Le lipopolysaccharide (LPS) a un métabolisme complexe, et n’a pas encore pu être utilisé efficacement comme biomarqueur ou cible thérapeutique au cours du sepsis. Nous avons utilisé une méthode de quantification du LPS par le 3-hydroxy myristate (3HM), un composé spécifique, qui permet de détecter le LPS associé aux lipoprotéines dans le plasma. Nous avons dosé les taux de 3HM dans une cohorte de patients admis en réanimation et chez des sujets volontaires sains. Les patients septiques avaient des taux plus élevés de 3HM, de même que les patients non-survivants au sepsis. Des taux substantiels de 3HM étaient aussi retrouvés chez les sujets sains, suggérant que le LPS est étroitement intégré au métabolisme humain. Ensuite, nous avons démontré que l’adsorption des lipoprotéines LDL (low density lipoproteins) par LDL aphérèse réduit les taux circulants de LPS, in vitro sur du plasma de sujets septiques, mais aussi in vivo chez des patients hypercholestérolémiques. Globalement, ces résultats apportent de informations importantes pour la compréhension de l’endotoxémie, et ouvrent des perspectives thérapeutiques d’épuration extracorporelle au cours du sepsis. / Lipopolysaccharide’s (LPS) metabolism is complex, and it is still not used as a consistent biomarker or therapeutic target during sepsis. We used a novel LPS quantification method, able to detect lipoprotein-associated LPS in plasma, using 3-hydroxy myristate (3HM), a specific compound. We measured the 3HM levels in a cohort of patients admitted to intensive care and healthy volunteers. Septic patients had higher levels of 3HM, as did non-survivors of sepsis. Substantial levels of 3HM were also found in healthy subjects, suggesting that LPS is tightly integrated with human metabolism. Then, we demonstrated that the adsorption of LDL (low density lipoproteins) lipoproteins by LDL apheresis reduces the circulating levels of LPS, in vitro in the plasma of septic subjects, but also in vivo in hypercholesterolemic patients. Overall, these results provide important information for the understanding of endotoxemia, and open therapeutic perspectives for extracorporeal treatment during sepsis.

Summary The group of E. coli strains is a highly heterogeneous group of strains, including pathogenic and non-pathogenic strains. The classification of these strains is made upon specific virulence factors of these bacteria. Some of these pathogenic strains are not capable to cross over the intestinal border and are responsible for intestinal disorders associated with diarrhoea. Other strains can cross the intestinal border and produce septicaemia and other complications depending on the infected organs. These pathogenic strains of E. coli interact with the host in a typical manner and produce specific lesions. These specific interactions are observed after the colonisation of the gut by pathogenic bacteria and are the results of the presence of specific virulence factors. The colonisation of the gut is mediated by specific adhesins, which are specific for each group of pathogenic E. coli. It seems that these adhesins are not only responsible to initiate the interaction between the pathogen and the host but are also responsible for the host specificity shown by some pathogenic strains. Its for that that a better understanding of these adhesins would permit a better understanding of the host specificity and a better evaluation of the zoonotic potential of these strains. The aim of this work was to identify bacterial structures involved in the intestinal adhesion step and in the intestinal colonisation of the enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and verotoxigenic E. coli (VTEC) bovine isolates, focusing mainly (but not exclusively) on strains of serogroup O26. In order to achieve this objective, two different approaches have been followed: i) the immunologic approach and ii) the genetic approach. The immunologic approach is using the benefits of the 2F3 monoclonal antibody (MAb), which was obtained against an outer membrane protein extract from a human O26 EHEC strain. The work on this approach focused on the study of this MAb as epidemiological tool and on the identification of the epitope recognised by this antibody and of its genetic basis. The work on the genetic approach involves the screening by PCR and by colony hybridization of a collection of EHEC, VTEC and EPEC of human and bovine isolates for the presence of homologues for gene clusters coding for fimbriae of type I, II, III and IV: (i) putative adhesins of human EPEC and EHEC strains like LifA, Iha, LpfA, BfpA; (ii) fimbriaires adhesins (CS31A et F17) and afimbriaire adhesins of Afa family (Afa III, Afa VII, Afa VIII and F1845) described for other groups of bovine pathogenic E. coli. The accomplished work allowed, first of all, to confirm that the 2F3 MAb is specific for the O26 EPEC and O26 EHEC strains without being capable to distinguish between human and animal isolates, that means without being capable to distinguish them depending on theirs host. Moreover, it has been shown that: i) the epitope recognised by the 2F3 MAb is component of the O antigen from the O26 EPEC and O26 EHEC strains; ii) the genetic basis of this epitope is located inside the O antigen gene cluster of O26 EPEC and O26 EHEC strains; and iii) the 2F3 + and O26 + characters are two characters that can be dissociated by random insertional mutagenesis in a bovine EHEC strain. Nevertheless, the results of this work not allowed us to explain the specificity of the 2F3 MAb for the O26 EPEC and O26 EHEC strains. Actually, by sequencing the O-antigen gene cluster (the rfb locus) of an O26 non-EPEC and non-EHEC strain and comparing the obtained sequence with the already published sequence of the O-antigen gene cluster of an O26 EHEC strain no major differences (which could explain the specificity of the 2F3 MAb for the O26 EPEC or EHEC strains) were observed. The obtained results in the genetic approach have been shown that: i) all O145 and O157 EPEC and EHEC strains were positive for the LpfA probe (lpfA gene is coding for the type IV pili) and all other human and bovine strains belonging to other serogroups (O5, O26, O103, O111 and O118) have no homologue sequences for this probe; ii) a big majority of EPEC and EHEC strains belonging