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African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

Made available in DSpace on 2016-12-06T17:06:59Z (GMT). No. of bitstreams: 1 Alexandra Amin Lineburger.pdf: 1015080 bytes, checksum: 1d7fd8b623689b7bd6fd0e3efda64391 (MD5) Previous issue date: 2014-05-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Heart failure (HF) is a disease with systemic involvement, because in addition to cardiac involvement it causes cardiorenal, hemodynamic and neurohormonal dysfunctions contributing to aggravate the catabolism and exercise intolerance and impacting on quality of life. In this context, the high prevalence of hypotestosteronemia is associated with increased cardiovascular events and mortality in men with HF. Regular physical exercise, especially high intensity, and testosterone supplementation are therapeutic options in the management of the disease due to the reduction of risk factors such as dyslipidemia and low-grade inflammation. The aim of this study was to analyze the effects of high intensity exercise and testosterone supplementation therapy (TST) on lipid and inflammatory profile of men with HF. The study is characterized as a randomized controlled trial, with 17 men participants of cardiopulmonary rehabilitation program, HF functional class II and III (NYHA) with ejection fraction <45% and testosterone levels <400ng/dl, with 8 patients undergoing high intensity exercise (EAI) and 9 patients who received hormonal supplementation with testosterone undecanoate (Nebido 1000mg) and performed high-intensity exercise (EAIT group). The physical exercise protocol was carried out for 12 weeks and testosterone supplementation was applied in the first and sixth week. Lipid profile (LDL-c, HDL-c, triglycerides and total cholesterol) and plasma levels of inflammatory mediators (TNF-&#945; and CRP) were analyzed before and after the intervention. The results showed significant increase of 26.7% from the level of HDL-C (p = 0.04) in the EAI group. In comparing the two groups, there were statistically significant differences for the variations in the levels of triglycerides (p = 0.03). Both the EAI group as EAIT decreased plasma levels of TNF-&#945;, respectively 47.4% (p = 0.02) and 43.7% (p = 0.04) with no statistically significant difference between groups. Based on these results it is suggested that high intensity exercise positively influence lipid and inflammatory profile without additional effects associated with supplementation of testosterone therapy. / A insuficiência cardíaca (IC) é uma doença de acometimento sistêmico, pois além do comprometimento cardíaco causa disfunções cardiorrenais, hemodinâmicas e neurohormonais que contribuem para agravar o catabolismo e a intolerância ao exercício impactando na qualidade de vida. Neste contexto, a alta prevalência de hipotestosteronemia está associada ao aumento de eventos cardiovasculares e mortalidade em homens com IC. A prática regular de exercícios físicos, especialmente o de alta intensidade, e a suplementação de testosterona são opções terapêuticas no manejo da doença, devido à redução de fatores de risco como a dislipidemia e a inflamação de baixo grau. O objetivo do presente estudo foi analisar os efeitos do exercício de alta intensidade e terapia de suplementação de testosterona (TST) no perfil lipídico e inflamatório de homens com IC. O estudo é caracterizado como ensaio clínico controlado randomizado, com 17 homens participantes de um programa de reabilitação cardiopulmonar, portadores de IC classe funcional II e III (NYHA), com frequência de ejeção < 45 % e níveis de testosterona < 400 ng/dl, sendo 8 pacientes submetidos a exercício de alta intensidade (EAI) e 9 pacientes que receberam suplementação hormonal com undecanoato de testosterona (Nebido 1000 mg) e realizaram exercício de alta intensidade (grupo EAIT). O protocolo de exercícios físicos foi realizado por 12 semanas e a suplementação de testosterona foi aplicada na primeira e na sexta semana. Foi analisado o perfil lipídico (LDL-c, HDL-c, triglicerídeos e colesterol total) e os níveis plasmáticos de mediadores inflamatórios (TNF-&#945; e PCR) antes e após a intervenção. Os resultados mostraram aumento significativo de 26,7% do nível de HDL-c (p=0,04) no grupo EAI. Na comparação entre os dois grupos, houve diferença estatística para as variações dos níveis de triglicerídeos (p = 0,03). Tanto o grupo EAI quanto o EAIT apresentaram redução nos níveis plasmáticos de TNF- &#945;, respectivamente de 47,7% (p=0,02) e 43,7% (p=0,04) sem diferença estatística entre os grupos. Com base nestes resultados sugere-se que o exercício físico de alta intensidade influenciou positivamente o perfil lipídico e inflamatório, sem efeitos adicionais quando associado à terapia de suplementação de testosterona.

