCR reaction all samples carried the blaKPC-2 57

gene (100%) and no samples were detected in the blaNDM, blaOXA-48, blaVIM and mcr-1 genes.

For CT-M-type ESBL, we found CTX-M-1 13 (22.8%), CTX-M-15 12 (21.1%), CTX-M-26 (10.5%), CTX -M-8

18 (31.6%), CTX-M-932 (56.1%) and CTX-M-25 (no positive sample). The ERIC-PCR technique

demonstrated great genetic variability among the isolates. The analysis of the charts was evaluated

26 (45,6%) patients were hospitalized in the ICU at the beginning of the infection. In the previous use

of antibiotic therapy, 27 (47.4%) already used polymyxins, followed by carbapenems and piperacillin

tazobactam 20 (35.1%), tigecycline 14 (24.6), aminoglycoside and sulfamethoxazole trimethoprim 7

(12.3%), quinolone 5 (8.8%), third generation cephalosporin 4 (7.0%) and fourth generation

cephalosporin 1 (1.8%). We also assessed Charlson's comorbidity index and (59.8%) obtained higher

(> 2) data classified as severe. Of the 57 patients studied, 32 (56.1%) died.

Conclusion: This study provided important epidemiological information and identification of

resistance to polymyxins, on the current scenario of infections caused by carbapenem resistant.

Keywords: Klebsiella pneumoniae, polymyxin; carbapenens.

135

MANGIFERIN CREAM EXHIBITS ACTIVITY AGAINST HERPES SIMPLEX VIRUS RESISTANT TO ACYCLOVIR

Daniele Zendrini Rechenchoski1; Karoline Fontana Agostinho1; Ananda Marques de Godoi1; Ligia

Carla Faccin-Galhardi1; Audrey Alesandra Stinghen Garcia Lonni2; Arcelina Pacheco Cunha3;

Nágila Maria Pontes Silva Ricardo3; Carlos Nozawa1; Rosa Elisa Carvalho Linhares1

1Universidade Estadual de Londrina, Department of Microbiology, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil 3Universidade Federal do Ceará, Department of Organic and Inorganic Chemistry, Fortaleza, CE,

Brazil

[email protected]

Introduction and objectives: Herpes simplex virus (HSV) is a common human pathogen, causal agent

of remarkable diseases in the public health. Considering the high prevalence of the HSV infection

and the emergence of resistant strains to acyclovir (ACV), the development of new drugs has been

highly encouraged. In this context, the natural products represent an excellent source of bioactive

compounds with complex structure and chemical diversity, capable of presenting multiple

mechanisms of action and, in general, with low toxicity. Mangiferin is a glycosylated xanthone found

in many plant species, among them the mango tree (Mangifera indica). Pharmacological

properties, such as, antioxidant, antimicrobial, antiallergic, anticancer, anti-inflammatory,

antinociceptive, immunomodulatory and adjuvant in dermatology have been suggested. Herein,

we investigated the activity of formulation containing mangiferin against ACV-resistant HSV-1 AR-29

strain.

Material and methods: Oil/water emulsion was prepared by the phase-inversion emulsification

method in two steps. Organoleptic properties of the formulation, as well as, pH, spreadability and

stability were carried out. In vivo experimental procedure was granted by the UEL Animal Ethics

Committee (CEUA nº 24414.2015.49). The antiviral activity of the cream containing 0.7% (w/w)

mangiferin was performed in Balb/c mice infected by skin scarification and treated with the

formulation, 4 h after infection, 5 times a day, for 10 days. The development of skin lesions and

mortality were monitored and the severity of the lesions scored.

Results: Some animals of control group and treated with ACV developed zosteriform lesions on day

7 post infection (pi). By this time most of the infected and treated animals were already presenting

significant difference (p < 0.05) either by retrieved skin or with minor lesions. The results demonstrated

that mice treated with the cream presented a significant delay in the development and progression

of skin lesions compared with the control.

Conclusion: We therefore concluded that the formulation is a valuable candidate in antiherpetic

therapy including cases of ACV-resistant infections.

Keywords: Mangiferin; Herpes simplex virus; antiviral; resistance

Grants: CNPq, CAPES (Finance Code 001) and Fundação Araucária

136

POSSIBLE ASSOCIATION BETWEEN Paracoccidioides brasiliensis and Trichosporon asahii IN SOIL

Rafaela Macagnan1; Igor Massahiro de Souza Suguiura1; Eiko Nakagawa Itano1 Mario Augusto

Ono1

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil Email

[email protected]

Introduction and objectives: Fungi of the Paracoccidioides genus cause Paracoccidioidomycosis

(PCM), a systemic mycosis that affects mainly agricultural workers from Latin American countries. Its

habitat is not yet completely defined although infection probably occurs by inhalation of fungal

propagules from soil. Soil is a complex niche, highly colonized by different fungal species. Therefore,

the objective of this study was to identify a fungus similar to Paracoccidioides isolated from soil

samples obtained and analyzed macro-and microscopically in preliminary studies.

Material and methods: The identification by Molecular Biology was performed by PCR using primers:

ITS-1/4, Pb ITS-R/E, and -TubF/R; The amplicons were purified with ammonium acetate and

sequenced by Sanger method. The sequences were analyzed and the contigs were assembled

through the BioEdit program and compared to the database of the National Center for

Biotechnology Information (NCBI) using the BLAST nucleotide tool.

Results: The sequences obtained using the set of Pb-ITS-E/R and -tubulin-F/R pimers, showed 99%

similarity to P. brasiliensis in GenBank (BLAST analysis). However, the yeast isolate 20 showed 100%

similarly to Trichosporon asahii, using the ITS-1/4 primers. When the ITS regions were compared to

sequences deposited in GenBanK it was possible to observe the region amplified by the primers PB-

ITS-E/R and α-TubF/R were distinct from the sequences of T. asahii. The positivity observed in PCR for

P. brasiliensis and T. asahii suggests that isolate 20 is a co-culture of both fungi. Attempts to isolate P.

brasiliensis from this co-culture were unsuccessful because the growth of T. asahii is faster than P.

brasiliensis. The isolate 20 has been cultured for several months and the PCR positivity for P. brasiliensis

continues indicating its viability even in co-cultivation with T. asahii.

Conclusion: Although Paracoccidiodomycosis was discovered more than a century ago there are

few reports of P. brasiliensis isolation from soil samples. The difficulty in isolation is due to several

factors.The results of the present study suggest an association between P.

brasiliensis and T. asahii in soil, the probable habitat of Paracocidioides spp.

Keywords: Environmental samples; Paracoccidioides; PCM;

Grants: Fundação Araucária and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

137

PRESENCE OF VIRULENCE FACTORS AND PATHOGENICITY ISLANDS IN EXTRAINTESTINAL PATHOGENIC

ESCHERICHIA COLI ISOLATES FROM DIFFERENT CLINICAL MATERIALS

Daga, A.P.1; Tizura, A.T.1; Soncini, J.G.M.1; Soares, L.1; Pelisson, M.1; Perugini, M.R.E.1.; Vespero, E.C.1;

Koga, V.L2.; Kobayashi, R.K.T2.;

1Universidade Estadual de Londrina, Postgraduate program in Clinical and Laboratory

Physiopathology, Londrina, PR, Brazil. ²Universidade Estadual de Londrina, Postgraduate program in microbiology, Londrina, PR, Brazil.

[email protected]

Introduction and objectives: Escherichia coli is an extremely common and complex human

pathogen. It is both a common commensal inhabitant of the gastrointestinal tract and one of the

most important pathogens in humans. Extraintestinal Pathogenic E. coli (ExPEC) is descriptor for all

non-commensal E. coli isolates capable of causing extra-intestinal disease. The aim of this study was

to investigate and compare the virulence determinants of ExPEC from different clinical materials.

Material and methods: This study included 183 E. coli samples, 48 isolated from the bloodstream, 42

isolated from liquid and secretion, 44 isolated of inpatients with urinary tract infection (UTI), and 49 E.

coli of outpatients with UTI. Through PCR, we investigated the presence of virulence factors (VFs),

pathogenicity islands (PAIs), and phylogenetic classification (A, B1, B2, C, D, E and F). The study was

approved by the Ethics and Research Committee of the State University of Londrina CAAE

43013315.8.0000.5231.

Results: The most prevalent gene in all clinical materials was fimH and the prevalence was statistically

higher in blood and liquid/secretion (non-urinary) samples, as well as papC gene. E. coli isolated from

blood and liquids/secretions showed statistically higher prevalence of genes related to serum

resistance (traT, iss and ompT) compared to urinary samples. However, K5 capsule was more

prevalent in urinary samples. In general, isolates of UTI of outpatients presented higher prevalence of

VFs when compared to inpatients UTI isolates. In addition to the K5 capsule, KpsMT II (synthesis k1 and

k5), adhesin FimH and salmochelin receptor (iroN) were also statistically more prevalent in outpatients

UTI samples. The prevalence of PAIs in the different clinical materials was similar, with exception PAI

IV536, that was higher prevalence in blood than inpatients UTI isolates. The phylogroup B2 was the

most prevalent in isolates from all clinical materials and the second phylogroup more prevalent was

B1 for blood and HA-UTI, and E for liquids/secretion and outpatients UTI. There was a significant

prevalence of phylogroup A in urinary that non-urinary isolates.

Conclusion: In summary, the virulence capability is determined by a combination of distinctive

accessory traits, as VFs, in conjunction with their distinctive phylogenetic background. Many different

combinations of VFs occur in different ExPEC strains and can be associated with specific infectious

diseases.

Keywords: : Extraintestinal Pathogenic Escherichia coli; Virulence Factors, Pathogenic Islands;

Phylogenetic Classification

138

REDUCTION OF GOBLET CELLS IN HAMSTERS INFECTED BY Leishmania (Viannia) braziliensis

Beatriz Fonseca Ferreira1; Daniele Stefanie Sara Lopes Lera2, Maria Valdrinez Campana Lonardoni3,

Débora de Mello Gonçales Sant’Ana4, Gessilda Alcantara Nogueira- Melo3

1 Undergraduate student (Master degree) in Biosciences and Pathophysiology, State University of

Maringá, Maringá, PR, Brazil.

2 Post-graduate student (PhD) in Health Sciences, State University of Maringá, Maringá, PR, Brazil.

3 Professor at Post-graduate Program in Biosciences and Physiopathology, State University of



Introduction and objectives: The goblet cells (GC) are mucus-producing cells in the small intestine.

This mucus represents, along with the epithelial cells, the first defense barrier of the intestine. The

species Leishmania (Viannia) braziliensis (LVB) response for mucus-cutaneous form of leishmaniasis

because of its affinity for skin and mucous membranes, however recent studies report that the

parasite may also spread to other organs, such as the intestine. The objective of this study was to

quantitatively analyze goblet cells from ileum of hamsters after LVB infection.

Material and methods: Nine male hamsters (Mesocricetus auratus) were divided into four groups:

Control group (CG) and groups infected with strain LVB MHOM/2003/2314 by three different times of

infection. The infected hamsters group were inoculated with promastigotes of LVB (5x106 parasites)

and euthanized at 3, 5 and 8 weeks of infection (groups 3S, 5S and 8S). Histological sections of 4μm

thickness were prepared in paraffin and stained with the 2.5 Alcian-Blue technique. GCs were

counted among 2560 epithelial cells and the ratio GC/100 epithelial cells was calculated. The

distribution of the data was considered non parametric by the Shapiro-Wilk test. Next, Kruskall-Wallis

test was performed with post-test of Dunns, considering p <0.05 (median and percentiles). The Ethics

Committee on the Use of Animals of State University of Maringá did approve this study

experimentation under No. 306206916.

Results: Significant reduction of CC in the ileum of the infected groups was observed in the three

infection times: 3S 6.4 (5,8; 7,6), 5S 4.4 (3,1; 5,6) and 8S 5.5 (4,2; 7,1), when compared to the CG 11

(9,4; 12,6). This represents, therefore, a reduction of on average 50% of the number of goblet cells in

the small intestine.

Conclusion: LVB strain 2314 significantly reduced the number of GC in the ileum of hamsters at 3, 5

and 8 weeks of infection.

Keywords: Ileum; Intestinal mucous; Leishmaniasis.

139

REDUCTION OF INTRAEPITHELIAL LYMPHOCYTES IN HAMSTERS INFECTED BY Leishmania (Viannia)

braziliensis

Felipe Steinmacher Batista1; Beatriz Fonseca Ferreira2; Daniele Stefanie Sara Lopes Lera3, Maria

Valdrinez Campana Lonardoni4, Débora de Mello Gonçales Sant’Ana4 Gessilda Alcantara

Nogueira de Melo4

1 Undergraduate student in Medicine, State University of Maringá, Maringá, PR, Brazil.

2 Post-graduate student (Master degree) in Biosciences and Pathophysiology, State University of


3 Post-graduate student (PhD) in Health Sciences, State University of Maringá, Maringá, PR, Brazil.

4 Professor at Post-graduate Program in Biosciences and Physiopathology, State University of



Introduction and objectives: American Cutaneous Leishmaniasis (ACL) is a zoonosis caused by the

protozoan specie Leishmania (Viannia) braziliensis (LVB). Recent studies have demonstrated that the

capacity of the disease to spread to the organs, causing damage similar to those observed in cases

of Visceral Leishmaniasis (VL). Since the intestine is an organ widely affected by LV, it is important to

investigate the impact of the infectioncan cause to the intestinal tissue, which can be evaluated by

the quantitative analysis of intraepithelial lymphocytes (IEL), cells with important immunological

function in the gut. The objective of this study was to quantitatively analyze intraepithelial

lymphocytes from the ileum of infected hamsters.

Material and methods: Nine male hamsters (Mesocricetus auratus) were divided into four groups:

control group (CG) and groups infected with the LVB MHOM/2003/2314 strain at three different

infection times. Into the hind paw of GC hamsters were injected 100μL phosphatebuffered saline

(PBS), while infected animals received promastigotes of LVB (5×106 parasites) and were euthanized

at 3, 5 and 8 weeks after infection (groups 3W, 5W and 8W, respectively). Histological sections of 4μm

thickness were prepared in paraffin, stained with HematoxylinEosin technique. The counts of IELs

present among 2560 epithelial cells were calculated and the IEL/100 epithelial cells ratio was

calculated. Next, ANOVA test was performed with posttest of Tukey, considering p<0.05 (mean ±

standard deviation). The Ethics Committee on the Use of Animals of State University of Maringá

approved this study experimentation under No.306206916.

Results: Significant reduction of IELs was observed in the ileum of the infected groups 3W (6.5 ± /0.8),

5W (5.9 ± 0.4) and 8W (4.6 ± 0.4) when compared to the CG (10.5 ± 1.3). Therefore, there was an

average reduction of 40% of IELs in the infected groups when compared to the control group.

Conclusion: LVB infection significantly reduced the number of IEL in the ileum of hamsters at 3, 5 and

8 weeks of infection.

Keywords: ileum; mucosal immunity; leishmaniasis.

140

Staphylococcus aureus CA-MRSA EMERGING AS PATHOGEN IN HOSPITAL ENVIRONMENT.

Duarte, Felipe Crepaldi1; Danelli, Tiago1; da Silva, Raquel Soares1, Casonatto, Alexandre1; da Silva,

Alisson Santana1, De Oliveira, Thilara Alessandra1, Carraro, Diogo1; De Oliveira, Caio Ferreira2,

Bodnar, Giovana Carolina2, Pelisson, Marsileni1, Vespero, Eliana Carolina1, Nakazato, Gerson2;

Yamauchi, Lucy Megumi2; Yamada-Ogatta, Sueli Fumie2; Perugini, Marcia Regina Eches1

1Universidade Estadual de Londrina, Departamento de Patologia, Análise Clínicas e

Toxicológicas, Setor de Microbiologia, Londrina – PR, Brazil. 2Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de

Microbiologia, Londrina – PR, Brazil.

[email protected]

Introduction and objectives: Staphylococcus aureus are extremely versatile pathogens, surviving in

several environments under different conditions. These microorganisms have adapted well to the

environment, and their epidemiology has been changing over the years. It was believed that the

clone prevalent in Brazil was the isolate that carried SCCmec type III, however, recent studies have

demonstrated that clone type II has taken this place, at least in the south and southeast of Brazil. In

addition, SCCmec type IV and V were commonly associated with community-based infections, but

besides his frequency has been increasing in hospital-acquired infections, their antimicrobial profile

has change, becoming a MR (Multi Drug Resistant) pathogen. The objective of this work is to show

the increase of the frequency of clones related to community infections emerging as pathogen in

the hospital environment.

Material and methods: Between 2010 and 2017, 245 isolates of S. aureus isolated from clinical samples

collected from patients admitted at a university hospital in southern Brazil was evaluated. The isolates

were stored in TSB broth plus 30% glycerin in freezer (-20oC) until the analysis time. The antimicrobial

susceptibility profile and the identification of the isolates were performed using the vitek 2 compact

and Phoenix automated systems at appropriate times. The mecA gene search as well as the

SCCmec typing was performed by multiplex-PCR methodology. This study was submitted to the

research ethics committee involving human subjects, and was approved under the number CEP/UEL

- CAAE: 78657317.0.0000.5231.

Results: The samples were divided into two periods: the first one comprises collections between 2010-

2013 and the second between 2014 - 2017. When analyzing the molecular biology data, a

percentage of 6.87% (9/131) of samples were found carrying SCCmec type IV in first period. In the

second period of analysis this number increased to 15.45% (19/123). When the union of the

community types was performed, SCCmec type IV and V, this number is even higher, leaving the

same 6.87% of the first period to 17.07% (21/123) in the second. In addition, 30 isolates belonging to

community class 17 (56.66%) present as MR microorganisms, that is, resistant to three or more classes

of antimicrobials.

Conclusion: Therefore, with this study, it was possible to observe an increase in the prevalence of S.

aureus clones related to community infections as an emerging infectious agent in the hospital

environment. In addition, there is a change in the antimicrobial susceptibility profile of these isolates,

which are becoming MR.

Keywords: Staphylococcus aureus; SCCmec typing; Perfil changing; Community Infections

141

THE INFECTION BY DIFFERENT STRAINS OF THE Leishmania (Viannia) braziliensis CAUSE ALTERATIONS

IN THE QUANTITY OF INTESTINAL MAST CELLS

Pastre, M. J. ¹; Lima, L. L.2; Santos, A. G. A. 3; Silveira, T. G. V. 3; Nogueira-Melo, G. A. 3; Sant’Ana, D. M.

G.1

1 Universidade Estadual de Maringá, Department of Morphological Sciences, Postgraduate

Program in Biosciences and Pathophysiology – PBF Maringá, PR, Brazil

2 Universidade Estadual de Maringá, Department of Morphological Sciences, Postgraduate

Program in Comparative Biology – PGB Maringá, PR, Brazil

3 Universidade Estadual de Maringá, Clinical Analyzes and Biomedicine, Postgraduate Program

in Biosciences and Pathophysiology – PBF Maringá, PR, Brazil

[email protected]

Introduction and objectives: Leishmaniasis is a disease of high incidence worldwide, reaching up to

98 countries. In the infected human, it can attack secondary organs beyond the place of the

phlebotomine bite, like the gastrointestinal tract. The literature shows that mast cells located in the

lamina propria of the intestine, close to blood vessels, nerves and epithelial surfaces, have an

important role in immunoregulation. Thus, the aim was to analyze the effects of infection by different

strains of Leishmania (Viannia) braziliensis on the total number of mast cells in the jejunum of hamsters

(Mesocricetus auratus).

Material and methods: The study was approved by the Comissão de Ética no uso de Animais of the

Universidade Estadual de Maringá (protocol 7587260416). It were used 24 female hamsters (n=4)

randomly distributed into six groups: the control group (CG) and five infected groups (1655, 2311,

2314, 2903 and 3476). The infected groups received 2x107 promastigotes forms of different strains of

Leishmania (Viannia) braziliensis (MHOM/BR/2000/1655, MHOM/BR/2003/2311, MHOM/BR/2003/2314,

MHOM/BR/1975/M2903 e MHOM/BR/2009/3476) in the left hind paw. After 120 days were submitted

to euthanasia by anesthetic deepening, the jejunum was collected, washed and fixed. Semi-serial

cross-sections of 4 μm thickness were performed and subsequently stained by Toluidine Blue and

Fucsin G. All mast cells present in 100 fields (1 mm²) were counted in the 100x objective. Statistical

analysis was performed using ANOVA, followed by Tukey's post-test (p <0.05) and the results were

presented by mean ± standard error.

Results: The groups 1655, 2311 e 2314 (501.0±4.88; 503.4±15.49 e 490.6± 6.07, respectively), presented

an increased in the amount of mast cells present in 1 mm² when compared with the CG

(379.0±23.41).

