Setor 08. Imunofarmacologia

08.001 Inhaled JMF2-1, a lidocaine derivative, prevents allergic airway inflammation and hyperresponsiveness in mice by causing apoptosis of lymphocytes. Olsen, P. C.1; Serra, M. F.1; Ferreira, T. P. T.1; Farias-Filho, F. A.1; Costa, J. C. S.2; Fonseca, B.3; J. P. Viola3; Cordeiro, R. S. B.1; Silva, P. M. R. e1; Martins, M. A.1 - 1FIOCRUZ - Inflamação; 2FIOCRUZ - Far-manguinhos; 3INCa – Biologia Celular

Introduction: We have recently reported that the non anesthetic lidocaine derivative compound JMF2-1 is able to inhibit (i) allergen-evoked eosinophil and Th2 cell infiltration into the lung, (ii) generation of pro-inflammatory cytokines in lung explants, as well as (iii) airway hyperresponsiveness (AHR), measured non invasively in unrestrained sensitized mice. In the current study, we confirm the effectiveness of JMF2-1 on allergic AHR using a more conservative invasive methodology. We also provide evidence that apoptosis is underlying the effect of JMF2-1 upon Th2 cells. Methods: Sensitized BALB/c mice were exposed to aerosolized ovalbumin from day 19 to 21 post-sensitization. JMF2-1 (0.5-2%,), lidocaine (2%) were aerosolized for 30 min twice a day, being administered concomitantly and 8 h post-challenge. Dexamethasone (1 mg/kg, intraperitoneal) was administered 1 h before challenge. Airway function in anesthetized, tracheostomized and mechanically ventilated mice was monitored 24 h after the last provocation. At the same time point, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) was used for the quantification of apoptotic cells in lung sections. DO11.10 lymph node cells exposed to allergen stimulation in the presence or absence of treatments were stained with propidium iodide and annexin V-FITC, before being subjected to flow cytometry analyses. A pan-caspase inhibitor, ZVAD, was used to confirm the apoptosis pathway. Results: We found that as well as lidocaine and dexamethasone, JMF2-1 abolished allergen-induced AHR, as attested by impairment of both increased lung resistance and reduced dynamic compliance following exposure to methacholine aerosol. The rate of apoptotic infiltrating cells over viable cells was significantly higher in lung sections from animals treated with JMF2-1 (27 ± 4 %) (mean ± SEM, n=4), as compared to those from saline (5 ± 1 %), lidocaine (17 ± 4%) or dexamethasone (17 ± 4%). JMF2-1 (300-600 μM) induced apoptosis of allergen-challenged T cells in vitro, in a mechanism clearly sensitive to the caspase inhibitor Z-VAD. Conclusion: These results confirm the capacity of nebulized JMF2-1 to inhibit cardinal features of asthma, including AHR. They also strongly suggest that the protective effect of JMF2-1 is, at least in part, accounted for by a direct pro-apoptotic action upon Th2 lymphocytes. Apoio Financeiro: CAPES; PDTIS; CNPq; FAPERJ.

08.002 Contribuição da iNOS na inibição da síntese de citocinas pelo linfócito Th2 em camundongos alérgicos. Santos, K. L.; Toledo, A.P.; Nogueira, J. S.; Pelaquini, E. H.; Benetti, L. R.; Pallis, F. R.; Ferreira, H. H. A. USF - Inflamação

Introdução: O óxido nítrico (NO), importante mediador fisiológico pulmonar, é sintetizado a partir da L-arginina pela família das óxido nítrico sintases (NOS) constitutivas, neuronal (bNOS) e endotelial (eNOS), e induzível (iNOS). Na asma alérgica, a migração de eosinófilos (EOs) para o pulmão é influenciada pelas interleucinas produzidas pelo linfócito Th2, como a IL-4, IL-5, IL-10 e IL-13, além do INF-g e TNF-α liberados pela Th1. Estas citocinas podem regular a expressão e/ou função das moléculas de adesão do EO e do endotélio vascular (Kelly, Curr. Rev. Allergy Clin. Imm. 120:3, 2007). Existem sugestões de que o NO pode modular a resposta alérgica pulmonar, desde que uma redução do número de EO e dos níveis de TNF-α, IL-13 e eotaxina foi observado em ratos alérgicos tratados com L-NAME (inibidor não-seletivo das NOS). Por outro lado, o tratamento com a L-arginina provocou aumento dos níveis de IL-5 e da infiltração de EO pulmonar (Ferreira, Anti-Inflam. Anti-All. Ag. Med. Chem. 5:45, 2006). No entanto, a identificação da NOS envolvida na secreção de citocinas não está totalmente elucidada. O presente estudo teve como objetivo pesquisar a influência da iNOS na secreção de citocinas em camundongos alérgicos. Metodologia: Camundongos BALB/C foram sensibilizados, 100 mg - via subcutânea, e desafiados com ovalbumina (OVA), 10 mg - via intranasal, 2 vezes ao dia. Um grupo de camundongos recebeu tratamento agudo por injeção intraperitoneal do inibidor seletivo da iNOS, 1400W (1 mg/kg de camundongo) duas horas antes e 4 e 12 horas após o desafio. Os controles receberam apenas salina. Os camundongos foram sacrificados 48h após o desafio. Os pulmões foram retirados e congelados a -80ºC e após homogeneização, os níveis de eotaxina, IL-4, IL-5, IL-10, INF-g e TNF-α foram verificados utilizando-se kits comerciais de ELISA (R&D Systems). Resultados: Os resultados estão expressos como média ± EPM de 12 animais por grupo. Em comparação ao grupo controle, verificamos que o tratamento 1400W reduziu os níveis de IL-4 (137±23 pg/ml para 69±20), IL-5 (32±3,5 pg/ml para 21±2), IL-10 (9±1.5 pg/ml para 5±1) e eotaxina (334±39 pg/ml para 225±13). Os níveis de IFN-g e TNF-α não foram modificados pelo tratamento (11,2±0,8 pg/ml para 10.9±0,9 e 24,6±2.2 pg/ml para 24.3±2.7, para controles e tratados, respectivamente). Discussão: Nossos resultados sugerem que o NO influencia a liberação de citocinas pelo linfócito Th2 e não pela Th1 e que a iNOS está envolvida no processo. Portanto, a inibição da migração de EO para o pulmão de camundongos tratados com 1400W descrito em trabalho anterior (Sbfte, 2007) pode ser decorrente da inibição das citocinas envolvidas neste processo. Agradecimento: Fapesp. Apoio Financeiro: FAPESP

08.003 Avaliação do pré-condicionamento físico aeróbio moderado sobre a inflamação pulmonar aguda induzida por LPS. Nadur-Andrade, N.; Ramos, D. S.; Ribeiro, W.; Pires, F. S.1; Gouvêa, H. A.; Ferrari, E. F. – UNIVAP - Fisiologia e Farmacodinâmica

Introdução: Dentre os principais fatores que determinam se a atividade física aeróbia (AFA) estimulará ou deprimirá o sistema imune (SI) encontram-se a intensidade, a freqüência e a duração [1]. Nosso objetivo foi avaliar os efeitos da AFA moderada durante 6 semanas sobre a inflamação pulmonar aguda (IPA) induzida por LPS. Material e Métodos: Camundongos Balb/c machos foram divididos em 4 grupos: Controle; AFA50%; LPS e AFA50% + LPS. Os animais foram submetidos a uma AFA moderada (natação), com intensidade de 50% da carga máxima, durante 60 min, 4 vezes/semana durante 6 semanas. Foi instilado dose única de LPS intranasal (1 mg/kg), sendo o seu tempo de reação de 24horas, onde após esse tempo foi realizada avaliação da migração celular, total e diferencial, do lavado broncoalveolar (LBA). Resultados: No grupo AFA50% não foi observado alterações no número de células totais do LBA. A administração intranasal com LPS elevou em 3 vezes mais o número de células totais doLBA, neste caso a AFA50% reduziu em torno de 50% no número de células totais.O número de neutrófilos também reduziu em torno de 60% com a AFA50% + LPS, ouve uma redução de 50% no número de macrófagos no grupo submetido a AFA50%+LPS (p< 0.05). Em relação à migração de linfócitos, somente o grupo AFA apresentou um pequeno aumento, porém não significativo. Discussão: O modelo de Inflamação Pulmonar aguda (IPA) por LPS provoca característica liberação de citocinas e neutrofilia [2]. Os macrófagos, em geral, representam na contagem celular do LBA 90% das células aspiradas [3].A redução do número total das células e também a neutrofilia ocasionada pela sensibilização com LPS, sugere que a AFA quando praticada dentro de limites fisiológicos pode exercer um importante papel na IPA.Apesar da incidência da hiperreatividade das vias aéreas, existem poucos estudos avaliando os efeitos da AFA em pacientes com IPA. A AFA parece trazer benefícios importantes para o processo inflamatório, entretanto seu mecanismo ainda é pouco compreendido. Atualmente a explicação mais facilmente utilizada, é que a AFA melhora a capacidade física das pessoas, modulando o Sistema Imune, além de acarretar benefícios para todos os sistemas orgânicos [4]. Referências Bibliográficas: 1. Fagard R. H.; Cornelissen V. A. Eur.Cardiovasc Prev Rehabil., 14, 12-17, 2007. 2. Kline J.N. et al. Am. J. Respir. Crit. Care Med., 160, 297–303, 1999. 3- Rufino, R.; Silva J. R. L. J B Pneumology,. 32, 241-248, 2006. 4. Petersen, A. M.; Pedersen, B. K. J Appl Physiol., 98, 1154-1162, 2005. Apoio Financeiro: Universidade do Vale do Paraíba

08.004 ATL-1, an aspirin-triggered lipoxin A4 synthetic analog, prevents the inflammatory and fibrotic effects of bleomycin-induced pulmonary fibrosis. Martins,V.1; Valença, S. S.2; Farias-Filho, F. A.3; Silva, P. M. R. e3; Hogaboam, C.4; Kunkel, S. L.4; Fierro, I. M.5; Canetti, C.6; Benjamim, C. F.7 - 1UFRJ - Farmacologia; 2IBRAG-UERJ - Histologia e Embriologia; 3FIOCRUZ - Fisiologia e Farmacodinâmica; 4University of Michigan Medical School, USA - Pathology; 5UERJ - Farmacologia; 6IBCCF-UFRJ; 7UFRJ - Farmacologia Básica e Clínica