PhD (Microbiology) / Department of Microbiology / Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity. / NRF

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a devastating disease in the channel catfish industry. Our research group has developed several E. ictaluri live attenuated vaccine (LAV) candidates (EiΔevpB, EiΔevpBΔfur, EiΔevpBΔhfq, EiΔevpBΔfurΔhfq), which were able to stimulate an immune response in vaccinated channel catfish and reduce ESC. However, innate, and adaptive immune responses in the lymphoid tissues of channel catfish to these LAVs are not known well. The overall goal of the project is to determine the role of adaptive and innate immune responses in catfish after vaccination with LAVs. Analysis of innate and adaptive immune-related gene expressions showed that the LAVs induced expression of adaptive immune-related genes in lymphoid tissues with less inflammation compared to wild type control. Also, the LAVs induced the expression of IgM in the sera of catfish.

Résumé : Dans les maladies respiratoires chroniques, les propriétés biochimiques et mécaniques des muscles lisses des voies respiratoires (MLVR) ont été analysées, mais les modes d’action des médiateurs lipidiques endogènes dérivés des oméga-3 (époxy- ou polyhydroxy- éicosanoïdes) restent à clarifier. Mon travail de recherche a pour but de caractériser le rôle potentiel de monoacyglycéride de l’acide éicosapentaénoϊque (MAG-EPA) et de monoacylglycéride de l’acide docosahexaénoϊque (MAG-DHA) ainsi que leurs métabolites (acide 17,18-époxyéicosatétraénoϊque : 17,18-EpETE, résolvine D1 : RvD1), sur le statut inflammatoire et l’activité contractile des voies respiratoires mises en culture organoϊde avec des cytokines pro-inflammatoires. Sur des trachées de cobayes (TC) natives précontractées au U-46619 (agoniste du récepteur thromboxane prostanoϊde), le 17,18-EpETE relaxe les tissus de manière plus importante que son précurseur, le MAG-EPA. Dans les TC mises en culture pendant 3 jours, les niveaux de TNF-α ont augmenté dans les fractions microsomales par rapport aux trachées natives. Sur ces tissues cultivés et traités avec 0.3 µM 17,18-EpETE, une réduction de la sensibilité au Ca2+ a été démontrée. De plus, une diminution de niveaux de détection des P-p65-NFκB, c-fos et c-Jun a été quantifiée en présence de 17,18-EpETE et des inhibiteurs de PKC ou Rho kinase lorsque les TC sont préalablement traités par 10 ng/ml TNF-α. Les cytokines pro-inflammatoires (IL-13 et TNF-α, etc…) jouent un rôle majeur dans la physiopathologie de l’asthme. Le projet de bronchioles humaines évalue l’effet de la RvD1 et de ses précurseurs (MAG-DHA, MAG-DPA et 17(S)-HpDoHE) sur le statut inflammatoire et la bronchoréactivité, in vitro. Dans les bronchioles stimulées par l’IL-13 pendant 48 h, le MAG-DHA ainsi que ces métabolites diminuent