Conclusion: We conclude that subcutaneous infection with different strains of L. (V.) braziliensis may

lead to the increase in the amount of mast cells present in the jejunum of infected hamsters.

Keywords: Leishmaniasis; Gastrointestinal tract, GALT

Grants: CNPq and Fundação Araucária

142

THE INFECTION BY Toxoplasma gondii PROMOTE QUANTITATIVE AND MORPHOMETRIC ALTERATIONS

IN PANETH CELLS IN THE INTESTINE OF C57BL/6 MICE

Lucas Antônio da Silva Guerra¹; Mariana Sacchi Silva2; Lainy Leiny de Lima3; Lucas Casagrande4;

Maria José Pastre4; Aline Rosa Trevizan4; Débora Mello Gonçales Sant'Ana5; Gessilda Alcântara de

Nogueira de Melo 4

1 Universidade Estadual de Maringá, Undergraduate student in Biological Sciences, Maringá,

PR, Brazil 2 Universidade Estadual de Maringá, Undergraduate student in Biomedicine, Maringá, PR,

Brazil 3 Universidade Estadual de Maringá, Department of Morphology Sciences, Postgraduation Program

in Comparative Biology - PGB, Maringá, PR, Brazil 4 Universidade Estadual de Maringá, Department of Clinical Analyzes and

Biomedicine, Postgraduate Program in Biosciences and Pathophysiology - PBF, Maringá, PR, Brazil 5 Universidade Estadual de Maringá, Department of Morphology Sciences, Postgraduate

Program in Biosciences and Pathophysiology - PBF, Maringá, PR, Brazil

[email protected]

Introduction and objectives: Toxoplasma gondii is an obligate intracellular parasite. The main via of

infection is the ingestion of contaminated food and water. The parasite invades intestinal cells, which

can lead to morphofunctional changes in the digestive system. Among the cells found in the

intestine, the Paneth cells (PC) in the crypts are responsible by the secretion of granules containing

antimicrobial peptides, mainly α-defensins. Thus, the aim of this work was to analyze quantitatively

and morphometrically the PC present in the jejunum of C57BL/6 mice after acute oral infection by T.

gondii.

Material and methods: This work was approved by the Comissão de Ética no Uso de Animais from

Universidade Estadual de Maringá under protocol number 4092040517. We used 14 C57BL/6 female

mice, that were randomly separated in two groups (n=7), the control group (CG) and the infected

group (IG). The IG received by gavage 1,000 sporulated oocysts of T. gondii (ME49). After five days,

the animals underwent to euthanasia and 2 cm of the jejunum were collected and submitted to

histological routine. Semi-serial cuts of 4µm was obtained and stained with hematoxylin and eosin

(HE). The quantification of PC was performed by counting all the PC present in 64 crypts/animal. In

addition, it were measured 160 PC (by animal) from images obtained with the objective of 100x.

Statistical analysis was performed using Student's T-Test (p <0.05) and the results were presented by

mean ± standard error.

Results: There was an increase (p <0.05) in the number and in the area of the PC in the IG (311.90 ±

31.03 PC/animal, 323.70 ± 27.36 μm², respectively) when compared with the CG (163.70 ± 6.04

PC/animal, 188.10 ± 12.86 μm², respectively).

Conclusion: The acute infection by T. gondii was able to promote changes in PC of the jejunum of

C57BL/6 mice. We believe that the antimicrobial peptides secreted by these cells, contribute to a

better protection of the host against T. gondii.

Keywords: Toxoplasmosis; Gastrointestinal tract, GALT

Grants: CNPq and Fundação Araucária

143

THE OCCURRENCE OF ANTIGENS OF CANINE DISTEMPER VIRUS IN THE OVARY AND UTERUS OF DOGS:

IMPLICATIONS OF A POSSIBLE REPRODUCTIVE DISEASE PATHOGEN

Pelaquim I. F.1, Viana N. E.1, Pinho F.1, Martins M. I.2, Saut, J. P.E.3, Headley S. A.1

1Laboratory Animal Pathology, Department of Veterinary Preventive Medicine, Universidade

Estadual de Londrina, Paraná, Brazil. 2 Small Animal Reproduction, Department of Veterinary Clinics, Universidade Estadual de Londrina,

Paraná, Brazil. 3Animal Health Laboratory, Faculty of Veterinary Medicine, Universidade Federal de Uberlândia,

Minas Gerais, Brazil


Introduction and objective: Canine distemper virus (CDV) occurs worldwide, is one of the most

important infectious disease agent of canine populations, with most terrestrial carnivores being

considered susceptible to natural infections. CDV is a pantropic pathogen and produces systemic,

respiratory, and neurological manifestations in dogs. Although CDV has been identified in several

organs, there are only three reports describing this agent in the female reproductive system of dogs.

These studies have demonstrated viral particles of CDV in the uterus and ovaries of dogs and in fetal

tissues and have suggested that CDV may be a possible reproductive disease pathogen.

Consequently, the aim of this study was to investigate the possible occurrence of antigens of CDV in

the ovaries and uterus of female dogs by immunohistochemistry and determine the possible

association of this virus with histopathologic patterns of disease.

Material and methods: This study utilized sections of the uterus and ovary from 30 female dogs

submitted to elective ovary-hysterectomy at the Veterinary Teaching Hospital, UEL, between January

2017 and December 2018. Formalin-fixed paraffin-embedded (FFPE) tissue fragments of the ovaries

and uteri of these dogs (n=30) were routinely processed to identify possible histopathologic patterns

of disease. Additionally, selected FFPE tissues sections of the uterus and ovaries were used in

immunohistochemical (IHC) assays designed to identify antigens of CDV using a monoclonal

commercial antibody.

Results: The principal histopathologic findings in the uteri were hemorrhagic (44.4%; 4/9) and fibrotic

endometritis (33.3%; 3/9), followed by endometrial edema (22.2%; 2/9), and lymphocytic endometritis

(22.2%; 2/9). Histopathologic alterations of the ovaries included edema (66.6%; 6/9), fibrotic oophoritis

(44.4%; 4/9), hemorrhagic oophoritis (22.2%; 2/9) and necrotizing oophoritis (22.2%; 2/9). There was

positive intracytoplasmic and intralesional immunoreactivity to antigens of CDV within the epithelial

cells of the uterus and ovary of all dogs evaluated.

Conclusion: The immunohistochemical findings confirmed the presence of CDV in the uterus and

ovaries of these dogs and add to the few investigations that have documented this virus in the

reproductive tract of the dog. The finding of intralesional positive immunoreactivity to CDV within the

uterus and ovaried of these dogs suggest that this viral pathogen was associated with these lesions;

similar histopathologic findings were described in experimental and natural cases of transmission of

CDV in dogs. Additionally, previous studies have demonstrated CDV particles in aborted fetuses and

placenta of dogs experimentally exposed and spontaneously infected by CDV. Collectively, these

results suggest that CDV may contribute to the development of reproductive disease in the female

reproductive of dogs. Additional studies are being done to confirm the active participation of CDV

in the reproductive tract of female dogs and the associated cytokine response.

Keywords: Reproductive pathology; female reproductive system; immunohistochemistry;

144

VENOM OF THE SCORPION Tityus bahiensis, IN LOW CONCENTRATIONS, REDUCES IL-8 AND THE

PROLIFERATION OF TACHIZOITES OF Toxoplasma gondii (CEPA RH) IN HeLa CELLS

Gabriela Alcântara Dalevedo¹, Raquel Arruda Sanfelice¹, João Paulo Assolini¹, Larissa Rodrigues

Bosqui¹, Taylon Felipe Silva¹*, Sara Semedo¹, Laís Machado¹, Italmar Teodorico Navarro², Fábio

Henrique Kwasniewski¹, Milena Menegazzo Miranda-Sapla¹, Francisco José Abreu Oliveira¹, Wander

Rogério Pavanelli¹, Ivete Conchon-Costa¹, Idessania Nazareth Costa¹*

¹Universidade Estadual de Londrina, Departamento de Ciências Patológicas, Londrina, PR, Brazil

²Universidade Estadual de Londrina, Departamento de Medicina Veterinária Preventiva, Londrina,

PR, Brazil

*Email: [email protected]; [email protected]

Introduction and objectives: Toxoplasma gondii is the protozoan that causes toxoplasmosis, an

infection that presents a serious character in immunosuppressed conditions, in cases of congenital

or ocular toxoplasmosis. The conventional drugs for the treatment of this infection present high

toxicity, being necessary the search for alternative compounds, such as the venom of the scorpion

Tityus bahiensis. Thus, the objective of this work was to evaluate the toxoplasmic and immunological

potential of T. bahiensis venom on tachyzoites of the T. gondii RH strain in HeLa cells.

Material and methods: For the experiment, it was evaluated by the MTT test, the cytotoxicity of the

venom of the scorpion Tityus bahiensis in the cells. HeLa cells (1x105) were infected with tachyzoites

of T. gondii (5x105); then treated with concentrations of 4μg/ml, 7.5μg/ml and 15μg/ml or 50 and

25 g/mL of sulfadiazine plus pyrimethamine respectively (positive control) or treated with medium

(negative control) for 24 hours. After, we assessed the number of infected cells and intracellular

tachyzoites. In addition, culture supernatants were collected for determination of IL-1b, IL-6, IL-8, IL-

10, IL-12p70 and TNFa by citometric bead array (CBA). One-way analysis of variance (ANOVA) was

performed to assess the differences between treatments and controls followed by a Tukey’s multiple

comparison tests using GraphPad Prism 5.0.

Results: The cytotoxicity of the venom in the cells, none of the concentrations tested showed cellular

cytotoxicity after 24 hours of treatment. Subsequently, the action of venom on the infection and cell

proliferation showed a statistically significant reduction in the number of infected cells in relation to

the negative control. All concentrations of T. bahiensis venom were statistically different from the

positive control. The treatments analyzed when compared to the negative control showed reduction

of intracellular proliferation with 17.92% in the concentration of 4ug / ml, 10.18% in the concentration

of 7.5ug / ml and 16.82% in the concentration of 15ug / ml. Among the cytokines dosed, IL-8

presented reduced rates in the concentration of 15ug / mL of the scorpion venom and also of the

positive control when compared to the negative control.

Conclusion: In view of the obtained results, it is possible to affirm the anti-Toxoplasma action of T.

bahiensis venom especially in the proliferation of tachyzoites.

Keywords: Toxoplasmosis, Scorpio venom, HeLa cells.

145

AMY WINEHOUSE’S DEATH: AN ALCOHOL INTOXICATION CASE STUDY

Francyne Baldo do Nascimento1, Anielle Aparecida Parmagnani1, André Demambre Bacchi2

1 Universidade Estadual de Londrina, Dentistry course, Londrina, PR, Brazil

²Universidade Federal de Mato Grosso, Department of Exact and Natural Sciences,

Rondonópolis, MT, Brazil

[email protected]

Introduction: The famous singer-songwriter Amy Winehouse, 27, was found dead in her bed in prone

position on July 23rd 2011.

Case Report: It is public knowledge that Amy Winehouse had several episodes of substance abuse.

Her history of illicit drug use includes marijuana, methamphetamine, ketamine and heroin. The use of

these drugs has always been accompanied by smoking cigarettes and the excessive consumption

of alcoholic beverages. Near the time of her death, Amy had stopped using illicit drugs and was

trying to stay abstinent of alcohol. This fact was confirmed by the absence of any illegal substances

in her toxicological examination results. To support her in the control of alcohol withdrawal symptoms,

she was taking chlordiazepoxide (Librium ®), a substance found in her blood in the postmortem

toxicological test, in suitable levels for ethanol withdrawal relief. Alcohol is a psychotropic depressant

of the CNS. Its action occurs in different neurotransmitters, including the stimulation of gamma-

aminobutyric acid (GABA) effect, the main inhibitory neurotransmitter of the CNS, and the inhibition

of glutamate effect, the main central excitatory neurotransmitter. Alcohol potentiates the effects of

GABA by acting directly on its receptors, enhancing their inhibitory effects. Alcohol also exerts direct

and indirect effects on the reward system through the opioid and dopaminergic systems. Studies also

report that alcohol can exert effects on other systems like serotoninergic, endocannabinoid, among

others. All of these actions end up contributing to both dosedependent central depression and

substance dependence. The chlordiazepoxide used by Amy was aimed to assist her in the

management of withdrawal symptoms, by acting mainly on GABAA receptors. However, in addition

to Librium, high ethanol levels were found in Amy’s blood. There were 416 milligrams of alcohol per

deciliter of blood, and above 300mg/dL, which is enough to cause alcoholic coma or even death.

It is important to note that alcohol tolerance varies according to individual genetics as well as the

degree of exposure to the drink. Frequent alcohol consumption rapidly leads to pharmacodynamic

and pharmacokinetic tolerance. Thus, higher doses of ethanol are required to produce a significant

central effect. Amy, who had been chronically taking large amounts of alcohol, has possibly

developed great tolerance and was able to consume large amounts of drinks daily. However, this

tolerance is a reversible process. By trying to abstain from drinking for a few months, her drinking

tolerance declined. When she returned drinking and consuming the same amount or more alcohol

than she consumed before, Winehouse ended up having an alcoholic overdosage. Intense inhibition

of the central nervous system, coupled with the fact that Amy laid in the ventral decubitus with her

face on the mattress, possibly led to fatal hypoxia.

Final consideration: It is possible to conclude that Amy Winehouse's death was probably due to acute

alcohol intoxication. It is therefore an interesting case study for learning pharmacology and drug

intoxication concepts. All information used in this article was taken from public documents of free

access.

Keywords: acute intoxication, toxicology, alcohol, drug abuse

146

ANTIPROLIFERATIVE ACTIVITY of 3,3',5,5'-tetramethoxybiphenyl-4,4'-diol ON THE HUMAN CELL LINE OF

NON-SMALL CELL LUNG CARCINOMA A549

Virginia Marcia Concato1, Fernanda Tomiotto-Pellissier2, Taylon Felipe Silva1, Manoela Daiele

Gonçalves3, Bruna Taciane da Silva Bortoleti2, Mariana Barbosa Detoni1, Ana Carolina Jacob

Rodrigues1, Elaine da Silva Siqueira1, Jéseka Gabriela Schirmann3, Tatiane Renata Fagundes1, Alex

Barbosa Alves1, Aneli de Melo Barbosa-Dekker3, Idessania Nazareth Costa1, Ivete Conchon-Costa1,

Milena Menegazzo Miranda-Sapla1, Mario Sergio Mantovani4, Wander Rogério Pavanelli1

1State University of Londrina, Department of General Pathology, Londrina, PR, Brazil 2 Post-graduate Program in Biosciences, Instituto Carlos Chagas/Fiocruz, PR, Brazil.Email

3State University of Londrina, Department of Chemistry, Londrina, PR, Brazil 4State University of Londrina, Department of Genetics, Londrina, PR, Brazil

[email protected]

Introduction and objectives: The 3,3', 5,5'-tetramethoxybiphenyl-4,4'-diol (TMBP) is described in the

literature as having antioxidant activity and can be obtained through enzyme-catalyzed processes.

Several studies have reported that biphenolic compounds belonging to the TMBP family have

inhibitory activity on the proliferation of HT-44 (melanoma) cells, were able to promote cell cycle

arrest in the G2/M phase in the MDA-MB-231 and MCF-7 (breast cancer) and induce death in HT-

1080 cells (fibrosarcoma). However, little is known about its action in lung cancer. Lung cancer is one

of the leading causes of cancer death worldwide. Overall, this disease causes about 1.7 to 1.8 million

deaths annually, in addition to having a reduced survival rate (17% in five years). The current

treatment available is through the use of cisplatin and carboplatin, medications that have several

side effects. Thus, the search for new compounds with antiproliferative potential, which respond

selectively to tumor cells, has been the focus of the development of many studies. In this context, this

work aims to evaluate in vitro the antiproliferative activity of TMBP on the human lineage of non-small

cell lung cancer A549 (adenocarcinoma lung).

Methodology: In the present study, culture of tumor cells (A549) and peritoneal macrophages were

incubated (10 4 ) in 96-well plates for 24 hours. After this period, the cultures were treated with TMBP

at concentrations of 12.5-200 μΜ, and incubated for 24, 48 and 72 hours in a 5% CO 2 and 37ºC

incubator. The MTT (3- (4,5 dimethylthiazol-2yl) - 2,5 diphenyl tetrazolium bromide) assay was

performed to evaluate mitochondrial activity in both strains. After the MTT assay, the IC 50 (148 μM ±

0.05) of the TMBP was defined on the A549 tumor cells at the 72 h time. From these data, we verified

the action of TMBP in the regulation of the cell cycle, by the labeling with PI, and later the induction

of death by Annexin/PI.

Results: When evaluating cell viability by MTT after 72 hours of treatment with TMBP, there was a

reduction of the proliferation of A549 cells at concentrations of 50, 100 and 200 μM and reduction of

macrophages only from the concentration of 200 μM. Subsequently, in treating A549 cells with IC 50

(148 μM ± 0.05), we observed that the treatment, after 72 h, was able to induce morphological

changes in the size of these cells. We also verified that the TMBP treatment, in 24 and 72 h, promoted

cell cycle arrest in G2/M (50.06%, p<0,0001) and induced apoptosis in 30.71% of A549 cells for IC50

treatment (p<0,0001).

Conclusion: Our results therefore suggest that treatment with TMBP on A549 cells contributed to the

reduction of proliferation inducing death by apoptosis.

Keywords: Lung cancer; apoptosis; tetramethoxibiphenil;

Grants: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

147

BRACHYDIN FLAVONOIDS DECREASE MIGRATION AND INVASION OF PROSTATE TUMOR CELLS DU-145

Oliveira, L.C.B.1, Nunes, H.L.1, Rocha, C.Q.2, Cólus, I.M.S.1, Serpeloni, J.M.1

1Department of General Biology, Center of Biological Sciences; State University of Londrina,

Londrina, PR, Brazil 2Department of Chemistry, Center of Science and Technology, Federal University of Maranhão, São

Luis, MA, Brazil.


Introduction and objectives: Cancer is one of the leading causes of mortality worldwide. As a result

of its increased occurrence in recent decades, high cost of treatment, and adverse effects caused

by traditional therapies, there is currently a relevant interest in the development of new derivatives

from natural sources that have an anti-cancer potential. Certain phytochemicals are considered

both chemopreventive and chemotherapeutic agents, with the advantage of being abundant and

capable of yielding medication that causes fewer adverse effects. The native plant from Brazilian

cerrado, Fridericia platyphylla (Cham.) L.G. Lohmann, is popularly known as "cervejinha do campo"

or "cipó-una," and the tea from its root is traditionally consumed in folk medicine as treatment of

kidney stones and arthritis. The aim of this study was to analyze the cytotoxic and antimetastatic

effect of the flavonoids isolated from these roots, brachydin A, brachydin B and brachydin C, which

have similar chemical structure, differing in only one radical.

Material and methods: Flavonoids brachydin A, B and C were evaluated by in vitro assays for their

cytotoxic effects through the MTT test in human prostate tumor cell line (DU-145) using nine previously

established concentrations (0.24 to 30.72 μM). The influence of these flavonoids on the migration and

invasion processes of DU-145 cells was evaluated using the concentrations of 6.00μL and 3.84μL in

the migration assay and 6.00μL in the invasion assay.

Results: In the MTT assay, brachydins A and C decreased viability at concentrations above 6.00μM

and brachydrin B from the concentration of 1.5μM. In the migration and invasion assays, it was

possible to observe that the 6.00μM concentration of brachydins B and C was able to reduce the

migration of the cells from the upper to the lower part of the insert. All three brachydins (6.00μM)

decreased the DU-145 cell invasion process.

Conclusion: In this study the flavonoids brachydins A, B and C decreased the migration and invasion

processes of prostate cancer cells (DU-145 lineage). Further studies are warrant to provide a

comprehensive understanding of the signaling pathways that are being altered by these

phytochemicals, leading to the observed antimetastatic activity.

Keywords: Phytochemicals, Brachydin, Cytotoxicity, Migration test, Invasion test, DU145.

148

CAN THE SALMON TROUT CONSUMPTION BE CONSIDERED A RISK FACTOR FOR HEPATIC STEATOSIS?

