M.Sc. / Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T14:01:31Z (GMT). No. of bitstreams: 1 Chiesa_WandersonMiguelMaia_D.pdf: 2308331 bytes, checksum: 52ef248e0f498c63a519acd9b0b14e9a (MD5) Previous issue date: 2011 / Resumo: Conteúdos dos canais radiculares podem ficar aderidos às limas endodônticas durante o preparo químico-mecânico, tais como endotoxinas, com grande potencial de desencadear resposta inflamatória. Instrumentos endodônticos são frequentemente reutilizados, existindo preocupação sobre a neutralização daqueles conteúdos pelos métodos de esterilização empregados em consultórios odontológicos. Os objetivos deste estudo foram: avaliar os efeitos de esterilização por estufa de calor seco ou autoclave, na detecção de DNA bacteriano e níveis de endotoxinas de limas endodônticas contaminadas in vivo, verificando correlações entre as espécies detectadas; investigar a produção de IL-1ß por macrófagos murinos estimulados in vitro por amostras coletadas de hastes metálicas contaminadas de limas endodônticas e esterilizadas por estufa de calor seco ou autoclave. Oitenta limas endodônticas manuais número 15 de aço inoxidável ficaram estéreis e apirogênicas, após esterilização pelo calor seco a 200ºC/4h. Vinte limas tiveram seus cabos removidos e as hastes metálicas destinadas ao Grupo I (Controle negativo), em frascos apirogênicos. Vinte pacientes apresentando necrose da polpa dental, lesão periapical e sem dor espontânea foram incluídos nesta pesquisa. Cavidades de acesso foram assepticamente preparadas e três limas apirogênicas sucessivamente introduzidas e removidas até o comprimento de trabalho de cada canal. Depois do uso, os cabos foram removidos, as hastes metálicas inseridas em frascos estéreis e apirogênicos e os outros grupos foram divididos (n=20): Grupo II (Controle positivo) - amostras sem esterilização; Group III - esterilização pela estufa de calor seco (170ºC/1h); Grupo IV - amostras autoclavadas. A técnica PCR (16S rDNA) foi usada para detectar DNA bacteriano, um teste turbidimétrico do Lisado dos Amebócitos de Limulus (LAL) foi usado para mensurar os níveis de endotoxinas e o método ELISA calculou as quantidades de IL-1ß liberadas por macrófagos murinos estimulados pelos conteúdos das limas contaminadas e esterilizadas. Prevotella nigrescens, Porphyromonas endodontalis e Treponema socranskii foram as bactérias-alvo mais frequentemente detectadas, a partir das amostras contaminadas e não esterilizadas. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia e Prevotella tannerae não foram identificadas. Correlação positiva estatisticamente significante (p<0.05) foi encontrada entre Porphyromonas endodontalis e Treponema denticola, e desta com Treponema socranskii. Depois de esterilização pela estufa de calor seco ou autoclave, o método PCR não detectou bactérias. O teste turbidimétrico indicou a mediana de 1,070 UE/mL, 0,875 UE/mL e 0,251 EU/mL, no grupo sem esterilização, esterilizado pela estufa de calor seco e autoclavado, respectivamente. Macrófagos estimulados pelas amostras das limas contaminadas e autoclavadas liberaram IL-1ß (mediana de 55,39 pg/mL). Conclusão: DNA bacteriano não foi detectado depois da esterilização pela estufa de calor seco ou autoclave, mas estes métodos não foram capazes de eliminar endotoxinas de limas endodônticas contaminadas e não foram encontradas diferenças estatisticamente significantes entre os dois métodos de esterilização empregados (p>0.05). Correlações positivas significantes foram detectadas entre micro-organismos colhidos de limas endodônticas contaminadas. Amostras autoclavadas estimularam a liberação de IL-1ß por macrófagos, mas não foram detectados níveis mensuráveis desta citocina depois da estimulação pelas amostras esterilizadas por calor seco / Abstract: Contents from root canal can be attached on endodontic files during chemomecanical preparation, such as endotoxin, with great potential to trigger inflammation. Endodontic instruments are often reused, and there is a concern about the neutralization of those contents by sterilization methods employed in dental offices. The objectives of this study were: to evaluate the effect of sterilization by dry heat oven or autoclave, in the bacterial DNA detection and endotoxin levels from in vivo contaminated endodontic files, verifying correlations between detected species; to investigate IL-1ß production by murine macrophage stimulated in vitro by samples from contaminated metal shafts of endodontic files and sterilized by dry heat oven or autoclave. Eighty size 15 stainless steel hand files were sterile and apyrogenic, after dry heat sterilization at 200ºC/4h. Twenty files had their handles cut off and metal shafts destined to