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IMMUNOREGULATION (IR)

A NEW DAIRY PROBIOTIC WITH ANTI-INFLAMMATORY PROPERTIES

CLARISSA SANTOS ROCHA1; ANA CRISTINA GOMES SANTOS

2; PHILIPPE LANGELLA

3; VASCO AZEVEDO

4;

TESSALIA LUERCE5; ANA MARIA CAETANO FARIA

6; MAARTEN VAN DE GUCHTE

7; ANDERSON MIYOSHI

8.

1,2,4,5,6,8.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 3,7.INRA, JOUY

EN JOSAS - FRANÇA.

CLARISSA SANTOS ROCHA (1); ANA CRISTINA GOMES-SANTOS (1); PHILIPPE LANGELLA (2); VASCO

AZEVEDO (1); TESSALIA LUERCE (2); ANA MARIA CAETANO FARIA (1); MAARTEN VAN DE GUCHTE (2);

ANDERSON MIYOSHI (1)

(1). Universidade Federal de Minas Gerais – Brazil; (2). INRA – France

Introduction: Inflammatory bowel diseases (IBD) are characterized by a chronic inflammation of the gastrointestinal

tract (GIT). Although the causes for IBD remain obscure, there is evidence that IBD results from abnormal immune

responses to the gut microbiota in individuals with genetic predisposition. The treatments available are accompanied

by side effects and recent studies have demonstrated the potential therapeutic use of probiotic bacteria, even though

their mechanisms of action are still largely unknown. Most of the probiotics studied are lactic acid bacteria, mainly

lactobacilli, which have been isolated from the human GIT, however, fermented food are important sources of

transiting bacteria, such as Lactobacillus delbrueckii. While research on probiotics with immune modulation effects is

mostly focused on GIT isolates, relatively little is known about the effects of the dairy bacteria that make part of our

diet. In this context, we studied the anti-inflammatory effects of a collection of L. delbrueckii strain as well as

the mechanisms underlying their effects.

Methods and Results: 60 L.delbrueckii strains were screened for anti-inflammatory effects in vitro, measuring their

effect on NF-κB activation by TNF-α. For this purpose, an NF-kB dependent reporter gene was used in human gut

epithelial HT29 cells. All the strains tested reduced TNFa-induced NFkB activation in a strain-dependent manner. The

best-performing strain (Lb CNRZ327) inhibited the phosphorylation of IκB. Lb CNRZ327 was then tested in a mice

model of IBD and was able to prevent inflammation damages. Its effect was correlated with the maintenance of the

normal levels of IL-12, IL-6 and TGF-β in colonic tissue and that TGF-β in the spleen. The strain also increases the

population of Foxp3 regulatory T cells in the colon and spleen.

Conclusion: We showed that dairy lactobacilli can modulate immune responses and may thus affect health more

than generally thought. This is the first demonstration that orally administered dairy lactobacilli can not only modulate

mucosal but also systemic immune responses.

Financial support: FAPEMIG; CNPq.

A NEW ROLE FOR TNF IN SEPSIS: DEVELOPMENTAL REGULATION OF SEPSIS-INDUCED

IMMUNOSUPPRESSION

PAULO HENRIQUE MELO; DANIELE C.B. NASCIMENTO; RAPHAEL G. FERREIRA; GABRIELA T.

SCORTEGAGNA; FERNANDO DE QUEIRÓZ CUNHA; JOSÉ CARLOS ALVES-FILHO.

USP, RIBEIRÃO PRETO - SP - BRASIL.

Introduction: During sepsis, TNF is an important inflammatory mediator acting through its two cognate receptor

isotypes: TNFR1 and TNFR2. In its acute phase systemic TNF release leads to dysregulated local inflammatory

responses and organ damage. Recent results show in patients and mouse models who survived severe sepsis that

they are more susceptible to secondary infection. An important change encountered by these survivors is a large

expansion of Tregs cells. It has shown that TNF plays an important role in the activity of Tregs cells, inducing

proliferation, stabilization of phenotype and increasing their immunosuppressive activity. Aim: To evaluate the specific

roles of TNF receptors in developing sepsis-induced immunosuppression. Methods and Results: WT, TNFR1-/-

and

TNFR1/2-/-

mice underwent cecal ligation and puncture. They subsequently were treated with antibiotics. During acute

phase of sepsis, survival increased in both groups of TNF receptor deficient mice due to declines in bacterial load

and organ damage. The sepsis survivor’s mice were induced with a secondary infection (1 x 107

L. pneumophilla via

i.n.). More of the TNFR1-/-

survivors were more susceptible and had a higher bacterial load in their lungs and

spleens. In contrast, TNFR1/2-/-

sepsis survival mice were more resistant to infection and had lower bacterial loads

than their WT counterpart. In vivo BrdU incorporation measurements showed that CD4+ T cells proliferation in the

spleen was less in the WT and TNFR1-/-

than in TNFR1/2-/-

sepsis survival mice. This decline in TNFR1-/-

mice was

associated with a larger expansion of Tregs cells (15% ±2). On the other hand, compared to WT Treg levels (10%

±1)., in TNFR1/2-/-

mice 15 days after sepsis they did not undergo expansion(5% ±1).. The FACS assay results

indicated that TNF receptor density on Tregs was greater than on conventional T cells. Following recovery from sepsis

their Treg cells receptor density became even greater. Such an increase after sepsis also occurred in TregsTNFR2+,

but not in TregsTNFR1+.We also demonstrated in vitro that TNF expanded the Tregs cells population possibly due to

an increase in Treg cells proliferation. Conclusion: These results suggest that TNF receptors activation by sepsis has

opposing effects on Treg cells expansion. TNFR1 can be a negative regulator, whereas TNFR2 may play a role in

Treg cells up regulation during development of sepsis-induced imunossupression. Financial Support: CNPq,

FAEPA and FAPESP

ABSENCE IN IL-33 SIGNALING RESULTS IN A MORE SEVERE EXPERIMENTAL NEPHROPATHY

VANESSA OLIVEIRA DOS REIS1; BRUNO CAMOLESE VIVANCO

2; ANA PAULA FERNANDES S. MONTEIRO

3;

DEBORA TAVARES R. S. ABATE4; DAVID ANIBAL GARRIDO

5; ALEXANDRE CASTRO KELLER

6.

1,2,3,5,6.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO - SP - BRASIL; 4.UNIVERSIDADE DE SÃO

PAULO, SÃO PAULO - SP - BRASIL.

Introduction: Focal segmental glomerulosclerosis (FSGS) is a major cause of chronic kidney disease. Several

studies suggest an association between Th2 and the pathogenesis of FSGS. Reinforcing Th2/FSGS relationship, we

showed in a FSGS adriamycin-induced model that the Th1/Th2 balance is associated with the modulation of the

disease. Therefore, we can assume that inflammatory diseases associated with Th2 immune responses expression

can be a major factor for the development of FSGS. These diseases may be an important factor but not the only one.

Immune system molecules are widely associated with Th1/Th2 balance. In light of this, we chose allergic asthma and

the shaft IL-33/ST2 participation as the object of study, both related to Th2 immune responses and kidney damage. To

determine the role of the shaft IL-33/ST2 at FSGS, we used BALB/c WT and ST2-/-

animals, intravenously injected

with a single dose of 10mg/kg of adriamycin.

Methods and results: The groups were euthanized at different points of the disease development and analysis of

renal function and tissue were made through biochemical assay, western blot analysis, electrophoresis, flow

cytometry, ELISA and histology. Since ST2-deficient animals exhibit more severe kidney damage when induced by

adriamycin, the absence of this receptor signaling is intimately related to induced loss of renal function. Observing the

changes of these animals, it is clear the increase in serum creatinine levels and elevated protein / creatinine curve.

Nevertheless, these animals show more severe tissue damage and decreased expression of nephrin protein, STAT3

and Smad2/3. In addition, there was an increase in PAI-1 and nephrin phosphorylation, Prha, pTyr. Serum levels of IL-

33 are also higher in ST2-/-

animals. In the cellular context, we observed an increase in the percentage of TCD4pos

splenic cells in these ST2-/-

animals. However, CD4pos

/ FOXp3pos

cells are less present when compared to wild type

animals and similar to control animals. We also observed that the effect found in ST2-/-

animals was mitigated by the

transfer of TCD4pos

wildtype lymphocytes. The same effect was obtained by its treatment with recombinant IL-33.

Conclusion: Considering all results, we can conclude so far that cytokine IL-33 plays an important role in the

development and control of FSGS. Therefore, the data presented are extremely important to clarify the complex

mechanisms involved in FSGS.

Financial supports: FSPESP, processes number: 2012/04152-7

ADENOSINE SIGNALLING VIA A2A RECEPTOR IS INVOLVED IN DEVELOPMENT OF SEPSIS-INDUCED

IMMUNOSUPPRESSION

DANIELE CARVALHO BERNARDO NASCIMENTO1; PAULO HENRIQUE MELO

2; RAPHAEL GOMES FERREIRA

3;

RAPHAEL SANCHES PERES4; DARIO SIMÕES ZAMBONI

5; FERNANDO QUEIROZ CUNHA

6; JOSÉ CARLOS

FARIAS ALVES-FILHO7.

1,3,6,7.DEPARTMENT OF PHARMACOLOGY, SCHOOL OF MEDICINE OF RIBEIRÃO PRETO, UNIVERSITY OF

SÃO PAULO-SP, RIBEIRÃO PRETO - SP - BRASIL; 2,4.PROGRAM OF GRADUATE STUDIES ON APPLIED AND

BASIC IMMUNOLOGY, SCHOOL OF MEDICINE OF RIBEIRÃO PRETO, U, RIBEIRÃO PRETO - SP - BRASIL;

5.SCHOOL OF MEDICINE OF RIBEIRÃO PRETO, UNIVERSITY OF SÃO PAULO-SP, BRAZIL, RIBEIRÃO PRETO -

SP - BRASIL.

Introduction and Aim: Sepsis is regularly accompanied by the development of a pronounced immunosuppression,

which is characterized by predominance of regulatory T cells (Treg), reduced proliferation and function of effector T

cells and increased susceptibility to secondary infection (Crit Care Med. 38:1718-25, 2010). The adenosine 2a

receptor (A2aR) activation is firmly implicated in modulating the immune response. Recently, it was demonstrated that

the adenosine promotes expansion of Treg via A2aR (Front Immunol. 3:190, 2012). In the present study, we

investigated the role of A2aR in the development of sepsis-induced immunosuppression. Methods and Results: WT

mice were subjected to severe sepsis induced by cecal ligation and puncture (CLP–100% of mortality without

treatment) and treated with antibiotic (30mg/kg of ertapenem sodium by 3 days), resulting in 50% of survival (all

experiment n=5-10). Then, sepsis-surviving mice were treated with A2aR antagonist [8-(3-chloro-styryl) caffeine; 3

mg/kg/day; i.p.] or vehicle (veh) beginning at third day after CLP and then every day thereafter up to day 12. At 15th

day after CLP (CLP15d), mice were challenged intranasally with a nonlethal inoculum of L. pneumophila. All CLP15d

mice showed higher bacterial load in lung (naive: 4200±3178; CLP: 212786±57441) and spleen (naive: 4.6±2.2; CLP:

558.6±63.4) then naïve mice and succumbed to the infection with L. pneumophila. Interestingly, CLP15d mice treated

with A2aR antagonist showed reduced levels of bacteria in lung (naive: 18250±6539; CLP: 8965±7487) and spleen

(naive: 8.6±5.6; CLP: 63±27.8) and improved survival (40% of survival rate) after L. pneumophila infection. Moreover,

an increased of Tregs numbers were observed in the spleen from CLP15d mice (naive: 5.3±0.4; CLP: 9.4±0.7) was

not present in mice treated with A2aR antagonist (naive: 6.4±0.3; CLP: 6.9±0.1). In parallel, we also observed that

A2aR antagonist treatment markedly improved the T cells proliferative responses in vivo in CLP15d mice (naive+veh:

9.0±0.6 and CLP+veh: 6.2±0.2; naive+A2aR antagonist: 7.2±0.1 and CLP+A2aR antagonist: 8.6±0.4) similar to those

observed in naive mice. Conclusion: These results point to an important role of A2aR in the establishment of

immunosuppression post-sepsis. Financial support: FAPESP.

ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS INDUCE BIOGENESIS OF LIPID BODIES IN

REGULATORY MACROPHAGE

LUCIANA DE SOUZA MOREIRA1; ELENA GONZALEZ-REY

2; MARIO DELGADO

3; PATRÍCIA TORRES BOZZA

4.

1,4.INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL; 2,3.INSTITUTO DE PARASITOLOGIA Y

BIOMEDICINA LOPEZ NEYRA, GRANADA - ESPANHA.

Introduction: Mesenchymal stromal cells (MSCs) have emerged as a potential therapy for a range of inflammatory

insults. Recent evidences have demonstrated that MSCs re-educate a variety of immune cells to promote tolerance in

inflammatory and autoimmune conditions. Prostanoids metabolites are key mediators in several immunomodulatory

effects of MSC. Lipid bodies are functionally active organelles found in all cell types. These organelles can be induced

in response to different inflammatory conditions and are sites for synthesis and storage of lipid inflammatory

mediators. Our objective is investigating the biogenesis of lipid bodies in regulatory macrophages induced by factors

derived from adipose-derived MSCs (ASCs).

Methods and Results: We differentiated macrophages from bone marrow cells, then co-culture with ASCs or in the

presence of ASCs conditioned medium (ASCcm). Lipid bodies were analyzed by Oil Red O or Bodipy stain (n=3-5),

and the production of lipoxin A4 were measured by EIA (n=2). We have showed previously that ASCm induce a

distinct regulatory activation state of macrophage (high expression of arginase 1 and elevated IL-10 release) which

possess therapeutic potential in inflammatory diseases. Here, we demonstrated that ASCs induce significantly lipid

bodies biogenesis in macrophages. Importantly, indomethacin partially suppressed lipid bodies formation in co-culture,

but did not showed any effects in macrophage treated with ASCcm. Moreover, neutralizing antibodies against M-CSF,

but not against IL-6, selectively inhibited this process. Trying to understand the increased formation of lipid bodies in

regulatory macrophages, we measured an anti-inflammatory lipid mediator, Lipoxin A4. Previous results demonstrated

that macrophage pre-treated with ASCcm showed an increased release of Lipoxin A4.

Conclusions: Our results suggest that ASCcm generate lipid bodies formation in macrophage by M-CSF production,

and probably, the induction of these organelles could be related with increased production of Lipoxin A4 and others

anti-inflammatory lipid mediators. However more studies are necessary to understanding the role of these organelles

in the therapeutics effects of these macrophages, which participate in beneficial effects and a cell-based therapy with

ASC

Financial Support: CNPq & FAPERJ

ALTERATION IN GUT MICROBIOTA COMPOSITION AND IN GUT-ASSOCIATED LYMPHOID TISSUE OF DIET-

INDUCED OBESE MICE AFTER TRIBUTYRIN TREATMENT

ANDREA CARICILLI1; ANGELA CASTOLDI

2; VINICIUS ANDRADE

3; SUSAN SILVA

4; TIAGO SOUZA

5; MARCO

VINOLO6; PATRICIA GAMA

7; RUI CURI

8; NIELS CÂMARA

9.

1,2,3,4,5,7,8,9.UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 6.STATE UNIVERSITY OF CAMPINAS,

CAMPINAS - SP - BRASIL.

Introduction. Low-grade inflammation, found in obese individuals, is associated with impaired intestinal barrier and

increased lipopolysaccharide serum concentration. Obesity is also associated with alteration in gut microbiota

composition. Short-chain fatty acids, such as butyrate, are fermentation products of some types of bacteria and until

now, only in vitro studies have shown that they may interfere with the immune system. We aim to investigate the

modulation of the immune system found in the gastrointestinal tract of diet-induced obese mice and correlate it with

possible changes in intestinal barrier function after treatment with butyrate prodrug, tributyrin (TB). Methods. C57BL/6

male mice fed a standard chow or high-fat diet (HFD) were treated with TB (2g/kg, by gavage) during 10 weeks and

evaluated for metabolic and immune cell profile, gut microbiota composition and intestinal tight-junction protein

expression. Results.TB-treated mice fed a HFD gained less weight and presented increased glucose tolerance than

saline-treated mice. TB-treated mice on a HFD showed increased expression of T regulatory cells and decreased

TNF-α and IFN-δ producing cells in mesenteric limph nodes. There was also a decrease in Firmicutes and increase in

Verrucomicrobia phyla accompanied of increased expression of ZO-1 and occluding after TB treatment. Conclusion.

Our results suggest that TB treatment leads to an improvement of the intestinal barrier by reducing proinflammatory

cytokine production and increasing regulatory T cells, which may be related to changes in the gut microbiota

composition. Thus, tributyrin may present itself as a potential drug to be used in the treatment of obesity. Grant

#2011/19247-0, São Paulo Research Foundation (FAPESP)

ANALYSIS OF THE ACTIVATION OF MONOCITIC CELL LINE BY BJCUL

MARCELA FERREIRA DIAS-NETIPANYJ1; LIDIANE MARIA BOLDRINI-LEITE

2; ANDRÉA NOVAIS MORENO-

AMARAL3; SELENE ELÍFIO-ESPOSITO

4.

1,3,4.NÚCLEO DE INVESTIGAÇÃO MOLECULAR AVANÇADA-PUCPR, CURITIBA - PR - BRASIL; 2.NÚCLEO DE

TECNOLOGIA CELULAR-PUCPR, CURITIBA - PR - BRASIL.

Introduction: Lectins are non-immune glycoproteins that interact with membrane carbohydrates in a specific and

reversible manner, and are able to induce different cell responses. BJcuL is a lectin purified from Bothrops

jararacussu venom, known to have antitumoral activity and recently as an immune response inductor as it was able to

induce migration, polarization and adhesion of human neutrophils. The aim of this study was to identify the action of

BJcuL as an activator of macrophages using U937 monocytic cell line as a model. Methodology: Differentiation cell

line was achieved with 20 nM PMA, followed by labeling with CD14. The cytotoxicity of BJcuL was analyzed by MTT

colorimetric assay. The expression of cell surface markers CD80, CD86, HLA-DR and HLA-ABC was determined by

flow cytometry. Cell activation was evaluated by production of ROS through the probe DCFH-DA also by flow

cytometry. Results: Cells treated with 20 nM PMA, showed increased cytoplasmic volume accompanied by cell

adhesion and spreading as seen by light microscopy. Analysis by flow cytometry showed an increase in granulosity

and an auto-fluorescence rase of approx. 30 ± 3.1% when compared to undifferentiated cell, however no increase in

CD14 expression on the cell surface was observed. BJcuL at different concentrations did not affect cell morphology,

maintaining the phenotype of the untreated cells. Our results showed that BJcuL has no cytotoxicity against

macrophages. The analysis of cell activation markers showed that BJcuL was able to induce increased expression of

all the markers and that this increase was even higher when the lectin was combined with LPS, except for HLA-DR.

When analyzing ROS production, there was an increase of approximately 10% of the cells previously stimulated with

LPS and BJcuL compared to control. Conclusion: The lectin showed no cytotoxicity and was able to induce activation

of the cells by increased ROS production and also induction of cellular surface molecules responsible for effective

immune response, suggesting its action as immunomodulator.

ANTI-INFLAMMATORY ACTIVITY OF CHENOPODIUMAMBROSIOIDES L. AQUEOUS EXTRACT AFTER

REPLANTED RAT TEETH: A HISTOMORPHOMETRICAL ANALYSIS

FRANCISCO DE ASSIS SOUZA JUNIOR; FRANCISCO NEUREMBERG FERNANDES FILHO; JOAO NILTON

LOPES DE SOUZA; MARIA GORETTI FREIRE DE CARVALHO; REJANE ANDRADE DE CARVALHO; CIRO

DANTAS SOARES; IRENE VALÉRIO DA SILVA; ROSÉLIA DE SOUSA LEAL; TIAGO VICTO ANDRADE DE

CARVALHO; MONISA MARTINS NÓBREGA; MARIA DAS DORES AMORIM MELO.

UNIVERSIDADE POTIGUAR, NATAL - RN - BRASIL.

In Brazilian folk medicine, Chenopodium ambrosioides(Ca) popularly known as “Mastruz”, have gained reputation as

effective antimicrobial and anti-inflammatory agent. This study aimed to evaluate the effect of topical aqueous extract

(CaAE) from Ca 0.5% (v/w) in experimental tooth avulsion (ETA) in rats. Wistar rats were subjected to teeth avulsion

on the upper incisors. Animals were divided in two groups with nine animals each. One treated topically with saline

solution (S) and other treated with Ca Aqueous Extract (CaAE), one hour after ETA induction. The rats were sacrificed

in two time points, on third and 15th day. The periodontum and the surrounding gingivae were examined at

histopathology, as well as the neutrophil counting using optical microscopy method. The replanted teeth were

evaluated on the third and 15th day. The specimens were stained with hematoxylin-eosin and examined by optical

microscopy. The data was subjected to Kruskal-Wallis (p<0.05) and post-test Dunn. The results suggest that the

mastruz aqueous extract was not able to prevent root resorption. However, the aqueous extract was able to stimulated

alveolar bone repair and reduced intensity of polymorphonuclear (PMNs) migration. The treatment with CaAe was

able to prove beneficial effects on periodontal tissues, favoring the replantation process compared to the saline control

group.

ARYL HYDROCARBON RECEPTOR MODULATES PATHOLOGY DEVELOPMENT DURING EXPERIMENTAL

SEVERE MALARIA

FATIMA BRANT1; ALINE S MIRANDA

2; LISIA ESPER

3; ANDREIA BARROSO

4; LUCAS M KANGUSSU

5; CYNTHIA H

VAL6; BRUNO C OLIVEIRA

7; RONAN R S ARAUJO

8; MILENE A RACHID

9; HERBERT B TANOWITZ

10; MAURO M

TEIXEIRA11

; ANTONIO L TEIXEIRA12

; FABIANA S MACHADO13

.

1,2,3,4,5,6,7,8,9,11,12,13.UFMG, BH - MG - BRASIL; 10.ALBERT EINSTEIN COLLEGE OF MEDICINE, NY -

ESTADOS UNIDOS.

Introduction: Malaria is an important cause of death and illness especially in tropical countries. Severe malaria(SM)

is the most important complication of Plasmodium falciparum infection in children and adults. Mice infected with

P.berghei ANKA(PbA) faithfully recapitulate many of the characteristics of human SM and allows the analysis of the

immune response and pathological changes. The disease affect several organs include: brain, liver and spleen.

Although the pathogenesis of malaria is not completely understood, the events that result in the development of SM

are multi-factorial and could not be explained by one mechanism. The Aryl Hydrocarbon receptor(AhR) is an

intracellular receptor activated by several ligands and is important to modulate the inflammatory response. However,

the involvement of AhR in severe malaria is not known.Objective: Investigate the role of AhR in the regulation of

immune response and the pathogenesis malaria. Methods: C57Bl/6 (WT) and AhR-/-

mice were infected with PbA and

the parasitemia, survival and body weight were monitored periodically. The production of cytokines (TNF-α, IL-1β,

TGF-β, IL6, IFN-γ, IL12, IL17 and IL10) in the serum, brain and spleen was assessed by ELISA and flow cytometry.

The expression of SOCS1,2 and 3 was assessed by qPCR. Leukocyte recruitment in the brain was evaluated by

intravital microscopy. Nitric oxide(NO) was assessed by the Griess method in the brain. Histopathology analysis was

also performed in brain and liver. Results: The parasitemia was dramatically higher in PbA-infected AhR-/-

mice when

compared with WT. In the brain and spleen of PbA-infected AhR-/-

mice there was a significant decreased expression

of TNF-α, IL-1β, IFN-γ, IL12 and IL10 when compared with WT counterparts. Additionally, there was an increased

levels of TGF-β, IL6, IL17 and high expression of FOXP3 in the brain of infected AhR-/-

mice when compared with WT

infected mice. In the absence of AhR, there was decrease expression of SOCS3 in brain after infection. AhR

deficiency also resulted in greater inflammation in brain and liver and increased level of alanine aminotransferase

indicates a greater degeneration in liver, as well as lower hematocrit during the infection. Moreover, PbA-infected AhR-

/- mice had increased brain blood barrier permeability. Conclusions: These findings indicate, for the first time, the role

for AhR in the immunopathogenesis of PbA-associated severe malaria.Financial support: CAPES, CNPq, FAPEMIG.

B-1 CELL-DERIVED MONONUCLEAR PHAGOCYTE (B1CDP) FACILITATES LEISHMANIA MAJOR INFECTION

IN VITRO VIA IL-10 AND PGE-2 SECRETION.

A F ARCANJO1; R A COSTA

2; IF LA ROQUE DE FREITAS

3; J D ROCHA

4; M P NUNES

5; A F POPI

6; M MARIANO

7;

C BANDEIRA-DE-MELO8; G A DS REIS

9; D DECOTE-RICARDO

10; C FREIRE-DE-LIMA

11.

1,2,4,5,8,9,11.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL;

3,10.UNIVERSIDADE FEDERAL RURAL DO RIO DE JANEIRO, SEROPEDICA - RJ - BRASIL; 6,7.UNIVERSIDADE

FEDERAL DE SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: B-1 cells are a small fraction of the population of B lymphocytes of the spleen and they represent the

major population of B lymphocytes in the pleural and peritoneal cavity. It has been demonstrated that B-1 cells secrete

IL-10 and thereby they modulate the phagocytic activity of macrophages, as well as suppress the immune system.