to O5, O26, O103, O111 or O118 serogroups were positives for the LifA probe (the lifA gene is involved in the synthesis of adhesins not very well characterised) but none of those belonging to the O145 et O157 serogroups; iii) different proportions of these strains belonging to various serogroups were positives for the Iha probe (the iha gene is involved in the synthesis of an adhesin not very well characterised); and iv) ten out of twelve bovine O26 EPEC strains were positive for the ClpE probe and all other strains (human EPEC and EHEC strains and bovines EHEC strains) were negative for the ClpE probe. The clpE gene is involved in the synthesis of the chaperon protein of the CS31A fimbria, which was described for the enterotoxigenic and septicaemic bovine and porcine E. coli isolates. Since the O145 and O157 EHEC strains show the same pathotype, it looks like that the presence of the lpfA gene and the absence of the lifA gene is linked more to a specific pathotype than to a specific serotype. Concerning the results obtained results with the ClpE probe for the O26 EPEC strains, it is possible that these strains have not an entire clp gene cluster since all the strains positive for the ClpE probe were negative for the ClpG (clpG gene is involved in the biosynthesis of the major unit of the CS31A fimbriae). Nevertheless, it is possible that the clpE-like gene of these strains belongs to an entire gene cluster for which the major unit is different from the ClpG. Finally, the screening of a collection of O8 and O20 VTEC bovine isolates for the presence of sequences homologues to the genes involved in the biosynthesis of adhesins of the Afa family (Afa III, Afa VII, Afa VIII and F1845), adhésines produced mainly by human uropathogenic isolates and by some septicaemic strains isolated from animals, allowed the identification of two O8 VTEC bovine strains harbouring the afa-VIII D/afa-VIII E genes and expressing the Afa VIII E adhesin. During this work, a lot of progresses, that could be useful for a better understanding of the pathogenesis of the EPEC, EHEC and VTEC strains, have been done. Despite that, the general objective of this project description of new factors involved in the initial adhesion stage and/or in the intestinal colonisation by the EPEC, EHEC and VTEC bovine strains in order to allow an easy and reliable diagnostic of these strains have been not accomplished. In perspective of this work, complementary works should focus: i) to identify the epitope recognised by the 2F3 MAb; and ii) to investigate which is the role of the adhesins, for which homologues sequences have been described in the human or bovine EPEC and EHEC isolates of different serogroups, in the adhesion to eukaryotic cells like bovine and human enterocytes. Résumé Lespèce Escherichia coli (E. coli) est hétérogène, contenant des souches pathogènes et des souches inoffensives. La classification des différentes souches pathogènes est basée sur leurs propriétés spécifiques de virulence. Les souches pathogènes dE. coli sont en effet associées à des pathologies variées. Certaines souches ne franchisent pas la barrière intestinale produisant des lésions dentérite, associées à de la diarrhée et dautres souches pathogènes dE. coli peuvent franchir la barrière intestinale, produisant de la septicémie, avec des complications variables selon les organes infectés. Ces souches interagissent avec lhôte dune manière particulière produisant des lésions typiques au niveau cellulaire et tissulaire. Ces interactions spécifiques sont dues aux propriétés de virulence spécifiques après la colonisation de lintestin. Létape de colonisation de lintestin se fait grâce à des adhésines particulières, spécifiques pour chaque groupe de souches pathogènes dE. coli. Il semble que ces adhésines nont pas seulement le rôle dinitialiser linteraction entre lhôte et le pathogène mais quelles soient responsables aussi de la spécificité dhôte présentée par certaines souches pathogènes. Ainsi la connaissance de ces adhésines permet la reconnaissance de la véritable spécificité dhôte de ces souches et lévaluation de leur potentiel zoonotique. Lobjectif général du travail était didentifier des structures bactériennes impliquées dans ladhérence aux entérocytes et, donc, dans la colonisation intestinale par des souches entérohémorragiques (EHEC), entéropathogènes (EPEC) et vérotoxinogènes (VTEC) bovines, et qui représenteraient une base de la spécificité dhôte de ces souches, en ciblant plus particulièrement le sérogroupe somatique O26. Pour accomplir lobjectif de ce travail, deux approches ont été suivies : immunologique et génétique. Lapproche immunologique a utilisé lanticorps monoclonal 2F3 qui a été dérivé contre des extraits protéiques de membrane dune souche EPEC humaine du sérogroupe O26. Le travail a consisté à identifier l'antigène reconnu par l'anticorps monoclonal 2F3, à déterminer sa base génétique et à lutiliser en tant qu'outil d'épidémiologie moléculaire. Le travail dans lapproche génétique a consisté à rechercher, par des épreuves de PCR et d'hybridation sur colonies sur une collection de souches EHEC, EPEC et VTEC bovines et humaines, la présence de gènes dopérons qui codent pour différents fimbriae de type I, II, III et IV : (i) des adhésines potentielles des souches EHEC et EPEC humaines, telle que LifA, Iha, LpfA, BfpA; (ii) des adhésines fimbriaires (CS31A et F17) et afimbriaires la famille Afa (Afa III, Afa VII, Afa VIII et F1845) présentes chez dautres catégories de souches dE. coli pathogènes pour le bovin. Les travaux effectués ont tout dabord permis de confirmer que lanticorps monoclonal 2F3 est spécifique pour les souches EPEC et EHEC du sérogroupe O26 et ne reconnaît pas les souches non-EHEC non-EPEC, sans cependant quil puisse faire la différence entre les souches EHEC et EPEC dorigine humaine et animale, cest-à-dire en fonction de leur hôte. Dans la partie des recherches consacrée à létude de cet anticorps, il a été aussi démontré que : i) lépitope reconnu par lanticorps monoclonal 2F3 est une composante du lipopolysaccharide (LPS) des souches EPEC et EHEC du sérogroupe O26 ; ii) la base génétique de cet épitope est situé dans lopéron codant pour lantigène O26 ; et iii) le caractère 2F3+ peut néanmoins être dissocié du caractère O26+ par mutagenèse