Lenzi-Almeida, K.C.1; Igor Jenkins Paim Oliveira2; Isabella Gonçalves de Oliveira Vilela2; Marina

Matos Souto2; Xiaoxin Wu2; Latini, Juliana Tomaz Pacheco3

1Universidade Federal do Rio de Janeiro, Professor at School of Medicine, Macaé, Rio de Janeiro,

Brazil

2Universidade Federal do Rio de Janeiro, Undergraduate student at School of Medicine, Macaé,

Rio de Janeiro, Brazil 3Universidade Federal do Rio de Janeiro, Professor at School of Pharmacy, Macaé, Rio de Janeiro,

Brazil


Introduction and objectives: Canthaxanthin is a lipophilic carotenoid with antioxidant and

immunomodulatory properties, being widely used in salmon trout rearing in order to provide a

salmon-like coloration. The present study aims to demonstrate the deleterious effects of

canthaxanthin consumption on the liver, additionally to explaining the possible pathophysiological

mechanisms involved.

Material and methods: The project was approved by the Animal Research Ethics Committee of UFRJ

/ Macaé Campus under protocol MAC034.Twenty-four female Swiss mice were divided into four

groups (n = 6 each): Control Group (CG); Canthaxanthin Group (CXG); Salmon Trout Group (STG)

and Wild Salmon Group (WSG). CG received casein diet (standard) as a control (equivalent to 14,22

g/kg/day of casein), CXG received standard ration plus 0.6% canthaxanthin (corresponding to 785

mg/kg/day), STG received casein diet plus 21,7% of salmon trout, and WSG was fed with casein diet

plus 21,7% salmon (both groups corresponding to 28,39 g/kg/day of salmon trout and salmon,

respectively). The mice were mated, originating the offspring, which received the same rations

offered to their mothers after weaning. The animals were euthanized at 70 days of age, their livers

were removed and routinely processed for inclusion in paraffin. The morphometry was performed

from the blades stained with H & E. They were photographed using iVm 5000 camera, through

ProgRes Capture Pro 2.7 program. Measurements were performed using Image J.

The Kruskal-Wallis test was applied, followed by Dunn's post-test, with significance at the p ≤ 0.05 level

in GraphPad Prism 5.0.

Results: About the number of cell nuclei, there was a statistical difference between the groups, since

STG (36,43±19,49 - P value: 0,0120) presented a reduced nuclei number when compared to CG

(88,0±7,07 - P value: 0,0120) and WSG (72,0±14,82 - P value: 0,0120). The Kupffer Cells number had

statistical significance, as CXG (64,0±23,08 - P value 0,0530) showed higher values in comparison to

the other groups (CG: 43,0±28,28; STG: 25,25±11,30 e WSG: 30,50±1,29 - P value 0,0530). Cell fat

droplets results revealed that STG (1430±655,33 - P value 0,0123) confirmed to be statistically superior

when compared to the other groups (CG: 68,50±51,62; CXG: 585,40±151,89 e WSG: 377,57±201,40 - P

value 0,0123). Finally, the hepatocytes number was lower in STG (29,0±10,86 - P value 0,0073) than

other groups CG (83,5±17,68 - P value 0,0073) and WSG (55,14±12,29 - P value 0,0073).

Conclusion: The consumption of salmon trout can be considered as a risk factor for hepatic

steatosis.

Keywords: Hepatic steatosis, canthaxanthin, salmon trout.

Grants: CNPQ, FAPERJ, PIBIC/UFRJ.

149

CHANGES IN THE SPERMATOZOIDS OF MICE EXPOSED TO SIBUTRAMINE

Mestre, V. F.1; Menezes, E. V.2; Brito, L. V.; 2; Martins, C. C. N.2; Ezequiel, B. S.2; Sestario, C. S1.; Salles,

M. J. S1,.

1 State University of Londrina, Department of Health Sciences, Center of Health Sciences Londrina,

PR, Brazil

2 Universidade Estadual de Londrina, Department of General Biology, Center for Biological

Sciences, Londrina City, Brazil


Introduction and objectives: : Sibutramine is a drug used to treat obesity. It is a reuptake inhibitor of

noradrenaline and serotonin increasing levels of these substances in the synaptic clefts and helping

to increase satiety. Sibutramine also stimulates the adrenergic system, which induces a mild

thermogenic effect, promoting the reduction of fat absorption by the intestine. This study aimed to

investigate the possible toxic effects, changes in the morphology of sperm and the level of the

hormone testosterone.

Material and methods: Material and methods: Male Swiss mice were used, divided into treated

group (G1) with sibutramine 15 mg / kg and the control group (G0) with saline. Treatment was via

gavage for 45 days. For toxicity analysis body mass during the treatment period was monitored and

clinical signs were observed such as: presence of piloerection, red eyes, diarrhea, motor

coordination and death. On the 46th day, males were euthanized with collection of organs: heart,

lungs, liver, kidneys, testes and epididymis that were externally analyzed and weighed. For

morphological evaluation the sperm were collected from the cauda epididymis and 400 cells per

animal were evaluated under a light microscope at 1000x resolution. Testosterone levels were

measured by chemiluminescence. After checking the normality of the samples with Shapiro Wilk test,

used for the quantitative analyzes, as organ weight, testosterone dosage, histological analysis of the

testes the Student's T test for parametric data and Mann-Whitney test for non-parametric data. In

qualitative analyzes such as sperm morphology, we used Chi-Square. The statistical program used

was GraphPad Prism 5. The level of significance was 5%. This work was approved by the Ethics

Committee on the use of animals of the State University of Londrina in the number: 19079.2016.56.

Results: There was no change in heart weight (G0: 0.2084 ± 0.0317; G1: 0.2071 ± 0.0059), lung (G0:

0.2934 ± 0.0345; G1: 0.3346 ± 0.0881), liver (G0: 2,339 ± 0.4678; G1: 2.099 ± 0.2182) and kidneys (G0:

0.5819 ± 0.07752; G1: 0.6423 ± 0.05243) between the treated group when compared to the control

group. No clinical signs of toxicity were identified. The comparative study of testis weight found a

significant difference between the groups (G0: 0.2257 ± 0.0079; G1: 0.2525 ± 0.0085). Morphological

analysis of sperm showed head and tail changes (G0: 3.15%, G1: 11.16%). The testosterone dosage

was normal (G0: 646.4 ± 469,5 ng/dL; G1: 315.4 ± 402.0 ng/dL).

Conclusion: It can be concluded that the Sibutramine promoted morphological changes in sperm.

Male infertility and sterility in several species have been associated with morphological abnormalities

of spermatozoa. Given the results of this study, it is suggested that men of childbearing age who

make use of Sibutramine receive greater attention and monitoring. Further studies are needed to

better understand the effects of sibutramine under male fertility.

Keywords: Sibutramine, sperm alterations, Spermatogenesis, Obesity

150

CHIKUNGUNYA VIRUS INDUCES ARTICULAR HYPERALGESIA IN MICE

Amanda Martins Dionisio1; Carina Z. Segato-Vendrameto1, Amanda Z. Zucoloto1, Victor

Fattori1, Larissa Staurengo-Ferrari1, Stephanie Badaro-Garcia1, Tiago H. Zaninelli1, Mariana M.

Bertozzi1, Camila Zanluca2, Rubia Casagrande1, Cláudia N. Duarte dos Santos2, Waldiceu A. Verri,

Jr1.

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2 Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil


Introduction and objectives: Chikungunya virus (CHIKV), which is transmitted by mosquitoes of the

genus Aedes, is an arthritogenic alphaviruses responsible for causing Chikungunya fever disease and

debilitating joint pain. CHIKV has an incubation period that lasts about 2-4 days (range 1-12 days)

and then the course of the disease can be divided into acute and chronic stages, though not all of

the infected individuals develop the symptoms. The acute stage is characterized by crippling joint

pain, high fever, rash, headaches, myalgia and arthralgia; being easily confused with dengue. The

polyarthralgia is usually symmetric, bilateral and intense; affecting mostly the extremities as

phalanges and wrists but also the large joints as knees and shoulders. The chronic stage is marked by

severe arthralgia and/or arthritis which persist even after the clearance of the virus from blood. The

treatment consists in lessening the symptoms with non-salicylate analgesics and non-steroidal anti-

inflammatory, once there is no effective antiviral specific for Chikungunya. Since there have been

studies relating the envelop E2 protein ability to stimulating the immune response against the virus,

and its potential as a therapeutic target, we investigated the participation of the E2 protein in the

hyperalgesia induction after the infection and the possibility of monoclonal antibodies as a treatment

to control pain.

Material and methods: The use of male Swiss mice at this experiment was properly approved by The

Ethics Committee for Animal Research of the Universidade Estadual de Londrina (process number

13216.2017.97). The mice received intra-articular (i.a.) injection of β-propiolactone inactivated

Chikungunya virus (iCHIKV: 1, 10, 100 and 1000 FFU, 10ul) or Mock (noninfected control, 10ul) and the

mechanical hyperalgesia was evaluated 1–7 hours after injection and daily during seven days.

Subsequently, three different types of monoclonal antibodies (AbM) were tested to evaluate if the

neutralization of E2 protein affects the hyperalgesia induction. AbM1, AbM2 and AbM3 (1 or 5 µg, 5

µL) were co-injected (i.a) with iCHIKV (1000 FFU, 5 µL) or Mock (control, 10 µL) and the mechanical

hyperalgesia was evaluated 1 – 7 hours after injection and daily during seven days.

Results: Inactivated Chikungunya virus was able to induce significant mechanical hyperalgesia

starting at the 3rd hour after the stimulus and the highest doses (100 and 1000FFU) maintained its levels

until day 2. Once there was no significant difference between these two doses, the lower one was

picked to continue the experiments. The highest dose of all three monoclonal antibodies was

capable to inhibit the iCHIKV induced-hyperalgesia after de 3rd hour maintaining it until day 7 and

also confirms the participation of E2 in the pain process.

Conclusion: Our results have demonstrated that Chikungunya virus is capable of inducing

mechanical hyperalgesia in mice and that the envelop E2 protein is involved in this process. We have

also showed the possibility of therapeutic use of monoclonal antibodies to control CHIKV induced

articular pain.

Keywords: Chikungunya fever, joint pain, monoclonal antibody, hyperalgesia

151

CHIKUNGUNYA VIRUS RECOMBINANT E2 PROTEIN-INDUCED ARTICULAR HYPERALGESIA IS INHIBITED BY

MONOCLONAL ANTIBODIES IN MICE.

Fernanda Soares Rasquel de Oliveira1; Carina Z. Segato-Vendrameto1, Amanda Z. Zucoloto1,

Victor Fattori1, Larissa Staurengo-Ferrari1, Stephanie Badaro-Garcia1, Tiago H. Zaninelli1, Mariana M.

Bertozzi1, Camila Zanluca2, Rubia Casagrande1, Cláudia N. Duarte dos Santos2, Waldiceu A. Verri

Jr.1

1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 2

Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil

[email protected]

Introduction and objectives: Chikungunya virus (CHIKV) is an arthritogenic alphaviruses transmitted

by mosquitoes of the genus Aedes, which is responsible for causing the Chikungunya fever. The

disease can be divided in acute and chronic stages with an abrupt clinical onset of crippling joint

pains, high fever and rash, being easily confused with dengue. Currently, there is no effective antiviral

specific for Chikungunya and patient’s treatment is purely symptomatic and based on non-salicylate

analgesics and non-steroidal antiinflammatory drugs. The virus genome consists in a single-strand

RNA molecule encoding four nonstructural proteins (nsP1-4) that are required for virus replication,

and three structural proteins (C, E1, E2). The use of the structural proteins as targets to immunization

and therapy against CHIKV infection have received attention over the last few years and E2 protein

has been related to induce joint swelling and inflammation. Once the envelop proteins are capable

of stimulating immune response against CHIKV becoming important therapeutic target, we

investigate the participation of E2 recombinant protein in hyperalgesia induction, as well as the

possibility of treatment with monoclonal antibodies to control pain.

Material and methods: Male Swiss mice received intra-articular (i.a.) injection of recombinant E2

protein (rE2: 0,03 or 0,1 μg, 10ul) or Mock (control, 10ul) and the mechanical hyperalgesia was

evaluated 1 – 7 hours after injection and daily during seven days. Subsequently, three different types

of monoclonal antibodies were tested to evaluate if the neutralization of E2 protein affects the

hyperalgesia induction. AbM1, AbM2 and AbM3 (1 or 5 μg, 5 μL) were co-injected (intra-articular)

with rE2 (0,1 μg, 5 μL) or Mock (control, 10 μL) and the mechanical hyperalgesia was evaluated 1 –

7 hours after injection and daily during seven days. All the procedures using animals were properly

approved by The Ethics Committee for Animal Research of the Universidade Estadual de Londrina

(process number 13216.2017.97).

Results: Significant mechanical hyperalgesia was induced by CHIKV recombinant E2 protein starting

at the 3rd hour after injection, and the highest doses maintained its levels until day 3 post stimulation.

The highest doses of the three used monoclonal antibodies showed efficacy in inhibiting the rE2

induced hyperalgesia in mice after the 3rd hour of treatment.

Conclusion: Our results have demonstrated that Chikungunya recombinant protein E2 is capable of

inducing mechanical hyperalgesia in mice, revealing its participation in CHIKV induced articular

pain. We also have showed the possibility of therapeutic use of monoclonal antibodies to control rE2

induced hyperalgesia.

Keywords: Chikungunya virus, E2 protein, hyperalgesia, monoclonal antibody.

152

CONTRACEPTIVE POTENTIAL OF CYCLOSPORIN A IN SHORT-TERM TREATMENT

Costa, I. R1; Siervo, G. E. M. L.1; Silva, A. A. S2; Mariani, N. A. P. 2; Andrade, A. D. 2; Silva, E. J. R.2;

Fernandes, G. S. A.¹

1 Universidade Estadual de Londrina, Departmentof General Pathology, Londrina, PR, Brazil.

2 Universidade Estadual Paulista, Department of Pharmacology, Botucatu, SP, Brazil.

[email protected]

Introduction and objectives: Currently available contraceptive drugs are targeted primarily at the

female audience and have several adverse effects. Hence, the need for a drug targeting the male

audience is evident. Cyclosporin A (CsA) is an immunosuppressive drug that acts by inhibiting

calcineurin. Calcineurin is an enzyme that regulates the expression of interleukin 2 (IL-2) in the immune

system and there are studies that indicate that calcineurin participates in spermatogenesis and

spermiogenesis in the male reproductive system. Recently, its contraceptive implication has been

reported in mice. Thus, the objective of the present study was to evaluate whether cyclosporin A has

contraceptive potential in adult male mice and whether these effects are reversible in a short-period

treatment.

Material and methods: Adult Swiss male mice were distributed into 4 groups. Two treated groups

received Cyclosporin A (SandimunnNeoral®, Novartis - 10mg / kg) for 10 days. The Control groups

received only the vehicle for the same period. One CsA group and one Control group underwent a

10-day recovery period. At the end of the experimental periods animals were euthanized and the

right testis and epididymis were collected for analysis of sperm count and sperm transit time.

Spermatozoa from the vas deferens were collected and separated into aliquots for analysis of sperm

morphology. Sperm from cauda epididymis was used to motility assay, acrosomal integrity analysis

and mitochondrial sperm activity. This research was approved by the Committee of Ethics in Animal

Use of the State University of Londrina (CEUA/UEL), protocol #10952.2017.79.

Results: The analysis of sperm count, sperm transit time and morphological analysis did not show

significant changes, since the spermatogenesis process lasts approximately 35 days in mice.

Acrosomal integrity analysis showed a decrease of the intact acrosomes (p<0.05) and the analysis

of mitochondrial activity showed a reduction of functional mitochondria (p<0.05) at CsA-treated

mice, but both alterations normalized after the recovery period. However, the hyperactivated

motility at CsAgroup was altered after the recovery period (p<0.05), indicating that the proteins

involved in this process probably was modified at testicular level.

Conclusion: Cyclosporin A, in a short period of time, does not affect spermatogenesis process.

However, it affects sperm function, as evidenced by hyperactivated motility impairment, which can

also impair sperm reach to the egg. Nevertheless, a fertility test is necessary to prove the

contraceptive effect of this drug.

Keywords: calcineurin, sperm, acrosome, testis, epididymis

153

CYTOTOXICITY OF THE STEAM PHASE FROM Jasminum officinale (JASMINE) AND Pogostemon cablin

(PATCHOULI) ESSENTIAL OILS ON LUNG CANCER CELLS

Daniele de Fatima Kosmo1; Liane Emi Maeda 2; Jacqueline Gonçalves dos Santos 3

1 Universidade Estadual de Ponta Grossa, Department of General Biology, Ponta Grossa, PR, Brazil

2Universidade Estadual de Ponta Grossa, Department of Pharmaceutical Sciences, Ponta Grossa,

PR, Brazil 3Universidade Estadual de Ponta Grossa, Department of General Biology, Ponta Grossa, PR, Brazil


Introduction and objectives: Among different types of cancer, lung cancer is the one with the

highest mortality rate, as it is also one of the most common malignant tumors worldwide. In that way,

it is necessary to research for new therapeutic intervention, especially by means of substances with

less toxicity to the non-tumor cells. Essential oils (EOs) are an abundant source of compounds with

pharmacological activities, including antitumor action. The EOs have an interesting characteristic for

lung cancer; because the oil is volatile, the components can be delivered to the lung tissue directly

by inhalation, which would probably minimize the side effects. Patchouli and many species of

jasmine have significant medicinal properties and have been used as traditional medicines for years.

In fact, some studies have demonstrated the antitumor action of jasmine and patchouli EOs;

however, none of them evaluated the vapor phase of EO on tumor cells. In this context, the vapor

phase of EOs may present some pharmacological properties, that is why this work aimed to evaluate

the cytotoxicity of the steam phase from jasmine and patchouli EOs on lung cancer cells.

Material and methods: Both EOs were acquired commercially in a house of essences, then one

sample of each EO was sent to the Central Analysis of the Biotechnology Institute of the University of

Caxias do Sul to perform the analysis of the EOs chemical composition by GC/MS analysis. The MTT

reduction assay and Sulforhodamine B colorimetric assay (SRB) were used to evaluate the

cytotoxicity of both EOs against Calu-3 cancer cells. Cells were incubated at 37ºC and 5% CO2 for

72h for dose-response studies; and for time-response studies cells were incubated for 24, 48 and 72h

with the EO concentration that reduced the cell viability in 50%.

Results: The major components of patchouli EO were patchoulol (32,92%) and α-guaieno (16,90%),

which is in accordance with other studies. For jasmine EO the major compounds were benzyl acetate

(31,43%) and α-hexylcinnamaldehyde (30,49%); the first one is a natural constituent in jasmine EO but

the second one is a semisynthetic compound with floral aroma, widely used in cosmetic and

pharmaceutical industries. The vapor phase of patchouli EO had higher cytotoxicity than jasmine;

the first one presented dose and time-dependent effects on Calu-3 cells with IC50 of 225 µg.mL-1 and

the jasmine EO presented the IC50 of 1495, 57 μg/mL-1.

Conclusion: This study demonstrated that the vapor phase of both EO present a cytotoxic effect

against lung cancer cells (Calu-3). However, patchouli EO had higher cytotoxicity than jasmine.

Furthermore, the jasmine EO used in this work was adulterated, thus we cannot affirm that jasmine

EO has antiproliferative activity on cancer cells. Evidence that vapor phase of patchouli EO is toxic

to lung cancer cells may refer to it as a potential agent for the development of new inhaled or

nebulized therapy for lung cancer treatment; however, further studies are needed to elucidate the

mechanism involved in this process as well as in vivo studies.

Keywords: cytotoxic activity, Calu-3 cells, jasmine essential oil, patchouli essential oil

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

154

EFFECT AND ROLE OF 15-EPI-LIPOXIN A4 IN ASPIRIN MECHANISM OF ACTION ON UVB-IRRADIATION

INDUCED SKIN INFLAMMATION AND OXIDATIVE STRESS IN HAIRLESS MICE

Saito, P.1; Martinez, R.M.1; Pinto, I.C. 1; Rodrigues, C.C.A.1; Melo, C.P.B.1; Kumagai, C.M.1; Baracat,

M.M.1; Georgetti, S.R.1; Verri, W.A.2; Casagrande, R.1*

1 Universidade Estadual de Londrina, Department of Sciences Pharmaceutical, Londrina, PR, Brazil 2 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil


Introduction and objectives: Skin exposure to UVB irradiation increased significantly over the last years

and represents the main cause of many skin diseases. Targeting the physiopathological mechanisms

of UVB irradiation-induced skin inflammation and oxidative damage is a promising approach to

reduce skin damage. Lipoxins are anti-inflammatory and proresolution lipid mediators, however, the

therapeutic potential of 15-epi-lipoxin A4 (an aspirin triggered lipoxin, ATL) in UVB irradiation-induced

skin inflammation and oxidative stress remains to be determined. Thus, the present study aimed to

evaluate the effect and role of 15-epi-lipoxin A4 (ATL) in aspirin mechanism of action on UVB-induced

skin inflammation and oxidative stress.

