Plantas do gênero Hypericum (Hypericaceae) são reconhecidas fontes de moléculas com fins terapêuticos. Para espécies nativas do sul do Brasil, atividades como antifúngica e antinociceptiva já foram relatadas, atribuídas principalmente a compostos extraídos em suas frações lipofílicas como derivados de floroglucinol, benzopiranos e benzofenonas. Neste estudo, o potencial sinérgico entre frações lipofílicas de H. carinatum e o fármaco fluconazol, frente a fungos leveduriformes emergentes, foi avaliado por duas metodologias distintas: checkerboard e isobolograma. Para isolados de Candida krusei e C. famata o efeito da associação foi superior ao do fármaco isolado. Dessa forma, o perfil de suscetibilidade observado sugere que a fração esteja auxiliando a ação do fármaco. Ainda abordando o potencial terapêutico de espécies de Hypericum, a investigação da atividade antinociceptiva (via oral) do benzopirano HP1 de H. polyanthemum, quando incorporado em nanoemulsões, foi avaliada. Os resultados demonstraram que HP1 pode ser adequadamente incorporado em nanoemulsões, dada sua solubilidade no núcleo oleoso. Em relação ao efeito antinociceptivo, nanoemulsões contendo HP1 demonstraram o mesmo efeito do composto livre, em magnitude, porém em dose inferior. A redução da dose ativa sugere que uma melhor solubilização do composto possa ter ocorrido quando o mesmo está inserido em nanoemulsões. Nesse contexto, estudos de permeabilidade intestinal ex vivo (Ussing chambers) de HP1, na sua forma livre e incorporado em nanoemulsões, foram realizados. Os resultados demonstraram que a permeabilidade intestinal do benzopirano HP1, quando incorporado em nanoemulsões, foi cerca de 4 vezes maior em relação a forma livre. Além disso, experimentos de lipólise in vitro mostraram que enzimas presentes no trato gastrointestinal são hábeis em hidrolisar nanoemulsões a espécies coloidais, mais solúveis e facilmente absorvíveis pelas células intestinais. Ainda, a permeabilidade intestinal do benzopirano HP1, na sua forma livre, no sentido absortivo foi maior que no sentido secretório indicando que transportadores ativos estão, ao menos em parte, auxiliando a absorção deste composto pelas células intestinais. Dessa forma, com vistas a elucidar o provável transportador ativo de HP1, dada a semelhança estrutural deste benzopirano com moléculas canabinoides e a relação existente entre os sistemas opioide e canabinoide, a influência deste último na absorção de HP1 foi investigada. Os resultados demonstraram que o benzopirano HP1 pode estar relacionado ao sistema canabinoide, mas a natureza dessa ligação, seja de transporte, agonismo/antagonismo ou físico-química, não foi possível de ser elucidada. Os resultados obtidos nesta tese são relevantes à medida que espécies de fungos leveduriformes emergentes se mostram cada vez mais resistentes aos fármacos comumente utilizados. Além disso, a importância destes resultados se dá pela viabilidade de incorporação do benzopirano HP1 em nanoemulsões e a capacidade desses sistemas em reduzir a dose ativa no benzopirano HP1 por uma maior solubilização do composto e assim, melhor absorção. Dessa maneira, os resultados deste trabalho representam o alto potencial biológico de espécies de Hypericum e abrem possibilidade para mais estudos utilizando estas plantas. / Plants from genus Hypericum (Hypericaceae) are recognized as a source of therapeutical agents. To south Brazil species, acitivities like antifungal and antinociceptive had already been demonstrated, attributed mainly to compounds from lipophilic fractions as phloroglucinol derivatives, benzophenones and benzopyrans. In this study, antifungal potential of lipophilic fractions of H. carinatum and fluconazole against emerging yeasts was evaluated by two methodologies for multiple dose-response analyzes: checkerboard and isobologram. To Candida krusei and C. famata isolates the effect of association was higher than the effect of fluconazole alone. Thus, the susceptibility profile observed for these species suggests that, somehow, the fractions are facilitating the action of drug. Still on therapeutical potential of Hypericum species, the antinociceptive study of a benzopyran (HP1) isolated from H. polyanthemum, incorporate in nanoemulsions, was evaluated. The results demonstrated that HP1 could be incorporated in a nanoemulsion system, given the high solubility in the oil core. Regarding the antinociceptive effect, HP1 loaded in nanoemulsions showed the same effect of free form, in magnitude, at lower doses. These results suggest a better solubilization of HP1 when loaded in nanoemulsions, and, thus, better absorption by organism. In this context, ex vivo intestinal permeability studies (Ussing chambers) of HP1 free form and loaded in nanoemulsions were performed. The results showed that the intestinal permeability of HP1 loaded in nanoemulsions were about 4 times higher than HP1 free form. Besides, the intestinal permeability of HP1 free form in absorptive direction was higher than secretory direction indicating that active transporters are, at least in part, involved in HP1 intestinal absorption. Thus, in order to elucidate the probable active transporter of HP1 and since its structure looks like a cannabinoid molecule and there is a relation between the opioid and cannabinoid pathways, the influence of intestinal cannabinoid system in HP1 absorption was investigated. The results indicated that the benzopyran HP1 may be related to cannabinoid system, but the nature of this interaction: transport, agonism/antagonism or physico-chemical is still unknown. The outcomes obtained are relevant since the resistance of emerging yeast species to available drugs, used for a variety of fungal infections, is increasing. The importance of these findings lies also in the feasibility of incorporating HP1 into nanoemulsions, and the capacity of these systems in reduce the antinociceptive active doses, by higher solubilization, and thus, absorption. Then, together the results represent the high biological potential of Hypericum species and open new possibilities to further studies with these plants.