Introduction: Pulmonary fibrosis is an interstitial disease characterized by diffuse chronic interstitial inflammation, increased fibroblast proliferation, and enhanced extracellular matrix synthesis and deposition (Gross, T. J. N Engl J Med 345:517.2001). Lipoxins (LX) are endogenously produced eicosanoids via lipoxygenase-lipoxygenase interactions and by aspirin-triggered acetylation of cyclooxygenase-2 and activation of 5-lipoxygenase forming 15-epimer LX or aspirin-triggered LX (ATL). Among the various ATL analogs studied, 15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATL-1) and related molecules have been shown to be active in vivo in several models of inflammatory disease (Levy, B. D. Drugs Today (Barc) 39:373. 2003). Methods: For induction of pulmonary fibrosis, groups of mice C57Bl/6 (n=5-6) were administered by intratracheal (i.t.) route with bleomycin (BLM) (0.1U/mouse) or sterile saline (30 µl) as control. ATL-1 (1µg/mouse) was injected concomitantly with BLM (0.1U/mouse), or four days after BLM inoculation. Treatment with ATL-1 was boosted i.v. in the 7th and 14 th days. For analysis, mice were sacrificed on day 21 after BLM administration and lungs removed for preparation of histological analysis, morphometry and immunohistochemistry. Results: Bleomycin-induced lung fibrosis was prevented by ATL-1, which inhibited inflammation, matrix deposition and decreased α-actin expression. Furthermore, ATL-1 post-treatment (4 days after BLM) showed similar inhibitory effects on inflammation, matrix deposition, and fibroblast differentiation. Discussion: The present results elucidate the antifibrotic effect of an aspirin-triggered LX analog using a relevant in vivo model of lung fibrosis. It is noteworthy that in this report we showed the therapeutic effect of ATL-1 in addition to the early namely preventive effect, given that in the clinical use, the therapeutic effect is often more important when the fibrotic changes of various etiology are already apparent in the patient. Apoio Financeiro: Conselho Nacional de Pesquisa (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

08.005 Effects of 3 different chemokine blockers, on inflammation in mouse antigen-induced arthritis. Coelho, F. M.1; Costa, V. V.1; Amaral, F. A.1; Sousa, L. F. C.1; Pinho, V.1; Teixeira, M. M.1; Sachs, D.2; Souza, D. da G. de1 1UFMG - Bioquímica e Imunologia; 2FMRP-USP - Farmacologia

Introduction and Objectives: Rheumatoid Arthritis is a complex systemic disease that ultimately leads to the progressive destruction of articular and periarticular structures. In the present work, we investigated the effects of Reparixin (a noncompetitive allosteric blocker of the CXCR2 receptor), Evasin-3 (a new chemokine binding protein) and PA 401 (novel class of protein based GAG antagonists) on inflammation as well as leukocyte-endothelial cell interaction. Methods: AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated by using an electronic pressure-meter. Neutrophil accumulation in tissues was measured by counting neutrophils in the joint and assaying myeloperoxidase (MPO) activity. TNF-a and Chemokines (KC and MIP-2) were quantified by ELISA. Histological analysis was performed to evaluate the severity of arthritis and leukocyte infiltration. Results: Antigen challenge of immunized mice induced TNF-a and MIP-2 production, neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Pretreatment with all compounds (Reparixin -30 mg/Kg; Evasin-3 -1mg/mice; PA 401 -1mg/mice) decreased neutrophil recruitment, an effect associated with marked inhibition of neutrophil adhesion. Drug treatment was also associated with inhibition of the production of TNF-a, hypernociception and overall tissue pathology. Using intravital microscopy, we showed that repertaxin reduced the number of adherent leukocytes in synovial microvessels and the number of rolling leukocytes. Pretreatment of animals with Evasin-3 decreased significantly the number of adherent leukocytes in synovial microvessels but had no effect on the number of rolling leukocytes. Post-treatment with PA 401 and intravital microscopy will be done soon. Conclusion: This study shows that the compounds appear to act via blockade of leukocyte adhesion, with consequent inhibition of neutrophil migration to the site of joint inflammation. In conclusion, this study shows that treatment with different chemokine blockers prevented 3 major aspects of arthritis, namely neutrophil recruitment, TNF-a production and inflammatory hypernociception. Apoio Financeiro: CNPq, CAPES e Fapemig

08.006 Evaluation of a chemokine-binding protein in experimental models of a delayed-type hypersensitivity. Alessandri, A. L.1; Pinho, V.1; Vieira A. T.1; Russo, R. C.1; Castor, M. G. M.2; Silva, A. F. C.3; Proudfoot, A. E. I.3; Teixeira, M. M.1 - 1UFMG - Bioquímica e Imunologia; 2UFMG - Fisiologia e Farmacologia; 3Serono Pharmaceutical Research Institute

Introduction: Evasins are a new family of chemokine binding proteins (CHBPs) from tick saliva which have potent anti-inflammatory activity. Evasin-1 binds to MIP-1a/CCL3, MIP-1b/CCL4 and PARC/CCL18. CCL3 plays a role in driving leukocyte accumulation into inflamed sites. We evaluated the ability of an Evasin-1 to modify leukocyte recruitment in a murine model of a Th1-predominant delayed-type hypersensitivity and a Th2-predominant, late phase reaction. Methods: To induce Th1-predominant delayed-type hypersensitivity, Balb/c mice were immunized on day 1. On day 14, the animals were pretreated systemically with Evasin-1 and challenged with methylated BSA (mBSA). The cells present in the pleural cavity were harvested after a further 48 h. To induce Th2-predominant, late phase reaction, Balb/c mice were immunized i.p. with 2500 isolated Schistosoma mansoni eggs at days 0 and 7. On day 20, the animals were pretreated systemically with Evasin-1 and challenged with soluble egg antigens (SEA). The cells present in the lungs were harvested after a further 24 or 48 h. Results: In the Th1 sensitization model using mBSA, recruitment of leukocytes into the pleural cavity was significantly impaired. Whilst the inhibition of neutrophil recruitment was most striking, inhibition of CD3+ lymphocytes was also observed, but not of CD11b+ monocytes. In the Th2 sensitization model, Evasin-1 inhibited eosinophil recruitment induced by antigen challenge into the lungs of mice immunized with S. mansoni eggs. Conclusion: The Evasin-1 decreased the cellular recruitment in delayed type hypersensitivity models. The Evasins are distinguished from viral CHBPs in terms of sequence, specificity, size and structure. It may prove to be therapeutically useful as novel anti-inflammatory agents in the future. Apoio Financeiro: FAPEMIG e Merck-Serono

08.007 Monosodium urate crystals-induced joint hypernociception in mice: the participation of 5-lipoxigenase pathway and neutrophils in an experimental model of articular acute gout. Costa, V. V.1; Amaral F. A.1; Sachs, D.2; Coelho, F. M.3; Fagundes, C. T.4; Rezende C. B.4; Silva, T. A.5; Teixeira, M. M.3; Souza, D. G.1 1UFMG - Microbiologia; 2FMRP - USP - Farmacologia; 3UFMG - Bioquímica e Imunologia; 4UFMG - Biologia Celular; 5UFMG - Patologia, Clínica e Cirurgia odontológicas

Introduction: Gout arthritis is characterized by precipitation of uric acid crystals (UAC) in several tissues, mainly in articular cavity, leading to local inflammation that can compromise the life quality of the patient. The basic mechanism of this inflammatory response is the intense neutrophil recruitment, with the release of cytokines/chemokines, arachdonic acid metabolites, proteases, and oxygen radicals. However, there are no efficient drugs against the symptoms of gout. Here, our aim was to set up a new animal model of acute gout and to evaluate the role of 5-lipoxigenase (5-LO) metabolites and of neutrophils in its pathogenesis. Methods: Wild type male C57/BL6 mice (WT) and 5-LO knock-out (5-LO-/-) SV129 mice were used. Treatment with MK886 [5LO-activating protein inhibitor] and CP105696 (BLT1 antagonist) were used to interfere with the 5-LO pathway. To access the role of neutrophil migration, mice received: CXCR2 receptor antagonists (Reparixin and DF2162) and Fucoidan. UAC were prepared by precipitation of the crystals after addition of uric acid in borate buffer solution (pH 8.5). For the experiments, a dose of 100µg of UAC was diluted and a volume of 10 µL was injected in the knee joint. Hypernociception was measured by a digital analgesimeter (EFF-301). Samples of the periarticular tissue were collected for cytokine analysis (ELISA) and neutrophil quantification (MPO). A lavage of the joint (BSA 3%; 10 µL) was made to evaluate the cell infiltration on articular space. Results: Using C57/BL6 mice, we made a temporal curve (3, 6, 9, 15, and 24 hours after UAC injection) to evaluate the adequate time-points to access the inflammatory response induced by UAC. The time-points chosen were 6 and 15 hours, where there was an increase in all parameters analyzed, as compared to vehicle mice. All strategies used to block the action of the metabolites of 5-LO pathway decreased the inflammatory parameters analyzed when compared to vehicle-treated mice. Similarly, administration of the CXCR2 receptor antagonists or fucoidan lead to reduced cell influx, MPO activity and inhibited production of IL-1β and MIP-2 after UAC injection. Discussion: Our data point presents an interesting animal model of acute gout due to a direct injection of UAC on articular joint, as well as the measurement of the hypernociception in these mice. Furthermore, the reduced inflammatory response observed in 5-LO-/- ,MK 886-treated, CP105696-treated and CXCR2 antagonists/fucoidan-treated mice suggest an important role of the 5-LO pathway and neutrophil in this model. Apoio Financeiro: CNPq, CAPES, FAPEMIG

08.008 Effect of a new molecule from tick’s saliva with potent anti-inflammatory activity by biding to CCL11 in experimental model of ulcerative colitis. Vieira, A. T.1; Fagundes, C. T.1; Alessandri, A. L.1; Borges, V.O.1; Proudfoot, A. E. I.2; Sousa, L. P.1; Teixeira, M. M.1 1UFMG - Bioquímica e Imunologia; 2UFMG - Bioquímica e Imunologia; 3UFMG - Bioquímica; 2Serono Pharmaceutical Research Institute