This information is based on studies showing that, in mice and human, B-1 cells can generate offspring phagocytic,

exhibiting similar characteristics to those of macrophages. The B-1 cells in primary cultures proliferate and become a

mononuclear phagocyte. These cells were called B-1 cell-derived mononuclear phagocyte (B1CDP). This evidence

motivated us to study the actions of B1CDP cells in infectious and inflammatory processes.

Methods and Results: For this propose we used the model of experimental infection with Leishmania major. Our

data demonstrated that B1CDP are more susceptible to infection by L. major in vitro and this effect is blocked when

added doses of neutralizing anti-IL-10 but not TGF-b. Also, we observed the presence of a large number of lipid

bodies in B1CDP and PGE-2 production. B1CDP cells are defective in IL-10 production in response to COX-2

inhibitors. The IL-10 production was shown to be significantly dependent on the PGE2 production.

Conclusion: These data strongly suggest that B1CDP cells are cellular components that might compromise host

immune responses to infection diseases.

Financial support: CNPq e FAPERJ

BENEFICIAL EFFECTS OF NONI (MORINDA CITRIFOLIA L.) FRUIT JUICE ON DEXTRAN SODIUM SULFATE-

INDUCED COLITIS

ANA LUIZA POVEDA CASSIANO1; BEATRIZ COUTINHO DE SOUSA

2; THIAGO ALVARES COSTA

3; JAVIER

EMILIO LAZO CHICA4; CRISTINA RIBEIRO DE BARROS CARDOSO

5; CARLO JOSE FREIRE OLIVEIRA

6.

1,2,3,4,6.FEDERAL UNIVERSITY OF TRIÂNGULO MINEIRO, UBERABA - MG - BRASIL; 5., SCHOOL OF

PHARMACEUTICAL SCIENCES OF RIBEIRAO PRETO/USP, RIBEIRÃO PRETO - SP - BRASIL.

Introduction: Plants have been used for medicinal purposes for as long as history has been recorded. Among the

diseases in which the use of plants has been indicated, we may call attention to the main forms of inflammatory bowel

diseases (IBD): Crohn's disease and ulcerative colitis. Crohn's disease and Ulcerative colitis are characterized by Th1

and Th17 or Th2 atypical immune response with production of distinct cytokines including IFN-γ, TNF-α and IL-17 or IL-

4 and IL-10, respectively. Products derived from the fruit of the plant Morinda citrifolia L., commonly known as Noni,

has been suggested for the treatment of IBD although works demonstrating its activity or mechanism of action are still

unknown. Thus, we aim to identify if compounds present in the Noni fruit juice have protective and immunomodulatory

effects on Dextran Sodium Sulfate (DSS)-induced colitis. Methods and Results: Colitis was triggered in C57Bl/6 mice

by exposure to DSS (3%) dissolved in water for 9 uninterrupted days. Two groups of mice with DSS-induced colitis

were treated daily with 40 µl of Noni fruit juice i.p., pure or diluted 1:10 in saline. During the 9 days of the experiment

we evaluated the weight, feed and water intake as well as the clinical score. At the ninth day we collect the blood and

colon for assessment of cell count and histopathology. The colon was also collected to assess tissue cytokines TNF-

α, IFN-γ, IL-17, IL-4 and IL-10. We didn’t find any statistical differences in weight, water and feed consumption or

blood cell count in all tested groups. Mice with colitis treated with Noni juice (pure or diluted 1:10), had a significant

improvement in clinical score (p<0.01) and a reduced mortality (5%) when compared with mice that received only DSS

(20%). Both mice treated with Noni juice reduced (p<0.01,) in a dose-dependent manner, the production of Th1 (TNF-

α: reduction of 72.68% and 65.25%; IFN-γ: reduction of 92.28% and 58.96%) and Th17 (IL-17: reduction of 82.76%)

cytokines. IL-4 and IL-10 remained unchanged. Despite the improvement of the score and inhibition of cytokine

production mice treated with Noni juice presented a greater disruption of gut architecture. Conclusion: We show that

Noni fruit juice has a protective role on the development of colitis in an experimental murine model. Our results also

demonstrate the immunomodulatory properties of Noni juice and may justify its usage in traditional medicine.

Financial support: FAPEMIG, UFTM, CNPq, CAPES.

BETA2 ADRENERGIC RECEPTOR SIGNALING IN CD4+ FOXP3+ REGULATORY T CELLS ENHANCES THEIR

SUPPRESSIVE FUNCTION AND ITREG GENERATION IN VITRO

MARCIA GRANDO GUERESCHI; LEANDRO PIRES ARAÚJO; JULIANA TERZI MARICATO; MAISA CARLA

TAKENAKA; VANESSA DE MENDONÇA NASCIMENTO; BRUNO CAMOLESE VIVANCO; VANESSA OLIVEIRA

REIS; ALEXANDRE DE CASTRO KELLER; ALEXANDRE SALGADO BASSO.

IMMUNOLOGY DIVISION, DEPARTMENT OF MICROBIOLOGY, IMMUNOLOGY AND PARASITOLOGY -

UNIFESP, SÃO PAULO - SP - BRASIL.

Considering modulation of CD4+ T cell responses, beta2 adrenergic receptor (B2AR) signaling is known to impair Th1

differentiation and function in a cAMP-dependent way, leading to inhibition of cell proliferation and decreased

production of IL-2 and IFN-g. Foxp3+ CD4+ regulatory T (Treg) cells play a key role in the regulation of immune

responses and are essential for maintenance of self-tolerance. Nevertheless, very little is known on adrenergic

receptor expression in Treg cells or either on the influence of noradrenaline on their function. Objective: The aim of

this study was to investigate the effect of B2AR stimulation on Treg function. Methods and Results: Treg and CD4+

CD62L+ GFP- naïve T cells from Foxp3gfp

mice were sorted in a FACSAria II. We first confirmed B2AR expression in

Treg cells by qPCR, western blot and immunofluorescence. We found that B2AR activation by specific agonist in Treg

cells leads to increased intracellular cAMP levels and to PKA-dependent CREB phosphorylation. For suppression

assays, sorted naïve T cells and Treg cells were cultured in a proportion of 1:0,25 with irradiated splenocytes and

soluble anti-CD3. Before setting up the cultures, Treg cells were treated or not with B2AR agonist, or with B2AR

agonist plus B2AR antagonist. After 3 days, naïve T cell proliferation was analyzed by proliferation dye dilution in a

flow cytometer and quantified using FlowJo. We found that B2AR agonist was able to increase suppressive activity of

Treg cells since naïve T cell proliferation was about 40% lower than the one observed in control cultures. Pre-

treatment with B2AR antagonist reverted B2AR agonist-mediated increase in Treg suppression. We also found

increased CTLA-4 expression in Treg cells that were pre-treated with B2AR agonist. Moreover, Treg cells treated with

B2AR agonist were able to induce more iTreg cells from naïve T cells than non-treated Treg cells. Since cAMP

activates PKA, we investigated if B2AR agonist-mediated increase in Treg suppression and CTLA-4 expression could

be reverted by inhibiting PKA. We found that pre-treatment with PKA inhibitor prevented the effect of the B2AR

agonist. Conclusion: Our data suggest that sympathetic fibers could be able to regulate Treg suppressive activity in a

positive manner through B2AR signaling. This increased Treg suppressive function is dependent on the cAMP-PKA

pathway.

Financial support: FAPESP, CNPq

BONE MARROW SOLUBLE HLA-G AND CYTOKINE PROFILES IN CHILDHOOD ACUTE MYELOID LEUKEMIA

NORMA LUCENA-SILVA1; ROSSANA SANTOS

2; RENAN GARCIA GOMES

3; ALESSANDRA MARIA LUNA

RAMOS4; ESTER VINHAS

5; TERESA FONSECA

6; FRANCISCO PEDROSA

7; EDUARDO ANTÔNIO DONADI

8.

1,2,3.AGGEU MAGALHÃES RESEARCH CENTER/FIOCRUZ, RECIFE - PE - BRASIL; 4,5,6,7.PEDIATRIC

ONCOLOGY OF THE IMIP HOSPITAL, RECIFE - PE - BRASIL; 8.FACULTY OF MEDICINE OF THE UNIVERSITY

OF SÃO PAULO, RIBEIRÃO PRETO - SP - BRASIL.

Introduction: HLA-G proteins bind to several inhibitory receptors on the surface of NK, CD4+ and CD8+ T cells,

inhibiting their lytic activity and the proliferation response. Soluble HLA-G (sHLA-G) stimulates the release of IL-4 and

IL-10, inducing Th2 cytokine profile that in turn up-regulates HLA-G expression at the tumor site. Little is known about

the level of sHLA-G in bone marrow of healthy or leukemia children. We aimed to determine the level of sHLA-G and

the cytokine profile in the bone marrow of childhood myeloid leukemia.

Methods and Results: The level of sHLA-G was determined by ELISA in bone marrow of 34 children with myeloid

leukemia, and 10 without hematological disease. Cytokines were measured by flow cytometer using Th1/Th2/Th17

CBA kit (Becton-Dickinson). Normal bone marrow presented an average of 195 +/- 24 pg/uL of sHLA-G, whereas in

the majority of sick children (24) the levels were low, but normal and higher levels were still found in five cases each.

Three cytokine profiles were observed: 15 cases with TNF levels lower than the median obtained from the normal

bone marrow associated with low level of others cytokines; and 19 cases of high expression of TNF with low or no

expression of others cytokines (8 cases) or associated with high expression of IL-4 and IL-2, independent of being IL-

10 positive or not (11 cases). The five cases presenting high sHLA-G expression overexpressed also TNF, IL-4, IL-2

and IL-10, except one case of secondary LMA that presented low level of IL-10. Expression levels of sHLA-G and

cytokines were not associated with cell phenotype, presence of mutation, leucocyte counts or other clinic or laboratory

variable.

Conclusion: The level of sHLA-G expression in leukemic environmental is highly variable in relation to the normal

bone marrow. Further experiments should be done to confirm whether the highly expressed HLA-G levels modulate

the immune response at bone marrow in myeloid leukemia.

Financial Support: CNPq #150572/2011-5, BD do Brasil (Pesquisador SBI2012-BD Prize) and Fiocruz.

BRADYKININ B1 RECEPTOR MEDIATES CISPLATIN-INDUCED NEPHROTOXICITY

ANA PAULA F S MONTEIRO1; VANESSA O REIS

2; NATHÁLIA A KHALED

3; DAVID A G ANDRADE

4; NIELS O S

CAMARA5; ALEXANDRE CASTRO KELLER

6.

1,2,3,4,6.UNIFESP, SÃO PAULO - SP - BRASIL; 5.USP, SÃO PAULO - SP - BRASIL.

Abstract: Cisplatin is an effective chemotherapeutic agent commonly used for treatment for a wide range of tumors. A

major side effect of cisplatin is nephrotoxicity, leading to acute renal failure (ARF). Cisplatin activates multiple

signaling pathways, resulting in necrosis and apoptosis of renal tubular cells. Because Bradykinin 1 receptor (B1R)

has been implicated in the progress of chronic kidney disease, we hypothesized that a similar pathway could be

involved in the development of acute kidney injury (AKI). Thus, we took advantage of the pharmacologic modulation of

B1R to determine its implication in cisplatin nephrotoxicity. Objective: To study the involvement of B1R in the cisplatin-

induced AKI. Methodology: AKI was induced by a single peritoneal injection of cisplatin (20mg/Kg) in C57Bl/6 mice. In

order to block B1R signaling, animals received DALBK (10mg/Kg) for three consecutive days. The renal function was

determined by the proteinuria/creatininuria ratio, and q-PCR analysis was performed to establish the transcription of

pro-inflammatory genes, as IFN- and TNF-α. Results: The treatment with DALBK improved the renal function in

comparison to cisplatin non-treated mice, indicating that blockage of B1R signaling protects renal tissue against

cisplatin cytotoxicity. In concordance, histological analysis revealed that DALBK treatment inhibited cisplatin-mediated

tubular cell death. In addition, DALBK injection decreased the transcription of IFN- and TNF-α genes. Conclusion:

Ours data supports the hypothesis that bradykinin and B1R receptor influence the cisplatin nephrotoxicity and suggest

that BR1 antagonists can be used to avoid kidney injury during chemotherapy. Financial Support: CNPq

BUTYRATE IMPROVES ATHEROSCLEROSIS BY REDUCING INFLAMMATION, FOAM CELL FORMATION AND

INCREASING FIBROUS CAP STABILITY.

EDENIL COSTA AGUILAR1; ALDA JUSCELINE LEONEL

2; ANALINA RAQUEL DA SILVA

3; JOSIANE FERNANDES

DA SILVA4; JULIANA MARIA NAVIA PELAEZ

5; LUCIANO DOS SANTOS AGGUM CAPETTINE

6; LÍLIAN

GONÇAVES TEIXEIRA7; ROBSON AUGUSTO SOUZA DOS SANTOS

8; JACQUELINE ISAURA ALVAREZ-LEITE

9.

1,2,3,4,5,6,8,9.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL;

7.UNIVERSIDADE FEDERAL DA GRANDE DOURADOS, DOURADOS - MS - BRASIL.

Introduction: Inflammation is an important component of atherosclerosis. Since butyrate (BUT) presents well-

established anti-inflammatory properties, we studied the effects of butyrate on atherogenesis.

Methods and Results: ApoE-deficient mice were fed on control (CT group, n=8) or butyrate-supplemented (1%; BUT

group, n=8) diets for 10 weeks. Atherosclerosis lesion area was evaluated on aorta and aortic valve. The presence of

macrophages, collagen, CCL2, VCAM-1 and matrix metalloproteinase-2 (MMP-2) in aortic valve were evaluated by

immunofluorescence. Results showed that BUT supplementation reduced lesion area in aorta (CT=4.1±0.6%,

BUT=1.8±0.5% of total area), but not in aortic valve (CT=3.6±6.7 and BUT=4.1±1.2 x105 µm

2). However, percentage

of macrophage migration (foam cells) was lower in BUT (0.22±0.04 FI) compared to CT group (0.05±0.02; FI).

Nonetheless, collagen content was higher in BUT (0.4±0.1; FI) than in CT (0.2±0.01-FI) group. CCL2 (CT=0.3±0.1,

BUT=1.2±0.03; FI) and VCAM-1 (CT=0.4±0.1, BUT=0.13±0.03; FI) as well as MMP2 (CT=0.5±0.1, BUT=0.13±0.03;

FI) were also reduced in BUT group. This result suggests that BUT reduces macrophage migration and induces

fibrous cap stabilization. To confirm those data, cultures of human endothelial cells (E.A. Hy926) were pre-treated

without (CT) or with BUT (0.5 mM) for 2h and stimulated with oxidized LDL (oxLDL) for 4h. Data of ELISA and western

blot showed that BUT pre-treatment reduced TNF (CT=17.9±1.2, BUT=11.4±0.6 pg/mL) and IL-1β (CT=72.5±2.2,

BUT=54.2±2.5 pg/mL) secretions (ELISA) associated to reductions on p65 NF-kB production (CT=1.2±0.2,

BUT=0.5±0.1; p65/βactin). Furthermore, it was demonstrated by immunofluorescence that the translocation of p65

into the nucleus was also reduced in cells pre-treated with butyrate (CT=3.0±0.1 and BUT=2.6±0.1 x105 AU).

Conclusion: Butyrate supplementation improves atherogenesis mainly due to anti-inflammatory properties associated

to NF-kB reduction.

SUPPORTING: FAPEMIG (APQ-# 01062-10) and CNPq.

COMBINATION OF LOW LEVEL LASER (LLL) AND HUMAN TUBAL MESENCHYMAL STEM CELLS (HTMSCS)

AMELIORATES CIGARETTE-INDUCED LUNG INFLAMMATION IN MICE.

ANA PAULA PAULA LIGEIROD DE OLIVEIRA1; VANESSA ROZA DA SILVA

2; JEAN PIERRE SCHATZMANN

PERON3; MAYRA PELATTI

4; CARLOS EDUARDO CZERESNIA

5; PAULO PERIN

6; MARIANGELA MALUF

7; LUCILA

EVANGELISTA8; MARCELO GIL NISENBAUM

9; SILVIO HALPERN

10; MAYANA ZATZ

11.

1,2.PROGRAMA DE PÓS-GRADUAÇÃO EM BIOFOTÔNICA APLICADA À CIÊNCIAS DA SAÚDE, UNIVERSIDADE

NOVE DE JULHO –, SAO PAULO - SP - BRASIL; 3.NEURO-IMMUNE INTARACTIONS LABORATORY,

UNIVERSITY OF SÃO PAULO, SÃO PAULO – BRAZIL., SÃO PAULO - SP - BRASIL; 4,11.DIVISION OF HUMAN

GENOME RESEARCH CENTER, UNIVERSITY OF SÃO PAULO - SÃO PAULO- BRAZIL., SAO PAULO - SP -

BRASIL; 5,8,9,10.DIVISION OF REPRODUCTIVE MEDICINE, CÉLULA MATER - SÃO PAULO, SP – BRAZIL., SAO

PAULO - SP - BRASIL; 6,7.DIVISION OF REPRODUCTIVE MEDICINE – SPECIALIZED CENTER FOR HUMAN

REPRODUCTION – CEERH - SÃO PAULO, S, SAO PAULO - SP - BRASIL.

Introduction: Chronic Obstructive Pulmonary Disease (COPD) is poorly managed with currently available therapies.

In this context, new therapeutic approaches are of great interest, and the use of Mesenchymal Stem Cells (MSCs) or

low level laser therapy (LLL) may be innovative and promising approaches. Thus we investigated the effect of LLL

(670 nm) and human tubal-derived MSCs (htMSCs) in the treatment of cigarette-induced pulmonary inflammation in

mice. Methods and Results: To induce COPD C57Bl/6 mice were submitted to cigarette smoke for 75 days (2

times/day). Next, animals were treated with LLL (COPD+LLL group), htMSCs intraperitoneal route (COPD+htMSCs

group) 7 and 15 days before the experiment or both (COPD+LLL+htMSCs group), on day 76th when mice were

sacrified for morphologic and functional analysis of the lung. Bronchoalveolar lavage (BAL) analysis showed that

COPD+LLL, COPD+htMSCs and COPD+LLL+htMSCs groups had a significant decrease in neutrophils, macrophages

and lymphocytes when compared to COPD group. This was associated with reduced mucous secretion, collagen

deposition and alveolar enlargement. Moreover, LLL pus htMSC therapies significantly reduced IL-6 and TNF-a in

BAL which was not observed after LLL or htMSCs alone. Conclusions: Our results indicate that the treatment with

LLL and htMSC greatly reduces lung inflammation (as demonstrated through BAL cell counting and

histomorphometric analysis in lung parenchyma) and pro-inflammatory cytokines release (BAL IL-1β and IL-6).

Furthermore, these therapies also reduced airway mucus secretion and collagen deposition, beyond to promote re-

alveolization of lung parenchyma. Thus, the association of both therapies improve the main features of COPD,

becoming a potential therapeutic strategy for lung disease treatment.

Financial Support: UNINOVE, FAPESP, INCT,CNPq.

CONJUGATED LINOLEIC ACID AMELIORATES COLITIS

THAIS GARCIA MOREIRA1; ANA CRISTINA GOMES-SANTOS

2; LAILA SAMPAIO HORTA

3; BERNARDO COELHO

HORTA4; ARCHIMEDES CASTRO

5; NINA QUEIROZ

6; JOANA FERREIRA AMARAL

7; JULIANA LAUAR

8; ANA

MARIA CAETANO FARIA9.

1,2,4,5,6,7,8,9.UFMG, BELO HORIZONTE - MG - BRASIL; 3.PUC MINAS, BELO HORIZONTE - MG - BRASIL.

Moreira, T.G1; Gomes-Santos, A.C

2; Horta, L.S

2 ; Horta, B.C

2; Castro, A.B

2; Queiroz, N.M.G.P

2; Lauar, J.G

2; Amaral,

J.F2; Faria, A.M.C.

2

1- Departamento de Alimentos, FAFAR; 2- Departamento de Bioquímica e Imunologia,ICB,UFMG Brasil.

Introduction: Inflammatory bowel diseases (IBD) include ulcerative colitis and Crohn's disease, and they are

characterized by an intense inflammatory response in the gastrointestinal tract.

There is strong evidence that IBD result, in part, from an imbalance between inflammatory and regulatory immune

responses to the commensal bacterial microbiota. Among the proposed therapeutic and preventive treatments for IBD,

is conjugated linoleic acid (CLA), which consists of a mixture of short-chain fatty acid isomers. Several actions linked

to human health have been described as associated with CLA administration. Studies with in vitro cultured lymphoid

cells and with animal models have shown that CLA can modulate immune function and inflammatory responses.

Objetive: This study aims to test the effect of 1% CLA (50:50 isomers) administration as dietary supplementation for 4

weeks in mice with ulcerative colitis induced by a 7-day treatment with 1.5% dextran sodium sulphate

(DSS). Results: Supplementation of CLA in the diet before the induction of colitis decreased both mucosal damage

at colonic mucosa and weight loss. Inflammatory alterations such as increased levels of IL-4 and decreased levels of

IL-10 in colonic mucosa of DSS-treated mice were prevented by CLA supplementation. CLA-fed animals had also

lower levels of IFN-γ in mesenteric lymph nodes, and of IL-17 and MCP-1 in colonic mucosa. Serum and secretory IgA

levels that were reduced by colitis were restored by CLA treatment. Activation of peroxisome proliferator-activated

receptor (PPAR) by CLA supplementation on macrophages may be one important pathway involved in the

mechanism. Conclusion: CLA mediated protection against experimental colitis and might represent a novel

therapeutic tool for IBD.

Financial support: FAPEMIG, CNPq and CAPES, Brazil.

CROTOXIN FROM CROTALUS DURISSUS TERRIFICUS MODULATES THE MACROPHAGE AND LYMPHOCYTE

POPULATIONS IN ACUTE INTESTINAL INFLAMMATION IN MICE

CAROLINE ALMEIDA1; JACQUELINE JACYSYN

2; CRISTIANO DE MORAES

3; ELIANA FAQUIM

4.

1,4.BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL; 2,3.SÃO PAULO UNIVERSITY, SÃO PAULO - SP -

BRASIL.

Introduction: Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases (IBDs) possible due to

an abnormal activation of the immune response against constituents of the luminal flora. Infiltrated lymphocytes and

macrophages have a key role in exacerbation of pathogenesis on inflammatory bowel disease. Crohn’s disease is

classically regarded as a Th1 mediated inflammatory disorder in lamina propria. Recently, it was verified that Th17

cells are also involved in this inflammatory intestinal response. Macrophages, including ‘classical’ (M1) and

‘alternatively’ activated (M2) have been studied as a target for the treatment of inflammatory bowel diseases such as

Crohn’s disease. Crotoxin (CTX) is the main component of the Crotalus durissus terrificus rattlesnake venom and it

has immunosuppressive effect. Here, we evaluated the modulatory effect of CTX on the murine model of colitis

induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS).

Methods and Results: Male BALB/c mice (n=20) were anesthetized and 0.1 mL of TNBS in 45% ethanol was

administered by intrarretal route to induce acute colitis. The control mice received only 0.1mL of 45% ethanol. After 18

h, the mice received the CTX or PBS ip and 4 days after induction they were sacrificed. Entire colon was quickly

removed and gently cleared of feces for measurement of myeloperoxidase (MPO) enzyme activity and cytokines

secretion by ELISA. Macrophages and lymphocytes were isolated from lamina propria for flow cytometry analysis.

Clinical and histological scores showed that the CTX-administration decreased the disease progression in TNBS-

colitis induced mice. MPO activity was significantly higher in the TNBS-group than in TNBS group that received CTX

which indicates a reduction of the index of tissue inflammation by the toxin administration. High IL-17, IL-6, IL-1, IFN-g

and TNF-a secretion was verified in cell supernatants from TNBS-mice. In contrast, these cytokines secretion was

lower in mice that received the CTX. Higher percentages of CD11b+CD68

+ (M1), CD4

+RORg

+ and CD4

+Tbet

+ cells

were observed in lamina propria of TNBS-colitis mice, however when CTX was administered in TNBS-group these cell

populations were decreased as observed in ethanol control groups. Conclusion: Our preliminary results suggest a

modulatory role of CTX in the macrophages and lymphocytes differentiation in acute intestinal inflammation induced

by TNBS in mice.

Financial Support: FAPESP 2010/05701-9 and CNPq.

DEVELOPMENT OF PEPTIDE MIMETICS INTERLEUKIN 10 (IL-10) TARGETED THERAPEUTIC

EMÍLIA REZENDE VAZ; LARISSA PRADO MAIA; PATRÍCIA TIEMI FUJIMURA; LUIZ RICARDO GOULART;

CARLOS UEIRA-VIEIRA.

FEDERAL UNIVERSITY OF UBERLANDIA, UBERLÂNDIA - MG - BRASIL.

Introduction: IL-10 is é expressed by various immune cells including T and B-lymphocytes and macrophages. The

immunosuppressive effects of this interleukin include the inhibition of T lymphocytes cytokines secretion, inhibition of

antigen presentation, inhibition of co-stimulatory molecules expression on antigen-presenting cells. Moreover, it is

important role in controlling inflammation in target tissues, since, IL-10 acts like antagonist of IFN-γ. Due to the

important regulatory role in the immune system, the deregulation of the gene encoding IL-10 is a frequent

characteristic in autoimmune diseases such as rheumatoid arthritis and lupus erythematosus.