Plantas do gênero Hypericum (Hypericaceae) são reconhecidas fontes de moléculas com fins terapêuticos. Para espécies nativas do sul do Brasil, atividades como antifúngica e antinociceptiva já foram relatadas, atribuídas principalmente a compostos extraídos em suas frações lipofílicas como derivados de floroglucinol, benzopiranos e benzofenonas. Neste estudo, o potencial sinérgico entre frações lipofílicas de H. carinatum e o fármaco fluconazol, frente a fungos leveduriformes emergentes, foi avaliado por duas metodologias distintas: checkerboard e isobolograma. Para isolados de Candida krusei e C. famata o efeito da associação foi superior ao do fármaco isolado. Dessa forma, o perfil de suscetibilidade observado sugere que a fração esteja auxiliando a ação do fármaco. Ainda abordando o potencial terapêutico de espécies de Hypericum, a investigação da atividade antinociceptiva (via oral) do benzopirano HP1 de H. polyanthemum, quando incorporado em nanoemulsões, foi avaliada. Os resultados demonstraram que HP1 pode ser adequadamente incorporado em nanoemulsões, dada sua solubilidade no núcleo oleoso. Em relação ao efeito antinociceptivo, nanoemulsões contendo HP1 demonstraram o mesmo efeito do composto livre, em magnitude, porém em dose inferior. A redução da dose ativa sugere que uma melhor solubilização do composto possa ter ocorrido quando o mesmo está inserido em nanoemulsões. Nesse contexto, estudos de permeabilidade intestinal ex vivo (Ussing chambers) de HP1, na sua forma livre e incorporado em nanoemulsões, foram realizados. Os resultados demonstraram que a permeabilidade intestinal do benzopirano HP1, quando incorporado em nanoemulsões, foi cerca de 4 vezes maior em relação a forma livre. Além disso, experimentos de lipólise in vitro mostraram que enzimas presentes no trato gastrointestinal são hábeis em hidrolisar nanoemulsões a espécies coloidais, mais solúveis e facilmente absorvíveis pelas células intestinais. Ainda, a permeabilidade intestinal do benzopirano HP1, na sua forma livre, no sentido absortivo foi maior que no sentido secretório indicando que transportadores ativos estão, ao menos em parte, auxiliando a absorção deste composto pelas células intestinais. Dessa forma, com vistas a elucidar o provável transportador ativo de HP1, dada a semelhança estrutural deste benzopirano com moléculas canabinoides e a relação existente entre os sistemas opioide e canabinoide, a influência deste último na absorção de HP1 foi investigada. Os resultados demonstraram que o benzopirano HP1 pode estar relacionado ao sistema canabinoide, mas a natureza dessa ligação, seja de transporte, agonismo/antagonismo ou físico-química, não foi possível de ser elucidada. Os resultados obtidos nesta tese são relevantes à medida que espécies de fungos leveduriformes emergentes se mostram cada vez mais resistentes aos fármacos comumente utilizados. Além disso, a importância destes resultados se dá pela viabilidade de incorporação do benzopirano HP1 em nanoemulsões e a capacidade desses sistemas em reduzir a dose ativa no benzopirano HP1 por uma maior solubilização do composto e assim, melhor absorção. Dessa maneira, os resultados deste trabalho representam o alto potencial biológico de espécies de Hypericum e abrem possibilidade para mais estudos utilizando estas plantas. / Plants from genus Hypericum (Hypericaceae) are recognized as a source of therapeutical agents. To south Brazil species, acitivities like antifungal and antinociceptive had already been demonstrated, attributed mainly to compounds from lipophilic fractions as phloroglucinol derivatives, benzophenones and benzopyrans. In this study, antifungal potential of lipophilic fractions of H. carinatum and fluconazole against emerging yeasts was evaluated by two methodologies for multiple dose-response analyzes: checkerboard and isobologram. To Candida krusei and C. famata isolates the effect of association was higher than the effect of fluconazole alone. Thus, the susceptibility profile observed for these species suggests that, somehow, the fractions are facilitating the action of drug. Still on therapeutical potential of Hypericum species, the antinociceptive study of a benzopyran (HP1) isolated from H. polyanthemum, incorporate in nanoemulsions, was evaluated. The results demonstrated that HP1 could be incorporated in a nanoemulsion system, given the high solubility in the oil core. Regarding the antinociceptive effect, HP1 loaded in nanoemulsions showed the same effect of free form, in magnitude, at lower doses. These results suggest a better solubilization of HP1 when loaded in nanoemulsions, and, thus, better absorption by organism. In this context, ex vivo intestinal permeability studies (Ussing chambers) of HP1 free form and loaded in nanoemulsions were performed. The results showed that the intestinal permeability of HP1 loaded in nanoemulsions