par transposition aléatoire dans une souche EHEC bovine. Néanmoins, les résultats de ces travaux nont pas permis dexpliquer la raison pour laquelle lanticorps monoclonal 2F3 reconnaît les souches EPEC et EHEC O26, mais non les souches non-EPEC et non-EHEC O26. En effet, le séquençage de lopéron codant pour lantigène O26 (locus rfb) dune souche non-EHEC non-EPEC et sa comparaison avec la séquence publiée de lopéron rfb dune souche O26 EHEC na pas permis de reconnaître une différence pouvant expliquer la reconnaissance par lanticorps 2F3. Les résultats obtenus dans le cadre des travaux selon lapproche génétique ont montré que : i) toutes les souches EHEC bovines et humaines appartenant aux sérogroupes O145 et O157 étaient positives pour la sonde LpfA (les gènes lpfA codent pour des pili de type IV), mais aucune souche appartenant aux autres sérogroupes (O5, O26, O103, O111 et O118); ii) une grande majorité des souches EHEC et EPEC bovines et humaines aux sérogroupes O5, O26, O103, O111 et O118 étaient positives pour la sonde LifA (les gènes lifA codent pour des adhésines encore peu caractérisées), mais pas celles appartenant aux sérogroupes O145 et O157; iii) des proportions variables de souches appartenant à ces différents sérogroupes étaient positives pour la sonde Iha (les gènes iha codent aussi pour des adhésines peu caractérisées); et iv) dix souches EPEC bovines O26 sur les douze étudiées étaient positives pour la sonde ClpE (dérivée du gène codant pour la protéine chaperone du fimbria CS31A produit par des souches entérotoxinogènes et septicémiques bovines et porcines dE. coli), alors que les souches EHEC bovines et humaines du même sérogroupe, ainsi que toutes les souches des autres sérogroupes, étaient négatives pour cette sonde. Comme les souches EHEC O157 et O145 présentent le même pathotype, il semblerait que la présence des gènes lpfA et labsence des gènes lifA soient associées plus à un pathotype particulier quà un, ou quelques sérogroupes. En ce qui concerne les résultats avec la sonde ClpE sur les souches EPEC bovines O26, il est possible que ces souches ne possèdent pas lopéron clp complet puisque toutes les souches étaient négatives pour la sonde clpG (codant pour la sous-unité majeure du fimbria CS31A). Il est cependant aussi possible que le gène clpE-like de ces souches appartienne à un opéron complet dont la sous-unité majeure soit différente de ClpG. Enfin, la recherche des gènes intervenant dans la biosynthèse dadhésines de la famille Afa (Afa III, Afa VII, Afa VIII and F1845), surtout produites par des souches uropathogènes humaines et sur certaines souches septicémiques animales, dans la collection des souches VTEC dorigine bovine de sérogroupe O8 et O20 a permis la mise en évidence de deux souches VTEC de sérogroupe O8 qui possèdent les gènes afa-8D/afa-8E et qui expriment ladhésine Afa 8E. Au cours de nos travaux une série de résultats, qui pourront amener à une meilleure compréhension des mécanismes de pathogénicité et des souches EPEC, EHEC et VTEC ont été réalisés. Cependant, lobjectif général du projet - la description de facteurs spécifiques de lhôte impliqués dans le processus dadhérence initiale des souches EPEC, EHEC et VTEC bovines permettant la colonisation intestinale, afin de pouvoir les diagnostiquer et de les typer de manière spécifique - na pas été atteint. A court terme, les compléments de travaux à envisager sont la poursuite des essais didentification de lépitope reconnu par lanticorps monoclonal 2F3 et le rôle dans ladhérence aux cellules eucaryotes, comme les entérocytes bovines et humains, des adhésines dont les gènes ont été détectés dans les souches EHEC et EPEC bovines et humaines appartenant à différentes sérogroupes.

Background: Airways diseases comprise mainly of COPD and asthma. There is a need to develop both new models and improve methodologies of existing models these diseases. LPS challenges in smokers would be an excellent model to study the drugs directed against TLR4 mediated inflammation in COPD. In asthma allergen challenges are established models of disease and extensively used in clinical trials. Back to back reproducibility of two bolus dose allergen challenges has not been studied; this would provide intra subject standard deviations which are useful for accurate power calculations for bolus allergen challenge studies. Aims: 1. To investigate Inhaled LPS Challenges in healthy smokers as a model of inflammation in COPD; study systemic and sputum biomarkers for use in such studies and use LPS challenge as a model to study corticosteroid insensitivity 2. Investigate LPS Challenges in HNS as a model to study neutrophil chemotaxis mechanisms 3. Study Reproducibility of bolus dose allergen challengeMethods 1. HNS and HS were recruited and underwent inhaled LPS challenges. Safety, airway and systemic inflammation was studied. 2. Mild atopic asthmatics underwent two bolus allergen challenges, reproducibility of EAR and LAR was studied and intrasubject SD was used for power calculationsResult LPS Challenges were safe in both HNS and HS and led to increase in sputum neutrophil% in both these populations with maximum effect at 6hours post 30µg LPS inhalation. The resulting airway neutrophilic inflammation was reproducible in HS. LPS challenge in HS also leads to increase in systemic biomarkers and upregulation of NFĸB pathways in induced sputum. There was moderate corticosteroid insensitivity in airway inflammation in HS which didnot increase post LPS challenge. In HNS sputum supernatants post LPS challenge increase chemotaxis of blood neutrophils which is related to CXCL8 levels and mediated by both CXCR1 and 2 receptors. Bolus allergen challenges in mild asthmatics show good reproducibility for both EAR and LAR; I have also presented intrasubject SD which maybe used for accurate power calculations for future studies.Conclusions LPS Challenges lead to neutrophilic airway inflammation in HS which is reproducible and mediated by upregulation of TLR4 signalling making this a good model to study anti-inflammatory drugs for COPD in clinical trials. Additionally, LPS challenges in HNS provide a model to study neutrophil chemotaxis mechanisms. Bolus allergen challenges show good reproducibility and accurate power calculations are presented in this thesis.

