Epidemiological observations have linked increased host iron with malaria susceptibility. At the same time, blood-stage malaria infection is associated with potentially life-threatening anemia. To improve our understanding of these relationships, this work presents an examination of the mechanisms controlling the upregulation of the hormone hepcidin, the master regulator of iron metabolism, in malaria infection. Chapter 2 presents data from a mouse model of malaria infection which indicate that hepcidin upregulation in malaria infection is associated with increased activity of the sons of mothers against decapentaplegic (Smad) signaling pathway. Although the canonical Smad pathway activators, bone morphogenetic proteins (Bmp) are not increased at the message level following infection, activin B, which has been recently shown to increase hepcidin through the Smad signaling pathway in conditions of inflammation and infection, is upregulated in the livers of malaria-infected mice. Chapter 3 shows that both activin B and the closely related protein activin A upregulate hepcidin in vitro and in vivo. Chapter 3 also explores the effects of the activin-binding protein follistatin in both systems and in the same malaria-infected mouse model as presented in Chapter 2. The work presented in Chapter 4 extends these studies to human infections by demonstrating that activin A protein co-increases with hepcidin in human serum during malaria infection. Taken together, these findings are consistent with a novel role for activin proteins in controlling hepcidin upregulation in the context of malaria infection. This work may form a basis for the development of novel therapeutics that speed recovery from malarial anemia by inhibiting activins’ actions. Chapter 5 examines the role of infected red blood cell-derived microparticles in the initial recognition of a P. falciparum malaria infection, and subsequent hepcidin upregulation. Microparticles stimulate production of cytokines from peripheral blood mononuclear cells (PBMC), which also upregulate activin A message in response to both microparticles and whole infected red blood cells. These data are consistent with a model in which malaria-derived stimuli such as microparticles trigger the systemic release of activin proteins, which then act on the liver to upregulate hepcidin. Evidence has shown that cytokine levels at birth are related to malaria risk. In Chapter 6, hepcidin is measured in cord blood samples from participants in a large-scale clinical study in a malaria-endemic area, and shown to be elevated in cord blood from neonates with a clinical history of placental malaria. Cord blood hepcidin is also compared to birth levels of iron markers and other cytokines, and future clinical outcomes. Finally, the contributions of DNA methylation levels to cord hepcidin and cytokine levels are assessed by comparison of CpG methylation, at sites in genes encoding hepcidin and cytokines, to the serum concentrations of the genes’ protein products. Several intriguing associations are noted which indicate a possible novel role for DNA methylation in the determination of birth cytokine and hepcidin levels. Chapter 7 synthesizes the data presented in this thesis, interprets the possible significance of the major findings, and offers suggestions for future work.

Double stranded RNA (dsRNA), the genomic material of some viruses and a replication intermediate in others, is recognized by multiple signaling receptors that initiate the anti-viral response1. Viruses have developed mechanisms to circumvent the anti-viral response by targeting components of the signaling pathway. An example of one such pathway is the TLR3 signaling pathway, which contains a kinase complex that activates interferon regulatory factor 3 (IRF3), leading to production of type I interferons. The kinase complex consists of a scaffold protein, NAK-associated protein 1 (NAP1), and two kinases, TANK-binding kinase 1 (TBK-1) and IκB kinase epsilon (IKKε). A fourty residue sequence in NAP1 was discovered that mediated its interaction with TBK1 and IKKε, termed the kinase binding domain (KBD)1. However, the function of NAP1 in mediating kinase activation is unknown and understanding this is the long-term goal of this project. The goal of this thesis was to test the dependency of NAP1’s dimeric structure on mediating interactions with the kinases. Biochemical characterization of recombinant targets was completed using size-exclusion chromatography (SEC) and NAP1 KBD WT eluted as a dimeric species. CFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 KBD and scaffold binding motif (SBM) of the kinases, TBK-1 and IKKε, were generated to assess interactions using fluorescence resonance energy transfer (FRET). NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis was attempted to identify the residues necessary for NAP1 dimerization and any effect dimerization may have on kinase recognition. This thesis shows data to support that NAP1 KBD forms stable homo-oligomers and directly interacts with a small C-terminal portion of TBK1 and IKKε.