were about 4 times higher than HP1 free form. Besides, the intestinal permeability of HP1 free form in absorptive direction was higher than secretory direction indicating that active transporters are, at least in part, involved in HP1 intestinal absorption. Thus, in order to elucidate the probable active transporter of HP1 and since its structure looks like a cannabinoid molecule and there is a relation between the opioid and cannabinoid pathways, the influence of intestinal cannabinoid system in HP1 absorption was investigated. The results indicated that the benzopyran HP1 may be related to cannabinoid system, but the nature of this interaction: transport, agonism/antagonism or physico-chemical is still unknown. The outcomes obtained are relevant since the resistance of emerging yeast species to available drugs, used for a variety of fungal infections, is increasing. The importance of these findings lies also in the feasibility of incorporating HP1 into nanoemulsions, and the capacity of these systems in reduce the antinociceptive active doses, by higher solubilization, and thus, absorption. Then, together the results represent the high biological potential of Hypericum species and open new possibilities to further studies with these plants.

Introdução: o estresse psicológico aumenta a permeabilidade intestinal em roedores e humanos, potencialmente levando a inflamação de baixo grau e aos sintomas em distúrbios gastrintestinais funcionais. No entanto, o efeito do estresse agudo combinado ao estresse da vida crônica, que mimetiza potencialmente melhor a situação humana, é desconhecido. Além disso, há poucos dados disponíveis sobre os efeitos do estresse em intestino delgado versus cólon. Métodos: ratos Wistar foram alocados em quatro protocolos de estresse: 1/ controles; 2/ estresse agudo (isolamento e movimentos limitados); 3/ Crowding stress:crônico e 4/ estresse agudo + estresse crônico. Amostras de jejuno e cólon foram colhidas para estudar a permeabilidade em câmaras deUssing, a expressão gênica de moléculas de junção firmes e a densidade de mastócitos. Níveis de corticosterona no plasma foram medidos. Principais resultados:corticosterona plasmática foi avaliada nas três condições de estresse, teve níveis mais altos na condição de estresse combinado. Permeabilidade do jejuno foi aumentada em todas as condições de estresse e correlacionada com os níveis de corticosterona. O aumento da expressão das claudinas 1, 5 e 8, daocludina e da ZO-1 foi detectado no estado de estresse agudo no jejuno. Em contraste, a permeabilidade do cólon foi aumentada no protocolo de estresse combinado, e a expressão de moléculas das junção firmes permaneceu inalterada. O aumento da densidade de mastócitos foi observado no cólon nos ratos submetidos aos estresses crônico e combinado. Conclusão e inferências:os estresses agudo, crônico e combinado influenciam diferentemente a permeabilidade intestinal, a expressão de moléculas de junção firmes e a atividade dos mastócitos, no jejuno e no cólon. Estes resultados fornecem uma visão mais aprofundada dos mecanismos de hiperpermeabilidade intestinal relacionadas ao estresse. / Background: Psychological stress increases intestinal permeability in rodents and humans, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders through disturbances in brain-gut axis. However, the effect of acute stress on the background of Crhonic life stress, potentially better approaching the human situation, is unknown. Moreover, only limited information is available on the effects in small intestine versus colon in animal model. Methods: Wistar rats were allocated to 4 stress protocols: 1/ sham; 2/ acute stress (isolation and limited movement); 3/ Crhonic crowding stress and 4/ acute + Crhonic stress (n = 8 per group). Jejunum and colon were harvested to study permeability in Ussing chambers, gene expression of tight junction molecules and mast cell density. Plasma corticosterone levels were measured. Key Results: Plasma corticosterone was elevated in all three stress conditions, with the highest levels in the combined stress condition. Permeability of the jejunum was increased in all stress conditions and correlated with corticosterone levels. Increased expression of claudin 1, 5 and 8, occludin and ZO-1 was detected in the acute stress condition in the jejunum. In contrast, colonic permeability was increased in the acute on Crhonic stress protocol only and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the Crhonic and acute on Crhonic stress condition in the colon only. Conclusion and Inferences: Acute, Crhonic and combined stress differentially affect intestinal permeability, expression of tight junction molecules and mast cells in the jejunum and the colon. These findings provide further insight in the mechanisms of stress-related intestinal hyperpermeability and barrier.