l’activation de la voie TNF-α/NFκB et la sensibilité au Ca2+ des tissus prétraités avec l’IL-13 vers des niveaux proches des conditions contrôles. Dans les bronchioles prétraitées par le TNF-α, l’inflammation et l’hypersensibilité au Ca2+ sont abolies par 1 µM MAG-DPA. De plus, l’aspirine combinée au MAG-oméga-3 potentialise les effets inhibiteurs de ce dernier sur l’inflammation et l’hyperréactivité bronchique induite par les cytokines, tout en régulant à la hausse les niveaux de détection du GPR-32 (le récepteur de RvD1). En conclusion, les dérivés des oméga-3 à longue chaîne pourraient résoudre l’inflammation et contrer les causes de l’hyperréactivité bronchique (HRB). / Abstract : In chronic respiratory diseases, the biochemical and mechanical properties of airway smooth muscle were analyzed, but the mode of action of omega-3 derivatives (epoxy or polyhydroxy-eicosanoids) remains to be clarified. My research work aims to characterize the potential role of eicosapentaenoic acid monoacyglyceride (MAG-EPA) and docosahexaenoic acid monoacylglyceride (MAGDHA) and their bioactive metabolites (17,18-epoxyeicosatetraenoic acid: 17 18-EpETE, resolvin D1: RvD1) on the inflammatory status and the contractile activity of organcultured airway explants with pro-inflammatory cytokines. On 30 nM U-46619 (Thromboxane prostanoid receptor agonist) pre-contracted fresh guinea pig trachea, 17,18 EpETE displays a greater ability than its precursor, MAG-EPA to relax airways. In 72-h-cultured tracheal rings, TNF-α levels increase in the microsomal fractions when compared to native trachea. In cultured and pre-treated tracheal rings with 0.3 µM 17,18-EpETE, the Ca2+ hypersensitivity is alleviated in comparaison to 3 day cultured tracheal rings. In addition, the detection levels of P-NFκB, c-fos and c-Jun were abolished in the presence of 17,18-EpETE and PKC or Rho kinase inhibitors in short term-TNF-α- incubated tracheal rings. Pro-inflammatory cytokines (IL-13, TNF-α, etc…) play a major role in asthma pathophysiology. The human bronchi project evaluates the effect of RvD1 and its precursors (MAG-DHA, MAG-DPA and 17 (S)-HpDoHE) on the inflammatory status and bronchial reactivity, in vitro. In IL-13 stimulated human bronchi for 48 h, the MAG-DHA and its metabolites decrease the activation of TNF-α / NFκB pathway and blunt the Ca2+ hypersensitivity triggered by IL-13. In TNF-α-pretreated human bronchi, airway inflammation and Ca2+ hypersensitivity are reversed by 1 µM MAG-DPA. Hence, aspirin combined with MAG-omega-3 potentiate the inhibitory effects of MAG-DHA on inflammation and bronchial hyperrresponsiveness triggered by pro-inflammatory cytokines, while upregulating the GPR-32 detection levels (RvD1 receptor). In conclusion, omega-3 derivatives could counteract the causes of airway hyperrresponsiveness (AHR).