Introduction and Objectives: CCL11 (Eotaxin) is a well-characterized chemokine with potent and selective chemotactic activity for eosinophils. Previous studies indicating that eosinophils accumulate and become activated in Ulcerative colitis (UC) led us to hypothesize that eotaxin is potentially involved in the pathophysiology of this disease. Thus, we assessed the effect of “Evasin-4”, a recombinant protein cloned from tick´s saliva that interferes with eotaxin activity, for the treatment of colitis. Methods: Colitis was induced by Dextran Sulfate Sodium – DSS administration. Female BALB/c mice received DSS 4% (wt/vol) in water, ad libitum, for 7 days and, after this time point, lethality was evaluated. Clinical score (diarrhea and occult bleeding in feces) and weight loss were monitored at every 2 days. At day 7, mice were sacrificed and colon samples were excised and submitted to histological analysis and to quantitative determination of neutrophils and eosinophils infiltration (through MPO and EPO activity assay, respectively). Results: All mice evolved to death after DSS-induced Colitis has been installed (100% of mortality), while Evasin-treated mice did not. The severity of disease, as evaluated by the clinical score, was milder in Evasin-treated mice than in mice that received only vehicle (p<0.01). At the end of DSS treatment, there was any loss of the initial weight in Evasin-treated mice, while in vehicle-treated mice the weight loss was about 20% (p<0.001). Furthermore, eosinophil accumulation in the colon was reduced in Evasin-treated mice, but not in vehicle-treated group (p< 0.001). Neutrophil accumulation was also significantly decreased in Evasin-treated mice when compared to vehicle-treated group. Of note, control animals received water and all these parameters remained unchanged. Conclusion: Administration of recombinant Evasin- 4 resulted in potent anti-inflammatory activities in the animal model of UC. This novel molecule, designed by nature, may be therapeutically useful as a novel anti-inflammatory agent for patients with ulcerative colitis in the future. Apoio Financeiro: CNPq,FAPEMIG,CAPES

08.009 Effects of tumor necrosis factor alpha (TNF-alpha), interleukine-1 beta (IL-1beta), interleukine-6 (IL-6) and interferon gamma (IFN-gamma) on pineal gland function. Fernandes, P. A. C. M.; Cecon, E.; Monteiro A. W. A.; Ferreira, Z. S.; Markus, R. P. IB-USP - Fisiologia

Introduction: We have shown that the pineal gland plays an important role in the mediation of an inflammatory response. Nocturnal melatonin production is suppressed by pro-inflammatory cytokines, while corticosterone enhances the noradreanaline-induced production. TNF-alpha inhibits the transcription of the arylalkylamine N-acetyltransferase gene (Aa-nat), while corticosterone potentiates. As TNF-alpha is known to activates and corticosterone to inhibits the nuclear translocation of the nuclear transcriptional factor kappa B (NF-kappaB), it was suggested that this factor might regulates Aa-nat transcription. In the present work we evaluated the effect of IL-1beta, IL-6, IFN-gamma and TNF-alpha on the noradrenaline-induced melatonin production and of NF-kappaB nuclear accumulation. Methodology: Pineal glands obtained from 2-3 month-old male rats were incubated in BGJB medium for 30 min before being exposed to IL-1beta, IL-6, IFN-gamma (10 ng/ml) or TNF-alpha (30 ng/ ml) for 30 min and to noradrenaline (NA, 10 or 100 nM) for 5 h, still in the presence of the cytokines. Controls were obtained by incubating the glands with vehicle. Melatonin was measured by HPLC and NF-kappaB nuclear content by EMSA. Results: NA-induced melatonin production was not changed by IL-1beta or IL-6 but, on one hand, was enhanced by IFN-gamma (13.56 ± 1.79 x 19.47 ± 0.93 ng/well, n=4, p<0.05) and, in the other hand, was decreased by TNF-alpha (34.79 ± 6.73 x 16.84 ± 2.41, n=4, p<0.05). At the higher NA concentration IFN-gamma was not able to enhance melatonin production. NF-kappaB nuclear accumulation was not changed by IL-1beta or TNF-alpha after the treatment, but was reduced by IFN-gamma and IL-6. Conclusions: Our data show that pineal gland melatonin production could be modulated by the inflammatory agents IFN-gamma and TNF-alpha but not by IL1-beta and IL-6. The fact that IFN-gamma and IL-6 reduce NF-kappaB accumulation but only the first increases melatonin production suggests the participation of others transcriptional factors in the process. One possibility is the STAT 3 pathway. Both cytokines, IFN-gamma and IL-6, are known to activate this transcriptional factor and, depending on the stimulus given, STAT 3 can modulate the quality of the nuclear NF-kappaB and lead to different responses. The data indicate that pineal gland works as a sensor and respond selectively to the modulators of the inflammatory process reinforcing our group idea that there is a pineal-immune axis important to the control of physiopathologic processes. Apoio Financeiro: FAPESP, CAPES and CNPq

08.010 Anandamide administration previously to an immunization increases cell-mediated immunity: possible role for corticosterone. Ribeiro, A.1; Ferraz de Paula, V.1; Sakai, M.2; Pinheiro, M. L.1; Palermo-Neto, J.3 1FMVZ-USP - Patologia; 2FMVZ-USP-VPT; 3USP - Neuroimunomodulação

Introduction: The endocannabinoid system is known to control many physiological functions, including movement, learning and memory, cognition, appetite, emesis, body temperature, pain, behavior, neuroendocrine system, and immune systems. The knowledge about these functions raised some questions about a possible role of the endocannabinoid system in the neuroendocrine-immune relationship. Therefore, the aim of our work was to search for neuroimmune interactions of anandamide (AEA) previously administrated of an immunization with ovalbumine (OVA). Methodology: 60 C57BL/6 male mice were used and divided randomly in 3 groups, saline (S), vehicle (V), and AEA 0.1 mg/kg (E), treated i.p. 90 min before each test. Corticosterone: blood samples were collected and the serums were analyzed by RIA. Cell-mediated immunity: each mouse was immunized s.c. with OVA (50 µg/mouse). After 7 days, they received an injection of a suspension of aggregated-OVA (4%) on the left paw. The measurement of the size of the paws was done 1h, 6h, 24h, and 48h to determine the Delay Type Hypersensitivity (DTH). After the 48h, mice spleens were collected to analyze lymphocyte proliferation (CD4+ and CD19+) using CFSE in vitro by flow cytometry. Results: AEA increased the corticosterone serum levels (S:229.89±57.2; V:454.96±116.8; E:710.76±135.7). AEA treatment previously to the immunization increased the DTH (S:0.0687±0.02; V:0.0375±0.02; E:0.094±0.03) and the antigen-specific T CD4+ lymphocyte proliferation (S:34.72±3.4; V:36.97±7.8; E:45.72±3.4). Discussion: For the first time, we show a neuroendocrine-immune interaction using an endocannabinoid, anandamide. We showed that AEA 0.1mg/kg increased the serum levels of corticosterone. Additionally, we showed an increase in the DTH and in the lymphocyte proliferation. There are some works showing that increased levels of corticosterone increases the cell-mediated immunity. Therefore, the acutely increased levels of corticosterone could be responsible for the increased cell-mediated immunity observed in this work. Apoio Financeiro: CNPq and FAPESP

08.011 PRÊMIO INOVAÇÃO

08.012 CCL3/MIP-1α chemokine biding protein interfere in the cells migration to inflammatory sites in murine graft versus host disease. Castor, M. G. M.; Resende, B. C.; Rezende, B.; Teixeira, M. M.; Pinho, V. ICB-UFMG - Bioquímica e Imunonologia e Morfologia

Introduction: Graft-versus-host disease is a major complication of allogeneic bone marrow transplantation, leading to significant morbidity and mortality in humans. CCL3 is a protein of subfamily CC of the chemokines involved in acute and chronic inflammations in the sites of lesion or infection and is also involved in graft-versus-host disease (GVHD). CCL3 affects the inflammatory cells trafficking and proliferation to specific sites of inflammation in GVHD and may act in an additive or synergistic manner improving the number theses cells in the target organs, including liver, spleen and lung, of the recipient animals. The present studies examined the role of CCL3 the cells migration to target organs utilizing the CCL3 chemokine binding protein (Evasin-1) in acute GVHD in mice. Methods: For induction of GVHD the recipient mice, B6D2F1, were exposed to a sublethal dose (400 cGy) of gamma radiation. Two days after, they received intravenously 3 x 107 splenocytes of parental mice, C57BL6. Approximately 20 days after transplant, the animals were sacrificed and their organs removed for evaluation of the inflammatory reaction caused by GVHD. To verify the role of CCL3 in GVHD, the animals were treated subcutaneously with Evasin-1 (10 µg/animal in 200 µL of PBS) at each 12 hours. Results: Mice treated with Evasin-1 showed prevention of mortality and lower note in the graduation clinical previously observed. Additionally, presented a lower percentage of CD8+ T cells in the spleen and lower relative number of neutrophils and macrophages in the lung and liver. Discussion: Evasin-1 diminished the cells recruitment to GVHD target organs. Additionally it diminished the occurrence and the intensity of GVHD clinical signs and also increased the animals survival. Chemokine binding proteins can be potentially useful in the treatment of inflammatory diseases, by exhibiting immunomodulatory and anti-inflammatory activities. Apoio Financeiro: CNPq, Fapemig, CAPES

08.013 Role of phosphoinositide 3-kinase gamma (PI3Kγ) in graft versus host disease. Castor, M. G. M.; Resende, B. C.; Rezende, B.; Teixeira, M. M.; Pinho, V. 1ICB-UFMG - Bioquímica e Imunologia e Morfologia

Introduction: Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation leading to significant morbidity and mortality in humans. This disease is characterized with a severe immunossupression and target organ injury by leukocytes. Mice animal models have helped much in the understanding of the pathophysiological mechanisms of this disease. The enzyme phosphoinositide 3-kinase gamma modulates cell responses including mitogenic signaling, survival, cell growth, metabolic control, degranulation, rearrangement of the cytoskeleton and migration. An isoform of this enzyme, PI3Kγ, is involved in regulation of inflammation and allergy by modulating the chemotaxis of leukocytes and degranulation of mast cells. The inhibition of PI3Kγ has been the subject of many studies emerging as a potential new therapeutic strategy for control of inflammatory diseases. The aim of the study was to verify role of PI3Kγ in the acute GVHD in mice. Methods: For induction of GVHD the recipient mice, B6D2F1, were exposed to a sublethal dose (4 Gy) of gama radiation. Two days after, they received intravenously 3 x 107 splenocytes of parental mice, C57BL/6J (WT) or C57BL/6J PI3k-/-. After transplant, body weight and lethality was monitoring every 2 days. A second experiment was conducted for the analysis of inflammatory response caused by GVHD. In this experiment the disease target organs were removed around the 15th day after the transplant. Results: Mice that received splenocytes PI3K-/ - have a higher rate of survival (62.5%) compared to mice that received cells from WT (28.5%). It was observed a decrease in the body weight and a reduced cells relative number (neutrophils and macrophages) in the lung and liver in comparison with mice that received WT cells. Discussion: The absence of PI3Kγ isoform of the donor cells resulted in a decreased mortality and smaller variation of body weight of GVHD mice. The recruitment of inflammatory cells to disease target organs was also reduced. Further experiments must be conducted to clarify how this protection occurs. Observação: B6D2F1 é a linhagem do camundongo. Este camundongo é resultante do cruzamento das linhagens C57BL6/J x DBA. Estou a disposição para maiores esclarecimentos. Apoio Financeiro: CNPq, Fapemig, CAPES