Heat stress (HS) is one of the consequential important problems facing the swine industry. The negative effects of HS include reduced growth performance, reproductive efficiency, and carcass quality as well as increased morbidity and mortality. Although, the swine industry has developed several abatement strategies (i.e., fans, cooling pads, sprinklers, etc.), these approaches may be ineffective in the future as global temperatures continue to rise and the frequency of more severe heat waves increases in regions where animal agriculture is prevalent. These extreme heat events put pigs (especially those approaching market weight) at risk for acute hyperthermia that can lead to death unless body temperature is rapidly returned to euthermia and thermoregulatory function is restored.Therefore, evaluating mitigation strategies to promote recovery from acute hyperthermia is of utmost importance for improving pigs’ health and well-being and ensuring profitability and food security. In four experiments, the existence of microclimates in grow-finish barns during late summer was ascertained and a rapid cooling technique using cold water dousing and feed removal to promote recovery from acute hyperthermia in pigs was evaluated. In the first study, it was determined that microclimates exist in grow-finish barns and that pigs raised in pens that were not located directly below air inlets and ventilation fans had greater body temperature and reduced feed efficiency despite similarities in the in-barn ambient temperature and relative humidity. These data exemplifythe importance of adequate ventilation systems in swine barns and the impact of microclimates on pigs’ health and productivity during warm summer months. In the second study, grow-finish pigs that did not have feed access were exposed to acute HS and then rapidly or gradually cooled. Following the acute HS and recovery phase, all pigs were maintained under thermoneutral conditions and then euthanized over three days to determine the temporal effects of the cooling treatment on body temperature and intestinal integrity. The results showed that rapid cooling following acute hyperthermia in pigswas effective in returning body temperature to euthermia more rapidly compared to gradual cooling and rapid cooling prevented further intestinal damage. Based on these results, it was hypothesized that feed removal may have played a role in the effectiveness of rapid cooling. Therefore, a third experiment was conducted in which grow-finish pigs with or without access to feed were exposed to an acute HS challenge and then rapidly cooled. This study concluded that feed access was a determinant factor in the cooling outcome, as the gastrointestinal temperature returned to euthermia during the rapid cooling period more rapidly when feed was removed. Finally, a fourth study was conducted to evaluate the effects of feed removal in the absence of rapid cooling on the systemic inflammatory response and short-term growth performance of grow-finish pigs. However, it was determined that feed removal alone did not reduce the inflammatory response as expected. Overall, these studies demonstrate the risk forgrow-finish pigs during summer heat events and the potential use of rapid cooling in combination with feed removal for promoting recovery from acute hyperthermia in pigs.

No description available.

L’épithélium intestinal, siège de l’absorption des (micro)-nutriments est aussi le premier système de défense de l’organisme. Un déséquilibre dans l’homéostasie peut être à l’origine d’une réaction inflammatoire associée à des défauts de la barrière intestinale et de la fonction immunitaire, ainsi qu’une malabsorption des nutriments, comme rencontré dans les MICI (Maladies Inflammatoires Chroniques de l’Intestin), dans les stratégies de fortification en micronutriments et les pathologies non transmissibles (obésité). Il est donc important de trouver des moyens d’action, via l’alimentation par exemple, pour prévenir ou au minima réduire, les conséquences nutritionnelles et pathologiques de l’inflammation intestinale, et de comprendre les mécanismes impliqués. Parmi les modèles d’études de l’intestin, les modèles in vitro de culture cellulaire sont de plus en plus utilisés et permettent d'évaluer les mécanismes moléculaires d'une manière simple et reproductible et de réduire l'expérimentation animale.Dans ce contexte et dans le but d’étudier l’interaction de composés bioactifs de l’alimentation avec l’intestin en état d’inflammation, le premier objectif de ce travail de thèse a été la mise au point d’un modèle in vitro d’intestin enflammé associant en co-culture deux lignées intestinales humaines : les Caco-2 TC7 (entérocytes) et HT29-MTX (cellules caliciformes) et une lignée immunitaire de macrophages (THP1). Plusieurs marqueurs d’inflammation ont été évalués et nous avons pu montrer que le modèle de tri-culture répondait à un stimulus inflammatoire (LPS/IFNγ), par une augmentation de la production de cytokines pro-inflammatoires (TNF-α, IL6 et IL8) et d’enzymes (INOS et COX2) ainsi que l’expression de leurs gènes. Par ailleurs, une augmentation de la perméabilité épithéliale via une altération des jonctions serrées (TJs) a également pu être mise en évidence ainsi qu’une surproduction de mucus, lesquels sont des caractéristiques reconnus d’inflammation.Le deuxième objectif était d’étudier l’interaction de la β-cryptoxanthine (BCX), caroténoïde des agrumes, lipophile et anti-oxydant, avec le modèle enflammé. Nous avons utilisé pour solubiliser la BCX deux types de micelles (artificielles et physiologiques) et étudié les marqueurs d’inflammation. Bien qu’il semble d’après les résultats préliminaires que les micelles de BCX montrent une tendance à diminuer la production de certaines cytokines (IL6 et IL8), le rôle des constituants des micelles (Tween 40 ou sels biliaires/phospholipides) dans