the Group I (Negative control), in apyrogenic vials. Twenty patients presenting dental pulp necrosis, periapical lesion and without spontaneous pain were included in this research. Access cavities were aseptically prepared and three apyrogenic files were successively introduced and removed at the working length of each canal. After use, handles were cut off, metal shafts inserted in apyrogenic vials and the other groups were divided (n=20): Group II (Positive control) - samples with no sterilization; Group III - sterilization by dry heat oven (170ºC/1h); Group IV- autoclaved samples. PCR technique (16S rDNA) was used to detect bacterial DNA, Limulus Amebocyte Lysate (LAL) turbidimetric test measured endotoxin levels and ELISA method calculated amounts of IL-1ß released by murine macrophage stimulated by contents from the contaminated and sterilized files. Prevotella nigrescens, Porphyromonas endodontalis and Treponema socranskii were the most frequently detected target bacteria, from contaminated and non sterilized samples. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia and Prevotella tannerae were not identified. Statistically significant positive correlation (p<0.05) was found between Porphyromonas endodontalis and Treponema denticola, and between Treponema denticola and Treponema socranskii. After sterilization by dry heat oven or autoclave, PCR method detected no bacteria. Turbidimetric test indicated median of 1.070 EU/mL, 0.875 EU/mL and 0.251 EU/mL, in the group without sterilization, sterilized by dry heat oven and autoclaved, respectively. Macrophage stimulated by samples from contaminated and autoclaved files released IL-1ß (median of 55.39 pg/mL). Conclusion: no bacterial DNA was detected (PCR technique - 16S rDNA) after sterilization by dry heat oven or autoclave, but these methods were not able to eliminate endotoxin from contaminated endodontic files and no statistically significant differences were found between the two methods of sterilization employed (p>0.05). Significant positive correlations were detected between microorganisms collected from contaminated endodontic files. Autoclaved samples stimulated IL- 1ß releasing by macrophage, but no measurable levels of this cytokine were detected after stimulation by dry heat sterilized samples / Doutorado / Endodontia / Doutor em Clínica Odontológica

A implantação embrionária determina eventos biológicos de fundamental importância para o desenvolvimento do embrião e para o sucesso da gestação. O fator de inibição de migração de macrófagos (MIF) é uma das muitas citocinas que atuam durante a gestação, desempenhando múltiplas funções biológicas e atividades pró-inflamatórias, em resposta a infecções ou à presença de toxinas bacterianas e estresse. Este estudo investigou a expressão de MIF na interface materno-placentária em condições infecto-inflamatórias simuladas no organismo materno pela administração de LPS de Escherichia coli, período imediatamente após a implantação embrionária. Na primeira etapa foi administrado LPS nas doses de 0,06, 0,1, 0,2 e 0,3 <font face=\"Symbol\">mg/g de peso corporal aos 7,5 dias de gestação e o perfil gestacional foi analisado. Na segunda etapa a dose mais adequada (de 0,1 <font face=\"Symbol\">mg/g) foi administrada e a gestação interrompida 30 minutos, 1, 3 e 6 após. Com esta dose observou-se diminuição o padrão de expressão protéica de MIF durante as 6 horas seguintes ao estímulo com LPS, enquanto que a expressão gênica permaneceu estável, aumentando significativamente apenas após 6 horas de tratamento. / The embryo implantation determines biological events essential for embryo development and the success of pregnancy. The macrophage migration inhibitory factor (MIF) is one of many cytokines that act during pregnancy, playing multiple biological functions and pro-inflammatory activities in response to infection or the presence of bacterial toxins and stress. This study investigated the expression of MIF in the maternal-placental interface in infectious and inflammatory conditions simulated in the mother by the administration of LPS from Escherichia coli, on the period immediately after embryo implantation. In the first step LPS was administered at doses of 0.06, 0.1, 0.2 and 0.3 <font face=\"symbol\">mg / g body weight at 7.5 day gestation and pregnancy profile was analyzed. In the second step the more appropriate dose (0.1 <font face=\"symbol\">mg / g) was administered 30 minutes and stopped pregnancy, 1, 3 and 6 after. At this dose there was a decrease in the pattern of protein expression of MIF during the 6 hours of stimulation with LPS, while the gene expression remained stable, significantly increasing only after 6 hours of treatment.