08.014 Papel do fator ativador de plaquetas (PAF) na doença do enxerto versus hospedeiro (GVHD). Resende, B. C.1; Castor, M. G. M.1; Rezende, B.1; Teixeira, M. M.1; Pinho, V.2 1ICB-UFMG - Bioquímica e Imunologia; 2ICB-UFMG - Morfologia

Introdução: A doença do enxerto-versus-hospedeiro (GVHD) é uma das maiores complicações do transplante de medula óssea alogênico e está associada a uma grande mortalidade de indivíduos transplantados. A GVHD caracteriza-se por processo inflamatório grave em diversos órgãos, acarretando sinais e sintomas sistêmicos que comprometem o funcionamento do organismo. O Fator Ativador de Plaquetas (PAF) é um mensageiro lipídico, pró-inflamatório, envolvido no recrutamento e ativação de leucócitos. Este estudo verifica a participação do PAF na doença do GVHD. Métodos: Utilizamos camundongos C576BL/6J, (C57BL/6J x DBA/2)B6D2F1 e C57BL/6J PAFR -/- com idade de 8 a 12 semanas. Para indução da GHVD aguda os camundongos receptores, B6D2F1, foram irradiados subletalmente com 4 Gy de radiação gama. Dois dias após a irradiação eles receberam as células dos doadores parentais, C57BL/6J ou C57BL/6J PAFR-/-, a partir de um “pool” de células do baço. Um total de 3 x 107

células foram injetadas i.v. nos camundongos receptores B6D2F1. Camundongos do grupo controle receberam células isogênicas (B6D2F1) e não desenvolveram o GVHD. Resultados: Após a indução da doença, a mortalidade e os sinais clínicos foram observados em todos os grupos. Os animais com GVHD aguda tiveram 100% de mortalidade em 14 dias e todos os camundongos que receberam células PAFR-/- sobreviveram durante o período analisado. Além disso, os camundongos do grupo PAFR -/- apresentaram menor perda de peso e sinais clínicos mais brandos, quando comparado com o grupo GVHD. Discussão e conclusão: O PAF parece estar envolvido na patogênese da GVHD, pois a ausência do seu receptor nas células transplantadas evitou a morte e diminuiu os sinais da doença nos camundongos com GVHD. Perspectivas: Estudar a participação do PAF nos mecanismos biológicos envolvidos na instalação e gravidade da GVHD. A partir dos resultados, procurar intervir farmacologicamente na ação do Fator Ativador de Plaquetas. Apoio Financeiro: CNPq, CAPES e Fapemig.

08.015 Evaluation of chemokine-binding proteins in experimental models of inflammation. Alessandri, A. L.2; Castor, M. G. M.2; Vieira, A. T.2; Coelho, F. M.2; Silva, A. F. C.1; Russo, R. C.1; Pinho, V.1; Proudfoot, A. E. I.3; Teixeira, M. M.1 1UFMG - Bioquímica e Imunologia; 2UFMG - Fisiologia e Farmacologia; 3Serono Pharmaceutical Research Institute

Introduction: Evasins are a new family of chemokine binding proteins (CHBPs) from tick saliva which have potent anti-inflammatory activity. The Evasin-3 binds to IL-8/CXCL8 and GRO-a/CXCL1 and their murine homologous (KC/CXCL1-3 and MIP-2/CXCL1-2). The Evasin-4 binds to eotaxin/CCL11 and RANTES/CCL5. We evaluated the ability of an Evasin-3 and Evasin-4 to modify leukocyte recruitment induced by chemothatic factors or by antigen in sensibilized animals. Methods: Balb/c mice were pretreated systemically with Evasin-3 or Evasin-4 and received an injection of different chemothatic factors in the left knee joint or pleural cavity. The cells present in the cavities were assessed at different time points. Animals were immunized on days 1 and 8. On day 14, the animals were pretreated systemically with Evasin-3 or Evasin-4 and challenged with OVA. The cells present in the pleural cavity were harvested after a further 8 or 48 h. Results: Evasin-3 inhibited KC induced neutrophil recruitment into the knee joint in a dose dependent manner. Evasin-3 was also tested in a mouse model of antigen-induced pleurisy. Administration of Evasin-3 resulted in a decrease in the total number of leukocytes in the pleural cavity. Evasin-4 inhibited CCL11 induced eosinophil recruitment into the pleural cavity. Similarly Evasin-3, Evasin-4 decreased the eosinophil influx into the pleural cavity induced by antigen. Conclusion: Evasin-3 and Evasin-4 prevented the neutrophil and eosinophil influx, respectively, induced by chemothatic factors or by antigen in sensibilized animals. These chemokines binding proteins have pharmacological activity in vivo and are effective in reducing inflammation in animal models. The evasins could be relevant anti-inflammatory strategies in the future. Apoio Financeiro: FAPEMIG and Merck-Serono

08.016 O Papel dos leucotrienos na imunossupressão pós-sepse severa. Antunes, C. A. C.1; Brogliato, A. R.2; Kunkel, S. L.3; Bozza, M.4; Canetti, C.5; Benjamim, C. F.2 1UFRJ - Microbiologia; 2UFRJ - Farmacologia Básica e Clinica; 3University of Michigan - Pathology; 4UFRJ - Imunologia; 5IBCCF-UFRJ

Introdução: Os leucotrienos são conhecidos por sua capacidade quimiotática para neutrófilos (LTB4) e envolvimento em processos patológicos como anafilaxia e asma (LTC4, LTD4, LTE4), entretanto seus papéis em um quadro de imunossupressão pós-sepse utilizando-se o modelo de ligação e perfuração do ceco (CLP) e desafio com conídios de Aspergillus fumigatus não são conhecidos. Assim, objetivamos verificar a importância desses mediadores lipídicos utilizando como ferramenta a comparação entre animais selvagens e deficientes para a enzima 5 lipoxigenase (5-LO-/-), abolindo assim a produção de leucotrienos. Métodos: Camundongos 5-LO-/- e o controle selvagem foram submetidos ao modelo de CLP, o ceco foi parcialmente ligado e perfurado nove vezes com agulha de 21G. Os falso-operados (SHAM) foram usados como controles. Camundongos submetidos ao CLP ou SHAM foram tratados com uma dose de antibiótico (80mg/kg) nos tempos de 6, 24 e 48 horas após a cirurgia, injetando-se conídios de A. fumigatus intratraquealmente no 3º dia após o CLP. A sobrevida foi avaliada durante uma semana. Também realizamos o desafio com A. fumigatus em animais sem realizar previamente o CLP e avaliamos a sobrevida durante uma semana. Para avaliar a fagocitose 3 dias após o CLP, os macrófagos alveolares (4x105) foram coletados incubados em câmaras de 8 poços por 1h a 37ºC em estufa de CO2 e em seguida procedeu-se a incubação por 1 hora com conídios de A. fumigatus na proporção de 1:10 (célula/conídio), sendo posteriormente corado com H&E para mensurar o índice fagocítico. Resultados: O grupo de animais 5 LO-/- que foi submetido ao CLP e posteriormente desafiado com A fumigatus apresentaram 100% de mortalidade 48h após o desafio, enquanto que os animais WT apresentaram mortalidade até o último dia avaliado, sendo que todos morreram. Em contrapartida, no grupo que somente sofreu desafio com A. fumigatus houve morte de 50% dos camundongos 5 LO-/-, enquanto que não houve morte no WT. O índice fagocítico dos camundongos WT submetidos ao CLP foi cerca de 35, enquanto que os 5-LO-/- submetidos ao CLP foi cerca de 8. Os animais Sham de cada grupo foram usados como controle e obtiveram níveis mínimos de índice fagocítico. Conclusão: Com o que fora mostrado acima é notório que os leucotrienos desempenham um papel de grande importância na proteção dos animais no quadro de imunossupressão pós-sepse severa. Suporte financeiro: FAPERJ.

08.017 TLR2 on dendritic cells plays a role on the imunossupresion followed severe sepsis? Secca, C.1; Molinaro, R.C.2; Kunkel, S. L.3; Bozza, M.1; Bellio, M.1; Benjamim, C. F.2 1UFRJ - Imunologia; 2UFRJ -; Farmacologia Básica e Clinica; 3University of Michigan - Pathology

Introduction and Objectives: Studies reveal that dendritic cells (DCs) are important for driving immune responses towards Th1 or Th2 profile. During severe sepsis, DCs are altered and present Th2 response. Interestingly, TLR2 is a toll-like receptor that initiates a Th1 or Th2 response depending on antigens bound and/or the cytokine environment. So, our aim is to study the role of TLR2 on bone marrow-derived DCs (BMDC) during imunossupression followed severe sepsis. Preliminary data from our group showed that pulmonary DCs from septic mice presented more TLR2 expression, which correlates with high mortality after fungal secondary infection. Methods and Results: C57/BL6 or TLR2KO mice were subjected to cecal ligation and puncture (CLP) sepsis model. After surgery, mice were treated with antibiotic since 6h after and until 3 days. TLR2KO mice subjected to CLP were more resistant to mortality (83% survival) compared to wild mice (40% survival). BMDCs obtained 15 days after CLP from wild-type mice, but not from TLR2KO mice, produced less IL-10 when stimulated in vitro with LPS. Interestingly, the cells obtained from CLP group and stimulated in vitro with LPS or Pam3Cys presented a lower expression of MHC II and an increased expression of TLR2 on the cell surface, compared to BMDCs obtained from Sham group mice. When the BMDCs were obtained 3 days after CLP surgery a reduction in the number of TLR2 expressing DCs was observed, and these cells expressed less MHCII, compared with the DCs obtained from Sham group of wild type mice. Conclusion: These data corroborates with our preliminary data suggesting that TLR2 plays an important role favoring the susceptibility observed in septic mice, since bone marrow precursor DCs after severe sepsis presented already prevalence for Th2 imune response and have high and long-term TLR2 expression. Financial support: CNPq, FAPERJ.