Une des difficultés majeures dans le traitement des cancers est l’acquisition de résistance par les cellules tumorales vis-à-vis de la mort induite par les différentes chimiothérapies. Au sein du laboratoire, nous nous intéressons aux propriétés anti-tumorales d’un donneur de monoxyde d’azote (NO), le Glycéryl TriNitrate (GTN), fréquemment utilisé dans le traitement de l’angine de poitrine. Au cours de ce travail, nous avons étudié les mécanismes moléculaires par lesquels le GTN sensibilise les cellules tumorales de plusieurs types de cancer (colique, mammaire, prostatique) à la mort impliquant des voies de signalisation régulées par des cytokines telles que le TNFα, l’IL-6 ou encore le GDF-15.Une meilleure compréhension des mécanismes sous-jacents à l’action anti-tumorale du GTN permettrait de potentialiser son utilisation comme nouvelle thérapie anti-cancéreuse.Modèle colique : le GTN, en présence de la cytokine pro-inflammatoire TNFα, sensibilise les cellules cancéreuses coliques et mammaires à l’apoptose. Du point de vue mécanistique, le GTN induit la S-nitrosylation de cIAP1, inhibant ainsi son activité ubiquitine E3 ligase. Ce qui abroge la voie de signalisation classique NF-кB de survie cellulaire activée par la voie TNFα/TNFR1 en faveur d’une voie de signalisation pro-apoptotique.Modèle mammaire : le GTN intervient au niveau de la migration cellulaire en altérant la voie de signalisation Jak2/STAT3 activée par la cytokine pro-inflammatoire IL-6, dans un modèle de cancer du sein triple négatif. En présence de dérivés du platine (carboplatine) générant de l’IL-6, le GTN freine la migration des cellules en induisant la S-nitrosylation, et probablement l’inactivation, de la kinase Jak-2, indispensable pour l’activation de la voie.Modèle prostatique : le GTN sensibilise à la mort les cellules cancéreuses prostatiques résistantes au docétaxel en modulant le taux de deux marqueurs de résistance à cette chimiothérapie : la clusterine (CLU) et le growth differentiation factor 15 (GDF-15). Au niveau moléculaire, le GTN diminue le taux de l'isoforme cytoprotectrice soluble de la CLU (sCLU) et augmente le taux de l'isoforme cytotoxique nucléaire (nCLU) dans les cellules prostatiques résistantes au docétaxel. Plus particulièrement, en présence de GTN, nous avons établi un lien entre le GDF-15 et la modulation du taux des isoformes de la CLU. / One of the main difficulties in the treatment of cancers is the acquisition of resistance by the tumor cells vis-à-vis the death induced by the different chemotherapies. In the laboratory, we are interested in the anti-tumor properties of a nitric oxide (NO) donor, Glyceryl TriNitrate (GTN), frequently used in the treatment of angina pectoris. In this work, we investigated the molecular mechanisms by which GTN sensitizes tumor cells of several types of cancer (colonic, mammary, prostate) to death involving signaling pathways regulated by cytokines such as TNFα, IL-6 or GDF-15.A better understanding of the mechanisms underlying the GTN's anti-tumor action would make it possible to use it as a new anti-cancer therapy.Colon model: GTN, in the presence of the pro-inflammatory cytokine TNFα, sensitizes colon and mammary cancer cells to apoptosis. From a mechanistic point of view, GTN induces S-nitrosylation of cIAP1, thus inhibiting its ubiquitin E3 ligase activity. This abrogates the classical NF-κB signaling pathway of TNFα / TNFR1 activated cell survival in favor of a pro-apoptotic signaling pathway.Mammary model: GTN intervenes at the level of cell migration by altering the Jak2 / STAT3 signaling pathway activated by the pro-inflammatory cytokine IL-6, in a model of triple negative breast cancer. In the presence of platinum (carboplatin) derivatives generating IL-6, GTN inhibits cell migration by inducing S-nitrosylation, and probably inactivation, of Jak-2 kinase, essential for the activation of the way.Prostate model: GTN sensitizes prostatic prostate cancer cells to death by modulating the level of two markers of resistance to this chemotherapy: clusterin (CLU) and growth differentiation factor 15 (GDF-15). At the molecular level, GTN decreases the level of the soluble cytoprotective isoform of CLU (sCLU) and increases the level of nuclear cytotoxic isoform (nCLU) in prostatic cells resistant to docetaxel. More particularly, in the presence of GTN, we have established a link between GDF-15 and the modulation of the isoform rate of the CLU.