Dans le cadre de l’évaluation du risque immunotoxique de composés glycolypidiques d’origine bactérienne, cette thèse a portée sur l’évaluation de toxicité d’endotoxines d’E.coli de deux sérotype différents : LPS O55:B5 utilisé couramment en comme immunostimulant et le LPS O157:H7 dont la réalité environnementale a soulevé notre questionnement scientifique quant à son impact sur le système immunitaire du poisson et son caractère potentiellement pro-inflammatoire. Les différents procédés employés ont compris l’évaluation des paramètres cellulaires (production d’espèces réactives d’oxygène et phagocytose) ainsi que la caractérisation et la quantification de l’expression de gènes de cytokines (TGFβ et IL-10) et facteurs immuno-associés (MARCO, HSP60 et vitellogénine) chez le modèle gardon (Rutilus rutilus).Les approches expérimentales se sont déroulées tout d’abord en ex vivo sur des leucocytes isolés d’organes lymphoïdes (Rein antérieur, rate et sang) de gardon et ont montré une tolérance endotoxique vis-à-vis de ces LPS à de 1µg/mL même combinés au diclofénac à 0,1 µM. ce travaill a été suivi par une évaluation du risque potentiel d’autres composés glycolipidiques d’origine bactérienne (rhamnolipides).Les approches in vivo qui ont suivies ont été réalisées sur : (i) sur modèle poisson-zèbre (Danio rerio) au laboratoire et (ii) sur modèle gardon par encagement sur terrain. Les résultats obtenus sur danios au laboratoire ont montré une toxicité du sérotype O157 :H7 et une influence sur les paramètres comportementaux par les LPS (Sickness behaviour). Sur terrain, l’approche in vivo par encagement a révélé – au niveau de la rate et du rein antérieur – des réponses cellulaires et moléculaires, sérotype, organe et sexe dépendante avec une immunomodulation prédominante chez les mâles d’autant plus que la période d’étude s’est déroulée durant la maturation sexuelle des gardons. Ce travail fait état du caractère inflammatoire et toxique du LPS peu étudié d’E.coli O157 :H7, évalué par des immunomarqueurs cellulaires bien maitrisés et moléculaires néo-développés. / In the context of immunotoxic risk evaluation of glycolypidic compounds of bacterial origin, this thesis focused on the evaluation of E.coli endotoxin toxicity of two different serotypes: LPS O55: B5 commonly used as an immunostimulant et LPS O157: H7, whose environmental reality has raised our scientific questioning about its impact on the fish's immune system et its potentially pro-inflammatory nature. The various methods used included the evaluation of cellular parameters (production of reactive oxygen species et phagocytosis) as well as the characterization of cytokines (TGFβ et IL-10) et immune-realted factors (MARCO, HSP60 et vitellogenin) genes et the quantification of their expression in the roach model (Rutilus rutilus).The experimental approaches were first carried out ex vivo on leukocytes isolated from lymphoid organs (anterior kidney, spleen et blood) roach et showed an endotoxic tolerance at 1μg / mL even combined with 0.1 μM diclofenac. This work was followed by an evaluation of the potential risk of other glycolipidic compounds of bacterial origin (rhamnolipids).The in vivo approaches that followed were performed on: (i) zebrafish (Danio rerio) model in the laboratory et (ii) on roach model by field caging. The results obtained on danios in the laboratory showed a toxicity of the serotype O157: H7 et an influence on the behavioral parameters by the LPS (Sickness behavior). On the field, the caging approach revealed - at spleen et the anterior kidney level - cellular et molecular responses, serotype, organ et sex-dependent with a predominant immunomodulation in males, especially since the study period took place during the sexual maturation of roaches. This work reports the inflammatory et toxic nature of the less studied E.coli O157: H7 LPS serotype, evaluated by well-mastered cellular et neo-developed molecular immunomarkers.

Lipopolysaccharide or LPS is localized to the outer leaflet of the outer membrane and serves as the major surface component of the bacterial cell envelope. This remarkable glycolipid is essential for virtually all Gram-negative organisms and represents one of the conserved microbial structures responsible for activation of the innate immune system. For these reasons, the structure, function, and biosynthesis of LPS has been an area of intense research. The LPS of a number of bacteria is composed of three distinct regions - lipid A, a short core oligosaccharide, and the O-antigen polysaccharide. The lipid A domain, also known as endotoxin, anchors the molecule in the outer membrane and is the bioactive component recognized by TLR4 during human infection. Overall, the biochemical synthesis of lipid A is a highly conserved process; however, investigation of the lipid A structures of various organisms shows an impressive amount of diversity. These differences can be attributed to the action of latent enzymes that modify the canonical lipid A molecule. Variation of the lipid A domain of LPS serves as one strategy utilized by Gram-negative bacteria to promote survival by providing resistance to components of the innate immune system and helping to evade recognition by TLR4. This review summarizes the biochemical machinery required for the production of diverse lipid A structures of human pathogens and how structural modification of endotoxin impacts pathogenesis.

No description available.