Methods and Results: This research aimed to select bioactive peptides agonists and antagonists of the IL-

10receptor(IL-10R) by Phage Display. The methodology was performed by incubation of a commercial library of

random peptides Ph-D.12 (NEW ENGLAND BioLabs®Inc.) with a macrophage J774-1 cells. The specific peptides

binding in IL-10Rwere eluted by competition with recombinant human IL-10 and selected peptides were submitted to

enzyme immunoassay (ELISA) on macrophages. Subsequently, we used flow cytometry method to confirm the

binding of selected peptides in peripheral blood mononuclear cells (PBMC).

Conclusion: Our results showed that the 60 peptides tested, two were able to bind to receptors present on

macrophages as well in PBMC.The both compounds from innate immune and adaptive responses must be considered

as potential targets for developing new drugs immune modulators. Thus, the manipulation of regulatory cytokines like

IL-10 is an attractive strategy for immunotherapy and, the use of peptide mimetics IL-10 can be adopted to decrease

the consequences of severe response autologous, creating a complementary therapy for autoimmunity.

Financial support: FAPEMIG, CNPq, CAPES, UFU.

EARLY CHANGES ON CARDIOVASCULAR FUNCTION OF ANALBUMINEMIC RATS CHALLENGED BY

LIPOPOLYSACCHARIDES

HERMES VIEIRA BARBEIRO1; CLARA BATISTA LORIGADOS

2; SERGIO CATANOZI

3; DENISE FREDIANI

BARBEIRO4; EDNA R NAKANDAKARE

5; ANTONIO DOS SANTOS FILHO

6; FERNANDA B FUSCO

7; FRANCISCO

GARCIA SORIANO8.

1,2,4,6,8.UNIVERSITY OF SÃO PAULO -LIM 51, SÃO PAULO - SP - BRASIL; 3,5,7.UNIVERSITY OF SÃO PAULO

LIM-10, SÃO PAULO - SP - BRASIL.

Introduction: Albumin is the major plasma protein responsible for maintaining the trans-endothelial oncotic pressure

gradient and regulating the transport of fatty acids, hormone and amino acids (Mani AR et al Biochemica Parmacology

84, 2012, 1062-69). This work came with intention to evaluate cardiovascular function in analbuminemics rats

challenged by lipopolysaccharides (LPS).Methods and results: Adult (300–350g) male rats Sprague-Dawley (SD)

and Nagase Analbuminemic Rats (NAR) were studied (minimum of 6 animals per group): both groups received saline

i.p. (SD and NAR) or LPS (E coli serotype 026:B6) 10 mg/kg, i.p. (SD+LPS and NAR+LPS). Animals were sacrificed

4h after injection. Intact thoracic aortic ring (5-6 mm) of each animal was kept in organ baths (37°C - 95% O2, 5%

CO2) with Krebs solution. Cardiovascular function was performed by catheter in left ventricule (LV- dP/dT max) and

carotid (Mean Arterial Pressure - MAP). Pulmonar cytokines were measured by ELISA. After LPS stimuli was

observed enhanced quantification of IL-10, TNF-alpha and IL-6 in SD+LPS (273±0,2; 69,6±31,6; 23860±3078

respectively) when compared with SD group (2,7±0,1; 3,6±0,2; 26,1±2,6 respectively). IL-6 on NAR+LPS group was

enhanced (15096±3274) when compared with NAR group (28,7±1,3). Despite the lower cytokines levels on NAR, this

group showed a higher mortality (65%) than SD (20%). Greater vasoconstrictor response to noradrenaline (NE) was

observed on NAR (4,3±0,4) under basal conditions when compared with SD (2,29±0,29) which is not maintained

after LPS injection. On the other hand MAP of NAR+LPS (64,14±1,7) was decreased when compared with NAR

(80,66±4,64). Cardiac dysfunction was observed between SD (6359±604) and NAR (4693±326) at baseline, after LPS

injection group SD increases dP / dT max (8394±299) and group NAR decreases (3059±222), so the NAR group has

already had a lower systolic pressure wich got worse after LPS injection, acting diversely to the SD group which, after

LPS, increases systolic pressure. Conclusion: Prevalence of higher response to NE and high heart contraction

(dP/dT max) on physiological conditions are essential to NAR survival and the difference observed after LPS injection

in systolic pressure, reflects in a systemic pressure (MAP) decrease. It indicates the possible cause of early mortality

observed in analbuminemics animals challenged by LPS.

EFFECT OF ADIPOSE TISSUE-DERIVED STEM CELL TREATMENT IN AN ANIMAL MODEL OF TUBULE-

INTERSTITIAL NEPHRITIS

CASSIANO DONIZETTI OLIVEIRA1; DANILO CÂNDIDO ALMEIDA

2; CRISTHIANE FAVERO AGUIAR

3; MARCOS

ANTÔNIO CENEDEZE4; ALVARO PACHECO SILVA-FILHO

5; NIELS OLSEN SARAIVA CÂMARA

6.

1,2,4,5.FEDERAL UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 3,6.UNIVERSITY OF SÃO PAULO,

SÃO PAULO - SP - BRASIL.

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are

similar to those of bone marrow stem cells. Tubule-interstitial nephritis (NTI) can be defined by inflammatory

involvement of the renal interstitium affecting tubules, with interstitial edema and acute tubular injury. If the offending

agent persists there are the formation of interstitial fibrosis and tubular atrophy. Studies in animals show that intake of

adenine mimics the long-term damage of NTI. Thus, in this study we intend to evaluate the protective role of ASC in

an animal model of tubule-interstitial nephritis induced by adenine. The animals were divided into three groups

wherein two groups received a diet containing 0.25% adenine for 10 days: sham group (n=5), which no received the

diet; adenine group (n=6), which received only the diet and adenine + ASC group (n=6) that received the diet plus

administration of ASC. After 10 days, the animals were sacrificed and kidney fragments and blood were collected for

analysis. The group of animals treated with ASC showed better renal function compared with the untreated group, with

lower levels of creatinine (0,95±0,14 vs 1,15±0,19) and urea (137,3±42,2 vs 170,5±38,3). Flow cytometric show us a

minor inflammatory cell infiltration in animals treated with ASC (37,53±1,52 vs 27,43±3,8). The decrease of the

inflammation may also be seen by high renal expression of TGF-β by ELISA (520±92,3 vs 1252,43±130). Following

this, we also found less renal fibrosis in adenine + ASC group by analysis of picrosirius staining and

immunohistochemical analysis of collagen type I and IV. So, we conclude that treatment with ASC in NTI model can

promote functional improvement as well as prevent the formation of tubule-interstitial fibrosis. Fapesp 2011/22568-3.

EFFECT OF AEDES AEGYPTI’S SALIVARY GLAND EXTRACT IN PHENOTHYPIC MODULATION OF

MACROPHAGES

DANIEL OLIVEIRA PATRICIO1; ANDERSON SA-NUNES

2; DANIEL SANTOS MANSUR

3.

1,3.UNIVERSIDADE FEDERAL DE SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL; 2.UNIVERSIDADE DE

SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: Dengue virus (DENV) is a member of the Flaviviridae family that is transmitted among humans mostly by

infected Aedes aegypti’s saliva released during blood feeding. Systemic dissemination of virus occurs by infecting

monocytes, macrophages and dendritic cells (J Infect Dis. 189:1411-18, 2004; Nat Rev Immunol. 11:532-43, 2011). It

is known that Dengue virus’ E protein can interact with mannose receptors (MR) on the cell surface, facilitating virus

entry in host cells (Plos Pathog. 4:e17, 2008). Alternatively activated macrophages (M2) display high mannose

receptor (MR) expression; therefore, our hypothesis is that Aedes aegypti’s saliva is a macrophage alternative

polarization mediator, which assists DENV during early infection.

Methods and Results: Using Real Time PCR we show that Aedes aegypti Salivary Gland Extract (SGE) increases MR

expression in human mononuclear cells and inhibit IL-10 and IL-12/IL-23p40 expression in activated cells treated with

IFN-γ and LPS. Also, SGE and DENV do not induce IL-12/IL-23p40 release from human mononuclear cells as shown

by ELISA.

Conclusion: SGE is able to modulate human mononuclear cells phenotype and cytokine response, which led us to

speculate that alternative activation of macrophages may take place during A. aegypti blood feeding.

Financial support: CNPq.

EFFECT OF CHLOROQUINE ON TH1 AND TH17 CITOKINES IN HEALTHY VOLUNTEERS

MARDONNY BRUNO DE OLIVEIRA CHAGAS; JULIANA CRUZ DA SILVA; HENRIQUE ATAIDE MARIZ; LAURINDO

FERREIRA DA ROCHA JUNIOR; PRISCILLA STELA SANTANA DE OLIVEIRA; ANDREA TAVARES DANTAS;

ANGELA LUZIA BRANCO PINTO DUARTE; IVAN DA ROCHA PITTA; SUELY LINS GALDINO; MAIRA GALDINO DA

ROCHA PITTA.

UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL.

Introduction: Chloroquine (CQ) is an antimalarial agent that has been used for many years in inflammatory diseases

treatments. Cytokines characteristics of lymphocyte subsets Th1 (IFN-γ) and Th17 (IL-6, IL-17, IL-22) play an

important role in immune response. In this context, the aim of this study is to evaluate the immunomodulatory activity

of CQ on Th1 and Th17 cytokines secretion in healthy volunteers. Methods and Results: For this study, peripheral

blood mononuclear cells (PBMCs) from healthy volunteers (n=5) were obtained and purified. The PBMCs (1x106

cells/ml) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. The cells were stimulated, in vitro,

with PMA+Ionomycin (PMA/Iono) in the presence or absence of different concentrations of CQ (25, 50, and 100 µM)

and incubated at 37°C in a humidified 5% CO2 incubator for 48h. Methylprednisolone (MP) (5µM) was used as

positive control. The levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA) of the

culture supernatants. Statistical significance was assessed by t-test paired (p<0.05). It was found that CQ100µM

induced significantly lower levels of IL-6 (p=0.0122), IL-17 (p=0.0388) and IFN-γ (p=0.0074) in comparison with

(PMA/Iono). Chloroquine induced the decrease in levels of IL-22 at all concentrations, but was not significant (p>0.05).

Conclusion: Our in vitro results demonstrated that chloroquine inhibits IL-6, IL-17 and IFN-γ production and

contributes to a better understanding of the mechanism of action of this drug. Financial support: CAPES; INCT-if;

FACEPE

EFFECT OF FLAVONOIDS RUTIN AND QUERCETIN ON ACTIVATION OF MACROPHAGES/MICROGLIAL

CELLS

ALESSANDRA BISPO DA SILVA; KARINA COSTA DA SILVA; MONA NEVES DAS OLIVEIRA; RAMON SANTOS

DOS EL-BACHÁ; MARIA DE FATIMA DIAS COSTA; SILVIA LIMA COSTA.

UFBA, SALVADOR - BA - BRASIL.

Introduction: Microglial cells have the potential to develop into full-blown macrophages but their morphological and

functional spectrum is highly regulated in vivo resulting in various intermediate states of activation. Adapted to their

CNS environment, the functions of microglia differ from those of macrophages in other organs.(Acta Neuropathol

119:89–105,2010.) Our previous study demonstrated that rutin, a flavonoid extracted from seeds of the Brazilian plant

Dimorphandra mollis, act as modulator of imumodulatory agents (TNFα and NO) in glial cells cultures. In this study we

investigated the effects of flavonoides rutin and its derivative aglicone quercetin on stimulation of cortical rat microglial

cells.

Methods and Results: Effetcs of flavonoids (50µM) on viability and proliferation of microglial cells derived from the

cortex of newborn rats were tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;

Sigma, St Louis, MO) after 24 h exposure. When compared with control (DMSO 0.1%) treatment with flavonoids

induced significant increase (p<0.05) on mitochondrial function, indicating an increase on cellular metabolic activity

and proliferation. On the other hand exposure to LPS/INF didn’t altered mitochondrial function. Moreover, flavonoids

(50µM) also induced chemotaxis of microglial cells after 24 h exposure in a model of transwell insert and counting

after 0.2% crystal violet staining.

Conclusions: These findings show that the flavonoids tested are capable of stimulates microglia cells altering its

metabolism and migration.

Supported by: CNPq, CAPES and FAPESB.

EFFECT OF PURIFIED FRACTION OF AGARICUS BLAZEI MURRILL ON EXPERIMENTAL SEVERE MALARIA

DEVELOPMENT

CYNTHIA HONORATO VAL1; FÁTIMA CALDEIRA BRANT

2; FLÁVIA RODRIGUES

3; ALINE SILVA MIRANDA

4;

BRUNO DE OLIVEIRA CABRAL5; ELÂNDIA DOS SANTOS

6; BRUNO COSTA

7; MILENE ALVARENGA RACHID

8;

DIEGO RODNEY9; WILIAM CÉSAR BENTO RÉGIS

10; FABIANA SIMÃO MACHADO

11.

1,2,3,4,5,6,7,8,9,11.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL;

10.PONTIFÍCIA UNIVERSIDADE CATÓLICA, BELO HORIZONTE - MG - BRASIL.

Introduction: The Agaricus blazei Murrill (AbM) is a Brazilian originated mushroom, and it is being used as a drug in

alternative medicine and functional food, to possess several bioactive compounds, mainly immunomodulatory,

antioxidants and antiparasitic, associated in preventing various diseases, such as inflammatory, allergy, cancer,

diabetes, cholesterol and parasitic disease. Malaria is a parasitic disease caused by Plasmodium species reaching

104 countries, affecting approximately 219 million people and leading to deaths of 660,000. The species more

pathogenic and deathly is the P. falciparum of which may lead the severe malaria, whose the major complication of

infection in human is the development of cerebral malaria (CM). CM has been exacerbated by the emergence of drug-

resistant parasites, faults in access to treatment, complex therapeutic, and that do not prevent the development of

neuronal damage. Therefore, it looks for complementary therapies aimed at rescuing patients of death and/or

cognitive impairments triggered by disease report pressing. Here, we investigated the effect of crude extract of AbM

and its purified fraction, fraction C, in modulation of immune response and development of CM in mice infected with

Plasmodium berghei ANKA (PbA), parasite strain faithfully recapitulate many of the characteristics of human CM.

Methods and Results: C57BL/6 mice were pretreated (3 days) with extract or fraction C of AbM and then infected

with 105 parasited red blood cells (pRBCs), followed by treatment with AbM or chloroquine (antimalarial), and the

parasitemia, survival, body weight, development of CM and immune response were evaluated. Mice treated with

crude extract or fraction C of AbM demonstrated lower parasitemia, longer survival, reduced weight loss and

protection against CM development. There was also a reduction pro- and anti-inflammatory cytokines production,

when compared with untreated PbA-infected mice. In addition, the pre-treatment of pRBCs with fraction C of AbM in

vitro following to infection in vivo, resulted in lower parasitemia, longer survival, and protection against CM compared

with the crude extract and even with untreated PbA-infected mice. Conclusion: These findings indicate that fraction C

of AbM has an important protective role in the development of experimental CM and modulation of immune response

during PbA infection presenting a good therapeutic option for severe malaria. Financial support: CNPq and

FAPEMIG.

EFFECTS OF ACETYLSALICYLIC ACID DURING TRYPANOSOMA CRUZI EXPERIMENTAL INFECTION: ROLE

OF AHR/SOCS2 MODULATORY PATHWAY

ISABELA DE AVELLAR COSTA; JÚLIA TEIXEIRA DE CASTRO; RICARDO MANOEL OLIVEIRA RODRIGUES;

RONAN RICARDO SABINO ARAÚJO; FÁTIMA BRANT; ANDRÉIA BARROSO; BRUNO CABRAL DE LIMA

OLIVEIRA; POLLYANA MARIA DE OLIVEIRA PIMENTEL; LÍSIA ESPER; FABIANA S. MACHADO.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Chagas’ disease is caused by the protozoan Trypanosoma cruzi (Tc), approximately, 40% of the cases

results in chagasic cardiomyopathy. Production of inflammatory mediators is essential for infection control. However,

an unbalanced inflammatory response could result in development of pathology. Our recent studies demonstrated that

Suppressor of cytokine signaling 2 (SOCS2) modulates immune response, cardiomyocytes and heart function during

Tc infection. Moreover, the Aryl hydrocarbon Receptor (AhR) was related in the modulation of SOCS2 expression in

dentric cells (DC) stimulated with Lipoxin (LXA) in vitro and upon Acetylsalicylic acid (ASA, a well known anti-

inflammatory mediator) treatment in vivo. We also demonstrated that ASA treatment during the chronic phase of

Chagas’ disease inhibit the development of cardiomyopathy. The Interleukin 10 (IL-10) stimulates SOCS1 and SOCS3

expression in DC, but not SOCS2. Here our goal was investigated the action of ASA during Tc infection and the

participation of AhR and SOCS2 on it.

Methods and results: Wild Type (WT), AhR knockout (KO) and SOCS2 KO mice were infected with Tc (Y strain) and

treated or not with ASA (5mg/Kg/day) during 15 days. Parasitemia, inflammatory mediators expression (in spleen and

heart) and histology was analyzed. ASA treatment resulted in an increased parasitemia in WT compared with WT

untreated. The deficiency of AhR or SOCS2 resulted in higher resistance against infection when compared with WT.

Moreover, ASA treatment partially increased the parasitemia in SOCS2 KO mice, which remained lower than the

levels found in WT treated or not. In the spleen, treatment with ASA demonstrated an increased expression of IL-10

and SOCS3, but not of IFN- and SOCS1, in Tc-infected SOCS2 KO mice when compared with WT. In absence of

AhR, ASA induced an increased expression of SOCS1, but not SOCS2 and SOCS3. In the heart of SOCS2 KO-

infected mice the expression of IL-10 was reduced when compared with WT. Treatment with ASA inhibited the IL-10

expression in WT and SOCS1 in SOCS2KO infected mice. However, the treatment increased IL-10 and SOCS1

levels, but not SOCS2 and SOCS3, in the heart of AhR KO when compared with WT.

Conclusion: In summary, our results suggests that AhR and SOCS2 molecules are triggered during ASA treatment of

Tc-infected mice participating in the modulation of immune response and development of myocarditis.

Financial Support: CNPq and FAPEMIG.

EFFECTS OF HSP65-PRODUCING LACTOCOCCUS LACTIS IN SPINAL CORD OF MICE WITH EXPERIMENTAL

AUTOIMMUNE ENCEPHALOMYELITIS

MAURO ANDRADE DE FREITAS GUIMARÃES1; RAFAEL MACHADO REZENDE

2; NATÁLIA PINHEIRO ROSA

3;

RAFAEL PIRES OLIVEIRA4; SAMARA RABELO MEDEIROS

5; ANA MARIA CAETANO FARIA

6.

1,3,4,5,6.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 2.BRIGHAM AND

WOMEN'S HOSPITAL; HARVARD MEDICAL SCHOOL, BOSTON - ESTADOS UNIDOS.

Introduction: Multiple sclerosis (MS) is an autoimmune disease associated with a dysfunctional operation of the

immune system of an individual. Experimental autoimmune encephalomyelitis (EAE) is a rodent model of MS in which

immunization with antigens derived from the myelin sheath leads to a specific immune response and disease

manifestations similar to those in humans. Methods and results: Using the strategy of oral tolerance induced by the

continuous intake of a specific antigen protein in low doses, we conducted a study to better understand the effect of

continuous feeding of HSP65-producing Lactococcus lactis in preventing EAE. Herein we focused the analysis in the

events taking place at the spinal cord and on cell migration. After a 5-day oral treatment with either HSP65-producing

L.lactis (HSP65+EAE) or water only as a control (water + EAE), C57BL/6 mice were immunized with MOG and CFA

for disease induction. On days 4, 10 and 14 after immunization, animals were killed. We observed a decreased in

inflammatory infiltrated and injured areas on spinal cord 14 days after the immunization only in HSP65 + EAE group. A

study of regulatory CD4+ T lymphocytes showed that, at this time point, a higher frequency of Foxp3-LAP+ cells

occurred in HSP65 + EAE group. Regarding the parameters related to cell migration studied on the 4th day after EAE

induction, we studied frequencies of T cells expressing the chemokines receptors CCR6 and CXCR3. We observed a

reduced frequency of effector T cells expressing CCR6 and an increased frequency of those espressing CXCR3 in

HSP65 + EAE group. We also evaluate, on the 14th day, frequencies of T cells expressing the integrins α4β1 and

αLβ2, and frequency of CD4+ effector T cells expressing α4β1 in spleen was reduced.

Conclusion: Ingestion of L.lactis producing of HSP65 leads to many effects in the spinal cord of mice that may be

involved in the prevention of EAE. Although we could not define the mechanism by which regulatory CD4+ T cells

induced by L.lactis HSP65 reach the spinal cord yet, the possibilities presented are plausible and will be further

explored.

Financial support: CNPq, CAPES, FAPEMIG.

EFFECTS OF NANOPARTICLES CURCUMIN ON GENE EXPRESSION IN VITRO AND LPS-INDUCED

PERIODONTAL DISEASE IN VIVO

FERNANDA REGINA GODOY ROCHA1; MORGANA RODRIGUES GUIMARÃES

2; FABIANA ALMEIDA

CURYLOFO3; DEBORA MIYAZAKI

4; ANTONIO CLAUDIO TEDESCO

5; CARLOS ROSSA JUNIOR

6.

1,2,3,4,6.SCHOOL OF DENTISTRY - UNESP, ARARAQUARA - SP - BRASIL; 5.CHEMISTRY DEPARTMENT - USP,

RIBEIRÃO PRETO - SP - BRASIL.

Introduction: Curcumin, pigment obtained from rhizome of turmeric (Curcuma longa L., Zingiberaceae) has been

confirmed to have various biological and pharmacological activities, including antiinflammatory properties. In previous

studies, our group has demonstrated the beneficial effects of the oral administration of curcumin in wound healing.

Nanoparticles have been widely studied for increasing treatment efficacy and specificity of treatment approaches,

which could improve the pharmacological and biological properties of curcumin. Objectives: In this study, we

evaluated the effects of nanoparticles as a vehicle for the delivery of curcumin in vitro, assessing regulation of

inflammatory gene expression by LPS-stimulated macrophages and in vivo, by quantifying bone resorption in a LPS-

induced periodontal disease model in rats. Methods: We used RAW 264.7 cells stimulated with 1 ug/mL of LPS, and

treated with 2.5, 5 and 10 ug of curcumin nanoparticles. After 6 h, we determined gene expression of IL-6, PTSE2

and TNFa, by RT-qPCR. In vivo, we injected 50 µg of LPS of Eschericia coli (LPS group n=8) or the same volume of

the vehicle (PBS n=8), plus injection of 4 uL of nanoparticled curcumin, or nanoparticled vehicle without curcumin, in

the same day. These injections were performed three times a week in 4 weeks, into the palatal gingiva, between the

first and second molars of male rats (Holtzman). Quantitation of bone resorption was performed by uCT. Results: IL6,

PTSE2 and TNFa mRNA expression by macrophages were attenuated by treatment with 5ug of nanoparticled

curcumin. The µCT showed significantly (p=0.01) less bone resorption in the animals treated with nanoparticled

curcumin. Conclusion: Nanoparticles were an effective delivery vehicle, supporting the biological effects of curcumin

in the attenuation of inflammatory gene expression by LPS-stimulated macrophages in vitro, and inhibiting

inflammation-associated bone loss in the LPS-induced periodontal disease model in vivo.

EFFECTS OF ORALLY-ADMINISTERED CURCUMIN ON PERIODONTAL REPAIR IN VIVO

MORGANA RODRIGUES GUIMARAES STABILI; FABIANA ALMEIDA CURYLOFO; SABRINA GARCIA AQUINO;

FERNANDA REGINA GODOY ROCHA; CAMILLA OLGA TARSO; JOSE PAULO PIZZOL JUNIOR; PAULO SERGIO

CERRI; CARLOS ROSSA JUNIOR.

FACULDADE DE ODONTOLOGIA DE ARARAQUARA, ARARAQUARA - SP - BRASIL.

Introduction: Curcumin is a yellow pigment found in the rhizome of turmeric (Curcuma longa L., Zingiberaceae) with

a wide range of pharmacological and biological activities. Previous studies of our group have clearly substantiated the

beneficial effects of the oral administration of curcumin in the acceleration of wound healing. In this study, we

assessed the effects of orally-administered curcumin in a repair model of experimental periodontal disease. Since

there is evidence that piperine, an alkaloid contained in black pepper, improves the absorption of curcumin in the

digestive trait, we also assessed the association of curcumin and piperine. Methods: Periodontal disease was

induced by ligature placement. 15 days after the induction of disease the ligatures were removed and Curcumin

(400mg/kg) and/or Piperine (20mg/kg) started to be administered daily by oral gavage for 5 and 15 days. The

expression of a marker of periodontal repair, TGF-β, was evaluated by ELISA. The extent of bone and tissue repair

and the inflammatory status of gingival tissue were analyzed by μCT, picrosirius stain and stereometric analysis,

respectively. Results: Curcumin increased TGF-β protein expression 5 and 15 days after removal of ligatures.

Curcumin and curcumin / piperine-treated animals presented a marked reduction on the inflammatory cell infiltrate (5

days: Not treated x Curc and Not treated x Curc/Pip (p<0,0001); 15 days: Not treated x Curc (p=0,0021); Not treated x

Curc/Pip (p=0,0010)) and increased collagen content (5 days: Not treated x Curc/Pip (p=0,0129); 15 days: Not treated

x Curc (p=0,0081), Not treated x Curc/Pip (p=0,0035)). Both curcumin and curcumin/piperine treatments increased

bone volume significantly (p=0,0081 and p<0,0001, respectively) in comparison to control group at 5 days, and there

was an additional improvement by the association with piperine. Conclusion: In conclusion, oral administration of

curcumin improves soft tissue healing and accelerates bone formation in the periodontal.