Material and methods: Hairless mice were randomly designed to different groups with 5 mice each:

non-irradiated control, irradiated control (irradiation session of 4.14 J/cm2), treated with aspirin (30,

100 or 300 mg/kg, via oral) 1 h before the irradiation, treated with BOC-2 (a lipoxin receptor

antagonist, 3, 10 or 30 µg/kg, via intraperitoneal) 30 min before the treatment with aspirin (100 mg/kg),

treated only BOC-2 (30 µg/kg) 1 h and 30 min before the irradiation, treated with aspirin (100 mg/kg)

and not irradiated, treated with ATL (0.3, 1 or 3 ng/mice, via intraperitoneal) 1 h before the irradiation,

treated with BOC-2 (30 µg/kg) 30 min before the treatment with ATL (3 ng/mice), and treated with

ATL (3 ng/mice) and not irradiated. Samples of skin were collected 12 h after the end of the

irradiation. The skin edema was measured as an increase in dorsal skin weight. The neutrophil

migration was evaluated by myeloperoxidase (MPO) activity. The ferric reducing ability (FRAP assay),

the ABTS radical scavenging ability, and reduced glutathione (GSH) levels were determined by

colorimetric assays. The expression of the lipoxin A4 receptor (ALXR) was determined by reverse

transcriptase and quantitative polymerase chain reaction. Data were statistically analyzed by one-

way ANOVA followed by Tukey’s test, p<0.05. The Animal Ethics Committee (CEUA process

1447.2015.10) of the Londrina State University approved this study.

Results: Twelve hours after exposure to UVB irradiation, there was an increase in the expression of the

receptor ALXR, thus, the use of molecules that control inflammation via activation of this receptor,

can be promising. Treatments with aspirin and ATL inhibited UVB irradiation-induced skin edema,

MPO activity, and depletion of antioxidant capacity (ferric and ABTS reducing abilities and GSH

levels) of dose-dependent manner. The pre-treatment with BOC-2, inhibited significantly anti-

inflammatory and antioxidants effects of aspirin and ATL. The treatment only with BOC-2 before the

irradiation did not induce significant changes in comparison to the irradiated control group.

Moreover, the treatment only with aspirin or ATL in animals not exposed to irradiation did not induce

significant changes in comparison to the non-irradiated control group.

Conclusion: The anti-inflammatory and antioxidants effects of aspirin and ATL in the skin damage

induced by UVB irradiation are mediated by activation of the receptor ALXR. These results to propose

novel approaches to control skin diseases related to UVB irradiation based on lipoxin receptor

agonists, such as 15-epi-lipoxin A4 (an aspirin triggered lipoxin).

Keywords: skin damage, radiation UV, lipoxins, BOC-2, lipoxin receptor antagonist.

Grants: CNPq, CAPES, Fundação Araucária and UEL.

155

EFFECT OF ACUTE RESTRAINT STRESS IN THE RECRUITMENT OF NEUTROPHILS TO THE PERITONEAL CAVITY

INDUCED BY CARRAGEENAN

Scacco, G.1; Oliveira, C.H.B.1; Lucchetti, B.F.C.1 ; Pinge-Filho, P.2; Freitas, A.3

1,3 Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil 2 Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Introduction and objectives: Stressful events activates autonomic and neuroendocrine pathways

that modulate the emotional, behavioral and immune system. An increasing number of studies have

focused on the importance of the activation of beta -adrenergic receptors (b-AR) in the control of

immune response, since the activation of these receptors is related to the anti-inflammatory actions.

Since, the relationship between acute stress, sympathetic nervous system activation and neutrophil

recruitment to the site of inflammation remains not fully elucidated, the present study aims to

determine whether acute stress induced by restriction modulates the neutrophil migration induced

by carrageenan and the mechanisms involved in this process.

Material and methods: Male Swiss adult mice were used in this study (CEUA: 14993.2018.74). To induce

the acute restraint stress the animal was placed in the 50 mL conical centrifuge tube with ventilation

for 2 hours. The inflammatory process was induced by intraperitoneal (i.p.) administration of

carrageenan 500μg/ cavity. Propranolol 5mg/kg, subcutaneously (s.c.), was used to block beta-AR1

and 2. Atenolol 20 mg/kg, s.c., to block beta-AR1. Mice were randomly divided into eight groups:

Control (n = 5): mice were not subjected to acute stress and received saline via i.p.; Carrageenan (n

= 5): the animals were not submitted to acute stress and received carrageenan i.p.; Stress + Saline (n

= 5): animals were restrained for 2 hours and then received saline via i.p.; Stress + Carrageenan (n =

5): animals were subjected to acute stress for 2 hours and then received carrageenan ip.; Propranolol

+ Carrageenan (n = 5): mice were pretreated with propranolol, s.c., and remained without restraint

stress for two hours, after two hours carrageenan was administered i.p.; Propranolol + Stress +

Carrageenan (n = 5): propranolol was administered to mice 30 minutes before the induction of

restraint stress and carrageenan was injected two hours after stress induction; Atenolol +

Carrageenan: (n=5) mice were pretreated with atenolol, s.c., and remained without restraint stress

for two hours, after two hours carrageenan was administered i.p.; Atenolol + Stress + Carrageenan

(n = 5): atenolol was administered to mice 30 minutes before the induction of restraint stress and

carrageenan was injected two hours after stress induction. After 4 hours of saline or carrageenan

administrations, the leukocyte/ neutrophil migration were determined from peritoneal exudate. Data

were analyzed statistically using the one-way ANOVA followed by Tukey’s.

Results: Acute restraint stress decreased the migration of total leukocytes and neutrophils to the

peritoneal cavity induced by carrageenan and this reduction is reversed by the pretreatment of

stressed mice with propranolol. On the other hand, atenolol was not able to reverse the reduction of

the neutrophil migration induced by stress.

Conclusion: Conclusion: The data from the present study shows that the acute restraint stress reduces

the neutrophil migration to the inflammatory site, and this effect is mediated through the activation

of beta-AR2.

Keywords: Inflammation; Neutrophils; Stress; Restraint.

Grants: CAPES

156

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED COLLAGEN DEGRADATION, AND

PRODUCTION OF CYTOKINES IN THE SKIN OF MICE, AND EFFECT OF THE BOC-2 IN THE EFFICACY OF

BML-111 IN THIS MODEL

Martinez, R.M.1; Saito, P.1; Mantovani, G.L.1; Pinto, I.C. 1; Rodrigues, C.C.A.1; Melo, C.P.B.1; Baracat,


1 Universidade Estadual de Londrina, Department of Sciences Pharmaceutical, Londrina, PR, Brazil



Introduction and objectives: Excessive skin exposure to ultraviolet B (UVB) irradiation induces skin

damage per se but also triggers inflammation that boosts tissue destruction. Targeting the

physiopathological mechanisms in UV radiation-induced skin inflammation are promising strategies

to reduce skin damage. Studies have demonstrated the effectiveness of pro-resolution lipid

mediators in UVB irradiation-induced skin damage, however, these mediators are rapidly

inactivated. In this sense, analogues have been developed, such as the BML-111, an agonist of the

lipoxin A4 receptor (ALXR), but the therapeutic potential of BML-111 in UVB irradiation-induced skin

inflammation remains to be determined. Thus, the present study aimed to evaluate the effects of

administration with BML-111 on UV radiationinduced collagen degradation, and production of

cytokines in the skin of mice, and effect of the BOC-2 (a lipoxin A4 receptor antagonist, ALXR) in the

efficacy of BML-111 in this model.


non-irradiated control, irradiated control (irradiation session of 4.14 J/cm2), treated with BML-111 (0.1

mg/kg, via intraperitoneal) 1 h before the irradiation, treated with BOC-2 (30 g/kg, via

intraperitoneal) 30 min before the treatment with BML-111 (0.1 mg/kg, 1 h before the beginning of

UV irradiation), and treated only BOC-2 (30 g/kg) 1 h and 30 min before the irradiation. Samples of

skin were collected 4 h (to measure the cytokine levels by an enzyme-linked immunosorbent assay

according to manufacturer’s instructions) or 12 h (to measure the collagen fiber intensity bundles

with Masson's trichrome stain showed in blue and analyzed by ImageJ Program). Data were

statistically analyzed by one-way ANOVA followed by Tukey’s test, p<0.05. The Animal Ethics

Committee (CEUA process 1447.2015.10) of the Londrina State University approved this study.

Results: UV can damage collagen fibers and thereby accelerate aging of the skin. The collagen

fibers in blue staining in the BML-111 treated group showed lower levels of damage (maintenance of

blue staining), compared with those in the irradiated control group. UV irradiation-induced

production of cytokines can to contribute to collagen degradation. Indeed, treatment with BML-111

inhibited UV irradiation-induced production of cytokines IL-1β, IL-6 and TGF-β. However, the pre-

treatment with BOC-2, significantly inhibited the effects of BML-111 in collagen degradation, and

production of cytokines. The treatment only with BOC-2 before the irradiation did not induce

significant changes in comparison to the irradiated control group.

Conclusion: BML-111 activity was related to inhibiting cytokine production. Moreover, the inhibition

of inflammation by BML-111 in the skin damage induced by UVB irradiation are mediated by

activation of the receptor ALXR, since the pre-treatment with BOC-2, (a lipoxin A4 receptor

antagonist, ALXR) inhibited the efficacy of BML-111 in this model. Thus, lipoxin receptor agonist, such

as BML-111 is promising candidate as therapeutic agent providing protection against UV irradiation-

induced skin inflammation.

Keywords: skin damage, radiation UV, lipoxin, BOC-2, lipoxin receptor agonist.


157

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED MAST CELL, IN THE EFFICACY OF BML-

111 IN THIS MODEL SUNBURN CELL, AND TNF-α LEVELS IN THE SKIN OF MICE, AND EFFECT OF THE BOC2

Martinez, R.M.1; Saito, P.1; Sagae, B.N. 1; Pinto, I.C. 1; Rodrigues, C.C.A.1; Melo, C.P.B.1; Baracat,


1 State University of Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil 2 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil


Introduction and objectives: Solar ultraviolet (UV) irradiation is the main cause for the vast majority of

cutaneous malignancies. UVB is the most genotoxic agent that is capable of causing cell damage

and eventually leading to skin cancer. Therefore, targeting the physiopathological mechanisms of

UVB irradiation-induced skin damage might contribute to reduce skin diseases. Studies have

demonstrated the effectiveness of pro-resolution lipid mediators in UVB irradiationinduced skin

damage, however, these mediators are rapidly inactivated. In this context, analogues have been

developed, such as the BML-111, an agonist of the lipoxin A4 receptor (ALXR), but the therapeutic

potential of BML-111 in UVB irradiation-induced skin inflammation remains to be determined. Thus,

the present study aimed to evaluate the effects of administration with BML-111 on UV irradiation-

induced inflammation in the skin of mice, and effect of the BOC-2 (a lipoxin A4 receptor antagonist,

ALXR) in the efficacy of BML-111 in this model.



mg/kg, via intraperitoneal) 1 h before the irradiation, treated with BOC-2 (30 µg/kg, via


UV irradiation), and treated only BOC-2 (30 µg/kg) 1 h and 30 min before the irradiation. Samples of

skin were collected 12 h (histology) and 4 h (cytokine) after the end of the irradiation. Tissue sections

were stained with hematoxylin and eosin, and sunburn cells were counted throughout the epidermis

(x100 magnification). Tissue sections were also stained with toluidine, and mast cells were quantified

(x40 magnification). Samples were used to measure the cytokine levels by Enzyme linked

immunosorbent assay according to manufacturer’s instructions (eBioscience). Data were statistically

analyzed by one-way ANOVA followed by Tukey’s test, p<0.05. The Animal Ethics Committee (CEUA

process 1447.2015.10) of the Londrina State University approved this study.

Results: Treatment with BML-111 significantly inhibited UVB irradiation-induced increase of mast cell

and sunburn cell counts in the skin of mice. Moreover, treatment with BML-111 significantly repressed

the production of the pro-inflammatory cytokine TNF-α. Cytokines such as TNF-α activate the

apoptosis of keratinocytes ending up in the formation of sunburn cells. Therefore, reduced mast cell

and sunburn cell counts might be related to the inhibition of pro-inflammatory cytokine production

by BML-111. On the other hand, the pre-treatment with BOC-2, significantly attenuated, the anti-

inflammatory effects of BML-111 in mast cell and sunburn cell counts, and in cytokine TNF-α levels. The

treatment only with BOC-2 before the irradiation did not induce significant changes in comparison

to the irradiated control group.

Conclusion: The anti-inflammatory effects of BML-111 in the skin damage induced by UVB irradiation

are mediated by activation of the receptor ALXR, since the pre-treatment with BOC-2, (a lipoxin A4

receptor antagonist, ALXR) inhibited the efficacy of BML-111 in this model. These results to propose


agonist, such as BML-111.

Keywords: inflammation, cytokine, lipoxin, BOC-2, lipoxin receptor agonist.


158

EFFECTIVENESS OF THE BML-111 ON UV IRRADIATION-INDUCED OXIDATIVE STRESS IN THE SKIN OF

MICE, AND EFFECT OF THE BOC-2 IN THE EFFICACY OF BML-111 IN THIS MODEL

Martinez, R.M.1; Saito, P.1; Baptista, G.G. 1; Pinto, I.C. 1; Rodrigues, C.C.A.1; Melo, C.P.B.1; Baracat,


1 State University of Londrina, Department of Sciences Pharmaceutical, Londrina, PR, Brazil



Introduction and objectives: The skin, is a physical barrier between the organism and the

environmental deleterious factors such as ultraviolet (UV) radiation, the major risk factors for

dermatologic disorders, as cancer and premature skin aging. Pathological levels of reactive oxygen

species (ROS) are formed as a consequence of exposure to UV radiation. Skin cells are equipped

with antioxidants, nevertheless, flooding of ROS causes antioxidant depletion and further formation

of reactive products resulting in oxidative stress. In this context, targeting oxidative stress might

contribute to reduce skin diseases. Studies have demonstrated the effectiveness of pro-resolution

lipid mediators in UVB irradiation-induced skin damage, however, these mediators are rapidly

inactivated. In this sense, analogues have been developed, such as the BML-111, an agonist of the

lipoxin A4 receptor (ALXR), but the therapeutic potential of BML111 in UVB irradiation-induced skin

oxidative stress remains to be determined. Thus, the present study aimed to evaluate the effects of

administration with BML-111 on UV radiation-induced oxidative stress in the skin of mice, and effect

of the BOC-2 (a lipoxin A4 receptor antagonist, ALXR) in the efficacy of BML-111 in this model.



mg/kg, via intraperitoneal) 1 h before the irradiation, treated with BOC-2 (30 g/kg, via


UV irradiation), and treated only BOC-2 (30 g/kg) 1 h and 30 min before the irradiation. Samples of

skin were collected 2 h (catalase activity spectrophotometer assay), 4 h [mRNA expression by reverse

transcriptase (RT) and quantitative polymerase chain reaction (qPCR) test] or 12 h [ferric reducing

(FRAP assay) and ABTS radical scavenging abilities by spectrophotometer assays] after the end of

the irradiation. Data were statistically analyzed by one-way ANOVA followed by Tukey’s test, p<0.05.

The Animal Ethics Committee (CEUA process 1447.2015.10) of the Londrina State University approved

this study.

Results: Treatments with BML-111 significantly inhibited UVB irradiation-induced depletion of

antioxidant capacity, such as catalase activity, ferric reducing and ABTS scavenging abilities,

nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression, and its downstream target

nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase (Nqo1) mRNA expression.

On the other hand, the pre-treatment with BOC-2, significantly attenuated, the effects of BML-111 in

catalase activity, ferric reducing and ABTS scavenging abilities, Nrf2 and Nqo1 mRNA expression. The

treatment only with BOC-2 before the irradiation did not induce significant changes in comparison

to the irradiated control group.

Conclusion: The inhibition of oxidative stress by BML-111 in the skin damage induced by UVB

irradiation are mediated by activation of the receptor ALXR, since the pre-treatment with BOC-2, (a

lipoxin A4 receptor antagonist, ALXR) inhibited the efficacy of BML-111 in this model. Thus, lipoxin

receptor agonist, such as BML-111 is promising candidate as therapeutic agent providing protection

against UV radiation-induced skin oxidative stress.



159

EFFECTS OF PTEROSTILBENE ON THE HEPATIC OXIDATIVE STRESS OF RATS SUBMITTED TO SUCROSERICH

DIET.

de Morais, J.M.B¹; Souza Cruz1, E.M.; Cremer1, M; Ferreira, F.B1; Rosa, C.V.D1; Seiva, F.R.F1

1 Universidade Estadual do Norte do Paraná, Department of Biology and Technology,

Bandeirantes, PR, Brazil.

[email protected]

Introduction and objectives: Obesity is considered an epidemic and is related to several pathologies,

such as metabolic syndrome, type 2 diabetes, cancer and liver disease. Excess sugar consumption

is one of the main risk factors for obesity and overweight development. Pteroestilbene is a

phytonutrient that exhibits antioxidant, anti-inflammatory and anticancer activities. The objective of

the present work was to determine the effects of pterostilbene on oxidative parameters of the liver

of rats that consumed sucrose solution.

Material and methods: After approval by the animal ethics committee of the Universidade Estadual

do Norte do Paraná (CEUA 05/2017), 24 male Wistar rats were initially allocated in two groups: 12

received 40% sucrose solution for 150 days and 12 received common water. Both groups were fed

standard chow. After that period, the rats were separated into four groups (n=6): C+PL, C+Ptero,

S+PL, S+Ptero, receiving water as placebo, pterostilbene (40 mg/kg), 40% sucrose solution + water,

and 40% sucrose solution + pterostilbene, respectively, for 45 days. Throughout the experiment the

weight was monitored. Monthly the glycemia of the animals was determined by the caudal vein.

During euthanasia and under anesthesia, liver samples were collected for further evaluation of the

following oxidative parameters: reduced (GSH) and oxidized (GSSG) glutathione and its ratio;

carbonylated proteins; activities of superoxide dismutase (SOD) and catalase. The data were

analyzed with ONE-WAY ANOVA and the Newman-Keuls post-hoc test was applied with a

significance level of 5% (p<0.05).

Results: The rats that received sucrose solution for five months showed higher final body weight and

glucose levels. After pterostilbene treatment, both groups that received sucrose solution showed

reduction on all the glutathione parameters compared to the control group. C+Ptero group has

lower levels of oxidized, reduced and total glutathione versus C+PL group. However, the GSH/GSSG

ratio was elevated in the C+Ptero group which is related to the reduction of GSSG. Treatment with

pterostilbene in the sucrose-supplemented group caused no change in GSH levels and total amount

of glutathione compared to the sucrose-only supplemented group, while the GSSG level was

reduced. The GSH/GSSG ratio of the S+Ptero group increased when compared to the S+PL. The levels

of carbonylated proteins were not altered by the treatments. SOD activity of the C+Ptero and S+PL

groups was reduced in relation to the control group, whereas catalase activity was increased only

in C+Ptero group.

Conclusion: Sucrose consumption promotes general reduction of glutathione system and in the

activity of SOD which are related to the protection against reactive oxygen species, indicating

metabolic imbalance and oxidative stress. Pterostilbene altered the activity of SOD and catalase in

animals fed with standard chow and elevated SOD activity in rats that received 40% sucrose solution

but could not avoid the fall of glutathione values.

Keywords: Antioxidant; Sucrose; Liver; Pterostilbene.

Grants: CNPq

160

EFFICACY OF Rosmarinus officinalis EXTRACT-LOADED FORMULATION AGAINST UV-INDUCED

OXIDATIVE STRESS

Oliveira, J.1; Takayama, K.S. 1; Couto, R.O2. Saito, P.1; Monteiro, M.1; Verri, W. A.3; Baracat, M. M.1;

Casagrande, R.1 Georgetti, S. R.1*

1 State University of Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil

2 School of Pharmacy, Campus Centro-Oeste Dona Lindu, Federal University of São João del-Rei



Introduction and objectives: Oxidative stress and inflammatory responses induced by ultravioleta

radiation (UVR) can cause a variety of harmful effects in skin, including the induction of premature

photoaging, immunosuppression, and skin carcinogenesis. Medicinal plants have served as rich

sources of pharmacologically active substances. Rosemary (Rosmarinus officinalis), a member of the

family Lamiaceae, is a well-reputed aromatic and medicinal herb. Known for their antioxidant

activity, the leaves of R. officinalis are not only used in the food industry but also have been shown

to be safe and antitoxic in animal tests. The present study investigated the potential use of topical

formulation (gel cream) containing acetone 80% (FA) or ethanol 80% (FE) extract of Rosmarinus

officinalis against ultraviolet (UV)B irradiation induced skin damage.