Les grands prématurés sont particulièrement vulnérables aux lésions inflammatoires de la substance blanche (WMI) qui augmentent le risque de troubles cognitifs et neurodéveloppementaux à long terme dans cette population. L’utilisation de l’imagerie par résonance magnétique (IRM) dans cette population a permis une évaluation non invasive de la progression des WMI et une meilleure compréhension de la pathologie. Les WMI sont associées une activation de la microglie et des astrocytes et la production de facteurs pro-inflammatoires, dont l’interleukine 1 (IL-1). En utilisant un modèle de WMI induite par injection intracérébrale de lipopolysaccharides (LPS), nous avons évalué dans un premier temps les changements de méthylation de l’ADN durant la phase aigüe (24 h) et la phase chronique (21 jours) de l’inflammation. Par la suite, nous avons déterminé la capacité de l’IRM multimodale de détecter la lésion et la réponse thérapeutique à un antagoniste du récepteur de l’IL-1. Finalement, par le biais d’un antagoniste et d’un modulateur allostérique du récepteur à l’IL-1, nous avons évalué in vitro le rôle de la signalisation IL-1 durant la phase aigüe de la modulation de l’activation de la microglie et des astrocytes par le LPS. Nous avons démontré la présence d’une altération du méthylome cérébral dans divers mécanismes liés au neurodéveloppement et à la réponse immunitaire. De plus, l’application de l’IRM multimodale dans notre modèle a permis d’évaluer in vivo la lésion et le début de la réponse thérapeutique durant la phase aigüe (24 h) de l’inflammation. L’évaluation à l’IRM corrèle aux changements observés par immunomarquage post mortem. In vitro, le LPS induit une réponse mixte de la microglie et des astrocytes qui évoluent dans le temps vers une réponse pro-inflammatoire et neurotoxique. Bien que l’IL-1 est hautement exprimée par la microglie et les astrocytes, son inhibition a un effet limité sur la modulation de l’activation gliale dû à la multitude de voies activées par le LPS durant la phase aigüe de l’inflammation. / Very premature infants are particularly vulnerable to inflammatory white matter injury (WMI) which increases the risk of long-term cognitive and neurodevelopmental disorders in this population. The use of magnetic resonance imaging (MRI) in this population has allowed non-invasive assessment of the progression of WMI and a better understanding of the pathology. WMI is associated with activation of microglia and astrocytes and the production of pro-inflammatory mediators, including interleukin 1 (IL-1). Using a model of inflammatory WMI induced by intracerebral injection of lipopolysaccharides (LPS), we first evaluated the changes in DNA methylation during the acute phase (24 h) and the chronic phase (21 days) of inflammation. We then determined the ability of multimodal MRI to detect the lesion and the therapeutic response to an IL-1 receptor antagonist. Finally, using an antagonist and an allosteric modulator of the IL-1 receptor, we evaluated in vitro the contribution of IL-1 signaling during the acute phase of the modulation of microglia and astrocytes activation by LPS. We have shown the presence of persistent alteration DNA methylation profile in the brain that was associated with pathways involved in neurodevelopment and immune response. In addition, the application of multimodal MRI in our model made it possible to evaluate in vivo the lesion and the therapeutic response during the acute phase (24 h) of the inflammation. The changes at the MRI correlated to post-mortem evaluation by immunostaining. In vitro, LPS induce a mixed response of microglia and astrocytes which evolved over time toward a pro-inflammatory and neurotoxic phenotype. Although IL-1 is highly expressed by microglia and astrocytes, its inhibition has a limited effect on the modulation of glial activation due to the multitude of pathways activated by LPS during the acute phase of inflammation.

Introdução: As citocinas de perfil Th17 parecem exercer um papel importante na fisiopatogenia da inflamação pulmonar alérgica crônica, embora sua exata influência seja incerta. Estudos demonstram uma influência destas citocinas em modular a resposta inflamatória e infecciosa. É importante enfatizar que a infecção é um dos responsáveis por perpetuar a resposta inflamatória crônica na asma, particularmente durante as exacerbações. Muitos dos efeitos desta via ainda não foram investigados em modelos de inflamação alérgica pulmonar crônica (asma) exarcebada pelo lipopolissacarídeos (LPS). Objetivos: Avaliar os efeitos do tratamento com anti-IL-17 em um modelo de inflamação pulmonar alérgica crônica exarcebada pelo LPS. Métodos: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina ou salina e com posterior desafios inalatórios por 28 dias. Nos 1° e 14° dias um grupo (grupo OVA) recebeu ovoalbumina 50 mg em hidróxido de alumínio 6mg em um volume total de 0,2 ml por via intraperitoneal. Do 22° ao 28° dia, os animais receberam em dias alternados, inalação de aerossol de OVA diluída em NaCl 0,9% (soro fisiológico) na concentração de 10 mg/ml (1%) por 30 minutos. Ao mesmo tempo, o grupo controle (grupo SAL) recebeu solução salina (NaCl 0,9%) e hidróxido de alumínio (6 mg) por via intraperitoneal e nos dias dos desafios inalatórios foram expostos ao aerossol de solução salina 0,9% também por 30 minutos. Uma hora antes da inalação de ovoalbumina ou de salina, o anticorpo neutralizador anti-IL17 foi administrado por via intraperitoneal (ip) (grupos OVA-antiIL17 e SAL-antiIL17). Vinte e