Aim: The aim of this study is to determine the frequency of single nucleotide polymorphisms (SNPs) in various pattern recognition receptor (PRR) genes, including Toll like receptors (TLR) -2, -4, -9, and CD14 in chronic (CP), localized (LAP) and generalized aggressive (GAP) periodontitis and periodontally healthy (NP) patients in an African American population. Methods: A total of 205 subjects were involved in the study. The LAP group consists of 25 subjects, the GAP group 50 subjects, the CP group 73 subjects and the NP group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. Results: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between diseased-groups versus reference group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in healthy group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD 14 polymorphism. Conclusion: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to the periodontal disease susceptibility in African Americans. Additionally, presence of C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD 14 genes are not related to periodontitis in African Americans. However, low copy number of certain alleles warrants further investigations with increased sample size to explore the role of SNPs in periodontal disease. This study was supported by the Alexander Fellowship.

Introduction: Diabetes mellitus is a polygenic disease and its development is influenced to some extent by environmental factors as well. Innate immunity triggers nonspecifically first defense reactions after penetration of the pathogen into the body, while overstimulation components of innate immunity may give rise to autoimmune diseases, including diabetes type 1. The components of innate immunity are, among others, Toll-like receptors (TLRs) belonging to a group of the structures recognizing preserved molecular structures characteristic of pathogens. Toll-like receptors are abundantly expressed by monocytes which produce prolactin (PRL) having an immunostimulatory function. To clarify the role of innate immunity in the pathogenesis of diabetes, we focused on the expression of mRNA and protein expression of TLR2 and TLR4. The expression of PRL was studied only at the level of mRNA. Monocytes were separated by flow cytometry into classical (CD14++) and nonclassical (CD14+). We monitored their percentages and the degree of expression of CD14 antigen on their surface.The operational objective of this dissertation was to optimize the stimulation of monocytes for the planned study of the function of non-pituitary prolactin in vitro and determine the appropriateness of the use of healthy donors' buffy...

La truite arc-en-ciel (Oncorhynchus mykiss), espèce d'élevage majeure en Europe et notamment en France, est l'une des espèces de poisson les mieux connues dans un grand nombre de domaines, y compris l'immunologie. Les virus qui l'infectent ont aussi été bien caractérisés, en particulier deux Novirhabdovirus, le virus de la septicémie hémorragique virale (VSHV) et le virus de la nécrose hématopoïétique infectieuse (VNHI), tous deux connus pour provoquer des pertes importantes dans les élevages aquacoles. Quelques travaux, conduits notamment à l'INRA, ont mis en évidence l'existence d'une variabilité génétique de la résistance à ces infections chez la truite (Quillet et al., 2007). Une approche combinant analyse génétique et étude des réponses cellulaires a été développée pour tenter de mieux caractériser la réponse de la truite contre le VSHV. L'objectif est de développer des outils d'amélioration de la santé dans les élevages piscicoles et de mieux comprendre les mécanismes de résistance antivirale chez les vertébrés. Tout d'abord, une démarche de cartographie de QTL (quantitative trait locus) a permis de détecter un QTL majeur de résistance au VSHV dans la région télomérique du groupe de liaison 31 de la truite arc-en-ciel. Ce QTL contrôle la survie des poissons et la croissance in vitro du virus sur explants de nageoire (VREFT), ce qui suggère fortement l'implication de mécanismes innés dans la résistance. Le QTL est retrouvé dans des croisements impliquant des reproducteurs de résistance variée, et peut expliquer jusqu'à 65% (survie) et 49% (VREFT) de la variance phénotypique observée. Enfin, l'effet du QTL est conservé quel que soit le mode d'infection employé (balnéation ou injection intrapéritonéale), suggérant que la résistance n'est pas liée à des particularités des tissus superficiels (peau, mucus), premiers sites de contact entre le virus et son hôte. En parallèle, des lignées cellulaires ont été dérivées à partir d'ovaires de truites appartenant à des lignées isogéniques présentant des niveaux de résistance variable à l'infection par le VSHV. Une corrélation remarquable est observée entre la résistance à l'infection des lignées cellulaires et la survie des poissons dont elles sont