08.018 Estudo do perfil de imunossupressão pós-sepse grave em camundongos Balb/c: avaliação da participação da prostaglandina E2. Brogliato, A. R.1; Antunes, C. A. C.1; Carvalho, R. S.2; Monteiro, A. P. T.3; Canetti, C.4; Vianna-Jorge, R.1; Benjamim, C. F.1; Macedo,P.R.1 1UFRJ - Farmacologia Básica e Clinica; 2INCa - Farmacologia; 3UERJ - Farmacologia e Psicobiologia; 4IBCCF-UFRJ;

Introdução: Sepse é definida como a síndrome da resposta inflamatória sistêmica oriunda de um foco infeccioso. A sepse grave apresenta uma taxa de mortalidade de 46,9% e pode levar a imunossupressão. Os mediadores envolvidos na imunossupressão após sepse grave não são totalmente caracterizados. Objetivo: Avaliar a participação da Prostaglandina E2 (PGE2), um importante mediador da resposta imune inata e adquirida, na imunossupressão pós-sepse grave. Métodos: Para a indução da sepse grave utilizamos o modelo de ligação e perfuração do ceco (CLP) em camundongos BALb/c onde o ceco é perfurado 2 vezes com agulha 21G. Os animais são submetidos a tratamento antibiótico ertapenem (75mg/Kg nos tempos 5, 24 e 48h após a cirurgia) para recuperação do quadro de sepse e, em seguida, desafiados com conídeos de A. fumigatus (4x106 por via i.t.) para avaliação da imunossupressão. O grupo controle (Sham) foi formado por animais submetidos a cirurgia sem a realização de CLP e tratados com o antibiótico. A PGE2 foi dosada no lavado broncoalveolar (BAL) usando-se a técnica de ELISA. A quantidade de RNAm para COX-2 e para Prostaglandina E sintase (PGES) foi determinada por PCR-tempo real e a expressão da proteína COX-2 foi determinada por Western-blotting. Resultados e discussão: Quando os animais, após o CLP, são desafiados com 4x106 conídios, observamos uma sobrevida de 25% em 7 dias, em comparação com 100 % no grupo Sham. Observamos também que a quantidade de PGE2 no BAL 24h após o desafio é 3 vezes menor em animais do grupo CLP quando comparados com o grupo Sham e 12 vezes maior quando comparados ao grupo naive. O recrutamento de neutrófilos para o BAL e a capacidade fagocítica dos macrófagos não foram diferentes entre os grupos CLP e Sham. A quantidade de RNAm para COX-2 e PGES em macrófagos alveolares foi 3 vezes maior no grupo Sham do que no grupo CLP. No entanto, observamos que a expressão da proteína COX-2 foi 1,4 vezes maior no grupo CLP em relação ao grupo Sham. Para avaliarmos a contribuição da PGE2 para o quadro de imunossupressão após sepse grave, utilizamos um inibidor não seletivo de COX (cetoprofeno, via i.p. 10 mg/Kg). Na presença de cetoprofeno, a sobrevida dos animais após a infecção secundária com A. fumigatus aumentou de 25% para 75%, enquanto o recrutamento de neutrófilos para o BAL e a capacidade fagocítica de macrófagos residentes foram de 2 a 3 vezes maiores. Nossos dados indicam que a PGE2 contribui para o quadro de imunossupresão pós-sepse grave e sugerem que a transcrição da COX-2 e PGES estão sendo diferentemente moduladas numa situação de sepse. Apoio Financeiro: FAPERJ, CAPES, CNPQ, FAF, MS-INCA

08.019 Ativação do eixo HPA induzida pelo MDMA (Ecstasy) altera a distribuição de leucócitos em diferentes compartimentos imunes e diminui a atividade de neutrófilos. Ferraz de Paula, V.1; Ribeiro, A.1; Pinheiro, M. L.1; Sakai, M.2; Moreau, R. L. M.3; Palermo-Neto, J.1 1FMVZ-USP - Patologia; 2VPT-FMVZ-USP; 3FCF-USP - Análises Clínicas e Toxicológicas

Introdução: O MDMA é uma anfetamina alucinógena amplamente utilizada por jovens como droga de abuso. O MDMA altera diversos parâmetros relacionados à esfera imunológica, tanto inata quanto adquirida, entretanto, pouco se sabe a respeito dos mecanismos pelos quais essas alterações são induzidas. Resultados preliminares mostraram o MDMA in vivo, ser capaz de diminuir a atividade de neutrófilos e uma correlação do eixo HPA nesses efeitos. Diante desses resultados, o presente trabalho buscou pelos efeitos do MDMA in vitro sobre a atividade de neutrófilos e alterações na distribuição de leucócitos em diferentes compartimentos imunes. Materiais: Foram utilizados camundongos Balb/C machos. No 1o experimento, 32 animais naive foram submetidos à eutanásia e o sangue foi incubado com solução salina 0.9% controle (C) e com solução experimental (E) de MDMA nas concentrações 66,7ng/mL (E1), 667ng/mL (E2) e 6670ng/mL (E3) a 37ºC por 1 hora e avaliada por citometria de fluxo para a análise da atividade de neutrófilos e morte celular. No 2º experimento, 20 animais foram divididos em 2 grupos: (C) tratados com salina 0,9% e Experimental (E) tratados com MDMA 10,0 mg/kg, via i.p. Após 60 min os animais foram submetidos à eutanásia, foram coletados o baço e a medula óssea, para determinação da celularidade total, e o sangue, para a realização do hemograma e leucograma completo. Resultados: A análise dos resultados mostrou que não houve alterações na morte e na atividade de neutrófilos avaliados in vitro, tanto pelo burst oxidativo quanto pela fagocitose. No 2º experimento, os animais experimentais apresentaram um aumento da porcentagem de neutrófilos (C=38,4±7,58; E=47,40±4,88), do número de eritrócitos (C=8,29±0,71; E=9,32±1,01), da quantidade de hemoglobina (C=13,43±0,53; E=14,85±0,49), da porcentagem do hematócrito (C=47,80±1,13; E=51,60±1,43), e, uma diminuição da porcentagem de linfócitos sanguíneos (C=60,50±6,04; E=50,80±3,73). Diminuição do número total de leucócitos na medula óssea (C=150,0±20,67; E=132,51±6,96) e aumento dos mesmos no baço (C=197,77±31,95; E=238,05±42,31) e diminuição do peso relativo do baço (C=0,48±0,02; E=0,45±0,02) nos animais tratados com MDMA. Discussão: O MDMA in vitro não foi capaz de induzir morte ou alteração na atividade de neutrófilos. Além disso, o MDMA in vivo induziu uma alteração na distribuição de leucócitos nos diferentes compartimentos imunes analisados, diminuindo estes na medula óssea e aumentando no baço. No sangue, aumentou o número de neutrófilos, seguido de uma diminuição de linfócitos. Estes resultados tomados em seu conjunto apóiam a hipótese de que as alterações observadas in vivo são decorrentes da ativação do eixo HPA induzida pelo MDMA, que já havia sido observada em nossos trabalhos. Apoio Financeiro: FAPESP e CNPq

08.020 Células mononucleares do colostro humano produzem melatonina quando ativadas por zimosan não-opsonizado (ZNO) via ativação de NFkB. Lapa, M. A. P. C.1; Pontes, G. N.2; Markus, R. P.1 1IB-USP - Fisiologia; 2USP - Fisiologia; 3IB-USP- Fisiologia

Introdução: A melatonina é produzida de forma rítmica pela glândula pineal e de maneira tônica por tecidos extra-pineais (Markus. Neuroimmunomodul 14:126, 2007). Os fagócitos do colostro humano produzem melatonina quando ativados por bactérias ou por zimosan opsonizado (Pontes. J Pineal Res 41:136, 2006). Esta produção cessa assim que as bactérias são mortas, e é mantida na presença de zimosan. Esta molécula, proveniente da parede celular de S. cerevisiae, quando não-opsonizada ativa receptores Toll 2 (TLR-2) e desencadeia a via de sinalização NF-kB (AKIRA. J Biol Chem 278: 38105, 2003). Neste trabalho testamos a hipótese que ZNO induz a produção de melatonina ativando a via de sinalização NF-kB em fagócitos mononucleares (MN) de colostro humano. Métodos: O colostro de puérperas (48-72 horas pós-parto) foi coletado no Hospital Universitário-USP. Fagócitos MN isolados foram ressuspendidos em RPMI 1640 (2.106 cel/ml) e ativados por 90 min com 10 mg/ml de ZNO na presença ou ausência de bloqueadores da atividade do NFkB (pré-incubação por 30 min; PDTC, ditiocarbamato de pirrolidina: 25 mM; ALLN, n-acetil-leucina-leucinil-norleucina-h: 50 mM). Melatonina do sobrenadante foi dosada por ELISA (IBL Hamburg, Germany). Os dados foram comparados por ANOVA seguida de teste de Newman-Keuls, aceitando-se a probabilidade de 5%. Resultados: O sobrenadante de células controle ou apenas incubadas com PDTC e ALLN não continha melatonina. No sobrenadante de células ativadas com ZNO foi detectada a concentração de 247,1 ± 66,0 (n = 3) pg/ml de melatonina. A pré-incubação com PDTC ou ALLN reduziu a concentração de melatonina no sobrenadante de células incubadas com ZNO em 40,6% ± 1,3% e 45,6 %±7,8 %, respectivamente. Discussão: Nossos dados mostram que fagócitos mononucleares do colostro ativados com ZNO produzem melatonina apenas quando a via NF-kB está disponível. Este mecanismo de ação sugere que ZNO ative, também em células do colostro, o receptor TLR-2. Considerando o efeito protetor exercido pela melatonina em diferentes sistemas, nossos dados sugerem que estas células ativadas poderiam levar à produção de melatonina diretamente no trato gastrointestinal do recém-nascido. Apoio Financeiro: CAPES

08.021 PRÊMIO INOVAÇÃO

08.022 Estudo dos efeitos biológicos do veneno da serpente Bothrops lutzi em rim isolado de rato. Jorge, A. R. C.1; Alves, R. S.1; Norões, T. B. S1; Sousa, D. F.1; Ximenes, R. M.1; Oliveira, I. M. S.1; Silva Neto, A. G.12; Araujo Lima, A. L. G. C.13; Nojosa, D. M. B.2; Monteiro, H. S. A.1 1UFC - Fisiologia e Farmacologia; 2UFC - Biologia