O canabidiol (CBD), o principal canabinóide não psicotrópico extraído da marijuana (Cannabis sativa), é reconhecido por sua potente propriedade imunosupressora e anti-inflamatória. A injúria pulmonar aguda (ALI) é uma doença inflamatória para qual ainda não foi desenvolvida terapias específicas e a única alternativa de tratamento é meramente de suporte em UTI. Desta forma, foi proposta uma investigação sobre os efeitos anti-inflamatórios do CBD em um modelo murino de ALI, induzida pela instilação intra-nasal de LPS (lipopolissacarídeo), dentro de uma perspectiva imune-neuro-endocrinológica. Para a análise do potencial anti-inflamatório do CBD, avaliou-se a contagem total e diferencial de células do lavado broncoalveolar (LBA) (análise da migração de leucócitos para os pulmões), a atividade de mieloperoxidase (MPO) no tecido pulmonar (análise indireta da atividade de neutrófilos), a produção de citocinas e quimicionas no sobrenadante do LBA (análise do perfil inflamatório pulmonar), a concentração de proteínas (albumina) no sobrenadante do LBA (análise indireta da permeabilidade vascular pulmonar) e a expressão de moléculas de adesão (ICAM-1 e VLA-4) em leucócitos do LBA. Analisou-se, ainda, o mecanismo farmacológico dos efeitos anti-inflamatórios do CBD no modelo de ALI, utilizando-se de um antagonista altamente seletivo para o receptor de adenosina A2A (ZM241385). Por fim, avaliou-se os efeitos neuroendócrinos do CBD na vigência da inflamação pulmonar; analisou-se a atividade geral dos animais no campo aberto (análise do comportamento doentio) e os níveis séricos de corticosterona (análise da ativação do eixo Hipotálamo-Hipófise-Adrenal (HPA)). Mostrou-se que tanto o tratamento prolifático (antes da indução da inflamação) como o tratamento terapêutico (depois da indução da inflamação), com uma dose única de CBD (20 ou 80 mg/kg) apresenta um efeito anti-inflamatório prolongado em camundongos submetidos ao modelo de ALI (principalmente 1 e 2 dias após a indução da inflamação). Mostrou-se que o CBD diminuiu a migração de leucócitos para os pulmões (neutrófilos, macrófagos e linfócitos), diminuiu a produção de citocinas (TNF e IL-6) e quimicinas (MCP-1 e MIP-2) no LBA, diminuiu a atividade MPO no tecido pulmonar, diminuiu a concentração de albumina no LBA e diminuiu a expressão de moléculas de adesão (ICAM-1) em neutrófilos do LBA. Mostrou-se, ainda, que o receptor de adenosina A2A está envolvido nos efeitos anti-inflamatórios do CBD na ALI, uma vez que o tratamento com o ZM241385 aboliu todos os efeitos anti-inflamatórios descritos previamente. Por fim, mostrou-se que o CBD apresentou poucos efeitos comportamentais no campo aberto e que não ativou o eixo HPA. Desta forma, mostrou-se pela primeira vez que o tratamento profilático e, também, o tratamento terapêutico com CBD (20 ou 80 mg/kg) tem um efeito anti-inflamatório prolongado em um modelo murino de ALI, muito provavelmente em decorrência de um aumento da sinalização via receptor de adenosina A2A. Por esta razão, acredita-se que o CBD possa ser considerado, no futuro, uma ferramenta terapêutica útil no tratamento de doenças inflamatórias pulmonares. / Cannabidiol (CBD), the major non-psychotropic plant (Cannabis sativa)-derived cannabinoid, is recognized for its immunossupressant and anti-inflammatory properties. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive (ICU patients), because effective therapies have not been developed. Therefore, it was proposed an investigation in order to address the anti-inflammatory effects of CBD in a murine model of LPS-induced ALI, within an immune-neuro-endocrine perspective. To analyze the potential anti-inflammatory effect of CBD, it was evaluated total and differencial cell count of leukocytes present in the bronchoalveolar lavage (BAL) (migration of leukocytes into the lungs), myeloperoxidase activity in the lung tissue (indirect analysis of neutrophil activity), production of cytokines and chemokines in the BAL (analysis of the pulmonar inflammatory profile), protein (albumin) concentration in the BAL (indirect analysis of