ENVOLVIMENTO DA SINALIZAÇÃO DE RECEPTORES DA IMUNIDADE INATA NOD-LIKE E TOL-LIKE NO

EFEITO ANTIINFLAMATÓRIO DE LACTOBACILLUS DELBRUECKII

MARCELA SANTIAGO PACHECO AZEVEDO1; CLARISSA SANTOS ROCHA

2; VANESSA BASTOS

3; TESSALIA

SARAIVA DINIZ4; MARTEEN VAN-DE-GAUCHE

5; VASCO AZEVEDO

6; SERGIO COSTA OLIVEIRA

7; ANDERSON

MIYOSHI8.

1,2,3,4,6,7,8.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 5.INRA, JOUY

EN JOSAS - FRANÇA.

As doenças inflamatórias intestinais (do inglês, Inflammatory Bowel Disease – IBD) constituem um problema médico

em todo o mundo como também no Brasil. Descobertas há cerca de 100 anos, a etiologia dessas doenças ainda não

é completamente entendida. Sabe-se que pacientes acometidos com IBDs apresentam um desequilíbrio das

interações entre as bactérias autóctones da microbiota intestinal com o sistema imune, gerando assim uma

inflamação crônica no trato gastrointestinal (TGI). Os tratamentos, atualmente disponíveis, acarretam em sérios

efeitos colaterais aos acometidos. Diante disso, a necessidade de um tratamento alternativo, mais eficaz e que não

apresente tais efeitos colaterais aos pacientes com IBD, tornou-se uma necessidade. Estudos recentes vêm

demonstrando o potencial terapêutico de bactérias probióticas no tratamento destas doenças e, inúmeros probióticos

isolados do TGI humano, já vêm gerando resultados promissores. Ainda assim, ainda há uma grande falta de

conhecimento a respeito dos mecanismos moleculares,microbiológicos e imunológicos envolvidos em tais efeitos.

Nesse contexto, nosso grupo de pesquisa está interessado em estudar os efeitos e os mecanismos imuno

modulatórios de Lactobacillus delbrueckii, uma bactéria isolada de produtos fermentados e que está em constante

trânsito no nosso TGI através da alimentação. Assim, em um trabalho prévio, foi demonstrado em experimentos in

vitro que algumas linhagens de L. delbrueckii são capazes de inibir a via NF-kB, um fator de transcrição crucial na

geração de respostas inflamatórias, de uma maneira linhagem dependente. Após administração em camundongos, L.

delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327) foi capaz de reduzir significantemente a inflamação em um modelo

de IBD induzido pelo composto Dextran Sulfato de Sódio (DSS). Esse resultado reforça o fato de que a administração

de certas linhagens probióticas pode representar uma alternativa promissora no tratamento de IBD. Diante disso,

tivemos o intresse de identificar os receptores da imunidade inata (TLR) e as vias de sinalização envolvidas em seus

efeitos antiinflamatórios. Esse estudo irá também contribuir para o melhor entendimento dos mecanismos envolvidos

nas relações probiótico/hospedeiro.

EVALUATION OF CHEMOKINES AND CYTOKINES PLASMA LEVELS IN MICE GENETICALLY DEFICIENT IN

THE ANGIOTENSIN-(1-7) RECEPTOR MAS DURING EXPERIMENTAL ENDOTOXEMIA

ONÉSIA CRISTINA OLIVEIRA LIMA1; JOHAN DUCHENE

2; MAURO CUNHA XAVIER PINTO

3; NATALIA ALENINA

4;

MICHAEL BADER5; ROBSON AUGUSTO SOUZA DOS SANTOS

6; JULIANA CARVALHO TAVARES

7.

1,3,6,7.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 2,4,5.MAX DELBRÜCK

CENTER, BERLIN - ALEMANHA.

Introduction: Several available evidence supports the involvement of renin-angiotensin system on

inflammation, emphasizing the pro-inflammatory effects of AT1 receptor activation by Ang-2. However, the

relevance of Ang-(1-7)/receptor Mas on inflammatory response remains to be elucidated. In this

study we evaluated the involvement of Ang-(1-7)/receptor Mas on chemokines and cytokines production during

experimental endotoxemia. Methods and Results The

experimental endotoxemia was induced in 8-12 week-old C57Bl/6 wild type mice (WT) and Mas deficient mice (Mas-

/-) by intraperitoneal injection of lipopolysaccharides (LPS, 5mg/Kg) (Escherichia coli O111:B4). Plasma levels of

monocyte chemotactic protein-1 (MCP-1); chemokine (C-X-C motif) ligand 1 (CXCL-1/KC), granulocyte colony-

stimulating fator (GCSF) and Interleukin 1-β (IL-1β) were evaluated by ELISA at 3 and 24 hours after LPS injection (n=

at least 5 animals per group). The results are reported as the means and standard errors of the means. The absence

of Mas receptor did not affect the plasma levels of MCP-1, CXCL-1, GCSF and IL-1 β plasma levels at 3 hrs after LPS

injection. In contrast, 24hs after LPS injection Mas-/- mice presented elevated plasma levels of MCP-1 (31.32 ± 7.78

pg/mL); CXCL-1 (11.31 ± 4.76 pg/mL) and GCSF (20.06 ± 15.43 pg/mL) compared to WT mice (MCP-1:3.921

±1.91pg/mL;CXCL1:0.02±0.02pg/mL;GCSF:73.11±14.31pg/mL).

Conclusion: These results suggest that absence of Mas

receptor leads to a delayed modulation of chemokines associated with impairment on resolution of the systemic

inflammatory process induced by LPS.

Financial support: CAPES, CNPQ, FAPEMIG

EVALUATION OF THE MECHANISMS DERIVED FROM TLR2 ACTIVATION INVOLVED IN THE INHIBITORY

EFFECT OF HIV-1 REPLICATION

BRUNO CISTER ALVES; PEDRO LOURENÇO CÂMARA FERREIRA; JAIRO RAMOS TEMEROZO; DUMITH

CHEQUER BOU-HABIB.

INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL.

Introduction: HIV-1 infected patients have increased intestinal permeability, which grants microorganisms passage to

the blood, event known as microbial translocation. Some microorganism’s components are recognized by TLRs, that

when activated induce the synthesis of cytokines and chemokines, modulating HIV-1 replication. Studies from our lab

show that TLR2 activation results in HIV-1 replication inhibition in lymphocytes and macrophages. This phenomenon

is mediated by IL-10 and β-chemokines. However, based in the complex response triggered by TLR2 activation, it is

possible that other factors may be involved in this inhibition. Thus, we intend to evaluate if other mechanisms take part

in the TLR2 mediated anti-HIV-1 effect.

Methods and Results: Human primary macrophages, obtained from healthy donors, were treated with different TLR2

agonists in order to analyze the production of IL-27 and other mediators by ELISA. Preliminary results suggest that

uninfected human primary macrophages exposed to TLR2 ligands for 24 hours presented increased production of IL-

27 (200,8 ± 114,9 pg/mL; n=4 for Pam3CSK4 and 216,4 ± 123,9 pg/mL; n=4 for Pam2CSK4), a cytokine capable of

potent inhibition of HIV-1 replication. In the same way, IL-10, another cytokine with anti-HIV-1 properties, had it’s

synthesis increased (201,1 ± 91,3 pg/mL; n=4 for Pam3CSK4; 82,3 ± 13,7 pg/mL for Zymosan and 128,4 ± 40,8

pg/mL for Pam2CSK4) by the same stimulus. In similar fashion, our results also show that CCL5, a β-chemokine that

blocks HIV-1 entry, had increased concentrations (245,3 ± 84,2; n=4 for Pam3CSK4; 112,7 ± 22,3 pg/mL for Zymosan

and 268,7 ± 138,5 pg/mL for Pam2CSK4) after the treatment. In other experiments, macrophages were treated with

TLR2 ligands for 4 hours and had their RNA extracted and analyzed for the IL-27 subunits by qPCR. Pam2CSK4-

treated cells showed a (145 ± 79; n=4) fold increase in EBI3 expression, while Pam3CSK4 increased EBI3’s

expression by (125 ± 9; n=4). Accordingly, IL-27’s p28 subunit had it’s expression increased by approximately (2 ±

0,5; n=4) by Pam3CSK4. No significant increase in p28 expression was observed by Pam2CSK4 stimuli.

Conclusion: It’s probable that the TLR2 mediated HIV-1 inhibition is total or partially dependent on these factors. We

intend to perform functional assays to evaluate the role of these molecules in the HIV-1 inhibition mediated by TLR2

activation.

Funding: CNPq, Faperj, POM/IOC

EX VIVO-EXPANDED HUMAN TREGS INHIBIT HUMAN ALLOGENEIC SKIN GRAFT INFLAMMATION IN A

HUMANIZED-SCID MOUSE MODEL

VIVIAN LEITE DE OLIVEIRA1; MALOU PEPPELMAN

2; ESTHER FASSE

3; ESTHER VAN RIJSSEN

4; PETER C.M.

VAN DER KERKHOF5; PIET VAN ERP

6; IRMA JOOSTEN

7; HANS J.P.M. KOENEN

8.

1,2,3,4,7,8.DEPT OF MEDICAL IMMUNOLOGY, RADBOUD UNIVERSITY NIJMEGEN MEDICAL CENTRE,

NIJMEGEN - HOLANDA; 5,6.DEPT OF DERMATOLOGY, RADBOUD UNIVERSITY NIJMEGEN MEDICAL CENTRE,

NIJMEGEN - HOLANDA.

Introduction: Treg cell-therapy is of interest for therapeutic intervention in transplant rejection and autoimmunity.

Although clinical Treg-therapy trials have started, the in vivo behavior of ex-vivo expanded human Treg is unclear.

Methods and Results: We investigated the effect of ex-vivo expanded CD4+CD25+CD127low human Treg-cells on

the inflammatory response of human skin allograft in a humanized-SCID mouse model. First, we demonstrated that

ex-vivo expanded human Treg-cells maintained suppressive capacity in vitro. Next, these expanded Treg were

studied in vivo in our humanized mouse model, in brief human skin-grafts were transplanted on immunodeficient

SCID/beige mice, after healing mice were infused (i.p.) with allogeneic hu-PBMC with/without Treg-cells. After 3-

weeks inflamed skin-grafts, spleen and peripheral blood were harvested and analyzed by histology and/or

flowcytometry. Analysis of inflamed human skin-grafts revealed that Treg infusion restored the inflammation related

aberrant K10/K16 epidermal marker expression and influx of hu-CD8 T-cells. The Infusion of Treg also affected the

systemic response, they inhibited human CD4+ and CD8+ T cell activation and proliferation as indicated by reduced

Ki67. Impact on local and systemic cytokine profile is under investigation.

Conclusion: We showed that under the conditions tested, ex-vivo expanded Treg-cells reduce but do not fully prevent

hu-PBMC induced skin inflammation in vivo. The observed reduction of skin transplant inflammation by human

Treg encourages the use of Treg-therapy in transplantation.

Financial support: Vanderes Research Foundation Grant – The Netherlands, LEO Pharma Research Foundation -

Denmark

EXERCISE AND CALORIC RESTRICTION ALTER THE IMMUNE SYSTEM OF MICE SUBMITTED TO A HIGH FAT

DIET

FREDERICK WASINSKI1; REURY FRANK BACURAU

2; MILTON ROCHA MORAES

3; GABRIEL RUFINO ESTRELA

4;

EDGAR PAREDES GAMERO5; CARLOS CASTILHO BARROS

6; SANDRO SOARES ALMEIDA

7; NIELS SARAIVA

CAMARA8; RONALDO CARVALHO ARAUJO

9.

1,3,4,5,7,9.FEDERAL UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 2,8.UNIVERSITY OF SAO

PAULO, SÃO PAULO - SP - BRASIL; 6.FEDERAL UNIVERSITY OF PELOTAS, PELOTAS - RS - BRASIL.

As the size of adipocytes increases during obesity, the establishment of resident immune cells in adipose tissue

becomes important as a source of pro-inflammatory mediators. Exercise and caloric restriction are two important, non-

pharmacological tools against body mass increase. To date, their effects on the immune cells of adipose tissue in

obese organisms, specifically when a high-fat diet is consumed, have been poorly investigated. Thus, after consuming

a high-fat diet, mice were submitted to chronic swimming training or a 30% caloric restriction in order to investigate the

effects of both interventions on resident immune cells in adipose tissue. Our results indicated that both exercise and

caloric restriction were able to reduce body mass in animals consuming a high-fat diet. These strategies resulted in

changes in the number of resident immune cells in the adipose tissue and in the levels of cytokines/chemokines in

serum. While exercise increased the number of NK cells in adipose tissue and serum levels of IL-6 and RANTES,

caloric restriction increased the CD4+/CD8+ cell ratio and MCP-1 levels. Together, these data demonstrated that

exercise and caloric restriction modulates resident immune cells in adipose tissues differently in spite of a equivalent

body weight reduction. Additionally, the results also reinforce the idea that a combination of both strategies is better

than either individually for combating obesity.

GENE EXPRESSION PROFILE OF T CELL TREATED WITH RECOMBINANT ANTI-CD3 ANTIBODIES

KELLY CRISTINA RODRIGUES SIMI; MARYANI ANDRESSA GOMES BEZERRA; ISABEL GARCIA SOUSA;

ANDREA QUEIROZ MARANHÃO; MARCELO DE MACEDO BRIGIDO.

UNIVERSIDADE DE BRASILIA, BRASILIA - DF - BRASIL.

GENE EXPRESSION PROFILE OF T CELL TREATED WITH RECOMBINANT ANTI-CD3 ANTIBODIES.

KELLY CRISTINA RODRIGUES SIMI1,2

; MARYANI ANDRESSA GOMES BEZERRA1,2

, ISABEL GARCIA SOUSA1,2

;

ANDREA QUEIROZ MARANHÃO1,2

; MARCELO DE MACEDO BRIGIDO1,2

1Laboratório de Imunologia Molecular, IB, UnB;

2Instituto de Investigação em Imunologia/INCT

Introduction: The induction of immune tolerance to treat autoimmune disease and allograft rejection is of a

considerable interest in many different areas of immunology and pharmacology. Antibodies against CD3 molecule of T

cell have potential to induce and maintain peripheral tolerance. Studies have been suggesting that the mechanism of

anti-CD3 action involves induction of T regulatory cells. These cells are components of the immune system that

suppress other cells. The first antibody approved to clinical treatment of acute transplant rejection was a mouse

monoclonal antibody anti-CD3, OKT3. However, the clinical use of OKT3 has serious side effects linked to its

immunogenic and mitogenic potentials. Our group developed two humanized versions of OKT3 (TVL and RVL). The

proposal of this work is to analyze the transcriptome of human T cell treated with humanized versions anti-CD3.

Methods and Results: The humanized fragments of the antibody were purified using affinity chromatography from

the CHO-K1 culture supernatant. Flow cytometry was performed using an assay of direct binding to peripheral blood

mononuclear cells (PBMC). The different versions of antibodies showed a similar binding capacity to CD3 molecule.

To investigate the mechanism of humanized anti-CD3 action total RNA from white blood cells (WBC) incubated with

250 ng of the purified anti-CD3 for 72h at 37°C was extracted using Trizol reagent (Invitrogen). RNA was reverse-

transcribed using SuperScript II reverse transcriptase and qPCR was performed using SybrGreen according

manufacturer’s recommendations. The expression of IL10, FOXP3, CD4, CD25, TGFB, and CTLA4 was investigated

revealing the induction of some immune regulatory genes as IL10, FOXP3, and CTLA4.

Conclusion: We produced and purified two different versions of the recombinant fragments of humanized antibodies

against CD3 molecule. These proteins were able to bind to the human lymphocyte CD3. These antibodies are

potential tools for therapies in immune regulation.

Financial support: CNPq; FAP/DF

HOW M. LEPRAE HSP65 INFLUENCES THE IMMUNE RESPONSE IN GENETICALLY SELECTED AGED MICE?

ESTEVAM JOSÉ BALDON1; ELIANA BLINI MARENGO

2; VALQUIRIA BUENO

3; MARCELO DE FRANCO

4;

OSVALDO AUGUSTO SANT'ANNA5.

1,4,5.INSTITUTO BUTANTAN, SÃO PAULO - SP - BRASIL; 2.HOSPITAL ISRAELITA ALBERT EINSTEIN, SÃO

PAULO - SP - BRASIL; 3.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: The immunosenescence process affects both humoral and cellular responses leading to an imbalance

of the individual homeostasis. Heat shock proteins may trigger innate immune responses and are involved in

immunosenescence and autoimmunity. Previous data showed that the Hsp65 of M. leprae interfere with the mean

survival time in HIII female mice, not affecting males. Methods and results: We characterized by flow cytometry some

cellular and humoral alterations after intraperitoneal administration of 2.5µg M. leprae Hsp65 in genetically selected

mice for High (HIII) or Low (LIII) antibody production (9-months-old) and in its F1 hybrids. Aged HIII female injected with

Hsp65 presented a survival decrease of 42% when compared to untreated group (control); no changes in IgG1 or

IgG2a anti-Hsp production were observed in HIII and LIII mice. Regarding the cellular changes, aged HIII female Hsp65-

group presented amplified frequency in CD4+CD154

+CD28

+ cells (p<0.01) and reduced percentage of B and activated

CD11c+ cells (p<0.01) in the spleen, and increased percentage of CD11c

+ and NKG1A/C/E

+ cells (p<0.01) in the

blood compared to control. Hsp65 acts like an imbalance trigger: post-injection, the aged F1H female Hsp65-group

died 2 months after the first death, as observed in aged HIII females; however, there was no statistically significance

compared with F1H control group. Furthermore, aged F1H and F1L female showed amplified frequency of naïve T cells

and CD11c cells in spleen (p<0.001). Conclusion: our results confirm the sex dichotomy (sex effect) of the Hsp65

interference in the immunity of aged mice, becoming evident in females. Next, we will characterize innate immune

cells in peritoneal cavity after Hsp65 inoculation; in addition, the role of myeloid-derived suppressor cells will be

investigated as these cells increase during ageing process and have been associated with attenuation of experimental

autoimmune diseases. Financial support: CNPq, FAPESP, INCT-TOX.

IMMUNOMODULATORY EFFECT OF METHYLPREDNISOLONE ON TH17 CYTOKINES PRODUCED BY PBMC

COLLECTED FROM ASTHMATIC CHILDREN

THIAGO UBIRATAN LINS LINS; ADRIANA AZOUBEL ANTUNES; MOACYR JESUS BARRETO DE MELO RÊGO;

MARIANA BRAYNER CAVALCANTI; EMANUEL SÁVIO CAVALCANTI SARINHO; MAIRA GALDINO DA ROCHA

PITTA.

UFPE, RECIFE - PE - BRASIL.

Introduction: Asthma is anti-inflammatory airway disease characterized by variable airway obstruction. The recent

Th17 cells discovery and further Th cell subtypes, promote a better understanding of immune allergic response. In this

disease, glucocorticoids like methylprednisolone are the most potent anti-inflammatory therapy commonly used. This

compound bind to a specific intracellular receptor to inhibit activation of T cells by various stimuli. In this work, it was

evaluated the immunomodulatory effect of methylprednisolone by measurement of IFNγ, IL-6, IL-17A and IL-22

cytokines levels in peripheral blood mononuclear cells (PBMCs) of children with moderate persistent asthma (MPA)

and severe persistent asthma (SPA). Methods and Results: The PBMCs (1x106 cells/ml) of 18 patients were

obtained and stimulated with PMA (phorbol 12-myristate 13-acetate) (50 ng/ml) and Ionomycin (1 μg/ml) for 48 hours.

Methylprednisolone (100 µM) was used to inhibit the cytokine production. The related cytokines in the culture

supernatant obtained was assayed by ELISA. Wilcoxon’s signed rank test was used for statistical analysis. P<0,05

was considered significant. The expression of IFNγ (p=0,0313), IL-6 (p=0,0223) and IL-17 (p=0,0313) in SPA patients

and IFNγ (p=0,0078), IL-6 (p=0,0078) and IL-17 (p=0,0156) in MPA patients was decreased significantly when in

presence of methylprednisolone. However, methylprednisolone could not inhibit significantly the IL-22 production. It

was even realized comparison among SPA and MPA groups but not statistically significant differences were founded.

Conclusion: The immunomodulatory effect of methylprednisolone was demonstrated through the decrease of

cytokines levels, in accordance with the cytokine production inhibitory properties of glucocorticoids. The poor

response observed in IL-22 levels could indicate a possible role of IL-22 in steroid-resistant (SR) asthmatic profile.

Financial support: CAPES, FACEPE and INCT-if.

IMMUNOMODULATORY EFFECTS OF HSP65-PRODUCING LACTOCOCCUS LACTIS MHSP65 IN A MURINE

MODEL OF ARTHRITIS

GUILHERME GUSMÃO SILVA; DANIELA SILVA DOS REIS; MAURO ANDRADE FREITAS GUIMARÃES; NATÁLIA

PINHEIRO ROSA; SAMARA RABELO MEDEIROS; RAFAEL OLIVEIRA PIRES; ANDERSON MIYOSHI; ANA MARIA

CAETANO FARIA.

UFMG, BELO HORIZONTE - MG - BRASIL.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease associated with joint damage, progressive

disability, systemic complications causing decrease in lifespan. There is no prevention or cure for RA, being the

treatment based on conventional anti-inflammatory, analgesic and antirheumatic drugs. The autoimmune component

of RA is complex and diverse. Type II collagen (CII) is one of the most known autoantigen in the pathology of RA.

Both in animal models and in human disease, reactivity to antigens derived from Mycobacteria heat shock protein

HSP65 is a hallmark, and was once considered the major antigen of RA.

Tolerance by oral administration of autoantigens has been reported to prevent disease development in several animal

models, including RA. The aim of the present study was to test the therapeutic potential of oral tolerance induced to

Mycobaterium leprae HSP65 produced by Lactococcus lactis in acute and chronic murine models of autoimmune

arthritis. In the acute model of Antigen-induced Arthritis (AIA) the C57BL/6 strain was immunized with mBSA

emulsified in complete Freund adjuvant (CFA). And in the chronic model BALB/c mice were immunized several times

with CII and ovalbumin (OVA) in CFA. Fifteen days before the first immunization, animals received HSP65 L. lactis in

their drinking bottle for four days. Wild type (WT) L. lactis was used as a control.

BALB/c mice treated with HSP65 L. lactis, but not with control L.lactis, had a swelling score similar to the value found

in non-manipulated mice. These mice had higher levels of IL-10 in mesenteric lymph nodes, lower levels of IL-17 in

spleen and mesenteric lymph nodes and lower IFN-γ in the inguinal lymph nodes when compared to other groups.

Cell counts in the articular lavage were also significantly decreased. Therefore, oral treatment with HSP65 L. lactis

had a positive effect on antigen-induced arthritis. Further studies are needed to clarify the mechanisms of HSP65 L.

lactis action in arthritis.

Financial support: CNPq, FAPEMIG and CAPES

IMMUNOREGULATORY ROLE OF SUPPRESSOR OF CYTOKINE SIGNALING 2 (SOCS2) IN ANIMAL MODEL OF

MULTIPLE SCLEROSIS

BRUNO CABRAL DE LIMA OLIVEIRA; DAVID HENRIQUE RODRIGUES; FATIMA BRANT; DIEGO RODNEY

RODRIGUES DE ASSIS; BRUNO COSTA SILVA; ANDREIA BARROSO; JULIA TEIXEIRA DE CASTRO; POLLYANA

MARIA DE OLIVEIRA PIMENTEL; FABIOLA MARA RIBEIRO; ANTONIO LUCIO TEIXEIRA JUNIOR; FABIANA

SIMÃO MACHADO.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Multiple sclerosis (MS) is an inflammatory disease of central nervous system(CNS), which affect

considering number of people around the world. The experimental autoimmune encephalomyelitis (EAE) is a

commonly employed model for the study of the immunopathogenesis of human MS. During EAE, wild type(WT)

animals present Th17 immunological response in its initial phase, following by a Th1 profile during later phases.

Suppressors of Signaling Cytokines (SOCS) proteins family regulate the production of several cytokines in various

models of inflammation. However, their involvement in EAE is still unclear. The goal of this study was to analyze and

characterize the involvement of SOCS2 in the immunopathogenesis of EAE. Method: EAE was induced both in

C57Bl/6 and SOCS2-/- mice using Myelin Oligodendrocyte Glycoprotein (MOG) and Freund adjuvant and the clinical

scores and body weight were monitored. In addition, at 14 and 28 days after injection (dpi), the inflammatory levels in

the spinal cord samples were analyzed.The production of cytokines and chemokines (IFN-GAMMA, IL17, IL6, IL10,

IL17, TGF-BETA, TNF-ALPHA, RANTES and MCP1) in the brain, serum and spleen were assessed by ELISA.

Analysis of IFN producing – cells was performed by flow cytometry. Interferon Factor Regulatory 1 (IRF1) expression

analysis was acquired by Western Blotting (WB).Results: SOCS2-/- mice displayed decreased severity of EAE at

14dpi, the peak phase of the disease, when compared to WT, which correlated with a lower percentage of weight loss.

However, KO mice showed lower remission rates at the late phase. Histopathology of the spinal cord of deficient

animals demonstrated a reduction in inflammation level. Proinflammatory cytokines and chemokines were reduced in

the brains of SOCS2-/- mice at the early and late phase when compared with WT. At peak phase of EAE,

CD3+CD4+IFN-GAMMA+ and splenic cells was reduced in SOCS2-/- animals when compared with WT. Moreover,

IFN-GAMMA level was significantly lower in spleen of SOCS2-/- mice on 14 dpi when compared with WT

counterparts. The IRF1, an important regulator factor of IFN-GAMMA and chemokines production decreased in

SOCS2-/- EAE brain when compared with WT. Conclusions: In summary these results indicate that SOCS2 protein

has an important role in the modulation of EAE pathophysiology development.