Sabe-se que a desnutrição modifica a resposta imune específica e inespecífica do organismo frente a agentes infecciosos comprometendo a produção e a função de células linfo-hemopoéticas, estando associada a modificações da resposta imune, resultando em maior suscetibilidade à infecções, porém os mecanismos exatos que comprometem o sistema imune em estados de desnutrição ainda estão para serem esclarecidos. A literatura relata que aproximadamente 60% das infecções que evoluem para sepse são adquiridas no ambiente hospitalar, envolvendo geralmente bactérias Gram negativas e incidindo especialmente em indivíduos com nutrição inadequada. Considerando tais aspectos e em função da complexidade da interação do estado nutricional e resposta do organismo frente a agentes patogênicos, envolvendo controles celulares e moleculares múltiplos ainda pouco conhecidos, propusemo-nos a estudar alguns aspectos da resposta inflamatória em desnutrição. Camundongos Swiss machos adultos, submetidos a desnutrição protéica-energética, após perda de aproximadamente 25% do peso corpóreo, foram inoculados com lipopolissacarideo de Escherichia coli: Hemograma, mielograma, esplenograma e dosagens sistêmicas de citocinas, foram realizadas. Células coletadas da cavidade peritonial de animais que não foram estimulados com LPS foram utilizadas para as determinações de citocinas e NO in vitro, bem como para quantificação dos receptores CD14 e TLR-4/MD-2 e do fator de transcrição NFκB. Células da cavidade peritonial foram usadas, também, para realização dos testes de espraiamento, fagocitose e atividade cida com Candida albicans. Animais desnutridos apresentaram anemia, leucopenia; severa redução na celularidade da medula óssea, do baço e da cavidade peritonial. A capacidade de espraiamento, fagocitose, atividade cida e síntese de citocinas e NO foram significativamente menores nos animais dos grupo desnutrido. O número de receptores de CD14 e TLR-4/MD-2 e do fator de transrição NFκB, também foram significativamente menores nos animais desnutridos. Estes achados sugerem que. animais desnutridos apresentam resposta deficiente frente ao LPS. A menor expressão de receptores CD14 e TLR-4/MD-2 pode ser responsável, em parte pela imunodepressão observada. Os dados nos levam a inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dos animais. / Malnutrition modifies the specific and non-specific immune response of the organism to infectious agents, hampering the production and function of Iympho-hemopoietic cells leading to a higher susceptibility of the orgonism to infections. However, the exact mechanisms by which the immune system is undermined has not yet been fully elucidated. Approximately 60% of infections that evolve to bacteremia are nosocomial, and usually involve Gram negative bacteria in individuais that have inadequate nutrition. Taking this into consideration, and in view of the complexity of the interaction between nutritional state and the organism\' s response to infection, which involves poorly known multiple cellular and molecular controls, we proposed to study a few aspects of the inflammatory response in protein malnutrition. Male, outbred Swiss mice were sumbitted to protein malnutrition and after the loss of about 25% of total body weight they were inoculad whith lipopolissacharide of Escherichia coli: Hemogram, mielogram, splenogram and the determination of systemic production of cytokines were used to evaluate. Cells from the peritoneal cavity of animais who were not inoculated were collected for determination of cytokines and NO production in vitro and to evaluate the expression of NFκB and CD14 and TLR-4/MD-2 receptors. Cells from the peritoneal cavity were used too for the spreading, phagocytosis and killing tests with Candida albicans. Malnourished animais presented anemia, leucopenia a severe reduction on bone marrow, spleen and peritoneal cavity cellularity. The spreading, phagocytosis, killing and the production of cytokines and NO was significantly lower in malnourished animais. The number of CD14 and TLR-4/MD-2 receptors and NFκB was found to be significantly lower in malnourished animais. These findings suggest that malnourished mice present a deficient response to LPS. The smaller expression of CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed. These data lead us to infer that the nutritional state interferes in the activation of macrophages and in the immune response capacity.