quatro horas antes do final do protocolo experimental (28° dia) um grupo de animais (grupo OVA-LPS) recebeu instilação traqueal de LPS (20 ?l de PBS + 0,1 mg / ml de Escherichia coli 0127: B8). O grupo sensibilizado que recebeu LPS e anti-IL17uma hora antes da exposição ao LPS foi chamado de OVA-LPS-antiIL17. No 29° dia do protocolo, os animais foram eutanasiados e inicialmente foram avaliadas as respostas máximas à metacolina da resistência e elastância do sistema respiratório. Os pulmões foram retirados e realizados as análises histológicas e morfométricas para avaliar a expressão celular de CD4+, CD8+, células dendríticas e células reguladoras T FOXP3; RT-PCR para arginase 1, transportador vesicular de acetilcolina (VAChT) e IL-17; ativação do fator de transcrição nuclear ?B (NF-?B); o número de células positivas para ROCK 1 e ROCK 2; resposta inflamatória de perfis Th1 (IL-2, IL-6, e TNF-alfa), Th2 (IL-4, IL-5, IL-10 e IL-13), Th-17 (IL-17), quimiocinas CCL17/TARC e CCL11/Eotaxina; resposta de remodelamento da matriz extracelular (conteúdo de fibras colágenas tipos I e III, decorina, lumican, biglicano, fibronectina, actina, TGFbeta1, o número de células positivas para MMP-9, MMP-12 e TIMP-1), e a resposta de estresse oxidativo (o número de células positivas para iNOS e conteúdo de isoprostano PGF2alfa e Arginase 1). Além disso, avaliamos por reação em cadeia da polimerase em tempo real (Real-time PCR) a expressão gênica de IL-17 e mRNA para VAChT As análises estatísticas foram realizada por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um p < 0,05 será considerado estatisticamente significativo. Resultados: O grupo OVA-LPS-antíIL17 apresentou uma diminuição na resposta máxima pós metacolina de elastância e resistência do sistema respiratório, NO exalado, o número de células positivas para CD4+ e CD8+, células dendríticas, células T reguladoras FOXP3, mRNA para VAChT, NF-?B, ROCK1 e 2, quimiocinas CCL17 / TARC, e CCL11 / eotaxina, IL2 IL4, IL5, IL6, IL10, IL13 e IL17 nas paredes das vias aéreas comparado aos grupos OVA e OVA-LPS (p < 0,05). Alem disso, o tratamento com anti-IL17 reduziu o remodelamento brônquico (conteúdo de fibras colágenas I e III, decorina, lumican, biglican e fibronectina nas paredes das vias aéreas) assim como o número de células positivas para TGFbeta1, MMP-9, MMP12 e TIMP-1 ao redor das vias aéreas em comparação ao grupo SAL (p < 0.05). Houve também a atenuação de todos os parâmetros exceto no grupo OVA-antiIL17 em comparação ao grupo OVA (p < 0.05). Quanto ao estresse oxidativo o tratamento com o anti-IL17 também foi efetivo. Observamos redução de expressão celular de iNOS, conteúdo de PGF2alfa e expressão gênica de arginase1 em comparação com os grupos OVA e OVA-LPS (p < 0,05). Ainda, observamos uma atenuação do antí-IL17 na expressão gênica de IL17 e VAChT. Conclusões: A inibição da IL17 contribuiu para o controle da hiperresponsividade brônquica, da inflamação Th1/Th2/Th17, da expressão de quimiocinas, do remodelamento da matriz extracelular e da resposta da via NO-arginase, do sistema colinérgico sinalizado pela avaliação do VAChT e de estresse oxidativo no modelo de asma experimental. No modelo de asma experimental exarcebado pelo LPS houve potencialização das respostas de hiperresponsividade das vias aéreas distais, assim como da maioria dos marcadores de resposta inflamatória, de remodelamento da matriz extracelular e da ativação das vias via NO-arginase e do estresse oxidativo. O tratamento com anti-IL17 nesses animais foi capaz de controlar a maior parte das alterações citadas, exceto o conteúdo de actina. Dentre os mecanismos envolvidos no controle pelo tratamento com anti-IL17 das alterações estudadas no modelo de asma experimental exarcebado pelo LPS observamos a importância da expressão do fator de transcrição NFkb e de Rho quinase 1, e expressão gênica mRNA para VAChT. Embora outros mecanismos possam estar envolvidos e doses repetidas de anti-IL17 nos animais com asma exarcebado pelo LPS também necessitem ser testadas, o tratamento com anti-IL17 se mostrou uma ferramenta farmacológica importante para o tratamento das alterações, que à semelhança do observado nestes modelos experimentais, também se observam em pacientes com asma grave mesmo associada a quadros infecciosos agudos / Introduction: Th17 cytokines appear to play an important role in the pathophysiology of chronic allergic pulmonary inflammation, although their exact role is uncertain. Studies have demonstrated an influence of these cytokines in modulating the inflammatory and infectious response. It is important to emphasize that infection is one of the factors responsible for perpetuating the chronic inflammatory response in asthma, particularly during exacerbations. Many of the effects of this pathway have not yet been investigated in models of chronic allergic lung inflammation (asthma) exacerbated by lipopolysaccharide (LPS). Objectives: To evaluate the effects of anti-IL17 treatment on a model of chronic allergic pulmonary inflammation exacerbated by LPS. Methods: BALB/c mice were submitted to protocol of chronic allergic induction of the airways with ovalbumin or saline and with subsequent inhaled challenges for 28 days. On days 1 and 14 a group (OVA group) received ovalbumin 50 mg in 6 mg aluminum hydroxide in a total volume of 0.2 ml intraperitoneally. From the 22 to the 28 day, the animals received, on alternate days, aerosol inhalation of OVA diluted in NaCl 0.9% (saline solution) at a concentration