Introdução: O acidente ofídico com serpentes do gênero Bothrops constitui 87,5% dos casos notificados no Brasil (FUNASA,2001), estudos anteriores com o veneno de Bothrops jararacussu e Bothrops insularis em rim isolado de rato mostraram alterações nos parâmetros renais, sugerindo necrose tubular direta em rim. Bothrops lutzi apesar de sua ampla distribuição no nordeste brasileiro, não há registro na literatura caracterizando seu veneno e seus principais efeitos. O objetivo deste trabalho foi avaliar os efeitos causados pelo veneno da serpente B. lutzi em rim isolado de rato. Métodos: Os rins de ratos Wistar (n=6) foram perfundidos com solução de Krebs-Henseileit contendo 6g/dL de albumina por 120 minutos, o veneno bruto da serpente B. lutzi (VBl) – 10 µg/ml - foi fornecido pela Profa. Dra. Diva Borges-Nojosa das serpentes obtidas do Núcleo Regional de Ofiologia (NUROF-UFC) liofilizado, sendo resuspendido em solução de Krebs e adicionado aos 30 minutos após o início do experimento. O controle interno (CI) corresponde aos 30 minutos iniciais. Os dados foram analisados por ANOVA e t de Student (*p<0,05). Resultados: O veneno de B. lutzi diminuiu a pressão de perfusão (PP) - (CIPP=111,5±2,5; VBlPP60=81,0±5,2* mmHg/ mL/g); o fluxo urinário (FU) – (CIFU=0,159±0,019; VBlFU60=0,091±0,022* mL/g/min) e o ritmo de filtração glomerular (RFG) – (CIRFG=0,754±0,089; VBlRFG60=0,457±0,105* mL/g/min) aos 60 minutos, porém o veneno aumentou o fluxo urinário (FU) – (CIFU=0,159±0,019; VBlFU90=0,466±0,093*; VBlFU120=0,981±0,103 mL/g/min) e o ritmo de filtração glomerular (RFG) – (CIRFG=0,754±0,089; VBlRFG90=1,804±0,347*;VBlRFG120=3,152±0,342* mL/g/min) aos 90 e 120 minutos. O veneno também interfere no transporte de eletrólitos, pois se observa uma redução do percentual de sódio transportado (%TNa+) – (CI%TNa+=85,41±1,17; VBl%TNa+90=73,85±2,07*; VBl%TNa+120=67,64±3,60*) e do percentual de cloreto transportado (%TCl-) – (CI%TCl-=81,65±1,46; CVBl%TCl-90=73,82±2,11*; VBl%TCl-120 =67,30±3,70*) aos 90 e 120 minutos. O percentual de potássio transportado aumentou aos 90 minutos (CI%TK+=63,59±1,73; VBl%TK+90=73,80±2,09*). DISCUSSÃO: O veneno de B. lutzi apresentou efeitos semelhantes aos da B. jararacussu, porém com um efeito diurético bem mais pronunciado, sugerindo nefrotoxiciade tubular direta em rim isolado. Apoio Financeiro: CNPq

08.023 Effects of phosphatidylserine liposomes administration in the inflammatory response associated with intestinal ischemia reperfusion in mice. Perucci, L. O.1; Fagundes, C. T.1; Souza, D. da G. de1; Cisalpino, D.2; Madeira, M. F. M.2; Teixeira, M. M.2; Assreuy, J.3 1UFMG - Bioquímica e Imunologia; 2UFMG - Microbiologia; 3UFSC - Farmacologia

Introduction: Ischemia is a common injury and reperfusion in the injured area is, usually, the first choice of treatment. However, this procedure leads to an acute inflammatory response which frequently exceeds the original ischemic insult. Some studies suggest that prolonged intestinal ischemia reperfusion (IRI) periods may cause bacteria translocation through damaged tissues. A recent study, suggest that phosphatidylserine liposomes (PS) exert anti-inflammatory effects in vivo, due to its mimicry of apoptotic cells. Then, we assessed the effects of PS treatment in intestinal IR model in mice. Methods: We developed a prolonged IRI model, in which reperfusion of the superior mesenteric artery (SMA), obstructed for 30 minutes, lead to 100% lethality during the following 48 hours of reperfusion. PS was given 10 minutes before reperfusion (50 mg/kg, i.v.), and its effects in the inflammatory response present in the intestine and in the peritoneal cavity of reperfused mice were analyzed. For this, we assessed the leukocyte recruitment to the peritoneal cavity and gut after 8 and 24 hours of reperfusion. We also measured bacterial translocation to the peritoneal cavity and the level of the cytokines TNF-α, IL-1β and IL-10 in the intestine and in the peritoneal wash fluid. The control group received the same dose of Phosphatidilcoline liposomes (PC). Results: The neutrophil migration to the intestine was enhanced only after 24 hours of reperfusion in PC-treated group. However, the PS treated group showed an early increase in neutrophil content, that is, higher MPO activity at 8 hours of reperfusion. There was significative bacterial translocation to the peritoneum cavity 8 hours after reperfusion in PC-treated group, which was followed by a huge increase in leukocyte (both neutrophil and mononuclear cells) migration to this cavity at 24 hours of reperfusion. PS treatment was able to reduce both, the bacterial translocation at 8 hours and the leukocyte influx to peritoneum after 24 hours of reperfusion. Its administration also leads to reduce production of the evaluated cytokines in both intestine and peritoneal wash fluid at 8 and 24 hours of reperfusion. Discussion: PS administration promotes an earlier inflammatory response after IRI, abolishing bacterial translocation to peritoneal cavity and probably leading to resolution of the inflammatory process before any more severe injury occurs to the reperfused tissue. Then, PS-treatment seems to be beneficial in the inflammatory response to reperfusion injury.

08.024 PRÊMIO INOVAÇÃO

08.025 The role of macrophage migration inhibitory factor (MIF) in an experimental periodontal disease by Aggregatibacter actinomycetemcomitans in a murine model. Madeira, M. F. M.1; Souza, D. da G. de2; Silva, T. A.3; Guimarães, M. O.1; Mitre, G. C.1; Campi, P. S.1; Cisalpino, P. S.1

1UFMG - Microbiologia; 2UFMG - Bioquímica e Imunologia; 3UFMG - Clínica, Patologia e Cirurgia Odontológicas

Introduction: Periodontal disease (PD) is a chronic inflammatory disease of the attachment structures of the teeth. The bacterial biofilm attached to the surface of the tooth in close association with periodontal tissues, is the etiologic factor of this disease. Aggregatibacter actinomycetemcomitans is a small nonmotille periodontophatogen, a gram-negative coccobacillus with a range of potential virulence factors. MIF (Macrophage Migration Inhibitory Factor) is a potent proinflammatory cytokine that may induce TNF-α release and play an important role in innate immune and inflammatory response. Objective: In the present work, our aim was to assess the role of MIF in the experimental model of periodontal disease induced by A. actinomycetemcomitans. Methods: BALB/c wild type (WT) or MIF deficient mice (MIF-/-) received a direct injection of 1 x 109 CFU/mL of A. actinomycetemcomitans strain FDC Y4 (diluted in 10 µL of PBS) into palatal gingival tissue of second molar for the establishment of periodontal disease. Immediately after this injection, it was performed an oral administration of the same inoculum (diluted in 100 µL of PBS with 1,5% of carboxymethylcellulose) and the administration was repeated 48 and 96 hours later. Negative controls received only PBS (NI). Tissues were analyzed by ELISA, myeloperoxidase content, and histology, in order to evaluate the levels of cytokines and chemokines, neutrophil influx, inflammatory infiltrate and alveolar bone loss, respectively. Results: WT mice presented increase of TNF-α production after 15 days of infection (NI: 17.59±5.05; 84.45±7.09 pg/100mg of tissue) and of neutrophil recruitment after 45 days (NI: 0.05±0.004; 0.17±0.016 relative unit). Furthermore, this infection was followed by important alveolar bone loss, leukocytes migration to periodontal tissues and gingival epithelium hyperplasia. Interestingly, there was an increase of MIF production after 10 days of infection (112±10.5; NI: 86±6.22). MIF-/- mice presented increase of neutrophil influx (WT: 0.10±0.02; MIF-/-: 0.15±0.02 relative unit) and CXCL1 production (NI: 358.2±16; 430.4±30.6) when compared to WT mice (NI: 362.7±11; 331.2±12). The increase of the inflammatory response was associated with the enhancement of bone loss in MIF-/- mice (NI: 0.56±0.02; 0.84±0.02 mm2), when compared to WT mice (NI: 0.81±0.03; 0.99±0.01). Conclusion: BALB/c mice submitted to oral inoculation of A. actinomycetemcomitans represent a useful experimental model of periodontal disease since it is characterized by several signals of human disease and MIF plays an important role in the modulation of the disease. Key words: Aggregatibacter actinomycetemcomitans, periodontal disease, MIF, bone loss, inflammation Apoio Financeiro: CNPq and FAPEMIG

08.026 PRÊMIO INOVAÇÃO

08.027 Rhodiola rosea aumenta o número de progenitores mielóides em LTBMCs de camundongos infectados com Listeria monocytogenes. Torello, C. O.1; Ramos, A.L.1; Queiroz, M. L. S.2

1UNICAMP- Farmacologia; 2UNICAMP – Farmacologia – Hemocentro

Objetivos: Estudos com a Rhodiola rosea (ERR) têm demonstrado uma ação adaptógena devido a sua habilidade em aumentar a resistência do organismo a agentes estressores. Neste estudo, utilizamos a cultura líquida de longa duração de células hematopoiéticas (LTBMC) de camundongos infectados com Listeria monocytogenes (LM), um agente estressor, para investigar os efeitos do ERR sobre a manutenção da hematopoese in vitro. Métodos e Resultados: As LTBMCs foram estabelecidas a partir da medula óssea de camundongos BALB/C machos (n=5), infectados IP com 1,5x104 bactérias/animal, pré-tratados (via oral) durante 7 dias com ERR. As culturas foram mantidas por 9 semanas a 37°C na presença de 5% de CO2. Em intervalos semanais, metade do volume do sobrenadante foi removido e substituído por igual volume de meio de cultura. A partir do sobrenadante coletado, verificamos a produção de IL-1α pelo método de ELISA (DY400; R&D) e utilizamos as células não aderentes para ensaios clonogênicos e de viabilidade. Na 9ª semana, o estroma foi tripsinizado e acessamos a viabilidade das células aderentes. Nas LTBMCs dos animais infectados ocorreu uma redução (P<0,05) dos progenitores mielóides (198±25; 34±9; 29±8; 18±5; 7±4) em relação ao controle (330±11; 98±8; 60±7; 42±9; 20±6) da 5ª a 9ª semanas experimentais, respectivamente. O tratamento desses animais com ERR reverteu esta mielossupressão (290±17; 125±10; 63±13; 52±10; 42±4). Com relação aos níveis de IL-1α, não foi observada nenhuma alteração no sobrenadante das LTBMCs dos animais tratados quando comparados com os grupos controle ou infectados. Não observamos diferenças na viabilidade das células não-aderentes e aderentes. Discussão e Conclusões: Nossos resultados demonstram que o ERR previne a mielossupressão induzida pela LM nas LTBMCs. Isto corrobora com nossos achados anteriores, nos quais o ERR foi capaz de promover um aumento in vivo na reserva de progenitores mielóides e nos níveis de TNF-α e IFN-γ dos animais infectados, prolongando em cerca de 60% a sobrevida destes animais. Estes resultados são promissores e sugerem que o ERR promove um aumento da resistência dos animais contra uma infecção com LM por melhorar a capacidade clonogênica dos progenitores mielóides. Apoio Financeiro: Capes