Indivíduos diabéticos apresentam incidência elevada de doenças infecciosas. Isto pode estar relacionado às alterações na capacidade da resposta destes indivíduos aos agentes agressores. Em animais diabéticos, algumas destas alterações já foram descritas, assim como sua reversão pela administração de insulina. Este hormônio regula o metabolismo celular, modulando a atividade de proteínas e mediadores inflamatórios envolvidos neste processo. Sabemos que o lipopolissacarídeo (LPS) estimula, em macrófagos alveolares (MA) de animais não-diabéticos, a liberação do fator de necrose tumoral (TNF)-&#945; e do óxido nítrico (NO). O pré-tratamento deste MA com insulina inibiu todos estes efeitos. Assim, neste projeto, avaliamos o papel da insulina em MA e macrófagos peritoneais (MP) de camundongos, tornados diabéticos pela indução com aloxana (60 mg/kg, i.v.). Uma suspensão contendo 1x106 células foi estimulada com LPS (100 ng/mL) na presença ou não de insulina (1mU/mL). Realizamos a evolução temporal (0,5; 1; 3; 6; 24 horas) para a dosagem de NO, TNF-&#945; e interleucina (IL)-10. Nos tempos de maior produção destas citocinas (0,5 e 3 horas), também quantificamos IL-6, interferon (IFN)-&#947; e IL-4. Nossos resultados mostram que a produção/liberação dos mediadores imunes por MA e MP estimulados por LPS, quando tratados simultaneamente com insulina, tiveram uma redução. Assim, a produção/liberação de NO foi reduzida durante 0,5; 1; 3; 6, 24 horas, em MA e em MP; a liberação de TNF-&#945; foi reduzida durante 0,5 hora, em MP; a liberação de IL-6 foi reduzida durante 3 horas, em MA, e 0,5 hora, em MP. Estes dados mostram que a insulina reduziu a liberação destes mediadores inflamatórios, tanto para os MA quanto para os MP, durante o estímulo com LPS, de animais diabéticos já nos primeiros minutos de estímulo com LPS, com um pico de redução, na maioria das vezes, em 3 horas. / Diabetic patients exhibit high incidence of infectious diseases, at least in part, by due impaired immune response against aggressive agents. Lipopolysaccharide (LPS) triggers the releasing of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) by alveolar macrophages (AM) of non-diabetic animals. The pretreatment using insulin inhibited of these cytokine release. The aim in the present study was to evaluate the insulin role on releasing of cytokines by MA and peritoneal macrophages (MP) of diabetic mouse. Resident AM and MP from diabetic (alloxan 60 mg/kg, i.v.) male C57BL/6 mice (CEUA/FCF/USP-339) stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) insulin treatment was simultaneously with stimulus by LPS. Cytokines were measured by ELISA, and NO by Griess reaction. We performed a time course (0,5; 1; 3; 6; 24 hours), and measured NO, TNF-alpha and interleukin (IL)-10. We also measured IL-6, interferon (IFN)-&#947;, and IL-4 in 0.5 and 3 hours. Results were evaluated by analysis of variance (ANOVA) followed by multiple comparison Tukey-Kramer or the nonparametric Kruskal-Wallis test. P value were considered when <0.05. Before the induction of diabetes by alloxan injection in day 0 animals showed: weight (26±0,3 g) and blood glucose concentration (164±2,6 mg/dL) - and 10 days after the onset of the disease, diabetic mice presented a lower weight gain (24 ± 0,4 g*) and elevated blood glucose concentration (546±9,7 mg/dL*). An increase was seen NO release levels and TNF-alpha in the time course (Figure 1); an increase of IL-6 in 3 hours (Figure 3), and a decrease of IL-4 in 3 hours (Figure 5) after LPS stimulation of MA in diabetic animals. In MP, there was an increase of NO levels, and TNF-alpha in the time course (Figure 2); in 3 hours increased IL-6 levels (Figure 4) - under the same conditions. Insulin treatment under stimulation by LPS in MA reduced the levels of NO, IL-6, and TNF-alpha compared to the group stimulated by LPS. Regarding MP, insulin treatment also decreased NO levels, TNF-alpha, and IL-6. IL-4 levels produced by MP, and IFN-&#947; levels were not be detected in MA and MP under stimulation by LPS, or in the presence of hormone. These findings suggest that insulin reduce the release of these inflammatory mediators by both resident macrophages of diabetic animals concomitantly stimulation by LPS

O sistema canabinóide endógeno tem uma importante função modulatória em muitos processos neurobiológicos, incluindo a neuroproteção, a plasticidade neuronal e a neuroinflamação. Neste estudo nós pré-tratamos os camundongos com um agonista inverso do receptor CB1, para avaliarmos se ocorria a neuroproteção através de respostas adaptativas. Camundongos machos adultos (C57BL/6J) de 3 meses de idade foram pré-tratados durantes 4 dias com injeções diearias de 3 mg/Kg de AM251 (agonista inverso CB1) ou com veículo, intraperitonealmente (i.p.). Vinte e quatro horas depois da última injeção de AM251/Veículo, os animais foram injetados com LPS i.p. (500 µg/kg) ou salina. Foram realizados testes comportamentais e testes bioquímicos com estruturas encefálicas. Nossa hipótese era que o pré-tratamento com o agonista inverso de CB1 iria funcionar como um estressor moderado, levando a uma neuroadaptação. Contudo, o pré-tratamento não ativou vias neuroprotetoras, e sim como atuou como um estresse deletério. / The endogenous cannabinoid system seems to play a modulatory function in many neurobiological processes, including neuroprotection, neuronal plasticity and neuroinflammation. In this study we pretreated the mice with an inverse agonist of the receptor CB1, in order to evaluate this neuroprotection through adaptive responses. Three months-old male mice (C57BL/6J) were pretreated during 4 days with daily injections of 3mg/Kg of AM251 (CB1 inverse agonist) or with vehicle, intraperitoneally (i.p.). Twenty-four hours after the last AM251/Vehicle injection, animals were injected i.p. with LPS (500 µg/kg) or saline. Behavioural and bichoemical assays were performed. Our hypothesis was that the pretreatment with the inverse agonist of CB1 was going to act as a moderate stressor, leading to a neuroadaptation. However CB1 pretreatment potentiated or sensitized the pro-inflammatory signaling pathway. Our pre-treatment worked not as a moderate stressor but as a deleterious stressor.