pulmonar vascular permeability), and expression of adhesion molecules (ICAM-1 and VLA-4) in leukocytes of the BAL. It was also analyzed the pharmacologic mechanism of the anti-inflammatory effects of CBD in the model of ALI, by using a highly selective antagonist of the adenosine A2A receptor (ZM241385). Finally, it was analyzed the neuro-endocrine effects of CBD in the context of lung inflammation; it was analyzed the general activity of the mice in the open field (analysis of sickness behavior) and the seric levels of corticosterone (activation of HPA (Hypothalamus-Hypophysis-Adrenal) axis). It was shown that both prophylactic (before the induction of inflammation) and therapeutic (after the induction of inflammation) protocols of treatment, with a sigle dose of CBD (20 or 80 mg/kg), has a long-term anti-inflammatory effect in mice submitted to the model of ALI (specially, after 1 and 2 days of the induction of inflammation). It was shown that CBD decreased leukocyte (neutrophil, macrophage, and lymphocytes) migration into the lungs, decreased cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the BAL, decreased MPO activity in the lung tissue, decreased albumin concentration in the BAL, and decreased adhesion molecule expression (ICAM-1) in neutrophils of the BAL. It was also shown that adenosine A2A receptor is involved in the anti-inflammatory effects of CBD on LPS-induced ALI, because ZM241385 abrogated all of the anti-inflammatory effects of cannabidiol previously described. Finally, it was shown that CBD has discreet behavioral effects in the open field and was not able to activate the HPA axis. Thus, it was shown for the first time that both prophylactic and also therapeutic treatment with CBD (20 or 80 mg/kg) has a long-term anti-inflammatory effect in a murine model of ALI, most likely associated with an increase in the signaling through the adenosine A2A receptor. Hence, it is possible that in the future CBD may prove useful as a therapeutical tool in the treatment of pulmonar inflammatory conditions.

O canabidiol (CBD), o principal canabinóide não psicotrópico extraído da marijuana (Cannabis sativa), é reconhecido por sua potente propriedade imunosupressora e anti-inflamatória. A injúria pulmonar aguda (ALI) é uma doença inflamatória para qual ainda não foi desenvolvida terapias específicas e a única alternativa de tratamento é meramente de suporte em UTI. Desta forma, foi proposta uma investigação sobre os efeitos anti-inflamatórios do CBD em um modelo murino de ALI, induzida pela instilação intra-nasal de LPS (lipopolissacarídeo), dentro de uma perspectiva imune-neuro-endocrinológica. Para a análise do potencial anti-inflamatório do CBD, avaliou-se a contagem total e diferencial de células do lavado broncoalveolar (LBA) (análise da migração de leucócitos para os pulmões), a atividade de mieloperoxidase (MPO) no tecido pulmonar (análise indireta da atividade de neutrófilos), a produção de citocinas e quimicionas no sobrenadante do LBA (análise do perfil inflamatório pulmonar), a concentração de proteínas (albumina) no sobrenadante do LBA (análise indireta da permeabilidade vascular pulmonar) e a expressão de moléculas de adesão (ICAM-1 e VLA-4) em leucócitos do LBA. Analisou-se, ainda, o mecanismo farmacológico dos efeitos anti-inflamatórios do CBD no modelo de ALI, utilizando-se de um antagonista altamente seletivo para o receptor de adenosina A2A (ZM241385). Por fim, avaliou-se os efeitos neuroendócrinos do CBD na vigência da inflamação pulmonar; analisou-se a atividade geral dos animais no campo aberto (análise do comportamento doentio) e os níveis séricos de corticosterona (análise da ativação do eixo Hipotálamo-Hipófise-Adrenal (HPA)). Mostrou-se que tanto o tratamento prolifático (antes da indução da inflamação) como o tratamento terapêutico (depois da indução da inflamação), com uma dose única de CBD (20 ou 80 mg/kg) apresenta um efeito anti-inflamatório prolongado em camundongos submetidos ao modelo de ALI (principalmente 1 e 2 dias após a indução da inflamação). Mostrou-se que o CBD diminuiu a migração