Material and methods: Mice received topical treatment on the dorsal surface with 0.5 g of the each

formulation. The reduced glutathione (GSH) levels was determined by the 5,5’dithiobis (2

nitrobenzoic acid) spectrophotometric assay. The ferric reducing antioxidant power (FRAP) and 2,2’-

azinobis-(3-ethyl benzothiazoline-6-sulphonic acid) (ABTS) assays were used to determine the skin

antioxidant capacity. The statistical analysis was performed using by one-way ANOVA followed by

the Bonferroni's test. Values were significant when p<0.05. This study was approved by the Animal

Ethics Committee (CEUA) of the State University of Londrina (process number 2974.2017.14).

Results: F1 and F2 were able to significantly inhibited depletion of GSH and iron-reducing power of

the skin. Only FE showed the ability to inhibit the reduction of the ABTS scavenging capacity of the

skin.

Conclusion: This study indicates for the first time that topical formulations containing R. officinalis

extract as possible treatment of skin-oxidative stress

Keywords: UVB irradiation, Rosmarinus officinalis, oxidative stress, antioxidant, skin damage.

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária.

161

EFFICACY OF THE ASPIRIN AND 15-EPI-LIPOXIN A4 IN SKIN DAMAGE INDUCED BY UVB IRRADIATION,

AND EFFECT OF THE BOC-2 IN THE EFFICACY OF ASPIRIN AND 15-EPILIPOXIN A4 IN THIS MODEL

Saito, P.1; Martinez, R.M.1; Bussmann, A.J.C. 2; Staurengo-Ferrari, L. 2; Pinto, I.C. 1; Rodrigues, C.C.A.1;

Melo, C.P.B.1; Baracat, M.M.1; Georgetti, S.R.1; Verri, W.A.2; Casagrande, R.1*

1 Universidade Estadual de Londrina, Department of Sciences Pharmaceutical, Londrina, PR, Brazil 2 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil


Introduction and objectives: UVB irradiation may cause oxidative stress-dependent skin cancer and

premature aging. Targeting the physiopathological mechanisms of UVB irradiation-induced skin

oxidative damage and inflammation is a promising approach to reduce skin damage. Lipoxins are

anti-inflammatory and pro-resolution lipid mediators, however, the therapeutic potential of 15-epi-

lipoxin A4 (an aspirin triggered lipoxin, ATL) in UVB irradiation-induced skin oxidative stress and

inflammation remains to be determined. Thus, the present study aimed to evaluate the effect and

role of 15-epi-lipoxin A4 (ATL) in aspirin mechanism of action on UVBinduced skin oxidative stress and

inflammation.


non-irradiated control, irradiated control (irradiation session of 4.14 J/cm2), treated with aspirin (100

mg/kg, via oral) 1 h before the irradiation, treated with BOC-2 (a lipoxin A4 receptor (ALXR)

antagonist, 30 µg/kg, via intraperitoneal) 30 min before the treatment with aspirin (100 mg/kg),

treated only BOC-2 (30 µg/kg) 1 h and 30 min before the irradiation, treated with aspirin (100 mg/kg)

and not irradiated, treated with ATL (3 ng/mice, via intraperitoneal) 1 h before the irradiation, treated

with BOC-2 (30 µg/kg) 30 min before the treatment with ATL (3 ng/mice), and treated with ATL (3

ng/mice) and not irradiated. Samples of skin were collected 2 h (catalase activity, and superoxide

anion production spectrophotometer assays), 4 h [mRNA expression by reverse transcriptase (RT) and

quantitative polymerase chain reaction (qPCR) test] or 12 h [zymography to measure matrix

metalloproteinase-9 (MMP-9) activity, and histology by Masson's trichrome stain for collagen fiber

analysis] after the end of the irradiation. Data were statistically analyzed by one-way ANOVA

followed by Tukey’s test, p<0.05. The Animal Ethics Committee (CEUA process 1447.2015.10) of the

Londrina State University approved this study.

Results: Treatments with aspirin and ATL inhibited UVB irradiation-induced depletion of antioxidant

capacity, such as catalase activity, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA

expression, and its downstream target nicotinamide adenine dinucleotide (phosphate) quinone

oxidoreductase (Nqo1) mRNA expression. Moreover, aspirin and ATL treatments reduced UV

irradiation-induced superoxide anion production, gp91phox [nicotinamide adenine dinucleotide

phosphate (NADPH) oxidase 2 (NOX2) subunit] mRNA expression, MMP-9 activity, and collagen fiber

damage. The pre-treatment with BOC-2, inhibited significantly these antiinflammatory and

antioxidants effects of aspirin and ATL. The treatment only with BOC-2 before the irradiation did not

induce significant changes in comparison to the irradiated control group. Moreover, the treatment

only with aspirin or ATL in animals not exposed to irradiation did not induce significant changes in

comparison to the non-irradiated control group.

Conclusion: The antioxidants and anti-inflammatory effects of aspirin and ATL in the skin damage

induced by UVB irradiation are mediated by activation of the receptor ALXR. Thus, lipoxin receptor

agonists, such as 15-epi-lipoxin A4 (an aspirin triggered lipoxin) are promising candidates as

therapeutic agents providing protection against UV radiation-induced skin oxidative stress and

inflammation.

Keywords: inflammation, oxidative stress, radiation UV, lipoxins, BOC-2, lipoxin receptor antagonist.


162

EFFICACY OF THE BML-111 ON UV IRRADIATION-INDUCED INFLAMMATION IN THE SKIN OF MICE, AND

EFFECT OF THE BOC-2 IN THE EFFICACY OF BML-111 IN THIS MODEL

Martinez, R.M.1; Saito, P.1; Bezerra, J.R.1; Pinto, I.C. 1; Rodrigues, C.C.A.1; Melo, C.P.B.1; Baracat,





Introduction and objectives: UV irradiation-induced inflammation contribute to the development of

skin diseases. Therefore, targeting inflammation might contribute to reduce skin diseases. Studies

have demonstrated the effectiveness of pro-resolution lipid mediators in UVB irradiation-induced skin

damage, however, these mediators are rapidly inactivated. In this context, analogues have been

developed, such as the BML-111, an agonist of the lipoxin A4 receptor (ALXR), but the therapeutic

potential of BML-111 in UVB irradiationinduced skin inflammation remains to be determined. Thus, the

present study aimed to evaluate the effects of administration with BML-111 on UV radiation-induced

inflammation in the skin of mice, and effect of the BOC-2 (a lipoxin A4 receptor antagonist, ALXR) in

the efficacy of BML-111 in this model.


non-irradiated control, irradiated control (irradiation session of 4.14 J/cm2), treated with BML-111

(0.001, 0.01, 0.1 or 1 mg/kg, via intraperitoneal) 1 h before the irradiation, treated with BML-111 (0.1

mg/kg, via intraperitoneal) 1, 24, 48 or 72 h before the beginning of UV irradiation, treated with BOC-

2 (3, 10 or 30 µg/kg, via intraperitoneal) 30 min before the treatment with BML-111 (0.1 mg/kg, 1 h

before the beginning of UV irradiation), and treated only BOC-2 (30 µg/kg) 1 h and 30 min before the

irradiation. Samples of skin were collected 12 h after the end of the irradiation. The neutrophil

migration was evaluated by myeloperoxidase (MPO) activity. The skin edema was measured as an

increase in dorsal skin weight. Histology for epidermal thickness was analyzed using Infinity Analyze

(Lumenera® Software) in samples stained with hematoxylin and eosin. Data were statistically

analyzed by one-way ANOVA followed by Tukey’s test, p<0.05. The Animal Ethics Committee (CEUA

process 1447.2015.10) of the Londrina State University approved this study.

Results: Treatment with BML-111 significantly inhibited UVB irradiation-induced MPO activity of dose-

dependent manner. A consistent inhibition of MPO activity was observed only with the 0.1 and 1

mg/kg of BML-111 and pretreatment the 1 and 24 h before the beginning of UV radiation. Moreover,

treatment with BML-111 significantly reduced the UV irradiation-induced skin edema, and UV-

induced increased epidermal thickness. On the other hand, the pre-treatment with BOC-2,

significantly attenuated, in a dose-dependent manner, the anti-inflammatory effects of BML-111 in

MPO activity, skin edema, and epidermal thickness. The treatment only with BOC-2 before the

irradiation did not induce significant changes in comparison to the irradiated control group.

Conclusion: The anti-inflammatory effects of BML-111 in the skin damage induced by UVB irradiation

are mediated by activation of the receptor ALXR, since the pre-treatment with BOC-2, (a lipoxin A4

receptor antagonist, ALXR) inhibited the efficacy of BML-111 in this model. These results to propose


agonist, such as BML-111.



163

ESCITALOPRAM INDUCES CHANGES IN MICE ODONTOGENESIS

Sestario, C.S1, Menezes, E.V1, Ezequiel, B.S1, Brito, L.V1, Martins, C.C.N1, Yokoyama, M.F1, Salles,

M.J.S1

1 Department of General Biology, Center for Biological Sciences, State University of Londrina,

Londrina, Brazil [email protected]

Introduction and objectives: The prescription of antidepressants for the anxiety control and

depression during pregnancy became frequent. Antidepressants which have fewer side effects are

used. Because it is considered safer, Escitalopram (ESC) is the first-choice drug used in pregnant

women. With the lack of information regarding the influence of the drug on odontogenesis, the

objective of this study was to investigate whether the administration of ESC during pregnancy

promotes changes in tooth formation.

Material and methods: With the approval of the Ethical Committee for the use of animals, number:

17937.2017.35, thirty pregnant female mice were treated with ESC at the dose of 20 mg / kg, via

gavage (treated group) or saline (control group) from 5th to 17th day of gestation. For the

odontogenesis histological evaluation, the first fetus of the right uterine horn of each female was

separated and had its head removed. The pieces were placed in previously identified containers

containing bouin for 48 hours. Then, they were processed for histological procedure and stained by

hematoxylin and eosin, and soon after, 25 sagittal cuts of each piece were made, with a thickness

of 7 micrometers. The cut plane chosen was parallel to the longitudinal axis of the molar,

demonstrating the region of the enamel and dental papilla. Three cuts of each animal were selected

so that the analysis of the beginning, middle and end of dental germs could be performed. The

analysis was performed on 3 slides of each piece and, on each slide, 5 fields were analyzed. The

slides were photographed in a digital camera coupled to an optical microscope (Moticam, Motic

Co., Xiamen, China) and analyzed in Motic Image Plus 2.0 software (Motic Co, Xiamen, China) with

10 and 20X magnification. For the analysis of the association between the dependent variables and

the groups (control or treatment), chi-square and Fisher's exact tests were used.

Results: The parameters that prevailed were: greater absence of mitotic figures in the ameloblasts

layer in the treated group (15%) in relation to the control (0), lower polarization index of the

ameloblasts layer in the treated group (22.5%) when compared to the control group 5%),

malformation in the stellate reticulum, with a higher rate of malformations in the treated group

(27.5%) than in the control group (7.5%). Highest rate of malformations in the Hertwing sheath in the

treated group (35%) in relation to the control (7.5%). Higher frequency of matrix secretion by

odontoblasts in the treated group (52.5%) than in the control group (22.5%). Highest frequency of

advanced development in the treated group (47.5%) in relation to the control (22.5%). The abnormal

tooth morphology obtained a higher index of the treated group (35%) on the control (2.5%).

Conclusion: The results suggest that ESC is a teratogenic drug for causing changes in tooth formation.

Medication administration during the first trimester of pregnancy can affect the development,

differentiation, and growth of dental germs. The dentist is responsible for advising pregnant women

about medications that may affect fetal development.

Keywords: Pregnancy, Mice, Antidepressants, Odontogenesis

164

EVALUATION OF AORTA REACTIVITY IN MALE RATS TREATED WITH TOPIRAMATE DURING ADOLESCENCE

Moura, K.F. 1; Vidigal, C.B.1; Silva, D.G.1; Borges, L.I.1; Gerardin, D.C.C.1; Ceravolo, G.S.1

¹Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina,

Londrina/PR. [email protected]

Introduction and objectives: Topiramate (TOP) is neurotherapeutic agent currently indicated for the

treatment of epilepsy. In 2006, TOP was approved by Food and Drug Administration (FDA) for the

prophylaxis of migraine in adolescents. The efficacy and adverse effects of this drug are not

completely established. It has been described that treatment with TOP at important stages of

development, such as adolescence, increases markers of vascular risk. However, the effect of TOP

after treatment during adolescence in the vascular reactivity has not been evaluated yet. The

present study aimed to evaluate the vascular reactivity of TOP-treated rats during adolescence.

Material and methods: The study was approved by the Ethics Committee of the State University of

Londrina (9379.2018.26). Wistar rats were treated with TOP 41.0 mg/kg/day (TOP, n=10) or water (CTR,

n=10) by gavage during adolescence (from the postnatal day (PND) 28 to 50). It was evaluated in

male (PND 51) the thoracic aorta reactivity to phenylephrine (Phenyl) in the presence (E+) or

absence of endothelium (E-), acetylcholine (ACh) and sodium nitroprussiate (SNP). The comparison

between groups was performed using the maximal response (maxR) and using pD2 (-log of

concentration that causes 50% of maxR). Statistical analyses were performed using Prism and the

variables analyzed by One way ANOVA (vascular reactivity to Phenil) and test t Student (vascular

reactivity to ACh and NPS), being results presented as mean ± standard error of the mean (SEM).

Differences were considered statistically significant if *p<0.05.

Results: In aortic rings with endothelium, the maxR [Endo+: CTR 1,948±0,058 (n=9) vs TOP 2,343 ± 0,097

(n=10)] and pD2 [Endo+: CTR 6.741±0.08 (n=9) vs TOP 6.801±0.07 (n=10)] to Phenyl was similar between

the groups. The removal of endothelium increased the maxR in all groups. In Endo- rings, the values

of maxR [Endo-: CTR 2.901±0.14 (n=9) vs TOP 2.939±0,12 (n=10)] and pD2 [Endo-: CTR 7.866±0.05 (n=9)

vs TOP 8.059±0.10 (n=10)] to Phenyl were similar between the groups. Regarding the relaxation

curves, there were no differences in maxR [CTR 94.97±1.79 (n=8) vs TOP 91.94±1.20 (n=8)] and pD2

[CTR 7.049±0.10 (n=8) vs TOP 7.041± 0.08 (n=8)] to Ach, even as there were no differences in the maxR

[CTR 98.86±1.42 (n=8) vs TOP 98.08±0.77 (n=11)] and pD2 [CTR 7.477±0.08 (n=8) vs TOP 7.4861± 0.05

(n=11)] to NPS between CTR and TOP.

Conclusion: These results suggest that treatment with topiramate during adolescence did not

interfere with the aortic reactivity in male rats.

Keywords: anticonvulsant; aorta; prepuberty; DOHAD.

165

EVALUATION OF THE HYPOGLYCEMIC ACTIVITY OF THE BARK ETHANOLIC EXTRACT FROM Spondias

dulcis AND Spondias purpurea IN ALOXAN-INDUCED DIABETIC RATS

Nogueira, F. A.¹; Ramos, D. M. M.¹; Evangelista, L. C.²; D’Andrade, M. R. P.²; Zocoler, M. A.³; Kerche,

L. E.²

¹ Universidade do Oeste Paulista, Faculdade de Artes, Ciências, Letras e Educação, Presidente

Prudente, SP, Brazil

² Universidade do Oeste Paulista, Faculdade de Medicina, Presidente Prudente, SP, Brazil

³ Universidade do Oeste Paulista, Faculdade de Farmácia, Presidente Prudente, SP, Brazil


Introduction and objectives: Diabetes mellitus is a metabolic disturb of high complexity and

prevalence in the population. The use of plants with hypoglicemic potential has increased

considerably in the last years, since they are easy to access and have low cost, and also by the belief

that they have beneficial effects and are spared of collateral effects. Among plants used by man

as food and medicine source, the Spondias dulcis Forst. F. and Spondias purpurea L. species produce

edible fruits that are used as remedy for different diseases. Although these plants have diverse

applications, no study relating the hypoglycemic activity of their bark is currently available.

Therefore, in this study the hypoglicemic effects of S. dulcis and S. purpurea bark ethanolic extracts

were investigated in aloxan-induced diabetic rats.

Material and methods: For the induction of diabetes, the animals were treated intraperitoneally with

one single dose of aloxan in the concentration of 120 mg/kg b.w. These animals were then divided

in six groups and treated via gavage with three concentrations of each extract (500, 1000 and 1500

mg/kg b.w.) for 21 days. The negative control group was treated with distilled water. The evaluated

parameters were blood glucose, water and food intake, body weight, triglycerides, total cholesterol

and glycated hemoglobin. The results of these animals were compared to normal animals treated

under the same conditions. The project was accepted by the animal ethics committee by protocol

4000.

Results: The treatment of the aloxan-induced diabetic rats with bark ethanolic extracts of S. dulcis

and S. purpurea reduced the levels of blood glucose to normal levels and the concentration of 1500

mg/kg b.w. of S. purpurea extract was able to reduce the blood glucose levels in 63%. Diabetic rats

treated with the extracts diminished their water and food intake to levels similar to the non-diabetic

rats. The extracts were able to reduce the levels of triglycerides in the blood, and the concentration

of 1500 mg/kg b.w. of S. dulcis was able to reduce triglycerides levels in 79.91%. The extracts of S.

dulcis were more efficient to reduce total cholesterol levels. The extracts were able to reduce the

levels of glycated hemoglobin, and the concentration of 500 mg/kg b.w. of S. dulcis was able to

reduce the glycated hemoglobin in 96.64%.

Conclusion: In conclusion, this works shows that the bark ethanolic extracts of S. dulcis and S.

purpurea presented significant hypoglycemic and hypolipidemic activities in aloxan-induced

diabetic rats. More studies are being accomplished to understand the mechanisms by which the

extract can act reducing the levels of these molecules in the blood.

Keywords: Diabetes, aloxan, Spondias dulcis, Spondias purpurea

Grants: The authors would like to acknowledge Universidade do Oeste Paulista for supporting this

work.

166

EXPOSURE OF ZOLPIDEM DURING SPERMATOGENESIS PROMOTES CONGENITAL MALFORMATION IN

PROLE

Ezequiel, B. S.1; Brito, L.V1; Sestário, C.S1; Menezes, E.V1; Martins, C.C.N1; Mestre, V. F.1; Motomura, V.

N.1; Costa, E. M.1; Oliveira, A. L. M1; Yokoyama, M.F1; Salles, M.J.S1

1 Department of General Biology, Center for Biological Sciences, State University of Londrina,

Londrina, Brazil

[email protected]

Introduction and objectives: Zolpidem is a hypnotic agent and one non-benzodiazepine receptor

agonist preferred subclass omega 1 (BZD1) on GABA receptors A. This selectivity gives predominantly

hypnotic properties and relative absence of anticonvulsant and muscle relaxant effects. It´s effective

in inducing and maintaining sleep in adults. Due to the fact it has better side effects of

benzodiazepines, its use is very frequent. Studies that evaluate the effects of zolpidem on male

reproductive performance and embryofetal development are scarce in the literature. Considering

the widespread use of this drug for men of reproductive age, the objective of this study was to

evaluate the effects of Zolpidem in the intrauterine development and the potential to cause

malformations in the offspring of adult mice treated with Zolpidem during spermatogenesis.

Material and methods: With the approval of the Ethical Commission of the use of animals, number:

264.2018.20, Swiss male mice were used, distributed in treated group (G1) with Zolpidem 10mg / kg

and control group (G0) with distilled water. The treatment was via gavage for 35 days. On the 36th

day the animals were placed to mate with untreated females, at the rate of 1 male for each female.

The vagina of the females was examined daily for confirmation of pregnancy. On the 46th day the

males were submitted to euthanasia. On the 18th day of pregnancy, females were submitted to

euthanasia for evaluation of intrauterine development, with: the uterus weight, presence and rate

of resorption, number of live and dead fetuses in the litter, fetal weight and length, placental weight

and index, fetal viability rate and presence of external, visceral and skeletal congenital

malformations.

Results: Data analysis was performed using Student's t-tests, for parametric data (mean and standard

deviation), Mann-Whitney U, for non-parametric data (median and quartiles) and Fisher exact test

for frequency data (in%), with a significance level of 5% (GraphPad Prism 5). G1 was lower in relation

to G0: uterine weight (G0: 20,14 ± 4,14, G1: 14,93 ± 4,42, P = 0,0141), fetal viability rate (G0: 93.54 [89.72

- 100], G1: 58.81 [52.50 - 90.0], P =

0.0132). G1 were higher for the number of resorptions (G0: 0.70 ± 0.48, G1: 4.80 ± 3.68, P = 0.0132),

post implantation loss (G0: 6.46 [0.0 - 10,28], G1: 41.19 [10.0 - 47.50], P = 0.0132), rate of resorption (G0:

6.46 [0.0-10.28], G1: 37,86 [10,0 – 47,50]; P= 0,0133), when compared to the control group (G0).