08.028 Odontoblastos estimulados por lipopolissacarídeos expressam fator derivado de célula progenitora. Santos, V. A. C.1; Gomes, A. C.2; Oliveira, S. H. P.3 1UNESP - Ciências Básicas; 2UNESP - Ciências Básicas- Farmacologia; 3Odontologia de Araçatuba-UNESP - Ciências Básicas

Bactérias gram-negativas e seus produtos tal como lipopolissacarídeos (LPS), provenientes de cárie dental podem levar a uma resposta inflamatória pulpar. Os odontoblastos situados na interface polpa/dentina são as primeiras células a encontrar estas bactérias e podem desempenhar um papel importante no controle na inflamação. Um dos pontos que compõe o processo inflamatório é a angiogênese, que pode ser iniciada por diversos fatores de crescimento tal como, o Fator derivado de célula progenitora (SCF) que poderia regular a neovascularização ativando as células residentes da polpa dental de dentes com cárie profunda levando a produção de mediadores inflamatórios que podem auxiliar a formação de um tecido novo. Dessa forma, o objetivo deste estudo foi avaliar a expressão de SCF por linhagem de odontoblastos estimulados por LPS. Para tanto, utilizamos odontoblastos da linhagem murina MDPC-23. As células foram estimuladas com 0,1; 1 e 100 ug/ml de LPS em diferentes períodos de tempo (1, 6 e 24 h). A expressão de SCF nos odontoblastos foi avaliada pela técnica da reação de polimerase em cadeia (RT-PCR) e os resultados são dados como expressão do RNA mensageiro em relação à β-actina. Os resultados demonstraram que as células estimuladas por LPS após 1 hora já são capazes de expressar SCF na concentração de 0,1 ug/ml (relação SCF/β-actina=1); 10 ug/ml (relação SCF/β-actina=0,9) e 100 ug/ml (relação SCF/β-actina=0,85). Após 6 horas da estimulação das células por LPS, observamos uma expressão mais significativa com a concentração de 10 ug/ml (relação SCF/β-actina=3,89), diminuindo com a dose de 100 ug/ml (relação SCF/β-actina=0,77). Após 24 horas da estimulação por LPS, a dose de 1 ug/ml (relação SCF/β-actina=1) induziu uma expressão significativa diminuindo com as doses de 10 ug/ml (relação SCF/β-actina=0,63) e 100 ug/ml (relação SCF/β-actina=0,48). Estes resultados demonstram que os odontoblastos quando estimulados por LPS expressam SCF e o pico de expressão foi 6 horas após com a dose LPS (10 µg/ml). Os dados em conjunto podem sugerir que SCF liberado pelos odontoblastos pode está relacionado com o processo de iniciação da angiogênese, após a inflamação, podendo assim regular a neovascularização, levando a produção de mediadores químicos que participam da formação de um tecido pulpar novo. Apoio Financeiro: Fapesp

08.029 PRÊMIO INOVAÇÃO

08.030 Protection against flu infection through the blockade of PAFR. Garcia, C. C.1; Russo, R. C.1; Guabiraba, R.1; Fagundes, C. T.1; Barbosa, R. P. A.1; Gazzinelli, R. T.2; Machado, A. de M. V.3; Teixeira, M. M.1 1UFMG - Bioquímica e Imunologia; 2UFMG/FIOCRUZ - Bioquímica e Imunologia; 3UNIFESP - Bioquímica; 6UFMG -

Introduction: Flu is a respiratory illness of great world-wide relevance, causing annual epidemics and great number of deaths and hospitalizations. Strategies to combat the Influenza virus face challenges because of the development of strains resistant to vaccines and antiviral drugs. It is wide known that the activation of the host immune system against this virus may cause clinical symptoms and mortality. Our hypothesis was that blocking the action of the platelet activating factor (PAF), a phospholipid mediator related to inflammatory and immune responses, could prevent transmigration and leukocyte activation during Influenza infection and hence be a useful tool to decrease the severity of the disease. Methods: To evaluate the effect of PAF receptor blockade in experimental infection with the Influenza virus WSN/33, PAFR deficient mice (KO) and C57 BL6J (WT) were infected with 104 or 106 PFU of the Influenza virus; treatment with PAFR antagonist (PCA 4248) was given, alone or in combination with the antiviral Oseltamivir Phosphate (Tamiflu®), for 7 days after the third day of 106 PFU infection in WT animals. Lethality and inflammatory parameters, as cellular recruitment to alveolar space and lungs, chemokine and cytokine levels in serum and lungs were evaluated, as well as viral titer. Interstitial lung inflammatory cell profile in lungs under infection was also studied through flow cytometry in PAFR KO and WT mice. Results: PAFR KO animals showed increased survival, reduction in neutrophilic transmigration to the alveolar space and similar levels of the main cytokines and chemokines involved in the inflammatory response, except for the increased levels of IL-1β in lungs, compared to WT animals. The viral titers in the lungs were lower or similar to the wild type animals. The amount of lung interstitial activated neutrophils, expressing GR1 and CXCR2 was also lower. The treatment with the PAFR antagonist resulted in similar protection and the combination with low doses of the antiviral, increased this protection. The treatment resulted in decrease in the systemic production of IL-6, but it did not modify levels of other cytokines and chemokines studied. Moreover, there was a decline of the viral titers similar to the reduction caused by the administration of the antiviral drug. Discussion: Therefore, PAFR blockade confers protection to the experimental infection with Influenza virus, probably due to inhibition of neutrophil recruitment and activation as well as the reduction of IL-6 in the blood. The maintenance in the levels of the main symptoms-related cytokine leads us to suppose that PAFR blockade acts modifying the cellular response to these cytokines, reducing leukocyte activation and systemic damage. Apoio Financeiro: CNPq e CAPES

08.031 TBXA2 PLAys an important role in the characteristic vascular and inflammatory alterations induced by DV infection. Cisalpino, D.1; Fagundes, C. T.2; Souza, R. S.2; Sousa, L. P.2; Teixeira, M. M.2; Souza, D. da G. de2 1UFMG - Bioquímica e Imunologia Microbiologia; 2UFMG - Bioquímica e Imunologia

Introduction: Dengue virus (DV) has emerged as the most relevant arthropod-borne viral disease in tropical areas, reaching millions of cases worldwide each year. During the viral infection, there is a characteristic development of vascular instability, as increased vascular permeability, elevated haemoconcentration and thrombocytopenia are observed. Eicosanoids are a major class of lipid mediators involved in several homeostatic and inflammatory processes, largely associated with platelet activity and endothelial behavior. Thromboxane A2 (TBXA2) and its receptors are some of the main molecules involved in these mechanisms. Our group is evaluating the importance of TBXA2 in an experimental model of Dengue virus serotype 2 (DEN-2) infection. This model of DEN-2 infection is followed by great inflammatory response as assessed by cytokine/chemokine production and neutrophil recruitment and severe circulatory dysfunction. Methods: Balb/c mice were infected intra-peritoneally with 1 PFU of DEN-2, and, from the 5th day of infection, received the following treatments: Acetyl Salicylic Acid (AAS) (doses of 100, 10 and 1 mg/kg,), BMS 180291 (20 mg/kg, selective antagonist of TP receptors) and Indomethacin (INDO) (2mg/kg). Non-infected mice, and animals treated only with drug vehicle were used as controls. After discreet intervals (7th day of infection for inflammatory parameters, 14th for lethality assessment) lethality, hematocrit and platelet number, viral titers, cytokine/chemokine production and neutrophil influx were evaluated. Results and Discussion: Treated animals showed an improved clinical condition as indicated by reduced haemoconcentration and higher platelet count. Inflammatory parameters, such as production of TNF-α, IL-6, IFN-γ in the liver and the spleen, production of CXCL1 in the liver and the lungs and MPO activity in these organs, were also significantly decreased in the treated groups. The observed effects were dose dependent for AAS, and the higher dose-treated group (100 mg/kg) presented similar results to that found in BMS and INDO-treated animals. This suggests that the reduced inflammatory response in the treated groups studied were due to blockade of TBXA2 action. However, any of the treated groups showed smaller lethality rates that vehicle-treated ones. This can be explained, partially, by the fact that treated animals showed similar viral titers in the spleen to those found in the vehicle treated groups. Conclusion: TBXA2 plays a significant role in the characteristic vascular alterations induced by DV infection, and its blockade results in reduced inflammatory response after disease onset. However, TBXA2 inhibition does not interfere in host ability to control such infection. Apoio Financeiro: CNPq e FAPEMIG

08.032 IL-12 and IL-18 act in synergism to induce IFN-g production and to improve host resistance to Dengue virus primary infection. Fagundes, C. T.1; Cisalpino, D.1; Amaral, F. A.1; Souza, R. S.1; Sousa, L. P.2; Guabiraba, R.1; Teixeira, M. M.1; Souza, D. da G. de3 1UFMG - Bioquímica e Imunologia; 2UFMG-COLTEC; 3UFMG - Microbiologia