of 10 mg/ml (1%) for 30 minutes. At the same time, the control group (SAL group) received saline solution (NaCl 0.9%) and aluminum hydroxide (6 mg) intraperitoneally and on the days of the inhalation challenges were exposed to aerosol 0.9% saline solution for 30 minutes. One hour prior to inhalation of ovalbumin or saline, the anti-IL17 neutralizing antibody was administered i.p. (OVA-anti-IL-17 and SAL-anti-IL17 groups). Twenty-four hours before the end of the experimental protocol (28th day) one group of animals (OVA-LPS group) received LPS tracheal instillation (20 ul PBS + 0.1 mg/ml Escherichia coli 0127: B8). The sensitized group that received LPS and anti-IL17 an hour before exposure to LPS was called OVA-LPS-anti-IL-17. On the 29th day of the protocol, the animals were euthanized and initially the maximum methacholine responses of resistance and elastance of the respiratory system were evaluated. The lungs were removed and the histological and morphometric analysis were performed to evaluate the cell expression of CD4+, CD8+, dendritic cells and T regulatory cells FOXP3; RT-PCR for arginase 1, vesicular acetylcholine transporter (VAChT) and IL-17; activation of nuclear transcription factor KappaB (NFkb); the number of positive cells for ROCK 1 and ROCK 2; Th1 (IL-2, IL-6, and TNF-alpha), Th2 (IL-4, IL-5, IL-10 and IL-13), Th-17 (IL-17), CCL17 chemokines/TARC and CCL11 / Eotaxin; (content of collagen fibers types I and III, decorin, lumican, biglican, fibronectin, actin, TGFbeta1, number of cells positive for MMP-9, MMP-12 and TIMP-1), and the response of oxidative stress (the number of positive cells for iNOS and isoprostane content PGF2alpha and Arginase 1). In addition, we evaluated the gene expression of IL-17 and mRNA for VAChT by real-time PCR. Statistical analysys were performed using the SigmaStat program (Jandel Scientific, San Rafael, CA), where a p < 0.05 were considered statistically significant. Results: The OVA-LPS-anti-IL-17 group showed a decrease in post-methacholine maximal response of elastance and respiratory system resistance, exhaled NO, number of CD4 + and CD8 + positive cells, dendritic cells, FOXP3 regulatory T cells, mRNA for VAChT, (p < 0.05), and in the presence of a significant increase in the expression of IL-1, IL-6, IL-10, IL-13 and IL17 in the airway walls compared to OVA and OVA-LPS (p < 0,05). In addition, anti-IL17 treatment reduced bronchial remodeling (content of collagen fibers I and III, decorin, lumican, biglican and fibronectin in the airway walls) as well as the number of TGFbeta1, MMP-9, MMP-12 and TIMP-1 around the airways compared to the SAL group (p < 0.05). There was also attenuation of all parameters except in the OVA-antiIL17 group compared to the OVA group (p < 0.05). As for oxidative stress, treatment with anti-IL17 was also effective. We observed reduction of iNOS cell expression, PGF2alpha content and arginase1 gene expression in comparison to OVA and OVA-LPS groups (p < 0.05). Furthermore, we observed an attenuation of the anti-IL17 in the IL17 and VAChT gene expression. Conclusions: Inhibition of IL-17 contributed to the control of bronchial hyperresponsiveness, Th1/Th2/Th17 inflammation, chemokine expression, extracellular matrix remodeling and NO-arginase pathway response, cholinergic system signaled by VAChT and of oxidative stress in the experimental asthma model. In the model of experimental asthma exacerbated by LPS there was a potentation of hyperresponsiveness responses of the distal airways, as well as of the majority of inflammatory response markers, extracellular matrix remodeling and activation of the pathways via NO-arginase and oxidative stress. Treatment with anti-IL-17 in these animals was able to control most of the above-mentioned changes, except the actin content. Among the mechanisms involved in the control by anti-IL17 treatment of the alterations studied in the model of experimental asthma exacerbated by LPS, we observed the importance of the expression of the transcription factor NF-Kappa B and Rho kinase 1, and gene expression mRNA for VAChT. Although other mechanisms may be involved and repeated doses of anti-IL17 in animals with LPS-exacerbated asthma also need to be tested, treatment with anti-IL17 has proved to be an important pharmacological tool for the treatment of the changes, which similar to those observed in these experimental models, are also seen in patients with severe asthma associated with acute infectious conditions

Bdellovibrionaceae sind außergewöhnliche Bakterien, da sie als die kleinsten bekannten räuberischen Organismen gelten. Seit ihrer Entdeckung in Bodenproben im Jahr 1962 durch Stolp und Starr konnte eine weite Verbreitung in der Natur nachgewiesen werden. Besonderes Interesse erlangten Bdellovibrionaceae durch ihre Fähigkeit bakterielle Erreger wie Escherichia coli, Salmonella und Yersinia zu attackieren. Der bestuntersuchte Vertreter der Familie Bdellovibrionaceae ist Bdellovibrio bacteriovorus. Er durchläuft einen komplexen Lebenszyklus, der nur partiell verstanden ist. In der Attack-Phase werden durch einen noch nicht aufgeklärten Mechanismus potentielle Beutebakterien erkannt und der interzelluläre Kontakt hergestellt. Nachdem eine Pore in der Zellwand der ausschließlich Gram-negativen Beutebakterien erzeugt wurde, dringt B. bacteriovorus in den periplasmatischen Raum ein. Nach etwa 30 Minuten ist der Invasionsprozess abgeschlossen und man kann die Ausbildung sphärischer Bdelloblasten beobachten. Im