issues, confirmant définitivement le rôle déterminant de mécanismes innés dans la résistance. Ce modèle cellulaire a également permis de montrer que le contrôle précoce de la prolifération virale était une étape clé de la résistance. Le parallélisme entre résistance in vitro et in vivo semble conservé lors de l'infection par un second rhabdovirus, le VNHI, bien qu'aucune corrélation dans la résistance à ces deux infections n'ait été observée dans cette étude. Par ailleurs, le QTL à effet fort identifié pour la résistance au VSHV ne joue pas un rôle majeur dans la variabilité de résistance au VNHI. Ceci suggère que, même si ils concourent à l'activation de voies de signalisation communes, les facteurs clés de la résistance aux deux virus sont différents, et leur expression contrôlée par des zones génomiques distinctes. Les résultats obtenus dans cette étude ont permis de démontrer sans équivoque le rôle clé des mécanismes innés dans la résistance de la truite à l'un de ses principaux virus, et l'existence d'une forte variabilité génétique sous-tendant l'expression des facteurs impliqués. En proposant des bases nouvelles pour aborder l'analyse des interactions hôte-virus chez la truite, ils ouvrent la voie à la découverte de mécanismes potentiellement nouveaux dans la réponse des poissons à ces infections et à une meilleure compréhension de ces mécanismes chez les vertébrés. / The rainbow trout (Oncorhynchus mykiss) is one of most significant fish model in many scientific fields, including immunology. Due to its importance in aquaculture, viruses that can infect this species have been well characterized. Two well-known Novirhabdoviruses, the viral haemorrhagic septicemia virus (VHSV) and the infectious hematopoietic necrosis virus (IHNV) cause serious damage in fish farms and represent a significant threat for aquaculture in a number of countries. Our laboratories have previously reported a wide range of susceptibility to these infections in rainbow trout depending on the host genetic background (Quillet et al., 2007). In this work, we undertook a dual approach to better characterize the antiviral response in fish. A without a priori approach led to the detection of a major QTL (quantitative trait locus) for resistance to VHSV in the telomeric region of the rainbow trout linkage group 31. This QTL controls both fish survival and viral replication in excised fin tissue (VREFT), suggesting the involvement of innate mechanisms in the resistance, and can explain up to 65% (survival) and 49% (VREFT) of the observed phenotypic variation. Additionally, this major locus was retrieved in a number of genetic backgrounds, and regardless of the infection route (waterborne infection or injection), suggesting that the virus entry in fish is not the main factor of resistance. In parallel, cell lines were derived from ovaries of several rainbow trout isogenic lines with various levels of susceptibility to infection with VHSV. Resistance of cell lines to infection by the virus was remarkably correlated with the survival of fish from which they were derived, confirming the importance of innate factors for the resistance. This model also showed that the early stage response is critical for the cellular fate after infection. The parallelism between resistance in vitro and in vivo has finally been observed after infection by a second rhabdovirus, IHNV, although no correlation in resistance to these two viruses could be detected. Moreover, no major QTL for IHNV resistance was found in the region of the VHSV QTL. This observation suggests that the key factors of resistance are different, even if they contribute to the activation of common signaling pathways. The expression of these factors is in any case controlled by distinct regions of the genome. Our work demonstrates a strong genetic determinism of resistance to a major virus in rainbow trout, based on innate mechanisms. We believe that these results pave the way for the discovery of new host response mechanisms against viruses, leading to a better understanding of antiviral immunity in vertebrates.

Doctor of Philosophy / Department of Biochemistry / Ramaswamy Krishnamoorthi / Insect [beta]-1,3-glucan recognition protein ([beta]GRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Delineation of mechanistic details of these processes may help develop strategies to control insect-borne diseases and economic losses. Multi-dimensional nuclear magnetic resonance (NMR) techniques were employed to solve the solution structure of the Indian meal moth (Plodia interpunctella) [beta]GRP N-terminal domain (N-[beta]GRP), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. This is the first determined three-dimensional structure of N-[beta]GRP, which adopts an immunoglobulin fold. Addition of laminarin, a [beta]-1,3 and [beta]-1,6 link-containing glucose