Financial support: CAPES, CNPq, FAPEMIG

IMPACT OF L-CITRULLINE ON HISTOLOGY, INFLAMMATORY SCORE, MYELOPEROXIDASE (MPO) ACTIVITY

AND INTESTINAL PERMEABILITY IN AN 5-FLUOROURACIL-INDUCED MUCOSITIS IN MICE.

MAÍSA MOTA ANTUNES; PAOLA LACERDA LEOCÁDIO; LÍLIAN GONÇALVES TEIXEIRA; ALDA JUSCELINE

LEONEL; JACQUELINE ALVAREZ LEITE; DENISE CARMONA MACHADO; VALBERT CARDOSO NASCIMENTO;

SIMONE VASCONCELOS GENEROSO; MARIA ISABEL DAVISSON CORREIA.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Rationale: Mucositis is a common side effect in 40% of patients undergoing radio and chemotherapy. It is associated

with pain, dehydration, malnutrition and prolonged time of hospitalization, resulting in worsening of well-being and

quality of life. Treatment options to alleviate the suffering of the patient are crucial for a successful treatment. The use

of immunonutrients has been evaluated as a potential option. The aim of this study was to evaluate the impact of L-

citrulline (citrulline) on intestinal mucositis in mice, in a model of mucositis induced by administration of 5-FU.

Methods and Results: Swiss male were divided into four groups: control ALA (alanine), control CIT, 5FU ALA and

5FU CIT. The animals were fed with commercial chow and oral solution of ALA (control group ALA and 5FU ALA) or

CIT solution (control group CIT and 5FU CIT). Both solutions were isoproteic (1g/kg/day). On the seventh day, the

animals received an intraperitoneal injection of PBS (control group ALA and control CIT) or 5-FU (200mg/kg, single

dose) (group 5FU ALA and 5FU CIT) for induction of mucositis. On the tenth day, the animals underwent euthanasia,

and blood as well as the small intestine were removed for assessment of intestinal histopathology, enzyme assay

(NAG, MPO, EPO) and permeability. This study was approved by the Ethics Committee for Animal Experimentation at

the Federal University of Minas Gerais (CETEA/UFMG). The animals in the 5FU CIT group had lower dietary intake

and greater weight loss than the others. However, these animals had smaller areas of injury, lower score of

inflammation and an increase in MPO activity in the proximal jejunum, as well as reduction in intestinal permeability

when compared with the 5FU ALA group (p <0.05).

Conclusion: CIT was able to attenuate damage to the mucosal architecture of the small intestine, decreasing areas

of injury, and promoting decreased intestinal permeability.

Disclosure of Interest: None Declared.

Keywords: Citrulline, Mucositis.

IN VIVO TREATMENT WITH MK801, AN IONOTROPIC NMDAR ANTAGONIST, REDUCES EXPERIMENTAL

AUTOIMMUNE ENCEPHALOMYELITIS.

JEAN PIERRE PIERRE SCHATZMANN PERON; WESLEY NOGUEIRA BRANDÃO; ANDIRA FICKINGER;

CRISTIANO ROSSATO; MATHEUS CORREA COSTA; DANIEL MAY OLIVEIRA; NIELS OLSEN SARAIVA

CÂMARA.

USP, SAO PAULO - SP - BRASIL.

Introduction: Glutamate is the most abundant and perhaps the most important neurotransmitter. It acts through

different receptors known as metabotropic and ionotropic. Recent reports demonstrated the expression of many

glutamate receptors on immune cells, such as macrophages and T cells. Its role however is still a matter of debate.

Thus, we used MK801, a specific NMDAR antagonist to evaluate the role of this receptor during the immune

response. Methods and Results: To evaluate the pathways involved, DO11.10 T cell clones were activated in vitro

with or without MK801 or N-methyl D-Aspartate. Total and phospho ERK 1-2 were evaluated by western blot. Our

data demonstrated that NMDAR activation increases ERK phosphorylation, reversed by MK801. Further, we

performed in vitro Treg differentiation and the presence of MK801 increased the percentage of CD4+Foxp3+ cells. For

in vivo experiments, C57BL/6 mice were immunized with 150 mg of MOG35-55 and treated or not with 0,3 mg/Kg of

MK801 on days 0,1,2 and 3. At the peak of disease animals were sacrificed and Th1, Th17, Treg cells were analyzed

by flow cytometry. MK801 treated animals had milder disease when compared to control. Interestingly, animals

showed a decreased frequency of Th1 and Th17 cells infiltrating brain and spinal cord. In vitro experiments indicate

that this was possibly due to T cell death. Conclusions: Our results demonstrated that glutamate signaling boosts the

commitment of Th1 and Th17 cells. However, less- or non-encephalitogenic cells are being generated. These findings

contribute to the understanding of NMDAR on immune cells and also for a new point of view concerning its role in

neuroinflammatory diseases.

Financial support: CNPq - 480827/2010-9 and FAPESP 2011/18703-2.

INCREASE OF REGULATORY LYMPHOCYTES IN THE SPLEEN AND DRAINING LYMPH NODES DURING

REGENERATION OF MDX DYSTROPHIC MUSCLE

AMANDA BRUM; ANA PAULA LOUVERA; THEREZA QUIRICO-SANTOS; JUSSARA LAGROTA-CANDIDO; RITA

VASCONCELLOS.

UFF, NITEROI - RJ - BRASIL.

Introduction: Mdx mice, the animal model of Duchenne muscular dystrophy, develop a chronic inflammatory

myopathy characterized at early age (4-6 wks) by extensive myonecrosis, subsequent muscle regeneration (12 wks)

and persistent fibrosis (24 wks). Contrasting to the human disease, mdx mice present a benign phenotype apparently

due to a sustained capacity for muscle regeneration. We have previously shown that resolution of mdx muscle

inflammation was accompanied by increased lymphocyte activation and augment of immunoglobulin-secreting cells in

regional lymph nodes and bone marrow (Int J Exp Pathol. 2002 Jun; 83(3):121-32). The present study aimed to

analyze regulatory T and B cell subsets at different stages of mdx muscular dystrophy.

Methods and Results: Spleen and brachial draining lymph nodes from mdx and control C57BL/10 mice were tested

at the height (4 wks) of myonecrosis and prevalence of muscle regeneration (12 wks) for the presence of T and B

regulatory cells by flow cytometry and mRNA IL-10 levels by RT-PCR. This project was approved by the Animal

Welfare Commission-CEPA/UFF. Mdx mice at 12 weeks consistently showed increased numbers of

CD4+CD25+Foxp3+ regulatory T cells in the draining lymph node, and Transicional-2 Marginal Zone precursor and

marginal zone B cells in the spleen. The raise of regulatory cell populations was accompanied by high mRNA IL-10

content in the draining lymph node. Such pattern was not observed in mdx mice at the height of myonecrosis, and

neither in control nondystrophic C57BL/10 mice.

Conclusion: The results indicate that regulatory T and B cells capable to produce IL-10 may be contributing to mdx

muscle regeneration by providing an anti-inflammatory milieu.

Financial support: FAPERJ, FOPESQ/UFF

INFLUENCE OF DIETARY FOLIC ACID IN THE IMMUNE SYSTEM

ADNA LUCIANA SOUZA1; ANA CRISTINA GOMES-SANTOS

2; DANIELA SILVA DOS REIS

3; LUISA LEMOS

4;

SARAH FIORINI AGUIAR5; JEAN GUY LEBLANC

6; ANA MARIA CAETANO FARIA

7.

1,2,3,4,5,7.UFMG, BELO HORIZONTE - MG - BRASIL; 6.CERELA-CONICET, CENTRO DE REFERENCIA PARA

LACTOBACILOS, SAN MIGUEL DE TUCUMÁN - ARGENTINA.

Introduction: Vitamin B9 (folate) is a water soluble vitamin, derived from the diet and also produced by commensal

bacteria, which is involved in essential functions of cellular metabolism such as DNA replication, repair and

methylation. It has been shown that consumption of vitamin B9-deficient diet reduces proliferative responses of

lymphocytes and NK cell activity. In addition, folate is important for maintenance of regulatory T cells (Tregs) in the

intestine, and folate receptor 4 (FR4) is expressed on natural and induced CD4+CD25+Foxp3+ Tregs. In conditions of

regulatory disturbances, such as intestinal inflammation, dietary folate may play an important role. However, the

influence of folate in the immune system and in gut inflammatory conditions is not fully understood yet. Objective: to

characterize the influence of folate deficiency in the immune system in physiological conditions and during intestinal

inflammation in mice. Methods: Folic-acid-deficient diet was offered to C57BL/6 mice over 12 weeks (period of

depletion). Hematological and immunological parameters were evaluated after a repletion period. Results: The dietary

deficiency did not cause megaloblastic anemia, but lead to a reduced production of proinflammatory and anti-

inflammatory cytokines such as IL-10, TGF-beta, IL-4, IL-17, INF-gamma, TNF-alfa, IL-2 in the jejunum but not in the

other parts of the intestine. Levels of secretory IgA were reduced in the small intestine after folic acid depletion.

Moreover, administration of folic-acid-deficient diet during 8 weeks worse spontaneously colitis in IL-10 deficient mice.

Conclusion. Dietary deficiency of folic acid caused suppression of gut immune responses and was associated with

increased susceptibility to intestinal inflammation.

Financial support: CNPq, FAPEMIG, CAPES.

INTERDISCIPLINARY THERAPY EFFECTS IN BODY COMPOSITION AND INFLAMMATION IN OBESE ADULT

WOMEN

RICARDO BADAN SANCHES1; STEPHAN GARCIA ANDRADE SILVA

2; SUZANA ROSSI

3; AMANDA DOS SANTOS

MORAES4; VANESSA SCHOENARDIE POLI

5; RONALDO THOMATIELI DOS SANTOS

6; DANIELLE ARISA

CARANTI7.

1,4,5.POST GRADUATE PROGRAM OF INTERDISCIPLINARY HEALTH SCIENCES, FEDERAL UNIVERSITY OF

SÃO PAULO/UNIFESP, SANTOS - SP - BRASIL; 2,3.OBESITY STUDY GROUP (GEO) - FEDERAL UNIVERSITY

OF SÃO PAULO/UNIFESP, SANTOS - SP - BRASIL; 6,7.DEPARTMENT OF BIOSCIENCES, FEDERAL

UNIVERSITY OF SÃO PAULO/UNIFESP, SANTOS - SP - BRASIL.

Introduction: Obesity is a chronic and multifactorial disease that has been increasing towards epidemic proportions. It

affects millions of people worldwide and is currently considered a major public health problem. In Brazil, it is estimated

that 50% of the adult population are either overweight or obese. Obesity is associated with many deleterious

outcomes such as type 2 diabetes, hypercholesterolemia, hypertension, hyperleptinemia, and heart disease, and is

directly related to increased mortality and reduced life expectancy. Sedentary lifestyle with inappropriate food habits is

considered the main factors for those modifications by promoting adipose tissue accumulation, causing imbalance in

cytokine concentrations and therefore low-grade chronic inflammatory status. Leptin and TNF-α concentrations are

increased in obesity. Recent our research studies show that interdisciplinary therapy promotes several health benefits

such as decreased body mass, improved body composition, better inflammatory status and improving the quality of

life in obesity population. The aim of this study was to analyze the effect of an interdisciplinary therapy (35 weeks) on

the anthropometric measurements, the body composition and the inflammation in obese adults women.

Methods and Results: A total of twenty (n=20) obese adult women (age 43.28 ± 5.8 years), were enrolled for 35 weeks

on interdisciplinary therapy consisted of physical exercise sessions three times a week and nutritional and

psychological intervention once a week at the Federal University of São Paulo – UNIFESP - Brazil. An improvement

in body composition and a decrease in body mass 93.7 ±10.2 vs. 88.1 ±8.3 (kg) (p < 0.001), waist circumference

108.6 ±10.1 vs. 99.6 ±7.5 (cm) (p < 0.001), on leptin levels 31.7 ±18.4 vs. 22.3 ±11.9 (ng/ml) (p < 0.001) and TNF-α

levels 9.53 ±5.8 vs. 6.50 ±3.0 (pg/mL) (p < 0.05) were showed after the interdisciplinary therapy. No significant

difference effect was found for in the adiponectin serum 9.53 (±8.0) vs. 9.10 (±7.9) (μg/l).

Conclusion: The highlights results after short term interdisciplinary therapy demonstrated an important improvement in

anthropometric measurements and body composition. In addition, this benefits effect may be associated with a

decreased in leptin and TNF-α levels, which suggests that the interdisciplinary therapy was effective improving

inflammatory pathways.

Financial support: FAPESP 11/51723-7; CNPq 471108/2011-1.

INVARIANT NATURAL KILLER T CELLS IN THE MODULATION OF ACUTE KIDNEY INJURY

NATHÁLIA AMATO KHALED; ANA PAULA FERNANDES S MONTEIRO; VANESSA OLIVEIRA DOS REIS; DAVID

ANIBAL GARRIDO ANDRADE; ALEXANDRE DE CASTRO KELLER.

FEDERAL UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: Acute kidney injury (AKI) is a common consequence of chemotherapy. Although the involvement of the

immune system in AKI complications, the role of invariant Natural Killer T lymphocytes (iNKT) in this process is still

neglected. In this sense, we decided to study the role of iNKT cells in the modulation of acute renal failure induced by

cisplatin injection.

Methods and Results: C57BL/6 WT or iNKT deficient (Jα18-/-

) mice (n = 4 to 6) were injected intraperitoneally with

cisplatin (20mg/kg).The injection of cisplatin led to an increase of the protein/creatinine ratio, which characterizes AKI

development. In animals Jα18-/-

(0h: 4,37 ± 0,37; 24h: 19,32 ± 0,52; 48h: 25,36 ± 0,48; 72h: 22,08 ± 1,07), there was a

significant increase of proteinuria compared to WT mice (0h: 3,17 ± 0,52; 24h: 10,67 ± 0,42; 48h: 19,10 ± 0,55; 72h:

16,03 ± 1,05), indicating that in the absence of the invariant NKT cells renal damage is more severe.The RT-PCR

analyses of the renal tissue revealed that mRNA for pro-inflammatory molecules as IFN-γ (Jα18-/-

control:1,34 ± 0,09;

Jα18-/-

AKI:8,80 ± 0,20; WT control: 1,22 ± 0,09; WT AKI:6,65 ± 0,27), T-bet (WT control:1,10 ± 0,09; WT AKI: 4,11 ±

0,15; Jα18-/-

control: 1,32 ± 0,06; Jα18-/-

AKI: 6,43 ± 0,17), TNF-α (WT control: 1,10 ± 0,12; WT AKI: 6,89 ± 0,17; Jα18-/-

control: 1,11 ± 0,09; Jα18-/-

AKI: 7,63 ± 0,23) and IL-6 (WT control: 1,15 ± 0,09 ; WT AKI: 7,24 ± 0,23; Jα18-/-

control:1,17 ± 0,09; Jα18-/-

AKI: 8,79 ± 0,14), was increased in WT mice and more even in the Jα18-/-

. In concordance,

histological analyses of kidney tissue showed that renal injury was more severe in Jα18-/-

, in comparison to WT group.

Finally, we found that repletion of Jα18-/-

with iNKT cells attenuated AKI severity. The reconstitution of animals Jα18-/-

(0h: 6,20 ± 0,90; 24h: 14,94 ± 0,52; 48h: 17,55± 0,59; 72h: 16,67 ± 0,35) iNKT cells maintained with the loss of renal

function at levels similar to that observed in WT animals (0h: 4,57 ± 0,66; 24h: 9,66 ± 0,31; 48h: 15,87± 0,87; 72h:

15,28 ± 1,05).

Conclusion: In conclusion, our data support the idea that during cisplatin-induced AKI the iNKT cells attenuate kidney

injury.

Financial Support: CNPQ/CAPES, FAPESP(2007/07120-1)

LECTIN–COMPLEMENT PATHWAY AS A PROBABLE IMMUNOMODULATOR MECHANISM INDUCED BY

BABASSU MESOCARP

CAROLINE SILVA COSTA DE ALMEIDA; ELZA MARIA MORAES DE ARAÚJO; GRACIOMAR CONCEIÇÃO

COSTA; JOSEMAR MARCELINO FERREIRA GODINHO JUNIOR; JULIANA LUCENA DOS SANTOS; LETÍCIA

PRINCE PEREIRA PONTES; FLÁVIA RAQUEL FERNANDES DO NASCIMENTO; ANA PAULA SILVA AZEVEDO

DOS SANTOS.

IMMUNOPHYSIOLOGY LABORATORY / FEDERAL UNIVERSITY OF MARANHÃO, UFMA, SÃO LUIS - MA -

BRASIL.

Introduction: Babassu (Attalea speciosa Mart.), palm tree that represents the most important product on Maranhão’s

extractive industry, produces a fruit, babassu coconut, which is composed mainly of carbohydrates and has

immunomodulatory effects. The aim of the study was to investigate the hemagglutinating effects of saccharides

obtained from babassu mesocarp.

Methods and Results: The extract was obtained from the maceration of babassu mesocarp in phosphate buffered

saline (PBS) 40:1 and filtered after 24 hours. The measurement of protein concentration was performed by the

colorimetric method of Bradford. In the hemagglutination assay was used a suspension of erythrocytes 1% treated or

not for one hour with extract concentrations 40, 20, 10, 5, 2.5 and 1.25 mg/mL in triplicate, using the suspension of

erythrocytes in PBS as control. Agglutination was observed macroscopically and microscopically by counting rosettes.

The hemolysis assay was performed using the suspension of erythrocytes treated or not with extract concentration 40

mg/mL, incubated for two hours with serum at 37 º or inactivated at 56º to check protein denaturation. Hemoglobin

concentration in the culture supernatant represented hemolyzing activity. The results showed that hemagglutination

assay demonstrated no macroscopic difference between the groups. Microscopically, the presence of rosettes

followed a dose-dependent standard, 53.3 ± 5.77 (40 mg/mL), 15.3 ± 6.35 (20 mg/mL), 7.6 ± 5.03 (10 mg/mL), 4 ±

3.00 (5 mg/mL), 1.3 ± 1.15 (2.5 mg/mL), 1 ± 1.73 (1.25 mg/mL), suggesting the presence of lectin in the extract, even

so protein measurement showed low concentrations, less than 1% per mL extract. In hemolysis assay, the

concentration of hemoglobin was higher in culture of erythrocytes treated with extract (0,37 ± 0,014) than the control

(0,13 ± 0,014). To confirm the action of complement proteins, erythrocytes treated with extract were incubated with

inactivated serum at 56 º, being observed a reduction of hemolysis (0,13 ± 0,028) similar to control (0,14 ± 0,014).

Conclusion: The results show that the extract of babassu mesocarp is able to induce hemagglutination and

hemolysis probably by protein lectin type with carbohydrates presence, suggesting that the complement system

activation plays a unique role on the immunomodulatory effect of the babassu mesocarp, possibly by lectins’ pathway.

Financial support: FAPEMA/CNPq/UFMA

LIPOXIN NUCLEAR RECEPTOR IS AN ESSENTIAL “TIME MACHINE” MODULATOR OF IMMUNE RESPONSE

AND PATHOLOGIES DEVELOPMENT DURING EXPERIMENTAL CHAGAS’ DISEASE

ANDRÉIA BARROSO; LÍSIA ESPER; FÁTIMA BRANT; RONAN RICARDO SABINO ARAÚJO; THIAGO VINÍCIUS

AVILA; MATHEUS BATISTA HEITOR CARNEIRO; ISABELA DE AVELLAR COSTA; DANIELE DA GLÓRIA DE

SOUZA; LEDA QUERCIA VIEIRA; MILENE ALVARENGA RACHID; MAURO MARTINS TEIXEIRA; FABIANA SIMÃO

MACHADO.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Trypanosoma cruzi infection requires immune response balance to control parasite growth and

pathology development. Activity of 5-lipoxygenase enzyme results in lipoxin (LXA)4 production that is a known anti-

inflammatory eicosanoid and it is an important regulator of inflammatory cytokines production during this infection. The

role of LXA nuclear receptor, aryl hydrocarbon receptor (AhR), during experimental Chagas’ disease is

unclear.Methods and Results: Herein, wild type (WT) and AhR KO mice were infected with the Y strain of T. cruzi

and the AhR expression, parasitemia and immune response was assessed. The spleens and hearts were harvested

at different days post-infection (dpi) for histology, cytokines analyses by RT-PCR and ELISA and flow cytometry. We

found that AhR expression is up-modulated in spleen and heart during T. cruzi infection. Deficiency of AhR resulted in

higher resistance to infection, related with precocious increased numbers of macrophages and dendritic cells

producing IL-12 and T CD4 and T CD8 cells releasing IFN-γ in spleen at 10dpi. The reduction levels of pro-

inflammatory cytokine were observed in T. cruzi-infected AhR KO mice at 15dpi, which was similar or lower to the

levels found in infected WT counterparts. In absence of AhR the modulation of immune response in heart “mirror” the

phenotype described in spleen, where a precocious inflammation was found at 10dpi and it was partially reversed at

the time point of myocarditis fully developed in WT mice. We found that, beside cytokines, an increased reactive

oxygen species, but not nitric oxide and peroxynitrite production, could be also a factor responsible for the increased

efficiency to control the parasite grown in infected AhR KO mice. Conclusion: Collectively, our data suggests that

AhR activity is responsible to modulate innate and adaptative immune response and development of myocarditis

during experimental T. cruzi infection. Thus, AhR is an essential “time machine” tool capable to control “where and

when” the effectively immune response must acts to avoid pathology development due to parasite or inflammatory

undertakings.

Supported by CNPq and FAPEMIG

MODULATION OF CARDIOMYOCYTE FUNCTION BY 5-LIPOXYGENASE ENZYME DURING TRYPANOSOMA

CRUZI INFECTION: EFFECT OF ZYFLO® TREATMENT

RONAN RICARDO SABINO ARAÚJO1; DANILO ROMAN CAMPOS

2; ANDRÉIA BARROSO

3; FÁTIMA BRANT

4;

LÍSIA ESPER5; ALLYSSON THIAGO CRAMER

6; ROSA M. E. ARANTES

7; DANIELE GLÓRIA SOUZA

8; JADER

SANTOS CRUZ9; HERBERT BERNARD TANOWITZ

10; MAURO MARTINS TEIXEIRA

11; FABIANA SIMÃO

MACHADO12

.

1,3,4,5,6,7,8,9,11,12.UFMG, BELO HORIZONTE - MG - BRASIL; 2.USP, RIBEIRÃO PRETO - SP - BRASIL;

10.ALBERT EINSTEIN, NEW YORK - ESTADOS UNIDOS.

Introduction. Trypanosoma cruzi is a protozoan responsible for the American tripanosomiasis, a high neglected

illness known as Chagas’ disease. The infection can lead digestive and/or cardiac complications; however, there are

not effective treatments against it after the acute phase. 5-lipoxygenase (5-LO) enzyme synthesizes lipidic mediators

like lipoxins (LXs) and leukotrienes (LTs) that exert strong influence in immune response balance. LTs have pro-

inflammatory actions while LXs have anti-inflammatory actions. Because the “power” of this mediators in the immune

system, and, since 5-LO is active during all inflammatory processes, 5-LO is a possible target for therapeutic actions

in infectious diseases. Zileuton is a 5-LO enzime inhibitor marketed with the name Zyflo®. Zyflo® is a drug that acts

inhibiting 5-LO enzyme, thereby inhibiting your products synthesis. Therefore, Zyflo® can be a great tool in therapeutic

of Chagas’ disease. Here we study the effects of Zyflo® treatment in T. cruzi experimental infection.

Methods. C57BL/6 mice were infected with T. cruzi (Y strain) and treated via oral with Zyflo®(30mg/Kg) in different

time points after infection. The parasitemia was assessed, and the heart, spleen and liver were removed for mRNA

expression of cytokines and histological analysis. Serum was analyzed for cytokine levels, and splenic cells were

analyzed by flow cytometry. Cardiomyocyte were harvest and analyzed by patch-clamp.

Results. We found less parasite burden in treated mice when compared with non-treated. The IL-12 and IFN-g serum

levels were decrease in acute phase of infection in treated mice when compared with non-treated mice. In the same

way, the IFN-g mRNA expression in spleen were lower in treated mice, which was associated with reduced

percentage of IFN-g expression by T CD4+ and CD8

+ cells in treated mice when compared with non-treated during

acute phase of the infection. Moreover there was a decrease inflammation in cardiac tissue harvested from infected-

chronic mice treated with Zyflo® compared to non-treat mice. The treatment with Zyflo® also prevented the increase

of repolarization time found in infected mice, which remained similar to uninfected mice.

Conclusion. The manipulation of 5-LO activity by Zyflo® shown be a great strategy for therapeutic use against

infectious diseases like T. cruzi infection improving several parameters as parasitemia, inflammatory infiltrate and

repolarization time. Supported by CNPq and FAPEMIG.

OBESITY EFFECTS IN SILICA-INDUCED LUNG FIBROSIS IN MICE.

JAQUELINE PEREIRA LANA; GABRIEL AUGUSTO OLIVEIRA LOPES; DÉBORA FERANDES RODRIGUES; LAÍS

BHERING MARTINS; MARINA CHAVES DE OLIVEIRA; ANDIARA CARDOSO PEIXOTO; MAURO MARTINS

TEIXEIRA; REMO CASTRO RUSSO; ADALIENE VERSIANI MATOS FERREIRA.

UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction

The survival of species depends on their ability to resist to starvation by energy storage. However, in the presence of

food excess, the metabolic state can configure a scenario of excessive adiposity and associated co-morbidities.

Several evidences have showing the interaction between adipose tissue and immune system, which suggests that

obesity may play a role in the pathogenesis of several immunopathologies, including pulmonary diseases, through

mechanisms that may involve pro-inflammatory mediators produced by fat pad. The objective of this study was to

evaluate the lung inflammatory response of silica-induced fibrosis in obese mice.

Methods and Results

The experimental protocol was approved by the “Ethics Committee in Animal Experimentation at the Universidade

Federal de Minas Gerais” (protocol N°: 374 / 2012). Male BALB/c mice (8 animals per group) were first divided into

two groups: fed a high carbohydrate (HC) or a chow diet during 8w. For exposure, mice were anesthetized and

instilled intranasally with 10 mg SiO2, suspended in 40 μl sterile saline. After 28 days, mice were euthanized and

samples of blood, bronchoalveolar lavage (BAL), lung, adipose tissue and liver were collected. Obese animals with

silicosis showed a decrease in BAL leukocytes in BAL (Silicosis: 76,78 ± 29,29 / Obese+silicosis : 53,11±12,47), as

well as a decrease in macrophage (Silicosis: 52,95 ±19,46 / Obese+silicosis: 28,11± 9,54) and low levels of protein

in BAL (Silicosis:0,05± 0,01 / Obese + Silicosis: 0,03± 0,01). It was showed a decrease in EPO and MPO activity in

the lung of obese mice (Silicosis: 0,07±0,01 / Obese+ Silicosis: 0,04±0,01) (Silicosis: 342,8±128,2 / Obese+ Silicosis:

242,7± 75,61), respectively.

Conclusion

Our data suggest that the increased adiposity led to an improvement in silica induced lung fibrosis due to, at least in

part, low inflammatory response in lung.

ORAL TOLERANCE INDUCED BY BROWN SPIDER DERMONECROTIC RECOMBINANT TOXINS

KATIA SABRINA PALUDO1; MURILO DELGOBO

2; MAICON RAMOS PINTO

3; OLGA MEIRI CHAIM

4; ANA

CAROLINA MATINS WILLE5; SILVIO SANCHES VEIGA

6; GIOVANI MARINO FAVERO

7.

1,2,3,7.STATE UNIVERSITY OF PARANÁ, PONTA GROSSA - PR - BRASIL; 4,5,6.FEDERAL UNIVERSITY OF

PARANÁ, CURITIBA - PR - BRASIL.

Introduction: Oral Tolerance refers to a physiologic tolerance in gut associated lymphoid tissues and at other

mucosal surfaces such as respiratory tract. Oral tolerance can provide a clinical applicable physiologic and non-toxic

therapy for autoimmune inflammatory diseases or allergy. In present work we proposed an oral tolerance protocol

using Swiss mice and oral administration of brown spider Loxosceles intermedia recombinant toxins: LiRecDT1,

dermonecrotic, and its mutated and non-toxic form, LiRecDT1H12A.

Methods and Results: Swiss mice were orally treated with 1, 5 and 10µg of LiRecDT1 or LiRecDT1H12A three times

a week, for three weeks. At end of protocol, animals were immunized with mutated toxin (LiRecDT1H12A). At the

fourteenth day, animals were boosted with mutated toxin and serum was collected. To access tolerance induction,

levels of IgG anti-dermonecrotic toxin were measured via ELISA in tolerized and immunized animals. Paw edema was

performed in tolerized and control groups injecting 6µg of dermonecrotic toxin subcutaneously in plantar surface.

After, paw edema was measured and results were expressed in millimeters. Mice mortality study was developed by

injecting 50µg of dermonecrotic toxin intraperitoneally in control group and tolerized animals. Tolerized group had a

decrease in IgG toxin levels production: 48,3±20% for 1µg; 55,1±20% for 5 µg and 75,3±21% for 10 µg – LiRecDT1,

and 47,9±17% for 1µg; 71,5±8,6% for 5µg, 52,6±22% for 10µg - LiRecDT1H12A. Oral treatment with LiRecDT1H12A

induced paw edema reduction: 55,4±4% for 10µg. Mice mortality reduction was not statistically significant but showed

a strong tendency (p<0,068). All statistical analysis were performed using ANOVA following Bonferroni’s pos hoc test.

Conclusion: Our results demonstrated evidences of tolerance induction through decrease in IgG anti-dermonecrotic

toxin levels, paw edema reduction and increased survey in 24h after challenge in tolerized animals. L. intermedia

recombinant toxins have potential to be used as tools for Tolerance Oral induction and its study.

Supported by grants of Fundação Araucária, CAPES and CNPq.

ORAL TOLERANCE UNDER THE LIGHT OF COMPLEX NETWORK THEORY

MURILO DELGOBO; PEDRO JEFERSON MIRANDA; KATIA SABRINA PALUDO; SANDRO ELY DE SOUZA PINTO;

GIOVANI MARINO FAVERO.

UEPG, PONTA GROSSA - PR - BRASIL.

Introduction: Oral tolerance refers to a local and systemic state of tolerance, induced in the gut associated lymphoid

tissues (GALT) after its exposure to innocuous antigens such as food proteins. The modulation of immune response

by the ingestion of immunogenic proteins had more attention in the mid of 1970s, where was observed as a powerful

mechanism that maintains the immune homeostasis. While recent findings shed light in the cellular and molecular

basis of oral tolerance, the network of interactions between the components mediating oral tolerance has not been

investigated yet. Our work brings a complex systems theory approach, aiming to identify the contribution of each

element in an oral tolerance network, through topological and dynamic parameters. Methods and Results:

Interactions network were built from the available literature and adapted to our system. Immune components were

represented as nodes; interactions between these nodes were described as directed edges, starting from the source

node and ending in the target node. Topological quantities were assessed in order to characterize the network.

Random and direct attacks were performed on the nodes, and their impact was calculated by the decrease of giant

component’s size. For dynamical analysis, a random-walk algorithm was implemented to assess the topological

dependence which biological interactions may display. Nodes and edges were individually knocked out and dynamic

flow was performed. The degree distribution showed an exponential decay. The size of giant component was reduced

to 76% after 20% of the nodes being randomly removed, while removing 20% of the nodes with higher betweenness

centrality reduced the giant component’s size to 32%. The knockout of respective nodes iTregFoxP3+, Tr1, DC

CD103+, IL-10 and TGF-β caused great impact in the network flow, as the knockout of respective edges

naïveCD4+FoxP3

- -> iTregFoxP3

+, IL-27 -> Tr1 and TH3 -> TGF-β. Conclusion: Although most of the biological

networks are scale-free, our model of oral tolerance did not demonstrated it, given it represents a partition of the

whole immunological network (i. e. organismal). Nevertheless, our results demonstrate network robustness to random

attacks as the presence of hubs; features of biological networks. The results of knockouts correspond to biological

data, where in the absence of these elements oral tolerance was impaired.

Capes and CNPq provided support to this work.

PARADOXICAL CONTRIBUTION OF INVARIANT NATURAL KILLER T CELLS TO THE DEVELOPMENT OF

EXPERIMENTAL ASTHMA

DAVID ANIBAL GARRIDO ANDRADE; JOES NOGUEIRA NETO; NATHÁLIA AMATO KHALED; ALEXANDRE DE

CASTRO KELLER.

FEDERAL UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: invariant "Natural Killer" T cells (iNKT) represent a distinct subpopulation of T lymphocytes with the

ability to rapidly produce pro-Th2 cytokines, such as IL-4. Therefore, these cells has been extensively associated with

the pathogenesis of allergic asthma; however its contribution to the development of allergic responses remains

unclear.

Objective: Investigate the contribution of iNKT cells to asthma development.

Methods: to induction of allergic asthma, C57Bl/6 deficient in iNKT cells (Jα18-/-

) mice were immunized

subcutaneously with ovalbumin (OVA) adsorbed onto aluminum hydroxide at days 0 and 7, and challenged via

intranasal administration of OVA, at days 14 and 21. At day 22, animals were euthanized; the bronchoalveolar lavage

(BAL), draining lymph nodes, lungs and spleen were collected and analyzed by flow cytometry. In order to study the

contribution of iNKT cells to asthma development, 1x105

DN3A4-1.4 iNKT cells were injected in Jα18-/-

mice 1h before

immunizations or airway challenges.

Results: Reconstitution of Jα18-/-

animals with iNKT cells during the immunization phase increased the influx of

inflammatory cells to the airways, in comparison to Jα18-/-

. In contrast, iNKT repletion during airway challenge inhibited

the development of antigen-specific inflammatory responses.

Conclusion: our results suggest that the contribution of iNKT cells to asthma pathology depends on the context that

occurs its activation.

Financial Support: FAPESP 2011/09868-8

PGE2 AS A REGULATOR OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

LUCIANA PARONETO MEDINA1; MARIA EMÍLIA ZENTENO

2; LEANDRO PIRES ARAUJO

3; ALEXANDRE

SALGADO BASSO4; JOÃO GUSTAVO PESSINI AMARANTE-MENDES

5.

1,2,5.USP, SÃO PAULO - SP - BRASIL; 3,4.UNIFESP, SÃO PAULO - SP - BRASIL.

Introduction: During the adaptive immune response antigen-specific CD4 T lymphocytes (LTCD4+) undergo

activation, differentiation and proliferation. To reestablish homeostasis at the end of the response, the majority of

these cells are eliminated by either AICD (activation-induced cell death) or ACAD (activated T cell autonomous cell

death). Data published by our group (Weinlich et al., 2008) demonstrated that TLR-mediated release of PGE2 by APC

modulates in vitro LTCD4+ survival by preventing TcR/CD3-mediated FASL upregulation and consequent AICD. To

evaluate whether similar regulation occurs in vivo, we asked whether interference with the production of PGE2 during

the establishment of an immune response would impact on the frequency of antigen-specific LTCD4+ and the

amplitude/efficiency of the immune response. We chose a murine model of experimental autoimmune

encephalomyelitis (EAE) to address our question. We hypothesize that physiological levels of PGE2 achieved during

the initiation of specific autoimmune LTCD4+ activation is important to prevent TcR/CD3-mediated FASL upregulation

and consequent apoptosis, thereby favoring the severity of EAE. Methods and Results: To test this, we induced EAE

in wild-type female C57BL/6 mice with approximately 8 weeks and treated the experimental groups with indomethacin,

an inhibitor of cycloxigenase and consequent PGE2 production, and/or misoprostol, an PGE1 analogue, which binds to

the PGE2 receptors, EP2 and EP4. The control, vehicle-treated mice, developed normal EAE, while the indomethacin-

treated mice showed a significant reduction of disease progression. Importantly, treatment of mice misoprostol

reverted indomethacin-induced amelioration of EAE. Finally, preliminary results suggest that neither indomethacin nor

misoprostol affect lymphocyte proliferation, but rather, they have an impact on cell death, as we proposed.

Conclusion: Our results suggest that endogenous levels of PGE2 produced during the initiation of an (auto)immune

response positively impact its amplitude/efficiency and influence, in the case of EAE, the progression of the disease.

Perspectives: Further experiments were designed to estimate the actual frequency of specific autoimmune LTCD4+

cells produced in the control versus experimental groups, and to determine the relative contribution of FAS/FASL

levels. Financial support: FAPESP, CNPq.

POTENTIAL ANTI-LEISHMANIA DRUG DOWNREGULATES NITRIC OXIDE

GABRIELLE BARCELLOS BEZERRA; RAPHAELA DA SILVA; MAURÍCIO VERÍCIMO; ALICE BERNARDINO;

VERONICA DO AMARAL.

UNIVERSIDADE FEDERAL FLUMINENSE, NITERÓI - RJ - BRASIL.

Introduction: Studies demonstrated that Pyrazole Carbohydrazide Derivatives (PCD) with substituents X=Br/

Y=NO2 and X =NO2 / Y=Cl showed anti-parasitic activity in vitro against Leishmania amazonensis with no toxicity to

macrophages. These derivatives were tested in mice infected with L. amazonensis and showed a significant

therapeutic effect (Charret et al., 2009; 2013). Oral treatment with BrNO2 and NO2Cl controlled dissemination of

parasites in draining lymph nodes. Nitric oxide (NO) was observed in supernatants of lymph nodes cells from infected

treated mice. The PCD also promoted a small decrease in blood neutrophils. Moreover, epidermis and dermis had

lower levels of inflammatory infiltration compared to the infected untreated control mice. Drugs containing PCD have

showed some important biological effects as analgesic, antiplatelets and anti-inflammatory activities. Objective of this

study is to investigate immunomodulatory in vitro effect of PCD on murine cells stimulated with Lipopolysaccharide

(LPS) to drive L-arginine metabolism toward NO production.Methods and Results: peritoneal cells from BALB/c and

C57BL/6 mice were stimulated with PCD BrNO2 and NO2Cl respectively (5, 10, 20 and 40 μg/mL) cultivated at 37°C,

5% CO2 in 96-well plate. After one hour, cells were stimulated with LPS (5μg/mL) and IFN-ɤ (50U/mL), and then were

incubated for 24 hours. NO released in supernatants of cells cultures was measured by Griess reaction. Indomethacin

(1.25, 2.5 and 5μg/mL) were used as control. Results: Cells obtained from BALB/c and C57BL/6 stimulated with

LPS/IFN-ɤ or LPS alone were able to produce NO. However, there was inhibition of NO production by the LPS-

stimulated cells with BrNO2 (5 and 20μg/mL) or NO2Cl (10, 20 and 40μg/mL). The inhibition by these compounds was

comparable to nitric oxide synthase inhibitor (L-NAME). In C57BL/6 model, the compounds BrNO2 at concentrations of

5, 10, 20, 40μg/mL reduced NO production in a dose dependent manner. The compound NO2Cl also promoted NO

inhibition at concentrations of 20 and 40μg/mL. Conclusion: L-arginine metabolic pathways are decisive for parasite

growth,host defense and in the regulation of other immune components, like B cells, T cells and neutrophils. These

pathways may be investigated as therapeutic approaches for the control of Leishmania infection. The PCD are

interesting for treatment of leishmaniasis, since modulate NO production by peritoneal cells.

Financial support: PROPPI/UFF, FAPERJ, CNPq

PRE-CONCEPTIONAL IMMUNIZATION IN MICE: REGULATION OF OFFSPRING ALLERGY IS POSSIBLE

MEDIATED BY BREG AND TH17 CELLS

JEFFERSON RUSSO VICTOR; LUANA DE MENDONÇA OLIVEIRA; ALINE LIMA LIRA; MARILIA GARCIA DE

OLIVEIRA; ALBERTO JOSÉ DA SILVA DUARTE; MARIA NOTOMI SATO.

FM-USP, SÃO PAULO - SP - BRASIL.

Introduction:Previously we have been observed that pre-conceptional immunization with Ovalbumin (OVA) is able to

prevent the development of allergy in offspring.

Methods and Results: Femalemice immunized with OVA were mated with non-immunized males. Offspring were

evaluated at 3 and 20 days-old (d.o.) or immunized with OVA at 3 d.o. and assessed at 20 d.o for B and TCD4+

cytokines secreting cells.

Maternal immunization up-regulates the expression of FcgRIIb on offspring B cells at 3 and 20 d.o. This effect was

maintained even when neonates were immunized. It was in parallel to decreased sera IL-6 levels. Although offspring

from immune mothers showed similar levels of IL-10+, IL-4+ and IFN-g+ B cell frequency, but median fluorescence

intensity analysis revealed higher IL-10 intensity, regardless neonatal immunization. Moreover, newborns from

immune mothers showed an increased number of TCD4+-secreting IL-17 as well as IFN-g. After OVA-immunization

only IFN-g levels were augmented on offspring from immune mothers.

Conclusion: Up-regulation of FcgRIIb on offspring B cells occurred concomitantly to the B-cell IL-10 response and

TCD4-secreting IL-17 suggesting that maternal immunization could induce the generation of B regulatory and Th17

cells in offspring. The role of these populations on offspring in the allergy development must be investigated.

Financial support: FAPESP/ LIM-56 HCFMUSP

PROINFLAMMATORY MEDIATORS INCREASE IN LUNG AFTER FIVE DAYS OF INTESTINAL

ISCHEMIA/REPERFUSION

LUANA BEATRIZ VITORETTI1; JOÃO ANTONIO GIMENES-JUNIOR

2; BEATRIZ GALEGÃ ACCETURI

3; HELORI

VANNI DOMINGOS4; ANA CRISTINA BREITHAUPT-FALOPPA

5; ADRIANA LINO-DOS-SANTOS-FRANCO

6;

WOTHAN TAVARES-DE-LIMA7.

1,2,3,4,7.INSTITUTE OF BIOMEDICAL SCIENCE, DEPARTMENT OF PHARMACOLOGY, SAO PAULO - SP -

BRASIL; 5.SCHOOL OF MEDICINE, MEDICAL INVESTIGATION LABORATORY-11, SAO PAULO - SP - BRASIL;

6.FACULTY OF PHARMACEUTICAL SCIENCES, DEPARTMENT OF CLINICAL AND TOXICOLOGICAL

ANALYSES, SAO PAULO - SP - BRASIL.

Experimental and clinical studies have reported that intestinal ischemia/reperfusion (i-I/R) induces acute lung

inflammation (ALI). It is known that inflammatory mediators, such as TNF-a, IL-1b, LTB4 e TXB2, are generated at the

site of i-I/R and transported by lymphatic and circulatory systems, reaching the lungs and interfering with the

homeostasis of this organ. Furthermore, endogenous signals, called danger associated molecular patterns (DAMPS),

originated from cells under stress, injury or necrosis are also seen as a danger to the body and can activate the

immune system after trauma. Male Wistar rats (8 per group) were subjected to 45 min of ischemia of the superior

mesenteric artery and then to 2 h, 3, 5 or 20 days of reperfusion. After these times, small pieces of lung parenchyma

were cultured for 4 or 24 h (explant) and the levels of some inflammatory mediators quantified in the medium and in

the serum. Additionally, we also evaluated the levels of HMGB-1 and lactate dehydrogenase (LDH) in serum. Lung

explants culture from reperfused rats revealed that while the levels of IL-1b increased during the days of reperfusion,

the levels of IL-10 decreased. Furthermore, the amount of IL-17A, TNF-a and IL-4 were higher after 5 days of

reperfusion than in the others times. In serum, we noted increase of IL-12 after 5 days and of IL-10 after 3 days when

compared to basal. We did not observed differences in the others cytokines analyzed. The levels of HMGB-1 in serum

decreased after 20 days of reperfusion when compared to basal and I/R 2 h. On the other hand, the levels of LDH

were higher after 5 and 20 days of reperfusion than in the others times. In conclusion, the inflammation observed after

5 days of reperfusion may be related with the large amount of proinflammatory mediators seen in this period.

PROSTAGLANDIN E2 INHIBITS PROLIFERATION AND DIFFERENTIATION OF TH17 CELLS

NAIARA NAIANA DEJANI1; FELIPE FORTINO VERDAN

2; LUIS GUSTAVO SILVA MONNAZZI

3; VICTORIA

EUGENIA NINO4; FERNANDA NUZZI DIAS

5; ANA CAROLINA G SALINA

6; ALEXANDRA IVO MEDEIROS

7.

1,2.UNIVERSIDADE DE SAO PAULO, RIBEIRAO PRETO - SP - BRASIL; 3,4,5,6,7.DEPARTMENT OF BIOLOGICAL

SCIENCES, SCHOOL OF PHARMACEUTICAL SCIENCES - UNESP, ARARAQUARA - SP - BRASIL.

Introduction: The role of prostaglandin E2 (PGE2) in adaptive immunity has been investigated as lymphocyte

differentiation, activation and suppression of T helper cells. Some studies have shown that PGE2-cAMP signaling

inhibits almost all pathways downstream of TCR stimulation and suppresses T cell activation, proliferation and

cytokine production. Our study aimed to evaluate the role of PGE2 during the Th17 cells differentiation and

proliferation. Methodology and Results: Naïve CD4+ T cells were isolated from spleen (MACS CD4

+CD62L

+ beads)

of C57BL/6 mice. Cells were labeled with CFSE, activated with anti-CD3/anti-CD28 and cultured for 4 days in the

presence or absence of IL-6 (25 ng/mL), TGF-β (2.5 ng/mL) and PGE2 (10 - 100 nM) in IMDM 10% FBS.

Measurement of T-cell proliferation was analyzed by CFSE dilution and the Th17 cell differentiation was confirmed by

intracellular IL-17 staining after PMA /Ionomycin/Brefeldin A stimulation. TGF-β and IL-6 in the presence of exogenous

PGE2 (10 or 100 nM) were able to inhibit T cells proliferation by ~ 80% compared to positive control (IL-6, TGF-β

without PGE2). Moreover, the addition of PGE2 to Th17-polarizing cytokines was also able to inhibit the expression of

IL-17 by ~ 40% when compared to positive control. Conclusion: Some studies have shown that PGE2 is able to

enhanced Th17 responses by increasing the expression of IL-23R, however we demonstrated that when naïve CD4+

T cells are exposed direct to PGE2 plus IL-6 and TGF-β, this lipid mediator is able to inhibit the proliferation and

differentiation of Th17 cells. Financial Support: FAPESP 2011/17611-7; 2011/20199-0; 2011/23788-7; CAPES,

CNPq.

PROTECTIVE ROLE OF IL-4 DURING INTESTINAL INFLAMMATION INDUCED BY TOXOPLASMA GONDII

INFECTION

RODRIGO PEREIRA DE ALMEIDA RODRIGUES; MURILO SOLANO DIAS; MARIA DO CARMO SOUZA; MARCELA

DAVOLI FERREIRA; LUCIANA BENEVIDES; JOAO SANTANA DA SILVA; DENISE MORAIS DA FONSECA.

USP, RIBEIRAO PRETO - SP - BRASIL.

Introduction. The oral infection with Toxoplasma gondii induces an intestinal inflammation in susceptible mice, which

succumb to the disease due to the exacerbated Th1 response. Much has been studied about the pathogenic role of

Th1 cells in the infection and, recently, the protective role of regulatory T cells (Tregs). However, the involvement of

Th2 lymphocytes in the regulation of gut inflammation or in blocking the Th1 microbicide mechanisms is not

completely understood. Here we evaluated the contribution of IL-4 to the intestinal inflammation established after T.

gondii infection. Methods and Results:

Initially, we detected the IL-4 expression in the guts of C57BL/6 (susceptible) and BALB/c (resistant) mice at days 0, 3,

5 and 7 after oral infection with 5 cysts of T. gondii (ME49 strain). We found a transient expression of mRNA for IL-4 in

the susceptible C57BL/6 mice, but not in BALB/c mice, at the early points of the infection. In order to evaluate the role

of this early IL-4 production, IL-4-/-

mice, in both BALB/c and C57BL/6 backgrounds, were infected with 40 or 5 cysts of

T. gondii and mortality was monitored daily or mice were euthanized at day 8 post-infection. Surprisingly, we found

that IL-4-/-

mice, in both C57BL/6 and BALB/c backgrounds, were more susceptible to infection and presented lower

survival rate than the respective wild type (WT) mice. The protective role of IL-4 was not STAT6-mediated, since

STAT6-/-

was even more resistant than WT mice. In parallel, IL-4-/-

mice exhibited increased frequency of Th1cells

(CD3+CD4

+Tbet

+) in the lamina propria (LP) and higher expression of Tbet mRNA in the ileum. However, despite the

increased intestinal inflammation, IL-4-/-

mice presented higher frequency of Tregs in the LP, mesenteric lymph nodes

and spleens compared to C57BL/6 WT mice. Interestingly, IL-4-/-

exhibited an change in the expression of chemokine

gradient, and expressed lower levels of CCR4 and CCR2 mRNA compared to WT infected mice. Conclusion: Taken

together, these data show that IL-4 may participate in the regulation of pro-inflammatory response and, subsequently,

in the susceptibility to T. gondii infection. We suggest that in the absence of IL-4 the Th1 cells could be more activated

or less suppressed despite the presence of Tregs. Further experiments are undergoing to evaluate the influence of IL-

4 in the recruitment and suppressive activity of Tregs during T. gondii infection.

Financial support: CAPES, CNPq, FAPESP and INCTV.

PROTEIN PHOSPHATASE 2C∂ (PP2C∂) PROBABLY REGULATES THE ACTIVITY OF PHOSPHO- P90 RSK2 IN

THE LESIONAL PSORIATIC SKIN.

MADS KIRCHHEINER RASMUSSEN; LARS IVERSEN; RASMUS BOYE KJELLERUP; STINE MARIE ANDERSEN;

ANNE HALD; CLAUS JOHANSEN; BORBALA GESSER.

DEPT. OF DERMATOLOGY, AARHUS - DINAMARCA.

Introduction: The p90 ribosomal S6 kinase 1 and 2 (RSK1, 2) are serine/threonine protein kinases, activated through

the ERK1/2 signalling pathway, regulating cell proliferation. Macrophage migration inhibitory factor (MIF) and

epidermal growth factor (EGF) is elevated in the skin and serum in patients with active psoriasis compared with

normal skin and serum. The level of phospho-ERK ½ is increased in the lesional psoriatic skin. We hypothesize that

the expression phospho-RSK1, 2 might be activated in psoriasis by these growth factors, similarly to skin cancer.

Methods: The expression of phospho-RSK (T573 and S380) was investigated in paired biopsies, by phospho-specific

antibodies for the conserved sites in RSK1, 2 isoforms. We studied also the expression of Protein phosphate 2C∂

(PP2C∂) / Wound-induced protein 1 (Wip1), by Western blotting and by immune-precipitation. The expression of

PP2C∂ was studied in normal human keratinocytes with Dimethylfumarate (DMF) and MIF or EGF.