Skeletal malformations were found in the supra and exoccipital (G0: (8%), G1: (35%), P = 0,0008), in

the sternum (G0: (12%), G1: (53%), P = <0.0001), and visce malformations (G0: (2%), G1: (19%), P =

0.0035), bone marrow (G0: (3%); G1: (17%); P = 0.0317), and trachea (G0: (2%), G1: (31%), P = <0.0001)

were more frequent in the treated group.

Conclusion: The results show that zolpidem has the potential toxic effects and teratogenic for

embryo-fetal development, serving as a warning to men of childbearing age who use the drug.

Keywords: Zolpidem, embryofetal development, malformations.

167

EXPOSURE TO BUPROPION HYDROCHLORIDE IN PREGNANCY MICE COMPROMISES FERTILITY AND

PROMOTES CONGENITAL MALFORMATIONS

Brito, L.V.¹; Ezequiel, B.S.¹; Sestario, C.S.¹; Mestre, V.F.¹; Menezes, E.V.¹; Salles, M.J.S.¹

¹ State University of Londrina, Department of General Biology, Center for Biological Sciences,

Londrina, Brazil.


Introduction and objectives: Bupropion hydrochloride (BUP) is considered an atypical antidepressant

with a mixed neuropharmacological profile, belonging to the class of aminoketones. They play an

important role in the treatment of depression and are also used as smoking cessation agents. Its

mechanism of action is the inhibition of the reuptake of dopamine and norepinephrine. The available

studies on the consequences of exposure to the drug on uterine development are still unclear. Thus,

this study aimed to investigate the possible toxic effects on intrauterine development of pregnant

mice and congenital malformations in the offspring subjected to administration of BUP during

pregnancy.

Material and methods: Thirty pregnant Swiss females were equally divided into treated group (G1)

and control group (G0), which was administered by gavage 40 mg / kg of BUP solution and distilled

water, respectively, in the period from the 5th to 17th gestation. On day 18, the mice were subjected

to euthanasia laparotomy and hysterectomy, and analysis of intrauterine development were

analyzed: uterus weight, presence and resorption rate, number of live and dead fetuses litter, weight

and fetal length, weight and placental index and fetal viability rate. The evaluation of malformations

in the offspring was through the analysis of the external, visceral and skeletal malformations of the

fetuses under stereoscope microscope. All procedures of this study were approved by the

Committee of Ethics in Use of Experimental Animals of the State University of Londrina, under the

number 8722.2016.33. Data analysis was performed using Student's t-tests for parametric data, Mann-

Whitney U, for non-parametric data, and Fisher's exact test for frequency data using GraphPad Prism

5.

Results: The BUP treated group showed a significant increase in the number of resorptions (G0: [0,3333

± 0,1869]; G1: [1,133 ± 0,3217*]) and resorption rate (G0: [3,311 ± 1,795]; G1: [11,33 ± 3,246*]) in relation

to the control group. There were no significant changes in the other parameters evaluated. In the

visceral analyzes of the groups, a significant increase in the palate malformation (GO: [0%], G1:

[11,76% **]) and nasal septum (G0: [0%]; 76% **]) when compared to the control group, the presence

of malformation in the skull (incomplete ossification of the supraoccipital) (GO: [6.15%], G1: [25% **]

and presence of reduced sternum (GO: [23.07%]; G1: [52.94% ***]) when compared to the control

group. It can be concluded that BUP exposure compromised fertility causing an increase in abortions

and also affected fetal development leading to congenital malformations.

Conclusion: The results demonstrate that BUP has teratogenic potential. Thus, until prospective studies

emerge, greater care is required in the prescription of the drug for pregnant women.

Keywords: Bupropion, mice, pregnancy, malformations.

168


HISTORY OF MULTIPLE ALLERGIES: A CASE STUDY

Francyne Baldo do Nascimento1, Anielle Aparecida Parmagnani1, André Demambre Bacchi2

1 Universidade Estadual de Londrina, Dentistry course, Londrina, PR, Brazil

²Universidade Federal de Mato Grosso, Department of Exact and Natural Sciences,

Rondonópolis, MT, Brazil [email protected]

Introduction: Patient K.M., female, 69 years old. Must be submitted to bridectomy surgery and must

be made prosthetic device. In the anamnesis, she reports being allergic to Dipirone, Norfloxacin and

Berotec.

Case Report: All drugs are capable of triggering both beneficial and harmful reactions. Adverse drug

reactions depend on the drug, dose administered, pharmacokinetics, pharmacodynamics and

individual predisposition. One possible reaction is hypersensitivity reaction, which varies according to

the intensity of its response, can be either localized or generalized, from skin erythema to anaphylaxis

reaction. Hypersensitivity reactions are grouped according to the classification of Gell & Coombs:

Type I (immediate or anaphylactic), triggers the production of IgE that attaches to the mast cells;

Type II (cytotoxic or antibody-dependent) mediated by IgM and IgG cytotoxic antibodies; Type III

(complex mediated); and Type IV (contact dermatitis), mediated by T cells, dendritic cells,

macrophages and cytokines. Treatment of these reactions involves immediate withdrawal of the

drug and, when necessary, hypersensitivity reaction treatment according to the clinical condition.

Immediate reactions (type I) usually have good response with oral anti-H1-histamines. More severe

reactions, such as anaphylaxis, require urgent treatment, such as: airway maintenance,

intramuscular adrenaline, anti-H1 and anti-H2 antihistamines, beta-adrenergic drugs and

corticosteroids. Corticosteroids are also required in cytotoxicity (type II) reactions. Immunocomplex

reactions (type III) usually ends after antigens clearance, although corticosteroids and systemic

antihistamines can be required in some cases. For late reactions (type IV), the drug of choice is

always the corticosteroid, topical or systemic, depending on the lesions extent. Antihistamines are

indicated only for the relief of pruritus. Drugs to which the patient is allergic, such as dipyrone

(analgesic and antipyretic) and norfloxacin (a fluoroquinolone antibiotic) are widely used in dentistry

to prevent or cure symptoms triggered by inflammatory reactions and infections, respectively.

Accordingly, professionals must have wide knowledge about the indications, contraindications,

mechanism of action and interaction with other medicines for an appropriate prescription. Allergic

reactions to these types of drugs directly affect the treatment of the patient in the pre, trans and

postoperative periods. Therefore, the surgeon must make changes in medication scheme to avoid

and eliminate possible risks during the treatment of the patient, without impairing the effectiveness

of the therapy. Thus, in the case described, it is possible to replace dipyrone with ibuprofen and

norfloxacin with amoxicillin, for example.

Final Consideration: Knowing the patient's medical history before any dental procedure is critical.

The correct diagnosis will dictate the success of the treatment and will avoid possible disorders like

allergies. The pharmacological and pharmacotherapeutic knowledge provides adequate repertoire

for the dentist to make the necessary substitutions, in order to avoid hypersensitivity reactions,

maintaining therapeutic efficacy.

Keywords: hypersensitivity, drug allergy, adverse reactions, dentistry

169

LOW DOSES OF MALATHION CAUSE ATROPHY OF JEJUNAL WALL IN RATS

Basso1, Camila Regina; Pupim1, Andréia Carla Eugenio; Machado1, Camila Cristina Alves; Dionisio1,

Joyce Hellen Ribeiro; Machado1, Cecília; Fernandes2, Glaura Scantamburlo Alves; Araújo1, Eduardo

José de Almeida1,2

1 State University of Londrina; Department of Histology, Londrina, PR, Brazil; State University of

Londrina; Department of General Biology, Londrina, PR; Brazil.


Introduction and Objective: Malathion is a broad spectrum organophosphate insecticide widely

used by public health to combat disease vectors such as the transmitting mosquito of dengue and

Chikungunya. In cases of acute intoxication, malathion causes inhibition of the enzyme

acetylcholinesterase and may trigger cholinergic syndrome. In cases of chronic exposure, malathion

presents late symptoms and involves neurobehavioral abnormalities. Studies with cholinesterase

inhibitors in the small intestine of rats showed a reduction in mucosal thickness and in the height of

the villi. However, there are no reports in the literature of investigations about the effect of malathion

on the morphological features of the jejunum wall and this, therefore, was the objective of this study.

Methods: The experimental protocols were approved by the Animal Research Ethics

Committee of State University of Londrina, Brazil (Approval number 137/2016). Fifteen male Wistar rats

were assigned into three groups treated for 40 days with either saline (control, n=5) or 10 mg/kg

malathion (M10, n=5) or 50 mg/kg malathion (M50, n=5) via gavage. The doses correspond to 0.5%

and 2.5% of the LD50 for rats (2000 mg/kg). After euthanasia, jejunum was removed, measured and

the number of Peyer patches was counted in the whole length. Jejunal sections were stained with

haematoxylin and eosin to evaluate the thickness of mucosa, submucosa and muscle layers. For

that, 28 images from each animal were captured using a high resolution camera and a

photomicroscope. The thickness of layers was measured in three points of each image using the

Motic Image Plus software. Sections stained with Periodic Acid Schiff (PAS) were used for counting

goblet cell in 40 intestinal crypts of each animal.

Results: Malathion caused atrophy of the mucosa in the M10 vs control and M50 vs M10 (p<0.05). The

villi height was reduced in the M10 and M50 vs control (p<0.05). The crypt deep was reduced in M10

vs control (p<0.05). Submucosa and external muscle were also atrophied in M10 and M50 vs control

(p<0.05). The jejunal wall thickness was reduced in M10 vs control and M50 vs M10 (p<0.05). The

number of Peyer patches and goblet cells was not changed due malathion exposure.

Conclusions: Low doses of malathion caused intense jejunal wall atrophy, but did not change the

number of Peyer patches and goblet cells.

Keywords: histology, malathion, small intestine, toxicology.

Grants: CAPES.

170

MARESIN-1 ATTENUATES INFLAMMATION IN TITANIUM DIOXIDE (TIO2)-INDUCED CHRONIC ARTHRITIS IN

MICE.

Anelise Francosi1; Telma Saraiva dos Santos 1; Nayara Anitelli Artero 1; Larissa Staurengo- Ferrari1;

Rubia Casagrande1 and Waldiceu Aparecido Verri Junior1.

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil (e.g)

[email protected]

Introduction and objectives: The partial or total joint replacement, procedure called arthroplasty

aims to improve the quality of life of patients suffering with inflammatory chronic joint conditions.

However, the prosthesis can introduce harmful effect through the release of metallic nanoparticles,

including titanium dioxide (TiO2). TiO2 induces a pro-inflammatory microenvironment, can still

stimulate the osteoclastogenesis and osteolysis, culminating in prosthetic rejection. The current

treatment is focused on low-efficiency or deleterious effects drugs. Pro-resolution lipids (SPMs) derived

from docosahexaenoic acid can contribute to the resolution of inflammation. Among these lipids,

Maresin-1 (MaR1) is SMP produced by macrophages of pro-resolutive phases (Mres) that present

microbicide capacity and analgesic activity. Thus, this aims to investigate a new alternative therapy

for arthritis induced by titanium dioxide (TiO2), through treatment with low doses of MaR1.

Material and methods: Male Swiss mice (20-25 g), were treated with MaR1 in doses of 110 ng

(i.p./100ul/animal) after a single stimulus of 0.3 mg/joint of TiO2. The treatment was every 3 days during

the 30 days after the stimulus. The therapeutic effect of MaR1-induced arthritis model TiO2 was

evaluated by assessing the articular mechanical hyperalgesia, knee joint edema and leukocyte

recruitment. To determine the safety of therapeutic treatment, hepatic and renal toxicity was

measured in serum samples collected 30 days after treatment.

Results: The treatment with MaR1 significantly reduced knee joint edema and mechanical

hyperalgesia induced by TiO2 in a dose-dependent manner over the 30 days of evaluations.

Importantly, no alterations in the levels of ALT/AST, urea and creatinine, markers of hepatic and renal

toxicity, respectively, were detected. The dose of 10ng/animal also reduced the recruitment of total

of leukocytes, mononuclear and polymorphonuclear cells to the knee joint at the 30th day.

Conclusion: In this work, it is possible to conclude that the MaR1 was able to decrease the pain and

edema induced by TiO2, as well as decrease the amount of recruted cells to the inflammatory foci

without inducing any potential toxicity.

Keywords: arthritis, titanium dioxide, maresin-1.

Grants: This work was supported by Programa para o Sistema Único de Saúde (PPSUS) grant

intermediated by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil)

and supported by Fundação Araucária and Secretaria Estadual de Saúde, Paraná (SESA-PR, Brazil)

and, Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

171

ACUTE AND LATE METABOLIC EFFECTS OF TOPIRAMATE TREATMENT DURING CHILDHOOD

Vidigal, C.B.1; Moura, K.F. 1; Borges, L.I.1; Silva, D.G.1; Gerardin, D.C.C.1; Ceravolo, G.S.1

¹Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina

(UEL), Londrina/PR.

Introduction: It has been recognized that adverse conditions during early life may later affect the

health of the individual, developmental origin of health and disease. Topiramate (TOP) is used for the

treatment of epilepsy in children from 2 years of age. It has been described that treatment with TOP

can lead to loss of weight gain and adiposity. However, it is unclear whether these effects are similar

between male and female children. Moreover, the long-term effects of treatment with TOP in

childhood have not been investigated. Aim: The present study evaluated, in male and female rats

treated with TOP during childhood some metabolic parameters. The evaluation was performed in

two different times, 24h after the last dose administrated and in adult life (57 (female rats) and 92

days (male rats) after the last TOP administration).

Methods: The study was approved by the Ethics Committee of the UEL (9379.2018.26). Male and

female Wistar rats were treated with TOP 41.0 mg/kg/day or water (CTR) by gavage during childhood

(from the postnatal day (PND) 16 to 28). It was evaluated in male (PND 29 and 120, n=44) and female

(PND 29 and 85, n=50) the Lee index (body weight1/3(g)/nasal-anal length (cm)×100); weight of

retroperitoneal, perigonadal and brown adipose tissue; weight of right and left adrenal and liver (all

values expressed as weight per 100g of body). Statistical analyses were performed using SPSS and

the variables analyzed by test t, being results presented as mean ± standard error of the mean.

Differences were considered statistically significant if *p<0.05.

Results: 24 hours after the last treatment, TOP male showed significant decreased in Lee Index [CTR

32.227 ± 0.262 vs TOP 31.305 ± 0.197] and in retroperitoneal adipose tissue [CTR 0.125 ± 0.158 vs TOP

0.073 ± 0.007] when compared with male CTR. Adult male treated with TOP during childhood had

lower Lee Index [CTR 31.362 ± 0.236 vs TOP 30.702 ± 0.147], decreased retroperitoneal adipose tissue

[CTR 0.998 ± 0.065 vs TOP 0.695 ± 0.064] and increased right [CTR 0.005 ± 0.000 vs TOP 0.007 ± 0.000]

and left [CTR 0.005 ± 0.000 vs TOP 0.007 ± 0.000] adrenal when compared with CTR adult male rat.

Regarding the female TOP showed increased of this tissue 24 hours after the last treatment [CTR 0.106

± 0.030 vs TOP 0.146 ± 0.005] and decreased in the brown adipose tissue in adult life [CTR 0.084 ± 0.002

vs TOP 0.071 ± 0.004]. The other parameters evaluated, in male and female, were similar between

CTR and TOP.

Conclusion: Treatment with TOP during childhood resulted in acute and late changes in metabolic

parameters in females and males. More studies are needed to understand the consequences of this

alteration to body health.

Keywords: anticonvulsant, disease programming, body index, adipose tissue.

Financial Support: CAPES.

172

MORPHOMETRIC ANALYSIS OF MYENTERIC NEURONS IMMUNOREACTIVE TO HUC/D PROTEIN OF

JEJUNUM FROM ARTHRITIC RATS TREATED WITH QUERCETINLOADED MICROCAPSULES

Bruna Thais da Silva1; Gustavo Henrique Doná Rodrigues Almeida2; Gleison Daion Piovezana

Bossolani2; Andressa Comelli Ballem Bessa4; Mariana Machado Lima4; Natália Pecin Bagon3;

Mariana Rodrigues Sanches4; Flávia Cristina Vieira Frez4; Ana Paula de Oliveira5; Fabiana Galvão

da Motta Lima5; Sabrina Silva Sestak5; Ciomar Aparecida BersaniAmado6; Juliana Vanessa

Colombo Martins Perles2; Jacqueline Nelisis Zanoni2

1 State University of Maringá, Department of Biology, Maringá, Brazil 2 State University of Maringá, Department of Morphological Sciences, Maringá, PR, Brazil

3 State University of Maringá, Department of Clinical Analysis and Biomedicine, Maringá, Brazil 4 State University of Maringá, Department of Pharmacy, Maringá, Brazil

5 State University of Maringá, Department of Physiological Sciences, Maringá, Brazil 6 State University of Maringá, Department of Pharmacology and Therapeutics, Maringá, Brazil

[email protected]

Introduction and objectives: Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects cartilage,

bones, tendons and muscles. Its development and chronicity interfere in a systemic way in the organism, due to

the intense formation of reactive species of oxygen and free radicals, inducing the development of oxidative

stress. Thus affecting, among other structures, the enteric nervous system, this may compromise the physiological

functions of the gastrointestinal tract. The most common treatment for RA is the use of non-steroidal

antiinflammatory drugs. But due to reports of adverse gastrointestinal effects in the treatment of RA, natural

compounds may be included as an alternative treatment to the disease, for example flavonoids such as

quercetin in order to reduce the damage caused by RA in the gastrointestinal tract and consequent

improvement of the quality of life of the arthritic organism. This study aimed to evaluate the morphometry of the

myenteric neurons of arthritic rats induced by complete adjuvant of Freund treated with quercetin-loaded

microcapsules.

Material and methods: We used 30 Holtzman rats (CEUA protocol 4462180216) were divided into 5 groups: C

(control), CQ (control treated with quercetin), AIA (arthritic), AI (arthritic treated with ibuprofen) and AQ (arthritic

treated with quercetin). Arthritic animals were induced by intradermal administration of Freund's complete

adjuvant, whereas controls only received an injection of mineral oil. Microencapsulated quercetin was

administered at the dosage of 10 mg / kg per gavage in each animal daily, for 60 days and the animals also

treated with ibuprofen the dose was 17.5 mg / kg per gavage daily. After this period, the animals were

euthanized, the jejunum was collected. The material prepared to perform the immunohistochemical technique

for labeling the general neuronal population by the HuC / D protein. Morphometric analysis of the neurons was

performed in Image Pro Plus 4® program and the results were analyzed by the GraphPad Prism 6 and Statística

8.0 programs.

Results: A significant statistically increase in the neuronal area of the CG group neurons in relation to the control

was observed; the neuronal areas of the AIA group presented values very similar to the control, possibly to the

adaptation of the arthritic animals to the pathology, which tends to regress and stabilize according to the time.

Both treatments (AI and AQ groups) showed an increase in neuronal area in relation to the control group,

revealing that quercetin acted similarly to the reference anti-inflammatory drug tested (ibuprofen), possibly

decreasing oxidative stress in the tissue, avoiding the atrophy of the neurons.

Conclusion: The treatment with quercetin was effective in preventing neuronal atrophy and similar action to the

reference drug tested, showing that it may have reduced the impact of oxidative stress on the population of

myenteric neurons.

Keywords: Enteric neuron system, Arthritis, Histology.

Grants: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

- Brasil (CAPES) - Finance Code 001.

173

MORPHOPHYSIOLOGICAL ALTERATIONS OF RAT COLON EXPOSED TO LOW DOSES OF MALATHION

Pupim1, Andréia Carla Eugenio; Basso1, Camila Regina; Watanabe1, Paulo da Silva; Machado1,

Camila Cristina Alves; Sodré1, Giovanni Bruno Clivati; Erthal2, Rafaela Pires; Fernandes2, Glaura

Scantamburlo Alves, Blackshaw3, L. Ashley; Araújo1, Eduardo José de Almeida¹

1State University of Londrina; Department of Histology, Londrina, PR, Brazil; 2State University of Londrina; Department of General Biology, Londrina, PR; Brazil;

3Queen Mary University of London, London, United Kingdom


Introduction and objectives: Dengue is one of the major public health problems in the world. Current

epidemiological data on dengue in Brazil show a wide distribution of Aedes aegypti in all regions,

leading to the occurrence of epidemics in the main urban centres. In addition to dengue, the

mosquito can transmit other diseases such as chikungunya fever and acute illness by Zika virus.