polysaccharide (∼6 kDa) that activates the proPO pathway, to N-[beta]GRP results in the loss of NMR cross-peaks from the backbone [subscript]1[subscript]5N-[subscript]1H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-[beta]GRP:laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (∼102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-[beta]GRP:laminarin complex in solution differs from the one in which a single N-[beta]GRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of a X-ray crystal structure of the N-[beta]GRP:laminarihexaose complex. AUC studies and phenoloxidase activation measurements made with designed mutants of N-[beta]GRP indicate that electrostatic interactions between the ligand-bound protein molecules contribute to the stability of the N-[beta]GRP:laminarin complex and that a decreased stability results in a reduction of proPO activation. These novel findings suggest that ligand-induced self-association of the [beta]GRP:[beta]-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the pathogen recognition signal. In the case of the homolog of GNBPA2 from Anopheles gambiae, the malaria-causing Plasmodium carrier, multiligand specificity was characterized, suggesting a functional diversity of the immunoglobulin domain structure.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

Em ouriços, os esferulócitos vermelhos são responsáveis pela biossíntese do equinocromo, um pigmento naftaquinônico considerado antioxidante e bactericida, no entanto seu papel na resposta imune permanece pouco elucidado. O presente trabalho avaliou a reposta imune inata de ouriços-do-mar Lytechinus variegatus, através da atividade fagocítica frente a diferentes concentrações de equinocromo (50 e 100 µg/ml). Para tanto, o equinocromo foi extraído e purificado por RP-HPLC. Nossos resultados demonstraram que o equinocromo em ambas as concentrações modula positivamente a fagocitose, aumentando a quantidade de células fagocitando. A concentração de 50 µg/ml foi capaz de ativar os amebócitos fagocíticos (AF), e aumentar a quantidade de AF com quatro ou mais leveduras fagocitadas. Já na concentração de 100 µg/ml, além da ativação dos AF, aumentou também, a quantidade de AF com uma, duas, quatro ou mais leveduras fagocitadas, sugerindo uma atuação dose-dependente. Desta forma, os dados apresentados demonstram que o equinocromo exerce um importante papel na resposta imune. / The biosynthesis of echinochrome is mediated by red sphere cell. This naphthoquinonic e pigment presents antioxidant and bactericidal characteristics. However, the echinochrome role in immune response remains unclear. In this study, we evaluated the innate immune response of the sea urchin Lytechinus variegatus. To this purpose, the echinochrome was extracted and purified by RP-HPLC. Finally, phagocytic amoebocytes were exposed to different concentrations of echinochrome (50 and 100 mg/ml), when phagocytic activity was analysed. Here, we showed that echinochrome positively modulate phagocytosis, increasing the number of phagocytizing cells. The concentration of 50 mg/ml activated phagocytic amoebocytes (AF), and increased the number of AF containing four or more phagocytosed yeasts. For the other hand, at 100 mg/ml exposure, the activation of AF also increased the number of AF with one, two, four or more yeast phagocytosed, suggesting a dose-dependent activity. Thus, the data presented demonstrated that echinochrome plays an important role in the immune response.

O diabetes mellitus (DM) é caracterizado por hiperglicemia associada à falta ou à ineficiência da insulina. DM também é marcado por alterações em diversos processos celulares que precisam ser mais bem entendidos. Estudou-se a via da autofagia em diferentes macrófagos, verificando se o tratamento com insulina é capaz de modular esse processo. Foram estudados macrófagos derivados da medula óssea (BMM), do lavado broncoalveolar (LBA) e do tecido esplênico de ratos Wistar, machos, diabéticos (aloxana, 42 mg/kg, i.v., 10 dias), ratos diabéticos tratados (insulina 4UI, s.c.) e respectivos controles. Para caracterização do modelo e avaliação do efeito da insulina sobre o processo autofágico, as seguintes análises foram realizadas: (a) glicemia, número de leucócitos no sangue periférico, número de células do LBA; (b) concentrações de citocinas: interleucina (IL)-1beta, fator de necrose tumoral (TNF)-alfa, IL-6, IL-4, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-1 e CINC-2 no sobrenadante do LBA pela técnica de ELISA; (c) caracterização de macrófago alveolar (MA) do LBA quanto a antígenos de superfície (MHCII, pan-macrophage KiM2R, CD11b) e marcadores autofágicos (proteína de cadeia leve associada a microtúbulo (LC)3 , gene/proteína relacionado à autofagia (ATG) pela técnica de citometria de fluxo e microscopia confocal ; (d) estudo dos macrófagos diferenciados, a