ce phénomène observé et dans la perméabilité épithéliale reste à clarifier par la suite. / The intestinal epithelium, main place of the absorption of (micro)-nutrients is also the first body's defense system. An imbalance in homeostasis can lead to an inflammatory reaction associated with defects in the intestinal barrier and immune function as well as malabsorption of nutrients, as seen in IBD (Inflammatory Bowel Diseases), in micronutrient fortification strategies and noncommunicable diseases (obesity). It is therefore important to find ways of action, for example through diet, to prevent or at least reduce the nutritional and pathological consequences of intestinal inflammation, and to understand the mechanisms involved. Among intestinal models, in vitro cell culture models are increasingly used and allow to evaluate the molecular mechanisms in a simple and reproducible way and to reduce animal experimentation.In this context and in order to study the interaction of dietary bioactive compounds with the intestine in state of inflammation, the first objective of this work was the development of an in vitro model of inflamed intestine combining in co-culture two human intestinal cell lines: Caco-2 TC7 (enterocytes) and HT29-MTX (goblet cells) and an immune cell line of macrophages (THP1). Several inflammation markers were evaluated and we were able to show that the tri-culture model responded to an inflammatory stimulus (LPS / IFNγ), by increasing the production of pro-inflammatory cytokines (TNF-α, IL6 and IL8) and enzymes (INOS and COX2) as well as the expression of their genes. In addition, an increase of epithelial permeability via tight junctions (TJs) alteration has also been demonstrated, as well as overproduction of mucus, which are recognized inflammation characteristics.The second objective was to study the interaction of β-cryptoxanthin (BCX), a lipophilic and antioxidant carotenoid of citrus, with the inflamed model. To solubilize BCX, we used two types of micelles (artificial and physiological) and studied markers of inflammation. Although it appears from the preliminary results that BCX micelles show a tendency to decrease the production of some cytokines (IL6 and IL8), the role of micelle constituents (Tween 40 or bile salts / phospholipids) in the phenomenon observed and in the epithelial permeability remains to be therefore clarified.

Chez les rongeurs rendus obèses par un régime hyper-lipidique, un changement du microbiote est associé à une altération de la perméabilité intestinale, augmentant le passage d’antigènes alimentaires ou bactériens et contribuant à une inflammation chronique de bas grade et une insulinorésistance. Cependant chez l’homme, les modifications de perméabilité intestinale, son impact sur les altérations métaboliques, inflammatoires systémiques et tissulaires sont peu documentées. L’objectif de ce travail est de caractériser la perméabilité intestinale (i.e. jéjunum) et les mécanismes impliqués dans sa régulation dans l’obésité humaine sévère en conditions de jeûne (basal) et après un apport aigu de lipides selon des approches complémentaires in vivo (biomarqueurs), ex vivo (chambre de Ussing, étude des protéines de jonctions serrées en immunofluorescence) et in vitro (lignée cellulaire Caco-2/TC7). A l’état basal nous avons observé une diminution de la localisation de l’occludine et de la tricelluline dans les jonctions serrées au niveau du jéjunum et des taux circulants en zonuline et LPS binding protéine plus élevés chez les obèses. La perméabilité Jéjunale basale mesurée ex vivo en chambre de Ussing était comparable entre obèses et non obèses avec cependant des liens entre ces mesures et les paramètres de l’inflammation systémique chez les patients obèses (CRP et Haptoglobine). Une charge unique en lipides alimentaires, entrainait une augmentation rapide et significative de la perméabilité aux macromolécules (FITC-Dextran 4 kDa) in vitro et ex vivo, démontrant ainsi l’effet direct des lipides postprandiaux sur la barrière épithéliale. La perméabilité aux macromolécules après exposition aux lipides était plus élevée chez les patients obèses à fortiori diabétiques de type 2 et était associée à l’inflammation systémique (CRP) et intestinale (calprotectine fécale). Ainsi, nos résultats mettent en évidence un défaut de la barrière intestinale dans l'obésité caractérisée par une hyperperméabilité jéjunale démasquée par les lipides alimentaires et associée à l’inflammation et aux troubles métaboliques. / Intestinal barrier damage is associated with low-grade inflammation and metabolic impairment in rodent models of obesity. Whether intestinal permeability is altered in human metabolic disorders remains poorly investigated. Using a large cohort of well-characterized obese subjects and a human enterocyte model, we examined intestinal permeability in the basal state and after a challenge by a lipid load. We showed a reduction of occludin and tricellulin at jejunal tight junctions and increased serum levels of zonulin and LPS-Binding Protein in obese subjects. Jejunal permeability, directly measured in Ussing chambers in the fasting condition, was not significantly increased compared to non-obese subjects. Nevertheless, within the obese cohort, high permeability was associated with systemic inflammation (CRP and haptoglobin). A single lipid load increased permeability both in Caco-2/TC7 cells and ex vivo in human jejunum, demonstrating dietary lipids’ direct effects on the epithelial barrier. Permeability after the lipid load was significantly higher in the jejunum of obese subjects and associated with systemic and intestinal inflammation (CRP and fecal calprotectin) and type 2 diabetes. Thus, our results highlight an intestinal barrier defect in obesity, with a jejunal permeability increased by a lipid challenge and linked to inflammatory and metabolic impairments.