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-14T12:01:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) / Made available in DSpace on 2016-07-14T12:01:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) Previous issue date: 2015-07-29 / FACEPE / O Sildenafil é um inibidor potente e seletivo da fosfodiesterase-5 (PDE5). Este fármaco foi aprovado para uso terapêutico na disfunção erétil e, atualmente, vem sendo usado também no tratamento da hipertensão pulmonar. Embora mantenha um excelente nível de segurança e perfil de tolerabilidade, poucos estudos avaliaram os possíveis efeitos colaterais do tratamento crônico com Sildenafil sobre o sistema reprodutor masculino, especialmente na próstata visando o relaxamento da uretra e alívio dos Sintomas do Trato Urinário Inferior (STUI). Desta forma, avaliamos o efeito do tratamento, em camundongos C57Bl/6, através de análise morfológica, ultraestrutural e expressão molecular de guanilato ciclase solúvel-sGC, Óxido nítrico sintase endotelial-eNOS, Antígeno prostático específico-PSA e Fator de crescimento transformador beta-TGF- no tecido prostático. Foi-se observado que o tratamento com Sildenafil não induz danos evidentes na próstata. Além disso, tem sido demonstrado que Sildenafil tem eficácia terapêutica em doenças inflamatórias crônicas, podendo apresentar uma eficácia terapêutica potencial em diferentes doenças. Entre elas, uma atenção especial tem sido dada para as patologias relacionadas ao trato urogenital masculino, como a Hiperplasia Prostática Benigna (HPB), Câncer de próstata e Prostatites. A inflamação tem sido considerada como um fator etiológio da HPB e STUI. Assim, foi proposto um modelo de lesão prostática com injeção intrauretral de LPS (1mg/ml), em camundongos machos Swiss e C57Bl/6, desenvolvido durante 3, 7, 10 e 14 dias. A análise dos resultados mostrou que a indução intrauretral com lipopolisacarideo-LPS atua como importante agente da HPB, além de promover o aumento de fatores de crescimento (FGF-7 e FGF- β), α-actina e citocinas pró-inflamatórias (IL-1, IL-6, IL-17), tanto no estroma como no epitélio. Uma vez que o Sildenafil tem potencial anti-inflamatório, este estudo se propôs a analisar a ação do Sildenafil em um modelo de lesão prostática experimental, induzido por injeção intrauretral de LPS em camundongos C57BL/6. O tratamento com Sildenafil (25mg/kg) dos animais com prostatite apresentaram redução significativa de -actina, COX-2, NFK- B, IL-6, IL-17 e FGF-7. Por não induzir danos na próstata , o Sildenafil, por não induzir a longo prazo danos evidentes na próstata pode representar uma estratégia farmacológica para o tratamento de doenças inflamatórias crônicas do trato urogenital. / Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5). This drug has been approved for therapeutic use in erectile dysfunction and currently has been also used to treat pulmonary hypertension. While maintaining an excellent level of safety and tolerability profile, few studies have evaluated the possible side effects of chronic treatment with Sildenafil on the male reproductive system, especially in prostate aimed at relaxing the urethra and relief of symptoms of Lower Urinary Tract (LUTS). Therefore, we assessed the treatment effect in C57Bl/6 mice, using morphological analysis, ultrastructural and molecular expression of soluble guanylate cyclase-sGC, endothelial nitric oxide synthase, eNOS, prostate-specific antigen PSA and transforming growth factor beta TGF- in prostate tissue. It is observed that the treatment with sildenafil does not induce apparent damage to the prostate. Furthermore, Sildenafil has been shown to have therapeutic efficacy in chronic inflammatory diseases, which have a potential therapeutic efficacy in various diseases. Among them, special attention has been given to the pathologies related to the male urogenital tract, such as Benign Prostatic Hyperplasia (BPH), prostate cancer and Prostatitis. Inflammation has been considered as a factor etiológio of BPH and LUTS. Thus, it has been proposed a prostatic intraurethral injection injury model of LPS (1mg/ml) in male Swiss mice and C57Bl /6 developed for 3, 7, 10 and 14 days. The results showed that the intraurethral induction with lipopolysaccharide-LPS acts as an important agent of HPB, and to promote the increase of growth factors (FGF-7 and FGF- ), -actin and proinflammatory cytokines (IL- 1, IL-6, IL-17), both in the stroma and the epithelium. Since Sildenafil has potential anti-inflammatory, this study aimed to analyze the action of Sildenafil in an experimental prostate injury model induced by intraurethral injection of LPS in C57Bl/6 mice. Treatment with sildenafil (25 mg/kg) of animals with prostatitis showed a significant reduction of -actin, COX-2 NFK-kB, IL-6, IL-17 and FGF-7. By not induce damage to the prostate, Sildenafil, not to induce long term damage evident in the prostate may represent a pharmacologic strategy for the treatment of chronic inflammatory diseases of the urogenital tract.