de leucócitos para os pulmões (neutrófilos, macrófagos e linfócitos), diminuiu a produção de citocinas (TNF e IL-6) e quimicinas (MCP-1 e MIP-2) no LBA, diminuiu a atividade MPO no tecido pulmonar, diminuiu a concentração de albumina no LBA e diminuiu a expressão de moléculas de adesão (ICAM-1) em neutrófilos do LBA. Mostrou-se, ainda, que o receptor de adenosina A2A está envolvido nos efeitos anti-inflamatórios do CBD na ALI, uma vez que o tratamento com o ZM241385 aboliu todos os efeitos anti-inflamatórios descritos previamente. Por fim, mostrou-se que o CBD apresentou poucos efeitos comportamentais no campo aberto e que não ativou o eixo HPA. Desta forma, mostrou-se pela primeira vez que o tratamento profilático e, também, o tratamento terapêutico com CBD (20 ou 80 mg/kg) tem um efeito anti-inflamatório prolongado em um modelo murino de ALI, muito provavelmente em decorrência de um aumento da sinalização via receptor de adenosina A2A. Por esta razão, acredita-se que o CBD possa ser considerado, no futuro, uma ferramenta terapêutica útil no tratamento de doenças inflamatórias pulmonares. / Cannabidiol (CBD), the major non-psychotropic plant (Cannabis sativa)-derived cannabinoid, is recognized for its immunossupressant and anti-inflammatory properties. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive (ICU patients), because effective therapies have not been developed. Therefore, it was proposed an investigation in order to address the anti-inflammatory effects of CBD in a murine model of LPS-induced ALI, within an immune-neuro-endocrine perspective. To analyze the potential anti-inflammatory effect of CBD, it was evaluated total and differencial cell count of leukocytes present in the bronchoalveolar lavage (BAL) (migration of leukocytes into the lungs), myeloperoxidase activity in the lung tissue (indirect analysis of neutrophil activity), production of cytokines and chemokines in the BAL (analysis of the pulmonar inflammatory profile), protein (albumin) concentration in the BAL (indirect analysis of pulmonar vascular permeability), and expression of adhesion molecules (ICAM-1 and VLA-4) in leukocytes of the BAL. It was also analyzed the pharmacologic mechanism of the anti-inflammatory effects of CBD in the model of ALI, by using a highly selective antagonist of the adenosine A2A receptor (ZM241385). Finally, it was analyzed the neuro-endocrine effects of CBD in the context of lung inflammation; it was analyzed the general activity of the mice in the open field (analysis of sickness behavior) and the seric levels of corticosterone (activation of HPA (Hypothalamus-Hypophysis-Adrenal) axis). It was shown that both prophylactic (before the induction of inflammation) and therapeutic (after the induction of inflammation) protocols of treatment, with a sigle dose of CBD (20 or 80 mg/kg), has a long-term anti-inflammatory effect in mice submitted to the model of ALI (specially, after 1 and 2 days of the induction of inflammation). It was shown that CBD decreased leukocyte (neutrophil, macrophage, and lymphocytes) migration into the lungs, decreased cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the BAL, decreased MPO activity in the lung tissue, decreased albumin concentration in the BAL, and decreased adhesion molecule expression (ICAM-1) in neutrophils of the BAL. It was also shown that adenosine A2A receptor is involved in the anti-inflammatory effects of CBD on LPS-induced ALI, because ZM241385 abrogated all of the anti-inflammatory effects of cannabidiol previously described. Finally, it was shown that CBD has discreet behavioral effects in the open field and was not able to activate the HPA axis. Thus, it was shown for the first time that both prophylactic and also therapeutic treatment with CBD (20 or 80 mg/kg) has a long-term anti-inflammatory effect in a murine model of ALI, most likely associated with an increase in the signaling through the adenosine A2A receptor. Hence, it is possible that in the future CBD may prove useful as a therapeutical tool in the treatment of pulmonar inflammatory conditions.