Beutebakterium verdaut B. bacteriovorus makromolekulare Bestandteile des Wirtes und wächst zu einem langen spiralförmigen Stäbchen aus. Es setzt schließlich die Zellteilung ein bei der 5 bis 30 Tochterzellen in Abhängigkeit zur Größe des Beutebakteriums freiwerden. Mit der Reproduktion ist der parasitäre Lebenszyklus nach etwa 3 Stunden beendet. Die komplexe Regulation der Expression von Proteinen konnte für die einzelnen Wachstumsphasen durch ein- sowie zweidimensionale Gelelektrophorese nachgewiesen werden. In der Vergangenheit haben verschiedene Studien die Komplexität der Wechselwirkung mit den Beutebakterien belegt. Hierbei wurde, neben dem offensichtlichen Abbau makromolekularer Bestandteile der Beutebakterien, ein Recycling und Einbau von Membranbestandteilen wie Lipopolysacchariden (LPS) und Outer Membrane Proteine (OmP) in das Membransystem von B. bacteriovorus diskutiert. Die biologische Interpretation dieses Phänomens ist eine erhöhte Effizienz in der Reproduktion von B. bacteriovorus wenn es Bestandteile des Wirtsbakteriums wiederverwertet im Gegensatz zur Eigensynthese. Trotz der morphologischen Ähnlichkeit und des ungewöhnlichen Lebensstils wurde festgestellt, dass die Familie der Bdellovibrionaceae phylogenetisch sehr heterogen ist. Es werden zur Zeit zwei Gattungen unterschieden Bdellovibrio und Bacteriovorax. Aufgrund von 16S rRNA Untersuchungen konnten auch innerhalb der Gattungen eine Vielzahl von phylogenetisch distinkten Arten nachgewiesen werden. In der Literatur wird eine regulierende Wirkung auf pathogene Gram-negative Bakterien für aquatische Systeme durch Bdellovibrionaceae beschrieben. Weiterhin konnten sie bei verschiedenen Nutztieren im Verdauungstrakt mikrobiologisch nachgewiesen werden. Ein positiver Einfluss auf die Gesundheit der Tiere wurde bei Vorkommen von B. bacteriovorus festgestellt. Für eine Charakterisierung von Umweltisolaten werden in der vorliegenden Arbeit genetische Methoden vorgestellt. Hierfür wurden Hybridisierungsmethoden und PCR-methoden entwickelt, die es ermöglichen aus der Umwelt isolierte Bdellovibrionaceae phylogenetisch einzuordnen. Es konnte gezeigt werden, das sowohl B. bacteriovorus als auch Bacteriovorax stolpii im Verdauungstrakt von Nutztieren vorkommen. Es ist weiterhin gelungen eine PCR-methode für Kotproben zu entwickeln, die einen direkten Nachweis ermöglicht ohne mikrobiologische Anzucht. B. bacteriovorus ist ein Gram-negatives Bakterium, allein dieser Sachverhalt spiegelt die Schwierigkeit wider, wenn der enzymattische Abbau von Zellwandbestandteilen der Beutebakterien Ziel der Untersuchung ist, da auch die Beutebakterien Gram-negativ sind. Es ergibt sich aufgrund eines ähnlichen Zellwandaufbaus ein immenses analytisches Problem die makromolekularen Bestandteile von Wirtsbakterium und Jäger zu trennen. Um dem Lebensstil angepasst Modifikationen von B. bacteriovorus zu untersuchen, wurde in dieser Arbeit LPS, charakteristischer Bestandteil der Äußeren Membran, von Wildtypstamm B. bacteriovorus HD100 und seiner wirtsunabhängigen Mutante HI100 isoliert und das Lipid A strukturell aufgeklärt. Die Isolation gelang durch die Ausnutzung unterschiedlicher Fällungseigenschaften des LPS von B. bacteriovorus HD100 gegenüber des E. coli K-12 LPS aus dem Extraktionsmittel. Weiterhin konnte nachgewiesen werden, das B. bacteriovorus S-Form LPS besitzt. Die außergewöhnliche Struktur des Lipid A von B. bacteriovorus wurde im Detail mit massenspektrometrischen Methoden, ein- und zweidimensionalen NMR Methoden sowie mikrochemischer Methoden in Kombination mit der GC/MS charakterisiert. Der Fettsäureanker besteht aus a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. Dies stellt eine neuartige Struktur dar, da an allen bisher bekannten Lipid A´s sich Brönstedtsäuren am hydrophilen Fettsäureanker befinden, die in physiologischer Lösung durch Protonenabgabe negative Ladungen tragen. Im Lipid A von B. bacteriovorus sind diese Substituenten durch den Neutralzucker Mannose ersetzt. Weiterhin wurden als Fettsäuren nur 3-Hydroxyfettsäuren nachgewiesen, wobei sich auf die 6 gebunden Fettsäuren etwa 1,5 Doppelbindungen verteilen. Dieses Lipid A zeigt eine wesentlich reduzierte endotoxische Aktivität im Vergleich zu E. coli Lipid A und weist als biophysikalische Besonderheit eine erhöhte Fluidität über einen weiten Temperaturbereich auf. Die vorgestellte 16S rRNA Analyse und die Strukturanalyse des Lipid A von B. bacteriovorus belegen seine besondere Stellung in der Welt der Bakterien. / Bdellovibrionaceae are extraordinary bacteria known as the smallest predatory organism so far. Since their discovery in soil samples by Stolp and Starr 1963 they have been detected in a wide range of other natural habitats. Bdellovibrionaceae became the focus of attention concerning their ability to attack pathogens like Escherichia coli, Salmonella and Yersinia. For Bdellovibrio bacteriovorus the most detailed studies are available. The up to now only partially understood lifecycle consists of several complex phases. In the attack phase B. bacteriovorus is motile possessing flagella and the preys are recognized by an unknown mechanism. After attachment on the cell wall within 15 to 30 minutes a pore is formed which is used as entrance to the periplasmatic space of the Gram-negative prey bacteria. The completion of the invasion process can be observed by the change of the prey s shape to spherical bdelloblasts. Inside of the prey B. bacteriovorus degrades macromolecular compounds and