Indivíduos diabéticos apresentam incidência elevada de doenças infecciosas. Isto pode estar relacionado às alterações na capacidade da resposta destes indivíduos aos agentes agressores. Em animais diabéticos, algumas destas alterações já foram descritas, assim como sua reversão pela administração de insulina. Este hormônio regula o metabolismo celular, modulando a atividade de proteínas e mediadores inflamatórios envolvidos neste processo. Sabemos que o lipopolissacarídeo (LPS) estimula, em macrófagos alveolares (MA) de animais não-diabéticos, a liberação do fator de necrose tumoral (TNF)-&#945; e do óxido nítrico (NO). O pré-tratamento deste MA com insulina inibiu todos estes efeitos. Assim, neste projeto, avaliamos o papel da insulina em MA e macrófagos peritoneais (MP) de camundongos, tornados diabéticos pela indução com aloxana (60 mg/kg, i.v.). Uma suspensão contendo 1x106 células foi estimulada com LPS (100 ng/mL) na presença ou não de insulina (1mU/mL). Realizamos a evolução temporal (0,5; 1; 3; 6; 24 horas) para a dosagem de NO, TNF-&#945; e interleucina (IL)-10. Nos tempos de maior produção destas citocinas (0,5 e 3 horas), também quantificamos IL-6, interferon (IFN)-&#947; e IL-4. Nossos resultados mostram que a produção/liberação dos mediadores imunes por MA e MP estimulados por LPS, quando tratados simultaneamente com insulina, tiveram uma redução. Assim, a produção/liberação de NO foi reduzida durante 0,5; 1; 3; 6, 24 horas, em MA e em MP; a liberação de TNF-&#945; foi reduzida durante 0,5 hora, em MP; a liberação de IL-6 foi reduzida durante 3 horas, em MA, e 0,5 hora, em MP. Estes dados mostram que a insulina reduziu a liberação destes mediadores inflamatórios, tanto para os MA quanto para os MP, durante o estímulo com LPS, de animais diabéticos já nos primeiros minutos de estímulo com LPS, com um pico de redução, na maioria das vezes, em 3 horas. / Diabetic patients exhibit high incidence of infectious diseases, at least in part, by due impaired immune response against aggressive agents. Lipopolysaccharide (LPS) triggers the releasing of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) by alveolar macrophages (AM) of non-diabetic animals. The pretreatment using insulin inhibited of these cytokine release. The aim in the present study was to evaluate the insulin role on releasing of cytokines by MA and peritoneal macrophages (MP) of diabetic mouse. Resident AM and MP from diabetic (alloxan 60 mg/kg, i.v.) male C57BL/6 mice (CEUA/FCF/USP-339) stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) insulin treatment was simultaneously with stimulus by LPS. Cytokines were measured by ELISA, and NO by Griess reaction. We performed a time course (0,5; 1; 3; 6; 24 hours), and measured NO, TNF-alpha and interleukin (IL)-10. We also measured IL-6, interferon (IFN)-&#947;, and IL-4 in 0.5 and 3 hours. Results were evaluated by analysis of variance (ANOVA) followed by multiple comparison Tukey-Kramer or the nonparametric Kruskal-Wallis test. P value were considered when <0.05. Before the induction of diabetes by alloxan injection in day 0 animals showed: weight (26±0,3 g) and blood glucose concentration (164±2,6 mg/dL) - and 10 days after the onset of the disease, diabetic mice presented a lower weight gain (24 ± 0,4 g*) and elevated blood glucose concentration (546±9,7 mg/dL*). An increase was seen NO release levels and TNF-alpha in the time course (Figure 1); an increase of IL-6 in 3 hours (Figure 3), and a decrease of IL-4 in 3 hours (Figure 5) after LPS stimulation of MA in diabetic animals. In MP, there was an increase of NO levels, and TNF-alpha in the time course (Figure 2); in 3 hours increased IL-6 levels (Figure 4) - under the same conditions. Insulin treatment under stimulation by LPS in MA reduced the levels of NO, IL-6, and TNF-alpha compared to the group stimulated by LPS. Regarding MP, insulin treatment also decreased NO levels, TNF-alpha, and IL-6. IL-4 levels produced by MP, and IFN-&#947; levels were not be detected in MA and MP under stimulation by LPS, or in the presence of hormone. These findings suggest that insulin reduce the release of these inflammatory mediators by both resident macrophages of diabetic animals concomitantly stimulation by LPS

Sabe-se que a desnutrição modifica a resposta imune específica e inespecífica do organismo frente a agentes infecciosos comprometendo a produção e a função de células linfo-hemopoéticas, estando associada a modificações da resposta imune, resultando em maior suscetibilidade à infecções, porém os mecanismos exatos que comprometem o sistema imune em estados de desnutrição ainda estão para serem esclarecidos. A literatura relata que aproximadamente 60% das infecções que evoluem para sepse são adquiridas no ambiente hospitalar, envolvendo geralmente bactérias Gram negativas e incidindo especialmente em indivíduos com nutrição inadequada. Considerando tais aspectos e em função da complexidade da interação do estado nutricional e resposta do organismo frente a agentes patogênicos, envolvendo controles celulares e moleculares múltiplos ainda pouco conhecidos, propusemo-nos a estudar alguns aspectos da resposta inflamatória em desnutrição. Camundongos Swiss machos adultos, submetidos a desnutrição protéica-energética, após perda de aproximadamente 25% do peso corpóreo, foram inoculados com lipopolissacarideo de Escherichia coli: Hemograma, mielograma, esplenograma e dosagens sistêmicas de citocinas, foram realizadas. Células coletadas da cavidade peritonial de animais que não foram estimulados com LPS foram utilizadas para as determinações de citocinas e NO in vitro, bem como para quantificação dos receptores CD14 e TLR-4/MD-2 e do fator de transcrição NF&#954;B. Células da cavidade peritonial foram usadas, também, para realização dos testes de espraiamento, fagocitose e atividade cida com Candida albicans. Animais desnutridos apresentaram anemia, leucopenia; severa redução na celularidade da medula óssea, do baço e da cavidade peritonial. A capacidade de espraiamento, fagocitose, atividade cida e síntese de citocinas e NO foram significativamente menores nos animais dos grupo desnutrido. O número de receptores de CD14 e TLR-4/MD-2 e do fator de transrição NF&#954;B, também foram significativamente menores nos animais desnutridos. Estes achados sugerem que. animais desnutridos apresentam resposta deficiente frente ao LPS. A menor expressão de receptores CD14 e TLR-4/MD-2 pode ser responsável, em parte pela imunodepressão observada. Os dados nos levam a inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dos