Introduction: Dengue is a mosquito-borne infection which has become a major international public health concern. Recently, we showed that IFN-g is essential for host ability to control Dengue infection. However, the factors involved in host control of IFN-g production during Dengue infection remain obscure. Then, we have evaluated the course of Dengue-infection in the absence of host production of IL-12 and IL-18, two cytokines showed to be able to induce IFN-g production during infection processes. Methods: IL-12p40-deficient (IL-12p40-/-), IL-18-deficient (IL-18-/-), and their respective wild type (WT) control mice were infected with Den-2 (10 PFU, i.p.) and after various periods of time, the lethality rates, haematocrit and platelets number, cytokine production in spleen and serum, and DENV2 titers in spleen, were evaluated. Furthermore, intending to block production of IL-12 and IL-18 simultaneously, IL-12p40-/- mice (with their adequate control groups) were infected with DENV2 (10 PFU, i.p.) and received a daily injection of an IL-18-neutralizing protein (IL-18bp – 250 mg/day, i.p, from day 1 until day 5 of infection) to interfere with the action of this cytokine. In the latter experiment, the same parameters were assessed in the 5th day of infection, except for lethality rates. Results and Discussion: The production of IFN-g after DENV2 infection was preceded by production of IL-12 and IL-18, in the spleen. After DENV2-infection, IL-12p40-/- mice showed a discreet IFN-g production, if compared with WT mice. This was followed by increased haemocontration, elevated IL-6 production and enhanced lethality rates after infection. However, the viral titers in spleen of IL-12p40-/- mice were similar to the levels found in WT mice after DENV2-infection. In parallel, infected IL-18-/- mice showed reduced, but not absent, production of IFN-g. Again, this reduced IFN-g production was followed by enhanced thrombocytopenia, increased haemoconcentration, and higher production of the cytokines IL-6 and TNF-a and lead to higher lethality rates in DENV2-infected IL-18-/- mice. Although, the viral titers in IL-18-/- and WT mice were similar after seven days DENV2-infection. Interestingly, the blockade of IL-18 action in IL-12p40-/- resulted in complete inhibition of IFN-g production and loss in control of DENV2-replication. This was followed by enhanced disease manifestation (as assessed by increased haemoconcentration and thrombocytopenia) and increased pro-inflammatory cytokine production. Conclusion: IL-12 and IL-18 seem to act in conjunct to induce IFN-g production after Dengue virus infection. In the absence of these molecules, there is reduced IFN-g production, and the infection evolves to a more severe manifestation, resulting in enhanced lethality. Strategies that promote IL-12 and IL-18 production by the host could be useful during the control of primary infection by Dengue virus. Apoio Financeiro: CNPq and FAPEMIG

08.033 A Potential major role for the CCR5 receptor in mediating the infection by dengue virus serotype-2 and its physiopathological manifestations in mice. Guabiraba, R.1; Fagundes, C. T.1; Hirako, I. C.1; Cisalpino, D.2; Souza, R. S.1; Ianzer, D.1; Souza, D. G.2; Teixeira, M. M.1 1UFMG - Bioquímica e Imunologia; 2UFMG - Microbiologia

Introduction: Dengue virus (DENV) is a mosquito-borne virus in the family Flaviviridae, consisting of four antigenically related serotypes. Infection with these viruses can lead to dengue hemorrhagic fever/dengue shock syndrome and also to a huge inflammatory response. The chemokine receptor CCR5 is a crucial host factor exploited by HIV for cell entry and is also important for HCV physiopathological manifestations. Here, we´re investigating how the infection by DENV serotype-2 in mice works in the absence of the CCR5 receptor. Methods: C57/BL6 (WT) or CCR5 -/-- mice were inoculated i.p. with 10 or 100 LD50 of a mouse adopted DENV-2 strain. An infected group received met-RANTES, a CCR1/CCR5 antagonist. Lungs, liver and spleen were removed at day 7 for MPO activity assay, histological analysis and cytokines measurement by ELISA. Liver, spleen and serum were also used for viral titration. Blood was collected for serum cytokine levels analysis, platelet count and hematocrit evaluation. Results: WT infected animals showed significant increase in neutrophil recruitment and higher levels of pro-inflammatory cytokines/chemokines (TNF-a, IFN-g and IL-12, CCL3/MIP-1a, CCL2/JE and CCL5/RANTES) in the spleen, liver and lungs at day 7 post-infection. IFN-g is also increased in serum. They also presented a markedly reduction in platelet count. No CCR5 -/- infected mice died after 14 days, while at least 60% of WT infected mice died after day 7. No differences in hemoconcentration were observed among the groups. CCR5 -/- also showed similar levels of MPO activity, platelet count, cytokines and chemokines to those observed in non-infected WT mice. Regarding viremia, CCR5 -/- showed a notably reduction or total absence in liver, spleen and serum viral titration when compared to WT infected mice. Histological analysis in the liver, lungs and spleen showed a mild hemorrhagy and leukocyte infiltrate in WT infected mice, what is not observed in CCR5 -/- infected mice. Treatment with met-RANTES leaded to the same phenotype observed for WT mice, with higher levels of MPO activity, cytokine and chemokines and platelet count, including discrete tissue damage. This result might be attributed to its effects on CCR1 receptor, since CCL3/MIP-a -/- infected mice have 100% of lethality before day 7, or even to its well-known conformational-change effects on the CCR5 receptor. Conclusion: From these data we showed, for the first time, that CCR5 receptor should be a main protein involved in DENV-2 infection and in its pathophysiological manifestations, and further cellular and molecular studies may clarify the real role of this receptor in this intriguing viral disease. Apoio Financeiro: CNPq

08.034 Caspase-1 deficient mice did not present failure of neutrophil migration in severe polymicrobial sepsis. Sonego, F.1; Alves-Filho, J. C.1; Freitas, A.1; Nascimento, D. C. B.2; Zamboni, D. S.3; Cunha, F. de Q.1 1FMRP-USP - Farmacologia; 2FMRP-USP - Imunologia e Bioquímica Aplicada; 3FMRP-USP - Biologia Celular, Molecular e Bioagentes Patogênicos

Background: Sepsis is characterized by an uncontrolled production of inflammatory mediators that culminate in a marked reduction in neutrophil migration in severe sepsis. Failure of leukocytes migration leads to an unsuccessful control of local infection and is associated to poor prognostic. Caspase-1 plays a crucial role in inflammatory response by cleaving of inactive in active forms of key cytokines in this process. In this study, we evaluated the role of caspase-1 on inflammatory response during polymicrobial severe sepsis. Methods: C57Bl/6 (WT) and caspase-1 knockout (caspase-1-/-) mice (20-25 g) were underwent to severe sepsis by cecum ligation and puncture (CLP) method. The animals were killed 18 hours after CLP surgery and peritoneal neutrophil migration, lung neutrophil sequestration and bacterial count in peritoneal cavity and blood were evaluated. Even, survival was observed during 7 days post CLP. Results: Caspase-1-/- mice presented approximately 75% of survival post severe sepsis in contrast to 0% of WT. In fact, the failure of neutrophil migration into peritoneal cavity observed in WT underwent to CLP-induced severe sepsis was not observed in Caspase-1-/- mice, (Caspase-1-/-= 9.5 ± 3x106 and WT= 0.8 ± 0.02x106 neutrophils/cavity). As consequence, caspase-1-/- mice presented a more efficient control of the infection in the peritoneal cavity, since bacteremia was lower in knockout mice than in WT (5.2 and 6.6 Log CFU/mL, respectively). Moreover, systemic inflammatory mediators and lung injury were lower in caspase-1-/- than WT mice. Discussion: Altogether, these results suggest that caspase-1 is involved in the mechanism of failure of neutrophil migration and consequently impaired bacterial clearance, leading to dissemination of infection to systemic circulation during polymicrobial severe sepsis. Apoio Financeiro: CNPq and FAPESP

08.035 Expression of CCR2 in neutrophils leads to detrimental tissue infiltration during sepsis: role of toll-like receptors. Souto, F. O.1; Alves-Filho, J. C.2; Freitas, A.2; Martins, A.1; Basile-Filho, A.1; Cunha, F. de Q.2 1FMRP-USP – Cirurgia e Anatomia; 2FMRP-USP - Farmacologia

Introduction: Chemokines display central role in mediating neutrophil migration. Neutrophils respond to CXC chemokines, such as CXCL2, but are usually unresponsive to CC chemokines, such as CCL2. It is known that chemokine responsiveness in neutrophils can be modulated by inflammatory stimuli. Here, we investigate the role of Toll-like receptors (TLRs) on the modulation of CCR2 expression and responsiveness in neutrophil and the consequence of this regulation during sepsis. Methods: Neutrophils purified from blood (healthy subjects) or bone marrow (WT, CCR2-/-, TLR2-/- and MyD88-/- mice) were incubated with lipoteichoic acid (LTA, TLR2-agonist, 10μg/mL) and lipopolysaccharide (LPS, TLR4-agonist, 10μg/mL) for 2 h. In some experiments, neutrophils were treatment with pyrrolidine dithiocarbamate (PDTC, NF-kB inhibitor, 10μM/mL) or cyclohexemide (CHX, protein synthesis inhibitor, 30μM/mL) 30 min before LTA/LPS incubation. Next, CCR2 expression was evaluated by FACS and immunofluorescence. Moreover, chemotaxis and F-actin polymerization in response to CCL2 were evaluated by Boyden chamber and fluorescence microscopy, respectively. These parameters were also evaluated in neutrophils purified from blood of septic patients or WT mice underwent cecal ligation and puncture (CLP) sepsis model. Finally, neutrophil infiltration in organs, determined by myeloperoxidase activity, and survival was evaluated in CCR2-/- mice underwent CLP. Results: Resting murine or human neutrophils do not present expression of CCR2 receptor or do not respond to CCL2 chemokine. However, after LTA or LPS incubation, high expression of CCR2 was detected and CCL2 was able to induce neutrophil chemotaxis and F-actin polymerization. CCL2 response was blocked by treatment with PDTC and CHX or by using CCR2-/- neutrophil, suggesting that CCL2 responsiveness require NF-kB-dependent CCR2 synthesis. Moreover, LTA or LPS-induced CCL2 chemotaxis was not observed in TLR2-/- or MyD88-/- neutrophils. Interestingly, neutrophils from septic patients and septic mice presented high CCR2 expression and CCL2 responsiveness. Finally, we found that CCR2-/- mice exhibited reduced neutrophil infiltration in heart, lung and kidney and enhanced survival rate compared to WT mice. Discussion: Our findings demonstrate that TLRs activation induces CCR2 expression and responsiveness in human and murine neutrophils, which is involved in the detrimental infiltration of these cells in organs during sepsis. Considering these results, the CCR2 blockade may be a potential strategy for treatment of human sepsis. Apoio Financeiro: FAEPA/ FAPESP/ CNPq /