Quattro diversi esperimenti sono stati eseguiti per investigare i fattori che influenzano la fermentazione ruminale delle diete altamente fermentescibili e per sviluppare un modello per studiare la permeabilità gastro-intestinale nei ruminanti. Nello studio 1, quattro ibridi di mais sono stati valutati per la digeribilità ruminale delle loro frazioni. I risultati suggeriscono che i genotipi e le fasi di maturità influenzano fortemente la digeribilità della sostanza secca e dell’amido nel rumine. Nello studio 2, i risultati della fermentazione in vitro di diversi zuccheri ha rivelato che il lattulosio può essere uno zucchero indicatore per studiare la permeabilità intestinale nei ruminanti, grazie alla sua bassa fermentescibilità ruminale. Nello studio 3, per la prima volta un modello di enteropatia indotta da indometacina è stato utilizzato per valutare nei ruminanti la permeabilità dell’intestino tenue attraverso il test del lattulosio. I risultati mostrano che il lattulosio è passato dall'intestino al sangue modificando alcuni parametri metabolici e dell'infiammazione. Nello studio 4, una acidosi acuta è stata indotta in pecore per testare la permeabilità gastro-intestinale con il test del lattulosio. I risultati hanno dimostrato che l'acidosi acuta compromette il funzionamento della barriera gastro-intestinale consentendo l'assorbimento e la traslocazione di LPS e altre sostanze nocive e incrementa l'infiammazione. / Four different experiments were performed for the better understanding of the factors that affect rumen fermentation of highly fermentable diets and to develop a model to study GI permeability in ruminants. In study 1, four corn hybrids recommended for corn silage were evaluated for ruminal starch digestibility of their grain fractions. Results suggest that the genotypes and maturity stages greatly influenced the DM and starch digestibility in rumen. In study 2, the results of the in vitro rumen fermentation of different naturally occurring and synthetic sugars revealed that lactulose can be a good probe sugar to study GI tract permeability in ruminants due to its low and slow fermentation rate in rumen. In study 3, for the first time an indomethacin-induced enteropathy model was used in ruminants to assess small intestinal permeability by the lactulose test. The results established that lactulose passed from the intestine to blood with perturbation of some metabolic parameters and inflammation. In study 4, acute acidosis was induced in sheep to test GI permeability during acidosis condition by lactulose test. The results demonstrated that acute acidosis impair the GI barrier function which allow absorption and translocation of LPS and other harmful substances and increase inflammation.