animais. / Malnutrition modifies the specific and non-specific immune response of the organism to infectious agents, hampering the production and function of Iympho-hemopoietic cells leading to a higher susceptibility of the orgonism to infections. However, the exact mechanisms by which the immune system is undermined has not yet been fully elucidated. Approximately 60% of infections that evolve to bacteremia are nosocomial, and usually involve Gram negative bacteria in individuais that have inadequate nutrition. Taking this into consideration, and in view of the complexity of the interaction between nutritional state and the organism\' s response to infection, which involves poorly known multiple cellular and molecular controls, we proposed to study a few aspects of the inflammatory response in protein malnutrition. Male, outbred Swiss mice were sumbitted to protein malnutrition and after the loss of about 25% of total body weight they were inoculad whith lipopolissacharide of Escherichia coli: Hemogram, mielogram, splenogram and the determination of systemic production of cytokines were used to evaluate. Cells from the peritoneal cavity of animais who were not inoculated were collected for determination of cytokines and NO production in vitro and to evaluate the expression of NF&#954;B and CD14 and TLR-4/MD-2 receptors. Cells from the peritoneal cavity were used too for the spreading, phagocytosis and killing tests with Candida albicans. Malnourished animais presented anemia, leucopenia a severe reduction on bone marrow, spleen and peritoneal cavity cellularity. The spreading, phagocytosis, killing and the production of cytokines and NO was significantly lower in malnourished animais. The number of CD14 and TLR-4/MD-2 receptors and NF&#954;B was found to be significantly lower in malnourished animais. These findings suggest that malnourished mice present a deficient response to LPS. The smaller expression of CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed. These data lead us to infer that the nutritional state interferes in the activation of macrophages and in the immune response capacity.

Subclinical endotoxemia (low levels of circulating bacterial endotoxin) has been observed in patients suffering from chronic inflammatory diseases such as atherosclerosis, diabetes, and obesity. However, the link between this condition and chronic inflammation is poorly understood. Previous work from our lab has shown that chronic exposure to super-low doses of bacterial endotoxin (LPS) aggravates atherosclerosis resulting in increased plaque size and instability in a macrophage-dependent manner in a mouse model of atherosclerosis. Further, we showed that super-low dose LPS (SLD-LPS) treatment was able to inhibit lysosomal fusion in immortalized macrophages. However, this was done under more acute treatment conditions. The aim of this project was to examine the molecular mechanisms by which chronic SLD-LPS may polarize macrophages to a non-resolving pro-inflammatory state consistent with chronic inflammation. This was carried out in two projects, the first a more broad phenotypic paper showing the disruption in homeostasis by chronic SLD-LPS in immortalized macrophages, while the second uses primary bone marrow-derived mouse macrophages to identify specific molecular signaling pathways used by chronic SLD-LPS. Here we show that chronic SLD-LPS led to the novel upregulation of pro-inflammatory mediators p62 and ccl2 with simultaneous downregulation of homeostatic mediators Nrf2 and slc40a1 in immortalized wild-type mouse macrophages. Further we showed this effect was reversed using the homeostatic restorative agent sodium phenylbutyrate (4-PBA), a newly reported activity for this reagent in mouse macrophages. This indicated that a disruption in homeostasis, possibly involving autophagy, may be responsible for the non-resolving pro-inflammatory polarization of macrophages. Therefore, in our second project, we further explored the effect of chronic SLD-LPS treatment on the homeostatic arm of the response by focusing on the Nrf2 inhibitor Keap1. Here we show that chronic SLD-LPS results in an accumulation of Keap1 in mouse bone marrow-derived macrophages, an effect specific to chronic SLD-LPS, as high doses of LPS failed to induce Keap1. We suggest that this effect may be related to a disruption in lysosomal fusion as evidenced by accumulation of autophagy flux markers MLKL and p62. Further, we show that these effects are dependent on the non-traditional TLR4 adaptor TRAM, suggesting an alternative dose-dependent signaling pathway for LPS. Together this work identifies novel signaling mechanisms involved in non-resolving pro-inflammatory polarization of murine macrophages, providing new insight behind how chronic super-low dose LPS exposure may lead to chronic inflammation. / Doctor of Philosophy / Inflammation is the body's natural response to injury or insult and can be beneficial in certain contexts such as pathogen clearance. However, left un-checked, chronic inflammation can exacerbate or even lead to disease pathology, such as is the case with modern diseases such as atherosclerosis, obesity, diabetes, etc. Despite the high prevalence of these diseases, effective treatments and therapies are still lacking. Recently it was discovered that many patients suffering from chronic inflammatory diseases had low levels bacterial endotoxin (LPS) in their circulation, a condition referred to as subclinical endotoxemia. However, possible links between this condition and chronic inflammatory disease remain poorly understood. Using a mouse model of atherosclerosis, previous research from our lab showed that persistent exposure to super-low doses of bacterial endotoxin (similar to those observed in humans) lead to aggravated atherosclerosis with both increased plaque size and instability. Further, we showed that this effect was primarily mediated by pro-inflammatory polarized immune cells called macrophages, but the molecular mechanism behind this polarization is still unclear. Further research into these molecular mechanisms may provide better targets for the development of future chronic inflammatory disease treatments. Here using a combination of mouse cell line and primary cell cultures, we discuss how chronic exposure to super-low doses of bacterial endotoxin leads to the chronic non-resolving pro-inflammatory polarization of macrophage immune cells, with particular emphasis on the distinct molecular signaling mechanisms induced by chronic super-low dose LPS.