Results: We demonstrated increased level of phospho-RSK (T573) in the lesional compared with the non lesional

skin, while phospho-RSK (S380) was expressed less in the lesional compared with the non lesional skin. After co-

immuno-precipitation with monoclonal Ab for RSK2, we showed that PP2C∂ was highly associated with RSK2 in the

lesional skin. In opposition to this the total expression of the PP2C∂ /WiP1 67 kDa was higher in the non lesional and

normal skin than in the lesional skin. RSK2 was not activated in biopsies from normal skin. Stimulation with EGF

induced PP2C∂ after 24 hours which was inhibited by pre-incubation with DMF. The PP2C∂ /ILKAP isoform 46 kDa

was only expressed in the lesional skin.

Conclusion: The co-expression of PP2C∂ with RSK2 may contribute to the dephosphorylation of p-RSK2 at (S380) in

the lesional skin and reduce the activity of RSK2. As RSK2 is highly activated in psoriatic skin, compared to normal

skin, PP2C∂ may therefore interfere with keratinocytes proliferation and play a significant role in the pathogenesis of

psoriasis.

Financial support: Aage Bangs fond.

REGULATION OF CUTANEOUS TISSUE REMODELING: ROLE OF UNSATURATED FATTY ACIDS PRESENT IN

PUMPKIN OIL

BELISE MARIA OLIVEIRA BEZERRA; LUANA OLIVEIRA LEITE; ANA CAROLINA HENRIQUE DE SOUZA; MARIA

LIDUÍNA MAIA DE OLIVEIRA; DIANA CÉLIA SOUSA NUNES PINHEIRO.

UNIVERSIDADE ESTADUAL DO CEARÁ-UECE, FORTALEZA - CE - BRASIL.

Vegetable oils from medicinal plants have been used in different therapeutic protocols of inflammation and wound

healing. In response to skin lesion, a complex process involving cellular, tissue, physiological and biochemical events

is triggered. This wound healing process is characterized by proliferation and migration of epithelial cells and

fibroblasts, which are responsible for collagen production. Pumpkin seeds (Cucurbita pepo;Cucurbitaceae) provide an

oil rich in unsaturated fatty acids (UFA), which exert major functions on inflammatory responses and cicatrization. The

aim of this study was to evaluate the effect of pumpkin oil (PO) on the remodeling phase of wound healing process.

PO was obtained commercially and analyzed by gas chromatography coupled to mass spectrometry. The wound

healing effect was evaluated by excision wound model skin in Swiss mice (n=6/group), 25-30g, as follow: G1: PO in

nature; G2: sunflower oil (reference); G3: saline 0.9% (negative control). The treatments were applied topically on skin

lesions (20 μL) once a day until complete healing. On days 7 and 10 of experiment, the animals were anesthetized

(ketamine-100 mg/kg; xilazine-10mg/kg IM) and planimetry was performed. Then, wound fragments were collected

using a punch biopsy instrument. Skin samples were fixed in 10% buffered formalin (pH 7.0) and processed by

conventional histological procedures. The sections (5 µm) were cut and stained with hematoxylin-eosin and eight

areas of dermis were selected for quantification of fibroblasts under light microscope (400x). Results were analyzed

using ANOVA followed by SNK test or Kruskall-Wallis/Dunn test (p<0.05) and expressed as mean±S.D.M. The

protocol was approved by CEUA/UECE (n° 103401806/2010). PO presented linoleic (ω-6), oleic (ω-9), palmitic and

stearic acids as main constituents. Other fatty acids as linolenic (ω-3) and palmitoleic (ω-7) acids were also identified.

On day 7, the wound contractions in G1, G2 and G3 groups were 95.12±2.07, 91.20±2.84 and 80.80±5.60,

respectively, and significant differences were observed between G1 and G3, but the number fibroblasts was similar

among groups (p>0.05). On day 10, although the wounds were completely healed by macroscopic examination in all

groups, the wounds treated with PO in nature showed a higher number of fibroblasts (8.3±0.8) when compared with

reference (6.3±0.9) and negative (5.4±1.0) groups (p<0.05). These data suggest a regulation of cutaneous tissue

remodeling by PO.

RETINOIC ACID DOWN-MODULATES PRO-INFLAMMATORY CYTOKINE AND CELLULAR INFILTRATION ON

AIR POUCH EXUDATES TRIGGERED BY TLR7/TLR8 ACTIVATION

LUANA DE MENDONÇA OLIVEIRA; ALINE APARECIDA DE LIMA LIRA; MARÍLIA GARCIA DE OLIVEIRA; BRUNO

PACOLA MUNIZ; JEFFERSON RUSSO VICTOR; ALBERTO JOSÉ DA SILVA DUARTE; MARIA NOTOMI SATO.

USP, SÃO PAULO - SP - BRASIL.

RETINOIC ACID DOWN-MODULATES PRO-INFLAMMATORY CYTOKINE AND CELLULAR INFILTRATION ON

AIR POUCH EXUDATES TRIGGERED BY TLR7/TLR8 ACTIVATION

LUANA DE MENDONÇA OLIVEIRA; ALINE APARECIDA DE LIMA LIRA; MARILÍA GARCÍA DE OLIVEIRA;

BRUNO PACOLA MUNIZ, JEFFERSON RUSSO VICTOR; ALBERTO JOSÉ DA SILVA DUARTE; MARIA NOTOMI

SATO.

Laboratory of Dermatology and Immunodeficiencies (LIM-56)

University of São Paulo, School of Medicine - USP – São Paulo – Brazil.

Introduction: Vitamin A comprises a group of components, including retinoic acid (RA), an essential factor for

regulation of the immune response. RA has broad activity on the immune system, as generation of regulatory T CD4 +

CD25 + Foxp3 cells and Th2 responses, as well as to inhibit Th1 and Th17 responses. The aim of this study is to

evaluate the effect of RA in the acute inflammatory response induced by TLR7/TLR8 activation in air pouch exudates

model, analyzing cellular infiltration and cytokines secretions.

Methods and Results: Air pouche was induced on the back of BALB/c mice (4-5 weeks old), and reinforced after 4

days. On the 6th day, RA (50 µg) was injected in the air pouche, and after 1 h they received a ligand of TLR7 and

TLR8, CL097 (12 µg/mL). After 3 hrs, cells were harvested from exudate and supernatants were assessed for

cytokines measurements by cytometry bead array. Mice groups receiving only vehicle, or RA or CL097 were

performed. Each group was performed with 5-8 mice, in at least 4 different experiments. The results showed that RA

is able to trigger cells infiltration composed of macrophages, and minor frequency of eosinophils and neutrophils,

compared to vehicle. Moreover, RA down-modulates TNF-α secretion in the exudate. To induce an inflammatory

milieu, we stimulated via TLR7/8, which induced an intense inflammatory infiltrate composed by neutrophils, followed

by macrophages, eosinophils. Notably, RA was able to inhibit the inflammatory cell influx-induced by CL097. Also, RA

decreased IFN-g secretion-induced by CL097, but not interfere in the IL-4, IL-6, and IL-5 secretions levels.

Conclusion: The findings showed that RA has anti-inflammatory role on TNF-α secretion. RA was also able to control

the influx of inflammatory cells in the exudates, induced by activation though TLR7/8 signaling pathway. The results

suggest that RA may inhibit, partially, the inflammatory effect mediated by an adjuvant of innate immune response.

Financial support: CNPq/FAPESP/LIM-56/HCFMUSP.

ROLE OF TNF-ALPHA RECEPTOR1 IN THE DEVELOPMENT OF METABOLIC ALTERATIONS IN MICE FED

HIGH REFINED CARBOHYDRATE-CONTAINING DIET

LAÍS BHERING MARTINS; MARINA CHAVES DE OLIVEIRA; DÉBORA FERNANDES RODRIGUES; JAQUELINE

PEREIRA LANA; ZÉLIA MENEZES-GARCIA; LEDA QUERCIA VIEIRA; MAURO MARTINS TEIXEIRA; ADALIENE

VERSIANI MATOS FERREIRA.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction

The Tumor Necrosis Factor-alpha (TNF-α) is a proinflammatory cytokine produced mainly by macrophages. This

cytokine modulates the metabolism of nutrients by stimulating lipolysis, regulating adipocyte function and insulin

signaling. It has been shown that this cytokine is also involved in the pathophysiology of many liver diseases. The aim

of this study was to verify whether the lack of TNF-α receptor 1 protects from metabolic and liver dysfunction in mice

fed a high refined carbohydrate-containing diet (HC).

Methods and Results

The experimental protocol was approved by the Ethics Committee in Animal Experimentation at the Universidade

Federal de Minas Gerais (protocol N°: 67/2012). Wild-type (WT) (n=5-7) and TNF-α receptor 1 knockout (TNF-αR1-/-

)

(n=5-11) mice were divided into groups according to experimental diets: chow diet (C) or high refined carbohydrate-

containing diet (HC). After 16 weeks of dietary treatment, the animals were submitted to an oral glucose tolerance test

(OGTT) (2 g of D-glucose/ kg body weight). Three days after, mice were killed and the epididymal, retroperitoneal and

mesenteric adipose tissues were weighed to determine the adiposity index. The liver was removed and total fat and

cholesterol were measured. The TNF-αR1-/- mice showed higher body weight gain over the treatment compared to

the WT (WT-C: 5.73 ± 2.56 g; WT-HC: 6.10 ± 3.07 g; TNF-αR1-/-

-C: 7.89 ± 3.41 g; TNF-αR1-/-

-HC: 7.89 ± 3.43 g). Mice

fed HC diet showed increased adiposity in both groups (TNF-αR1-/-

and WT), and TNF-αR1-/-

C had higher adiposity

compared to WT that fed the same diet (WT-C: 1.23 ± 0.36 %fat; WT-HC: 3.18 ± 0.77 %fat; TNF-αR1-/-

-C: 2.41 ± 0.15

%fat; TNF-αR1-/-

-HC: 2.93± 0.38%fat). WT-HC mice showed lower glucose tolerance when compared to the WT-C

and TNF-αR1-/-

HC. TNF-αR1-/-

HC mice showed similar glucose tolerance when compared to mice fed chow

diet. Furthermore, TNF-αR1-/-

-HC mice showed lower total fat and cholesterol in the liver when compared to WT-HC

(WT- HC: 32.86 ± 3.02 mg/100mg; 25.95 ± 2.70 mg/dL/100mg, respectively; TNF-αR1-/-

-HC: 28.00± 6.94 mg/100mg;

17.57± 7.53 mg/dL/100 mg, respectively).

Conclusion

Although the absence of TNF-alpha receptor 1 may be associated with increased adiposity, our results suggest that it

might protects against the development of glucose intolerance and liver dysfunction in mice fed HC diet.

Financial support: CNPq, FAPEMIG, CAPES.

SACCHAROMYCES BOULARDII AND BIFIDOBACTERIUM BREVE DOWNREGULATION INFLAMMATORY

RESPONSE AND ATTENUATE THE EXPERIMENTAL ULCERATIVE COLITIS

RENATA LACERDA LIMA; RAQUEL DUQUE ARIFA; ZÉLIA MENEZES GARCIA; TALLES PROSPERI DE PAULA;

LUANA ANTUNES DOURADO; FLAVIANO DOS SANTOS MARTINS; DANIELLE DA GLÓRIA SOUZA.

UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction: Inflammatory bowel diseases (IBD) such as ulcerative colitis are chronic inflammatory disorders of the

gastrointestinal tract. These IBDs result from an immune response to changes in intestinal microbiota in patients with

a genetic predisposition. Objetive: Then, the aim of this study was to investigate the effect of Saccharomyces

boulardii e Bifiddobacterium breve in experimental colitis induced by DSS. Methods: C57BL6 male mice were used

and the colitis was induced by dextran sulphate sodium (DSS) 3% in water from day 0 to day 5. The animals received

pre-treated with S. Boulardii (108 CFU) or B. breve (10

8 CFU) or PBS 1x ten days before and during the induction of

colitis. We analyzed the weight loss, the consistency and the presence of blood in the stool, the clinical signs of

disease and the colon length. We also evaluated the neutrophils, eosinophils and macrophages influx into the colon

through the activity of MPO, EPO and NAG, respectively. In addition, we measured hematocrit and leukocyte count in

the blood and the colon concentration of IL-1β and CXCL1 by ELISA. Results: The vehicle group has shown greater

weight loss and greater clinical score than control group. These mice also have shown shortening of the colon, lower

hematocrit, higher total number of leukocytes, monocytes and neutrophils in the blood when compared to controls.

However, the animals treated with the microorganisms have been protected against these parameters. Furthermore,

colitis has induced higher IL-1β and CXCL1 concentration in the colon when compared to control group. But only the

group that received the B. breve has shown lower IL-1β concentration. Animals with colitis have shown greater activity

of MPO and EPO compared to control, and the animals treated with probiotics have not interfered in these

parameters. There was no difference between groups in the activity of NAG. Conclusion: We conclude that S.

boulardii and B. breve downregulation inflammatory response and attenuate the clinical sings in experimental colitis

induced by DSS.

SALIVA FROM AMBLYOMMA CAJENNENSE TICKS MODULATES MATURATION OF MURINE DENDRITIC

CELLS

TAMIRES MARIELEM CARVALHO-COSTA; MARIA TAYS MENDES; ANA CAROLINA BORELLA MARFIL ANHÊ;

THIAGO ALVARES COSTA; CARLO JOSÉ FREIRE OLIVEIRA.

FEDERAL UNIVERSITY OF TRIÂNGULO MINEIRO, UBERABA - MG - BRASIL.

Introduction: Ticks are blood-sucking arthropods. To feed successfully in the host skin, ticks inject, via saliva, a wide

array of pharmacologically active compounds with vasodilatory, anti-inflammatory and immunomodulatory activities.

Dendritic cells (DCs) play a key role in initiation and regulation of inflammation and adaptive immunity against endo-

and ectoparasites. Although there are a growing number of molecules already described in some tick species

modulating DCs, studies showing the effect of A. cajennense tick saliva on these cells are unknown. Thus, we aim to

identify if compounds present in the saliva of A. cajennense ticks are able to modulate DCs maturation. Methods and

Results: The saliva-collection procedure was performed using fully engorged female ticks by inoculation of a solution

of dopamine in PBS. Bone Marrow-derived DCs were generated from C57BL/6 mice, as previously described (J Biol

Chem. 286:10960-9, 2011). DCs were pre-incubated with saliva (diluted 1:10, 1:30, 1:100, 1:300, 1:1000 v/v) for 1

hour and then stimulated with LPS, a TLR-4 ligand (100 ng/mL), for 18 hours. The expression of surface markers

(CD11c, CD40, CD86, MHC-II) were determined by flow cytometry. The production of TNF-α, IL-12p40 and IL-10 were

determined by ELISA. Tick saliva at remarkably diluted concentrations (<1:1000 and 1:300, respectively) was able to

inhibit TNF-α and IL-12p40 (56.2% and 37.2% respectively) while increased IL-10 cytokine production up to 147%

(<1:100) by DCs stimulated by LPS. Tick saliva (<1:30 and 1:100, respectively) was also able to inhibit (p<0.05) the

expression of CD40 (100%) and CD86 (80.8%) in DCs stimulated with the same ligand. We also examined whether

the effect of saliva could be caused by dopamine, because this mediator was used to induce tick salivation and is

detected in pooled samples. Dopamine had no effect on the expression of CD86 and MHC-II expression and IL-10

production by DCs. Conclusion: These results are particularly important because TNF-α and IL-12 cytokine secretion

and CD40 and CD86 expression are major characteristics of maturing DCs, cytokines and molecules on DC surfaces

important in initiating inflammation and adaptive immunity against pathogens. Moreover, the increase in the IL-10

suggests that ticks are able to promote an anti-inflammatory milieu in the skin site facilitating their feeding and

survival.

Financial support: FAPEMIG, UFTM, CNPq, CAPES.

SENSITIZATION WITH HUMAN TUMOR CELLS TREATED WITH BABASSU MESOCARP INDUCE THE MURINE

SPLENOCYTES PROLIFERATION IN VITRO

JULIANA LUCENA DOS SANTOS; JOSEMAR MARCELINO FERREIRA GODINHO JUNIOR; CAROLINE SILVA

COSTA DE ALMEIDA; ELZA MARIA MORAES DE ARAÚJO; LETÍCIA PRINCE PEREIRA PONTES; THIARE SILVA

FORTES DA CUNHA; DIÊGO DE SOUSA ARRUDA LOPES; GRACIOMAR CONCEIÇÃO COSTA; FLÁVIA RAQUEL

FERNANDES DO NASCIMENTO; ANA PAULA SILVA AZEVEDO DOS SANTOS.

IMMUNOPHYSIOLOGY LABORATORY / FEDERAL UNIVERSITY OF MARANHÃO (UFMA), SÃO LUÍS - MA -

BRASIL.

Introduction: The immunotherapy is a promising strategies for the cancer treatment. Previous studies have shown

that some natural products are able to induce a specific immune response and they destroy tumor cells. The Babassu

(Attalea speciosa Mart.) coconut mesocarp is composed mainly of carbohydrates and it has immunomodulatory

properties. The aim of this study was to evaluate the murine splenocytes proliferation in vitro after sensibilization with

MCF7 and SKBR3 human tumor line treated with an aqueous extract of babassu mesocarp (BM) in vivo.

Methods and Results: The babassu mesocarp powder (BM) was macerated in water at a concentration of 20mg/mL

for 24 hours and filtered. Swiss mice (n = 18) were sensitized on day 0, 5 and 15 with saline (control), extract (BM),

1x105 tumor cells (MCF7 or SKBR3) and 1x10

5 tumor cells pretreated with BM (BM+MCF7 or MB+SKBR3), by

subcutaneous route. On the 16th day of the animals were euthanized and the spleen was removed and triturated to

obtain the splenocytes. These cells were incubated during 2 hours for separation of adherent and nonadherent cells.

The non-adherent cells were labeled with carboxyfluorescein diacetate (CFSE) and maintained in co-culture with

MCF7 or SKBR3 for 5 days at a ratio of 30:1 spleen cells to tumor cells. After 5 days of co-culture was analyzed the

CFSE median fluorescence intensity. Splenocytes obtained from stimulated groups showed higher proliferation in

MCF7 (45,2±3,2), MB (47±7,4) and MB+MCF7 (47,1±0,1) than the control group (65±6,7). Similar, the groups SKBR3

(51,05±0,05), MB (61±10,3) and MB+SKBR3 (48,2±3,1) showed higher proliferation when compared control group

(94,05±21,9).

Conclusion: The results showed that sensitization with tumor and MB induced the splenocyte proliferation when in

co-culture with tumor cells. However, the MB group showed high proliferative response, without tumor treatment. This

data suggesting an immunomodulatory effect of babassu mesocarp.

Financial support: Foundation of Scientific Research and Development of Maranhão (FAPEMA), National Council of

Scientific and Technological Development (CNPq) and Federal University of Maranhão (UFMA).

SOCS-2 PARTICIPATES OF INFLAMMATORY RESPONSE FOLLOWING ISCHEMIA AND REPERFUSION

RAQUEL DUQUE NASCIMENTO ARIFA; RENATA LACERDA LIMA; TALLES PROSPERI DE PAULA; ZELIA

MENEZES GARCIA; FABIANA SIMÃO MACHADO; DANIELLE GLÓRIA SOUZA.

UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction: Intestinal ischemia is a severe abdominal emergency. During ischemia occurs hypoxia and absence of

the input of nutrients that result in cellular death and tissular damage. The delay in ischemia diagnosis and

treatment can lead to bowel infarction and death, being necessary the reperfusion to restore tissular functions.

However the reperfusion can lead increased ROS levels, inflammatory mediators release, recruitment and activation

of leukocytes which exacerbate intestinal injury. Several studies have demonstrated the importance of pro-

inflammatory cytokines in reperfusion injury. SOCS are negative regulators of some cytokines. Recently works have

shown SOCS2 as negative regulator of inflammatory response in Sepsis and toxoplasmosis infection, but still the

moment is not demonstrate the role of SOCS2 in intestinal inflammation. Objective: Thus, the aim of this present

study was investigate the role of SOCS2 in intestinal ischemia and reperfusion in mice. Methods and Results:

Intestinal ischemia was induced by superior mesenteric artery occlusion for 30 minutes in WT and SOCS2-/-

C57BL/6

mice following by four hours of reperfusion. After this period, mice were euthanized and intestines were removed and

homogenized for analysis of leukocytes infiltrate, cytokines production, western blotting and histology. Animal

procedures were performed in accordance with the Animal Care and Use Committee of Universidade Federal de

Minas Gerais. After ischemia and reperfusion intestinal (IRI) there was increase of SOCS2 expression in intestine of

WT mice. Interestingly, the lethality rate and intestinal injury increases in SOCS2-/-

mice compared WT mice. The

increase of the severity reperfusion injury in this former group was associated with neutrophils influx, vascular

permeability, hemoglobin and IL-6 increases. Interestingly, LXA4 and IL-10 production were increased in WT mice

after IRI, however these increased were abolished in SOCS2-/-

mice. Both LXA4 and IL-10 have anti-inflammatory

roles. The abolishment of the increase of these cytokines in SOCS2-/-

mice can be partly responsible for the disease

exacerbation in this group (P <0,05). Conclusion: These results suggest that SOCS2 down regulation the intestinal

inflammatory response and is involved in LXA4 production after IRI.

Supported by Fapemig and CNPq

SUPPLEMENTATION WITH CARBOHYDRATE INFLUENCE THE CITOKINES AFTER THE EXERCISE IN

HYPOXIA CONDITION

IRENE GUARIDO LUZ BITTAR; EDGAR TAVARES DA SILVA; SAMILE AMORIM DOS SANTOS; ALINE

VENTICINQUE CARIS; FABIO SANTOS LIRA; EDUARDO DA SILVA ALVES; MARCO TULIO DE MELLO;

RONALDO VAGNER DOS SANTOS THOMATIELI.

UNIFESP, SÃO PAULO - SP - BRASIL.

Introduction: The exercise realized in hypoxia equivalent to 4300 m is associated with elevated inflammatory

mediators in humans. On the other hand, the supplementation with carbohydrate has anti-inflammatory effect once the

exercise is executed at sea level. The aim this study is evaluates the supplementation with carbohydrate on cytokines

after an exercise in hypoxia condition at 4 200 m. Methods and Results: Seven male adult volunteers exercised for

60 min at intensity of 50% VO2Peak were evaluated into three randomly and distinct conditions: normoxia, hypoxia

and hypoxia+carbohydrate suplementation. Blood samples were collected in rest, immediately after the end of

exercise and after one hour of recovery. The solution of carbohydrate was administered to 6% (60g maltodextrina.L-1

)

60 minutes before (200ml), during the begin and every 20 minutes the volunteer drank 50ml until complete 200 ml in

each exercise sessions. In normoxia and hypoxia conditions observed significant increase of concentration of IL -6

immediately after the end of exercise (3.15 ± 1.64 and 3.30 ± 0.85, respectively) and after one hour of recovery (3.52

± 0.5 and 2.87 ± 0.99, respectively) when compared with the rest (1.13 ± 0.5 and 1.17 ± 0.14). In the hypoxia

condition with carbohydrate observed significant increase of concentration of IL -6, and TNFα after the exercise (2.21

± 0.59 and 6.22 ± 1.89, respectively) compared with the rest (0.66 ± 0.32 and 2.65 ± 1.25, respectively) (p<0,05). In

addition observed that the TNFα (5.36 ± 5.59) and the balance of IL-2/ IL-4 (11.84 ± 15.38) increased in the one hour

recovery compared with the rest (2.65 ± 1.25 and 0.21 ± 0.22, respectively) (p<0,05). Conclusion: The carbohydrate

supplementation can influence the balance of cytokines during and after the acute exercise, attenuating the effects of

hypoxia.

Financial support: CEPE, CEMSA, CNPq, AFIP and FAPESP.

THE CYCLIC NITROXIDE TEMPOL INHIBITS MURINE HEPATITIS VIRUS, STRAIN A59 (MHV-A59)

PROLIFERATION BY INHIBITORY EFFECT ON CYCLOOXYGENASE 2 PRODUCTION

MARIA HELOISA TSUHAKO1; OHÁRA AUGUSTO

2.

1.INSTITUTO BUTANTAN, LABORATÓRIO DE IMUNOLOGIA VIRAL, SÃO PAULO - SP - BRASIL; 2.INSTITUTO

DE QUÍMICA, DEPARTAMENTO DE BIOQUÍMICA, USP, SÃO PAULO - SP - BRASIL.