partir da medula óssea, por citometria de fluxo e microscopia confocal; (e) estudo da arquitetura do baço pela técnica de imuno-histoquímica associada à microscopia confocal. Avaliando os resultados em conjunto, a via autofágica parece ser de extrema importância e de capacidade inovadora para alvo terapêutico. Observou-se que a insulina exerceu efeitos antagônicos sobre os macrófagos de tecidos diferentes: aumentou a expressão LC3 nos macrófagos recuperados por LBA e não conseguiu alterar a atividade autofágica em macrófagos da polpa vermelha do baço em ratos diabéticos. Os BMM originários de ratos diabéticos comportaram-se de maneira contrária aos do animal controle: os BMM tipo M1 tiveram o conteúdo de LC3 diminuído, enquanto os M2 tiveram o conteúdo autofágico aumentado. O tratamento com insulina nos ratos diabéticos não alterou o nível do conteúdo LC3 dos BMM, mesmo após uma semana de cultura in vitro. Concluímos, assim, que o tratamento com dose única de insulina foi capaz de induzir a autofagia em macrófagos alveolares, mas insuficiente para resgatar os níveis basais da autofagia em macrófagos da medula óssea e da polpa vermelha do baço / Diabetes mellitus (DM) is characterized by hyperglycemia associated to a lack or ineffectiveness of insulin. DM is marked by changes in several cellular processes that need to be better understood. Autophagy pathway in macrophages from different tissues was studied with the purpose to verify whether treatment with insulin is capable of modulating this process. Bone marrow derived macrophages (BMM), bronchoalveolar lavage (BAL), and splenic tissue macrophages from Wistar rats, diabetic (alloxan, 42 mg/kg, iv, 10 days) and diabetic rats treated with insulin were studied. To characterize the model and evaluate the effect of insulin upon the autophagic process, the following analyzes were performed: (a) glucose levels, number of leukocytes in peripheral blood, BAL cell number; (b) concentrations of cytokines (interleukin (IL)-1beta, tumor necrosis factor (TNF)-alfa, IL-6, IL-4, IL-10, cytokineinduced neutrophil chemoattractant (CINC)-1 and CINC-2 in the supernatant of BAL fluid by ELISA; (c) characterization of BAL alveolar macrophage (AM) surface antigens (MHCII, pan macrophage marker KiM2R, CD11b) and autophagic markers (microtubule-associated protein light chain (LC)3, gene/protein associated to autophagy (ATG) by flow cytometry and confocal microscopy (d) study of macrophages diferenciated from bone marrow by flow cytometry and confocal microscopy; (e) the architecture of spleen and macrophages from red pulp by immunohistochemical techniques associated to confocal microscopy. Evaluating these results together, the autophagic pathway appears to be innovative for therapeutic target. In this study, it was observed that insulin exerted diverse effects on macrophages from different tissues: increased expression of LC3 in AM recovered from BAL and was unable to change the autophagic activity of macrophages from the red pulp of the spleen in diabetic rats. BMM from diabetic rats behaved in an antagonistic way compared to control animals: BMM M1 type decreased their autophagy content while M2 macrophages increased autophagic levels and insulin treatment did not alter the level of LC3 expression. In conclusion, treatment with a single dose of insulin was able to induce autophagy in alveolar macrophages, but insufficient for recovering baseline levels of autophagy in bone marrow derived macrophages and macrophages from the red pulp of the spleen

Estudos em nosso laboratório caracterizaram camundongos B10.A e A/J, respectivamente, como susceptíveis e resistentes à infecção pulmonar pelo fungo Paracoccidioides brasiliensis. A imunidade inata desempenha papel fundamental no controle inicial dos patógenos e na regulação da resposta imune adquirida. Como os macrófagos alveolares são as primeiras células do hospedeiro a interagir com o fungo, propusemo-nos a estudar a capacidade fungicida e secretora dos macrófagos alveolares de camundongos susceptíveis e resistentes ao P. brasiliensis para melhor compreender a PCM pulmonar. Camundongos B10.A e A/J normais (n: 10-15) foram submetidos a lavagem bronco-alveolar (LBA) e a suspensão celular obtida (2x105 células/poço) foi pré-ativada durante a noite com IFN-&#947;, IL-12 ou a combinação destas duas citocinas (50.000, 10.000 e 2.000 pg/rnL) e desafiados in vitro com leveduras viáveis de P. brasiliensis (relação fungo-macrófagos de 1:50). Após 72h de incubação a atividade fungicida dos macrófagos foi avaliada através da contagem do número de fungos viáveis pelo método de unidades formadoras de colônias (UFC). A produção de nitrito e de citocinas foi avaliada no sobrenadante de cultivo através da reação de Griess e ELISA, respectivamente. Os dados foram expressos como média &#177; EP e analisados pelo teste