nÃo hà / Baixos nÃveis sangÃÃneos de drogas antituberculose podem ser causa de resistÃncia do Mycobacterium tuberculosis. Este estudo objetivou avaliar a absorÃÃo intestinal transcelular e paracelular e verificar possÃvel repercussÃo nas concentra-ÃÃes sÃricas de de rifampicina (RMP) e isoniazida (INH) em pacientes com tuberculose multirresistente (TBMR). Realizou-se estudo caso-controle no AmbulatÃrio de Tisiologia do Hospital de Messejana, em Fortaleza-CearÃ, entre agosto de 2006 e abril de 2007. TBMR foi definida como o caso de portador de bacilo resistente a pelo menos RMP+INH, de acordo com o teste de sensibilidade realizado pelo mÃtodo das proporÃÃes. Foram formados dois grupos para controle, o dos portadores de tuberculose sensÃvel (TBS) e o dos voluntÃrios sÃos (VS). Realizaram-se exames hematolÃgicos e bioquÃmicos, o teste da lactulose / manitol (L/M) (para avaliar a absorÃÃo intestinal) e coleta de dados clÃnicos e sociais de todos os voluntÃrios. Para a avaliaÃÃo das concentraÃÃes sÃricas foi coletado sangue duas e seis horas apÃs a ingestÃo observada da RMP+INH. A tÃcnica utilizada para a quantificaÃÃo da L e M na urina e dosagem sÃrica de RMP e INH foi a cromatografia lÃquida de alta pressÃo. O total de componentes dos grupos com TBMR, TBS e de sadios foi, respectivamente, 41, 33 e 41, emparelhados por gÃnero e idade. Na anÃlise univariada, encontrou-se mediana / variaÃÃo do percentual de excreÃÃo urinÃria da L e M menor no grupo com TBMR em relaÃÃo aos sadios (p<0,05). Ao se corrigir para a associaÃÃo alcoolismo + tabagismo ou Ãndice de massa corporal (IMC), desapareceu a significÃncia da menor excreÃÃo de lactulose nos portadores de TBMR. ApÃs a anÃlise multivariada, a mÃdiaÂdesvio-padrÃo (dv) do percentual de excreÃÃo urinÃria do M foi menor no grupo com TBMR em relaÃÃo ao grupo de VS (p=0,0291) e em relaÃÃo ao de TBS (p=0,0369). A relaÃÃo L/M foi semelhante entre os grupos (p=0,4747). A concentraÃÃo sÃrica mÃxima de INH (CHX) mÃdiaÂdesvio-padrÃo foi maior no grupo com TBMR (3,82Â1,18) em relaÃÃo ao VS (2,79Â1,19), p<0,01, nÃo havendo diferenÃa entre TBS e VS nem entre TBMR e TBS. ApÃs a anÃlise multivariada, a CHX aumentou no grupo VS (3,07Â0,24), mas continuou a ser maior no grupo com TBMR e, agora, com diferenÃa significante em relaÃÃo apenas à TBS. Houve CHX < 3 Âg/ml em 18,8% (6/32) dos casos e 56,7% (17/30) dos sadios (p<0,05), nÃo havendo diferenÃa entre TBS, 39,3% (11/28) e sadios. ApÃs a anÃlise multivariada, a mediaÂdp da concentraÃÃo sÃrica mÃxima de RMP (CRX) foi menor no grupo com TBMR do que nos sadios (p<0,05) e no grupo com TBS do que nos sadios (p<0,001), nÃo havendo diferenÃa entre TBMR e TBS. Houve (CRX) < 8 Âg/ml em 90,6% (29/32) dos portadores de TBMR e 66,7% (20/30) dos sadios (p<0,05) e em 82,1% (23/28) do grupo com TBS (em relaÃÃo aos sadios, p<0,05). Em conclusÃo, observou-se reduÃÃo na absorÃÃo transcelular intestinal em pacientes com TBMR versus TBS ou sadios, e os dados sugerem significante participaÃÃo do alcoolismo+tabagismo e IMC na reduÃÃo do transporte paracelular em portadores de TBMR. A CRX foi mais baixa em portadores de TBMR e TBS do que em sadios, com altas proporÃÃes de nÃveis subterapÃuticos de RMP e INH nos trÃs grupos, principalmente para CRX, mas, tambÃm preocupante para CHX. / Reduced antituberculosis drugs concentrations are associated with Mycobacterium tuberculosis resistance. This study aims to evaluate intestinal permeability and serum concentrations of rifampin (RIF) and isoniazid (INH) in patients with multidrug-resistant tuberculosis (MDR-TB). A case-control was conducted with outpatients who attended Messejanaâs Hospital in Fortaleza-Cearà from August 2006 to April 2007. MDR-TB (case) was defined as resistance to at least RIF+INH according to the susceptibility test by the proportion method. Two control groups were formed. The drug sensible TB (DS-TB) group defined so when the isolate was sensible to RIF, INHH, streptomycin and ethambutol and the healthy control group (HC). The final MDR-TB, DS-TB and health control groups composition was 41, 33 and 41 respectively, matched by sex and age. Biochemical and haematological examinatios, lactulose:mannitol (L/M) test (to access intestinal absorption) were performed as well as social and clinical interview in all volunteers. To access the serum concentrations two blood samples were collect at two and six hours after RIF and INH ingestion in 32 MDR-TB and 28 DS-TB patients and 30 HC. The drug serum concentrations and L/M test in urine were performed by HPLC. After univariate analysis the median/range of the L and M urinary excretion percentage was significantly lower in MDR-TB patients comparing to HC (p<0.05). Adjusting for alcoholism+tabagism association or Body Mass Index (BMI), this difference disappeared for lactulose. After multivariate analysis the mean  standard (sd) deviation