Introduction: Recently, we showed that tempol greatly attenuates multiple sclerosis in an experimental model

of the disease induced by the murine hepatitis virus, neurotropic strain MHV-A59. In parallel, tempol

decreased the viral titers in the CNS of treated mice. Considering the lack of knowledge about the effects of

tempol on viral replication, we investigated the phenomenon in cell cultures. Methods and Results: Tempol

treatment was performed in astrocytoma DBT cells before and after virus adsorption. The cultures were

infected with MHV-A59 (MOI 0.04), treated with 500 µM tempol and incubated at 37°C for 8h. The number of

MHV-infected cells was determined by counting the number of syncytia. The expression level of the N gene of

MHV-A59 was determined by Taqman RT-PCR. The cytotoxicity of tempol was checked by the Trypan Blue

Exclusion Test. Significant reduction of viral production (54 ± 6.8%, p ˂0.007, n=3-4) and of viral RNA

synthesis (45,67 ± 6,50%, p<0.029, n=3-4) were observed when the cells were treated with tempol after viral

adsorption. Marginal differences were observed when the cells were treated before viral adsorption as shown

by the relative % of infected cells (90,31 ± 3.71%, p = 0,2, n=3-4 ) and the % of viral RNA (91,33 ± 13.22%, p =

0.38, n=3-4). Since prostaglandins, generated by cyclooxygenases, have been shown to participate in the

regulation of virus replication and in the modulation of inflammatory responses following infection, we

analyzed the levels of COX-2, COX-1, iNOS and Nrf2 proteins by western blotting. At 8 hpi, these experiments

showed a markedly inhibitory effect on COX-2 expression, a decrease in iNOS and a marginal increase of Nrf2

levels in lysates of cells treated with tempol compared with mock-treated MHV-A59 infected cells. The COX-1

protein levels remained unchanged. Conclusions: These results suggest that tempol at a non cytotoxic

concentration interferes with viral RNA synthesis and with production of infection particles when added after

virus adsorption by its inhibitory effect on COX-2 expression. Our findings also shown that nitroxides have

potential antiviral activity and that tempol may be an usefull adjuvant in the treatment of viral infection.

Financial support: Fapesp

THE EXPRESSION OF TH17 ASSOCIATED CYTOKINES IN CERVICAL INTRAEPITELIAL NEOPLASIA AND

CERVICAL CANCER

LIDYANE NEVES MIRANDA1; INGRYD CAMARA MORAIS

2; FERNANDA PRISCILA SANTOS REGINALDO

3; MARIA

DO PERPÉTUO SOCORRO NOBRE4; CERISE MARIA CORTEZ GOMES

5; CARLOS ANDRÉ NUNES JATOBÁ

6;

JOANLISE MARCO LEON ANDRADE7; EDUARDO ANTÔNIO DONADI

8; ANA KATHERINE SILVEIRA

GONÇALVES9; JANAÍNA CRISTIANA OLIVEIRA CRISPIM

10.

1,2,3,10.DEPARTMENT OF TOXICOLOGY AND CLINICAL ANALYSIS, SCHOOL OF PHARMACEUTICAL

SCIENCES, UFRN, NATAL - RN - BRASIL; 4,5.HOSPITAL DR. LUÍS ANTÔNIO-LIGA, NATAL - RN - BRASIL;

6.DEPARTMENT OF PATHOLOGY, SCHOOL OF MEDICINE, UFRN, NATAL - RN - BRASIL; 7.DEPARTMENT OF

STATISTICS, UFRN, NATAL - RN - BRASIL; 8.DIVISION OF CLINICAL IMMUNOLOGY, FACULDADE DE

MEDICINA DE RIBEIRÃO PRETO (FMRP), RIBEIRÃO PRETO - SP - BRASIL; 9.DEPARTMENT OF TOCO

GYNECOLOGY, SCHOOL OF MEDICINE, UFRN, NATAL - RN - BRASIL.

Introduction: Cervical intraepithelial neoplasia is one of the most frequent causes of cancer among women

worldwide. The development of such lesions is defined through well-known steps: human papilloma virus (HPV), viral

genomic integration, persistence, progression of gradually infected cells to neoplasia, and invasion. In the past few

years, Th17 cells and their effector cytokines are increasingly being recognized as key players in inflammation,

autoimmunity and allergic reactions. However, the nature of Th17 cells is elusively understood in cancer patients (Clin

Chim Acta. 413:23-24, 2012). The purpose of this study was explore the role of Th17 cells in both the cervical

microenvironment as at a systemic level, associating with clinicopathological and prognostic data involved with

intraepithelial cervical neoplasia.

Methods and results: In the present study, we have evaluated the expression of the IL-17 (eBioscience, San Diego,

CA,USA) and IL-23 (Abcam, Cambridge, MA, USA) by immunohistochemistry in 121 cervical histological samples

including 22 cases with chronic cervicitis(CC), 28 CIN I, 36 CIN II/III and 35 cases with cervical cancer. The detect

expression levels of IL-6 (RB, eBioscience, San Diego, CA, USA) and IL-17 (RB, eBioscience, San Diego, CA, USA)

were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that expressions of IL-17 and

IL-23 were increased in group CC and CIN I (p-value= 0.003) when compared to other groups. Meanwhile, the

cytokine IL-6 and IL-17concentrations were significantly higher in CIN I patients than those in other groups.

Conclusion: Interestingly, the levels of Th17 cells were positively correlated with early stages of cervical

intraepithelial neoplasia. We conclude that the Th17 cells and the changes of Th17-associated cytokines could be the

indicators of the disease and promising candidates of prognostic biomarkers of intraepithelial cervical neoplasia.

Financial support: CNPq, CAPES and PROPESQ

THE MODULATORY EFFECT OF HIGH MOLECULAR WEIGHT COMPONENTS FROM ASCARIS SUUM

EXTRACT IS DEPENDENT OF DC-SIGN AND MR RECEPTORS

BRUNA CRISTINA FAVORETTO1; JOSÉ ANTONIO PORTES JUNIOR

2; JACQUELINE FÁTIMA JACYSYN

3; ELIANA

FAQUIM DE LIMA MAURO4.

1,2,4.INSTITUTO BUTANTAN, SÃO PAULO - SP - BRASIL; 3.FACULDADE DE MEDICINA LIM-62, SÃO PAULO -

SP - BRASIL.

Introduction: Dendritic cells (DCs) recognize compounds of pathogens through distinct receptors and acquire the

ability to induce cellular response. C-type lectin receptors (CLRs) in DCs interact with carbohydrate structures and are

involved in activation or homeostasis of the immune system. We showed that high molecular weight components (PI)

from Ascaris suum down-modulate the DCs activities. Here, it was analyzed the glycosylated contents in PI, the

binding to CLRs on DCs and its ability to modulate these cells maturation. It was also evaluated the role of the CLRs

in the PI modulatory effect on the DCs ability to induce T cell proliferation.

Methods: N-glycosylated components of PI were obtained by affinity to Concanavalin A-Sepharose (ConA). The

modulatory effect of these N-glycosylated components was evaluated in DCs incubated with LPS for 18h. Purified

DCs from OVA-primed mice were pulsed with OVA, OVA+PI, OVA+PI+mannan or OVA+PI+anti-CLRs mAbs for 18h.

After this, the DCs were incubated with T cells from OVA-primed mice and the proliferation evaluated. The PI binding

to CLRs on DCs was analyzed by confocal microscopy. For this, DCs were incubated at 4°C with PI-Alexa, BSA-

Alexa, PI-Alexa+mannan or PI-Alexa+anti-DC-SIGN or MR for 30 min.

Results: The flow cytometry analysis showed that the ConA-linked components inhibited the expression of class II

MHC, CD40, CD80 and CD86 molecules on DCs incubated with LPS compared with those observed on DCs

incubated only with LPS. Low proliferation was verified in cultures of T cells and DCs pulsed with OVA+PI. In contrast,

the T cell proliferation was partially restored when the DCs were incubated with mannan or anti-DC-SIGN or MR

followed incubation with OVA+PI. Higher fluorescence was observed in DCs incubated with PI-Alexa. On the other

hand, the PI-binding on DCs was inhibited when the cells were previously incubated with mannan, anti-DC-SIGN or

anti-MR.

Conclusion: The results indicate that N-glycosylated components of the PI mediate the down-modulation of the DCs

activity. The CLRs, as DC-SIGN and MR, are involved in this process. Financial support: FAPESP (2010/10393-1 and

2011/23735-0).

THE ROLE OF T-BET IN IEL HOMEOSTASIS AND FUNCTION

BERNARDO SGARBI REIS1; FREDERICO COSTA-PINTO

2; DANIEL MUCIDA

3.

1,3.THE ROCKEFELLER UNIVERSITY, NEW YORK - ESTADOS UNIDOS; 2.USP, SAO PAULO - SP - BRASIL.

Intraepithelial lymphocytes can be divided in 5 groups based on surface marker expression; TCRab–CD8aa

+,

TCRgd+CD8aa

+, TCRab

+CD8ab

+, TCRab

+CD8aa

+ and TCRab

+CD4

+. The last can be further divided in CD4

+CD8aa

and CD4+CD8aa

+. CD8aa expression by intestinal intraepithelial CD4

+ T cells is accompanied by loss of the CD4 T

cell developmental transcription factor ThPOK in a TGF-b and RA dependent manner. We have previously showed

that in vitro stimulation of TCR transgenic OTII CD4 cells in the presence of TGF-B and RA lead to the development of

CD4+CD8aa

+ cells and Treg FOXP3+ cells. Interesting these two cells have different levels of T-bet expression,

suggesting that T-bet may be a molecular clue for CD4+CD8aa

+ cell vs Treg cell development decision. Here we

demonstrate that IFN-g, a potent T-bet inducer, can induce higher frequency of CD4+CD8aa

+ cells in detrimental of

Treg FOXP3+ cell development in the presence of TGF-b and RA. Depletion of Runx3 or T-bet abolished CD8aa

induction in OTII CD4 cells. In vivo data shows that loss of ThPOK and acquisition of CD8aa expression by intestinal

CD4 cells is partially regulated by T-bet. Intravital imaging shows that changes in CD4 cells induced by the

CD4+CD8aa

+ pathway is required for migration to the intraepithelial compartment. Moreover, absence of T-bet

expression renders less TCRgd cells and no CD4+CD8aa

+ cells. Taken together these data suggest that T-bet

modulates intestinal CD4 cells to acquire intraepithelial characteristics and permission to migrate to the epithelial

compartment.

THE USE OF ADIPOSE TISSUE DERIVED STEM CELLS (ASC) AS A METHOD FOR CONTROL OF THE

GRANULOMATOUS REACTION IN SCHISTOSOMA MANSONI CHRONIC INFECTION

ADRIANA BOZZI1; ANA THEREZA CHAVES

2; KELLY ALVES BICALHO

3; TALITA ROCHA GOMES

4; FÁBIO

CASSIRER COSTA5; GUILHERME OLIVEIRA E SILVA

6; JULIANA LOTT CARVALHO

7; ANDREA TEIXEIRA-

CARVALHO8; OLINDO ASSIS MARTINS-FILHO

9; DENISE CARMONA CARA MACHADO

10; ALFREDO MIRANDA

GOES11

; RODRIGO CORREA-OLIVEIRA12

.

1,3,4,5,6,8,9,12.CENTRO DE PESQUISAS RENÉ RACHOU - FIOCRUZ MINAS, BELO HORIZONTE - MG - BRASIL;

2.FACULDADE DE MEDICINA DA UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG -

BRASIL; 7,10,11.INSTITUTO DE CIÊNCIAS BIOLÓGICAS DA UNIVERSIDADE FEDERAL DE MINAS GERAIS,

BELO HORIZONTE - MG - BRASIL.

Introduction: Mesenchymal stem cells (MSC) have the ability of self-renewal and differentiation into various

mesodermal cell lineages. Recently, it has been observed that MSC have potent anti-proliferative and anti-

inflammatory effects in autoimmune and inflammatory diseases as a new strategy for immunosuppression. The control

of inflammation in infectious and parasitic diseases by MSC is yet to be evaluated. The present study is aimed to

evaluate if the ASC could regulate the inflammation in the mouse experimental S. mansoni infection, a major cause of

granulomatous reaction and tissue fibrosis.

Methods and Results: The ASC were isolated from C57BL/6 mice, expanded in vitro and characterized phenotypic

and functionally. These cell were injected by the tail vein into infected C57BL/6 mice (n=5) during the chronic infection

and after treatment with praziquantel (200mg/kg). Fifteen, thirty and sixty days after the ASC therapy, the splenocytes

were obtained and T cells activation evaluated by the expression of CD25, CD69, CD28 and CTLA-4 molecules. The

liver granulomatous reaction was analyzed by histological staining. The results showed a decrease (P<0.05) in TCD4+

regulation as determined by CD25 (25,7±4,95), CD69 (36,4±5,65) and CTLA-4 (43,9±21,4), after ASC therapy. We did

not observe significant modifications in molecules expression by TCD8+ subset. The granuloma analysis reveled a

reduction (P<0.05) in the number (1±1) and size area (4583,3±4222,96), after sixty days of ASC therapy.

Conclusion: our results suggest a potential use of ASC to modulate the immune response and their use for cellular

therapy in severe chronic S. mansoni infection.

Financial support: CNPq and FAPEMIG

THERAPEUTIC USES OF SOLUBLE FIBER IN ACUTE AND CHRONIC DSS-INDUCED COLITIS IN MICE

ANA LETÍCIA MALHEIROS SILVEIRA; ANGELICA THOMAZ VIEIRA; MARINA CHAVES OLIVEIRA; ADALIENE

VERSIANI MATOS FERREIRA; ANA CRISTINA GOMES-SANTOS; MILENE ALVARENGA RACHID; MAURO

MARTINS TEIXEIRA.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Inflammatory Bowel Diseases (IBD), including Crohn’s disease and Ulcerative Colitis, have an

important impact on global health and their incidence has increased considerably around the world. It is believed that

an imbalance of gut microbiota is associated with the development of IBD in susceptible individuals. Consumption of

prebiotics could be an alternative to modulate intestinal bacterial communities which could lead to amelioration of

inflammatory responses in the gut. Pectin is a dietary soluble fiber which is fermented at the colon and has prebiotic

properties. The aim of this study was to evaluate the effect of a pectin-rich diet (high fiber – HF), used as a pre-

treatment and as therapy in murine models of colitis induced by sulfate sodium dextran (DSS). Method and results:

Female BALB/c mice were fed chow or HF diet for 40 days prior to colitis induction. Acute colitis was induced by

adding DSS to drinking water for 7 days (control received water). Pre-treatment with HF diet, in animals with acute

colitis, substantially improved clinical signs of disease, colon length, reduced neutrophil recruitment and mucosal

epithelial lesion, when compared to mice fed with chow diet. This improvement was reflected in survival. 100% of mice

pre-treated with HF diet survived against only 40%animals fed with chow diet. The HF diet had similar effect on clinical

and histopathological parameters to pre-treatment with acetate (a short chain fatty acid with anti-inflammatory

property). In a chronic model of colitis induced by cycles of DSS (3%), animals pre-treated with the HF diet improved

clinically and maintained colon length, reduced eosinophil peroxidase activity (EPO) and had greater levels of

interleukin (IL)-6 and IL-10 in the colon. When treatment with HF diet was started after disease was established (at

cycles 3, 4 or 5 of DSS), there was only partial effect on EPO and colon weight compared to animals fed with chow

diet. Other clinical and laboratory parameters were unaltered. Conclusion: HF diet had major effects on the outcome

of colitis induced by DSS and the protective effects are mimicked by acetate, suggesting a possible generation of this

molecule in the beneficial effects of the HF diet. However, beneficial effects are mostly seen when diet is given as a

pre-treatment, suggesting that HF diet alone may not be sufficient to alter disease in IBD. Financial Support: Capes,

CNPq and Fapemig.

THERAPUTIC POTENTIAL OF LIPOXIN IN OBESTIY-INDUCED ADIPOSE INFLAMMATION AND CHRONIC

KIDNEY DISEASE

EMMA BORGESON1; CATHERINE GODSON

2; KUMAR SHARMA

3.

1,3.CENTER FOR RENAL TRANSLATIONAL MEDICINE, DIVISION OF NEPHROLOGY-HYPERTENSION, UC SAN

DIEGO, SAN DIEGO - ESTADOS UNIDOS; 2.DIABETES COMPLICATIONS RESEARCH CENTRE, CONWAY

INSTITUTE, UNIVERSITY COLLEGE DUBLIN, DUBLIN - IRLANDA.

Introduction: Lipid molecules likely play an important role in linking adipose inflammation to obesity-related chronic

kidney disease (CKD). Indeed, impairment of specialized pro-resolving mediators (SPMs) may underlie development

of obesity-related pathology (Diabetes 62:1945-56, 2013). SPMs, including lipoxins (LXs), actively promote the

resolution of inflammation. A potential beneficial effect of LXs may be via enhancing the protective hormone,

adiponectin. We have explored the therapeutic potential of LXs in experimental obesity-induced CKD in wild type and

adiponectin KO mice.

Methods and Results: C57BL/6 mice were subjected to 12 wk standard (10% fat) or high-fat diet (HFD, 60% fat);

n=7. HFD mice developed obesity and CKD, as evidenced by impaired glucose tolerance and albuminuria. As

interventional therapeutics, LXA4 (5ng/g) and benzo-LXA4 analogue (1.7ng/g) were given 3 times weekly, between wk

5-12. LXA4 attenuated obesity-induced CKD, reducing glomerular expansion (3430±127µm2 vs 2916±116µm

2,

p<0.01), mesangial matrix accumulation (1932±61µm2 vs 1669±58µm

2, p<0.01) and urine H2O2 (228±23µM vs

133±15µM, p<0.05), although albuminuria was not reduced. As expected, obesity shifted adipose macrophage (MΦ)

phenotype from M2 (CD206+) to M1 (CD11c

+). Interestingly, LXs did not alter total number of adipose MΦ, but acted in

a pro-resolving manner by increasing M2 MΦ (SFD 58±4%; HFD 28±1%; LXA4 43±4% p<0.05). LXs did not affect

renal MΦ infiltration in this model, suggesting that the LX-mediated reno-protective effect occurred via attenuation of

adipose inflammation. We evaluated the role of the reno-protective adiponectin, by comparing LX-mediated effects in

WT and Adiponectin-KO mice. LXs restored insulin-stimulated glucose uptake in the obese KO strain (p<0.05),

although not in the WT animals, suggesting that adiponectin may mask some of the protective LX effect. As predicted,

KO mice displayed augmented renal injury and increased H2O2 (WT 228±23µM, KO 326±58µM). Surprisingly LX-

induced reduction of renal H2O2 was augmented in the KO strain (167±12µM, p<0.01). Throughout the study Benzo-

LXA4 induced comparable significant trends to LXA4 and importantly neither altered weight gain.

Conclusion: Collectively the data suggests that LXs, through adiponectin-independent reduction of inflammation and

MΦ phenotype, may have therapeutic potential in obesity-induced CKD.

Financial support; Marie Curie IOF, National Institute of Health, Science Foundation Ireland

TNF-RECEPTOR 1 - ANNEXIN A1 AXIS IN THE DEVELOPMENT AND RESOLUTION OF MOUSE COLITIS

ANGELA A S SENA; LUCIANO P PEDROTTI; BIBIANA E BARRIOS; SILVIA G CORREA.

UNIVERSIDAD NACIONAL DE CÓRDOBA, CÓRDOBA - ARGENTINA.

Introduction: AnxA1 is a potent anti-inflammatory protein that plays critical regulatory roles in innate and adaptive

immune responses. Since alteration of mucosal immunity and TNF-alpha activity are crucial to the development of

inflammatory bowel diseases, we explored the expression and role of AnxA1 in DSS colitis using TNFR1-/- and WT

C57BL/6 mice. Methods and Results: Oral administration of DSS for 5 days provoked ulcerative colitis in both WT

and TNFR1-/- mice (n=9/group) that was clinically evaluated (weight loss, stool consistency and blood content) during

18 days. Interestingly, TNFR1-/- mice developed attenuated colitis and early recovery of clinical signs together with a

remarkable decrease of PMN infiltration and preservation of the histoarchitecture on day 7. We quantified the CD4,

CD8 and CD11b subsets and their AnxA1 expression on days 5, 10 and 18 by flow cytometry in peripheral blood and

colonic lamina propria (CLP). Until day 5, both mouse strains presented similar frequency of cell subsets and clinical

score, but already on day 10 TNFR1-/- group showed higher infiltrate of CD4 (20±2.4 vs 7.4±0.5) and CD8 (11±1.8 vs

5±1.7) in CLP comparing with WT mice, which was maintained until the colitis resolution on day 18. Since colitis onset

until the resolution phase, only KO mice showed a gradual increase of AnxA1 protein in CD4 (1.6-27%) and CD8 CLP

cells (1.5-9.3%). In contrast, in WT animals the AnxA1+ CLP cells augmented on day 10, but dropped by day 18 when

clinical signs were still evident. Regarding CLP CD11b cells, KO mice presented a striking AnxA1+ frequency (41±10

vs 3.4±0.1) before DSS treatment that diminished significantly (p=0.01) at day 5 of colitis, followed by gradual

recovery of the AnxA1+ cells at resolution phase. IL-6 detection in supernatants of cultured colon explants (ELISA)

showed that KO mice released lower levels during colitis course than WT mice. Then, TNFR1-/- mice reported a

negative correlation of blood AnxA1+ T cells and mucosal IL-6 levels (R=0.62,p=0.01) and we also found a negative

correlation between % of blood AnxA1+ T cells and clinical score in both strains (R=0.7,p<0.01). Conclusion: Our

data suggest that the suppression of the TNF-alpha pathway that attenuates DSS colitis could also favor the

modulatory activity of the AnxA1 on the innate and adaptive immune cells during the intestinal inflammation outcome.

Financial support: CAPES (SwF Program/Brazil), SECyT-UNC/CONICET/FONCyT (Argentina)

TREG AND TH17 IN SEPSIS

MARIANA MACEDO COSTA ANDRADE; SUELY CUBO ARIGA; ISABELA CASAGRANDE JEREMIAS;

ROSANGELA NASCIMENTO PIMENTEL; VANESSA JACOB VICTORINO; FRANCISCO GARCIA SORIANO.

FMUSP, SÃO PAULO - SP - BRASIL.

Introduction: Sepsis, which has a mortality rate of 40 to 60%, is a disease triggered by the presence of bacteria and /

or bacterial products such as lipopolysaccharide (LPS) by activating the immune response of the host. The

immunoparalysis or immunosuppression is reported as one of the causes of death in patients. The profile

characterization of lymphocytes acting in response to LPS tolerance is an extremely important contribution to the

study of immunosuppression in sepsis. The resistance to LPS can be genetically determined or acquired.

Experimental animals submitted to prior exposure to sub-lethal doses of LPS can induce a state of acquired

resistance and the animals become resistant to lethal doses, Metabolic and pyrogenic to LPS. The orientation of the

immune response to Th1, Th2, Treg and Th17 cells plays an important role in the defense against pathogens and

tumors. The aim of this study was evaluate the Treg and Th17 cells in CLP models with tolerance induced by LPS.

Methods and Results: After using subsequent doses of LPS (tolerance) and posterior cecal ligation (CLP) in 10

C57BL/6 mouse, spleen cells were removed, conducted to cell membrane permeabilization, after that labeled with

specific antibodies. Then they were analyzed in flow cytometer for quantification.According to the table above, there

was a significant increase in splenic T regulatory and Th17 cells in CLP animals compared to SHAM control animals

(#p<0,05) and CLP control animals (*p<0,05), demonstrating that tolerance can be responsible for the increase of

those cells, suggesting that they may be contributing to the decline in mortality previously seen in tolerance.

celulas (106 ) Sham controle Sham tolerante CLP controle CLP tolerante

CD3+CD4

+ 1,31 ±0,43 2,11±1,25 0,90±0,27 2,67±0,90

CD3+CD8

+ 1,05±0,28 1,38±0,75 0,59±0,174 5,29±2,08

CD3+CD19

+ 1,05±0,32 0,58±0,174 0,76±0,18 5,87±2,03

CD3+CD25

+ 0,31±0,11 1,42±0,68 0,23±0,16 3,56±1,35

#*

CD3+IL17

+ 1,77±0,58 7,55±4,12 0,89±0,31 16,84±6,46

#*

Conclusion: Therefore, it was confirmed that Treg and Th17 cells actively participate in the maintenance of immune

activity.

Financial support: CNPQ

UNBALANCED INNATE AND ADAPTIVE IMMUNITY RESPONSE IN ABSENCE OF DENDRITIC CELLS DURING

EXPERIMENTAL TRYPANSOMA CRUZI INFECTION

LISIA MARIA ESPER; JULIA TEIXEIRA CASTRO; ISABELA AVELLAR COSTA; FATIMA BRANT; ANDREIA

BARROSO; RONAN RICARDO SABINO ARAUJO; MAURO MARTINS TEIXEIRA; FABIANA SIMÃO MACHADO.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Background. During Trypanosoma cruzi infection, many cells types participate in the innate immune response, which

is important to control pathogen growth and avoid the development of pathology. Dendritic cells (DC) are specialized

antigen presenting cell (APC) population responsible for the critical role in the primary immune response against many

infections. Diphtheria toxin (DT) injection causes transient DC depletion in a transgenic mouse expressing Simian DT

receptors under the control of the CD11c promoter, allowing us to investigate the effects of dendritic cells depletion in

the innate and adaptive immune response against T. cruzi infection. Methods and results. CD11cDTR mice were

injected with DT (1ug/mouse) (DP mice) or with PBS (control group, CT mice) and 6 hs later were infected with

Trypanosoma cruzi (Y strain). Parasitemia, survival and development of immune response in the spleen were

investigated by flow cytometry. Depletion of DCs resulted in increased susceptibility to T. cruzi infection, which was

demonstrated by dramatically enhanced of parasite number circulating in the blood of DP mice. There was an

increased expression of the innate immune cells CD11b+GR1

+IL12

+ and F4/80

+CD11b

+IL-12

+ in the DP mice when

compared with CT mice. Also, we found decreased generation/expansion of CD3+CD4

+IFN-g

+ and CD3

+CD8

+IFN-g

+,

critical cells against T. cruzi infection in DP mice during the infection. Conclusions. Taken together, the results

indicated that DCs depletion cause a highly susceptibility and are critical for the development and regulation of

protective immune responses during the acute phase of T. cruzi infection. Keywords: Dendritic cells, Trypanosoma

cruzi. Supported by: CNPq and FAPEMIG