Prevention against these diseases depends on the control of vector mosquitoes by means of

chemical insecticides of the class of organophosphates which are inhibitors of acetylcholinesterase

like malathion. Oral absorption is quite important in environmental intoxications. Several symptoms

are observed due to the accumulation of acetylcholine such as abdominal pain, vomiting,

diarrhoea. The objective of this study was to evaluate the low dose toxicity of malathion on colonic

wall histology and in the colonic myenteric plexus morphophysiology of rats.

Material and methods: The experimental protocols were approved by the Animal Research Ethics

Committee of State University of Londrina, Brazil (Approval number 137/2016). Male Wistar rats were

assigned into three groups treated for 40 days with either saline (control) or 10 mg/kg malathion

(M10) or 50 mg/kg malathion (M50) via gavage. The doses correspond to 0.5% and 2.5% of the LD50

for rats (2000 mg/kg). During the exposition to malathion, faecal output was evaluated. After

euthanasia, colon was removed and measured. Colonic sections were stained with haematoxylin

and eosin to evaluate the thickness of muscle layers. Sections stained with Periodic Acid Schiff (PAS)

and alcian blue (pH 1.0 and 2.5) were used for goblet cell counting. Besides, the number and cell

body area of of general, nitrergic and cholinergic myenteric neurones were evaluated. Colon

intestinal motility using a multilumen perfusion manometry apparatus was analysed.

Results: Despite no changing in the colonic area, the muscular layers were atrophied in both

malathion groups (p<0.05). Acid mucin-producing goblet cells (alcian blue pH 2.5 positive) were

reduced in M10 and M50 vs control (p<0.05). Malathion exposure did not change the number of

neutralproducing goblet cells (PAS positive). Malathion did not change the number of general,

nitrergic and cholinergic myenteric neurones; however, neuronal cell body atrophy was observed in

the M10 groups compared to control and M50 (p<0.05). M10 showed few changes on the faecal

pellet output. These parameters increased significantly in M50. The manometric analysis revealed

stronger contractions on the proximal colon of M10 rats in comparison with control (p<0.05). The

colonic migrating motor complex (CMMC) had tendency to become faster on M50 rats in

comparison with control.

Conclusion: Malathion presented toxicity to colonic mucosa, muscle layers and morphophysiology

of the colonic myenteric neurones of rats, which impacted the faecal pellet output.

Keywords: Enteric nervous system, large intestine, malathion,toxicology.

Grants: CAPES

174

IN UTERO AND LACTATIONAL EXPOSURE TO TRICLOCARBAN DID NOT ALTER SPERM PARAMETERS IN

MALE RATS

Cavalcanti, L. F1; Costa, N. O. 1; Pereira, M. R. F.1; Ferreira, S. F.1; Gerardin, D. C. C.1

1State University of Londrina, Departament of Physiological Sciences, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Triclocarban (TCC) is an antimicrobial compound widely used in

personal care products, like soaps, toothpaste, and shampoo. This agent is incompletely removed

by wastewater treatment and represents an environmental contaminant. Studies shown that TCC

has been associated with some endocrine disruptions. In vitro, TCC demonstrated a potent

androgen-augmenting activity. In this sense, we studied if TCC exposures during critical period of

development (gestation and lactation) could lead to some adverse health outcomes in the

offspring, based on the concept of the Developmental Origins of Health and Disease. Therefore, the

purpose of this study was to evaluate if TCC exposure during peri and post-natal period could

adversely affect the sperm parameters in male pups.

Material and methods: Pregnant female Wistar rats were divided into four groups (n=811/group):

Control (CTR); TCC 0.3 mg/kg (TCC 0.3); TCC 1.5 mg/kg (TCC 1.5); TCC 3.0 mg/kg (TCC 3.0). The

females were treated daily by oral gavage from gestational day (GD) 0 to lactational day (LD) 21.

The male pups were weaned on post-natal day (PND) 21 and used for the study, no litter-mates were

used for the same group. After PND 120, all animals were weighed, euthanized and the reproductive

organs were removed. The testis and epididymis (caput/corpus and cauda segments) were

homogenized for sperm counting. The right vas deferens content was collected to perform sperm

morphology, and the left vas deferens was used to perform the sperm concentration, viability and

motility. Data were compared by factorial ANOVA and Fisher’s exact test, p<0.05 (CEUA-UEL nº

130.2016.24).

Results: There was no statistical difference in the percentage of abnormal sperm (CTR: 27% [8]; TCC

0,3: 26% [10]; TCC 1,5: 26% [11]; TCC 3,0: 29% [11]), percentage of viable sperm (CTR: 78% ;TCC 0,3:

78%; TCC 1,5: 80%; TCC 3,0: 73%), percentage of mobile sperm (CTR: 75% ;TCC 0,3: 75%; TCC 1,5: 74%;

TCC 3,0: 66%), concentration in the vas deferens (106/ml) (CTR: 38.21 ± 4.53; TCC 0,3: 34.90 ± 4.56;

TCC 1,5: 48.36 ± 5.05; TCC 3,0: 33.77 ± 3.5) and in any sperm counting parameters.

Conclusion: The present study reveals that peri and post-natal treatment with TCC did not alter sperm

parameters in male rat offspring.

Key-words: sperm count, antimicrobial, endocrine disrupter

Grants: Fundação Araucária, Capes.

175

PERIPUBERAL EXPOSURE OF MALE RATS TO LOW DOSES OF MALATHION IMPAIRS EPIDIDYMAL

MORPHOLOGY AND PHYSIOLOGY

Rafaela Pires Erthal1; Glaucia Eloisa Munhoz de Lion Siervo1; Glaura Scantamburlo Alves Fernandes2

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Department of General Biology, Londrina, Brazil

[email protected]

Introduction and objectives: Diseases currently considered as a public health problem, such as

dengue, chikungunya and zica, are caused by the Aedes aegypt mosquito. Prevention of such

diseases depends on the control of vector mosquitoes through the use of chemical insecticides being

the organophosphates used in large scale. From this insecticides class, an insecticide used on a large

scale is malathion, which has been shown to cause damage to reproductive parameters. Knowing

the importance of peripuberty as a critical period for postnatal development of organs of the male

genital system, and the importance of the epididymis in ensuring the motility and the ability to

acrosome reaction of spermatozoa, the present study aims to evaluate whether exposure to low

doses of malathion during the peripubertal period can cause damage to the epididymal

development.

Material and methods: Juvenile male Wistar rats were divided into 3 experimental groups: control

(saline), malathion 10 (saline + malathion 10 mg/kg) (M10) and malathion 50 (saline + malathion 50

mg/kg) (M50). The rats were exposed to malathion or vehicle from postnatal day (PND) 25 until PND

65 by gavage (oral route). At PND 65, the rats were anesthetized and euthanized. The protocol was

approved by the Ethics Committee on Animal Use of State University of Londrina (protocol number:

12305.2016.65 - CEUA/UEL). The right epididymis was collected and submitted to morphometrical and

histopathological analysis (n=5). The sperm was collected from vas deferens for following sperm

motility analysis (n=10) and measurement of acrosome reaction (n=6). The data were compared

using ANOVA followed by Dunnet’s post-hoc test. Differences were considered significant for p<0.05.

Statistical analyses were performed using GraphPad Prism (version 7.00).

Results: Histopathological analysis in epididymis revealed that both malathion doses significantly

induced abnormalities in epididymal duct when compared to the control group. The main alterations

observed were the presence of vacuoles, immature germ cells and multi-nucleated cells in the lumen

of tubules. Furthermore, exposure to malathion 50 mg/kg provoked tissue remodeling in caput and

cauda epididymis. The M10 group showed an increase in the percentage of immobile spermatozoa

and the M50 group showed an increase in the percentage of abnormal acrosome reaction.

Conclusion: These results appoints to an alteration in epididymal postnatal development for

morphological and histopathological parameters. The alterations observed reflected in impairment

observed in sperm capacitation, which can lead to reproductive damage.

Keywords: malathion, development, epididymis, sperm maturation

Grants: CAPES

176

PHOTOCHEMOPROTECTIVE EFFECTS OF TOPICAL FORMULATION WITH 15-DEOXY-Δ12,14-

PROSTAGLANDIN J2 IN UVB-INDUCED INFLAMMATION AND OXIDATIVE STRESS IN HAIRLESS MICE

Kumagai, C1; Saito, P.1; Mantovani, G.L.1; Martinez, R.M. 1; Melo, C.P.B.1; Pinto, I. C.1; Verri, W.A.2;

Casagrande, R.1*

1 Universidade Estadual de Londrina, Department of General Pharmaceutical Sciencies,

Londrina, PR, Brazil 2 Universidade Estadual de Londrina, Department of Pathology, Londrina, PR, Brazil


Introduction and objectives: The skin is the first line of defense against the environment and it is

consider an important marker to signal the aggressions suffered as it is continually exposed to harmful

factors that affect the integrity of its cellular structures. UV irradiation is an external aggressor and the

main cause of skin damage. UVB irradiation may lead of skin damage, resulting in precancerous,

cancerous lesions and accelerated skin aging. Skin disease by UVB radiation occur due to the

formation of reactive oxygen species (ROS), reduction of endogenous antioxidants, production of

proinflammatory molecules such as cytokines, and recruitment of leukocytes and neutrophils. 15d-

PGJ2 is an active lipid compound derived from arachidonic acid, that influences multiple signaling

pathways. There have been several studies published showing that use of 15-deoxy-Δ12,14-

prostaglandin J2 as anti-inflammatory, antiangiogenic, and anti-metastatic abilities, as well as a

significant anticancer effect. However, to date there is no evidence of the therapeutic effect in vivo

topically active formulation containing 15-d-PGJ2 to prevent or reduced UVB irradiationinduced skin

inflammation and oxidative stress. Thus, the aim of this study was to investigate the photoprotective

effect of 15-d-PGJ2.

Material and methods: Inflammation was induced in hairless mice by UVB irradiation (4.14 J/cm2).

Hairless mice were randomly designed to different groups with 6 mice each: non-irradiated control,

irradiated control, treated with vehicle and treated with formulation containing 15d-PGJ2.

Pretreatment with topical formulation containing prostaglandin J2 (30, 90 and 300ng/mice) was given

to mice 1 hour before, 5 minutes before and 5 min after the irradiation session. The skin edema was

measured as an increase in dorsal skin weight. The ferric reducing ability (FRAP assay), the ABTS

radical scavenging ability, and reduced glutathione (GSH) levels were determined by colorimetric

assays. The hydroperoxide production was evaluated by the chemiluminescence and catalase

activity by spectrophotometer assay.

Results: Prostaglandin J2 reduced the UVB irradiation-induced skin edema and depletion of

antioxidant capacity (ferric and ABTS reducing abilities and GSH levels). Finally, we demonstrated

that formulation containing 15dPGJ2 inhibited UVB-induced reduction of catalase activity, and UVB-

induced hydroperoxide formation, and thereby promote the resolution of inflammation.

Conclusion: These results suggest formulation containing 15d-PGJ2 as a promising treatment of skin

photoinflammation which merits further pre-clinical and clinical investigation.

Keywords: Antioxidant, oxidative stress, inflammation

Grants: CAPES, CNPq Fundação Araucária and Universidade Estadual de Londrina

177

PLATELET ACTIVATING FACTOR INVOLVEMENT IN ACUTE LUNG INJURY CAUSED BY SCORPION VENOM

Andrade, F.B.¹; Miyamoto, J.G2.; Victorino, V.J.3; Cândido, D.M.4; Pinge-Filho, P.¹; Ferraz, C. R.¹; Verri

Jr, W.¹; Kwasniewski, F.H.1

1Universidade Estadual de Londrina, Departamento de Ciências Patológicas, Londrina, PR, Brazil. 2Universidade Federal de São Paulo, Departamento de Biologia Molecular, São Paulo, SP, Brazil.

3Instituto Federal do Rio de Janeiro, Engenheiro Paulo de Frontin, RJ, Brazil. 4Instituto Butantan, Laboratório de Artrópodes, São Paulo, SP, Brazil.

[email protected]

Introduction and objectives: Acute lung injury (ALI) with participation of inflammatory mediators as

platelet activating factor (PAF) and nitric oxide (NO) can be triggered by venoms from scorpions of

medical importance. In Brazil, T. serrulatus and T. bahiensis are included in this group. We have

reported that despite important differences, T. serrulatus venom (Tsv) and T. bahiensis venom (Tbv)

caused ALI in rats. Although controversial, the participation of PAF it was studied early in some

aspects of ALI induced by Tsv, but concerning Tbv-induced ALI, the participation of inflammatory

mediators, including PAF, is unknown. Here the participation of PAF was investigated in ALI induced

by Tsv and Tbv.

Material and methods: Male rats (200 – 250 g) received Tsv or Tbv (200 µg/kg, iv), following the

indicated times the animals were killed by CO2 inhalation, lung circulation was perfused with isotonic

NaCl (50 mL, 25 mL/min) via a cannula inserted in the pulmonary artery and trachea, upper and

inner bronchi, and lungs were dissected. Edema (in 30 minutes, n=6 to 11) and hemorrhage (in 60

minutes, n=8 to 13) were investigated by Evans blue (EB) dye extravasation and

cyanometahemoglobin concentration using Drabkin's solution, respectively. After 4 hours of

envenomation the protein content (by Bradford method, n=4 to 7) and leukocyte influx (total and

differential counts, n=5 to 11) in brochoalveolar lavage (BAL), activity of myeloperoxidase (MPO, n=5

to 7) and nitrite as estimative of NO (by an adapted Griess method, n=5 to 7) production in lung

homogenates were analyzed. To evaluate the impact of PAF, the animals were pretreated with a

PAF antagonist receptor WEB2170 (WEB, 5 mg/kg, iv) 30 minutes before the envenomation. Venoms

and WEB were diluted in apirogenic NaCl 0.9%. Control data were obtained with rats injected with

apirogenic NaCl 0.9%. The procedures were approved by the institutional ethics committee (nº

16583.2013.29). Data were compared by one-way ANOVA and Tuckey’s post-hoc, or Kruskal-Wallis

and Dunn’s post-hoc. All analysis were performed in the GraphPad Prism (5.0) software (San Diego,

CA, USA) considering a significance level of α = 0.05. Results were expressed as mean ± standard

error of means (SEM).

Results: Tsv and Tbv induced ALI characterized by increased vascular permeability (30 and 240 min),

hemorrhage, alveolar influx of neutrophils, MPO activity and NO production into lungs. Early vascular

permeability (30 min, EB extravasation) was not modified by WEB, but it inhibited hemorrhage in Tsv

affected airways. Neutrophil influx towards alveoli (harvested with BAL) was inhibited by PAF-

antagonism in ALI induced by both venoms, but in Tsv envenomed animals this inhibitory effect of

WEB was also observed on total leukocyte and mononuclear counts. In contrast, late vascular

permeability (4 h, protein content in BAL) and MPO activity in lung homogenates were increased by

WEB in ALI induced by venoms. In envenomed groups, NO production in lungs was inhibited by WEB.

Conclusions: PAF seems to have a role not only in ALI induced by Tsv as well as by Tbv, but promoting

broader effect in Tsv envenomed rats.

Keywords: Tityus serrulatus, Tityus bahiensis, acute lung injury, platelet activating factor.

Grants: Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico -

CNPQ (Process 457512/2014-8).

178

PROTECTIVE EFFECT OF TOPICAL FORMULATION CONTAINING VANILLIC ACID AGAINST UVB-INDUCED

OXIDATIVE STRESS IN HAIRLESS MICE

Rodrigues, C.C.A.1; Baptista, G. G1; Melo, C.P.B.1; Martinez, R.M.1; Pinto, I.C. 1; Saito, P.1; Baracat,

M.M.1,Georgetti, S.R.1; Verri, W.A.2; Casagrande, R.1




Introduction and objectives: Ultraviolet B (UVB) radiation is known to cause damage to the skin by

mechanisms involving oxidative stress, DNA damage and apoptosis. Targeting the

pathophysiological mechanisms of UVB irradiation might reduce its deleterious effects in the skin,

which include oxidative damage. Vanillic acid (VA) is a phenolic compound derived from

dihydroxybenzoic acid, an oxidized form of vanillin present in vanilla extracts, mostly used as a

flavoring agent. VA presents varied activities such as antibacterial, antimicrobial, hepatoprotective,

chemopreventive, neuroprotective, antioxidant and anti-inflammatory. However, the therapeutic

potential of the VA-containing formulation in reducing oxidative stress by UVB radiation in hairless

mice has not yet been determined. We evaluated the physical-chemical and functional stability of

a formulation containing VA as well as the antioxidant parameters of this formulation in vivo context.

Material and methods: A formulation containing VA was prepared and subjected to accelerated

physicochemical tests, which stored the formulation containing VA at temperatures of 4°C, ambient

temperature and 40°C during six months. Functional stability testing was developed using the 2,2-

azinobisodium (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS) test. In vivo proof-of-concept

was also investigated. Mice were divided in 4 groups of 5 mice each as follows: non-irradiated

control, irradiated control (irradiation session of 4.14J/cm2), irradiated and treated with control

formulation (0.5g) and irradiated and treated with formulation containing VA (0.5%) (0.5 g) via

topical 1 h before, 5 minutes before and 5 minutes after irradiation section. Skin samples were

collected 2 and 12 hours after irradiation for the ABTS, reduced glutathione (GSH), catalase (CAT)

and reduction of iron ion (FRAP) tests, and the histological sections for epidermal thickness, apoptotic

cells and levels of dermal collagen. Data were analyzed statistically by one-way ANOVA followed

by the Tukey test, p <0.05. The Ethics Committee on Animals of the State University of Londrina

approved this study under the process #8225.2017.67.

Results: The formulation was stable at different temperatures and storage conditions as per visual

test, centrifugation and pH of the formulation during six months as well as in the evaluation of the

functional stability using the ABTS test. VA formulation inhibited the in vivo UVB irradiation-induced

depletion of the antioxidant capacity (e.g. ABTS, FRAP, GSH and CAT tests) and histological

alterations (e.g. epidermal thickness, apoptotic cells and collagen). Control unloaded formulation

presented no effect per se.

Conclusion: The present study demonstrate that a formulation containing VA remained stable during

6 months as per physical-chemical and functions stability tests, and that treatment with VA

containing formulation inhibited the deleterious effects of UVB irradiation in the skin.

Keywords: Vanillic acid; Formulation; Oxidative stress; Catalase; Grants: CNPq,

CAPES and Fundação Araucária.

179

RESOLVIN D2 ATTENUATES UVB RADIATION-INDUCED SKIN OXIDATIVE DAMAGE IN MICE

Pinto, I. C.1; Sagae, B.N.1; Saito, P.1; Rodrigues, C. C. A.1; Martinez, R. M.1; Melo, C. P. B.1; Kumagai, C.

M.1; Vale, D. L.1; Baracat, M. M.1; Georgetti, S. R.1; Verri, W. A.2; Casagrande, R.1*

1 State University of Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil 2 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil


Introduction and objectives: UVB irradiation is the main cause of the harmful effects of sunlight,

because it causes DNA direct damage and induces cutaneous damage by indirect mechanisms

that involve the excessive production of reactive oxygen species (ROS). For this reason, the

improvement of the endogenous antioxidant system become promising approach to prevent and

control UVB irradiation-induced skin oxidative stress. Resolvin D2 (RvD2) is pro-resolution lipid mediator

identified in the resolution phase of inflammation from omega-3 polyunsaturated fatty acids. Studies

have shown that RvD2 interacts with the receptor present in leukocytes denominated G protein-

coupled receptor 18 (GPR18), promoting resolution of inflammation and consequently decreasing

additional production of ROS. The aim of this study was evaluated RvD2 therapeutic potential in the

UVB irradiationinduced cutaneous lesion model.

Material and methods: Hairless mice were treated with different concentrations of RvD2 (0.3; 1.0 and

3.0 ng/mouse, intraperitoneally) 1 hour before the irradiation (irradiation dosage of 4.14 J/cm2).

Oxidative stress parameters were evaluated by measuring spectrophotometric assays such as ferric

reduction ability (FRAP assay) and the ABTS radical, reduced glutathione (GSH) levels, catalase

activity and superoxide anion production (NBT assay). In addition, lipid hydroperoxide formation was

determined by a chemiluminescence method initiated by tert-butyl hydroperoxide. The statistical

analysis was performed using by one-way ANOVA followed by the Tukey’s test. Values were

significant when p<0.05. This study was approved by the Animal Ethics Committee (CEUA) of the

State University of Londrina (process number 1447.2015.10).