T -Student. Nossos resultados mostraram que os macrófagos de camundongos B 10.A pré-ativados com IFN-&#947;, IL-12 ou ambas as citocinas nas diferentes concentrações ensaiadas apresentaram elevada capacidade fungicida (51-97%) acompanhada de produção aumentada de NO, IL-12 e MCP-l e baixa síntese de IL-10 e GM-CSF. Por outro lado, somente o tratamento com a mais alta concentração de IFN-&#947; foi capaz de induzir atividade fungicida em macrófagos alveolares de animais A/J. A síntese de NO ocorreu sempre em baixos níveis e nos sobrenadantes dos co-cultivos observamos altos níveis de IL-10 e GM-CSF associados a baixas concentrações de IL-12 e MCP-l. A inibição da síntese de NO por aminoguanidina demonstrou que a atividade fungicida dos macrófagos de animais B10.A, mas não de A/J, era mediada por este composto tratamento dos macrófagos com anticorpo monoclonal anti-IL-10 não alterou a capacidade fungicida dos macrófagos de ambas as linhagens, entretanto induziu franca produção de NO que, para animais A/J, não se traduziu em atividade microbicida aparente. Por outro lado. a neutralização de TGF-&#946; induziu alta capacidade fungicida das células de animais A/J níveis aumentados de NO e TNF-&#945; aliados a níveis reduzidos de IL-10. O mesmo tratamento não alterou a já elevada capacidade fungicida dos macrófagos alveolares de animais B10.A mas aumentou a produção de NO, IL-12 e TNF-&#945;. Em conclusão, os macrófagos alveolares de camundongos B10.A são facilmente ativáveis por 1FN-&#947; e IL-12, apresentam elevada capacidade fungicida, NO-dependente, sobre leveduras do P. brasiliensis associada com a síntese de níveis elevados de NO e IL-12. Por outro lado, os macrófagos de camundongos A/J são pobremente ativados por 1FN-&#947; e IL-12, produzem baixos níveis de NO, a sua capacidade fungicida, que parece ser NO-independente, pode ser aumentada pela neutralização do TGF-&#946; endógeno, mas não pelo tratamento com anti-IL-10. / Previous studies in our laboratory characterized B10.A and A/J mice as susceptible and resistant strains to pulmonary Paracoccidioides brasiliensis infection. Innate immunity plays a fundamental role on the control of the initial growth of pathogens as well as in the acquired immunity that subsequently develops. As alveolar macrophages are the first host cells to interact with P. brasiliensis we decided to study the fungicidal and secretory ability of alveolar macrophages from resistant and susceptible mice to P. brasiliensis to better understand the pulmonary model of paracoccidioidomycosis. Normal B10.A and A/J mice (n=10-15) were submitted to bronchoalveolar lavage (BAL) and cell suspensions (2x105 cells/well) were pre-activated ovemight with IFN-&#947;, IL-12 or the combination of these two cytokines (50,000, 10,000 and 2,000 pg/rnL). After that, macrophages were in vitro challenged with P. brasiliensis yeasts (1:50 fungus: macrophage ratio) and 72h later fungicidal activity was determined by colony forming units counts (CFU). Nitrite and cytokines production were determined in culture supematants by Griess reaction and ELISA, respectively. Data were expressed as means &#177; SE and analyzed by Student\'s t test. Our results showed that B10.A macrophages pre-activated with the different assayed concentrations of IFN&#947;, IL-12 or both cytokines presented elevated fungicidal ability (51¬-97%) concomitant with the presence of high levels of NO, IL-12 and MCP-l and low amounts of IL-10 and GM-CSF. NO synthesis occurred in low levels but high concentrations of IL-10 and GM-CSF associated to low amounts of IL-12 and MCP-1 were detected in the co-cultures supematants. NO synthesis inhibition by aminoguanidine clearly showed that the fungicidal ability of B10.A but not of A/J macrophages was NO¬ dependent. Treatment with anti-IL-10 monoclonal antibodies did not alter the fungicidal ability of macrophages from both mouse strains but enhanced NO synthesis which, however, did not alter the absent microbicidal ability of A/J macrophages. On the contrary, anti-TGF-&#946; treatment induced an increased fungicidal ability of A/J cells associated with enhanced levels of NO and TNF-&#945; besides diminished amounts of IL-10. The same treatment did not alter the high fungicidal ability of B10.A alveolar macrophages but increased NO, IL-12 and TNF-&#945; production. In conclusion, alveolar macrophages from susceptible mice are easily activated by IFN&#947; and IL-12, present a high and NO-dependent ability to kill P. brasiliensis yeasts and secrete elevated levels of NO and IL-12. In contrast, alveolar macrophages from resistant mice are poorly activated by IFN&#947; and IL¬12, secrete low amounts of NO and high of IL-10. Their fungicidal ability which appears to be NO-independent can be restored by neutralization of endogenous TGF-&#946; but not by anti-IL-10 treatment.