M urinary excretion percentage was lower in MDR-TB than in HC (p=0.0291) group or DS-TB (p=0.0369) group. The L:M ratio did not differ between the groups (p=0.4747). The meanÂsd of the INH maximum serum concentration (HCmax) was higher in MDR-TB (3.82Â1.18) than in HC (2.79Â1.19) group, p<0.01 and there was no difference between DS-TB and HC nor between MDR-TB and DS-TB groups. After multivariate analysis the HCmax increased in HC (3.07Â0.24), but, remained to be higher in MDR-TB group, and now, significantly higher only than DS-TB group. There was HCmax < 3 Âg/ml in 18.8% (6/32) of the cases and 56.7% (17/30) of the HC (p<0.05) and no difference between DS-TB (39.3%, 11/28) and HC. After multivariate analysis the meanÂsd RIF maximum serum concentration (RCmax) was lower in MDR-TB than in HC(p,0.05) and in DS-TB than in HC (p<0.001), with no difference between MDR-TB and DS-TB groups. The RCmax was < 8 Âg/ml in 90.6% (29/32) of the cases and 66.7% (20/30) of HC (p<0.05) and in 82.1% (23/28) of the DS-TB patients (comparing to HC, p<0.05). In conclusion there was reduction in transcellular intestinal absorption in MDR-TB versus DS-TB or HC and the data suggest that alcoholism+tabagism association and BMI have an important role in the reduction of paracellular transport in MDR-TB patients. The RCmax was low in MDR-TB and DS-TB patients with high proportions of subtherapeutic levels in theses groups, mainly for RCmax, but also worrying for HCmax.

Inflammatory bowel disease (IBD), which includes the subtypes Crohn's disease (CD) and ulcerative colitis (UC), is a common disease particularly in the Western world. IBD is characterised by inflammation of the small intestine and/or colon. The two subtypes affect different gut locations but both show an increased intestinal permeability or the 'leaky gut syndrome'. This led to the hypothesis that tight junction (TJ) proteins expressed in the epithelium may affect the intestinal permeability as a cause or effect of IBD.Initially, variants in the CARD15, IL23R and ATG16L1 genes, previously associated with an increased risk of IBD, were genotyped in a cohort of 500 IBD (295 CD and 205 UC) patients and 877 matched controls. These variants were significantly associated in our cohort. A random effects meta-analysis was undertaken on all previously reported CD associations with the variant rs2241880 from ATG16L1 (n=25, p=0.0017, OR: 1.36 95% CI 1.12-1.66) and with rs11209026 from IL23R (n=26, p=0.0006, OR: 0.37 95% CI 0.21-0.67), showing pooled odds ratios consistent with those reported in our cohort. Individuals carrying >1 CARD15 mutant variant were found to have a 2.5 fold increased risk of CD (p=0.0001). Candidate TJ proteins were chosen on the basis of previous reported associations and through the investigation of the claudin proteins which are abundant at TJs. Twenty one candidate genes were selected and 79 variants successfully genotyped in up to 1063 IBD (502 CD and 478 UC) and 870 control patients. Significant associations were detected with variants in the CLDN1, CLDN5 and CDH1 genes with CD; CLDN5, CLDN8 and CDH1 variants were associated to IBD; and the rs7791132 variant (between CLDN4 and ELN) and a CDH1 variant were associated to UC. The CLDN1 rs6809685 variant trended towards association in a Toronto ascertained IBD replication cohort (genotypic p=0.04, allelic p=0.06) suggesting this may be a novel IBD susceptibility variant. Small intestinal biopsies from CD patients with known rs6809685 genotypes showed a dose dependent reduced immunohistochemical staining of claudin 1 with carriage of the mutant G allele. Claudin 1 helps seal TJs and reduced levels may increase risk of CD.Peroxisome proliferator activator receptors (PPARs) can directly affect TJ proteins and could therefore affect intestinal permeability. Twelve PPARγ variants were genotyped in up to 1050 IBD (502 CD and 467 UC) and 725 control patients. Significant genotypic associations were found with the rs2067819 variant in CD (p=0.05) and IBD (p=0.02), and also the rs13099634 variant in UC (P=0.02). There was a strong gender difference particularly for rs2067819 and rs4135247, where allelic associations were highly significant and increased risk of IBD in men (p=0.01 and p=0.007 respectively). However no significant associations were found in the female cohort. Troglitazone a PPARα agonist increased Caco2 cell transepithelial electrical resistance (TEER), a marker of TJ integrity, and increased expression of claudins -3 and -4. In contrast, the PPARα antagonist GW6471 reduced the TEER without causing cell death and PPARγ ligands did not affect TEER measurements. In summary, using a robust cohort of cases and controls the data indicates that variants in genes encoding TJ proteins may affect susceptibility to IBD and that PPARs can regulate these proteins altering intestinal permeability.