transforms into a long spirally shaped rod. At the end of the lifecycle the rod divides yielding 5 up to 30 daughter cells depending on the size of the prey bacteria. This reproduction phase is completed within 3 hours. The complex regulation of expression of a certain number of proteins was observed by one and two dimensional gelelectrophoresis. The complexity of the interaction between predator and prey were examined in several studies. Besides the obvious degradation of macromolecular compounds of the prey, reutilising of lipopolysaccharides (LPS) and Outer Membrane Proteins (OmP) into the membrane system of B. bacteriovorus was discussed. The biological interpretation of such behaviour was that it is more efficient for reproduction to recycle components of the prey than to perform de novo synthesis. Unexpectedly, despite the unique predatory lifecycle and common morphological features, Bdellovibrionaceae show a great phylogenetical diversity based on 16S rRNA analyses. Bdellovibrionaceae are divided into the three species Bdellovibrio bacteriovorus, Bacteriovorax stolpii, Bacteriovorax starrii and some strains yet to be assigned. In two studies Bdellovibrionaceae were found as part of regulation processes for decreasing the number of pathogenic Gram-negative bacteria in aquatic systems. Furthermore, B. bacteriovorus were found in the intestinal tract of several domestic animals showing a positive influence on the state of health. For the phylogenetic characterization of environmental isolates techniques were developed based on hybridisation methods and the PCR. In this work we detected B. bacteriovorus and B. stolpii strains in the gut of animals. Furthermore, a PCR method for direct detection of Bdellovibrionaceae in fecal samples was developed. B. bacteriovorus are Gram-negative bacteria. This fact complicates the study of the degradation of the prey s cell wall as it possesses the architecture of Gram-negative bacteria also. Furthermore, the search for important modifications of the cell wall of B. bacteriovorus concerning the predatory lifestyle becomes an analytical problem. In this study we isolated LPS of the wild type strain B. bacteriovorus HD100 and its host independent mutant strain HI100. For the isolation of pure B. bacteriovorus HD100 S-form LPS we took advantage of different precipitation properties in the extraction solvent of E. coli K-12 LPS and the predator s LPS. The structure of the lipid A was examined in detail by mass spectrometric methods, one- and two-dimensional NMR and chemical analytical techniques. The novel structure consists of backbone built of a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. This is the first known natural lipid A without negatively charged substituents in physiological solution. The lipid A of B. bacteriovorus carries the neutral sugar mannose instead of Brönstedt acids. Furthermore, the lipid A exclusively consists of 3-hydroxy fatty acids with approximately 1.5 double bounds distributed on six bounded fatty acids. This lipid A shows a significant decreased endotoxic activity in comparison to E. coli lipid A and revealed increased fluidity over broad temperature range as further remarkable biophysical property. The 16S rRNA analysis and the structural analysis of the lipid A of B. bacteriovorus document the unique position in the world of bacteria.

A administração de Ags solúveis via mucosas, antes da sensibilização com o mesmo Ag, leva a tolerância. Na presente tese estudou-se o efeito do LPS i.n. durante a indução de tolerância nasal a ovalbumina (OVA). A maioria dos modelos murinos de asma utilizam a OVA como alérgeno, desse modo também utilizamos o extrato de Blomia tropicalis (Bt), um ácaro prevalente em pacientes asmáticos. Características da Bt impedem o estabelecimento de tolerância e evidências experimentais indicam que a tolerância a um Ag pode induzir tolerância a um Ag não relacionado (tolerância cruzada). Assim, avaliamos se a tolerância a OVA ou lizozima de ovo (HEL) poderia induzir tolerância cruzada a Bt. Observou-se que o LPS i.n. durante a indução de tolerância a OVA previne o estabelecimento da tolerância, resultando em neutrofilia pumonar, IgG2a, diminuição de IgE com aumento de IgG1 anafilática. Observou-se também que a tolerância nasal, a HEL ou OVA, não é eficaz em induzir tolerância cruzada para as respostas contra Bt. Já, a tolerância oral com OVA induziu tolerância cruzada para Bt. / Mucosal administration of soluble Ags, before the sensitization with the same Ag, leads to mucosal tolerance. We studied the effect of i.n. LPS during the induction of ovalbumin (OVA) nasal tolerance. The majority of murine models of asthma used OVA as allergen,thus, we also used Blomia tropicalis (Bt) extract, a mite more common in asthmatic patients. Features of Bt block the nasal tolerance establishment and experimental data indicates that tolerance towards an Ag can promote tolerance to another not related Ag (cross-tolerance). Therefore, we evaluated if OVA or hen-egg white lisozyme (HEL) tolerance could result in cross-tolerance to the Bt. Our data showed that i.n. LPS during induction of tolerance to OVA prevents the establishment of this tolerance, resulting in neutrophil migration, IgG2a production, decreased levels of IgE and increased anaphylactic IgG1. We verify that nasal tolerance to HEL or OVA is not capable in induce cross-tolerance against the Bt. We also observed that OVA oral tolerance is able to induce cross-tolerance against Bt.