Results: Systemic treatment with RvD2 was able to significantly decrease oxidative damage induced

by UVB radiation by reducing the oxidative parameters of superoxide anion production and lipid

hydroperoxides. In addition, the antioxidant capacity of the skin was recovered according to the

results of the reduced glutathione levels (GSH), ferric reducing antioxidant power (FRAP), reductive

capacity of 2,2’-azino-bis (3-ethylbenzothiazoline-6sulfonic acid) radical (ABTS) and catalase activity.

Conclusion: This study indicates for the first time that RvD2 has antioxidant activity in the model of skin

lesion induced by UVB irradiation. Therefore, RvD2 is a promising molecule for the prevent of UVB-

induced oxidative damage.

Keywords: UVB irradiation, resolvin, oxidative stress, antioxidant, skin damage.

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária.

180

RESOLVIN D5 REDUCES FORMALIN OVERT PAIN-LIKE BEHAVIOR IN MICE.

Souza, J.B.1; Staurengo-Ferrari, L. 1; Artero, N. A.1.; Sagato-Vendrameto, C. Z. 1; Carvalho, T.T.1;

Casagrande, R.2; Verri, W.A.1;

1 Universidade Estadual de Londrina, Department of General Pathology, Department, City,

Country 2 Universidade Estadual de Londrina, Department of Health Science, Londrina, PR, Brazil

[email protected]

Introduction and objectives: The resolution of the acute inflammatory process was recently

demonstrated to be an active process regulated by omega-3 fatty acidderived molecules, the

specialized pro-resolving lipid mediators (SPMs). These molecules include: resolvins, maresins and

protectins. Resolvins are a bioactive metabolite generated during inflammation to actively

orchestrate the resolution of inflammation. The compounds derived from eicosapentaenoic acid are

designated E series, given their EPA precursor, and denoted as Resolvins of the E series (RvEs), and

those biosynthesized from the precursor docosahexaenoic acid are Resolvins of the D series (RvDs).

The therapeutic potential of RvD5 in pain models, such as the formalin model, remains undetermined.

Herein, the objective of this study was to evaluate the analgesic effect of the specialized pro-

resolving lipid mediator RvD5 in the formalin model.

Material and methods: All experiments were performed with the approval of the Londrina State

University Ethics Committee on Animal Research (process number 1145.2016.54). To assess the effect

of RvD5, Swiss male mice (20-25g) were treated with RvD5 (with 0,3; 1 or 3 ng/animal) or vehicle (10%

ethanol plus saline) via intrathecal route (between L4-L6 spinal, 10 μL) before intraplantar injection of

formalin. Thirty minutes after the treatment, the formalin test was performed by counting the number

of paw flinches and the time of spent licking of the paw over 0–

30 min after intraplantar injection of 25 μL of formalin 1.5%. Results were obtained for both the first (0–

5 min) and second (15–30 min) phases. At the end of the experiment, paw edema was determined

by the observation of changes in paw volume using a caliper.

Results: RvD5 significantly decreased formalin-induced overt pain-like behavior. RvD5 inhibited paw

licking and flinching in the doses of 1 and 3 ng in the first phase (0-5 minutes). The treatment with all

doses of RvD5 decreased flinching behavior in both phases (0-5 minutes and 10-30 minutes).

However, only the 1 ng dose of RvD5 decreased the paw edema induced by formalin.

Conclusion: The treatment with 1 ng of RvD5 was the best dose to reduce pain and inflammation

induced by formalin. We demonstrated that RvD5 displays an analgesic effect in a nanogram range-

dose by inhibiting the neurogenic and inflammatory phases of formalin test. Importantly, to

determinate analgesic mechanisms of RvD5 more experiments have to be done, conversely, it might

represent in the future a new treatment of inflammatory pain.

Keywords: RvD5. Formalin. Pain.

181

SUCROSE AND PTEROSTILBENE ALTER THE HEPATIC MORPHOLOGY OF RATS

Souza Cruz, E.M.¹; de Morais, J.M.B.1;Ferreira,F.B.1; Cremer,M.1;Rosa,C.V.D.1; Seiva, F.R.F.1

1 Universidade Estadual do Norte do Paraná, Department of Biology and Technology,

Bandeirantes, PR, Brazil.

[email protected]

Introduction and objectives: Liver diseases are commonly associated with obesity, type 2 diabetes

mellitus and metabolic syndrome. The number of obese and overweight people continues to grow

around the world, being considered public health problems. Sucrose consumption through sugar-

sweetened beverages can contribute to metabolism impairment. Phytonutrients, such as

pteroestilbene can produce beneficial effects on glucose and lipid metabolism so, in thesis, it could

be used for therapeutic purposes. The objective of this work was to evaluate the effects of

pterostilbene on morphological and blood parameters related to the liver of rats supplemented with

sucrose solution.

Material and methods: After approval by the animal ethics committee of the Universidade Estadual

do Norte do Paraná (CEUA 05/2017), 24 male Wistar rats were initially allocated in two groups: 12

received 40% sucrose solution for 150 days and 12 received common water. Both groups were fed

standard chow. After that period, rats were divided into four groups (n=6): C + PL, C + Ptero, S + PL, S

+ Ptero, receiving water as placebo, pterostilbene (40 mg/kg), 40% sucrose solution + water, and 40%

sucrose solution + pterostilbene, respectively, for 45 days. Throughout the experiment, body weight,

body weight gain, food and liquid intake were monitored. Under anesthesia and after euthanasia

blood samples were collected for the evaluation of the enzymes Aspartate aminotransferase (AST)

and Alanine aminotransferase (ALT). The liver was weighed and samples were collected and fixed in

Bouin. Samples were processed histologically, obtaining semi-serial cuts that were stained in

Hematoxylin-Eosin. Under microscope, the number of hepatocytes in 30 microscopic fields (objective

of 20x) and the size of 100 hepatocytes per animal was evaluated. Parametric data were analyzed

with one-way ANOVA and Newman-Keuls post- test, while non-parametric data were analyzed using

the Kruskal-Wallis test with Dunns post-hoc test, with a significance level of 5% (p<0,05).

Results: Before being separated into four groups, animals that received sucrose solution had higher

body weight and during all the experimental period animals from sucrose groups showed reduced

food consumption, but liquid intake was similar among groups. Body mass of animals did not vary

significantly between groups after treatment. AST and ALT levels were increased in the S + PL group,

relative to the control, indicating liver damage. Pterostilbene supplementation minimized this

increase in the S + Ptero group. Supplementation of sucrose and/or with pterostilbene did not alter

the total or the relative mass of the liver. Both sucrose and pterostilbene promoted a decrease in the

number and size of hepatocytes compared to C + PL. The reduction caused by pterostilbeno was

more pronounced than the effect generated by sucrose, even in the C + Ptero group.

Conclusion: Sucrose and pterostilbene affect the liver differently. Sucrose promotes tissue damage,

affecting the histology of hepatocytes, while pterostilbene attenuates the liver damage caused by

sucrose supplementation. On the other hand, pterostilbene affects the hepatocytes in a way not yet

elucidated. Reduction in the amount and size of hepatocytes without hepatic mass change may

indicate a picture of tissue fibrosis.

Keywords: Pterostilbene; liver damage, sugar, morphology.


182

THE PRO-RESOLUTION 17-R-RVD1 LIPID MEDIATOR REDUCES PAIN, JOINT EDEMA, LEUKOCYTE

RECRUITMENT, PROINFLAMMATORY CYTOKINES PRODUCTION, AND OXIDATIVE STRESS IN A MURINE

MODEL OF GOUTY ARTHRITIS

Andrade, K.C.1; Zaninelli, T.1; Santos, T. S. 1, Rossaneis, C.1, Verri, W. A.1

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Gouty arthritis (GA) or gout is defined by intense inflammatory response and severe

pain. It is characterized by an imbalance in uric acid metabolism, due to an increase in its production and/or

low excretion leading to hyperuricemia leading to the crystallization and deposition of monosodium urate

crystals (MSU) in articular and periarticular tissues. Currently, the therapeutic approaches used in the treatment

of gout are mainly given by the control of inflammation and hyperuricemia, but are little effective in reducing

pain and have numerous adverse effects and high cost. 17-R-Resolvin D1 (17-R-RvD1) is a pro-resolution lipid

mediator derived from ω-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). This mediator has anti-

inflammatory and analgesic properties. Therefore, the objective of the present study was to evaluate the effect

and mechanisms of action of this molecule on pain and inflammation in murine model of gout.

Material and methods: For this study male swiss mice were used. Animal handling and procedures were

approved by animal welfare and ethics committee of the Londrina State University (CEUA nº 1415.2018.99). Mice

were treated with 17-R-RvD1 (0.03, 0.3 or 3 ng/animal/intraperitoneal [i.p.]) 30’ before intra-articular injection of

MSU (100 μg/ 10μL saline/animal) and mechanical hyperalgesia was measured at intervals of 1, 3, 5, 7 and 15h

after the stimulus with the objective of discovering what would be the best dose-response with analgesic action.

In other experiments, the mice were treated with 17-R-RvD1 (3 ng / animal / ip), 72, 48, 24h or 30 'prior to MSU

injection and mechanical hyperalgesia was performed with the purpose of finding the pre-treatment time-more

effective treatment of the mediator. With the dose and time-response found (3 ng/animal, 30'), thermal

hyperalgesia and joint edema formation were measured and molecular analyzes (leukocyte profile and

recruitment, cytokine production and parameters of oxidative stress, gastric, renal toxicity and hepatic). The

results are presented as ±SEM (standard error of the mean) of n = 6 animals per group. The data obtained from

the behavioral analysis and edema were analyzed by Anova two-way with a Tukey post-test and the molecular

analyzes, by Anova one way, followed by post Tukey test.

Results: 17-R-RvD1 at a dose of 3 ng/animal and 30’ before stimulus reduced MSUinduced mechanical

hyperalgesia in a dose-dependent manner, recruitment of leukocytes (total leukocytes, neutrophils and

mononuclear cells), joint edema, thermal hyperalgesia, oxidative stress (reestablished GSH levels and reverted

lipid peroxidation [TBARS]), production of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in addition to

increasing the release of the anti-inflammatory cytokine TGF-β. These effects were achieved without causing

renal damage (serum urea and creatinine levels), liver damage (aspartate aminotransferase [AST] and alanine

aminotransferase [ALT]) or gastric damage (myeloperoxidase [MPO] activity).

Conclusion: Thus, it has been demonstrated that 17-R-RvD1 has anti-inflammatory and analgesic properties in

the animal model of GA induced by MSU.

Keywords: Gouty arthritis, Mediator lipid, 17-R-RvD1, Inflammatory resolution, Analgesic

Grants: 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.

183

THE ROLE OF ESTROGEN IN VASCULAR REACTIVITY OF FEMALE PROGENY EXPOSED TO FLUOXETINE

DURING EARLY DEVELOPMENT

Higashi CM, Moura KF, Moreira EG, Ceravolo GS

Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil

[email protected]

Introduction and objectives: Selective serotonin reuptake inhibitors (SSRIs), due safety profile and lower toxicity,

have been largely prescribed to women of childbearing age, throughout pregnancy and lactation. Fluoxetine

(FLX) is the major representative drug of this class, however, little is known about the effects of this drug on

vascular development or function in fetuses. In a previous study, we verified that FLX exposure leads to vascular

reduced response to contractive agent in the presence of endothelium just in female progeny. It is well

described that female sex hormones can present cardiovascular protection by direct signalling in the

vasculature through ERα, ERβ and GPR30 receptors activation. Therefore, the study aimed to elucidate the role

of ovarian hormones in the endothelial modulation of aortic contractile response in female progeny intrauterine

and lactationally exposed to fluoxetine.

Material and methods: Ethics Committee of the State University of Londrina (CEUA/UEL 176/2016, 192/2016).

Wistar dams were gavaged daily with FLX (5mg/kg/day, n=14) or water (CTL, n=17) during pregnancy and

lactation. CTL and FLX female offspring at 28 days were divided into two protocols. Protocol 1: thoracic aorta

was isolated from prepubertal rats (before vaginal opening – 29 days of life) and concentration-effect curves

to phenylephrine (Phe) in the presence (E+) or absence of endothelium (E-) were performed. Protocol 2: The

offspring with 28 days were ovariectomized or had the incisions sutured without removal of the ovaries (false

operated rats - sham). Thus, following experimental groups: CTL-sham; FLX-sham; CTL-OVX and FLX-OVX. After

47 days of surgery, at 75 days of age, the thoracic aorta reactivity was evaluated with Phe in the presence or

absence of endothelium. The comparison between groups was performed using the maximal response (Rmax -

g) to Phe. The results were expressed as mean±SEM. Statistical analysis: Unpaired t-test for prepubertal reactivity

and one-way ANOVA followed by Tukey test for reactivity in adult offspring. Values were statistically significant

when p<0.05.

Results: In prepubertal female offspring, the Rmax of CTL and FLX was similar in aortic rings E+: (CTL=1.19±0.09,

n=13 vs FLX=1.14±0.08, n=13) and in E- (CTL=1.48±0.13, n=9 vs FLX=1.35±0.09, n=14). In female adult offspring, the

Rmax to Phe in E+ was reduced in FLX-sham rats (1.13±0.11, n=8) when compared to CTL-sham (2.62±0.10, n=10)

(p<0.05) and ovariectomy partially corrected this response (CTL-sham=2.71±0.13, n=8 vs FLXOVX=2.13±0.14, n=9).

In the absence of endothelium Rmax were similar between de groups [(CTL-sham=3.81±0.19, n=17); (FLX-

sham=3.63±0.16, n=13); (CTL-OVX=4.25±0.20, n=12);

(FLX-OVX=3.93±0.38, n=13)]. The pharmacological inhibition of ERα (MPP) or ERβ (PHTPP) did not interfere in the

aortic contractile response in the CTL group (CTLsham=2.56±0.21, n=10; MPP=2.28±0.4, n=10; PHTPP=2.35±0.17,

n=10). However, the inhibition of ERβ in FLX group corrected the reduced contraction in the aorta

(FLXsham=1.34±0.10 n=14 vs PHTPP=1.93±0.16, n=9 or vs MPP=1.53±0.10, n=8).

Conclusion: : FLX exposure during gestation and lactation cause aortic hypocontraction in adult female

progeny probably by mechanisms involving ovarian hormones, since there is no alteration in the vascular

reactivity before the puberty onset and in rats ovariectomized in prepubertal age. Further, ERβ has a role in this

reduced contraction.

Keywords: antidepressant; fetal programming; ovarian hormones; pregnancy; depression; endothelium

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES; Conselho Nacional de

Desenvolvimento Científico e Tecnológico – CNPq

184

THE ROLE OF SPINAL ASTROCYTES, MICROGLIAL CELLS AND NF-κB ON THE TRYPANOSOMA CRUZI

EXPERIMENTAL INFECTION-INDUCED PAIN

Andrade, K. C.1; Zaninelli, T. 1; Borghi, S.1; Verri, W. A. 1

1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil Email:

[email protected]

Introduction and objectives: Chagas disease (CD) is an infection caused by the protozoa

Trypanosoma cruzi and is transmitted to humans through the bite of Triatoma spp. hematofagous

insects. CD usually presents an acute and chronic phase. In the acute phase the levels of blood

parasites are increased and an intense inflammatory response is detected. A substantial decrease

of parasitemia and complications in neurologic and gastrointestinal systems can occur during the

chronic phase. Inflammatory pain results from the interaction between damaged tissue and

peripheral nociceptive sensory neurons through the participation of inflammatory mediators, which

culminates in hyperalgesia as a result of functional modifications in primary nociceptive afferent

neurons. Until now, there is no literature evidences of the participation of spinal components in the T.

cruzi experimental infection-induced pain. Therefore, this study aimed to evaluate the participation

of glial cells (microglia and astrocytes) and the production or activation of pain-related mediators

involved on T. cruzi infection-induced hyperalgesia.

Material and methods: For this study male C57Bl/6 mice, isogenic lineage susceptible to T. cruzi

infection (strain Y), were used. Animal handling and procedures were approved by animal welfare

and ethics committee of the Londrina State University (CEUA nº 1067.2015.64). The infection was

conduced by injection of 2x103 metacyclic promastigotes intraperitoneally (i.p.). The infected and

non-infected animals were assessed for behavioral parameters (mechanical and thermal

hyperalgesia) analyzed every 2 days for 28 days after infection and, molecular parameters (RT-qPCR,

immunoflorescence and NF-κB activity) analyzed at the 7th day post infection. The role of spinal cord

cell were determined by the intrathecally (i.t.) treatments with glial cell inhibitors (α-aminoadipate

[30 or 100 nmol] or minocycline [50 or 150 μg]) and the inhibitor of the NFκB transcription factor (PDTC

[30 or 300 μg]) performed on day 7 after infection. The results are presented as ±SEM (standard error

of the mean) of n=10 animals per group, analyzed by ANOVA followed by Tukey posttest. In addition,

comparative statistical analyzes between two groups were performed using the T-test.

Results: The T. cruzi infection induced mechanical and thermal hyperalgesia for 28 days and

increased the activation of glial cells and NF-κB. The peach of systemic parasitemia was on the 7th

day after infection and this parameter decreased with the infection chronicity. Minocycline, α-

aminoadipate and PDTC treatments significantly inhibited mechanical and thermal hyperalgesia as

well reduced astrocytes and microglial cells activation. Importantly, the treatments did not change

the systemic parasitemia. All treatments also reduced the activation of NF-κB.

Conclusion: Therefore, these data suggest that there is a close involvement of microglial and spinal

astrocytes in the T. cruzi experimental infection-induced pain, moreover that the mainly transcription

factor related the pain outcome is the NF-κB.

Keywords: Chagas disease, Glial cells, NF-κB

Grants: 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.

185

TREK-1 IS INVOLVED IN BRAIN CHANGES ON A DEPRESSION ANIMAL MODEL

José Francis-Oliveira1; Ianê Carvalho Shieh1; Matheus Azevedo Barbosa 1; Roberto De

Pasquale1.

1 São Paulo University, Department of Physiology and Biophysics, São Paulo, Brazil [email protected]

Introduction and objectives: Stress is related to the etiology of several neuropsychiatric diseases, like

anxiety and depression. Changes in the central nervous system, like serotonin or cortisol release

modulation, can occur after stress exposure. During the first periods of life (perinatal period and

infancy), stress can have an impact that yields permanent effects to be seen even on the adulthood,

which could be related to vulnerability to development of neuropsychiatric illnesses. Potassium

channels called TREK-1 have been related to depression, and knockout animals to these channels

showed a depression-resilient phenotype. Thus, this channel has been considered a new target for

the development of new antidepressant drugs, as well as a new player on the complex

physiopathology of depression and other related diseases. This work aimed to do an exploratory

investigation on possible changes in TREK-1 on a depression animal model that involves exposure to

stress in early-life (the maternal separation model) in rats, and to see if these changes would be

related to changes in the serotonergic system (evaluating the 5HT1A receptor), since serotonin is

clearly involved in some way on depression. As a second objective, we studied if manipulation on

these molecules could have an impact on synaptic plasticity, which changes are also implicated on

some neuropsychiatric illnesses. The main goal of this study was to establish new molecules

implicated on the physiopathology of depression.

Material and Methods: Male and female Wistar rats were submitted to a maternal separation

protocol, consisting in separate the dam from the pups for 3h/day from postnatal day (PND) 2

through PND14. At PND25, behavioral testing (forced swim, open field and novelty-suppressed

feeding test) were performed, and separate groups were perfused and the brains were collect to

do immunohistochemistry for TREK-1 and 5HT1A. Electrophysiological experiments on brain slices from

PND25 animals were executed in order to evaluate changes in synaptic plasticity. All methods were

approved by the Committee of Animal Use in Experimentation (CEUA) from the Sao Paulo University

under Protocol 54/2016.

Results: Only female rats showed anhedonia and behavioral despair in the behavioral testing.

Immunohistochemistry revealed an increase in the labeling for TREK-1 neurons for female rats in the

amygdala and prefrontal cortex, but not for males. Labeling for 5HT1A was increased in the amygdala

of males, but not females. Application of serotonin on brain slices were able to induce long term

depression on the prefrontal cortex, and also blockage of TREK-1 induced the same effect. Theta

burst stimulation showed the same effect for all conditions analyzed.

Conclusion: This exploratory work demonstrate some maternal separation induced changes in TREK-

1 and 5HT1A receptors in some key brain areas related to the stress circuitry, suggesting that TREK-1

can be involved in the physiopathology of depression. Importantly, TREK-1 also appear to have an

impact on synaptic plasticity on the prefrontal cortex, which is a very important area on the

elaboration and sustenance of a healthy stress response and behavioral modulation.

Keywords:

TREK-1; Depression; Serotonin; Maternal Separation; Synaptic Plasticity.

Grants:

FAPESP 2016/09116-0. CAPES - This study was financed in part by the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

186

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AND SUPPORT

187

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